CN109280046B - Benzimidazole derivative, preparation method thereof and application thereof in medicine - Google Patents

Abstract

The invention relates to benzimidazole derivatives, a preparation method and medical application thereof. Specifically, the invention relates to benzimidazole derivatives shown in a general formula (I), a preparation method thereof, pharmaceutically acceptable salts thereof and application thereof as a therapeutic agent, particularly as a bromodomain protein inhibitor, wherein the definition of each substituent in the general formula (I) is the same as that in the specification.

Description

Benzimidazole derivative, preparation method thereof and application thereof in medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel benzimidazole derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, and application of the benzimidazole derivative as a therapeutic agent, particularly as a bromodomain protein inhibitor.

Background

In recent years, tumors have become one of the leading causes of human death worldwide. The tumor has the characteristics of low overall cure rate, high recurrence rate and the like generally, so that the prevention, treatment and inhibition of tumor recurrence have important scientific research values, and the prevention and cure of the tumor are quite urgent and challenging. Abnormalities in epigenetic regulation are one of the important factors leading to tumorigenesis.

Bromodomain (bromodomain) is a protein domain that recognizes N-acetylated lysine residues. The BET family of bromodomain-containing proteins includes four members (BRD2, BRD3, BRD4, and BRDt). Each member of the BET family is identified using two bromodomains, with the N-acetylated lysine residue being found predominantly (but not exclusively) at the amino-terminal tail (amino-terminal tail) of histone proteins. Gene expression is regulated by recruitment of transcription factors to specific genomic locations within chromatin. For example, histone-linked BRD4 recruits the transcription factor P-TEFb to the promoter, resulting in the expression of a gene set involved in cell cycle progression (Yang et al, mol. cell. biol.28:967-976 (2008)). BRD2 and BRD3 also function as transcriptional regulators of growth promoting genes. Recent studies have demonstrated the targeting of BET bromodomains for the treatment of various cancers ((Zuber et al, Nature 478:524-528 (2011); Mertz et al, Proc. Natl. Acad. Sci.108:16669-16674 (2011); Delmore et al, Cell 146:1-14 (2011); Dawson et al, Nature 478:529-533(2011)), atherosclerosis, inflammation (Huang et al, mol. Cell. biol.29:1375-1387(2009)) and HIV infection.

Recent studies have found that the apparent genetic abnormality mediated by BRD4 protein is closely related to the overexpression of oncogenes and to the growth and proliferation of cancer cells. BRD4 is a member of the bromodomain-containing and extra terminal domain family of proteins (BET) and has attracted considerable attention from various pharmaceutical companies and research institutes due to its potential value in anti-tumor applications. It has also recently been discovered that BRD4 plays an important role in the transcriptional regulation of viral genes and has been linked to the pathogenesis of viral tumors. The research results show that BRD4 has close relation with various tumors, especially has important role in some tumors which are difficult to cure or have no effective treatment means so far, and the research on the relation of BRD4 and tumors provides a new strategy for treating tumors. The small molecular compound acting on the bromodomain of the BRD4 protein interferes the specific binding of the bromodomain and the acetylated lysine, influences the transcription regulation and other cell processes in tumor cells, and can realize the targeted therapy of tumors. Therefore, the BRD4 protein is a new epigenetic target with a very promising prospect, and the small molecule inhibitor acting on the bromodomain of the BRD4 protein also has a wide application prospect in tumor research, and a novel antitumor drug is possibly developed from the small molecule inhibitor.

A series of bromodomain protein inhibitor patents have been disclosed, including WO2011054846, WO2008092231, WO2012075383, and WO2016139292, among others, wherein WO2016139292 discloses the example 1 compound. At present, the medicine in the clinical stage III is Apabetalone, the medicine in the clinical stage II comprises GSK-525762A, INCB-54329, BMS-986158 and the like, and a plurality of medicines are in the clinical stage I. However, these studies on anti-tumor are far from sufficient, and there is still a need to research and develop new bromodomain protein inhibitors.

Disclosure of Invention

The invention provides a benzimidazole derivative shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof:

wherein:

X¹is selected from-CH or-N;

X²is selected from-CH or-N;

and X¹And X²Is not equal to-N at the same time;

R¹selected from alkyl groups; preferably methyl;

R²selected from a hydrogen atom or an alkyl group; preferably methyl;

R³selected from alkyl groups, wherein said alkyl groups are further substituted with one or more alkoxy groups;

R⁴selected from halogens;

R⁵selected from hydrogen atom, alkyl group, cyano group, halogen, alkoxy group, cycloalkyl group, heterocyclic group, aryl group, heteroaryl group, -OR¹⁰、-NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-C(O)OR¹⁰or-NR⁸C(O)R⁹Wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-C(O)OR¹⁰or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁶selected from the group consisting of hydrogen, alkyl, cyano, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR¹⁰、-NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-C(O)OR¹⁰or-NR⁸C(O)R⁹Wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl、＝O、-NR⁸R⁹、-C(O)NR⁸R⁹、-C(O)R¹⁰、-C(O)OR¹⁰or-NR⁸C(O)R⁹Substituted with the substituent(s);

R⁷selected from a hydrogen atom or an alkyl group; preferably a hydrogen atom;

R⁸、R⁹and R¹⁰Each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally further substituted by one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

or, R⁸And R⁹Together with the N atom to which they are attached form a 4-to 8-membered heterocyclic group containing one or more N, O, S (O) atoms in the 4-to 8-membered heterocyclic group_pAn atom, and optionally further substituted on the 4-to 8-membered heterocycle by one or more groups selected from hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

