CN108084193A - A kind of BRD4 protein inhibitors - Google Patents

Abstract

The invention belongs to pharmaceutical technology fields, and in particular to the BRD4 protein inhibitors shown in logical formula (I), pharmaceutically acceptable salt, the ester of its facile hydrolysis, its isomers and intermediate and these compounds and its preparation method of intermediate.The BRD4 protein inhibitors of the present invention can be used in preparing the drug for the treatment of and/or pre- preventing tumor.Wherein R¹、R²、R³、R⁴、R⁵, A, B, C, D, Q, m and n be defined as in the description.

Description

BRD4 protein inhibitor

1. Field of the invention

The invention belongs to the technical field of medicines, and particularly relates to a BRD4 protein inhibitor, pharmaceutically acceptable salts, easily hydrolyzed esters, isomers and intermediates thereof, a preparation method of the compounds and the intermediates thereof, and a pharmaceutical preparation containing the compounds. The BRD4 protein inhibitor can be used for treating cancer diseases including lung cancer, breast cancer, leukemia, skin cancer, lymphoma and the like, and autoimmune diseases including rheumatoid arthritis, psoriasis, Crohn's disease and ulcerative colitis, and preventing the diseases.

2. Background of the invention

Epigenetic inheritance is a hotspot in recent years. Epigenetic can be understood as that different gene expressions appear under different environments and conditions to finally achieve different physiological phenotypes, but the basic gene structure is not changed. The mechanism of this selectivity lies in the utilization of gene methylation (methylation) and the improvement of histone (histone) after gene translation. This phenomenon of improvement of histone (histone) after gene translation is mainly methylation (methylation) and acetylation (acetylation) on exposed lysine of histone (histone).

The Bromodomains (BRD) protein family is an important histone-modified transcription recognition system. Its main work is to recognize acetylated lysines in exposed histones and then to perform subsequent transcription, including transcription and assimilation of protein macromolecules, transcription of translation factors (transcription factors), and activation of RNA polymerase.

In one embodiment, the bromodomain of a BET family member polypeptide comprises about 110 amino acids and shares a conserved fold comprising a left-handed bundle of four α helices connected by distinct loop regions that interact with chromatin.

By "BET family polypeptide" is meant a polypeptide or fragment thereof comprising two bromo domains and an additional terminal (ET) domain, which has transcriptional regulatory activity or acetylated lysine binding activity, BET family members including BRD2, BRD3, BRD4 and BRDT.

There is now increasing evidence that the pathogenesis of many diseases, such as cancer, may be due to a dysregulation in gene transcription rather than to gene mutation. Thus, effective control of gene transcription mechanisms, such as BRD inhibitors, would be a very effective treatment for cancer and immune diseases.

Among the protein family of BRDs, BRD4 is particularly well-linked with cancer and immune diseases. JQ1 is a recognized highly selective BRD4 inhibitor. A number of papers have documented that JQ1 has a good therapeutic effect in a variety of animal models associated with cancer and immune diseases. However, the JQ1 compound has not been clinically studied in humans because of its poor drug metabolism and short patented protection period. The BRD4 inhibitor CPI-0610 is used in the treatment of myelodysplastic syndromes, and bone marrow and extramedullary proliferation in phase I clinical studies. The BRD (BRD2/3/4) inhibitor MK-8628 is in phase II clinical studies for the treatment of recurrent glioma, solid tumors.

3. Summary of the invention

The technical scheme of the invention is as follows:

a compound represented by the general formula (I), a pharmaceutically acceptable salt thereof, an easily hydrolysable ester thereof or an isomer thereof:

wherein,

A. b, C and D represent C or N, respectively;

R¹and R²Respectively represent halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy radical C_1-6Alkyl radical, C_1-6Alkoxy radical C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, hydroxy C_1-6Alkyl, carboxyl C_1-6Alkyl, amino C_1-6Alkyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkyl carbonyl oxy, C_1-6Alkyl oxycarbonyl radical, C_1-6Alkylamido, carbamoyl, C_1-6Alkylaminocarbonyl, di (C)_1-6Alkyl) carbamoyl, aminosulfonyl, C_1-6Alkylaminosulfonyl, di (C)_1-6Alkyl) aminosulfonyl, aminosulfonyl C_1-6Alkyl radical, C_1-6An alkylsulfonyl or guanidino group;

q represents saturated, partially saturated, unsaturated, having 1 to 4 substituents selected from N, S, O and/or SO₂A 3-to 14-membered heterocyclic group of the heteroatom (a);

R³、R⁴and R⁵Respectively represent hydrogen atom, OH, OR⁶-O-aryl, -OCH₂-aryl, C_1-6Alkyl, 3-to 8-membered cycloalkyl, -CF₃、-OCHF₂、-OCF₃Halogen, -CN, -NR^6aR^6bCarbonyl group, -COOR⁶、-COOH、-COR⁶、-CH(OH)R⁶、-CH(OR⁶)R⁶、-CONR^6aR^6b、-NHCOR⁶、-NHSO₂R⁶、-NHSO₂-aryl, -SR⁶、-SOR⁶、-SO₂R⁶、-SO₂Aryl or containing 1-4 of N, O, S, SO and/or SO₂A 3-to 14-membered heterocyclic ring of the heteroatom (a);

R⁶、R^6aand R^6bEach represents a hydrogen atom or C_1-6An alkyl group;

m is 0,1 or 2;

n is 1 or 2.

Preferred compounds are:

wherein,

A. b, C and D represent C or N, respectively;

R¹and R²Respectively represent halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy radical C_1-6Alkyl radical, C_1-6Alkoxy radical C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, hydroxy C_1-6Alkyl, carboxyl C_1-6Alkyl, amino C_1-6Alkyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkyl carbonyl oxy, C_1-6Alkyl oxycarbonyl radical, C_1-6Alkylamido, carbamoyl, C_1-6Alkylaminocarbonyl, di (C)_1-6Alkyl) carbamoyl, aminosulfonyl, C_1-6Alkylaminosulfonyl, di (C)_1-6Alkyl) aminosulfonyl, aminosulfonyl C_1-6Alkyl radical, C_1-6An alkylsulfonyl or guanidino group;

q represents saturated, partially saturated, unsaturated, having 1 to 4 substituents selected from N, S, O and/or SO₂A 5-to 10-membered heterocyclic group of the heteroatom (a);

R³、R⁴and R⁵Respectively represent hydrogen atom, OH, OR⁶、C_1-6Alkyl, -CF₃、-OCHF₂、-OCF₃Halogen, -CN, -NR^6aR^6bCarbonyl group, -COOR⁶、-COOH、-COR⁶、-CH(OH)R⁶、-CH(OR⁶)R⁶、-CONR^6aR^6b、-NHCOR⁶、-NHSO₂R⁶-SR⁶、-SOR⁶or-SO₂R⁶；

R⁶、R^6aAnd R^6bEach represents a hydrogen atom or C_1-6An alkyl group;

m represents 1;

n represents 1.

