Patent Application
20240408085
The present invention relates to a compound of general formula (I) or a stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or co-crystal thereof, and an intermediate thereof and a preparation method therefor, as well as the use thereof in Bcl-2 family proteins-related diseases such as cancer.
Apoptosis is an autonomous and orderly death process controlled by genes for most cells at certain developmental stages in the organism. It plays an important role in tissue evolution, organ development and the maintenance of the body's stability. Anti-apoptotic effects are considered a key feature in malignant tumors. Therefore, specifically targeting the anti-apoptotic pathways shows potential for cancer treatment applications. The Bcl-2 protein family consists of both pro-apoptotic and anti-apoptotic proteins, which can regulate the intrinsic apoptotic pathways of cancer cells. Bcl-2 protein family members Bcl-2, Bcl-xL and Mcl-1 have been identified as anti-tumor targets. Inhibition of these proteins can promote Bax/Bak oligomerization and ultimately induce mitochondrial outer membrane permeabilization, leading to release of cytochrome C and activation of caspases, which subsequently execute cancer cell apoptosis.
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 the 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.
Therefore, it is necessary to develop a novel Bcl-xL/Bcl-2 inhibitor and a PROTAC drug of E3 ubiquitin ligase for the treatment of apoptosis-related tumor diseases.
An objective of the present invention is to provide a compound with a novel structure, good efficacy, high bioavailability and higher safety that can inhibit and degrade Bcl-2 family proteins (e.g., Bcl-xL or Bcl-2), for use in the treatment of a disease related to Bcl-2 family proteins (e.g., Bcl-xL or Bcl-2), such as cancer.
The present invention provides a compound or a stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or co-crystal thereof, wherein the compound is selected from a compound of general formula (I),
B-L-K (I);
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;
is selected from
is selected from
the B1 is optionally further substituted with 0 to 4 RB1, and the Z is optionally further substituted with 0 to 4 RQ;
and Rw1 and Rw2 are selected from methyl, B3 is selected from
As a first embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein
As a second embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein
As a third embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein Ak1, Ak2, Ak3, Ak4, Ak5, Ak6, Ak7, Ak8 and Ak9 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 substituents selected from H, F, Cl, Br, I, OH, CN, NH2, CF3, hydroxymethyl, C1-4 alkyl, or C1-4 alkoxy; each Cy1, Cy2, Cy3, Cy4 or Cy5 is independently selected from a bond, a 4- to 7-membered nitrogen-containing mono-heterocyclic ring, a 4- to 10-membered nitrogen-containing fused-heterocyclic ring, a 5- to 12-membered nitrogen-containing spiro-heterocyclic ring, a 7- to 10-membered nitrogen-containing bridged-heterocyclic ring, 3- to 7-membered monocycloalkyl, 4- to 10-membered fused cycloalkyl, 5- to 12-membered spiro cycloalkyl, 7- to 10-membered bridged cycloalkyl, 5- to 10-membered heteroaryl or 6- to 10-membered aryl, wherein the mono-heterocyclic ring, fused-heterocyclic ring, bridged-heterocyclic ring, spiro-heterocyclic ring, cycloalkyl, aryl or heteroaryl is optionally further substituted with 0 to 4 substituents selected from H, F, Cl, Br, I, OH, COOH, CN, NH2, ═O, C1-4 alkyl, halogen-substituted C1-4 alkyl, hydroxyl-substituted C1-4 alkyl or C1-4 alkoxy, and the mono-heterocyclic ring, fused-heterocyclic ring, bridged-heterocyclic ring, spiro-heterocyclic ring or heteroaryl contains 1 to 4 heteroatoms selected from O, S or N, and is optionally further substituted with 0, 1 or 2 ═O when the heteroatom is selected from S; each RL is independently selected from H or C1-4 alkyl;
As a fourth embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein
which, when substituted, is optionally further substituted with 0 to 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;
the B1 is optionally further substituted with 0 to 4 RB1, and the Z is optionally further substituted with 0 to 4 RQ;
As a fifth embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein
which, when substituted, is optionally further substituted with 0 to 4 substituents selected from H, F, CF3, methyl, ═O, hydroxymethyl, COOH, CN or NH2,
As a sixth embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein
As a seventh embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein
As an eighth embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein K is selected from one of the following structural fragments:
As a ninth embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein the compound of general formula (I) is selected from the compound of general formula (Ia)
and Rw1 and Rw2 are selected from methyl, B3 is selected from
As a tenth embodiment of the present invention, provided is the above-mentioned compound of general formula (I) or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof, wherein the compound of general formula (I) is selected from the compound of general formula (Ib),
The present invention 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 one of the structures shown in Table P-1:
The present invention relates to a pharmaceutical composition, comprising the above-mentioned compound or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof according to the present invention, and a pharmaceutically acceptable carrier.
The present invention relates to the use of the above-mentioned compound or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof according to the present invention in the preparation of a medicament for treating a disease related to Bcl-2 family protein activity or expression level.
The present invention relates to the use of the above-mentioned compound or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof according to the present invention in the preparation of a medicament for treating a disease related to the inhibition or degradation of Bcl-2 family proteins.
The present invention relates to the use of the above-mentioned compound or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof according to the present invention, wherein the disease is selected from cancer.
The present invention relates to a pharmaceutical composition or pharmaceutical preparation comprising a therapeutically effective amount of the compound, or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof according to the present invention, and a pharmaceutically acceptable excipient. The pharmaceutical composition can be in a unit preparation form (the amount of the active drug in the unit preparation is also referred to as the “preparation specification”).
The present invention further provides a method for treating a disease in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the compound, or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof or the pharmaceutical composition according to the present invention. In some embodiments, the mammal according to the present invention comprises humans.
The term “effective amount” or “therapeutically effective amount” according to the present application refers to a sufficient amount of the compound disclosed in the present application that is administered to ameliorate, to some extent, one or more symptoms of a disease or condition (such as cancer) being treated. In some embodiments, the outcome is the reduction and/or remission of signs, symptoms or causes of the disease, or any other desired change in the biological system. For example, an “effective amount” in terms of the therapeutic use is an amount of the composition comprising the compound disclosed in the present application that is required to provide clinically significant reduction of the symptoms of the disease. Examples of the therapeutically effective amount include, but are limited to 1-1500 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 2-600 mg, 3-600 mg, 4-600 mg, 5-600 mg, 6-600 mg, 10-600 mg, 20-600 mg, 25-600 mg, 30-600 mg, 40-600 mg, 50-600 mg, 60-600 mg, 70-600 mg, 75-600 mg, 80-600 mg, 90-600 mg, 100-600 mg, 200-600 mg, 1-500 mg, 2-500 mg, 3-500 mg, 4-500 mg, 5-500 mg, 6-500 mg, 10-500 mg, 20-500 mg, 25-500 mg, 30-500 mg, 40-500 mg, 50-500 mg, 60-500 mg, 70-500 mg, 75-500 mg, 80-500 mg, 90-500 mg, 100-500 mg, 125-500 mg, 150-500 mg, 200-500 mg, 250-500 mg, 300-500 mg, 400-500 mg, 5-400 mg, 10-400 mg, 20-400 mg, 25-400 mg, 30-400 mg, 40-400 mg, 50-400 mg, 60-400 mg, 70-400 mg, 75-400 mg, 80-400 mg, 90-400 mg, 100-400 mg, 125-400 mg, 150-400 mg, 200-400 mg, 250-400 mg, 300-400 mg, 1-300 mg, 2-300 mg, 5-300 mg, 10-300 mg, 20-300 mg, 25-300 mg, 30-300 mg, 40-300 mg, 50-300 mg, 60-300 mg, 70-300 mg, 75-300 mg, 80-300 mg, 90-300 mg, 100-300 mg, 125-300 mg, 150-300 mg, 200-300 mg, 250-300 mg, 1-200 mg, 2-200 mg, 5-200 mg, 10-200 mg, 20-200 mg, 25-200 mg, 30-200 mg, 40-200 mg, 50-200 mg, 60-200 mg, 70-200 mg, 75-200 mg, 80-200 mg, 90-200 mg, 100-200 mg, 125-200 mg, 150-200 mg, 80-1500 mg, 80-1000 mg, and 80-800 mg;
The present invention further provides a method for treating a disease in a mammal, the method comprising administering to a subject a therapeutically effective amount of the compound, or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or co-crystal thereof according to the present invention, wherein the therapeutically effective amount is preferably 1-1500 mg, and the disease is preferably cancer.
The present invention further provides a method for treating a disease in a mammal, the method comprising administering to a subject a medicament, i.e., the compound, or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or co-crystal thereof according to the present invention in a daily dose of 1-1500 mg/day, wherein the daily dose can be a single dose or divided doses; in some embodiments, the daily dose includes, but is not limited to 10-1500 mg/day, 10-1000 mg/day, 10-800 mg/day, 25-800 mg/day, 50-800 mg/day, 100-800 mg/day, 200-800 mg/day, 25-400 mg/day, 50-400 mg/day, 100-400 mg/day, or 200-400 mg/day; in some embodiments, the daily dose includes, but is not limited to 10 mg/day, 20 mg/day, 25 mg/day, 50 mg/day, 80 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 160 mg/day, 200 mg/day, 300 mg/day, 320 mg/day, 400 mg/day, 480 mg/day, 600 mg/day, 640 mg/day, 800 mg/day, 1000 mg/day, or 1500 mg/day.
The present invention relates to a kit, wherein the kit can comprise a composition in the form of a single dose or multiple doses and comprises the compound, or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or co-crystal thereof according to the present invention, and the amount of the compound or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or co-crystal thereof according to the present invention is identical to the amount of same in the above-mentioned pharmaceutical composition.
In the present invention, the amount of the compound, or the stereoisomer, deuterated compound, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal thereof according to the present invention is calculated in the form of a free base in each case.
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 invention all comprise their isotopes, and the carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds of the present invention 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 or 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, piperidyl, imidazolidinyl, oxazolidinyl, oxazinanyl, morpholinyl, hexahydropyrimidyl or piperazinyl. Heterocycloalkyl can be monovalent, divalent, trivalent or tetravalent.
“Alkenyl” refers to a substituted or unsubstituted linear or 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, ethenyl, 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 or 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, a benzene ring, a 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 3- to 8-membered monocyclic ring, 4- to 12-membered bicyclic ring or 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, pyrimidyl, 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, benzopyrimidyl, 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.
“Fused-heterocyclic ring”, “fused-heterocyclic ring group”, “fused ring heterocyclyl” or “fused-heterocyclic ring group” refers to a “fused ring” containing a heteroatom. The definition of the “fused-heterocyclic ring”, “fused-heterocyclic ring group”, “fused ring heterocyclyl” or “fused-heterocyclic 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
and “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, pyridyl, pyranyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl, benzopyrazole, benzoimidazole, 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 a 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)mC(═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 can form a five- or six-membered cycloalkyl or heterocyclyl.
“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 with 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 according to the present invention, 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 invention, 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.
The term “preparation specification” refers to the weight of the active drug contained in each vial, tablet or other unit preparation.
“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.
“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 in 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 invention will be described in detail below in conjunction with examples, but the protection scope of the present invention includes but is not limited thereto.
To achieve the objectives of the present invention, 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.
References and monographs in the art introduce in detail the synthesis of reactants that can be used to prepare the compounds described herein, or provide articles describing the preparation method for reference. The references and monographs include: “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., New York; S. R. Sandler et al., “Organic Functional Group Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O. House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-Interscience, New York, 1992; Fuhrhop, J. and Penzlin G. “Organic Synthesis: Concepts, Methods, Starting Materials”, Second, Revised and Enlarged Edition (1994) John Wiley & Sons ISBN: 3 527-29074-5; Hoffman, R. V. “Organic Chemistry, An Intermediate Text” (1996) Oxford University Press, ISBN 0-19-509618-5; Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations” 2nd Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. “Advanced Organic Chemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) John Wiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) “Modern Carbonyl Chemistry” (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. “Patai's 1992 Guide to the Chemistry of Functional Groups” (1992) Interscience ISBN: 0-471-93022-9; Solomons, T. W. G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J. C., “Intermediate Organic Chemistry” 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; “Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann's Encyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; “Organic Reactions” (1942-2000) John Wiley & Sons, in over 55 volumes; and “Chemistry of Functional Groups”, John Wiley & Sons, in 73 volumes.
Specific and similar reactants can be selectively identified by the indexes of known chemicals prepared by the Chemical Abstracts Service of the American Chemical Society, wherein the indexes are available in most public libraries or university libraries and online. Chemicals that are known but not commercially available in the catalog are optionally prepared by custom chemical synthesis plants, wherein many of standard chemical supply plants (such as those listed above) provide custom synthesis services. Reference document for the preparation and selection of the pharmaceutically acceptable salts of the compounds described herein is P. H. Stahl & C. G. Wermuth “Handbook of Pharmaceutical Salts”, Verlag Helvetica Chimica Acta, Zurich, 2002.
The technical solutions of the present invention will be described in detail by the following examples, but the scope of protection of the present invention includes but is not limited thereto.
The compounds used in the reactions described herein are prepared 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. “Commercially available chemicals” are obtained from regular commercial sources, and suppliers include: Titan Technology Co., Ltd., Energy Chemical Co., Ltd., Shanghai Demo Co., Ltd., Chengdu Kelong Chemical Co., Ltd., Accela ChemBio Co., Ltd., PharmaBlock Sciences (Nanjing), Inc., WuXi Apptec Co., Ltd., J&K Scientific Co., Ltd., etc.
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 determined with Agilent 6120B (ESI) and Agilent 6120B (APCI);
HPLC is determined with Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18 100×4.6 mm, 3.5 μM);
Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate is used as a thin layer chromatography silica plate, and the silica gel plate for the thin layer chromatography (TLC) is of the specification of 0.15 mm-0.20 mm, and the specification when separating and purifying a product by thin layer chromatography is 0.4 mm-0.5 mm;
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; Troc: 2,2,2-trichloroethoxycarbonyl; DMAP: 4-dimethylaminopyridine;
(2S,4R)-1-((2S)-2-(7-(3-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 1)
1a (0.85 g, 4.0 mmol) was dissolved in 20 mL of dichloromethane and triethylamine (0.51 g, 5.0 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (1.0 g, 4.7 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 2 h. After the reaction was completed, 50 mL of dichloromethane and 50 mL of water were added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 1b (1.5 g).
Crude 1b (1.5 g) was added to 50 mL of 2 mol/L ethyl acetate hydrogen chloride solution and the mixture was reacted at room temperature for 3 h. The reaction system was filtered and 50 mL of dichloromethane was added to the filter cake. Saturated sodium bicarbonate solution (50 mL) was added and the mixture was stirred until the solid was completely dissolved. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 1b (0.9 g).
LCMS m/z=287.0 [M+1]+.
The above-mentioned crude 1c (0.9 g) was added to 50 mL of 1,2-dichloroethane. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (see Nature Communications, 2020, 11, 1996 for the synthetic method) (0.95 g, 3.2 mmol) was added and the mixture was reacted at room temperature for 1 h. Sodium triacetoxyborohydride (0.72 g, 3.4 mmol) was added and the resulting mixture was reacted at room temperature for 12 h. Dichloromethane (50 mL) was added to dilute the reaction solution. Saturated sodium bicarbonate solution was used to adjust the reaction mixture to pH 9.0. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (petroleum ether:ethyl acetate (v/v)=1:1) to afford 1d (1.6 g, total yield over three steps from compound 1a: 71%).
LCMS m/z=566.1 [M+1]+.
1d (1.6 g, 2.83 mmol) was added to 50 mL of 2 mol/L ethyl acetate hydrogen chloride solution and the mixture was reacted at room temperature for 3 h. The reaction system was filtered and 50 mL of dichloromethane was added to the filter cake. Saturated sodium bicarbonate solution (50 mL) was added and the mixture was stirred until the solid was completely dissolved. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 1e (1.3 g).
LCMS m/z=466.0 [M+1]+.
The above-mentioned crude 1e (1.3 g) was added to 30 mL of acetonitrile. Triethylamine (4.0 g, 4.0 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl) benzenesulfonamide (1.0 g, 3.3 mmol) were added and the mixture was refluxed and reacted at 85° C. for 3 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (petroleum ether:ethyl acetate (v/v)=2:1) to afford 1f (1.8 g, yield over two steps from compound 1d: 84%).
LCMS m/z=753.0 [M+1]+.
1f (1.8 g, 2.4 mmol) was added to 100 mL of dichloromethane. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (see WO 2017101851 for the synthetic method) (1.31 g, 3.0 mmol), DMAP (0.6 g, 4.9 mmol) and EDCI (0.96 g, 5.0 mmol) were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction system was added 100 mL of water. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (ethyl acetate) to afford 1g (2.0 g, yield: 71.0%).
LCMS m/z=588.1 [M/2+1]+.
1g (2.0 g, 1.7 mmol) was added to 40 mL of tetrahydrofuran. Zinc powder (6.5 g, 100.0 mmol) and 1.2 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 5 h. The reaction system was filtered and the filtrate was concentrated under reduced pressure. The residue was then diluted with 100 mL of dichloromethane and then 50 mL of saturated sodium bicarbonate solution was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 1h (1.5 g).
LCMS m/z=500.3 [M/2+1]+.
The above-mentioned crude 1h (1.5 g) was added to 30 mL of DMF. Intermediate 1 (0.96 g, 1.6 mmol), triethylamine (0.6 g, 4.9 mmol) and HATU (0.78 g, 2.1 mmol) were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction system was added water (200 mL), and a solid was precipitated. The system was filtered and the filter cake was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). To the preparative solution were added 100 mL of dichloromethane and 60 mL of saturated sodium bicarbonate solution and the mixture was stirred for 1 h. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 1 (0.7 g, yield over two steps from compound 1g: 26%).
1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 8.28 (s, 1H), 8.04 (d, 1H), 7.69-7.59 (m, 2H), 7.44-7.08 (m, 12H), 6.96-6.87 (m, 2H), 6.85-6.76 (m, 1H), 6.68 (d, 2H), 6.61-6.49 (m, 1H), 6.32-6.17 (m, 1H), 5.07-4.95 (m, 1H), 4.70-4.58 (m, 1H), 4.56-4.36 (m, 3H), 4.08-3.70 (m, 3H), 3.56-3.45 (m, 1H), 3.28-3.12 (m, 4H), 3.10-3.00 (m, 1H), 2.98-2.85 (m, 1H), 2.84-2.74 (m, 2H), 2.68-2.49 (m, 2H), 2.48-1.86 (m, 23H), 1.78-1.15 (m, 15H), 1.05-0.80 (m, 15H).