R¹¹、R¹²and R¹³Each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group is optionally further substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl group, aryl group, heteroaryl group, carboxyl group, or carboxylate group; and is

p is 0, 1 or 2.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof is a compound represented by the general formula (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein:

wherein:

R^aand R^bEach independently selected from a hydrogen atom or an alkoxy group, and R^aAnd R^bNot being hydrogen atoms at the same time; the alkoxy is preferably methoxy;

m is 1,2, 3 or 4; preferably 1 or 2;

n is 1,2, 3 or 4; preferably 1 or 2; and is

R¹、R²、R⁴～R⁷The definition of (A) is described in the general formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof is a compound represented by the general formula (III) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

wherein:

R^ais selected as a hydrogen atom;

R^bselected from alkoxy, preferably methoxy;

m is 1,2, 3 or 4; preferably 1 or 2;

n is 1,2, 3 or 4; preferably 1 or 2; and is

R¹、R²、R⁴～R⁷The definition of (A) is described in the general formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof is a compound represented by the general formula (IV) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

wherein:

R^ais a hydrogen atom;

R^bselected from alkoxy, preferably methoxy;

m is 1,2, 3 or 4; preferably 1 or 2;

n is 1,2, 3 or 4; preferably 1 or 2; and is

R¹、R²、R⁴～R⁷The definition of (A) is described in the general formula (I).

In a preferred embodiment of the present invention, a compound of formula (I), (II), (III) or (IV) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein R is⁴Selected from fluorine, chlorine or bromine; preferably fluorine.

In a preferred embodiment of the present invention, a compound of formula (I), (II), (III) or (IV) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein R is⁶Is selected from heterocyclic groups, preferably morpholinyl.

Typical compounds of the invention include, but are not limited to:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

Further, the present invention provides a process for the preparation of a compound of formula (I), which process comprises:

general formulae (IA) and R⁶H, reacting to obtain a compound shown in a general formula (I);

wherein:

x is selected from halogen, preferably chlorine or bromine;

R⁶is selected from heterocyclic radical; and is

R¹～R⁵、R⁷、X¹And X²The definition of (A) is described in the general formula (I).

The invention provides a compound shown as a general formula (IA) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof:

wherein:

x is selected from halogen, preferably chlorine or bromine; and is

R¹～R⁵、R⁷、X¹And X²The definition of (A) is described in the general formula (I).

Typical compounds of formula (IA) include, but are not limited to:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

Further, the present invention provides a process for the preparation of a compound of formula (IA), which process comprises:

reacting a compound of formula (Ib) with a compound of formula (Ic) to give a compound of formula (IA);

wherein:

x is selected from halogen, preferably chlorine or bromine; and is

R¹～R⁵、R⁷、X¹And X²The definition of (A) is described in the general formula (I).

Further, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I), (II), (III) or (IV), or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or combination thereof.

The present invention provides a method of inhibiting a bromodomain protein, comprising contacting the receptor with a compound of formula (I), (II), (III) or (IV), or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof, wherein the bromodomain protein is preferably BRD2, BRD3 and BRD4, more preferably BRD 4.

The present invention provides the use of a compound of formula (I), (II), (III) or (IV) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof, wherein the bromodomain proteins are preferably BRD2, BRD3 and BRD4, more preferably BRD4, in the manufacture of a medicament for the manufacture of a bromodomain protein inhibitor.

The invention provides a compound of general formula (I), (II), (III) or (IV) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, wherein the disease is preferably cancer or inflammation, and the inflammation is preferably rheumatoid arthritis, Crohn's disease, eczema, giant cell arteritis, hepatitis, inflammatory bowel disease, osteoarthritis, pancreatitis, pneumonia, psoriasis, psoriatic arthritis, systemic lupus erythematosus, glomerulonephritis, lupus nephritis, membranous glomerulonephritis or myocarditis; the inflammation is more preferably rheumatoid arthritis; wherein the cancer is preferably small cell lung cancer, non-small cell lung cancer, breast cancer, colorectal cancer, prostate cancer, melanoma, pancreatic cancer, glioma, brain tumor, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, gastric cancer, bladder cancer, liver cancer, testicular nucleoprotein-derived cancer, multiple myeloma, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, or chronic myelogenous leukemia.

The invention provides an application of a compound shown in a general formula (I), (II), (III) or (IV) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a medicament for treating diabetic nephropathy, hypertensive nephropathy, HIV-related nephropathy, polycystic kidney disease, obesity, dyslipidemia, hypercholesterolemia, Alzheimer's disease, metabolic syndrome, fatty liver, type II diabetes, insulin resistance, diabetic retinopathy or diabetic neuropathy.

Detailed description of the invention

Unless stated to the contrary, some of the terms used in the specification and claims of the present invention are defined as follows:

"alkyl" when taken as a group or part of a group means including C₁-C₂₀Straight-chain or branched aliphatic hydrocarbon groups. Preferably C₁-C₁₀Alkyl, more preferably C₁-C₆An alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. Alkyl groups may be substituted or unsubstituted.

"cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged, and spiro carbocyclic rings. Preferably C₃-C₁₂Cycloalkyl, more preferably C₃-C₈Cycloalkyl, most preferably C₃-C₆A cycloalkyl group. Examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, with cyclopropyl, cyclohexenyl being preferred.