Preferred compounds are:

wherein,

A. b, C and D represent C or N, respectively;

R¹and R²Respectively represent halogen and C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkylamino, hydroxy, carboxyl, amino or amino C_1-6An alkyl group;

q represents a saturated, partially saturated, unsaturated, 5-to 10-membered heterocyclic group having 1-2 heteroatoms selected from N, S or O;

R³、R⁴and R⁵Respectively represent hydrogen atom, OH, C_1-6Alkyl, -CF₃、-OCHF₂、-OCF₃Halogen, -CN, -NR^6aR^6bCarbonyl or-COOH;

R^6aand R^6bEach represents a hydrogen atom;

m represents 1;

n represents 1.

Preferred compounds are:

wherein,

A. b, C and D represent C or N, respectively;

R¹and R²Each represents halogen or C_1-6An alkyl group;

q represents a saturated, partially saturated, unsaturated, 5-to 10-membered heterocyclic group having 1 to 2 members selected from N; r³、R⁴And R⁵Each represents a hydrogen atom or a carbonyl group;

m represents 1;

n represents 1.

Further preferred compounds are:

A. b, C and D represent C or N, respectively;

R¹represents halogen;

R²represents C_1-6An alkyl group;

R³、R⁴and R⁵Each represents a hydrogen atom or a carbonyl group;

m represents 1;

n represents 1.

Particularly preferred compounds are:

Detailed Description

The "halogen atom" in the present invention includes a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and preferably a fluorine atom and a chlorine atom.

The "C1-6 alkyl" refers to a straight or branched alkyl group derived from an alkane having 1 to 6 carbon atoms by removing one hydrogen atom from the alkane moiety, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3-dimethylbutyl, 1-ethylbutyl, n-pentyl, 2-dimethylpropyl, neopentyl, 2-pentyl, 2-dimethylpropyl, 2-dimethylbutyl, 2-ethylbutyl, 1, 2-trimethylpropyl, 1,2, 2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl. Preferably C_1-4Alkyl radical, C_1-3An alkyl group.

Said "C" of the present invention_1-6Alkoxy "means the term" C_1-6Alkyl "a group attached to another structure through an oxygen atom, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentyloxy, neopentyloxy, hexyloxy, and the like.

Said "C" of the present invention_1-6Alkylcarbonyl "refers to the term" C_1-6Alkyl "a group attached to another structure through a carbonyl group, such as methylcarbonyl, ethylcarbonyl, propylcarbonyl, isopropylcarbonyl, butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, sec-butylcarbonyl, pentylcarbonyl, neopentylcarbonyl, hexylcarbonyl, and the like.

Said "C" of the present invention_1-6Alkoxycarbonyl "is the term" C_1-6Alkoxy "a group bonded to another structure through a carbonyl group, such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, sec-butoxycarbonyl, pentyloxycarbonyl, neopentyloxycarbonyl, hexyloxycarbonyl, etc.

The aryl refers to a cyclic aromatic group with 6-14-membered carbon atoms as ring atoms, and comprises 6-8-membered monocyclic aryl and 8-14-membered fused-ring aryl. The 6-to 8-membered monocyclic aryl group means an all-unsaturated aryl group such as phenyl, cyclooctatetraenyl and the like. The 8-to 14-membered fused ring aryl group is a cyclic group having two or more cyclic structures sharing two adjacent carbon atoms and having at least one ring being an all unsaturated aromatic ring, and includes 8-to 14-membered all unsaturated fused ring aryl, naphthyl, anthryl, phenanthryl and the like, and also includes 8-to 14-membered partially saturated fused ring aryl groups, such as benzo 3-to 8-membered saturated monocyclic cycloalkyl, benzo 3-to 8-membered partially saturated monocyclic cycloalkyl, and specific examples thereof include 2, 3-dihydro-1H-indenyl, 1,2,3, 4-tetrahydronaphthyl, 1, 4-dihydronaphthyl and the like.

The number of the carbon atoms is 1-4 selected from N, S, O, SO and/or SO₂The 3-14 membered heterocyclic ring of the heteroatom(s) is a monocyclic, bicyclic, spiro or bridged ring, wherein one of the carbon atoms which is not shared is N, S, O, SO and/or SO₂The heteroatom(s) in place of the fused heterocycle, spiroheterocycle, bridged heterocycle formed.

The heterocyclic group is a fused ring structure containing 6 to 14 ring atoms (wherein at least one heteroatom is contained) and formed by connecting two or more ring structures sharing two adjacent atoms, and comprises a 6 to 14-membered unsaturated heterocyclic group, a 6 to 14-membered partially saturated heterocyclic group and a 6 to 10-membered saturated heterocyclic group. The 6-14-membered unsaturated heterocyclic group refers to a condensed ring structure in which all rings are unsaturated, such as a structure formed by a benzo 3-8-membered unsaturated monocyclic heterocyclic group, a structure formed by a 3-8-membered unsaturated monocyclic heterocyclic group and a 3-8-membered unsaturated monocyclic heterocyclic group, and the like, and specific examples include but are not limited to: benzofuranyl, benzisofuranyl, benzothienyl, indolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, acridinyl, phenanthridinyl, pyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenazinyl, pteridinyl, purinyl, naphthyridinyl, indolyl, and mixtures thereof,And the like. A 6-to 14-membered partially saturated heterocyclic group, which means that the heterocyclic group contains at least one moietyRing-fused structures of rings, such as structures formed by a 3-8 membered partially saturated heteromonocyclic group, structures formed by a 3-8 membered partially saturated heteromonocyclic group and a 3-8 membered partially saturated heteromonocyclic group, and the like, specific examples include, but are not limited to: 1, 3-dihydrobenzofuranyl, benzo [ d ]][1.3]Dioxolyl, isoindolinyl, chromanyl, 1,2,3, 4-tetrahydropyrrolo [3,4-c ]]Pyrrole, pyrrole, And the like. The 6-10 membered saturated heterocyclic group refers to a fused ring structure in which all rings are saturated, such as a structure formed by a 3-8 membered saturated monocyclic heterocyclic group and a 3-8 membered saturated monocyclic heterocyclic group, and specific examples include but are not limited to: cyclobutane tetrahydropyrrole, cyclopentane tetrahydropyrrole, azetidine imidazolidine radical,And the like.

