Bromodomain selective inhibitors of the dihydroquinoxaline class and methods of making and uses thereof

Abstract

The application discloses a kind of dihydroquinoxaline selective bromine domain recognition protein inhibitor and its preparation method and purposes. According to the application, the compound represented by the following general formula (I), its stereoisomer, prodrug, functional molecule of PROTAC technology, pharmaceutically acceptable salt or solvate is provided, and the substituent group is defined as described in the specification. According to the dihydroquinoxaline compound of the application, as BET inhibitor, relative to prior art, the selectivity of kinase is higher, can be used for the treatment of polycystic kidney, cancer and BET expression abnormality related disease, and has good safety.

Description

Technical Field

[0001] This invention relates to a class of dihydroquinoxaline-based selective bromine domain recognition protein inhibitors, their compounds, compositions, preparation, and applications. Background Technology

[0002] In 1992, Tamkun et al. discovered a common domain in Drosophila and female sterility homologous proteins and named it bromodomains (BRDs). Bromodomains are a class of evolutionarily conserved proteins and important epigenetic "readers." They can recognize and bind acetylated lysine residues at the tail of histones, recruiting chromatin regulation-related proteins, transcription factors, chromatin remodeling factors, and other related proteins to accumulate at specific gene transcription sites. By regulating the activity of RNA polymerase II (RNA pol-II), they influence gene transcription regulation. Sequence analysis has identified 61 bromodomains in 46 different proteins in the human genome. Based on sequence homology, these 61 bromodomains can be divided into eight families, with the bromodomain and extra-terminal (BET) family being the most extensively studied. They play a crucial role not only in malignant transformation of cells but also in tumor cell invasion and metastasis. Studies have confirmed that many human diseases are inextricably linked to BET proteins, such as cancer, inflammation, atherosclerosis, hematological diseases, and viral infections, with the BRD4 target being of particular interest. Most reported BET inhibitors currently possess partial kinase activity, thus exhibiting some toxicity. These multi-target inhibitors are often used in cancer treatment because some kinases are associated with tumor development and progression, and cancer drugs generally have a higher tolerance for compound toxicity compared to other diseases. However, diseases such as inflammation, cardiovascular disease, and polycystic kidney disease require even higher drug safety standards, thus necessitating BET inhibitors with higher kinase selectivity, while also achieving safer efficacy in cancer treatment. Summary of the Invention

[0003] This invention provides a class of selective BET inhibitors based on the dihydroquinoxaline nucleus, which can be used to treat polycystic kidney disease, cancer, and diseases related to abnormal BET expression, and has good safety.

[0004] According to one aspect of the present invention, a compound represented by the following general formula (I) is provided, including its stereoisomers, prodrugs, functional molecules of PROTAC technology, pharmaceutically acceptable salts or solvates.

[0005]

[0006] in,

[0007] X and Y are each independently CH or N;

[0008] R 1 Selected from substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted 3- to 8-membered cycloalkyl, substituted or unsubstituted 3- to 10-membered heterocycloalkyl, substituted or unsubstituted C6-C 20 Aryl, substituted or unsubstituted 5- to 20-membered heteroaryl groups, wherein the heteroaryl and heterocyclic alkyl groups each contain at least one heteroatom selected from nitrogen, oxygen or sulfur, and the substituents used for substitution are selected from C1-C6 alkyl, halogenated C1-C6 alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, 3- to 8-membered cycloalkyl, cyano, halogen, hydroxyl, nitro, amino;

[0009] In particular, R 1 Selected from substituted or unsubstituted C1-C4 alkyl groups, substituted or unsubstituted C4 alkyl groups. 1-4 Alkoxy, substituted or unsubstituted 3- to 6-membered cycloalkyl, substituted or unsubstituted 3- to 6-membered heterocycloalkyl, substituted or unsubstituted C6-C 10 The aryl, substituted, or unsubstituted 5- to 10-membered heteroaryl groups, wherein the heteroaryl and heterocyclic alkyl groups each contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, or sulfur, and the substituents used for substitution are selected from C1-C4 alkyl, halo-C1-C4 alkyl, C1-4 alkoxy, halo-C 1-4 Alkoxy, 3- to 6-membered cycloalkyl, cyano, halogen, hydroxyl, nitro, amino;

[0010] More specifically, R 1 Selected from substituted or unsubstituted C1-C4 alkyl groups, substituted or unsubstituted C4 alkyl groups. 1-4 The alkyl group comprises an alkoxy group, a substituted or unsubstituted 3- to 6-membered cycloalkyl group, a substituted or unsubstituted 3- to 6-membered heterocycloalkyl group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted naphthyl group, a substituted or unsubstituted indazole group, a substituted or unsubstituted indole group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted pyridyl group, a substituted or unsubstituted oxazolyl group, or a substituted or unsubstituted isoxazolyl group, wherein the heterocycloalkyl group contains at least one heteroatom selected from nitrogen, oxygen, or sulfur, and the substituents used for substitution are selected from C1-C4 alkyl groups, halogenated C1-C4 alkyl groups, and C1-C4 alkyl groups. 1-4 Alkoxy, halogenated C 1-4 Alkoxy, 3- to 6-membered cycloalkyl, cyano, halogen, hydroxyl, nitro, amino, for example, the substituents used for substitution are selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, fluorine, chlorine, bromine, methoxy, ethoxy, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, nitro, amino;

[0011] For example, R 1 It can be a group selected from the following structures:

[0012]

[0013] in,

[0014] W is independently for -CR 6 Alternatively, -N; V is independently -CR 6 -NR 7 Or -O;

[0015] R 6 Each is independently selected from H, C1-C6 alkyl, C 1-6 Alkoxy, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, C5-C 20 Aryl, 5 to 20-membered heteroaryl, halogen, cyano, trifluoromethyl, trifluoromethoxy, hydroxy, nitro or -NR 8 R 9 The heteroaryl and heterocycloalkyl groups each contain at least one heteroatom selected from nitrogen, oxygen, or sulfur, and the C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, C5-C 20 The aryl group, or 5 to 20-membered heteroaryl group, may be optionally substituted with one or more of the following groups: C 1-6 Alkyl, C 1-6 Alkoxy, 3-8 membered cycloalkyl, 3-8 membered heterocycloalkyl, C 1-6 Alkyl-substituted 3-8 membered cycloalkyl, C 1-6 Alkyl substitution of 3-8 membered heterocyclic alkyl groups, halogens, cyano groups, trifluoromethyl groups, trifluoromethoxy groups, hydroxyl groups, nitro groups, and amino groups;

[0016] In particular, R 6 Each is independently selected from H, C1-C4 alkyl, C 1-4 Alkoxy, 3- to 6-membered cycloalkyl, 3- to 6-membered heterocycloalkyl, C6-C 10 Aryl, 5- to 10-membered heteroaryl, halogen, cyano, trifluoromethyl, trifluoromethoxy, hydroxy, nitro or -NR 8 R 9 The heteroaryl and heterocycloalkyl groups each contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, or sulfur, and the C 1-4 Alkyl, C 1-4 Alkoxy, 3- to 6-membered cycloalkyl, 3- to 6-membered heterocycloalkyl, C6-C 10 The aryl group, or 5- to 10-membered heteroaryl group, may be optionally substituted with one or more of the following groups: C 1-4 Alkyl, C 1-4 Alkoxy, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 1-4Alkyl substitution of 3-6 membered heterocyclic alkyl groups, halogens, cyano groups, trifluoromethyl groups, trifluoromethoxy groups, hydroxyl groups, nitro groups, and amino groups;