"spirocycloalkyl" refers to a 5 to 18 membered polycyclic group having two or more cyclic structures with single rings sharing a single carbon atom (called the spiro atom) with each other, containing 1 or more double bonds within the ring, but no ring has a completely conjugated pi-electron aromatic system. Preferably 6 to 14, more preferably 7 to 10. Spirocycloalkyl groups are classified according to the number of spiro atoms shared between rings into mono-spiro, di-spiro, or multi-spiro cycloalkyl groups, preferably mono-spiro and di-spiro cycloalkyl groups, preferably 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered. Non-limiting examples of "spirocycloalkyl" include, but are not limited to: spiro [4.5] decyl, spiro [4.4] nonyl, spiro [3.5] nonyl, spiro [2.4] heptyl.

"fused cycloalkyl" refers to a 5 to 18 membered all carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms with each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, preferably 6 to 12, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic fused alkyl groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicycloalkyl groups. Non-limiting examples of "fused ring alkyl" include, but are not limited to: bicyclo [3.1.0] hexyl, bicyclo [3.2.0] hept-1-enyl, bicyclo [3.2.0] heptyl, decalinyl or tetradecaphenanthryl.

"bridged cycloalkyl" means a 5 to 18 membered all carbon polycyclic group containing two or more cyclic structures sharing two non-directly attached carbon atoms with each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, preferably 6 to 12, more preferably 7 to 10. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic bridged cycloalkyl groups according to the number of constituent rings, and are preferably bicyclic, tricyclic or pyridone, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" groups include, but are not limited to: (1s,4s) -bicyclo [2.2.1] heptyl, bicyclo [3.2.1] octyl, (1s,5s) -bicyclo [3.3.1] nonyl, bicyclo [2.2.2] octyl, and (1r,5r) -bicyclo [3.3.2] decyl.

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring to which the parent structure is attached is cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted.

"Heterocyclyl", "heterocycle" or "heterocyclic" are used interchangeably herein and all refer to non-aromatic heterocyclic groups in which one or more of the ring-forming atoms is a heteroatom, such as oxygen, nitrogen, sulfur, and the like, including monocyclic, fused, bridged, and spiro rings. Preferably having a 5 to 7 membered monocyclic ring or a 7 to 10 membered bi-or tricyclic ring, which may contain 1,2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1, 1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, and piperazinyl. The heterocyclic group may be substituted or unsubstituted.

"spiroheterocyclyl" refers to a 5-to 18-membered polycyclic group having two or more cyclic structures wherein the individual rings share an atom with one another and which contains 1 or more double bonds within the ring, but none of the rings have a fully conjugated pi-electron aromatic system wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_p(wherein p is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. The spirocycloalkyl group is classified into a single spiroheterocyclic group, a double spiroheterocyclic group or a multiple spiroheterocyclic group, preferably a single spiroheterocyclic group and a double spiroheterocyclic group, according to the number of spiro atoms shared between rings. More preferred are 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered mono spiroheterocyclic groups. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to: 1, 7-dioxaspiro [4.5]]Decyl, 2-oxa-7-azaspiro [4.4]Nonyl, 7-oxaspiro [3.5]]Nonyl and 5-oxaspiro [2.4]]A heptyl group.

"fused heterocyclyl" refers to an all-carbon polycyclic group containing two or more cyclic structures sharing a pair of atoms with each other, one or more of which rings may contain one or more double bonds, but none of which rings has a fully conjugated pi-electron aromatic system, wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O)_p(wherein p is selected from 0, 1 or 2),the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic fused heterocyclic groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: octahydropyrrolo [3,4-c]Pyrrolyl, octahydro-1H-isoindolyl, 3-azabicyclo [3.1.0]Hexyl, octahydrobenzo [ b ]][1,4]Dioxins (dioxines).

"bridged heterocyclyl" means a 5-to 14-membered, 5-to 18-membered polycyclic group containing two or more cyclic structures sharing two atoms not directly attached to each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system in which one or more ring atoms are selected from nitrogen, oxygen, or S (O)_p(wherein p is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic bridged heterocyclic groups according to the number of constituent rings, preferably bicyclic, tricyclic or pyridone, more preferably bicyclic or tricyclic. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: 2-azabicyclo [2.2.1]Heptyl, 2-azabicyclo [2.2.2]Octyl and 2-azabicyclo [3.3.2]A decyl group. The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl. The heterocyclic group may be optionally substituted or unsubstituted.

"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused fashion. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably aryl is C₆-C₁₀Aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl. The aryl group may be substituted or unsubstituted. The "aryl" may be fused to a heteroaryl, heterocyclyl or cycloalkyl group, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples include, but are not limited to:

"heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 9-to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1, 2-dihydropyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2, 3-thiadiazolyl, benzodioxolyl, benzimidazolyl, indolyl, isoindolyl, 1, 3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, and benzisoxazolyl. Heteroaryl groups may be substituted or unsubstituted. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples include, but are not limited to:

"alkoxy" refers to a radical of (alkyl-O-). Wherein alkyl is as defined herein. C₁-C₆Alkoxy groups of (4) are preferred. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"hydroxy" refers to an-OH group.

"halogen" refers to fluorine, chlorine, bromine and iodine.

"amino" means-NH₂。

"cyano" means-CN.

"nitro" means-NO₂。

"benzyl" means-CH₂-phenyl.

"carboxy" refers to-C (O) OH.

"carboxylate" refers to-C (O) O (alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

"Bn" refers to benzyl.