LCMS m/z=523.5 [M/3+1]+.
Example 2
(2S,4R)-1-((2S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 2)
2a (0.8 g, 4.0 mmol) was dissolved in 20 mL of dichloromethane and triethylamine (0.51 g, 5.0 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (1.0 g, 4.7 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 2 h. After the reaction was completed, the reaction solution was diluted with dichloromethane (50 mL) and then 50 mL of water was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 2b (1.4 g).
The above-mentioned crude 2b (1.4 g) was added to 50 mL of 2 mol/L ethyl acetate hydrogen chloride solution and the mixture was reacted at room temperature for 3 h. The reaction system was filtered and 50 mL of DCM was added to the filter cake. Saturated sodium bicarbonate solution (50 mL) was added and the mixture was stirred until the solid was completely dissolved. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 2c (0.85 g).
LCMS m/z=273.0 [M+1]+.
The above-mentioned crude 2c (0.85 g) was added to 50 mL of 1,2-dichloroethane. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (0.95 g, 3.2 mmol) was added and the mixture was reacted at room temperature for 1 h. Sodium triacetoxyborohydride (0.72 g, 3.4 mmol) was added and the resulting mixture was reacted at room temperature for 12 h. DCM (50 mL) was added to dilute the reaction solution. Saturated sodium bicarbonate solution was used to adjust the reaction mixture to pH 9.0. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (petroleum ether:ethyl acetate (v/v)=1:1) to afford 2d (1.3 g, total yield over three steps from compound 2a: 59%).
LCMS m/z=552.2 [M+1]+.
2d (1.3 g, 2.36 mmol) was added to 50 mL of 2 mol/L ethyl acetate hydrogen chloride solution and the mixture was reacted at room temperature for 3 h. The reaction system was filtered and 50 mL of DCM was added to the filter cake. Saturated sodium bicarbonate solution (50 mL) was added and the mixture was stirred until the solid was completely dissolved. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 2e (1.0 g).
LCMS m/z=452.0 [M+1]+.
The above-mentioned crude 2e (1.0 g) was added to 30 mL of acetonitrile. Triethylamine (4.0 g, 4.0 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl) benzenesulfonamide (0.76 g, 2.6 mmol) were added and the mixture was refluxed and reacted at 85° C. for 3 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (petroleum ether:ethyl acetate (v/v)=2:1) to afford 2f (1.5 g, yield over two steps from compound 2d: 86%).
LCMS m/z=739.0 [M+1]+.
2f (1.5 g, 2.0 mmol) was added to 100 mL of dichloromethane. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (0.87 g, 2.0 mmol), DMAP (0.5 g, 4.1 mmol) and EDCI (0.76 g, 4.0 mmol) were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction system was added 100 mL of water. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (ethyl acetate) to afford 2g (1.6 g, yield: 69%).
LCMS m/z=581.2 [M/2+1]+.
2g (1.6 g, 1.38 mmol) was added to 40 mL of tetrahydrofuran. Zinc powder (6.5 g, 100.0 mmol) and 1.2 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 5 h. The reaction system was filtered and the filtrate was concentrated under reduced pressure. The residue was then diluted with 100 mL of DCM and then 50 mL of saturated sodium bicarbonate solution was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 2h (1.1 g).
LCMS m/z=493.2 [M/2+1]+.
The above-mentioned crude 2h (1.1 g) was added to 30 mL of DMF. Intermediate 1 (0.96 g, 1.6 mmol), triethylamine (0.6 g, 4.9 mmol) and HATU (0.78 g, 2.1 mmol) were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction system was added water (200 mL), and a solid was precipitated. The system was filtered and the filter cake was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). To the preparative solution were added 100 mL of dichloromethane and 60 mL of saturated sodium bicarbonate solution and the mixture was stirred for 1 h. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 2 (0.05 g, yield over two steps from compound 2g: 2%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.46-8.34 (m, 1H), 8.12-7.92 (m, 1H), 7.83-7.63 (m, 2H), 7.44-7.05 (m, 13H), 7.04-6.93 (m, 2H), 6.90-6.65 (m, 3H), 6.48-6.30 (m, 1H), 5.16-4.94 (m, 1H), 4.86-4.42 (m, 4H), 4.15-3.97 (m, 2H), 3.70-2.95 (m, 10H), 2.94-1.95 (m, 24H), 1.62-1.20 (m, 14H), 1.10-0.95 (m, 15H).
LCMS m/z=518.9 [M/3+1]+.
Example 3
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(1-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperidin-4-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 3)
4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-carbaldehyde (see WO 2020041406 for the synthetic method) (350 mg, 1.41 mmol) was dissolved in 15 mL of tetrahydrofuran. 3a (see WO 2020212530 for the synthetic method) (309 mg, 1.41 mmol) and 1 mL of tetraisopropyl titanate were sequentially added and the mixture was stirred at room temperature for 4 h. Then sodium triacetoxyborohydride (1.49 g, 7.03 mmol) was added and the resulting mixture was reacted at room temperature for 16 h. To the reaction solution was slowly added 20 mL of saturated sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate (60 mL×2). The organic phase was washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 3b (400 mg, yield: 63%).
LCMS m/z=452.3 [M+1]+.
3b (0.40 g, 0.88 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.15 g, 3.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction system was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in 10 mL of water and washed with 20 mL of methyl tert-butyl ether. The aqueous phase was separated and adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 3c (0.20 g).
LCMS m/z=438.2 [M+1]+.
The above-mentioned crude 3c (200 mg) was dissolved in 15 mL of DCM. Intermediate 2 (330 mg, 0.45 mmol), DMAP (110 mg, 0.9 mmol) and EDCI (180 mg, 0.95 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 30 mL of water and the mixture was extracted with DCM (60 mL×2). The organic phase was washed with 50 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 3d (260 mg, yield over two steps from compound 3b: 26%).
LCMS m/z=574.5 [M/2+1]+.
3d (260 mg, 0.23 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (0.6 mL) and zinc powder (960 mg, 14.8 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (10 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (250 mg). The above-mentioned crude (250 mg) was dissolved in 10 mL of DCM. Triethylamine (0.2 mL), intermediate 1 (154 mg, 0.26 mmol) and HATU (142 mg, 0.37 mmol) were sequentially added and the mixture was reacted at room temperature for 1 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was washed with 20 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). To the preparative solution were added 100 mL of dichloromethane and 60 mL of saturated sodium bicarbonate solution and the mixture was stirred for 1 h. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 3 (40 mg, yield from compound 3d: 11%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.32 (s, 1H), 8.05-7.97 (m, 1H), 7.95-7.84 (m, 2H), 7.64-7.55 (m, 1H), 7.48-7.12 (m, 12H), 7.09-6.96 (m, 3H), 6.86-6.78 (m, 1H), 6.65-6.57 (m, 1H), 6.34-6.22 (m, 1H), 5.16-5.03 (m, 1H), 4.71-4.61 (m, 1H), 4.50-4.39 (m, 2H), 4.12-3.96 (m, 1H), 3.96-3.79 (m, 1H), 3.70-3.45 (m, 2H), 3.40-2.88 (m, 9H), 2.58-1.98 (m, 23H), 1.77-1.38 (m, 14H), 1.36-1.18 (m, 2H), 1.08-0.82 (m, 15H).
LCMS m/z=770.8 [M/2+1]+.
Example 4
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((4′-cyano-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 4) trifluoroacetate
4a (see WO 2020041406 for the synthetic method) (1.00 g, 4.61 mmol) and (4-cyanophenyl)boronic acid (1.01 g, 6.87 mmol) were dissolved in 20 mL of dioxane and 2 mL of water. Potassium acetate (1.36 g, 13.8 mmol) and Pd(dppf)Cl2 (0.33 g, 0.45 mmol) were sequentially added and the mixture was reacted at 90° C. for 4 h. The reaction solution was cooled to room temperature and to the reaction solution was slowly added 100 mL of water. The mixture was extracted with ethyl acetate (60 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:10) to afford 4b (0.95 g, yield: 86%).
LCMS m/z=240.3 [M+1]+.
4b (510 mg, 2.13 mmol) was dissolved in 15 mL of tetrahydrofuran. Ethyl 4-(piperazin-1-yl)benzoate (500 mg, 2.13 mmol) was added, followed by 1 mL of tetraisopropyl titanate. The mixture was stirred at room temperature for 4 h and then sodium triacetoxyborohydride (1.35 g, 6.37 mmol) was added. The resulting mixture was reacted at room temperature for 16 h. To the reaction solution was slowly added 20 mL of saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted with 60 mL of ethyl acetate twice and the organic phase was washed with 50 mL of saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 4c (550 mg, yield: 56%).
LCMS m/z=458.3 [M+1]+.
4c (0.55 g, 1.20 mmol) was dissolved in 20 mL of methanol. Water (2 mL) was added, followed by sodium hydroxide (0.24 g, 6.0 mmol) and the mixture was stirred at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.35 g). The above-mentioned crude (350 mg) was dissolved in 20 mL of DCM. Intermediate 2 (590 mg, 0.81 mmol), DMAP (200 mg, 1.64 mmol) and EDCI (310 mg, 1.62 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 30 mL of water and the mixture was extracted with 60 mL of DCM twice. The organic phase was washed with 50 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 4d (310 mg, yield: 23%).
4d (310 mg, 0.27 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (0.6 mL) and zinc powder (960 mg, 14.8 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (20 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (250 mg). The above-mentioned crude (250 mg) was dissolved in 10 mL of DCM. Triethylamine (0.2 mL), intermediate 1 (150 mg, 0.26 mmol) and HATU (142 mg, 0.37 mmol) were sequentially added and the mixture was reacted at room temperature for 1 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of dichloromethane. The organic phase was washed with 20 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). Lyophilization was performed to afford the trifluoroacetate of compound 4 (20 mg).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.30 (s, 1H), 8.13-8.00 (m, 1H), 7.90-7.75 (m, 2H), 7.70-7.52 (m, 3H), 7.40-7.28 (m, 6H), 7.26-7.12 (m, 5H), 6.94-6.80 (m, 1H), 6.75-6.55 (m, 3H), 6.35-6.15 (m, 1H), 5.15-4.99 (m, 1H), 4.70-4.58 (m, 1H), 4.47-4.33 (m, 2H), 4.09-3.99 (m, 1H), 3.98-3.82 (m, 1H), 3.68-3.40 (m, 2H), 3.36-2.93 (m, 9H), 2.80-2.66 (m, 2H), 2.50 (s, 3H), 2.40-1.97 (m, 21H), 1.65-1.42 (m, 10H), 1.32-1.20 (m, 2H), 1.10-0.90 (m, 15H).
LCMS m/z=767.4 [M/2+1]+.
Example 5
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((4-(4-chlorophenyl)-5,6-dihydro-2H-pyran-3-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 5)
Under nitrogen protection, 5a (see European Journal of Medicinal Chemistry, 2018, 149, 79-89 for the synthetic method) (2 g, 10.47 mmol) and (4-chlorophenyl)boronic acid (2.47 g, 15.80 mmol) were dissolved in 20 mL of dioxane and 2 mL of water. Potassium acetate (3.11 g, 31.69 mmol) and Pd(dppf)Cl2 (0.2 g, 0.27 mmol) were sequentially added and the mixture was reacted at 90° C. for 4 h. The reaction solution was cooled to room temperature and to the reaction solution was slowly added 100 mL of water. The mixture was extracted with ethyl acetate (60 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:10) to afford 5b (1.76 g, yield: 75%).
1H NMR (400 MHz, CDCl3) δ 9.51 (s, 1H), 7.46-7.36 (m, 2H), 7.26-7.18 (m, 2H), 4.50-4.40 (m, 2H), 3.92 (t, 2H), 2.67-2.59 (m, 2H).
5b (1.67 g, 7.50 mmol) was dissolved in 15 mL of tetrahydrofuran. Ethyl 4-(piperazin-1-yl)benzoate (1.64 g, 7.00 mmol) and 5 mL of tetraisopropyl titanate were sequentially added and the mixture was stirred at room temperature for 4 h. Then sodium triacetoxyborohydride (3.17 g, 14.96 mmol) was added and the resulting mixture was reacted at room temperature for 16 h. To the reaction solution was slowly added 20 mL of saturated sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate (60 mL×2). The organic phase was washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 5c (2.01 g, yield: 61%).
5c (0.40 g, 0.91 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.15 g, 3.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.20 g). The above-mentioned crude (200 mg) was dissolved in 15 mL of DCM. Intermediate 2 (330 mg, 0.45 mmol), DMAP (110 mg, 0.9 mmol) and EDCI (180 mg, 0.94 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 30 mL of water and the mixture was extracted with DCM (60 mL×2). The organic phase was washed with 50 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 5d (260 mg, yield: 25%).
5d (260 mg, 0.23 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (0.6 mL) and zinc powder (960 mg, 14.8 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (10 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (250 mg). The above-mentioned crude (250 mg) was dissolved in 10 mL of DCM. Triethylamine (0.2 mL), intermediate 1 (154 mg, 0.27 mmol) and HATU (142 mg, 0.37 mmol) were sequentially added and the mixture was reacted at room temperature for 1 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was washed with 20 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). To the preparative solution were added 100 mL of dichloromethane and 60 mL of saturated sodium bicarbonate solution and the mixture was stirred for 1 h. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 5 (40 mg, yield: 11%).
1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.35 (d, 1H), 8.09 (dd, 1H), 7.67 (d, 2H), 7.50-7.20 (m, 12H), 7.15-7.01 (m, 3H), 6.83-6.60 (m, 3H), 6.30-6.20 (m, 1H), 5.14-5.02 (m, 1H), 4.78-4.69 (m, 1H), 4.62-4.56 (m, 1H), 4.52-4.45 (m, 1H), 4.33-4.24 (m, 2H), 4.18-4.04 (m, 1H), 4.00-3.80 (m, 3H), 3.76-3.54 (m, 2H), 3.53-3.20 (m, 7H), 3.17-2.90 (m, 4H), 2.60-2.02 (m, 21H), 1.70-1.40 (m, 9H), 1.37-1.15 (m, 2H), 1.07-1.00 (m, 9H).
LCMS m/z=758.5 [M/2+1]+.
Example 6
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 6) trifluoroacetate
Under nitrogen protection, 6a (see WO 2020041406 for the synthetic method) (2 g, 9.85 mmol) and (4-chlorophenyl)boronic acid (2.47 g, 15.80 mmol) were sequentially dissolved in 20 mL of dioxane and 2 mL of water. Potassium acetate (3.11 g, 31.69 mmol) and Pd(dppf)Cl2 (0.2 g, 0.27 mmol) were sequentially added and the mixture was reacted at 90° C. for 4 h. The reaction solution was cooled to room temperature and to the reaction solution was slowly added 100 mL of water. The mixture was extracted with ethyl acetate (60 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:10) to afford 6b (2.0 g, yield: 87%).
1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 7.38-7.30 (m, 2H), 7.18-7.10 (m, 2H), 2.78-2.68 (m, 2H), 2.66-2.56 (m, 2H), 1.95-1.80 (m, 2H), 1.78-1.65 (m, 2H), 1.60-1.47 (m, 2H).
6b (1.75 g, 7.52 mmol) was dissolved in 15 mL of tetrahydrofuran. Ethyl 4-(piperazin-1-yl)benzoate (1.64 g, 7.00 mmol) and 5 mL of tetraisopropyl titanate were sequentially added and the mixture was stirred at room temperature for 4 h. Then sodium triacetoxyborohydride (3.17 g, 14.96 mmol) was added and the resulting mixture was reacted at room temperature for 16 h. To the reaction solution was slowly added 20 mL of saturated sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate (60 mL×2). The organic phase was washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 6c (2.0 g, yield: 63%).
1H NMR (400 MHz, CDCl3) δ 7.93-7.86 (m, 2H), 7.30-7.20 (m, 2H), 7.03-6.94 (m, 2H), 6.86-6.74 (m, 2H), 4.31 (q, 2H), 3.33-3.19 (m, 4H), 2.81 (s, 2H), 2.55-2.26 (m, 8H), 1.90-1.75 (m, 2H), 1.67-1.48 (m, 4H), 1.36 (t, 3H).
6c (0.40 g, 0.88 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.15 g, 3.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.20 g). The above-mentioned crude (200 mg) was dissolved in 15 mL of DCM. Intermediate 2 (330 mg, 0.45 mmol), DMAP (110 mg, 0.9 mmol) and EDCI (180 mg, 0.94 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 30 mL of water and the mixture was extracted with DCM (60 mL×2). The organic phase was washed with 50 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 6d (260 mg, yield: 26%).
LCMS m/z=568.2 [M/2+1]+.
6d (260 mg, 0.23 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (0.6 mL) and zinc powder (960 mg, 14.8 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (10 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (250 mg). The above-mentioned crude (250 mg) was dissolved in 10 mL of DCM. Triethylamine (0.2 mL), intermediate 1 (154 mg, 0.27 mmol) and HATU (142 mg, 0.37 mmol) were sequentially added and the mixture was reacted at room temperature for 1 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was washed with 20 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). Lyophilization was performed to afford the trifluoroacetate of compound 6 (20 mg).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.43-8.30 (m, 1H), 8.15-8.05 (m, 1H), 7.74-7.62 (m, 2H), 7.47-7.22 (m, 12H), 7.16-7.05 (m, 1H), 7.02-6.92 (m, 2H), 6.84-6.71 (m, 2H), 6.70-6.55 (m, 1H), 6.34-6.18 (m, 1H), 5.14-5.00 (m, 1H), 4.80-4.67 (m, 1H), 4.64-4.55 (m, 1H), 4.55-4.43 (m, 1H), 4.16-4.04 (m, 1H), 4.00-3.84 (m, 1H), 3.75-3.55 (m, 2H), 3.54-3.20 (m, 7H), 3.17-2.85 (m, 4H), 2.65-2.00 (m, 24H), 1.90-1.75 (m, 2H), 1.66-1.50 (m, 9H), 1.50-1.42 (m, 3H), 1.40-1.25 (m, 2H), 1.04 (s, 9H).
LCMS m/z=764.4 [M/2+1]+.
