Patent Application
20240269298
The present disclosure relates to a compound shown in general formula (I) or a stereoisomer, a deuterated compound, a solvate, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal thereof, and an intermediate thereof and a preparation method therefor, as well as the use thereof in EGFR-related diseases such as cancer.
Epidermal growth factor receptor (EGFR) is a transmembrane protein tyrosine kinase that can act as a receptor for EGF family members to trigger the EGFR signaling pathway in human epithelial cells, thereby regulating cell proliferation, invasion, metastasis, apoptosis and angiogenesis (Nat. Rev. Cancer, 2007, 7, 169-181; Expert Opin. Ther. Targets, 2012, 16, 15-31). The overexpression, mutation or amplification of EGFR genes in the human body leads to an abnormal increase in EGFR activity, which may cause many malignant tumors such as esophageal cancer, glioblastoma, anal cancer, head and neck epithelial cancer, breast cancer, and lung cancer, especially non-small cell lung cancer (NSCLC) (Cells, 2019, 8, 350-361).
PROTAC (proteolysis targeting chimera) molecules are a class of bifunctional compounds that can simultaneously bind targeting proteins and E3 ubiquitin ligases. Such compounds can be recognized by proteasomes of cells, causing the degradation of targeting proteins, and can effectively reduce the content of targeting proteins in the cells. By introducing a ligand capable of binding to various targeting proteins into PROTAC molecules, it is possible to apply the PROTAC technology to the treatment of various diseases, and this technology has attracted extensive attention in recent years (ACS Chem. Biol. 2017, 12, 892-898; Drug Discovery Today Technol. 2019, 31, 15-27).
The development of novel PROTAC drugs that bind EGFR proteins and E3 ubiquitin ligases for the treatment of EGFR protein-related diseases will have great application prospects.
An objective of the present disclosure is to provide a compound with a novel structure, good efficacy, high bioavailability and higher safety that can inhibit and degrade EGFR, for use in the treatment of EGFR-related diseases such as cancer.
The present disclosure provides a compound or a stereoisomer, a deuterated compound, a solvate, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal thereof, wherein, the compound is selected from a compound shown in general formula (I),
B—L—K (I)
in some embodiments, Ak1, Ak2, Ak3, Ak4 and Ak5 are each independently selected from —(CH2)q—, —(CH2)q—O—, —O—(CH2)q—, —(CH2)q—NRL—, —NRL—(CH2)q—, —(CH2)q—NRLC(═O)—, —(CH2)q—C(═O)NRL—, —C(═O)—, —C(═O)—(CH2)q—NRL—, —(C≡C)q— or a bond, wherein the —CH2— is optionally further substituted with 0 to 2 (such as 0, 1 or 2) substituents selected from H, halogen, OH, CN, NH2, C1-4 alkyl, C1-4 alkoxy, halogen-substituted C1-4 alkyl, hydroxyl-substituted C1-4 alkyl or cyano-substituted C1-4 alkyl;
in some embodiments, Ak1, Ak2, Ak3, Ak4 and Ak5 are each independently selected from —O—, —OCH2—, —CH2O—, —OCH2CH2—, —CH2CH2O—, —C═C—, —C(CH3)2—, —CH2—, —CH2CH2—, —CH2CH2CH2—, —N(CH3)—, —NH—, —CH2N(CH3)—, —CH2NH—, —NHCH2—, —CH2CH2N(CH3)—, —CH2CH2NH—, —NHCH2CH2—, —C(═O)—, —C(═O)CH2NH—, —CH2C(═O)NH—, —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH— or —NHC(═O)—;
which, when substituted, is optionally further substituted with 0 to 4 (such as 0, 1, 2, 3 or 4) substituents selected from H, F, Cl, Br, I, OH, NH2, COOH, CN, ═O, C1-4 alkyl, halogen-substituted C1-4 alkyl, hydroxyl-substituted C1-4 alkyl or C1-4 alkoxy;
which, when substituted, is optionally further substituted with 0 to 4 (such as 0, 1, 2, 3 or 4) substituents selected from H, F, CF3, methyl, ═O, hydroxymethyl, COOH, CN or NH2;
wherein represents a ring selected from an aromatic ring or a non-aromatic ring;
C(CH3)2, CO, CH2, SO2,
in some embodiments, each Rk6 is independently selected from CO, CH, SO, SO2, CH2 or N;
C(CH3)2, CO, CH, N, CH2, O, S, N(CH3), N(CH2CH3), N(cyclopropyl) or NH;
C(CH3)2, CH2, O, N(CH3), N(CH2CH3), N(cyclopropyl) or NH;
C(CH3)2, CO, CH2, CH2CH2 or SO2;
is selected from
As a first embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
B—L—K (I),
As a second embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, L is selected from -Cy1-Ak1-Cy2-Ak2-Cy3-Ak3-Cy4-Ak4-Cy5-Ak5-, -Cy1-Cy2-Cy3-Cy4-Ak1-Ak2-Ak3-Ak4-Ak5-, -Cy1-Ak1-Cy2-Ak2-Cy3-Ak3-Cy4-Ak4-Ak5-, -Ak1-Cy1-Ak2-Cy2-Ak3-Cy3-Ak4-Cy4-Ak5-, -Cy1-Ak1-Cy2-Ak2-Cy3-Cy4-Ak3-Ak4-Ak5-, -Cy1-Ak1-Cy2-Ak2-Ak3-Cy3-Cy4-Ak4-Ak5-, -Cy1-Ak1-Ak2-Ak3-Ak4-Ak5-Cy2-Cy3-Cy4-, -Cy1-Cy2-Ak1-Ak2-Ak3-Ak4-Ak5-Cy3-Cy4-, -Cy1-Cy2-Cy3-Ak1-Ak2-Ak3-Ak4-Ak5-Cy4-, -Cy1-Cy2-Cy3-Cy4-Ak1-Ak2-Ak3-Ak4-Ak5-, -Cy1-Ak1-Cy2-Cy3-Cy4-Ak2-Ak3-Ak4-Ak5-, -Cy1-Cy2-Ak1-Cy3-Cy4-Ak2-Ak3-Ak4-Ak5-, -Cy1-Cy2-Cy3-Ak1-Cy4-Ak2-Ak3-Ak4-Ak5-, -Cy1-Ak1-Ak2-Cy2-Cy3-Cy4-Ak3-Ak4-Ak5-, -Cy1-Cy2-Ak1-Ak2-Cy3-Cy4-Ak3-Ak4-Ak5-, -Cy1-Cy2-Cy3-Ak1-Ak2-Cy4-Ak3-Ak4-Ak5-, -Cy1-Ak1-Ak2-Ak3-Cy2-Cy3-Cy4-Ak4-Ak5-, -Cy1-Cy2-Ak1-Ak2-Ak3-Cy3-Cy4-Ak4-Ak5-, -Cy1-Cy2-Cy3-Ak1-Ak2-Ak3-Cy4-Ak4-Ak5-, -Cy1-Ak1-Ak2-Ak3-Ak4-Cy2-Cy3-Cy4-Ak5-, -Cy1-Cy2-Ak1-Ak2-Ak3-Ak4-Cy3-Cy4-Ak5-, -Cy1-Cy2-Cy3-Ak1-Ak2-Ak3-Ak4-Cy4-Ak5-, -Ak1-Ak2-Ak3-Ak4-Ak5-Cy1-Cy2-Cy3-Cy4-, -Ak1-Cy1-Cy2-Cy3-Cy4-Ak2-Ak3-Ak4-Ak5-, -Ak1-Ak2-Cy1-Cy2-Cy3-Cy4-Ak3-Ak4-Ak5-, -Ak1-Ak2-Ak3-Cy1-Cy2-Cy3-Cy4-Ak4-Ak5-, -Ak1-Ak2-Ak3-Ak4-Cy1-Cy2-Cy3-Cy4-Ak5-, -Ak1-Cy1-Ak2-Ak3-Ak4-Ak5-Cy2-Cy3-Cy4-, -Ak1-Cy1-Cy2-Ak2-Ak3-Ak4-Ak5-Cy3-Cy4-, -Ak1-Cy1-Cy2-Cy3-Ak2-Ak3-Ak4-Ak5-Cy4-, -Ak1-Ak2-Cy1-Ak3-Ak4-Ak5-Cy2-Cy3-Cy4-, -Ak1-Ak2-Cy1-Cy2-Ak3-Ak4-Ak5-Cy3-Cy4-, -Ak1-Ak2-Cy1-Cy2-Cy3-Ak3-Ak4-Ak5-Cy4-, -Ak1-Ak2-Ak3-Cy1-Ak4-Ak5-Cy2-Cy3-Cy4-, -Ak1-Ak2-Ak3-Cy1-Cy2-Ak4-Ak5-Cy3-Cy4-, -Ak1-Ak2-Ak3-Cy1-Cy2-Cy3-Ak4-Ak5-Cy4-, -Ak1-Ak2-Ak3-Ak4-Cy1-Ak5-Cy2-Cy3-Cy4-, -Ak1-Ak2-Ak3-Ak4-Cy1-Cy2-Ak5-Cy3-Cy4-, or -Ak1-Ak2-Ak3-Ak4-Cy1-Cy2-Cy3-Ak5-Cy4-Ak1, Ak2, Ak3, Ak4 and Ak5 are each independently selected from —(CH2)q—, —(CH2)q—O—, —O—(CH2)q—, —(CH2)q—NRL—, —NRL—(CH2)q—, —(CH2)q—NRLC(═O)—, —(CH2)q—C(═O)NRL—, —C(═O)—, —C(═O)—(CH2)q—NRL—, —(C≡C)q— or a bond, wherein the —CH2— is optionally further substituted with 0 to 2 (such as 0, 1 or 2) substituents selected from H, halogen, OH, CN, NH2, C1-4 alkyl, C1-4 alkoxy, halogen-substituted C1-4 alkyl, hydroxyl-substituted C1-4 alkyl or cyano-substituted C1-4 alkyl;
As a third embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
C(CH3)2, CO, CH2, SO2,
C(CH3)2, CO, CH, N, CH2, O, S, N(CH3), N(CH2CH3), N(cyclopropyl) or NH;
C(CH3)2, CO, CH2, CH2CH2 or SO2;
As a fourth embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
which, when substituted, is optionally further substituted with 0 to 4 (such as 0, 1, 2, 3 or 4) substituents selected from H, F, Cl, Br, I, OH, NH2, COOH, CN, ═O, C1-4 alkyl, halogen-substituted C1-4 alkyl, hydroxyl-substituted C1-4 alkyl or C1-4 alkoxy;
is selected from
C(CH3)2, CH2, O, N(CH3), N(CH2CH3), N(cyclopropyl) or NH;
As a fifth embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
which, when substituted, is optionally further substituted with 0 to 4 (such as 0, 1, 2, 3 or 4) substituents selected from H, F, CF3, methyl, ═O, hydroxymethyl, COOH, CN or NH2;
As a sixth embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
As a seventh embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
As an eighth embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
As a ninth embodiment of the present disclosure, provided is the above-mentioned compound shown in general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
The present disclosure relates to a compound as described below or a stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof,
The present disclosure relates to a compound as described below or a stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein the compound is selected from a compound of example 1 to example 173.
The present disclosure relates to a compound as described below or a stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein the compound is selected from
wherein K is the same as that in Table K-2.
The present disclosure relates to a pharmaceutical composition, comprising the above-mentioned compound in the present disclosure or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, and a pharmaceutically acceptable carrier, optionally comprising one or more other chemotherapeutic agents.
The present disclosure relates to the use of the above-mentioned compound in the present disclosure or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, or the pharmaceutical composition in the preparation of a medicament for treating a disease related to EGFR activity or expression level.
The present disclosure relates to the use of the above-mentioned compound in the present disclosure or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, or the pharmaceutical composition in the preparation of a medicament for treating a disease related to the inhibition or degradation of EGFR.
The present disclosure relates to the use of the above-mentioned compound in the present disclosure or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein the disease is selected from cancer, preferably non-small cell lung cancer.
Unless stated to the contrary, the terms used in the description and claims have the following meanings.
The carbon, hydrogen, oxygen, sulfur, nitrogen or F, Cl, Br, I involved in the groups and compounds of the present disclosure all comprise their isotopes, and the carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds of the present disclosure is optionally further substituted with one or more of their corresponding isotopes, wherein the isotopes of carbon comprise 12C, 13C and 14C, the isotopes of hydrogen comprise protium (H), deuterium (D, also known as heavy hydrogen), tritium (T, also known as superheavy hydrogen), the isotopes of oxygen comprise 16O, 17O and 18O, the isotopes of sulfur comprise 32S, 33S, 34S and 36S, the isotopes of nitrogen comprise 14N and 15N, the isotopes of fluorine comprise 17F and 19F, the isotopes of chlorine comprise 35Cl and 37Cl, and the isotopes of bromine comprise 79Br and 81Br.
“Halogen” refers to F, Cl, Br or I.
“Halogen-substituted” refers to F, Cl, Br or I substitution, including but not limited to a substitution with 1 to 10 substituents selected from F, Cl, Br or I, a substitution with 1 to 6 substituents selected from F, Cl, Br or I, or a substitution with 1 to 4 substituents selected from F, Cl, Br or I. “Halogen-substituted” is referred to simply as “halo”.
“Alkyl” refers to a substituted or unsubstituted linear or branched saturated aliphatic hydrocarbyl group, including but not limited to an alkyl group of 1 to 20 carbon atoms, an alkyl group of 1 to 8 carbon atoms, an alkyl group of 1 to 6 carbon atoms, or an alkyl group of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isoamyl, neopentyl, n-hexyl and various branched isomers thereof. The definition of the “alkyl” herein is consistent with this definition. Alkyl can be monovalent, divalent, trivalent or tetravalent.
“Hydrocarbyl” refers to a substituted or unsubstituted linear or branched saturated or unsaturated group consisting of carbon and hydrogen atoms. Hydrocarbyl can be monovalent, divalent, trivalent or tetravalent.
“Heteroalkyl” refers to a substituted or unsubstituted alkyl group in which one or more (including but not limited to 2, 3, 4, 5 or 6) carbon atoms are replaced by heteroatoms (including but not limited to N, O or S). Non-limiting examples include —X(CH2)v-X(CH2)v-X(CH2)v-H (v is an integer from 1 to 5; each X is independently selected from a bond or a heteroatom, which includes but is not limited to N, O or S; at least one X is selected from a heteroatom; and N or S in the heteroatom can be oxidized to various oxidation states). Heteroalkyl can be monovalent, divalent, trivalent or tetravalent.
“Alkylene” refers to a substituted or unsubstituted linear and branched divalent saturated hydrocarbyl group, including —(CH2)v— (v is an integer from 1 to 10), and examples of alkylene include, but are not limited to, methylene, ethylene, propylene, butylene, etc.
“Heteroalkylene” refers to a substituted or unsubstituted alkylene group in which one or more (including but not limited to 2, 3, 4, 5 or 6) carbon atoms are replaced by heteroatoms (including but not limited to N, O or S). Non-limiting examples include —X(CH2)v-X(CH2)v-X(CH2)v-, wherein v is an integer from 1 to 5, each X is independently selected from a bond, N, O or S, and at least one X is selected from N, O or S.
“Cycloalkyl” refers to a substituted or unsubstituted saturated carbocyclic hydrocarbyl group, usually having from 3 to 10 carbon atoms, and non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. The “cycloalkyl” herein is as defined above. Cycloalkyl can be monovalent, divalent, trivalent or tetravalent.
“Heterocycloalkyl” refers to a substituted or unsubstituted saturated heteroatom-containing cyclic hydrocarbyl group, including but not limited to 3 to 10 atoms, 3 to 8 atoms, or 1 to 3 heteroatoms selected from N, O or S. N and S selectively substituted in the heterocycloalkyl ring can be oxidized to various oxidation states. Heterocycloalkyl can be connected to a heteroatom or a carbon atom; heterocycloalkyl can be connected to an aromatic ring or a non-aromatic ring; and heterocycloalkyl can be connected to a bridged ring or a spiro ring. Non-limiting examples include oxiranyl, azacyclopropyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydro-2H-pyranyl, dioxolanyl, dioxanyl, pyrrolidinyl, piperidinyl, imidazolidinyl, oxazolidinyl, oxazinanyl, morpholinyl, hexahydropyrimidinyl or piperazinyl. Heterocycloalkyl can be monovalent, divalent, trivalent or tetravalent.
“Alkenyl” refers to a substituted or unsubstituted linear and branched unsaturated hydrocarbyl group having at least 1, usually 1, 2 or 3 carbon-carbon double bonds, with a main chain including but not limited to 2 to 10, 2 to 6, or 2 to 4 carbon atoms. Examples of alkenyl include, but are not limited to vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 2-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 1-octenyl, 3-octenyl, 1-nonenyl, 3-nonenyl, 1-decenyl, 4-decenyl, 1,3-butadiene, 1,3-pentadiene, 1,4-pentadiene, 1,4-hexadiene, etc. The definition of the “alkenyl” herein is consistent with this definition. Alkenyl can be monovalent, divalent, trivalent or tetravalent.
“Alkynyl” refers to a substituted or unsubstituted linear and branched monovalent unsaturated hydrocarbyl group having at least 1, usually 1, 2 or 3 carbon-carbon triple bonds, with a main chain including but not limited to 2 to 10 carbon atoms, 2 to 6 carbon atoms or 2 to 4 carbon atoms. Examples of alkynyl include but are not limited to ethynyl, propargyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-1-butynyl, 2-methyl-1-butynyl, 2-methyl-3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-1-pentynyl, 2-methyl-1-pentynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 1-octynyl, 3-octynyl, 1-nonynyl, 3-nonynyl, 1-decynyl, 4-decynyl, etc. Alkynyl can be monovalent, divalent, trivalent or tetravalent.
“Alkoxy” refers to a substituted or unsubstituted —O-alkyl group. Non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexyloxy, cyclopropoxy and cyclobutoxy.
“Carbocyclyl” or “carbocycle” refers to a substituted or unsubstituted saturated or unsaturated aromatic ring or non-aromatic ring, wherein the aromatic ring or non-aromatic ring can be a 3- to 8-membered monocyclic ring, a 4- to 12-membered bicyclic ring or a 10- to 15-membered tricyclic ring system. Carbocyclyl can be connected to an aromatic ring or a non-aromatic ring, wherein the aromatic ring or non-aromatic ring is optionally a monocyclic ring, a bridged ring or a spiro ring. Non-limiting examples include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexenyl, benzene ring, naphthalene ring,
“Carbocyclyl” or “carbocycle” can be monovalent, divalent, trivalent or tetravalent.
“Heterocyclyl” or “heterocycle” refers to a substituted or unsubstituted saturated or unsaturated aromatic ring or non-aromatic ring, wherein the aromatic ring or non-aromatic ring can be a 3- to 8-membered monocyclic ring, a 4- to 12-membered bicyclic ring or a 10- to 15-membered tricyclic ring system, and contains one or more (including but not limited to 2, 3, 4 or 5) heteroatoms selected from N, O or S, and the selectively substituted N and S in the heterocyclyl ring can be oxidized to various oxidation states. Heterocyclyl can be connected to a heteroatom or a carbon atom; heterocyclyl can be connected to an aromatic ring or a non-aromatic ring; and heterocyclyl can be connected to a bridged ring or a spiro ring. Non-limiting examples include oxiranyl, azacyclopropyl, oxetanyl, azetidinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, azacycloheptyl, pyridyl, furyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidyl, morpholinyl, thiomorpholinyl, 1,3-dithianyl, dihydrofuryl, dihydropyranyl, dithiolanyl, tetrahydrofuryl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzoimidazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuryl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, pyrazinyl, indazolyl, benzothienyl, benzofuryl, benzopyrrolyl, benzoimidazolyl, benzothiazolyl, benzoxazolyl, benzopyridyl, benzopyrimidinyl, benzopyrazinyl, piperazinyl, azabicyclo[3.2.1]octanyl, azabicyclo[5.2.0]nonanyl, oxatricyclo[5.3.1.1]dodecyl, azaadamantyl, oxaspiro[3.3]heptanyl,
“Heterocyclyl” or “heterocycle” can be monovalent, divalent, trivalent or tetravalent.
“Spiro ring” or “spiro ring group” refers to a polycyclic group that shares one atom (called a spiro atom) between substituted or unsubstituted monocyclic rings. The number of ring atoms in the spiro ring system includes but is not limited to 5 to 20, 6 to 14, 6 to 12, or 6 to 10, wherein one or more rings may contain 0 or more (including but not limited to 1, 2, 3 or 4) double bonds, and can optionally contain 0 to 5 heteroatoms selected from N, O or S(═O)n.
“Spiro ring” or “spiro ring group” can be monovalent, divalent, trivalent or tetravalent.
“Fused ring” or “fused ring group” refers to a polycyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, wherein one or more rings may contain 0 or more (including but not limited to 1, 2, 3 or 4) double bonds, and may be substituted or unsubstituted, and each ring in the fused ring system may contain 0 to 5 heteroatoms or groups containing heteroatoms (including but not limited to N, S(═O)n or O, wherein n is 0, 1 or 2). The number of ring atoms in the fused ring system includes but is not limited to 5 to 20, 5 to 14, 5 to 12, or 5 to 10. Non-limiting examples include:
“Fused ring” or “fused ring group” can be monovalent, divalent, trivalent or tetravalent.
“Bridged ring” or “bridged ring group” refers to a substituted or unsubstituted polycyclic group containing any two atoms that are not directly connected, and may contain 0 or more double bonds. Any ring in the fused ring system may contain 0 to 5 groups selected from heteroatoms or groups containing heteroatoms (including but not limited to N, S(═O)n or O, wherein n is 0, 1 or 2). The number of ring atoms includes but is not limited to 5 to 20, 5 to 14, 5 to 12 or 5 to 10. Non-limiting examples include
cubane or adamantane. “Bridged ring” or “bridged ring group” can be monovalent, divalent, trivalent or tetravalent.
“Carbospiro ring”, “spiro ring carbocyclyl”, “spirocarbocyclyl” or “carbospiro ring group” refers to a “spiro ring” with a ring system consisting only of carbon atoms. The definition of the “carbospiro ring”, “spiro ring carbocyclyl”, “spirocarbocyclyl” or “carbospiro ring group” herein is consistent with that of a spiro ring.
“Carbo-fused ring”, “fused ring carbocyclyl”, “fused carbocyclyl” or “carbo-fused ring group” refers to a “fused ring” with a ring system consisting only of carbon atoms. The definition of the “carbo-fused ring”, “fused ring carbocyclyl”, “fused carbocyclyl” or “carbo-fused ring group” herein is consistent with that of a fused ring.
“Carbo-bridged ring”, “bridged ring carbocyclyl”, “bridged carbocyclyl” or “carbo-bridged ring group” refers to a “bridged ring” with a ring system consisting only of carbon atoms. The definition of the “carbo-bridged ring”, “bridged ring carbocyclyl”, “bridged carbocyclyl” or “carbo-bridged ring group” herein is consistent with that of a bridged ring.
“Mono-heterocyclic ring”, “monocyclic heterocyclyl” or “mono-heterocyclic ring group” refers to “heterocyclyl” or “heterocycle” with a monocyclic system. The definition of the “heterocyclyl”, “monocyclic heterocyclyl” or “mono-heterocyclic ring group” herein is consistent with that of heterocycle.
“Hetero-fused ring”, “hetero-fused ring group”, “fused ring heterocyclyl” or “hetero-fused ring group” means a “fused ring” containing a heteroatom. The definition of hetero-fused ring, “hetero-fused ring group”, “fused ring heterocyclyl” or “hetero-fused ring group” herein is consistent with that of a fused ring.
“Spiro-heterocyclic ring”, “spiro-heterocyclic ring group”, “spiro ring heterocyclyl” or “spiro-heterocyclic ring group” refers to a “spiro ring” containing a heteroatom. The definition of the “spiro-heterocyclic ring”, “spiro-heterocyclic ring group”, “spiro ring heterocyclyl” or “spiro-heterocyclic ring group” herein is consistent with that of a spiro ring.
“Bridged-heterocyclic ring”, “bridged-heterocyclic ring group”, “bridged ring heterocyclyl” or “bridged-heterocyclic ring group” refers to a “bridged ring” containing a heteroatom. The definition of the “bridged-heterocyclic ring”, “bridged-heterocyclic ring group”, “bridged ring heterocyclyl” or “bridged-heterocyclic ring group” herein is consistent with that of a bridged ring.
“Aryl” or “aromatic ring” refers to a substituted or unsubstituted aromatic hydrocarbyl group with a monocyclic ring or a fused ring, wherein the number of ring atoms in the aromatic ring includes but is not limited to 6 to 18, 6 to 12 or 6 to 10 carbon atoms. The aryl ring can be fused to a saturated or unsaturated carbocycle or heterocycle, wherein the ring connected to the parent structure is an aryl ring. Non-limiting examples include a benzene ring, a naphthalene ring, or
“Aryl” or “aromatic ring” can be monovalent, divalent, trivalent or tetravalent. When divalent, trivalent or tetravalent, the point of connection is on the aryl ring.
“Heteroaryl” or “heteroaromatic ring” refers to a substituted or unsubstituted aromatic hydrocarbyl group containing 1 to 5 heteroatoms or groups containing heteroatoms (including but not limited to N, O or S(═O)n, wherein n is 0, 1 or 2), wherein the number of ring atoms in the heteroaromatic ring includes but is not limited to 5-15, 5-10 or 5-6. Non-limiting examples of heteroaryl include, but are not limited to pyridyl, furyl, thienyl, pyridinyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, benzopyrazole, benzimidazolyl, benzopyridine, pyrrolopyridine, etc. The heteroaryl ring may be fused to a saturated or unsaturated carbocycle or heterocycle, wherein the ring connected to the parent structure is an heteroaryl ring. Non-limiting examples include
The definition of the “heteroaryl” herein is consistent with this definition. Heteroaryl can be monovalent, divalent, trivalent or tetravalent. When divalent, trivalent or tetravalent, the point of connection is on the heteroaryl ring.
“5-membered ring fused 5-membered heteroaromatic ring” refers to a 5 fused 5-membered fused heteroaromatic ring, wherein at least one of the two fused rings contains at least one heteroatom (including but not limited to O, S or N), and the entire group is aromatic. Non-limiting examples include a pyrrolopyrrole ring, a pyrazolopyrrole ring, a pyrazolopyrazole ring, a pyrrolofuran ring, a pyrazolofuran ring, a pyrrolothiophene ring and a pyrazolothiophene ring.
“5 fused 6-membered heteroaromatic ring” refers to a 5 fused 6-membered fused heteroaromatic ring, wherein at least one of the two fused rings contains at least one heteroatom (including but not limited to O, S or N), and the entire group is aromatic. Non-limiting examples include a benzo 5-membered heteroaryl and 6-membered heteroaromatic ring fused 5-membered heteroaromatic ring.
“Substitution” or “substituted” refers to a substitution with 1 or more (including but not limited to 2, 3, 4 or 5) substituents including but not limited to H, F, Cl, Br, I, alkyl, cycloalkyl, alkoxy, haloalkyl, mercaptan, hydroxyl, nitro, mercapto, amino, cyano, isocyano, aryl, heteroaryl, heterocyclyl, bridged ring group, spiro ring group, fused ring group, hydroxyalkyl, ═O, carbonyl, aldehyde, carboxylic acid, carboxylate, —(CH2)m—C(═O)—Ra, —O—(CH2)m—C(═O)—Ra, —(CH2)m—C(═O)—NRbRc, —(CH2)mS(═O)nRa, —(CH2)m-alkenyl —Ra, ORd or —(CH2)m-alkynyl —Ra (wherein m and n are 0, 1 or 2), arylthio, thiocarbonyl, silyl, —NRbRc, etc., wherein Rb and Rc are independently selected from H, hydroxyl, amino, carbonyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, sulfonyl, or trifluoromethylsulfonyl. Alternatively, Rb and Rc may form a five- or six-membered cycloalkyl or heterocyclyl; and Ra or Rd is independently selected from aryl, heteroaryl, alkyl, alkoxy, cycloalkyl, heterocyclyl, carbonyl, an ester group, a bridged ring group, a spiro ring group or a fused ring group.
“Containing 1 to 5 heteroatoms selected from O, S or N” means containing 1, 2, 3, 4 or 5 heteroatoms selected from O, S or N.
“Substituted with 0 to X substituents selected from . . . ” means substituted with 0, 1, 2, 3 . . . X substituents selected from . . . , wherein X is selected from any integer between 1 and 10. For example, “substituted with 0 to 4 substituents selected from . . . ” means substituted with 0, 1, 2, 3 or 4 substituents selected from . . . For example, “substituted with 0 to 5 substituents selected from . . . ” means substituted with 0, 1, 2, 3, 4 or 5 substituents selected from . . . For example, “bridged-heterocyclic ring is optionally further substituted with 0 to 4 substituents selected from H or F” means that the bridged-heterocyclic ring is optionally further substituted with 0, 1, 2, 3 or 4 substituents selected from H or F.
An X- to Y-membered ring (X is selected from an integer less than Y and greater than or equal to 3, and Y is selected from any integer between 4 and 12) includes X+1-, X+2-, X+3-, X+4-, . . . , Y-membered rings. Rings include heterocycle, carbocycle, an aromatic ring, aryl, heteroaryl, cycloalkyl, a mono-heterocyclic ring, a fused heterocyclic ring, a spiro-heterocyclic ring or a bridged-heterocyclic ring. For example, a “4- to 7-membered mono-heterocyclic ring” refers to a 4-, 5-, 6- or 7-membered mono-heterocyclic ring, and a “5- to 10-membered fused heterocyclic ring” refers to a 5-, 6-, 7-, 8-, 9- or 10-membered fused heterocyclic ring.
The term “optional” or “optionally” refers to that the events or circumstances subsequently described may but not necessarily occur, and the description includes the occasions where the events or circumstances occur or do not occur. For example, “alkyl optionally substituted with F” means that the alkyl may but not necessarily be substituted by F, and the description includes the case where the alkyl is substituted with F and the case where the alkyl is not substituted with F.
“Pharmaceutically acceptable salt” or “pharmaceutically acceptable salt thereof” refers to a salt of the compound of the present disclosure, which salt maintains the biological effectiveness and characteristics of a free acid or a free base, and is obtained by reacting the free acid with a non-toxic inorganic base or organic base, or reacting the free base with a non-toxic inorganic acid or organic acid.
“Pharmaceutical composition” refers to a mixture of one or more compounds of the present disclosure, or stereoisomers, tautomers, deuterated compounds, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or co-crystals thereof and other chemical components, wherein “other chemical components” refer to pharmaceutically acceptable carriers, excipients and/or one or more other therapeutic agents.
Pharmaceutical compositions are administered in a manner suitable for a disease to be treated (or prevented). Appropriate dosage and suitable duration and frequency of administration will be determined by factors such as patient conditions, the type and severity of the patient's disease, particular forms of active ingredients and administration methods. Optimal dosages can be determined by using experimental models and/or clinical trials.
In some embodiments, the present method involves administering about 0.1 μg to about 500 mg of at least one compound of the present disclosure/kg body weight of a subject, and more generally using a dosage of about 10 μg to about 200 mg of the compound disclosed in the present application, depending on the physiological response of the subject. For example, the dosage of the compound described in the present application for the treatment and/or prevention of diseases as described in the present application is about 0.001 to about 1 mg/kg body weight of a subject/day, such as about 0.001 mg, about 0.002 mg, about 0.005 mg, about 0.010 mg, 0.015 mg, about 0.020 mg, about 0.025 mg, about 0.050 mg, about 0.075 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.5 mg, about 0.75 mg or about 1 mg/kg body weight/day. In some embodiments, the dosage of the compound described in the present application for use in the described methods is about 1 to about 1000 mg/kg body weight of a subject being treated/day, such as about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 500 mg, about 750 mg or about 1000 mg/day.
“Carrier” refers to a material that does not cause significant irritation to an organism and does not eliminate the biological activity and characteristics of a compound administered.
“Excipient” refers to an inert substance added to a pharmaceutical composition to facilitate the administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugar, starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, adhesives and disintegrants.
“Prodrug” refers to a compound that can be converted into the compound of the present disclosure with the biological activity by metabolism in vivo. The prodrug of the present disclosure is prepared by modifying an amino or carboxyl group in the compound of the present disclosure, and the modification can be removed by conventional operations or in vivo to obtain a parent compound. When the prodrug of the present disclosure is administered to a mammalian individual, the prodrug is split to form a free amino or carboxyl group.
The term “co-crystal” refers to a crystal formed by the combination of active pharmaceutical ingredient (API) and co-crystal former (CCF) under the action of hydrogen bonds or other non-covalent bonds. The pure state of API and CCF are both solid at room temperature, and there is a fixed stoichiometric ratio between various components. The co-crystal is a multi-component crystal, which includes both a binary co-crystal formed between two neutral solids and a multi-element co-crystal formed between a neutral solid and a salt or solvate.
“Animal” is meant to include mammals, such as humans, companion animals, zoo animals, and domestic animals, preferably humans, horses, or dogs.
The term “stereoisomer” refers to an isomer produced as a result of different spatial arrangement of atoms in molecules, including cis-trans isomers, enantiomers and conformational isomers.
“Tautomer” refers to a functional group isomer produced by the rapid movement of an atom between two positions in a molecule, such as keto-enol isomerization and amide-imino alcohol isomerization.
“IC50” refers to the concentration of a medicament or inhibitor required to inhibit half of a given biological process (or a component of the process such as an enzyme, a receptor and a cell).
The technical solutions of the present disclosure will be described in detail below in conjunction with examples, but the protection scope of the present disclosure includes but is not limited thereto.
To achieve the objectives of the present disclosure, according to organic synthesis techniques known to those skilled in the art, and starting from commercially available chemicals and/or compounds described in chemical documents, the prepared compounds, “commercially available chemicals”, for use in the reactions described herein are obtained from standard commercial sources, including Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai Macklin Biochemical Co., Ltd., Sigma-Aldrich, Alfa Aesar (China) Chemical Co., Ltd., Tokyo Chemical Industry (Shanghai) Co., Ltd., Energy Chemical Co., Ltd., Shanghai Titan Scientific Co., Ltd., Kelong Chemical Co., Ltd., J&K Scientific and the like.
The structures of the compounds are determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS). The NMR shift (δ) is given in the unit of 10−6 (ppm). NMR is determined with (Bruker Avance III 400 and Bruker Avance 300) nuclear magnetic resonance instrument; the solvents for determination are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3) and deuterated methanol (CD3OD); and the internal standard is tetramethylsilane (TMS).
MS is measured with (Agilent 6120B(ESI) and Agilent 6120B(APCI));
For the column chromatography, Yantai Huanghai silica gel of 200-300 mesh silica gel is generally used as a carrier.
SEM:
Boc: tert-butoxycarbonyl; Ms:
MTBE: methyl tert-butyl ether; Bn:
DIPEA: N,N-diisopropylethylamine; DMAc: N,N-dimethylacetamide; DMSO: dimethyl sulfoxide; DCM: dichloromethane; Cbz:
NMP: N-methylpyrrolidone; DMF: N,N-dimethylformamide; TFA: trifluoroacetic acid
Unless otherwise specified, the preparative HPLC of the present disclosure involves waters 2767 (preparative liquid phase chromatographic instrument) as an instrument, and XBridge@ Prep C18 (30 mm×150 mm) as a chromatographic column.
Unless otherwise specified, in the preparative purification of the present disclosure, the solvent IPA refers to isopropanol, CAN refers to acetonitrile, and DEA refers to diethylamine.
Step 1: tert-butyl 2-((1-((benzyloxy)carbonyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (1A)
Benzyl 4-formylpiperidine-1-carboxylate (2.47 g, 10 mmol) and tert-butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate (2.54 g, 10 mmol) were mixed in dichloromethane (100 mL); acetic acid (1.2 g, 20 mmol) and sodium triacetoxyborohydride (4.24 g, 20 mmol) were successively added; and the mixture was stirred overnight at room temperature. 100 mL of dichloromethane and 50 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=10/1-1/1) to obtain 1A (4.1 g, yield: 84.5%).
Step 2: tert-butyl 2-(piperidin-4-ylmethyl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (1B)
1A (4.1 g, 0.85 mmol) was dissolved in methanol (30 mL); palladium on carbon (wt %=10%, 410 mg) was added; the mixture was subjected to hydrogen replacement 3 times, stirred overnight under hydrogen atmosphere (balloon pressure) at room temperature and filtered; and the filtrate was concentrated under reduced pressure to obtain 1B (2.96 g).
Step 3: 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C)
4-bromo-5-fluoro-2-nitrophenol (1 g, 4.24 mmol) was dissolved in 10 mL of DMF; potassium carbonate (1.76 g, 12.72 mmol) and iodomethane (1.2 g, 8.48 mmol) were added; and the mixture was reacted at 45° C. for 2 h, and cooled to room temperature. The reaction solution was diluted by adding 30 mL of water and subjected to suction filtration under reduced pressure; and the filter cake was 1C (0.95 g, yield: 90%).
Step 4: 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D)
Under nitrogen protection, 1C (500 mg, 2 mmol) and N-methylpyrazole-4-boronic acid (CAS: 847818-55-7, 450 mg, 3.57 mmol) were added to a 50 mL single-necked flask and dissolved in 10 mL of dioxane and 2 mL of water; Pd(dppf)Cl2·DCM (160 mg, 0.2 mmol) and potassium carbonate (560 mg, 4 mmol) were added; and the mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 2 h and cooled to room temperature. The reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 1D (400 mg, yield: 80%).
1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, 1H), 8.18 (s, 1H), 7.92 (s, 1H), 7.40 (d, 1H), 3.95 (s, 3H), 3.89 (s, 3H).
Step 5: tert-butyl 2-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (1E)
1B (351 mg, 1 mmol), 1D (301 mg, 1.2 mmol) and potassium carbonate (414 mg, 3 mmol) were mixed and dissolved in DMSO (10 mL), and the mixture was stirred at 120° C. for 16 h, cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 1E (407 mg, yield: 70%).
Step 6: 2-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecane (1F)
1E (407 mg, 0.7 mmol) was dissolved in DCM (10 mL); TFA (3 mL) was added at room temperature; and the mixture was stirred for 3 h and concentrated under reduced pressure. 20 mL of DCM was added, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 1F (337 mg).
Step 7: 2-(2,6-dioxopiperidin-3-yl)-5-(2-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)isoindoline-1,3-dione (1G)
1F (337 mg, 0.7 mmol) was dissolved in DMSO (5 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (276 mg, 1 mmol) and DIPEA (0.31 g, 2.4 mmol) were successively added; and the mixture was stirred at 90° C. for 3 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 1G (306 mg, yield: 59.2%).
Step 8: 5-(2-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1H)
1G (306 mg, 4.15 mmol), iron powder (200 mg, 3.57 mmol) and ammonium chloride (200 mg, 3.77 mmol) were dissolved in ethanol (30 mL) and water (10 mL), and the mixture was stirred at 80° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated to obtain 1H (250 mg, 86.9%).
Step 9: (6-((2,5-dichloropyrimidin-4-yl)amino)quinoxalin-5-yl)dimethylphosphine oxide (1I)
(6-aminoquinoxalin-5-yl)dimethylphosphine oxide (2.80 g, 12.66 mmol, see patent WO 2020147838) and 2,4,5-trichloropyrimidine (4.64 g, 25.32 mmol) were dissolved in NMP (15 mL); DIPEA (1.96 g, 5.19 mmol) was added; and under nitrogen protection, the mixture was reacted at 130° C. for 6 h, cooled to room temperature and extracted by adding 60 mL of ethyl acetate and 60 mL of water. The organic layer was washed once with 30 mL of saturated brine, dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V)=1/2-1/0) to obtain 1I (3 g, yield: 64%).
1H NMR (400 MHz, CDCl3) δ 13.28 (s, 1H), 9.22-9.16 (m, 1H), 8.82 (d, 1H), 8.76 (d, 1H), 8.37-8.22 (m, 2H), 2.15 (s, 3H), 2.12 (s, 3H).
Step 10: 5-(2-((1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 1)
1H (180 mg, 0.25 mmol) and 1I (0.12 g, 0.33 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (142 mg, 0.75 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative product, which was alkalized with saturated aqueous sodium bicarbonate solution to obtain compound 1 (53 mg, yield: 20.1%).
1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 11.04 (s, 1H), 9.03-8.91 (m, 1H), 8.86-8.73 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.71-7.44 (m, 3H), 7.33-7.25 (m, 1H), 7.20 (dd, 1H), 6.83 (s, 1H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.55-3.39 (m, 4H), 3.14-3.04 (m, 2H), 2.94-2.82 (m, 1H), 2.72-2.51 (m, 6H), 2.37-2.31 (m, 1H), 2.25-2.13 (m, 3H), 2.08-1.94 (m, 7H), 1.81-1.72 (m, 2H), 1.70-1.47 (m, 7H), 1.39-1.28 (m, 4H).
Step 1: tert-butyl 4-(1-[(benzyloxy)carbonyl]piperidin-4-yl)piperazine-1-carboxylate (2A)
Tert-butyl piperazine-1-carboxylate (20.00 g, 107.38 mmol), 1-Cbz-4-piperidone (20.05 g, 107.38 mmol) and anhydrous sodium sulfate (15.25 g, 107.38 mmol) were mixed in dichloromethane (500 mL); acetic acid (12.90 g, 214.76 mmol) and sodium triacetoxyborohydride (45.52 g, 214.76 mmol) were successively added; and the mixture was reacted at room temperature for 2 h, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=10/1-1/1) to obtain 2A (39.6 g, yield: 82%).
LCMS m/z=404.2 [M+H]+.
Step 2: benzyl 4-(piperazin-1-yl)piperidine-1-carboxylate (2B)
2A (20.00 g, 44.61 mmol) was dissolved in dichloromethane (120 mL); trifluoroacetic acid (40 mL) was slowly added; and the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, and slurried by adding methyl tert-butyl ether to obtain a white solid, which was then dissolved by adding 200 mL of dichloromethane. The resulting mixture was adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2B (10.0 g, yield: 74%), which was directly used in the next step.
LCMS m/z=304.2 [M+H]+.
Step 3: benzyl 4-(4-[(1-[(tert-butoxy)carbonyl]piperidin-4-yl)methyl]piperazin-1-yl) piperidine-1-carboxylate (2C)
2B (10.00 g, 32.96 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (7.03 g, 32.96 mmol) were dissolved in dichloromethane (120 mL); acetic acid (3.96 g, 65.92 mmol) and sodium triacetoxyborohydride (13.97 g, 65.92 mmol) were successively added; and the mixture was reacted at room temperature for 1 h, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 2C (16.14 g, yield: 98%).
LCMS m/z=501.1 [M+H]+.
Step 4: tert-butyl 4-[(4-(piperidin-4-yl)piperazin-1-yl)methyl]piperidine-1-carboxylate (2D)
2C (6.00 g, 11.98 mmol) was dissolved in a mixed solvent of isopropanol (100 mL) and ammonia methanol solution (25 mL); palladium on carbon (wt %=10%, 2.93 g) was added; and the mixture was subjected to 1 atm hydrogen replacement 3 times, reacted at room temperature for 1.5 h and filtered over celite. The filter cake was washed with dichloromethane/methanol (V/V=10/1), and the filtrate was concentrated under reduced pressure to obtain the crude of 2D (4.3 g), which was directly used in the next step.
LCMS m/z=367.3 [M+H]+.
Step 5: 1-cyclopropyl-2-fluoro-4-methoxy-5-nitrobenzene (2E)
Under nitrogen protection, 1C (3.0 g, 12.0 mmol), cyclopropyl boronic acid (2.06 g, 24.0 mmol), potassium carbonate (4.98 g, 36.0 mmol) and Pd(dppf)Cl2·DCM (880 mg, 1.2 mmol) were added to a round bottom flask; 1,4-dioxane (40 mL) and water (5 mL) were added; and the mixture was reacted at 100° C. for 3 h. The reaction solution was cooled to room temperature and subjected to suction filtration over celite. The filtrate was extracted with ethyl acetate (30 mL×3), dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (PE:EA=20:1) to obtain 2E (1.8 g, yield: 70%).
1H NMR (400 MHz, CDCl3) δ 7.55 (d, 1H), 6.75 (d, 1H), 3.93 (s, 3H), 2.07-1.92 (m, 1H), 1.07-0.92 (m, 2H), 0.76-0.64 (m, 2H).
Step 6: tert-butyl 4-[(4-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl) methyl]piperidine-1-carboxylate (2F)
2E (0.3 g, 1.42 mmol) and 2D (0.78 g, 2.13 mmol) were dissolved in DMSO (10 mL); potassium carbonate (0.59 g, 4.26 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed three times with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 2F (0.5 g, yield: 63%).
LCMS m/z=558.5 [M+H]+.
Step 7: 1-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)-4-[(piperidin-4-yl)methyl]piperazine (2G)
2F (0.5 g, 0.9 mmol) was dissolved in dichloromethane (6 mL); trifluoroacetic acid (3.06 g, 26.84 mmol) was added at room temperature; and the mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH>10 with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 2G (0.38 g).
Step 8: 5-(4-[(4-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl]piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (2H)
2G (0.38 g, 0.83 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (0.25 g, 0.91 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.66 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 20 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane methanol (V/V)=100/1-30/1) to obtain 2H (0.46 g, yield: 77%)
Step 9: 5-(4-[(4-(1-(4-amino-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl]piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione (2I)
2H (0.2 g, 0.28 mmol) was dissolved in ethanol (12 mL) and water (3 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (0.22 g, 4.05 mmol) and iron powder (0.23 g, 4.05 mmol) was added, and the resulting mixture was stirred at 80° C. for 0.5 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 21 (190 mg), which was directly used in the next step.
LCMS m/z=684.5 [M+H]+.
Step 10: 5-(4-((4-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 2)
2I (0.19 g, 0.28 mmol) and (6-((5-bromo-2-chloropyrimidin-4-yl)amino)quinoxalin-5-yl)dimethylphosphine oxide (2J) (0.12 g, 0.28 mmol, see patent WO 2020147838) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 2 (100 mg, yield: 33%).
LCMS m/z=530.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 11.05 (s, 1H), 8.92-8.72 (m, 3H), 8.34-8.19 (m, 2H), 7.87 (d, 1H), 7.68-7.58 (m, 1H), 7.36-7.15 (m, 2H), 6.87 (s, 1H), 6.74 (s, 1H), 5.06 (dd, 1H), 4.03 (d, 2H), 3.76 (s, 3H), 3.38-3.18 (m, 2H), 3.02-2.82 (m, 3H), 2.70 (t, 2H), 2.64-2.51 (m, 6H), 2.44-2.25 (m, 5H), 2.18-2.06 (m, 3H), 2.06-1.95 (m, 7H), 1.93-1.74 (m, 5H), 1.69-1.55 (m, 2H), 1.21-1.06 (m, 2H), 0.78-0.66 (m, 2H), 0.47-0.31 (m, 2H).
Step 1: tert-butyl 9-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate(3A)
2E (0.3 g, 1.42 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (0.43 g, 1.69 mmol) were dissolved in DMSO (10 mL); potassium carbonate (0.59 g, 4.26 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (ethyl acetate: petroleum ether (V/V)=10/1-2/1) to obtain 3A (0.6 g, yield: 95%).
LCMS m/z=446.3 [M+H]+.
Step 2: 3-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane (3B)
3A (0.6 g, 1.35 mmol) was dissolved in dichloromethane (6 mL); trifluoroacetic acid (3.06 g, 26.84 mmol) was added at room temperature; and the mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH>10 with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 3B (0.4 g).
Step 3: tert-butyl 4-((9-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidine-1-carboxylate (3C)
3B (0.4 g, 1.16 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (0.3 g, 1.39 mmol) were dissolved in dichloromethane (10 mL); acetic acid (0.14 g, 2.32 mmol) and sodium triacetoxyborohydride (0.49 g, 2.32 mmol) were successively added; and the mixture was reacted at room temperature for 1 h, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 3C (0.52 g, yield: 82%).
LCMS m/z=543.3 [M+H]+.
Step 4: 3-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-9-(piperidin-4-ylmethyl)-3,9-diazaspiro[5.5]undecane (3D)
3C (0.52 g, 0.96 mmol) was dissolved in dichloromethane (6 mL); trifluoroacetic acid (3.06 g, 26.84 mmol) was added at room temperature; and the mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH>10 with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 3D (0.4 g).
Step 5: 5-(4-((9-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-3,9-diazaspiro [5.5]undecan-3-yl)methyl) piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (3E)
3D (0.4 g, 0.90 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (0.25 g, 0.91 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.66 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 20 mL of water was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-30/1) to obtain 3E (0.45 g, yield: 71%).
LCMS m/z=699.3 [M+H]+.
Step 6: 5-(4-((9-(4-amino-2-cyclopropyl-5-methoxyphenyl)-3,9-diazaspiro [5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (3F)
3E (0.13 g, 0.19 mmol) was dissolved in ethanol (10 mL) and water (2 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (100 mg, 1.90 mmol) and iron powder (106 mg, 1.90 mmol) was added. The resulting mixture was stirred at 80° C. for 1 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 3F (120 mg), which was directly used in the next step.
LCMS m/z=669.3 [M+H]+.
Step 7: 5-(4-((9-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 3)
3F (0.12 g, 0.18 mmol) and 2J (74 mg, 0.18 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (102 mg, 0.54 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain the crude (0.10 g), which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 3 (58 mg, yield: 31%).
LCMS m/z=522.9 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 11.05 (s, 1H), 8.89-8.71 (m, 3H), 8.32-8.17 (m, 2H), 7.88 (d, 1H), 7.65 (d, 1H), 7.37-7.16 (m, 2H), 6.90-6.74 (m, 2H), 5.06 (dd, 1H), 4.12-3.97 (m, 2H), 3.77 (s, 3H), 3.04-2.81 (m, 7H), 2.68-2.52 (m, 2H), 2.44-2.30 (m, 4H), 2.21-2.06 (m, 3H), 2.06-1.97 (m, 7H), 1.89-1.73 (m, 3H), 1.69-1.46 (m, 8H), 1.21-1.07 (m, 2H), 0.80-0.60 (m, 2H), 0.51-0.27 (m, 2H).
Step 1: tert-butyl 4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidine-1-carboxylate (4A)
1D (0.30 g, 1.19 mmol) and 2D (0.44 g, 1.19 mmol) were dissolved in DMSO (15 mL); potassium carbonate (0.33 g, 2.38 mmol) was added; and the mixture was reacted at 120° C. for 4 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase dichloromethane/methanol (V/V)=50/1-15/1) to obtain 4A (0.28 g, yield: 40%).
LCMS m/z=598.5 [M+H]+.
Step 2: 1-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)-4-(piperidin-4-ylmethyl)piperazine (4B)
4A (0.30 g, 0.50 mmol) was dissolved in dichloromethane (12 mL); trifluoroacetic acid (4 mL) was slowly added; and the mixture was reacted at room temperature for 1.5 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, redissolved by adding 50 mL of dichloromethane, and adjusted to a basic pH with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 4B (0.24 g), which was directly used in the next step.
LCMS m/z=498.4 [M+H]+.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (4C)
4B (0.22 g, 0.45 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (0.12 g, 0.45 mmol) were dissolved in DMSO (10 mL); DIPEA (0.17 g, 1.35 mmol) was added dropwise; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 4C (0.13 g, yield: 34%).
LCMS m/z=754.4 [M+H]+.
Step 4: 5-(4-((4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4D)
4C (0.12 g, 0.15 mmol) was dissolved in ethanol (9 mL); reduced iron powder (0.04 g, 0.75 mmol) was added, and then an aqueous solution (3 mL) of ammonium chloride (0.04 g, 0.75 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 3 h. The reaction solution was cooled to room temperature. 5 mL of water was added, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 4D (0.11 g).
LCMS m/z=724.5 [M+H]+.
Step 5: 5-(4-((4-(1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 4)
4D (0.11 g, 0.16 mmol) and 1I (0.06 g, 0.16 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid hydrate (0.09 g, 0.48 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain the crude, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 4 (36 mg, yield: 21%).
LCMS m/z=528.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.05 (s, 1H), 9.02-8.93 (m, 1H), 8.88-8.78 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.79 (s, 1H), 7.65 (d, 1H), 7.61-7.50 (m, 2H), 7.33-7.28 (m, 1H), 7.23 (dd, 1H), 6.84 (s, 1H), 5.06 (dd, 1H), 4.03 (d, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.17-3.10 (m, 2H), 3.01-2.93 (m, 2H), 2.90-2.83 (m, 1H), 2.68-2.51 (m, 8H), 2.44-2.34 (m, 4H), 2.27-2.21 (m, 1H), 2.17-2.10 (m, 2H), 2.08-1.95 (m, 7H), 1.88-1.77 (m, 5H), 1.62-1.53 (m, 2H), 1.18-1.08 (m, 2H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1)
4,5-difluorophthalic acid (500 mg, 2.47 mmol) was dissolved in 25 mL of acetonitrile; 3-aminopiperidine-2,6-dione hydrochloride (0.41 g, 2.49 mmol) and N,N′-carbonyldiimidazole (0.8 g, 4.94 mmol) were added; and the reaction was refluxed for 4 h. The reaction system was concentrated under reduced pressure, and then the residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=2:1) to obtain 5A-1 (0.4 g, yield: 55.0%).
LCMS m/z=295.2 [M+H]+.
Step 2: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (5A)
4B (0.16 g, 0.54 mmol) and 5A-1 (0.27 g, 0.54 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.62 mmol) was added dropwise; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase dichloromethane/methanol (V/V)=50/1-12/1) to obtain 5A as a yellow solid (0.22 g, yield: 53%).
LCMS m/z=772.4 [M+H]+.