[0017] R 7 Selected from H, C 1-6 Alkyl, or 3- to 8-membered cycloalkyl, particularly, R 7 Selected from H, C 1-4 Alkyl, or 3- to 6-membered cycloalkyl;

[0018] R 8 and R 9 Each is independently selected from H, C1-C6 alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, 5- to 10-membered aryl, or 5- to 10-membered heteroaryl; the C 1-6 Alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, 5- to 10-membered aryl, or 5- to 10-membered heteroaryl may optionally be further substituted with one or more of the following groups: C 1-6 Alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, halogen, hydroxyl, nitro or amino; or R 8 and R 9 They can form 5-6 membered rings with the N atoms attached to them; the heteroaryl and heterocycloalkyl groups each contain at least one, for example, 1, 2 or 3 heteroatoms selected from nitrogen, oxygen or sulfur;

[0019] For example, R 1 The following structures can be selected:

[0020]

[0021] R 6 Each is independently selected from H, C1-C6 alkyl, C 1-6 Alkoxy, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, C5-C 20 Aryl, 5 to 20-membered heteroaryl, halogen, cyano, trifluoromethyl, trifluoromethoxy, hydroxy, nitro or -NR 8 R 9 The C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, C5-C 20 The aryl group, or 5 to 20-membered heteroaryl group, may be optionally substituted with one or more of the following groups: C 1-6 Alkyl, C 1-6 Alkoxy, 3-8 membered cycloalkyl, 3-8 membered heterocycloalkyl, C 1-6 Alkyl-substituted 3-8 membered cycloalkyl, C 1-6 Alkyl-substituted 3-8 membered heterocyclic alkyl groups, halogens, cyano groups, trifluoromethyl groups, trifluoromethoxy groups, hydroxyl groups, nitro groups, and amino groups; wherein the heteroaryl and heterocyclic alkyl groups each contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0022] In particular, R 6 Each is independently selected from H, C1-C4 alkyl, C 1-4 Alkoxy, 3- to 6-membered cycloalkyl, 3- to 6-membered heterocycloalkyl, C6-C 10 Aryl, 5- to 10-membered heteroaryl, halogen, cyano, trifluoromethyl, trifluoromethoxy, hydroxy, nitro or -NR 8 R 9 The C 1-4 Alkyl, C 1-4 Alkoxy, 3- to 6-membered cycloalkyl, 3- to 6-membered heterocycloalkyl, C6-C 10 The aryl group, or 5- to 10-membered heteroaryl group, may be optionally substituted with one or more of the following groups: C 1-4 Alkyl, C 1-4 Alkoxy, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, C 1-4 Alkyl-substituted 3-6 membered cycloalkyl, C 1-4 Alkyl-substituted 3-6 membered heterocyclic alkyl groups, halogens, cyano groups, trifluoromethyl groups, trifluoromethoxy groups, hydroxyl groups, nitro groups, and amino groups; wherein the heteroaryl and heterocyclic alkyl groups each contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, or sulfur;

[0023] R 7 Selected from H, C 1-6 Alkyl, or 3- to 8-membered cycloalkyl, particularly, R 7 Selected from H, C 1-4 Alkyl, or 3- to 6-membered cycloalkyl;

[0024] R 8 and R 9 Each is independently selected from H, C1-C6 alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, 5- to 10-membered aryl, or 5- to 10-membered heteroaryl; the C 1-6 Alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, 5- to 10-membered aryl, or 5- to 10-membered heteroaryl may optionally be further substituted with one or more of the following groups: C 1-6 Alkyl, 3- to 8-membered cycloalkyl, 3- to 8-membered heterocycloalkyl, halogen, hydroxyl, nitro or amino; or R 8 and R 9 It can form 5-6 membered rings with the N atoms it is connected to;

[0025] n1 is 1, 2, 3, 4, or 5; n2 is 1, 2, 3, or 4; n3 is 1, 2, or 3; n4 is 1 or 2; n5 is 1, 2, 3, 4, 5, or 6; n6 is an integer from 1 to 9; n7 is 1, 2, 3, 4, or 5; n8 is 1, 2, 3, or 4; n9 is an integer from 1 to 8; and when there are multiple R... 6 At that time, multiple R 6They are the same or different from each other;

[0026] R 2 Selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3- to 8-membered cycloalkyl, wherein the substituents used for substitution are selected from C1-C6 alkyl, 3- to 8-membered cycloalkyl, cyano, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, nitro, amino, methoxy; particularly, R 2 Selected from H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted 3- to 6-membered cycloalkyl, the substituent used for substitution is selected from C1-C4 alkyl, 3- to 6-membered cycloalkyl, cyano, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, nitro, amino, methoxy;

[0027] R 3 R 4 R 5 Each is independently selected from substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted 3- to 8-membered cycloalkyl groups, wherein the substituents used for substitution are selected from C1-C6 alkyl groups and 3- to 8-membered cycloalkyl groups; in particular, R 3 R 4 R 5 Each is independently selected from substituted or unsubstituted C1-C4 alkyl groups, substituted or unsubstituted 3- to 6-membered cycloalkyl groups, wherein the substituents used for substitution are selected from C1-C4 alkyl groups and 3- to 6-membered cycloalkyl groups.

[0028] When X is CH, Y can be CH or N; when X is N, Y can be CH or N; that is, the compounds represented by general formula (I) can be represented by the following general formulas (II-1) to (II-4):

[0029]

[0030] Among them, R 1 -R 5 The definitions are as described above.

[0031] Furthermore, the compounds represented by general formula (I) can be represented by the following general formulas (III-1) to (III-7):

[0032]

[0033] Among them, X, Y and R 2 -R 7 The definitions are as described above.

[0034] Furthermore, the compounds according to the present invention may be selected from the following compounds:

[0035]

[0036]

[0037] In this invention The fragment has a common kinase inhibitor structure and can bind to the hinge region of a kinase protein to inhibit kinase activity. This invention is based on R... 2 Introducing steric hindrance groups at the junction can prevent the compound from binding to the hinge region, thereby preventing it from inhibiting the kinase and obtaining a highly selective BET inhibitor with higher safety in diseases related to BET target abnormalities.

[0038] According to another aspect of the present invention, a method for preparing the above-mentioned compound is also provided, said method being carried out via either Route 1 or Route 2.

[0039] Route 1 includes the following steps:

[0040]

[0041] (a) 3-aminoindazole (m1) and phthalic anhydride react under heating conditions to give intermediate m2;

[0042] (b) Intermediate m2 undergoes nucleophilic substitution with a halide, or undergoes a coupling reaction with a borate ester or borate reagent to obtain intermediate m3;

[0043] (c) Deprotection under alkaline conditions yields m4;

[0044] (d) 4-Bromo-2-fluoro-1-nitrobenzene (m5) undergoes a substitution reaction with D-alanine under the action of a base to give intermediate m6;

[0045] (e) Intermediate m6 can be cyclized to give intermediate m7 under the action of alkali and sodium dithionite;

[0046] (f) Intermediate m7 reacts with acetone, benzylsilane and dibutyltin dichloride to give intermediate m8;

[0047] (g) Intermediate m8 reacts with iodomethane under the action of sodium hydride to form intermediate m9;

[0048] (h) Intermediate m9 and m-chloroaniline are coupled together in the presence of a palladium catalyst to obtain intermediate m10;

[0049] (i) Intermediate m10 and intermediate m4 can be coupled together with palladium to yield the final product.

