Patent Application
20240308992
The present invention belong to the field of pharmaceutical chemistry, specifically relate to Drak2 inhibitor, and preparation method therefor and use thereof.
Drak2 (Death-associated protein-related apoptotic kinase-2) is a member of death related protein kinases DAPK family, which is abundantly expresses in the thymus, spleen, and lymph nodes, and mainly participates in the regulation of cell apoptosis.
It has been reported that both gene and protein levels of Drak2 were dramatically increased in pancreatic β-cells treated with free fatty acid (FFA) and inflammatory cytokine (IL-1β, IFNγ, TNFα), along with anti-apoptotic factors such as Bcl-2, Bcl-xL, and Flip were increased. Up-regulations of these genes and protein levels promote an increase of apoptosis of pancreatic β-cells and reduces insulin secretion. Meanwhile, Drak2 transgenic mice showed worse glucose tolerance after obesity being induced by high-fat diet, confirming that Drak2 is not conducive to pancreatic β-cell survival under simulated inflammatory and high-fat pathological conditions. Therefore, Drak2 may become a novel target for preventing apoptosis of pancreatic β-cells and restoring the function thereof to achieve the fundamental treatment of type 2 diabetes.
In addition to participating in the regulation of cell apoptosis, Drak2 also has an immunomodulatory function, which is responsible for the negative regulation of T cell activation, and is closely related to T cell survival and differentiation, tumor immune regulation, and autoimmunity and the like.
Therefore, it is necessary to develop new Drak2 inhibitors.
The purpose of the present invention is to provide a novel Drak2 inhibitor with broad clinical application prospects.
In the first aspect of the present invention, provided is a compound of formula I, or a stereoisomer or optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof,
In another preferred embodiment, the compound, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure as represented by formula II
In another preferred embodiment, Ring A is selected from: phenyl, C5-C6 cycloalkyl, 5-6-membered heteroaryl and 5-6-membered heteroaryl, preferably phenyl.
In another preferred embodiment, ring B is 5-6-membered heteroaryl, preferably thienyl or thiazolyl.
In another preferred embodiment, the compound, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure as represented by formula III
In another referred embodiment,
preferably,
In another preferred embodiment, the compound, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure as represented by formula IV
In another preferred embodiment, R1 is H.
In another preferred embodiment, R4 is H.
In another preferred embodiment, R5 is H.
In another preferred embodiment, R1 is H, R4 is H and R5 is H.
In another preferred embodiment, R2 is C1-6 alkoxy (preferably, methoxy).
In another preferred embodiment, R3 is C1-C6 alkoxy (preferably methoxy), C(O)NRm6Rm7, or S(O)2NRm6Rm7; wherein, Rm6 and Rm7 are described as above.
In another preferred embodiment, R3 is C1-C6 alkoxy-3-6 membered heterocyclyl (preferably, —O—CH2-3-6-membered heterocyclyl, —O—CH2CH2-3-6-membered heterocyclyl, etc.), wherein C1-C6 alkoxy-3-6 membered heterocyclyl is substituted with 1 to 4 substituents selected from halogen and C1-C6 alkyl.
In another preferred embodiment, the compound, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure as represented by formula A
In another preferred embodiment, R2 and R3 together with the adjacent C atoms a 5-6-membered heterocyclyl; preferably, R2 and R3 together with the adjacent C atoms form
In another preferred embodiment, the compound, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure as represented by formula B
In another preferred embodiment, R7 (or Rp) is selected from methyl, ethyl, n-propyl, n-butyl n-pentyl, n-hexyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl,
In another preferred example,
is selected from:
In another preferred embodiment, the compound, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure as represented by formula C
In another preferred embodiment, X1 is N or CH.
In another preferred embodiment, X2 is N or CH.
In another preferred embodiment, X3 is N, C—C(═O)NH2 or C—CN.
In another preferred embodiment, X5 is N or CH.
In another preferred embodiment,
In another preferred embodiment, L is bond.
In another preferred embodiment, L is NH.
In another preferred embodiment, ring A is C6-C10 aryl or 3-12 membered heterocyclyl; wherein, the 3-12 membered heterocyclyl is preferably 5-7 membered heterocyclyl, and more preferably 6-membered heterocyclyl; the definition of substituents on ring A is the same as that of Rm mentioned above (the number of substituents is the same as n).
In another preferred embodiment, ring A is phenyl
the definition of substituents on ring A (the position of substituent can be any position of H, including H in —NH—) is the same as that of above-mentioned Rm (the number of substituents is defined as n).
In another preferred embodiment, ring B is 5-12 membered heteroaryl or C3-C12 cycloalkyl, preferably 5-membered heteroaryl or 6-membered cycloalkyl; the definition of substituents on ring B is the same as that of R mentioned above.
In another excellent embodiment, ring B is
the definition of substituents on ring B is the same as that of R mentioned above.
In another preferred embodiment, Rm is methoxy, F, Cl, cyano, hydroxyl, carboxyl, formamide,
In another preferred embodiment, Rn is
H, acetyl
isobutyryl
cyclopropylcarbonyl
cyclobutylcarbonyl
cyclopentyl carbonyl
cyclohexyl carbonyl
phenyl carbonyl
cyclopropyl methylene carbonyl
cyclobutyl methylene carbonyl
cyclopentyl methylene carbonyl
cyclohexyl methylene carbonyl
In another preferred embodiment, the compound, or a stereoisomer or optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure of formula D
In another preferred embodiment, L2 is selected from: bond, C1-C6 alkylene, C1-C6 alkylene-O—, C1-C6 alkylene-NH—, C3-C6 cycloalkylene, 3-7 membered heterocyclylene, C6 arylene and 5-7 membered heteroarylene; wherein the C1-C6 alkylene, C1-C6 alkylene-O—, C1-C6 alkylene-NH—, C3-C6 cycloalkylene, 3-7 membered heterocyclylene, C6 arylene and 5-7 membered heteroarylene are optionally substituted with 1 to 3 substituents selected from the group consisting of: D, halogen, cyano, hydroxyl, carboxyl, NH2, oxo-(═O), C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C(O)OC1-C6 alkyl, S(O)2C1-C6 alkyl, C3-C6 cycloalkyl, 3-7 membered heterocyclyl.
In another preferred example, L2 is selected from the group consisting of: bond, C1-C6 alkylene, C1-C6 alkylene-O—, C1-C6 alkylene-NH—; wherein the C1-C6 alkylene, C1-C6 alkylene-O—, and C1-C6 alkylene-NH— are optionally substituted with 1 to 3 substituents selected from the group consisting of; hydroxyl, oxo (═O), D, halogen, cyano, carboxyl, NH2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino.
In another preferred embodiment, R11 is selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C10 cycloalkyl, 3-9 membered heterocyclyl, C6-C10 aryl and 5-9 membered heteroaryl; wherein the C1-C6 alkyl, C1-C6 alkoxy, C1-C6alkylamino, C3-C10 cycloalkyl, 3-9 membered heterocyclyl, C6-C10 aryl and 5-9 membered heteroaryl are optionally substituted with 1 to 3 substituents selected from the group consisting of: D, halogen, cyano, hydroxyl, carboxyl, NH2, oxo-(═O), C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C(O)OC1-C6 alkyl, S(O)2C1-C6 alkyl, C3-C6 cycloalkyl, 3-7 membered heterocyclyl, 3-7 membered heterocyclyl substituted with C1-C6 alkyl, C6 aryl, 5-7 membered heteroaryl and 5-7 membered heteroaryl substituted with C1-C6 alkyl.
In another preferred embodiment, R11 is selected from: H, C1-C6 alkyl, 3-9 membered heterocyclyl, and 5-9 membered heteroaryl; wherein the C1-C6 alkyl, 3-9 membered heterocyclyl, and 5-9 membered heteroaryl are optionally substituted with 1 to 3 substituents selected from the group consisting of: D, halogen, cyano, hydroxyl, carboxyl, NH2, oxo-(═O), C1-C6 alkyl, C1-C6 alkoxy, 3-7 membered heterocyclyl, 3-7 membered heterocyclyl substituted with C1-C6 alkyl, 5-7 membered heteroaryl and 5-7 membered heteroaryl substituted with C1-C6 alkyl.
In another preferred embodiment, RP is selected from: C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, C6-C10 aryl and 5-7 membered heteroaryl; wherein the C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, C6-C10 aryl and 5-7 membered heteroaryl are optionally substituted with 1 to 3 substituents selected from the group consisting of: D, halogen, cyano, hydroxyl, carboxyl, NH2, oxo-(═O), C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C(O)OC1-C6 alkyl, S(O)2C1-C6 alkyl, C3-C6 cycloalkyl, 3-7 membered heterocyclyl, C6-C10 aryl, and 5-7 membered heteroaryl; wherein the C3-C6 cycloalkyl, 3-7 membered heterocyclyl, C6-C10 aryl, and 5-7 membered heteroaryl are optionally substituted with 1 to 3 substituents selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, —O—C1-4 alkylene-phenyl.
In another preferred embodiment, the compound, or a stereoisomer or optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, has a structure of formula E:
In another preferred embodiment, ring A is 5-10-membered heteroaryl, preferably 5 membered heteroaryl, or 9-10 membered heteroaryl.
In another preferred embodiment, ring A is selected from pyrazole, furan, thiophene, thiazole, benzene ring, pyridine, pyrimidine, pyrazine, indole, indazole, benzopyrazole, pyrrolo [2,3-b] pyridine.
In another preferred embodiment, each Rm is independently selected from: H, D, halogen, cyano, hydroxyl, carboxyl, —C(O)OC1-C6 alkyl, —(CH2)qC6 aryl, —C1-C6 alkyl, —C1-C6 hydroxyalkyl, —C1-C6 haloalkyl, —C1-C6 alkoxy, —C1-C6 alkylamino, C3-C6 cycloalkyl, 3-7-membered heterocyclyl, C6 aryl and 5-7-membered heteroaryl; wherein q is 1, 2, 3, 4, 5, or 6.
In another preferred embodiment the compound has a structure as represented b formula I′:
In another preferred embodiment,
is ORp; wherein, Rp is selected from C1-C15 alkyl (preferably C1-C6 alkyl); wherein, C1-C15 alkyl (preferably C1-C6 alkyl) is optionally substituted with 1, 2, or 3 R; R is selected from C1-C15 alkylamino; the C1-C15 alkylamino is optionally substituted with 1, 2, or 3 substituents selected from group consisting of: halogen, cyano, hydroxyl, carboxyl, C1-C6 alkyl, halogenated C1-C6 alkyl, C6-C10 aryl substituted with halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C6-C10 aryl substituted with halogenated C1-C6 alkoxy, C(O)C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino. C(O)OC1-C6 alkyl, and S(O)2C1-C6 alkyl; preferably,
In another preferred embodiment, the compound satisfies one or more of the following conditions:
the definition of substituents on ring A (the position of substituents can be any position where H locates, including H in —NH—) is the same as that of Rm mentioned above (the number of substituents is the same as n).
In another preferred embodiment, X1, X2, X3, X5, ring A, ring B, Rm, Rp, n, R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are corresponding groups in each specific compound of the examples.
In another preferred embodiment, R is selected from: C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C(O)OC1-C6 alkyl, S(O)2C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, 3-12 membered heterocyclyl, C6-C10 aryl and 5-12 membered heteroaryl; wherein, C1-C6 alkyl, C1-C6 alkoxy, C3-C12 cycloalkyl, 3-12 membered heterocyclyl, C6-C10 aryl and 5-12 membered heteroaryl are optionally substituted with 1 to 3 substituents selected from the group consisting of: halogen, cyano, hydroxyl, carboxyl, C1-C6 alkyl, halogenated C1-C6 alkyl, C6-C10 aryl substituted with halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C6-C10 aryl substituted with halogenated C1-C6 alkoxy, C(O)C1-C6 alkyl, C1-C6 alkylamino, C(O)OC1-C6 alkyl, and S(O)2C1-C6 alkyl.
In another preferred example, Rn is selected from: C(O)Rp; wherein, Rp is selected from: C1-C15 alkyl (preferably C1-C6 alkyl); wherein, C1-C15 alkyl (preferably C1-C6 alkyl) is optionally substituted with 1, 2, or 3 R; R is selected from: C3-C12 cycloalkyl; wherein, C3-C12 cycloalkyl is optionally substituted with 1, 2, or 3 substituents selected from the group consisting of: C1-C6 alkyl, halogenated C1-C6 alkyl, C6-C10 aryl substituted with halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C6-C10 aryl substituted with halogenated C1-C6 alkoxy, and C(O)C1-C6 alkyl.
In another preferred example, Rn is selected from: ORp; wherein, Rp is selected from: C1-C15 alkyl; wherein, C1-C15 alkyl is optionally substituted with 1, 2, or 3 R; R is selected from: C1-C15 alkylamino; C1-C15 alkylamino is optionally substituted with 1, 2, or 3 substituents selected from the group consisting of: halogen, cyano, hydroxyl, carboxyl, C1-C6 alkyl, halogenated C1-C6 alkyl, C6-C10 aryl substituted with halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C6-C10 aryl substituted with halogenated C1-C6 alkoxy, C(O)C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C(O)OC1-C6 alkyl, and S(O)2C1-C6 alkyl.
In another referred embodiment, ring A is selected from
the substituents on ring A (the position of substituent can be any position of H, including H in —NH—) is defined as above-mentioned Rm (the number of substituents is defined as n).
In another preferred example,
is selected from
wherein, Rm is defined as above.
In another preferred embodiment, ring B selected from
the substituents on ring B is defined as R mentioned above.
In another preferred embodiment Rm is selected from:
In another preferred embodiment, Rn is selected from:
In another preferred embodiment, n is 0.
In another preferred embodiment, the compound is selected from the group consisting of:
In the second aspect of the present invention, provided is a pharmaceutical composition, comprising the compound of formula I as described in the first aspect, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, and pharmaceutically acceptable carriers.
In another preferred embodiment, provided is a preparation method for a pharmaceutical composition, comprising a step of: mixing pharmaceutically acceptable carriers with the compound as described in the first aspect of the present invention, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug, or a solvate thereof, thereby forming the pharmaceutical composition.
In another preferred example, the compound of the present invention can be prepared into powders, tablets, granules, capsules, solutions, emulsions, suspensions, external application ointment, external application hydrogel, etc.
In the third aspect of the present invention, provided is a use of the compound as described in the first aspect, or a stereoisomer, an optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, or the pharmaceutical composition as described in the second aspect, in the preparation of a drug for treating or preventing a disease associated with the activity or expression of Drak2 kinase, or in the preparation of a drug for inhibiting the activity of Drak2 kinase.
It should be understood that, within the scope of the present invention, each of the above technical features of the present invention and each of the technical features specifically described in the following (such as the embodiments) can be combined with each other to constitute a new or preferred technical solution. Due to space limitations, It will not be repeated herein.
The inventor, through extensive and in-depth research, unexpectedly discovered for the first time a new Drak2 inhibitor with a novel structure and excellent biological activity and safety. The present invention was completed on this basis.
As used herein, unless otherwise specified, the terms used have a general meaning known to those skilled in the art.
When a substituent is described by a conventional formula written from left to right, the substituent likewise includes the chemically equivalent substituent obtained when the formula is written from right to left. For example, —CH2O— is equivalent to —OCH2—.
As used herein, term “about” means that the value may change by no more than 1% from the enumerated value when used in reference to a specific enumerated value. For example, as used herein, the expression “about 100” includes all values between 99 and 101 and (e. g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, term “contain” or “include (comprise)” may be open, semi-closed, and closed. In other words, said term also includes “consisting essentially of” or “consisting of”.
As used herein, the term “alkyl” includes a linear or branched alkyl. For example, C1-C6 alkyl represents a straight or branched alkyl group with 1-6 carbon atoms. The alkyl group is preferably C1-C15 alkyl, more preferably C1-C10 alkyl, more preferably C1-C6 alkyl, more preferably C1-C4 alkyl, and more preferably C1-C3 alkyl. Alkyl includes but are not limited to methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, tert-butyl, etc. As used herein, alkyl may be optionally substituted or unsubstituted. Substituted alkyl includes halogenated alkyl, benzyl, etc.
As used herein, the term “alkenyl” includes a linear or branched alkenyl. For example, C2-C6 alkenyl refers to a straight or branched alkenyl with 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups.
As used herein, the term “alkynyl” includes a linear or branched alkynyl. For example, C2-C6 alkynyl refers to a straight or branched alkynyl containing 2-6 carbon atoms, for example ethynyl, propynyl, butynyl, or similar groups.
As used herein, the term “cycloalkyl” refers to a cyclic alkyl group having a specified number of carbon atoms. Such as, “C3-C10 cycloalkyl” refers to a cycloalkyl group having 3-10 carbon atoms (preferably 3, 4, 5, 6, 7 or 8). It may be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may be bicyclic, such as a bridged ring or a spiro ring. In the present invention, the cycloalkyl group is intended to include substituted cycloalkyl group.
As used herein, the term “C1-C6 alkoxy” refers to a linear or branched alkoxy group having 1-6 carbon atoms, with a formula represented by C1-C6 alkyl-O—, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutyloxy, tert-butoxy, etc.
As used in this article, the term “cycloalkyl” refers to a cyclic alkyl group containing a specified number of C atoms, for example, “C3-C12 cycloalkyl” refers to a cycloalkyl group with 3-12 (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms. It may be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may be bicyclic, such as a bridged ring or a spiro ring. The cycloalkyl group can also be fused with aryl, heteroaryl, or heterocyclyl, wherein the ring connected to the parent structure is the cycloalkyl group, such as
etc. As used herein, the cycloalkyl group is intended to include substituted cycloalkyl group.
As used herein, “heterocyclyl” refers to a saturated or partially saturated cyclic group with heteroatoms selected from N, S, and O, and “3-12 membered heterocyclyl” refers to a saturated or partially saturated cyclic group with 3-12 (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) atoms, and wherein 1-3 (such as 1, 2, or 3) atoms are heteroatoms selected from the group consisting of N, S, and O. It can be a monocyclic ring, or can also be a bicyclic ring, such as a bridged ring or a spiro ring. The 3-12-membered heterocyclyl is preferably 3-8-membered heterocyclyl, and more preferably 3-6 membered or 6-8-membered heterocyclyl. Specific examples may be oxetanyl, azetidinyl, tetrahydro-2H-pyranyl, piperidinyl, piperazinyl, tetrahydrofuryl, morpholinyl and pyrrolidinyl, etc. The heterocyclyl can be fused with heteroaryl aryl, or cycloalkyl rings, wherein the ring connected to the parent structure is heterocyclyl, such as
As used herein, “aryl” refers to an aromatic ring group which does not contain heteroatoms on the ring, and “C6-C10 aryl” means an aromatic ring group with 6 to 10 carbon atoms which does not contain heteroatoms on the ring. Said aryl can be fused with heteroaryl, heterocyclyl, and cycloalkyl rings, wherein the ring connected to the parent structure is the aryl ring. Such as phenyl (i.e. six membered aromatic ring), naphthyl, etc., wherein six membered aromatic ring is also intended to include six membered aromatic group fused with 5-6-membered cycloalkyl and six membered aromatic group fused 5-6-membered heterocyclic alkyl. Aryl may be optionally substituted or unsubstituted.
As used herein, the term “heteroaryl” refers to a cyclic aromatic group having 1-3 (e.g., 1, 2 or 3) heteroatoms selected from the group consisting of: N, S and O; “5-12 membered heteroaryl” refers to a cyclic aromatic group having 5-12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) atoms and wherein 1-3 (e.g., 1, 2 or 3) atoms are heteroatoms selected from N, S and O. It can be monocyclic ring, or it can also be fused. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl, and (1,2,4)-triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, etc. Heteroaryl may be fused with an aromatic, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. Heteroaryl may be optionally substituted or unsubstituted. When substituted, the substituent may be one or more of the following groups, and independently selected from the group consisting of: alkyl, deuterated alkyl, halogenated alkyl, alkoxyl, halogenated alkoxyl, alkenyl, alkenyl, alkylthiol, alkylamino, halogen, amino, nitro, hydroxyl, thiol, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylthiol, oxo, amide, sulfonamido, formyl, formamido, carboxyl, and carboxylate, etc.
As used herein, “halogen” or “halogen atom” refers to F, Cl, Br and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br.
As used herein, “amino” refers to —NH2.
As used herein, “carboxyl” refers to —COOH.
As used herein, the term “amido” refers to a group having a structure of —CONRR′, wherein, R and R′ can independently represent hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, as defined above. R and R′ any be the same or different in dialkylamino fragments.
As used herein, the term “sulfonamido” refers to a group having a structure of —SO2NRR′, wherein, R and R′ can independently represent hydrogen, alkyl, cycloalkyl, aryl, and heterocyclyl, as defined above. R and R′ can be the same or different in dialkylamino fragments.
As used herein, the term “formyl” refers to a group containing —CHO.
As used herein, the term “formamido” refers to a group containing
and formamido is also intended to include substituted formamido having a structure represented by formula
wherein R independently represents hydrogen, alkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclyl, as defined above. Each R can be the same or different.
In the present invention, “alkylamino” refers to a structure having —N(R)(R′) or -alkyl-N(R)(R′), wherein R and R′ each independently represent hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, as defined above, and R and R′ may be the same or different. “C1-C6 alkylamino” includes but is not limited to: NHCH3, N(CH3)2, NHCH2CH3, N(CH2CH3)2, N(CH3)(CH2CH3), CH2NHCH3, CH2CH2NHCH3, CH2CH2CH2NHCH3, CH2N(CH3)2, CH2CH2N(CH3)2, CH2CH2CH2N(CH3)2, CH2N(CH3)(CH2CH3), CH2CH2N(CH3)(CH2CH3), CH2CH2CH2N(CH3)(CH2CH3).
As used herein, “sulfoxide group” refers to a structure having —S(O)—R, wherein R independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, as defined above.
As used herein, “sulfonyl” refers to a structure having —S(O)2—R, wherein R independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, as defined above.
As used herein, “ester group” refers to a structure having —C(O)—O—R or R—C(O)—O—, wherein, R each independently represent hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, as defined above.