"DMSO" refers to dimethyl sulfoxide.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

As used herein, "substituted" or "substituted," unless otherwise specified, means that the group may be substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, ═ O, -OR¹⁰、-SR¹⁰、-C(O)NR⁸R⁹、-C(O)R¹⁰、-C(O)OR¹⁰or-NR⁸C(O)R⁹；

R⁸、R⁹And R¹⁰Each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally further substituted by one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

or, R⁸And R⁹Together with the N atom to which they are attached form a 4-to 8-membered heterocyclic group containing one or more N, O, S (O) atoms in the 4-to 8-membered heterocyclic group_pAtomic, and 4 &The 8-membered heterocycle is further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -NR¹¹R¹²、-C(O)NR¹¹R¹²、-C(O)R¹³、-C(O)OR¹³or-NR¹¹C(O)R¹²Substituted with the substituent(s);

R¹¹、R¹²and R¹³Each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group is optionally further substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl group, aryl group, heteroaryl group, carboxylic acid, or carboxylic acid ester; and is

p is 0, 1 or 2.

"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain their biological activity and are suitable for pharmaceutical use. Pharmaceutically acceptable salts of the compounds of formula (I) may be metal salts, preferably alkali metal, alkaline earth metal salts, amine salts formed with suitable acids including inorganic and organic acids.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the invention relates to a preparation method of a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, which comprises the following steps:

compounds of the general formula (Ia) and R³NH₂Reacting to obtain a compound of a general formula (Ib); reacting a compound of formula (Ib) with a compound of formula (Ic) to give a compound of formula (IA); compounds of the general formula (IA) and R⁶H, reacting to obtain a compound shown in a general formula (I);

wherein:

x is selected from halogen, preferably chlorine or bromine;

R⁶is selected from heterocyclic radical; and is

R¹～R⁵、R⁷、X¹And X²Is as defined in formula (I);

drawings

FIG. 1 is a graph of the change in mean tumor volume of the example 1 compound of WO2016139292, the examples 1 and the example 4 compounds of the invention in test example 2 against the transplanted tumor of BALB/c nude mice bearing the tumor MV 4-11.

FIG. 2 is a graph of the mean relative change in tumor volume of the example 1 compound of WO2016139292, the examples 1 and the example 4 compounds of the invention in test example 2 versus transplanted tumors of BALB/c nude mice bearing MV4-11 tumors.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Examples

The examples show the preparation of representative compounds represented by formula (I) and the associated structural identification data. It must be noted that the following examples are intended to illustrate the invention and are not intended to limit the invention.¹The H NMR spectrum was obtained using a Bruker instrument (400MHz) and the chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00ppm) was used.¹Method for H NMR expression: s is singlet, d is doublet, t is triplet, m is multiplet, br is broadened, dd is doublet of doublet, dt is doublet of triplet. If a coupling constant is provided, it is in Hz.

The mass spectrum is measured by an LC/MS instrument, and the ionization mode can be ESI or APCI.

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

In the following examples, all temperatures are in degrees Celsius unless otherwise indicated, and unless otherwise indicated, the various starting materials and reagents are commercially available or synthesized according to known methods, and none of the commercially available materials and reagents are used without further purification, and unless otherwise indicated, commercially available manufacturers include, but are not limited to, Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Prov Chemical science Inc. and Sci Chemical science Inc., and the like.

CD₃OD: deuterated methanol.

CDCl₃: deuterated chloroform.

DMSO-d₆: deuterated dimethyl sulfoxide.

The argon atmosphere means that the reaction flask is connected with an argon balloon having a volume of about 1L.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

Purifying the compound by silica gel column chromatography using an eluent system selected from the group consisting of: a: petroleum ether and ethyl acetate systems; b: dichloromethane and methanol systems; c: dichloromethane: ethyl acetate; the volume ratio of the solvent is different according to the polarity of the compound, and a small amount of acidic or basic reagent such as acetic acid or triethylamine can be added for adjustment.

Example 1

5- (1- (1, 3-Dimethoxypropan-2-yl) -7-fluoro-5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

First step of

2- (dibenzylamino) propane-1, 3-diol

2-aminopropane-1, 3-diol 1a (10.0g,109.7mmol) and potassium carbonate (47.7g,345.6mmol) were dissolved in 300mL of ethanol, stirred at room temperature, and benzyl bromide (56g,329mmol) was added dropwise to the reaction mixture, followed by reflux reaction for 12 hours. Filtration was carried out, the filtrate was concentrated under reduced pressure, the obtained residue was dissolved in 200mL of methylene chloride, washed with water (100mL), the aqueous layer was separated, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: System A) to obtain 2- (dibenzylamino) propane-1, 3-diol 1b (20g, white solid) in yield: 67%.

¹H NMR(400MHz,CDCl₃):δ7.40-7.25(m,10H),3.74(s,4H),3.69(m,2H),3.59(m,2H),2.99(m,3H).

Second step of

N, N-dibenzyl-1, 3-dimethoxy propyl-2-amine

2- (dibenzylamino) propane-1, 3-diol 1b (5g,18.4mmol) and potassium hydroxide (8.3g,147.6mmol) were dissolved in 80mL of dimethyl sulfoxide under an argon atmosphere and stirred at room temperature for 10 minutes. Methyl iodide (10.4g,73.6mmol) was added dropwise to the reaction mixture, and the reaction was carried out at room temperature for 1 hour. 100mL of water was added to the reaction mixture to quench the reaction. Extraction with ethyl acetate (100mL × 3), combination of the organic phases, washing with saturated aqueous sodium chloride (100mL × 1), drying over anhydrous sodium sulfate, concentration under reduced pressure and purification of the residue obtained by silica gel column chromatography (eluent: system a) gave N, N-dibenzyl-1, 3-dimethoxypropan-2-amine 1c (5g, white solid) in yield: 90.9 percent.