After the reaction in step 4, the system was quenched with water and extracted with ethyl acetate. The organic phase was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was subjected to preparative liquid phase chromatography to afford free-form compound 6.
instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18. Preparation method: the crude was dissolved with acetonitrile and water to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 10 mmol/L ammonium bicarbonate). Gradient elution method: gradient elution with acetonitrile from 50% to 70% (elution time: 20 min).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.40-8.31 (m, 1H), 8.13-8.04 (m, 1H), 7.78-7.63 (m, 2H), 7.46-7.18 (m, 12H), 7.12-7.02 (m, 1H), 7.02-6.92 (m, 2H), 6.80-6.68 (m, 2H), 6.67-6.56 (m, 1H), 6.43-6.31 (m, 1H), 5.13-4.99 (m, 1H), 4.78-4.67 (m, 1H), 4.66-4.55 (m, 1H), 4.54-4.44 (m, 1H), 4.14-4.02 (m, 1H), 3.98-3.82 (m, 1H), 3.71-3.54 (m, 2H), 3.50-3.18 (m, 7H), 3.16-2.85 (m, 4H), 2.59-1.96 (m, 24H), 1.90-1.75 (m, 2H), 1.75-1.50 (m, 9H), 1.50-1.40 (m, 3H), 1.40-1.16 (m, 2H), 1.04 (s, 9H).
LCMS m/z=764.4 [M/2+1]+.
Example 7
6-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(7-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)nicotinamide (Compound 7)
4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-carbaldehyde (370 mg, 1.49 mmol) was dissolved in 15 mL of tetrahydrofuran. 7a (see CN 105985321 for the synthetic method) (350 mg, 1.49 mmol) and 1 mL of titanium isopropoxide were sequentially added and the mixture was stirred at room temperature for 4 h. Then sodium triacetoxyborohydride (947 mg, 4.47 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. To the reaction solution was slowly added 20 mL of saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted with 60 mL of ethyl acetate twice and the organic phase was washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 7b (320 mg, yield: 46%).
LCMS m/z=468.2 [M+1]+.
7b (0.320 g, 0.68 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.11 g, 2.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction system was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The resulting mixture was adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.23 g). The above-mentioned crude (230 mg) was dissolved in 15 mL of DCM. Intermediate 2 (390 mg, 0.53 mmol), DMAP (200 mg, 1.64 mmol) and EDCI (130 mg, 0.68 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 30 mL of water and the mixture was extracted with 60 mL of DCM twice. The organic phase was washed with 50 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 7c (260 mg, yield: 33%).
LCMS m/z=575.8 [½M+1]+.
7c (260 mg, 0.23 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (0.6 mL) and zinc powder (960 mg, 14.8 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (10 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (190 mg). The above-mentioned crude (190 mg) was dissolved in 10 mL of DCM. Triethylamine (0.2 mL), intermediate 1 (120 mg, 0.21 mmol) and HATU (110 mg, 0.29 mmol) were sequentially added and the mixture was reacted at room temperature for 1 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was washed with 20 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). To the preparative solution were added 100 mL of DCM and 60 mL of saturated sodium bicarbonate solution and the mixture was stirred for 1 h. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 7 (60 mg, yield: 17%).
1H NMR (400 MHz, CDCl3) δ 8.74-8.62 (m, 2H), 8.38-8.32 (m, 1H), 8.08-7.99 (m, 1H), 7.93-7.84 (m, 1H), 7.53-7.20 (m, 12H), 7.11-7.00 (m, 1H), 6.99-6.92 (m, 2H), 6.67-6.58 (m, 1H), 6.53-6.44 (m, 1H), 6.44-6.35 (m, 1H), 5.15-5.02 (m, 1H), 4.80-4.68 (m, 1H), 4.65-4.55 (m, 1H), 4.53-4.45 (m, 1H), 4.20-4.05 (m, 1H), 3.97-3.80 (m, 1H), 3.75-3.53 (m, 6H), 3.45-3.20 (m, 3H), 3.18-2.80 (m, 4H), 2.55-1.95 (m, 24H), 1.75-1.40 (m, 10H), 1.40-1.20 (m, 2H), 1.04 (s, 9H), 1.00-0.90 (m, 6H).
LCMS m/z=771.8 [M/2+1]+.
Example 8
cis-(2S,4R)-1-((2S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 8)
8a (1.88 g, 8.86 mmol) was dissolved in 10 mL of dichloromethane and triethylamine (0.98 g, 9.68 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (2.06 g, 9.72 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 2 h. After the reaction was completed, the reaction solution was diluted with 50 mL of dichloromethane and then 50 mL of water was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=10:1-2:1) to afford 8b (2.4 g, yield: 70%).
8b (0.7 g, 1.81 mmol) was dissolved in 2 mL of dichloromethane. Trifluoroacetic acid (1 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure. To the residue were added 20 mL of dichloromethane and 10 mL of water. The mixture was adjusted to pH 9 with saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 8c (0.5 g).
LCMS m/z=287.0 [M+1]+.
The above-mentioned crude 8c (0.5 g) was added to 10 mL of 1,2-dichloroethane. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (1.03 g, 3.49 mmol) and glacial acetic acid (0.1 mL) were added and the mixture was reacted at room temperature for 0.5 h. Sodium triacetoxyborohydride (0.74 g, 3.49 mmol) was added and the resulting mixture was reacted at room temperature for 19 h. Dichloromethane (50 mL) was added to dilute the reaction solution. Saturated sodium bicarbonate solution was used to adjust the reaction mixture to pH 9. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 8d (0.64 g, yield over two steps from compound 8b: 62%).
LCMS m/z=566.1 [M+1]+.
Step 4: Preparation of trifluoroacetate of 8e
8d (0.64 g, 1.13 mmol) was dissolved in 3 mL of dichloromethane. Trifluoroacetic acid (1 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to afford crude trifluoroacetate of 8e (0.6 g).
The above-mentioned crude trifluoroacetate of 8e (0.64 g) was added to 4 mL of acetonitrile. Triethylamine (0.78 mL, 5.61 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl)benzenesulfonamide (0.35 g, 1.14 mmol) were added and the mixture was refluxed and reacted at 80° C. for 3 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 8f (0.88 g, yield over two steps from compound 8d: 97%).
LCMS m/z=753.0 [M+1]+.
8f (0.88 g, 1.17 mmol) was added to 10 mL of dichloromethane. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (0.51 g, 1.16 mmol), DMAP (0.29 g, 2.37 mmol) and EDCI (0.45 g, 2.35 mmol) were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction system was added 20 mL of water. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 8g (0.85 g, yield: 62%).
LCMS m/z=588.2 [M/2+1]+.
8g (0.44 g, 0.37 mmol) was added to 30 mL of tetrahydrofuran. Zinc powder (1.26 g, 19.27 mmol) and 0.78 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 5 h. The reaction system was filtered and the filtrate was concentrated under reduced pressure. The residue was then diluted with 100 mL of DCM and then 50 mL of saturated sodium bicarbonate solution was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.11 g). The above-mentioned crude (0.11 g) was added to 8 mL of DCM. Intermediate 1 (65 mg, 0.11 mmol), triethylamine (0.15 mL, 1.08 mmol) and HATU (63 mg, 0.166 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 20 mL of water and the mixture was extracted with 30 mL of DCM twice. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 m, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). The preparative solution was lyophilized and 2 mL of ethyl acetate and 1 mL of saturated sodium bicarbonate solution were added to the lyophilized solid. The mixture was stirred for 1 min and the organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 8 (18 mg, yield: 10%).
1H NMR (400 MHz, CDCl3) δ 8.76-8.62 (m, 1H), 8.54-8.38 (m, 1H), 8.06-7.68 (m, 3H), 7.43-6.90 (m, 15H), 6.87-6.64 (m, 3H), 6.56-6.34 (m, 1H), 5.15-4.87 (m, 1H), 4.85-4.65 (m, 2H), 4.56-4.44 (m, 1H), 4.22-3.84 (m, 2H), 3.75-3.41 (m, 3H), 3.40-2.95 (m, 8H), 2.95-2.55 (m, 6H), 2.55-1.95 (m, 22H), 1.76-1.40 (m, 8H), 1.40-1.20 (m, 4H), 1.15-0.90 (m, 15H).
LCMS m/z=784.5 [M/2+1]+.
Example 9
(2S,4R)-1-((S)-2-(7-(2-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,7-diazaspiro[3.5]nonan-7-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 9) trifluoroacetate
9a (5.00 g, 22.09 mmol) was dissolved in 25 mL of dichloromethane and triethylamine (2.46 g, 24.30 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (5.15 g, 24.31 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 3 h. After the reaction was completed, the reaction solution was diluted with dichloromethane (50 mL) and then 50 mL of water was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 9b (8.00 g).
The above-mentioned crude 9b (8.00 g) was dissolved in 60 mL of dichloromethane. Trifluoroacetic acid (20 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure and the residue was dissolved in 50 mL of dichloromethane. The mixture was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution. Liquid separation was performed. The aqueous phase was extracted with 100 mL of dichloromethane twice, and the organic phases were combined and dried over anhydrous sodium sulfate. The reaction system was filtered and 50 mL of dichloromethane was added to the filter cake. Saturated sodium bicarbonate solution (50 mL) was added and the mixture was stirred until the solid was completely dissolved. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 9c (3.50 g).
LCMS m/z=301.1 [M+1]+.
The above-mentioned crude 9c (0.51 g) was dissolved in 20 mL of DCM. The mixture was cooled to 0° C. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (0.50 g, 1.69 mmol) and triethylamine (0.68 g, 6.72 mmol) were added and the mixture was reacted at 0° C. for 15 min. Sodium triacetoxyborohydride (0.54 g, 2.55 mmol) was added and the resulting mixture was reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=20:1) to afford 9d (0.60 g, yield: 61%).
LCMS m/z=580.2 [M+1]+.
9d (0.60 g, 1.03 mmol) was dissolved in 6 mL of dichloromethane. Trifluoroacetic acid (2 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to afford crude 9e (0.5 g).
LCMS m/z=480.1 [M+1]+.
The above-mentioned crude 9e (0.5 g) was added to 10 mL of acetonitrile. DIPEA (0.54 g, 4.18 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl) benzenesulfonamide (0.32 g, 1.04 mmol) were added and the mixture was refluxed and reacted at 85° C. for 12 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 9f (0.2 g, yield over two steps from compound 9d: 25%).
LCMS m/z=767.0 [M+1]+.
9f (0.18 g, 0.23 mmol) was added to 6 mL of DCM. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (0.10 g, 0.23 mmol), DMAP (56 mg, 0.46 mmol) and EDCI (88 mg, 0.46 mmol) were sequentially added and the mixture was reacted at 40° C. for 12 h. The reaction mixture was cooled to room temperature. To the reaction system was added 5 mL of water. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 9g (0.2 g, yield: 73%).
LCMS m/z=1189.0 [M+1]+.
Step 7: Preparation of trifluoroacetate of Compound 9
9g (0.15 g, 0.13 mmol) was added to 12 mL of tetrahydrofuran. Zinc powder (0.56 g, 8.56 mmol) and 0.36 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction solution was added 5 mL of water to quench the reaction. The mixture was filtered over diatomaceous earth and the filtrate was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution and extracted with 6 mL of dichloromethane twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was diluted with 100 mL of dichloromethane and 50 mL of saturated aqueous sodium bicarbonate solution was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v/v)=100:10:1) to afford a crude (0.06 g). The above-mentioned crude (0.06 g) was added to 3 mL of dichloromethane. Intermediate 1 (0.042 g, 0.071 mmol), triethylamine (0.03 g, 0.3 mmol) and HATU (0.027 g, 0.071 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water and liquid separation was performed. The aqueous phase was extracted with 6 mL of dichloromethane twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). Lyophilization was performed to afford the trifluoroacetate of compound 9 (0.02 g).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.30 (s, 1H), 8.15-7.97 (m, 1H), 7.90-7.70 (m, 2H), 7.67-7.50 (m, 1H), 7.43-7.26 (m, 7H), 7.25-7.12 (m, 4H), 7.05-6.86 (m, 3H), 6.80-6.60 (m, 3H), 6.28-6.05 (m, 1H), 5.16-5.03 (m, 1H), 4.74-4.60 (m, 1H), 4.51-4.36 (m, 2H), 4.16-4.05 (m, 1H), 3.94-3.80 (m, 1H), 3.58-3.47 (m, 1H), 3.45-2.70 (m, 16H), 2.60-2.45 (m, 5H), 2.40-2.15 (m, 10H), 2.12-1.95 (m, 6H), 1.70-1.40 (m, 13H), 1.38-1.25 (m, 2H), 1.10-0.90 (m, 15H).
LCMS m/z=791.6 [M/2+1]+.
Example 10
(2S,4R)-1-((S)-2-(7-((1R,4R)-5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 10)
10a (1.90 g, 9.58 mmol) was dissolved in 10 mL of dichloromethane and triethylamine (1.07 g, 10.57 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (2.24 g, 10.57 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 3 h. After the reaction was completed, the reaction was quenched with 15 mL of water and liquid separation was performed. The aqueous phase was extracted with 15 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 10b (3.25 g).
The above-mentioned crude 10b (3.25 g) was dissolved in 20 mL of DCM. Trifluoroacetic acid (7 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure and the residue was dissolved with 50 mL of DCM and adjusted to pH 8 with saturated aqueous sodium bicarbonate solution. Liquid separation was performed. The aqueous phase was extracted with 100 mL of DCM twice and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 10c (2.30 g).
The above-mentioned crude 10c (0.93 g) was dissolved in 20 mL of DCM. The mixture was cooled to 0° C. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (1.00 g, 3.38 mmol) and triethylamine (1.37 g, 13.54 mmol) were added and the mixture was reacted at 0° C. for 15 min. Sodium triacetoxyborohydride (1.08 g, 5.10 mmol) was added and the resulting mixture was reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=20:1) to afford 10d (1.20 g, yield: 64%).
LCMS m/z=552.1 [M+1]+.
10d (1.20 g, 2.18 mmol) was dissolved in 9 mL of DCM. Trifluoroacetic acid (3 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to afford crude trifluoroacetate of 10e (1.48 g).
The above-mentioned crude trifluoroacetate of 10e (1.48 g) was added to 10 mL of acetonitrile. DIPEA (1.40 g, 10.83 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl)benzenesulfonamide (0.67 g, 2.18 mmol) were added and the mixture was refluxed and reacted at 85° C. for 12 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 10f (1.46 g, yield over two steps from compound 10d: 91%).
LCMS m/z=739.0 [M+1]+.
10f (0.66 g, 0.89 mmol) was added to 6 mL of DCM. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (0.39 g, 0.90 mmol), DMAP (0.22 g, 1.81 mmol) and EDCI (0.34 g, 1.78 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. The reaction mixture was cooled to room temperature. To the reaction system were added 5 mL of water and 3 mL of DCM. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 10g (0.77 g, yield: 75%).
LCMS m/z=581.2 [M/2+1]+.
10g (0.77 g, 0.66 mmol) was added to 60 mL of tetrahydrofuran. Zinc powder (2.28 g, 34.85 mmol) and 1.42 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 4 h. To the reaction solution was added 25 mL of water to quench the reaction. The mixture was filtered over diatomaceous earth and the filtrate was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution and extracted with 6 mL of DCM twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v/v)=100:10:1) to afford a crude (0.33 g). The above-mentioned crude (0.33 g) was added to 5 mL of DCM. Intermediate 1 (0.21 g, 0.36 mmol), triethylamine (0.17 g, 1.68 mmol) and HATU (0.19 g, 0.5 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water and liquid separation was performed. The aqueous phase was extracted with 6 mL of DCM twice and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was preliminarily separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water containing 10 mmol/L NH4HCO3. Gradient elution method: gradient elution with acetonitrile from 51% to 81% (elution time: 15 min). Lyophilization was performed to afford compound 10 (0.13 g, yield: 13%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.41-8.32 (m, 1H), 8.14-7.92 (m, 1H), 7.80-7.62 (m, 2H), 7.44-7.26 (m, 10H), 7.26-7.10 (m, 3H), 7.04-6.92 (m, 2H), 6.84-6.70 (m, 2H), 6.68-6.53 (m, 1H), 6.40-6.20 (m, 1H), 5.15-5.00 (m, 1H), 4.82-3.80 (m, 6H), 3.67-2.95 (m, 10H), 2.93-2.55 (m, 4H), 2.55-1.88 (m, 20H), 1.85-1.40 (m, 12H), 1.38-1.20 (m, 2H), 1.10-0.90 (m, 15H).
LCMS m/z=518.8 [M/3+1]+.
Example 11
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((6-(4-chlorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 11)
11a (see WO 2013185202 for the synthetic method) (350 mg, 0.8 mmol) was dissolved in 15 mL of DCM. Intermediate 2 (583 mg, 0.79 mmol), DMAP (195 mg, 1.6 mmol) and EDCI (306 mg, 1.6 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 100 mL of water and the mixture was extracted with 60 mL of DCM twice. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 11b (550 mg, yield: 60%).
LCMS m/z=574.1 [M/2+1]+.
11b (260 mg, 0.23 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (0.6 mL) and zinc powder (960 mg, 14.8 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (10 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by chromatographic column on silica gel (methanol/dichloromethane/triethylamine (v/v)=19:1:0-89:10:1) to afford a crude (160 mg). The above-mentioned crude (160 mg) was dissolved in 15 mL of DCM. Triethylamine (0.22 mL), intermediate 1 (95 mg, 0.17 mmol) and HATU (110 mg, 0.29 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 100 mL of water. The mixture was extracted with 50 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water containing 10 mmol/L NH4HCO3. Gradient elution method: gradient elution with acetonitrile from 58% to 88% (elution time: 10 min)). Lyophilization was performed to afford compound 11 (50 mg, yield: 14%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.37-8.30 (m, 1H), 8.09 (dd, 1H), 7.71 (d, 2H), 7.44-7.19 (m, 12H), 7.13-6.97 (m, 3H), 6.75 (d, 2H), 6.62 (d, 1H), 6.39 (d, 1H), 5.15-5.00 (m, 1H), 4.80-4.67 (m, 1H), 4.61 (d, 1H), 4.55-4.43 (m, 1H), 4.14-4.02 (m, 1H), 3.99-3.82 (m, 1H), 3.75-3.53 (m, 2H), 3.50-3.17 (m, 7H), 3.16-2.95 (m, 2H), 2.89 (s, 2H), 2.57-1.97 (m, 24H), 1.76-1.40 (m, 10H), 1.38-1.22 (m, 2H), 1.04 (s, 9H), 0.35 (s, 4H).
LCMS m/z=770.8 [M/2+1]+.
Example 12
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((4′-chloro-2′-fluoro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 12)
Under nitrogen protection, 12a (see WO 2021066873 for the synthetic method) (2 g, 9.21 mmol) and (4-chloro-2-fluorophenyl)boronic acid (2.41 g, 13.82 mmol) were dissolved in 20 mL of dioxane and 2 mL of water. Potassium acetate (2.71 g, 27.61 mmol) and Pd(dppf)Cl2 (0.2 g, 0.27 mmol) were sequentially added and the mixture was reacted at 90° C. for 4 h. The reaction solution was cooled to room temperature and to the reaction solution was slowly added 100 mL of water. The mixture was extracted with ethyl acetate (60 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:10) to afford 12b (2.0 g, yield: 81%).