As used herein, “C3-C12 cycloalkyl-C1-C6 alkyl” refers to —C3-C12 cycloalkyl-C1-C6 alkyl or C3-C12 cycloalkyl-C1-C6 alkyl-. “3-12 membered heterocyclyl-C1-C6 alkyl” has a similar meaning.
As used herein, “optional” refers to that the event or condition subsequently described may, but is not must occur, and the description includes situations in which said event or condition occurs, and situations in which said event or condition does not occur.
As used herein, the term “substituted” indicates that one or more hydrogen atoms on a specific group are replaced by specific substituents. The specific substituent is the substituent described above, or the substituent appeared in each example. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of that group, and the substituent may be the same or different in each position. It should be understood by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically achievable.
Unless the groups described in the present invention being specifically stated as “substituted or unsubstituted”, the groups of the present invention can be substituted by substituents selected from the group consisting of deuterium, halogen, cyano, nitro, hydroxyl, amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, 3-10 membered heterocycloalkyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl.
As used herein, the term “more” independently refers to 2, 3, 4, and 5.
Unless otherwise specified, the structural formula described in the present invention is intended to include all isomeric forms (e. g., enantiomeric, diastereomeric, and geometric (or conformational) isomers): for example, R and S configurations of asymmetric centers, (Z) and (E) isomers of double bonds, etc. Thus, a single stereochemical isomer of the compound of the invention or a mixture of its enantiomers, diastereomers or geometric isomers (or conformational isomers) is within the scope of the invention.
As used herein, the term “tautomeric isomer” indicates that structural isomers with different energies can exceed low energy barriers and thus transform into each other. For example, proton tautomers (i.e. proton shift) include tautomerism through proton transfer, such as 1H-indazole and 2H-indazole. Valence tautomers include mutual transformations through the recombination of some bonding electrons.
As used herein, the term “solvate” refers to complexes formed of the compound of the present invention and solvent molecules in any specific ratio.
As used herein, “the compound of the present invention” refers to a compound of formula I, and also includes a stereoisomer, a pharmaceutically acceptable salt, a prodrug or a solvate of the compound of formula I.
The compound of the present invention may contain one or more chiral carbon atoms and may therefore produce enantiomers, diastereomers, and other stereoisomeric forms. Each chiral carbon atom may be defined as (R)- or (S)-based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. Racemates, diastereomers or enantiomers can be selected as starting materials or intermediates in the preparation of the compounds of the present invention. Isomers with optical activity can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.
Conventional techniques for the preparation/separation of individual optical isomers (i.e., enantiomers and diastereomers) include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or racemate of salts or derivatives) using, for example, chiral high performance liquid chromatography, see, for example, Gerald Gübitz and Martin G. Schmid (Eds.), Chiral Separations, Methods and Protocols, Methods in Molecular Biology, Vol. 243, 2004; A. M. Stalcup, Chiral Separations. Annu. Rev. Anal. Chem. 3:341-63, 2010; Fumiss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup.TH ED., Longman Scientific and Technical Ltd., Essex, 1991, 809-816; Heller, Acc. Chem. Res. 1990, 23, 128.
The present invention also includes isotopically labeled compounds (i.e. isotopic derivatives), equivalent to the original compounds disclosed herein. However, in reality, it is common for one or more atoms to be replaced by atoms with different atomic weights or mass numbers. Examples of isotopes in the isotopic derivatives of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes, such as 2H, 3H, 13C, 11C, 14C, 15N, 19O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively. The isotopic derivatives of the compound of the present invention are all within the scope of protection of the present invention. In this article, compounds labeled with 3H and compounds labeled with 14C are useful in tissue distribution experiments of drugs and substrates. The preparation and detection of tritium (i.e. 3H) labeled compounds and carbon-14 (i.e. 14C) labeled compounds are relatively easy, making them the preferred isotopes. In addition, substitution of heavier isotopes such as deuterium, also known as 2H, has advantages in certain therapies due to good metabolic stability, such as increasing half-life in vivo or reducing dosage. Therefore, in some cases, they can be given priority consideration. Isotope labeled compounds can be prepared using general methods by replacing non isotope reagents with readily available isotope labeled reagents, using the scheme disclosed in the example.
As used herein, the term “pharmaceutically acceptable salt” includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
The term “pharmaceutically acceptable acid addition salts” refers to salts formed with inorganic or organic acids that retain bioavailability of the free base without exerting other adverse effects. The inorganic acid salts include but are not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; the organic acid salts include but are not limited to formate, acetate, 2,2-dichloro acetate, trifluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, lauroleate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzene sulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.
The term “pharmaceutically acceptable base addition salts” refers to salts formed with inorganic or organic bases that retain bioavailability of the free acid without exerting other adverse effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, ferric salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines; substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resin, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.
If a synthesis of a specific enantiomer of the compound of the present invention is to be designed, it can be asymmetrically synthesized or derivatized using chiral excipients, whereby the resulting diastereoisomeric mixture is isolated, and then the chiral excipients is removed to obtain the pure enantiomer. In addition, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as carboxyl group, a suitable optically active acid or base can be used to form a diastereoisomeric salt with it, which can then be separated by conventional means, such as separation crystallization or chromatography to obtain pure enantiomer.
As used herein, the compound of the present invention can be substituted by any number of substituents or functional groups to expand the scope of coverage thereof. Usually, the term “substitution” refers to the substitution of hydrogen radicals with a specified substituent. When a plurality of positions in a particular structure are replaced by a plurality of particular substituents, the substituents at each position may be the same or different. The term “substitution” used herein includes all allowed organic compound substitutions. Broadly speaking, the allowed substituents include non-cyclic, cyclic, branched, non-branched, carbocyclic, and heterocyclic, aromatic, and non aromatic organic compounds. In this article, heteroatoms such as nitrogen may have hydrogen substituents or any allowed organic compounds as described above to complement its valence. Furthermore, the present invention is not intended to limit in any way the permissible substitution of organic compounds. The present invention considers that the combination of substituents and variable groups in the form of stabilized compounds is good in the treatment of diseases. The term “stabilized” herein refers to a compound with stability which is sufficient to maintain the integrity of the compound structure when being detected over a sufficient long period of time, preferably is effective over a sufficient long period of time, and is used herein for the foregoing purposes.
The metabolites of the compound shown in formula I and its pharmaceutically acceptable salt, as well as prodrugs that can convert in vivo into the compound shown in formula I and its pharmaceutically acceptable salt are also included in the scope of protection of the present invention.
The method for preparing the compound shown in formula I is described in the following schemes. In some cases, the order of steps for executing the reaction scheme can be changed to promote the reaction or avoid unnecessary side reaction products. The compound of the present invention can also be conveniently prepared by combining various synthesis methods described in this specification or known in the art, and such combinations can be easily carried out by those skilled in the art to which the present invention belongs.
Usually, in the preparation process, each reaction is carried out in an inert solvent at from room temperature to reflux temperature (such as 0° C.-150° C., preferably 10° C.-100° C.). The reaction time is usually 0.1-60 hours, preferably 0.5-48 hours.
Preferably, the compound of the present invention can be prepared by the following methods:
In the above reaction steps, the solvent, reaction temperature, reaction time, and catalyst, etc. can be selected according to specific reactants.
Raw materials and intermediates are commercially available, or prepared using known steps, or using methods well-known in the art.
Due to the excellent inhibitory activity of the compound of the present invention on Drak2 kinase, a pharmaceutical composition with the compound of the present invention as the main active ingredient can be used for the prevention and/or treatment (stabilization, alleviation, or cure) of Drak2 kinase related diseases.
The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. Wherein “safe and effective amount” refers to the amount of compound which is sufficient to significantly improve the condition, and not to generate severe side effects. Generally, the pharmaceutical composition contains 1-2000 mg the compound of present invention per dose, preferably, 10-200 mg the compound of present invention per dose. Preferably, the “one dose” is one capsule or one pill.
“Pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, or gelatinous materials which are suitable for human use and should be of sufficient purity and sufficiently low toxicity. “Compatible” used herein refers to the ability of each component of a pharmaceutical composition can be mixed with the compound of the present invention and with each other without appreciably reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carrier include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifier (such as Tween®), wetting agent (such as lauryl sodium sulfate), colorant, flavoring, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
There is no special limitation on the administration mode of the compound or pharmaceutical compositions of the present invention, and the representative administration mode includes (but is not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous) administration.
The solid dosage forms used for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the compound of the present invention are mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with any of the following components: (a) fillers or compatibilizer, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and arabic gum; (c) humectant, such as, glycerol; (d) disintegrating agents such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain composite silicates, and sodium carbonate; (e) dissolution-retarding agents, such as paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants such as talc, stearin calcium, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or the mixtures thereof. In capsules, tablets, and pills, the dosage form may also include buffers.
Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coating and shell materials, such as casings and other materials well-known in the art. They can contain opacifiers, and the compound of the present invention in the pharmaceutical composition can be released in a delayed mode in a certain part of the digestive tract. Examples of embedding components that may be employed are polymeric substances and waxes. If necessary, the compound of the present invention may also be formed into a microcapsules with one or more of the above excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable lotion, solutions, suspensions, syrups or tinctures. In addition to the compound of the present invention, the liquid dosage forms may contain any conventional inert diluents known in the art such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethyl formamide, as well as oil, in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or the combination thereof.
In addition to these inert diluents, the pharmaceutical composition can also include additives such as wetting agents, emulsifiers and suspensions, sweeteners, correctors, and spices.
In addition to the compound of the present invention, suspensions can include suspending agents such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, methanol aluminum and agar, or mixtures of these substances.
Pharmaceutical compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersion liquid, suspensions or lotions, and sterile powders for re-dissoluting into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and their suitable mixtures.
The compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compound (such as EGFR inhibitor).
When co-administered, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds (such as Drak2 inhibitor). One or more (2, 3, 4, or more) of other pharmaceutically acceptable compounds (such as Drak2 inhibitor) may be used simultaneously, separately, or sequentially with the compounds of the present invention for preventing and/or treating diseases related with the activity or expression level of Drak2 kinase.
When the pharmaceutical compositions are used, a safe and effective amount of compound of the present invention is applied to a mammal (such as human) in need thereof, wherein the dose of administration is a pharmaceutically effective dose. For a person weighed 60 kg, the daily dose is usually 1-2000 mg, preferably 20-500 mg. Of course, the particular dose should also depend on various factors, such as the route of administration, patient healthy status, which are within the skills of an experienced physician.
The present invention was further described hereafter in combination with specific embodiments. It should be understood that these examples are only used to illustrate the and not to limit the scope of the invention. The experimental methods without specific conditions in the following examples generally follow the conventional conditions or the conditions suggested by the manufacturer. Unless otherwise stated, percentages and parts are by weight.
As used herein, all temperatures were represented by ° C., unless otherwise specified.
As used herein, percentages for yield were all mass percentages.
As used herein, all portions were by volume, and all percentages were by volume, unless otherwise specified.
In this article, the preparative chromatography of PTLC or TLC (thin layer chromatography) was carried out on a 20×20 cm plate (500 μm thick silica gel); Biotage rapid chromatography system was used for silica gel chromatography.
In this article, 1H NMR (hydrogen spectrum) was performed using a Bruker Ascend™400 spectrometer at 400 MHz, 298° K, and the chemical shift (ppm) of residual protons in deuterated reagents was given as a reference: δ (Chemical shift) for CDCl3 was 7.26 ppm, δ for CD3OD was 3.31 ppm, δ for DMSO-d6 was 2.50 ppm.
In this article, in LCMS (liquid chromatography-mass spectrometry) testing, Agilent Technologies 1200 series or 6120 quadrupole spectrometers were used for liquid chromatography. For liquid chromatography, the mobile phase consists of acetonitrile (A), water (B), and 0.01% formic acid. The eluent gradient is 5-95% A for 6.0 minutes, 60-95% A for 5.0 minutes, 80-100% A for 5.0 minutes, and 85-100% A for 10 minutes, a SBC1850 mm×4.6 mm×2.7 μm capillary column is used; mass spectrometry (MS) is determined by electrospray ionization mass spectrometry (ESI).
As used herein, the analysis conditions for high-performance liquid chromatography (HPLC)-mass spectrometry (MS) were as follows:
As used herein, the meaning of each abbreviation was as follows:
In the above reaction steps, the solvent, reaction temperature, reaction time, and catalyst, etc. can be selected according to specific reactants.
Raw materials and intermediates are commercially available, or prepared using known steps, or using methods well-known in the art.
3,4-Dimethoxybromobenzene (910 mg, 4.20 mmol), potassium acetate (1.03 g, 10.50 mmol), 1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)(154 mg, 0.21 mmol), Bis(pinacolato)diboron (1.28 g, 5.00 mmol) and, 1,4-dioxane (20 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. for 5 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The reaction solution and the washing solution were combined, and spin dried to obtain 3,4-dimethoxyphenyl boronic acid pinacol ester (A1-1, crude, 4.20 mmol), which was directly used in the next reaction without any purification.
2,6-Dibromopyrazine (1.0 g, 4.2 mmol), 3,4-dimethoxyphenyl boronic acid pinacol ester (A1-1, crude, 4.20 mmol), potassium carbonate (1.38 g, 10.00 mmol), Tetrakis(triphenylphosphine)palladium (243 mg, 0.21 mmol), 1,4-dioxane (40 mL), and water (10 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. for 5 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and re-dissolved with ethyl acetate (50 mL). The organic phase was extracted successively with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-bromo-6-(3,4-dimethoxyphenyl) pyrazine (A1-2, 900 mg, 72.6%). 1HNMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 8.55 (s, 1H), 7.63 (d, J=2.0 Hz, 1H), 7.60 (dd, J=8.4, 2.1 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 4.02 (s, 3H), 3.98 (s, 3H).
2-bromo-6-(3,4-dimethoxyphenyl) pyrazine (A1-2, 488 mg, 1.65 mmol), (4-bromothiophen-2-yl) boronic acid (377 mg, 1.82 mmol), tetrakis(phosphorus)palladium (38 mg, 0.033 mmol), potassium carbonate (524 mg, 3.79 mmol), 1,4-dioxane (16 mL), and water (2.5 mL) were successively added to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. for 4 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and then added with dichloromethane (30 mL) and water (10 mL), and fractioned. The aqueous phase was extracted with dichloromethane (20 mL*2), and the organic phases were combined, washed with saturated salt water (20 mL*1), dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-(4-bromothiophen-2-yl)-6-(3,4-dimethoxyphenyl) pyrazine (A1-3, 463 mg, 74.4%). 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.78 (s, 1H), 7.74 (d, J=1.9 Hz, 1H), 7.67 (dd, J=8.4, 2.0 Hz, 1H), 7.64 (d, J=1.2 Hz, 1H), 7.40 (d, J=1.1 Hz, 1H), 7.02 (d, J=8.4 Hz, 1H), 4.04 (s, 3H), 3.99 (s, 3H).
2-(4-bromothiophen-2-yl)-6-(3,4-dimethoxyphenyl) pyrazine (A1-3, 106 mg, 0.28 mmol), benzophenone imine (76.4 mg, 0.42 mmol), 1.1′-binaphthyl-2.2′-diphenyl phosphine (17.4 mg, 0.028 mmol), Tris(dibenzylideneacetone)dipalladium (12.8 mg, 0.014 mmol), cesium carbonate (136.8 mg, 0.42 mmol), and toluene (10 mL) were sequentially added to a reaction flask. The mixture was reacted under nitrogen protection at 110° C. for 16 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford N-(5-(6-(3,4-dimethoxyphenyl) pyrazin-2yl) thiazol-3yl)-1,1-diphenylimine (A1-4, 59 mg, 43.9%). 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J=9.7 Hz, 1H), 8.56 (s, 1H), 7.82 (d, J=7.3 Hz, 2H), 7.74 (d, J=1.8 Hz, 1H), 7.65 (dd, J=8.4, 1.9 Hz, 1H), 7.57-7.49 (m, 1H), 7.43 (dt, J=9.2, 5.8 Hz, 5H), 7.29 (m, 3H), 7.20 (d, J=1.3 Hz, 1H), 7.01 (d, J=8.4 Hz, 1H), 4.03 (s, 3H), 3.98 (s, 3H).
N-(5-(6-(3,4-dimethoxyphenyl) pyrazin-2yl) thiazol-3yl)-1,1-diphenylimine (A1-4, 59 mg, 0.12 mmol) was dissolved in tetrahydrofuran (10 mL), to the reaction solution was added 1M concentrated hydrochloric acid (0.5 mL) dropwise and the mixture was reacted at room temperature for 4 hours. White solid precipitated. The solid was collected by filtration, and dried to obtain 5-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl)thiazol-3-amine hydrochloride (A1-5, 34 mg, 78.5%). 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.74 (s, 1H), 7.77 (s, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.33 (s, 1H), 7.02 (d, J=8.4 Hz, 1H), 6.38 (s, 1H), 4.05 (s, 3H), 3.99 (s, 3H).
5-(6-(3,4-Dimethoxyphenyl) pyrazin-2-yl)thiazol-3-amine hydrochloride (A1-5, 32 mg, 0.09 mmol) was dissolved in dichloromethane (5 mL), and the solution was cooled under an ice water bath and added with triethylamine (18.5 mg, 0.183 mmol). Additionally, pentanoyl chloride (14.9 mg, 0.123 mmol) was taken and dissolved in dichloromethane (0.5 mL) and the resulting mixture was slowly dropped into the above reaction system. Upon addition, the reaction system was warmed to room temperature and reacted for 16 hours. LCMS detection showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford N-(5-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiophen-3-yl) pentanamide (A1, 19 mg, 53.1%). 1H NMR (400 MHz, DMSO) δ 10.39 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 7.86 (d, J=1.2 Hz, 1H), 7.80 (dd, J=8.4, 1.9 Hz, 1H), 7.77 (d, J=1.8 Hz, 1H), 7.71 (d, J=1.2 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 3.90 (s, 3H), 3.83 (d, J=19.3 Hz, 3H), 2.32 (t, J=7.4 Hz, 2H), 1.69-1.51 (m, 2H), 1.41-1.29 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
Compounds A2-A34 of Examples 2-34 were Prepared Using Experimental Steps Similar to Those in Example 1 Above, and Compounds A1-A34 were Summarized in Table 1.
3-(4-bromo-2-methoxyphenyl) oxetan-3-ol (400 mg, 1.54 mmol), potassium acetate (379 mg, 3.86 mmol), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (57 mg, 0.08 mmol), and bis(pinacolato)diboron (471 mg, 1.85 mmol) were added to 1,4-dioxane (15 mL) and the mixture was reacted under nitrogen protection at 80° C. for 5 hours. TLC monitoring showed that the reaction was complete. The reaction solution was filtered and the filter cake was washed with ethyl acetate. The reaction solution and the washing solution were combined, and spin dried to obtain 3-(2-methoxy-4-borate-phenyl)oxetan-3-ol (A35-1, crude, 1.54 mmol in theory), which was directly used in the next reaction without any purification.
3-(2-Methoxy-4-borate-phenyl)oxetan-3-ol (A35-1, crude, 1.54 mmol), potassium carbonate (512 mg, 3.70 mmol), 2,6-dibromopyrazine (551 mg, 2.32 mmol), and tetrakis(triphenylphosphine)palladium (89 mg, 0.08 mmol) were added to 1,4-dioxane/water (4:1, 15 mL) and the mixture was reacted under nitrogen protection at 80° C. for 5 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 45 mL of water and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated salt water (20 mL*1), dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 3-(4-(6-bromopyrazin-2-yl)-2-methoxyphenyl)oxetan-3-ol (A35-2, 310 mg, 59.6%). MS (ESI) m/z: calcd 337.01 (M+H+). found 337.00.
3-(4-(6-bromopyrazin-2-yl)-2-methoxyphenyl)oxetan-3-ol (A35-2, 310 mg, 0.92 mmol), (4-bromothiophen-2-yl) boronic acid (191 mg, 0.93 mmol), tetrakis(triphenylphosphine)palladium (55 mg, 0.05 mmol) and potassium carbonate (305 mg, 2.21 mmol) were added to 1,4-dioxane/water (4:1, 15 mL) and the mixture was reacted under nitrogen protection at 60° C. for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with water (45 mL) and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated saline (20 mL*1), dried over anhydrous sodium sulfate, and spin-dried. The residue was purified by column chromatography to afford 3-(4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-2-methoxyphenyl) oxetan-3-ol (A35-3, 160 mg, 41.6%). MS (ESI) m/z: calcd 419.29 (M+H). found 419.00.
3-(4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-2-methoxyphenyl) oxetan-3-ol (A35-3, 160 mg, 0.38 mmol), cuprous iodide (15 mg, 0.08 mmol), L-proline (35 mg, 0.31 mmol), dimethyl sulfoxide (3 mL), and ammonia (25% wt, 360 mg, 2.57 mmol) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain 3-(4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-2-methoxyphenyl) oxetan-3-ol (A35-4, crude, theoretical 0.38 mmol), which was directly used in the next reaction step without any purification.
3-(4-(6-(4-Aminothiophen-2-yl) pyrazin-2-yl)-2-methoxyphenyl) oxetan-3-ol (A35-4, crude, 0.38 mmol) was dissolved in dichloromethane (10 mL). The solution was cooled under an ice water bath, and added with triethylamine (117 mg, 1.16 mmol). Pentanoyl chloride (93 mg, 0.77 mol) was taken and dissolved in dichloromethane (0.5 mL) and the resulting mixture was slowly dropped into the above reaction system. Upon addition, the reaction continued for 1 hour under an ice water bath. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford N-(5-(6-(4-(3-hydroxyoxetan-3-yl)-3-methoxyphenyl)pyrazin-2-yl) thiophen-3-yl) pentanamide (A35, 32 mg, 19.1%). MS (ESI) m/z: calcd 440.16 (M+H+). found 440.10 1H NMR (400 MHz, DMSO) δ 10.39 (s, 1H), 9.19 (s, 1H), 9.06 (s, 1H), 7.89 (d, J=1.4 Hz, 1H), 7.76 (dd, J=13.5, 7.3 Hz, 2H), 7.72 (d, J=1.3 Hz, 1H), 7.42 (d, J=7.8 Hz, 1H), 5.97 (s, 1H), 5.02 (d, J=6.9 Hz, 2H), 4.67 (d, J=6.9 Hz, 2H), 3.94 (d, J=6.9 Hz, 3H), 2.36-2.29 (m, 2H), 1.64-1.56 (m, 2H), 1.38-1.30 (m, 2H), 0.92 (t, J=7.3 Hz, 3H).