MS m/z(ESI)：300.0[M+1]

The third step

1, 3-Dimethoxypropan-2-amine

N, N-dibenzyl-1, 3-dimethoxypropan-2-amine 1c (5g, 16.7mmol) was dissolved in 50mL of methanol under hydrogen protection, and 10% palladium on carbon catalyst (500mg, 10%) was added to replace hydrogen for 3 times, followed by reaction at room temperature for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave crude 1, 3-dimethoxypropan-2-amine 1d (1.99g, colorless oil), yield: 100 percent. MS m/z (ESI): 120.1[ M +1]

¹H NMR(400MHz,CDCl₃):δ5.72(s,2H),3.44(m,4H),3.32(m,1H)3.28(s,6H).

The fourth step

4-bromo-N- (1, 3-dimethoxypropan-2-yl) -2-fluoro-6-nitroaniline

5-bromo-1, 2-difluoro-3-nitrobenzene 1e (500mg,2.11mmol) and 1, 3-dimethoxypropan-2-amine 1d (276mg,2.32mmol) were dissolved in 20mL acetonitrile, and potassium carbonate (437mg,3.16mmol) was added to react at 80 ℃ for 6 hours. Concentrated under reduced pressure, the solvent was removed, 200mL of water was added, extraction was performed with ethyl acetate (300mL × 3), the organic phases were combined, washed with a saturated aqueous sodium chloride solution (100mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to give 4-bromo-N- (1, 3-dimethoxypropan-2-yl) -2-fluoro-6-nitroaniline 1f (560mg, yellow solid) in yield: 78.7 percent.

MS m/z(ESI)：337.9[M+1]

The fifth step

5- (5-bromo-1- (1, 3-dimethoxypropan-2-yl) -7-fluoro-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

4-bromo-N- (1, 3-dimethoxypropan-2-yl) -2-fluoro-6-nitroaniline 1f (186mg,0.55mmol) and 1g (100mg,0.66mmol, prepared according to the procedure for the preparation of intermediate 2 disclosed in patent application WO2016146738 (A1)) of 1, 5-dimethyl-6-oxo-1, 6-dihydropyridine-3-carbaldehyde were dissolved in 5mL of ethanol, 2mL of water and sodium hydrosulfite (287mg,1.66mmol) were added in succession and reacted at 90 ℃ for 3 hours. The reaction solution was diluted with 10mL of water and 10mL of ethyl acetate, extracted with ethyl acetate (10 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system B) to give 5- (5-bromo-1- (1, 3-dimethoxypropan-2-yl) -7-fluoro-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 1H (150mg, yellow solid), yield: and (4) 64.9%.

MS m/z(ESI)：438.9[M+1]

The sixth step

5- (1- (1, 3-Dimethoxypropan-2-yl) -7-fluoro-5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

5- (5-bromo-1- (1, 3-dimethoxypropan-2-yl) -7-fluoro-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 1H (50mg,0.12mmol), morpholine (21mg,0.24mmol), tris (dibenzylideneacetone) dipalladium (11mg,0.012mmol), 2-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (11mg,0.024mmol) and potassium tert-butoxide (34mg,0.36mmol) were dissolved in 5mL1, 4-dioxane under argon and reacted at 100 ℃ for 1 hour with microwave. The reaction mixture was diluted with 10mL of water and 10mL of ethyl acetate, extracted with ethyl acetate (10 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developer: system B) to give 5- (1- (1, 3-dimethoxypropan-2-yl) -7-fluoro-5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 1(10mg, white solid) with a yield: 18.5 percent.

MS m/z(ESI)：444.5[M+1]

¹H NMR(400MHz,CDCl₃):δ7.98(s,1H),7.78(s,1H),7.07(s,1H),6.75-6.71(m,1H),4.88-4.83(m,1H),3.90-3.83(m,4H),3.80-3.75(m,4H),3.62(s,3H),3.26(s,6H),3.18-3.16(t,J＝4.0Hz,4H),2.22(s,3H).

Example 2

5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

First step of

4-bromo-2-fluoro-N- (1-methoxybut-2-yl) -6-nitroaniline

5-bromo-1, 2-difluoro-3-nitrobenzene 1e (600mg,2.52mmol) and 1-methoxybutan-2-amine 2a (260mg,2.52mmol) were dissolved in 20mL acetonitrile, potassium carbonate (521mg,3.78mmol) was added, and the reaction was carried out at 80 ℃ for 6 hours. Concentrated under reduced pressure, the solvent was removed, 50mL of water was added, extraction was performed with ethyl acetate (30mL × 3), the organic phases were combined, washed with a saturated aqueous sodium chloride solution (100mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to give 4-bromo-2-fluoro-N- (1-methoxybut-2-yl) -6-nitroaniline 2b (600mg, yellow solid), yield: 74.2 percent.

MS m/z(ESI)：322.8[M+1]

Second step of

5- (5-bromo-7-fluoro-1- (1-methoxybut-2-yl) -1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

4-bromo-2-fluoro-N- (1-methoxybut-2-yl) -6-nitroaniline 2b (600mg,1.87mmol) and 1g (282mg,1.87mmol) of 1, 5-dimethyl-6-oxo-1, 6-dihydropyridine-3-carbaldehyde were dissolved in 10mL of ethanol, and 2mL of water and sodium dithionite (813mg,4.67mmol) were sequentially added to react at 100 ℃ for 3 hours. Concentrated under reduced pressure, 50mL of ethyl acetate was added to the residue, stirred, filtered, and insoluble solid was removed, the filtrate was concentrated under reduced pressure, the residue was dissolved in toluene (10mL × 3), and further concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to obtain 5- (5-bromo-7-fluoro-1- (1-methoxybut-2-yl) -1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 2c (650mg, yellow solid), yield: 82.2 percent.