Step 3: 5-(4-((4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (5B)
5A (0.21 g, 0.27 mmol) was dissolved in ethanol (15 mL); reduced iron powder (0.07 g, 1.35 mmol) was added, and then an aqueous solution (5 mL) of ammonium chloride (0.07 g, 0.75 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 3 h, and cooled to room temperature. 5 mL of water was added, and then the mixture was extracted 3 times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 5B (0.19 g).
LCMS m/z=742.3 [M+H]+.
Step 4: 5-(4-((4-(1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 5)
5B (0.21 g, 0.28 mmol) and 1I (0.10 g, 0.28 mmol) were dissolved in DMF (15 mL); p-toluenesulfonic acid hydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain the crude, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 5 (50 mg, yield: 17%).
LCMS m/z=537.3 [(M+2H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.08 (s, 1H), 9.02-8.93 (m, 1H), 8.86-8.80 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.79 (s, 1H), 7.69 (d, 1H), 7.62-7.51 (m, 2H), 7.44 (d, 1H), 6.84 (s, 1H), 5.10 (dd, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.61 (d, 2H), 3.14 (d, 2H), 2.94-2.83 (m, 3H), 2.70-2.52 (m, 8H), 2.47-2.34 (m, 4H), 2.30-2.24 (m, 1H), 2.22-2.13 (m, 2H), 2.07-1.98 (m, 7H), 1.89-1.79 (m, 4H), 1.77-1.69 (m, 1H), 1.63-1.53 (m, 2H), 1.33-1.19 (m, 2H).
Step 1: benzyl 4-(4-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)methyl)piperazin-1-yl)piperidine-1-carboxylate (6A)
2B (0.65 g, 2.14 mmol) and 1-Boc-3-pyrrolidinecarbaldehyde (0.51 g, 2.57 mmol) were dissolved in dichloromethane (10 mL); acetic acid (0.26 g, 4.28 mmol) and sodium triacetoxyborohydride (0.91 g, 4.28 mmol) were successively added; and the mixture was reacted at room temperature for 1 h, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution, extracted by adding 20 mL of dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 6A (1 g, yield: 96%).
Step 2: tert-butyl 3-((4-(piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidine-1-carboxylate (6B)
6A (1.00 g, 2.05 mmol) was dissolved in a mixed solvent of isopropanol (10 mL) and 7 N ammonia methanol solution (2.5 mL); palladium on carbon (wt %=10%, 0.4 g) was added; and the mixture was subjected to 1 atm hydrogen replacement 3 times, reacted at room temperature for 2 h and filtered over celite. The filter cake was washed with dichloromethane/methanol (V/V=10/1), and the filtrate was concentrated under reduced pressure to obtain 6B (0.73 g).
LCMS m/z=353.3 [M+H]+.
Step 3: tert-butyl 3-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidine-1-carboxylate (6C)
6B (0.73 g, 2.08 mmol), 1D (0.35 g, 1.39 mmol) and potassium carbonate (0.58 g, 4.17 mmol) were mixed and dissolved in DMSO (10 mL), and the mixture was stirred at 120° C. for 6 h, cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase dichloromethane/methanol (V/V)=100/1-20/1) to obtain 6C (530 mg, yield: 65%).
LCMS m/z=584.3 [M+H]+.
Step 4: 1-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)-4-(pyrrolidin-3-ylmethyl)piperazine (6D)
6C (0.53 g, 0.91 mmol) was dissolved in dichloromethane (6 mL); trifluoroacetic acid (3.06 g, 26.84 mmol) was added at room temperature; and the mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH>10 with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 6D (0.4 g), which was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(3-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidin-1-yl)isoindoline-1,3-dione (6E)
6D (0.4 g, 0.83 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (0.25 g, 0.91 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.66 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 20 mL of water was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 6E (0.47 g, yield: 76%).
Step 6: 5-(3-((4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (6F)
6E (0.2 g, 0.27 mmol) was dissolved in ethanol (12 mL) and water (3 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (0.22 g, 4.05 mmol) and iron powder (0.23 g, 4.05 mmol) was added. At this temperature, the resulting mixture was stirred for 1 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 6F (190 mg), which was directly used in the next step.
LCMS m/z=710.3 [M+H]+.
Step 7: 5-(3-((4-(1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 6)
6F (0.19 g, 0.27 mmol) and 1I (99 mg, 0.27 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.15 g, 0.81 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 6 (60 mg, yield: 21%).
LCMS m/z=521.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.05 (s, 1H), 9.06-8.77 (m, 3H), 8.37 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.79 (s, 1H), 7.70-7.47 (m, 3H), 6.94-6.74 (m, 3H), 5.05 (dd, 1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.59-3.36 (m, 3H), 3.18-3.06 (m, 3H), 2.95-2.81 (m, 1H), 2.72-2.52 (m, 9H), 2.49-2.29 (m, 6H), 2.28-2.19 (m, 1H), 2.17-2.07 (m, 1H), 2.07-1.93 (m, 7H), 1.91-1.68 (m, 3H), 1.66-1.49 (m, 2H).
Step 1: 7-azaspiro[3.5]nonan-2-one (7B)
7A (13.0 g, 54.32 mmol) was dissolved in dichloromethane (100 mL); trifluoroacetic acid (61.94 g, 543.2 mmol) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. To the residue were added 50 mL of dichloromethane and a sodium bicarbonate solid (70 g). The mixture was subjected to suction filtration, and the filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 7B (7.50 g, yield: 99%).
LCMS m/z=140.1 [M+H]+.
Step 2: 2-(2,6-dioxopiperidin-3-yl)-5-(2-oxo-7-azaspiro[3.5]nonan-7-yl) isoindoline-1,3-dione (7C)
7B (7.50 g, 53.88 mmol) was dissolved in dimethyl sulfoxide (100 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (22.32 g, 80.81 mmol) and DIPEA (13.93 g, 107.78 mmol) were successively added; and the mixture was stirred at 100° C. for 3 h, and cooled to room temperature. 100 mL of water was added, and the mixture was stirred for 5-10 min and subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 7C as a yellow solid (11.40 g, yield: 54%).
LCMS m/z=521.3 [M+H]+.
Step 3: tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylate (7D-1)
To a 100 mL reaction flask were successively added 2A (4.91 g, 12.16 mmol), isopropanol/ammonia methanol solution (v/v=4/1, 60 mL) and palladium on carbon (2.5 g); and the mixture was subjected to hydrogen replacement three times, reacted at room temperature for 2 h and subjected to suction filtration over celite. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=25/1-10/1) to obtain 7D-1 (1.26 g, yield: 38%).
LCMS m/z=270.1 [M+H]+.
Step 4: tert-butyl 4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)piperazine-1-carboxylate (7D)
7D-1 (1.23 g, 4.57 mmol), 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (0.96 g, 3.80 mmol) and potassium carbonate (1.58 g, 11.41 mmol) were mixed and dissolved in DMSO (20 mL), and the mixture was stirred overnight at 120° C., cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 7D (970 mg, yield: 62%).
LCMS m/z=501.5 [M+H]+.
Step 5: 1-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazine (7E)
7D (0.31 g, 0.62 mmol) was dissolved in dichloromethane (10 mL); trifluoroacetic acid (0.71 g, 6.2 mmol) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. To the residue were added 20 mL of dichloromethane and saturated aqueous sodium bicarbonate solution. The mixture was adjusted to pH=8-9 and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 7E (0.24 g, yield: 97%).
LCMS m/z=501.5 [M+H]+.
Step 6: 2-(2,6-dioxopiperidin-3-yl)-5-(2-(4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)isoindoline-1,3-dione (7F)
7E (0.24 g, 0.60 mmol) and 7C (0.71 g, 1.80 mmol) were dissolved in 1,2-dichloroethane (10 mL), and a 4A molecular sieve (200 mg) and acetic acid (0.05 g, 0.90 mmol) were successively added. The mixture was stirred at room temperature for 2 h; sodium triacetoxyborohydride (0.38 g, 1.79 mol) was added; and the resulting mixture was reacted overnight at room temperature. After the reaction was completed, the reaction system was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 7F (0.23 g, yield: 49%).
LCMS m/z=780.4 [M+H]+.
Step 7: 5-(2-(4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (7G)
7F (0.23 g, 0.29 mmol) was dissolved in ethanol (5 mL) and water (1 mL); an ammonium chloride solid (0.16 g, 2.99 mmol) and iron powder (0.16 g, 2.86 mmol) were added; and the mixture was subjected to N2 replacement 3 times and stirred at 80° C. for 1 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane and concentrated under reduced pressure. The resultant obtained from the concentration under reduced pressure was dissolved in dichloromethane and extracted. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 7G (0.18 g, yield: 83%).
LCMS m/z=750.4 [M+H]+.
Step 8: 5-(2-(4-(1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 7); 2,2,2-trifluoroacetic acid
7G (0.18 g, 0.24 mmol) and 1I (0.09 g, 0.24 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.14 g, 0.74 mol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 7 (40 mg).
LCMS m/z=1081.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 11.05 (s, 1H), 9.02-8.78 (m, 3H), 8.52 (s, 1H), 8.22 (s, 1H), 8.00 (s, 1H), 7.82 (s, 1H), 7.69-7.48 (m, 3H), 7.34 (d, 1H), 7.26 (dd, 1H), 6.82 (s, 1H), 5.06 (dd, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.72-3.37 (m, 9H), 3.28-3.11 (m, 5H), 2.95-2.81 (m, 2H), 2.74-2.52 (m, 5H), 2.26-2.16 (m, 2H), 2.14-1.90 (m, 11H), 1.87-1.57 (m, 6H).
Step 1: tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl) piperazine-1-carboxylate (8B)
8A (5.0 g, 26.84 mmol) was dissolved in dimethyl sulfoxide (50 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (11.12 g, 40.26 mmol) and DIPEA (6.94 g, 53.69 mmol) were successively added; and the mixture was stirred at 100° C. for 3 h, and cooled to room temperature. 50 mL of water was added, and the mixture was stirred for 5-10 min and subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase:dichloromethane/methanol (V/V)=100/1-10/1) to obtain 8B (8.08 g, yield: 68%).
LCMS m/z=443.2 [M+H]+.
Step 2: 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (8C)
8B (3 g, 6.78 mmol) was dissolved in dichloromethane (30 mL); trifluoroacetic acid (7.73 g, 67.8 mmol) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. To the residue were added 20 mL of dichloromethane and a sodium bicarbonate solid (20 g); and the mixture was subjected to suction filtration. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 8C (2.09 g), which was directly used in the next step.
LCMS m/z=343.1 [M+H]+.
Step 3: tert-butyl 4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl) piperazin-1-yl)methyl)piperidine-1-carboxylate (8D)
8C (2.09 g, 6.10 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (3.91 g, 18.33 mmol) were dissolved in DMAc (40 mL); and a 4A molecular sieve (300 mg) and acetic acid (0.55 g, 9.16 mmol) were successively added. The mixture was stirred at room temperature for 2 h; sodium triacetoxyborohydride (3.88 g, 18.31 mmol) was added; and the resulting mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was extracted with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase dichloromethane/methanol (V/V)=100/1-10/1) to obtain 8D (1.53 g, yield: 46%).
LCMS m/z=540.3 [M+H]+.
Step 4: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(piperidin-4-ylmethyl)piperazin-1-yl) isoindoline-1,3-dione (8E)
8D (1.53 g, 2.84 mmol) was dissolved in dichloromethane (15 mL); trifluoroacetic acid (3.23 g, 28.33 mmol) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. To the residue were added 20 mL of dichloromethane and a sodium bicarbonate solid (15 g); and the mixture was subjected to suction filtration. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 8E (1.13 g), which was directly used in the next step.
LCMS m/z=440.2 [M+H]+.
Step 5: tert-butyl 4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl) piperazin-1-yl)methyl)-[1,4′-bipiperidine]-1′-carboxylate (8F)
8E (1.13 g, 2.57 mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (1.54 g, 7.73 mmol) were dissolved in DMAc (20 mL); and a 4A molecular sieve (200 mg) and acetic acid (0.23 g, 3.83 mmol) were successively added. The mixture was stirred at room temperature for 2 h; sodium triacetoxyborohydride (1.63 g, 7.69 mmol) was added; and the resulting mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was extracted with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase dichloromethane/methanol (V/V)=100/1-10/1) to obtain 8F (1.27 g, yield: 79%).
LCMS m/z=623.2 [M+H]+.
Step 6: 5-(4-([1,4′-bipiperidin]-4-ylmethyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (8G)
8F (1.27 g, 2.04 mmol) was dissolved in dichloromethane (15 mL); trifluoroacetic acid (2.33 g, 20.43 mmol) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. To the residue were added 20 mL of dichloromethane and a sodium bicarbonate solid (20 g); and the mixture was subjected to suction filtration. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 8G (0.98 g), which was directly used in the next step.
LCMS m/z=523.3 [M+H]+.
Step 7: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-dione (8H)
8G (980 mg, 1.87 mmol) and 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (0.31 g, 1.23 mmol) were dissolved in DMSO (10 mL); a sodium bicarbonate solid (0.32 g, 3.81 mmol) was added; and the mixture was reacted overnight at 100° C. After the reaction was completed, the mixture was cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-10/1) to obtain 8H (0.24 g, yield: 17%).
LCMS m/z=754.4 [M+H]+.
Step 8: 5-(4-((1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (8I)
8H (0.24 g, 0.32 mmol) was dissolved in ethanol (5 mL) and water (1 mL); an ammonium chloride solid (0.17 g, 3.18 mmol) and iron powder (0.18 g, 3.22 mmol) were added; and the mixture was subjected to N2 replacement 3 times and stirred at 80° C. for 1 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane and concentrated under reduced pressure. The resultant obtained from the concentration under reduced pressure was dissolved in dichloromethane and extracted. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 81 (0.12 g, yield: 52%).
LCMS m/z=724.4 [M+H]+.
Step 9: 5-(4-((1′-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 8)
8I (0.12 g, 0.17 mmol) and 1I (0.06 g, 0.16 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.095 g, 0.50 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 8 (60 mg, yield: 35%).
LCMS m/z=1055.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.06 (s, 1H), 9.06-8.77 (m, 3H), 8.37 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.80 (s, 1H), 7.67 (d, 1H), 7.62-7.49 (m, 2H), 7.33 (d, 1H), 7.24 (dd, 1H), 6.83 (s, 1H), 5.07 (dd, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.48-3.38 (m, 4H), 3.18-3.09 (m, 2H), 2.98-2.82 (m, 3H), 2.69-2.52 (m, 4H), 2.48-2.43 (m, 3H), 2.39-2.27 (m, 1H), 2.22-2.11 (m, 4H), 2.07-1.95 (m, 7H), 1.85-1.41 (m, 8H), 1.19-1.05 (m, 2H).
Step 1: tert-butyl 9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (9A)
1D (0.4 g, 1.59 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (0.61 g, 2.40 mmol) were dissolved in DMSO (10 mL); potassium carbonate (0.88 g, 6.37 mmol) was added; and the mixture was reacted at 130° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed three times with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 9A (0.35 g, yield: 45%).
LCMS m/z=486.2 [M+H]+.
Step 2: 3-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane (9B)
9A (350 mg, 0.72 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (2 N, 10 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 M aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 9B (264 mg), which was directly used in the next step.
Step 3: tert-butyl 4-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro [5.5]undecan-3-yl)methyl)piperidine-1-carboxylate (9C)
9B (264 mg, 0.68 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (217.5 mg, 1.02 mmol) were dissolved in DMAc (10 mL), and acetic acid (40.8 mg, 0.68 mmol) was added. The mixture was stirred at room temperature for 30 min, and then sodium triacetoxyborohydride (215.3 mg, 1.02 mmol) was added; and the resulting mixture was reacted overnight at room temperature, and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase DCM/MeOH (V/V)=100/1-20/1) to obtain 9C (260 mg, yield: 66%).
LCMS m/z=583.3 [M+H]+.
Step 4: 3-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-9-(piperidin-4-ylmethyl)-3,9-diazaspiro[5.5]undecane (9D); HCl
9C (260 mg, 0.45 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (2 N, 6 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (9E)
The crude hydrochloride of 9D from the previous step was dissolved in DMSO (8 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (187.8 mg, 0.68 mmol) and DIPEA (232.7 mg, 1.8 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 9E (174 mg, yield: 52%).
LCMS m/z=739.3 [M+H]+.
Step 6: 5-(4-((9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9F)
9E (174 mg, 0.24 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (70 mg, 1.25 mmol) and ammonium chloride (66.9 mg, 1.25 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 9F (150 mg, yield: 88%).
LCMS m/z=709.4 [M+H]+.
Step 7: 5-(4-((9-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 9)
9F (150 mg, 0.21 mmol) and 1I (117.8 mg, 0.32 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (119.8 mg, 0.63 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain compound 9 (60 mg, yield: 27%).
LCMS m/z=1040.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 11.05 (s, 1H), 9.01-8.89 (m, 1H), 8.87-8.76 (m, 2H), 8.38 (s, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.65 (d, 1H), 7.61-7.48 (m, 2H), 7.30 (s, 1H), 7.27-7.18 (m, 1H), 6.89 (s, 1H), 5.06 (dd, 1H), 4.10-3.97 (m, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.04-2.77 (m, 7H), 2.70-2.53 (m, 2H), 2.42-2.28 (m, 4H), 2.24-2.09 (m, 2H), 2.06-1.95 (m, 7H), 1.90-1.72 (m, 3H), 1.66-1.46 (m, 8H), 1.21-1.09 (m, 2H).
Step 1: tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (10B)
10A (2.0 g, 7.86 mmol) was dissolved in DMSO (15 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (2.5 g, 9.05 mmol) and DIPEA (2.77 g, 21.43 mmol) were successively added; and the mixture was stirred at 90° C. for 3 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 10B as a yellow solid (3.0 g, yield: 75%).
LCMS m/z=511.3 [M+H]+.
Step 2: 2-(2,6-dioxopiperidin-3-yl)-5-(2,9-diazaspiro[5.5]undecan-2-yl) isoindoline-1,3-dione (10C); 2,2,2-trifluoroacetic acid
10B (3.0 g, 5.88 mmol) was dissolved in dichloromethane (60 mL); trifluoroacetic acid (4.6 g, 40 mmol) was added; and the mixture was stirred at room temperature for 3 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: tert-butyl 4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro[5.5]undecan-9-yl)methyl)piperidine-1-carboxylate (10D)
The trifluoroacetate of 10C (0.6 g) and tert-butyl 4-formylpiperidine-1-carboxylate (0.4 g, 1.87 mol) were mixed in DMAc (20 mL); acetic acid (0.12 g, 2 mmol) and sodium triacetoxyborohydride (0.42 g, 2 mmol) were successively added; and the mixture was stirred overnight at room temperature. 100 mL of dichloromethane and 50 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=10/1-1/1) to obtain 10D (0.44 g, yield: 50%).
LCMS m/z=750.4 [M+H]+.
Step 4: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(piperidin-4-ylmethyl)-2,9-diazaspiro [5.5]undecan-2-yl)isoindoline-1,3-dione (10E); 2,2,2-trifluoroacetic acid
Compound 10D (0.44 g, 0.72 mmol) was dissolved in dichloromethane (10 mL); trifluoroacetic acid (2.3 g, 20 mmol) was added; and the mixture was stirred at room temperature for 3 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (10F)
The crude trifluoroacetate of 10E from the previous step was dissolved in DMSO (15 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (260 mg, 1.03 mmol) and sodium bicarbonate (250 mg, 3.0 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase dichloromethane/methanol (V/V)=100/1-20/1) to obtain 10F (230 mg, yield: 43%).
LCMS m/z=739.3 [M+H]+.
Step 6: 5-(9-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10G)
10F (230 mg, 0.31 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (53 mg, 0.95 mmol) and ammonium chloride (50 mg, 0.93 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 10G as a yellow solid (150 mg, yield: 68%).
LCMS m/z=709.3 [M+H]+.
Step 7: 5-(9-((1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 10)
10G (150 mg, 0.21 mmol) and 1I (95.7 mg, 0.26 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (97 mg, 0.51 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain compound 10 (30 mg, yield: 14%).
LCMS m/z=1040.6 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.05 (s, 1H), 9.06-8.89 (m, 1H), 8.87-8.76 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.79 (s, 1H), 7.67-7.46 (m, 3H), 7.29 (d, 1H), 7.23 (dd, 1H), 6.83 (s, 1H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.51-3.41 (m, 2H), 3.36 (s, 2H), 3.14-3.01 (m, 2H), 2.95-2.80 (m, 1H), 2.69-2.53 (m, 4H), 2.45-2.20 (m, 6H), 2.08-1.93 (m, 7H), 1.81-1.70 (m, 2H), 1.69-1.45 (m, 7H), 1.45-1.20 (m, 4H).
Step 1. tert-butyl 4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro[5.5]undecan-9-yl)piperidine-1-carboxylate (11A)
The trifluoroacetate of 10C (0.25 g) and N-tert-butoxycarbonyl-4-piperidone (183 mg, 0.92 mmol) were dissolved in DMAc (10 mL), and acetic acid (39 mg, 0.65 mmol) was added. The mixture was stirred at room temperature for 30 min, and then sodium triacetoxyborohydride (192 mg, 0.91 mmol) was added; and the resulting mixture was reacted overnight at room temperature, and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 11A (250 mg, yield: 69%).
LCMS m/z=594.3 [M+H]+.
Step 2: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(piperidin-4-yl)-2,9-diazaspiro[5.5] undecan-2-yl)isoindoline-1,3-dione (11B); HCl
11A (250 mg, 0.42 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (2 N, 6 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (11C)
The crude hydrochloride of 11B from the previous step was dissolved in DMSO (5 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (158 mg, 0.63 mmol) and sodium bicarbonate (353 mg, 4.2 mmol) were successively added; and the mixture was reacted at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase dichloromethane/methanol (V/V)=100/1-20/1) to obtain 11C (140 mg, yield: 46%).
LCMS m/z=725.3 [M+H]+.
Step 4: 5-(9-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11D)
11C (140 mg, 0.19 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (53 mg, 0.95 mmol) and ammonium chloride (51 mg, 0.95 mmol) were successively added; and under nitrogen protection, the mixture was reacted at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 11D (120 mg, yield: 91%).
LCMS m/z=695.3 [M+H]+.
Step 5: 5-(9-(1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 11)
11D (120 mg, 0.17 mmol) and (6-((2,5-dichloropyrimidin-4-yl)amino)quinoxalin-5-yl)dimethylphosphine oxide (1I) (96 mg, 0.26 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (97 mg, 0.51 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain compound 11 (40 mg, yield: 23%).
LCMS m/z=1026.5 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 11.05 (s, 1H), 9.04-8.89 (m, 1H), 8.88-8.76 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.79 (s, 1H), 7.65-7.49 (m, 3H), 7.34-7.27 (m, 1H), 7.24 (dd, 1H), 6.83 (s, 1H), 5.05 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.51-3.42 (m, 2H), 3.39-3.34 (m, 2H), 3.19-3.09 (m, 2H), 2.95-2.82 (m, 1H), 2.69-2.53 (m, 6H), 2.38-2.26 (m, 1H), 2.08-1.94 (m, 8H), 1.89-1.78 (m, 2H), 1.70-1.34 (m, 11H).
Step 1: benzyl 4-(1-(tert-butoxycarbonyl)piperidin-4-yl)piperazine-1-carboxylate (12B)
12A (6.6 g, 30 mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (6.0 g, 30 mmol) were mixed in DMAc (220 mL); acetic acid (1.2 g, 20 mmol) and sodium triacetoxyborohydride (8.4 g, 40 mmol) were successively added; and the mixture was stirred overnight at room temperature. 500 mL of dichloromethane and 250 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=10/1-1/1) to obtain 12B (8 g, yield: 66%).
LCMS m/z=404.3 [M+H]+.
Step 2: benzyl 4-(piperidin-4-yl)piperazine-1-carboxylate (12C)
12B (8.0 g, 19.8 mmol) was dissolved in dichloromethane (120 mL); trifluoroacetic acid (40 mL) was slowly added; the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, and slurried by adding methyl tert-butyl ether to obtain a white solid, which was then dissolved by adding 200 mL of dichloromethane; and the resulting mixture was adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 12C (5.0 g, yield: 83%), which was directly used in the next step.
LCMS m/z=304.3 [M+H]+.
Step 3: benzyl 4-(1-((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)piperidin-4-yl)piperazine-1-carboxylate (12D)
12C (5.0 g, 16.5 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (3.5 g, 16.4 mmol) were mixed in DMAc (100 mL); acetic acid (0.3 g, 5 mmol) and sodium triacetoxyborohydride (4.2 g, 20 mmol) were successively added; and the mixture was stirred overnight at room temperature. 500 mL of dichloromethane and 250 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=10/1-1/1) to obtain 12D (4.8 g, yield: 58%).
LCMS m/z=501.4 [M+H]+.
Step 4: tert-butyl 4-((4-(piperazin-1-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate (12E)
12D (4.8 g, 9.6 mmol) was dissolved in methanol solution (100 mL); palladium on carbon (wt %=10%, 2.93 g) was added; and the mixture was subjected to 1 atm hydrogen replacement 3 times and reacted at room temperature for 1.5 h. The solution was filtered over celite. The filter cake was washed with dichloromethane/methanol (V/V=10/1), and the filtrate was concentrated under reduced pressure to obtain the crude of 12E (3.2 g), which was directly used in the next step.
LCMS m/z=367.3 [M+H]+.
Step 5: tert-butyl 4-((4-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate (12F)
12E (0.8 g, 2.18 mmol) and 1D (0.63 g, 2.5 mmol) were dissolved in DMSO (20 mL); potassium carbonate (0.59 mg, 4.26 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 12F (0.4 g, yield: 31%).
LCMS m/z=598.4 [M+H]+.
Step 6: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4-(1-(piperidin-4-ylmethyl) piperidin-4-yl)piperazine (12G)
12F (0.4 g, 0.67 mmol) was dissolved in dichloromethane (6 mL); trifluoroacetic acid (3.06 g, 26.84 mmol) was added at room temperature; and the mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH>10 with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 12G (0.30 g), which was directly used in the next step.
Step 7: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((4-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (12H)
12G (0.3 g, 0.60 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (0.25 g, 0.91 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.62 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 20 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-15/1) to obtain 12H (0.28 g, yield: 62%).
LCMS m/z=754.3 [M+H]+.
Step 7: 5-(4-((4-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12I)
12H (0.28 g, 0.37 mmol) was dissolved in ethanol (12 mL) and water (3 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (0.22 g, 4.11 mmol) and iron powder (0.23 g, 4.12 mmol) was added. At this temperature, the resulting mixture was stirred for 0.5 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 121 (0.19 g), which was directly used in the next step.
LCMS m/z=724.3 [M+H]+.
Step 9: 5-(4-((4-(4-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 12)
12I (0.19 g, 0.26 mmol) and 1I (0.12 g, 0.33 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC ((instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized to obtain the trifluoroacetate of compound 12 (32 mg).
LCMS m/z=528.3 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H), 10.60 (s, 1H), 8.95-8.68 (m, 4H), 7.95 (s, 1H), 7.91 (s, 1H), 7.67 (d, 1H), 7.63-7.50 (m, 3H), 7.25-7.22 (m, 1H), 7.08-7.01 (m, 1H), 6.70 (s, 1H), 4.96 (dd, 1H), 3.97-3.90 (m, 2H), 3.87-3.70 (m, 9H), 3.37-3.20 (m, 6H), 3.07-2.57 (m, 13H), 2.47-2.34 (m, 2H), 2.21-1.94 (m, 10H), 1.47-1.32 (m, 2H).
Step 1: tert-butyl 1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[4,4′-bipiperidine]-1-carboxylate (13A)
1D (1.0 g, 3.98 mmol) and 1-(tert-butoxycarbonyl)-4,4′-bipiperidine (1.28 g, 4.77 mmol) were dissolved in DMSO (50 mL); potassium carbonate (1.10 mg, 7.96 mmol) was added; and the mixture was reacted at 120° C. for 12 h, cooled to room temperature and extracted by adding 100 mL of ethyl acetate and 100 mL of water. The organic layer was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (ethyl acetate: petroleum ether (V/V)=10/1-2/1) to obtain 13A (1.3 g, yield: 65%).
LCMS m/z=500.3 [M+H]+.
Step 2: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4,4′-bipiperidine (13B)
To a 50 mL round bottom flask were successively added 13A (1.3 g, 2.60 mmol) and a solution of hydrogen chloride in 1,4-dioxane (30 mL, 4 mol/L); and the mixture was reacted at room temperature for 2 h and concentrated under reduced pressure. 1,4-dioxane (20 mL) and ammonia water (5 mL) were added; and the mixture was stirred at room temperature for 20 min and concentrated to dryness under reduced pressure at 50° C. to obtain 13B, which was directly used in the next reaction.
LCMS m/z=400.3 [M+H]+.
Step 3: tert-butyl 4-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidine-1-carboxylate (13C)
13B (0.5 g, 1.25 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (0.54 g, 2.53 mmol) were dissolved in DMAc (10 mL); a drop of acetic acid was added; and then the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (0.79 g, 3.73 mmol) was added, and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed sequentially with water (30 mL×2) and saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 13C (0.62 g, yield: 83%).
LCMS m/z=597.4 [M+H]+.
Step 4: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-1′-(piperidin-4-ylmethyl)-4,4′-bipiperidine (13D)
To a 50 mL round bottom flask were successively added 13C (0.47 g, 0.79 mmol) and a solution of hydrogen chloride in 1,4-dioxane (20 mL, 4 mol/L); and the mixture was reacted at room temperature for 2 h and concentrated under reduced pressure. 1,4-dioxane (20 mL) and ammonia water (5 mL) were added; and the mixture was stirred at room temperature for 20 min and concentrated to dryness under reduced pressure at 50° C. to obtain 13D, which was directly used in the next reaction.
LCMS m/z=497.3 [M+H]+.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (13E)
13D (387 mg, 0.78 mmol) was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (258 mg, 0.93 mmol) and DIPEA (504 mg, 3.90 mmol) were added at room temperature; and the mixture was reacted at 100° C. for 5 h. 30 mL of water was added to the reaction solution. The aqueous phase was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with water (30 mL×2), dried over anhydrous sodium sulfate and concentrated. The residue was separated and purified by silica gel column chromatography (DCM:MeOH=15:1) to obtain 13E (377 mg, yield 64%).
LCMS m/z=753.4 [M+H]+.
Step 6: 5-(4-((1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (13F)
13E (377 mg, 0.5 mmol), iron powder (140 mg, 2.5 mmol) and ammonium chloride (134 mg, 2.5 mmol) were dissolved in ethanol (10 mL) and water (5 mL); and the mixture was stirred at 85° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated to obtain 13F (236 mg, 65%), which was directly used in the next step.
LCMS m/z=362.3 [M+H]+.
Step 7: 5-(4-((1′-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 13); 2,2,2-trifluoroacetic acid
13F (236 mg, 0.33 mmol) and 1I (0.18 g, 0.49 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (188 mg, 0.99 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 13 (100 mg).
LCMS m/z=750.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 11.06 (s, 1H), 9.05-8.81 (m, 1H), 8.89-8.74 (m, 3H), 8.51 (s, 1H), 8.21 (s, 1H), 7.98 (s, 1H), 7.83 (s, 1H), 7.68 (d, 1H), 7.64-7.49 (m, 2H), 7.36 (d, 1H), 7.28 (dd, 1H), 6.83 (s, 1H), 5.07 (dd, 1H), 4.17-4.04 (m, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.65-3.51 (m, 2H), 3.20-3.09 (m, 2H), 3.09-2.80 (m, 7H), 2.68-2.52 (m, 4H), 2.24-2.10 (m, 1H), 2.10-1.90 (m, 9H), 1.90-1.80 (m, 2H), 1.80-1.68 (m, 2H), 1.61-1.15 (m, 8H).
Step 1: tert-butyl 2-(1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)-7-azaspiro[3.5]nonane-7-carboxylate (14A)
13B (0.5 g, 1.25 mmol) and tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate (0.54 g, 2.27 mmol) were dissolved in DMAc (10 mL); a drop of acetic acid was added; and then the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (0.79 g, 3.73 mmol) was added; and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed sequentially with water (30 mL×2) and saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 14A (0.49 g, yield: 63%).
LCMS m/z=623.4 [M+H]+.
Step 2: 2-(1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)-7-azaspiro[3.5]nonane (14B)
To a 50 mL round bottom flask were successively added 14A (0.49 g, 0.79 mmol) and a solution of hydrogen chloride in 1,4-dioxane (20 mL, 4 mol/L); and the mixture was reacted at room temperature for 2 h and concentrated under reduced pressure. 1,4-dioxane (20 mL) and ammonia water (5 mL) were added; and the mixture was stirred at room temperature for 20 min and concentrated to dryness under reduced pressure at 50° C. to obtain 14B, which was directly used in the next reaction.
LCMS m/z=523.4 [M+H]+.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(2-(1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)-7-azaspiro[3.5]nonan-7-yl)isoindoline-1,3-dione (14C)
14B (402 mg, 0.77 mmol) from the previous step was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (255 mg, 0.92 mmol) and DIPEA (498 mg, 3.85 mmol) were added at room temperature; and the mixture was stirred at 100° C. for 5 h. 30 mL of water was added to the reaction solution. The aqueous phase was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with water (30 mL×2), dried over anhydrous sodium sulfate and concentrated. The residue was separated and purified by silica gel column chromatography (DCM:MeOH=15:1) to obtain 14C (350 mg, yield 58%).
LCMS m/z=779.4 [M+H]+.
Step 4: 5-(2-(1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[4,4′-bipiperidin]-1-yl)-7-azaspiro[3.5]nonan-7-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (14D)
14C (350 mg, 0.45 mmol), iron powder (126 mg, 2.26 mmol) and ammonium chloride (134 mg, 2.50 mmol) were dissolved in ethanol (10 mL) and water (5 mL); and the mixture was stirred at 85° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated to obtain 14D (144 mg, 43%), which was directly used in the next step.
LCMS m/z=749.6 [M+H]+.
Step 5: 5-(2-(1′-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[4,4′-bipiperidin]-1-yl)-7-azaspiro[3.5]nonan-7-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 14); 2,2,2-trifluoroacetic acid
14D (144 mg, 0.19 mmol) and 1I (105 mg, 0.29 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (165 mg, 0.87 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 14 (80 mg).
LCMS m/z=540.8 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 11.06 (s, 1H), 9.38-9.17 (m, 1H), 9.05-8.91 (m, 1H), 8.89-8.77 (m, 2H), 8.49 (s, 1H), 8.21 (s, 1H), 7.99 (s, 1H), 7.83 (s, 1H), 7.67 (d, 1H), 7.63-7.48 (m, 2H), 7.34 (d, 1H), 7.30-7.21 (m, 1H), 6.83 (s, 1H), 5.06 (dd, 1H), 3.86-3.59 (m, 7H), 3.57-3.30 (m, 6H), 3.22-3.11 (m, 2H), 2.96-2.82 (m, 1H), 2.81-2.65 (m, 2H), 2.65-2.53 (m, 3H), 2.30-2.15 (m, 2H), 2.09-1.90 (m, 10H), 1.83-1.56 (m, 7H), 1.53-1.14 (m, 7H).
Step 1: tert-butyl 4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro [5.5]undecan-9-yl)-[1,4′-bipiperidine]-1′-carboxylate (15A)
11B (475 mg, 0.96 mmol) and N-tert-butoxycarbonyl-4-piperidone (287 mg, 1.44 mmol) were dissolved in DMAc (10 mL), and acetic acid (58 mg, 0.96 mmol) was added. The mixture was stirred at room temperature for 60 min, and then sodium triacetoxyborohydride (304 mg, 1.43 mmol) was added; and the resulting mixture was reacted overnight at room temperature, and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 15A (320 mg, yield: 49%).
LCMS m/z=677.3 [M+H]+.
Step 2: 5-(9-([1,4′-bipiperidin]-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (15B); HCl
Compound 15A (320 mg, 0.47 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (2 N, 6 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (15C)
The crude hydrochloride of compound 15B from the previous step was dissolved in DMSO (8 mL); 1D (163 mg, 0.65 mmol) and sodium bicarbonate (144 mg, 1.71 mmol) were successively added; and the mixture was reacted at 100° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 15C (160 mg, yield: 42%).
LCMS m/z=808.4 [M+H]+.
Step 4: 5-(9-(1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (15D)
15C (160 mg, 0.20 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (56 mg, 1 mmol) and ammonium chloride (53 mg, 1 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 15D as a yellow solid (120 mg, yield: 77%).
LCMS m/z=778.8 [M+H]+.
Step 5: 5-(9-(1′-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 15)
15D (120 mg, 0.15 mmol) and 11 (85 mg, 0.23 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (86 mg, 0.45 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain compound 15 (30 mg, yield: 18%).
LCMS m/z=555.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.05 (s, 1H), 9.03-8.91 (m, 1H), 8.89-8.77 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.80 (s, 1H), 7.67-7.46 (m, 3H), 7.33-7.26 (m, 1H), 7.22 (dd, 1H), 6.82 (s, 1H), 5.05 (dd, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.50-3.41 (m, 2H), 3.34 (s, 2H), 3.18-3.07 (m, 2H), 3.05-2.82 (m, 3H), 2.69-2.53 (m, 4H), 2.46-2.39 (m, 2H), 2.37-2.07 (m, 5H), 2.07-1.94 (m, 7H), 1.86-1.70 (m, 4H), 1.69-1.32 (m, 13H).
Step 1: tert-butyl 4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro [5.5]undecan-9-yl)methyl)-[1,4′-bipiperidine]-1′-carboxylate (16A)
The trifluoroacetate of 10E (0.6 g) and tert-butyl 4-oxopiperidine-1-carboxylate (0.4 g, 2.0 mmol) were mixed in DMAc (20 mL); acetic acid (0.12 g, 2 mmol) and sodium triacetoxyborohydride (0.42 g, 2 mmol) were successively added; and the mixture was stirred overnight at room temperature. 100 mL of dichloromethane and 50 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase:dichloromethane/methanol (V/V)=10/1-1/1) to obtain 16A (0.40 g).
LCMS m/z=691.3 [M+H]+.
Step 2: 5-(9-([1,4′-bipiperidin]-4-ylmethyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (16B); 2,2,2-trifluoroacetic acid
Compound 16A (0.40 g, 0.58 mmol) was dissolved in dichloromethane (10 mL); trifluoroacetic acid (2.3 g, 20 mmol) was added; and the mixture was stirred at room temperature for 3 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(9-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (16C)
The crude trifluoroacetate of 16B from the previous step was dissolved in DMSO (15 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (260 mg, 1.03 mmol) and sodium bicarbonate (250 mg, 3.0 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase dichloromethane/methanol (V/V)=100/1-10/1) to obtain 16C (230 mg, yield: 48%).
LCMS m/z=822.3 [M+H]+.
Step 4: 5-(9-((1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (16D)
16C (230 mg, 0.28 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (53 mg, 0.95 mmol) and ammonium chloride (50 mg, 0.93 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 16D (160 mg, yield: 72%).
LCMS m/z=792.4 [M+H]+.
Step 5: 5-(9-((1′-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 16)
16D (160 mg, 0.20 mmol) and 1I (95.7 mg, 0.26 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (97 mg, 0.51 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1). The product obtained from the column chromatography was further purified by preparative HPLC ((instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized, and then saturated sodium bicarbonate solution (50 ml) was added. The mixture was extracted with dichloromethane (50 ml). The organic phase was concentrated under reduced pressure to obtain compound 16 (20 mg, yield: 9%).
LCMS m/z=562.4 [M+2H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 11.06 (s, 1H), 9.01-8.91 (m, 1H), 8.88-8.77 (m, 2H), 8.53-8.42 (m, 1H), 8.22 (s, 1H), 8.01-7.82 (m, 2H), 7.70-7.48 (m, 3H), 7.44-7.20 (m, 2H), 6.82 (s, 1H), 5.11-5.02 (m, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.69-3.58 (m, 2H), 3.56-2.82 (m, 18H), 2.75-2.53 (m, 4H), 2.16-1.96 (m, 10H), 1.93-1.57 (m, 10H), 1.54-1.45 (m, 2H).
Step 1: tert-butyl 2-((1′-((benzyloxy)carbonyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-di azaspiro [5.5]undecane-9-carb oxyl ate (17A)
1B (702 mg, 2 mmol) and benzyl 4-oxopiperidine-1-carboxylate (699 mg, 3 mmol) were mixed in dichloromethane (100 mL); acetic acid (180 mg, 3 mmol) and sodium triacetoxyborohydride (636 mg, 3 mmol) were successively added; and the mixture was stirred overnight at room temperature. 30 mL of dichloromethane and 10 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=10/1-1/1) to obtain 17A (512 mg, yield: 45%).
LCMS m/z=569.4 [M+H]+.
Step 2: tert-butyl 2-([1,4′-bipiperidin]-4-ylmethyl)-2,9-diazaspiro[5.5] undecane-9-carboxylate (17B)
17A (512 mg, 0.9 mmol) was dissolved in methanol (10 mL); palladium on carbon (wt %=10%, 51 mg) was added; the mixture was subjected to hydrogen replacement 3 times, stirred overnight under hydrogen atmosphere (balloon pressure) at room temperature and filtered; and the filtrate was concentrated under reduced pressure to obtain 17B (390 mg), which was directly used in the next step.
Step 3: tert-butyl2-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (17C)
17B (390 mg, 0.9 mmol), 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (301 mg, 1.2 mmol) and potassium carbonate (414 mg, 3 mmol) were mixed and dissolved in DMSO (10 mL), and the mixture was stirred at 120° C. for 16 h, cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 17C (310 mg, yield: 52%).
LCMS m/z=666.5 [M+H]+.
Step 4: 2-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecane (17D)
17C (310 mg, 0.47 mmol) was dissolved in DCM (10 mL); trifluoroacetic acid (3 mL) was added at room temperature; and the mixture was stirred for 3 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 17D (262 mg), which was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(2-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)isoindoline-1,3-dione (17E)
17D (262 mg, 0.46 mmol) was dissolved in DMSO (5 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (276 mg, 1 mmol) and DIPEA (0.31 g, 2.4 mmol) were successively added; and the mixture was stirred at 90° C. for 3 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 17E (210 mg, yield: 55%).
Step 6: 5-(2-((1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (17F)
Compound 17E (210 mg, 0.26 mmol), iron powder (200 mg, 3.57 mmol) and ammonium chloride (200 mg, 3.77 mmol) were dissolved in ethanol (30 mL) and water (10 mL); and the mixture was stirred at 80° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated to obtain 17F (150 mg, 74.3%), which was directly used in the next step.
Step 7: 5-(2-((1′-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 17)
17F (70 mg, 0.09 mmol) and (6-((2,5-dichloropyrimidin-4-yl)amino)quinoxalin-5-yl)dimethylphosphine oxide (1I) (0.44 g, 0.12 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (57 mg, 0.3 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative product, which was alkalized with saturated aqueous sodium bicarbonate solution to obtain compound 17 (23 mg, yield: 23.3%).
LCMS m/z=562.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 11.15-10.89 (m, 1H), 9.10-8.89 (m, 1H), 8.88-8.77 (m, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 7.98 (s, 1H), 7.80 (s, 1H), 7.67-7.46 (m, 3H), 7.31-7.19 (m, 2H), 6.82 (s, 1H), 5.05 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.57-3.35 (m, 6H), 3.17-3.06 (m, 2H), 2.99-2.80 (m, 3H), 2.71-2.52 (m, 6H), 2.37-1.93 (m, 14H), 1.87-1.42 (m, 11H), 1.31-1.02 (m, 4H).
5-(4-((4-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 18)
4D (0.22 g, 0.31 mmol) and 2J (0.13 g, 0.31 mmol) were dissolved in DMF (15 mL); p-toluenesulfonic acid hydrate (0.18 g, 0.93 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 18 as a yellow solid (150 mg, yield: 44%).
LCMS m/z=550.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 11.05 (s, 1H), 8.89-8.76 (m, 3H), 8.35 (s, 1H), 8.27 (s, 1H), 7.96 (s, 1H), 7.77 (s, 1H), 7.65 (d, 1H), 7.62-7.50 (m, 2H), 7.31 (s, 1H), 7.23 (d, 1H), 6.82 (s, 1H), 5.06 (dd, 1H), 4.04 (d, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.17-3.09 (m, 2H), 3.01-2.94 (m, 2H), 2.91-2.84 (m, 1H), 2.72-2.51 (m, 8H), 2.46-2.30 (m, 4H), 2.29-2.22 (m, 1H), 2.20-2.10 (m, 2H), 2.06-1.96 (m, 7H), 1.87-1.74 (m, 5H), 1.65-1.49 (m, 2H), 1.22-1.05 (m, 2H).
5-(4-((1′-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 19); 2,2,2-trifluoroacetic acid
8I (0.27 g, 0.37 mmol) and 2J (0.153 g, 0.37 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.213 g, 1.12 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 19 (50 mg, yellow solid).
LCMS m/z=1099.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H), 11.08 (s, 1H), 10.13-9.45 (m, 2H), 8.90-8.73 (m, 3H), 8.45 (s, 1H), 8.29 (s, 1H), 7.96 (s, 1H), 7.84-7.75 (m, 2H), 7.62 (s, 1H), 7.57-7.49 (m, 2H), 7.38 (dd, 1H), 6.81 (s, 1H), 5.10 (dd, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.70-3.59 (m, 4H), 3.48-2.81 (m, 14H), 2.74-2.52 (m, 4H), 2.22-1.96 (m, 11H), 1.93-1.74 (m, 3H), 1.59-1.43 (m, 2H).
5-(4-((9-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 20); 2,2,2-trifluoroacetic acid
9F (150 mg, 0.21 mmol) and 2J (132 mg, 0.32 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (120 mg, 0.63 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 20 (44 mg).
LCMS m/z=542.8 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H), 11.06 (s, 1H), 8.91-8.72 (m, 3H), 8.56-8.47 (m, 1H), 8.29 (s, 1H), 7.99 (s, 1H), 7.80 (s, 1H), 7.68 (d, 1H), 7.62-7.49 (m, 2H), 7.37 (d, 1H), 7.28 (dd, 1H), 6.89 (s, 1H), 5.07 (dd, 1H), 4.17-4.04 (m, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 3.48-3.35 (m, 2H), 3.16-2.76 (m, 10H), 2.68-2.51 (m, 4H), 2.21-2.09 (m, 1H), 2.07-1.91 (m, 7H), 1.90-1.50 (m, 8H), 1.37-1.21 (m, 3H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (21A)
The crude of 9D (470 mg, 0.97 mmol) was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1) (430 mg, 1.46 mmol) and sodium bicarbonate (326 mg, 3.88 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 21A as a yellow solid (600 mg, yield: 82%).
LCMS m/z=757.3 [M+H]+.
Step 2: 5-(4-((9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (21B)
21A (200 mg, 0.26 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (73 mg, 1.3 mmol) and ammonium chloride (70 mg, 1.3 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 21B as a yellow solid (180 mg, yield: 95%).
Step 3: 5-(4-((9-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 21)
21B (180 mg, 0.25 mmol) and 2J (157 mg, 0.38 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (143 mg, 0.75 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain compound 21 (85 mg, yield: 31%).
LCMS m/z=551.8 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 11.08 (s, 1H), 8.87-8.72 (m, 3H), 8.36 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.79 (s, 1H), 7.69 (d, 1H), 7.63-7.48 (m, 2H), 7.43 (d, 1H), 6.88 (s, 1H), 5.10 (dd, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.67-3.55 (m, 2H), 2.96-2.76 (m, 7H), 2.68-2.52 (m, 2H), 2.43-2.29 (m, 4H), 2.24-2.14 (m, 2H), 2.06-1.94 (m, 8H), 1.88-1.69 (m, 3H), 1.62-1.50 (m, 7H), 1.32-1.25 (m, 2H).
Step 1: tert-butyl 1′-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-[4,4′-bipipenidine]-1-carboxylate (21A)
The crude of 2E (450 mg, 2.13 mmol) was dissolved in DMSO (10 mL); tert-butyl [4,4′-bipiperidine]-1-carboxylate (859 mg, 3.2 mmol) and sodium bicarbonate (716 mg, 8.52 mmol) were successively added; and the mixture was reacted at 100° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 22A (800 mg, yield: 82%).
LCMS m/z=460.3 [M+H]+.
Step 2: 1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-4,4′-bipiperidine (22B)
22A (800 mg, 1.74 mmol) was dissolved in methanol (4 mL); a solution of hydrochloric acid in dioxane (2 M, 15 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 M aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 22B, which was directly used in the next step.
Step 3: tert-butyl 4-((1′-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidine-1-carboxylate (22C)
22B (550 mg, 1.53 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (491 mg, 2.30 mmol) were dissolved in DMAc (15 mL), and acetic acid (92 mg, 1.53 mmol) was added. The mixture was stirred at room temperature for 30 min, and then sodium triacetoxyborohydride (485 mg, 2.29 mmol) was added; and the resulting mixture was reacted overnight at room temperature, and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1) to obtain 22C as a yellow solid (650 mg, yield: 76%).
LCMS m/z=557.5 [M+H]+.
Step 4: 1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-1′-(piperidin-4-ylmethyl)-4,4′-bipiperidine (22D); HCl
22C (650 mg, 1.17 mmol) was dissolved in methanol (3 mL); a solution of hydrochloric acid in dioxane (2 M, 10 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 5: 5-(4-((1′-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (22E)
The crude hydrochloride of compound 22D from the previous step was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (453 mg, 1.64 mmol) and sodium bicarbonate (366 mg, 4.36 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 22E as a yellow solid (600 mg, yield: 72%).
LCMS m/z=713.4 [M+H]+.
Step 6: 5-(4-((1′-(4-amino-2-cyclopropyl-5-methoxyphenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (22F)
22E (200 mg, 0.28 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (78 mg, 1.40 mmol) and ammonium chloride (75 mg, 1.40 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 22F (150 mg, yield: 78%).
LCMS m/z=342.3 [M+H]+.
Step 7: 5-(4-((1′-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 22); 2,2,2-trifluoroacetic acid
22F (150 mg, 0.22 mmol) and 2J (136 mg, 0.33 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (125 mg, 0.66 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 22 (83 mg).
LCMS m/z=529.8 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H), 11.06 (s, 1H), 8.94-8.69 (m, 4H), 8.50-8.23 (m, 2H), 7.94-7.81 (m, 1H), 7.68 (d, 1H), 7.36 (s, 1H), 7.31-7.24 (m, 1H), 6.94-6.73 (m, 2H), 5.07 (dd, 1H), 4.16-4.05 (m, 2H), 3.77 (s, 3H), 3.63-3.51 (m, 2H), 3.39-3.28 (m, 2H), 3.08-2.53 (m, 11H), 2.19-1.75 (m, 15H), 1.60-1.25 (m, 8H), 0.78-0.65 (m, 2H), 0.44-0.34 (m, 2H).
Step 1: 3-iodo-[1,1′-biphenyl]-4-amine (23B)
[1,1′-biphenyl]-4-amine (23A) (846 mg, 5.0 mmol), iodine (1.27 g, 5.0 mmol) and sodium bicarbonate (1.26 g, 15.0 mmol) were added to a mixed solution of dichloromethane (20 mL) and water (10 mL), and the mixture was stirred overnight at room temperature. After the reaction was completed, the organic layers were separated, and the aqueous layer was extracted with dichloromethane (3×20 mL). The organic layers were combined, washed sequentially with saturated Na2S2O3 solution (2×20 mL) and saturated brine (2×20 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V)=1/20-1/5) to obtain 23B (530 mg, yield: 36%).
LCMS m/z=296.0 [M+H]+.
Step 2: (4-amino-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23C)
Under nitrogen protection, 23B (401 mg, 1.36 mmol), dimethylphosphine oxide (117 mg, 1.50 mmol), tripotassium phosphate (318 mg, 1.50 mmol), palladium acetate (30 mg, 0.13 mmol) and 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (79 mg, 0.14 mmol) were added to anhydrous DMF (20 mL), and the mixture was reacted at 150° C. for 3 h. After the reaction was completed, the mixture was cooled to room temperature. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The organic phase was separated, washed sequentially with water (20 mL×2) and saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V)=1/10-1/3) to obtain 23C (170 mg, yield: 51%).
LCMS m/z=246.2 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 7.52-7.44 (m, 3H), 7.45-7.37 (m, 2H), 7.34-7.22 (m, 2H), 6.80 (dd, 1H), 1.82 (d, 6H).
Step 3: (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl) dimethylphosphine oxide (23D)
23C (170 mg, 0.69 mmol) and 5-bromo-2,4-dichloropyrimidine (315 mg, 1.38 mmol) were dissolved in NMP (10 mL); DIPEA (107 mg, 0.83 mmol) was added; and under nitrogen protection, the mixture was stirred at 130° C. for 6 h, cooled to room temperature and extracted by adding 60 mL of ethyl acetate and 60 mL of water. The organic layer was washed with 30 mL of saturated brine, dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (V/V)=1/2-1/0) to obtain 23D (240 mg, yield: 80%).
LCMS m/z=436.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 11.37 (s, 1H), 8.66 (dd, 1H), 8.35 (s, 1H), 7.85-7.76 (m, 1H), 7.57-7.35 (m, 6H), 1.89 (d, 6H).
Step 4: 5-(4-((4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 23); 2,2,2-trifluoroacetic acid
4D (87 mg, 0.12 mmol) and 23D (58 mg, 0.13 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (68 mg, 0.36 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 23 (30 mg).
LCMS m/z=562.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.05 (s, 1H), 8.43-8.34 (m, 1H), 8.27 (s, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.85 (s, 1H), 7.73-7.61 (m, 2H), 7.61-7.50 (m, 3H), 7.45 (t, 2H), 7.40-7.33 (m, 1H), 7.33-7.29 (m, 1H), 7.27-7.13 (m, 2H), 6.83 (s, 1H), 5.06 (dd, 1H), 4.10-3.98 (m, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.14-3.04 (m, 2H), 3.04-2.81 (m, 4H), 2.65-2.52 (m, 6H), 2.44-2.30 (m, 4H), 2.25-1.95 (m, 5H), 1.92-1.73 (m, 11H), 1.61-1.45 (m, 2H), 1.21-1.08 (m, 2H).