Compounds A36-A45 of Examples 36-45 were Prepared Using Experimental Steps Similar to Those in Example 35 Above, and Compounds A35-A45 were Summarized in Table 2.
Methyl 4-Bromo-2-methoxybenzoate (10 g, 40.8 mmol), potassium acetate (10.1 g, 103 mmol), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.51 g, 2.06 mmol), bis(pinacolato)diboron (12.44 g, 49.0 mmol) and 1,4-dioxane (100 mL) were successively added to a reaction flask and the mixture was reacted under nitrogen protection at 80° C. for 5 hours. TLC monitoring showed that the reaction was complete. The reaction solution was dried by rotary dryer directly, and purified by column chromatography to afford 3-methoxy-4-methoxycarbonylphenyl boronic acid pinacol ester (B4-1, 11.0 g, 92.3%). 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J=7.6 Hz, 1H), 7.43 (d, J=7.6 Hz, 1H), 7.40 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 1.37 (s, 12H).
2,6-dibromopyrazine (11.6 g, 48.8 mmol), 3-methoxy-4-methoxycarbonylphenyl boronic acid pinacol ester (B4-1, 11.0 g, 37.6 mmol), potassium carbonate (13.52 g, 98.0 mmol), tetrakis(triphenylphosphine)palladium(2.37 g, 2.05 mmol), 1,4-dioxane (120 mL), and water (10 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 100° C. for 5 hours, and TLC monitoring showed that the reaction was complete. The most of 1,4-dioxane was removed by rotary evaporation, the residue was added with water (50 mL), and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed successively with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain methyl 2-methoxy-4-(6-bromopyrazin-2-yl) benzoate (B4-2, 6.67 g, 54.7%). MS (ESI) m/z: calcd 323.00 (M+H+). found 322.90: 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 1H), 8.68 (s, 1H), 7.94 (d, J=8.1 Hz, 1H), 7.71 (d, J=1.4 Hz, 1H), 7.59 (dd, J=8.1, 1.6 Hz, 1H), 4.05 (s, 3H), 3.95 (s, 3H).
Methyl 2-methoxy-4-(6-bromopyrazin-2-yl) benzoate (B4-2, 6.67 g, 20.8 mmol), (4-bromothiophen-2-yl) boronic acid (4.30 g, 20.8 mmol), tetrakis(triphenylphosphorus)palladium (1.3 g, 0.86 mmol), potassium carbonate (10.25 g, 74.3 mmol), 1,4-dioxane (120 mL), and water (2.5 mL) were successively added to a reaction flask. The mixture was reacted under nitrogen protection at 60° C. for 0.5 hour, and TLC monitoring showed that the reaction was complete. The most of 1,4-dioxane was removed by rotary evaporation, the residue was added with dichloromethane (60 mL) and water (30 mL), and fractioned. The organic phases were combined, washed with saturated salt water (30 mL*1), dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain methyl 2-methoxy-4-(6-bromothiophen-2-yl) pyrazin-2-yl)benzoate (B4-3, 3.96 g, 47.3%). MS (ESI) m/z: calcd 404.98 (M+H+). found 404.90: 1H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H), 8.90 (s, 1H), 7.97 (d, J=8.1 Hz, 1H), 7.82 (s, 1H), 7.68 (d, J=6.5 Hz, 1H), 7.44 (s, 1H), 6.98 (s, 1H), 4.08 (s, 3H), 3.96 (s, 3H).
Methyl 2-methoxy-4-(6-bromothiophen-2-yl) pyrazin-2-yl)benzoate (B4-3, 400 mg, 0.99 mmol), cuprous iodide (47 mg, 0.25 mmol), L-proline (57 mg, 0.50 mmol), dimethyl sulfoxide (25 mL), and ammonia (25% wt, 1.0 g, 5.92 mmol) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with water (100 mL), and extracted with ethyl acetate (40 mL*3). The organic phases were combined, washed with saturated ammonium chloride (40 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain methyl 2-methoxy-4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)benzoate (B4-4, 450 mg crude, theoretical 0.99 mmol), which was directly used in the next reaction without any purification. MS (ESI) m/z: calcd 342.08 (M+H+). found 342.10.
Methyl 2-methoxy-4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)benzoate (B4-4, 450 mg crude, theoretical 0.99 mmol) was dissolved in dichloromethane (5 mL). The solution was cooled under an ice water bath, and added with triethylamine (666 mg, 6.59 mmol). Additionally, pentanoyl chloride (318 mg, 2.64 mmol) was taken and dissolved in dichloromethane (1 mL) and the resulting mixture was slowly dropped into the above system. Upon addition, the reaction was warmed to room temperature and reacted for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain methyl 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)benzoate (B1, 295 mg, 70.0%). MS (ESI) m/z: calcd 426.14 (M+H+). found 426.10. 1H NMR (400 MHz, DMSO) δ 10.42 (s, 1H), 9.27 (s, 1H), 9.13 (s, 1H), 7.91 (d, J=3.2 Hz, 2H), 7.85 (s, 2H), 7.74 (s, 1H), 3.98 (s, 3H), 3.84 (s, 3H), 2.32 (t, J=7.4 Hz, 2H), 1.67-1.54 (m, 2H), 1.39-1.30 (m, 2H), 0.92 (t, J=7.3 Hz, 3H).
Methyl 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)benzoate (B1, 295 mg, 0.69 mmol), lithium hydroxide monohydrate (146 mg, 3.48 mmol), tetrahydrofuran (4 mL) and water (2 mL) 50 were added successively to a reaction flask and the mixture was reacted at room temperature for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was evaporated by rotary dryer to remove tetrahydrofuran, diluted by adding 5 mL of water, acidified to pH=3-4 using 1M hydrochloric acid and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)benzoic acid (B2, 258 mg, 90.4%). MS (ESI) m/z: calcd 412.13 (M+H+). found 412.10.
2-Methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)benzoic acid (B4-6, 258 mg, 0.63 mmol), 5-aminotetrazole (56 mg, 0.66 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (241 mg, 1.26 mmol), 1-hydroxybenzotriazole (170 mg, 1.26 mmol), N,N-diisopropylethylamine (324 mg, 2.51 mmol), and N, N-dimethylformamide (5 mL) were sequentially added to a reaction flask and the mixture was reacted at room temperature for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)-N-(1H-tetrazol-5-yl) benzamide (B4, 115 mg, 38.3%). MS (ESI) m/z: calcd 477.15 (M−H+). found 477.10: 1H NMR (400 MHz, DMSO) δ 10.40 (s, 1H), 9.29 (s, 1H), 9.13 (s, 1H), 8.05-7.82 (m, 3H), 7.73 (d, J=1.3 Hz, 1H), 4.05 (s, 3H), 2.36-2.26 (m, 2H), 1.64-1.57 (m, 2H), 1.44-1.28 (m, 2H), 0.92 (t, J=7.3 Hz, 3H).
-4-(6-(4-(2-
-2-
-2-
)-N- (1H-
-5-
3,5-dibromo-4-cyanopyridine (1.5 g, 5.73 mmol), 3,4-dimethoxyphenylboronic acid (1.15 g, 6.30 mmol), tetrakis(triphenylphosphine)palladium(132 mg, 0.12 mmol), potassium carbonate (1.82 g, 13.17 mmol), 1,4-dioxane (20 mL), and water (5 mL) were added to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. for 14 hours, and TLC monitoring showed that the reaction was complete. The most of 1,4-dioxane was removed by rotary evaporation, the residue was diluted by adding 25 mL of water, and extracted with dichloromethane (15 mL*3). The organic phases were combined, washed with saturated salt water (15 mL*1), dried over anhydrous sodium sulfate, and purified by column chromatography after rotary drying to obtain 3-bromo-5-(3,4-dimethoxyphenyl)isonicotinonitrile (C1-1, 676 mg, 37.0%). 1H NMR (400 MHz, DMSO) δ 9.01 (s, 1H), 8.89 (s, 1H), 7.34 (d, J=2.0 Hz, 1H), 7.26 (dd, J=8.3, 2.0 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H).
3-Bromo-5-(3,4-dimethoxyphenyl)isonicotinonitrile (C1-1, 470 mg, 0.48 mmol), (4-bromothiophen-2-yl) boronic acid (335.1 mg, 1.62 mmol), tetrakis(triphenylphosphine)palladium (34 mg, 0.029 mmol), potassium carbonate (610 mg, 4.42 mmol), 1,4-dioxane (10 mL), and water (2.5 mL) were added to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. for 14 hours, and TLC monitoring showed that the reaction was complete. The most of 1,4-dioxane was removed by rotary evaporation, the residue was diluted by adding water (15 mL), and extracted with dichloromethane (10 mL*3). The organic phases were combined, washed with saturated salt water (5 mL*1), dried over anhydrous sodium sulfate, and purified by column chromatography after rotary drying to obtain 3-(4-bromothiophen-2-yl)-5-(3,4-dimethoxy) isonicotinonitrile (C1-2, 190.5 mg, 30.8%). 1H NMR (400 MHz, DMSO) δ 8.94 (s, 1H), 8.90 (s, 1H), 8.02 (d, J=1.3 Hz, 1H), 7.77 (d, J=1.4 Hz, 1H), 7.36 (d, J=2.0 Hz, 1H), 7.29 (dd, J=8.3, 2.0 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H).
3-(4-bromothiophen-2-yl)-5-(3,4-dimethoxy) isonicotinonitrile (C1-2, 100 mg, 0.238 mmol), benzophenone imine (64.83 mg, 0.358 mmol), 1.1′-binaphthyl-2.2′-diphenyl phosphine (29.70 mg, 0.048 mmol), tris(dibenzylideneacetone)dipalladium (21.84 mg, 0.024 mmol), sodium tert-butoxide (68.76 mg, 0.715 mmol), and toluene (5 mL) were sequentially added to a reaction flask. The mixture was reacted under nitrogen protection at 100° C. for 16 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford 3-(3,4-Dimethoxyphenyl)-5-(4-((diphenylmethylene) amino) thiophen-2-yl) isonicotinonitrile (C1-3, 110 mg, 92.1%). 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 8.69 (s, 1H), 7.82 (d, J=6.2 Hz, 2H), 7.47-7.40 (m, 10H), 6.82 (d, J=3.5 Hz, 1H), 6.77 (s, 1H), 6.65 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H).
3-(3,4-Dimethoxyphenyl)-5-(4-((diphenylmethylene) amino) thiophen-2-yl) isonicotinonitrile (C1-3, 110 mg, 0.219 mmol) was dissolved in dichloromethane. Two drops of concentrated hydrochloric acid were added and the mixture was reacted at room temperature for 4 hours. White solid precipitated, the reaction solution was filtered and the solid was collected, and dried to obtain 3-(4-aminothiophen-2-yl)-5-(3,4-dimethoxyphenyl) isonicotinonitrile hydrochloride (C1-4, 47 mg, 57.4%). MS (ESI) m/z: calcd 338.09 (M+H+). found 338.10.
3-(4-aminothiophen-2-yl)-5-(3,4-dimethoxyphenyl) isonicotinonitrile hydrochloride (C1-4, 30 mg, 0.08 mmol) was dissolved in 3 mL of dichloromethane. The system was cooled under an ice water bath, and added with triethylamine (40.4 mg, 0.40 mmol). Additionally, cyclobutylcarbonyl chloride (11.61 mg, 0.098 mmol) was taken and dissolved in dichloromethane (0.5 mL) and the resulting mixture was slowly dropped into the reaction system. Upon addition, the reaction was warmed to room temperature and reacted for 3 hours. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford N-(5-(4-cyano-5-(3,4-dimethoxyphenyl) pyridin-3-yl) thiophen-3-yl) cyclobutylformamide (C2, 32 mg, 95%). 1H NMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 8.75 (s, 1H), 7.91 (s, 1H), 7.61 (s, 1H), 7.48 (s, 1H), 7.19 (dd, J=8.3, 2.0 Hz, 1H), 7.14 (d, J=1.9 Hz, 1H), 7.06 (d, J=8.3 Hz, 1H), 3.99 (s, 6H), 3.25-3.13 (m, 1H), 2.51-2.37 (m, 2H), 2.31-2.25 (m, 2H).
N-(5-(4-cyano-5-(3,4-dimethoxyphenyl) pyridin-3-yl) thiophen-3-yl) cyclobutylformamide (C2, 2 mg, 0.0048 mmol), sodium tert-butoxide (1.68 mg, 0.015 mmol), and tert butanol (3 mL) were added to a reaction flask, heated to 50° C. and reacted for 2 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, quenched by adding ice water (10 mL) and extracted with ethyl acetate (5 mL*3). The organic phases were combined, washed successively with saturated salt water, dried over anhydrous sodium sulfate, and spin-dried to obtain 3-(4-(cyclobutylformamido) thiophen-2-yl)-5-(3,4-dimethoxyphenyl) isonicotinamide (C1, 1.8 mg,). 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.64 (s, 1H), 7.78 (s, 1H), 7.60 (s, 1H), 7.12 (d, J=1.5 Hz, 1H), 7.09 (dd, J=8.0, 1.7 Hz, 1H), 6.97 (d, J=8.3 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.20-3.12 (m, 1H), 2.44-2.36 (m, 2H), 2.28-2.20 (m, 2H).
Example 123-124 were Prepared Using Experimental Steps Similar to Those in Example 122 Above, and Compounds C1-C4 were Summarized in Table 4.
2-bromo-6-(3,4-dimethoxyphenyl) pyrazine (A1-2, 50 mg, 0.17 mmol), 1,4-cyclohexanediamine (97 mg, 0.85 mmol), sodium tert-butoxide (33 mg, 0.34 mmol), 1,1′-binaphthyl-2.2′-diphenyl phosphine (10.6 mg, 0.017 mmol), tris(dibenzylideneacetone)dipalladium (7.3 mg, 0.008 mmol), and toluene (10 mL) were sequentially added to a reaction flask. The mixture was reacted under nitrogen protection at 110° C. for 4 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, spin-dried to remove solvent and purified by column chromatography to afford N1-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) cyclohexan-1,4-diamine (D1, 25 mg, 45.0%). 1H NMR (400 MHz, DMSO) δ 8.23 (s, 1H), 7.78 (s, 1H), 7.64 (s, 1H), 7.61 (d, J=8.3 Hz, 1H), 7.05 (d, J=8.4 Hz, 1H), 6.96 (d, J=7.0 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.04 (dt, J=13.9, 7.0 Hz, 2H), 2.05 (s, 2H), 1.87 (s, 2H), 1.26 (s, 4H).
N1-(6-(3,4-Dimethoxyphenyl) pyrazin-2-yl) cyclohexan-1,4-diamine (D1, 14 mg, 0.043 mmol) was dissolved in dichloromethane (5 mL) and then cyclobutylcarbonyl chloride (6.1 mg, 0.051 mmol) was added. The mixture was reacted at room temperature for 2 hours. TLC monitoring showed that the reaction was complete. The reaction system was quenched by adding water (10 mL) and fractioned. The aqueous phase was extracted again with dichloromethane (5 mL). The organic phases were combined, washed with saturated salt water, dried over anhydrous sodium sulfate, and purified by column chromatography after rotary drying to obtain N-(4-((6-(3,4-dimethoxyphenyl) pyrazin-2-yl) amino) cyclohexyl) cyclobutylformamide (D2, 8 mg, 45.3%). MS (ESI) m/z: calcd 411.23 (M+H+). found 411.57: 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.80 (s, 1H), 7.60 (d, J=1.8 Hz, 1H), 7.55 (dd, J=8.3, 1.9 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 5.20 (d, J=7.6 Hz, 1H), 4.70 (s, 1H), 4.00 (s, 3H), 3.96 (s, 3H), 3.37-3.26 (m, 1H), 3.03-2.94 (m, 1H), 2.34-2.31 (m, 4H), 2.21-2.07 (m, 4H), 1.93-1.86 (m, 1H), 1.35-1.22 (m, 4H), 0.96-0.77 (m, 2H).
2-bromo-6-(3,4-dimethoxyphenyl) pyrazine (A1-2, 520 mg, 1.76 mmol), cuprous cyanide (189 mg, 2.11 mmol), and N-methylpyrrolidone (14 mL) were added successively to a reaction flask and the mixture was reacted at 180° C. for 1 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, diluted with water (70 mL) and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate to obtain 6-(3,4-dimethoxyphenyl) pyrazin-2-nitrile (D4-1, 268 mg, 63.1%). 1H NMR (400 MHz, DMSO) δ 9.58 (s, 1H), 9.09 (s, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.73 (s, 1H), 7.16 (d, J=8.5 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H).
6-(3,4-Dimethoxyphenyl) pyrazin-2-nitrile (D4-1, 268 mg, 1.11 mmol) was dissolved in trifluoroacetic acid/concentrated sulfuric acid (4/1, 10 mL) and reacted at 50° C. for 3 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled under an ice water bath, and alkalized to pH=8-11 using 2 M sodium hydroxide solution. Solid precipitates were observed; the reaction solution was filtered, and the solid was collected and dried to obtain 6-(3,4-dimethoxyphenyl) pyrazin-2-formamide (D4-2, 201 mg, 69.8%). 1H NMR (400 MHz, DMSO) δ 9.44 (s, 1H), 9.04 (s, 1H), 8.48 (s, 1H), 8.07-7.87 (m, 3H), 7.12 (d, J=8.4 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H).
6-(3,4-Dimethoxyphenyl) pyrazin-2-formamide (D4-2, 201 mg, 0.775 mmol), lawesson's reagent (188 mg, 0.465 mmol), and acetonitrile (10 mL) were added to a reaction flask and the mixture was reacted at 80° C. for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, spin-dried and purified by column chromatography to afford 6-(3,4-dimethoxyphenyl) pyrazin-2-thioamide (D4-3, 157 mg, 73.5%). 1H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 10.18 (s, 1H), 9.44 (d, J=6.2 Hz, 2H), 8.09-7.83 (m, 2H), 7.11 (d, J=8.5 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H).
6-(3,4-Dimethoxyphenyl) pyrazin-2-thioamide (D4-3, 142 mg, 0.52 mmol), methyl bromopyruvate (140 mg, 0.62 mmol), and methanol (15 mL) were added successively to a reaction flask and the mixture was reacted at 70° C. for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature. Solid precipitates were observed; the reaction solution was filtered, and the solid was collected and dried to obtain methyl 2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiazol-4-carboxylate (D4-4, 82 mg, 44.1%). 1H NMR (400 MHz, DMSO) δ 9.42 (s, 1H), 9.20 (s, 1H), 8.80 (s, 1H), 7.87 (dd, J=8.5, 1.9 Hz, 1H), 7.82 (d, J=1.9 Hz, 1H), 7.18 (d, J=8.5 Hz, 1H), 3.92 (s, 6H), 3.87 (s, 3H).
Methyl 2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiazol-4-carboxylate (D4-4, 80 mg, 0.224 mmol), lithium hydroxide monohydrate (47 mg, 1.12 mmol), and tetrahydrofuran/water (3/1, 4 mL) were added successively to a reaction flask and the mixture was reacted at 40° C. for 2 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled under an ice water bath, acidified with 2 M hydrochloric acid, and adjusted to pH=3-4. Solid precipitates were observed; the reaction solution was filtered, and the solid was collected and dried to obtain 2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiazol-4-carboxylic acid (D4-5, 75.0 mg, 97.5%). 1H NMR (400 MHz, DMSO) δ 9.41 (s, 1H), 9.19 (s, 1H), 8.70 (s, 1H), 7.87 (dd, J=8.4, 1.9 Hz, 1H), 7.82 (d, J=1.9 Hz, 1H), 7.18 (d, J=8.5 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H).
2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiazol-4-carboxylic acid (D4-5, 50.0 mg, 0.146 mmol), triethylamine (44.3 mg, 0.438 mmol), and tert butanol (5 mL) were added successively to a reaction flask and diphenyl azidophosphate (80.1 mg, 0.292 mmol, diluted with 0.5 mL tert butanol) was added dropwise. Upon addition, the reaction system was heated to 85° C. and reacted for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford tert-butyl 2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) 50 thiazol-4-carbamate (D3, 25 mg, 41.3%). 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 9.02 (s, 1H), 7.77 (d, J=1.9 Hz, 1H), 7.73-7.61 (m, 2H), 7.47 (s, 1H), 7.04 (d, J=8.4 Hz, 1H), 4.06 (s, 3H), 4.00 (s, 3H), 1.59 (s, 9H).
Tert-Butyl 2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiazol-4-carbamate (D3, 18 mg, 0.043 mmol) and hydrogen chloride ethyl acetate solution (3 M, 5 mL) were sequentially added to a reaction flask, and the mixture was reacted at room temperature for 1 hour. Solid precipitates were observed, and the solid was collected by filtration and transferred to a reaction flask. Dichloromethane (2 mL) and triethylamine (27.3 mg, 0.27 mmol) were added. Additionally, N,N-diethylchloroformamide (14.9 mg, 0.11 mmol) was taken and dissolved in dichloromethane (0.5 mL) and added dropwise to the above system. Upon addition, the reaction was carried out at room temperature for 2 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford 3-(2-(6-(3,4-dimethoxyphenyl) pyrazin-2-yl) thiazol-4-yl)-1,1-diethylurea (D4, 0.8 mg, 4.5%). MS (ESI) m/z: calcd 414.15 (M+H+). found 414.10: 1H NMR (400 MHz, CDCl3) δ 9.16 (s, 1H), 9.01 (s, 1H), 7.77 (d, J=1.9 Hz, 1H), 7.70 (dd, J=8.3, 1.9 Hz, 1H), 7.64 (s, 1H), 7.44 (s, 1H), 7.04 (d, J=8.4 Hz, 1H), 4.06 (s, 3H), 4.00 (s, 3H), 3.46 (q, J=7.2 Hz, 4H), 1.31 (t, J=7.2 Hz, 6H).