MS m/z(ESI)：421.8[M+1]

The third step

5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

5- (5-bromo-7-fluoro-1- (1-methoxybut-2-yl) -1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 2c (220mg,0.52mmol), morpholine (90.6mg,1.0mmol), tris (dibenzylideneacetone) dipalladium (47.6mg,0.052mmol), 2-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (47.6mg,0.1mmol) and potassium tert-butoxide (149.9mg,1.56mmol) were dissolved in 5mL1, 4-dioxane under argon and reacted at 100 ℃ for 2 hours with microwave. The reaction mixture was diluted with 50mL of water and 30mL of ethyl acetate, extracted with ethyl acetate (30 mL. times.2), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system B) to give 5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 2(221mg, white solid) in yield: 99.1 percent.

MS m/z(ESI)：429.0[M+1]

¹H NMR(400MHz,CDCl₃)δ8.31(s,1H),7.80(s,1H),7.19(s,1H),6.81(d,J＝15.1Hz,1H),4.58(s,1H),4.05(t,J＝10.3Hz,1H),3.92-3.85(m,4H),3.81(d,J＝9.1Hz,1H),3.68(s,3H),3.36(s,3H),3.26-3.19(m,4H),2.23(s,3H),2.03-1.92(m,1H),1.90-1.79(m,1H),0.61(t,J＝7.3Hz,3H).

Example 3 and example 4

(S) -5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

(R) -5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

First step of

(S) -5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

(R) -5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d ] imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one

Reacting 5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d]Imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 2(210mg,0.49mmol) was further prepared by resolving the chiral isomer using high performance liquid chromatography and a chiral column using Supercritical Fluid Chromatography (SFC) (chiral column ChiralPak AD,300 × 50mm i.d.,10 μm; 80 mL/min; mobile phase A for CO₂and B for ETOH(0.1％NH₃.H₂O)) to give (S) -5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d)]Imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 3(57.09mg, white solid), yield: 27.2%, 100% ee, retention time 4.590 min; (R) -5- (7-fluoro-1- (1-methoxybut-2-yl) -5-morpholinyl-1H-benzo [ d]Imidazol-2-yl) -1, 3-dimethylpyridin-2 (1H) -one 4(60.38mg, white solid), yield: 28.8%, 100% ee, retention time 2.863 min.

3

MS m/z(ESI)：429.0[M+1]

¹H NMR(400MHz,CDCl₃)δ8.31(s,1H),7.80(s,1H),7.19(s,1H),6.81(d,J＝15.1Hz,1H),4.58(s,1H),4.05(t,J＝10.3Hz,1H),3.92-3.85(m,4H),3.81(d,J＝9.1Hz,1H),3.68(s,3H),3.36(s,3H),3.26-3.19(m,4H),2.23(s,3H),2.03-1.92(m,1H),1.90-1.79(m,1H),0.61(t,J＝7.3Hz,3H).

4

MS m/z(ESI)：429.0[M+1]

¹H NMR(400MHz,CDCl₃)δ8.31(s,1H),7.80(s,1H),7.19(s,1H),6.81(d,J＝15.1Hz,1H),4.58(s,1H),4.05(t,J＝10.3Hz,1H),3.92-3.85(m,4H),3.81(d,J＝9.1Hz,1H),3.68(s,3H),3.36(s,3H),3.26-3.19(m,4H),2.23(s,3H),2.03-1.92(m,1H),1.90-1.79(m,1H),0.61(t,J＝7.3Hz,3H).

Biological evaluation

Test example 1 determination of BRD4 protein Activity of Compounds of the present invention

Preferred compounds of the invention are tested for biological activity against the BRD4 protein by the following method.

The test method adopts EPIGENEOUS of Cisbio assay company^TMBROMODOMAIN ASSAY the biological activity of compounds against BRD4 was expressed by testing the effect of compounds on FRET (fluorescence resonance energy transfer) -based interactions between recombinant human BRD4 protein and acetylated histone polypeptide substrates under in vitro conditions. Wherein the GST-labeled recombinant humanized BRD4(1/2) protein is derived from BPS Bioscience, and acetylated histone polypeptide substrate [ Lys (5,8,12,16) Ac]H4(1-21) -biotin peptide was purchased from Anaspec.

The specific experimental method and process can refer to EPIGENEOUS^TMThe BROMODOMAIN ASSAY kit instruction and the experimental process are briefly described as follows:

the compounds of the invention were first dissolved in DMSO and then diluted to the concentration required for the assay (final concentration range 10. mu.M-0.1 nM) in the buffer provided in the kit. mu.L of the compound was added to 384-well microplates, followed by 4uL of GST-labeled recombinant human BRD4(1/2) protein diluted in buffer and 4. mu.L of acetylated histone polypeptide substrate, respectively, and finally to the wellsAdding 5uL of each anti-GST antibody coupled with europium element compounds and FRET receptor d2 coupled with streptavidin, sealing with a sealing plate film after shaking and mixing uniformly, and shaking and incubating for 3-5 hours at room temperature. Duplicate well controls were set for each concentration of compound, and 2 μ L DMSO was added to control and blank wells. The fluorescence intensity of each well at the excitation wavelength of the corresponding europium-based element, the emission wavelength of 620nM and 665nM is then measured and read on a microplate reader compatible with TF-FRET mode. Calculating the inhibition rate of the compound at each concentration point by comparing with the fluorescence intensity value of a control group, and further performing nonlinear regression analysis on the inhibition rate by the compound concentration logarithm in GraphPad Prism 5 software to obtain the IC of the compound for inhibiting the interaction between the BRD4 protein and the acetylated histone polypeptide substrate₅₀The value is obtained.