[0050] Among them, R 7 The definition is as described above.

[0051] In particular,

[0052] In step (a), the heating conditions can be 80-120℃;

[0053] In step (c), the alkaline conditions are provided by hydrazine hydrate.

[0054] In steps (d) and (e), the alkali is selected from potassium carbonate, potassium hydroxide, sodium hydroxide, and cesium carbonate.

[0055] Route 2 includes the following steps:

[0056]

[0057] (j) m11 undergoes a nucleophilic reaction with sodium methoxide to give intermediate m12;

[0058] (k) Intermediate m12 and intermediate m9 in route 1 are coupled together under the action of palladium catalyst to obtain intermediate m13;

[0059] (l) Intermediate m13 is demethylated under the action of acid to generate intermediate m14;

[0060] (m) Intermediate m14 reacts with phosphorus oxychloride to generate intermediate m15;

[0061] (n) Intermediate m15 and amine R 1 -NH2 undergoes nucleophilic substitution or palladium-catalyzed coupling to yield the final product.

[0062] Among them, R 1 and R 2 The definition is as described above.

[0063] Specifically, in step (l), the acid is selected from aqueous HCl solution or organic HCl solution.

[0064] In this invention,

[0065] “C 1-6 "Alkyl" refers to a straight-chain or branched alkyl group containing 1 to 6 (1, 2, 3, 4, 5, 6) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0066] Halogenated C 1-6 "Alkyl" indicates that one or more hydrogen atoms are replaced by halogens, such as C as defined herein. 1-6Alkyl groups, and when more than one hydrogen atom is replaced by a halogen atom, the halogen atoms may be the same or different from each other. Examples include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, 1,1-difluoroethyl, 1,1,-difluoropropyl, and 1,1,1-trifluoropropyl;

[0067] “C 1-6 "Alkoxy" refers to "C 1-6 alkyl-O-" group, wherein C 1-6 Alkyl groups are defined as described above and include, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, 1,1-dimethylethoxy, pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, hexoxy, 1-methylpentoxy, 2-methylpentoxy, 3-methylpentoxy, 4-methylpentoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, 1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy, and 1-ethyl-2-methylpropoxy.

[0068] Halogenated C 1-6 "Alkoxy" indicates that one or more hydrogen atoms, such as 1, 2, or 3 hydrogen atoms, are replaced by a halogen as defined herein. 1-6 Alkyl groups, and when more than one hydrogen atom is replaced by a halogen atom, the halogen atoms may be the same as or different from each other. Examples include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2-fluoroethoxy, and 2,2,2-trifluoroethoxy.

[0069] "Cycloalkyl" refers to a saturated, non-aromatic carbon ring that does not contain cyclic heteroatoms. For example, 3- to 8-membered cycloalkyl groups refer to cycloalkyl groups containing 3 to 8 cyclic carbon atoms, including C3, C4, C5, C6, C7, and C8 cycloalkyl groups. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0070] "Heterocyclic alkyl" refers to a saturated non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, or sulfur as a ring member. For example, 3- to 10-membered heterocyclic alkyl groups refer to groups containing 3 to 10 ring atoms (a ring carbon atom and a ring heteroatom). Exemplary monocyclic heterocyclic alkyl groups include, but are not limited to, azirrobutyl, oxobutyl, dioxanepentyl, etc.

[0071] "Aryl" refers to a monocyclic or bicyclic aromatic cyclic group that does not contain heteroatoms in its ring atom, such as phenyl or naphthyl. For example, C5-C... 20 Aryl refers to a monocyclic or bicyclic aromatic cyclic group with 5-20 carbon atoms and no heteroatoms.

[0072] "Heteroaryl" refers to a monocyclic or bicyclic aromatic ring group containing at least one heteroatom selected from nitrogen, oxygen, or sulfur as a ring member; "5 to 20-membered heteroaryl" indicates a heteroaryl group with 5 to 20 ring atoms, examples of which include, but are not limited to, the following groups: pyrrole, imidazolyl, pyrazolyl, pyridinyl, pyridinyl, aziridine, aziridine, aziridine, aziridine, 1,2-diazaheptatrienyl, furanyl, thiophene, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, benzimidazolyl, inzolyl, benzofuranyl, benzothiophene, etc.

[0073] Halogens include fluorine, chlorine, bromine, and iodine.

[0074] The compounds according to the present invention can be prepared as prodrugs or functional molecules using PROTAC technology. The formation of the prodrugs or functional molecules using PROTAC technology can be in accordance with conventional linkage modes of the prior art, such as linking a cleavable fragment, such as a peptide chain, phosphate ester, protac (ubiquitin linker), etc., to the R2 region, which will not be described in detail in this application.

[0075] According to another aspect of the invention, a composition is provided comprising the above-described compound, its stereoisomer, a prodrug, a functional molecule of PROTAC technology, a pharmaceutically acceptable salt or solvate, and optionally a pharmaceutically acceptable excipient.

[0076] According to another aspect of the present invention, there is provided the use of the above-mentioned compound, its stereoisomer, prodrug, PROTAC-technical functional molecule, pharmaceutically acceptable salt or solvate, or said composition in the preparation of a medicament for the prevention or treatment of diseases associated with polycystic kidney disease, abnormal cell proliferation, morphological changes, and hypermotility disorders related to bromine domain protein and / or kinase disorders in vivo.

[0077] According to the present invention, the drug may be a BRD4 inhibitor; it may be used for the treatment of polycystic kidney disease, anti-tumor therapy, and treatment of diseases related to abnormal BET expression.

[0078] According to another aspect of the present invention, a treatment method is provided, comprising the steps of: applying to a subject an effective amount of the above-described compound, its stereoisomer, a pharmaceutically acceptable salt or solvate, or the prodrug, or the functional molecule of the above-described PROTAC technology, or the composition.

[0079] According to the present invention, the treatment method is used to treat polycystic kidney disease, abnormal cell proliferation, morphological changes, and hypermotility-related diseases associated with bromodomain protein and / or kinase disorders in organisms.

[0080] The dihydroquinoxaline-based BET inhibitor of the present invention has higher selectivity for kinases and can be used to treat polycystic kidney disease, cancer and diseases related to abnormal BET expression, and has good safety. Attached Figure Description

[0081] Figure 1 The in vitro cyst-inhibiting effect of compound 7 was demonstrated. Detailed Implementation

[0082] Example 1

[0083]

[0084] (a) Compound 1A (25 g, 113.64 mmol), D-alanine (11.12 g, 125.00 mmol), and potassium carbonate (17.25 g, 125.00 mmol) were dissolved in 500 mL of a mixed solvent of ethanol:water = 3:1. The mixture was heated to reflux at 80 °C for 8 hours, and the reaction was monitored by TLC plate. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, and the solid was dissolved in water. The pH was adjusted to 1-2 with 1N HCl, and a large amount of yellow solid precipitated. The solid was filtered, washed with 200 mL of petroleum ether (PE), and dried in a vacuum drying oven to obtain 28.7 g of yellow solid, which is compound 1B, with a yield of 88%.

[0085] 1 H NMR (400MHz, CDCl3) δ8.35(d,J=6.9Hz,1H),8.06(d,J=9.1Hz,1H),6.90(s,1H),6.85(d,J=9.2Hz,1H),4.33(p,J=7.0Hz,1H),1.67(d,J=7.0Hz,3H).