2,4-dibromopyrimidine (100 mg, 0.42 mmol), (4-bromothiophen-2-yl) boronic acid (87 mg, 0.42 mmol), tetrakis(triphenylphosphine)palladium (24 mg, 0.021 mmol), potassium carbonate (87 mg, 0.63 mmol), 1,4-dioxane (1.6 mL), and water (0.4 mL) were added sequentially. The mixture was reacted under nitrogen protection at 80° C. for 4 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain 2-bromo-4-(4-bromothiophen-2-yl) pyrimidine (D5-1, 90 mg, 67.0%). MS (ESI) m/z: calcd 318.85 (M+H+). found 318.90.
2-bromo-4-(4-bromothiophen-2-yl) pyrimidine (D5-1, 90 mg, 0.28 mmol), 3-methoxy-4-methoxycarbonylphenylborate pinacol ester (B4-1, 98 mg, 0.34 mmol), tetrakis(triphenylphosphine)palladium (32 mg, 0.028 mmol), potassium carbonate (58 mg, 0.42 mmol), 1,4-dioxane (2 mL), and water (0.5 mL) were added. The mixture was reacted under nitrogen protection at 80° C. for 2 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to obtain methyl 4-(4-(4-bromothiophen-2-yl) pyrimidin-2-yl)-2-methoxybenzoate (D5-2, 101 mg, 88.9%). 1H NMR (400 MHz, DMSO) δ 9.00 (d, J=5.3 Hz, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 8.08 (d, J=8.1 Hz, 1H), 8.04 (d, J=6.7 Hz, 2H), 7.84 (d, J=8.0 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 3H).
Methyl 4-(4-(4-bromothiophen-2-yl) pyrimidin-2-yl)-2-methoxybenzoate (D5-2, 100 mg, 0.247 mmol), cuprous iodide (9.4 mg, 0.049 mmol), L-proline (11.4 mg, 0.10 mmol), dimethyl sulfoxide (2 mL), and ammonia (25% wt, 200 mg, 1.48 mmol) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with water (10 mL), and extracted with ethyl acetate (5 mL*3). The organic phases were combined, washed with saturated ammonium chloride (5 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain methyl 4-(4-(4-aminothiophen-2-yl) pyrimidin-2-yl)-2-methoxybenzoate (D5-3, 105 mg crude, theoretical 0.247 mmol), which was directly used in the next reaction without any purification. MS (ESI) m/z: calcd 342.08 (M+H+). found 342.10.
Methyl 4-(4-(4-aminothiophen-2-yl) pyrimidin-2-yl)-2-methoxybenzoate (D5-3, 105 mg crude, theoretical 0.247 mmol) was dissolved in dichloromethane (5 mL). The reaction system was cooled under an ice water bath, and added with triethylamine (31 mg, 0.31 mmol). Additionally, pentanoyl chloride (56 mg, 0.46 mmol) was taken and dissolved in dichloromethane (1 mL) and the resulting mixture was slowly dropped into the reaction system. Upon addition, the reaction was warmed to room temperature and reacted for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain methyl 2-methoxy-4-(4-(4-pentamidothiophen-2-yl) pyrimidin-2-yl) benzoate (D5-4, 67 mg, 63.8%). MS (ESI) m/z: calcd 426.14 (M+H+). found 426.10.
Methyl 2-methoxy-4-(4-(4-pentamidothiophen-2-yl) pyrimidin-2-yl) benzoate (D5-4, 67 mg, 0.158 mmol), lithium hydroxide monohydrate (33 mg, 0.786 mmol), tetrahydrofuran (4 mL) and water (1 mL) were added successively to a reaction flask and the mixture was reacted at room temperature for 16 hours. TLC monitoring showed that the reaction was complete. Tetrahydrofuran was removed by rotary evaporation, the residue was diluted with water (5 mL), acidified to pH=3-4 using saturated citric acid and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-methoxy-4-(4-(4-pentamidothiophen-2-yl) pyrimidin-2-yl)benzoic acid (D5-5, 56 mg, 86.1%). MS (ESI) m/z: calcd 412.13 (M+H+). found 412.10.
2-Methoxy-4-(4-(4-pentamidothiophen-2-yl) pyrimidin-2-yl)benzoic acid (D5-5, 10 mg, 0.024 mmol), 1-methyl-4-(methylamino) piperidine (4.7 mg, 0.036 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.3 mg, 0.048 mmol), 1-hydroxybenzotriazole (6.6 mg, 0.048 mmol), N, N-diisopropylethylamine (9.4 mg, 0.073 mmol), and N,N-dimethylformamide (1 mL) were sequentially added to a reaction flask and the mixture was reacted at room temperature for 5 hours. TLC monitoring showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (5 mL*3). The organic phase phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-methoxy-4-(4-(4-pentamidothiophen) pyrimidin-2-yl)-N-methyl-N-(1-methylpiperidin-4-yl) benzamide (D5, 5 mg, 39.4%). MS (ESI) m/z: calcd 522.25 (M+H+). found 522.40: 1H NMR (400 MHz, DMSO) δ 10.42 (s, 1H), 8.92 (dd, J=5.3, 2.2 Hz, 1H), 8.16-8.06 (m, 2H), 8.01 (dd, J=5.6, 1.4 Hz, 1H), 7.84 (dd, J=5.3, 2.6 Hz, 1H), 7.78 (dd, J=3.2, 1.3 Hz, 1H), 7.38 (dd, J=10.3, 8.2 Hz, 1H), 3.93 (s, 3H), 3.52 (d, J=8.0 Hz, 2H), 3.19 (d, J=12.6 Hz, 2H), 2.99 (s, 1H), 2.92-2.79 (m, 3H), 2.66 (d, J=7.1 Hz, 3H), 2.32 (t, J=7.4 Hz, 2H), 2.08-1.77 (m, 4H), 1.60 (dt, J=15.0, 7.5 Hz, 2H), 1.34 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
1-tert butyl carboxylate piperazine (300 mg, 1.61 mmol), 2,6-dibromopyrazine (460 mg, 1.93 mmol), potassium carbonate (445 mg, 3.22 mmol), and dimethyl sulfoxide (9 mL) were added, and the mixture was reacted under nitrogen protection for 5 hours at 120° C. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, diluted with 50 mL of water and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated salt water (20 mL*1), dried over anhydrous sodium sulfate and spin-dried to obtain Tert butyl 4-(6-bromopyrazin-2-yl) piperazin-1-carboxylate (D6-1, 250 mg, 45.2%). MS (ESI) m/z: calcd 343.07 (M+H). found 343.10.
Tert butyl 4-(6-bromopyrazin-2-yl) piperazin-1-carboxylate (250 mg, 0.73 mmol), (4-bromothiophen-2-yl) boronic acid (200 mg, 0.97 mmol), tetrakis(triphenylphosphine)palladium (42 mg, 0.036 mmol), potassium carbonate (242 mg, 1.75 mmol) and 1,4-dioxane/water (4:1, 15 mL) were successively added to a reaction flask, and the mixture was reacted under nitrogen protection at 60° C. for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was added with water (25 mL) and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated saline, dried over anhydrous sodium sulfate, and spin-dried. The residue was purified by column chromatography to afford tert butyl 4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl) piperazin-1-carboxylate (D6-2, 105 mg, 33.8%). 1H NMR (400 MHz, DMSO) δ 8.46 (s, 1H), 8.26 (s, 1H), 7.91 (d, J=1.3 Hz, 1H), 7.81 (d, J=1.3 Hz, 1H), 3.64-3.60 (m, 4H), 3.48-3.45 (m, 4H), 1.44 (s, 9H).
Tert butyl 4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl) piperazin-1-carboxylate (105 mg, 0.25 mmol), cuprous iodide (10 mg, 0.052 mmol), L-proline (23 mg, 0.2 mmol), dimethyl sulfoxide (2 mL), and ammonia (25% wt, 59 mg, 1.69 mmol) were sequentially added to a sealed tube, and the reaction system was sealed and reacted at 80° C. for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with water (10 mL), and extracted with ethyl acetate (5 mL*3). The organic phases were combined, washed with saturated ammonium chloride (5 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain tert butyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl) piperazin-1-carboxylate (D6-3, 90 mg, theoretical 0.25 mmol), which was directly used in the next reaction without any purification. MS (ESI) m/z: calcd 362.16 (M+H). found 362.10.
Tert butyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl) piperazin-1-carboxylate (D6-3, 90 mg, theoretical 0.25 mmol) was dissolved in dichloromethane (5 mL). The reaction system was cooled under an ice water bath, and added with triethylamine (76 mg, 0.75 mmol). Additionally, pentanoyl chloride (60 mg, 0.498 mmol) was taken and dissolved in dichloromethane (0.5 mL) and the resulting mixture was slowly dropped into the above reaction system. The reaction system was reacted under ice water bath for 1 hour. TLC monitoring that the reaction was complete. The reaction solution was purified by column chromatography to afford tert butyl 4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) piperazin-1-carboxylate (D6-4, 65 mg, 58.3%). MS (ESI) m/z: calcd 446.21 (M+H+). found 446.30.
Tert butyl 4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) piperazin-1-carboxylate (65 mg, 0.146 mmol) was dissolved in methanol (1 mL). To the system then was added with hydrogen chloride methanol solution (3 M, 1 mL), and the reaction was carried out at room temperature for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin dried without any treatment to afford N-(5-(6-(piperazin-1-yl) pyrazin-2-yl) thiophen-3-yl) pentanamide (D6-5, theoretical 0.146 mmol). MS (ESI) m/z: calcd 346.21 (M+H+). found 346.30.
N-(5-(6-(piperazin-1-yl) pyrazin-2-yl) thiophen-3-yl) pentanamide (D6-5, theoretical 0.146 mmol) was dissolved in dichloromethane (5 mL). The reaction system was cooled under an ice water bath, and added with triethylamine (73 mg, 0.72 mmol). Additionally, methanesulfonyl chloride (25 mg, 0.22 mmol) was taken and dissolved in dichloromethane (0.5 mL) and the resulting mixture was slowly dropped into the above system. Upon addition, the reaction was warmed to room temperature and reacted for 0.5 hour. TLC monitoring that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford N-(5-(6-(4-(methylsulfonyl) piperazin-1-yl) pyrazin-2-yl) thiophen-3-yl) pentanamide (D6, 21 mg, 34.0%). MS (ESI) m/z: calcd 423.55 (M+H+). found 424.30; 1H NMR (400 MHz, DMSO) δ 10.31 (s, 1H), 8.31 (s, 1H), 8.27 (s, 1H), 7.69 (d, J=1.4 Hz, 1H), 7.62 (s, 1H), 3.77 (s, 4H), 3.26 (d, J=4.9 Hz, 4H), 2.92 (s, 3H), 2.31-2.27 (m, 2H), 1.58 (d, J=7.4 Hz, 2H), 1.35-1.30 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
N-Boc-1,2,5,6-tetrahydropyridin-4-boronic acid pinacol ester (500 mg, 1.62 mmol), 2,6-dibromopyrazine (385 mg, 1.62 mmol), sodium carbonate (429 mg, 4.05 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (60 mg, 0.082 mmol), and 1,4-dioxane/water (5:1, 12 mL) were sequentially added to a reaction flask. The reaction was carried out at 100° C. for 5 hours under nitrogen protection. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain tert butyl 4-(6-bromopyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-1, 310 mg, 56.3%). 1H NMR (400 MHz, DMSO) δ 8.88 (s, 1H), 8.72 (s, 1H), 6.88 (s, 1H), 4.10 (d, J=2.3 Hz, 2H), 3.60-3.49 (m, 2H), 2.54 (d, J=1.7 Hz, 2H), 1.43 (s, 9H).
Tert butyl 4-(6-bromopyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-1, 220 mg, 0.647 mmol), (4-bromothiophen-2-yl) boronic acid (134 mg, 0.647 mmol), tetrakis(triphenylphosphine)palladium (75 mg, 0.065 mmol), potassium carbonate (214 mg, 1.75 mmol) and 1,4-dioxane/water (5:1, 6 mL) were successively added to a reaction flask, and the mixture was reacted under nitrogen protection at 60° C. for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was added with water (25 mL) and extracted with ethyl acetate (20 mL*3). The organic phase phases were combined, washed with saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain tert butyl 4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-2, 158 mg, 57.8%). 1H NMR (400 MHz, DMSO) δ 9.12 (s, 1H), 8.78 (s, 1H), 8.07 (d, J=1.4 Hz, 1H), 7.89 (d, J=1.4 Hz, 1H), 6.90 (s, 1H), 4.12 (s, 2H), 3.66-3.52 (m, 3H), 2.62 (s, 2H), 1.44 (s, 9H).
Tert butyl 4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-2, 158 mg, 0.375 mmol), cuprous iodide (14 mg, 0.074 mmol), L-proline (17 mg, 0.148 mmol), dimethyl sulfoxide (2 mL), and ammonia (25% wt, 315 mg, 2.25 mmol) were sequentially added to a sealed tube, and the reaction system was sealed and reacted at 80° C. for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with water (10 mL), and extracted with ethyl acetate (5 mL*3). The organic phases were combined, washed with saturated ammonium chloride (5 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain tert butyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-3, theoretical 0.375 mmol), which was directly used in the next reaction without any purification. MS (ESI) m/z: calcd 359.15 (M+H+). found 359.10.
Tert butyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-3, theoretical 0.375 mmol) was dissolved in dichloromethane (5 mL). The reaction system was cooled under an ice water bath, and added with triethylamine (73 mg, 0.72 mmol). Additionally, pentanoyl chloride (134 mg, 1.11 mmol) was taken and dissolved in dichloromethane (0.5 mL) and the resulting mixture was slowly dropped into the above system. Upon addition, the reaction system was warmed to room temperature and reacted for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford tert butyl 4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-4, 35 mg, 21.1%). MS (ESI) m/z: calcd 443.20 (M+H+). found 443.10.
Tert butyl 4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)-3,6-dihydropyridin-1(2H)-carboxylate (D7-4, 35 mg, 0.079 mmol) was dissolved in methanol (2 mL), the solution was added palladium carbon (35 mg), and the reaction was carried out at room temperature under hydrogen atmosphere (1 atm) for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was filtered to remove palladium carbon and spin-dried to afford tert butyl 4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)piperidin-1-carbonate (D7-5, 35 mg, 100%). MS (ESI) m/z: calcd 445.22 (M+H+). found 445.20.
Tert butyl 4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)piperidin-1-carbonate (D7-5, 35 mg, 0.079 mmol) was dissolved in methanol (4 mL), the solution was added with hydrogen chloride methanol solution (3 M, 4 mL), and the mixture was reacted at room temperature for 1 hour. LCMS detection showed that the reaction was complete. The reaction solution was spin-dried and used directly in the next reaction to afford N-(5-(6-(piperidin-4-yl) pyrazin-2-yl) thiophen-3-yl) pentanamide hydrochloride (D7-6, 30 mg, 100%). MS (ESI) m/z: calcd 345.17 (M+H+). found 345.10.
N-(5-(6-(piperidin-4-yl) pyrazin-2-yl) thiophen-3-yl) pentanamide hydrochloride (D7-6, 30 mg, 0.079 mmol) was dissolved in dichloromethane (5 mL). The solution was added with triethylamine (24 mg, 0.238 mmol) and cooled under an ice water bath. Additionally, methylsulfonyl chloride (13.6 mg, 0.119 mmol) was taken and dissolved in dichloromethane (2 mL) and the resulting mixture was slowly dropped into the above system. Upon addition, the reaction system was warmed to room temperature and reacted for 0.5 hour. LCMS detection showed that the reaction was complete. The reaction solution was directly spin-dried and purified by column chromatography to afford N-(5-(6-(1-(methylsulfonyl) pyridin-4-yl) pyrazin-2yl) thiophen-3-yl) pentanamide (D7, 1.6 mg, 4.8%). MS (ESI) m/z: calcd 423.14 (M+H+). found 423.10: 1H NMR (400 MHz, DMSO) δ 10.35 (s, 1H), 8.94 (s, 1H), 8.51 (d, J=3.7 Hz, 1H), 7.81 (d, J=1.4 Hz, 1H), 7.66 (d, J=1.3 Hz, 1H), 3.70 (d, J=11.9 Hz, 2H), 3.01-2.83 (m, 5H), 2.34-2.28 (m, 2H), 2.08-2.01 (m, 2H), 1.88-1.77 (m, 2H), 1.63-1.55 (m, 2H), 1.38-1.30 (m, 2H), 1.24 (s, 1H), 0.91 (t, J=7.3 Hz, 3H).
Example 130 was Prepared Using Experimental Steps Similar to Those in Examples 125-129 Above, and Compounds D1-D9 were Summarized in Table 5.
Examples 131-152 (Compounds B79-100) were Prepared Using Experimental Steps Similar to Those in Example 46 Above, and Compounds B79-100 were Summarized in Table 6.
Cuprous chloride (20 mg, 0.20 mmol) was dissolved in water (30 mL), and the reaction system was cooled to 0-10° C. under iced-water bath. Dichlorosulfoxide (8.0 g, 67.24 mmol) was slowly added dropwise to the reaction system and the reaction system was naturally returned to room temperature and reacted for 16 hours to form a stable system A. 2-Methoxy-4-bromoaniline (3.0 g, 14.85 mmol) was dissolved in acetic acid (15 mL), and the system was cooled to around 10° C. under ice water bath. In addition, sodium nitrite (1.12 g, 16.23 mmol) was taken and dissolved in water (5 mL) and then added dropwise to the reaction system. Upon addition, the reaction continued at 0° C. for 1 hour to form a stable system B. The stable system A was cooled to 0-10° C., and the stable system B was slowly added to A. Upon addition, the mixture was naturally warmed to room temperature and continued to react for 16 hours. TLC detection showed that the reaction was complete. The reaction was extracted with ethyl acetate (50 mL*3), and the organic phase were combined and washed with saturated saline (50 mL*1), dried over anhydrous sodium sulfate, and spin-dried. The residue was purified by column chromatography to afford 4-bromo-2-methoxybenzenesulfonyl chloride (B103-1, 671 mg, 15.8%). 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J=8.4 Hz, 1H), 7.30-7.27 (m, 2H), 4.09 (s, 3H).
4-bromo-2-methoxybenzenesulfonyl chloride (B103-1, 200 mg, 0.7 mmol) was dissolved in dichloromethane (8 mL), and 1-methyl-4-(methylamino) piperidine (99 mg, 0.77 mmol), and triethylamine (142 mg, 1.40 mmol) were sequentially added to the above reaction system. The reaction was carried out at room temperature for 14 hours, and TLC detection showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain 4-bromo-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-2, 264 mg, 100%). MS (ESI) m/z: calcd 377.05 (M+H+). found 377.40.
4-bromo-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-2, 264 mg, 0.70 mmol) and triisopropyl borate (396 mg, 2.10 mmol) were added to tetrahydrofuran (20 mL), and the mixture was cooled to −78° C., and n-butyl lithium (0.84 mL, 2.10 mmol, 2.5 M in THF) was slowly added dropwise. Upon addition, the reaction was naturally warmed to room temperature and reacted for another 14 hours, and LCMS detected that the reaction was complete. An appropriate amount of water (1 mL) was added to the system to quench the reaction, and the reaction was filtered to remove most of the inorganic salts, spin-dried, and then used directly in the next reaction without any purification. MS (ESI) m/z: calcd 343.14 (M+H+). found 343.60.
(3-Methoxy-4-(N-methyl-N-(1-methylpiperidin-4-yl) aminosulfonyl) phenyl) boronic acid (B103-3, crude, theoretical 0.70 mmol), 2,6-dibromopyrazine (200 mg, 0.84 mmol), and tetrakis(triphenylphosphine)palladium (40 mg, 0.035 mmol) were added to 1,4-dioxane/water (4:1, 10 mL), and the mixture reacted under nitrogen protection at 80° C. for 5 hours. LCMS monitored that the reaction is complete. The reaction solution was directly spin dried and purified by column chromatography to obtain 4-(6-bromopyrazin-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-4, 216 mg, 47.9%). MS (ESI) m/z: calcd 455.07 (M+H+). found 454.90.
4-(6-bromopyrazin-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-4, 216 mg, 0.48 mmol), (4-bromothiophene-2-yl) boronic acid (98.5 mg, 0.48 mmol), tetrakis(triphenylphosphine)palladium (27 mg, 0.023 mmol) and potassium carbonate (158 mg, 1.14 mmol) were added to 1,4-dioxane/water (4:1, 10 mL), and the mixture reacted under nitrogen protection at 60° C. for 0.5 hour. TLC detected the completion of reaction. The reaction solution was directly spin-dried and purified by column chromatography to obtain 4-(6-(4-bromothiophene-2-yl) pyrazine-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-5, 182 mg, 71.4%). MS (ESI) m/z: calcd 537.06 (M+H). found 536.90.
4-(6-(4-bromothiophene-2-yl) pyrazine-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-5, 182 mg, 0.34 mmol), cuprous iodide (13 mg, 0.07 mmol), L-proline (16 mg, 0.14 mmol), dimethyl sulfoxide (2 mL), and ammonia (25% wt, 286 mg, 2.04 mmol) were sequentially added to the sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. LCMS monitored that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried with anhydrous sodium sulfate, and spin-dried to obtain 4-(6-(4-aminothiophene-2-yl) pyrazine-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-6, crude, theoretical 0.34 mmol), which was directly used in the next reaction without any purification. MS (ESI) m/z: calcd 474.16 (M+H). found 474.10.