TABLE 1 IC inhibition of BRD4 protein Activity by preferred Compounds of the invention₅₀Data of

Compound numbering	IC50(nM)/BRD4(1/2)
		Example 1 Compounds of WO2016139292	27
1	8.5
		4	13

And (4) conclusion: the preferred compounds of the present invention have a better inhibitory effect on BRD4 protein.

Remarking: the compound of example 1 of WO2016139292 has the formula:

see example 1 of WO2016139292 for a method of preparation thereof.

Test example 2 inhibition of prostate cancer cell (LNCaP) proliferation assay by the compounds of the present invention

The cell level activity of the preferred compounds of the invention is measured by the absorbance method of CCK-8(Dojindo, Donkeyi Chemie). Prostate cancer cells LNCaP (purchased from cell resource center, shanghai life science institute of china academy of sciences) in logarithmic growth phase were seeded into 96-well culture plates at a density of 4000 per well, and after overnight culture, different concentrations of test compounds (final concentration ranging from 30 μ M to 0.1nM) were added. Cells were cultured at 37 ℃ for 72 hours under 5% CO 2. After incubation, an appropriate volume of CCK-8 reagent (available from Donnell chemical technologies, Inc., Shanghai, cat # CK04) was added to each well and incubation was continued at 37 ℃ for 1-5 hours, followed by reading absorbance values at 450nM for each well on a microplate reader. Calculating the inhibition rate of the compound at each concentration point by comparing the absorbance value with the control group, and performing nonlinear regression analysis on the inhibition rate of the compound concentration logarithm in GraphPad Prism 5 software to obtain the IC of the compound for inhibiting cell proliferation₅₀The value is obtained.

TABLE 2 IC inhibition of LNCaP proliferation by preferred compounds of the invention₅₀Data of

Compound numbering	IC50(nM)/LNCaP
		Example 1 Compounds of WO2016139292	76
1	16
		4	43

And (4) conclusion: the preferred compound of the invention has better inhibition effect on the proliferation of prostate cancer cells LNCaP. Test example 3 test of growth inhibitory Effect of the Compound of the present invention on human acute monocytic leukemia cell MV4-11 nude mouse transplanted tumor

1. Purpose of experiment

This test was used to evaluate the growth inhibitory effect of orally administered compound of example 1, example 4 and compound of example 1 of WO2016139292 on human acute monocytic leukemia cells MV4-11 transplanted tumors in nude mice twice daily for 22 consecutive days.

2. Test article preparation

2.1 preparation of blank drug administration preparation

Appropriate volumes of test solutions containing 90% PEG300 and 10% ethanol (10% TPGS, w/v) were prepared as blanks for dosing.

2.2 formulation of the compound of example 1 of WO2016139292 for administration

An appropriate amount of the compound of example 1 of WO2016139292 is weighed, an appropriate volume of a solvent containing 90% PEG300 and 10% ethanol (10% TPGS, w/v) is added, and the mixture is vortexed and shaken uniformly to prepare an administration preparation with the concentration of 0.5 mg/mL.

2.3 formulation for oral administration of the Compound of example 1

An appropriate amount of the compound of example 1 was weighed, an appropriate volume of a solvent containing 90% PEG300 and 10% ethanol (10% TPGS, w/v) was added, and the mixture was vortexed and shaken uniformly to prepare an administration preparation with a concentration of 0.5 mg/mL.

2.4 formulation for oral administration of the Compound of example 4

An appropriate amount of the compound of example 4 was weighed, an appropriate volume of a vehicle containing 90% PEG300 and 10% ethanol (10% TPGS, w/v) was added, and the mixture was vortexed and shaken uniformly to prepare an administration preparation with a concentration of 0.5 mg/mL.

3. Laboratory animal

Variety and strain: BALB/c nude mice, SPF, female, 6-7 weeks old (16-22 g), good health condition, 32 mice, and 5-7 days of environment adaptation time. Certificate number: 1140070017310, available from Experimental animals technology, Inc. of Weitonglihua, Beijing.

MV-4-11 cell culture

At 5% CO₂MV4-11 cells (purchased from American Type Culture Collection (ATCC)) were routinely cultured in IMDM medium containing 10% fetal bovine serum under 37 ℃ culture conditions. Carrying out digestion and passage by 0.25% of pancreatin according to the growth condition of the cells, wherein the passage ratio is 1:3 or 1: 4.

5. Animal vaccination and grouping

On day 0 of the experiment, MV4-11 cells were harvested in the logarithmic growth phase, counted and resuspended in 50% PBS (phosphate buffered saline) (pH7.4, 0.01M) and 50% Matrigel (Matrigel), and the cell concentration was adjusted to 7.0X 10⁷cell/mL; the cells were placed in an ice box, the cell suspension was aspirated by a 1-mL syringe and injected subcutaneously into the right axilla of nude mice, 200. mu.L (14X 10) per animal⁶Cell/cell), MV4-11 transplantation tumor model was established. On day 12 after inoculation, tumors grew to a volume of 100-300 mm³On the left and right sides, mice with similar tumor volume and better shape (the shape is a single round sphere as much as possible, no irregular shape or a plurality of tumors are gathered together) are selected, and each group is divided into 4 groups.