[0086] (b) Compound 1B (28.7 g, 99.31 mmol) and potassium carbonate (27.41 g, 198.62 mmol) were dissolved in 500 mL of water. Sodium dithionite (86.45 g, 496.55 mmol) was slowly added in portions. The reaction was carried out at 60 °C for 8 h, yielding a colorless, clear, and transparent solution containing a large amount of white precipitate. The reaction was monitored by TLC plate. After the reaction was completed, the solution was cooled to room temperature, filtered, and the solid was washed with 200 mL of water and dried in a vacuum drying oven to obtain 9 g of white solid, namely compound 1C, with a yield of 38%.

[0087] 1H NMR (400MHz, DMSO-d6) δ10.31(s,1H),6.82(d,J=2.1Hz,1H),6.74(dd,J=8.2,2.1Hz ,1H),6.65(d,J=8.3Hz,1H),6.29(s,1H),3.87–3.77(m,1H),1.25(d,J=6.6Hz,3H).

[0088] (c) Compound 1C (9 g, 37.34 mmol), phenylsilane (11.90 g, 113.14 mmol), acetone (8.3 mL, 112.02 mmol), and dibutyltin dichloride (17.02 g, 56.01 mmol) were dissolved in 100 mL THF and reacted overnight at room temperature. The reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated to dryness, and the sample was purified by rapid chromatography using silica gel. The sample was eluted with a gradient of ethyl acetate (EA) / PE = 10-20% to give 8.8 g of a colorless oily liquid, namely compound 1D, with a yield of 87%.

[0089] 1 H NMR (400MHz, CDCl3) δ10.03(s,1H),6.96(d,J=1.9Hz,1H),6.88(dd,J=8.3,2.0Hz,1H),6.73(d,J=8.3Hz,1H), 4.13(q,J=6.8Hz,1H),3.95–3.80(m,1H),1.33(d,J=6.7Hz,3H),1.26(d,J=6.1Hz,3H),1.20(d,J=6.7Hz,3H).

[0090] (d) Compound 1D (8.8 g, 31.08 mmol) was dissolved in 20 mL of anhydrous DMF. NaH (1.25 g, 46.62 mmol) was added in portions in an ice-water bath. The mixture was stirred at 0 °C for 30 min. Iodomethane (2.9 mL, 46.62 mmol) was slowly added. The mixture was reacted at room temperature for 2 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was quenched in 100 mL of ice water. The mixture was extracted with 100 mL of dichloromethane (DCM). The organic layers were combined and washed once with 150 mL of saturated saline solution. The mixture was dried over anhydrous sodium sulfate. The solvent was evaporated. The organic phase was mixed with silica gel and purified by rapid chromatography using an EA / PE gradient of 10-20%. 8.8 g of a colorless oily liquid, namely compound 1E, was obtained, with a yield of 95%.

[0091] 1H NMR (400MHz, DMSO-d6) δ7.04–6.93(m,3H),4.15(q,J=6.7Hz,1H),3.90(hept,J=6.5Hz ,1H),3.25(s,3H),1.24(d,J=6.6Hz,3H),1.17(d,J=6.6Hz,3H),0.97(d,J=6.7Hz,3H).

[0092] (e) Compound 1E (200 mg, 0.673 mmol), 3-chloroaniline (172 mg, 1.35 mmol), and sodium tert-butoxide (130 mg, 1.35 mmol) were dissolved in 5 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (62 mg, 0.0673 mmol) and Xphos (64 mg, 0.1346 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 50 mL of water and extracted with 50 mL of 2 DCM. The organic layers were combined, washed once with 100 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by passing it through a rapid chromatography column with silica gel. The elution was performed using an EA / PE gradient of 20-50%, yielding 170 mg of a black oily liquid, namely compound 1F, with a yield of 73.5%.

[0093] 1 H NMR (400MHz, CDCl3) δ7.15(d,J=8.1Hz,1H),7.10–7.03(m,1H),6.99(t,J=2.3Hz,1H),6.92–6.79(m,2H),6.62(d,J=7.7Hz,2H ),5.72(s,1H),4.18(q,J=6.8Hz,1H),3.81(p,J=6.7Hz,1H),3.36(s,3H),1.26(dd,J=14.6,6.6Hz,6H),1.12(d,J=6.8Hz,3H).

[0094] (f) Compound 1F (20 mg, 0.0673 mmol), 1-methyl-1H-indazole-3-amine (10 mg, 0.074 mmol), and sodium tert-butoxide (13 mg, 0.135 mmol) were dissolved in 1 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (6 mg, 0.00673 mmol) and Xphos (6 mg, 0.01346 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water and extracted with 10 mL of DCM twice. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by passing it through a rapid chromatography column with silica gel. The elution was performed using a gradient of EA / PE = 20-50%, yielding 27 mg of a yellow solid, namely compound 1, with a yield of 95.07%.

[0095] 1 H NMR (400MHz, CDCl3) δ7.58 (dd, J=8.2, 1.0Hz, 1H), 7.38 (dd, J=8.3, 6.9Hz, 1H), 7.31–7.27 (m, 1H) ,7.15(d,J=8.0Hz,1H),7.11(dd,J=5.0,2.8Hz,1H),7.05(dd,J=8.0,6.9Hz,1H),6.85–6.75(m,2 H),6.63–6.56(m,3H),6.25(s,1H),4.15(q,J=6.8Hz,1H),3.95(s,3H),3.78(p,J=6.7Hz,1H),3. 34(s,3H),1.25(d,J=3.3Hz,3H),1.21(d,J=6.6Hz,3H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 455.26.

[0096] Example 2

[0097]

[0098] (a) Compound 2A (500 mg, 3.76 mmol) and phthalic anhydride (723 mg, 4.88 mmol) were dissolved in 5 mL of 1,4-dioxane and reacted overnight at 110 °C. The reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated to dryness, and the mixture was stirred in 10 mL of anhydrous diethyl ether for 30 min. The mixture was then filtered, the filter cake was washed with anhydrous diethyl ether, and dried under vacuum to give 1 g of yellow solid, namely compound 2B, with a yield of 101.16%.

[0099] 1H NMR (400MHz, DMSO-d6) δ13.47(s,1H),8.04(dd,J=5.5,3.1Hz,2H),7.97(dd,J=5.5,3.1Hz,2H),7. 70(d,J=8.2Hz,1H),7.66–7.55(m,1H),7.49–7.41(m,1H),7.17(t,J=7.5Hz,1H).LC-MS(ESI)[M+H] + 264.26.

[0100] (b) Compound 2B (50 mg, 0.190 mmol) and potassium carbonate (53 mg, 0.380 mmol) were dissolved in 2 mL of DMF, and iodoethane (45 mg, 0.285 mmol) was added dropwise. The reaction was carried out overnight at room temperature, and the reaction was monitored by TLC plate. After the reaction was completed, the reaction solution was poured into 15 mL of water and extracted with 10 mL * 2 DCM. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The sample was mixed with silica gel and purified by rapid chromatography column using EA / PE gradient elution of 20-50% to give 42 mg of yellow solid, namely compound 2C, with a yield of 75.91%.

[0101] 1 H NMR (400MHz, CDCl3) δ7.99 (s, 2H), 7.81 (s, 2H), 7.56 (d, J = 8.1Hz, 1H), 7.45 (s, 2H), 7 .20(d,J=7.5Hz,1H),4.49(q,J=7.5Hz,2H),1.57(t,J=7.4Hz,3H).LC-MS(ESI)[M+H] + 291.12.