4-(6-(4-aminothiophene-2-yl) pyrazine-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzenesulfonamide (B103-6, crude, theoretical 0.17 mmol) was added to DMF (3 mL). The flask was sequentially added with cyclopropyl acetic acid (25.4 mg, 0.25 mmol), EDCI (65 mg, 0.34 mmol), HOBT (46 mg, 0.34 mmol), and DIEA (109 mg, 0.84 mmol), and the mixture was reacted at room temperature for 14 hours. LCMS detection showed that the reaction was complete. The reaction solution was diluted with sufficient water and extracted with ethyl acetate (20 mL*3), and the ethyl acetate phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried. The residue was purified by column chromatography to afford 2-cyclopropyl-N-(5-(6-(3-methoxy-4-(N-methyl-N-(1-methylpiperidin-4-yl) sulfamoyl) phenyl) pyrazin-2-yl) thiophene-3-yl) acetamide (B103, 25 mg, 26.4%). MS (ESI) m/z: calcd 556.20 (M+H+). found 556.00. 1H NMR (400 MHz, DMSO) δ 10.35 (s, 1H), 9.27 (s, 1H), 9.15 (s, 1H), 8.01-7.86 (m, 4H), 7.75 (t, J=2.4 Hz, 1H), 4.03 (s, 3H), 3.64-3.57 (m, 1H), 2.85-2.74 (m, 5H), 2.22 (d, J=7.0 Hz, 2H), 2.12 (s, 3H), 1.89 (t, J=11.0 Hz, 2H), 1.76-1.66 (m, 2H), 1.36 (d, J=13.0 Hz, 2H), 1.29-1.20 (m, 1H), 0.55-0.46 (m, 2H), 0.26-0.17 (m, 2H).
Examples 153-157 (Compounds B101-105) were Prepared Using Experimental Steps Similar to Those in Example 155 Above, and Compounds B101-105 were Summarized in Table 7.
2-Amino-5-bromophenol (500 mg, 2.66 mmol), 1,2-dibromoethane (1 g, 5.32 mmol), potassium carbonate (735 mg, 5.32 mmol), and acetonitrile (35 mL) were taken and placed sequentially in a reaction flask and the mixture was reacted at 80° C. for 16 hours. LCMS monitoring showed that the reaction was completed. The reaction solution was concentrated, added with water and extracted with ethyl acetate. The aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by column chromatography to afford 7-bromo-3,4-dihydro-2H-benzo[b] [1,4]oxazine (E2-1, 190 mg, 33.4%). MS (ESI) m/z: calcd 216.1 (M+H). found 216.10.
7-bromo-3,4-dihydro-2H-benzo[b] [1,4]oxazine (E2-1, 190 mg, 0.89 mmol), bis(pinacolato)diboron (271 mg, 1.07 mmol), potassium acetate (218 mg, 2.22 mmol), Pd (dppf)2Cl2 (33 mg, 0.05 mmol), and 1,4-dioxane (10 mL) were taken and placed sequentially in a reaction flask. The mixture was reacted at 80° C. for 6 hours under nitrogen protection. LCMS monitoring showed that the reaction was complete. The reaction solution was filtered, and the solid was washed with ethyl acetate. The filtrate was subjected to concentration. The residue was purified by column chromatography to afford 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-benzo [b] [1,4] oxazine (E2-2, 243 mg, 100%) MS (ESI) m/z: calcd 262.15, (M+H). found 262.10.
7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-benzo [b] [1,4] oxazine (E2-2, 243 mg, 0.93 mmol), 2,6-dibromopyrazine (266 mg, 1.12 mmol), potassium carbonate (309 mg, 2.24 mmol), Pd (dppf)2 (54 mg, 0.05 mmol), and 1,4-dioxane:water=4:1 (15 mL) were taken and placed sequentially in a reaction flask. The mixture was reacted at 80° C. for 6 hours under nitrogen protection. LCMS monitoring showed that the reaction was completed. The reaction solution was diluted with water and extracted with EA, and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by column chromatography to afford 7-(6-bromopyrazine 2-yl)-3,4-dihydro-2H-benzo [b][1,4] oxazine (E2-3, 120 mg, 44.12%). MS (ESI) m/z: calcd 292.00 (M+H). found 293.90.
7-(6-bromopyrazin-2-yl)-3,4-dihydro-2H-benzo [b] [1,4] oxazine (E2-3, 120 mg, 0.41 mmol), (4-bromothiophene-2-yl) boronic acid (85 mg, 0.41 mmol), potassium carbonate (136 mg, 0.98 mmol), Pd(PPh3)4 (24 mg, 0.02 mmol), and 1,4-dioxane:water=4:1 (10 mL) were taken and added sequentially in a reaction flask. The mixture was reacted at 60° C. for 40 min under nitrogen protection. LCMS monitoring showed that the reaction was completed. The reaction solution was extracted with water and EA and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by column chromatography to afford 7-(6-(4-bromothiophene-2-yl) pyrazine-2-yl)-3,4-dihydro-2H-benzo [b] [1,4] oxazine (E2-4, 85 mg, 55.19%). MS (ESI) m/z: calcd 373.99 (M+H). found 376.00.
1-Methylpiperidin-4-carboxylic acid (150 mg, 1.05 mmol) was taken and placed in a flask. Dichloromethane (10 mL) was added and oxalyl chloride (266 mg, 2.10 mmol) was added slowly dropwise. Upon addition, N,N-dimethylformamide (0.5 mL) was added dropwise and the resulting solution was reacted at room temperature for 3 hours. The reaction solution was spin-dried directly to obtain 1-methylpiperidin-4-carbonyl chloride (E2-5, 167 mg, 99.82%).
7-(6-(4-bromothiophene-2-yl) pyrazine-2-yl)-3,4-dihydro-2H-benzo [b] [1,4] oxazine (E2-4, 85 mg, 0.23 mmol), 1-methylpiperidin-4-carbonyl chloride (E2-5, 55 mg, 0.34 mmol), and 1,2-dichloroethane (8 mL) were taken and added sequentially to a reaction flask. The mixture was reacted at 85° C. for 2 hours, and LCMS monitoring showed that the reaction was complete. The reaction solution was diluted with water and extracted with DCM, and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, and dried over anhydrous sodium sulfate, and concentrated to afford (7-(6-(4-bromothiophene-2-yl) pyrazin-2-yl)-2,3-dihydro-4H-benzo [b] [1,4]oxazin-4-yl) (1-methylpiperidin-4-yl) ketone (E2-6, 60 mg, 53.1%). MS (ESI) m/z: calcd 499.07 (M+H). found 501.20.
(7-(6-(4-bromothiophene-2-yl) pyrazin-2-yl)-2,3-dihydro-4H-benzo [b] [1,4] oxazin-4-yl) (1-methylpiperidin-4-yl) ketone (E2-6, 60 mg, 0.12 mmol), cuprous iodide (5 mg, 0.03 mmol), L-proline (6 mg, 0.18 mmol), potassium carbonate (25 mg, 0.18 mmol), dimethyl sulfoxide (4 mL), and ammonia (42 mg, 1.2 mmol) were taken and added sequentially to a sealed tube, and the mixture was reacted at 80° C. for 10 hours. LCMS monitoring showed that the reaction was complete. The reaction solution was added with water and extracted with EA, and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, dried over anhydrous sodium sulfate and concentrated to afford crude product (7-(6-(4-aminothiophene-2-yl) pyrazin-2-yl)-2,3-dihydro-4H-benzo [b] [1,4]oxazin-4-yl) (1-methylpiperidin-4-yl) ketone (E2-7, 20 mg, 38.5%). MS (ESI) m/z: calcd 436.17 (M+H). found 436.30.
1-Methylenecyclopropanoic acid (7 mg, 0.07 mmol), EDCI (18 mg, 0.09 mmol), HOBT (12 mg, 0.09 mmol), N, N-dimethylformamide (5 mL) were taken and placed in a reaction flask and the mixture was reacted at room temperature for 5 minutes. Then, the reaction was added with N, N-diisopropylethylamine (30 mg, 0.23 mmol), and (7-(6-(4-aminothiophene-2-yl) pyrazin-2-yl)-2,3-dihydro-4H-benzo [b] [1,4] oxazin-4-yl) (1-methylpiperidin-4-yl) ketone (E2-7, 20 mg, 0.05 mmol), and then reacted overnight at room temperature. MS detection showed that the reaction was complete. The reaction solution was added with water and extracted with EA, and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography to afford 2-cyclopropyl-N-(5-(6-(4-(1-methylpiperidin-4-carbonyl)-3,4-dihydro-2H-benzo [b] [1,4] oxazin-7-yl) pyrazin-2-yl) thiophen-3-yl) acetamide (E2, 5.18 mg, 21.7%). MS (ESI) m/z: calcd 518.21 (M+H). found 518.50. 1H NMR (400 MHz, DMSO) δ 10.33 (s, 1H), 9.12 (s, 1H), 9.02 (s, 1H), 7.89 (d, J=1.4 Hz, 1H), 7.79-7.71 (m, 3H), 4.38-4.31 (m, 2H), 4.01-3.93 (m, 2H), 2.92-2.79 (m, 3H), 2.22 (d, J=7.1 Hz, 2H), 2.20 (s, 2H), 2.12 (d, J=7.0 Hz, 1H), 2.02-1.92 (m, 2H), 1.82-1.65 (m, 4H), 1.11-1.05 (m, 1H), 0.52-0.43 (m, 2H), 0.23-0.19 (m, 1H), 0.14-0.08 (m, 1).
Methyl 4-(6-(4-bromothiophen-2-yl) pyrazine-2-yl-2-methoxybenzoate (B4-3, 200 mg, 0.49 mmol), cuprous iodide (19 mg, 0.1 mmol), L-proline (23 mg, 0.2 mmol), and potassium carbonate (102 mg, 0.74 mmol) were added to dimethyl sulfoxide (4 mL) followed by the addition of ammonia (1 mL). The tube was sealed, and heated to 80° C. and stirred for 8 hours. After TLC confirmed completion of the reaction, the reaction was poured into water and extracted with ethyl acetate. The organic phase was washed three times with deionized water, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate to obtain methyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl-2-methoxybenzoate (F1-1, 150 mg, 89%). MS (ESI) m/z: calcd 342.08 (M+H). found 342.1.
Methyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl-2-methoxybenzoate (F1-1, 148 mg, 0.43 mmol) was dissolved in dichloromethane (10 mL). The solution was added with triethylamine (53 mg, 0.52 mmol) and phenyl chloroformate (75 mg, 0.48 mmol) and the mixture was stirred at room temperature for 4 hours. LCMS monitoring showed that the reaction was completed. The reaction solution was washed for three times by adding water; the organic phase was dried over anhydrous sodium sulfate and concentrated. The product and cyclobutylamine (34 mg, 0.48 mmol) were added to N,N-dimethylformamide (8 ml), followed by the addition of N,N-diisopropylethylamine (181 mg, 1.4 mmol). The mixture was stirred at room temperature for 4 hours. LCMS monitoring showed that the reaction was completed. The reaction solution was poured into water, and extracted with ethyl acetate. The organic phase was washed three times with deionized water, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate. The residue was purified by column chromatography to afford methyl 4-(6-(4-(3-cyclobutylurea) thiophen-2-yl-pyrazin-2-yl)-2-methoxybenzoate (F1-2, 75 mg, 39.5%). MS (ESI) m/z: calcd 439.14 (M+H). found 349.3.
Methyl 4-(6-(4-(3-cyclobutylurea) thiophen-2-yl-pyrazin-2-yl)-2-methoxybenzoate (F1-2, 75 mg, 0.17 mmol) was added to a mixed solvent of tetrahydrofuran and water (8 mL, 2 mL). Then the mixture was added with lithium hydroxide (29 mg, 0.69 mmol), and stirred at room temperature for 16 hours. LCMS monitoring showed that the reaction was completed. The reaction solution was adjusted to weak acidity with 2M HCl and extracted with ethyl acetate three times. The organic phases were combined and dried over anhydrous sodium sulfate, and concentrated to afford 4-(6-(4-(3-cyclobutylurea) thiophen-2-yl-pyrazin-2-yl)-2-methoxybenzoic acid (F1-3, 40 mg, 55.3%). MS (ESI) m/z: calcd 425.12, (M+H). found 452.2.
4-(6-(4-(3-cyclobutylurea) thiophen-2-yl-pyrazin-2-yl)-2-methoxybenzoic acid (F1-3, 40 mg, 0.094 mmol) and 1-methyl-4 (methylamino) piperidine (15 mg, 0.12 mmol), and o-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (72 mg, 0.19 mmol) were added to dichloromethane. The mixture was then added with N, N-diisopropylethylamine (49 mg, 0.38 mmol) and stirred at room temperature for 1 hour. TLC detection showed that the reaction was completed. The reaction solution was washed with water and the organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography to afford 4-(6-(4-(3-cyclobutylurea) thiophen-2-yl-pyrazin-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl) benzamide (F1, 25 mg, 49.8%). MS (ESI) m/z: calcd 535.24, (M+H). found 535.4. 1H NMR (400 MHz, DMSO) δ 9.21 (d, J=10.7 Hz, 1H), 9.10 (d, J=2.6 Hz, 1H), 8.69 (s, 1H), 7.84 (d, J=8.2 Hz, 3H), 7.40 (s, 1H), 7.34 (t, J=7.3 Hz, 1H), 6.51 (d, J=7.8 Hz, 1H), 4.21-4.07 (m, 1H), 3.93 (d, J=2.7 Hz, 3H), 3.16 (s, 1H), 2.95-2.61 (m, 6H), 2.22-2.17 (m, 2H), 2.09-1.94 (m, 4H), 1.92-1.75 (m, 4H), 1.69-1.54 (m, 4H).
Methyl 4-(6-(4-bromothiophen-2-yl) pyrazin-2-ylmethoxybenzoate (B4-3, 200 mg, 0.49 mmol), bis(pinacolato)diboron (150 mg, 0.59 mmol), Pd (dppf)2Cl2 (18 mg, 0.02 mmol), potassium acetate (121 mg, 1.23 mmol), and dioxane (10 mL) were placed sequentially into a reaction flask. The mixture was reacted at 80° C. for 6 hours under nitrogen protection. MS monitoring showed that the reaction was completed. The reaction solution was filtered. The solid was washed with ethyl acetate and the filtrate was concentrated and purified by column chromatography to afford methyl 2-methoxy-4-(6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) thiophen-2-yl) pyrazin-2-ylbenzoate (G1-1, 197 mg, 89.8%). MS (ESI) m/z: calcd 453.16 (M+H). found 453.50.
2-Methoxy-4-(6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) thiophen-2-yl) pyrazin-2-ylbenzoate (G1-1,197 mg, 0.44 mmol), sodium periodate (189 mg, 0.88 mmol), sodium acetate (171 mg, 2.21 mmol), and tetrahydrofuran:water=3:1 (10 mL) were sequentially added to a reaction flask and the mixture was reacted at room temperature for 14 hours. LCMS detection showed that the reaction was completed. The reaction solution was filtered and the solid was collected, washed with water and dried to afford yellow product (5-(6-(3-methoxy-4-(methoxycarbonyl) phenyl) pyrazin-2-yl) thiophen-3-yl) boronic acid (G1-2, 110 mg, 67.6%). MS (ESI) m/z: calcd 371.08 (M+H). found 371.30.
To a flask containing (5-(6-(3-methoxy-4-(methoxycarbonyl) phenyl) pyrazin-2-yl) thiophen-3-yl) boronic acid (G1-2, 110 mg, 0.30 mmol) was added tetrahydrofuran (10 mL). The reaction was placed in an ice bath and added slowly with hydrogen peroxide (35%, 230 mg, 2.37 mmol), followed by the addition of a 2 mol/L sodium hydroxide solution (5 mg). Upon addition, the ice bath was removed and the reaction was carried out at room temperature for 14 hours. LCMS monitoring showed that the reaction was complete. The reaction solution was filtered and the solid was collected, washed with water and dried to afford methyl 4-(6-(4-hydroxythiophen-2-yl)pyrazin-2-yl)-2-methoxybenzoate (G1-3, 54 mg, 48.9%). MS (ESI) m/z: calcd 342.07 (M+H). found 343.30.
(R)-(4-hydroxy-2-methylbutyl) carbamate (100 mg, 0.49 mmol) was taken and placed in a flask. Dichloromethane (10 mL) was added and the reaction was placed in an ice bath. Then the reaction was added with triethylamine (149 mg, 1.47 mmol) and slowly added with methylsulfonyl chloride (68 mg, 0.59 mmol). Upon addition, the ice bath was removed and the reaction was carried out at room temperature for 30 min. LCMS monitoring showed that the reaction was completed. The reaction was quenched with water and extracted with dichloromethane, and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to afford butyl (R)-4-((tert butoxycarbonyl) amino)-3-methyl methanesulfonate (Gi-4, 131 mg, 94.93%). MS (ESI) m/z: calcd 282.13 found 282.40.
Methyl 4-(6-(4-hydroxythiophen-2-yl)pyrazin-2-yl)-2-methoxybenzoate (G1-3, 54 mg, 0.16 mmol), butyl (R)-4-((tert butoxycarbonyl) amino)-3-methyl methanesulfonate (G1-4, 53 mg, 0.19 mmol), cesium carbonate (103 mg, 0.32 mmol), and N, N-dimethylformamide (10 mL) were sequentially added to a flask and the mixture was reacted at 100° C. for 14 hours. MS monitoring showed that the reaction was completed. The reaction solution was added with water and extracted with ethyl acetate. The aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by column chromatography to afford methyl (S)-4-(6-(4-(4-(tert butoxycarbonyl) amino)-3-methylbutoxy) thiophen-2-yl) pyrazin-2-yl-2-methoxybenzoate (G1-5, 40 mg, 48.2%). MS (ESI) m/z: calcd 528.21 (M+H). found 528.40.
Methyl (S)-4-(6-(4-(4-(tert butoxycarbonyl) amino)-3-methylbutoxy) thiophen-2-yl) pyrazin-2-yl-2-methoxybenzoate (G1-5, 40 mg, 0.08 mmol), lithium hydroxide (13 mg, 0.31 mmol) and tetrahydrofuran:water=4:1 (10 mL) were sequentially added to a reaction flask and the mixture was reacted at room temperature for 14 hours. MS monitoring showed that the reaction was completed. The reaction solution was adjusted to around pH 6 with citric acid, added with water, and extracted with ethyl acetate. The aqueous phase was extracted again. The organic phase was combined, washed with saturated salt water, dried over anhydrous sodium sulfate, and concentrated to obtain crude (S)-4-(6-(4-(4-(tert butoxycarbonyl) amino)-3-methylbutoxy) thiophen-2-yl) pyrazin-2-yl-2-methoxybenzoic acid (G1-6, 45 mg). MS (ESI) m/z: calcd 514.19 (M+H). found 514.40.
(S)-4-(6-(4-(4-(tert butoxycarbonyl) amino)-3-methylbutoxy) thiophen-2-yl) pyrazin-2-yl-2-methoxybenzoic acid (G1-6, 45 mg, 0.09 mmol) was mixed with 2-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (67 mg, 0.18 mmol) and dichloromethane (10 mL). The mixture was reacted at room temperature for 5 min and then added with 1-methyl-4-(methylamino) piperidine (14 mg, 0.11 mmol), and N, N-diisopropylethylamine (45 mg, 0.35 mmol). The resulting solution was reacted at room temperature for 2 hours. LCMS detection showed that the reaction was completed. The reaction solution was added with water and extracted with DCM, and the aqueous phase was extracted again. The organic phases were combined, washed with saturated saline, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by column chromatography to afford tert-butyl (S)-(4-(5-(6-(3-methoxy-4-(methyl (1-methylpiperidin-4-ylcarbamoyl) phenyl) pyrazin-2-yl) thiophen-3-yl-oxy)-2-methylbutyl) carbamate (G1-7, 21 mg, 38.2%). MS (ESI) m/z: calcd 624.31, (M+H). found 624.50.
Tert-butyl (S)-(4-(5-(6-(3-methoxy-4-(methyl (1-methylpiperidin-4-ylcarbamoyl) phenyl) pyrazin-2-yl) thiophen-3-yloxy)-2-methylbutyl) carbamate (G1-7, 21 mg, 0.03 mmol) was taken and added with hydrochloric acid ethyl acetate solution (3 mol/L, 3 mL) and ethyl acetate (1 mL). The mixture was reacted at room temperature for 30 min. LCMS monitoring showed that the reaction was complete. The reaction solution was directly dried by rotary evaporation to afford yellow solid product (S)-4-(6-(4-(4-amino-3-methylbutoxy) thiophen-2-yl) pyrazin-2-yl)-2-methoxy-N-methyl-N-(1-methylpiperidin-4-yl)benzamide hydrochloride (Gi, 12 mg, 66.67%). MS (ESI) m/z: calcd 524.26 (M+H). found 524.50; 1H NMR (400 MHz, DMSO) δ 9.21 (dd, J=15.2, 7.4 Hz, 2H), 8.10 (s, 3H), 7.95-7.74 (m, 3H), 7.46-7.33 (m, 1H), 6.96-6.88 (m, 1H), 4.12-4.06 (m, 1H), 3.94 (s, 3H), 3.65-3.53 (m, 2H), 3.47 (d, J=11.2 Hz, 1H), 3.41-3.23 (m, 2H), 3.23-3.07 (m, 2H), 2.91-2.81 (m, 3H), 2.74-2.67 (m, 3H), 2.59 (d, J=4.6 Hz, 2H), 2.38-2.17 (m, 2H), 2.08-1.96 (m, 1H), 1.95-1.87 (m, 1H), 1.80 (t, J=12.1 Hz, 1H), 1.70-1.56 (m, 2H), 1.02 (d, J=6.7 Hz, 3H).
Examples 162-164 (Compounds A46-A48) were Prepared Using Experimental Steps Similar to Those in Example 35 Above, and Compounds A46-A48 were Summarized in Table 9.
6-bromo-1H-indazole (600 mg, 3.04 mmol), tert butyl 4-(hydroxymethyl) piperidin-1-carboxylate (1.0 g, 3.6 mmol), and cesium carbonate (1.98 g, 6.08 mmol) were added to N,N-dimethylformamide (10 ml), and the mixture was heated to 60° C. and stirred for 3 hours. After LCMS and TLC confirmed completion of the reaction, the reaction solution was poured into water, and extracted with ethyl acetate. The organic phase was washed three times with water, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate. The residue was purified by column chromatography to afford tert butyl 4-(6-bromo-2H-indazol-2-yl) methyl) piperidin-1-carboxylate (A50-1, 630 mg, 52.3%). MS (ESI) m/z: calcd 394.11 (M+H). found 338.2.