6. Animal dosing and observation

The subjects were given 1 day 1 (QD), orally (po) on a fixed time per day basis based on animal body weight for each group of animals, on the day of the group (12 days post-inoculation), the first dose was started for 22 consecutive days, and the animal body weight per day was recorded.

A blank solvent is given to the group 1 by intragastric administration, the administration volume is 10mL/kg, QD and PO; group 2 the compound of example 1 of WO was administered by gavage at a dose of 5mg/kg, QD, PO; group 3 the compound of example 1 was administered at the following dose: 5mg/kg, QD and PO in 12-19 days; day 20-33 is 2mg/kg, QD, PO; group 4 the compound of example 4 was administered at the following dose: 5mg/kg, QD and PO in 12-19 days; day 20-33 is 2mg/kg QD, PO.

The formation of tumors at the inoculated sites of the animals in each group was observed, the tumor size was measured 2 times per week after the start of the experiment, and the tumor volume was calculated while the animal body weight was weighed and recorded.

Tumor Volume (TV) calculation formula is as follows:

TV(mm³)＝l×w²/2

wherein l represents the tumor major axis (mm); w represents the tumor minor diameter (mm).

Evaluation indexes of antitumor activity: tumor growth inhibition ratio TGI (%), relative tumor proliferation ratio T/C (%).

The formula for the Relative Tumor Volume (RTV) is:

RTV＝100×TV_t/TV_initial

wherein, TV_initialTumor volume measured when administered in groups; TV (television)_tThe tumor volume at each measurement during dosing.

The calculation formula of the relative tumor proliferation rate T/C (%) is:

T/C＝100％×(RTV_T/RTV_C)

wherein, RTV_TRepresenting treatment group RTV; RTV_CRepresenting the solvent control RTV.

The calculation formula of the tumor growth inhibition rate TGI (%) is as follows:

TGI＝100％×[1－(TV_t(T)－TV_initial(T))/(TV_t(C)－TV_initial(C))]

wherein, TV_t(T)Represents the tumor volume for each measurement in the treatment group; TV (television)_initial(T)Represents the tumor volume of the treatment group when administered in groups; TV (television)_t(C)Represents the tumor volume of each measurement of the solvent control group; TV (television)_initial(C)The tumor volume of the solvent control group at the time of group administration is indicated.

The calculation formula of the tumor weight inhibition rate IR (%) is as follows:

IR＝100％×(W_C－W_T)/W_C

wherein, W_CRepresenting tumor weight of control group; w_TIndicates the tumor weight of the treated group.

7. Results

The graph of the change in mean tumor volume of the example 1 compound of WO2016139292, the examples 1 and the example 4 compounds of the present invention against the transplanted BALB/c tumor of MV4-11 nude mice is shown in FIG. 1;

the mean relative change in tumor volume of the example 1 compound of WO2016139292, the examples 1 according to the invention and the example 4 compound against the transplanted BALB/c tumor of MV4-11 nude mice is shown in FIG. 2.

TABLE 3 growth inhibition ratio (TGI%)

TABLE 4 relative tumor proliferation rate T/C (%) (for M.V. 4-11 bearing BALB/C nude mice transplanted tumors) of the compounds of the present invention

TABLE 5 tumor weight and tumor inhibition rate of each group of animals at the end of the experiment

As can be seen from tables 3 to 5 and FIGS. 1 to 2, the concentration of the compound is 5 mg/kg; at a dose of 2mg/kg (PO, QD), the compounds of examples 1 and 4 of the present invention had a significant growth inhibitory effect on MV-411 cells in establishing a mouse in vivo tumor model within 22 days of administration, and both activities were superior to the compound of example 1 of WO 2016139292.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (12)

1. A compound, or a pharmaceutically acceptable salt thereof, wherein said compound is:

2. a pharmaceutical composition comprising an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 2 in the manufacture of a medicament for a bromodomain protein inhibitor.

4. The use according to claim 3, wherein the bromodomain protein is selected from the group consisting of BRD2, BRD3 and BRD 4.

5. The use according to claim 3, wherein the bromodomain protein is BRD 4.

6. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 2, in the manufacture of a medicament for the treatment of a disease associated with a bromodomain protein.

7. The use according to claim 6, wherein the disease is cancer or inflammation.

8. The use according to claim 7, wherein the inflammation is rheumatoid arthritis, crohn's disease, eczema, giant cell arteritis, hepatitis, inflammatory bowel disease, osteoarthritis, pancreatitis, pneumonia, psoriasis, psoriatic arthritis, systemic lupus erythematosus, glomerulonephritis, lupus nephritis, or myocarditis.

9. The use according to claim 7, wherein the inflammation is rheumatoid arthritis.

10. The use of claim 7, wherein the cancer is small cell lung cancer, non-small cell lung cancer, breast cancer, colorectal cancer, prostate cancer, melanoma, pancreatic cancer, glioma, brain tumor, cervical cancer, ovarian cancer, renal cell carcinoma, gastric cancer, bladder cancer, liver cancer, testicular nucleoprotein-midline cancer, multiple myeloma, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, or chronic myelogenous leukemia.

11. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 2, in the manufacture of a medicament for the treatment of diabetic nephropathy, hypertensive nephropathy, HIV-associated nephropathy, polycystic kidney disease, obesity, dyslipidemia, hypercholesterolemia, alzheimer's disease, metabolic syndrome, fatty liver, type II diabetes, insulin resistance, diabetic retinopathy or diabetic neuropathy.

12. The use according to claim 7, wherein the inflammation is membranous glomerulonephritis.

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