12b (2.4 g, 9.0 mmol) was dissolved in 15 mL of DCE. Ethyl 4-(piperazin-1-yl)benzoate (2.10 g, 9.0 mmol) and 5 mL of tetraisopropyl titanate were sequentially added and the mixture was stirred at room temperature for 3 h. Then sodium triacetoxyborohydride (3.81 g, 17.98 mmol) was added and the resulting mixture was reacted at room temperature for 16 h. To the reaction solution was slowly added 20 mL of saturated sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate (60 mL×2). The organic phase was washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 12c (1.0 g, yield: 23%).
LCMS m/z=485.2 [M+1]+.
12c (1.4 g, 2.89 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.35 g, 8.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then washed with 20 mL of methyl tert-butyl ether. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.8 g). The above-mentioned crude (0.4 g) was dissolved in 15 mL of DCM. Intermediate 2 (0.64 g, 0.87 mmol), DMAP (320 mg, 2.62 mmol) and EDCI (340 mg, 1.77 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 30 mL of water and the mixture was extracted with DCM (60 mL×2). The organic phase was washed with 50 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=9:1) to afford 12d (0.6 g, yield: 59%).
LCMS m/z=584.1 [M/2+1]+.
12d (0.5 g, 0.43 mmol) was dissolved in 20 mL of tetrahydrofuran. Acetic acid (1.6 g, 26.64 mmol) and zinc powder (1.6 g, 24.67 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (10 mL). The mixture was then extracted with 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.2 g). The above-mentioned crude (0.2 g) was dissolved in 10 mL of DCM. Triethylamine (0.2 mL), intermediate 1 and HATU (0.15 g, 0.4 mmol) were sequentially added and the mixture was reacted at room temperature for 1 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was washed with 20 mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water containing 10 mmol/L NH4HCO3. Gradient elution method: gradient elution with acetonitrile from 54% to 74% (elution time: 15 min). Lyophilization was performed to afford compound 12 (80 mg, yield: 12%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.38-8.32 (m, 1H), 8.09 (dd, 1H), 7.70 (d, 2H), 7.45-7.18 (m, 10H), 7.15-6.87 (m, 4H), 6.84-6.70 (m, 2H), 6.62 (d, 1H), 6.34 (d, 1H), 5.15-5.00 (m, 1H), 4.80-4.68 (m, 1H), 4.66-4.56 (m, 1H), 4.54-4.44 (m, 1H), 4.15-4.03 (m, 1H), 3.98-3.82 (m, 1H), 3.73-3.53 (m, 2H), 3.51-3.20 (m, 7H), 3.16-2.93 (m, 2H), 2.75 (s, 2H), 2.55-1.95 (m, 24H), 1.80-1.43 (m, 10H), 1.40-1.22 (m, 2H), 1.10-0.94 (m, 15H).
LCMS m/z=520.8 [M/3+1]+.
Example 13
(2S,4R)-1-((S)-2-(7-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 13)
13a (1.90 g, 9.58 mmol) was dissolved in 10 mL of DCM and triethylamine (1.07 g, 10.57 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (2.24 g, 10.57 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 3 h. After the reaction was completed, the reaction was quenched with 15 mL of water and liquid separation was performed. The aqueous phase was extracted with 15 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 13b (3.25 g).
The above-mentioned crude 13b (3.0 g) was dissolved in 20 mL of DCM. Trifluoroacetic acid (7 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure and the residue was dissolved with 50 mL of DCM and adjusted to pH 8 with saturated aqueous sodium bicarbonate solution. Liquid separation was performed. The aqueous phase was extracted with 100 mL of DCM twice and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 13c (2.0 g).
The above-mentioned crude 13c (1.00 g) was dissolved in 20 mL of DCM. The mixture was cooled to 0° C. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (1.08 g, 3.65 mmol) and triethylamine (1.37 g, 13.54 mmol) were added and the mixture was reacted at 0° C. for 15 min. Sodium triacetoxyborohydride (1.08 g, 5.10 mmol) was added and the resulting mixture was reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=20:1) to afford 13d (1.2 g, yield: 59%).
13d (1.20 g, 2.18 mmol) was dissolved in 9 mL of DCM. Trifluoroacetic acid (3 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to afford crude trifluoroacetate of 13e (1.48 g).
The above-mentioned crude trifluoroacetate of 13e (1.48 g) was added to 10 mL of acetonitrile. DIPEA (1.40 g, 10.83 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl)benzenesulfonamide (0.67 g, 2.18 mmol) were added and the mixture was refluxed and reacted at 85° C. for 12 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 13f (1.46 g, yield over two steps from compound 13d: 91%).
LCMS m/z=739.0 [M+1]+.
13f (0.7 g, 0.95 mmol) was added to 6 mL of DCM. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (0.42 g, 0.97 mmol), DMAP (0.35 g, 2.88 mmol) and EDCI (0.36 g, 1.88 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. The reaction mixture was cooled to room temperature. To the reaction system were added 5 mL of water and 3 mL of DCM. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 13g (0.5 g, yield: 45%).
LCMS m/z=581.2 [M/2+1]+.
13g (0.5 g, 0.43 mmol) was added to 60 mL of tetrahydrofuran. Zinc powder (1.6 g, 24.46 mmol) and 1.6 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 4 h. To the reaction solution was added 25 mL of water to quench the reaction. The mixture was filtered over diatomaceous earth and the filtrate was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution and extracted with 6 mL of DCM twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v)=100:10:1) to afford a crude (0.2 g). The above-mentioned crude (0.2 g) was added to 5 mL of DCM. Intermediate 1 (0.12 g, 0.21 mmol), triethylamine (0.061 g, 0.6 mmol) and HATU (0.15 g, 0.39 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water and liquid separation was performed. The aqueous phase was extracted with 6 mL of DCM twice and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was preliminarily separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water. Gradient elution method: gradient elution with acetonitrile from 60% to 90% (elution time: 15 min). Lyophilization was performed to afford compound 13 (0.1 g, yield: 15%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.45-8.32 (m, 1H), 8.06-7.93 (m, 1H), 7.82-7.66 (m, 2H), 7.43-7.11 (m, 13H), 7.03-6.92 (m, 2H), 6.83-6.56 (m, 3H), 6.53-6.32 (m, 1H), 5.15-4.90 (m, 1H), 4.84-4.17 (m, 4H), 4.14-3.86 (m, 2H), 3.68-2.91 (m, 10H), 2.88-2.70 (m, 3H), 2.70-1.75 (m, 22H), 1.75-1.22 (m, 13H), 1.12-0.88 (m, 15H).
LCMS m/z=518.8 [M/3+1]+.
Example 14
cis-(2S,4R)-1-((S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-2′-fluoro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl) sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl) hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl) pyrrolidine-2-carboxamide (Compound 14)
12c (1.4 g, 2.89 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.35 g, 8.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then washed with 20 mL of methyl tert-butyl ether. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.8 g). The above-mentioned crude (240 mg) was dissolved in 15 mL of DCM. 8f (400 mg, 0.53 mmol), DMAP (130 mg, 1.06 mmol) and EDCI (200 mg, 1.04 mmol) were added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 100 mL of water and the mixture was extracted with DCM (100 mL×2). The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane:methanol (v/v)=0:1-9:1) to afford 14a (500 mg, yield: 48%).
LCMS m/z=597.1 [M/2+1]+.
14a (500 mg, 0.42 mmol) was dissolved in 40 mL of tetrahydrofuran. Acetic acid (1.2 mL) and zinc powder (1.8 g, 27.75 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by chromatographic column on silica gel (methanol/dichloromethane/triethylamine (v/v)=1:19:0-10:89:1) to afford a crude (330 mg). The above-mentioned crude (150 mg) was dissolved in 15 mL of DCM. Triethylamine (0.21 mL), intermediate 1 (88 mg, 0.15 mmol) and HATU (86 mg, 0.23 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 50 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was first separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water. Gradient elution method: gradient elution with acetonitrile from 59% to 89% (elution time: 15 min). Lyophilization was performed to afford compound 14 (35 mg, yield: 12%).
1H NMR (400 MHz, CDCl3) δ 8.75-8.60 (m, 1H), 8.55-8.43 (m, 1H), 7.96-7.68 (m, 3H), 7.46-7.15 (m, 10H), 7.15-6.89 (m, 4H), 6.86-6.63 (m, 3H), 6.56-6.30 (m, 1H), 5.15-4.64 (m, 3H), 4.57-4.44 (m, 1H), 4.26-3.85 (m, 2H), 3.80-3.40 (m, 3H), 3.39-2.92 (m, 8H), 2.92-1.95 (m, 28H), 1.85-1.20 (m, 12H), 1.18-0.90 (m, 15H).
LCMS m/z=793.8 [M/2+1]+.
Example 15
cis-(2S,4R)-1-((S)-2-(5-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-5-oxopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 15)
8g (0.44 g, 0.37 mmol) was added to 30 mL of tetrahydrofuran. Zinc powder (1.26 g, 19.27 mmol) and 0.78 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 5 h. The reaction system was filtered and the filtrate was concentrated under reduced pressure. The residue was then diluted with 100 mL of DCM and then 50 mL of saturated sodium bicarbonate solution was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.11 g). The above-mentioned crude (101 mg) was added to 2 mL of DCM. 5-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (see WO 2020163823 for the synthetic method) (58 mg, 0.104 mmol), triethylamine (0.14 mL, 1.01 mmol) and HATU (57 mg, 0.15 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of DCM twice. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 60% to 90% (elution time: 15 min). Lyophilization was performed to afford compound 15 (35 mg, yield: 7%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.45 (s, 1H), 7.95-7.70 (m, 3H), 7.46-7.14 (m, 12H), 7.10-6.90 (m, 4H), 6.85-6.63 (m, 3H), 5.12-4.92 (m, 1H), 4.82-4.40 (m, 3H), 4.22-3.85 (m, 2H), 3.72-3.38 (m, 3H), 3.35-2.57 (m, 14H), 2.56-1.77 (m, 24H), 1.75-1.55 (m, 1H), 1.53-1.30 (m, 5H), 1.16-0.87 (m, 15H).
LCMS m/z=770.8 [M/2+1]+.
Example 16
cis-(2S,4R)-1-((S)-2-(9-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-9-oxononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 16)
8g (0.44 g, 0.37 mmol) was added to 30 mL of tetrahydrofuran. Zinc powder (1.26 g, 19.27 mmol) and 0.78 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 5 h. The reaction system was filtered and the filtrate was concentrated under reduced pressure. The residue was then diluted with 100 mL of DCM and then 50 mL of saturated sodium bicarbonate solution was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.11 g). The above-mentioned crude (101 mg) was added to 2 mL of DCM. 9-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoic acid (see WO 2020163823 for the synthetic method) (61 mg, 0.099 mmol), triethylamine (0.14 mL, 1.01 mmol) and HATU (57 mg, 0.15 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 30 mL of water and the mixture was extracted with 20 mL of DCM twice. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 60% to 90% (elution time: 15 min). Lyophilization was performed to afford compound 16 (85 mg, yield: 16%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.52-8.34 (m, 1H), 8.07-7.87 (m, 1H), 7.83-7.67 (m, 2H), 7.50-7.16 (m, 12H), 7.11-6.90 (m, 3H), 6.85-6.55 (m, 3H), 6.40-6.25 (m, 1H), 5.18-5.00 (m, 1H), 4.80-4.58 (m, 2H), 4.56-4.45 (m, 1H), 4.18-4.05 (m, 1H), 4.00-3.85 (m, 1H), 3.70-3.42 (m, 3H), 3.35-2.95 (m, 8H), 2.94-2.64 (m, 5H), 2.63-1.95 (m, 23H), 1.80-1.36 (m, 10H), 1.35-1.15 (m, 6H), 1.10-0.90 (m, 15H).
LCMS m/z=798.2 [M/2+1]+.
Example 17
cis-(2S,4R)-1-((S)-2-(8-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 17)
8g (0.30 g, 0.25 mmol) was added to 300 mL of tetrahydrofuran. Zinc powder (1.36 g, 20.8 mmol) and ammonium chloride (0.41 g, 7.66 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate, followed by extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v)=95:5:0-89:10:1) to afford a crude (0.26 g). The above-mentioned crude (120 mg) was added to 15 mL of DCM. Intermediate 3 (75 mg, 0.12 mmol), triethylamine (0.17 mL, 1.2 mmol) and HATU (68 mg, 0.18 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 200 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water. Gradient elution method: gradient elution with acetonitrile from 59% to 89% (elution time: 15 min). Lyophilization was performed to afford compound 17 (90 mg, yield: 49%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.52-8.39 (m, 1H), 8.05-7.86 (m, 1H), 7.85-7.70 (m, 2H), 7.58-7.14 (m, 12H), 7.07-6.92 (m, 3H), 6.86-6.60 (m, 3H), 6.54-6.29 (m, 1H), 5.15-4.92 (m, 1H), 4.84-4.60 (m, 2H), 4.58-4.42 (m, 1H), 4.19-3.85 (m, 2H), 3.68-3.40 (m, 3H), 3.35-2.94 (m, 8H), 2.94-1.95 (m, 28H), 1.78-1.15 (m, 14H), 1.15-0.95 (m, 15H).
LCMS m/z=791.8 [M/2+1]+.
Example 18
cis-(2S,4R)-1-((S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((6-(4-chlorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (compound 18)
Step 1: Preparation of 18a
11a (170 mg, 0.39 mmol) was dissolved in 15 mL of DCM. 8f (300 mg, 0.40 mmol), DMAP (100 mg, 0.82 mmol) and EDCI (150 mg, 0.78 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 100 mL of water and the mixture was extracted with 100 mL of DCM. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=0:1-9:1) to afford 18a (300 mg, yield: 66%).
LCMS m/z=587.2 [M/2+1]+.
18a (300 mg, 0.26 mmol) was dissolved in 5 mL of tetrahydrofuran and 15 mL of methanol. Ammonium chloride (0.41 g, 7.66 mmol) and zinc powder (1.36 g, 20.9 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate, followed by extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v)=95:5:0-89:10:1) to afford a crude (220 mg). The above-mentioned crude (120 mg) was dissolved in 15 mL of DCM. Triethylamine (0.17 mL), intermediate 1 (70 mg, 0.12 mmol) and HATU (68 mg, 0.18 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 50 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 40% to 70% (elution time: 15 min). Lyophilization was performed to afford compound 18 (35 mg, yield: 16%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.42-8.32 (m, 1H), 8.15-7.90 (m, 1H), 7.80-7.60 (m, 2H), 7.44-6.96 (m, 15H), 6.83-6.47 (m, 3H), 6.43-6.16 (m, 1H), 5.15-5.02 (m, 1H), 4.85-3.83 (m, 6H), 3.65-2.68 (m, 15H), 2.68-1.17 (m, 35H), 1.04 (s, 9H), 0.36 (s, 4H).
LCMS m/z=783.9 [M/2+1]+.
Example 19
(2S,4R)-1-((S)-2-(7-((1R,4R)-5-((R)-3-((4-(N-(4-(4-((6-(4-chlorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 19)
11a (150 mg, 0.34 mmol) was dissolved in 10 mL of DCM. 10f (250 mg, 0.34 mmol), DMAP (83 mg, 0.68 mmol) and EDCI (130 mg, 0.68 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 100 mL of water and the mixture was extracted with 100 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=0:1-9:1) to afford 19a (220 mg, yield: 56%).
LCMS m/z=580.0 [M/2+1]+.
19a (220 mg, 0.19 mmol) was dissolved in 3 mL of tetrahydrofuran and 12 mL of methanol. Ammonium chloride (0.32 g, 5.98 mmol) and zinc powder (1.05 g, 16 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate for extraction, followed by liquid separation. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v/v)=95:5:0-89:10:1) to afford a crude (0.16 g). The above-mentioned crude (120 mg) was dissolved in 15 mL of DCM. Triethylamine (0.17 mL), intermediate 1 (70 mg, 0.12 mmol) and HATU (68 mg, 0.18 mmol were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 50 mL of dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min). Lyophilization was performed to afford compound 19 (40 mg, yield: 18%).
1H NMR (400 MHz, CDCl3) δ 8.75-8.61 (m, 1H), 8.54-8.44 (m, 1H), 7.97-7.70 (m, 3H), 7.48-6.87 (m, 15H), 6.87-6.64 (m, 3H), 6.55-6.32 (m, 1H), 5.17-4.89 (m, 1H), 4.87-4.62 (m, 2H), 4.59-4.45 (m, 1H), 4.25-3.80 (m, 3H), 3.79-2.58 (m, 18H), 2.58-1.95 (m, 21H), 1.63-1.20 (m, 9H), 1.04 (s, 9H), 0.37 (s, 4H).
LCMS m/z=776.5 [M/2+1]+.
Example 20
(2S,4R)-1-((S)-2-(7-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((4′-chloro-2′-fluoro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 20)
12c (1.4 g, 2.89 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.35 g, 8.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then washed with 20 mL of methyl tert-butyl ether. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.8 g). The above-mentioned crude (250 mg) was dissolved in 10 mL of DCM. 13f (410 mg, 0.55 mmol), DMAP (130 mg, 1.07 mmol) and EDCI (210 mg, 1.10 mmol) were added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 100 mL of water and the mixture was extracted with 50 mL of DCM. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=1:0-9:1) to afford 20a (310 mg, yield: 48%).
LCMS m/z=590.2 [M/2+1]+.
20a (300 mg, 0.25 mmol) was dissolved in 3 mL of tetrahydrofuran and 12 mL of methanol. Ammonium chloride (0.4 g, 7.5 mmol) and zinc powder (1.31 g, 20.15 mmol) were added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate, followed by extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v/v)=95:5:0-89:10:1) to afford a crude (0.22 g). The above-mentioned crude (27 mg) was dissolved in 5 mL of DCM. Triethylamine (0.037 mL), intermediate 1 (16 mg, 0.027 mmol) and HATU (15 mg, 0.04 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 20 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 40% to 70% (elution time: 15 min). Lyophilization was performed to afford compound 20 (18 mg, yield: 37%).