Tert butyl 4-(6-bromo-2H-indazol-2-yl) methyl) piperidin-1-carboxylate (A50-1, 630 mg, 1.59 mmol), bis(pinacolato)diboron (487 mg, 1.90 mmol), [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (58 mg, 0.08 mmol), and potassium acetate (392 mg, 3.98 mmol) were added to dioxane (10 mL), and the mixture was heated to 80° C. under nitrogen protection, and stirred for 14 hours. LCMS and TLC confirmed completion of the reaction, and the crude product was used directly in the next step to afford tert butyl 4-((6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)-2H-indazol-2-ylmethyl) piperidin-1-carboxylate (A50-2, 705 mg Crude, theoretical 1.59 mmol). MS (ESI) m/z: calcd 442.28 (M+H). found 442.5.
Tert butyl 4-((6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)-2H-indazol-2-ylmethyl) piperidin-1-carboxylate (A50-2, 705 mg Crude, theoretical 1.59 mmol), 2,6-dibromopyrazine (456 mg, 1.90 mmol), tetrakis(triphenylphosphine)palladium (92 mg, 0.08 mmol), and potassium carbonate (530 mg, 3.80 mmol) were added to a mixed solvent of dioxane/water (12 mL/3 mL), and the mixture was heated to 80° C. under nitrogen protection, and stirred for 8 hours. After MS and TLC confirmed completion of the reaction, the reaction solution was poured into water, and extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography to afford tert butyl 4-((6-(6-bromopyrazin-2-yl)-2H-indazol-2-ylmethyl) piperidin-1-carboxylate (A50-3, 280 mg, 37.1%). MS (ESI) m/z: calcd 472.13 (M+H). found 374.2.
Tert butyl 4-((6-(6-bromopyrazin-2-yl)-2H-indazol-2-ylmethyl) piperidin-1-carboxylate (A50-3, 280 mg, 0.59 mmol), (4-bromothiophen-2-yl) boronic acid (122 mg, 0.59 mmol), tetrakis(triphenylphosphine)palladium (35 mg, 0.03 mmol) and potassium carbonate (197 mg, 1.44 mmol) were added to dioxane/water (12 mL/3 mL), and the mixture was heated to 60° C. and stirred for 0.5 h under nitrogen protection. After MS and TLC confirmed completion of the reaction, the reaction solution was poured into water, and extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography to afford tert butyl 4-(6-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-2H-indazol-2-yl) methylpiperidin-1-carboxylate (A50-4, 240 mg, 83.0%). MS (ESI) m/z: calcd 554.11 (M+H). found 456.2.
Tert butyl 4-(6-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-2H-indazol-2-yl) methylpiperidin-1-carboxylate (A50-4, 240 mg, 0.49 mmol), cuprous iodide (16 mg, 0.10 mmol), L-proline (19 mg, 0.20 mmol), and potassium carbonate (85 mg, 1.18 mmol) were added to dimethyl sulfoxide (4 mL), and the ammonia (1 mL) was added. The tube was sealed, and heated to 80° C. and stirred for 8 hours. TLC confirmed completion of the reaction. The reaction solution was poured into water, and extracted with ethyl acetate. The organic phase was washed three times with water, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate to afford tert butyl 4-(6-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-2H-indazol-2-yl) methyl) piperidin-1-carboxylate (A50-5, 220 mg crude, 0.49 mmol theoretical), which was used directly in the next step. MS (ESI) m/z: calcd 491.22 (M+H). found 491.6.
Tert butyl 4-(6-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-2H-indazol-2-yl) methyl) piperidin-1-carboxylate (A50-5, 220 mg crude, 0.49 mmol theoretical), 2-cyclobutyl acetic acid (62 mg, 0.54 mmol), 1-hydroxybenzotriazole (122 mg, 0.90 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (172 mg, 0.90 mmol) were added to dichloromethane (12 mL), and then the mixture was added with N, N-diisopropylethylamine (232 mg, 1.80 mmol) and stirred at room temperature for 4 hours. LCMS and TLC confirmed completion of the reaction. The reaction solution was poured into water, and extracted with ethyl acetate. The organic phase was washed three times with water, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate. The residue was purified by column chromatography to afford tert butyl 4-(6-(6-(4-(2-cyclobutylacetamido) thiophen-2-yl)pyrazin-2-yl)-2H-indazol-2-yl)methyl)piperidin-1-carboxylate (A50-6, 70 mg, 20.4%). MS (ESI) 50 m/z: calcd 586.27 (M+H). found 487.6.
Tert butyl 4-(6-(6-(4-(2-cyclobutylacetamido) thiophen-2-yl)pyrazin-2-yl)-2H-indazol-2-yl)methyl)piperidin-1-carboxylate (A50-6, 70 mg, 0.1 mmol) was dissolved in 2 mL of ethyl acetate. The mixture was added with 4 mL of 3M hydrogen chloride in ethyl acetate and then stirred at room temperature for 10 hours. TLC detection showed that the reaction was completed. The solvent was dried by rotary evaporation to afford 2-cyclobutyl-N-(5-(6-(2-(2-piperidin-4-ylmethyl)-2H-indazol-6-yl) pyrazin-2-yl) thiophen-3-yl) acetamide (A50-7, 90 mg crude product, theoretical 0.1 mmol) and the crude product was directly used in the next step. MS (ESI) m/z: calcd 486.22 (M+H). found 487.5.
2-cyclobutyl-N-(5-(6-(2-(2-piperidin-4-ylmethyl)-2H-indazol-6-yl) pyrazin-2-yl) thiophen-3-yl) acetamide (A50-7, 90 mg crude product, theoretical 0.1 mmol), formaldehyde (55 mg, 1.90 mmol) and acetic acid (34.2 mg, 0.57 mmol) were added to methanol (12 mL). After stirring at room temperature for 0.5 hours, the mixture was added with sodium triacetoxyborohydride (196 mg, 0.93 mmol) and stirred at room temperature for 14 hours. After LCMS and TLC confirmed completion of the reaction, the reaction was dried to remove solvent. The residue was added with water, and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate. The residue was purified by column chromatography to afford 2-cyclobutyl-N-(5-(6-(2-(1-methylpiperidin-4-yl) methyl)-2H-indazol-6-yl) pyrazin-2-yl) thiophene-3-yl) acetamide (A50, 20 mg, 39.9%). MS (ESI) m/z: calcd 501.24 (M+H). found 501.40. 1H NMR (400 MHz, DMSO) δ 10.40 (s, 1H), 9.23 (s, 1H), 9.03 (s, 1H), 8.50 (s, 1H), 8.45 (s, 1H), 7.90 (dd, J=5.0, 1.1 Hz, 3H), 7.71 (d, J=1.2 Hz, 1H), 4.37 (d, J=7.2 Hz, 2H), 2.79-2.69 (m, 2H), 2.69-2.65 (m, 1H), 2.44 (d, J=7.6 Hz, 2H), 2.13 (s, 2H), 2.10-2.05 (m, 1H), 2.02-1.93 (m, 1H), 1.91-1.82 (m, 2H), 1.80 (s, 3H), 1.78-1.69 (m, 2H), 1.45 (d, J=10.8 Hz, 2H), 1.35-1.26 (m, 3H).
Examples 165-168 (Compounds A49-A52) were Prepared Using Experimental Steps Similar to Those in Above Example A50, and Compounds A49-A52 were Summarized in Table 10.
To a solution of 4-bromo-2-methoxyphenol (500 mg) in DMF (10 mL) was added BnBr (450 mg), and potassium carbonate (350 mg). The mixture was reacted at room temperature for 14 hours. TLC detection showed that the reaction was completed. The reaction solution was extracted with ethyl acetate, and washed with water. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 1-benzyloxy-4-bromo-2-methoxybenzene (700 mg, yield 97%). LCMS (m/z): 293.1 [M+H]+.
1-Benzyloxy-4-bromo-2-methoxybenzene (100 mg), potassium acetate (840 mg), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (125 mg), bis(pinacolato)diboron (1100 mg) and 1,4-dioxane (40 mL) were successively added to a reaction flask and the mixture was reacted under nitrogen protection at 80° C. overnight. TLC monitoring showed that the reaction was complete. The reaction solution was filtered and the filtrate was concentrated, and purified by thin-layer chromatography to obtain the product 2-(4-(benzyloxy)-3-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.16 g, yield 99%). LCMS (m/z): 341.2 [M+H]+.
2-(4-(benzyloxy)-3-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.16 g), 2,6-dibromopyrazine (1210 mg), potassium carbonate (1130 mg), tetrakis(triphenylphosphine)palladium (198 mg), 1,4-dioxane (16 mL), and water (4 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. overnight, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and re-dissolved with ethyl acetate (50 mL). The organic phase was washed successively with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-[4-(benzyloxy)-3-methoxyphenyl]-6-bromopyrazine (690 mg, yield 48%). LCMS (m/z): 371.1 [M+H]+.
2-[4-(benzyloxy)-3-methoxyphenyl]-6-bromopyrazine (600 mgl), (4-bromothiophen-2-yl) boronic acid (332 mg), tetrakis(triphenylphosphorus)palladium (94 mg), potassium carbonate (537 mg), 1,4-dioxane (16 mL), and water (4 mL) were successively added to a reaction flask. The mixture was reacted under nitrogen protection at 60° C. for 0.5 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, then added with dichloromethane (30 mL) and water (30 mL), and fractioned. The aqueous phase was extracted with dichloromethane (20 mL*2) and the organic phases were combined, washed with saturated salt water (20 mL*1), dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-[4-(benzyloxy)-3-methoxyphenyl]-6-(4-bromothiophen-2-yl)pyrazine (500 mg, yield 69%). LCMS (m/z): 453.1 [M+H]+
2-[4-(benzyloxy)-3-methoxyphenyl]-6-(4-bromothiophen-2-yl)pyrazine (250 mg), cuprous iodide (22 mg), L-proline (28 mg), dimethyl sulfoxide (3 mL), and ammonia (25% wt, 1 mL) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain 5-(6-(4-(benzyloxy)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-amine (crude, 200 mg), which was directly used in the next reaction without any purification. LCMS (m/z): 390.1 [M+H]+
5-(6-(4-(benzyloxy)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-amine (crude, 200 mg) was added with DMF (10 mL). The flask was sequentially added with cyclobutyl acetic acid (88 mg), EDCI (246 mg), HOBT (174 mg), and DIEA (332 mg), and the mixture was reacted at room temperature for 14 hours. LCMS detection showed that the reaction was complete. The reaction solution was washed with water and the organic phase was dried over anhydrous sodium sulfate and spin-dried. The residue was purified by column chromatography to obtain N-(5-(6-(4-(benzyloxy)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-yl)-2-cyclobutyl acetamide (90 mg, yield 36%). LCMS (m/z): 486.2 [M+H]+.
To a solution of (5-(6-(4-(benzyloxy)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-yl)-2-cyclobutyl acetamide (90 mg) in methanol (10 mL) was added palladium carbon (10 mg) and the mixture was reacted at room temperature overnight. MS monitoring showed that the reaction was completed. The reaction solution was filtered and concentrated to afford 2-cyclobutyl-N-(5-(6-(4-hydroxy-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-yl) acetamide (70 mg, yield 93%). LCMS (m/z): 396.1 [M+H]+.
To a mixture of 2-cyclobutyl-N-(5-(6-(4-hydroxy-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-yl) acetamide (20 mg) in acetonitrile (10 mL) was added cesium carbonate (32 mg) and the mixture was reacted at 75° C. for 2 h. MS monitoring showed that the reaction was completed. The reaction solution was filtered, concentrated and purified by thin layer chromatography to afford 2-cyclobutyl-N-(5-(6-(3-methoxy-4-(2-morpholinylethoxy) phenyl) pyrazin-2-yl) thiophen-3-yl) acetamide (5 mg, yield 20%). LCMS (m/z): 509.2[M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.16 (s, 1H), 9.00 (s, 1H), 7.89-7.78 (m, 3H), 7.69 (d, J=1.1 Hz, 1H), 7.26 (d, J=8.1 Hz, 1H), 4.47-4.44 (m, 2H), 4.02 (s, 2H), 3.93 (s, 3H), 3.75 (s, 2H), 3.68-3.52 (m, 4H), 3.28 (s, 2H), 2.69 (dq, J=15.3, 7.7 Hz, 1H), 2.43 (d, J=7.5 Hz, 2H), 2.13-2.01 (m, 2H), 1.94-1.67 (m, 4H).
Examples 170-198 were prepared using the experimental steps similar to those in Example 169 above.
Yellow solid (2.23 mg), LCMS (ESI): [M+H]+=507.50; 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.14 (s, 1H), 8.97 (s, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.83-7.73 (m, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.17 (d, J=9.0 Hz, 1H), 4.16 (t, J=5.6 Hz, 2H), 3.91 (s, 3H), 3.59 (t, J=5.5 Hz, 2H), 3.52 (t, J=7.0 Hz, 2H), 2.70 (dt, J=15.4, 7.6 Hz, 1H), 2.43 (d, J=7.6 Hz, 2H), 2.24 (t, J=8.1 Hz, 2H), 2.12-2.04 (m, 2H), 1.98-1.69 (m, 6H).
Yellow solid (2.86 mg), LCMS (ESI): [M+H]+=539.40: 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.10 (s, 1H), 8.98 (s, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.81-7.72 (m, 2H), 7.68 (d, J=1.3 Hz, 1H), 7.32 (d, J=8.6 Hz, 1H), 5.76 (s, 1H), 4.17 (td, J=11.8, 3.3 Hz, 1H), 3.90 (s, 3H), 3.84 (dd, J=11.3, 4.2 Hz, 1H), 3.59 (t, J=5.6 Hz, 2H), 3.56-3.47 (m, 1H), 3.38 (ddd, J=13.3, 12.8, 3.9 Hz, 3H), 2.67 (dt, J=15.5, 7.8 Hz, 1H), 2.40 (t, J=7.3 Hz, 2H), 2.11-1.99 (m, 2H), 1.89-1.78 (m, 2H), 1.78-1.65 (m, 2H).
Yellow solid (2.47 mg), LCMS (ESI): [M+H]+=501.50: 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 8.53 (d, J=4.8 Hz, 1H), 7.85 (d, J=1.3 Hz, 1H), 7.82-7.65 (m, 4H), 7.41 (d, J=7.8 Hz, 1H), 7.26 (dd, J=7.0, 5.3 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 4.45 (t, J=6.8 Hz, 2H), 3.85 (s, 3H), 3.25 (t, J=6.7 Hz, 2H), 2.69 (dt, J=15.3, 7.8 Hz, 1H), 2.43 (d, J=7.6 Hz, 2H), 2.15-1.99 (m, 2H), 1.95-1.79 (m, 2H), 1.78-1.66 (m, 2H)
Yellow solid (2.63 mg), LCMS (ESI): [M+H]+=522.50: 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 7.85 (d, J=1.3 Hz, 1H), 7.83-7.73 (m, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.17 (d, J=9.0 Hz, 1H), 4.15 (t, J=5.9 Hz, 2H), 3.89 (s, 3H), 2.80-2.67 (m, 3H), 2.47-2.39 (m, 2H), 2.37 (d, J=20.0 Hz, 3H), 2.17 (s, 3H), 2.14-2.03 (m, 2H), 1.93-1.80 (m, 2H), 1.80-1.66 (m, 2H).
Yellow solid (2.17 mg), LCMS (ESI): [M+H]+=502.50: 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 8.70 (d, J=1.4 Hz, 1H), 8.61 (dd, J=2.5, 1.6 Hz, 1H), 8.53 (d, J=2.5 Hz, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.77 (dt, J=5.9, 2.0 Hz, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 4.47 (t, J=6.5 Hz, 2H), 3.84 (s, 3H), 3.32-3.29 (m, 2H), 2.76-2.63 (m, 1H), 2.43 (d, J=7.6 Hz, 2H), 2.15-2.01 (m, 2H), 1.92-1.80 (m, 2H), 1.80-1.70 (m, 2H).
Yellow solid (1.74 mg), LCMS (ESI): [M+H]+=546.40: 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.14 (s, 1H), 8.97 (s, 1H), 7.92 (s, 1H), 7.85 (d, J=1.3 Hz, 1H), 7.82-7.75 (m, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.21 (d, J=9.0 Hz, 1H), 4.28 (t, J=5.5 Hz, 2H), 4.15 (t, J=5.5 Hz, 2H), 3.97-3.79 (m, 5H), 3.19-3.11 (m, 2H), 3.05 (t, J=5.5 Hz, 2H), 2.70 (dt, J=15.6, 7.8 Hz, 1H), 2.43 (d, J=7.6 Hz, 2H), 2.12-2.03 (m, 2H), 1.90-1.80 (m, 2H), 1.78-1.68 (m, 2H).
Yellow solid (2.36 mg), LCMS (ESI): [M+H]+=536.50: 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.14 (s, 1H), 8.99 (s, 1H), 7.86 (d, J=1.3 Hz, 1H), 7.79 (d, J=1.9 Hz, 1H), 7.74 (dd, J=8.5, 2.0 Hz, 1H), 7.69 (d, J=1.2 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 4.99 (s, 2H), 4.43 (s, 1H), 4.10 (s, 1H), 3.93 (s, 3H), 3.49 (s, 2H), 3.07 (d, J=50.0 Hz, 4H), 2.86 (s, 3H), 2.70 (dt, J=15.4, 7.8 Hz, 1H), 2.43 (d, J=7.6 Hz, 2H), 2.15-2.01 (m, 2H), 1.96-1.80 (m, 2H), 1.80-1.64 (m, 2H).
Yellow solid (2.44 mg), LCMS (ESI): [M+H]+=605.60: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.16 (s, 1H), 9.00 (s, 1H), 7.87 (d, J=1.1 Hz, 1H), 7.81 (d, J=7.2 Hz, 2H), 7.70 (d, J=1.1 Hz, 1H), 7.23 (d, J=8.8 Hz, 1H), 4.38 (s, 2H), 3.92 (s, 3H), 3.60-3.34 (m, 6H), 3.30-3.00 (m, 5H), 2.95 (d, J=9.6 Hz, 3H), 2.77 (s, 4H), 2.69 (dd, J=15.4, 7.7 Hz, 2H), 2.43 (d, J=7.6 Hz, 2H), 2.19-1.96 (m, 4H), 1.93-1.56 (m, 6H).
Yellow solid (1.76 mg), LCMS (ESI): [M+H]+=534.60: 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.96 (s, 1H), 7.86 (d, J=1.3 Hz, 1H), 7.83-7.74 (m, 2H), 7.68 (d, J=1.3 Hz, 1H), 7.17 (d, J=8.3 Hz, 1H), 4.42 (d, J=23.8 Hz, 6H), 4.24 (s, 4H), 3.93 (s, 3H), 3.62 (s, 2H), 2.80 (s, 3H), 2.72-2.59 (m, 1H), 2.41 (d, J=7.6 Hz, 2H), 2.12-1.98 (m, 2H), 1.92-1.60 (m, 4H).
Yellow solid (4.22 mg), LCMS (ESI): [M+H]+=497.70: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.78 (dd, J=6.4, 2.0 Hz, 2H), 7.70 (d, J=1.3 Hz, 1H), 7.17 (d, J=9.1 Hz, 1H), 4.17 (t, J=5.9 Hz, 2H), 3.91 (d, J=7.2 Hz, 3H), 3.65-3.50 (m, 4H), 2.74 (t, J=5.9 Hz, 2H), 2.32 (t, J=7.4 Hz, 2H), 1.67-1.53 (m, 2H), 1.34 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.05 mg), LCMS (ESI): [M+H]+=545.70: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 7.86 (d, J=1.4 Hz, 1H), 7.79 (dd, J=6.8, 2.0 Hz, 2H), 7.70 (d, J=1.4 Hz, 1H), 7.18 (d, J=9.1 Hz, 1H), 4.17 (t, J=5.7 Hz, 2H), 3.90 (s, 3H), 3.10 (d, J=4.0 Hz, 8H), 2.98 (t, J=5.7 Hz, 2H), 2.32 (t, J=7.4 Hz, 2H), 1.60 (dt, J=15.0, 7.5 Hz, 2H), 1.41-1.28 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.65 mg), LCMS (ESI): [M+H]+=384.50: 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.59 (s, 1H), 9.08 (s, 1H), 8.93 (s, 1H), 7.84 (d, J=1.3 Hz, 1H), 7.76 (d, J=2.0 Hz, 1H), 7.72-7.66 (m, 2H), 6.95 (d, J=8.3 Hz, 1H), 3.90 (s, 3H), 2.31 (t, J=7.4 Hz, 2H), 1.68-1.51 (m, 2H), 1.45-1.27 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (3.65 mg), LCMS (ESI): [M+H]+=398.30: 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 9.09 (s, 1H), 8.42-8.31 (m, 2H), 8.29 (d, J=1.3 Hz, 1H), 7.82 (dd, J=8.4, 2.1 Hz, 1H), 7.78 (d, J=2.0 Hz, 1H), 7.15 (d, J=8.5 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.31-3.21 (m, 2H), 1.58-1.47 (m, 2H), 1.43-1.30 (m, 2H), 0.92 (t, J=7.3 Hz, 3H).
Yellow solid (2.30 mg), LCMS (ESI): [M+Na]+=418.20: 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.08 (s, 1H), 8.93 (s, 1H), 7.84 (d, J=1.4 Hz, 1H), 7.76 (d, J=2.0 Hz, 1H), 7.68 (td, J=4.5, 2.1 Hz, 2H), 6.95 (d, J=8.3 Hz, 1H), 3.90 (s, 3H), 2.43 (d, J=7.6 Hz, 2H), 2.13-2.03 (m, 2H), 1.94-1.66 (m, 4H).
Yellow solid (4.61 mg), LCMS (ESI): [M+H]+=507.40: 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.14 (s, 1H), 8.98 (s, 1H), 7.85 (s, 1H), 7.79 (d, J=5.2 Hz, 2H), 7.69 (s, 1H), 7.18 (s, 1H), 3.97 (d, J=5.5 Hz, 2H), 3.91 (s, 3H), 3.48 (d, J=11.6 Hz, 2H), 3.00 (d, J=12.5 Hz, 2H), 2.78 (d, J=4.8 Hz, 3H), 2.43 (d, J=7.5 Hz, 2H), 2.33 (s, 1H), 2.03 (s, 1H), 1.86 (dd, J=11.1, 6.6 Hz, 4H), 1.77-1.71 (m, 4H), 1.46 (s, 2H).