The bridged heterocyclic group is a bridged ring structure formed by 5 to 12 ring atoms (wherein at least one heteroatom is contained). The 5-12 membered bridged heterocyclic ring includes a 5-12 membered saturated bridged heterocyclic ring and a 5-12 membered partially saturated bridged heterocyclic ring.

The 5-12 membered saturated bridged heterocyclic ring refers to a saturated cyclic group in all rings in the bridged heterocyclic ring, and is preferably a 7-8 membered saturated bridged heterocyclic ring, and specific examples include but are not limited to:

and the like.

5-12 membered partially saturated bridged heterocycle, meaning the bridgeAmong the heterocyclic rings, there is a cyclic group in which at least one ring is unsaturated, preferably a 7-to 8-membered partially saturated bridged heterocyclic ring, and specific examples include, but are not limited to: and the like.

The spiroheterocyclic group is a spiro ring structure formed by 5 to 12 ring atoms (wherein at least one heteroatom is contained). The 5-12 membered spiroheterocycle includes a 5-12 membered saturated spiroheterocycle and a 5-12 membered partially saturated spiroheterocycle.

The 5-12 membered saturated spiroheterocycle refers to a cyclic group in which all rings in the spiroheterocycle are saturated, and specific examples include but are not limited to: and the like.

The 5-12 membered partially saturated spiroheterocycle refers to a cyclic group in which at least one ring of the spiroheterocycle is unsaturated, and specific examples include but are not limited to: a step of,And the like.

Heterocyclic compounds containing one N: such as aziridine (aziridine), azetidine, 1, 2-diazetidine, pyrrolidine, 2, 5-dihydropyrrole, 2H-pyrrole or 3H-pyrrole (isoxazole), piperidine, dihydropyridine, tetrahydropyridine, azepine, and the like;

heterocyclic compounds containing one O: for example ethylene oxide, propylene oxide (azetidine), tetrahydrofuran, dihydrofuran, 2H-pyran, 4H-pyrazinePyran, tetrahydropyran, 3, 4-dihydro-2H-pyran,Heptins, etc.;

heterocyclic compounds containing one S: for example, thietane (tetrahydrothiophene), 2, 5-dihydrothienyl, thiane (hexahydrothiopyran), thiacyclohexane, thiacycloheptane, etc.;

heterocyclic compounds containing two N: such as imidazolidine, imidazoline (4, 5-dihydroimidazole), pyrazolidine, pyrazoline (4, 5-dihydropyrazole), piperazine, etc.;

heterocyclic compounds containing two O: such as dioxirane, 1, 2-dioxetane, dioxolane (1, 3-dioxolane), dioxane (1, 3-dioxane, 1, 4-dioxane), dioxepane, etc.;

heterocyclic compounds containing two S: such as 1, 3-dithiolane, 1, 3-dithiane, etc.;

heterocyclic compounds containing three O: for example trioxane (tricyclo)Alkyl), etc.;

heterocyclic compounds containing one N — O: for example oxaziridines, tetrahydro-heterocyclesAzole, 4, 5-dihydroOxazole, tetrahydroisoquinolineAzole, 4, 5-dihydro-isoAzole, 2, 3-dihydro-isoOxazole, morpholine, tetrahydroOxazine and dihydroOxazine,Oxazines, 5, 6-dihydro-4H-1, 3-Oxazines and the like;

heterocyclic compounds containing one N and one S: for example, thiazoline (4, 5-dihydrothiazole), thiazolidine, thiomorpholine, 2H-1, 3-thiazine, 4H-1, 3-thiazine, 5, 6-dihydro-4H-1, 3-thiazine, 6H-1, 3-thiazine, 2H-1, 4-thiazine, 4H-1, 4-thiazine, etc.;

heterocyclic compounds containing one O — S: for example oxathiolane, 1, 3-oxathiane, oxathianeAlkyl), etc.;

heterocyclic compounds containing two N and one O: for exampleDiazines (oxadiazines), etc.;

heterocyclic compounds containing one N — O — S: e.g. N₁O₁S₁：Thiazines, and the like.

The invention further claims a preparation method of the compound shown in the general formula (I).

The preparation method of the compound of the general formula (I) comprises the steps of enabling the compound of the general formula (IV), pharmaceutically acceptable salt thereof or easily hydrolyzed ester thereof to react with the compound of the general formula (V), pharmaceutically acceptable salt thereof, easily hydrolyzed ester thereof or isomers thereof,

wherein R is¹、R²、R³、R⁴、R⁵A, B, C, D, Q, m and n are as defined above.

The above compounds of the present invention can be synthesized using the methods described in the schemes below and/or other techniques known to those of ordinary skill in the art, but are not limited to the methods below.

When n is 1, the reaction scheme is as follows:

the reaction steps are as follows:

step 1 preparation of Compound c

Dissolving the raw material a in acetonitrile, adding calcium hydroxide, controlling the temperature to be less than 30 ℃, dropwise adding the raw material b (210g, 1.2mol), and standing overnight at normal temperature. TLC monitored the reaction complete. Filtering, spin-drying the filtrate, adding water, extracting with EA, washing the organic phase with saturated sodium bicarbonate, and drying to obtain a yellow solid compound c which is directly used in the next step.

Step 2 preparation of Compound d

Compound c was added to phosphorus oxychloride and refluxed overnight. TLC monitored the reaction complete. Most of phosphorus oxychloride is removed by spinning, the residual oily matter is poured into an ice-water mixture to be stirred, yellow solid is separated out, and a yellow solid compound d is obtained by filtering and drying.