[0102] (c) Compound 2C (40 mg, 0.137 mmol) was dissolved in 2 mL of DMF, and hydrazine hydrate (34 mg, 0.687 mmol) was added dropwise. The reaction was carried out overnight at room temperature, and the reaction was monitored by TLC plate. After the reaction was completed, the reaction solution was poured into 15 mL of water and extracted with 10 mL * 2 DCM. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The sample was mixed with silica gel and purified by rapid chromatography column elution using EA / PE = 20-50% gradient to give 22 mg of brown oil, namely compound 2D, with a yield of 99.39%.

[0103] 1H NMR (400MHz, CDCl3) δ7.51(d,J=8.1Hz,1H),7.31(t,J=7.6Hz,1H),7.27–7.21(m,1H),7.02( t,J=7.4Hz,1H),4.20(q,J=7.1Hz,2H),3.68(s,2H),1.43(t,J=6.9Hz,3H).LC-MS(ESI)[M+H] + 162.17.

[0104] (d) Compound 2 was prepared using the same method as in step (f) of Example 1, except that 2D was used instead of 1-methyl-1H-indazole-3-amine.

[0105] 1 H NMR (400MHz, CDCl3) δ7.57 (d, J=8.0Hz, 1H), 7.41–7.30 (m, 2H), 7.18–7.09 (m, 2H), 7.05 (ddd, J=7.9, 6.6,1.1Hz,1H),6.83(d,J=8.5Hz,1H),6.77(dd,J=8.0,2.1Hz,1H),6.66(dd,J=8.5,2.3Hz,1H),6.6 2–6.55(m,2H),6.17(s,1H),4.31(q,J=7.2Hz,2H),4.13(q,J=12.1,7.0Hz,1H),3.79(p,J=6.6Hz,1H ),3.35(s,3H),1.25(d,J=6.5Hz,3H),1.21(d,J=6.6Hz,3H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 469.33.

[0106] Example 3

[0107]

[0108] Compound 3 was prepared using the same method as in Example 2, except that 2-iodopropane was used instead of iodoethane in step (b) of Example 2.

[0109] 1H NMR (400MHz, CDCl3) δ7.57(d,J=8.1Hz,1H),7.34(d,J=3.7Hz,2H),7.14–7.10(m,2H),7.06–7.01( m,1H),6.83(d,J=8.5Hz,1H),6.76(dd,J=7.9,2.0Hz,1H),6.68(dd,J=8.5,2.3Hz,1H),6.60–6.54( m,2H),6.25(s,1H),4.73(q,J=6.6Hz,1H),4.20–4.07(m,1H),3.78(p,J=6.6Hz,1H),3.34(s,3H), 1.52(d,J=6.6Hz,6H),1.25(d,3H),1.20(d,J=6.5Hz,3H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 483.29.

[0110] Example 4

[0111]

[0112] Compound 4 was prepared using the same method as in Example 2, except that iodocyclopropane was used instead of iodoethane in step (b) of Example 2.

[0113] 1 H NMR (400MHz, CDCl3) δ7.57 (d, J=8.0Hz, 1H), 7.41–7.30 (m, 2H), 7.18–7.09 (m, 2H), 7.05 (ddd, J=7.9, 6.6,1.1Hz,1H),6.83(d,J=8.5Hz,1H),6.77(dd,J=8.0,2.1Hz,1H),6.66(dd,J=8.5,2.3Hz,1H),6.6 2–6.55(m,2H),6.17(s,1H),4.31(q,J=7.2Hz,2H),4.13(q,J=12.1,7.0Hz,1H),3.79(p,J=6.6Hz,1H ),3.35(s,3H),1.25(d,J=6.5Hz,3H),1.21(d,J=6.6Hz,3H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 469.33.

[0114] Example 5

[0115]

[0116] (a) Compound 1E (50 mg, 0.168 mmol), 2-amino-6-chloropyridine (24 mg, 0.185 mmol), and sodium tert-butoxide (33 mg, 0.336 mmol) were dissolved in 2 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (16 mg, 0.0168 mmol) and Xphos (16 mg, 0.0336 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 20 mL of water and extracted with 10 mL of DCM twice. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by passing it through a rapid chromatography column with silica gel. The elution was performed using a gradient of EA / PE = 20-50%, yielding 37 mg of brown solid, namely compound 5A, with a yield of 63.78%.

[0117] 1 H NMR (400MHz, CDCl3) δ7.40(t,J=7.9Hz,1H),6.96(d,J=2.3Hz,1H),6.89(d,J=8.5Hz,1H),6.76–6.68(m,2H),6.65(d,J=8.2Hz,1H),6.60(s,1H), 4.19(q,J=6.8Hz,1H),3.85(p,J=6.6Hz,1H),3.36(s,3H),1.31(d,J=6.7Hz,3H),1.26(d,J=6.6Hz,3H),1.12(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 345.26.

[0118] (b) Compound 5A (35 mg, 0.102 mmol), 1-methyl-1H-indazole-3-amine (17 mg, 0.112 mmol), and sodium tert-butoxide (20 mg, 0.203 mmol) were dissolved in 1 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (10 mg, 0.0102 mmol) and Xphos (10 mg, 0.0204 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 15 mL of water and extracted with 10 mL of 2 DCM. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by passing it through a rapid chromatography column with silica gel. The elution was performed using a gradient of EA / PE = 20-50%, yielding 27 mg of a yellow solid, namely compound 5, with a yield of 58.39%.

[0119] 1H NMR(400MHz, CDCl3)δ7.65(d,J=8.2Hz,1H),7.46–7.31(m,3H),7.21(dd,J=8.4,0.8 Hz,1H),7.11–6.96(m,2H),6.92–6.86(m,2H),6.82(dd,J=8.5,2.2Hz,1H),6.33(d, J=8.0Hz,1H),4.19(q,J=6.8Hz,1H),3.96(s,3H),3.85(p,1H),3.37(s,3H),1.31(d ,J=6.8,1.5Hz,3H),1.26(d,J=6.2Hz,3H),1.12(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 456.26.

[0120] Example 6

[0121]

[0122] Compound 6 was prepared using the same method as in Example 5, except that 2-amino-4-chloro-6-methylpyridine was used instead of 2-amino-6-chloropyridine in step (a) of Example 5.

[0123] 1 H NMR(400MHz, CDCl3)δ7.96(d,J=8.2Hz,1H),7.43–7.34(m,1H),7.28(d,J=12.5Hz,2H) ,7.18(s,1H),7.07(t,J=7.5Hz,1H),6.87–6.81(m,2H),6.76(dd,J=8.4,2.2Hz,1H),6. 71(d,J=2.2Hz,1H),4.16(q,J=6.8Hz,1H),3.90(s,3H),3.76(p,J=12.7,6.3Hz,1H),3. 31(s,3H),2.36(s,3H),1.18(t,J=6.5Hz,6H),1.06(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 470.32.

[0124] Example 7

[0125]

[0126] (a) Compound 7A (2 g, 14.37 mmol) was dissolved in 10 mL of phosphorus oxychloride and refluxed at 60 °C for 2 h. The reaction was monitored by TLC plate. After the reaction was completed, the reaction solution was slowly poured into ice water to quench it. The solution was neutralized to pH 7 with sodium hydroxide solid and extracted with 50 mL * 2 DCM. The organic layers were combined, washed once with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to give 1.43 g of white solid, namely compound 7B, with a yield of 63.13%.