1H NMR (400 MHz, CDCl3) δ 8.72-8.60 (m, 1H), 8.50-8.34 (m, 1H), 8.08-7.90 (m, 1H), 7.80-7.64 (m, 2H), 7.48-6.88 (m, 14H), 6.85-6.55 (m, 3H), 6.49-6.25 (m, 1H), 5.15-4.90 (m, 1H), 4.85-3.85 (m, 6H), 3.68-2.90 (m, 10H), 2.86-1.94 (m, 22H), 1.90-1.18 (m, 16H), 1.12-0.88 (m, 15H).
LCMS m/z=524.8 [M/3+1]+.
Example 21
cis-(2S,4R)-1-((S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((6-(4-chloro-2-fluorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 21)
21a (see WO 2013185202 for the synthetic method) (1.00 g, 5.86 mmol) and (4-chloro-2-fluorophenyl)boronic acid (1.02 g, 5.85 mmol) were dissolved in 30 mL of dioxane and 3 mL of water. After nitrogen replacement was carried out three times, potassium acetate (1.73 g, 17.63 mmol) and Pd(dppf)Cl2 (0.13 g, 0.18 mmol) were added under nitrogen protection and the mixture was reacted at 90° C. for 7 h. The reaction solution was cooled to room temperature and to the reaction solution was slowly added 40 mL of water. The mixture was extracted with 40 mL of ethyl acetate, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:10) to afford 21b (0.78 g, yield: 50%).
21b (780 mg, 2.95 mmol) was dissolved in 35 mL of tetrahydrofuran. Ethyl 4-(piperazin-1-yl)benzoate (1.11 g, 4.74 mmol), 2 mL of acetic acid and sodium triacetoxyborohydride (2.01 g, 9.48 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. To the reaction solution was slowly added 50 mL of saturated sodium bicarbonate solution. The resulting mixture was extracted with 60 mL of ethyl acetate twice and the organic phase was washed with 50 mL of saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v)=0:1-1:1) to afford 21c (1.27 g, yield: 89%).
LCMS m/z=483.3 [M+1]+.
Sodium hydroxide (0.26 g, 6.5 mmol) was dissolved in 5 mL of methanol and 3 mL of water. A solution of 21c (1.27 g, 2.63 mmol) in 10 mL of tetrahydrofuran was added and the resulting mixture was reacted at 80° C. for 6 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×3). The organic phase was washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.96 g). The above-mentioned crude (170 mg) was dissolved in 15 mL of DCM. 8f (300 mg, 0.40 mmol), DMAP (100 mg, 0.82 mmol) and EDCI (150 mg, 0.78 mmol) were added and the mixture was reacted at room temperature for 16 h. To the reaction system was slowly added 100 mL of water and the mixture was extracted with 100 mL of DCM. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v)=0:1-9:1) to afford 21d (350 mg, yield: 73%).
LCMS m/z=596.1 [M/2+1]+.
21d (350 mg, 0.294 mmol) was dissolved in 5 mL of tetrahydrofuran and 15 mL of methanol. Ammonium chloride (0.48 g, 8.97 mmol) and zinc powder (1.57 g, 24.15 mmol) were added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate, followed by extraction. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by chromatographic column on silica gel (methanol/dichloromethane/triethylamine (v/v/v)=1:19:0-10:89:1) to afford a crude (230 mg). The above-mentioned crude (100 mg) was dissolved in 15 mL of DCM. Triethylamine (0.14 mL), intermediate 1 (60 mg, 0.10 mmol) and HATU (57 mg, 0.15 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 50 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was first separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and subsequently, the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 40% to 70% (elution time: 15 min). Lyophilization was performed to afford compound 21 (35 mg, yield: 17%).
1H NMR (400 MHz, CDCl3) δ 8.73-8.60 (m, 1H), 8.55-8.45 (m, 1H), 7.95-7.70 (m, 3H), 7.44-7.14 (m, 10H), 7.13-6.90 (m, 4H), 6.86-6.62 (m, 3H), 6.52-6.32 (m, 1H), 5.15-4.88 (m, 1H), 4.87-4.63 (m, 2H), 4.57-4.43 (m, 1H), 4.27-3.82 (m, 2H), 3.80-3.36 (m, 3H), 3.35-2.93 (m, 8H), 2.91-1.95 (m, 25H), 1.68-1.39 (m, 11H), 1.38-1.20 (m, 4H), 1.15-0.95 (m, 9H), 0.37 (s, 4H).
LCMS m/z=528.9 [M/3+1]+.
Example 22
cis-(2S,4R)-1-((S)-2-(8-(5-((R)-3-((4-(N-(4-(4-((6-(4-chlorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 22)
18a (300 mg, 0.26 mmol) was dissolved in 5 mL of tetrahydrofuran and 15 mL of methanol. Ammonium chloride (0.41 g, 7.66 mmol) and zinc powder (1.36 g, 20.9 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate, followed by extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v/v)=95:5:0-89:10:1) to afford a crude (0.22 g). The above-mentioned crude (120 mg) was added to 15 mL of DCM. Intermediate 3 (70 mg, 0.11 mmol), triethylamine (0.17 mL, 1.2 mmol) and HATU (68 mg, 0.18 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 200 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 35% to 65% (elution time: 15 min). Lyophilization was performed to afford compound 22 (70 mg, yield: 31%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.54-8.38 (m, 1H), 8.04-7.70 (m, 3H), 7.57-7.14 (m, 12H), 7.10-6.92 (m, 3H), 6.90-6.59 (m, 3H), 6.52-6.18 (m, 1H), 5.16-4.92 (m, 1H), 4.85-4.62 (m, 2H), 4.59-4.42 (m, 1H), 4.20-3.82 (m, 2H), 3.68-3.40 (m, 3H), 3.38-2.93 (m, 8H), 2.93-1.95 (m, 26H), 1.80-1.42 (m, 11H), 1.41-1.16 (m, 5H), 1.12-0.94 (m, 9H), 0.36 (s, 4H).
LCMS m/z=790.5 [M/2+1]+.
Example 23
cis-(2S,4R)-1-((S)-2-(8-(5-((R)-3-((4-(N-(4-(4-((6-(4-chloro-2-fluorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 23)
21d (350 mg, 0.294 mmol) was dissolved in 5 mL of tetrahydrofuran and 15 mL of methanol. Ammonium chloride (0.48 g, 8.97 mmol) and zinc powder (1.57 g, 24.15 mmol) were added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate were added 10 mL of water and 20 mL of ethyl acetate, followed by extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by chromatographic column on silica gel (methanol/dichloromethane/triethylamine (v/v)=1:19:0-10:89:1) to afford a crude (230 mg). The above-mentioned crude (120 mg) was added to 15 mL of DCM. Intermediate 3 (70 mg, 0.11 mmol), triethylamine (0.17 mL, 1.2 mmol) and HATU (68 mg, 0.18 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 100 mL of water. The mixture was extracted with 200 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was separated and purified by silica gel column chromatography (methanol/dichloromethane (v/v)=0:1-1:9) and then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 35% to 65% (elution time: 15 min). Lyophilization was performed to afford compound 23 (65 mg, yield: 27%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.52-8.41 (m, 1H), 8.04-7.86 (m, 1H), 7.84-7.71 (m, 2H), 7.56-7.15 (m, 10H), 7.13-6.94 (m, 4H), 6.87-6.60 (m, 3H), 6.50-6.23 (m, 1H), 5.15-4.92 (m, 1H), 4.85-4.62 (m, 2H), 4.59-4.44 (m, 1H), 4.20-3.82 (m, 2H), 3.70-3.40 (m, 3H), 3.35-2.93 (m, 8H), 2.93-1.96 (m, 28H), 1.75-1.42 (m, 9H), 1.40-1.16 (m, 5H), 1.13-0.92 (m, 9H), 0.37 (s, 4H).
LCMS m/z=799.5 [M/2+1]+.
Example 24
(2S,4R)-1-((S)-2-(8-(4-((R)-3-((4-(N-(4-(4-((4′-chloro-2′-fluoro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 24)
12d (5 g, 4.3 mmol) was dissolved in 200 mL of tetrahydrofuran. Acetic acid (16 g, 266.4 mmol) and zinc powder (16 g, 246.7 mmol) were sequentially added and the mixture was reacted at room temperature for 16 h. The reaction system was filtered and to the filtrate was added water (100 mL). The mixture was then extracted with 200 mL of ethyl acetate. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (2 g). The above-mentioned crude (370 mg) was added to 8 mL of DCM. Intermediate 3 (220 mg, 0.35 mmol), triethylamine (0.51 mL, 3.6 mmol) and HATU (209 mg, 0.55 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 20 mL of water and the mixture was extracted with 20 mL of DCM twice. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 20% to 50% (elution time: 15 min). Lyophilization was performed to afford compound 24 (221 mg, yield: 18%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.38-8.32 (m, 1H), 8.09 (dd, 1H), 7.68 (d, 2H), 7.48-7.22 (m, 10H), 7.16-6.90 (m, 4H), 6.82-6.71 (m, 2H), 6.62 (d, 1H), 6.26 (d, 1H), 5.15-5.00 (m, 1H), 4.80-4.67 (m, 1H), 4.60 (d, 1H), 4.55-4.45 (m, 1H), 4.15-4.07 (m, 1H), 3.97-3.82 (m, 1H), 3.73-3.54 (m, 2H), 3.48-3.20 (m, 7H), 3.18-2.92 (m, 2H), 2.75 (s, 2H), 2.57-1.90 (m, 24H), 1.76-1.40 (m, 10H), 1.36-1.23 (m, 4H), 1.09-0.93 (m, 15H).
LCMS m/z=788.0 [M/2+1]+.
Example 25
(2S,4R)-1-((S)-2-(8-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 25)
13g (0.5 g, 0.43 mmol) was added to 60 mL of tetrahydrofuran. Zinc powder (1.6 g, 24.46 mmol) and 1.6 mL of acetic acid were sequentially added and the mixture was reacted at room temperature for 4 h. To the reaction solution was added 25 mL of water to quench the reaction. The mixture was filtered over diatomaceous earth and the filtrate was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution and extracted with 6 mL of dichloromethane twice. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol:triethylamine (v/v)=100:10:1) to afford a crude (0.2 g). The above-mentioned crude (0.20 g) was added to 5 mL of DCM. Intermediate 3 (0.12 g, 0.2 mmol), triethylamine (0.28 mL, 2.0 mmol) and HATU (0.11 g, 0.29 mmol) were sequentially added and the mixture was reacted at room temperature for 3 h. To the reaction system was added 15 mL of water and the mixture was extracted with 20 mL of DCM, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min). Lyophilization was performed to afford compound 25 (97 mg, yield: 14%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.44-8.32 (m, 1H), 8.07-7.95 (m, 1H), 7.80-7.67 (m, 2H), 7.54-7.16 (m, 13H), 7.03-6.94 (m, 2H), 6.83-6.70 (m, 2H), 6.69-6.55 (m, 1H), 6.39-6.25 (m, 1H), 5.15-4.96 (m, 1H), 4.80-4.18 (m, 4H), 4.16-4.05 (m, 1H), 4.03-3.87 (m, 1H), 3.63-3.53 (m, 1H), 3.53-3.35 (m, 2H), 3.33-2.72 (m, 10H), 2.70-1.95 (m, 21H), 1.95-1.20 (m, 16H), 1.15-0.86 (m, 15H).
LCMS m/z=785.0 [M/2+1]+.
Example 26
cis-(2S,4R)-1-((S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-3a,6a-dimethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 26)
26a (1.0 g, 4.16 mmol) was dissolved in 10 mL of DCM and triethylamine (0.88 g, 8.70 mmol) was added. The mixture was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (0.97 g, 4.58 mmol) was slowly added dropwise. Then the mixture was slowly warmed to room temperature and reacted for 2 h. After the reaction was completed, the reaction solution was diluted with 40 mL of DCM and then 50 mL of water was added. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=10:1-2:1) to afford 26b (1.7 g, yield: 98%).
26b (1.65 g, 3.97 mmol) was dissolved in 10 mL of DCM. Trifluoroacetic acid (2.9 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure. To the residue were added 20 mL of DCM and 20 mL of water. The mixture was adjusted to pH 9 with saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 26c (1.5 g).
The above-mentioned crude 26c (1.5 g) was added to 10 mL of 1,2-dichloroethane. Tert-butyl (R)-(4-oxo-1-(phenylthio)butan-2-yl)carbamate (0.94 g, 3.18 mmol) and glacial acetic acid (0.18 mL) were added and the mixture was reacted at room temperature for 0.5 h. Sodium triacetoxyborohydride (2.2 g, 10.38 mmol) was added and the resulting mixture was reacted at room temperature for 19 h. DCM (50 mL) was added to dilute the reaction solution. Saturated sodium bicarbonate solution was used to adjust the reaction mixture to pH 9. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 26d (0.68 g, yield over two steps from compound 26b: 29%).
26d (0.68 g, 1.14 mmol) was dissolved in 4 mL of DCM. Trifluoroacetic acid (2 mL) was added and the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to afford crude trifluoroacetate of 26e (0.6 g).
The above-mentioned crude trifluoroacetate of 26e (0.6 g) was added to 4 mL of acetonitrile. Triethylamine (0.79 mL, 5.67 mmol) and 4-fluoro-3-((trifluoromethyl)sulfonyl)benzenesulfonamide (0.35 g, 1.14 mmol) were added and the mixture was refluxed and reacted at 80° C. for 3 h. The reaction mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 26f (0.87 g, yield over two steps from compound 26d: 98%).
26f (0.87 g, 1.11 mmol) was added to 10 mL of DCM. 4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoic acid (0.49 g, 1.11 mmol), DMAP (0.27 g, 2.21 mmol) and EDCI (0.43 g, 2.25 mmol) were sequentially added and the mixture was reacted at room temperature for 12 h. To the reaction system was added 20 mL of water. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 26g (0.80 g, yield: 60%).
26g (0.25 g, 0.208 mmol) was added to 5 mL of mixed solvents of methanol/tetrahydrofuran ((v/v)=10:1). Zinc powder (1.1 g, 16.9 mmol) and ammonium chloride (0.34 g, 6.36 mmol) were sequentially added and the mixture was stirred at 30° C. for 19 h. The reaction system was filtered and the filtrate was concentrated under reduced pressure. The crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.08 g). The above-mentioned crude (0.08 g) was added to 8 mL of DCM. Intermediate 1 (50 mg, 0.085 mmol), triethylamine (0.12 mL, 0.86 mmol) and HATU (48 mg, 0.126 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction system was added 20 mL of water and the mixture was extracted with 30 mL of DCM twice. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Then the resulting crude was passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water. Gradient elution method: gradient elution with acetonitrile from 59% to 89% (elution time: 15 min). Lyophilization was performed to afford compound 26 (80 mg, yield: 24%).
1H NMR (400 MHz, CDCl3) δ 8.73-8.62 (m, 1H), 8.55-8.45 (m, 1H), 8.00-7.70 (m, 3H), 7.44-7.15 (m, 12H), 7.05-6.70 (m, 6H), 6.63-6.34 (m, 1H), 5.17-4.60 (m, 3H), 4.58-4.45 (m, 1H), 4.25-3.90 (m, 2H), 3.75-2.62 (m, 15H), 2.60-1.91 (m, 20H), 1.65-0.75 (m, 35H).
LCMS m/z=799.0 [M/2+1]+.
Example 27
(2S,4R)-1-((S)-2-(7-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((6-(4-chlorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 27)
13f (0.50 g, 0.68 mmol) was added to 6 mL of DCM. 11a (0.30 g, 0.687 mmol), DMAP (0.17 g, 1.39 mmol) and EDCI (0.26 g, 1.36 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. The reaction system was cooled to room temperature and 5 mL of water and 3 mL of dichloromethane were added, followed by liquid separation. The aqueous phase was extracted with dichloromethane (5 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 27a (0.30 g, yield: 38%).
LCMS m/z=1159.1 [M+1]+.
27a (0.30 g, 0.26 mmol) was added to 3 mL of tetrahydrofuran and 15 mL of methanol. Zinc powder (1.36 g, 20.9 mmol) and ammonium chloride (0.42 g, 7.85 mmol) were sequentially added and the mixture was stirred at 27° C. for 12 h. To the reaction solution was added 50 mL of ethyl acetate and 30 mL of water. The mixture was filtered over diatomaceous earth and the filtrate was extracted with ethyl acetate (15 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.23 g). The above-mentioned crude (0.13 g) was added to 5 mL of DCM. Intermediate 1 (0.076 g, 0.13 mmol), triethylamine (0.066 g, 0.65 mmol) and HATU (0.074 g, 0.195 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min). Lyophilization was performed to afford compound 27 (0.10 g, yield: 44%).
1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 8.39-8.28 (m, 1H), 8.02-7.84 (m, 1H), 7.74-7.56 (m, 2H), 7.40-6.90 (m, 15H), 6.79-6.51 (m, 3H), 6.42-6.20 (m, 1H), 5.06-4.85 (m, 1H), 4.75-4.10 (m, 4H), 4.10-3.80 (m, 2H), 3.61-2.70 (m, 12H), 2.70-1.86 (m, 22H), 1.75-1.15 (m, 14H), 1.07-0.91 (m, 9H), 0.29 (s, 4H).
LCMS m/z=776.5 [M/2+1]+.
Example 28
(2S,4R)-1-((S)-2-(8-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((6-(4-chlorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 28)
27a (0.30 g, 0.26 mmol) was added to 3 mL of tetrahydrofuran and 15 mL of methanol. Zinc powder (1.36 g, 20.9 mmol) and ammonium chloride (0.42 g, 7.85 mmol) were sequentially added and the mixture was stirred at 27° C. for 12 h. To the reaction solution was added 50 mL of ethyl acetate and 30 mL of water. The mixture was filtered over diatomaceous earth and the filtrate was extracted with ethyl acetate (15 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.23 g). The above-mentioned crude (0.10 g) was added to 5 mL of DCM. Intermediate 3 (0.060 g, 0.10 mmol), triethylamine (0.051 g, 0.50 mmol) and HATU (0.057 g, 0.15 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min). Lyophilization was performed to afford compound 28 (60 mg, yield: 34%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.38 (s, 1H), 8.10-7.92 (m, 1H), 7.80-7.66 (m, 2H), 7.49-7.12 (m, 13H), 7.07-6.98 (m, 2H), 6.83-6.58 (m, 3H), 6.38-6.24 (m, 1H), 5.15-4.97 (m, 1H), 4.83-4.20 (m, 4H), 4.16-3.90 (m, 2H), 3.68-2.75 (m, 13H), 2.75-1.97 (m, 21H), 1.97-1.17 (m, 16H), 1.14-0.90 (m, 9H), 0.36 (s, 4H).
LCMS m/z=783.5 [M/2+1]+.