Step 3 preparation of Compound e

And adding the compound d into ammonia water, stirring at room temperature until the solid is completely dissolved, spin-drying to obtain a brown solid, pulping the solid with EA, and performing suction filtration to obtain a light brown solid compound e.

Step 4 preparation of Compound g

The intermediate e is dissolved in dichloromethane, added with triethylamine and stirred. Dissolving the raw material f in dichloromethane, controlling the temperature to be 30 ℃, dropwise adding the mixture into the mixed solution, refluxing the mixture overnight after the dropwise adding is finished, and monitoring the reaction by TLC to be basically complete. Filtration, spin-drying of the filtrate, addition of the residue to acetic acid: 1, 2-dichloroethane (1: 1) was added to the mixed liquid and refluxed overnight. And (3) spin-drying the solution, pouring a saturated sodium carbonate solution, separating out solids, filtering, and recrystallizing with ethanol to obtain a yellow solid compound g.

Step 5 preparation of Compound h

And adding the intermediate g into toluene, adding HMPA and Lawesson's reagent, refluxing, and performing rotary drying on a solvent column for chromatography to obtain a yellow solid compound h.

Step 6 preparation of Compounds of formula I

And (5) dissolving the intermediate h in tetrahydrofuran, cooling to 0 ℃, adding hydrazine hydrate and stirring. And (3) maintaining the temperature, adding triethylamine, continuing stirring, dropwise adding acetyl chloride, heating to room temperature, stirring, adding acetic acid, heating to reflux overnight, monitoring by TLC (thin layer chromatography) to basically complete the reaction, and performing spin-drying solvent column chromatography to obtain a yellow solid compound of the formula (I).

In the reaction equation, R¹、R²、R³、R⁴、R⁵A, B, C, D, Q and m are as defined above.

The invention further claims intermediates of the compounds shown in the general formula (I) in the preparation process, namely the compounds shown in the general formulas (II) and (III), pharmaceutically acceptable salts thereof, easily hydrolyzed esters thereof or isomers thereof, wherein R¹、R²、R³、R⁴、R⁵A, B, C, D, Q, m and n are as defined above.

"pharmaceutically acceptable salts" of any of the above compounds of the invention include alkali metal salts, such as sodium, potassium, lithium, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, and the like; other metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts such as ammonium salts; organic base salts such as tert-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N' -dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, tris (hydroxymethyl) aminomethane salt; hydrohalic acid salts such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide and the like; inorganic acid salts such as nitrate, perchlorate, sulfate, phosphate and the like; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate and the like; aryl sulfonates such as benzenesulfonate, p-benzenesulfonate and the like; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.; amino acid salts such as glycinate, trimethylglycinate, arginate, ornithine, glutamate, aspartate and the like.

"readily hydrolyzable esters" of any of the above compounds of the present invention are those pharmaceutically acceptable esters which hydrolyze in vivo to form the parent compound. It will be apparent to those skilled in the art that readily hydrolyzable esters of the compounds of the present invention may be formed at the free carboxyl or hydroxyl group of the compound and may be prepared by conventional methods.

"isomers" of any of the above compounds of the invention include all epimeric, diastereomeric and tautomeric forms. When a key is represented by a wedge, this indicates that the key will come out of the paper in three dimensions, and when a key is shaded, this indicates that the key will come back into the paper in three dimensions.

The present invention further claims a pharmaceutical composition comprising any of the above-mentioned compounds, pharmaceutically acceptable salts thereof, easily hydrolysable esters thereof or isomers thereof, and other pharmaceutically active ingredients.

The invention also includes any compound, its pharmaceutically acceptable salt, its easily hydrolysable ester or its isomer, which can be prepared into any clinically or pharmaceutically acceptable dosage form by the known method in the field, and can be applied to the patients needing the treatment by oral, parenteral, rectal or pulmonary administration. For oral administration, it can be made into conventional solid preparations such as tablet, capsule, pill, granule, etc.; it can also be made into oral liquid, such as oral solution, oral suspension, syrup, etc. When the composition is formulated into oral preparations, appropriate filler, binder, disintegrating agent, lubricant, etc. can be added. For parenteral administration, it can be made into injection, including injection solution, sterile powder for injection and concentrated solution for injection. The injection can be prepared by conventional method in the existing pharmaceutical field, and can be prepared without adding additives or adding appropriate additives according to the properties of the medicine. For rectal administration, it can be made into suppository, etc. For pulmonary administration, it can be made into inhalant or spray. Each unit preparation contains 0.01g to 10g, 0.01g, 0.05g, 0.1g, 0.125g, 0.2g, 0.25g, 0.3g, 0.4g, 0.5g, 0.6g, 0.75g, 1g, 1.25g, 1.5g, 1.75g, 2g, 2.5g, 3g, 4g, 5g, 10g and the like of a physiologically effective amount of the compound represented by the formula (I).

The compound, the pharmaceutically acceptable salt thereof or the stereoisomer thereof has better BRD4 inhibition effect, and is a better medicament with excellent antitumor effect and autoimmune disease treatment effect. Meanwhile, the compound, the pharmaceutically acceptable salt thereof or the stereoisomer thereof plays an important role in preparing medicaments for treating cancer diseases (including lung cancer, breast cancer, leukemia, skin cancer, lymphoma and the like) and autoimmune diseases (including rheumatoid arthritis, psoriasis, Crohn's disease and ulcerative colitis).

Autoimmune and/or inflammatory diseases that may be affected using the compounds and compositions according to the invention include, but are not limited to: psoriasis, allergies, regional enteritis, irritable bowel syndrome, sjogren's disease, tissue graft rejection and hyperacute rejection of transplanted organs, asthma, systemic lupus erythematosus < and associated glomerulonephritis, dermatomyositis, multiple sclerosis, scleroderma, vasculitis (ANCA-associated and other vasculitides), autoimmune hemolytic and thrombocytopenic symptoms, goodpasture's syndrome < and associated glomerulonephritis and pulmonary hemorrhage >, atherosclerosis, rheumatoid arthritis, chronic Idiopathic Thrombocytopenic Purpura (ITP), addison's disease, parkinson's disease, alzheimer's disease, diabetes, septic shock and myasthenia gravis.