[0127] 1 H NMR (400MHz, CDCl3) δ6.53 (s, 1H), 5.41 (s, 2H), 2.57 (q, J = 7.6Hz, 2H), 1.23 (t, J = 7.5Hz, 3H). LC-MS (ESI) [M+H] + 158.16.

[0128] (b) Compound 7B (1.43 g, 9.07 mmol) was dissolved in 10 mL of methanol. Sodium methoxide (980 mg, 18.15 mmol) was added in portions in an ice-water bath. The mixture was refluxed at 65 °C for 6 h. The reaction was monitored by TLC plate. After the reaction was completed, the solvent was evaporated to dryness, 100 mL of water was added, and the mixture was extracted with 50 mL of 2 DCM. The organic layers were combined, washed once with 100 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness to give 1.35 g of white solid, which was 4-ethyl-6-methoxy-2-pyrimidinamine. No rapid column purification was required for the next step of the reaction.

[0129] 4-Ethyl-6-methoxy-2-pyrimidinamine (2.95 g, 19.26 mmol), compound 1E (6.87 g, 23.11 mmol), and sodium tert-butoxide (3.70 g, 38.52 mmol) were dissolved in 30 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (1.76 g, 1.926 mmol) and Xphos (1.83 g, 3.852 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 200 mL of water and extracted with 100 mL of DCM twice. The organic layers were combined, washed once with 200 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by rapid chromatography using silica gel with 20-50% gradient elution to give 4.95 g of yellow solid, compound 7C, with a yield of 69.94%.

[0130] 1H NMR (400MHz, CDCl3) δ7.50(d,J=2.3Hz,1H),7.03(s,1H),6.97(dd,J=8.6,2.3Hz,1H),6.87(d,J=8.6Hz,1H),6.05(s,1H),4.15 (p,J=7.0Hz,1H),3.94(s,4H),3.35(s,3H),2.59(q,J=7.6Hz,2H),1.34–1.21(m,9H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 370.33.

[0131] (c) Compound 7C (2.5 g, 6.80 mmol) was dissolved in 20 mL of 4 M HCl / dioxane. Concentrated HCl (24.8 g, 680.29 mmol) was added dropwise in an ice-water bath. The mixture was heated to reflux at 80 °C overnight. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was slowly poured into ice water to quench it. The solution was neutralized to pH 7 with sodium hydroxide solid. The mixture was extracted with 100 mL of 2 DCM. The organic layers were combined and washed once with 200 mL of saturated saline solution. The solution was dried over anhydrous sodium sulfate. The solvent was evaporated. The organic phase was mixed with silica gel and purified by rapid chromatography using a MeOH / DCM gradient of 2-10%. 1.8 g of brown solid, namely compound 7D, was obtained, with a yield of 74.86%.

[0132] 1 H NMR (400MHz, CDCl3) δ7.65(s,1H),6.90(d,J=8.5Hz,1H),5.80(d,J=3.6Hz,1H),4.19(q,J=6.5Hz,1H),3.94(p,J=6.7Hz,1H),3.36(d ,J=2.6Hz,3H),2.52(q,J=7.6Hz,2H),1.34(d,J=6.6Hz,3H),1.26(dd,J=12.0,6.7Hz,6H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M++H] + 356.36.

[0133] (d) Compound 7D (1.8 g, 5.09 mmol) was dissolved in 7 mL of phosphorus oxychloride and refluxed at 60 °C for 2 h. The reaction was monitored by TLC plate. After the reaction was completed, the reaction solution was slowly poured into ice water to quench it. The solution was neutralized to pH 7 with sodium hydroxide solid and extracted with 100 mL * 2 DCM. The organic layers were combined, washed once with 200 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The organic phase was mixed with silica gel and purified by rapid chromatography column elution using an EA / PE gradient of 20-50% to give 1.1 g of yellow foamy solid, namely compound 7E, with a yield of 58.08%.

[0134] 1 H NMR (400MHz, CDCl3) δ7.53(d,J=1.5Hz,1H),7.14(s,1H),6.88(d,J=1.3Hz,2H),6.62(s,1H),4.19(q,J=6.8Hz,1H),3.92(p,J=6 .6Hz,1H),3.36(s,3H),2.66(q,J=7.6Hz,2H),1.35(d,J=6.7Hz,3H),1.32–1.25(m,6H),1.12(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 372.16.

[0135] (e) Compound 7E (200 mg, 0.535 mmol) and 1-methyl-1H-indazole-3-amine (95 mg, 0.642 mmol) were dissolved in 3 mL of tert-butanol, and concentrated HCl (97.5 mg, 0.00267 mmol) was added dropwise. The mixture was refluxed at 80 °C for 2 h. The reaction was monitored by TLC plate. After the reaction was completed, 50 mL of saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted with 20 mL of 2 DCM, and the organic layers were combined. The mixture was washed once with 40 mL of saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was evaporated. The organic phase was mixed with silica gel and purified by rapid chromatography column elution using an EA / PE gradient of 20-50%. 190 mg of a yellow solid, namely compound 7, was obtained, with a yield of 73.29%.

[0136] 1H NMR(400MHz, CDCl3)δ7.90(s,1H),7.59(d,J=8.2Hz,1H),7.48–7.34(m,4H),7.11(t,J=7.4H z,1H),6.97(dd,J=8.5,2.3Hz,1H),6.84(d,J=8.5Hz,1H),6.69(s,1H),4.17(q,J=6.8Hz,1H ),4.01(s,3H),3.88(p,J=8.0,7.3Hz,1H),3.35(s,3H),2.59(q,J=7.6Hz,2H),1.32(d,J=6. 7Hz,3H),1.26(d,J=4.3Hz,3H),1.30–1.22(m,6H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 485.47.

[0137] Example 8

[0138]

[0139] Compound 8 was prepared using the same method as in Example 7, except that 2-amino-4-hydroxy-6-methylpyrimidine was used instead of 2-amino-4-hydroxy-6-ethylpyrimidine (7A) in step (a) of Example 7.

[0140] 1 H NMR (400MHz, CDCl3) δ7.68 (s, 1H), 7.58 (d, J = 8.2Hz, 1H), 7.47–7.32 (m, 3H), 7.16– 7.08(m,1H),6.98(dd,J=8.6,2.3Hz,1H),6.84(d,J=8.6Hz,1H),6.71(s,1H),4.17 (q,J=6.8Hz,1H),4.01(s,3H),3.86(p,J=6.6Hz,1H),3.35(s,3H),2.32(s,3H),1. 33(d,J=6.7Hz,3H),1.26(d,J=6.5Hz,3H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 485.47.

[0141] Example 9

[0142]

[0143] Compound 9 was prepared using the same method as in Example 7, except that aniline was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0144] 1 H NMR(400MHz, CDCl3)δ7.38–7.30(m,5H),7.14(ddt,J=8.5,5.4,2.8Hz,1H),7 .02–6.97(m,1H),6.85(d,J=8.5Hz,1H),6.76(s,1H),6.08(s,1H),4.16(q,J =6.8Hz,1H),3.86(p,J=6.6Hz,1H),3.35(s,3H),2.55(q,J=7.6Hz,2H),1.29 –1.25(m,6H),1.23(d,J=6.5Hz,3H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 431.39.