Example 29
(2S,4R)-1-((S)-2-(7-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((6-(4-chloro-2-fluorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 29)
Sodium hydroxide (0.26 g, 6.5 mmol) was added to 5 mL of methanol and 3 mL of water. A solution of 21c (1.27 g, 2.63 mmol) in 10 mL of tetrahydrofuran was added and the resulting mixture was reacted at 80° C. for 6 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×3). The organic phase was washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.96 g). 13f (0.50 g, 0.68 mmol) was added to 6 mL of DCM. The above-mentioned crude (0.31 g), DMAP (0.17 g, 1.36 mmol) and EDCI (0.26, 1.36 mmol) were sequentially added and the mixture was warmed to 35° C. and reacted for 12 h. The reaction system was cooled to room temperature and 5 mL of water and 3 mL of DCM were added, followed by liquid separation. The aqueous phase was extracted with DCM (5 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane methanol (v/v)=10:1) to afford 29a (0.35 g, yield: 44%).
LCMS m/z=1177.3 [M+1]+.
29a (0.35 g, 0.30 mmol) was added to 3 mL of tetrahydrofuran and 15 mL of methanol. Zinc powder (1.57 g, 24.15 mmol) and ammonium chloride (0.48 g, 8.97 mmol) were sequentially added and the mixture was stirred at 27° C. for 12 h. To the reaction solution was added 50 mL of ethyl acetate and 30 mL of water. The mixture was filtered over diatomaceous earth and the filtrate was extracted with ethyl acetate (15 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.24 g). The above-mentioned crude (0.14 g) was added to 5 mL of DCM. Intermediate 1 (0.082 g, 0.14 mmol), triethylamine (0.071 g, 0.70 mmol) and HATU (0.080 g, 0.21 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min). Lyophilization was performed to afford compound 29 (85 mg, yield: 31%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.45-8.35 (m, 1H), 8.08-7.92 (m, 1H), 7.84-7.64 (m, 2H), 7.44-6.94 (m, 14H), 6.82-6.58 (m, 3H), 6.50-6.30 (m, 1H), 5.17-4.90 (m, 1H), 4.85-4.15 (m, 4H), 4.15-3.87 (m, 2H), 3.80-2.92 (m, 10H), 2.90-1.80 (m, 24H), 1.80-1.20 (m, 14H), 1.04 (s, 9H), 0.37 (s, 4H).
LCMS m/z=785.5 [M/2+1]+.
Example 30
(2S,4R)-1-((S)-2-(8-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((6-(4-chloro-2-fluorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 30)
29a (0.35 g, 0.30 mmol) was added to 3 mL of tetrahydrofuran and 15 mL of methanol. Zinc powder (1.57 g, 24.15 mmol) and ammonium chloride (0.48 g, 8.97 mmol) were sequentially added and the mixture was stirred at 27° C. for 12 h. To the reaction solution was added 50 mL of ethyl acetate and 30 mL of water. The mixture was filtered over diatomaceous earth and the filtrate was extracted with ethyl acetate (15 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.24 g). The above-mentioned crude (0.10 g) was added to 5 mL of DCM. Intermediate 3 (0.060 g, 0.10 mmol), triethylamine (0.051 g, 0.50 mmol) and HATU (0.057 g, 0.15 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 35% to 65% (elution time: 15 min). Lyophilization was performed to afford compound 30 (75 mg, yield: 38%).
1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.41-8.36 (m, 1H), 8.09-7.94 (m, 1H), 7.78-7.66 (m, 2H), 7.48-6.95 (m, 14H), 6.82-6.58 (m, 3H), 6.35-6.24 (m, 1H), 5.15-4.97 (m, 1H), 4.81-4.20 (m, 4H), 4.16-3.90 (m, 2H), 3.65-2.95 (m, 10H), 2.90-1.97 (m, 24H), 1.80-1.15 (m, 16H), 1.12-0.95 (m, 9H), 0.37 (s, 4H).
LCMS m/z=792.5 [M/2+1]+.
Example 31
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((6-(4-chloro-2-fluorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 31)
Sodium hydroxide (0.26 g, 6.5 mmol) was added to 5 mL of methanol and 3 mL of water. A solution of 21c (1.27 g, 2.63 mmol) in 10 mL of tetrahydrofuran was added and the resulting mixture was reacted at 80° C. for 6 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×3). The organic phase was washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.96 g). Intermediate 2 (0.62 g, 0.85 mmol) was added to 6 mL of DCM. The above-mentioned crude (0.43 g), DMAP (0.21 g, 1.72 mmol) and EDCI (0.33 g, 1.73 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. The reaction system was cooled to room temperature and 5 mL of water and 3 mL of DCM were added, followed by liquid separation. The aqueous phase was extracted with DCM (5 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane methanol (v/v)=10:1) to afford 31a (0.70 g, yield: 71%).
LCMS m/z=583.2 [M/2+1]+.
31a (0.70 g, 0.6 mmol) was added to 5 mL of tetrahydrofuran and 15 mL of methanol. Zinc powder (3.14 g, 48.31 mmol) and ammonium chloride (0.96 g, 17.95 mmol) were sequentially added and the mixture was stirred at 27° C. for 12 h. To the reaction solution was added 50 mL of ethyl acetate and 30 mL of water. The mixture was filtered over diatomaceous earth and the filtrate was extracted with ethyl acetate (15 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.41 g). The above-mentioned crude (0.14 g) was added to 5 mL of DCM. Intermediate 1 (0.082 g, 0.14 mmol), triethylamine (0.071 g, 0.70 mmol) and HATU (0.080 g, 0.21 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by extraction using DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3), gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min)). Lyophilization was performed to afford compound 31 (50 mg, yield: 16%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.40-8.32 (m, 1H), 8.09 (dd, 1H), 7.73-7.65 (m, 2H), 7.46-7.20 (m, 10H), 7.16-6.96 (m, 4H), 6.81-6.71 (m, 2H), 6.68-6.59 (m, 1H), 6.36-6.25 (m, 1H), 5.15-5.00 (m, 1H), 4.80-4.68 (m, 1H), 4.65-4.44 (m, 2H), 4.15-4.03 (m, 1H), 4.00-3.83 (m, 1H), 3.75-2.70 (m, 13H), 2.60-1.95 (m, 24H), 1.77-1.20 (m, 12H), 1.04 (s, 9H), 0.37 (s, 4H).
LCMS m/z=779.5 [M/2+1]+.
Example 32
(2S,4R)-1-((S)-2-(8-(4-((R)-3-((4-(N-(4-(4-((6-(4-chloro-2-fluorophenyl)spiro[2.5]oct-5-en-5-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-8-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 32)
31a (0.70 g, 0.6 mmol) was added to 5 mL of tetrahydrofuran and 15 mL of methanol. Zinc powder (3.14 g, 48.31 mmol) and ammonium chloride (0.96 g, 17.95 mmol) were sequentially added and the mixture was stirred at 27° C. for 12 h. To the reaction solution was added 50 mL of ethyl acetate and 30 mL of water. The mixture was filtered over diatomaceous earth and the filtrate was extracted with ethyl acetate (15 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.41 g). The above-mentioned crude (0.10 g) was added to 5 mL of DCM. Intermediate 3 (0.060 g, 0.10 mmol), triethylamine (0.051 g, 0.504 mmol) and HATU (0.057 g, 0.15 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by extraction using DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3), gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min)). Lyophilization was performed to afford compound 32 (50 mg, yield: 22%).
1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.37-8.32 (m, 1H), 8.09 (dd, 1H), 7.74-7.65 (m, 2H), 7.46-7.20 (m, 10H), 7.14-6.96 (m, 4H), 6.82-6.70 (m, 2H), 6.66-6.56 (m, 1H), 6.36-6.24 (m, 1H), 5.15-5.00 (m, 1H), 4.80-4.43 (m, 3H), 4.16-4.04 (m, 1H), 3.98-3.82 (m, 1H), 3.73-3.54 (m, 2H), 3.50-3.20 (m, 7H), 3.17-2.92 (m, 2H), 2.78 (s, 2H), 2.60-1.95 (m, 24H), 1.78-1.40 (m, 10H), 1.36-1.20 (m, 4H), 1.04 (s, 9H), 0.36 (s, 4H).
LCMS m/z=786.5 [M/2+1]+.
Example 33
(2S,4R)-1-((S)-2-(7-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 33)
6c (4 g, 8.8 mmol) was dissolved in 100 mL of methanol. Water (10 mL) and sodium hydroxide (1.5 g, 37.5 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 100 mL of water and then extracted with 200 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (500 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (2.0 g). 13f (0.60 g, 0.82 mmol) was added to 10 mL of DCM. The above-mentioned crude (0.38 g), DMAP (0.20 g, 1.64 mmol) and EDCI (0.31 g, 1.62 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. To the reaction system were added 10 mL of water and 10 mL of DCM. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford 33a (0.85 g, yield: 90%).
LCMS m/z=1147.1 [M+1]+.
33a (0.85 g, 0.74 mmol) was added to 8 mL of tetrahydrofuran and 40 mL of methanol. Zinc powder (3.87 g, 59.54 mmol) and ammonium chloride (1.19 g, 22.25 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.46 g). The above-mentioned crude (0.22 g) was added to 10 mL of DCM. Intermediate 1 (0.15 g, 0.26 mmol), triethylamine (0.070 g, 0.69 mmol) and HATU (0.13 g, 0.34 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 40% to 70% (elution time: 15 min). Lyophilization was performed to afford compound 33 (165 mg, yield: 41%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.47-8.35 (m, 1H), 8.06-7.91 (m, 1H), 7.82-7.64 (m, 2H), 7.44-7.08 (m, 13H), 7.03-6.92 (m, 2H), 6.83-6.57 (m, 3H), 6.50-6.31 (m, 1H), 5.15-4.90 (m, 1H), 4.84-3.86 (m, 6H), 3.67-2.94 (m, 10H), 2.94-2.71 (m, 3H), 2.71-1.98 (m, 22H), 1.98-1.42 (m, 14H), 1.40-1.22 (m, 3H), 1.13-0.98 (m, 9H).
LCMS m/z=514.0 [M/3+1]+.
Example 34
(2S,4R)-1-((S)-2-(8-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 34)
33a (0.85 g, 0.74 mmol) was added to 8 mL of tetrahydrofuran and 40 mL of methanol. Zinc powder (3.87 g, 59.54 mmol) and ammonium chloride (1.19 g, 22.25 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.46 g). The above-mentioned crude (0.22 g) was added to 5 mL of DCM. Intermediate 3 (0.15 g, 0.25 mmol), triethylamine (0.070 g, 0.69 mmol) and HATU (0.13 g, 0.34 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3), gradient elution method: gradient elution with acetonitrile from 45% to 75% (elution time: 15 min)). Lyophilization was performed to afford compound 34 (158 mg, yield: 41%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.41-8.35 (m, 1H), 8.08-7.94 (m, 1H), 7.78-7.66 (m, 2H), 7.50-7.16 (m, 13H), 7.01-6.93 (m, 2H), 6.84-6.55 (m, 3H), 6.39-6.25 (m, 1H), 5.14-4.97 (m, 1H), 4.81-3.85 (m, 6H), 3.66-2.73 (m, 13H), 2.73-1.87 (m, 22H), 1.87-1.38 (m, 15H), 1.37-1.20 (m, 4H), 1.13-0.95 (m, 9H).
LCMS m/z=777.5 [M/2+1]+.
Example 35
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((2-(4-chloro-2-fluorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 35)
6a (2.20 g, 10.84 mmol) and (4-chloro-2-fluorophenyl)boronic acid (2.83 g, 16.23 mmol) were dissolved in 15 mL of 1,4-dioxane and 1.5 mL of water. Under nitrogen protection, potassium acetate (3.19 g, 32.51 mmol) and Pd(dppf)Cl2 (0.27 g, 0.36 mmol) were sequentially added and the mixture was reacted at 90° C. for 1 h. The reaction solution was cooled to room temperature. To the reaction solution was slowly added 20 mL of ethyl acetate and 20 mL of water, followed by liquid separation. The aqueous phase was extracted with ethyl acetate (25 mL×2). The organic phases were combined, washed with 25 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=1:0-20:1) to afford 35a (2.5 g, yield: 92%).
LCMS m/z=253.0 [M+1]+.
35a (0.60 g, 2.38 mmol), ethyl 4-(piperazin-1-yl)benzoate (0.83 g, 3.54 mmol) and acetic acid (0.94 g, 15.67 mmol) were added to 50 mL of tetrahydrofuran. Sodium triacetoxyborohydride (1.51 g, 7.13 mmol) was added and the mixture was reacted at room temperature for 16 h. To the reaction solution was added 50 mL of saturated aqueous sodium bicarbonate solution, followed by extraction using ethyl acetate (50 mL×2). The organic phases were combined, washed with 25 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=1:0-5:1) to afford 35b (0.80 g, yield: 71%).
LCMS m/z=471.2 [M+1]+.
35b (0.80 g, 1.70 mmol) was dissolved in mixed solvents of 8 mL of tetrahydrofuran, 4 mL of ethanol and 2 mL of water. Sodium hydroxide (0.27 g, 6.75 mmol) was added and the mixture was stirred at 65° C. for 16 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. To the residue was added 20 mL of water. Then the mixture was extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×2). The organic phases were combined, washed with 25 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.68 g). Intermediate 2 (0.60 g, 0.82 mmol) was added to 12 mL of DCM. The above-mentioned crude (0.40 g), DMAP (0.20 g, 1.64 mmol) and EDCI (0.31 g, 1.62 mmol) were sequentially added and the mixture was warmed to 35° C. and reacted for 12 h. To the reaction system were added 5 mL of water and 3 mL of DCM. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford 35c (0.62 g, yield: 66%).
LCMS m/z=1153.1 [M+1]+.
35c (0.62 g, 0.54 mmol) was added to 5 mL of tetrahydrofuran and 30 mL of methanol. Zinc powder (2.83 g, 43.54 mmol) and ammonium chloride (0.87 g, 16.26 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.34 g). The above-mentioned crude (0.17 g) was added to 15 mL of DCM. Intermediate 1 (0.11 g, 0.19 mmol), triethylamine (0.17 g, 1.70 mmol) and HATU (0.097 g, 0.26 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 5 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 40% to 70% (elution time: 15 min). Lyophilization was performed to afford compound 35 (24.02 mg, yield: 8%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.42-8.30 (m, 1H), 8.14-8.05 (m, 1H), 7.74-7.62 (m, 2H), 7.45-7.21 (m, 10H), 7.16-6.88 (m, 4H), 6.82-6.70 (m, 2H), 6.68-6.57 (m, 1H), 6.36-6.21 (m, 1H), 5.15-5.00 (m, 1H), 4.80-4.67 (m, 1H), 4.65-4.55 (m, 1H), 4.55-4.44 (m, 1H), 4.17-4.05 (m, 1H), 4.00-3.83 (m, 1H), 3.75-3.52 (m, 2H), 3.50-3.20 (m, 7H), 3.18-2.95 (m, 2H), 2.95-2.70 (m, 2H), 2.60-1.95 (m, 24H), 1.90-1.40 (m, 14H), 1.40-1.20 (m, 2H), 1.04 (s, 9H).
LCMS m/z=516.1 [M/3+1]+.
Example 36
(2S,4R)-1-((S)-2-(8-(4-((R)-3-((4-(N-(4-(4-((2-(4-chloro-2-fluorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 36)
35c (0.62 g, 0.54 mmol) was added to 5 mL of tetrahydrofuran and 30 mL of methanol. Zinc powder (2.83 g, 43.54 mmol) and ammonium chloride (0.87 g, 16.26 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.34 g). The above-mentioned crude (0.17 g) was added to 5 mL of DCM. Intermediate 3 (0.11 g, 0.18 mmol), triethylamine (0.17 g, 1.70 mmol) and HATU (0.097 g, 0.26 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by extraction using DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 42% to 72% (elution time: 15 min). Lyophilization was performed to afford compound 36 (25 mg, yield: 9%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.40-8.32 (m, 1H), 8.13-8.05 (m, 1H), 7.75-7.61 (m, 2H), 7.45-7.21 (m, 10H), 7.14-7.02 (m, 3H), 6.98-6.90 (m, 1H), 6.84-6.58 (m, 3H), 6.32-6.15 (m, 1H), 5.15-5.00 (m, 1H), 4.78-4.68 (m, 1H), 4.64-4.46 (m, 2H), 4.17-4.06 (m, 1H), 4.02-3.84 (m, 1H), 3.80-3.20 (m, 9H), 3.18-2.72 (m, 4H), 2.70-2.00 (m, 24H), 1.95-1.20 (m, 18H), 1.04 (s, 9H).
LCMS m/z=520.8 [M/3+1]+.
Example 37
(2S,4R)-1-((S)-2-(7-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((2-(4-chloro-2-fluorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 37)
35b (0.80 g, 1.70 mmol) was dissolved in mixed solvents of 8 mL of tetrahydrofuran, 4 mL of ethanol and 2 mL of water. Sodium hydroxide (0.27 g, 6.75 mmol) was added and the mixture was stirred at 65° C. for 16 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. To the residue was added 20 mL of water. Then the mixture was extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×2). The organic phases were combined, washed with 25 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.68 g). 13f (0.60 g, 0.82 mmol) was added to 12 mL of DCM. The above-mentioned crude (0.39 g), DMAP (0.20 g, 1.64 mmol) and EDCI (0.31 g, 1.62 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. To the reaction system were added 5 mL of water and 3 mL of DCM. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford 37a (0.75 g, yield: 79%).
LCMS m/z=1165.2 [M+1]+.
37a (0.75 g, 0.64 mmol) was added to 8 mL of tetrahydrofuran and 40 mL of methanol. Zinc powder (3.35 g, 51.54 mmol) and ammonium chloride (1.03 g, 19.26 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude (0.46 g). The above-mentioned crude (0.23 g) was added to 10 mL of DCM. Intermediate 1 (0.15 g, 0.26 mmol), triethylamine (0.23 g, 2.30 mmol) and HATU (0.13 g, 0.34 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 53% to 83% (elution time: 10 min). Lyophilization was performed to afford compound 37 (68 mg, yield: 17%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.45-8.35 (m, 1H), 8.08-7.92 (m, 1H), 7.80-7.65 (m, 2H), 7.45-7.10 (m, 11H), 7.09-7.01 (m, 2H), 6.98-6.87 (m, 1H), 6.82-6.58 (m, 3H), 6.51-6.32 (m, 1H), 5.14-4.92 (m, 1H), 4.84-3.85 (m, 6H), 3.68-2.92 (m, 10H), 2.87-1.95 (m, 25H), 1.92-1.42 (m, 14H), 1.40-1.21 (m, 3H), 1.09-0.99 (m, 9H).