Included herein are methods of treatment wherein at least one chemical entity provided herein is administered in combination with an anti-inflammatory agent. Anti-inflammatory agents include, but are not limited to: NSAIDs, nonspecific and COX-2 specific cyclooxygenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptor antagonists, immunosuppressants and methotrexate.

Examples of NSAIDs include, but are not limited to, ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, a combination of diclofenac sodium and misoprostol, sulindac, phenytopropionic acid, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, nabumetone sodium, sulfasalazine, tolmetin sodium, and hydroxychloroquine. Examples of NSAIDs also include COX-2 specific inhibitors such as celecoxib, valdecoxib, lumiracoxib and/or etoricoxib.

In some embodiments, the anti-inflammatory agent is a salicylate. Salicylates include, but are not limited to, acetylsalicylic acid or aspirin, sodium salicylate, and choline salicylate and magnesium salicylate.

The anti-inflammatory agent may also be a corticosteroid. For example, the corticosteroid can be cortisone, dexamethasone, methylprednisolone, prednisolone sodium phosphate, or prednisone.

In another embodiment, the anti-inflammatory agent is a gold compound such as disodium aurothioate or auranofin.

The invention also includes embodiments wherein the anti-inflammatory agent is a metabolic inhibitor such as a dihydrofolate reductase inhibitor, e.g., methotrexate, or a dihydroorotate dehydrogenase inhibitor, e.g., leflunomide.

Other embodiments of the invention relate to combinations wherein at least one of the anti-inflammatory compounds is an anti-monoclonal antibody (e.g., eculizumab or peclizumab), a TNF antagonist such as etanercept or infliximab, which is an anti-TNF α monoclonal antibody.

Other embodiments of the present invention relate to combinations wherein at least one of the active agents is an immunosuppressant compound such as an immunosuppressant compound selected from the group consisting of methotrexate, leflunomide, cyclosporin, tacrolimus, azathioprine and mycophenolate mofetil.

Compared with the closest prior art, the compound of the invention has the following advantages:

(1) the BRD4 protein inhibitor has better BRD4 inhibition effect;

(2) the BRD4 protein inhibitor has simple preparation process, high medicine purity and stable quality, and is easy to be produced in large scale.

4. Detailed description of the preferred embodiments

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

Example 1 6- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]Preparation of (E) -4-indolyl) methyl) piperidin-2-one (Compound 1)

Step 1: preparation of (4-chlorophenyl) - (2-hydroxy-1H-3-indolyl) methyl ketone (intermediate C)

Dissolving raw material A (133g, 1.0mol) in acetonitrile 1L, adding calcium hydroxide (148g, 2.0mol), controlling the temperature to be less than 30 ℃, dropwise adding raw material B (210g, 1.2mol), and standing overnight at normal temperature. TLC monitored the reaction complete. Filtration, spin-drying of the filtrate, addition of 2L of water, EA extraction (500ml x 3), washing of the organic phase with saturated sodium bicarbonate, drying to yield 210g of yellow solid, 77.8% yield, and direct use in the next step.

Step 2: preparation of (4-chlorophenyl) - (2-chloro-1H-3-indolyl) methyl ketone (intermediate D)

Intermediate C (210g, 0.78mol) was added to 1L of phosphorus oxychloride and refluxed overnight. TLC monitored the reaction complete. Most of phosphorus oxychloride is removed by spinning, the residual oily matter is poured into 2L of ice-water mixture and stirred for 1h, yellow solid is separated out, and the yellow solid is filtered and dried to obtain 160g of yellow solid with the yield of 71%.

And step 3: preparation of (4-chlorophenyl) - (2-amino-1H-3-indolyl) methyl ketone (intermediate E)

And adding the intermediate D (160g, 0.55mol) into 800ml of ammonia water, stirring at room temperature for 24 hours until all solids are dissolved, spinning to obtain brown solids, pulping the solids by using EA500ml, and performing suction filtration to obtain 125g of light brown solids with the yield of 83%.

And 4, step 4: preparation of (S) -5- (4-chlorophenyl) -3- (2-oxopiperidin-1-methyl) -3, 10-dihydro-1H- [1,4] azahepta [5,6-b ] indol-2-one (intermediate G)

Intermediate E (120g, 0.44mol) was dissolved in 600ml of dichloromethane and triethylamine (53g, 1.2mol) was added and stirred. Starting material F (225g, 1.1mol) was dissolved in 100ml of dichloromethane, added dropwise to the mixture at 30 ℃ and refluxed overnight with TLC monitoring of substantial completion of the reaction. Filtration, spin-drying of the filtrate, addition of the residue to acetic acid: 1, 2-dichloroethane was added to the 1:1 mixed liquid 1L and refluxed overnight. The solution was spin dried and saturated sodium carbonate solution was poured, solid precipitated, filtered and recrystallized from ethanol to yield 51g of yellow solid. The yield thereof was found to be 26%.

And 5: preparation of (S) -5- (4-chlorophenyl) -3- (2-oxopiperidine-1-methyl) -3, 10-dihydro-1H- [1,4] azahepta [5,6-b ] indole-2-thione (intermediate H)

Intermediate G (50G, 0.12mol) was added to 500ml of toluene, HMPA (24G, 0.14mol), Lawesson's reagent (59G, 0.14mol) were added, refluxed for 3h, and column chromatographed on dry solvent to give 41G of yellow solid in 79% yield. (eluent dichloromethane: methanol ═ 10:1)

Step 6: preparation of 6- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4] -triazolo [3',4':3,4] [1,4] -diaza [5,6-b ] -4-indolyl) methyl) piperidin-2-one (Compound 1)

Intermediate H (10g, 23mmol) was dissolved in 100ml tetrahydrofuran, cooled to 0 ℃ and added hydrazine hydrate (3.5g, 46mmol) and stirred for 2H. Triethylamine (4.6g, 46mmol) was added with the temperature maintained, stirring was continued for 1h, acetyl chloride (4g, 46mmol) was added dropwise, the temperature was raised to room temperature and stirred for 3h, 50ml acetic acid was added, the temperature was raised to reflux overnight, the reaction was monitored by TLC and 1.7g of a yellow solid was obtained by spin-drying solvent column chromatography. The yield thereof was found to be 12%.