Yellow solid (2.56 mg), LCMS (ESI): [M+H]+=545.50: 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.17 (s, 1H), 9.02 (s, 1H), 7.87 (d, J=1.3 Hz, 1H), 7.82 (d, J=8.1 Hz, 2H), 7.71 (d, J=1.2 Hz, 1H), 7.26 (d, J=8.2 Hz, 1H), 4.44 (s, 2H), 4.02 (s, 2H), 3.93 (s, 3H), 3.73 (s, 4H), 3.27 (s, 4H), 2.78-2.70 (m, 2H), 2.58 (d, J=6.7 Hz, 2H), 2.37 (dd, J=15.4, 5.7 Hz, 2H), 2.04-1.97 (m, 1H).
Yellow solid (2.63 mg), LCMS (ESI): [M+H]+=396.20: 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.16 (s, 1H), 9.00 (s, 1H), 7.89-7.78 (m, 3H), 7.69 (d, J=1.1 Hz, 1H), 7.26 (d, J=8.1 Hz, 1H), 3.93 (s, 3H), 3.28 (s, 2H), 2.69 (dq, J=15.3, 7.7 Hz, 1H), 2.13-2.01 (m, 2H), 1.94-1.67 (m, 4H).
Yellow solid (2.18 mg), LCMS (ESI): [M+H]+=504.40: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (d, J=18.7 Hz, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 7.85 (s, 1H), 7.78 (d, J=6.5 Hz, 2H), 7.69 (s, 1H), 7.60 (s, 1H), 7.38 (s, 1H), 7.16 (d, J=8.9 Hz, 1H), 4.16 (t, J=6.8 Hz, 2H), 3.91 (s, 3H), 3.79 (s, 3H), 2.89 (t, J=6.8 Hz, 2H), 2.69 (dt, J=15.2, 7.7 Hz, 1H), 2.43 (d, J=7.5 Hz, 2H), 2.08 (d, J=4.7 Hz, 2H), 1.90-1.67 (m, 4H).
Yellow solid (2.35 mg), LCMS (ESI): [M+H]+=383.20: 1H NMR (400 MHz, DMSO-d6) δ 9.07 (d, J=16.6 Hz, 2H), 7.90 (d, J=1.2 Hz, 1H), 7.82-7.74 (m, 2H), 7.38 (s, 1H), 7.14 (d, J=8.4 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 2.63 (t, J=7.6 Hz, 2H), 1.69-1.58 (m, 2H), 1.34 (d, J=17.5 Hz, 6H), 0.88 (dd, J=9.1, 4.8 Hz, 3H).
Yellow solid (2.40 mg), LCMS (ESI): [M+H]+=493.50: 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.76 (s, 1H), 7.72 (s, 2H), 7.67 (s, 1H), 7.62 (d, J=8.6 Hz, 2H), 7.01 (d, J=8.3 Hz, 1H), 4.39 (t, J=5.3 Hz, 2H), 4.00 (s, 3H), 3.32 (s, 2H), 3.12 (s, 4H), 2.90-2.76 (m, 1H), 2.53 (d, J=7.5 Hz, 2H), 2.27-2.21 (m, 2H), 2.02 (s, 4H), 1.97-1.89 (m, 2H), 1.86-1.77 (m, 2H).
Yellow solid (2.40 mg), LCMS (ESI): [M+H]+=525.40: 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 1H), 8.78 (s, 1H), 7.74 (dd, J=7.4, 1.6 Hz, 2H), 7.68-7.61 (m, 2H), 7.41 (s, 1H), 7.03 (d, J=8.4 Hz, 1H), 4.28 (s, 2H), 4.02 (s, 3H), 3.10-2.87 (m, 6H), 2.84 (d, J=7.8 Hz, 1H), 2.82-2.63 (m, 4H), 2.52 (d, J=7.6 Hz, 2H), 2.29-2.20 (m, 2H), 2.06-1.89 (m, 2H), 1.88-1.76 (m, 2H), 1.69-1.59 (m, 2H).
Yellow solid (2.60 mg), LCMS (ESI): [M+H]+=523.50: 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.12 (s, 1H), 8.97 (s, 1H), 7.85 (d, J=1.2 Hz, 1H), 7.78 (d, J=1.9 Hz, 1H), 7.74 (dd, J=8.4, 1.9 Hz, 1H), 7.68 (d, J=1.1 Hz, 1H), 7.65-7.52 (m, 1H), 7.09-7.01 (m, 1H), 4.93 (s, 2H), 3.91 (s, 3H), 3.61 (d, J=10.9 Hz, 4H), 2.69 (dt, J=15.5, 8.0 Hz, 1H), 2.42 (d, J=7.6 Hz, 2H), 2.12-2.00 (m, 2H), 1.91-1.79 (m, 2H), 1.77-1.67 (m, 2H), 1.23 (s, 2H), 0.84 (d, J=7.1 Hz, 1H).
Yellow solid (2.20 mg), LCMS (ESI): [M+H]+=619.60: 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.13 (s, 1H), 8.98 (s, 1H), 7.86 (s, 1H), 7.78 (s, 1H), 7.74 (d, J=8.4 Hz, 1H), 7.69 (s, 1H), 7.09 (d, J=8.5 Hz, 1H), 4.97 (s, 2H), 3.92 (s, 3H), 3.58 (d, J=10.7 Hz, 4H), 2.99 (s, 4H), 2.78 (s, 3H), 2.68 (dd, J=15.4, 7.7 Hz, 2H), 2.42 (d, J=7.6 Hz, 2H), 2.24 (s, 2H), 2.07 (d, J=4.7 Hz, 4H), 1.84 (dt, J=10.9, 7.7 Hz, 4H), 1.74 (dd, J=18.4, 8.8 Hz, 2H), 1.23 (s, 2H).
Yellow solid (2.31 mg), LCMS (ESI): [M+H]+=513.40: 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.13 (s, 1H), 8.96 (s, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.80-7.75 (m, 2H), 7.70 (d, J=1.3 Hz, 1H), 7.17 (d, J=9.0 Hz, 1H), 4.16 (s, 2H), 3.89 (s, 3H), 2.79 (s, 6H), 2.62 (s, 4H), 2.31 (t, J=7.4 Hz, 2H), 1.59 (dt, J=15.0, 7.5 Hz, 2H), 1.40-1.27 (m, 2H), 0.95-0.86 (m, 3H).
Yellow solid (2.25 mg), LCMS (ESI): [M+H]+=529.50: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.13 (s, 1H), 8.96 (s, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.82-7.75 (m, 2H), 7.70 (d, J=1.3 Hz, 1H), 7.18 (d, J=9.1 Hz, 1H), 4.17 (t, J=5.7 Hz, 2H), 3.89 (s, 3H), 3.04 (t, J=11.1 Hz, 2H), 2.95-2.68 (m, 8H), 2.31 (t, J=7.4 Hz, 2H), 1.59 (dt, J=15.0, 7.5 Hz, 2H), 1.38-1.28 (m, 2H), 0.94-0.87 (m, 3H).
Yellow solid (2.56 mg), LCMS (ESI): [M+H]+=368.40: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.15 (s, 1H), 9.05 (s, 1H), 7.88 (d, J=1.4 Hz, 1H), 7.77 (d, J=7.8 Hz, 1H), 7.74-7.72 (m, 1H), 7.71 (d, J=1.3 Hz, 1H), 7.49 (t, J=8.0 Hz, 1H), 7.11 (dd, J=8.2, 2.0 Hz, 1H), 3.87 (s, 3H), 2.31 (t, J=7.4 Hz, 2H), 1.64-1.52 (m, 2H), 1.33 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.71 mg), LCMS (ESI): [M+H]+=383.20: 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.02 (s, 1H), 8.84 (s, 1H), 7.81 (d, J=1.3 Hz, 1H), 7.65 (ddd, J=10.0, 8.0, 1.5 Hz, 3H), 6.80 (d, J=8.1 Hz, 1H), 3.90 (s, 3H), 2.30 (t, J=7.4 Hz, 2H), 1.59 (dt, J=15.0, 7.4 Hz, 2H), 1.33 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.11 mg), LCMS (ESI): [M+H]+=354.10: 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.15 (s, 1H), 9.05 (s, 1H), 7.86 (d, J=1.4 Hz, 1H), 7.77 (d, J=7.8 Hz, 1H), 7.74-7.72 (m, 1H), 7.71 (d, J=1.3 Hz, 1H), 7.49 (t, J=8.0 Hz, 1H), 7.11 (dd, J=8.2, 2.0 Hz, 1H), 2.31 (t, J=7.4 Hz, 2H), 1.64-1.52 (m, 2H), 1.33 (q, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.19 mg), LCMS (ESI): [M+H]+=363.50: 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.25 (s, 1H), 9.13 (s, 1H), 8.63 (t, J=1.5 Hz, 1H), 8.59-8.49 (m, 1H), 8.08-7.98 (m, 1H), 7.91 (d, J=1.4 Hz, 1H), 7.81 (t, J=7.8 Hz, 1H), 7.73 (d, J=1.4 Hz, 1H), 2.32 (t, J=7.4 Hz, 2H), 1.60 (dt, J=15.0, 7.5 Hz, 2H), 1.40-1.29 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
4-(6-(4-bromothiophene-2-yl) pyrazine-2-yl)-2-methoxybenzoate methyl ester (300 mg, synthesized using the same method as Example 46 in WO2023/083330), cuprous iodide (24 mg), L-proline (30 mg), dimethyl sulfoxide (3 mL), and ammonia (25% wt, 1 mL) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried to afford methyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-2-methoxybenzoate (crude, 180 mg), which was directly used in the next reaction without any purification. LCMS (ESI): [M+H]+=342.2.
A solution of methyl 4-(6-(4-aminothiophen-2-yl) pyrazin-2-yl)-2-methoxybenzoate (180 mg crude) in dichloromethane (5 mL) was cooled under an ice water bath, and added with triethylamine (107 mg). Additionally, pentanoyl chloride (112 mg) was taken and dissolved in dichloromethane (1 mL) and the resulting mixture was slowly dropped into the above system. Upon addition, the reaction system was warmed to room temperature for 0.5 hour. TLC monitoring showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain methyl 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) benzoate (180 mg, yield 80.3%). LCMS (ESI): [M+H]+=426.10.
Methyl 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) benzoate (180 mg), lithium hydroxide monohydrate (35 mg), tetrahydrofuran (4 mL) and water (2 mL) were added successively to a reaction flask and the mixture was reacted at room temperature for 16 hours. TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove tetrahydrofuran, diluted with water (5 mL), acidified to pH=3-4 using saturated citric acid and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 2-methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) benzoic acid (150 mg, yield 86.2%). LCMS (ESI): [M+H]+=412.10.
2-Methoxy-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) benzoic acid (25 mg), tert butyl 6-(methylamino)-2-azaspiro [3.3] heptane-2-carboxylate (27.12 mg), HATU (45.6 mg), N,N-diisopropylethylamine (16 mg), and N,N-dimethylformamide (1 mL) were sequentially added to a reaction flask and the mixture was reacted at room temperature for 5 hours. TLC monitoring showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (5 mL*3). The organic phases were combined, washed successively with water and saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain tert butyl 6-(2-methoxy-N-methyl-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) benzamido)-2-azaspiro [3.3] heptane-2-carboxylate (25 mg, yield 67.5%). LCMS (ESI): [M+H]+=620.2.
To a solution of tert butyl 6-(2-methoxy-N-methyl-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl) benzamido)-2-azaspiro [3.3] heptane-2-carboxylate (25 mg) in 10 mL of methanol was added TBAF (31 mg), and the mixture was reacted at room temperature overnight. TLC monitoring showed that the reaction was complete. The reaction solution was concentrated and the crude product was used in the next step. LCMS (ESI): [M+H]+=520.2.
To a solution of 2-methoxy-N-methyl-4-(6-(4-pentamidothiophen-2-yl) pyrazin-2-yl)-N-(2-azaspiro [3.3] heptane-6-yl) benzamide (21 mg) in 10 mL of methanol was added formaldehyde (12 mg) and then acetic acid (0.2 mL). The mixture was reacted at room temperature for 30 min. Sodium borohydride acetate (16 mg) was added and the mixture was reacted overnight at room temperature. TLC monitoring showed that the reaction was complete. The reaction solution was concentrated, and purified by thin-layer chromatography to afford 3.5 mg product with a yield of 14%. LCMS (ESI): [M+H]+=534.2. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.23 (d, J=6.7 Hz, 1H), 9.10 (d, J=3.6 Hz, 1H), 7.90 (dd, J=4.4, 1.3 Hz, 1H), 7.87-7.81 (m, 2H), 7.72 (d, J=1.1 Hz, 1H), 7.37-7.30 (m, 1H), 3.99 (dd, J=16.9, 8.5 Hz, 4H), 3.93 (d, J=4.9 Hz, 3H), 2.95 (s, 2H), 2.80 (s, 1H), 2.70 (dd, J=13.9, 5.3 Hz, 3H), 2.32 (t, J=7.4 Hz, 2H), 2.01 (dd, J=14.6, 7.0 Hz, 1H), 1.60 (dt, J=15.0, 7.4 Hz, 2H), 1.36-1.30 (m, 2H), 1.29-1.19 (m, 4H), 0.92 (t, J=7.3 Hz, 3H).
Examples 200-207 and example 223 were prepared using the experimental steps similar to those in Example 199 above.
Yellow solid (1.40 mg), LCMS (ESI): [M+H]+=524.50.
Yellow solid (2.06 mg), LCMS (ESI): [M+H]+=574.40.
Yellow solid (2.19 mg), LCMS (ESI): [M+H]+=582.30.
Yellow solid (1.75 mg), LCMS (ESI): [M+H]+=610.70.
Yellow solid (1.42 mg), LCMS (ESI): [M−H]−=501.30; 1H NMR (400 MHz, DMSO-d6) δ 12.05 (s, 1H), 10.34 (s, 1H), 9.25 (s, 1H), 9.12 (s, 1H), 8.11 (s, 1H), 7.90 (d, J=1.4 Hz, 1H), 7.86 (s, 2H), 7.73 (s, 1H), 7.58 (d, J=8.3 Hz, 1H), 3.91 (s, 3H), 2.33 (d, J=1.9 Hz, 2H), 1.78 (s, 6H), 1.27 (s, 1H).
Yellow solid (1.57 mg), LCMS (ESI): [M+H]+=562.60; 1H NMR (400 MHz, DMSO-d6) δ 10.40 (d, J=15.8 Hz, 1H), 9.23 (d, J=6.7 Hz, 1H), 9.09 (d, J=3.5 Hz, 1H), 7.90 (dd, J=4.4, 1.4 Hz, 1H), 7.84 (d, J=8.1 Hz, 2H), 7.71 (d, J=1.2 Hz, 1H), 7.32 (t, J=7.1 Hz, 1H), 4.91-4.79 (m, 1H), 4.05 (ddd, J=25.6, 21.1, 13.9 Hz, 4H), 3.92 (s, 3H), 3.89-3.85 (m, 2H), 2.95 (s, 2H), 2.81 (d, J=5.0 Hz, 1H), 2.72 (d, J=5.3 Hz, 3H), 2.53 (s, 1H), 2.46 (d, J=9.8 Hz, 1H), 2.43-2.31 (m, 4H), 2.14-1.95 (m, 2H), 1.55 (td, J=10.8, 2.7 Hz, 2H).
Yellow solid (3.51 mg), LCMS (ESI): [M+H]+=576.70; 1H NMR (400 MHz, DMSO-d6) δ 10.41 (d, J=12.3 Hz, 1H), 9.23 (d, J=6.7 Hz, 1H), 9.10 (d, J=3.6 Hz, 1H), 7.89 (d, J=2.9 Hz, 1H), 7.84 (d, J=7.6 Hz, 2H), 7.71 (s, 1H), 7.35-7.29 (m, 1H), 4.88-4.80 (m, 1H), 3.99 (d, J=6.8 Hz, 1H), 3.93 (d, J=4.3 Hz, 3H), 3.89 (s, 2H), 3.71 (dt, J=14.5, 7.2 Hz, 1H), 3.12 (d, J=4.1 Hz, 3H), 2.95 (s, 2H), 2.81 (s, 1H), 2.75-2.65 (m, 3H), 2.49-2.40 (m, 4H), 2.39-2.32 (m, 2H), 2.19 (dd, J=14.9, 7.5 Hz, 2H), 2.03 (dd, J=16.4, 8.7 Hz, 2H), 1.63-1.52 (m, 2H).
Yellow solid (2.21 mg), LCMS (ESI): [M+H]+=652.50; 1H NMR (400 MHz, DMSO-d6) δ 10.42 (d, J=12.5 Hz, 1H), 9.23 (d, J=6.6 Hz, 1H), 9.10 (d, J=3.4 Hz, 1H), 7.92-7.87 (m, 1H), 7.84 (d, J=8.3 Hz, 2H), 7.71 (s, 1H), 7.32 (dt, J=10.3, 5.5 Hz, 5H), 4.91-4.76 (m, 1H), 4.43-4.29 (m, 3H), 4.14-4.04 (m, 2H), 3.97 (d, J=8.6 Hz, 1H), 3.93 (d, J=5.6 Hz, 4H), 3.89 (d, J=7.1 Hz, 2H), 2.95 (s, 2H), 2.81 (d, J=5.0 Hz, 1H), 2.72 (d, J=4.2 Hz, 3H), 2.56 (s, 1H), 2.48-2.38 (m, 4H), 2.12 (ddd, J=31.5, 19.7, 5.9 Hz, 2H), 1.65 (dd, J=19.2, 9.4 Hz, 2H).
6-Benzyl-2,4-dichloro-5,6,7,8-tetrahydropyridino [4,3-D] pyrimidine (3.7 g) and zinc powder (6.78 g) were dissolved in anhydrous ethanol (20 mL), and then added with ammonia (2.19 g). The mixture was reacted overnight at 80° C. TLC detection showed that the reaction was completed. The reaction solution was filtered and the filter cake was washed with ethyl acetate. The reaction solution and the washing solution were combined and concentrated. The residue was purified by column chromatography to afford 6-benzyl-2-chloro-5,6,7,8-tetrahydropyrido [4,3-D] pyrimidine (1.34 g, yield 42%). LCMS (ESI): [M+H]+=260.0.
4-Bromo-3-methoxyaniline (3 g), di-tert-butyl dicarbonate (6.5 g), and triethylamine (3 g) were dissolved in tetrahydrofuran and the mixture was reacted at 70° C. for 8 hours. TLC monitoring showed that the reaction was complete. The reaction solution was concentrated and purified by column chromatography to afford product tert butyl (4-bromo-3-methoxyphenyl) carbamate (4.3 mg, yield 96%). LCMS (ESI): [M+H]+=302.1.
Tert butyl (4-bromo-3-methoxyphenyl) carbamate (2.5 g), potassium acetate (1.2 g), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (300 mg), bis(pinacolato)diboron (2.5 g) and 1,4-dioxane (40 mL) were successively added to a reaction flask and the mixture was reacted under nitrogen protection at 80° C. for 5 hours. TLC monitoring showed that the reaction was complete. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated salt water, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain tert butyl (3-Methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)carbamate (1.5 g, yield 52%). LCMS (ESI): [M+H]+=350.1.
6-benzyl-2-chloro-5,6,7,8-tetrahydropyrido [4,3-D] pyrimidine (500 mg), tert butyl (3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)carbamate (1.35 mg), tripotassium phosphate (1.05 g), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (140 mg), 1,4-dioxane (40 mL), and water (10 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 100° C. 14 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and re-dissolved with ethyl acetate (50 mL). The organic phase was washed successively with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain tert butyl (4-(6-benzyl-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidin-2-yl)-3-methoxyphenyl) carbamate (800 mg, yield 93%). LCMS (ESI): [M+H]+=447.1.
To a solution of tert butyl (4-(6-benzyl-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidin-2-yl)-3-methoxyphenyl) carbamate (800 mg) in methanol (10 mL) was added 3M Hydrogen chloride methanol solution (40 mL). The mixture was reacted at 50° C. for 14 hours. TLC detection showed that the reaction was completed. The reaction solution was concentrated to afford 700 mg crude product, which was directly used in the next step.
(4-(6-benzyl-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidin-2-yl)-3-methoxyaniline (700 mg) was dissolved in water (4 mL) and hydrobromic acid (2 mL), and the mixture was cooled to 0° C. under an ice-salt bath, added dropwise with sodium nitrite (330 mg), and then reacted under an ice-salt bath for 30 minutes. A solution of cuprous bromide (314 mg) in water (2 mL) and hydrobromic acid (2 mL) was added dropwise to the reaction solution, and the mixture was reacted overnight at room temperature. TLC detection showed that the reaction was completed. The reaction solution was diluted with water, and extracted with dichloromethane. The organic phase was washed with saturated salt water, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to obtain 6-benzyl-2-(4-bromo-2-methoxyphenyl)-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine (140 mg, yield 17%). LCMS (ESI): [M+H]+=410.0.
6-benzyl-2-(4-bromo-2-methoxyphenyl)-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine (220 mg), potassium acetate (127 mg), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (20 mg), bis(pinacolato)diboron (204 mg) and 1,4-dioxane (40 mL) were successively added to a reaction flask and the mixture was reacted under nitrogen protection at 80° C. overnight. TLC monitoring showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated and purified by thin-layer chromatography to obtain the product 6-benzyl-2-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine (200 mg, yield 83%). LCMS (ESI): [M+H]+=458.1.
6-benzyl-2-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine (200 mg), 2,6-dibromopyrazine (160 mg), potassium carbonate (145 mg), tetrakis(triphenylphosphine)palladium (26 mg), 1,4-dioxane (16 mL), and water (4 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. overnight, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and re-dissolved with ethyl acetate (50 mL). The organic phase was washed successively with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 6-benzyl-2-(4-(6-bromopyrazin-2-yl)-2-methoxyphenyl)-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine (110 mg, yield 51.4%). LCMS (ESI): [M+H]+=488.0.