LCMS m/z=520.2 [M/3+1]+.
Example 38
(2S,4R)-1-((S)-2-(8-((1S,4S)-5-((R)-3-((4-(N-(4-(4-((2-(4-chloro-2-fluorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 38)
37a (0.75 g, 0.64 mmol) was added to 8 mL of tetrahydrofuran and 40 mL of methanol. Zinc powder (3.35 g, 51.54 mmol) and solid ammonium chloride (1.03 g, 19.26 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude (0.46 g). The above-mentioned crude (0.23 g) was added to 10 mL of DCM. Intermediate 3 (0.15 g, 0.25 mmol), triethylamine (0.070 g, 0.69 mmol) and HATU (0.13 g, 0.34 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3), gradient elution method: gradient elution with acetonitrile from 53% to 83% (elution time: 10 min)). Lyophilization was performed to afford compound 38 (66 mg, yield: 17%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.42-8.35 (m, 1H), 8.07-7.94 (m, 1H), 7.78-7.66 (m, 2H), 7.50-7.14 (m, 11H), 7.11-7.00 (m, 2H), 6.98-6.88 (m, 1H), 6.83-6.57 (m, 3H), 6.35-6.23 (m, 1H), 5.15-4.97 (m, 1H), 4.83-3.88 (m, 6H), 3.65-2.95 (m, 10H), 2.95-2.03 (m, 24H), 2.01-1.38 (m, 16H), 1.35-1.20 (m, 4H), 1.10-0.98 (m, 9H).
LCMS m/z=524.8 [M/3+1]+.
Example 39
cis-(2S,4R)-1-((S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((2-(4-chloro-2-fluorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 39)
35b (0.80 g, 1.70 mmol) was dissolved in mixed solvents of 8 mL of tetrahydrofuran, 4 mL of ethanol and 2 mL of water. Sodium hydroxide (0.27 g, 6.75 mmol) was added and the mixture was stirred at 65° C. for 16 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. To the residue was added 20 mL of water. Then the mixture was extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×2). The organic phases were combined, washed with 25 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.68 g). 8f (1.00 g, 1.33 mmol) was added to 12 mL of DCM. The above-mentioned crude (0.65 g), DMAP (0.32 g, 2.62 mmol) and EDCI (0.51 g, 2.67 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. To the reaction system were added 10 mL of water and 5 mL of DCM. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=20:1) to afford 39a (0.90 g, yield: 57%).
LCMS m/z=1179.1 [M+1]+.
39a (0.90 g, 0.76 mmol) was added to 9 mL of tetrahydrofuran and 45 mL of methanol. Zinc powder (3.98 g, 61.23 mmol) and ammonium chloride (1.22 g, 22.81 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.70 g). The above-mentioned crude (0.35 g) was added to 15 mL of DCM. Intermediate 1 (0.23 g, 0.40 mmol), triethylamine (0.11 g, 1.09 mmol) and HATU (0.20 g, 0.526 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 70% (elution time: 20 min). Lyophilization was performed to afford compound 39 (280 mg, yield: 45%).
1H NMR (400 MHz, CDCl3) δ 8.69-8.64 (m, 1H), 8.53-8.46 (m, 1H), 7.96-7.70 (m, 3H), 7.45-7.16 (m, 9H), 7.12-6.87 (m, 5H), 6.85-6.65 (m, 3H), 6.52-6.31 (m, 1H), 5.15-4.88 (m, 1H), 4.86-4.65 (m, 2H), 4.57-4.45 (m, 1H), 4.25-3.86 (m, 2H), 3.78-3.41 (m, 3H), 3.40-2.94 (m, 8H), 2.94-1.98 (m, 26H), 1.92-1.40 (m, 14H), 1.39-1.22 (m, 4H), 1.13-0.98 (m, 9H).
LCMS m/z=786.6 [M/2+1]+.
Example 40
cis-(2S,4R)-1-((S)-2-(8-(5-((R)-3-((4-(N-(4-(4-((2-(4-chloro-2-fluorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 40)
39a (0.90 g, 0.76 mmol) was added to 9 mL of tetrahydrofuran and 45 mL of methanol. Zinc powder (3.98 g, 61.23 mmol) and ammonium chloride (1.22 g, 22.81 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.70 g). The above-mentioned crude (0.35 g) was added to 15 mL of DCM. Intermediate 3 (0.23 g, 0.38 mmol), triethylamine (0.11 g, 1.09 mmol) and HATU (0.20 g, 0.526 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3), gradient elution method: gradient elution with acetonitrile from 50% to 70% (elution time: 20 min)). Lyophilization was performed to afford compound 40 (290 mg, yield: 48%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.52-8.38 (m, 1H), 8.04-7.87 (m, 1H), 7.84-7.72 (m, 2H), 7.56-7.14 (m, 10H), 7.09-6.89 (m, 4H), 6.84-6.73 (m, 2H), 6.72-6.62 (m, 1H), 6.50-6.24 (m, 1H), 5.15-4.93 (m, 1H), 4.84-4.62 (m, 2H), 4.57-4.45 (m, 1H), 4.18-3.85 (m, 2H), 3.69-3.42 (m, 3H), 3.39-2.01 (m, 34H), 1.90-1.16 (m, 20H), 1.13-0.96 (m, 9H).
LCMS m/z=793.9 [M/2+1]+.
Example 41
cis-(2S,4R)-1-((S)-2-(7-(5-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 41)
6c (0.40 g, 0.88 mmol) was dissolved in 10 mL of methanol. Water (1 mL) and sodium hydroxide (0.15 g, 3.75 mmol) were added and the mixture was reacted at 80° C. for 10 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved with 10 mL of water and then extracted with 20 mL of methyl tert-butyl ether to remove the impurities. The aqueous phase was separated, adjusted to pH 6 with 1 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.20 g). 8f (1.00 g, 1.33 mmol) was added to 12 mL of DCM. The above-mentioned crude (0.62 g), DMAP (0.32 g, 2.62 mmol) and EDCI (0.51 g, 2.67 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. To the reaction system were added 10 mL of water and 5 mL of dichloromethane. Liquid separation was performed. The aqueous phase was extracted with 5 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=20:1) to afford 41a (1.00 g, yield: 65%).
LCMS m/z=1161.2 [M+1]+.
41a (1.00 g, 0.86 mmol) was added to 10 mL of tetrahydrofuran and 50 mL of methanol. Zinc powder (4.50 g, 69.23 mmol) and ammonium chloride (1.38 g, 25.80 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.80 g). The above-mentioned crude (0.40 g) was added to 15 mL of DCM. Intermediate 1 (0.26 g, 0.45 mmol), triethylamine (0.12 g, 1.19 mmol) and HATU (0.23 g, 0.605 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 45% to 75% (elution time: 20 min). Lyophilization was performed to afford compound 41 (150 mg, yield: 21%).
1H NMR (400 MHz, CDCl3) δ 8.70-8.62 (m, 1H), 8.52-8.44 (m, 1H), 7.96-7.70 (m, 3H), 7.44-6.92 (m, 15H), 6.83-6.65 (m, 3H), 6.55-6.35 (m, 1H), 5.15-4.90 (m, 1H), 4.85-4.65 (m, 2H), 4.55-4.45 (m, 1H), 4.22-3.86 (m, 2H), 3.80-3.40 (m, 3H), 3.37-1.96 (m, 34H), 1.90-1.20 (m, 18H), 1.10-0.98 (m, 9H). LCMS m/z=777.3 [M/2+1]+.
Example 42
cis-(2S,4R)-1-((S)-2-(8-(5-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 42)
41a (1.00 g, 0.86 mmol) was added to 10 mL of tetrahydrofuran and 50 mL of methanol. Zinc powder (4.50 g, 69.23 mmol) and ammonium chloride (1.38 g, 25.80 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction solution was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.80 g). The above-mentioned crude (0.40 g) was added to 15 mL of DCM. Intermediate 3 (0.27 g, 0.45 mmol), triethylamine (0.12 g, 1.19 mmol) and HATU (0.23 g, 0.605 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18, mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3), gradient elution method: gradient elution with acetonitrile from 45% to 75% (elution time: 20 min)). Lyophilization was performed to afford compound 42 (270 mg, yield: 38%).
1H NMR (400 MHz, CDCl3) δ 8.69-8.63 (m, 1H), 8.49-8.39 (m, 1H), 8.04-7.88 (m, 1H), 7.84-7.70 (m, 2H), 7.57-7.14 (m, 12H), 7.06-6.94 (m, 3H), 6.83-6.59 (m, 3H), 6.54-6.32 (m, 1H), 5.14-4.92 (m, 1H), 4.80-4.61 (m, 2H), 4.55-4.45 (m, 1H), 4.16-3.84 (m, 2H), 3.70-3.41 (m, 3H), 3.33-1.99 (m, 34H), 1.91-1.15 (m, 20H), 1.14-0.94 (m, 9H). LCMS m/z=784.5 [M/2+1]+.
Example 43
(2S,4R)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclooct-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 43)
DMF (17.12 g, 234.22 mmol) was added to 80 mL of dichloromethane. Under nitrogen protection, the temperature was controlled to be 0° C.-5° C. PBr3 (34.54 g, 127.6 mmol) was slowly added dropwise to the above-mentioned reaction solution. The ice-bath was removed and the mixture was slowly warmed to room temperature and stirred for 0.5 h. The reaction system was cooled to 0° C.-5° C. again. A solution of 43a (5.00 g, 39.62 mmol) in dichloromethane (40 mL) was added while the temperature was controlled in the range and then the mixture was warmed to room temperature and reacted for 16 h. The reaction solution was slowly added to 200 mL of ice water, followed by liquid separation. The aqueous phase was extracted with dichloromethane (100 mL×2). The organic phases were combined, washed with 50 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was separated and purified by chromatographic column on silica gel (pure petroleum ether) to afford 43b (2.20 g, yield: 26%).
43b (2.20 g, 10.13 mmol) and 4-chlorophenylboronic acid (2.38 g, 15.22 mmol) were added to 20 mL of 1,4-dioxane and 2 mL of water. Under nitrogen protection, potassium acetate (2.98 g, 30.36 mmol) and Pd(dppf)Cl2 (0.25 g, 0.34 mmol) were sequentially added and the mixture was reacted at 90° C. for 1 h. The reaction system was cooled to room temperature and 20 mL of ethyl acetate and 20 mL of water were added, followed by liquid separation. The aqueous phase was extracted with ethyl acetate (25 mL×2). The organic phases were combined, washed with 30 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=1:0-20:1) to afford 43c (1.60 g, yield: 64%).
LCMS m/z=249.2 [M+1]+.
43c (1.60 g, 6.45 mmol), ethyl (4-piperazin-1-yl)benzoate (2.26 g, 9.64 mmol) and acetic acid (2.56 g, 42.67 mmol) were added to 20 mL of tetrahydrofuran. Sodium triacetoxyborohydride (4.09 g, 19.30 mmol) was added and the mixture was reacted at room temperature for 16 h. To the reaction solution was added 100 mL of saturated sodium bicarbonate solution, followed by extraction using ethyl acetate (60 mL×2). The organic phases were combined, washed with 50 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v)=1:0-5:1) to afford 43d (2.20 g, yield: 73%).
LCMS m/z=467.6 [M+1]+.
43d (2.20 g, 4.71 mmol) was dissolved in mixed solvents of 32 mL of tetrahydrofuran, 16 mL of ethanol and 8 mL of water. Sodium hydroxide (0.75 g, 18.75 mmol) was added and the mixture was reacted at 65° C. for 16 h. The reaction system was cooled to room temperature and concentrated under reduced pressure and 20 mL of water and 20 mL of methyl tert-butyl ether were added. The aqueous phase was separated, adjusted to pH 5 with 1 mol/L hydrochloric acid and extracted with ethyl acetate (15 mL×2). The organic phases were combined, washed with 15 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a crude (0.80 g). Intermediate 2 (0.50 g, 0.68 mmol) was dissolved in 10 mL of dichloromethane. The above-mentioned crude (0.33 g), DMAP (0.17 g, 1.39 mmol) and EDCI (0.26 g, 1.36 mmol) were sequentially added and the mixture was reacted at 35° C. for 12 h. To the reaction system was added 10 mL of saturated aqueous potassium dihydrogen phosphate solution, followed by liquid separation. The aqueous phase was extracted with 10 mL of dichloromethane and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-20:1) to afford 43e (0.75 g, yield: 96%).
LCMS m/z=1149.3 [M+1]+.
43e (0.75 g, 0.65 mmol) was added to 8 mL of tetrahydrofuran and 40 mL of methanol. Zinc powder (3.42 g, 52.62 mmol) and ammonium chloride (1.04 g, 19.44 mmol) were sequentially added and the mixture was reacted at 27° C. for 12 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (0.29 g). The above-mentioned crude (0.24 g) was added to 15 mL of DCM. Intermediate 1 (0.16 g, 0.28 mmol), triethylamine (0.076 g, 0.75 mmol) and HATU (0.14 g, 0.37 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of water, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2) and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then passed through Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Mobile phase system: acetonitrile/water (containing 10 mmol/L NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 50% to 80% (elution time: 15 min). Lyophilization was performed to afford compound 43 (0.20 g, yield: 46%).
1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.40-8.30 (m, 1H), 8.13-8.04 (m, 1H), 7.73-7.63 (m, 2H), 7.45-7.21 (m, 12H), 7.12-7.02 (m, 1H), 7.00-6.90 (m, 2H), 6.78-6.66 (m, 2H), 6.66-6.57 (m, 1H), 6.40-6.29 (m, 1H), 5.15-5.00 (m, 1H), 4.80-4.42 (m, 3H), 4.16-4.02 (m, 1H), 3.97-3.82 (m, 1H), 3.75-3.52 (m, 2H), 3.50-3.20 (m, 7H), 3.17-2.80 (m, 4H), 2.60-2.00 (m, 24H), 1.80-1.20 (m, 18H), 1.04 (s, 9H).
LCMS m/z=514.8 [M/3+1]+.
Example 44
4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidin-3-yl)methyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (Compound 44)
6d (3.00 g, 2.64 mmol) was added to 150 mL of methanol and 30 mL of tetrahydrofuran. Ammonium chloride (4.24 g, 79.27 mmol) and zinc powder (13.81 g, 212.46 mmol) were sequentially added and the mixture was reacted at 27° C. for 19 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (2.0 g). The above-mentioned crude (0.20 g) was added to 10 mL of dichloromethane. 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidine-3-carbaldehyde (see CN114292270 for the synthetic method) (0.11 g, 0.32 mmol), acetic acid (0.05 g, 0.83 mmol) and sodium triacetoxyborohydride (0.13 g, 0.61 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of saturated aqueous sodium bicarbonate solution, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was preliminarily separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1). The resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). The preparative solution was adjusted to pH 7 with saturated aqueous sodium bicarbonate solution and extracted with DCM (20 mL×2). The organic phase was washed with 20 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 44 (68 mg, yield: 17%).
1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 8.18-8.01 (m, 1H), 7.84-7.45 (m, 3H), 7.40-7.15 (m, 7H), 7.03-6.90 (m, 3H), 6.80-6.55 (m, 4H), 6.50-6.35 (m, 1H), 4.95-4.80 (m, 1H), 4.20-4.00 (m, 2H), 3.98-3.79 (m, 1H), 3.76-3.55 (m, 2H), 3.30-2.90 (m, 6H), 2.85-2.57 (m, 6H), 2.55-2.25 (m, 18H), 2.15-1.95 (m, 3H), 1.90-1.75 (m, 3H), 1.67-1.45 (m, 5H).
LCMS m/z=1284.3 [M+1]+.
Example 45
4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (Compound 45)
6d (3.00 g, 2.64 mmol) was added to 150 mL of methanol and 30 mL of tetrahydrofuran. Ammonium chloride (4.24 g, 79.27 mmol) and zinc powder (13.81 g, 212.46 mmol) were sequentially added and the mixture was reacted at 27° C. for 19 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (2.0 g). The above-mentioned crude (0.20 g) was added to 10 mL of DCM. 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carbaldehyde (see CN 114292270 for the synthetic method) (0.12 g, 0.32 mmol), acetic acid (0.050 g, 0.83 mmol) and sodium triacetoxyborohydride (0.13 g, 0.61 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of saturated aqueous sodium bicarbonate solution, followed by liquid separation. The aqueous phase was extracted with DCM (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the crude was preliminarily separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1). The resulting crude was passed through Pre-HPLC (instrument and preparative column: using Glison GX-281 preparative liquid phase chromatographic instrument, preparative column model: Sunfire C18, 5 μm, inner diameter×length=30 mm×150 mm). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.1% TFA). Gradient elution method: gradient elution with acetonitrile from 5% to 60% (elution time: 15 min). The preparative solution was adjusted to pH 7 with saturated aqueous sodium bicarbonate solution and extracted with DCM (20 mL×2). The organic phase was washed with 20 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford compound 45 (0.13 g, yield: 31%).
1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 8.16-7.98 (m, 1H), 7.82-7.55 (m, 3H), 7.40-7.15 (m, 8H), 7.05-6.83 (m, 4H), 6.80-6.50 (m, 3H), 4.98-4.84 (m, 1H), 4.00-3.75 (m, 3H), 3.30-2.60 (m, 13H), 2.60-2.15 (m, 20H), 2.15-1.95 (m, 3H), 1.92-1.75 (m, 4H), 1.68-1.46 (m, 7H).
LCMS m/z=657.2 [M/2+1]+.
Example 46: Preparation of Compound 46
4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-((1-(4-(2,6-dioxopiperidin-3-yl)phenyl)azetidin-3-yl)methyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (Compound 46)
46a (15.00 g, 53.02 mmol) was added to 150 mL of DMSO and under nitrogen protection, (azetidin-3-yl)methanol hydrochloride (7.86 g, 63.60 mmol), L-proline (2.44 g, 21.19 mmol), potassium carbonate (21.98 g, 159.04 mmol) and CuI (2.02 g, 10.61 mmol) were added and the system was subjected to nitrogen replacement three times and reacted at 100° C. for 16 h. The reaction system was cooled to room temperature and 100 mL of water was added. The mixture was extracted with ethyl acetate (60 mL×3). The organic phase was washed with 50 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (petroleum ether:ethyl acetate (v/v)=20:1-5:1) to afford 46b (5.60 g, yield: 44%).
LCMS m/z=242.1 [M+1]+.