The molecular formula is as follows: C25H23N6ClO molecular weight: 458.16LC-MS (M + H) +:459

1H NMR(DMSO)δ1.51(2H,t),δ1.57(2H,m),δ2.18(2H,t),δ2.36-2.45(5H,m),δ3.74(1H,m),δ4.41(1H,t),δ7.11-7.13(2H,m)7.30(2H,s)，δ7.45(2H,s),δ7.89-7.93(2H,m),δ9.59(1H,br),δ10.83(1H,br).

Example 26- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]Preparation of (E) -4-indolyl) methyl) hexahydropyrimidin-2-one (Compound 2)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₂₂N₇Molecular weight of ClO: 459.16LC-MS (M + H)⁺:460

1H NMR(DMSO)δ1.56(2H,t),δ1.70(2H,m),,δ2.39-2.57(5H,m),δ3.90(1H,m),δ4.60(1H,t),δ7.32-7.67(2H,m)7.49(2H,s)，δ7.79(2H,s),δ7.99-8.12(2H,m),δ9.01(1H,br),δ10.32(2H,br).

Example 36- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]Preparation of (E) -4-indolyl) methyl) pyrrolin-2-one (Compound 3)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₂₁N₆Molecular weight of ClO: 444.15 LC-MS (M + H)⁺:445

1H NMR(DMSO)δ1.41-1.57(4H,m),,δ2.36-2.45(5H,m),δ3.74(1H,m),δ4.41(1H,t),δ7.11-7.13(2H,m)7.30(2H,s)，δ7.45(2H,s),δ7.89-7.93(2H,m),δ9.59(1H,br),δ10.83(1H,br).

EXAMPLE 46- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]Preparation of (E) -4-indolyl) methyl) imidazolin-2-one (Compound 4)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₃H₂₀N₇Molecular weight of ClO: 445.14 LC-MS (M + H)⁺:446

1H NMR(DMSO)δ1.79(2H,d),δ2.22(3H,s),δ2.36-2.45(2H,m),δ3.41(1H,m),δ4.01(1H,t),δ7.07-7.11(2H,m)7.25(2H,s)，δ7.34(2H,s),δ7.66-7.69(2H,m),δ8.87(1H,br),δ10.01(2H,br).

Example 5 6- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]Preparation of (E) -4-indolyl) methyl) pyrimidin-2-one (Compound 5)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₅H₁₉N₆Molecular weight of ClO: 454.13 LC-MS (M + H)⁺:455

1H NMR(DMSO)2.45(3H,s),δ3.41-3.57(3H,m),δ3.86(1H,m),δ7.07-7.11(2H,m)7.25(2H,s)，δ7.30(1H,s)δ7.34(2H,s),δ7.50(1H,t)δ7.66-7.69(2H,m),δ7.91(1H,d)δ9.45(1H,br),δ11.33(1H,br).

Example 66- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]Preparation of (E) -4-indolyl) methyl) pyrimidine-2, 4-dione (Compound 6)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₁₈N₇ClO₂Molecular weight: 471.12 LC-MS (M + H)⁺:472

1H NMR(DMSO)2.39(3H,s),δ3.12-3.35(3H,m),δ7.12(2H,m)7.25(2H,s)，δ7.30(1H,s)δ7.34(2H,s),δ7.66-7.69(2H,m),δ9.57(2H,br),δ12.33(1H,br).

EXAMPLE 78- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]-4-indolyl) methyl) -2-azaspiro [4, 4] e]Preparation of nonane-2-one (Compound 7)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₈H₂₇N₆Molecular weight of ClO: 498.19 LC-MS (M + H)⁺:499

1H NMR(DMSO)δ1.51-1.70(4H,m),δ1.85-2.03(6H,m),δ3.33(3H,s),δ3.46-3.65(3H,m),δ4.65(1H,t),δ7.11-7.20(4H,m)，δ7.45(2H,s),δ7.89-7.93(2H,m),δ10.34(1H,br),δ12.87(1H,br).

EXAMPLE 8 Octa9- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza [5,6-b]-4-indolyl) methyl) -2-azaspiro [4,5]Preparation of decan-2-one (Compound 8)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₉H₂₉N₆Molecular weight of ClO: 512.21LC-MS (M + H)⁺:513

1H NMR(DMSO)δ1.38(2H,m),δ1.55-1.83(8H,m),δ1.93(2H,d),δ(3.11,s)δ3.34(2H,m)δ3.65(1H,m),δ4.65(1H,t),δ7.11-7.20(4H,m)，δ7.77(2H,s),δ7.83-7.91(2H,m),δ9.56(1H,br),δ11.87(1H,br).

EXAMPLE 99- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazoles [3',4' ] 3,4] [1,4]-diaza [5,6-b]-4-indolyl) methyl) -2-azaspiro [5,4]Preparation of decan-2-one (Compound 9)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₉H₂₉N₆Molecular weight of ClO: 512.21LC-MS (M + H)⁺:513

1H NMR(DMSO)δ1.40(4H,m),δ1.50-1.83(4H,m),δ1.93(2H,s),δ(2.01,s)δ(3.01,s)δ3.44(2H,m)δ3.56(1H,m),δ4.24(1H,t),δ7.19(2H,d)，δ7.67(2H,d),δ7.83-7.91(4H,m),δ9.79(1H,br),δ11.56(1H,br).

Example 106- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [1,8 a-dihydro-imidazo [1,2-a [ ]]Pyridine) methyl) piperidin-2-one (Compound 10) Preparation of

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₂₂N₇Molecular weight of ClO: 459.16LC-MS (M + H)⁺:460

1H NMR(DMSO)δ1.51(2H,t),δ1.57(2H,m),δ2.18(2H,t),δ2.36-2.45(5H,m),δ3.74(1H,m),δ4.41(1H,t),δ7.10-7.17(2H,dd)7.30(2H,s)，δ7.45-7.60(2H,m),δ8.34-8.40(2H,m),δ11.37(1H,br)

Example 116- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [1,8 a-dihydro-imidazo [1,2-a [ ]]Pyridine) methyl) pyrrol-2-one (Compound 11) Preparation of

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₃H₂₀N₇Molecular weight of ClO: 445.14 LC-MS (M + H)⁺:446

1H NMR(DMSO)δ1.45-1.52(2H,m),δ1.55-1.64(2H,m),δ2.44(2H,m),2.89(3H,s)δ3.66(1H,m),δ4.05(1H,t),δ7.08-7.12(2H,m)7.30(2H,s)，δ7.45(2H,s),δ7.89-7.93(2H,m),δ11.63(1H,br).