[0145] Example 10

[0146]

[0147] Compound 10 was prepared using the same method as in Example 7, except that p-chloroaniline was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0148] 1 H NMR (400MHz, CDCl3) δ7.39–7.32(m,3H),7.32–7.26(m,3H),7.06–6.96(m,2H),6.86(dd,J=8.6,1.2Hz,1H),6.01(s,1H),4.17(q,J =6.8Hz,1H),3.83(p,J=6.7Hz,1H),3.36(s,3H),2.56(q,J=7.6Hz,2H),1.33–1.20(m,9H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 465.40.

[0149] Example 11

[0150]

[0151] Compound 11 was prepared using the same method as in Example 7, except that 3-chloroaniline was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0152] 1H NMR (400MHz, CDCl3) δ8.07 (dd, J=8.2, 1.5Hz, 1H), 7.41 (dt, J=5.5, 1.5Hz, 2H ),7.29–7.20(m,1H),7.12(s,1H),7.07–6.94(m,2H),6.87(t,J=8.0Hz,2H), 6.10(s,1H),4.17(q,J=6.8Hz,1H),3.86(p,J=6.6Hz,1H),3.36(s,3H),2.59 (q,J=7.6Hz,2H),1.29–1.18(m,9H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 465.42.

[0153] Example 12

[0154]

[0155] Compound 12 was prepared using the same method as in Example 7, except that o-chloroaniline was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0156] 1 H NMR(400MHz, CDCl3)δ7.57(q,J=1.4Hz,1H),7.29–7.20(m,3H),7.06(tt,J=5.2 ,2.1Hz,2H),6.97(s,1H),6.89(d,J=8.6Hz,1H),6.05(s,1H),5.30(s,1H),4.16 (q,J=6.8Hz,1H),3.84(p,J=6.7Hz,1H),3.36(s,3H),2.57(q,J=7.6Hz,2H),1. 31–1.23(m,6H),1.21(d,J=6.6Hz,3H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 465.43.

[0157] Example 13

[0158]

[0159] Compound 13 was prepared using the same method as in Example 7, except that o-methoxyaniline was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0160] 1H NMR(400MHz, CDCl3)δ8.07(dd,J=7.9,1.6Hz,1H),7.42(d,J=2.3Hz,1H),7.09(s,1H),7.06–6.83(m,6H),6.11(s,1H),4.1 6(q,J=6.8Hz,1H),3.88(s,4H),3.36(s,3H),2.56(q,J=7.6Hz,2H),1.31–1.18(m,9H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)

[0161] [M+H] + 461.39.

[0162] Example 14

[0163]

[0164] Compound 14 was prepared using the same method as in Example 7, except that m-aminoanisole was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0165] 1 H NMR(400MHz, CDCl3)δ7.36(d,J=2.3Hz,1H),7.29–7.20(m,1H),7.10(s,1H),7.04–6.95 (m,2H),6.94–6.88(m,1H),6.85(d,J=8.6Hz,1H),6.82(s,1H),6.68(ddd,J=8.3,2.5,0. 8Hz,1H),6.11(s,1H),4.16(q,J=6.8Hz,1H),3.86(p,J=6.6Hz,1H),3.80(s,3H),3.35(s ,3H),2.55(q,J=7.6Hz,2H),1.30–1.19(m,9H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 461.36.

[0166] Example 15

[0167]

[0168] Compound 15 was prepared using the same method as in Example 7, except that p-aminoanisole was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0169] 1H NMR(400MHz, CDCl3)δ7.38(d,J=2.3Hz,1H),7.27–7.20(m,2H),7.13(d,J=7.4H z,1H),6.97(dd,J=8.6,2.3Hz,1H),6.94–6.89(m,2H),6.84(d,J=8.7Hz,1H),5. 92(s,1H),4.16(q,J=6.8Hz,1H),3.86(p,J=6.6Hz,1H),3.82(s,3H),3.35(s,3 H),2.51(q,J=7.6Hz,2H),1.31–1.19(m,9H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)

[0170] [M+H] + 461.36.

[0171] Example 16

[0172]

[0173] Compound 16 was prepared using the same method as in Example 7, except that 3-amino-5-methylisoxazole was used instead of 1-methyl-1H-indazole-3-amine in step (e) of Example 7.

[0174] 1 H NMR (400MHz, CDCl3) δ7.55(s,1H),7.34(s,1H),7.03(dd,J=8.6,2.2Hz,1H),6.87(d,J=8.6Hz,1H),6.53(s,1H),6.28(s,1H),4.17(q,J=6.8 Hz,1H),3.90(p,J=6.6Hz,1H),3.35(s,3H),2.64(q,J=7.6Hz,2H),2.39(s,3H),1.32–1.23(m,9H),1.10(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 436.27.

[0175] Example 17

[0176]

[0177] Compound 7E (50 mg, 0.134 mmol), 3-methylisoxazol-5-amine (26 mg, 0.267 mmol), and sodium tert-butoxide (26 mg, 0.267 mmol) were dissolved in 1 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (12 mg, 0.0134 mmol) and Xantphos (16 mg, 0.0267 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 15 mL of water and extracted with 10 mL of DCM twice. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by rapid chromatography using silica gel, eluting with a MeOH / DCM gradient of 1-4%, to obtain 30 mg of a yellow solid, namely compound 17, with a yield of 51.98%.

[0178] 1 H NMR (400MHz, CDCl3) δ7.27(d,J=8.6Hz,1H),7.01(dd,J=8.6,2.3Hz,1H),6.88(d,J=8.6Hz,1H),6.25(s,1H),6.01(s,1H),4.19(q,J=6.8H z,1H),3.88(p,J=6.6Hz,1H),3.36(s,3H),2.62(q,J=7.6Hz,2H),2.23(s,3H),1.32–1.20(m,9H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 436.30.

[0179] Example 18

[0180]

[0181] Compound 7E (30 mg, 0.080 mmol), 2-aminopyridine (9 mg, 0.096 mmol), and cesium carbonate (52 mg, 0.160 mmol) were dissolved in 1 mL of toluene. After purging with N2 for 1 min, Pd2(dba)3 (7 mg, 0.0080 mmol) and Xphos (8 mg, 0.016 mmol) were added. After purging with N2 for another 3 min, the mixture was reacted overnight at 100 °C. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 15 mL of water and extracted with 10 mL of DCM twice. The organic layers were combined, washed once with 20 mL of saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was then purified by rapid chromatography using silica gel, eluting with a MeOH / DCM gradient of 1-4%, to obtain 50 mg of a yellow solid, namely compound 18, with a yield of 144.40%.

[0182] 1 H NMR (400MHz, CDCl3) δ8.34–8.28(m,1H),8.22–8.06(m,1H),7.73(d,J=8.3Hz,1H),7.61(ddd,J=8.6, 7.2,1.9Hz,1H),7.57–7.50(m,1H),7.40(dd,J=3.5,2.1Hz,1H),7.01(dd,J=8.6,2.3Hz,1H),6.94(dd d,J=7.3,5.0,1.0Hz,1H),6.87(d,J=8.6Hz,1H),6.57(s,1H),4.17(q,J=6.8Hz,1H),3.88(p,J=6.7Hz ,1H),3.36(s,3H),2.62(q,J=7.6Hz,2H),1.34–1.20(m,9H),1.11(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 432.30.

[0183] Example 19

[0184]

[0185] Compound 19 was prepared using the same method as in Example 18, except that 4-aminopyridine was used instead of 2-aminopyridine.