6-benzyl-2-(4-(6-bromopyrazin-2-yl)-2-methoxyphenyl)-5,6,7,8-tetrahydropyrido [4,3-d]pyrimidine (600 mgl), (4-bromothiophen-2-yl) boronic acid (52 mg), tetrakis(triphenylphosphine)palladium (12 mg), potassium carbonate (70 mg), 1,4-dioxane (16 mL), and water (4 mL) were successively added to a reaction flask. The mixture was reacted under nitrogen protection at 60° C. for 0.5 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and added with dichloromethane (30 mL) and water (10 mL), and fractioned. The aqueous phase was extracted with dichloromethane (20 mL*2), and the organic phases were combined, washed with saturated salt water (20 mL*1), dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 6-benzyl-2-(4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-2-methoxyphenyl)-5,6,7,8-tetrahydropyrido [4,3-d]pyrimidine (120 mg, yield 94%). LCMS (ESI): [M+H]+=569.0.
6-benzyl-2-(4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-2-methoxyphenyl)-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine (120 mg), cuprous iodide (8 mg), L-proline (10 mg), dimethyl sulfoxide (3 mL), and ammonia (25% wt, 1.0 mL) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain 5-(6-(4-(6-benzyl-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidin-2-yl)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-amine (crude, 95 mg), which was directly used in the next reaction without any purification.
5-(6-(4-(6-benzyl-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidin-2-yl)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-amine (crude, 95 mg) was added into DCM (10 mL). The flask was sequentially added with cyclobutyl acetic acid (26 mg), EDCI (72 mg), HOBT (51 mg), and DIEA (97 mg), and the mixture was reacted at room temperature for 14 hours. LCMS detection showed that the reaction was complete. The reaction solution was washed with water and the organic phase was dried over anhydrous sodium sulfate and spin-dried. The residue was purified by column chromatography to afford N-(5-(6-(4-(6-benzyl-5,6,7,8-tetrahydropyrido [4,3-d] pyrimidin-2-yl)-3-methoxyphenyl) pyrazin-2-yl) thiophen-3-yl)-2-cyclobutyl acetamide (20 mg, yield 18%). LCMS (ESI): [M+H]+=603.1. 1HNMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.84 (s, 1H), 8.53 (s, 1H), 7.86 (s, 1H), 7.81 (d, J=7.9 Hz, 1H), 7.78-7.72 (m, 2H), 7.67 (d, J=1.2 Hz, 1H), 7.49-7.38 (m, 5H), 4.01 (s, 3H), 3.78 (d, J=34.2 Hz, 4H), 3.16 (s, 2H), 2.99 (s, 2H), 2.82 (dd, J=15.6, 7.9 Hz, 1H), 2.52 (d, J=7.6 Hz, 2H), 2.28-2.21 (m, 2H), 2.01-1.91 (m, 2H), 1.86-1.78 (m, 2H).
To a solution of 4-bromo-1H-pyrrolo [2,3-b] pyridine (500 mg) in anhydrous tetrahydrofuran (10 mL) was added NaH (121 mg). The reaction was carried out under an ice water bath for 30 minutes, and then added with SEMCl. The reaction was carried out at room temperature for 14 hours. TLC detection showed that the reaction was completed. The reaction solution was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain product 4-bromo-1-((2-(trimethylsilyl) ethoxy) methyl)-1H pyrrolo [2,3-b] pyridine (530 mg, yield 64%). LCMS (ESI): [M+H]+=327.1.
4-bromo-1-((2-(trimethylsilyl) ethoxy) methyl)-1H pyrrolo [2,3-b] pyridine (530 mg), potassium acetate (400 mg), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (60 mg), bis(pinacolato)diboron (500 mg) and 1,4-dioxane (40 mL) were successively added to a reaction flask and the mixture was reacted under nitrogen protection at 80° C. overnight. TLC monitoring showed that the reaction was complete. The reaction solution was filtered and the filtrate was concentrated, and purified by thin-layer chromatography to obtain the product 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrrolo [2,3-b] pyridine (300 mg, yield 49.4%). LCMS (ESI): [M+H]+=375.1.
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrrolo [2,3-b] pyridine (300 mg), 2,6-dibromopyrazine (284 mg), potassium carbonate (266 mg), tetrakis(triphenylphosphine)palladium (46.5 mg), 1,4-dioxane (16 mL), and water (4 mL) were added successively to a reaction flask. The mixture was reacted under nitrogen protection at 80° C. overnight, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and re-dissolved with ethyl acetate (50 mL). The organic phase was washed successively with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 4-(6-bromopyrazin-2-yl)-1-((2-(trimethylsilyl) ethoxy) methyl)-1H pyrrolo [2,3-b] pyridine (270 mg, yield 83.1%). LCMS (ESI): [M+H]+=405.1.
4-(6-bromopyrazin-2-yl)-1-((2-(trimethylsilyl) ethoxy) methyl)-1H pyrrolo [2,3-b] pyridine (270 mgl), (4-bromothiophen-2-yl) boronic acid (150 mg), tetrakis(triphenylphosphine)palladium (40 mg), potassium carbonate (222 mg), 1,4-dioxane (16 mL), and water (mL) were successively added to a reaction flask. The mixture was reacted under nitrogen protection at 60° C. for 0.5 hours, and TLC monitoring showed that the reaction was complete. The reaction solution was evaporated to remove the most of 1,4-dioxane, and then added with dichloromethane (30 mL) and water (10 mL), and fractioned. The aqueous phase was extracted with dichloromethane (20 mL*2), and the organic phases were combined, washed with saturated salt water (20 mL*1), dried over anhydrous sodium sulfate, spin-dried and purified by column chromatography to obtain 4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrrolo [2,3-b] pyridine (140 mg, yield 43%). LCMS (ESI): [M+H]+=487.1.
4-(6-(4-bromothiophen-2-yl) pyrazin-2-yl)-1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrrolo [2,3-b] pyridine (140 mg), cuprous iodide (11 mg), L-proline (14 mg), dimethyl sulfoxide (3 mL), and ammonia (25% wt, 1 mL) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 10 hours. TLC monitoring showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain 5-(6-(1-(2-(trimethylsilyl) ethoxy) methyl)-1H-indol-4-yl) pyrazine-2-yl) thiophen-3-amine (crude, 95 mg), which was directly used in the next reaction without any purification. LCMS (ESI): [M+H]+=423.1.
5-(6-(1-(2-(trimethylsilyl) ethoxy) methyl)-1H-indol-4-yl) pyrazine-2-yl) thiophen-3-amine (crude, 95 mg) was added to DMF (10 mL). The flask was sequentially added with cyclobutyl acetic acid (26 mg), EDCI (72 mg), HOBT (51 mg), and DIEA (97 mg), and the mixture was reacted at room temperature for 14 hours. LCMS detection showed that the reaction was complete. The reaction solution was washed with water and the organic phase was dried over anhydrous sodium sulfate and spin-dried. The residue was purified by column chromatography to afford 2-cyclobutyl-N-(5-(6-(1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrrolo [2,3-b] pyridin-4-yl) pyrazin-2-yl) thiophen-3-yl) acetamide (60 mg, yield 53%). LCMS (ESI): [M+H]+=520.1.
To a solution of 2-cyclobutyl-N-(5-(6-(1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrrolo [2,3-b] pyridin-4-yl) pyrazin-2-yl) thiophen-3-yl) acetamide (20 mg) in DCM (10 mL) was added TFA (1 mL) and the mixture was reacted overnight at room temperature. The reaction solution was concentrated, then added with ethanol (10 mL) and amine in methanol (2 mL). The mixture was reacted overnight at room temperature. LCMS monitoring showed that the reaction was complete. The reaction solution was concentrated and purified by thin-layer chromatography to obtain 3 mg of the product. LCMS (ESI): [M+H]+=390.1. 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 10.39 (s, 1H), 9.27 (s, 1H), 9.16 (s, 1H), 8.40 (d, J=5.0 Hz, 1H), 7.92 (d, J=1.4 Hz, 1H), 7.80 (d, J=5.1 Hz, 1H), 7.74 (d, J=1.3 Hz, 1H), 7.71-7.66 (m, 1H), 7.25 (dd, J=3.4, 1.9 Hz, 1H), 2.70 (dd, J=13.6, 5.9 Hz, 1H), 2.44 (d, J=7.6 Hz, 2H), 2.14-2.02 (m, 2H), 1.91-1.80 (m, 2H), 1.75 (dd, J=17.9, 8.3 Hz, 2H).
Examples 210-215 and example 224 were prepared using the experimental steps similar to those in Example 209 above.
Yellow solid (2.05 mg), LCMS (ESI): [M+H]+=432.50; 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.26 (s, 1H), 9.16 (s, 1H), 8.43 (d, J=5.0 Hz, 1H), 7.92 (d, J=1.4 Hz, 1H), 7.89 (d, J=3.6 Hz, 1H), 7.81 (d, J=5.0 Hz, 1H), 7.75 (t, J=2.7 Hz, 1H), 7.25 (d, J=3.6 Hz, 1H), 5.26-5.13 (m, 1H), 2.81-2.63 (m, 2H), 2.44 (d, J=7.6 Hz, 2H), 2.15-2.02 (m, 2H), 1.94-1.65 (m, 4H), 1.52 (d, J=6.8 Hz, 6H).
Yellow solid (2.08 mg), LCMS (ESI): [M+H]+=480.50; 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.27 (s, 1H), 9.16 (s, 1H), 8.46 (d, J=5.0 Hz, 1H), 7.92 (d, J=1.4 Hz, 1H), 7.85 (dd, J=5.3, 4.4 Hz, 2H), 7.74 (d, J=1.3 Hz, 1H), 7.33-7.22 (m, 6H), 5.58 (s, 2H), 2.79-2.62 (m, 1H), 2.42 (dd, J=15.6, 7.6 Hz, 2H), 2.15-1.97 (m, 2H), 1.97-1.65 (m, 4H).
Yellow solid (3.77 mg), LCMS (ESI): [M+H]+=430.50; 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.29 (s, 1H), 9.09 (s, 1H), 8.52 (s, 1H), 8.11 (s, 1H), 8.01 (dd, J=8.5, 1.2 Hz, 1H), 7.92 (dd, J=13.3, 4.9 Hz, 2H), 7.72 (d, J=1.3 Hz, 1H), 3.94-3.85 (m, 1H), 2.79-2.64 (m, 1H), 2.44 (d, J=7.6 Hz, 2H), 2.16-2.01 (m, 2H), 1.92-1.68 (m, 4H), 1.23-1.16 (m, 4H).
Yellow solid (3.23 mg), LCMS (ESI): [M+H]+=432.50; 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.31 (s, 1H), 9.07 (s, 1H), 8.54 (s, 1H), 8.16 (s, 1H), 8.00 (dd, J=8.5, 1.2 Hz, 1H), 7.94 (d, J=8.5 Hz, 1H), 7.90 (d, J=1.4 Hz, 1H), 7.72 (d, J=1.3 Hz, 1H), 5.18 (dt, J=13.3, 6.6 Hz, 2H), 2.78-2.63 (m, 2H), 2.44 (d, J=7.6 Hz, 2H), 2.16-2.00 (m, 2H), 1.98-1.67 (m, 4H), 1.55 (d, J=6.6 Hz, 6H).
Yellow solid (2.09 mg), LCMS (ESI): [M+H]+=520.30; 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.12 (s, 1H), 9.02 (s, 1H), 7.88 (d, J=1.3 Hz, 1H), 7.74 (d, J=8.1 Hz, 2H), 7.71 (d, J=1.3 Hz, 1H), 4.42-4.28 (m, 2H), 3.96 (s, 3H), 2.93 (s, 3H), 2.30 (dd, J=16.9, 9.4 Hz, 5H), 2.09 (s, 2H), 1.75 (dd, J=35.0, 11.1 Hz, 4H), 1.60 (dt, J=15.0, 7.5 Hz, 2H), 1.34 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (3.19 mg), LCMS (ESI): [M+H]+=472.50; 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.28 (s, 1H), 9.18 (s, 1H), 8.50 (d, J=5.0 Hz, 1H), 7.92 (dd, J=5.4, 3.2 Hz, 2H), 7.81 (d, J=3.6 Hz, 1H), 7.75 (dd, J=5.7, 1.3 Hz, 1H), 7.36 (d, J=3.6 Hz, 1H), 5.28 (q, J=9.3 Hz, 2H), 2.79-2.60 (m, 1H), 2.43 (d, J=7.6 Hz, 2H), 2.15-2.00 (m, 2H), 1.89-1.79 (m, 2H), 1.79-1.66 (m, 2H).
1,3-dimethyl-1H-pyrazole-4-boronic acid, pinacol ester (200 mg), 2,6-dibromopyrazine (278 mg), and tetrakis(triphenylphosphine)palladium (26 mg) were added to 1,4-dioxane/water (4:1, 12.5 mL) and the mixture was reacted under nitrogen protection at 80° C. for 6 hours. TLC monitoring showed that the reaction was complete. The reaction solution was diluted with sufficient water and extracted with ethyl acetate. The organic phases were combined, washed with saturated saline, dried over anhydrous sodium sulfate, and spin-dried. The residue was purified by column chromatography to afford 2-bromo-6-(1,3-dimethylpyrazol-4-yl) pyrazine (160 mg, yield 70.2%). 1HNMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.47 (s, 1H), 7.93 (s, 1H), 3.94 (s, 3H), 2.58 (s, 3H).
2-bromo-6-(1,3-dimethylpyrazol-4-yl) pyrazine (160 mg), (4-bromothiophen-2-yl) boronic acid (157 mg), tetrakis(triphenylphosphine)palladium (37 mg), potassium carbonate (210 mg) were added to 1,4-dioxane/water (4:1, 10 mL). The mixture was reacted under nitrogen protection at 60° C. for 2 hours, and LCMS monitoring showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain 2-(4-bromothiophen-2-yl)-6-(1,3-dimethylpyrazol-4-yl) pyrazine (154 mg, yield 72.9%). LCMS (ESI): [M+H]+=334.90.
2-(4-bromothiophen-2-yl)-6-(1,3-dimethylpyrazol-4-yl) pyrazine (154 mg), cuprous iodide (35 mg), L-proline (42 mg), dimethyl sulfoxide (2.5 mL), and ammonia (25% wt, 386 mg) were sequentially added to a sealed tube, and the reaction was sealed and reacted at 80° C. for 14 hours. LCMS detection showed that the reaction was complete. The reaction solution was cooled to room temperature, added with 20 mL of water, and extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated ammonium chloride (10 mL*2), dried over anhydrous sodium sulfate, and spin-dried to obtain 5-[6-(1,3-dimethylpyrazol-4-yl) pyrazin-2-yl] thiophen-3-amine (DK415-03, crude, 35 mg), which was directly used in the next reaction without any purification. LCMS (ESI): [M+H]+=272.10.
5-[6-(1,3-dimethylpyrazol-4-yl) pyrazin-2-yl] thiophen-3-amine (crude, 35 mg), DCM (5 mL), and triethylamine (26 mg) were sequentially added to a reaction flask. The mixture was cooled under an ice water bath, and added with pentanoyl chloride (24 mg). The reaction was then continued under an ice water bath for 0.5 hours. LCMS detection showed that the reaction was complete. The reaction solution was directly spin dried and purified by column chromatography to obtain N-{5-[6-(1,3-dimethylpyrazol-4-yl) pyrazin-2-yl] thiophen-3-yl} pentanamide (8 mg, yield 17%). LCMS (ESI): [M+H]+=356.20. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.82 (s, 1H), 8.75 (s, 1H), 8.41 (s, 1H), 7.80 (d, J=1.4 Hz, 1H), 7.68 (d, J=1.3 Hz, 1H), 3.84 (s, 3H), 2.54 (s, 3H), 2.31 (t, J=7.4 Hz, 2H), 1.59 (dt, J=15.0, 7.5 Hz, 2H), 1.40-1.29 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
Examples 217-222 were prepared using the experimental steps similar to those in Example 216 above.
Yellow solid (2.46 mg), LCMS (ESI): [M+H]+=345.30: 1HNMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.29 (s, 1H), 9.19 (s, 1H), 9.01 (s, 1H), 8.81 (s, 1H), 7.87 (d, J=1.1 Hz, 1H), 7.73 (s, 1H), 2.31 (t, J=7.4 Hz, 2H), 1.68-1.49 (m, 2H), 1.43-1.26 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (1.96 mg), LCMS (ESI): [M+H]+=388.40: 1HNMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.02 (s, 1H), 8.87 (s, 1H), 7.83 (dd, J=3.6, 2.7 Hz, 2H), 7.70 (d, J=1.3 Hz, 1H), 7.00 (d, J=3.7 Hz, 1H), 4.89 (t, J=5.2 Hz, 1H), 3.68 (dd, J=11.8, 6.4 Hz, 2H), 2.99 (t, J=6.5 Hz, 2H), 2.37-2.26 (m, 2H), 1.60 (dt, J=15.1, 7.4 Hz, 2H), 1.38-1.30 (m, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.97 mg), LCMS (ESI): [M+H]+=344.20: 1HNMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.09 (s, 1H), 8.93 (s, 1H), 8.02 (dd, J=3.7, 1.1 Hz, 1H), 7.85 (d, J=1.4 Hz, 1H), 7.80 (dd, J=5.0, 1.0 Hz, 1H), 7.71 (d, J=1.4 Hz, 1H), 7.26 (dd, J=5.0, 3.7 Hz, 1H), 2.31 (t, J=7.4 Hz, 2H), 1.69-1.51 (m, 2H), 1.34 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (3.95 mg), LCMS (ESI): [M+H]+=328.20: 1HNMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.93 (s, 1H), 8.91 (s, 1H), 8.51 (dd, J=1.4, 0.8 Hz, 1H), 7.88 (t, J=1.7 Hz, 1H), 7.84 (d, J=1.4 Hz, 1H), 7.70 (d, J=1.4 Hz, 1H), 7.14 (dd, J=1.9, 0.8 Hz, 1H), 2.31 (t, J=7.4 Hz, 2H), 1.60 (dt, J=15.0, 7.5 Hz, 2H), 1.34 (dq, J=14.5, 7.3 Hz, 2H), 0.91 (t, J=7.3 Hz, 3H).
Yellow solid (2.14 mg), LCMS (ESI): [M+H]+=446.20.
Yellow solid (3.16 mg), LCMS (ESI): [M+H]+=402.40: 1HNMR (400 MHz, DMSO-d6) 10.43 (s, 1H), 9.21 (s, 1H), 9.05 (s, 1H), 8.09 (d, J=4.0 Hz, 1H), 7.90 (dd, J=5.6, 2.7 Hz, 2H), 7.74 (d, J=1.4 Hz, 1H), 3.88 (s, 3H), 2.34-2.29 (m, 2H), 2.04-1.97 (m, 2H), 1.59 (d, J=7.5 Hz, 2H), 1.34 (d, J=7.8 Hz, 3H).
Yellow solid (1.56 mg), LCMS (ESI): [M+H]+=535.60: 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.46 (d, J=10.0 Hz, 1H), 9.18 (d, J=1.6 Hz, 1H), 7.95-7.83 (m, 3H), 7.43-7.31 (m, 1H), 3.94 (d, J=2.4 Hz, 3H), 2.89 (s, 2H), 2.78 (s, 1H), 2.73-2.61 (m, 3H), 2.38-2.19 (m, 4H), 2.17-1.97 (m, 4H), 1.93-1.78 (m, 4H), 1.77-1.47 (m, 6H).
Yellow solid (2.07 mg), LCMS (ESI): [M+H]+=404.40: 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.28 (s, 1H), 9.16 (s, 1H), 8.45 (d, J=5.0 Hz, 1H), 7.92 (d, J=1.2 Hz, 1H), 7.84 (d, J=5.1 Hz, 1H), 7.74 (d, J=1.6 Hz, 2H), 7.25 (d, J=3.4 Hz, 1H), 3.91 (s, 3H), 2.77-2.62 (m, 1H), 2.44 (d, J=7.6 Hz, 2H), 2.15-2.02 (m, 2H), 1.92-1.80 (m, 2H), 1.80-1.68 (m, 2H).
The activity of Drak2 was detected using the ADP-Glo kinase assay kit from Promega. The reaction for this method was in a 384 white shallow well plate and the total volume of the reaction was 5 μL. Specifically, including 1 μL of the compound to be tested (2% DMSO), 2 μL of Drak2, and 2 μL of ATP. The reaction buffer system includes 50 mM Na3PO4, 0.02% NaN3, 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% (w/v) BSA, and 50 mM HEPES with pH value of 7.0 (the above reaction buffer system was used to dilute Drak2 and ATP, and the above concentrations were their respective concentrations in the total reaction volume of 5 μL). After incubating at room temperature for two hours, 5 μL of ADP Glo termination buffer (reagent in the kit) was added to terminate the kinase reaction and consume the remaining ATP. After one hour of reaction, a kinase detection reagent (reagent in the kit) was added and incubated for half an hour. It not only converted ADP to ATP, but also used a coupled luciferase/luciferin (reagent in the kit) reaction to detect newly synthesized ATP. An Envision multifunctional microplate enzyme-linked immunosorbent assay instrument was used to read the value and detect the effect of the compounds to be tested on Drak2 kinase activity. Envision parameter was set to Aperture, and the specific selection was 384 plate US Luminescence Aperture-In. Background pores without Drak2 and Drak2 full enzyme active pores without compounds were set up in the reaction.
The IC50 value of the compound inhibiting Drak2 kinase activity was calculated using Graphpad Prism 7.00 software, using the formula: Y=100/(1+10{circumflex over ( )}(Log IC50−X)*HillSlope). Activity range: A: <10 nM; B: 11-100 nM; C: 101-1000 nM; D: 1001-10000 nM; E: >10000 nM.
It can be concluded from the activity data from above table that the compound of the present invention possess Drak2 kinase inhibitory activity.
All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that, after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111342457.7 | Nov 2021 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/131513 | Nov 2022 | WO |
| Child | 18662284 | US |