Example 126- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [1,8 a-dihydro-imidazo [1,2-a [ ]]Pyridine) methyl) pyrimidin-2-one (compound 12) Preparation of

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₁₈N₇Molecular weight of ClO: 455.13 LC-MS (M + H)⁺:456

1H NMR(DMSO)2.45(3H,s),δ3.41-3.47(2H,m),δ3.68,(1H,m)δ3.89(1H,m),δ7.07-7.13(2H,m)7.25(2H,s)，δ7.32-7.45(3H,m)δ7.34(2H,s),,δ7.91-8.11(2H,d)δ8.17-8.22(2H,m)δδ12.19(1H,br).

Example 136- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [1,3 a-dihydro-pyrrolo [1,5-a ]]Pyridine) methyl) piperidin-2-one (Compound 13) Preparation of

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₂₂N₇Molecular weight of ClO: 459.16LC-MS (M + H)⁺:460

1H NMR(DMSO)δ1.53(2H,t),δ1.56(2H,m),δ2.32(2H,t),δ2.45-2.55(5H,m),δ3.81(1H,m),δ4.32(1H,t),δ7.15-7.18(2H,m)7.31(2H,s)，δ7.67(2H,s),δ8.11-8.32(2H,m),δ11.83(1H,br).

Example 146- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [1,3 a-dihydro-pyrrolo [1,5-a ]]Pyridine) methyl) pyrrol-2-one (Compound 14) Preparation of

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₃H₂₀N₇Molecular weight of ClO: 445.14 LC-MS (M + H)⁺:446

1H NMR(DMSO)δ1.59(2H,m),δ2.16(2H,m),,δ2.48-2.59(5H,m),δ3.86(1H,m),δ4.37(1H,t),δ7.12-7.15(2H,m)7.35(2H,s)，δ7.68(2H,s),δ8.12-8.22(2H,m),δ11.33(1H,br).

Example 156- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [1,3 a-dihydro-pyrrolo [1,5-a ]]Pyridine) methyl) pyrimidin-2-one (compound 15) Preparation of

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₁₈N₇Molecular weight of ClO: 455.13 LC-MS (M + H)⁺:456

1H NMR(DMSO)2.49(3H,s),δ3.44-3.58(3H,m),δ3.94(1H,m),δ7.15-7.21(2H,m)7.32(2H,s)，δ7.39(1H,s)δ7.45(2H,s),δ7.71(1H,t)δ8.01-8.12(2H,m),δ8.23(1H,d)δ11.33(1H,br).

Example 166- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-triazolo [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [ 1H-pyrrolo [2,3-c]Preparation of pyridine) methyl) piperidin-2-one (Compound 16)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₂₂N₇Molecular weight of ClO: 459.16LC-MS (M + H)⁺:460

1H NMR(DMSO)δ1.53(2H,t),δ1.59(2H,m),δ2.16(2H,t),δ2.33-2.43(5H,m),δ3.76(1H,m),δ4.42(1H,t),δ7.11-7.31(4H,m)，δ7.45(1H,s),δ7.56(1H,d),7.77(1H,d),δ9.37(1H,br),δ10.61(1H,br).

Example 176- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [ 1H-pyrrolo [2,3-c]Preparation of pyridine) methyl) pyrrol-2-one (Compound 17)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₃H₂₀N₇Molecular weight of ClO: 445.14 LC-MS (M + H)⁺:446

1H NMR(DMSO)δ1.50(2H,t),δ1.53(2H,m),δ2.11(2H,t),δ2.33(3H,d)δ2.46(2H,m),δ3.78(1H,m),δ4.43(1H,t),δ7.14-7.36(4H,m)，δ7.47(1H,s),δ7.57(1H,d),7.73(1H,d),δ10.17(1H,br),δ11.61(1H,br).

EXAMPLE 186- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [ 1H-pyrrolo [2,3-c]Preparation of pyridine) methyl) pyrimidin-2-one (Compound 18)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₁₈N₇Molecular weight of ClO: 455.13 LC-MS (M + H)⁺:456

1H NMR(DMSO)δ2.53(3H,d)δ2.70(2H,m),δ3.58(1H,m),δ4.23(1H,t),δ7.14-7.36(4H,m)，δ7.47-7.52(3H,m),δ7.60(1H,d),δ7.66(1H.d)7.75(1H,d),δ10.17(1H,br),δ11.61(1H,br).

Example 196- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [ 1H-pyrrolo [3,2-c]Preparation of pyridine) methyl) piperidin-2-one (Compound 19)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₂₂N₇Molecular weight of ClO: 459.16LC-MS (M + H)⁺:460

1H NMR(DMSO)δ1.49(2H,t),δ1.64(2H,m),δ2.19(2H,t),δ2.31-2.42(5H,m),δ3.73(1H,m),δ4.40(1H,t),δ7.11(2H,d)7.24(2H,d)，δ7.69(1H,s),δ7.76(1H,d),7.89(1H,d),δ10.17(1H,br),δ11.41(1H,br).

Example 206- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [ 1H-pyrrolo [3,2-c]Preparation of pyridine) methyl) pyrimidin-2-one (Compound 20)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₄H₁₈N₇Molecular weight of ClO: 455.13 LC-MS (M + H)⁺:456

1H NMR(DMSO)δ2.42(3H,s),δ3.53(1H,m),δ(2H,t)δ4.27(1H,t),δ7.08(2H,d)δ7.14(2H,d)，δ7.33-7.40(3H,m)δ7.59(1H,s),δ7.72(1H,d),7.89(1H,d),δ10.17(1H,br),δ11.41(1H,br).