The trifluoroacetate of compound 23 (60 mg) was dissolved in 20 mL of dichloromethane; 20 mL of water and 1 mL of saturated sodium bicarbonate solution were added. The organic layers were separated, and the aqueous layer was extracted with dichloromethane (20 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain compound 23 (42 mg).
LCMS m/z=562.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 11.05 (s, 1H), 8.31-8.22 (m, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.85 (s, 1H), 7.75-7.61 (m, 2H), 7.61-7.50 (m, 3H), 7.45 (t, 2H), 7.40-7.32 (m, 1H), 7.30 (s, 1H), 7.27-7.08 (m, 2H), 6.83 (s, 1H), 5.06 (dd, 1H), 4.10-3.93 (m, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.14-3.03 (m, 2H), 3.03-2.81 (m, 4H), 2.65-2.52 (m, 6H), 2.44-2.30 (m, 4H), 2.23-1.95 (m, 5H), 1.89-1.72 (m, 11H), 1.59-1.45 (m, 2H), 1.19-1.07 (m, 2H).
Step 1: 5-(4-((1′-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (24A)
8G (0.84 g, 1.61 mmol) and 1-cyclopropyl-2-fluoro-4-methoxy-5-nitrobenzene (2E) (226 mg, 1.07 mmol) were dissolved in DMSO (10 mL); a sodium bicarbonate solid (405 mg, 4.82 mmol) was added; and the mixture was reacted overnight at 100° C. After the reaction was completed, the mixture was cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-10/1) to obtain 24A as a yellow solid (0.61 g, yield: 53%).
LCMS m/z=714.4 [M+H]+.
Step 2: 5-(4-((1′-(4-amino-2-cyclopropyl-5-methoxyphenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (24B)
24A (0.61 g, 0.85 mmol) was dissolved in ethanol (5 mL) and water (1 mL); an ammonium chloride solid (455 mg, 8.5 mmol) and iron powder (476 mg, 8.5 mmol) were added; and the mixture was subjected to N2 replacement 3 times and stirred at 80° C. for 1 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane and concentrated under reduced pressure. The resultant obtained from the concentration under reduced pressure was dissolved in 50 mL of dichloromethane and extracted. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 24B as a yellow solid (0.32 g, yield: 55%).
LCMS m/z=684.4 [M+H]+.
Step 3: 5-(4-((1′-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 24)
24B (0.32 g, 0.468 mmol) and 2J (193 mg, 0.468 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (266 mg, 1.40 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C. and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 24 (60 mg, yield: 12%).
LCMS m/z=1059.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 11.06 (s, 1H), 8.90-8.74 (m, 3H), 8.31-8.22 (m, 2H), 7.87 (d, 1H), 7.67 (d, 1H), 7.32 (s, 1H), 7.27-7.21 (m, 1H), 6.86 (s, 1H), 6.74 (s, 1H), 5.07 (dd, 1H), 3.76 (s, 3H), 3.44-3.41 (m, 3H), 3.34-3.31 (m, 3H), 2.94-2.83 (m, 3H), 2.73-2.64 (m, 2H), 2.63-2.52 (m, 2H), 2.49-2.43 (m, 4H), 2.24-2.07 (m, 5H), 2.06-1.97 (m, 7H), 1.89 (s, 1H), 1.87-1.79 (m, 2H), 1.76-1.60 (m, 4H), 1.56-1.46 (m, 1H), 1.16-1.04 (m, 2H), 0.79-0.66 (m, 2H), 0.44-0.32 (m, 2H).
Step 1: 5-(9-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)-2,9-diazaspiro[0.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (25A)
The hydrochloride of 11B (728 mg) was dissolved in DMSO (10 mL); 1-cyclopropyl-2-fluoro-4-methoxy-5-nitrobenzene (2E) (350 mg, 1.66 mmol) and sodium bicarbonate (353 mg, 4.2 mmol) were successively added; and the mixture was reacted at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 25A (540 mg, yield: 60%).
LCMS m/z=685.3 [M+H]+.
Step 2: 5-(9-(1-(4-amino-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (25B)
25A (540 mg, 0.79 mmol) was dissolved in ethanol/water (16 mL, 3:1); iron powder (530 mg, 9.5 mmol) and ammonium chloride (510 mg, 9.5 mmol) were successively added; and under nitrogen protection, the mixture was reacted at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 25B (250 mg, yield: 48%).
LCMS m/z=655.4 [M+H]+.
Step 3: 5-(9-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 25)
25B (125 mg, 0.19 mmol) and 2J (87 mg, 0.21 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (110 mg, 0.57 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain compound 25 (40 mg, yield: 25.4%).
LCMS m/z=515.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.65 (s, 1H), 11.05 (s, 1H), 8.98-8.70 (m, 3H), 8.33-8.20 (m, 2H), 7.86 (d, 1H), 7.63 (d, 1H), 7.36-7.20 (m, 2H), 6.86 (s, 1H), 6.74 (s, 1H), 5.05 (dd, 1H), 3.76 (s, 3H), 3.50-3.43 (m, 2H), 3.39-3.32 (m, 3H), 2.95-2.82 (m, 1H), 2.76-2.53 (m, 7H), 2.14-1.82 (m, 11H), 1.76-1.37 (m, 12H), 0.74-0.64 (m, 2H), 0.44-0.27 (m, 2H).
Step 1: tert-butyl 4-((4-(4-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate (26A)
12E (0.8 g, 2.18 mmol) and 2E (0.50 g, 2.4 mmol) were dissolved in DMSO (20 mL); potassium carbonate (0.59 mg, 4.26 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 30 mL of ethyl acetate and 30 mL of water. The organic layer was washed three times with saturated brine (20 mL×3). dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 26A (0.6 g, yield: 49%).
LCMS m/z=558.4 [M+H]+.
Step 2: 1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)-4-(1-(piperidin-4-ylmethyl) piperidin-4-yl)piperazine (26B)
26A (0.6 g, 1.07 mmol) was dissolved in dichloromethane (6 mL); trifluoroacetic acid (3.06 g, 26.84 mmol) was added at room temperature; and the mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=10 with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 26B (0.5 g), which was directly used in the next step.
Step 3: 5-(4-((4-(4-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (26C)
26B (0.5 g) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (0.30 g, 1.08 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.62 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 20 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-15/1) to obtain 26C (0.40 g, yield: 52%)
LCMS m/z=562.3 [M+H]+.
Step 4: 5-(4-((4-(4-(4-amino-2-cyclopropyl-5-methoxyphenyl)piperazin-1-yl) piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (26D)
26C (0.40 g, 0.58 mmol) was dissolved in ethanol (12 mL) and water (3 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (0.22 g, 4.11 mmol) and iron powder (0.23 g, 4.12 mmol) was added, and the resulting mixture was stirred at 80° C. for 0.5 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 26D (0.31 g, yield: 78%).
LCMS m/z=562.3 [M+H]+.
Step 5: 5-(4-((4-(4-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 26)
26D (0.31 g, 0.45 mmol) and 1I (0.18 g, 0.5 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 26 (30 mg, yield: 7%).
LCMS m/z=508.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 11.06 (s, 1H), 9.06-8.91 (m, 1H), 8.90-8.80 (m, 2H), 8.33 (s, 1H), 8.17 (s, 1H), 7.89 (d, 1H), 7.65 (d, 1H), 7.31 (d, 1H), 7.23 (dd, 1H), 6.88 (s, 1H), 6.80 (s, 1H), 5.07 (dd, 1H), 4.10-3.98 (m, 2H), 3.77 (s, 3H), 3.09-2.83 (m, 9H), 2.76-2.53 (m, 6H), 2.30-2.09 (m, 4H), 2.08-1.96 (m, 7H), 1.94-1.71 (m, 7H), 1.55-1.39 (m, 2H), 1.22-1.08 (m, 2H), 0.82-0.71 (m, 2H), 0.49-0.40 (m, 2H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1l-yl)isoindoline-1,3-dione (27A)
The hydrochloride of 9D (0.27 g) and 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisodihydroindole-1,3-dione (5A-1) (0.16 g, 0.56 mmol) were dissolved in DMSO (15 mL); DIPEA (0.22 g, 1.68 mmol) was added dropwise; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 27A as a yellow solid (0.30 g, yield: 71%).
LCMS m/z=757.4 [M+H]+.
Step 2: 5-(4-((9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (27B)
27A (0.30 g, 0.40 mmol) was dissolved in ethanol (15 mL); reduced iron powder (0.11 g, 2.00 mmol) was added, and then an aqueous solution (5 mL) of ammonium chloride (0.11 g, 2.00 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 3 h. The reaction solution was cooled to room temperature. 5 mL of water was added, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 27B as a yellow solid (0.26 g, yield: 89%).
LCMS m/z=727.3 [M+H]+.
Step 3: 5-(4-((9-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 27)
27B (0.13 g, 0.18 mmol) and 1I (0.07 g, 0.18 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid hydrate (0.10 g, 0.54 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain compound 27 (44 mg, yield: 23%).
LCMS m/z=529.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 11.08 (s, 1H), 9.00-8.92 (m, 1H), 8.87-8.78 (m, 2H), 8.38 (s, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.82 (s, 1H), 7.69 (d, 1H), 7.57 (s, 2H), 7.44 (d, 1H), 6.89 (s, 1H), 5.10 (dd, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.66-3.55 (m, 2H), 2.95-2.80 (m, 7H), 2.68-2.53 (m, 2H), 2.41-2.31 (m, 4H), 2.22-2.15 (m, 2H), 2.08-1.97 (m, 7H), 1.88-1.78 (m, 2H), 1.78-1.69 (m, 1H), 1.62-1.50 (m, 8H), 1.33-1.24 (m, 2H).
Step 1: 5-(9-(1-(4-((5-chloro-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 28)
33F (100 mg, 0.14 mmol) and 1I (77 mg, 0.21 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (80 mg, 0.42 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the crude was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 28 (20 mg, yield: 14%).
LCMS m/z=1044.4 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.08 (s, 1H), 9.04-8.91 (m, 1H), 8.83 (dd, 2H), 8.37 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.80 (s, 1H), 7.71 (d, 1H), 7.63-7.51 (m, 2H), 7.47 (d, 1H), 6.83 (s, 1H), 5.10 (dd, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.23-3.10 (m, 4H), 3.06 (s, 2H), 2.95-2.82 (m, 1H), 2.70-2.52 (m, 6H), 2.41-2.28 (m, 1H), 2.10-1.94 (m, 7H), 1.90-1.41 (m, 12H), 1.34-1.20 (m, 2H).
Step 1: 5-(4-((4-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 29)
5B (0.16 g, 0.21 mmol) and 2J (0.09 g, 0.21 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid hydrate (0.12 g, 0.63 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain compound 29 (51 mg, yield: 22%).
LCMS m/z=559.4 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 11.08 (s, 1H), 8.90-8.71 (m, 3H), 8.36 (s, 1H), 8.27 (s, 1H), 7.97 (s, 1H), 7.78 (s, 1H), 7.70 (d, 1H), 7.64-7.49 (m, 2H), 7.44 (d, 1H), 6.82 (s, 1H), 5.10 (dd, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.66-3.57 (m, 2H), 3.20-3.10 (m, 2H), 2.94-2.84 (m, 3H), 2.70-2.51 (m, 8H), 2.47-2.10 (m, 7H), 2.07-1.95 (m, 7H), 1.90-1.68 (m, 5H), 1.66-1.43 (m, 2H), 1.31-1.25 (m, 2H).
Step 1: 5-(3-((4-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidin-1l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 30)
6F (0.11 g, 0.15 mmol) and 2J (0.06 g, 0.15 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid hydrate (0.09 g, 0.45 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain compound 30 (31 mg, yield: 19%).
LCMS m/z=543.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.67 (s, 1H), 11.04 (s, 1H), 8.90-8.75 (m, 3H), 8.35 (s, 1H), 8.27 (s, 1H), 7.98 (s, 1H), 7.76 (s, 1H), 7.68-7.46 (m, 3H), 6.93-6.79 (m, 3H), 5.05 (dd, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.58-3.48 (m, 2H), 3.44-3.36 (m, 1H), 3.19-3.06 (m, 3H), 2.95-2.81 (m, 1H), 2.75-2.52 (m, 9H), 2.47-2.08 (m, 8H), 2.07-1.96 (m, 7H), 1.91-1.69 (m, 3H), 1.66-1.49 (m, 2H).
Step 1: 5-(4-((4-(4-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 31)
12I (0.25 g, 0.34 mmol) and 2J (0.18 g, 0.44 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized to obtain a solid, which was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 31 (30 mg, yield: 8%).
LCMS m/z=550.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 11.05 (s, 1H), 8.88-8.75 (m, 3H), 8.37 (s, 1H), 8.27 (s, 1H), 7.97 (s, 1H), 7.78 (s, 1H), 7.65 (d, 1H), 7.62-7.50 (m, 2H), 7.30 (d, 1H), 7.23 (dd, 1H), 6.85 (s, 1H), 5.06 (dd, 1H), 4.09-3.98 (m, 2H), 3.81 (s, 3H), 3.75 (s, 3H), 3.03-2.82 (m, 9H), 2.69-2.52 (m, 6H), 2.24-2.08 (m, 3H), 2.07-1.96 (m, 7H), 1.93-1.73 (m, 7H), 1.51-1.37 (m, 2H), 1.21-1.08 (m, 2H).
Step 1: 5-(9-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 32)
11D (100 mg, 0.14 mmol) and 2J (0.21 g, 0.51 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (87 mg, 0.46 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 32 (22 mg, yield: 15%).
LCMS m/z=1070.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 11.05 (s, 1H), 8.88-8.73 (m, 3H), 8.34 (s, 1H), 8.26 (s, 1H), 7.95 (s, 1H), 7.76 (s, 1H), 7.67-7.46 (m, 3H), 7.34-7.16 (m, 2H), 6.81 (s, 1H), 5.05 (dd, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.51-3.35 (m, 4H), 3.18-3.05 (m, 2H), 2.96-2.81 (m, 1H), 2.69-2.52 (m, 6H), 2.34-2.24 (m, 1H), 2.08-1.94 (m, 8H), 1.89-1.74 (m, 2H), 1.70-1.34 (m, 11H).
Step 1: tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (33A)
2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dioneo(5A-1) (700 mg, 2.38 mmol) was dissolved in DMSO (10 mL); tert-butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate (786 mg, 3.09 mmol) and sodium bicarbonate (800 mg, 9.52 mmol) were successively added; and the mixture was stirred at 100° C. for 4 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 33A as a yellow solid (0.6 g, yield: 48%).
LCMS m/z=529.3 [M+H]+.
Step 2: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (33B)
33A (600 mg, 1.14 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (2 M, 6 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 M aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 33B (462 mg, yield: 95%).
Step 3: tert-butyl 4-(2-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,9-diazaspiro[5.5]undecan-9-yl)piperidine-1-carboxylate (33C)
33B (462 mg, 1.08 mmol) and N-tert-butoxycarbonyl-4-piperidone (279 mg, 1.40 mmol) were dissolved in DMAc (8 mL), and acetic acid (65 mg, 1.08 mmol) was added. The mixture was stirred at room temperature for 30 min, and then sodium triacetoxyborohydride (342 mg, 1.61 mmol) was added; and the resulting mixture was reacted overnight at room temperature, and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 33C (400 mg, yield: 61%).
LCMS m/z=612.3 [M+H]+.
Step 4: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(9-(piperidin-4-yl)-2,9-diazaspiro [5.5]undecan-2-yl)isoindoline-1,3-dione (33D); HCl
33C (400 mg, 0.65 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (2 N, 6 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (33E)
The crude hydrochloride of 33D from the previous step was dissolved in DMSO (5 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (196 mg, 0.78 mmol) and sodium bicarbonate (202 mg, 2.4 mmol) were successively added; and the mixture was reacted at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 33E (178 mg, yield: 37%).
LCMS m/z=562.3 [M+H]+.
Step 6: 5-(9-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (33F)
33E (178 mg, 0.24 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (67 mg, 1.20 mmol) and ammonium chloride (64 mg, 1.20 mmol) were successively added; and under nitrogen protection, the mixture was reacted at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 33F (100 mg, yield: 58%).
Step 7: 5-(9-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 33)
33F (100 mg, 0.14 mmol) and 2J (87 mg, 0.21 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (80 mg, 0.42 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 33 (30 mg, yield: 20%).
LCMS m/z=1088.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 11.09 (s, 1H), 8.90-8.73 (m, 3H), 8.34 (s, 1H), 8.26 (s, 1H), 7.95 (s, 1H), 7.77 (s, 1H), 7.70 (d, 1H), 7.62-7.49 (m, 2H), 7.46 (d, 1H), 6.81 (s, 1H), 5.10 (dd, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.24-2.99 (m, 7H), 2.95-2.82 (m, 1H), 2.68-2.51 (m, 7H), 2.36-2.25 (m, 1H), 2.09-1.94 (m, 7H), 1.89-1.39 (m, 12H).
Step 1: tert-butyl 5-((1-((benzyloxy)carbonyl)piperidin-4-yl)methyl) hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (34B)
2-tert-butoxycarbonyl-hexahydropyrrolo[3,4-c]pyrrole (34A) (1.06 g, 5 mmol) and benzyl 4-formylpiperidine-1-carboxylate (2.47 g, 10 mmol) were dissolved in 1,2-dichloroethane (18 mL), and acetic acid (600 mg, 10 mmol) was added. The mixture was stirred at room temperature for 30 min, and then sodium triacetoxyborohydride (3.18 g, 15 mmol) was added; and the resulting mixture was reacted overnight at room temperature, and extracted by adding 20 mL of aqueous sodium hydroxide solution (4 N) and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain the crude of 34B (3.55 g), which was directly used in the next step.
LCMS m/z=444.3 [M+H]+.
Step 2: tert-butyl 5-(piperidin-4-ylmethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (34C)
34B (3.55 g, 5 mmol) was dissolved in ammonia/methanol (30 mL); palladium on carbon (wt %=10%, 355 mg) was added; and the mixture was subjected to hydrogen replacement 3 times, stirred under hydrogen atmosphere (balloon pressure) at room temperature for 3 h and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 34C (474 mg, yield: 31%).
LCMS m/z=310.3 [M+H]+.
Step 3: tert-butyl 5-((1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (34D)
2E (356 mg, 1.69 mmol) and 34C (474 mg, 1.53 mmol) were dissolved in DMSO (10 mL); potassium carbonate (634 mg, 4.59 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 60 mL of ethyl acetate and 60 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain the crude of 34D (766 mg), which was directly used in the next reaction.
LCMS m/z=501.3 [M+H]+.
Step 4: 2-((1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl) octahydropyrrolo[3,4-c]pyrrole hydrochloride (34E); HCl
A solution of hydrochloric acid in dioxane (4 N, 6 mL, 24 mmol) was added to the crude of 34D (766 mg); and the mixture was stirred at room temperature for 5 min. (6 mL) was added. The reaction solution was treated until a clear solution was obtained, and the solution was stirred at room temperature for 5 min and subjected to LCMS for monitoring the reaction process. The reaction solution was concentrated to dryness under reduced pressure to obtain the crude hydrochloride of 34E, which was directly used in the next step.
LCMS m/z=401.3 [M+H]+.
Step 5: 5-(5-((1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl) hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (34F)
The crude hydrochloride of 34E from the previous step and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (422 m g, 1.53 mmol) were dissolved in DMSO (10 mL); DIPEA (593 mg, 4.59 mmol) was added; and the mixture was stirred at 100° C. for 5 h and cooled to room temperature. 20 mL of water was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-30/1) to obtain 34F (611 mg, yield: 61%).
LCMS m/z=657.3 [M+H]+.
Step 6: 5-(5-((1-(4-amino-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)methyl) hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (34G)
34F (611 mg, 0.93 mmol) was dissolved in methanol (12 mL) and water (3 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (611 mg, 11.5 mmol) and iron powder (611 mg, 11 mmol) was added. At this temperature, the resulting mixture was stirred for 0.5 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was subjected to preparative plate purification (DCM/MeOH=10/1) to obtain 34G (140 mg, yield: 24%).
LCMS m/z=627.3 [M+H]+.
Step 7: 5-(5-((1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino) pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)methyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 34)
34G (0.14 g, 0.22 mmol) and 2J (92 mg, 0.22 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (127 mg, 0.67 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 34 (35 mg, yield: 16%).
LCMS m/z=501.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 11.04 (s, 1H), 8.90-8.61 (m, 3H), 8.31-8.10 (m, 2H), 7.95-7.78 (m, 1H), 7.65 (d, 1H), 7.03-6.65 (m, 4H), 5.04 (dd, 1H), 3.79-3.63 (m, 5H), 3.29-3.13 (m, 4H), 3.06-2.94 (m, 2H), 2.93-2.80 (m, 1H), 2.74-2.59 (m, 4H), 2.59-2.48 (m, 4H), 2.38-2.27 (m, 2H), 2.12-1.92 (m, 8H), 1.86-1.76 (m, 2H), 1.63-1.53 (m, 1H), 1.37-1.24 (m, 2H), 0.73-0.58 (m, 2H), 0.43-0.20 (m, 2H).
Step 1: tert-butyl 4-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)piperazine-1-carboxylate (35A)
Tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylate (7D-1) (0.51 g, 1.89 mmol), 1-cyclopropyl-2-fluoro-4-methoxy-5-nitrobenzene (2E) (0.40 g, 1.89 mmol) and potassium carbonate (0.78 g, 5.67 mmol) were mixed and dissolved in DMSO (15 mL), and the mixture was stirred at 120° C. for 3 h, cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 35A (340 mg, yield: 39%).
LCMS m/z=461.3 [M+H]+.
Step 2: 1-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)piperazine (compound 35B)
35A (0.34 g, 0.74 mmol) was dissolved in DCM (15 mL); trifluoroacetic acid (5 mL) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. 30 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 35B (265 mg), which was directly used in the next step.
LCMS m/z=361.3 [M+H]+.
Step 3: tert-butyl 3-((4-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidine-1-carboxylate (compound 35C)
35B (0.27 g, 0.74 mmol) and tert-butyl 3-formylpyrrolidine-1-carboxylate (0.18 g, 0.89 mmol) were dissolved in dichloromethane (15 mL); acetic acid (0.09 g, 1.48 mmol) and sodium triacetoxyborohydride (0.31 g, 1.48 mmol) were successively added; and the mixture was reacted at room temperature for 2 h, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 for removing impurities, dichloromethane/methanol (V/V)=20/1 for collecting a product) to obtain 35C (400 mg, yield: 99%).
LCMS m/z=544.3 [M+H]+.
Step 4: 1-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl)-4-(pyrrolidin-3-ylmethyl)piperazine (35D)
35C (0.40 g, 0.74 mmol) was dissolved in DCM (15 mL); trifluoroacetic acid (5 mL) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. 30 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 35D (305 mg), which was directly used in the next step.
LCMS m/z=444.3 [M+H]+.
Step 5: 5-(3-((4-(1-(2-cyclopropyl-5-methoxy-4-nitrophenyl)piperidin-4-yl) piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (35E)
35D (0.31 g, 0.69 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (0.19 g, 0.69 mmol) were dissolved in DMSO (10 mL); DIPEA (0.27 g, 2.07 mmol) was added dropwise; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 35E (365 mg, yield: 76%).
LCMS m/z=700.3 [M+H]+.
Step 6: 5-(3-((4-(1-(4-amino-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl) piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (35F)
35E (0.18 g, 0.26 mmol) was dissolved in ethanol (15 mL); reduced iron powder (0.07 g, 1.30 mmol) was added, and then an aqueous solution (5 mL) of ammonium chloride (0.07 g, 1.30 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 3 h. The reaction solution was cooled to room temperature. 5 mL of water was added, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 35F (170 mg), which was directly used in the next step.
LCMS m/z=670.3 [M+H]+.
Step 7: 5-(3-((4-(1-(4-((5-bromo-4-((5-(dimethylphosphoryl)quinoxalin-6-yl)amino)pyrimidin-2-yl)amino)-2-cyclopropyl-5-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 35)
35F (0.17 g, 0.25 mmol) and 2J (0.10 g, 0.25 mmol) were dissolved in DMF (12 mL); p-toluenesulfonic acid hydrate (0.14 g, 0.75 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 35 (80 mg, yield: 31%).
LCMS m/z=523.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.65 (s, 1H), 11.04 (s, 1H), 8.89-8.74 (m, 3H), 8.26 (s, 1H), 8.24 (s, 1H), 7.87 (d, 1H), 7.64 (d, 1H), 6.94-6.83 (m, 2H), 6.81 (dd, 1H), 6.75 (s, 1H), 5.05 (dd, 1H), 3.76 (s, 3H), 3.59-3.45 (m, 2H), 3.44-3.35 (m, 1H), 3.34-3.22 (m, 2H), 3.17-3.09 (m, 1H), 2.93-2.83 (m, 1H), 2.76-2.52 (m, 9H), 2.48-2.27 (m, 7H), 2.17-2.07 (m, 2H), 2.06-1.95 (m, 7H), 1.93-1.82 (m, 2H), 1.80-1.71 (m, 1H), 1.68-1.57 (m, 2H), 0.79-0.64 (m, 2H), 0.46-0.31 (m, 2H).
Step 1: 5-(4-((1′-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 36)
13F (315 mg, 0.44 mmol) and 23D (224 mg, 0.51 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (251 mg, 1.32 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 36 (110 mg, yield: 22%).
LCMS m/z=561.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.05 (s, 1H), 8.44-8.33 (m, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.74-7.61 (m, 2H), 7.61-7.50 (m, 3H), 7.49-7.33 (m, 3H), 7.30 (d, 1H), 7.36 (s, 1H), 7.27-7.12 (m, 2H), 6.83 (s, 1H), 5.06 (dd, 1H), 4.09-3.97 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.15-3.03 (m, 2H), 3.03-2.81 (m, 5H), 2.64-2.52 (m, 3H), 2.18-2.07 (m, 2H), 2.05-1.96 (m, 1H), 1.89-1.75 (m, 10H), 1.75-1.65 (m, 2H), 1.34-1.04 (m, 10H).
Step 1: tert-butyl 9-((1-((benzyloxy)carbonyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (37A)
The compound benzyl 4-formylpiperidine-1-carboxylate (6.0 g, 24.26 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (8.0 g, 31.45 mmol) were dissolved in DMAC (100 mL); ten drops of acetic acid was added dropwise (by a 3 mL disposable plastic dropper); and then the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (7.7 g, 36.33 mmol) was added, and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed sequentially with water (200 mL×2) and saturated brine (200 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 37A (10 g, yield: 85%).
LCMS m/z=486.4 [M+H]+.
Step 2: tert-butyl 9-(piperidin-4-ylmethyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (37B)
37A (4.2 g, 8.65 mmol) and palladium on carbon (2.5 g, 10 wt %) were successively added and dissolved in 60 mL of methanol. The mixture was subjected to hydrogen replacement three times, reacted at room temperature for 2 h and subjected to suction filtration over celite. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=25/1-10/1) to obtain 37B (2.8 g, yield: 92%).
LCMS m/z=352.3 [M+H]+.
Step 3: tert-butyl 9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (37C)
37B (1.6 g, 4.55 mmol) and 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (1.49 g, 5.93 mmol) were dissolved in DMSO (20 mL); sodium bicarbonate (1.53 g, 18.2 mmol) was added; and the mixture was reacted at 100° C. for 6 h, cooled to room temperature and extracted by adding 50 mL of ethyl acetate and 30 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 37C (1.4 g, yield: 53%).
LCMS m/z=583.3 [M+H]+.
Step 4: 3-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecane(37D); HCl
37C (1.2 g, 2.06 mmol) was dissolved in methanol (4 mL); a solution of hydrochloric acid in dioxane (2 N, 15 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione (37E)
The crude hydrochloride of 37D from the previous step was dissolved in DMSO (20 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (683 mg, 2.47 mmol) and sodium bicarbonate (692 mg, 8.24 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 37E (1.4 g, yield: 92%).
LCMS m/z=739.3 [M+H]+.
Step 6: 5-(9-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (37F)
37E (1.0 g, 1.35 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (375 mg, 6.71 mmol) and ammonium chloride (361 mg, 6.75 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 37F (700 mg, yield: 73%).
LCMS m/z=709.4 [M+H]+.
Step 7: 5-(9-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 37)
37F (200 mg, 0.28 mmol) and 23D (148 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 37 (84 mg, yield: 27%).
LCMS m/z=1108.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.05 (s, 1H), 8.45-8.34 (m, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.86 (s, 1H), 7.74-7.62 (m, 2H), 7.61-7.49 (m, 3H), 7.48-7.39 (m, 2H), 7.38-7.27 (m, 2H), 7.25-7.10 (m, 2H), 6.85 (s, 1H), 5.06 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.53-3.39 (m, 4H), 3.12-2.99 (m, 2H), 2.95-2.80 (m, 1H), 2.64-2.52 (m, 4H), 2.43-2.28 (m, 4H), 2.27-2.17 (m, 2H), 2.07-1.95 (m, 1H), 1.83 (d, 6H), 1.76-1.66 (m, 2H), 1.62-1.43 (m, 9H), 1.35-1.23 (m, 2H).
Step 1: 5-(9-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 38)
10G (0.08 g, 0.11 mmol) and 23D (0.05 g, 0.11 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid hydrate (0.06 g, 0.33 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% trifluoroacetic acid)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 38 (30 mg, yield: 25%).
LCMS m/z=554.9 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.05 (s, 1H), 8.43-8.36 (m, 1H), 8.28 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.87 (s, 1H), 7.76-7.62 (m, 2H), 7.62-7.48 (m, 3H), 7.47-7.39 (m, 2H), 7.37-7.28 (m, 2H), 7.28-7.21 (m, 1H), 7.20-7.12 (m, 1H), 6.82 (s, 1H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.55-3.33 (m, 4H), 3.15-3.00 (m, 2H), 2.94-2.83 (m, 1H), 2.70-2.51 (m, 4H), 2.49-2.07 (m, 6H), 2.04-1.95 (m, 1H), 1.85 (s, 3H), 1.81 (s, 3H), 1.78-1.25 (m, 13H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (39A)
The hydrochloride of 9D (820 mg) and 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisodihydroindole-1,3-dione (5A-1) (557 mg, 1.89 mmol) were dissolved in DMSO (10 mL); N,N-diisopropylethylamine (413 mg, 3.2 mmol) was added; and the mixture was reacted at 90° C. for 6 h, cooled to room temperature and extracted by adding 60 mL of ethyl acetate and 60 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-15/1) to obtain 39A (0.91 g, yield: 76%).
LCMS m/z=757.3 [M+H]+.
Step 2: 5-(4-((9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro [5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (39B)
39A (910 mg, 1.2 mmol) was dissolved in methanol/water (8 mL, 3:1); iron powder (910 mg, 17 mmol) and ammonium chloride (66.9 mg, 17.8 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 39B (345 mg, yield: 39%).
LCMS m/z=727.4 [M+H]+.
Step 3: 5-(4-((9-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 39)
39B (154 mg, 0.21 mmol) and 23D (99 mg, 0.23 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (119.8 mg, 0.63 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain the crude. The resulting crude was separated and purified by preparative liquid phase chromatography to obtain compound 39 (35 mg, yield: 15%).
LCMS m/z=563.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.23-10.92 (m, 2H), 8.45-8.34 (m, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.74-7.62 (m, 2H), 7.60-7.49 (m, 3H), 7.48-7.39 (m, 3H), 7.39-7.31 (m, 1H), 7.26-7.11 (m, 1H), 6.89 (s, 1H), 5.09 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.66-3.54 (m, 2H), 2.99-2.69 (m, 7H), 2.65-2.51 (m, 2H), 2.40-2.25 (m, 4H), 2.22-2.12 (m, 2H), 2.07-1.98 (m, 1H), 1.90-1.64 (m, 9H), 1.62-1.39 (m, 8H), 1.32-1.23 (m, 2H).
Step 1: (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A)
23C (4.3 g, 17.5 mmol) and 2,4,5-trichloropyrimidine (6.4 g, 34.9 mmol) were dissolved in 50 mL of NMP; DIPEA (2.7 g, 20.9 mmol) was added; and the mixture was stirred at 130° C. for 3 h and cooled to room temperature. 80 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V)=10/1-1/10) to obtain 40A (5.7 g, yield: 83%).
LCMS m/z=392.0 [(M+2H)/2]+.
Step 2: 5-(4-((4-(4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 40)
12I (0.20 g, 0.28 mmol) and 40A (0.18 g, 0.46 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 40 (12 mg, yield: 4%).
LCMS m/z=540.0 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.05 (s, 1H), 8.54-8.45 (m, 1H), 8.26 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.74-7.68 (m, 1H), 7.67-7.60 (m, 2H), 7.59-7.52 (m, 2H), 7.49-7.42 (m, 2H), 7.39-7.33 (m, 1H), 7.30 (d, 1H), 7.23 (dd, 1H), 7.20-7.13 (m, 1H), 6.86 (s, 1H), 5.06 (dd, 1H), 4.09-3.98 (m, 2H), 3.80 (s, 3H), 3.75 (s, 3H), 3.02-2.81 (m, 9H), 2.69-2.53 (m, 6H), 2.26-2.09 (m, 3H), 2.06-1.97 (m, 1H), 1.93-1.73 (m, 13H), 1.49-1.41 (m, 2H), 1.17-1.11 (m, 2H).
5-(4-((1′-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[4,4′-bipiperidin]-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 41)
13F (235 mg, 0.33 mmol) and 40A (157 mg, 0.4 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (188 mg, 0.99 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain compound 41 (82 mg, yield: 23%).
LCMS m/z=539.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 11.05 (s, 1H), 8.54-8.42 (m, 1H), 8.27 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.87 (s, 1H), 7.71 (dd, 1H), 7.65 (d, 1H), 7.61-7.52 (m, 3H), 7.48-7.33 (m, 3H), 7.30 (d, 1H), 7.26-7.12 (m, 2H), 6.84 (s, 1H), 5.06 (dd, 1H), 4.10-3.97 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.15-3.04 (m, 2H), 3.03-2.79 (m, 5H), 2.64-2.52 (m, 4H), 2.17-1.97 (m, 3H), 1.89-1.65 (m, 14H), 1.37-1.05 (m, 9H).
5-(4-((4-(4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 42)
12I (0.20 g, 0.28 mmol) and 23D (0.17 g, 0.39 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 42 (12 mg, yield: 4%).
LCMS m/z=562.4 [(M+2H)/2]+.
5-(4-((1′-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 43)
8I (180 mg, 0.249 mmol) and 23D (109 mg, 0.25 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (142 mg, 0.747 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 43 (84 mg). 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 43, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 43 (60 mg, yield: 21%).
LCMS m/z=1123.4 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.05 (s, 1H), 8.42-8.35 (m, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.72-7.65 (m, 2H), 7.59-7.52 (m, 3H), 7.47-7.42 (m, 2H), 7.39-7.35 (m, 1H), 7.34-7.31 (m, 1H), 7.25 (dd, 1H), 7.22-7.15 (m, 1H), 6.83 (s, 1H), 5.07 (dd, 1H), 3.77 (d, 6H), 3.48-3.39 (m, 4H), 3.13-3.06 (m, 2H), 2.95-2.87 (m, 2H), 2.64-2.53 (m, 4H), 2.49-2.45 (m, 3H), 2.20-2.12 (m, 4H), 2.04-1.97 (m, 2H), 1.83 (d, 6H), 1.78-1.71 (m, 4H), 1.62-1.48 (m, 5H), 1.17-1.14 (m, 2H).
5-(2-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 44)
1H (142 mg, 0.2 mmol) and 23D (99 mg, 0.23 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane, and saturated sodium bicarbonate solution (50 ml) was added. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 44 (34 mg, yield: 15.3%).
LCMS m/z=554.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.04 (s, 1H), 8.47-8.33 (m, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.87 (s, 1H), 7.75-7.50 (m, 5H), 7.48-7.38 (m, 2H), 7.36-7.26 (m, 2H), 7.24-7.06 (m, 2H), 6.83 (s, 1H), 5.05 (dd, 1H), 3.77 (s, 3H), 3.75 (s, 3H), 3.56-3.39 (m, 4H), 3.14-3.00 (m, 2H), 2.97-2.81 (m, 1H), 2.75-2.51 (m, 6H), 2.41-2.11 (m, 4H), 2.04-1.96 (m, 1H), 1.83 (d, 6H), 1.76-1.44 (m, 9H), 1.40-1.23 (m, 4H).
Step 1: benzyl 4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)piperazine-1-carboxylate (45B)
45A (6.0 g, 27.24 mmol) and tert-butyl 4-formylpiperidine-1-carboxylate (11.62 g, 54.48 mmol) were dissolved in DMAc (50 mL); acetic acid (3.27 g, 54.45 mmol) was added; and the mixture was stirred at room temperature for 2 h. Sodium triacetoxyborohydride (17.32 g, 81.72 mmol) was added, and the mixture was reacted overnight at room temperature. After the reaction was completed, 100 mL of ethyl acetate and 200 mL of water were added. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 45B as a white solid (6.0 g, yield: 53%).
LCMS m/z=418.3 [M+H]+.
Step 2: benzyl 4-(piperidin-4-ylmethyl)piperazine-1-carboxylate (45C); HCl
45B (3.0 g, 7.18 mmol) was dissolved in 5 mL of methanol; a solution of hydrogen chloride in dioxane (1 M, 30 mL) was added to the reaction system; and the mixture was reacted at room temperature for 1 h. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the hydrochloride of 45C (2.24 g), which was directly used in the next reaction.
LCMS m/z=318.2 [M+H]+.
Step 3: benzyl 4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl) methyl)piperazine-1-carboxylate (45D)
The crude hydrochloride of 45C (2.24 g) was dissolved in DMSO (20 mL); solid potassium carbonate (8.13 g, 58.8 mmol) was added in batches; and the mixture was stirred for 5 min. The compound 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (1.48 g, 5.89 mmol) was added, and the mixture was reacted overnight at 100° C. After the reaction was completed, the mixture was cooled to room temperature and extracted with 50 mL of ethyl acetate and 200 mL of water. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 45D (2.28 g, yield: 58%).
LCMS m/z=549.9 [M+H]+.
Step 4: 2-methoxy-5-(1-methyl-1H-pyrazol-4-yl)-4-(4-(piperazin-1-ylmethyl) piperidin-1-yl)aniline (45E)
Compound 45D (2.28 g, 4.16 mmol) was dissolved in 20 mL of methanol; palladium on carbon (44 mg) was added; and the mixture was subjected to hydrogen replacement three times. Under hydrogen atmosphere, the mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was subjected to suction filtration, and the filtrate was concentrated under reduced pressure to obtain the crude of 45E (1.6 g).
LCMS m/z=385.3 [M+H]+.
Step 5: 5-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (45F)
The crude of 45E (800 mg) was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (1.14 g, 4.13 mmol) and DIPEA (538 mg, 4.16 mmol) were successively added; and the mixture was reacted at 100° C. for 3 h, and cooled to room temperature. 10 mL of water was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 45F as a yellow solid (1.0 g, yield: 75%).
LCMS m/z=641.3 [M+H]+.
1H NMR (400 MHz, CD3OD) δ 7.98 (s, 1H), 7.89 (s, 1H), 7.68 (d, 1H), 7.36 (d, 1H), 7.22 (dd, 1H), 6.87 (s, 1H), 6.74 (s, 1H), 5.06 (dd, 1H), 3.90 (s, 3H), 3.85 (s, 3H), 3.53-3.42 (m, 4H), 3.08-2.96 (m, 2H), 2.91-2.69 (m, 3H), 2.69-2.54 (m, 6H), 2.41-2.28 (m, 2H), 2.15-2.06 (m, 1H), 1.90-1.78 (m, 2H), 1.77-1.61 (m, 1H), 1.43-1.24 (m, 2H).
Step 6: 5-(4-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 45)
45F (200 mg, 0.31 mmol) and 23D (203 mg, 0.465 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (177 mg, 0.93 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 45 (60 mg). 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 45, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 45 (45 mg, yield: 14%).
LCMS m/z=1040.3 [M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1))δ 8.30 (s, 1H), 8.13-8.00 (m, 2H), 7.89-7.68 (m, 4H), 7.68-7.55 (m, 4H), 7.55-7.43 (m, 2H), 7.43-7.20 (m, 3H), 5.13 (dd, 1H), 4.25-4.09 (m, 2H), 4.06 (s, 3H), 3.94-3.64 (m, 6H), 3.61-3.46 (m, 5H), 3.39-3.12 (m, 4H), 2.96-2.85 (m, 2H), 2.83-2.67 (m, 1H), 2.57-2.38 (m, 1H), 2.30-2.11 (m, 3H), 2.09-1.85 (m, 8H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((4-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (46A)
12G (0.3 g, 0.60 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1) (0.25 g, 0.85 mmol) were dissolved in DMSO (10 mL); DIPEA (0.21 g, 1.62 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 20 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-15/1) to obtain 46A (0.31 g, yield: 67%)
LCMS m/z=772.4 [M+H]+.
Step 2: 5-(4-((4-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (46B)
46A (0.31 g, 0.40 mmol) was dissolved in ethanol (12 mL) and water (3 mL), and the mixture was warmed to 80° C. A mixture of ammonium chloride (0.22 g, 4.11 mmol) and iron powder (0.23 g, 4.12 mmol) was added. At this temperature, the resulting mixture was stirred for 0.5 h, cooled to room temperature and filtered. The filter cake was washed with 50 mL of dichloromethane. 10 mL of saturated brine was added to the filtrate. Liquid separation was performed. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude of 46B (0.19 g), which was directly used in the next step.
LCMS m/z=742.3 [M+H]+.
Step 3: 5-(4-((4-(4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 46)
The crude of 46B (0.15 g) and 23D (0.12 g, 0.27 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane, and saturated sodium bicarbonate solution (50 ml) was added. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 46 (26 mg, yield: 7%).
LCMS m/z=571.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.08 (s, 1H), 8.39 (s, 1H), 8.26 (s, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.73-7.70 (m, 1H), 7.68 (s, 1H), 7.59 (s, 1H), 7.55 (d, 2H), 7.48-7.43 (m, 3H), 7.36 (t, 1H), 7.18 (m, 1H), 6.85 (s, 1H), 5.10 (dd, 1H), 3.78 (d, 3H), 3.75 (s, 3H), 3.61 (d, 2H), 2.92-2.83 (m, 8H), 2.66-2.56 (m, 5H), 2.54-2.52 (m, 1H), 2.25-2.14 (m, 3H), 2.09-2.00 (m, 1H), 1.94-1.72 (m, 13H), 1.48-1.39 (m, 2H), 1.33-1.20 (m, 3H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione (47A)
The crude hydrochloride of 37D (333 mg) was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1) (265 mg, 0.90 mmol) and sodium bicarbonate (232 mg, 2.76 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 47A (420 mg).
Step 2: 5-(9-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (47B)
47A (420 mg, 0.55 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (154 mg, 2.75 mmol) and ammonium chloride (147 mg, 2.75 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 47B (380 mg, yield: 95%).
LCMS m/z=727.4 [M+H]+.
Step 3: 5-(9-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 47)
47B (200 mg, 0.28 mmol) and 23D (148 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 47 (48 mg, yield: 15%).
LCMS m/z=563.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.08 (s, 1H), 8.44-8.32 (m, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.86 (s, 1H), 7.75-7.64 (m, 2H), 7.60-7.50 (m, 3H), 7.49-7.39 (m, 3H), 7.39-7.30 (m, 1H), 7.27-7.10 (m, 1H), 6.85 (s, 1H), 5.10 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.28-3.19 (m, 4H), 3.14-2.99 (m, 2H), 2.95-2.82 (m, 1H), 2.68-2.53 (m, 4H), 2.42-2.30 (m, 4H), 2.28-2.16 (m, 2H), 2.08-1.97 (m, 1H), 1.83 (d, 6H), 1.77-1.65 (m, 2H), 1.65-1.44 (m, 9H), 1.34-1.23 (m, 2H).
Step 1: 5-(9-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 48)
37F (235 mg, 0.33 mmol) and 40A (157 mg, 0.4 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (188 mg, 0.99 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane, and saturated sodium bicarbonate solution (50 ml) was added. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 48 (82 mg, yield: 23%).
LCMS m/z=1064.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.05 (s, 1H), 8.49 (s, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.71 (dd, 1H), 7.65 (d, J=8.5 Hz, 1H), 7.62-7.50 (m, 3H), 7.44 (t, 2H), 7.39-7.31 (m, 1H), 7.33-7.26 (m, 1H), 7.26-7.12 (m, 2H), 6.85 (s, 1H), 5.06 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.55-3.40 (m, 4H), 3.07 (d, 2H), 2.98-2.81 (m, 1H), 2.70-2.53 (m, 4H), 2.36 (s, 4H), 2.29-2.17 (m, 2H), 2.07-1.95 (m, 1H), 1.84 (d, 6H), 1.72 (d, 2H), 1.64-1.45 (m, 9H), 1.38-1.20 (m 3H).
Step 1: 5-(2-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-9-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 49)
1H (142 mg, 0.2 mmol) and (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A) (90 mg, 0.23 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 49 (50 mg, yield: 23.5%).
LCMS m/z=532.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.05 (s, 1H), 8.56-8.40 (m, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.88 (s, 1H), 7.70 (d, 1H), 7.65-7.49 (m, 4H), 7.42 (t, 2H), 7.35-7.24 (m, 2H), 7.22-7.07 (m, 2H), 6.84 (s, 1H), 5.05 (dd, 1H), 3.77 (s, 3H), 3.75 (s, 3H), 3.58-3.35 (m, 4H), 3.14-3.02 (m, 2H), 2.94-2.82 (m, 1H), 2.73-2.52 (m, 4H), 2.42-2.11 (m, 6H), 2.05-1.93 (m, 1H), 1.84 (d, 6H), 1.78-1.46 (m, 9H), 1.45-1.17 (m, 4H).
Step 1: 5-(4-((4-(4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 50)
46B (0.15 g, 0.20 mmol) and 40A (0.13 g, 0.33 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 50 (30 mg, yield: 14%).
LCMS m/z=549.4 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.08 (s, 1H), 8.54-8.44 (m, 1H), 8.26 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.75-7.66 (m, 2H), 7.61 (s, 1H), 7.58-7.51 (m, 2H), 7.50-7.40 (m, 3H), 7.40-7.33 (m, 1H), 7.21-7.09 (m, 1H), 6.86 (d, 1H), 5.10 (dd, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.67-3.55 (m, 2H), 3.00-2.77 (m, 10H), 2.70-2.52 (m, 6H), 2.18-2.11 (m, 2H), 2.06-1.99 (m, 1H), 1.96-1.65 (m, 13H), 1.50-1.35 (m, 2H), 1.31-1.19 (m, 2H).
Step 1: 5-(4-((9-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 51)
39B (100 mg, 0.138 mmol) and (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A) (56 mg, 0.143 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (79 mg, 0.413 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain the crude. The resulting crude was separated and purified by preparative liquid phase chromatography to obtain compound 51 (18 mg, yield: 12%).
LCMS m/z=541.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.44-11.32 (m, 1H), 11.21-10.93 (m, 1H), 8.54-8.39 (m, 1H), 8.28 (s, 1H), 8.10 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.75-7.63 (m, 2H), 7.61-7.50 (m, 3H), 7.49-7.40 (m, 3H), 7.39-7.30 (m, 1H), 7.28-7.13 (m, 1H), 6.89 (s, 1H), 5.10 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.65-3.56 (m, 2H), 2.95-2.74 (m, 7H), 2.64-2.52 (m, 2H), 2.37-2.26 (m, 4H), 2.22-2.12 (m, 2H), 2.09-1.98 (m, 1H), 1.87-1.77 (m, 8H), 1.76-1.68 (m, 1H), 1.59-1.45 (m, 8H), 1.31-1.22 (m, 2H).
Step 1: 5-(9-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 52)
47B (200 mg, 0.28 mmol) and 40A (133 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane, and saturated sodium bicarbonate solution (50 ml) was added. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 52 (56 mg, yield: 18%).
LCMS m/z=1082.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.08 (s, 1H), 8.57-8.39 (m, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.76-7.64 (m, 2H), 7.63-7.50 (m, 3H), 7.50-7.40 (m, 3H), 7.40-7.32 (m, 1H), 7.23-7.11 (m, 1H), 6.85 (s, 1H), 5.10 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.28-3.16 (m, 4H), 3.13-3.03 (m, 2H), 2.95-2.82 (m, 1H), 2.68-2.52 (m, 4H), 2.44-2.29 (m, 4H), 2.29-2.17 (m, 2H), 2.08-1.98 (m, 1H), 1.84 (d, 6H), 1.79-1.68 (m, 2H), 1.63-1.47 (m, 9H), 1.35-1.25 (m, 3H).
Step 1: 5-(4-((1′-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 53)
8I (180 mg, 0.249 mmol) and 40A (98 mg, 0.249 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (142 mg, 0.747 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 53 (160 mg). 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 53, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 53 (120 mg, yield: 45%).
LCMS m/z=1079.5 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.05 (s, 1H), 8.52-8.45 (m, 1H), 8.26 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.87 (s, 1H), 7.74-7.66 (m, 2H), 7.61-7.53 (m, 3H), 7.48-7.43 (m, 2H), 7.39-7.36 (m, 1H), 7.35-7.33 (m, 1H), 7.25 (dd, 1H), 7.20-7.14 (m, 1H), 6.83 (s, 1H), 5.07 (dd, 1H), 3.77 (d, 6H), 3.48-3.40 (m, 4H), 3.14-3.09 (m, 2H), 2.99-2.91 (m, 2H), 2.63-2.55 (m, 3H), 2.49-2.46 (m, 3H), 2.21-2.13 (m, 4H), 2.03-1.96 (m, 2H), 1.84 (d, 6H), 1.80-1.72 (m, 4H), 1.66-1.42 (m, 6H), 1.17-1.13 (m, 2H).
Step 1: (2-amino-5-bromophenyl)dimethylphosphine oxide (54A)
4-bromo-2-iodoaniline (0.5 g, 1.68 mmol), dimethylphosphine oxide (0.13 g, 1.68 mmol), Xant-Phos (4,5-bisdiphenylphosphine-9,9-dimethylxanthene, 49 mg, 0.084 mmol) and anhydrous potassium phosphate (0.46 g, 2.18 mmol) were successively added to 10 mL of 1,4-dioxane, and palladium acetate (19 mg, 0.084 mmol) was added under stirring. The mixture was subjected to nitrogen replacement three times, stirred at 80° C. for 5 h, cooled to room temperature and filtered. The filter cake was washed with 20 mL of ethyl acetate. The filtrates were combined, and concentrated under reduced pressure. The residue was slurried by adding 5 mL of methyl tert-butyl ether and filtered. The filter cake was dried to obtain 54A (0.3 g, yield: 72%).
LCMS m/z=248.1 [M+H]+.
Step 2: (4-amino-2′-fluoro-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (54B)
54A (0.3 g, 1.21 mmol) and 2-fluorophenylboronic acid (0.34 g, 2.42 mmol) were dissolved in 10 mL of dioxane and 2 mL of water, and then Pd(dppf)Cl2·DCM (44 mg, 0.06 mmol) and potassium carbonate (670 mg, 4.84 mmol) were added. The mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 2 h and cooled to room temperature. The reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 54B (140 mg, yield: 44%).
LCMS m/z=264.1 [M+H]+.
Step 3: (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-2′-fluoro-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (54C)
54B (0.14 g, 0.53 mmol) and 5-bromo-2,4,-dichloropyrimidine (0.24 g, 1.06 mmol) were dissolved in 5 mL of NMP; DIPEA (82 mg, 0.64 mmol) was added; and the mixture was stirred at 120° C. for 2 h and cooled to room temperature. 8 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V)=10/1-1/10) to obtain 54C (0.16 g, yield: 66%).
1H NMR (400 MHz, CDCl3) δ 11.47 (s, 1H), 8.72-8.66 (m, 1H), 8.36 (s, 1H), 7.75 (d, 1H), 7.52-7.46 (m, 1H), 7.46-7.40 (m, 1H), 7.39-7.33 (m, 1H), 7.28-7.13 (m, 2H), 1.88 (d, 6H).
Step 4: 5-(4-((4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 54)
4D (250 mg, 0.35 mmol) and 54B (190 mg, 0.42 mmol) were dissolved in a mixed solution of 1,4-dioxane (4 mL) and NMP (1 mL); p-toluenesulfonic acid monohydrate (200 mg, 1.05 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 54. 20 mL of dichloromethane and 10 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 54, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 54 (120 mg, yield: 30%).
LCMS m/z=571.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 11.05 (s, 1H), 8.47-8.37 (m, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 7.98 (s, 1H), 7.82 (s, 1H), 7.71-7.57 (m, 2H), 7.53 (s, 1H), 7.48-7.37 (m, 2H), 7.35-7.02 (m, 5H), 6.80 (s, 1H), 5.06 (dd, 1H), 4.12-3.95 (m, 2H), 3.77 (s, 3H), 3.76 (s, 3H), 3.15-3.04 (m, 2H), 3.03-2.92 (m, 2H), 2.92-2.82 (m, 1H), 2.68-2.51 (m, 8H), 2.46-2.26 (m, 4H), 2.22-2.09 (m, 3H), 2.05-1.94 (m, 1H), 1.91-1.68 (m, 11H), 1.59-1.43 (m, 2H), 1.21-1.08 (m, 2H).