46b (5.10 g, 21.15 mmol) was added to 60 mL of 1,4-dioxane and 15 mL of water. Under nitrogen protection, 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (26.37 g, 63.19 mmol) (see WO 2021262812 for the synthetic method), cesium carbonate (20.59 g, 63.19 mmol) and Pd(dppf)Cl2 (0.77 g, 1.05 mmol) were added and the system was subjected to nitrogen replacement three times and reacted at 100° C. for 16 h. The reaction system was cooled to room temperature, filtered over diatomaceous earth, and washed with 50 mL of ethyl acetate. To the filtrate was added 50 mL of water and the mixture was extracted with ethyl acetate (50 mL×3). The organic phase was washed with 50 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (petroleum ether:ethyl acetate (v/v)=20:1-5:1) to afford 46c (9.10 g, yield: 95%).
LCMS m/z=453.3 [M+1]+.
46c (9.10 g, 20.12 mmol) was added to 100 mL of methanol. 10% Pd/C (5.00 g) was added and the system was subjected to nitrogen replacement three times and reacted at room temperature under the atmosphere of hydrogen balloon for 16 h. The reaction system was filtered over diatomaceous earth and then washed with mixed solvents of dichloromethane and methanol (v/v)=10:1. The filtrate was concentrated under reduced pressure and to the resulting crude were added 40 mL of ethyl acetate and 40 mL of petroleum ether. The mixture was stirred at room temperature for 0.5 h and then filtered. The filter cake was dried under reduced pressure to afford crude 46d (3.10 g).
LCMS m/z=275.2 [M+1]+.
The above-mentioned crude 46d (0.20 g) was added to 5 mL of DMSO. 2-iodoxybenzoic acid (0.33 g, 1.18 mmol) was added and the mixture was reacted at 50° C. for 1 h. The reaction system was cooled to room temperature and 10 mL of ethyl acetate and 10 mL of water were added, followed by liquid separation. The aqueous phase was extracted with ethyl acetate (5 mL×2). The organic phases were combined, washed with 10 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude 46e (0.15 g).
6d (3.00 g, 2.64 mmol) was added to 150 mL of methanol and 30 mL of tetrahydrofuran. Ammonium chloride (4.24 g, 79.27 mmol) and zinc powder (13.81 g, 212.46 mmol) were sequentially added and the mixture was reacted at 27° C. for 19 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (2.0 g). The above-mentioned crude (0.15 g) was added to 10 mL of dichloromethane. Crude 46e (0.15 g), acetic acid (0.038 g, 0.63 mmol) and sodium triacetoxyborohydride (0.10 g, 0.472 mmol) were sequentially added and the mixture was reacted at room temperature for 2 h. To the reaction solution was added 10 mL of saturated aqueous sodium bicarbonate solution, followed by liquid separation. The aqueous phase was extracted with dichloromethane (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then separated and purified by preparative liquid phase chromatography to afford compound 46 (0.13 g, yield over three steps from compound 46c: 8%).
1H NMR (400 MHz, CDCl3) δ 8.40-8.29 (m, 1H), 8.10 (dd, 1H), 7.93 (s, 1H), 7.69-7.60 (m, 2H), 7.41-7.20 (m, 7H), 7.10-6.92 (m, 5H), 6.84-6.72 (m, 2H), 6.63 (d, 1H), 6.46-6.37 (m, 2H), 4.05-3.83 (m, 3H), 3.74-3.65 (m, 1H), 3.57-3.47 (m, 2H), 3.32-3.22 (m, 4H), 3.14-2.89 (m, 3H), 2.84 (s, 2H), 2.77-2.03 (m, 25H), 1.88-1.47 (m, 7H).
LCMS m/z=608.3 [M/2+1]+.
Example 47: Preparation of Compound 47
4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (Compound 47)
6d (3.00 g, 2.64 mmol) was added to 150 mL of methanol and 30 mL of tetrahydrofuran. Ammonium chloride (4.24 g, 79.27 mmol) and zinc powder (13.81 g, 212.46 mmol) were sequentially added and the mixture was reacted at 27° C. for 19 h. The reaction system was filtered over diatomaceous earth. The filtrate was concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=10:1) to afford a crude (2.0 g). The above-mentioned crude (0.10 g) was added to 5 mL of dimethyl sulfoxide. 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (0.083 g, 0.30 mmol) and N,N-diisopropylethylamine (0.065 g, 0.50 mmol) were sequentially added and the mixture was reacted at 100° C. for 16 h. The reaction system was cooled to room temperature and 10 mL of water and 10 mL of ethyl acetate were added, followed by liquid separation. The aqueous phase was extracted with ethyl acetate (6 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure and the resulting crude was separated and purified by chromatographic column on silica gel (dichloromethane:methanol (v/v)=50:1-10:1) to afford a crude, which was then separated and purified by preparative liquid phase chromatography to afford compound 47 (20 mg, yield: 5%).
1H NMR (400 MHz, CDCl3) δ 8.40-8.35 (m, 1H), 8.13 (dd, 1H), 8.03-7.92 (m, 1H), 7.69 (d, 1H), 7.65-7.57 (m, 2H), 7.44-7.20 (m, 8H), 7.17-7.09 (m, 1H), 7.06-6.94 (m, 3H), 6.83-6.74 (m, 2H), 6.62 (d, 1H), 4.99-4.88 (m, 1H), 4.00-3.87 (m, 1H), 3.49-3.20 (m, 8H), 3.18-2.97 (m, 2H), 2.96-2.65 (m, 5H), 2.65-2.07 (m, 16H), 1.89-1.40 (m, 7H).
LCMS m/z=608.8 [M/2+1]+.
Example 48: Preparation of Compound 48
(3R,5S)-1-((S)-2-(7-(4-((R)-3-((4-(N-(4-(4-((2-(4-chlorophenyl)cyclohept-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl) pyrrolidin-3-yl acetate (Compound 48)
Free-form compound 6 (50 mg, 0.033 mmol) was dissolved in 3 mL of dichloromethane. 4-dimethylaminopyridine (8.1 mg, 0.066 mmol) and acetic anhydride (33 mg, 0.32 mmol) were added and the mixture was reacted at room temperature for 2 h. The reaction solution was concentrated under reduced pressure and the crude product was subjected to Pre-HPLC (instrument and preparative column: using SHIMADZU LC-20AP preparative liquid phase chromatographic instrument, preparative column model: Phenomenex C18). Preparation method: the crude was dissolved with methanol and dimethyl sulfoxide, and filtered with a 0.45 m filter membrane, to prepare into a sample liquid. Mobile phase system: acetonitrile/water (containing 0.05% NH4HCO3). Gradient elution method: gradient elution with acetonitrile from 60% to 90% (elution time: 15 min). Lyophilization was performed to afford compound 48 (30 mg, yield: 58%).
1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.40-8.32 (m, 1H), 8.16-8.06 (s, 1H), 7.69-7.58 (m, 2H), 7.46-7.20 (m, 12H), 7.17-7.08 (m, 1H), 7.02-6.93 (m, 2H), 6.84-6.71 (m, 2H), 6.64-6.54 (m, 1H), 6.18-6.08 (m, 1H), 5.43-5.27 (m, 1H), 5.15-4.97 (m, 1H), 4.77-4.67 (m, 1H), 4.62-4.54 (m, 1H), 4.13-4.00 (m, 1H), 3.97-3.56 (m, 3H), 3.54-3.17 (m, 7H), 3.16-2.95 (m, 2H), 2.94-2.65 (m, 3H), 2.55-1.97 (m, 26H), 1.90-1.30 (m, 16H), 1.02 (s, 9H).
LCMS m/z=785.5 [M/2+1]+.
Experimental Examples
MOLT-4 cells are acute lymphoblastic leukemia cell lines in humans. The cells were purchased from ATCC. Culture conditions: RPMI-1640+10% FBS+1% penicillin-streptomycin solution. The cells were cultured in a 37° C., 5% CO2 incubator. The cells were plated in a 12-well plate, with 2×105 cells/well. After plating, compounds at different concentrations were added and cultured in a 37° C., 5% CO2 incubator for 16 h. After the completion of culture, the cells were harvested and RIPA lysis buffer (beyotime, Cat. P0013B) was added. The cells were lysed on ice for 15 minutes and centrifuged at 12000 rpm at 4° C. for 10 minutes. The supernatant protein samples were collected and the protein was quantified by using the BCA kit (Beyotime, Cat. P0009). Then the protein was diluted to 0.8 mg/mL. The expressions of BCL-xL (CST, Cat. 2764S), BCL-2 (CST, Cat. 15071S) and the internal reference β-actin (CST, Cat. 3700S) were detected using a fully automated western blot quantitative analyzer (Proteinsimple). The expression level of BCL-xL or BCL-2 relative to the internal reference was calculated by using Compass software, and the DC50 value was calculated by using GraphPad Prism 8.0 software according to formula (1), wherein the Proteinadministration denoted the relative expression level of BCL-xL or BCL-2 in administration groups at different doses, and the Proteinvehicle denoted the relative expression level of BCL-xL or BCL-2 in the vehicle control group.
Protein %=Proteinadministration/Proteinvehicle×100% formula (1)
The results of DC50 values for the degradation of BCL-xL proteins in MOLT-4 cells are shown in Table 1.
Conclusion: the compounds of the present invention had a given degradation effect on the BCL-xL proteins in MOLT-4 cells.
MOLT-4 cells are acute lymphoblastic leukemia cell lines in humans. The cells were purchased from ATCC. Culture conditions: RPMI-1640+10% FBS+1% penicillin-streptomycin solution. The cells were cultured in a 37° C., 5% CO2 incubator. The cells were plated in a 96-well plate, with 5×103 cells/well. After plating, compounds at different concentrations were added and cultured in a 37° C., 5% CO2 incubator for 72 h. After the completion of culture, a reagent (Promega, G7573) for detecting the cell viability was added. The mixture was uniformly mixed for 2 minutes and then incubated at room temperature for 10 minutes. The luminescence signal was detected with a multimode plate reader (BMG, PHERAstar FSX). The luminescence readings were processed using GraphPad Prism 8.0 software and the IC50 values of the compounds for the inhibition of cell proliferation and the maximum inhibition rate were calculated according to formula (2) and formula (3), respectively, wherein Tadministration denotes the cell signal readings obtained after incubation with the compounds for 72 h, and Tvehicle denotes the cell signal readings obtained after incubation with the vehicle control for 72 h.
Calculation of Max inhibition %: after the processing according to formula (2), the inhibition rate of the compounds at the highest concentration was calculated.
The results of IC50 values for the inhibition of MOLT-4 cell proliferation are shown in Table 2.
MOLT-4 cells are acute lymphoblastic leukemia cell lines in humans. The cells were purchased from ATCC and cultured in RPMI-1640+10% FBS+1% o penicillin-streptomycin solution in a 37° C., 5% CO2 incubator. When the cells were in the exponential growth phase, the cells were harvested and counted. An equal volume of matrigel was added. Female SCID Beige mice (4-6 weeks old, 14-16 g, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were inoculated subcutaneously in the right flank with the cells (200 μL, containing 1×107 MOLT-4 cells and 50% matrigel). When the tumor volume reached 150-200 mm3, the mice were randomly grouped for administration according to the tumor volume and body weight of the mice. Three administration dosages were provided: 5 mpk, 15 mpk and 50 mpk. The compounds were injected intraperitoneally (ip) weekly (qw). The mice in all the experimental groups (10 mice in each group) were continuously administered for 4 weeks.
After inoculation, the body weights of the mice were measured three times a week and the long (a) and short (b) diameters of the tumors were measured. The tumor volume was calculated according to the formula: V=ab2/2. At day 28 after the administration, the experiment was terminated.
Experimental results: in MOLT-4 xenograft tumor models, compound 6 had significant tumor-suppressing efficacy on MOLT-4 xenograft tumors in mice relative to the vehicle control group, and with the increase of the administration dosage, the tumor-suppressing effects grew stronger. See
Experimental objective: in this test, a single dose of test compounds was administered to SD rats intragastrically, and the concentrations of the test compounds in plasma of the rats were measured to evaluate the pharmacokinetic characteristics of the test compounds in the rats.
Experimental animals: male SD rats, 200-250 g, 6-8 weeks old, 3 rats/compound, purchased from Chengdu Ddossy Experimental Animals Co., Ltd.
Experimental method: on the day of the test, 3 SD rats were randomly grouped according to their body weight. The animals were fasted with water available for 12 to 14 h one day before the administration of a test compound, and were fed 4 hours after the administration.
Sampling: before and after the administration, 0.1 mL of blood samples were drawn from the orbits of the animals 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 PO group: 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h.
Before analysis and detection, all samples were stored at −60° C. The samples were analyzed quantitatively by LC-MS/MS.
Conclusion: the compound of the present invention had good oral absorption in rats.
Experimental objective: in this test, a single dose of test compounds was administered to C57 mice intragastrically, and the concentrations of the test compounds in plasma of the mice were measured to evaluate the pharmacokinetic characteristics of the test compounds in the mice.
Experimental animals: male C57 mice, 20-25 g, 6-8 weeks old, 3 mice/compound. purchased from Chengdu Ddossy Experimental Animals Co., Ltd.
Experimental method: on the day of the test, 3 C57 mice were randomly grouped according to their body weight. The animals were fasted with water available for 12 to 14 h one day before the administration of a test compound, and were fed 4 hours after the administration.
Sampling: before and after the administration, 0.03 mL of blood samples were drawn from the orbits of the animals 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 PO group: 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 7 h, and 24 h.
Before analysis and detection, all samples were stored at −60° C. The samples were analyzed quantitatively by LC-MS/MS.
Conclusion: the compound of the present invention had good oral absorption in mice.
Experimental animals: male cynomolgus monkeys, 3-5 kg, 3-6 years old, 2 monkeys/compound. Purchased from Suzhou Xishan Biotechnology Inc.
Experimental method: on the day of the test, 2 monkeys were randomly grouped according to their body weights. The animals were fasted with water available for 14 to 18 h one day before the administration of a test compound, and were fed 4 hours after the administration.
Before and after the administration, 1.0 mL of blood samples were drawn from the limb veins 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. Blood collection time points for the intravenous group were: 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 48 h. Before analysis and detection, all samples were stored at −80° C., and a quantitative analysis of samples was performed using LC-MS/MS.
In this experiment, liver microsomes of five species, including human, monkey, dog, rat, and mouse, were used as in vitro models to evaluate the metabolic stability of the test compound.
At 37° C., 1 μM of the test compound was co-incubated with microsomal protein and coenzyme NADPH. At given time points of the reaction (5 min, 10 min, 20 min, 30 min, and 60 min), the reaction was terminated by adding ice-cold acetonitrile containing an internal standard. The LC-MS/MS method was used to measure the concentration of the test compound in the sample. Ti/2 was calculated using the natural logarithm (ln) of the residual rate of the drug in the incubation system and the incubation time. In addition, the intrinsic clearance in liver microsomes CLint(mic) and the intrinsic clearance in liver CLint(Liver) were further calculated.
Conclusion: the compound according to the present invention had good stability in liver microsomes.
The purpose of this study was to evaluate the effect of the test compound on the activity of five isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450 (CYP) by using an in vitro testing system. The specific probe substrates of CYP450 isoenzymes were incubated with human liver microsomes and test compounds of different concentrations, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added to initiate the reaction. After the completion of the reaction, the sample was treated and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively detect metabolites produced by specific substrates, changes in CYP enzyme activity were determined, and IC50 value was calculated to evaluate the inhibitory potential of the test compound on each CYP enzyme subtype.
Conclusion: the compound according to the present invention did not exhibit significant inhibitory effects on the five isoforms of human liver microsomal cytochrome P450.
9. Test for hERG Potassium Ion Channel
Experimental platform: electrophysiological manual patch-clamp system
Cell line: Chinese hamster ovary (CHO) cell lines stably expressing hERG potassium ion channel
Experimental method: in CHO (Chinese Hamster Ovary) cells stably expressing hERG potassium channel, whole cell patch-clamp technique was used to record hERG potassium channel current at room temperature. The glass microelectrode was made of a glass electrode blank (BF150-86-10, Sutter) by a puller. The tip resistance after filling the liquid in the electrode was about 2-5 MΩ. The glass microelectrode can be connected to the patch-clamp amplifier by inserting the glass microelectrode into an amplifier probe. The clamping voltage and data recording were controlled and recorded by the pClamp 10 software through a computer. The sampling frequency was 10 kHz, and the filtering frequency was 2 kHz. After the whole cell records were obtained, the cells were clamped at −80 mV, and the step voltage that induced the hERG potassium current (IhERG) was depolarized from −80 mV to +20 mV for 2 s, then repolarized to −50 mV, and returned to −80 mV after 1 s. This voltage stimulation was given every 10 s, and the administration process was started after the hERG potassium current was confirmed to be stable (at least 1 minute). The compound was administered for at least 1 minute at each test concentration, and at least 2 cells (n≥2) were tested at each concentration.
Data processing: data analysis processing was carried out by using pClamp 10, GraphPad Prism 5 and Excel software. The inhibition degree of hERG potassium current (peak value of hERG tail current induced at −50 mV) at different compound concentrations was calculated by the following formula:
wherein, Inhibition % represents the percentage of inhibition of hERG potassium current by the compound, and I and Io represent the amplitude of hERG potassium current after and before the administration, respectively.
Conclusion: the compound according to the present invention did not exhibit significant inhibitory activity on the hERG potassium current.
10. Effect on Platelet in Mice after Single Intravenous Injection
Experimental animal: 78 SD rats, with an equal number of males and females, male (♂): 290-380 g, female (♀): 220-290 g, 8-10 weeks old, purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.
Experimental protocol: a clear solution was formulated using a vehicle (500 DMS+95% (3O % Tween 80 in PBS)). The animals were dosed via tail vein a single time (administration volume: 20 mL/kg; bolus injection period: about 4 minutes; recovery period: 7 days).
The experimental results are shown in Table 7 and Table 8:
Table 7 Effect of Compound 6 and Control Compound on PLT in SD Rats after Single Intravenous Injection
Conclusion: at 4 h and D2 after the administration via single intravenous injection, compound 6 exhibited less platelet toxicity in rats at doses of 6 mg/kg and 20 mg/kg relative to the control compound. At D8, compound 6 exhibited less platelet toxicity in rats at the dose of 60 mg/kg relative to the control compound.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111000638.1 | Sep 2021 | CN | national |
| 202111220461.6 | Oct 2021 | CN | national |
| 202210003537.8 | Jan 2022 | CN | national |
| 202210091103.8 | Jan 2022 | CN | national |
| 202210508260.4 | May 2022 | CN | national |
| 202210950054.9 | Aug 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/116529 | 9/1/2022 | WO |