[0186] 1 H NMR (400MHz, CDCl3) δ8.41(d,J=5.6Hz,2H),7.52(d,J=5.5Hz,2H),7.33(s,1H),7.26(s,1H),7.10–6.99(m,2H),6.90(d,J=8.6Hz,1H),6.16(s,1 H),4.17(q,J=6.8Hz,1H),3.87(p,J=6.6Hz,1H),3.37(s,3H),2.61(q,J=7.6Hz,2H),1.33–1.20(m,9H),1.12(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 432.30.

[0187] Example 20

[0188]

[0189] Compound 20 was prepared using the same method as in Example 18, except that 3-aminopyridine was used instead of 2-aminopyridine.

[0190] 1H NMR(400MHz, CDCl3)δ8.67(s,1H),8.30(d,J=4.7Hz,1H),8.11–7.96(m,2H),7.32( s,1H),7.28–7.19(m,1H),7.06(dd,J=8.2,4.7Hz,1H),7.03–6.93(m,1H),6.85(d,J =8.6Hz,1H),6.04(s,1H),4.15(q,J=6.8Hz,1H),3.83(p,J=6.6Hz,1H),3.34(s,3H) ,2.55(q,J=7.6Hz,2H),1.29–1.17(m,9H),1.09(d,J=6.8Hz,3H).LC-MS(ESI)[M+H] + 432.33.

[0191] Biological evaluation

[0192] The affinity of the target compound for the BRD4BD1 / BD2 bromodomain protein was tested using fluorescence polarization (FP).

[0193] Experimental Procedure: The fluorescent substrate and BRD4 BD1 / BD2 protein were dissolved in a buffer solution (composed of 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 150 mM sodium chloride, and 0.5 mM 3-(3-cholamidopropyl dimethylamino)propanesulfonic acid inner salt, pH 7.4. The concentration of the BRD4 BD1 / BD2 bromine domain protein was 40 nM, and the concentration of the fluorescent substrate was 5 nM). The prepared solution and a series of concentration gradients of the target compound or DMSO were then added to Corning 384-well black low-volume plates and reacted at room temperature in the dark for 4 hours. After the reaction, the fluorescence polarization values ​​of each system were read using a BioTek Synergy2 multimode microplate reader.

[0194] Data Analysis:

[0195] (a) Calculation formula: Inhibition rate = (CF) / (CB) × 100%.

[0196] Where: C is the fluorescence polarization value at which the fluorescent substrate binds to the protein to achieve stability; B is the fluorescence polarization value of the fluorescent substrate itself; and F is the fluorescence polarization value after treatment with different concentrations of the target compound.

[0197] (b) Fitting the dose-response curve: By simulating the S-curve with the concentration of the target compound on the horizontal axis and the inhibition rate at the corresponding concentration on the vertical axis, the corresponding IC50 can be obtained. 50 value.

[0198] Table 1 below shows the IC50 values ​​of the inhibitory activity of the compounds of the present invention against BRD4 BD1 / BD2 proteins. 50 Value (nM).

[0199] Table 1

[0200] Compound numbering <![CDATA[BRD4 BD1 IC 50 (nM)]]> <![CDATA[BRD4 BD2 IC 50 (nM)]]> 1 + + 2 + + 3 + + 4 + + 5 +++ ++ 6 +++ +++ 7 +++ ++ 8 +++ +++ 9 ++ +++ 10 + ++ 11 + ++ 12 ++ ++ 13 +++ +++ 14 ++ +++ 15 ++ ++ 16 ++ ++ 17 ++ ++ 18 +++ +++ 19 +++ ++ 20 +++ +++

[0201] +++: Represents the IC of the compound. 50 Less than or equal to 100 nM

[0202] ++: Represents the IC of a compound. 50 Greater than 100 nM and less than or equal to 500 nM

[0203] +: Represents the IC of the compound. 50 Greater than 500 nM

[0204] Table 2 below shows the results of tests conducted by Eurofins, representing the selectivity of compound 7 against multiple kinases.

[0205] Table 2 shows the activities of various kinases after the addition of compound 7. A value of 100% or higher indicates that the compound has no inhibitory effect, and the kinases retain their original activity. The data in the table are mostly above 80%, indicating that compound 7 has a very small inhibitory effect on them. Therefore, the compounds of this invention are selective inhibitors of BRD4, exhibiting high selectivity for kinases.

[0206] Table 2

[0207]

[0208]

[0209] In vitro cyst-inhibiting effect of the compound

[0210] In the in vitro 3D-MDCK cyst model, the addition of compound 7 to the culture environment significantly reduced the number of cysts and the changes in vesicle size.

[0211] Experimental plan:

[0212] 1. Vesicle culture

[0213] Madin-Darby canine kidney cells (MDCK) were suspended in a three-dimensional matrix gel and cultured in a 37°C, 5% CO2 incubator. During culture, 10 μM forskolin was added to stimulate vesicle formation for 4 days.

[0214] 2. Grouping and Dosing

[0215] Starting from day 4, the vesicles were divided into two groups: a control group and a treatment group. The control group was continuously stimulated with 10 μM forskolin until day 12; the treatment group was additionally stimulated with 100 nM BRD4 inhibitor (compound 7) with 10 μM forskolin until day 12.

[0216] 3. Taking photos and compiling statistics

[0217] Starting on day 4 of culture, at least 10 vesicles (diameter greater than 50 nm) were randomly selected from each well, tracked, located, and photographed every two days. The experiment was terminated on day 12, and the vesicle diameter on day 12 was recorded to determine the efficacy of the administered drug.

[0218] Each group of the above experiment was repeated 3 times.

[0219] The addition of compound 7 to the culture environment significantly reduced both the number of cysts and the size of the vesicles. Figure 1 The changes in vesicles during the experiment are shown.

Claims

1. Compounds represented by the following general formulas (III-1) to (III-4) and (III-7), their stereoisomers or pharmaceutically acceptable salts: ， in, X， Y is always N; or X is CH and Y is N; or X is N and Y is CH. R 2 Selected from C1-C4 alkyl groups; R 3 R 4 R 5 Each is independently selected from C1-C6 alkyl groups; In III-1, R 6 Each is independently selected from H and C. 1-6 Alkoxy; In III-2 to III-4, R 6 Each is independently selected from H and C1-C6 alkyl groups; In III-7, R 6 Each is independently represented by H; R 7 Selected from H, C 1-6 alkyl.

2. The compound of claim 1, wherein its stereoisomer or pharmaceutically acceptable salt, wherein, The R 6 and R 7 C in 1-6 The alkyl group is selected from methyl, ethyl, n-propyl, and isopropyl; C 1-6 The alkoxy group is selected from methoxy and ethoxy groups; the halogen is selected from fluorine, chlorine, and bromine.

3. Among the compounds represented below, their stereoisomers or pharmaceutically acceptable salts, wherein, The compound is selected from the following compounds: 。 4. A composition comprising the compound according to any one of claims 1 to 3, its stereoisomer or pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable excipient.

5. Use of the compound, its stereoisomer, or pharmaceutically acceptable salt according to any one of claims 1 to 3, or the composition according to claim 4, in the preparation of a medicament for the prevention or treatment of diseases associated with polycystic kidney disease, abnormal cell proliferation, morphological changes, and hypermotility disorders related to bromine domain protein and / or kinase disorders in vivo.

6. The use according to claim 5, wherein the drug is a BRD4 inhibitor.

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