Step 1: (2-amino-5-cyclopropylphenyl)dimethylphosphine oxide (55A)
54A (2.3 g, 9.27 mmol) and cyclopropyl boronic acid (2.39 g, 27.81 mmol) were dissolved in 20 mL of dioxane and 4 mL of water, and then tricyclohexylphosphine (0.52 g, 1.85 mmol), palladium acetate (0.21 g, 0.93 mmol) and potassium phosphate (7.87 g, 37.08 mmol) were added. The mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 24 h and cooled to room temperature. The reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 55A (1.26 g, yield: 65%).
1H NMR (400 MHz, CDCl3) δ 6.94 (d, 1H), 6.88-6.80 (m, 1H), 6.63-6.57 (m, 1H), 1.82-1.71 (m, 7H), 0.91-0.83 (m, 2H), 0.59-0.51 (m, 2H).
Step 2: (2-((5-bromo-2-chloropyrimidin-4-yl)amino)-5-cyclopropylphenyl) dimethylphosphine oxide (55B)
Compound 55A (0.26 g, 1.24 mmol) and 5-bromo-2,4,-dichloropyrimidine (0.57 g, 2.48 mmol) were dissolved in 5 mL of NMP; DIPEA (190 mg, 1.49 mmol) was added; and the mixture was stirred at 120° C. for 2 h and cooled to room temperature. 8 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V)=10/1-0/10). The residue was slurried with a mixed solvent of ethyl acetate/petroleum ether (10 mL, V/V=1/2) and filtered. The filter cake was dried under reduced pressure to obtain 55B (0.28 g, yield: 56%).
1H NMR (400 MHz, CDCl3) δ 11.20 (s, 1H), 8.45-8.39 (m, 1H), 8.30 (s, 1H), 7.24-7.19 (m, 1H), 7.06-7.00 (m, 1H), 1.93-1.79 (m, 7H), 1.05-0.96 (m, 2H), 0.78-0.65 (m, 2H).
Step 3: 5-(4-((4-(1-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 55)
4D (200 mg, 0.28 mmol) and 55B (130 mg, 0.33 mmol) were dissolved in DMF (4 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 55. 20 mL of dichloromethane and 10 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 55, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 55 (90 mg, yield: 29%).
LCMS m/z=544.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.84 (s, 1H), 8.19-8.05 (m, 3H), 7.96 (s, 1H), 7.86 (s, 1H), 7.65 (d, 1H), 7.57 (s, 1H), 7.32-7.10 (m, 3H), 6.79 (s, 1H), 6.55-6.34 (m, 1H), 5.06 (dd, 1H), 4.11-3.95 (m, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 3.18-3.06 (m, 2H), 3.02-2.81 (m, 3H), 2.70-2.51 (m, 8H), 2.45-2.30 (m, 4H), 2.29-2.09 (m, 3H), 2.05-1.96 (m, 1H), 1.89-1.68 (m, 12H), 1.64-1.50 (m, 2H), 1.21-1.05 (m, 2H), 0.96-0.86 (m, 2H), 0.57-0.43 (m, 2H).
Step 1: Methyl 5-(4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)picolinate (56A)
4B (0.22 g, 0.44 mmol) and methyl 5-fluoropyridine-2-carboxylate (0.08 g, 0.52 mmol) were dissolved in DMSO (10 mL); potassium carbonate (0.15 mg, 1.09 mmol) was added; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 40 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was then extracted by adding 50 ml of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 56A (0.18 g, yield: 65%).
LCMS m/z=633.3 [M+H]+.
Step 2: 5-(4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)picolinic acid (56B)
56A (0.18 g, 0.28 mmol) was dissolved in a mixed solution of tetrahydrofuran (12 mL) and water (4 mL); lithium hydroxide monohydrate (0.05 g, 1.19 mmol) was added; and the mixture was reacted at room temperature for 4 h. After the reaction was completed, the reaction solution was adjusted to pH=7 by adding dilute hydrochloric acid (2 mol/L) and concentrated under reduced pressure to obtain the crude of 56B (0.18 g), which was directly used in the next step.
LCMS m/z=619.3 [M+H]+.
Step 3: N-(2,6-dioxopiperidin-3-yl)-5-(4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)picolinamide (56C)
56B (0.18 g) and 3-aminopiperidine-2,6-dione hydrochloride (0.06 g, 0.36 mmol) were dissolved in DMF (10 mL); DIPEA (0.10 g, 0.77 mmol) and HATU (0.14 g, 0.37 mmol) were added; and the mixture was reacted at room temperature for 3 h. After the reaction was completed, 40 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was then extracted by adding 50 ml of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-10/1) to obtain 56C (0.15 g, two-step yield: 74%).
LCMS m/z=729.4 [M+H]+.
Step 4: 5-(4-((4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (56D)
56C (0.15 g, 0.21 mmol) was dissolved in ethanol (9 mL) and water (3 mL); iron powder (0.04 g, 0.72 mmol) was added, and then ammonium chloride (0.04 g, 0.75 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 3 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. 20 ml of saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted twice with dichloromethane (20 ml). The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the crude of 56D as a white solid (0.12 g), which was directly used in the next step.
LCMS m/z=699.4 [M+H]+.
Step 5: 5-(4-((4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 56)
56D (0.12 g) and 23D (0.08 g, 0.18 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (0.09 g, 0.47 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 56 (8 mg, yield: 3%).
LCMS m/z=549.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 10.82 (s, 1H), 8.68 (d, 1H), 8.39 (s, 1H), 8.31-8.25 (m, 2H), 8.18 (s, 1H), 8.00 (s, 1H), 7.87-7.80 (m, 2H), 7.70 (dd, 1H), 7.60-7.51 (m, 3H), 7.49-7.33 (m, 4H), 7.23-7.14 (m, 1H), 6.83 (s, 1H), 4.82-4.65 (m, 1H), 3.93 (d, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.10 (d, 2H), 2.86 (t, 2H), 2.80-2.73 (m, 1H), 2.64-2.52 (m, 6H), 2.45-2.32 (m, 4H), 2.23-2.12 (m, 4H), 2.04-1.97 (m, 1H), 1.89-1.75 (m, 10H), 1.60-1.48 (m, 3H), 1.22-1.18 (m, 3H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (57A)
The hydrochloride of compound 9D (820 mg) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (523 mg, 1.9 mmol) were dissolved in DMSO (20 mL); N,N-diisopropylethylamine (612 mg, 4.74 mmol) was added; and the mixture was reacted at 90° C. for 6 h, cooled to room temperature and extracted by adding 60 mL of ethyl acetate and 60 mL of water. The organic layer was washed three times with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-15/1) to obtain 57A (0.39 g, yield: 33%).
LCMS m/z=739.4 [M+H]+.
Step 2: 5-(4-((9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (57B)
57A (390 mg, 0.53 mmol) was dissolved in methanol/water (8 mL, 3:1); iron powder (390 mg, 6.98 mmol) and ammonium chloride (390 mg, 7.29 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 57B (270 mg, yield: 72%).
LCMS m/z=709.3 [M+H]+.
Step 3: 5-(4-((9-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 57)
57B (134 mg, 0.19 mmol) and 23D (90 mg, 0.21 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (119.8 mg, 0.63 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain the crude, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 57 (22 mg, yield: 10.5%).
LCMS m/z=554.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.17-10.96 (m, 2H), 8.47-8.34 (m, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.73-7.62 (m, 2H), 7.60-7.49 (m, 3H), 7.44 (t, 2H), 7.36 (t, 1H), 7.33-7.10 (m, 3H), 6.89 (s, 1H), 5.06 (dd, 1H), 4.12-3.95 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.06-2.71 (m, 7H), 2.68-2.52 (m, 2H), 2.44-2.26 (m, 4H), 2.24-2.10 (m, 2H), 2.06-1.92 (m, 1H), 1.90-1.73 (m, 9H), 1.59-1.44 (m, 8H), 1.23-1.09 (m, 2H).
Step 1: 5-(4-((4-(1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione; (compound 58)
4D (87 mg, 0.12 mmol) and 40A (56 mg, 0.14 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (68 mg, 0.36 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated. Suction filtration was performed for collecting the solid. The residue was purified sequentially by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1) and preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a preparative solution, which was concentrated, and then 20 mL of dichloromethane and 3 mL of saturated sodium bicarbonate solution were added to the concentrated solution. The organic phases were separated. The aqueous phase was extracted with dichloromethane (10 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 58 (15 mg, yield: 11.6%).
LCMS m/z=540.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 11.05 (s, 1H), 8.54-8.44 (m, 1H), 8.27 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H), 7.71 (dd, 1H), 7.65 (d, 1H), 7.59 (s, 1H), 7.58-7.51 (m, 2H), 7.45 (t, 2H), 7.40-7.34 (m, 1H), 7.30 (s, 1H), 7.26-7.20 (m, 1H), 7.20-7.12 (m, 1H), 6.84 (s, 1H), 5.06 (dd, 5.4 Hz, 1H), 4.10-3.98 (m, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.11 (d, 3H), 3.03-2.82 (m, 5H), 2.68-2.53 (m, 6H), 2.25-2.08 (m, 4H), 2.07-1.95 (m, 2H), 1.88-1.74 (m, 10H), 1.63-1.45 (m, 3H), 1.21-1.07 (m, 3H).
5-(4-((9-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 59)
57B (134 mg, 0.19 mmol) and (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A) (81 mg, 0.21 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (119.8 mg, 0.63 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain the crude, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 59 (25 mg, yield: 12.4%).
LCMS m/z=532.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.20-10.75 (m, 1H), 8.61-8.39 (m, 1H), 8.29 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.76-7.61 (m, 2H), 7.59-7.50 (m, 3H), 7.45 (t, 2H), 7.36 (t, 1H), 7.32-7.12 (m, 3H), 6.89 (s, 1H), 5.06 (dd, 1H), 4.14-3.93 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.05-2.73 (m, 7H), 2.69-2.51 (m, 2H), 2.43-2.22 (m, 4H), 2.22-2.06 (m, 2H), 2.05-1.94 (m, 1H), 1.89-1.72 (m, 9H), 1.65-1.45 (m, 8H), 1.20-1.06 (m, 2H).
Step 1: benzyl 9-(1-(tert-butoxycarbonyl)piperidin-4-yl)-2,9-diazaspiro [5.5]undecane-2-carboxylate (60A)
Benzyl 2,9-diazaspiro[5.5]undecane-2-carboxylate (2.60 g, 9.02 mmol), N-BOC-piperidone (2.16 g, 10.82 mmol), anhydrous sodium sulfate (2.56 g, 18.04 mmol) and acetic acid (1.08 g, 18.04 mmol) were successively added to dichloromethane (20 ml), and sodium triacetoxyborohydride (3.82 g, 18.04 mmol) was added under stirring at room temperature. After the addition, the mixture was stirred overnight at room temperature, adjusted to pH>8 with 1 N aqueous sodium hydroxide solution and extracted by adding 30 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 60A (4.3 g, yield: 98%).
LCMS m/z=472.3 [M+1]+.
Step 2: benzyl 9-(piperidin-4-yl)-2,9-diazaspiro[5.5]undecane-2-carboxylate (60B)
60B (4.50 g, 9.54 mmol) was dissolved in DCM (30 ml); trifluoroacetic acid (15.30 g, 134.23 mmol) was added; and the mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was adjusted to pH=9-10 by adding 1 N aqueous NaOH solution. The organic layer was concentrated under reduced pressure to obtain a product, which was directly used in the next step.
Step 3: benzyl 9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)-2,9-diazaspiro[5.5]undecane-2-carboxylate (60C)
4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (1.70 g, 6.77 mmol), compound 60B (3.27 g, 8.80 mmol) and potassium carbonate (3.74 g, 27.08 mmol) were added to DMSO (20 ml), and the mixture was stirred at 120° C. for 6 h, cooled to room temperature and extracted by adding 100 mL of ethyl acetate and 100 mL of water. The organic layer was washed with saturated brine (50 mL×3), and concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 60C (2.3 g, yield: 56%).
LCMS m/z=603.3 [M+1]+.
Step 4: 9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)-2,9-diazaspiro[5.5]undecane (60D)
60C (2.20 g, 3.65 mmol) was dissolved in acetic acid (6 ml); a solution of hydrogen bromide in acetic acid (10 mL, wt %=33%) was added at room temperature; and the mixture was stirred for 0.5 h. 30 mL of MTBE was added, with a large amount of solids precipitated. The mixture was filtered. The filter cake was dissolved in 20 mL of water and extracted with 20 mL of ethyl acetate. The organic layer was discarded. The aqueous phase was extracted by adding 30 mL of dichloromethane and adjusted to pH>8 with 1 N aqueous sodium hydroxide solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 60D, which was directly used in the next step.
LCMS m/z=469.3 [M+1]+.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (60E)
60D (0.4 g, 0.85 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (0.26 g, 0.94 mmol) were dissolved in DMSO (5 ml); DIPEA (0.22 g, 1.7 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 10 mL of water was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography to obtain 60E (0.45 g, yield: 73%).
Step 6: 5-(9-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (60F)
60E (400 mg, 0.55 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (310 mg, 5.5 mmol) and ammonium chloride (290 mg, 5.5 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was extracted by adding 20 mL of dichloromethane and 20 mL of saturated aqueous sodium chloride solution. The organic layer was dried and then concentrated under reduced pressure to obtain 60F (380 mg), which was directly used in the next step.
Step 7: 5-(9-(1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro [5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 60)
60F (140 mg, 0.20 mmol) and 40A (78 mg, 0.20 mmol) were dissolved in a mixed solution of 1,4-dioxane (4 mL) and NMP (1 mL); p-toluenesulfonic acid monohydrate (110 mg, 0.60 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 60. 20 mL of dichloromethane and 10 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 60, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 60 (60 mg, yield: 28%).
LCMS m/z=525.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 11.05 (s, 1H), 8.65-8.32 (m, 1H), 8.26 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.71 (dd, 1H), 7.67-7.49 (m, 4H), 7.49-7.40 (m, 2H), 7.39-7.29 (m, 2H), 7.28-7.09 (m, 2H), 6.83 (s, 1H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.52-3.42 (m, 2H), 3.41-3.34 (m, 2H), 3.18-3.03 (m, 2H), 2.98-2.81 (m, 1H), 2.72-2.50 (m, 9H), 2.09-1.97 (m, 1H), 1.93-1.32 (m, 18H).
Step 1: 2-benzyl 9-(tert-butyl) 2,9-di azaspiro [5.5]undecane-2,9-di carboxylate (61A)
10A (5.00 g, 19.66 mmol) was dissolved in dichloromethane (50 mL); triethylamine (4.18 g, 41.29 mmol) was added; and then benzyl chloroformate (3.42 g, 20.05 mmol) was added dropwise under an ice bath. After the dropwise addition, the mixture was reacted at room temperature for 1 h. The reaction was quenched by adding water and extracted with 400 mL of ethyl acetate. The organic phases were collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=20/1-5/1) to obtain 61A (6.07 g, yield: 79%).
LCMS m/z=333.2 [M−55]+.
Step 2: benzyl 2,9-diazaspiro[5.5]undecane-2-carboxylate (61B)
61A (2.00 g, 5.15 mmol) was dissolved in dichloromethane (30 mL); trifluoroacetic acid (10 mL) was slowly added; and the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, redissolved by adding 50 mL of dichloromethane and adjusted to a basic pH with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 61B (1.35 g), which was directly used in the next step.
LCMS m/z=289.2 [M+H]+.
Step 3: benzyl 9-((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecane-2-carboxylate (61C)
61B (1.35 g) and tert-butyl 4-formylpiperidine-1-carboxylate (1.00 g, 4.68 mmol) were dissolved in dichloromethane (50 mL); acetic acid (0.56 g, 9.36 mmol) and sodium triacetoxyborohydride (1.98 g, 9.36 mmol) were successively added; and the mixture was reacted at room temperature for 2 h, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 for removing impurities, dichloromethane/methanol (V/V)=20/1 for collecting a product) to obtain 61C (2.07 g, yield: 91%).
LCMS m/z=486.4 [M+H]+.
Step 4: benzyl 9-(piperidin-4-ylmethyl)-2,9-diazaspiro[5.5]undecane-2-carboxylate (61D)
Compound 61C (2.07 g, 4.26 mmol) was dissolved in dichloromethane (30 mL); trifluoroacetic acid (10 mL) was slowly added; and the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, redissolved by adding 50 mL of dichloromethane and adjusted to a basic pH with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 61D (1.64 g), which was directly used in the next step.
LCMS m/z=386.3 [M+H]+.
Step 5: benzyl 9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl) methyl)-2,9-diazaspiro[5.5]undecane-2-carboxylate (61E)
61D (1.64 g, 4.25 mmol), 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (1.07 g, 4.25 mmol) and potassium carbonate (2.94 g, 21.25 mmol) were mixed and dissolved in DMSO (50 mL); and the mixture was stirred at 120° C. for 3 h, and cooled to room temperature. The reaction solution was diluted by adding 200 mL of ethyl acetate and washed 3 times with water. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 61E (640 mg, yield: 24%).
LCMS m/z=617.3 [M+H]+.
Step 6: 4-(4-((2,9-diazaspiro[5.5]undecan-9-yl)methyl)piperidin-1-yl)-2-methoxy-5-(1-methyl-1H-pyrazol-4-yl)aniline (61F)
61E (0.64 g, 1.04 mmol) was dissolved in a mixed solvent of isopropanol (20 mL) and ammonia methanol solution (5 mL); palladium on carbon (wt %=10%, 0.33 g) was added; and the mixture was subjected to 1 atm hydrogen replacement 3 times, reacted at room temperature for 1.5 h and filtered over celite. The filter cake was washed with dichloromethane/methanol (V/V=10/1), and the filtrate was concentrated under reduced pressure to obtain 61F (450 mg), which was directly used in the next step.
LCMS m/z=453.4 [M+H]+.
Step 7: 5-(9-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (61G)
61F (0.23 g, 0.50 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1) (0.15 g, 0.50 mmol) were dissolved in DMSO (10 mL); DIPEA (0.19 g, 1.50 mmol) was added dropwise; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-10/1) to obtain 61G (167 mg, yield: 46%).
LCMS m/z=727.3 [M+H]+.
Step 8: 5-(9-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 61)
61G (0.08 g, 0.11 mmol) and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (0.05 g, 0.11 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid hydrate (0.06 g, 0.33 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% trifluoroacetic acid)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 61 (15 mg, yield: 12%).
LCMS m/z=563.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.08 (s, 1H), 8.43-8.35 (m, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.85 (s, 1H), 7.75-7.62 (m, 2H), 7.60-7.51 (m, 3H), 7.50-7.39 (m, 3H), 7.33 (t, 1H), 7.25-7.07 (m, 1H), 6.84 (s, 1H), 5.10 (dd, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.24-3.13 (m, 2H), 3.12-2.97 (m, 4H), 2.94-2.80 (m, 1H), 2.69-2.52 (m, 4H), 2.45-2.14 (m, 6H), 2.08-1.95 (m, 1H), 1.83 (d, 6H), 1.76-1.63 (m, 4H), 1.63-1.38 (m, 7H), 1.35-1.24 (m, 2H).
Step 1: 5-(9-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro[5.5] undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 62)
60F (140 mg, 0.20 mmol) and 23D (87 mg, 0.20 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (110 mg, 0.60 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 62. 20 mL of dichloromethane and 10 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 62, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 62 (30 mg, yield: 14%).
LCMS m/z=547.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 11.05 (s, 1H), 8.47-8.32 (m, 1H), 8.26 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.86 (s, 1H), 7.69 (d, 1H), 7.63 (d, 1H), 7.60-7.48 (m, 3H), 7.45 (t, 2H), 7.40-7.07 (m, 4H), 6.82 (s, 1H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.56-3.37 (m, 4H), 3.17-3.04 (m, 2H), 2.95-2.81 (m, 1H), 2.72-2.51 (m, 9H), 2.05-1.95 (m, 1H), 1.92-1.74 (m, 8H), 1.74-1.35 (m, 10H).
Step 1: 5-(2-(4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 63)
7G (0.15 g, 0.20 mmol) and 23D (0.10 g, 0.23 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (0.09 g, 0.47 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-12/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 63 (30 mg, yield: 13%).
LCMS m/z=575.4 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (d, 1H), 11.05 (s, 1H), 8.39 (s, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.85 (s, 1H), 7.69 (dd, 1H), 7.64 (d, 1H), 7.59-7.52 (m, 3H), 7.45 (t, 2H), 7.39-7.34 (m, 1H), 7.31 (d, 1H), 7.23 (dd, 1H), 7.21-7.13 (m, 1H), 6.83 (s, 1H), 5.06 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.50-3.44 (m, 2H), 3.41-3.36 (m, 2H), 3.29-3.27 (m, 1H), 3.09 (d, 2H), 2.92-2.84 (m, 1H), 2.72-2.65 (m, 1H), 2.63-2.51 (m, 6H), 2.36-2.16 (m, 5H), 2.04-1.95 (m, 3H), 1.87-1.75 (m, 8H), 1.67-1.47 (m, 9H).
Step 1: 1-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)piperazine (64A)
45D (1.4 g, 2.55 mmol) was dissolved in 5 mL of acetic acid; 10 mL of HBr-AcOH was added; and the mixture was stirred at room temperature for 30 min. The reaction system was poured into 40 mL of water, with a solid precipitated, and the mixture was filtered. The filter cake was dissolved in 150 ml of dichloromethane, washed with aqueous sodium bicarbonate solution (100 mL×2), dried over anhydrous sodium sulfate and concentrated to obtain 64A (1.0 g, yield: 95.2%).
LCMS m/z=415.3 [M+H]+.
Step 2: methyl 5-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl) methyl) piperazin-1-yl)picolinate (64B)
64A (1.0 g, 2.41 mmol) was added to 30 mL of dimethyl sulfoxide; methyl 5-fluoropyridinecarboxylate (0.56 g, 3.61 mmol) and triethylamine (1.22 g, 12.08 mmol) were added; and the mixture was reacted at 90° C. for 3 h, and cooled to room temperature. 100 mL of water was added, with a solid precipitated, and the mixture was filtered. The filter cake was dissolved in dichloromethane, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to an oil, and the residue was purified by column chromatography (dichloromethane/methanol=10/1 (V/V) as a mobile phase) to obtain 64B (1.0 g, yield: 81%).
LCMS m/z=550.3 [M+1]+.
Step 3: 5-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)piperazin-1-yl)picolinic acid (64C)
64B (1.0 g, 1.82 mmol) was added to 40 mL of tetrahydrofuran and 4 mL of water; lithium hydroxide monohydrate (153 mg, 3.64 mmol) was added; and the mixture was reacted at room temperature for 18 h. The system was adjusted to a neutral pH with 1 N HCl and concentrated under reduced pressure to obtain 64C (1.2 g, crude).
LCMS m/z=536.3 [M+1]+.
Step 4: N-(2,6-dioxopiperidin-3-yl)-5-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)piperazin-1-yl)picolinamide (64D)
64C (1.2 g, crude) was added to 30 mL of DMF; 3-aminopiperidine-2,6-dione hydrochloride (0.36 g, 2.20 mmol), DIPEA (1.64 g, 12.7 mmol) and HATU (1.04 g, 2.74 mmol) were added; and the mixture was reacted at room temperature for 16 h. 120 mL of purified water was added, with a solid precipitated. The mixture was subjected to suction filtration and dried, and the residue was purified by column chromatography (dichloromethane/methanol=10/1 (V/V) as a mobile phase) to obtain 64D (1.0 g, yield: 87.5%)
LCMS m/z=646.2 [M+1]+.
Step 5: 5-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)piperazin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (64E)
64D (1.0 g, 1.55 mmol) was dissolved in a mixed solution of THE (10 mL), ethanol (30 mL) and water (6 mL); reduced iron powder (875 mg, 15.63 mmol) and ammonium chloride (830 mg, 15.63 mmol) were added; and the mixture was reacted at 85° C. for 2 h. The reaction solution was cooled to room temperature and concentrated in vacuo to remove a solvent. 50 mL of water and 50 mL of dichloromethane were added to the residue, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 64E (0.7 g, yield: 73%)
LCMS m/z=616.3 [M+H]+.
Step 6: 5-(4-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 64)
64E (350 mg, 0.57 mmol) and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (373 mg, 0.85 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (325 mg, 1.71 mmol) was added; and the mixture was stirred at 100° C. for 8 h, and cooled to room temperature. 200 mL of saturated aqueous sodium bicarbonate solution was added, with a white solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (100 ml×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (100 ml×3). The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude was subjected to prep-TLC (DCM: MeOH (V/V)=15:1) purification to obtain compound 64 (200 mg, yield: 35%).
LCMS m/z=1015.3 [M+1]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 10.84 (s, 1H), 8.72 (d, 1H), 8.46-8.24 (m, 3H), 8.19 (s, 1H), 8.01 (s, 1H), 7.88 (t, 2H), 7.70 (dd, 1H), 7.63-7.50 (m, 3H), 7.49-7.40 (m, 3H), 7.39-7.31 (m, 1H), 7.25-7.12 (m, 1H), 6.85 (s, 1H), 4.81-4.70 (m, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.44-3.33 (m, 4H), 3.14-3.02 (m, 2H), 2.86-2.73 (m, 1H), 2.71-2.51 (m, 7H), 2.29 (d, 2H), 2.25-2.13 (m, 1H), 2.07-2.00 (m, 1H), 1.83 (d, 6H), 1.79-1.70 (m, 2H), 1.69-1.54 (m, 1H), 1.39-1.24 (m, 2H).
Step 1: 5-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 65)
45F (250 mg, 0.39 mmol) and 40A (153 mg, 0.39 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (223 mg, 1.17 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 65 (63 mg, yellow solid). The trifluoroacetate of compound 65 was extracted by adding 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 65 as a yellow solid (45 mg, yield: 12%).
LCMS m/z=996.4 [M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.27-8.14 (m, 2H), 8.10 (s, 1H), 7.88-7.69 (m, 4H), 7.65-7.55 (m, 4H), 7.55-7.42 (m, 2H), 7.38-7.22 (m, 3H), 5.12 (dd, 1H), 4.22-4.08 (m, 2H), 4.05 (s, 3H), 3.89-3.69 (m, 6H), 3.61-3.47 (m, 5H), 3.37-3.13 (m, 4H), 2.95-2.85 (m, 2H), 2.82-2.65 (m, 1H), 2.56-2.39 (m, 1H), 2.30-2.13 (m, 3H), 2.08-1.86 (m, 8H).
Step 1: 5-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 66)
64E (350 mg, 0.57 mmol) and (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A) (336 mg, 0.85 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (325 mg, 1.71 mmol) was added; and the mixture was stirred at 100° C. for 8 h, and cooled to room temperature. 200 mL of saturated aqueous sodium bicarbonate solution was added, with a white solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (100 ml×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (100 ml×3). The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude was subjected to prep-TLC (DCM: MeOH (V/V)=15:1) purification to obtain compound 66 (230 mg, yield: 41%).
LCMS m/z=971.3 [M+1]+.
1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 10.84 (s, 1H), 8.72 (d, 1H), 8.58-8.42 (m, 1H), 8.33 (d, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 8.03 (s, 1H), 7.88 (d, 2H), 7.71 (dd, 1H), 7.64-7.50 (m, 3H), 7.49-7.39 (m, 3H), 7.39-7.29 (m, 1H), 7.27-7.05 (m, 1H), 6.86 (s, 1H), 4.82-4.67 (m, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.46-3.33 (m, 4H), 3.15-3.01 (m, 2H), 2.86-2.72 (m, 1H), 2.70-2.51 (m, 7H), 2.29 (d, 2H), 2.25-2.10 (m, 1H), 2.10-1.97 (m, 1H), 1.84 (d, 6H), 1.80-1.69 (m, 2H), 1.69-1.54 (m, 1H), 1.40-1.25 (m, 2H).
Step 1: tert-butyl 3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)methyl)pyrrolidine-1-carboxylate (67A)
The hydrochloride (1.0 g) of 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazine (compound 80B) and tert-butyl 3-formylpyrrolidine-1-carboxylate (0.8 g, 4.02 mmol) were dissolved in DMAc (50 mL); acetic acid (1.0 g, 16.65 mmol) was added; and the mixture was stirred at room temperature for 2 h. Sodium triacetoxyborohydride (1.0 g, 4.72 mmol) was added, and the mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was washed by adding 100 mL of ethyl acetate and 200 mL of saturated aqueous sodium bicarbonate solution. Liquid separation was performed. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 67A as a yellow solid (1.1 g, yield: 70%).
LCMS m/z=501.2 [M+H]+.
Step 2: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4-(pyrrolidin-3-ylmethyl)piperazine (67B); HCl
Compound 67A (1.1 g, 2.20 mmol) was dissolved in methanol (14 mL); a solution of hydrochloric acid in dioxane (4 N, 30 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)isoindoline-1,3-dione (67C)
The crude hydrochloride of 67B from the previous step was dissolved in DMSO (20 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (683 mg, 2.47 mmol) and sodium bicarbonate (692 mg, 8.24 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 67C (0.95 g, two-step yield: 66%).
LCMS m/z=657.3 [M+H]+.
Step 4: 5-(3-((4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (67D)
67C (0.95 g, 1.45 mmol) was dissolved in ethanol/water (20 mL, 3:1); iron powder (375 mg, 6.71 mmol) and ammonium chloride (361 mg, 6.75 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 67D (700 mg, yield: 77%).
LCMS m/z=627.3 [M+H]+.
Step 5: 5-(3-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 67)
67D (200 mg, 0.32 mmol) and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (148 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 67 (40 mg, yield: 12%).
LCMS m/z=513.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.04 (s, 1H), 8.45-8.34 (m, 1H), 8.28 (s, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.70 (dd, 1H), 7.65 (d, 1H), 7.60 (s, 1H), 7.55 (d, 2H), 7.45 (t, 2H), 7.33 (t, 1H), 7.23-7.11 (m, 1H), 6.91 (d, 1H), 6.87 (s, 1H), 6.82 (dd, 1H), 5.05 (dd, 1H), 3.78 (d, 6H), 3.64-3.48 (m, 2H), 3.48-3.37 (m, 1H), 3.18 (dd, 1H), 3.00-2.77 (m, 5H), 2.71-2.52 (m, 7H), 2.47-2.40 (m, 2H), 2.21-2.09 (m, 1H), 2.07-1.95 (m, 1H), 1.90-1.73 (m, 7H).
Step 1: 5-(3-((4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 68)
67D (200 mg, 0.32 mmol) and (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A) (180 mg, 0.46 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 68 (30 mg, yield: 10%).
LCMS m/z=491.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.05 (s, 1H), 8.54-8.45 (m, 1H), 8.27 (s, 1H), 8.12 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.71 (dd, 1H), 7.65 (d, 1H), 7.62 (s, 1H), 7.55 (d, 2H), 7.45 (t, 2H), 7.34 (t, 1H), 7.21-7.10 (m, 1H), 6.90 (d, 1H), 6.88 (s, 1H), 6.81 (dd, 1H), 5.05 (dd, 1H), 3.78 (d, 6H), 3.61-3.48 (m, 2H), 3.48-3.37 (m, 1H), 3.18 (dd, 1H), 2.97-2.82 (m, 5H), 2.69-2.52 (m, 7H), 2.47-2.40 (m, 2H), 2.21-2.10 (m, 1H), 2.05-1.96 (m, 1H), 1.90-1.73 (m, 7H).
Step 1: methyl 5-(9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)picolinate (69A)
37D (1.00 g, 2.07 mmol) was dissolved in DMSO (20 mL); methyl 5-fluoropyridine-2-carboxylate (388 mg, 2.50 mmol) and sodium carbonate (530 mg, 5.00 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 69A (0.95 g, yield: 74%).
LCMS m/z=618.3 [M+H]+.
Step 2: 5-(9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)picolinic acid (69B)
69A (0.95 g, 1.54 mmol) was dissolved in tetrahydrofuran (20 mL); water (2 mL) was added, and then lithium hydroxide monohydrate (126 mg, 3.00 mmol) was added. The mixture was heated to 50° C. and reacted for 4 h. After the reaction was completed, the mixture was adjusted to pH=7 by dropwise adding dilute hydrochloric acid (2 mol/L) and concentrated under reduced pressure, and the residue was directly used in the next step.
LCMS m/z=604.3 [M+H]+.
Step 3: N-(2,6-dioxopiperidin-3-yl)-5-(9-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)picolinamide (69C)
The crude of 69B from the previous step and 3-aminopiperidine-2,6-dione hydrochloride (0.30 g, 1.82 mmol) were dissolved in DMF (10 mL); DIPEA (0.50 g, 3.87 mmol) and HATU (0.70 g, 1.84 mmol) were added; and the mixture was reacted at room temperature for 3 h, and then extracted by adding 40 mL of saturated aqueous sodium bicarbonate solution and 50 ml of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-10/1) to obtain (69C) (0.73 g, two-step yield: 66%).
LCMS m/z=714.3 [M+H]+.
Step 4: 5-(9-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (69D)
69C (0.73 g, 1.02 mmol) was dissolved in ethanol/water (20 mL, 3:1); iron powder (375 mg, 6.71 mmol) and ammonium chloride (361 mg, 6.75 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 69D (600 mg, yield: 86%).
LCMS m/z=342.8 [(M+2H)/2]+.
Step 5: 5-(9-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 69)
69D (200 mg, 0.29 mmol) and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (148 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 69 (45 mg, yield: 14%).
LCMS m/z=542.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 10.82 (s, 1H), 8.68 (d, 1H), 8.42-8.35 (m, 1H), 8.30 (d, 2H), 8.18 (s, 1H), 8.01 (s, 1H), 7.87-7.82 (m, 2H), 7.69 (dd, 1H), 7.58-7.52 (m, 3H), 7.47-7.33 (m, 4H), 7.23-7.15 (m, 1H), 6.85 (s, 1H), 4.78-4.68 (m, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.42-3.32 (m, 4H), 3.06 (d, 2H), 2.84-2.73 (m, 1H), 2.64-2.52 (m, 3H), 2.36 (s, 4H), 2.24-2.13 (m, 3H), 2.05-1.98 (m, 1H), 1.83 (s, 3H), 1.81 (s, 3H), 1.71 (d 2H), 1.59-1.49 (m, 8H), 1.32-1.22 (m, 3H).
Step 1: 5-(9-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 70)
69D (200 mg, 0.29 mmol) and (4-((2,5-dichloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (40A) (180 mg, 0.46 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 70 (40 mg, yield: 13%).
LCMS m/z=520.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.82 (s, 1H), 8.68 (d, 1H), 8.51-8.45 (m, 1H), 8.32-8.27 (m, 2H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88-7.82 (m, 2H), 7.71 (dd, 1H), 7.60-7.53 (m, 3H), 7.47-7.32 (m, 4H), 7.21-7.10 (m, 1H), 6.85 (s, 1H), 4.78-4.68 (m, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.38-3.33 (m, 4H), 3.07 (d, 2H), 2.83-2.75 (m, 1H), 2.66-2.53 (m, 3H), 2.42-2.30 (m, 4H), 2.27-2.14 (m, 3H), 2.05-1.99 (m, 1H), 1.85 (s, 3H), 1.82 (s, 3H), 1.72 (d, 2H), 1.59-1.48 (m, 8H), 1.32-1.18 (m, 3H).
Step 1: 1-iodonaphthalen-2-amine (71B)
71A (2.00 g, 13.97 mmol) was dissolved in DMSO (30 mL), and NIS (3.14 g, 13.97 mmol) was added at room temperature. After the addition, the mixture was reacted at 20° C. for 2 h. The reaction solution was diluted by adding 50 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phases were collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=7/1) to obtain 71B (2.69 g, yield: 72%).
LCMS m/z=270.0 [M+H]+.
Step 2: (2-aminonaphthalen-1-yl)dimethylphosphine oxide (71C)
71B (2.68 g, 9.96 mmol) and dimethylphosphine oxide (0.78 g, 9.96 mmol) were dissolved in 1,4-dioxane (80 mL); 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (1.15 g, 1.99 mmol), palladium acetate (0.22 g, 1.00 mmol) and potassium phosphate (4.23 g, 19.92 mmol) were added; and the mixture was subjected to nitrogen replacement 3 times and reacted at 100° C. for 5 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain 71C (1.68 g, yield: 77%).
LCMS m/z=220.1 [M+H]+.
Step 3: (2-((5-bromo-2-chloropyrimidin-4-yl)amino)naphthalen-1-yl) dimethylphosphine oxide (71D)
71C (0.87 g, 3.97 mmol) and 5-bromo-2,4-dichloropyrimidine (1.81 g, 7.94 mmol) were dissolved in NMP (25 mL); DIPEA (1.03 g, 7.94 mmol) was added dropwise; and the mixture was reacted at 130° C. for 3 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain 71D (580 mg, yield: 36%).
LCMS m/z=410.0 [M+H]+.
Step 4: 5-(4-((4-(1-(4-((5-bromo-4-((1-(dimethylphosphoryl)naphthalen-2-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 71)
71D (0.17 g, 0.41 mmol) and 4D (0.30 g, 0.41 mmol) were dissolved in DMF (15 mL); p-toluenesulfonic acid hydrate (0.23 g, 1.23 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% trifluoroacetic acid)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 71 (30 mg, yield: 7%).
LCMS m/z=549.4[(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 11.05 (s, 1H), 8.33-8.24 (m, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 7.93 (d, 1H), 7.89-7.77 (m, 2H), 7.72-7.49 (m, 4H), 7.49-7.33 (m, 2H), 7.34-7.17 (m, 2H), 6.78 (s, 1H), 5.06 (dd, 1H), 4.15-3.99 (m, 2H), 3.79 (s, 3H), 3.72 (s, 3H), 3.16-3.02 (m, 2H), 3.03-2.81 (m, 3H), 2.69-2.51 (m, 8H), 2.45-2.18 (m, 5H), 2.14 (d, 2H), 2.09-1.92 (m, 7H), 1.92-1.73 (m, 5H), 1.63-1.43 (m, 2H), 1.22-1.06 (m, 2H).
Step 1: (2-((2,5-dichloropyrimidin-4-yl)amino)naphthalen-1-yl)dimethylphosphine oxide (72A)
71C (0.87 g, 3.97 mmol) and 2,4,5-trichloropyrimidine (1.46 g, 7.94 mmol) were dissolved in NMP (25 mL); DIPEA (1.03 g, 7.94 mmol) was added dropwise; and the mixture was reacted at 130° C. for 3 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain 72A (720 mg, yield: 50%).
LCMS m/z=366.0 [M+H]+.
Step 2: 5-(4-((4-(1-(4-((5-chloro-4-((1-(dimethylphosphoryl)naphthalen-2-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 72)
72A (0.15 g, 0.41 mmol) and 4D (0.30 g, 0.41 mmol) were dissolved in DMF (15 mL); p-toluenesulfonic acid hydrate (0.23 g, 1.23 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% trifluoroacetic acid)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 72 (40 mg, yield: 9%).
LCMS m/z=527.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 11.06 (s, 1H), 8.49-8.40 (m, 1H), 8.19-8.03 (m, 2H), 7.97-7.79 (m, 3H), 7.76-7.50 (m, 4H), 7.49-7.32 (m, 2H), 7.30 (d, 1H), 7.27-7.18 (m, 1H), 6.79 (s, 1H), 5.06 (dd, 1H), 4.11-3.97 (m, 2H), 3.80 (s, 3H), 3.72 (s, 3H), 3.14-2.81 (m, 5H), 2.70-2.51 (m, 8H), 2.48-2.18 (m, 5H), 2.14 (d, 2H), 2.10-1.93 (m, 7H), 1.90-1.69 (m, 5H), 1.66-1.45 (m, 2H), 1.21-1.08 (m, 2H).
Step 1: 2-iodo-3,4-dimethyl-1-nitrobenzene (73A)
To a reaction flask were added 2,3-dimethyl-6-nitroaniline (6.0 g, 36.14 mmol) and hydrochloric acid (30 mL, 360.14 mmol). At 0° C., a solution of potassium nitrate (2.99 g, 43.33 mmol) in water (15 mL) was slowly added dropwise. At this temperature, the mixture was stirred for 1 h, and then a solution of potassium iodide (9.0 g, 54.22 mmol) in water (30 mL) was added. After the addition, the mixture was naturally warmed to room temperature and stirred for another 1 h. After the reaction was completed, 60 mL of water was added, and the mixture was extracted with ethyl acetate (60 mL×3). The organic layer was washed sequentially with saturated sodium thiosulfate solution (40 mL×2) and saturated brine (40 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether: ethyl acetate (V/V)=10/1) to obtain 73A (5.3 g, yield: 53%).
Step 2: (2,3-dimethyl-6-nitrophenyl)dimethylphosphine oxide (73B)
Under nitrogen protection, 73A (5.29 g, 19.09 mmol), dimethylphosphine oxide (1.94 g, 24.82 mmol), potassium phosphate (8.1 g, 38 mmol), palladium acetate (429 mg, 1.91 mmol) and 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (2.21 g, 3.82 mmol) were added to 1,4-dioxane (150 mL), and the mixture was reacted at 100° C. for 16 h. After the reaction was completed, the mixture was cooled to room temperature. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phase was washed with water (50 mL×2) and saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (methanol/dichloromethane (V/V)=1/50-1/20) to obtain 73B (2.7 g, yield: 62.3%).
LCMS m/z=228.1 [M+H]+.
Step 3: (6-amino-2,3-dimethylphenyl)dimethylphosphine oxide (73C)
73B (2.7 g, 11.88 mmol) was dissolved in ethanol/water (30 mL/10 mL); iron powder (5.31 g, 95 mmol) and ammonium chloride (5.08 mg, 95 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 3 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 73C (1.3 g, yield: 98.2%).
LCMS m/z=198.1 [M+H]+.
Step 4: (6-((5-bromo-2-chloropyrimidin-4-yl)amino)-2,3-dimethylphenyl) dimethylphosphine oxide (73D)
(6-amino-2,3-dimethylphenyl)dimethylphosphine oxide (73C) (2.5 g, 12.7 mmol) and 5-bromo-2,4-dichloropyrimidine (4.3 g, 19.0 mmol) were dissolved in DMF (20 mL); potassium carbonate (3.5 mg, 25.4 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, cooled to room temperature and extracted by adding 80 mL of ethyl acetate and 80 mL of water. The organic layer was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/dichloromethane (V/V)=1/50-1/20) to obtain 73D (3.5 g, yield: 71.2%).
LCMS m/z=388.0 [M+H]+.
Step 5: 5-(4-((4-(1-(4-((5-bromo-4-((2-(dimethylphosphoryl)-3,4-dimethylphenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 73)
4D (250 mg, 0.35 mmol) and 73D (160 mg, 0.42 mmol) were dissolved in 1,4-dioxane and NMP (4 mL/1 mL); p-toluenesulfonic acid monohydrate (200 mg, 1.05 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 30 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 73 (70 mg, yield: 18.6%).
LCMS m/z=1075.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 11.05 (s, 1H), 8.11 (s, 1H), 8.03-7.84 (m, 3H), 7.75 (s, 1H), 7.70-7.53 (m, 2H), 7.30 (s, 1H), 7.22 (dd, 1H), 6.85-6.56 (m, 2H), 5.06 (dd, 1H), 4.13-3.94 (m, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 3.15-3.03 (m, 2H), 2.96 (t, 2H), 2.91-2.80 (m, 1H), 2.72-2.51 (m, 8H), 2.46-2.31 (m, 4H), 2.28-2.19 (m, 4H), 2.18-2.10 (m, 2H), 2.07 (s, 3H), 2.04-1.95 (m, 1H), 1.93-1.71 (m, 11H), 1.65-1.42 (m, 2H), 1.21-1.03 (m, 2H).
Step 1: tert-butyl 4-(3-((2,6-dioxopiperidin-3-yl)amino)benzyl)piperazine-1-carboxylate (74B)
74A (synthesized with reference to patent WO 2017018803, 3 g, 10.30 mmol) and 3-bromopiperidine-2,6-dione (3.96 g, 20.6 mmol) were dissolved in DMSO (30 mL); sodium bicarbonate (4.33 g, 51.5 mmol) was added; and the mixture was reacted at 100° C. for 6 h, cooled to room temperature and extracted by adding 100 mL of ethyl acetate and 100 mL of water. The organic layer was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (ethyl acetate: petroleum ether (V/V)=10/1-2/1) to obtain 74B (2.57 g, yield: 62%).
LCMS m/z=403.1 [M+H]+.
Step 2: 3-((3-(piperazin-1-ylmethyl)phenyl)amino)piperidine-2,6-dione (74C); HCl
To a 50 mL round bottom flask were successively added 74B (1.06 g, 2.63 mmol) and a solution of hydrogen chloride in 1,4-dioxane (30 mL, 4 mol/L); and the mixture was reacted at room temperature for 2 h and concentrated to dryness under reduced pressure to obtain the hydrochloride of 74C, which was directly used in the next reaction.
Step 3: 3-((3-((4-(2-bromo-5-methoxy-4-nitrophenyl)piperazin-1-yl)methyl) phenyl)amino)piperidine-2,6-dione (74D)
1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C) (0.6 g, 2.40 mmol), the hydrochloride of 74C from the previous step and sodium bicarbonate (1.01 g, 12 mmol) were dissolved in 20 mL of DMSO, and the mixture was reacted at 100° C. for 3 h. After the reaction was completed, the mixture was cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The aqueous layer was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed sequentially with water (30 mL×2) and saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 74D (1.0 g, yield: 71%).
Step 4: 3-((3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperazin-1-yl)methyl)phenyl)amino)piperidine-2,6-dione (74E)
Under nitrogen protection, 74D (202 mg, 0.38 mmol) and 1-methyl-1H-pyrazole-4-boronic acid (95.7 mg, 0.76 mmol) were added to a 50 mL single-necked flask and dissolved in 20 mL of dioxane and 4 mL of water; Pd(dppf)Cl2·DCM (31 mg, 0.04 mmol) and sodium bicarbonate (96 mg, 1.14 mmol) were added; and the mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 3 h and cooled to room temperature. The reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 74E (190 mg, yield: 94%).
LCMS m/z=534.2 [M+H]+.
Step 5: 3-((3-((4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)methyl)phenyl)amino)piperidine-2,6-dione (74F)
74E (190 mg, 0.36 mmol) was dissolved in methanol (10 mL); palladium on carbon 10% (190 mg) was added at room temperature; and the mixture was reacted under hydrogen atmosphere for 2 h. After the reaction was completed, the mixture was subjected to suction filtration over celite. The filtrate was concentrated to obtain 74F, which was directly used in the next reaction.
LCMS m/z=504.3 [M+H]+.
Step 6: 3-((3-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)phenyl)amino)piperidine-2,6-dione (compound 74)
74F (101 mg, 0.2 mmol) from the previous step and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (105 mg, 0.24 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (114 mg, 0.6 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated. 50 mL of DCM was added, and then the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. The dichloromethane layers were separated, and the aqueous layer was extracted with dichloromethane (2×30 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 74 (15 mg, yield: 8.3%).
LCMS m/z=452.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 10.76 (s, 1H), 8.46-8.33 (m, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.87 (s, 1H), 7.76-7.64 (m, 1H), 7.58 (s, 1H), 7.56-7.46 (m, 2H), 7.44-7.25 (m, 3H), 7.15 (s, 1H), 7.06 (t, 1H), 6.86 (s, 1H), 6.68 (s, 1H), 6.64-6.53 (m, 2H), 5.80 (d, 1H), 4.38-4.28 (m, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.43 (s, 2H), 2.97-2.82 (m, 4H), 2.81-2.69 (m, 1H), 2.67-2.52 (m, 5H), 2.18-2.07 (m, 1H), 1.94-1.75 (m, 7H).
Step 1: 8-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-1,4-dioxa-8-azaspiro[4.5]decane (75A)
1,4-dioxa-8-azaspiro[4.5]decane (1.37 g, 9.56 mmol), 1D (1.2 g, 4.78 mmol) and potassium carbonate (3.30 g, 23.90 mmol) were mixed and dissolved in DMSO (30 mL), and the mixture was stirred at 120° C. for 5 h, cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1) to obtain 75A (1 g, yield: 56%).
LCMS m/z=375.1 [M+H]+.
Step 2: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-one (75B)
To a 50 mL round bottom flask were successively added 75A (1 g, 2.67 mmol) and an aqueous solution of acetone (30 mL) in hydrochloric acid (3 mL, 4 mol/L); and the mixture was reacted overnight at room temperature. 50 mL of DCM was added, and the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted with DCM (2×30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain 75B, which was directly used in the next reaction.
1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.90 (s, 1H), 7.27 (s, 1H), 6.65 (s, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.31 (t, 4H), 2.53 (t, 4H).
Step 3: (1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methanol (75C)
75B (787 mg, 2.38 mmol) and piperidin-4-ylmethanol (329 mg, 2.86 mmol) were mixed in dichloroethane (50 mL); acetic acid (143 mg, 2.38 mmol) was added; and the mixture was reacted at 50° C. for 2 h and cooled to room temperature. Sodium triacetoxyborohydride (1 g, 4.72 mmol) was added, and the mixture was stirred overnight at room temperature. 50 mL of dichloromethane and 50 mL of saturated aqueous sodium bicarbonate solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 75C (430 mg, yield: 42%).
LCMS m/z=430.2 [M+H]+.
Step 4: 1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidine]-4-carbaldehyde (75D)
75C (430 mg, 1.00 mmol) and Dess-Martin periodinane (1.27 g, 3 mmol) were dissolved in 20 mL of dichloromethane; and the mixture was reacted at room temperature for 2 h, concentrated and purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 75D (150 mg, yield: 35%).
LCMS m/z=428.3 [M+H]+.
Step 5: 3-(4-(1-((1′-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperidin-4-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (75F)
75E (synthesized with reference to patent WO 2021127283, 156 mg, 0.46 mmol) and 75D (150 mg, 0.35 mmol) were dissolved in DMAC (10 mL); a 4A molecular sieve (200 mg) and acetic acid (21 mg, 0.35 mmol) were successively added; and the mixture was reacted at 50° C. for 2 h and then cooled to room temperature. Sodium triacetoxyborohydride (148 mg, 0.70 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was completed, the reaction was quenched by adding 50 ml of water, adjusted to a basic pH by adding saturated sodium bicarbonate solution, extracted with ethyl acetate (3×50 ml), dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (DCM: MeOH: Et3N=10:1:0.01) to obtain 75F (100 mg, yield: 38%).
Step 6: 3-(4-(1-((1′-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperidin-4-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (75G)
75F (100 mg, 0.13 mmol) was dissolved in methanol (10 mL); palladium on carbon 10% (100 mg) was added at room temperature; and the mixture was reacted under hydrogen atmosphere for 2 h. After the reaction was completed, the mixture was subjected to suction filtration over celite. The filtrate was concentrated to obtain 75G, which was directly used in the next reaction.
Step 7: 3-(4-(1-((1′-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-[1,4′-bipiperidin]-4-yl)methyl)piperidin-4-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (compound 75)
75G (85 mg, 0.12 mmol) and 23D (63 mg, 0.14 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (68 mg, 0.36 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated. 30 mL of DCM was added, and then the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. The dichloromethane layers were separated, and the aqueous layer was extracted with dichloromethane (2×30 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 75 (5 mg, yield: 3.7%).
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 8.45-8.33 (s, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.70 (d, 1H), 7.62-7.50 (m, 3H), 7.50-7.41 (m, 3H), 7.40-7.33 (m, 1H), 7.20 (s, 1H), 7.08 (s, 1H), 6.95-6.87 (m, 2H), 6.83 (s, 1H). δ 4.85 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.46 (s, 3H), 3.10 (d, 2H), 2.99-2.86 (m, 4H), 2.68-2.34 (m, 5H), 2.30-2.05 (m, 6H), 2.05-1.91 (m, 2H), 1.85 (s, 3H), 1.81 (s, 3H), 1.79-1.65 (m, 8H), 1.65-1.42 (m, 4H), 1.18-1.03 (m, 2H).
Step 1: 5-(3-((4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 76)
6F (200 mg, 0.28 mmol) and 23D (150 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 76 (40 mg, yield: 13%).
LCMS m/z=553.9 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.04 (s, 1H), 8.43-8.34 (m, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.70 (dd, 1H), 7.64 (d, 1H), 7.59-7.51 (m, 3H), 7.45 (t, 2H), 7.36 (t, 1H), 7.23-7.14 (m, 1H), 6.90 (d, 1H), 6.82 (dd, 2H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.59-3.37 (m, 4H), 3.19-3.03 (m, 3H), 2.94-2.83 (m, 1H), 2.65-2.51 (m, 10H), 2.47-2.32 (m, 5H), 2.25-2.09 (m, 2H), 2.05-1.99 (m, 1H), 1.85-1.73 (m, 8H), 1.60-1.49 (m, 2H).
Step 1: tert-butyl 4-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl) methyl)piperidine-1-carboxylate (77A)
The hydrochloride (1.0 g) of 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazine (80B) and 1-tert-butoxycarbonylpiperidine-4-carbaldehyde (0.8 g, 3.75 mmol) were dissolved in DMAc (50 mL); acetic acid (1.0 g, 16.65 mmol) was added; and the mixture was stirred at room temperature for 2 h. Sodium triacetoxyborohydride (1.0 g, 4.72 mmol) was added, and the mixture was reacted overnight at room temperature. After the reaction was completed, 100 mL of ethyl acetate and 200 mL of saturated aqueous sodium bicarbonate solution were added. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain compound (77A) as a yellow solid (1.2 g, yield: 74%).
LCMS m/z=515.3 [M+H]+.
Step 2: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4-(piperidin-4-ylmethyl)piperazine (77B); HCl
77A (1.2 g, 2.33 mmol) was dissolved in methanol (14 mL); a solution of hydrochloric acid in dioxane (4 N, 30 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (77C)
The crude hydrochloride of 77B from the previous step was dissolved in DMSO (20 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (683 mg, 2.47 mmol) and sodium bicarbonate (692 mg, 8.24 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 77C (0.90 g, two-step yield: 58%).
LCMS m/z=671.3 [M+H]+.
Step 4: 5-(4-((4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (77D)
77C (0.90 g, 1.34 mmol) was dissolved in ethanol/water (20 mL, 3:1); iron powder (375 mg, 6.71 mmol) and ammonium chloride (361 mg, 6.75 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 77D (650 mg, yield: 76%).
LCMS m/z=641.3 [M+H]+.
Step 5: 5-(4-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 77)
77D (200 mg, 0.31 mmol) and 23D (150 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 77 (50 mg, yield: 15%).
LCMS m/z=520.7 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.28 (s, 1H), 8.25-8.03 (m, 6H), 7.83 (s, 1H), 7.75 (d, 1H), 7.63-7.45 (m, 5H), 7.43-7.27 (m, 1H), 6.98 (s, 1H), 5.23 (dd, 1H), 4.00-3.90 (m, 5H), 3.90-3.71 (m, 5H), 3.71-3.59 (m, 2H), 3.44-3.07 (m, 8H), 2.97-2.89 (m, 2H), 2.87-2.71 (m, 1H), 2.66-2.48 (m, 1H), 2.48-2.32 (m, 2H), 2.32-2.21 (m, 1H), 2.21-2.07 (m, 2H), 2.02 (d, 6H).
Step 1: tert-butyl 5-((1-((benzyloxy)carbonyl)piperidin-4-yl)methyl) hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (78A)
Benzyl 4-formylpiperidine-1-carboxylate (2.5 g, 10.11 mmol) and tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (3.58 g, 16.88 mmol) were dissolved in dichloromethane (100 mL); acetic acid (610 mg, 10.11 mmol) was added; and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (4.29 g, 20.22 mmol) was added; and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with water (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 78A (3.7 g, yield: 82.5%).
LCMS m/z=444.3 [M+H]+.
Step 2: tert-butyl 5-(piperidin-4-ylmethyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (78B)
78A (3.7 g, 8.34 mmol) and palladium on carbon (1.5 g, 10 wt %) were successively added and dissolved in 60 mL of methanol, and then the mixture was subjected to hydrogen replacement three times, reacted at room temperature for 3 h and subjected to suction filtration over celite. The filtrate was concentrated under reduced pressure to obtain 78B (2.5 g, yield: 96.8%).
Step 3: tert-butyl 5-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl) methyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (78C)
78B (2.46 g, 7.96 mmol) and 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (2.0 g, 7.96 mmol) were dissolved in DMSO (10 mL); anhydrous potassium carbonate (3.3 g, 23.88 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 50 mL of ethyl acetate and 30 mL of water. The organic layer was washed three times with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 78C (0.8 g, yield: 18%).
LCMS m/z=541.3 [M+H]+.
Step 4: 2-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)octahydropyrrolo[3,4-c]pyrrole (78D); HCl
78C (800 mg, 1.48 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (4 N, 4 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
LCMS m/z=441.3 [M+H]+.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(5-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)isoindoline-1,3-dione (78E)
The crude hydrochloride of 78D from the previous step was dissolved in DMSO (6 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (410 mg, 1.48 mmol) and DIPEA (1.15 g, 8.88 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 78E (318 mg, yield: 30.8%).
LCMS m/z=697.3 [M+H]+.
Step 6: 5-(5-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (78F)
Compound 78E (318 mg, 0.46 mmol) was dissolved in ethanol/water (9 mL/3 mL); iron powder (210 mg, 3.68 mmol) and ammonium chloride (200 mg, 3.68 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 3 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-20/1) to obtain 78F (240 mg, yield: 78%).
Step 7: 5-(5-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 78)
Compound 78F (120 mg, 0.18 mmol) and 23D (86 mg, 0.20 mmol) were dissolved in 1,4-dioxane (2 mL) and NMP (0.5 mL); p-toluenesulfonic acid monohydrate (93 mg, 0.54 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 78 (20 mg, yield: 10.4%).
LCMS m/z=533.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.24-10.92 (m, 2H), 8.50-8.32 (m, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 7.98 (s, 1H), 7.83 (s, 1H), 7.77-7.60 (m, 2H), 7.59-7.45 (m, 3H), 7.39 (t, 2H), 7.25 (t, 1H), 7.22-7.06 (m, 1H), 6.97 (d, 1H), 6.88 (dd, 1H), 6.83 (s, 1H), 5.05 (dd, 1H), 3.86-3.65 (m, 8H), 3.30-3.19 (m, 2H), 3.12-2.93 (m, 4H), 2.93-2.82 (m, 1H), 2.71-2.51 (m, 8H), 2.34 (d, 2H), 2.05-1.96 (m, 1H), 1.82 (d, 6H), 1.76-1.63 (m, 2H), 1.60-1.44 (m, 1H), 1.36-1.24 (m, 2H).
Step 1: tert-butyl 9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (79A)
Tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (1.27 g, 4.98 mmol), 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (1.5 g, 5.98 mmol) and potassium carbonate (2.75 g, 19.92 mmol) were mixed and dissolved in DMSO (10 mL), and the mixture was stirred at 120° C. for 16 h, cooled to room temperature and extracted by adding 50 mL of water and 50 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain tert-butyl 9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (79A) (2.0 g, yield: 83%).
LCMS m/z=486.3 [M+H]+.
Step 2: 3-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane (79B)
Compound 79A (2.0 g, 4.12 mmol) was dissolved in DCM (20 mL); trifluoroacetic acid (6 mL) was added at room temperature; and the mixture was stirred for 3 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain the compound 3-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecane (79B) (1.5 g), which was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione (79C)
Compound 79B (1.5 g, 3.89 mmol) was dissolved in DMSO (5 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (1.29 g, 4.67 mmol) and DIPEA (1.01 g, 7.78 mmol) were successively added; and the mixture was stirred at 90° C. for 3 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 2-(2,6-dioxopiperidin-3-yl)-5-(9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione (79C) (1.4 g, yield: 56.1%).
LCMS m/z=642.3 [M+H]+.
Step 4: 5-(9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (79D)
Compound 79C (1.4 g, 2.18 mmol), iron powder (0.97 mg, 17.44 mmol) and ammonium chloride (0.93 mg, 17.44 mmol) were dissolved in ethanol (30 mL) and water (10 mL), and the mixture was stirred at 80° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated to obtain the title compound 5-(9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (79D) (1.0 g), which was directly used in the next step.
LCMS m/z=612.3 [M+H]+.
Step 5: 5-(9-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 79)
79D (180 mg, 0.29 mmol) and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (150 mg, 0.35 mmol) were dissolved in 1,4-dioxane (3 mL) and NMP (1 mL); p-toluenesulfonic acid monohydrate (165 mg, 0.87 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 79 (22 mg, yield: 7.5%).
LCMS m/z=506.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 11.06 (s, 1H), 8.45-8.36 (m, 1H), 8.32 (s, 1H), 8.18 (s, 1H), 8.04 (s, 1H), 7.88 (s, 1H), 7.75-7.64 (m, 2H), 7.59-7.50 (m, 3H), 7.50-7.41 (m, 2H), 7.41-7.29 (m, 2H), 7.30-7.16 (m, 2H), 6.92 (s, 1H), 5.07 (dd, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.54-3.39 (m, 4H), 2.96-2.78 (m, 5H), 2.69-2.52 (m, 2H), 2.06-1.96 (m, 1H), 1.83 (d, 6H), 1.71-1.51 (m, 8H).
Step 1: tert-butyl 4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperazine-1-carboxylate (80A)
1D (0.8 g, 3.18 mmol) was dissolved in DMSO (20 mL); potassium carbonate (1.3 g, 9.41 mmol) and tert-butyl piperazine-1-carboxylate (712 mg, 3.82 mmol) were successively added; and the mixture was reacted at 100° C. for 6 h. After the reaction was completed, the mixture was extracted with 50 mL of ethyl acetate and 200 mL of water. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 80A (650 mg, yield: 49%).
LCMS m/z=418.2 [M+H]+.
Step 2: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazine (80B); HCl
80A (400 mg, 0.96 mmol) was dissolved in methanol (2 mL); a solution of hydrochloric acid in dioxane (4 N, 6 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)isoindoline-1,3-dione (80C)
The crude hydrochloride of 80B from the previous step was dissolved in DMSO (8 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (318 mg, 1.15 mmol) and sodium bicarbonate (323 mg, 3.84 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 80C (284 mg, two-step yield: 52%).
LCMS m/z=574.2 [M+H]+.
Step 4: 5-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (80D)
80C (284 mg, 0.50 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (140 mg, 2.5 mmol) and ammonium chloride (134 mg, 2.5 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 80D (260 mg, yield: 96%).
LCMS m/z=544.2 [M+H]+.
Step 5: 5-(4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 80)
80D (130 mg, 0.24 mmol) and (4-((5-bromo-2-chloropyrimidin-4-yl)amino)-[1,1′-biphenyl]-3-yl)dimethylphosphine oxide (23D) (135 mg, 0.31 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (137 mg, 0.72 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 80 (77 mg, yield: 34%).
LCMS m/z=943.2 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 11.07 (s, 1H), 8.38 (s, 1H), 8.29 (s, 1H), 8.20 (s, 1H), 8.15 (s, 1H), 7.88 (s, 1H), 7.77-7.63 (m, 3H), 7.55 (d, 2H), 7.48-7.40 (m, 2H), 7.41-7.25 (m, 3H), 7.18 (s, 1H), 6.87 (s, 1H), 5.09 (dd, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.67-3.54 (m, 4H), 3.05-2.92 (m, 4H), 2.92-2.83 (m, 1H), 2.68-2.54 (m, 2H), 2.06-1.94 (m, 1H), 1.83 (d, 6H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)isoindoline-1,3-dione (81A)
7E (1.0 g, 2.50 mmol) was dissolved in DMSO (20 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (828 mg, 3.00 mmol) and sodium bicarbonate (692 mg, 8.24 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 81A (0.90 g, yield: 55%).
LCMS m/z=657.3 [M+H]+.
Step 2: 5-(4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (81B)
81A (0.90 g, 1.37 mmol) was dissolved in ethanol/water (20 mL, 3:1); iron powder (375 mg, 6.71 mmol) and ammonium chloride (361 mg, 6.75 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 81B (700 mg, yield: 82%).
LCMS m/z=627.3 [M+H]+.
Step 3: 5-(4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 81)
81B (200 mg, 0.32 mmol) and 23D (150 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 81 (55 mg, yield: 17%).
LCMS m/z=513.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.06 (s, 1H), 8.44-8.33 (m, 1H), 8.29 (s, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.75-7.65 (m, 2H), 7.61-7.52 (m, 3H), 7.46 (t, 2H), 7.40-7.33 (m, 2H), 7.32-7.24 (m, 1H), 7.23-7.12 (m, 1H), 6.84 (s, 1H), 5.08 (dd, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.52-3.39 (m, 4H), 3.18-3.05 (m, 2H), 2.95-2.80 (m, 1H), 2.75-2.52 (m, 8H), 2.36-2.23 (m, 1H), 2.08-1.99 (m, 1H), 1.88-1.75 (m, 8H), 1.65-1.51 (m, 2H).
Step 1: tert-butyl 9-(4-((benzyloxy)carbonyl)piperazin-1-yl)-3-azaspiro[5.5] undecane-3-carboxylate (82B)
Tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate (82A) (1.50 g, 5.61 mmol) and benzyl piperazine-1-carboxylate (1.23 g, 5.58 mmol) were dissolved in DMAc (50 mL); acetic acid (1.0 g, 16.65 mmol) was added; and the mixture was stirred at room temperature for 2 h. Sodium triacetoxyborohydride (2.0 g, 9.44 mmol) was added, and the mixture was reacted overnight at room temperature. After the reaction was completed, 100 mL of ethyl acetate and 200 mL of saturated aqueous sodium bicarbonate solution were added. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=100/1-3/1) to obtain 82B as a white solid (1.6 g, yield: 61%).
LCMS m/z=472.3 [M+H]+.
Step 2: benzyl 4-(3-azaspiro[5.5]undecan-9-yl)piperazine-1-carboxylate (82C); HCl
82B (1.6 g, 3.39 mmol) was dissolved in methanol (10 mL); a solution of hydrochloric acid in dioxane (4 N, 40 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: benzyl 4-(3-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3-azaspiro [5.5]undecan-9-yl)piperazine-1-carboxylate (82D)
The crude hydrochloride of 82C from the previous step was dissolved in DMSO (20 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (851 mg, 3.39 mmol) and potassium carbonate (690 mg, 5.00 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 82D (1.0 g two-step yield: 49%).
LCMS m/z=603.3 [M+H]+.
Step 4: 2-methoxy-5-(1-methyl-1H-pyrazol-4-yl)-4-(9-(piperazin-1-yl)-3-azaspiro [5.5]undecan-3-yl)aniline (82E)
82D (1.0 g, 1.66 mmol) was dissolved in methanol (40 mL). Under nitrogen protection, palladium on carbon (wt %=10%, 500 mg) was added, and the mixture was subjected to 1 atm hydrogen replacement 3 times and reacted at room temperature for 1.5 h. After the reaction was completed, the mixture was filtered over celite. The filter cake was washed with dichloromethane/methanol (V/V=10/1), and the filtrate was concentrated under reduced pressure to obtain 82E (650 mg, yield: 89%).
LCMS m/z=439.3 [M+H]+.
Step 5: 5-(4-(3-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3-azaspiro[5.5]undecan-9-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (82F)
82E (650 mg, 1.48 mmol) was dissolved in DMSO (20 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (552 mg, 2.00 mmol) and sodium bicarbonate (692 mg, 8.24 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 82F (400 mg, yield: 39%).
LCMS m/z=695.3 [M+H]+.
Step 6: 5-(4-(3-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3-azaspiro[5.5]undecan-9-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 82)
82F (200 mg, 0.29 mmol) and 23D (148 mg, 0.34 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 82 (30 mg, yield: 9%).
LCMS m/z=547.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 11.06 (s, 1H), 8.44-8.35 (m, 1H), 8.30 (s, 1H), 8.18 (s, 1H), 8.02 (s, 1H), 7.87 (s, 1H), 7.75-7.63 (m, 2H), 7.62-7.50 (m, 3H), 7.44 (t, 2H), 7.39-7.30 (m, 2H), 7.25 (dd, 1H), 7.22-7.12 (m, 1H), 6.89 (s, 1H), 5.07 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.50-3.35 (m, 4H), 2.96-2.72 (m, 5H), 2.69-2.51 (m, 6H), 2.34-2.22 (m, 1H), 2.09-1.99 (m, 1H), 1.93-1.71 (m, 8H), 1.68-1.51 (m, 4H), 1.49-1.31 (m, 4H), 1.19-1.03 (m, 2H).
Step 1: tert-butyl 2-(4-((benzyloxy)carbonyl)piperazin-1-yl)-7-azaspiro[3.5] nonane-7-carboxylate (83A)
Tert-butyl 2-oxo-7-azaspirocyclo[3.5]nonane-7-carboxylate (8.1 g, 33.84 mmol) and benzyl piperazine-1-carboxylate (5 g, 22.70 mmol) were dissolved in 1,2-dichloroethane (100 mL); ten drops of acetic acid was added; and then the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (9.6 g, 45.30 mmol) was added, and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (50 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=100/1-1/1) to obtain 83A (9.3 g, yield: 92%).
LCMS m/z=444.3 [M+H]+.
Step 2: benzyl 4-(7-azaspiro[3.5]nonan-2-yl)piperazine-1-carboxylate (83B); HCl
Compound 83A (3 g, 6.76 mmol) was dissolved in methanol (5 mL); a solution of hydrochloric acid in dioxane (4 N, 30 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 3: benzyl 4-(7-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-7-azaspiro[3.5] nonan-2-yl)piperazine-1-carboxylate (83C)
The hydrochloride of 83B from the previous step and 1D (1.49 g, 5.93 mmol) were dissolved in DMSO (20 mL); sodium bicarbonate (1.53 g, 18.2 mmol) was added; and the mixture was reacted at 100° C. for 6 h, cooled to room temperature and extracted by adding 50 mL of ethyl acetate and 30 mL of water. The organic layer was washed three times with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 83C (2 g, yield: 59%).
LCMS m/z=575.3 [M+H]+.
Step 4: 2-methoxy-5-(1-methyl-1H-pyrazol-4-yl)-4-(2-(piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)aniline (83D)
83C (1 g, 1.74 mmol) and palladium on carbon (1 g, 10 wt %) were successively added and dissolved in 60 mL of methanol, and then the mixture was subjected to hydrogen replacement three times, reacted at room temperature for 2 h and subjected to suction filtration over celite. The filtrate was concentrated under reduced pressure to obtain 83D (600 mg, yield: 84%)
LCMS m/z=411.3 [M+H]+.
Step 5: 5-(4-(7-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-7-azaspiro[3.5]nonan-2-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (83E)
83D (600 mg, 1.46 mmol) from the previous step was dissolved in DMSO (20 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (604 mg, 2.19 mmol) and DIPEA (942 mg, 7.29 mmol) were successively added; and the mixture was stirred at 100° C. for 3 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 83E (540 mg, yield: 55.5%).
LCMS m/z=667.3 [M+H]+.
Step 6: 5-(4-(7-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-7-azaspiro[3.5]nonan-2-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 83)
83E (270 mg, 0.40 mmol) and 23D (265 mg, 0.61 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (230 mg, 1.21 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane: methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a preparative solution, which was concentrated. 50 mL of dichloromethane was added, and the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 83 (20 mg, yield: 5%).
LCMS m/z=533.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 11.06 (s, 1H), 8.48-8.35 (m, 1H), 8.31 (s, 1H), 8.18 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.76-7.63 (m, 2H), 7.61-7.50 (m, 3H), 7.45 (t, 2H), 7.40-7.31 (m, 2H), 7.27 (dd, 1H), 7.22-7.09 (m, 1H), 6.83 (s, 1H), 5.07 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.59-3.40 (m, 4H), 2.94-2.83 (m, 1H), 2.82-2.67 (m, 5H), 2.66-2.53 (m, 2H), 2.47-2.25 (m, 4H), 2.10-1.95 (m, 3H), 1.83 (d, 6H), 1.75-1.51 (m, 6H).
Step 1: tert-butyl 9-(1-((benzyloxy)carbonyl)piperidin-4-yl)-3,9-diazaspiro [5.5]undecane-3-carboxylate (84A)
Benzyl 4-oxopiperidine-1-carboxylate (2.75 g, 11.79 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (3.0 g, 11.79 mmol) were dissolved in dichloromethane (100 mL); acetic acid (710 mg, 11.79 mmol) was added; and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (5.0 g, 23.58 mmol) was added, and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with water (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 84A (2.5 g, yield: 45.0%).
LCMS m/z=472.3 [M+H]+.
Step 2: tert-butyl 9-(piperidin-4-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (84B)
84A (2.5 g, 5.30 mmol) and palladium on carbon (1.5 g, 10 wt %) were dissolved in 60 mL of methanol, and then the mixture was subjected to hydrogen replacement three times, reacted at room temperature for 3 h and subjected to suction filtration over celite. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=25/1-10/1) to obtain 84B (1.5 g, yield: 83.9%).
Step 3: tert-butyl 9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (84C)
84B (1.5 g, 4.44 mmol) and 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (1.1 g, 4.44 mmol) were dissolved in DMSO (10 mL); anhydrous potassium carbonate (1.84 g, 13.32 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 50 mL of ethyl acetate and 30 mL of water. The organic layer was washed with saturated brine (20 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 84C (1.0 g, yield: 39.6%).
LCMS m/z=569.3 [M+H]+.
Step 4: 3-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)-3,9-diazaspiro[5.5]undecane (84D); HCl
84C (1.0 g, 1.76 mmol) was dissolved in methanol (3 mL); a solution of hydrochloric acid in dioxane (4 N, 15 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
LCMS m/z=469.2 [M+H]+.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)-3,9-diazaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione (84E)
The crude hydrochloride of 84D from the previous step was dissolved in DMSO (10 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (490 mg, 1.76 mmol) and DIPEA (1.14 g, 8.80 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 84E (600 mg, yield: 47.0%).
LCMS m/z=725.4 [M+H]+.
Step 6: 5-(9-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (84F)
84E (600 mg, 0.83 mmol) was dissolved in ethanol/water (9 mL/3 mL); iron powder (370 mg, 6.64 mmol) and ammonium chloride (360 mg, 6.64 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 3 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 84F (520 mg, yield: 90%).
Step 7: 5-(9-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 84)
84F (220 mg, 0.32 mmol) and 23D (150 mg, 0.35 mmol) were dissolved in DMF (3 mL); p-toluenesulfonic acid monohydrate (182 mg, 0.96 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 84 (100 mg, yield: 28.5%).
LCMS m/z=547.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.05 (s, 1H), 8.48-8.34 (m, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.76-7.62 (m, 2H), 7.61-7.49 (m, 3H), 7.45 (t, 2H), 7.36 (t, 1H), 7.30 (s, 1H), 7.25-7.08 (m, 2H), 6.83 (s, 1H), 5.06 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.55-3.39 (m, 4H), 3.18-3.01 (m, 2H), 2.97-2.77 (m, 1H), 2.70-2.50 (m, 8H), 2.31-2.16 (m, 1H), 2.06-1.95 (m, 1H), 1.91-1.71 (m, 8H), 1.65-1.41 (m, 10H).
Step 1: tert-butyl 9-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperazin-1-yl)-3-azaspiro[5.5] undecane-3-carboxylate (85C)
1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazine (80B) hydrochloride (1.05 g, crude) was added to 30 mL of THF; sodium bicarbonate (806 mg, 9.60 mmol) was added; and the mixture was stirred at room temperature for 20 min. 3-Boc-9-oxo-3-azaspiro[5.5]undecane (1.28 g, 4.80 mmol) and 0.3 mL of acetic acid were added, and then the mixture was stirred at room temperature for 2 h. Sodium triacetoxyborohydride (1.02 g, 4.80 mmol) was added, and the mixture was reacted at room temperature for 16 h, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted 3 times with ethyl acetate. The organic phases were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=50/1-20/1) to obtain 85C (1.1 g, yield: 82%).
LCMS m/z=569.3 [M+1]+.
Step 2: 9-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)-3-azaspiro[5.5]undecane (85D); 2,2,2-trifluoroacetate
85C (1.0 g, 1.76 mmol) was dissolved in 25 mL of DCM; 10 mL of trifluoroacetic acid was added; and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate of 85D (1.2 g, crude).
LCMS m/z=469.3 [M+1]+.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(9-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)-3-azaspiro[5.5]undecan-3-yl)isoindoline-1,3-dione (85E)
The trifluoroacetate of 85D (1.2 g, crude) was dissolved in 20 mL of DMSO; solid sodium bicarbonate (740 mg, 8.81 mmol) was added; and the mixture was stirred at room temperature for 10 min. 3 mL of DIPEA and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (585 mg, 2.12 mmol) were added, and the reaction was stirred at 80° C. for 5 h. The reaction solution was cooled to room temperature. 200 mL of water was added, and the mixture was filtered to collect the solid, which was washed with water, dissolved in DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=15/1) to obtain 85E (1.1 g, yield: 88%).
LCMS m/z=725.3 [M+1]+.
Step 4: 5-(9-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)-3-azaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (85F)
85E (1.1 g, 1.52 mmol) was dissolved in a mixed solution of THE (10 mL), ethanol (30 mL) and water (6 mL); reduced iron powder (827 mg, 14.77 mmol) and ammonium chloride (790 mg, 14.77 mmol) were added; and the mixture was reacted at 85° C. for 2 h. The reaction solution was cooled to room temperature and concentrated in vacuo to remove a solvent. 50 mL of water and 50 mL of dichloromethane were added to the residue, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 85F (0.71 g, yield: 66%).
LCMS m/z=695.3 [M+H]+.
Step 5: 5-(9-(4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-3-azaspiro [5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 85)
85F (320 mg, 0.46 mmol) and 23D (241 mg, 0.55 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (263 mg, 1.38 mmol) was added; and the mixture was stirred at 100° C. for 8 h, and cooled to room temperature. 100 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (100 ml×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (100 ml×3). The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude was subjected to prep-TLC (DCM: MeOH (V/V)=15:1) purification to obtain compound 85 (180 mg, yield: 33%).
LCMS m/z=1094.3 [M+1]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.05 (s, 1H), 8.51-8.34 (m, 1H), 8.27 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.88 (s, 1H), 7.75-7.62 (m, 2H), 7.62-7.50 (m, 3H), 7.45 (t, 2H), 7.36 (t, 1H), 7.33-7.10 (m, 3H), 6.85 (s, 1H), 5.06 (dd, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.57-3.40 (m, 4H), 2.94-2.77 (m, 5H), 2.70-2.52 (m, 6H), 2.30-2.20 (m, 1H), 2.08-1.98 (m, 1H), 1.89-1.73 (m, 8H), 1.72-1.64 (m, 2H), 1.63-1.52 (m, 2H), 1.50-1.32 (m, 4H), 1.21-1.06 (m, 2H).
Step 1: tert-butyl 4-fluoro-4-formylpiperidine-1-carboxylate (86B)
At 0° C., Dess-Martin periodinane (8.48 g, 20 mmol) was added in batches to a solution of 86A (2.33 g, 10 mmol) in dichloromethane, and the mixture was stirred at room temperature for 2 h and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: PE/EA (V/V)=10/1-1/1) to obtain 86B (2.1 g, yield: 90.9%).
1H NMR (400 MHz, CDCl3) δ 9.66 (d, 1H), 4.11-3.85 (m, 2H), 3.17-2.84 (m, 2H), 1.86-1.59 (m, 4H), 1.47-1.35 (m, 9H).
Step 2: tert-butyl 4-fluoro-4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidine-1-carboxylate (86C)
7E (400 mg, 1.0 mmol) and 86B (462 mg, 2.0 mmol) were dissolved in 1,2-dichloroethane (10 mL); a 4A molecular sieve (200 mg) and acetic acid (0.12 g, 2 mmol) were successively added. The mixture was stirred at room temperature for 2 h; sodium triacetoxyborohydride (0.38 g, 1.79 mol) was added; and the resulting mixture was reacted overnight at room temperature. After the reaction was completed, the reaction system was directly concentrated under reduced pressure, and then the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 86C (492 mg, yield: 80%).
LCMS m/z=616.3 [M+H]+.
Step 3: 1-((4-fluoropiperidin-4-yl)methyl)-4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazine (86D)
86C (492 mg, 0.8 mmol) was dissolved in DCM (15 mL); trifluoroacetic acid (5 mL) was added at room temperature; and the mixture was stirred for 1 h and concentrated under reduced pressure. 30 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain 86D (412 mg), which was directly used in the next step.
LCMS m/z=516.3 [M+H]+.
Step 4: 2-(2,6-dioxopiperidin-3-yl)-5-(4-fluoro-4-((4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (86E)
86D (412 mg, 0.8 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (276 mg, 1 mmol) were dissolved in DMSO (10 mL); DIPEA (0.27 g, 2.07 mmol) was added dropwise; and the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 86E (370 mg, yield: 60%).
LCMS m/z=772.3 [M+H]+.
Step 5: 5-(4-((4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)methyl)-4-fluoropiperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (86F)
86E (0.37 g, 0.48 mmol) was dissolved in ethanol (15 mL); reduced iron powder (0.07 g, 1.30 mmol) was added, and then an aqueous solution (5 mL) of ammonium chloride (0.07 g, 1.30 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 3 h. The reaction solution was cooled to room temperature. 5 mL of water was added, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 86F (170 mg, yield: 47.9%)
LCMS m/z=371.7 [(M+2H)/2]+.
Step 6: 5-(4-((4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)-4-fluoropiperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 86); 2,2,2-trifluoroacetic acid
86F (0.13 g, 0.18 mmol) and 23D (0.08 g, 0.18 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (0.09 g, 0.47 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 5 mL of water and 5 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized to obtain the trifluoroacetate of compound 86 (10 mg).
LCMS m/z=571.3 [(M+2H)/2]+.
1H NMR (400 MHz, CD3OD) δ 8.29-8.16 (m, 2H), 7.83-7.72 (m, 3H), 7.71-7.59 (m, 2H), 7.55-7.42 (m, 4H), 7.42-7.29 (m, 3H), 7.26 (dd, 1H), 6.83 (s, 1H), 5.06 (dd, 1H), 3.95-3.79 (m, 5H), 3.68 (s, 3H), 3.62-3.32 (m, 5H), 3.28-3.03 (m, 6H), 2.96-2.45 (m, 9H), 2.17-2.03 (m, 5H), 2.01-1.70 (m, 10H).
Step 1: 5-(4-((4-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 87)
5B (150 mg, 0.20 mmol) and 23D (96 mg, 0.22 mmol) were dissolved in DMIF (10 mL); p-toluenesulfonic acid monohydrate (114 mg, 0.6 mmol) was added; and the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-10/1) to obtain the crude, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a preparative solution, which was concentrated. 30 mL of dichloromethane was added, and the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 87 (25 mg, yield: 11%).
LCMS m/z=571.2 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.08 (s, 1H), 8.47-8.32 (m, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.85 (s, 1H), 7.78-7.63 (m, 2H), 7.62-7.40 (m, 6H), 7.36 (t, 1H), 7.29-7.05 (m, 1H), 6.83 (s, 1H), 5.10 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.68-3.51 (m, 2H), 3.15-3.01 (m, 2H), 2.98-2.80 (m, 3H), 2.71-2.51 (m, 8H), 2.46-2.29 (m, 4H), 2.27-2.11 (m, 3H), 2.10-1.96 (m, 1H), 1.91-1.68 (m, 11H), 1.63-1.45 (m, 2H), 1.35-1.22 (m, 2H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(9-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)isoindoline-1,3-dione (88A)
60D (0.4 g, 0.85 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1) (0.26 g, 0.94 mmol) were dissolved in DMSO (5 ml); DIPEA (0.22 g, 1.7 mmol) was added; and the mixture was stirred at 90° C. for 5 h and cooled to room temperature. 10 mL of water was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography to obtain 88A (0.5 g, yield: 79%).
Step 2: 5-(9-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (88B)
88A (500 mg, 0.67 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (370 mg, 6.7 mmol) and ammonium chloride (360 mg, 6.7 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was extracted by adding 20 mL of dichloromethane and 20 mL of saturated aqueous sodium chloride solution. The organic layer was dried and then concentrated under reduced pressure to obtain 88B (260 mg), which was directly used in the next step.
Step 3: 5-(9-(1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 88)
88B (140 mg, 0.20 mmol) and 23D (87 mg, 0.20 mmol) were dissolved in DMF (5 mL) solution; p-toluenesulfonic acid monohydrate (110 mg, 0.60 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 88. 20 mL of dichloromethane and 10 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 88, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 88 (40 mg, yield: 18%).
LCMS m/z=556.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.09 (s, 1H), 8.50-8.32 (m, 1H), 8.26 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.75-7.64 (m, 2H), 7.64-7.51 (m, 3H), 7.51-7.41 (m, 3H), 7.34 (t, 1H), 7.23-7.07 (m, 1H), 6.82 (s, 1H), 5.10 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.22-3.02 (m, 6H), 2.95-2.82 (m, 1H), 2.68-2.50 (m, 9H), 2.07-1.95 (m, 1H), 1.91-1.39 (m, 18H).
Step 1: 5-(9-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-2,9-diazaspiro[5.5]undecan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 89)
10G (0.20 g, 0.28 mmol) and 40A (0.11 g, 0.28 mmol) were dissolved in a mixed solvent of 1,4-dioxane (10 mL) and NMP (2 mL); p-toluenesulfonic acid hydrate (0.16 g, 0.84 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% trifluoroacetic acid)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 89 (30 mg, yield: 10%).
LCMS m/z=532.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.04 (s, 1H), 8.55-8.40 (m, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.86 (s, 1H), 7.70 (dd, 1H), 7.62 (d, 1H), 7.59-7.47 (m, 3H), 7.42 (t, 2H), 7.36-7.26 (m, 2H), 7.24 (dd, 1H), 7.20-7.05 (m, 1H), 6.85 (s, 1H), 5.05 (dd, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.55-3.34 (m, 4H), 3.11-3.00 (m, 2H), 2.95-2.79 (m, 1H), 2.71-2.51 (m, 4H), 2.44-2.16 (m, 6H), 2.04-1.95 (m, 1H), 1.83 (d, 6H), 1.74-1.35 (m, 11H), 1.32-1.25 (m, 2H).
Step 1: 5-(4-((4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 90)
77D (200 mg, 0.31 mmol) and 40A (180 mg, 0.46 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 90 (50 mg, yield: 16%).
LCMS m/z=498.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.32-8.03 (m, 7H), 7.86 (s, 1H), 7.72 (d, 1H), 7.60-7.45 (m, 5H), 7.40-7.23 (m, 1H), 6.98 (s, 1H), 5.21 (dd, 1H), 3.98-3.89 (m, 5H), 3.89-3.72 (m, 5H), 3.72-3.64 (m, 2H), 3.42-3.10 (m, 8H), 2.97-2.89 (m, 2H), 2.85-2.70 (m, 1H), 2.65-2.47 (m, 1H), 2.45-2.32 (m, 2H), 2.32-2.21 (m, 1H), 2.21-2.07 (m, 2H), 2.02 (d, 6H).
Step 1: 5-(2-(4-(1-(4-(5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 91)
7G (285 mg, 0.38 mmol) and 40A (179 mg, 0.46 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (217 mg, 1.14 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 91 (65 mg, yield: 15%).
LCMS m/z=1105.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.06 (s, 1H), 8.63-8.39 (m, 1H), 8.27 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H), 7.71 (dd, 1H), 7.64 (d, 1H), 7.60 (s, 1H), 7.58-7.50 (m, 2H), 7.45 (t, 2H), 7.41-7.34 (m, 1H), 7.33-7.26 (m, 1H), 7.27-7.08 (m, 2H), 6.84 (s, 1H), 5.06 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.52-3.35 (m, 4H), 3.18-3.03 (m, 2H), 2.97-2.79 (m, 1H), 2.76-2.50 (m, 8H), 2.44-2.11 (m, 6H), 2.10-1.91 (m, 3H), 1.91-1.71 (m, 8H), 1.69-1.48 (m, 8H).
Step 1: 5-(4-(1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 92)
81B (200 mg, 0.32 mmol) and 40A (180 mg, 0.46 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 92 (40 mg, yield: 13%).
LCMS m/z=491.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.06 (s, 1H), 8.55-8.41 (m, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.76-7.65 (m, 2H), 7.65-7.51 (m, 3H), 7.46 (t, 2H), 7.41-7.33 (m, 2H), 7.31-7.11 (m, 2H), 6.85 (s, 1H), 5.08 (dd, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.53-3.39 (m, 4H), 3.19-3.05 (m, 2H), 2.97-2.83 (m, 1H), 2.74-2.51 (m, 8H), 2.36-2.22 (m, 1H), 2.08-1.92 (m, 1H), 1.89-1.74 (m, 8H), 1.67-1.53 (m, 2H).
Step 1: 5-(4-((4-(1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 93)
5B (150 mg, 0.20 mmol) and 40A (94 mg, 0.24 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (114 mg, 0.6 mmol) was added; and the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-10/1) to obtain the crude, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a preparative solution, which was concentrated. 30 mL of dichloromethane was added, and the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 93 (20 mg, yield: 9%).
LCMS m/z=549.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 11.08 (s, 1H), 8.60-8.38 (m, 1H), 8.26 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.86 (s, 1H), 7.77-7.65 (m, 2H), 7.64-7.51 (m, 3H), 7.51-7.41 (m, 3H), 7.40-7.32 (m, 1H), 7.23-7.06 (m, 1H), 6.84 (s, 1H), 5.10 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.67-3.51 (m, 2H), 3.15-3.03 (m, 2H), 2.96-2.78 (m, 3H), 2.70-2.51 (m, 8H), 2.47-2.29 (m, 4H), 2.26-2.12 (m, 3H), 2.08-1.98 (m, 1H), 1.91-1.66 (m, 11H), 1.62-1.45 (m, 2H), 1.34-1.18 (m, 2H).
Step 1: tert-butyl 7-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate (94A)
1D (2 g, 7.96 mmol) and tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate (2.67 g, 11.80 mmol) were dissolved in DMSO (30 mL); potassium carbonate (3.26 mg, 23.59 mmol) was added; and the mixture was reacted at 120° C. for 6 h, cooled to room temperature and extracted by adding 50 mL of ethyl acetate and 50 mL of water. The organic layer was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol (V/V)=100/1-20/1) to obtain 94A (2.47 g, yield: 68%).
LCMS m/z=458.3 [M+H]+.
Step 2: 7-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-2,7-diazaspiro[3.5]nonane (94B)
94A (2.47 g, 5.40 mmol) was dissolved in methanol (5 mL); a solution of hydrogen chloride in dioxane (4 N, 20 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure. 20 mL of dioxane and 2 mL of ammonia water were added to the residue, and the mixture was concentrated under reduced pressure to obtain the crude of 94B, which was directly used in the next step.
Step 3: tert-butyl 4-((7-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-2,7-diazaspiro[3.5]nonan-2-yl)methyl)piperidine-1-carboxylate (94C)
The crude of 94B from the previous step and tert-butyl 4-formylpiperidine-1-carboxylate (2.30 g, 10.78 mmol) were dissolved in DMAC (30 mL), and acetic acid (323 mg, 5.37 mmol) was added. The mixture was stirred at room temperature for 30 min, and then sodium triacetoxyborohydride (3.43 g, 16.18 mmol) was added; and the resulting mixture was reacted overnight at room temperature. The reaction was quenched by adding 50 mL of saturated aqueous sodium bicarbonate solution and extracted wish ethyl acetate (50 mL×3). The organic layer was washed with saturated brine (3×30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1) to obtain 94C (2.3 g, yield: 77%).
LCMS m/z=555.4 [M+H]+.
Step 4: 7-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-2-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonane (94D); HCl
94C (2.3 g, 4.15 mmol) was dissolved in methanol (5 mL); a solution of hydrogen chloride in dioxane (4 N, 25 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((7-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-2,7-diazaspiro[3.5]nonan-2-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (94E)
The crude hydrochloride of 94D from the previous step was dissolved in DMSO (30 mL); 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (5A-1) (1.46 g, 4.96 mmol) and DIPEA (2.67 g, 20.7 mmol) were successively added; and the mixture was stirred at 100° C. for 5 h, and cooled to room temperature. 30 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 94E (1.8 g, yield: 60%).
LCMS m/z=729.3 [M+H]+.
Step 6: 5-(4-((7-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-2,7-diazaspiro[3.5]nonan-2-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (94F)
94E (700 mg, 0.96 mmol) was dissolved in ethanol/water (20 mL, 3:1); iron powder (280 mg, 5 mmol) and ammonium chloride (335 mg, 6.26 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 85° C. for 2 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated, diluted by adding 20 mL of water, extracted with dichloromethane (3×30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain 94F (600 mg, yield: 89%).
LCMS m/z=699.3 [M+H]+.
Step 7: 5-(4-((7-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-2,7-diazaspiro[3.5]nonan-2-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 94)
94F (150 mg, 0.21 mmol) and 40A (124 mg, 0.32 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (120 mg, 0.63 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification, lyophilization and silica gel preparative plate purification (dichloromethane/methanol (V/V)=15/1) to obtain compound 94 (15 mg, yield: 6.8%).
LCMS m/z=527.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 11.08 (s, 1H), 8.61-8.42 (m, 1H), 8.29 (s, 1H), 8.11 (s, 1H), 8.06 (s, 1H), 7.87 (s, 1H), 7.75-7.64 (m, 2H), 7.62-7.52 (m, 3H), 7.51-7.40 (m, 3H), 7.39-7.32 (m, 1H), 7.23-7.02 (m, 1H), 6.84 (s, 1H), 5.09 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.66-3.52 (m, 2H), 3.20-2.95 (m, 4H), 2.94-2.82 (m, 3H), 2.81-2.68 (m, 4H), 2.65-2.52 (m, 2H), 2.48-2.34 (m, 2H), 2.08-1.93 (m, 1H), 1.92-1.69 (m, 12H), 1.61-1.43 (m, 1H), 1.41-1.25 (m, 2H).
Step 1: 5-(9-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 95)
37F (1.7 g, 2.40 mmol) and 54C (1.09 g, 2.40 mmol) were dissolved in DMF (15 mL) solution; p-toluenesulfonic acid monohydrate (0.91 g, 4.8 mmol) was added; and the mixture was reacted overnight at 100° C., and cooled to room temperature. 30 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further subjected to medium-pressure preparative reversed-phase column purification (acetonitrile/water (containing 0.1% TFA)=3%-40%) to obtain the preparative solution of the trifluoroacetate of compound 95. 10 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the mixture was stirred for 5 min. Liquid separation and extraction were performed. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 95 (600 mg, yield: 22%).
LCMS m/z=563.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 11.05 (s, 1H), 8.48-8.36 (m, 1H), 8.33 (s, 1H), 8.19 (s, 1H), 7.99 (s, 1H), 7.83 (s, 1H), 7.69-7.56 (m, 2H), 7.52 (s, 1H), 7.46-7.34 (m, 2H), 7.34-7.03 (m, 5H), 6.81 (s, 1H), 5.06 (dd, 1H), 3.77 (s, 3H), 3.75 (s, 3H), 3.54-3.40 (m, 4H), 3.11-2.97 (m, 2H), 2.96-2.79 (m, 1H), 2.69-2.51 (m, 4H), 2.43-2.27 (m, 4H), 2.21 (d, 2H), 2.08-1.96 (m, 1H), 1.80 (d, 6H), 1.73-1.63 (m, 2H), 1.61-1.41 (m, 9H), 1.31-1.17 (m, 2H).
Step 1: 5-(4-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 96)
77D (3.5 g, 5.46 mmol) and 54C (2.98 g, 6.55 mmol) were dissolved in DMF (50 mL); p-toluenesulfonic acid monohydrate (3.1 g, 16.30 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 100 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further subjected to reversed-phase preparative column C18 purification (composition of mobile phases: mobile phase A: water (containing 0.1% TFA), mobile phase B: acetonitrile). The resulting solid was dissolved in 100 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 96 (1.5 g, yield: 26%).
LCMS m/z=529.7 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.33-8.06 (m, 7H), 7.76-7.62 (m, 2H), 7.52-7.37 (m, 2H), 7.37-7.21 (m, 3H), 7.00 (s, 1H), 5.22 (dd, 1H), 4.01-3.90 (m, 8H), 3.90-3.78 (m, 2H), 3.77-3.63 (m, 2H), 3.44-3.15 (m, 8H), 2.96-2.89 (m, 2H), 2.85-2.70 (m, 1H), 2.64-2.50 (m, 1H), 2.46-2.32 (m, 2H), 2.32-2.22 (m, 1H), 2.21-2.05 (m, 2H), 1.99 (d, 6H)
Step 1: (4-((2,5-dichloropyrimidin-4-yl)amino)-2′-fluoro-[1,1′-biphenyl]-3-yl) dimethylphosphine oxide (97A)
54B (4.5 g, 17.09 mmol) and 2,4,5-trichloropyrimidine (6.27 g, 34.18 mmol) were dissolved in 20 mL of NMP; DIPEA (2.65 g, 20.51 mmol) was added; and the mixture was stirred at 120° C. for 2 h and cooled to room temperature. 50 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure. The residue was slurried by adding 10 mL of ethanol and 30 mL of methyl tert-butyl ether for 1 h and filtered. The filter cake was washed with 10 mL of methyl tert-butyl ether and dried under reduced pressure to obtain 97A (4.5 g, yield: 64%).
1H NMR (400 MHz, CDCl3) δ 11.69 (s, 1H), 8.82-8.76 (m, 1H), 8.24 (s, 1H), 7.76 (d, 1H), 7.52-7.40 (m, 2H), 7.40-7.32 (m, 1H), 7.28-7.14 (m, 2H), 1.88 (d, 6H).
Step 2: 5-(4-((4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 97)
77D (3.5 g, 5.46 mmol) and 97A (2.69 g, 6.56 mmol) were dissolved in DMF (50 mL); p-toluenesulfonic acid monohydrate (3.1 g, 16.30 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 100 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a preparative solution, which was concentrated. 100 mL of dichloromethane was added, and the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 97 (1.2 g, yield: 22%).
LCMS m/z=507.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.37-8.23 (m, 3H), 8.23-8.07 (m, 4H), 7.78-7.62 (m, 2H), 7.52-7.37 (m, 2H), 7.37-7.20 (m, 3H), 7.01 (s, 1H), 5.22 (dd, 1H), 4.00-3.90 (m, 8H), 3.90-3.79 (m, 2H), 3.77-3.67 (m, 2H), 3.45-3.20 (m, 8H), 2.96-2.90 (m, 2H), 2.85-2.71 (m, 1H), 2.65-2.51 (m, 1H), 2.48-2.33 (m, 2H), 2.32-2.22 (m, 1H), 2.21-2.06 (m, 2H), 2.00 (d, 6H).
Step 1: 5-(4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 98)
80D (130 mg, 0.24 mmol) and 40A (122 mg, 0.31 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (137 mg, 0.72 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 98 (55 mg, yield: 25%).
LCMS m/z=899.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 11.07 (s, 1H), 8.64-8.36 (m, 1H), 8.28 (s, 1H), 8.17 (s, 1H), 8.13 (s, 1H), 7.89 (s, 1H), 7.76-7.64 (m, 3H), 7.61-7.51 (m, 2H), 7.50-7.24 (m, 5H), 7.23-7.04 (m, 1H), 6.88 (s, 1H), 5.09 (dd, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.69-3.47 (m, 4H), 3.06-2.83 (m, 5H), 2.70-2.52 (m, 2H), 2.10-1.97 (m, 1H), 1.84 (d, 6H).
Step 1: tert-butyl 4-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl) piperidine-1-carboxylate (99A)
The hydrochloride of 80B (1.05 g, crude) was added to 30 mL of THF; sodium bicarbonate (806 mg, 9.60 mmol) was added; and the mixture was stirred at room temperature for 20 min. (955 mg, 4.80 mmol) and 0.3 mL of acetic acid were added, and then the mixture was stirred at room temperature for 2 h. Sodium triacetoxyborohydride (1.02 g, 4.80 mmol) was added, and the mixture was reacted at room temperature for 16 h, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted 3 times with ethyl acetate. The organic phases were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=50/1-20/1) to obtain 99A (1.0 g, yield: 84.6%).
LCMS m/z=501.3 [M+1]+.
Step 2: 1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4-(piperidin-4-yl)piperazine (99B); 2,2,2-trifluoroacetate
99A (1.0 g, 2.0 mmol) was dissolved in 25 mL of DCM; 10 mL of trifluoroacetic acid was added; and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate of 99B (1.2 g, crude).
LC-MS m/z=401.3 [M+1]+.
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione (99C)
The trifluoroacetate of 99B (1.2 g, crude) was dissolved in 20 mL of DMSO; solid sodium bicarbonate (840 mg, 10.0 mmol) was added; and the mixture was stirred at room temperature for 10 min. 3 mL of DIPEA and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (662 mg, 2.40 mmol) were added, and the reaction was stirred at 80° C. for 5 h. The reaction solution was cooled to room temperature. 200 mL of water was added; and the mixture was filtered to collect the solid, which was washed with water, dissolved in DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=15/1) purification to obtain 99C (0.72 g, yield: 55.3%).
LCMS m/z=675.3 [M+1]+.
Step 4: 5-(4-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (99D)
99C (0.72 g, 1.10 mmol) was dissolved in a mixed solution of THE (10 mL), ethanol (30 mL) and water (6 mL); reduced iron powder (614 mg, 10.96 mmol) and ammonium chloride (587 mg, 10.97 mmol) were added; and the mixture was reacted at 85° C. for 2 h. The reaction solution was cooled to room temperature and concentrated in vacuo to remove a solvent. 50 mL of water and 50 mL of dichloromethane were added to the residue, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 99D (0.52 g, yield: 75.4%)
LCMS m/z=627.3 [M+H]+.
Step 5: 5-(4-(4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 99)
99D (250 mg, 0.40 mmol) and 23D (260 mg, 0.60 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (230 mg, 1.21 mmol) was added; and the mixture was stirred at 100° C. for 8 h, and cooled to room temperature. 100 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (100 mL×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (100 ml×3). The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude was subjected to prep-TLC (DCM: MeOH (V/V)=15:1) purification to obtain compound 99 (120 mg, yield: 28%).
LCMS m/z=1026.3 [M+1]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.06 (s, 1H), 8.45-8.33 (m, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.88 (s, 1H), 7.76-7.64 (m, 2H), 7.61-7.50 (m, 3H), 7.43 (t, 2H), 7.40-7.30 (m, 2H), 7.27 (dd, 1H), 7.22-7.08 (m, 1H), 6.85 (s, 1H), 5.07 (dd, 1H), 4.15-3.96 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.11-2.98 (m, 2H), 2.97-2.77 (m, 5H), 2.71-2.51 (m, 7H), 2.10-1.87 (m, 3H), 1.83 (d, 6H), 1.60-1.41 (m, 2H).
Step 1: 5-(4-(4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 100)
99D (250 mg, 0.40 mmol) and 40A (188 mg, 0.48 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (263 mg, 1.38 mmol) was added; and the mixture was stirred at 100° C. for 8 h, and cooled to room temperature. 100 mL of saturated aqueous sodium bicarbonate solution was added, with a white solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (100 ml×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (100 ml×3). The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude was subjected to prep-TLC (DCM: MeOH (V/V)=15:1) purification to obtain compound 100 (100 mg, yield: 25%).
LCMS m/z=982.3 [M+1]+.
1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 11.06 (s, 1H), 8.65-8.35 (m, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.89 (s, 1H), 7.78-7.65 (m, 2H), 7.64-7.48 (m, 3H), 7.44 (t, 2H), 7.40-7.29 (m, 2H), 7.27 (dd, 1H), 7.22-7.09 (m, 1H), 6.85 (s, 1H), 5.07 (dd, 1H), 4.19-3.96 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.11-2.96 (m, 2H), 2.95-2.73 (m, 5H), 2.73-2.51 (m, 7H), 2.10-1.88 (m, 3H), 1.84 (d, 6H), 1.61-1.41 (m, 2H).
5-(4-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 101)
45F (2.5 g, 3.90 mmol) and 54C (1.77 g, 3.90 mmol) were dissolved in DMF (20 mL) solution; p-toluenesulfonic acid monohydrate (1.48 g, 7.8 mmol) was added, and the mixture was reacted overnight at 100° C., and cooled to room temperature. 30 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further subjected to medium-pressure preparative reversed-phase column purification (acetonitrile/water (containing 0.1% TFA)=3%-40%) to obtain the preparative solution of the trifluoroacetate of compound 101. 10 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the mixture was stirred for 5 min. Liquid separation and extraction were performed. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 101 (1.5 g, yield: 36%).
LCMS m/z=529.7 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.28 (s, 1H), 8.19-8.07 (m, 1H), 7.93 (s, 1H), 7.87-7.78 (m, 3H), 7.76-7.65 (m, 1H), 7.53-7.41 (m, 3H), 7.40-7.21 (m, 5H), 5.12 (dd, 1H), 4.21-4.07 (m, 2H), 4.03 (s, 3H), 3.89-3.66 (m, 9H), 3.59-3.47 (m, 2H), 3.37-3.15 (m, 4H), 2.94-2.83 (m, 2H), 2.82-2.67 (m, 1H), 2.56-2.40 (m, 1H), 2.31-2.13 (m, 3H), 2.07-1.87 (m, 8H).
Step 1: 5-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 102)
45F (2.5 g, 3.90 mmol) and 97A (1.6 g, 3.90 mmol) were dissolved in DMF (20 mL) solution; p-toluenesulfonic acid monohydrate (1.48 g, 7.8 mmol) was added, and the mixture was reacted overnight at 100° C., and cooled to room temperature. 30 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further subjected to medium-pressure preparative reversed-phase column purification (acetonitrile/water (containing 0.1% TFA)=3%-40%) to obtain the preparative solution of the trifluoroacetate of compound 102. 10 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the mixture was stirred for 5 min. Liquid separation and extraction were performed. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 102 (1.5 g, yield: 38%).
LCMS m/z=507.7 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1))δ 8.28-8.21 (m, 1H), 8.19 (s, 1H), 7.98 (s, 1H), 7.91-7.77 (m, 3H), 7.74-7.64 (m, 1H), 7.54-7.40 (m, 3H), 7.40-7.21 (m, 5H), 5.12 (dd, 1H), 4.24-4.07 (m, 2H), 4.04 (s, 3H), 3.89-3.69 (m, 9H), 3.61-3.48 (m, 2H), 3.37-3.12 (m, 4H), 2.95-2.84 (m, 2H), 2.83-2.67 (m, 1H), 2.58-2.41 (m, 1H), 2.28-2.11 (m, 3H), 2.06-1.89 (m, 8H).
Step 1: tert-butyl 4-(2-bromo-5-methoxy-4-nitrophenyl)piperazine-1-carboxylate (103H)
1C (30 g, 120.48 mmol) and 1-tert-butoxycarbonylpiperazine (24.65 g, 132.52 mmol) were dissolved in DMSO (300 mL); potassium carbonate (50 g, 361.44 mmol) was added; and the mixture was reacted at 120° C. for 6 h. The reaction solution was poured into water under stirring, with a solid precipitated, and the mixture was subjected to suction filtration and washed 3 times with water. The filter cake was dried in vacuo to obtain 103H (50 g, yield: 99%).
LCMS m/z=360.0 [M-55]+.
Step 2: 1-(2-bromo-5-methoxy-4-nitrophenyl)piperazine (103I)
103H (33 g, 80 mmol) was dissolved in dichloromethane (100 mL); trifluoroacetic acid (50 mL) was slowly added; and the mixture was reacted at room temperature for 3 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, redissolved by adding 100 mL of dichloromethane and adjusted to a basic pH with 2 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1031 (24.5 g, yield: 98%), which was directly used in the next step.
LCMS m/z=316.0 [M+H]+.
Step 3: tert-butyl (R)-3-(((methylsulfonyl)oxy)methyl)pyrrolidine-1-carboxylate (103B)
103A (tert-butyl(R)-3-(hydroxymethyl)pyrrolidine-1-carboxylate) (5.00 g, 24.84 mmol) was dissolved in DCM (50 mL); triethylamine (7.54 g, 74.52 mmol) was added; and under an ice bath, methanesulfonyl chloride (5.69 g, 49.68 mmol) was added dropwise. After the addition, the mixture was reacted at 20° C. for 2 h, and the reaction was quenched by adding water. The reaction solution was diluted with 200 mL of dichloromethane, washed 3 times with 0.1 N dilute hydrochloric acid, and washed once with aqueous sodium bicarbonate solution and saturated sodium chloride. The organic phases were collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 103B (6.90 g, which was directly used in the next step).
LCMS m/z=224.1 [M−55]+.
Step 4: tert-butyl (S)-3-((4-(2-bromo-5-methoxy-4-nitrophenyl)piperazin-1-yl)methyl) pyrrolidine-1-carboxylate (103C)
103I (5.20 g, 16.45 mmol) and 103B (5.51 g, 19.74 mmol) were dissolved in DMF (50 mL); sodium iodide (0.99 g, 6.58 mmol) and potassium carbonate (6.82 g, 49.35 mmol) were added; and the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=2/1-pure ethyl acetate) to obtain 103C (3.30 g, yield: 40%).
LCMS m/z=499.1 [M+H]+.
Step 5: tert-butyl (S)-3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl) methyl)pyrrolidine-1-carboxylate (103D)
103C (3.30 g, 6.61 mmol) and 1-methyl-1H-pyrazole-4-boronic acid (1.33 g, 10.58 mmol) were dissolved in 1,4-dioxane (50 mL); Pd(dppf)Cl2·DCM (0.54 g, 0.66 mmol) and an aqueous solution (10 mL) of potassium carbonate (1.83 g, 13.22 mmol) were added; and the mixture was subjected to nitrogen replacement 3 times and reacted at 90° C. for 5 h. The reaction solution was cooled to room temperature, diluted by adding 300 mL of ethyl acetate, washed 3 times with water and washed once with saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=3/1-pure ethyl acetate) to obtain 103D (2.89 g, yield: 87%).
LCMS m/z=501.3 [M+H]+.
Step 6: (R)-1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4-(pyrrolidin-3-ylmethyl) piperazine (103E)
103D (2.89 g, 5.77 mmol) was dissolved in dichloromethane (30 mL); trifluoroacetic acid (10 mL) was slowly added; and the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, redissolved by adding 100 mL of dichloromethane and adjusted to a basic pH with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 103E (2.26 g, yield: 98%), which was directly used in the next step.
LCMS m/z=401.3 [M+H]+.
Step 7: 2-(2,6-dioxopiperidin-3-yl)-5-((S)-3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)isoindoline-1,3-dione (103F)
103E (2.26 g, 5.64 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (1.87 g, 6.77 mmol) were dissolved in DMSO (30 mL); DIPEA (2.19 g, 16.92 mmol) was added dropwise; and the mixture was reacted at 90° C. for 16 h. The reaction solution was cooled to room temperature. 20 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 103F (3.58 g, yield: 97%).
LCMS m/z=657.2 [M+H]+.
Step 8: 5-((S)-3-((4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (103G)
103F (3.58 g, 5.45 mmol) was dissolved in ethanol (60 mL); reduced iron powder (1.83 g, 32.70 mmol) was added, and then an aqueous solution (20 mL) of ammonium chloride (1.75 g, 32.70 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 90° C. for 3 h. The reaction solution was cooled to room temperature. 20 mL of water was added, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 103G (3.40 g, yield: 99%), which was directly used in the next step.
LCMS m/z=627.3 [M+H]+.
Step 9: 5-((S)-3-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 103)
103G (1.70 g, 2.71 mmol) and 23D (1.18 g, 2.71 mmol) were dissolved in DMF (40 mL); p-toluenesulfonic acid hydrate (1.55 g, 8.13 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 103 (580 mg, yield: 21%).
LCMS m/z=513.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.04 (s, 1H), 8.46-8.33 (m, 1H), 8.28 (s, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.74-7.50 (m, 5H), 7.45 (t, 2H), 7.33 (t, 1H), 7.26-7.07 (m, 1H), 6.97-6.85 (m, 2H), 6.82 (dd, 1H), 5.05 (dd, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.62-3.48 (m, 2H), 3.48-3.36 (m, 1H), 3.23-3.11 (m, 1H), 3.00-2.79 (m, 5H), 2.74-2.51 (m, 7H), 2.47-2.31 (m, 2H), 2.24-2.09 (m, 1H), 2.08-1.94 (m, 1H), 1.91-1.68 (m, 7H).
Step 1: tert-butyl (S)-3-(((methylsulfonyl)oxy)methyl)pyrrolidine-1-carboxylate (104B)
104A (5.00 g, 24.84 mmol) was dissolved in DCM (50 mL); triethylamine (7.54 g, 74.52 mmol) was added; and under an ice bath, methanesulfonyl chloride (5.69 g, 49.68 mmol) was added dropwise. The mixture was reacted at 20° C. for 2 h, and the reaction was quenched by adding water. The reaction solution was diluted with 200 mL of dichloromethane, washed 3 times with 0.1 N dilute hydrochloric acid, and washed once with aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution. The organic phases were collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 104B (7 g, which was directly used in the next step).
Step 2: tert-butyl (R)-3-((4-(2-bromo-5-methoxy-4-nitrophenyl)piperazin-1-yl)methyl) pyrrolidine-1-carboxylate (compound 104C)
103I (6.7 g, 21.19 mmol) and 104B (7 g, 25.40 mmol) were dissolved in DMF (50 mL); sodium iodide (0.32 g, 2.12 mmol) and potassium carbonate (8.79 g, 63.57 mmol) were added; and the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water, and washed once with saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=2/1-pure ethyl acetate) to obtain 104C (5 g, yield: 47%).
LCMS m/z=499.1 [M+H]+.
Step 3: tert-butyl (R)-3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperazin-1-yl)methyl)pyrrolidine-1-carboxylate (104D)
104C (5 g, 10.01 mmol) and 1-methyl-1H-pyrazole-4-boronic acid (2.52 g, 20.02 mmol) were dissolved in 1,4-dioxane (50 mL); Pd(dppf)Cl2·DCM (0.82 g, 1.00 mmol) and an aqueous solution (15 mL) of potassium carbonate (4.15 g, 30.03 mmol) were added; and the mixture was subjected to nitrogen replacement 3 times and reacted at 100° C. for 4 h. The reaction solution was cooled to room temperature, diluted by adding 300 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=3/1-pure ethyl acetate) to obtain 104D (5 g, yield: 99%).
LCMS m/z=501.3 [M+H]+.
Step 4: (S)-1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-4-(pyrrolidin-3-ylmethyl)piperazine (104E)
104D (5 g, 9.99 mmol) was dissolved in dichloromethane (30 mL); trifluoroacetic acid (10 mL) was slowly added; and the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, redissolved by adding 100 mL of dichloromethane and adjusted to a basic pH with 1 N aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted 3 times with dichloromethane. All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 104E (3.8 g, yield: 95%), which was directly used in the next step.
Step 5: 2-(2,6-dioxopiperidin-3-yl)-5-((R)-3-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)isoindoline-1,3-dione (104F)
104E (3.8 g, 9.49 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (2.88 g, 10.44 mmol) were dissolved in DMSO (30 mL); DIPEA (2.45 g, 18.98 mmol) was added dropwise; and the mixture was reacted at 90° C. for 1.5 h. The reaction solution was cooled to room temperature. 30 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-12/1) to obtain 104F (3.8 g, yield: 60%).
LCMS m/z=657.3 [M+H]+.
Step 6: 5-((R)-3-((4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl) phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (104G)
104F (3.8 g, 5.79 mmol) was dissolved in ethanol (60 mL); reduced iron powder (3.23 g, 57.9 mmol) was added, and then an aqueous solution (20 mL) of ammonium chloride (3.1 g, 57.9 mmol) was added dropwise; and under nitrogen protection, the mixture was reacted at 80° C. for 0.5 h. The reaction solution was cooled to room temperature and filtered. The filter cake was washed with 100 mL of dichloromethane. 50 mL of saturated brine was added, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 104G (3.8 g, yield: 100%), which was directly used in the next step.
Step 7: 5-((R)-3-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 104)
23D (1.32 g, 3.03 mmol) and 104G (1.9 g, 3.03 mmol) were dissolved in DMF (15 mL); p-toluenesulfonic acid hydrate (1.15 g, 6.06 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further subjected to medium-pressure preparative reversed-phase column purification (acetonitrile/water (containing 0.1% TFA)=3%-40%) to obtain the preparative solution of the trifluoroacetate of compound 104. 10 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added; and the mixture was stirred for 5 min. Liquid separation and extraction were performed. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 104 (1.0 g, yield: 32%).
LCMS m/z=513.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.04 (s, 1H), 8.50-8.34 (m, 1H), 8.28 (s, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.74-7.50 (m, 5H), 7.45 (t, 2H), 7.33 (t, 1H), 7.26-7.03 (m, 1H), 6.98-6.85 (m, 2H), 6.82 (dd, 1H), 5.05 (dd, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.63-3.47 (m, 2H), 3.47-3.36 (m, 1H), 3.22-3.07 (m, 1H), 2.97-2.76 (m, 5H), 2.71-2.51 (m, 7H), 2.44 (d, 2H), 2.23-2.06 (m, 1H), 2.06-1.93 (m, 1H), 1.91-1.67 (m, 7H).
Step 1: tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl) piperazine-1-carboxylate (105A)
3-(5-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (4.0 g, 12.4 mmol) and tert-butyl piperazine-1-carboxylate (4.0 g, 21.5 mmol) were dissolved in ultra-dry DMF (60 mL); cesium carbonate (8.1 g, 24.9 mmol) and RuPhosPdG3 (1.0 g, 1.2 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h. After the reaction was completed, 200 mL of ethyl acetate and 100 mL of aqueous solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 105A (1.3 g, yield: 24%).
LCMS m/z=429.2 [M+H]+.
Step 2: 3-(1-oxo-5-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione (105B); HCl
105A (1.3 g, 3.0 mmol) was dissolved in methanol (3 mL); a solution of hydrochloric acid in dioxane (4 N, 15 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
LCMS m/z=329.2 [M+H]+.
Step 3: tert-butyl 4-((4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl) piperazin-1-yl)methyl)piperidine-1-carboxylate (105C)
The crude hydrochloride of 105B (1.1 g) and tert-butyl 4-formylpiperidine-1-carboxylate (1.1 g, 5.16 mmol) were dissolved in DMAC (30 mL); 0.1 ml of acetic acid was added; and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (1.1 g, 5.2 mmol) was added, and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with water (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 105C (1.0 g, two-step yield: 63%).
LCMS m/z=526.3 [M+H]+.
Step 4: 3-(1-oxo-5-(4-(piperidin-4-ylmethyl)piperazin-1-yl)isoindolin-2-yl) piperidine-2,6-dione (105D); HCl
105C (1.0 g, 1.9 mmol) was dissolved in methanol (3 mL); a solution of hydrochloric acid in dioxane (4 N. 15 mL) was added; and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
LCMS m/z=426.3 [M+H]+.
Step 5: 3-(5-(4-((1-(2-bromo-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl) piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (105E)
The crude hydrochloride of 105D (900 mg) was dissolved in DMSO (25 mL); 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C) (500 mg, 2.0 mmol) and sodium bicarbonate (504 mg, 6.0 mmol) were successively added; and the mixture was stirred at 90° C. for 3 h, and cooled to room temperature. 80 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 105E (700 mg, two-step yield: 56%).
LCMS m/z=655.3 [M+H]+.
Step 6: 3-(5-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (105F)
Under nitrogen protection, 105E (300 mg, 0.46 mmol) and N-methylpyrazole-4-boronic acid (115 mg, 0.91 mmol) were added to a 50 mL single-necked flask and dissolved in 10 mL of dioxane and 2 mL of water, and then Pd(dppf)Cl2·DCM (81 mg, 0.10 mmol) and potassium carbonate (168 mg, 1.2 mmol) were added. The mixture was subjected to nitrogen replacement three times, reacted at 80° C. for 2 h and cooled to room temperature. The reaction solution was poured into 100 ml of water and extracted with 100 ml of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 105F (220 mg, yield: 73%).
LCMS m/z=657.3 [M+H]+.
Step 7: 3-(5-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (105G)
105F (220 mg, 0.33 mmol), iron powder (200 mg, 3.57 mmol) and ammonium chloride (200 mg, 3.77 mmol) were dissolved in ethanol (30 mL) and water (10 mL), and the mixture was stirred at 80° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated. 30 mL of water was added, and then the mixture was extracted with 30 ml of dichloromethane. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 105G (170 mg, yield: 81%), which was directly used in the next step.
LCMS m/z=627.3 [M+H]+.
Step 8: 3-(5-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (compound 105)
105G (170 mg, 0.27 mmol) and 40A (180 mg, 0.46 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 30 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 105 (25 mg, yield: 9.3%).
LCMS m/z=491.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.93 (s, 1H), 8.63-8.41 (m, 1H), 8.30 (s, 1H), 8.11 (s, 1H), 8.04 (s, 1H), 7.88 (s, 1H), 7.71 (d, 1H), 7.64-7.48 (m, 4H), 7.45 (t, 2H), 7.36 (t, 1H), 7.26-7.11 (m, 1H), 7.11-7.00 (m, 2H), 6.86 (s, 1H), 5.05 (dd, 1H), 4.41-4.17 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.39-3.22 (m, 4H), 3.17-3.00 (m, 2H), 2.98-2.81 (m, 1H), 2.69-2.52 (m, 6H), 2.43-2.16 (m, 4H), 2.02-1.92 (m, 1H), 1.89-1.72 (m, 8H), 1.69-1.53 (m, 1H), 1.45-1.26 (m, 2H).
Step 1: 5-(4-((1-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 106)
45F (256 mg, 0.4 mmol) and (2-((5-bromo-2-chloropyrimidin-4-yl)amino)-5-cyclopropylphenyl) dimethylphosphine oxide (55B) (200 mg, 0.5 mmol) were dissolved in DMF (5 mL): p-toluenesulfonic acid monohydrate (228 mg, 1.2 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 106. 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 106, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 106 (125 mg, yield: 31%).
LCMS m/z=502.7 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.23 (s, 1H), 7.96-7.75 (m, 5H), 7.46 (s, 1H), 7.35 (s, 1H), 7.31-7.15 (m, 2H), 6.91 (d, 1H), 5.12 (dd, 1H), 4.22-4.07 (m, 5H), 4.03 (s, 3H), 3.93-3.70 (m, 6H), 3.62-3.48 (m, 2H), 3.39-3.18 (m, 4H), 2.95-2.85 (m, 2H), 2.82-2.67 (m, 1H), 2.63-2.44 (m, 1H), 2.37-2.16 (m, 3H), 2.12-1.88 (m, 9H), 1.19-1.08 (m, 2H), 0.73-0.65 (m, 2H).
Step 1: (5-cyclopropyl-2-((2,5-dichloropyrimidin-4-yl)amino)phenyl) dimethylphosphine oxide (107A)
55A (10.7 g, 51.14 mmol) and 2,4,5-trichloropyrimidine (13.96 g, 76.71 mmol) were dissolved in NMP (40 mL); DIPEA (9.91 g, 76.71 mmol) was added; and under nitrogen protection, the mixture was stirred at 120° C. for 2 h, cooled to room temperature and extracted by adding 200 mL of ethyl acetate and 50 mL of water. The organic layer was washed 3 times with 50 mL of saturated brine, dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=1/100-1/20) to obtain 107A (15.6 g, yield: 86%).
LCMS m/z=356.1 [M+H]+.
Step 2: 5-(4-((1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 107)
45F (256 mg, 0.4 mmol) and 107A (178 mg, 0.5 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (228 mg, 1.2 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was filtered. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 107. 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 107, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 107 (120 mg, yield: 31.3%).
LCMS m/z=480.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.13 (s, 1H), 8.02-7.91 (m, 3H), 7.86 (s, 1H), 7.82 (d, 1H), 7.46 (s, 1H), 7.36 (s, 1H), 7.33-7.11 (m, 2H), 6.90 (d, 1H), 5.12 (dd, 1H), 4.23-4.07 (m, 5H), 4.04 (s, 3H), 3.93-3.74 (m, 6H), 3.63-3.48 (m, 2H), 3.39-3.20 (m, 4H), 2.95-2.85 (m, 2H), 2.82-2.65 (m, 1H), 2.63-2.45 (m, 1H), 2.37-2.17 (m, 3H), 2.14-1.88 (m, 9H), 1.18-1.07 (m, 2H), 0.71-0.63 (m, 2H).
Step 1: (2-((5-bromo-2-chloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (108B)
108A (5 g, 29.56 mmol) and 5-bromo-2,4-dichloropyrimidine (13.47 g, 59.12 mmol) were dissolved in NMP (50 mL); DIPEA (5.73 g, 44.34 mmol) was added; and under nitrogen protection, the mixture was stirred at 120° C. for 2 h, cooled to room temperature and extracted by adding 300 mL of ethyl acetate and 300 mL of water. The organic layer was washed with 150 mL of saturated brine, dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-10/1) to obtain 108B (8.6 g, yield: 80.6%)
LCMS m/z=360.0 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.61-8.47 (m, 1H), 8.37-8.21 (m, 1H), 7.76-7.45 (m, 2H), 7.31-7.13 (m, 1H), 1.81 (d, 6H).
Step 2: 5-(4-((1-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione; (compound 108)
45F (2 g, 3.12 mmol) and 108B (1.24 g, 3.43 mmol) were dissolved in DMF (30 mL); p-toluenesulfonic acid monohydrate (1.78 g, 9.36 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 100 mL of water and 100 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 108. 200 mL of dichloromethane and 100 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 108, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 108 (1.1 g, yield: 36.5%).
LCMS m/z=964.3 [M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.29 (s, 1H), 7.94 (s, 1H), 7.91-7.77 (m, 4H), 7.59 (dd, 1H), 7.46 (d, 1H), 7.39-7.23 (m, 3H), 7.19 (t, 1H), 5.12 (dd, 1H), 4.23-4.08 (m, 5H), 4.05 (s, 3H), 3.97-3.73 (m, 6H), 3.60-3.48 (m, 2H), 3.39-3.15 (m, 4H), 2.95-2.85 (m, 2H), 2.83-2.65 (m, 1H), 2.65-2.46 (m, 1H), 2.37-2.16 (m, 3H), 2.13-1.88 (m, 8H).
Step 1: (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (109B)
108A (5 g, 29.56 mmol) and 2,4,5-trichloropyrimidine (10.84 g, 59.12 mmol) were dissolved in NMP (50 mL); DIPEA (5.73 g, 44.34 mmol) was added; and under nitrogen protection, the mixture was stirred at 120° C. for 2 h, cooled to room temperature and extracted by adding 300 mL of ethyl acetate and 300 mL of water. The organic layer was washed with 150 mL of saturated brine, dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-10/1) to obtain 109B (8.3 g, yield: 88%)
LCMS m/z=316.0 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.49-8.36 (m, 2H), 7.70-7.56 (m, 2H), 7.33-7.18 (m, 1H), 1.82 (d, 6H).
Step 2: 5-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 109)
45F (3 g, 4.68 mmol) and 109B (1.63 g, 5.15 mmol) were dissolved in DMF (30 mL); p-toluenesulfonic acid monohydrate (2.67 g, 14.04 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 100 mL of water and 100 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was filtered. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 109 200 mL of dichloromethane and 100 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 109, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 109 (1.9 g, yield: 44.1%).
LCMS m/z=920.3 [M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.20 (s, 1H), 8.01-7.91 (m, 2H), 7.90-7.77 (m, 3H), 7.58 (dd, 1H), 7.46 (s, 1H), 7.39-7.24 (m, 3H), 7.17 (t, 1H), 5.12 (dd, 1H), 4.23-4.09 (m, 5H), 4.06 (s, 3H), 3.96-3.73 (m, 6H), 3.64-3.48 (m, 2H), 3.39-3.16 (m, 4H), 2.96-2.86 (m, 2H), 2.82-2.67 (m, 1H), 2.64-2.47 (m, 1H), 2.36-2.15 (m, 3H), 2.13-1.88 (m, 8H).
Step 1: tert-butyl 4-((1-((benzyloxy)carbonyl)piperidin-4-yl)methyl)piperazine-1-carboxylate (110A)
Benzyl 4-formylpiperidine-1-carboxylate (2.47 g, 10 mmol) and tert-butylpiperazine-1-carboxylate (1.86 g, 10 mmol) were mixed in dichloromethane (100 mL); acetic acid (1.2 g, 20 mmol) and sodium triacetoxyborohydride (4.24 g, 20 mmol) were successively added; and the mixture was stirred overnight at room temperature. 100 mL of dichloromethane and 50 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=10/1-1/1) to obtain 110A (3.54 g, yield: 85%).
LCMS m/z=418.2 [M+H]+.
Step 2: tert-butyl 4-(piperidin-4-ylmethyl)piperazine-1-carboxylate (110B)
110A (3.54 g, 8.49 mmol) was dissolved in methanol (50 mL); palladium on carbon (wt %=10%, 500 mg) was added; the mixture was subjected to hydrogen replacement 3 times, stirred overnight under hydrogen atmosphere (balloon pressure) at room temperature and filtered; and the filtrate was concentrated under reduced pressure to obtain the crude of 110B, which was directly used in the next step.
Step 3: 1-ethyl-4-(2-fluoro-4-methoxy-5-nitrophenyl)-1H-pyrazole (110C)
Under nitrogen protection, 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C) (2.49 g, 10 mmol) and 1-ethyl-1H-pyrazole-4-pinacolatodiboron (Cas: 847818-70-6, 3.33 g, 15 mmol) were added to a 500 mL single-necked flask and dissolved in 100 mL of 1,4-dioxane and 10 mL of water, and then Pd(dppf)Cl2·DCM (400 mg, 0.5 mmol) and potassium carbonate (4.14 g, 30 mmol) were added. The mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 16 h and cooled to room temperature. The reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 110C (2.1 g, yield: 80%).
LCMS m/z=266.1 [M+H]+.
Step 4: tert-butyl 4-((1-(2-(1-ethyl-1H-pyrazol-4-yl)-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate (110D)
110B (8.49 mmol), 110C (2.1 g, 7.92 mmol) and potassium carbonate (3.51 g, 25.5 mmol) were mixed and dissolved in DMSO (30 mL), and the mixture was stirred at 120° C. for 16 h, cooled to room temperature and extracted by adding 100 mL of water and 100 mL of ethyl acetate. The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 110D (2.5 g, yield: 60%).
LCMS m/z=529.1 [M+H]+.
Step 5: 1-((1-(2-(1-ethyl-1H-pyrazol-4-yl)-5-methoxy-4-nitrophenyl) piperidin-4-yl)methyl)piperazine (110E); 2,2,2-trifluoroacetate
110D (1.1 g, 2.0 mmol) was dissolved in 5 mL of dichloromethane; 3 mL of trifluoroacetic acid was added; and the mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the crude trifluoroacetate of 110E, which was directly used in the next step.
Step 6: 2-(2,6-dioxopiperidin-3-yl)-5-(4-((1-(2-(1-ethyl-1H-pyrazol-4-yl)-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-dione (110F)
The crude trifluoroacetate of 110E (2.08 mmol) was dissolved in 10 mL of DMSO; solid sodium bicarbonate (840 mg, 10.0 mmol) was added; and the mixture was stirred at room temperature for 10 min. DIPEA (3.45 mL, 20.8 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (662 mg, 2.40 mmol) were added, and the reaction was stirred at 80° C. for 5 h. The reaction solution was cooled to room temperature. 100 mL of water was added; and the mixture was filtered to collect the solid, which was washed with water, dissolved in DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude. The crude was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=15/1) to obtain 110F (0.73 g, yield: 51%).
LCMS m/z=685.3 [M+H]+.
Step 7: 5-(4-((1-(4-amino-2-(1-ethyl-1H-pyrazol-4-yl)-5-methoxyphenyl) piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (110I)
110F (0.73 g, 1.07 mmol) was dissolved in a mixed solution of ethanol (20 mL) and water (5 mL); reduced iron powder (480 mg, 8.56 mmol) and ammonium chloride (460 mg, 8.56 mmol) were added; and the mixture was reacted at 85° C. for 4 h. The reaction solution was cooled to room temperature and concentrated in vacuo to remove a solvent. 10 mL of water and 50 mL of dichloromethane were added to the residue, and then the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 1101 (620 mg, yield: 88%)
LCMS m/z=655.4 [M+H]+.
Step 8: 5-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-2-(1-ethyl-1H-pyrazol-4-yl)-5-methoxyphenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 110)
110I (310 mg, 0.47 mmol) and 40A (200 mg, 0.52 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (268 mg, 1.41 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 30 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (20 mL×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (30 ml×3), dried over anhydrous sodium sulfate and concentrated. Water was added, and the mixture was lyophilized to obtain compound 110 (120 mg, yield: 25%).
LCMS m/z=505.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.07 (s, 1H), 8.60-8.39 (m, 1H), 8.31 (s, 1H), 8.11 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.76-7.63 (m, 2H), 7.62-7.51 (m, 3H), 7.44 (t, 2H), 7.40-7.31 (m, 2H), 7.27 (d, 1H), 7.23-7.05 (m, 1H), 6.86 (s, 1H), 5.08 (dd, 1H), 4.06 (q, 2H), 3.80 (s, 3H), 3.56-3.40 (m, 4H), 3.17-3.01 (m, 2H), 2.96-2.80 (m, 1H), 2.70-2.50 (m, 8H), 2.29 (d, 2H), 2.11-1.97 (m, 1H), 1.91-1.72 (m, 8H), 1.72-1.47 (m, 1H), 1.41-1.25 (m, 5H).
Step 1: 5-(4-((1-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl) amino)pyrimidin-2-yl)amino)-2-(1-ethyl-1H-pyrazol-4-yl)-5-methoxyphenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 111)
110I (310 mg, 0.47 mmol) and 23D (230 mg, 0.52 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (268 mg, 1.41 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 30 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated. Suction filtration was performed for collecting the solid, and the filter cake was washed with water (20 ml×3) and redissolved in dichloromethane. The organic layers were separated, dried over anhydrous sodium sulfate and concentrated, and the crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (30 ml×3), dried over anhydrous sodium sulfate and concentrated. Water was added, and the mixture was lyophilized to obtain compound 111 (120 mg, yield: 24%).
LCMS m/z=527.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 11.07 (s, 1H), 8.50-8.35 (m, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.89 (s, 1H), 7.79-7.63 (m, 2H), 7.63-7.50 (m, 3H), 7.44 (t, 2H), 7.41-7.30 (m, 2H), 7.27 (d, 1H), 7.21-7.02 (m, 1H), 6.85 (s, 1H), 5.08 (dd, 1H), 4.06 (q, 2H), 3.79 (s, 3H), 3.57-3.38 (m, 4H), 3.16-3.00 (m, 2H), 2.97-2.79 (m, 1H), 2.71-2.51 (m, 8H), 2.28 (d, 2H), 2.10-1.96 (m, 1H), 1.90-1.69 (m, 8H), 1.69-1.52 (m, 1H), 1.39-1.25 (m, 5H).
Step 1: 5-(4-(1-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 112)
81B (2.3 g, 3.30 mmol) and 55B (1.45 g, 3.63 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (1.88 g, 9.90 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 100 mL of saturated aqueous sodium bicarbonate solution and 200 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 100 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (100 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 112 (1.0 g, yield: 31%).
LCMS m/z=495.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.84 (s, 1H), 8.25-8.07 (m, 3H), 7.98 (s, 1H), 7.88 (s, 1H), 7.69 (d, 1H), 7.57 (s, 1H), 7.41-7.13 (m, 3H), 6.80 (s, 1H), 6.57-6.34 (m, 1H), 5.07 (dd, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.60-3.40 (m, 4H), 3.20-3.06 (m, 2H), 2.97-2.82 (m, 1H), 2.75-2.55 (m, 8H), 2.40-2.29 (m, 1H), 2.09-1.97 (m, 1H), 1.95-1.85 (m, 2H), 1.85-1.70 (m, 7H), 1.69-1.54 (m, 2H), 0.95-0.81 (m, 2H), 0.57-0.39 (m, 2H).
Step 1: 5-(4-(1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 113)
81B (2.3 g, 3.30 mmol) and 107A (1.29 g, 3.63 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid monohydrate (1.88 g, 9.90 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution and 200 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 200 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (100 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 113 (1.1 g, yield: 32%).
LCMS m/z=473.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.07 (s, 1H), 8.31-8.19 (m, 1H), 8.15 (s, 1H), 8.07 (s, 1H), 8.00 (s, 1H), 7.89 (s, 1H), 7.69 (d, 1H), 7.59 (s, 1H), 7.41-7.15 (m, 3H), 6.81 (s, 1H), 6.54-6.31 (m, 1H), 5.07 (dd, 1H), 3.85 (s, 3H), 3.78 (s, 3H), 3.54-3.41 (m, 4H), 3.20-3.08 (m, 2H), 2.97-2.82 (m, 1H), 2.76-2.54 (m, 8H), 2.41-2.28 (m, 1H), 2.09-1.97 (m, 1H), 1.95-1.86 (m, 2H), 1.85-1.70 (m, 7H), 1.70-1.55 (m, 2H), 1.01-0.79 (m, 2H), 0.62-0.45 (m, 2H).
Step 1: 5-(4-(1-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 114)
81B (2.24 g, 3.57 mmol) and 108B (1.29 g, 3.57 mmol) were dissolved in DMF (30 mL); p-toluenesulfonic acid hydrate (2.04 g, 10.71 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 114 (1.1 g, yield: 32%).
LCMS m/z=475.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.94 (s, 1H), 8.40-8.23 (m, 1H), 8.24-8.08 (m, 2H), 7.95 (s, 1H), 7.84 (s, 1H), 7.69 (d, 1H), 7.59 (s, 1H), 7.55-7.44 (m, 1H), 7.39-7.20 (m, 2H), 7.06-6.86 (m, 2H), 6.80 (s, 1H), 5.07 (dd, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.53-3.42 (m, 4H), 3.20-3.05 (m, 2H), 2.96-2.81 (m, 1H), 2.76-2.53 (m, 8H), 2.40-2.27 (m, 1H), 2.07-1.97 (m, 1H), 1.93-1.83 (m, 2H), 1.76 (d, 6H), 1.68-1.50 (m, 2H).
Step 1: 5-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 115)
81B (2.24 g, 3.57 mmol) and 109B (1.13 g, 3.57 mmol) were dissolved in DMF (30 mL); p-toluenesulfonic acid hydrate (2.04 g, 10.71 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 115 (780 mg, yield: 24%).
LCMS m/z=453.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 11.07 (s, 1H), 8.55-8.39 (m, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 7.97 (s, 1H), 7.85 (s, 1H), 7.69 (d, 1H), 7.63-7.44 (m, 2H), 7.40-7.20 (m, 2H), 7.08-6.86 (m, 2H), 6.80 (s, 1H), 5.07 (dd, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.55-3.36 (m, 4H), 3.21-3.07 (m, 2H), 2.97-2.82 (m, 1H), 2.75-2.53 (m, 8H), 2.39-2.24 (m, 1H), 2.07-1.97 (m, 1H), 1.94-1.84 (m, 2H), 1.76 (d, 6H), 1.67-1.50 (m, 2H).
Step 1: 5-((S)-3-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 116)
103G (1.70 g, 2.71 mmol) and 54C (1.23 g, 2.71 mmol) were dissolved in DMF (40 mL); p-toluenesulfonic acid hydrate (1.55 g, 8.13 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 116 (750 mg, yield: 26%).
LCMS m/z=522.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 11.05 (s, 1H), 8.51-8.37 (m, 1H), 8.34 (s, 1H), 8.19 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H), 7.71-7.48 (m, 3H), 7.48-7.33 (m, 2H), 7.33-7.22 (m, 2H), 7.20-7.00 (m, 1H), 6.96-6.72 (m, 3H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.63-3.47 (m, 2H), 3.47-3.37 (m, 1H), 3.22-3.10 (m, 1H), 2.96-2.77 (m, 5H), 2.72-2.51 (m, 7H), 2.46-2.36 (m, 2H), 2.21-2.09 (m, 1H), 2.07-1.93 (m, 1H), 1.90-1.69 (m, 7H).
Step 1: 5-((R)-3-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-2′-fluoro-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 117)
54C (1.38 g, 3.03 mmol) and 104G (1.9 g, 3.03 mmol) were dissolved in DMF (15 mL); p-toluenesulfonic acid hydrate (1.15 g, 6.06 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further subjected to medium-pressure preparative reversed-phase column purification (acetonitrile/water (containing 0.1% TFA)=3%-40%) to obtain the preparative solution of the trifluoroacetate of compound 117. 10 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added; and the mixture was stirred for 5 min. Liquid separation and extraction were performed. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 117 (1.1 g, yield: 35%).
LCMS m/z=522.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 11.04 (s, 1H), 8.57-8.37 (m, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.85 (s, 1H), 7.73-7.48 (m, 3H), 7.47-7.34 (m, 2H), 7.34-7.22 (m, 2H), 7.19-7.00 (m, 1H), 6.96-6.69 (m, 3H), 5.05 (dd, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.63-3.48 (m, 2H), 3.47-3.38 (m, 1H), 3.21-3.10 (m, 1H), 3.00-2.76 (m, 5H), 2.73-2.51 (m, 7H), 2.45-2.35 (m, 2H), 2.21-2.09 (m, 1H), 2.08-1.96 (m, 1H), 1.93-1.67 (m, 7H).
Step 1: 5-((R)-3-((4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 118)
104G (250 mg, 0.40 mmol) and 40A (200 mg, 0.53 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 118 (60 mg, yield: 15%).
LCMS m/z=982.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.04 (s, 1H), 8.59-8.42 (m, 1H), 8.28 (s, 1H), 8.12 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.71 (dd, 1H), 7.67-7.59 (m, 2H), 7.59-7.50 (m, 2H), 7.45 (t, 2H), 7.33 (t, 1H), 7.22-7.06 (m, 1H), 6.98-6.86 (m, 2H), 6.82 (dd, 1H), 5.05 (dd, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.62-3.48 (m, 2H), 3.48-3.37 (m, 1H), 3.22-3.13 (m, 1H), 3.00-2.78 (m, 5H), 2.73-2.52 (m, 7H), 2.45 (d, 2H), 2.23-2.09 (m, 1H), 2.06-1.96 (m, 1H), 1.90-1.71 (m, 7H).
Step 1: 5-((R)-3-((4-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 119)
104G (250 mg, 0.40 mmol) and 107A (200 mg, 0.56 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 119 (100 mg, yield: 27%).
LCMS m/z=473.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.24-10.78 (m, 2H), 8.37-8.20 (m, 1H), 8.16 (s, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.65 (d, 1H), 7.59 (s, 1H), 7.23 (dd, 1H), 6.94-6.76 (m, 3H), 6.58-6.32 (m, 1H), 5.05 (dd, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 3.63-3.47 (m, 2H), 3.47-3.36 (m, 1H), 3.23-3.13 (m, 1H), 3.00-2.77 (m, 5H), 2.75-2.51 (m, 7H), 2.46 (d, 2H), 2.23-2.10 (m, 1H), 2.06-1.94 (m, 1H), 1.86-1.71 (m, 8H), 0.95-0.85 (m, 2H), 0.60-0.46 (m, 2H).
Step 1: 5-((R)-3-((4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 120)
104G (250 mg, 0.40 mmol) and 109B (200 mg, 0.63 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (160 mg, 0.84 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 20 mL of dichloromethane and extracted by adding saturated sodium bicarbonate solution (50 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 120 (120 mg, yield: 33%).
LCMS m/z=906.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 11.04 (s, 1H), 8.57-8.31 (m, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 7.70-7.57 (m, 2H), 7.56-7.44 (m, 1H), 7.05-6.93 (m, 2H), 6.92-6.86 (m, 1H), 6.86-6.73 (m, 2H), 5.05 (dd, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.63-3.46 (m, 2H), 3.46-3.35 (m, 1H), 3.22-3.11 (m, 1H), 3.01-2.79 (m, 5H), 2.73-2.52 (m, 7H), 2.45 (d, 2H), 2.21-2.08 (m, 1H), 2.06-1.93 (m, 1H), 1.86-1.66 (m, 8H).
Step 1: 5-((R)-3-((4-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 121)
55B (0.26 g, 0.64 mmol) and 104G (0.4 g, 0.64 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid hydrate (0.24 g, 1.28 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.05% ammonia water)) to obtain compound 121 (0.2 g, yield: 32%).
LCMS m/z=495.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.33-10.89 (m, 1H), 10.84 (s, 1H), 8.26-8.10 (m, 3H), 7.99 (s, 1H), 7.90 (s, 1H), 7.65 (d, 1H), 7.58 (s, 1H), 7.23 (dd, 1H), 6.98-6.76 (m, 3H), 6.48 (s, 1H), 5.05 (dd, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 3.64-3.36 (m, 3H), 3.25-3.14 (m, 1H), 2.99-2.80 (m, 5H), 2.75-2.51 (m, 7H), 2.48-2.37 (m, 2H), 2.22-2.08 (m, 1H), 2.07-1.93 (m, 1H), 1.88-1.67 (m, 8H), 0.98-0.78 (m, 2H), 0.62-0.37 (m, 2H).
Step 1: 5-((R)-3-((4-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 122)
108B (0.23 g, 0.64 mmol) and 104G (0.4 g, 0.64 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid hydrate (0.24 g, 1.28 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 122. 20 mL of dichloromethane and 20 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 122, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 122 (0.18 g, yield: 30%).
LCMS m/z=475.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 10.94 (s, 1H), 8.42-8.28 (m, 1H), 8.25-8.10 (m, 2H), 7.96 (s, 1H), 7.85 (s, 1H), 7.65 (d, 1H), 7.61 (s, 1H), 7.56-7.38 (m, 1H), 7.15-6.87 (m, 3H), 6.87-6.67 (m, 2H), 5.05 (dd, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.67-3.36 (m, 3H), 3.22-3.12 (m, 1H), 3.02-2.82 (m, 5H), 2.76-2.51 (m, 7H), 2.47-2.36 (m, 2H), 2.22-2.11 (m, 1H), 2.04-1.94 (m, 1H), 1.88-1.63 (m, 7H).
Step 1: methyl 5-(4-((4-(5-methoxy-2-(1-methyl-H-pyrazol-4-yl)-4-nitrophenyl) piperazin-1-yl)methyl)piperidin-1-yl)picolinate (123A)
The crude hydrochloride of 77B (prepared from 4.43 mmol of 77A) and methyl 5-fluoropyridine-2-carb oxyl ate (1.5 g, 9.66 mmol) were dissolved in DMSO (30 mL); potassium carbonate (3.34 g, 24.15 mmol) was added; and the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 100 mL of water was added, and the mixture was extracted by adding 100 mL of ethyl acetate. The organic layers were separated. The aqueous layer was extracted with ethyl acetate (3×30 mL). The organic phases were combined, washed sequentially with 50 mL of water and 50 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-19/1) to obtain 123A (1.66 g, yield: 68%).
LCMS m/z=550.3 [M+H]+.
Step 2: 5-(4-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperazin-1-yl)methyl)piperidin-1-yl)picolinic acid (123B)
123A (1.66 g, 3.02 mmol) was dissolved in a mixed solution of tetrahydrofuran (45 mL) and water (15 mL); lithium hydroxide monohydrate (0.44 g, 10.48 mmol) was added; and the mixture was reacted at room temperature for 16 h. After the reaction was completed, the reaction solution was adjusted to pH=7 by adding dilute hydrochloric acid (2 mol/L) and concentrated under reduced pressure to obtain the crude of 123B, which was directly used in the next step.
LCMS m/z=536.3 [M+H]+.
Step 3: N-(2,6-dioxopiperidin-3-yl)-5-(4-((4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)methyl)piperidin-1-yl)picolinamide (123C)
The crude of 123B from the previous step and 3-aminopiperidine-2,6-dione hydrochloride (0.75 g, 4.53 mmol) were dissolved in DMF (30 mL); DIPEA (1.95 g, 15.1 mmol) and HATU (1.72 g, 4.53 mmol) were added; and the mixture was reacted at room temperature for 3 h. After the reaction was completed, 100 mL of water was added, and the mixture was extracted by adding 100 ml of ethyl acetate. The organic layers were separated. The aqueous layer was extracted with ethyl acetate (3×30 mL). The organic phases were combined, washed sequentially with 50 mL of water and 50 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-19/1) to obtain 123C (1.48 g, two-step yield: 76.4%).
LCMS m/z=646.3 [M+H]+.
Step 4: 5-(4-((4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (123D)
123C (1.9 g, 2.94 mmol) was dissolved in ethanol (100 mL) and water (30 mL); iron powder (1.64 g, 29.4 mmol) was added, and ammonium chloride (1.57 g, 29.4 mmol) was then added; and under nitrogen protection, the mixture was reacted at 85° C. for 2 h. The reaction solution was cooled to room temperature and subjected to suction filtration over celite. The filtrate was extracted with dichloromethane (3×50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude of 123D as a white solid (1.27 g), which was directly used in the next step.
LCMS m/z=616.3 [M+H]+.
Step 5: 5-(4-((4-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 123)
123D (265 mg) and 23D (244 mg, 0.56 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (245 mg, 1.29 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with dichloromethane, concentrated in vacuo, and purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). 50 mL of dichloromethane was added to the preparative solution. The mixture was extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 123 (50 mg, yield: 8%).
LCMS m/z=1015.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 10.82 (s, 1H), 8.68 (d, 1H), 8.39 (s, 1H), 8.34-8.26 (m, 2H), 8.19 (s, 1H), 8.02 (s, 1H), 7.91-7.83 (m, 2H), 7.70 (dd, 1H), 7.62-7.51 (m, 3H), 7.49-7.33 (m, 4H), 7.17 (s, 1H), 6.86 (s, 1H), 4.81-4.64 (m, 1H), 3.95 (d, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 2.97-2.70 (m, 7H), 2.59-2.49 (m, 5H), 2.32-2.12 (m, 3H), 2.01-1.97 (m, 1H), 1.88-1.76 (m, 9H), 1.31-1.16 (m, 2H).
Step 1: 5-(4-((4-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)picolinamide (compound 124)
123D (265 mg) and 40A (219 mg, 0.56 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (245 mg, 1.29 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with dichloromethane, concentrated in vacuo, and purified by preparative HPLC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). 50 mL of dichloromethane was added to the preparative solution. The mixture was extracted by adding saturated sodium bicarbonate solution (50 ml). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 124 (80 mg, yield: 13%).
LCMS m/z=486.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.83 (s, 1H), 8.68 (d, 1H), 8.49 (s, 1H), 8.35-8.25 (m, 2H), 8.12 (s, 1H), 8.04 (s, 1H), 7.93-7.82 (m, 2H), 7.71 (dd, 1H), 7.61 (s, 1H), 7.56 (d, 2H), 7.50-7.34 (m, 4H), 7.16 (s, 1H), 6.87 (s, 1H), 4.84-4.68 (m, 1H), 3.95 (d, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.00-2.71 (m, 7H), 2.59-2.50 (m, 5H), 2.31-2.10 (m, 3H), 2.05-2.00 (m, 1H), 1.92-1.72 (m, 9H), 1.31-1.14 (m, 2H).
Step 1: 5-(4-((4-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 125)
77D (210 mg, 0.33 mmol) and 55B (158 mg, 0.40 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (188 mg, 0.99 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 40 mL of dichloromethane. The mixture was extracted by adding saturated sodium bicarbonate solution (20 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 125 (100 mg, yield: 30%).
LCMS m/z=1004.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.84 (s, 1H), 8.25-8.09 (m, 3H), 7.98 (s, 1H), 7.89 (s, 1H), 7.65 (d, 1H), 7.59 (s, 1H), 7.36-7.28 (m, 1H), 7.27-7.14 (m, 2H), 6.83 (s, 1H), 6.59-6.28 (m, 1H), 5.06 (dd, 1H), 4.11-3.97 (m, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.05-2.78 (m, 7H), 2.64-2.51 (m, 6H), 2.25 (d, 2H), 2.06-1.95 (m, 1H), 1.95-1.79 (m, 4H), 1.74 (d, 6H), 1.30-1.08 (m, 2H), 0.95-0.83 (m, 2H), 0.56-0.45 (m, 2H).
Step 1: 5-(4-((4-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 126)
77D (250 mg, 0.39 mmol) and 107A (170 mg, 0.47 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (223 mg, 1.17 mmol) was added; and under nitrogen protection, the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure. The concentrated solution was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The resultant was lyophilized. The resulting solid was dissolved in 40 mL of dichloromethane. The mixture was extracted by adding saturated sodium bicarbonate solution (20 mL). Liquid separation was performed. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 126 (80 mg, yield: 21%).
LCMS m/z=480.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.30-10.80 (m, 2H), 8.42-7.94 (m, 4H), 7.89 (s, 1H), 7.72-7.53 (m, 2H), 7.42-7.14 (m, 3H), 6.83 (s, 1H), 6.56-6.38 (m, 1H), 5.05 (dd, 1H), 4.13-3.96 (m, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.05-2.78 (m, 7H), 2.64-2.51 (m, 6H), 2.25 (d, 2H), 2.06-1.95 (m, 1H), 1.93-1.78 (m, 4H), 1.75 (d, 6H), 1.29-1.08 (m, 2H), 1.00-0.79 (m, 2H), 0.59-0.38 (m, 2H).
Step 1: 5-(4-((4-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 127)
77D (600 mg, 0.94 mmol) and 108B (372.83 mg, 1.03 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (536.42 mg, 2.82 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of water and 20 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 127. 60 mL of dichloromethane and 30 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 127, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 127 (260 mg, yield: 28.26%).
LCMS m/z=964.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.94 (s, 1H), 8.40-8.27 (m, 1H), 8.22-8.14 (m, 2H), 7.95 (s, 1H), 7.84 (s, 1H), 7.65 (d, 1H), 7.60 (s, 1H), 7.55-7.41 (m, 1H), 7.36-7.27 (m, 1H), 7.23 (dd, 1H), 7.04-6.90 (m, 2H), 6.82 (s, 1H), 5.06 (dd, 1H), 4.10-3.97 (m, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.07-2.79 (m, 7H), 2.68-2.50 (m, 6H), 2.28-2.17 (m, 2H), 2.09-1.97 (m, 1H), 1.92-1.79 (m, 3H), 1.76 (d, 6H), 1.27-1.08 (m, 2H).
Step 1: 5-(4-((4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 128)
77D (600 mg, 0.94 mmol) and 109B (329.84 mg, 1.04 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (536.42 mg, 2.82 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of water and 20 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 128. 60 mL of dichloromethane and 30 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 128, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 128 (200 mg, yield: 23.12%).
LCMS m/z=920.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 11.05 (s, 1H), 8.58-8.34 (m, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.97 (s, 1H), 7.85 (s, 1H), 7.65 (d, 1H), 7.62 (s, 1H), 7.57-7.40 (m, 1H), 7.31 (s, 1H), 7.28-7.14 (m, 1H), 7.07-6.90 (m, 2H), 6.83 (s, 1H), 5.06 (dd, 1H), 4.23-3.93 (m, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.05-2.78 (m, 7H), 2.67-2.50 (m, 6H), 2.24 (d, 2H), 2.08-1.94 (m, 1H), 1.93-1.80 (m, 3H), 1.76 (d, 6H), 1.29-1.08 (m, 2H).
Step 1: tert-butyl 9-(3-nitrobenzyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (129A)
Tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (3.0 g, 11.8 mmol) and 3-nitrobenzaldehyde (1.3 g, 8.6 mmol) were dissolved in DMAC (30 mL); 1 ml of acetic acid was added; and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (2.1 g, 10 mmol) was added, and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with water (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 129A (3.5 g, yield: 76%).
LCMS m/z=390.3[M+H]+.
Step 2: tert-butyl 9-(3-aminobenzyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (129B)
129A (3.5 g, 9.0 mmol), iron powder (5 g, 89.3 mmol) and ammonium chloride (5 g, 93.5 mmol) were dissolved in ethanol (60 mL) and water (20 mL); and the mixture was stirred at 80° C. for 2 h, cooled to room temperature and subjected to suction filtration. The filtrate was concentrated. 60 mL of water was added, and then the mixture was extracted with 60 ml of dichloromethane. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 129B (3.0 g, yield: 93%), which was directly used in the next step.
LCMS m/z=360.3 [(M+H]+.
Step 3: tert-butyl 9-(3-((2,6-dioxopiperidin-3-yl)amino)benzyl)-3,9-diazaspiro [5.5]undecane-3-carboxylate (129C)
129B (3.0 g, 8.36 mmol) and 3-bromopiperidine-2,6-dione (3.96 g, 20.6 mmol) were dissolved in DMSO (40 mL); sodium bicarbonate (4.0 g, 47.6 mmol) was added; and the mixture was reacted at 100° C. for 6 h, and cooled to room temperature and extracted by adding 100 mL of ethyl acetate and 100 mL of water. The organic layer was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (ethyl acetate: petroleum ether (V/V)=10/1-2/1) to obtain 129C (2.4 g, yield: 61%).
LCMS m/z=471.3 [M+H]+.
Step 4: 3-((3-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)phenyl)amino)piperidine-2,6-dione (129D); HCl
To a 50 mL round bottom flask were successively added 129C (1.0 g, 2.1 mmol) and a solution of hydrogen chloride in 1,4-dioxane (30 mL, 4 mol/L); and the mixture was reacted at room temperature for 2 h and concentrated to dryness under reduced pressure to obtain the hydrochloride of 129D, which was directly used in the next reaction.
Step 5: 3-((3-((9-(2-bromo-5-methoxy-4-nitrophenyl)-3,9-diazaspiro[5.5] undecan-3-yl)methyl)phenyl)amino)piperidine-2,6-dione (129E)
The compound 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C) (0.6 g, 2.40 mmol), the hydrochloride of 129D from the previous step and sodium bicarbonate (1.0 g, 11.9 mmol) were dissolved in 20 mL of DMSO; and the mixture was reacted at 100° C. for 3 h. After the reaction was completed, the mixture was cooled to room temperature. 50 mL of water and 50 mL of ethyl acetate were added. The ethyl acetate layer was separated. The aqueous layer was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 129E (0.8 g, two-step yield: 63%).
LCMS m/z=486.3 [M+H]+.
Step 6: 3-((3-((9-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)phenyl)amino)piperidine-2,6-dione (129F)
Under nitrogen protection, 129E (400 mg, 0.67 mmol) and 1-methyl-1H-pyrazole-4-boronic acid (95.7 mg, 0.76 mmol) were added to a 50 mL single-necked flask and dissolved in 20 mL of dioxane and 4 mL of water, and then Pd(dppf)Cl2·DCM (31 mg, 0.04 mmol) and sodium bicarbonate (96 mg, 1.14 mmol) were added. The mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 3 h and cooled to room temperature. The reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 129F (180 mg, yield: 45%).
LCMS m/z=602.3 [M+H]+.
Step 7: 3-((3-((9-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)phenyl)amino)piperidine-2,6-dione (129G)
129F (180 mg, 0.30 mmol) was dissolved in methanol (10 mL); palladium on carbon 10% (180 mg) was added at room temperature; and the mixture was reacted under hydrogen atmosphere for 2 h. After the reaction was completed, the mixture was subjected to suction filtration over celite. The filtrate was concentrated to obtain 129G (140 mg, yield: 82%).
LCMS m/z=572.3 [M+H]+.
Step 8: 3-((3-((9-(4-((5-bromo-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)phenyl)amino)piperidine-2,6-dione (compound 129)
129G (140 mg, 0.25 mmol) from the previous step and 23D (140 mg, 0.32 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (114 mg, 0.6 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated. 50 mL of DCM was added, and then the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. The dichloromethane layers were separated, and the aqueous layer was extracted with dichloromethane (2×30 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 129 (30 mg, yield: 13%).
LCMS m/z=486.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 10.75 (s, 1H), 8.48-8.35 (m, 1H), 8.30 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.86 (s, 1H), 7.68 (dd, 1H), 7.59-7.47 (m, 3H), 7.42 (t, 2H), 7.33 (t, 1H), 7.25-7.10 (m, 1H), 7.03 (t, 1H), 6.88 (s, 1H), 6.65 (s, 1H), 6.60-6.43 (m, 2H), 5.78 (d, 1H), 4.37-4.25 (m, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.39-3.32 (m, 2H), 2.86-2.70 (m, 5H), 2.69-2.54 (m, 1H), 2.41-2.22 (m, 4H), 2.17-2.04 (m, 1H), 1.98-1.78 (m, 7H), 1.62-1.37 (m, 8H).
Step 1: 3-((3-((9-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)phenyl)amino)piperidine-2,6-dione (compound 130)
129G (140 mg, 0.25 mmol) and 40A (140 mg, 0.74 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (140 mg, 0.81 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution and 50 mL of dichloromethane were added, and the layers were separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=100/1-20/1). The crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative solution, which was concentrated. 50 mL of DCM was added, and then the mixture was adjusted to a basic pH by adding saturated sodium bicarbonate solution. The dichloromethane layers were separated, and the aqueous layer was extracted with dichloromethane (2×30 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 130 (20 mg, yield: 8.8%).
LCMS m/z=464.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.75 (s, 1H), 8.52-8.43 (m, 1H), 8.30 (s, 1H), 8.10 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.69 (dd, 1H), 7.60-7.48 (m, 3H), 7.42 (t, 2H), 7.33 (t, 1H), 7.23-7.12 (m, 1H), 7.03 (t, 1H), 6.89 (s, 1H), 6.65 (s, 1H), 6.61-6.45 (m, 2H), 5.78 (d, 1H), 4.36-4.25 (s, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.40-3.30 (m, 2H), 2.88-2.69 (m, 6H), 2.65-2.54 (m, 1H), 2.42-2.25 (m, 4H), 2.17-2.07 (m, 1H), 1.96-1.77 (m, 7H), 1.62-1.42 (s, 7H).
Step 1: tert-butyl 4-(4-nitrophenyl)piperazine-1-carboxylate (131B)
131A (14.1 g, 10 mmol) and 1-tert-butoxycarbonylpiperazine (18.6 g, 10 mmol) were dissolved in DMF (100 mL); potassium carbonate (34.5 g, 25 mmol) was added; and the mixture was reacted at 50° C. for 3 h. The reaction solution was cooled to room temperature. 100 mL of water was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was slurried with petroleum ether (300 mL), subjected to suction filtration and concentrated under reduced pressure to obtain 131B (29.2 g, yield: 95%).
LCMS m/z=252.2 [M−55]+.
Step 2: tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (131C)
131B (11.9 g, 38.8 mmol) and palladium on carbon (1.2 g, 10 wt %) were dissolved in 60 mL of methanol and 60 mL of tetrahydrofuran, and then the mixture was subjected to hydrogen replacement three times, reacted at room temperature for 8 h and subjected to suction filtration over celite. The filtrate was concentrated under reduced pressure to obtain 131C (10.7 g, yield: 100%).
LCMS m/z=278.2 [M+H]+.
Step 3: tert-butyl 4-(4-((3-ethoxy-3-oxopropyl)amino)phenyl)piperazine-1-carboxylate (131D)
Lactic acid (7.2 g, 80 mmol) was added dropwise to a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (12.2 g, 80 mmol) in diethyl ether (100 ml), and the mixture was stirred overnight at room temperature, concentrated under reduced pressure and then dissolved in 1,4-dioxane (200 mL). 131C (10.7 g, 38.6 mmol) and ethyl acrylate (8 g, 80 mmol) were successively added, and the mixture was reacted at 90° C. for 24 h. After the reaction was completed, the mixture was cooled to room temperature and extracted with 300 mL of ethyl acetate and 200 mL of water. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=20/1-4/1) to obtain 131D (7.3 g, yield: 50%).
LCMS m/z=378.3 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 6.91-6.71 (m, 2H), 6.69-6.39 (m, 2H), 4.08 (q, 2H), 3.61-3.45 (m, 4H), 3.43-3.24 (m, 2H), 3.08-2.80 (m, 4H), 2.57-2.48 (m, 2H), 1.41 (s, 9H), 1.19 (t, 3H).
Step 4: tert-butyl4-(4-(N-(3-ethoxy-3-oxopropyl)cyanamido)phenyl)piperazine-1-carboxylate (131E)
131D (7.3 g, 19.4 mmol) and cyanogen bromide (4.24 g, 40 mmol) were dissolved in tetrahydrofuran (100 mL); sodium bicarbonate (10.08 g, 120 mmol) was added; and under nitrogen protection, the mixture was reacted at room temperature for 3 h. After the reaction was completed, the mixture was extracted with 200 mL of ethyl acetate and 100 mL of water. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 131E (6.5 g, yield: 83.5%).
LCMS m/z=403.3 [M+H]+.
Step 5: tert-butyl 4-(4-(1-(3-ethoxy-3-oxopropyl)ureido)phenyl)piperazine-1-carboxylate (131F)
131E (6.5 g, 16.2 mmol) was dissolved in toluene (100 mL); acetaldoxime (2.95 g, 50 mmol) and indium (III) chloride tetrahydrate (1.46 g, 5 mmol) were successively added; and the mixture was reacted at 130° C. for 3 h, and cooled to room temperature. 100 mL of ethyl acetate and 100 mL of saturated aqueous sodium bicarbonate solution were added, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-10/1) to obtain 131F (5.6 g, 82.5%).
LCMS m/z=421.2 [M+H]+.
Step 6: tert-butyl 4-(4-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl) piperazine-1-carboxylate (131G)
131F (5.6 g, 13.3 mmol) was dissolved in acetonitrile (50 mL); benzyltrimethylammonium hydroxide (40% in methanol) (8.3 g, 20 mmol) was added; and under nitrogen protection, the mixture was reacted at 90° C. for 15 min. After the reaction was completed, the mixture was cooled to room temperature and extracted with 150 mL of ethyl acetate and 60 mL of water. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 131G (3.7 g, yield: 74%).
LCMS m/z=375.2 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H), 7.11 (d, 2H), 6.86 (d, 2H), 3.74 (t, 2H), 3.63-3.45 (m, 4H), 3.16-3.02 (m, 4H), 2.74 (t, 2H), 1.41 (s, 9H).
Step 7: 1-(4-(piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (131H)
131G (3.7 g, 9.9 mmol) was dissolved in DCM (40 mL); trifluoroacetic acid (10 mL) was added at room temperature; and the mixture was stirred for 3 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=7-8 with 1 N aqueous NaOH solution and concentrated under reduced pressure to obtain 131H (2.7 g), which was directly used in the next step.
Step 8: tert-butyl 4-((4-(4-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl) piperazin-1-yl)methyl)piperidine-1-carboxylate (131I)
131H (1.37 g, 5 mmol) and 1-tert-butoxycarbonylpiperidine-4-carbaldehyde were mixed in dichloromethane (20 mL) and 2 ml of acetic acid, and the mixture was stirred at room temperature for 10 min. Sodium triacetoxyborohydride (2.12 g, 10 mmol) was added, and the mixture was stirred at room temperature for 0.5 h. 100 mL of dichloromethane was added. The mixture was adjusted to pH=7-8 with 1 N aqueous NaOH solution, and then the layers were separated. The organic layer was dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=20/1-10/1) to obtain 1311 (1.3 g, yield: 55%).
LCMS m/z=472.1 [M+H]+.
Step 9: 1-(4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (131J); Trifluoroacetate
131I (1.3 g, 2.76 mmol) was dissolved in dichloromethane (10 mL); trifluoroacetic acid (3 mL) was added; and the mixture was stirred at room temperature for 3 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 10: 1-(4-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (131K)
The crude trifluoroacetate of 131J from the previous step was dissolved in DMSO (25 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (828 mg, 3.3 mmol) and sodium bicarbonate (2.77 g, 33 mmol) were successively added; and the mixture was stirred at 100° C. for 12 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 131K (1.1 g, yield: 66%).
LCMS m/z=603.3 [M+H]+.
Step 11: 1-(4-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl) phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (131L)
131K (1.1 g, 1.83 mmol) was dissolved in ethanol/water (20 mL, 3:1); iron powder (530 mg, 9.5 mmol) and ammonium chloride (500 mg, 9.3 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 131L as a yellow solid (860 mg, yield: 82.3%).
LCMS m/z=573.4 [M+H]+.
Step 12: 1-(4-(4-((1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (compound 131)
131L (200 mg, 0.35 mmol) and 107A (178 mg, 0.5 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (228 mg, 1.2 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 131. 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 131, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 131 (80 mg, yield: 25.6%).
LCMS m/z=446.7 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.15 (s, 1H), 8.00-7.89 (m, 3H), 7.86 (s, 1H), 7.66-7.45 (m, 4H), 7.34 (s, 1H), 7.20 (d, 1H), 6.88 (d, 1H), 4.19-3.98 (m, 10H), 3.98-3.69 (m, 10H), 3.44-3.23 (m, 2H), 2.97-2.84 (m, 2H), 2.62-2.45 (m, 1H), 2.37-2.19 (m, 2H), 2.12-1.98 (m, 2H), 1.97-1.81 (m, 7H), 1.15-1.00 (m, 2H), 0.70-0.53 (m, 2H).
Step 1: 1-(4-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (compound 132)
131L (200 mg, 0.35 mmol) and 109B (158 mg, 0.5 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (228 mg, 1.2 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 132. 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 132, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 132 (60 mg, yield: 20.2%).
LCMS m/z=426.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.20 (s, 1H), 8.00-7.91 (m, 2H), 7.88-7.78 (m, 2H), 7.66-7.47 (m, 5H), 7.42-7.25 (m, 2H), 7.18 (t, 1H), 4.18 (s, 3H), 4.13-4.03 (m, 7H), 3.98-3.77 (m, 10H), 3.43-3.31 (m, 2H), 2.93 (t, 2H), 2.66-2.46 (m, 1H), 2.38-2.21 (m, 2H), 2.15-1.99 (m, 2H), 1.94 (d, 6H).
Step 1: 1-(4-(piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (133A)
131G (2.4 g, 6.41 mmol) was dissolved in DCM (20 mL); trifluoroacetic acid (20 mL) was added at room temperature; and the mixture was stirred for 3 h and concentrated under reduced pressure. 20 mL of dichloromethane was added to the residue, and the mixture was adjusted to pH=9-10 with 1 N aqueous NaOH solution. Liquid separation was performed. The organic layer was concentrated under reduced pressure to obtain the crude of 133A (1.5 g).
Step 2: tert-butyl 4-(4-(4-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl) piperazin-1-yl)piperidine-1-carboxylate (133B)
133A (1.5 g) and tert-butyl 4-oxopiperidine-1-carboxylate (1.3 g, 6.56 mmol) were dissolved in dichloromethane (20 mL); acetic acid (394 mg, 6.56 mmol) and sodium triacetoxyborohydride (1.7 g, 8.21 mmol) were successively added; and the mixture was reacted overnight at room temperature, adjusted to a basic pH by adding 1 N aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=20/1) to obtain 133B (1.2 g, yield: 48%).
LCMS m/z=458.3 [M+H]+.
Step 3: 1-(4-(4-(piperidin-4-yl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4 (1H,3H)-dione (133C); trifluoroacetic acid
133B (1.2 g, 2.62 mmol) was dissolved in DCM (15 mL); trifluoroacetic acid (15 mL) was added at room temperature; and the mixture was stirred at room temperature for 1 h and concentrated under reduced pressure, and the residue was directly used in the next step.
Step 4: 1-(4-(4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (133D)
The crude trifluoroacetate of 133C from the previous step was dissolved in DMSO (10 mL); 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (942 mg, 3.75 mmol) and sodium bicarbonate (1.7 g, 20 mmol) were successively added; and the mixture was stirred at 100° C. for 7 h, and cooled to room temperature. 10 mL of water was added, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 133D as a yellow solid (0.3 g, yield: 20%).
LCMS m/z=589.3 [M+H]+.
Step 5: 1-(4-(4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (133E)
133D (0.3 g, 0.51 mmol) was dissolved in ethanol/water (8 mL, 3:1); iron powder (142 mg, 2.55 mmol) and ammonium chloride (136 mg, 2.55 mmol) were successively added; and under nitrogen protection, the mixture was stirred at 80° C. for 1 h, cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was concentrated under reduced pressure, and then the crude was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 133E (220 mg, yield: 77%).
Step 6: 1-(4-(4-(1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (compound 133)
133E (90 mg, 0.16 mmol) and 107A (76 mg, 0.21 mmol) were dissolved in DMF (4 mL); p-toluenesulfonic acid monohydrate (61 mg, 0.32 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., cooled to room temperature and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1). The residue was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 133. The resulting solid was added to saturated aqueous sodium bicarbonate solution (20 mL). The mixture was extracted with dichloromethane (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 133 (15 mg, white solid, yield: 11%).
LCMS m/z=439.3 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1))δ 8.24 (s, 1H), 8.05 (s, 1H), 7.99 (s, 1H), 7.95 (dd, 1H), 7.64-7.54 (m, 3H), 7.52-7.45 (m, 2H), 7.17 (d, 1H), 6.98 (s, 1H), 6.79-6.71 (m, 1H), 4.14 (s, 3H), 4.10-4.00 (m, 4H), 3.97-3.81 (m, 9H), 3.62-3.52 (m, 1H), 3.49-3.37 (m, 2H), 3.14-3.00 (m, 2H), 2.93-2.83 (m, 2H), 2.45-2.32 (m, 2H), 2.19-2.05 (m, 2H), 1.93-1.78 (m, 7H), 1.08-0.97 (m, 2H), 0.55-0.46 (m, 2H).
Step 1: 1-(4-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)phenyl) dihydropyrimidine-2,4(1H,3H)-dione (compound 134)
133E (90 mg, 0.16 mmol) and 109B (76 mg, 0.24 mmol) were dissolved in DMF (4 mL); p-toluenesulfonic acid monohydrate (61 mg, 0.32 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. overnight, cooled to room temperature and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1). The residue was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 134. The resulting solid was added to saturated aqueous sodium bicarbonate solution (20 mL). The mixture was extracted with dichloromethane (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 134 (31 mg, white solid, yield: 23%).
LCMS m/z=419.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.23-8.19 (m, 1H), 8.13-8.05 (m, 3H), 7.73-7.67 (m, 1H), 7.67-7.58 (m, 2H), 7.59-7.47 (m, 3H), 7.33-7.25 (m, 1H), 7.21-7.12 (m, 1H), 7.08 (s, 1H), 4.19 (s, 3H), 4.16-4.04 (m, 4H), 4.03-3.88 (m, 7H), 3.74-3.63 (m, 1H), 3.59-3.49 (m, 1H), 3.30-3.17 (m, 1H), 3.04-2.87 (m, 4H), 2.52-2.38 (m, 1H), 2.30-2.13 (m, 1H), 1.95 (s, 3H), 1.91 (s, 3H), 1.89-1.76 (m, 2H), 0.68-0.58 (m, 2H).
Step 1: tert-butyl 4-(4-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (135A)
Under nitrogen protection, methyl 2-(4-bromophenyl)acetate (10.0 g, 43.65 mmol) and tert-butylpiperazine-1-carboxylate (12.2 g, 65.47 mmol) were added to a 500 mL single-necked flask and dissolved in 300 mL of 1,4-dioxane, and then S-(−)-1,1′-binaphthalene-2,2′-bisdiphenylphosphine (2.72 g, 4.37 mmol), palladium acetate (0.98 g, 4.37 mmol) and cesium carbonate (35.56 g, 109.13 mmol) were added. The mixture was subjected to nitrogen replacement three times, reacted at 100° C. for 16 h, and cooled to room temperature. The reaction solution was filtered, and the filtrate was concentrated. The residue was dissolved in ethyl acetate, and water was added. The mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 135A (9.5 g, yield: 65%).
LCMS m/z=335.1 [M+H]+.
Step 2: tert-butyl 4-(4-(4-cyano-1-methoxy-1-oxobutan-2-yl)phenyl) piperazine-1-carboxylate (135B)
Compound 135A (8.0 g, 23.95 mmol) was dissolved in toluene (80 mL); allyl cyanide (1.93 g, 28.7 mmol) and a solution of benzyltrimethylammonium hydroxide in methanol (wt %=40%, 501 mg, 1.2 mmol) were added; and the mixture was stirred overnight at room temperature. The mixture was extracted by adding 50 mL of water and 100 mL of ethyl acetate. Liquid separation was performed. The organic phases were collected and concentrated under reduced pressure. The residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=6/1-3/1) to obtain 135B (2.2 g, yield: 24%).
LCMS m/z=388.2 [M+H]+.
Step 3: tert-butyl 4-(4-(5-amino-1-methoxy-1,5-dioxopentan-2-yl)phenyl) piperazine-1-carboxylate (135C)
135B (2.2 g, 5.68 mmol) was dissolved in toluene (20 mL); acetaldoxime (1.0 g, 17.0 mmol) and indium chloride tetrahydrate (832 mg, 2.84 mmol) were added; and the mixture was stirred at 130° C. for 3 h, cooled to room temperature, and then extracted by adding 50 mL of ethyl acetate and 20 mL of water. Liquid separation was performed. The organic phases were collected, washed by adding saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain 135C (1.0 g, yield: 43%).
LCMS m/z=406.3 [M+H]+.
Step 4: tert-butyl 4-(4-(2,6-dioxopiperidin-3-yl)phenyl)piperazine-1-carboxylate (135D)
135C (1.0 g, 2.47 mmol) was dissolved in acetonitrile (20 mL), and the mixture was warmed to 60° C. A solution of benzyltrimethylammonium hydroxide in methanol (wt %=40%, 2.06 g, 4.94 mmol) was added dropwise to the reaction solution, and the mixture was stirred for 20 min, cooled to room temperature and extracted by adding 20 mL of water and 50 mL of dichloromethane. The organic layer was collected and washed by adding 20 mL of water. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-25/1) to obtain 135D (420 mg, yield: 46%).
LCMS m/z=374.3 [M+H]+.
Step 5: 3-(4-(piperazin-1-yl)phenyl)piperidine-2,6-dione (135E); HCl
135D (400 mg, 1.07 mmol) was dissolved in 1 mL of methanol; 4 N hydrochloric acid/dioxane solution (3 mL) was added; and the mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the crude hydrochloride of 135E, which was directly used in the next step.
Step 6: tert-butyl 4-((4-(4-(2,6-dioxopiperidin-3-yl)phenyl)piperazin-1-yl)methyl)piperidine-1-carboxylate (135F)
The crude hydrochloride of 135E was dissolved in 10 mL of methanol; a solution of sodium bicarbonate (180 mg, 2.14 mmol) in water (0.2 mL) was added; and the mixture was stirred at room temperature for 10 min, concentrated under reduced pressure to remove water, and dissolved in a mixed solution of dichloromethane (20 mL) and acetic acid (2 mL). 1-tert-butoxycarbonylpiperidine-4-carbaldehyde (227 mg, 1.07 mmol) was added, and the mixture was stirred at room temperature for 1 h. Sodium triacetylborohydride (677 mg, 3.21 mmol) was added, and the mixture was stirred at room temperature for another 2 h. 20 mL of dichloromethane and 5 mL of 1 N aqueous sodium hydroxide solution were added, and then the layers were separated. The organic layer was collected and dried under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 135F (300 mg, yield: 60%).
LCMS m/z=471.2 [M+H]+.
Step 7: 3-(4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (135G); HCl
135F (300 mg, 0.64 mmol) was dissolved in 0.5 mL of methanol; 4 N hydrochloric acid/dioxane solution (3 mL) was added; and the mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the crude hydrochloride of 135G, which was directly used in the next step.
Step 8: 3-(4-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl) piperidin-4-yl)methyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (135H)
The crude hydrochloride of 135G was dissolved in 10 mL of dimethyl sulfoxide; a sodium bicarbonate (538 mg, 6.4 mmol) solid was added; and the mixture was stirred at room temperature for 10 min. 4-(2-fluoro-4-methoxy-5-nitrophenyl)-1-methyl-1H-pyrazole (1D) (161 mg, 0.64 mmol) was then added, and the mixture was reacted overnight at 100° C. The reaction solution was cooled to room temperature, diluted by adding 30 mL of ethyl acetate and then washed with water (20 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude, which was purified by silica gel column chromatography (mobile phase: DCM/MeOH (V/V)=15/1) to obtain 135H (180 mg, two-step yield: 47%).
LCMS m/z=602.3 [M+H]+.
Step 9: 3-(4-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (135I)
135H (180 mg, 0.3 mmol) was dissolved in tetrahydrofuran (12 mL); a solution of ammonium chloride (80 mg, 1.5 mmol) in water (2 mL) was added; zinc powder (98 mg, 1.5 mmol) was added; and the mixture was reacted at room temperature for 20 min. The reaction solution was filtered over celite. 10 mL of saturated sodium bicarbonate solution was added to the filtrate, and the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 135I (130 mg, yield: 74%).
LCMS m/z=286.7 [(M+2H)/2]+.
Step 10: 3-(4-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (compound 135)
135I (130 mg, 0.23 mmol) and 109B (79 mg, 0.25 mmol) were dissolved in DMF (3 mL); p-toluenesulfonic acid monohydrate (131 mg, 0.69 mmol) was added; and the mixture was stirred at 100° C. for 16 h, cooled to room temperature and adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated. The aqueous phase was extracted with dichloromethane (10 ml×3), dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain compound 135 (19 mg, yield: 10%).
LCMS m/z=426.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 10.75 (s, 1H), 8.50-8.36 (m, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.96 (s, 1H), 7.84 (s, 1H), 7.60 (s, 1H), 7.57-7.44 (m, 1H), 7.29-7.12 (m, 1H), 7.11-7.03 (m, 2H), 7.02-6.96 (m, 1H), 6.93-6.86 (m, 2H), 6.81 (s, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.73 (dd, 1H), 3.18-3.03 (m, 6H), 2.73-2.57 (m, 3H), 2.54-2.40 (m, 5H), 2.36-2.22 (m, 2H), 2.20-2.06 (m, 1H), 2.06-1.99 (m, 1H), 1.83-1.71 (m, 8H), 1.69-1.60 (m, 1H), 1.39-1.27 (m, 2H).
Step 1: 1-(4-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (compound 136)
131L (200 mg, 0.35 mmol) and 40A (156 mg, 0.4 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (228 mg, 1.2 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of saturated aqueous sodium bicarbonate solution was added, and the mixture was subjected to suction filtration. The filter cake was dissolved in 50 mL of dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 136. 20 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 136, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 136 (100 mg, yield: 30.9%).
LCMS m/z=928.3 [M+]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.49-8.42 (m, 1H), 8.42-8.35 (m, 1H), 8.33-8.25 (m, 1H), 8.08-7.99 (m, 2H), 7.99-7.89 (m, 1H), 7.88-7.77 (m, 6H), 7.76-7.69 (m, 3H), 7.60-7.48 (m, 2H), 4.40-3.66 (m, 20H), 3.62-3.46 (m, 2H), 3.19-3.05 (m, 2H), 2.77-2.60 (m, 1H), 2.47-2.33 (m, 2H), 2.28-2.12 (m, 8H).
Step 1: 1-(4-(4-(1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (compound 137)
133E (70 mg, 0.13 mmol) and 40A (78 mg, 0.20 mmol) were dissolved in DMF (4 mL); p-toluenesulfonic acid monohydrate (49 mg, 0.26 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., cooled to room temperature and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1). The residue was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 137. The resulting solid was added to saturated aqueous sodium bicarbonate solution (20 mL). The mixture was extracted with dichloromethane (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 137 (17 mg, yield: 14%).
LCMS m/z=457.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1))δ 8.21 (s, 1H), 8.10 (d, 3H), 7.69 (s, 1H), 7.62 (d, 2H), 7.59-7.47 (m, 3H), 7.29 (t, 1H), 7.17 (t, 1H), 7.08 (s, 1H), 4.20 (s, 3H), 4.16-4.03 (m, 5H), 4.00-3.86 (m, 9H), 3.54 (d, 2H), 3.24 (t, 2H), 2.93 (t, 2H), 2.46 (d, 2H), 2.29-2.16 (m, 2H), 1.94 (d, 6H).
500 mg of compound 65 was subjected to preparative SFC separation and purification. Preparative SFC separation conditions: instrument: Waters 150 MGM; chromatographic column: DAICEL CHIRALCEL AD (250 mm×30 mm, 10 m); mobile phase: A for CO2 and B for IPA+ACN (0.1% NH3·H2O); gradient: 70% phase B isocratic elution; flow rate: 100 mL/min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 220 nm.
SFC analysis conditions: instrument: SHIMADZU LC-30AD sfc; chromatographic column: Chiralpak AD-3 50×4.6 mm I.D., 3 m; mobile phase: A for CO2 and B for IPA+ACN (0.05% DEA); gradient: B 60%; flow rate: 3 mL/min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 220 nm.
After preparative separation, the components with the same retention time were combined and concentrated under reduced pressure to obtain compound 138 (220 mg) and compound 139 (200 mg).
LCMS m/z=498.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.06 (s, 1H), 8.58-8.40 (m, 1H), 8.29 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.78-7.64 (m, 2H), 7.63-7.52 (m, 3H), 7.45 (t, 2H), 7.36 (t, 2H), 7.27 (dd, 1H), 7.22-7.10 (m, 1H), 6.86 (s, 1H), 5.07 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.57-3.39 (m, 4H), 3.15-3.00 (m, 2H), 2.97-2.77 (m, 1H), 2.72-2.51 (m, 8H), 2.35-2.21 (m, 2H), 2.07-1.94 (m, 1H), 1.91-1.71 (m, 8H), 1.70-1.56 (m, 1H), 1.40-1.26 (m, 2H).
LCMS m/z=498.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 11.06 (s, 1H), 8.58-8.41 (m, 1H), 8.29 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.88 (s, 1H), 7.75-7.65 (m, 2H), 7.62-7.51 (m, 3H), 7.45 (t, 2H), 7.36 (t, 2H), 7.28 (dd, 1H), 7.22-7.09 (m, 1H), 6.86 (s, 1H), 5.07 (dd, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.55-3.39 (m, 4H), 3.14-3.01 (m, 2H), 2.99-2.79 (m, 1H), 2.69-2.51 (m, 8H), 2.35-2.23 (m, 2H), 2.06-1.96 (m, 1H), 1.90-1.72 (m, 8H), 1.70-1.57 (m, 1H), 1.40-1.26 (m, 2H).
Step 1: 3-(5-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (compound 140)
105G (300 mg, 0.48 mmol) and 109B (228 mg, 0.72 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (182 mg, 0.96 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., cooled to room temperature and extracted by adding 20 mL of saturated aqueous sodium bicarbonate solution and 20 mL of dichloromethane. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1). The residue was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain the trifluoroacetate of compound 140. The resulting solid was added to saturated aqueous sodium bicarbonate solution (20 mL). The mixture was extracted with dichloromethane (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 140 (150 mg, yield: 35%).
LCMS m/z=906.3 [M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 11.21 (s, 1H), 10.93 (s, 1H), 8.44 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.96 (s, 1H), 7.85 (s, 1H), 7.61 (s, 1H), 7.56-7.45 (m, 2H), 7.10-7.04 (m, 2H), 7.04-6.90 (m, 2H), 6.81 (s, 1H), 5.05 (dd, 1H), 4.45-4.14 (m, 2H), 3.81 (d, 6H), 3.09 (d, 2H), 2.90 (ddd, 1H), 2.68-2.54 (m, 3H), 2.29 (d, 2H), 2.03-1.91 (m, 1H), 1.88-1.71 (m, 8H).
Step 1: (2-amino-5-fluorophenyl)dimethylphosphine oxide (141A)
4-fluoro-2-iodoaniline (5.00 g, 21.10 mmol), dimethylphosphine oxide (2.14 g, 27.43 mmol), potassium phosphate (6.72 g, 31.65 mmol), palladium acetate (0.24 g, 1.06 mmol), Xant-Phos (0.61 g, 1.06 mmol) and anhydrous magnesium sulfate (2.54 g, 21.1 mmol) were successively added to 1,4-dioxane (50 ml), and the mixture was subjected to nitrogen replacement three times, stirred at 100° C. for 8 h, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (ethyl acetate/petroleum ether (V/V)=1/1 to dichloromethane/methanol (V/V)=30/1) to obtain 141A (3 g, yield: 76%).
LCMS m/z=188.1 [M+H]+.
Step 2: (2-((5-bromo-2-chloropyrimidin-4-yl)amino)-5-fluorophenyl) dimethylphosphine oxide (141B)
141A (1.50 g, 7.97 mmol) and 5-bromo-2,4-dichloropyrimidine (3.63 g, 15.94 mmol) were dissolved in NMP (10 ml); DIPEA (1.24 g, 9.56 mmol) was added; and the mixture was stirred at 120° C. for 1 h, cooled to room temperature and extracted by adding 30 mL of water and 30 mL of ethyl acetate. The organic layer was washed twice with saturated brine, and concentrated under reduced pressure. The residue was slurried with 10 mL of MTBE, filtered and dried under reduced pressure to obtain 141B (1.5 g, yield: 50%).
LCMS m/z=378.0 [M+H]+.
Step 3: 5-(4-((1-(4-((5-bromo-4-((2-(dimethylphosphoryl)-4-fluorophenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 141)
45F (1.20 g, 1.87 mmol) and 141B (0.71 g, 1.87 mmol) were dissolved in DMF (10 mL); p-toluenesulfonic acid monohydrate (0.71 g, 3.74 mmol) was added; and the mixture was stirred overnight at 100° C., and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/3-100/5). 8 mL of acetonitrile was added to the resulting residue, and the mixture was stirred at 70° C. for 1 h and filtered. The filter cake was dried under reduced pressure to obtain compound 141 (0.44 g, yield: 24%).
LCMS m/z=491.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 10.78 (s, 1H), 8.30 (s, 1H), 8.17 (s, 1H), 8.16 (s, 1H), 7.98 (s, 1H), 7.82 (s, 1H), 7.68 (d, 1H), 7.61 (s, 1H), 7.44-7.36 (m, 1H), 7.34 (d, 1H), 7.26 (d, 1H), 6.81 (s, 1H), 6.68 (s, 1H), 5.11-5.03 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.49-3.40 (m, 4H), 3.13-3.02 (m, 2H), 2.95-2.81 (m, 1H), 2.70-2.45 (m, 8H), 2.29 (d, 2H), 2.06-1.97 (m, 1H), 1.78 (d, 6H), 1.84-1.60 (m, 3H), 1.39-1.27 (m, 2H).
Step 1: (2-((2,5-dichloropyrimidin-4-yl)amino)-5-fluorophenyl) dimethylphosphine oxide (142A)
141A (1.50 g, 7.97 mmol) and 2,4,5-trichloropyrimidine (2.92 g, 15.94 mmol) were dissolved in NMP (10 ml); DIPEA (1.24 g, 9.56 mmol) was added; and the mixture was stirred at 120° C. for 1 h, cooled to room temperature and extracted by adding 30 mL of water and 30 mL of ethyl acetate. The organic layer was washed twice with saturated brine, and concentrated under reduced pressure. The residue was slurried with 10 mL of MTBE, filtered and dried under reduced pressure to obtain 142A (1.4 g, yield: 52%).
LCMS m/z=334.0 [M+H]+.
Step 2: 5-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)-4-fluorophenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 142)
45F (1.20 g, 1.87 mmol) and 142A (0.62 g, 1.87 mmol) were dissolved in DMF (10 ml); p-toluenesulfonic acid monohydrate (0.71 g, 3.74 mmol) was added; and the mixture was stirred overnight at 100° C., and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/3-100/5). 8 mL of acetonitrile was added to the resulting residue, and the mixture was stirred at 70° C. for 1 h and filtered. The filter cake was dried under reduced pressure to obtain compound 142 (0.8 g, yield: 45%).
LCMS m/z=469.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.08-11.03 (m, 1H), 8.42 (s, 1H), 8.17 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.68 (d, 1H), 7.62 (s, 1H), 7.46-7.38 (m, 1H), 7.34 (d, 1H), 7.26 (d, 1H), 6.81 (s, 1H), 6.67 (s, 1H), 5.10-5.03 (m, 1H), 3.84 (s, 3H), 3.80 (s, 3H) 3.49-3.40 (m, 4H), 3.15-3.01 (m, 2H), 2.96-2.79 (m, 1H), 2.70-2.45 (m, 8H), 2.29 (d, 2H), 2.05-1.97 (m, 1H), 1.79 (d, 6H), 1.84-1.60 (m, 3H), 1.41-1.28 (m, 2H).
Step 1: tert-butyl4-(1-((benzyloxy)carbonyl)piperidine-4-carbonyl)piperazine-1-carboxylate (143A)
N-Cbz-piperidine-4-carboxylic acid (7.80 g, 29.63 mmol), N-Boc-piperazine (5.52 g, 29.63 mmol), HOBT (4.40 g, 32.59 mmol) and dichloromethane (80 mL) were added to a reaction flask. Under ice-water bath cooling, EDCI (8.52 g, 44.44 mmol) was added. After the addition, the mixture was reacted at room temperature for 3 h. The reaction solution was washed once with saturated sodium bicarbonate solution (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (eluent: DCM/CH3OH=50/1 to 20/1) to obtain 143A (10 g, yield: 78.2%).
Step 2: tert-butyl 4-(piperidine-4-carbonyl)piperazine-1-carboxylate (143B)
143A (10 g, 23.17 mmol), methanol (100 mL) and palladium on carbon (2 g) were added to a reaction flask, and the mixture was subjected to hydrogen replacement three times, reacted at room temperature for about 18 h and subjected to suction filtration over an appropriate amount of celite. The filter cake was washed with a small amount of methanol. The filtrate was concentrated to dryness under reduced pressure, stirred for 30 min under stirring by adding MTBE (50 mL) and subjected to suction filtration under reduced pressure. The filter cake was concentrated to dryness under reduced pressure to obtain 143B (6.30 g, yield: 91.4%).
Step 3: tert-butyl 4-(1-(2-bromo-5-methoxy-4-nitrophenyl)piperidine-4-carbonyl)piperazine-1-carboxylate (143C)
143B (6.30 g, 21.18 mmol), 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C, 5.30 g, 21.18 mmol), potassium carbonate (4.39 g, 31.77 mmol) and DMSO (70 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for 2-3 h. The reaction solution was cooled to room temperature. Methanol (35 mL) was added, and water (200 mL) was added dropwise. After the addition, the mixture was stirred for 1 h and subjected to suction filtration. The filter cake was washed with a small amount of water and subjected to suction filtration until dryness. The filter cake was transferred to a reaction flask. MTBE (100 mL) was added; under stirring, petroleum ether (50 mL) was added dropwise; and the mixture was slurried under stirring for 1 h and subjected to suction filtration. The filter cake was washed with a small amount of petroleum ether and concentrated to dryness under reduced pressure to obtain 143C (10 g, yield: 89.5%).
Step 4: tert-butyl 4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidine-4-carbonyl)piperazine-1-carboxylate (143D)
143C (10 g, 18.96 mmol), 1-methyl-1H-pyrazole-4-boronic acid (3.58 g, 28.44 mmol), Pd(dppf)Cl2·DCM (2.31 g, 2.84 mmol), potassium carbonate (8.14 g, 56.88 mmol), 1,4-dioxane (100 mL) and water (30 mL) were added to a reaction flask, and the mixture was subjected to nitrogen replacement three times, reacted at 100° C. for about 3 h, and cooled in a water bath. Ethyl acetate (100 mL) and water (50 mL) were added. The mixture was stirred, and the layers were separated. The organic layer was washed with saturated sodium chloride solution (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to obtain 143D (8.2 g, yield: 81.81%).
Step 5: (1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)(piperazin-1-yl)methanone (143E)
143D (8.2 g, 15.51 mmol) and DCM (40 mL) were added to a reaction flask; trifluoroacetic acid (16 mL) was added under stirring; and the mixture was reacted overnight at room temperature for about 16 h. The reaction solution was concentrated to dryness under reduced pressure. Dichloromethane (50 mL) was added to the residue, and the mixture was adjusted to pH 12-13 by dropwise adding 1 N sodium hydroxide solution. The layers were separated. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to obtain 143E (6.2 g, yield: 93.3%).
Step 6: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidine-4-carbonyl)piperazin-1-yl)isoindoline-1,3-dione (143F)
143E (4.0 g, 9.34 mmol), 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (2.58 g, 9.34 mmol), DIPEA (1.81 g, 14.01 mmol) and DMSO (40 mL) were added to a reaction flask, and the mixture was reacted at 90° C. for 3 h and cooled in an ice water. Ethyl acetate (150 mL) and water (100 mL) were added, and the mixture was stirred for 5 min and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 20/1 to obtain 143F (3 g, yield: 46.9%).
Step 7: 5-(4-(1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidine-4-carbonyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (143G)
143F (2 g, 2.92 mmol), zinc powder (0.95 g, 14.60 mmol), ammonium chloride (0.78 g, 14.60 mmol), tetrahydrofuran (20 mL) and water (8 mL) were added to a reaction flask, and the mixture was reacted at room temperature for 1 h. Dichloromethane (50 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred for 2 min, subjected to suction filtration over an appropriate amount of celite and washed with dichloromethane (30 mL×2). The layers in the filtrate were separated. The organic layer was washed with water (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to obtain 143G (yellow solid, 1.6 g, yield: 83.7%).
Step 8: 5-(4-(1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidine-4-carbonyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 143)
143G (0.4 g, 0.61 mmol), 107A (0.22 g, 0.61 mmol), p-toluenesulfonic acid monohydrate (0.23 g, 1.22 mmol) and DMF (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, dichloromethane (30 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to 20/1 to obtain compound 143 (0.23 g, yield: 38.7%).
LCMS m/z=487.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 11.07 (s, 1H), 8.29-8.21 (m, 1H), 8.14 (s, 1H), 8.07 (s, 1H), 7.96 (s, 1H), 7.91 (s, 1H), 7.71 (d, 1H), 7.62 (s, 1H), 7.36 (d, 1H), 7.29-7.20 (m, 2H), 6.82 (s, 1H), 6.53-6.33 (m, 1H), 5.12-5.04 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.76-3.63 (m, 4H), 3.58-3.45 (m, 4H), 3.17-3.07 (m, 2H), 2.95-2.67 (m, 4H), 2.64-2.45 (m, 2H), 2.07-2.00 (m, 1H), 1.85-1.72 (m, 11H), 0.95-0.88 (m, 2H), 0.54-0.48 (m, 2H).
Step 1: 5-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidine-4-carbonyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 144)
143G (0.4 g, 0.61 mmol), (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (109B, 0.22 g, 0.61 mmol), p-toluenesulfonic acid monohydrate (0.23 g, 1.22 mmol) and DMF (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, dichloromethane (30 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCMICH3OH=DCM to 50/1 to 30/1 to 20/1 to obtain compound 144 (0.26 g, yield: 45.55%).
LCMS m/z=467.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 11.07 (s, 1H), 8.50-8.39 (m, 1H), 8.19 (s, 1H), 8.11 (s, 1H), 7.93 (s, 1H), 7.87 (s, 1H), 7.71 (d, 1H), 7.63 (s, 1H), 7.55-7.46 (m, 1H), 7.36 (d, 1H), 7.28-7.22 (m, 1H), 7.05-6.91 (m, 2H), 6.81 (s, 1H), 5.12-5.04 (m, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 377-3.61 (m, 4H), 3.57-3.43 (m, 4H), 3.17-3.07 (m, 2H), 2.96-2.66 (m, 4H), 2.64-2.45 (m, 2H), 2.08-1.98 (m, 1H), 1.86-1.68 (m, 10H).
Step 1: 5-(4-(1-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidine-4-carbonyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 145)
143G (0.4 g, 0.61 mmol), 55B (0.24 g, 0.61 mmol), p-toluenesulfonic acid monohydrate (0.23 g, 1.22 mmol) and DMF (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, dichloromethane (30 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to 20/1. The resulting crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative product, which was alkalized with saturated aqueous sodium bicarbonate solution to obtain compound 145 (0.06 g, yield: 9.65%).
LCMS m/z=509.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 10.84 (s, 1H), 8.20-8.10 (m, 3H), 7.94 (s, 1H), 7.91 (s, 1H), 7.71 (d, 1H), 7.60 (s, 1H), 7.37 (d, 1H), 7.29-7.19 (m, 2H), 6.81 (s, 1H), 6.54-6.35 (m, 1H), 5.12-5.03 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.75-3.63 (m, 4H), 3.58-3.45 (m, 4H), 3.16-3.07 (m, 2H), 2.96-2.67 (m, 4H), 2.65-2.45 (m, 2H), 2.06-1.99 (m, 1H), 1.85-1.71 (m, 11H), 0.94-0.87 (m, 2H), 0.53-0.47 (m, 2H).
Step 1: 5-(4-(1-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidine-4-carbonyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 146)
143G (0.4 g, 0.61 mmol), 108B (0.22 g, 0.61 mmol), p-toluenesulfonic acid monohydrate (0.23 g, 1.22 mmol) and DMF (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, dichloromethane (30 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to 20/1. The resulting crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a product, which was alkalized with saturated aqueous sodium bicarbonate solution to obtain compound 146 (0.09 g, yield: 15%).
LCMS m/z=489.6 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 10.95 (s, 1H), 8.40-8.28 (m, 1H), 8.18 (s, 2H), 7.91 (s, 1H), 7.86 (s, 1H), 7.71 (d, 1H), 7.61 (s, 1H), 7.53-7.44 (m, 1H), 7.36 (d, 1H), 7.29-7.23 (m, 1H), 7.05-6.90 (m, 2H), 6.80 (s, 1H), 5.11-5.03 (m, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.75-3.63 (m, 4H), 3.57-3.45 (m, 4H), 3.16-3.07 (m, 2H), 2.94-2.66 (m, 4H), 2.65-2.45 (m, 2H), 2.05-1.97 (m, 1H), 1.83-1.70 (m, 10H).
Step 1: tert-butyl 4-fluoro-4-formylpiperidine-1-carboxylate (147A)
1-tert-butoxycarbonyl-4-fluoro-4-(hydroxymethyl)piperidine (14 g, 60.01 mmol) and dichloromethane (210 mL) were added to a reaction flask, and the mixture was stirred until a clear solution was obtained. Under ice water cooling, Dess-Martin periodinane (80.91 g, 120.02 mmol) was added, and the mixture was reacted at room temperature for about 3 h. Saturated sodium bicarbonate solution (300 mL) was added, and the mixture was stirred for 5 min and subjected to suction filtration over an appropriate amount of celite. The filter cake was washed with dichloromethane (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: PE/EA=1/1) to obtain 147A (5 g, yield: 36.03%).
Step 2: benzyl 4-((1-(tert-butoxycarbonyl)-4-fluoropiperidin-4-yl)methyl)piperazine-1-carboxylate (147B)
147A (5 g, 21.62 mmol), benzyl-1-piperazine carbonate (4.76 g, 21.62 mmol) and dichloromethane (100 mL) were added to a reaction flask; glacial acetic acid (2.59 g, 43.24 mmol) and sodium triacetoxyborohydride (11.45 g, 54.05 mmol) were added under stirring; and the mixture was reacted overnight at room temperature for about 18 h. Water (100 mL) was added, and the mixture was stirred for 5 min and allowed to stand for layer separation. The organic layer was washed with saturated sodium bicarbonate solution (100 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: PE/EA=2/1) to obtain 147B (6.2 g, yield: 65.84%).
Step 3: benzyl 4-((4-fluoropiperidin-4-yl)methyl)piperazine-1-carboxylate (147C)
147B (6.2 g, 14.24 mmol) and dichloromethane (30 mL) were added to a reaction flask, and the mixture was stirred and dissolved. Trifluoroacetic acid (12 mL) was added dropwise. After the addition, the mixture was reacted at room temperature for 3 h and concentrated to dryness under reduced pressure. DCM (50 mL) was added to the residue, and the mixture was adjusted to about pH 12 with 1 N sodium hydroxide solution and allowed to stand for layer separation. The aqueous layer was extracted once with DCM (30 mL). The organic layers were combined, washed with saturated sodium chloride solution (60 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to obtain 147C (4.2 g, yield: 87.93%).
Step 4: benzyl 4-((1-(2-bromo-5-methoxy-4-nitrophenyl)-4-fluoropiperidin-4-yl)methyl)piperazine-1-carboxylate (147D)
147C (4.2 g, 12.52 mmol), 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C, 3.13 g, 12.52 mmol), potassium carbonate (3.46 g, 25.04 mmol) and DMSO (40 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 3 h and cooled to room temperature. Ethyl acetate (100 mL) and water (100 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: PE/EA=PE to 5/1 to 3/1 to 2/1) to obtain 147D (5 g, yield: 70.63%).
Step 5: Benzyl 4-((4-fluoro-1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)piperazine-1-carboxylate (147E)
147D (5 g, 8.84 mmol), 1-methyl-1H-pyrazole-4-boronic acid (1.67 g, 13.26 mmol), Pd(dppf)Cl2·DCM (1.07 g, 1.33 mmol), potassium carbonate (2.44 g, 17.68 mmol), 1,4-dioxane (50 mL) and water (20 mL) were added to a reaction flask, and the mixture was subjected to nitrogen replacement three times, reacted at 100° C. for about 3 h and cooled in a water bath. Ethyl acetate (100 mL) and water (50 mL) were added. The mixture was stirred, and the layers were separated. The organic layer was washed with saturated sodium chloride solution (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to obtain 147E (4.1 g, yield: 81.85%).
Step 6: 4-(4-fluoro-4-(piperidin-4-ylmethyl)piperidin-1-yl)-2-methoxy-5-(1-methyl-1H-pyrazol-4-yl)aniline (147F)
147E (0.5 g, 0.88 mmol), Pd/C (0.1 g), methanol (5 mL) and glacial acetic acid (1 mL) were added to a reaction flask, and the mixture was subjected to hydrogen replacement three times, subjected to hydrogenation at room temperature for about 20 h and filtered over an appropriate amount of celite. The filter cake was washed with a small amount of methanol, and the filtrate was concentrated to dryness under reduced pressure to obtain 147F, which was directly used in the next step.
Step 7: 5-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-4-fluoropiperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (147G)
147F (0.35 g, 0.88 mmol), 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 g, 0.88 mmol), DIPEA (0.17 g, 1.32 mmol) and DMSO (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for 3 h and cooled in ice water. Ethyl acetate (30 mL) and water (20 mL) were added, and the mixture was stirred for 5 min and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (10 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to obtain 147G (0.10 g, yield: 17.45%).
Step 8: 5-(4-((1-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-4-fluoropiperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 147)
147G (0.10 g, 0.15 mmol), (2-((5-bromo-2-chloropyrimidin-4-yl)amino)-5-cyclopropylphenyl) dimethylphosphine oxide (55B, 0.06 g, 0.15 mmol), p-toluenesulfonic acid monohydrate (0.057 g, 0.30 mmol) and DMF (2 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, dichloromethane (30 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH3OH=DCM to 50/1 to 30/1 to obtain compound 147 (0.09 g, yield: 58.66%).
LCMS m/z=511.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 10.84 (s, 1H), 8.22-8.10 (m, 3H), 7.99 (s, 1H), 7.90 (s, 1H), 7.68 (d, 1H), 7.59 (s, 1H), 7.35 (s, 1H), 7.30-7.19 (m, 2H), 6.83 (s, 1H), 6.48 (s, 1H), 5.07 (dd, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 3.47 (s, 4H), 2.98-2.82 (m, 5H), 2.76-2.52 (m, 8H), 2.09-1.93 (m, 4H), 1.91-1.67 (m, 8H), 0.94-0.83 (m, 2H), 0.56-0.48 (m, 2H).
Step 1: tert-butyl 3-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)azetidine-1-carboxylate (148A)
80B (3.17 g, 9.99 mmol) and tert-butyl 3-oxoazetidine-1-carboxylate (1.71 g, 9.99 mmol) were dissolved in DCM (100 mL); acetic acid (1.71 mL, 29.97 mmol) was added; and the mixture was stirred at 40° C. for 1 h, and then cooled to room temperature. Sodium triacetoxyborohydride (4.23 g, 19.98 mmol) was added, and the mixture was reacted at room temperature for 3 h. After the reaction was completed, the mixture was washed by adding 50 mL of saturated aqueous sodium bicarbonate solution. Liquid separation was performed. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-20/1) to obtain 148A (2.42 g, yield: 51%).
LCMS m/z=473.2 [M+H]+.
Step 2: 1-(azetidin-3-yl)-4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazine (148B)
148A (2.40 g, 5.08 mmol) was added to a flask. Under stirring, a solution of hydrochloric acid in dioxane (4 N, 13 mL) was slowly added, and the mixture was reacted at room temperature for 1 h and concentrated under reduced pressure. 20 mL of dichloromethane and a sodium bicarbonate solid (2.13 g, 25.4 mmol) were added to the residue, and the mixture was stirred at room temperature for 10 min and filtered to remove excess sodium bicarbonate solids. The filtrate was concentrated to obtain 148B (1.5 g, yield: 79%).
Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-(3-(4-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperazin-1-yl)azetidin-1-yl)isoindoline-1,3-dione (148C)
148B (1.5 g, 4.03 mmol) was dissolved in DMSO (15 mL); 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (1.11 g, 4.03 mmol) and DIPEA (2.0 mL, 12.09 mmol) were successively added; and the mixture was stirred at 90° C. overnight, and cooled to room temperature. Saturated sodium bicarbonate solution was added, with a large amount of solids precipitated, and the mixture was filtered. The filter cake was dried under reduced pressure and then purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain 148C (1.35 g, yield: 53%).
LCMS m/z=629.2 [M+H]+.
Step 4: 5-(3-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (148D)
148C (1.3 g, 2.07 mmol) was dissolved in tetrahydrofuran (20 mL), and a solution of ammonium chloride (1.11 g, 20.7 mmol) in water (5 mL) and zinc powder (1.35 g, 20.7 mmol) were successively added. After the addition, the mixture was stirred at 40° C. for 0.5 h. After the reaction was completed, the mixture was cooled to room temperature and subjected to suction filtration under reduced pressure. The filtrate was extracted by adding dichloromethane (20 mL) and saturated aqueous sodium bicarbonate solution (10 mL). Liquid separation was performed. The organic phase was then dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to obtain 148D (900 mg, yield: 73%).
LCMS m/z=599.2 [M+H]+.
Step 5: 5-(3-(4-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 148)
148D (200 mg, 0.33 mmol) and 107A (120 mg, 0.33 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (190 mg, 0.99 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction solution, with a yellow solid precipitated, and the mixture was filtered to obtain a solid, which was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain a purified crude product. The crude product obtained from the column chromatography was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The collected preparative solution was adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and then concentrated to obtain compound 148 (60 mg, yield: 20%).
LCMS m/z=459.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 11.05 (s, 1H), 8.29-8.23 (m, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 8.00 (s, 1H), 7.89 (s, 1H), 7.66 (d, 1H), 7.62 (s, 1H), 7.22 (dd, 1H), 6.83-6.82 (m, 2H), 6.69 (dd, 1H), 6.51-6.40 (m, 1H), 5.08-5.03 (m, 1H), 4.16 (t, 2H), 3.91-3.88 (m, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.52-3.44 (m, 1H), 2.99-2.84 (m, 5H), 2.64-2.51 (m, 6H), 2.06-1.96 (m, 1H), 1.87-1.78 (m, 1H), 1.75 (d, 6H), 0.92-0.83 (m, 2H), 0.58-0.41 (m, 2H).
Step 1: 5-(3-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 149)
148D (200 mg, 0.33 mmol) and (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (109B) (100 mg, 0.33 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (190 mg, 0.99 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction solution, with a yellow solid precipitated, and the mixture was filtered to obtain a solid, which was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain a purified crude product. The crude product obtained from the column chromatography was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The collected preparative solution was adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and then concentrated to obtain compound 149 (50 mg, yield: 17%).
LCMS m/z=439.6 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 11.10-10.96 (m, 1H), 8.68-8.64 (m, 1H), 8.52-8.32 (m, 1H), 8.21 (s, 1H), 8.10 (s, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 7.66 (d, 1H), 7.60 (s, 1H), 7.54-7.42 (m, 1H), 7.08-6.94 (m, 2H), 6.85-6.76 (m, 2H), 6.68 (dd, 1H), 5.06 (dd, 1H), 4.15 (t, 2H), 3.96-3.86 (m, 2H), 3.81 (d, 6H), 3.50-3.40 (m, 1H), 2.97-2.80 (m, 5H), 2.59-2.52 (m, 4H), 2.06-1.97 (m, 1H), 1.76 (d, 6H).
Step 1: 5-(3-(4-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 150)
148D (200 mg, 0.33 mmol) and (2-((5-bromo-2-chloropyrimidin-4-yl)amino)-5-cyclopropylphenyl) dimethylphosphine oxide (55B) (130 mg, 0.5 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (190 mg, 0.99 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction solution, with a yellow solid precipitated, and the mixture was filtered to obtain a solid, which was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain a purified crude product. The crude product obtained from the column chromatography was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The collected preparative solution was adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and then concentrated to obtain compound 150 (50 mg, yield: 16%).
LCMS m/z=481.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.84 (s, 1H), 8.23-8.10 (m, 3H), 7.98 (s, 1H), 7.88 (s, 1H), 7.66 (d, 1H), 7.61 (s, 1H), 7.21 (dd, 1H), 6.85-6.78 (m, 2H), 6.68 (dd, 1H), 6.57-6.37 (m, 1H), 5.06 (dd, 1H), 4.16 (t, 2H), 3.93-3.87 (m, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 3.55-3.40 (m, 1H), 2.98-2.80 (m, 5H), 2.63-2.51 (m, 6H), 2.08-1.96 (m, 1H), 1.85-1.79 (m, 1H), 1.74 (d, 6H), 0.95-0.83 (m, 2H), 0.56-0.44 (m, 2H).
Step 1: 5-(3-(4-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 151)
148D (200 mg, 0.33 mmol) and (2-((5-bromo-2-chloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (108B) (120 mg, 0.33 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (190 mg, 0.99 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction solution, with a yellow solid precipitated, and the mixture was filtered to obtain a solid, which was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain a purified crude product. The crude product obtained from the column chromatography was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The collected preparative solution was adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and then concentrated to obtain compound 151 (50 mg, yield: 16%).
LCMS m/z=461.6 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.94 (s, 1H), 8.38-8.27 (m, 1H), 8.20 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 7.85 (s, 1H), 7.66 (d, 1H), 7.59 (s, 1H), 7.53-7.41 (m, 1H), 7.05-6.93 (m, 2H), 6.82 (s, 2H), 6.68 (dd, 1H), 5.06 (dd, 1H), 4.15 (t, 2H), 3.95-3.86 (m, 2H), 3.81 (d, 6H), 3.50-3.41 (m, 1H), 2.96-2.81 (m, 5H), 2.62-2.53 (m, 4H), 2.05-1.95 (m, 1H), 1.75 (d, 6H).
Step 1: 3-(4-(4-((1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (compound 152)
135I (165 mg, 0.29 mmol) and 107A (110 mg, 0.32 mmol) were dissolved in DMF (4 mL); p-toluenesulfonic acid monohydrate (170 mg, 0.87 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction solution, with a yellow solid precipitated, and the mixture was filtered to obtain a solid, which was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain a purified crude product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The collected preparative solution was adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and then concentrated to obtain compound 152 (50 mg, yield: 19%).
LCMS m/z=446.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.19-11.04 (m, 1H), 10.75 (s, 1H), 8.33-8.21 (m, 1H), 8.14 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.88 (s, 1H), 7.59 (s, 1H), 7.23 (dd, 1H), 7.05 (d, 2H), 6.89 (d, 2H), 6.82 (s, 1H), 6.45 (s, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.72 (dd, 1H), 3.18-3.03 (m, 6H), 2.68-2.57 (m, 3H), 2.55-2.51 (m, 5H), 2.34-2.26 (m, 2H), 2.20-2.07 (m, 1H), 2.04-1.94 (m, 1H), 1.85-1.78 (m, 3H), 1.75 (d, 6H), 1.70-1.60 (m, 1H), 1.42-1.26 (m, 2H), 0.93-0.85 (m, 2H), 0.53-0.46 (m, 2H).
Step 1: tert-butyl 4-(3-fluoro-4-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (153B)
153A (10.00 g, 40.48 mmol) and N-Boc-piperazine (11.31 g, 60.72 mmol) were dissolved in 1,4-dioxane (150 mL); palladium acetate (0.91 g, 4.05 mmol), (S)-BINAP (2.52 g, 4.05 mmol) and cesium carbonate (32.97 g, 101.20 mmol) were added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h, cooled to room temperature and filtered over celite. The filter cake was washed 3 times with dichloromethane. The organic phases were collected and concentrated under reduced pressure, and the residue was subjected to flash column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=3/1) to obtain 153B (12.30 g, yield: 86%)
LCMS m/z=297.2 [M−55]+.
Step 2: tert-butyl 4-(4-(4-cyano-1-methoxy-1-oxobutan-2-yl)-3-fluorophenyl) piperazine-1-carboxylate (153C)
153B (12.60 g, 35.75 mmol) and acrylonitrile (2.85 g, 53.63 mmol) were dissolved in toluene (100 mL); benzyltrimethylammonium hydroxide (0.30 g, 1.79 mmol) was added; and the mixture was reacted at room temperature for 16 h. The reaction solution was diluted with 500 mL of ethyl acetate and washed 3 times with water. The organic phase was dried over anhydrous sodium sulfate, and purified by flash column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=3/1) to obtain 153C (5.00 g, yield: 34%).
Step 3: tert-butyl 4-(4-(5-amino-1-methoxy-1,5-dioxopentan-2-yl)-3-fluorophenyl)piperazine-1-carboxylate (153D)
153C (8.70 g, 21.46 mmol) was dissolved in toluene (80 mL); acetaldoxime(3.80 g, 64.38 mmol) and indium (III) chloride tetrahydrate (3.15 g, 10.73 mmol) were added; and the mixture was reacted at 130° C. for 2 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-20/1) to obtain 153D (4.7 g, yield: 52%).
LCMS m/z=368.2 [M−55]+.
Step 4: tert-butyl 4-(4-(2,6-dioxopiperidin-3-yl)-3-fluorophenyl)piperazine-1-carboxylate (153E)
153D (4.70 g, 11.10 mmol) was dissolved in acetonitrile (50 mL); benzyltrimethylammonium hydroxide (9.28 g, 22.20 mmol) was added; and the mixture was reacted at 60° C. for 1 h. The reaction solution was cooled to room temperature, diluted by adding 500 mL of ethyl acetate, washed 3 times with water, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain 153E (3.4 g, yield: 78%).
LCMS m/z=336.1 [M−55]+.
Step 5: 3-(2-fluoro-4-(piperazin-1-yl)phenyl)piperidine-2,6-dione (153F)
153E (3.50 g, 8.94 mmol) was dissolved in methanol (30 mL); a solution of 1,4-dioxane in hydrochloric acid (10 mL, 4 mol/L) was added dropwise at room temperature; and the mixture was reacted at room temperature for 3 h and concentrated under reduced pressure to remove a solvent. The residue was then redissolved in 300 mL of dichloromethane, and adjusted to a basic pH with aqueous sodium bicarbonate solution. Liquid separation was performed. The organic phases were collected, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 153F (2.6 g), which was directly used in the next reaction.
LCMS m/z=292.2 [M+H]+.
Step 6: tert-butyl 4-((4-(4-(2,6-dioxopiperidin-3-yl)-3-fluorophenyl)piperazin-1-yl)methyl)piperidine-1-carboxylate (153G)
153F (2.60 g, 8.92 mmol) and 1-tert-butoxycarbonylpiperidine-4-carbaldehyde (1.90 g, 8.92 mmol) were dissolved in dichloromethane (40 mL); acetic acid (2.14 g, 35.68 mmol) and sodium triacetoxyborohydride (3.78 g, 17.84 mmol) were successively added; and the mixture was reacted at room temperature for 1 h, adjusted to a basic pH by adding aqueous sodium bicarbonate solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=20/1) to obtain 153G (3.48 g, yield: 80%).
Step 7: 3-(2-fluoro-4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (153H)
153G (3.48 g, 7.12 mmol) was dissolved in dichloromethane (20 mL); a solution of 1,4-dioxane in hydrochloric acid (6 mL, 4 mol/L) was added dropwise at room temperature; and the mixture was reacted at room temperature for 3 h, concentrated under reduced pressure to remove a solvent and dissolved in 200 mL of methanol. A potassium carbonate solid was added, and the mixture was stirred for 30 min, adjusted to a basic pH and subjected to suction filtration. The filter cake was washed with dichloromethane. The organic phases were combined and concentrated under reduced pressure to obtain 153H (2.7 g, yield: 98%).
LCMS m/z=389.2 [M+H]+.
Step 8: 3-(4-(4-((1-(2-bromo-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl) piperazin-1-yl)-2-fluorophenyl)piperidine-2,6-dione (compound 153I)
153H (2.00 g, 5.15 mmol) and 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C) (1.42 g, 5.67 mmol) were dissolved in DMSO (50 mL); potassium carbonate (1.42 g, 10.30 mmol) was added; and the mixture was reacted at 100° C. for 2 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-20/1) to obtain 1531 (2.4 g, yield: 75%).
LCMS m/z=618.1 [M+H]+.
Step 9: 3-(2-fluoro-4-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (153J)
153I (2.40 g, 3.88 mmol) and (1-methyl-1H-pyrazol-4-yl)boronic acid (0.78 g, 6.21 mmol) were dissolved in 1,4-dioxane (80 mL); Pd(dppf)Cl2·DCM (0.32 g, 0.39 mmol) and an aqueous solution (10 mL) of potassium carbonate (1.07 g, 7.76 mmol) were added; and the mixture was subjected to nitrogen replacement 3 times and reacted at 80° C. for 2 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=3/1-pure ethyl acetate) to obtain 153J (1.58 g, yield: 66%).
LCMS m/z=620.3 [M+H]+.
Step 10: 3-(4-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl) phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-fluorophenyl)piperidine-2,6-dione (153K)
153J (1.63 g, 2.63 mmol) was dissolved in tetrahydrofuran (30 mL); zinc powder (0.86 g, 13.15 mmol) was added, and then an aqueous solution (5 mL) of ammonium chloride (0.70 g, 13.15 mmol) was added dropwise; and the mixture was reacted at room temperature for 1 h. 5 mL of ammonia water and 20 mL of water were added to the reaction solution, and the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 153K (1.5 g), which was directly used in the next step.
LCMS m/z=590.3 [M+H]+.
Step 11: 3-(4-(4-((1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-fluorophenyl)piperidine-2,6-dione (compound 153)
153K (0.40 g, 0.68 mmol) and 107A (0.24 g, 0.68 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid hydrate (0.39 g, 2.04 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 153 (0.16 g, yield: 26%).
LCMS m/z=455.2[(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.78 (s, 1H), 8.30-8.22 (m, 1H), 8.14 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.88 (s, 1H), 7.59 (s, 1H), 7.23 (dd, 1H), 7.09 (t, 1H), 6.82 (s, 1H), 6.76-6.72 (m, 1H), 6.71 (s, 1H), 6.45 (s, 1H), 3.89 (dd, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.19-3.14 (m, 4H), 3.09 (d, 2H), 2.77-2.67 (m, 1H), 2.63 (t, 2H), 2.54-2.48 (m, 5H), 2.29 (d, 2H), 2.20-2.08 (m, 1H), 2.01-1.92 (m, 1H), 1.85-1.64 (m, 3H), 1.76 (s, 3H), 1.73 (s, 3H), 1.71-1.63 (m, 1H), 1.39-1.29 (m, 2H), 0.93-0.86 (m, 2H), 0.53-0.43 (m, 2H).
Step 1: 3-(4-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-fluorophenyl)piperidine-2,6-dione (compound 154)
153K (0.40 g, 0.68 mmol) and 109B (0.21 g, 0.68 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid hydrate (0.39 g, 2.04 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 154 (0.17 g, yield: 30%).
LCMS m/z=435.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 10.78 (s, 1H), 8.44 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.96 (s, 1H), 7.84 (s, 1H), 7.60 (s, 1H), 7.53-7.46 (m, 1H), 7.09 (t, 1H), 7.02-6.92 (m, 2H), 6.81 (s, 1H), 6.75-6.72 (m, 1H), 6.71 (s, 1H), 3.89 (dd, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.19-3.13 (m, 4H), 3.09 (d, 2H), 2.77-2.66 (m, 1H), 2.61 (t, 2H), 2.55-2.45 (m, 5H), 2.28 (d, 2H), 2.20-2.08 (m, 1H), 2.00-1.92 (m, 1H), 1.83-1.72 (m, 2H), 1.78 (s, 3H), 1.74 (s, 3H), 1.70-1.60 (m, 1H), 1.37-1.28 (m, 2H).
Step 1: 3-(4-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)piperidine-2,6-dione (compound 155)
135I (165 mg, 0.29 mmol) and 40A (130 mg, 0.32 mmol) were dissolved in DMF (4 mL); p-toluenesulfonic acid monohydrate (170 mg, 0.87 mmol) was added; and the mixture was stirred at 100° C. for 16 h, and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction solution, with a yellow solid precipitated, and the mixture was filtered to obtain a solid, which was purified by silica gel column chromatography (mobile phase: dichloromethane/methanol (V/V)=100/1-10/1) to obtain the crude, which was further purified by preparative SFC (instrument: waters 2767 preparative chromatographic column; SunFire@ Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)). The collected preparative solution was adjusted to a basic pH by adding dichloromethane and saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and then concentrated to obtain compound 155 (50 mg, yield: 19%).
LCMS m/z=464.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.75 (s, 1H), 8.56-8.41 (m, 1H), 8.29 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.71 (dd, 1H), 7.62-7.52 (m, 3H), 7.45 (t, 2H), 7.39-7.31 (m, 1H), 7.25-7.13 (m, 1H), 7.06 (d, 2H), 6.90 (d, 2H), 6.86 (s, 1H), 3.78 (d, 6H), 3.75-3.68 (m, 1H), 3.19-3.03 (m, 6H), 2.71-2.57 (m, 3H), 2.56-2.51 (m, 5H), 2.28 (d, 2H), 2.20-2.06 (m, 1H), 2.05-1.95 (m, 1H), 1.84 (d, 6H), 1.78-1.71 (m, 2H), 1.69-1.58 (m, 1H), 1.39-1.26 (m, 2H).
Step 1: 3-(4-(4-((1-(4-((5-chloro-4-((3-(dimethylphosphoryl)-[1,1′-biphenyl]-4-yl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-fluorophenyl)piperidine-2,6-dione (compound 156)
153K (0.40 g, 0.68 mmol) and 40A (0.27 g, 0.68 mmol) were dissolved in DMF (20 mL); p-toluenesulfonic acid hydrate (0.39 g, 2.04 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 156 (0.11 g, yield: 17%).
LCMS m/z=473.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.79 (s, 1H), 8.52-8.44 (m, 1H), 8.29 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.71 (dd, 1H), 7.58 (d, 2H), 7.55 (s, 1H), 7.45 (t, 2H), 7.36 (t, 1H), 7.25-7.14 (m, 1H), 7.09 (t, 1H), 6.86 (s, 1H), 6.77-6.73 (m, 1H), 6.72 (s, 1H), 3.89 (dd, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.20-3.15 (m, 4H), 3.09 (d, 2H), 2.77-2.66 (m, 1H), 2.62 (t, 2H), 2.54-2.48 (m, 5H), 2.28 (d, 2H), 2.20-2.08 (m, 1H), 2.01-1.92 (m, 1H), 1.85 (s, 3H), 1.82 (s, 3H), 1.79-1.72 (m, 2H), 1.67-1.58 (m, 1H), 1.36-1.26 (m, 2H).
Step 1: tert-butyl 4-(2-fluoro-4-nitrophenyl)piperazine-1-carboxylate (157B)
157A (10.00 g, 62.86 mmol) and N-Boc-piperazine (11.71, 62.86 mmol) were dissolved in DMF (50 mL); potassium carbonate (17.38 g, 125.72 mmol) was added; and the mixture was reacted at 50° C. for 12 h. The reaction solution was cooled to room temperature and poured into 200 mL of water, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in 300 mL of dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 157B (20.0 g), which was directly used in the next reaction.
LCMS m/z=270.1 [M−55]+.
Step 2: 1-(2-fluoro-4-nitrophenyl)piperazine (157C)
157B (20.00 g, 61.47 mmol) was dissolved in dichloromethane (50 mL); a solution of 1,4-dioxane in hydrochloric acid (20 mL, 4 mol/L) was added dropwise at room temperature; and the mixture was reacted at room temperature for 1 h, concentrated under reduced pressure to remove a solvent, dissolved by adding a small amount of water, adjusted to a basic pH with aqueous sodium hydroxide solution and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 157C (13.8 g, yield: 99%), which was directly used in the next reaction.
LCMS m/z=226.1 [M+H]+.
Step 3: tert-butyl 4-((4-(2-fluoro-4-nitrophenyl)piperazin-1-yl)methyl) piperidine-1-carboxylate (157D)
157C (12.8 g, 56.83 mmol) and 1-tert-butoxycarbonylpiperidine-4-carbaldehyde (12.12, 56.83 mmol) were dissolved in dichloromethane (130 mL); acetic acid (13.65 g, 227.32 mmol) and sodium triacetoxyborohydride (24.09 g, 113.66 mmol) were successively added; and the mixture was reacted at room temperature for 4 h. The reaction solution was adjusted to a basic pH with aqueous sodium hydroxide solution. Liquid separation was performed. The organic phases were collected. The aqueous phase was extracted 3 times with dichloromethane, combined with the previous organic phases, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=1/1 for removing small polar impurities, dichloromethane/methanol (V/V)=20/1) to obtain 157D (24.0 g, yield: 99%).
LCMS m/z=423.2 [M+H]+.
Step 4: tert-butyl 4-((4-(4-amino-2-fluorophenyl)piperazin-1-yl)methyl) piperidine-1-carboxylate (157E)
157D (12.00 g, 28.40 mmol) was dissolved in ethyl acetate (250 mL); palladium on carbon (wt %=10%, 5.00 g) was added; and the mixture was subjected to 1 atm hydrogen replacement 3 times, reacted at 30° C. for 4 h and directly filtered over celite to remove palladium on carbon. The filter cake was washed with dichloromethane. The organic phases were combined and concentrated under reduced pressure to obtain 157E (10.9 g, yield: 98%), which was directly used in the next step.
LCMS m/z=393.3 [M+H]+.
Step 5: tert-butyl 4-((4-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl) piperazin-1-yl)methyl)piperidine-1-carboxylate (157F)
In a 150 mL sealed tube, 157E (3.50 g, 8.92 mmol) and the compound 3-bromopiperidin-2,6-dione (5.14 g, 26.76 mmol) were dissolved in DMF (60 mL); sodium bicarbonate (4.50 g, 53.52 mmol) was added; and the mixture was reacted at 90° C. for 16 h under a sealed condition. The reaction solution was cooled to room temperature, diluted by adding 300 mL of ethyl acetate, washed 3 times with water and washed once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=1/1) to obtain 157F (2.7 g, yield: 60%).
LCMS m/z=504.3 [M+H]+.
Step 6: 3-((3-fluoro-4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)phenyl)amino) piperidine-2,6-dione (compound 157G)
157F (2.70 g, 5.36 mmol) was dissolved in dichloromethane (50 mL); a solution of 1,4-dioxane in hydrochloric acid (15 mL, 4 mol/L) was added dropwise at room temperature; and the mixture was reacted at room temperature for 0.5 h and concentrated under reduced pressure to remove a solvent. The residue was redissolved in 200 mL of methanol; a potassium carbonate solid was added; and the mixture was stirred for 30 min, adjusted to a basic pH and subjected to suction filtration. The filter cake was washed with dichloromethane. The organic phases were combined and concentrated under reduced pressure to obtain 157G (2.16 g, yield: 99%).
LCMS m/z=404.2[M+H]+.
Step 7: 3-((4-(4-((1-(2-bromo-5-methoxy-4-nitrophenyl)piperidin-4-yl)methyl) piperazin-1-yl)-3-fluorophenyl)amino)piperidine-2,6-dione (157H)
157G (2.06, 5.11 mmol) and 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (1C) (1.28 g, 5.11 mmol) were dissolved in DMSO (50 mL); sodium bicarbonate (2.15 g, 25.55 mmol) was added; and the mixture was reacted at 100° C. for 3 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-20/1) to obtain 157H (1.71 g, yield: 53%).
LCMS m/z=633.2 [M+H]+.
Step 8: 3-((3-fluoro-4-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)amino)piperidine-2,6-dione (compound 157I)
157H (1.81 g, 2.86 mmol) and (1-methyl-1H-pyrazol-4-yl)boronic acid (0.58 g, 4.58 mmol) were dissolved in 1,4-dioxane (40 mL); Pd(dppf)Cl2·DCM (0.23 g, 0.29 mmol) and an aqueous solution (5 mL) of potassium carbonate (0.79 g, 5.72 mmol) were added; and the mixture was subjected to nitrogen replacement 3 times and reacted at 80° C. for 3 h. The reaction solution was cooled to room temperature, diluted by adding 200 mL of ethyl acetate, washed 3 times with water and washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: petroleum ether/ethyl acetate (V/V)=3/1-pure ethyl acetate) to obtain 157I (0.43 g, yield: 24%).
LCMS m/z=635.3 [M+H]+.
Step 9: 3-((4-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-3-fluorophenyl)amino)piperidine-2,6-dione (157J)
157I (0.43 g, 0.67 mmol) was dissolved in tetrahydrofuran (18 mL); zinc powder (0.22 g, 3.35 mmol) was added, and then an aqueous solution (3 mL) of ammonium chloride (0.18 g, 3.35 mmol) was added dropwise; and the mixture was reacted at room temperature for 0.5 h. 2 mL of ammonia water and 10 mL of water were added to the reaction solution, and the mixture was extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 157J (0.41 g).
LCMS m/z=605.3 [M+H]+.
Step 10: 3-((4-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-3-fluorophenyl)amino)piperidine-2,6-dione (compound 157)
157J (0.13 g, 0.22 mmol) and 109B (0.07 g, 0.22 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid hydrate (0.13 g, 0.66 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 157 (10 mg, yield: 5%).
LCMS m/z=442.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 10.75 (s, 1H), 8.48-8.39 (m, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.97 (s, 1H), 7.84 (s, 1H), 7.60 (s, 1H), 7.50 (dd, 1H), 7.04-6.93 (m, 2H), 6.84 (d, 1H), 6.81 (s, 1H), 6.51 (dd, 1H), 6.43 (dd, 1H), 5.78 (d, 1H), 4.29-4.21 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.08 (d, 2H), 2.90-2.82 (m, 4H), 2.78-2.68 (m, 1H), 2.66-2.54 (m, 4H), 2.54-2.45 (m, 2H), 2.27 (d, 2H), 2.13-2.05 (m, 1H), 1.90-1.82 (m, 1H), 1.82-1.70 (m, 9H), 1.70-1.60 (m, 1H), 1.31 (d, 2H).
Step 1: 3-((4-(4-((1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-3-fluorophenyl)amino)piperidine-2,6-dione (compound 158)
157J (0.13 g, 0.22 mmol) and 107A (0.08 g, 0.22 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid hydrate (0.13 g, 0.66 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 158 (10 mg, yield: 5%).
LCMS m/z=462.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.75 (s, 1H), 8.30-8.20 (m, 1H), 8.14 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.88 (s, 1H), 7.59 (s, 1H), 7.23 (dd, 1H), 6.87-6.79 (m, 2H), 6.51 (dd, 1H), 6.48-6.38 (m, 2H), 5.79 (d, 1H), 4.29-4.19 (m, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.08 (d, 2H), 2.88 (s, 4H), 2.77-2.68 (m, 1H), 2.67-2.50 (m, 6H), 2.36-2.23 (m, 2H), 2.13-2.05 (m, 1H), 2.04-1.85 (m, 1H), 1.85-1.77 (m, 4H), 1.77 (s, 3H), 1.73 (s, 3H), 1.71-1.61 (m, 1H), 1.40-1.28 (m, 2H), 0.93-0.87 (m, 2H), 0.54-0.48 (m, 2H).
Step 1: 3-((4-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)-4-fluorophenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-3-fluorophenyl)amino)piperidine-2,6-dione (compound 159)
157J (0.13 g, 0.22 mmol) and 142A (0.07 g, 0.22 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid hydrate (0.13 g, 0.66 mmol) was added; and under nitrogen protection, the mixture was reacted at 100° C. for 16 h. The reaction solution was cooled to room temperature. 50 mL of saturated aqueous sodium bicarbonate solution was added, with a yellow solid precipitated, and the mixture was subjected to suction filtration. The filter cake was washed 3 times with water, redissolved in dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by column chromatography (mobile phase: dichloromethane/methanol (V/V)=50/1-15/1) to obtain a product, which was further purified by preparative HPLC (composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain a product, which was alkalized with aqueous sodium bicarbonate solution, extracted with dichloromethane, concentrated and dried to obtain compound 159 (12 mg, yield: 6%).
LCMS m/z=451.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 10.76 (s, 1H), 8.41 (s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.82 (s, 1H), 7.61 (s, 1H), 7.46-7.38 (m, 1H), 6.87-6.78 (m, 2H), 6.75-6.59 (m, 1H), 6.51 (dd, 1H), 6.42 (d, 1H), 5.79 (d, 1H), 4.31-4.20 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.12-3.04 (m, 2H), 2.91-2.82 (m, 4H), 2.78-2.66 (m, 1H), 2.65-2.52 (m, 4H), 2.49-2.43 (m, 2H), 2.28 (d, 2H), 2.14-2.04 (m, 1H), 2.04-1.73 (m, 10H), 1.70-1.59 (m, 1H), 1.35-1.28 (m, 2H).
Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-((1-(5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)-4-nitrophenyl)piperidin-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-di one (160A)
64A (1.5 g, 3.62 minol), 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (1.07 g, 3.62 minol), DIPEA (0.70 g, 5.43 minol) and DMSO (15 mL) were added to a reaction flask, and the mixture was reacted at 90° C. for 3 h and cooled in ice water. Ethyl acetate (100 mL) and water (100 mL) were added, and the mixture was stirred for 5 min and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH30H=DCM to 50/1 to 20/1) to obtain 160A (1.7 g, yield: 68.19%). Step 2: 5-(4-((1-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (160B)
160A (0.5 g, 0.73 mmol), zinc powder (0.24 g, 3.63 mmol), ammonium chloride (0.19 g, 3.63 mmol), tetrahydrofuran (6 mL) and water (2 mL) were added to a reaction flask. and the mixture was reacted at room temperature for 1 h. Dichloromethane (50 mL) and ammonia water (10 mL) were added, and the mixture was stirred for 2 min, subjected to suction filtration over an appropriate amount of celite and washed with an appropriate amount of dichloromethane. The layers in the filtrate were separated. The organic layer was washed with water (20 mL×1), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain 160B (0.40 g, yield: 83.18%). Step 3: 5-(4-((1-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl) phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 160)
160B (0.4 g, 0.61 mmol), 107A (0.22 g, 0.61 mmol), p-toluenesulfonic acid monohydrate (0.23 g, 1.22 mmol) and DMF (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, ethyl acetate (20 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH30H=50/1 to 30/1 to 20/1. The resulting crude was subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative product, which was alkalized with saturated aqueous sodium bicarbonate solution to obtain compound 160 (0.11 g, yield: 18.43%).
LCMS m/z=489.8 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.15-11.03 (m, 2H), 8.31-8.23 (m, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.72 (d, 1H), 7.60 (s, 1H), 7.46 (d, 1H), 7.24 (dd, 1H), 6.82 (s, 1H), 6.45 (s, 1H), 5.10 (dd, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.27 (s, 4H), 3.15-3.04 (m, 2H), 2.95-2.83 (m, 1H), 2.68-2.51 (m, 8H), 2.31 (d, 2H), 2.05-1.97 (m, 2H), 1.89-1.56 (m, 11H), 1.42-1.30 (m, 1H), 0.95-0.87 (m, 2H), 0.54-0.47 (m, 2H).
Step 1: 5-(4-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (compound 161)
160B (0.41 g, 0.64 mmol), 109B (0.2 g, 0.64 mmol), p-toluenesulfonic acid monohydrate (0.24 g, 1.28 mmol) and DMF (5 mL) were added to a reaction flask, and the mixture was reacted at 100° C. for about 18 h. Under ice water cooling, ethyl acetate (20 mL) and saturated sodium bicarbonate solution (20 mL) were added, and the mixture was stirred and allowed to stand for layer separation. The organic layer was washed with saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: DCM/CH30H=50/1 to 30/1 to 20/1. The resulting crude was further subjected to preparative HPLC (instrument: waters 2767 (preparative liquid phase chromatographic instrument); chromatographic column: XBridge@ Prep C18 (30 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) purification to obtain a preparative product, which was alkalized with saturated aqueous sodium bicarbonate solution to obtain compound 161 (0.22 g, yield: 36.63%).
LCMS m/z=469.7 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 11.09 (s, 1H), 8.52-8.36 (m, 1H), 8.28-8.14 (m, 1H), 8.14-8.07 (m, 1H), 8.02-7.90 (m, 1H), 7.90-7.78 (m, 1H), 7.76-7.40 (m, 4H), 7.08-6.90 (m, 2H), 6.86-6.73 (m, 1H), 5.16-5.05 (m, 1H), 3.93-3.73 (m, 6H), 3.33-3.17 (m, 4H), 3.15-3.00 (m, 2H), 2.98-2.79 (m, 1H), 2.70-2.45 (m, 8H), 2.40-2.20 (m, 2H), 2.08-1.99 (m, 1H), 1.90-1.50 (m, 9H), 1.42-1.22 (m, 2H).
Step 1: 5-(9-(4-((5-chloro-4-((2-(dimethylphosphoryl)-4-fluorophenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 162)
79D (397.60 mg, 0.65 mmol) and 142A (238.89 mg, 0.72 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (370.93 mg, 1.95 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of water and 20 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-15/1) to obtain compound 162 (140 mg, yield: 23.69%).
LCMS m/z=909.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 2H), 8.42 (br.s, 1H), 8.17 (s, 1H), 8.10 (s, 1H), 8.03 (s, 1H), 7.85 (s, 1H), 7.70-7.57 (m, 2H), 7.48-7.17 (m, 3H), 6.87 (s, 1H), 6.67 (br.s, 1H), 5.11-5.00 (m, 1H), 3.93-3.75 (m, 6H), 3.60-3.40 (m, 4H), 3.00-2.75 (m, 5H), 2.70-2.50 (m, 2H), 2.10-1.92 (m, 1H), 1.88-1.72 (m, 6H), 1.72-1.52 (m, 8H).
Step 1: 5-(9-(4-((5-bromo-4-((2-(dimethylphosphoryl)-4-fluorophenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 163)
79D (400 mg, 0.65 mmol) and 141B (270.67 mg, 0.72 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (370.93 mg, 1.95 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of water and 20 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in DCM and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-15/1) to obtain compound 163 (150 mg, yield: 24.19%).
LCMS m/z=953.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 10.78 (s, 1H), 8.30 (br.s, 1H), 8.20-8.13 (m, 2H), 8.01 (s, 1H), 7.84 (s, 1H), 7.69-7.57 (m, 2H), 7.45-7.19 (m, 3H), 6.87 (s, 1H), 6.69 (br.s, 1H), 5.11-5.01 (m, 1H), 3.92-3.75 (m, 6H), 3.58-3.42 (m, 4H), 2.97-2.77 (m, 5H), 2.65-2.50 (m, 2H), 2.10-1.95 (m, 1H), 1.85-1.72 (m, 6H), 1.72-1.54 (m, 8H).
Step 1: 5-(9-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 164)
79D (400 mg, 0.65 mmol) and 108B (257.81 mg, 0.72 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (370.93 mg, 1.95 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of water and 20 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-10/1) to obtain a product, which was further purified by preparative liquid phase chromatography (instrument: waters 2767 preparative chromatographic column; SunFire@Prep C18 (19 mm×150 mm); composition of mobile phases: mobile phase A: acetonitrile, and mobile phase B: water (containing 0.1% TFA)) to obtain the trifluoroacetate of compound 164. 60 mL of dichloromethane and 30 mL of saturated sodium bicarbonate solution were added to the trifluoroacetate of compound 164, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain compound 164 (120 mg, yield: 19.73%).
LCMS m/z=935.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.94 (s, 1H), 8.33 (br.s, 1H), 8.22-8.13 (m, 2H), 7.96 (s, 1H), 7.86 (s, 1H), 7.70-7.43 (m, 3H), 7.36-7.21 (m, 2H), 7.07-6.92 (m, 2H), 6.86 (s, 1H), 5.12-4.99 (m, 1H), 3.88-3.77 (m, 6H), 3.58-3.45 (m, 4H), 2.96-2.77 (m, 5H), 2.70-2.50 (m, 2H), 2.10-1.95 (m, 1H), 1.84-1.51 (m, 14H).
Step 1: 5-(9-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 165)
79D (397.60 mg, 0.65 mmol) and 55B (286.46 mg, 0.72 mmol) were dissolved in DMF (8 mL); p-toluenesulfonic acid monohydrate (370.93 mg, 1.95 mmol) was added; and under nitrogen protection, the mixture was reacted overnight at 100° C., and cooled to room temperature. 20 mL of water and 20 mL of saturated aqueous sodium bicarbonate solution were added, and the mixture was subjected to suction filtration. The filter cake was dissolved in dichloromethane and extracted. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/1-15/1) to obtain compound 165 (190 mg, yield: 29.95%).
LCMS m/z=975.3 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.85 (s, 1H), 8.24-8.08 (m, 3H), 8.00 (s, 1H), 7.90 (s, 1H), 7.72-7.50 (m, 2H), 7.38-7.16 (m, 3H), 6.88 (s, 1H), 6.48 (br.s, 1H), 5.12-5.01 (m, 1H), 3.96-3.72 (m, 6H), 3.60-3.44 (m, 4H), 3.00-2.77 (m, 5H), 2.70-2.50 (m, 2H), 2.10-1.50 (m, 16H), 1.00-0.80 (m, 2H), 0.60-0.40 (m, 2H).
Step 1: 5-(4-(4-amino-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl) piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (166A)
80C (10 g, 18.40 mmol) was placed in a 500 mL single-necked flask; ammonium chloride (14.76 g, 276 mmol), THE (100 ml), ethanol (100 ml), water (50 ml) and iron powder (15.41 g, 276 mmol) were successively added; and after the addition, the mixture was stirred at 80° C. for 2 h, cooled to room temperature and filtered. The filter cake was washed with a mixed solvent of dichloromethane/methanol (v/v=10/1). The organic phases were combined and washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 166A (2 g, yield: 20%).
Step 2: 5-(4-(4-((5-bromo-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 166)
166A (0.4 g, 0.74 mmol) and 55B (0.3 g, 0.74 mmol) were dissolved in DMF (5 ml); p-toluenesulfonic acid monohydrate (0.28 g, 1.48 mmol) was added; and the mixture was stirred overnight at 100° C., and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/3-100/5). 8 mL of acetonitrile was added to the resulting residue, and the mixture was stirred at 70° C. for 1 h and filtered. The filter cake was dried under reduced pressure to obtain compound 166 (0.2 g, yield: 30%).
LCMS m/z=454.3 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.83 (s, 1H), 8.27-8.07 (m, 4H), 7.89 (s, 1H), 7.71 (d, 1H), 7.65 (s, 1H), 7.45-7.39 (m, 1H), 7.36-7.29 (m, 1H), 7.25-7.14 (m, 1H), 6.84 (s, 1H), 6.57-6.44 (m, 1H), 5.19-4.99 (m, 1H), 3.83 (d, 6H), 3.66 (s, 4H), 3.01 (s, 4H), 2.95-2.82 (m, 1H), 2.67-2.55 (m, 2H), 2.06-1.99 (m, 1H), 1.82-1.68 (m, 7H), 0.94-0.82 (m, 2H), 0.60-0.46 (m, 2H).
Step 1: 5-(4-(4-((5-chloro-4-((4-cyclopropyl-2-(dimethylphosphoryl)phenyl) amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 167)
166A (0.4 g, 0.74 mmol) and 107A (0.26 g, 0.74 mmol) were dissolved in DMF (5 ml); p-toluenesulfonic acid monohydrate (0.28 g, 1.48 mmol) was added; and the mixture was stirred overnight at 100° C., and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/3-100/5). 8 mL of acetonitrile was added to the resulting residue, and the mixture was stirred at 70° C. for 1 h and filtered. The filter cake was dried under reduced pressure to obtain compound 167 (0.25 g, yield: 39%).
LCMS m/z=432.2 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 11.07 (s, 1H), 8.36-8.22 (m, 1H), 8.21-8.11 (m, 2H), 8.08 (s, 1H), 7.90 (s, 1H), 7.71 (d, 1H), 7.66 (s, 1H), 7.46-7.39 (m, 1H), 7.38-7.30 (m, 1H), 7.26-7.15 (m, 1H), 6.85 (s, 1H), 6.57-6.39 (m, 1H), 5.13-5.00 (m, 1H), 3.83 (d, 6H), 3.66 (s, 4H), 3.01 (s, 4H), 2.96-2.82 (m, 1H), 2.69-2.52 (m, 2H), 2.09-1.97 (m, 1H), 1.83-1.67 (m, 7H), 0.94-0.83 (m, 2H), 0.59-0.48 (m, 2H).
Step 1: 5-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 168)
166A (0.45 g, 0.83 mmol) and 109B (0.26 g, 0.83 mmol) were dissolved in DMF (5 mL); p-toluenesulfonic acid monohydrate (0.32 g, 1.66 mmol) was added; and the mixture was stirred overnight at 100° C., and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/3-100/5). 8 mL of acetonitrile was added to the resulting residue, and the mixture was stirred at 70° C. for 1 h and filtered. The filter cake was dried under reduced pressure to obtain compound 168 (0.3 g, yield: 44%).
LCMS m/z=412.2 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 11.07 (s, 1H), 8.48-8.39 (m, 1H), 8.22 (s, 1H), 8.13-8.06 (m, 2H), 7.86 (s, 1H), 7.71 (d, 1H), 7.66 (s, 1H), 7.56-7.46 m, 1H), 7.44-7.39 (m, 1H), 7.37-7.28 (m, 1H), 7.07-6.92 (m, 2H), 6.85 (s, 1H), 5.14-5.01 (m, 1H), 3.83 (d, 6H), 3.64 (s, 4H), 3.01 (s, 4H), 2.95-2.84 (m, 1H), 2.68-2.53 (m, 2H), 2.05-1.98 (m, 1H), 1.76 (d, 6H).
Step 1: 5-(4-(4-((5-bromo-4-((2-(dimethylphosphoryl)phenyl)amino) pyrimidin-2-yl)amino)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 169)
166A (0.4 g, 0.74 mmol) and 108B (0.27 g, 0.74 mmol) were dissolved in DMF (5 ml); p-toluenesulfonic acid monohydrate (0.28 g, 1.48 mmol) was added; and the mixture was stirred overnight at 100° C., and cooled to room temperature. 10 mL of saturated aqueous sodium bicarbonate solution was added, with a solid precipitated, and the mixture was filtered. The filter cake was dried and then purified by silica gel column chromatography (dichloromethane/methanol (V/V)=100/3-100/5). 8 mL of acetonitrile was added to the resulting residue, and the mixture was stirred at 70° C. for 1 h and filtered. The filter cake was dried under reduced pressure to obtain compound 169 (0.25 g, yield: 39%).
LCMS m/z=434.2 [(M+2H)/2]+.
1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.94 (s, 1H), 8.39-8.28 (m, 1H), 8.25-8.16 (m, 2H), 8.07 (s, 1H), 7.85 (s, 1H), 7.71 (d, 1H), 7.65 (s, 1H), 7.54-7.45 (m, 1H), 7.44-7.39 (m, 1H), 7.36-7.29 (m, 1H), 7.09-6.92 (m, 2H), 6.84 (s, 1H), 5.15-5.04 (m, 1H), 3.82 (d, 6H), 3.64 (s, 4H), 3.00 (s, 4H), 2.95-2.81 (m, 1H), 2.68-2.53 (m, 2H), 2.09-1.93 (m, 1H), 1.76 (d, 6H).
300 mg of compound 106 was subjected to preparative SFC separation and purification. Preparative SFC separation conditions: instrument: Waters 150 MGM; chromatographic column: Chiralpak Column; mobile phase: A for CO2 and B for IPA (isopropanol)+ACN (acetonitrile); gradient: 65% phase B isocratic elution; flow rate: 120 mL/min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 220 nm.
SFC analysis conditions: instrument: SHIMADZU LC-30AD sfc; chromatographic column: Chiralpak AD-3 50×4.6 mm I.D., 3 m; mobile phase: A for CO2 and B for IPA (isopropanol)+ACN (acetonitrile) (0.05% DEA (diethylamine)); gradient: B 60%; flow rate: 3 mL/min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 220 nm.
After preparative separation, the components with the same retention time were combined, lyophilized, redissolved in 20 mL of DCM and extracted by adding water (10 mL) and saturated sodium bicarbonate solution (1 mL). The organic layers were separated, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 170 (30 mg) and compound 171 (70 mg).
LCMS m/z=502.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.23 (s, 1H), 7.96-7.75 (m, 5H), 7.46 (s, 1H), 7.35 (s, 1H), 7.31-7.15 (m, 2H), 6.92 (d, 1H), 5.12 (dd, 1H), 4.22-4.07 (m, 5H), 4.03 (s, 3H), 3.93-3.70 (m, 6H), 3.62-3.48 (m, 2H), 3.39-3.18 (m, 4H), 2.95-2.85 (m, 2H), 2.82-2.67 (m, 1H), 2.63-2.44 (m, 1H), 2.37-2.16 (m, 3H), 2.12-1.88 (m, 9H), 1.19-1.08 (m, 2H), 0.73-0.65 (m, 2H).
LCMS m/z=502.8 [(M+2H)/2]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1))δ 8.23 (s, 1H), 7.96-7.75 (m, 5H), 7.46 (s, 1H), 7.35 (s, 1H), 7.31-7.15 (m, 2H), 6.92 (d, 1H), 5.12 (dd, 1H), 4.22-4.07 (m, 5H), 4.03 (s, 3H), 3.93-3.70 (m, 6H), 3.62-3.48 (m, 2H), 3.39-3.18 (m, 4H), 2.95-2.85 (m, 2H), 2.82-2.67 (m, 1H), 2.63-2.44 (m, 1H), 2.37-2.16 (m, 3H), 2.12-1.88 (m, 9H), 1.19-1.08 (m, 2H), 0.73-0.65 (m, 2H).
345 mg of compound 107 was subjected to preparative SFC separation and purification. Preparative SFC separation conditions: instrument: Waters 150 MGM; chromatographic column: Chiralpak Column; mobile phase: A for CO2 and B for IPA+ACN; gradient: 70% phase B isocratic elution; flow rate: 100 mL/min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 220 nm.
SFC analysis conditions: instrument: SHIMADZU LC-30AD sfc; chromatographic column: Chiralpak AD-3 50×4.6 mm I.D., 3 m; mobile phase: A for CO2 and B for IPA+ACN (0.05% DEA); gradient: B 60%; flow rate: 3 mL/min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 220 nm.
After preparative separation, the components with the same retention time were combined and concentrated under reduced pressure to obtain compound 172 (160 mg) and compound 173 (165 mg).
Compound 172: retention time under SFC analysis conditions: 1.5 min, LCMS m/z=960.2[M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.14 (s, 1H), 8.02-7.91 (m, 3H), 7.86 (s, 1H), 7.82 (d, 1H), 7.46 (s, 1H), 7.36 (s, 1H), 7.31-7.13 (m, 2H), 6.90 (d, 1H), 5.12 (dd, 1H), 4.25-4.08 (m, 5H), 4.04 (s, 3H), 3.95-3.73 (m, 6H), 3.63-3.45 (m, 2H), 3.39-3.18 (m, 4H), 2.94-2.85 (m, 2H), 2.83-2.67 (m, 1H), 2.63-2.44 (m, 1H), 2.37-2.19 (m, 3H), 2.13-1.88 (m, 9H), 1.20-1.06 (m, 2H), 0.72-0.64 (m, 2H).
Compound 173: retention time under SFC analysis conditions: 2.5 min, LCMS m/z=960.2[M+H]+.
1H NMR (400 MHz, D2O/CF3COOD(v/v=1:1)) δ 8.14 (s, 1H), 8.03-7.91 (m, 3H), 7.86 (s, 1H), 7.82 (d, 1H), 7.46 (s, 1H), 7.37 (s, 1H), 7.30-7.16 (m, 2H), 6.90 (d, 1H), 5.12 (dd, 1H), 4.25-4.08 (m, 5H), 4.04 (s, 3H), 3.94-3.75 (m, 6H), 3.62-3.45 (m, 2H), 3.38-3.15 (m, 4H), 2.94-2.85 (m, 2H), 2.83-2.66 (m, 1H), 2.63-2.45 (m, 1H), 2.35-2.17 (m, 3H), 2.12-1.89 (m, 9H), 1.18-1.06 (m, 2H), 0.74-0.64 (m, 2H).
NCI-H1975 (EGFR-L858R-T790M) and A431 (EGFR-WT) cells were purchased from ATCC and cultured in an incubator at 37° C. with 5% CO2, and the culture mediums were RPMI1640+10% FBS and DMEM+10% FBS, respectively. On the first day, NCI-H1975 (EGFR-L858R-T790M) and A431 (EGFR-WT) cells in the logarithmic phase were collected, and viable cells were counted using an automatic cell analyzer (countstar). The cell suspensions were adjusted with the culture mediums and plated on a 96-well cell culture plate at 1000 cells/well for NCI-H1975 (EGFR-L858R-T790M) cells and at 3000 cells/well for A431 cells. The next day, the culture mediums were aspirated, and 90 μL of fresh culture mediums and 10 μL of the compounds at different concentrations were added to each well, wherein the final concentration of DMSO in each well was 0.1%. The plate was cultured in the incubator at 37° C. with 5% CO2 for 72 hours. After 72 hours of drug treatment, 50 μL of CTG solution (promega, G7572), which was already pre-melted and equilibrated to room temperature, was added to each well, and the mixtures were uniformly mixed for 2 min using a microplate shaker. The plate was placed at room temperature for 10 min, and then fluorescence signal values were measured using a microplate reader (PHERAstar FSX).
Surviving cells % was calculated based on Vsample/Vvehicle control×100%, wherein Vsample was the readout of the drug-treated group, and Vvehicle control was the average value of the vehicle control group. Using the origin 9.2 software and nonlinear regression model, S-shaped dose-survival curves were plotted, and IC50 values were calculated.
Inhibitory activity of test compounds on proliferation of A431 (WT) cells:
Inhibitory activity of compounds 1, 2, 4 and 6, trifluoroacetate of compound 7, trifluoroacetate of compound 14, compound 18, trifluoroacetate of compound 23, compounds 24, 30, 34, 35, 36, 37, 38, 40, 41, 42, 45, 46, 47, 48, 50, 54, 55, 57, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 71, 72, 73, 76, 77, 78, 79, 80, 81, 82, 83, 84 and 85, trifluoroacetate of compound 86, and compounds 87, 88, 89, 90, 91, 92, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 116, 117, 118, 119, 121, 125, 126, 133, 137, 138, 139, 143, 145, 146, 147, 148, 150, 160, 162, 163, 164, 165, 166, 167, 170, 171, 172 and 173 on proliferation of A431 (EGFR-WT) cells showed IC50≥10 μM.
Conclusion: the compounds of the present disclosure have good inhibitory activity on proliferation of NCI-H1975 (EGFR-L858R-T790M) cells, and poor inhibitory activity on proliferation of and good selectivity for A431 (EGFR-WT) cells.
Ba/F3-TEL-EGFR-T790M-L858R-C797S cells were cultured in an incubator at 37° C. with 5% CO2. The cells in the logarithmic phase were collected, counted and plated in a 96-well plate at a density of 2000 cells/well. The compounds at different concentrations were added, and a vehicle control group (cells plus DMSO) and a blank control group (cell culture mediums plus DMSO) were provided, wherein the concentration of DMSO was both 0.1%. The cell culture plate was cultured in an incubator at 37° C. with 5% CO2 for 72 hours. After the incubation was completed, the cell culture plate was removed and allowed to equilibrate to room temperature for 10 min. According to operation instructions for a CellTiter-Glo kit (Promega, G7573), 50 μL of CTG solution, which was already pre-melted and equilibrated to room temperature, was added to each well, and the mixtures were uniformly mixed for 2 min using a microplate shaker. The plate was placed at room temperature for 10 min, and then fluorescence signal values were measured using a microplate reader (SpectraMax Paradigm). The cell proliferation inhibition rate data was processed based on formula (1). The inhibition rates corresponding to different concentrations of compounds were calculated, and using the GraphPad Prism software, the inhibition rate curves were plotted, and IC50 values were calculated. wherein RLUDrug was the readout of the drug-treated group, RLUMax was the readout of the control group, and RLUMin was the readout of the blank group.
Conclusion: the compounds of the present disclosure have good inhibitory activity on proliferation of Ba/F3-TEL-EGFR-T790M-L858R-C797S cells, for example, the IC50 of compound 106 is 15.6 nM, and the IC50 of compound 107 is 15.6 nM.
NCI-H1975 EGFR-L858R-T790M-C797S cells were cultured in an incubator at 37° C. with 5% CO2, and the culture medium was RPMI1640+10% FBS+100 μg/mL hygromycin. The cells in the logarithmic phase were collected, and the cell suspension was adjusted to an appropriate concentration with a hygromycin-free culture medium, and then plated on a 96-well plate at a density of 1500 cells/well and a volume of 90 μL. 10 μL of the compounds at different concentrations were added, and a vehicle control group (cells plus DMSO) was provided, wherein the concentration of DMSO was 0.1%. The cell culture plate was cultured in an incubator at 37° C. with 5% CO2 for 72 hours. After the incubation was completed, according to operation instructions for a CellTiter-Glo kit (Promega, G7572), 50 μL of CTG solution, which was already pre-melted and equilibrated to room temperature, was added to each well, and the mixtures were uniformly mixed for 2 min using a microplate shaker. The plate was placed at room temperature for 10 min, and then fluorescence signal values were measured using a microplate reader (Envision 2104). Surviving o data was processed based on formula (2), and using the GraphPad Prism 5.0 software and nonlinear regression model, S-shaped dose-survival curves were plotted, and IC50 values were calculated. wherein Vsample was the readout of the drug-treated group, and Vvehicle control was the readout of the control group.
Conclusion: the compounds of the present disclosure have good inhibitory activity on proliferation of NCI-H1975 EGFR-L858R-T790M-C797S cells, which is obviously better than that of the control compounds A and B.
Test objective: In this experiment, a single dose of test compounds was administered to ICR mice intravenously and intragastrically, the concentrations of the test compounds in plasma of mice were measured, and the pharmacokinetic characteristics and bioavailability of the test compounds in mice were evaluated.
Experimental animals: male ICR mice, 20-25 g, purchased from BEIJING HFK BIOSCIENCE CO., LTD. with the laboratory animal production license number of SCXK (JING) 2019-0008; or purchased from CHENGDU DDOSSY EXPERIMENTAL ANIMALS CO., LTD. (SCXK (CHUAN) 2020-030); or purchased from HUNAN SJA LABORATORY ANIMAL CO., LTD. (SCXK (XIANG) 2019-0004).
Experimental method: on the day of the experiment, ICR mice were randomly grouped according to their body weights. The mice were fasted with water available for 12 to 14 hours one day before the administration, and were fed 4 hours after the administration.
Sampling: before and after the administration, blood was taken from the orbits of the mice under isoflurane anesthesia, and placed in an EDTAK2 centrifuge tube. Centrifugation was carried out at 5000 rpm at 4° C. for 10 min, and the plasma was collected.
Time points for plasma collection in G1&G2 groups: 0, 5 min, 15 min, 30 min, 1h, 2h, 4h, 6h, 8h, and 24 h;
Conclusion: the compounds synthesized by using the technology of the present disclosure have a certain oral absorption performance in mice, which is better than that of the control compounds A and B.
Protein sample preparation: NCI-H1975 EGFR-L858R-T790M-C797S cells were cultured in an incubator at 37° C. with 5% CO2, and the culture medium was RPMI1640+10% FBS+100 μg/mL hygromycin. The cells in the logarithmic phase were collected, and the cell suspension was adjusted to an appropriate concentration with a hygromycin-free culture medium, plated on a 6-well plate at a density of 200000 cells/well and a volume of 2 mL, and cultured overnight in an incubator at 37° C. with 5% CO2. The next day, the compounds at different concentrations were added. DMSO control wells were provided, wherein the concentration of DMSO in all wells was 0.1%. The 6-well plate was cultured in an incubator at 37° C. with 5% CO2 for 72 h, and then 30 μL of RIPA lysates (Beyotime, Cat. P0013B) containing protease inhibitors (Beyotime, Cat. P0013B) were added. The mixtures were lysed on ice for 30 min. Centrifugation was carried out at 13000 rpm/min at 4° C. for 20 min to collect protein supernatant samples, and protein quantification was performed by the BCA method. Samples for western blot detection were prepared.
Western blot detection: 20 μg of protein samples were added to each well, subjected to polyacrylamide gel electrophoresis and transferred to a membrane, and then diluted anti-EGFR (CST, Cat. 4267S) and NADPH (Kangchen, Cat. KC-5G4) antibodies were added and incubated overnight at 4° C. After washing the membrane, diluted goat anti-rabbit (Licor, Cat. 926-32211) and goat anti-mouse (Licor, Cat. 926-68070) antibodies were added and incubated in the dark for 45 min. Scanning and detection at wavelengths of 700 nm and 800 nm were performed with a far-infrared imaging system (Odyssey).
According to formula (3), the expression level of EGFR protein in the cells relative to the DMSO control group was calculated after incubation with different concentrations of compounds. wherein EGFRcompound was the fluorescence value of EGFR protein after incubation with compounds, and EGFRvehicle was the fluorescence value of EGFR protein in the DMSO control group. Using the Origin 9.2 software, the drug concentration DC50 values when the expression level of EGFR protein was 50% relative to the DMSO control group were calculated.
Protein sample preparation: NCI-H1975 EGFR-L858R-T790M-C797S cells were cultured in an incubator at 37° C. with 5% CO2, and the culture medium was RPMI1640+10% FBS+100 μg/mL hygromycin. The cells in the logarithmic phase were collected, and the cell suspension was adjusted to an appropriate concentration with a hygromycin-free culture medium, plated on a 6-well plate at a density of 350000 cells/well and a volume of 2 mL, and cultured overnight in an incubator at 37° C. with 5% CO2. The next day, the compounds at different concentrations were added. DMSO control wells were provided, wherein the concentration of DMSO in all wells was 0.1%. The 6-well plate was cultured in an incubator at 37° C. with 5% CO2 for 48 h, and then 30 μL of RIPA lysates (Beyotime, Cat. P0013B) containing protease inhibitors (Beyotime, Cat. P0013B) were added. The mixtures were lysed on ice for 30 min. Centrifugation was carried out at 13000 rpm/min at 4° C. for 20 minutes to collect protein supernatant samples, and protein quantification was performed by the BCA method. Western Blot detection samples were prepared.
Western blot detection: 20 μg of protein samples were added to each well, subjected to polyacrylamide gel electrophoresis and transferred to a membrane, and then diluted anti-EGFR (CST, Cat. 4267S) and NADPH (Kangchen, Cat. KC-5G4) antibodies were added and incubated overnight at 4° C. After washing the membrane, diluted goat anti-rabbit (Licor, Cat. 926-32211) and goat anti-mouse (Licor, Cat. 926-68070) antibodies were added and incubated in the dark for 45 min. Scanning and detection at wavelengths of 700 nm and 800 nm were performed with a far-infrared imaging system (Odyssey).
According to formula (3), the expression level of EGFR protein in the cells relative to the DMSO control group was calculated after incubation with different concentrations of compounds. wherein EGFRcompound was the fluorescence value of EGFR protein after incubation with compounds, and EGFRvehicle was the fluorescence value of EGFR protein in the DMSO control group. Using the Origin 9.2 software, the drug concentration DC50 values when the expression level of EGFR protein was 50% relative to the DMSO control group were calculated.
Conclusion: the compounds of the present disclosure have good degradation effects on EGFR protein in H1975-EGFR-T790M-L858R-C797S cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110470748.8 | Apr 2021 | CN | national |
| 202110570092.7 | May 2021 | CN | national |
| 202110651028.1 | Jun 2021 | CN | national |
| 202110824204.7 | Jul 2021 | CN | national |
| 202111025788.8 | Sep 2021 | CN | national |
| 202111214457.9 | Oct 2021 | CN | national |
| 202210000254.8 | Jan 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/090243 | 4/29/2022 | WO |