Example 216- (((S) -6- (4-chlorophenyl) -1-methyl-4, 11-dihydro- [1,2,4]-a triazole [3',4':3, 4][1,4]-diaza- [5,6-b]-4- [ 1H-pyrrolo [3,2-c]Pyridine compound) Preparation of methyl) pyrrol-2-one (Compound 21)

Reference example 1 was made to the synthesis method.

The molecular formula is as follows: c₂₃H₂₀N₇Molecular weight of ClO: 445.14 LC-MS (M + H)⁺:446

1H NMR(DMSO)δ2.19-2.33(4H,m),δ2.42(3H,s),δ3.56-3.70(3H,m),δ4.26(1H,t),δ7.12(2H,d)7.25(2H,d)，δ7.39(1H,s),δ7.56(1H,d),7.69(1H,d),δ10.67(1H,br),δ11.48(1H,br).

EXAMPLE 22 in vitro anti-Bromo Domain-4 (BRD4) Activity assay for Compounds of the invention

A 384-well AlphaScreen screening assay was used to determine the ability of compounds to bind BRD 4.

Receptor: the recombinant human BRD4 protein expressed by Escherichia coli cells pNIC-28-Bsa4 is added with a 6 histidine tag at the N-terminal. The his-tagged BRD4 was extracted from e.coli cells and purified by nickel chelate column affinity chromatography, gradient eluted with 10-500mM imidazole and further purified by HiLoad 16/60Superdex 75 Size Exclusion Chromatography (SEC). Protein integrity was determined by polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray mass spectrometry using Agilent 1100LC/MSD TOF. The purified protein was stored at 50mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), pH 7.510mM, 500mM NaCl and 5% glycerol at-80 ℃.

Buffer solution: pH7.450mM HEPES, 0.1M NaCl, 0.05% 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt (Chaps).

The method comprises the following steps: BRD4 protein 4 μ L was added to the dishes containing the compound/control, followed by incubation at room temperature for 30min, followed by addition of 4 μ L H4KAc4 peptide (custom-labeled peptide), followed by incubation for 30min, followed by addition of 8 μ L AlphaScreen beads at 0.064 μ g beads per well. The dish was placed in the dark for 1h and then read on an EnVision microplate detector.

The experimental results are as follows: as shown in table 1 below.

TABLE 1 in vitro anti-Bromo Domain-4 (BRD4) Activity assay for Compounds of the invention

Wherein, + + + + represents IC₅₀(μM)<1 mu M; + represents IC₅₀(μM)<5 mu M; + represents IC₅₀(μM)<10μM

And (4) experimental conclusion:

as can be seen from Table 1, compounds 1,3 and 7 of the present invention all had strong inhibitory activity against BRD4 protein, and compounds 2,4,5,10,11,16,17 and 21 of the present invention all had inhibitory activity against BRD4 protein.

Claims (10)

1. A compound represented by the general formula (I), a pharmaceutically acceptable salt thereof, an easily hydrolysable ester thereof or an isomer thereof:

wherein,

A. b, C and D represent C or N, respectively;

R¹and R²Respectively represent halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy radical C_1-6Alkyl radical, C_1-6Alkoxy radical C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, hydroxy C_1-6Alkyl, carboxyl C_1-6Alkyl, amino C_1-6Alkyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkyl carbonyl oxy, C_1-6Alkyl oxycarbonyl radical, C_1-6Alkylamido, carbamoyl, C_1-6Alkylaminocarbonyl, di (C)_1-6Alkyl) carbamoyl, aminosulfonyl, C_1-6Alkylaminosulfonyl, di (C)_1-6Alkyl) aminosulfonyl, aminosulfonyl C_1-6Alkyl radical, C_1-6An alkylsulfonyl or guanidino group;

q represents saturated, partially saturated, unsaturated, having 1 to 4 substituents selected from N, S, O and/or SO₂A 3-to 14-membered heterocyclic group of the heteroatom (a);

R³、R⁴and R⁵Respectively represent hydrogen atom, OH, OR⁶-O-aryl, -OCH₂-aryl, C_1-6Alkyl, 3-to 8-membered cycloalkyl, -CF₃、-OCHF₂、-OCF₃Halogen, -CN, -NR^6aR^6bCarbonyl group, -COOR⁶、-COOH、-COR⁶、-CH(OH)R⁶、-CH(OR⁶)R⁶、-CONR^6aR^6b、-NHCOR⁶、-NHSO₂R⁶、-NHSO₂-aryl, -SR⁶、-SOR⁶、-SO₂R⁶、-SO₂Aryl or containing 1-4 of N, O, S, SO and/or SO₂A 3-to 14-membered heterocyclic ring of the heteroatom (a);

R⁶、R^6aand R^6bEach represents a hydrogen atom or C_1-6An alkyl group;

m is 0,1 or 2;

n is 1 or 2.

2. The compound of claim 1, a pharmaceutically acceptable salt thereof, a readily hydrolyzable ester thereof, or an isomer thereof:

wherein,

q represents saturated, partially saturated, unsaturated, having 1 to 4 substituents selected from N, S, O and/or SO₂A 5-to 10-membered heterocyclic group of the heteroatom (a);

R³、R⁴and R⁵Respectively represent hydrogen atom, OH, OR⁶、C_1-6Alkyl, -CF₃、-OCHF₂、-OCF₃Halogen, -CN, -NR^6aR^6bCarbonyl group, -COOR⁶、-COOH、-COR⁶、-CH(OH)R⁶、-CH(OR⁶)R⁶、-CONR^6aR^6b、-NHCOR⁶、-NHSO₂R⁶-SR⁶、-SOR⁶or-SO₂R⁶；

m represents 1;

n represents 1.

3. The compound of claim 2, a pharmaceutically acceptable salt thereof, a readily hydrolyzable ester thereof, or an isomer thereof:

wherein,

R¹and R²Respectively represent halogen and C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkylamino, hydroxy, carboxyl, amino or amino C_1-6An alkyl group;

q represents a saturated, partially saturated, unsaturated, 5-to 10-membered heterocyclic group having 1-2 heteroatoms selected from N, S or O;

R³、R⁴and R⁵Respectively represent hydrogen atom, OH, C_1-6Alkyl, -CF₃、-OCHF₂、-OCF₃Halogen, -CN, -NR^6aR^6bCarbonyl or-COOH;

R^6aand R^6bEach represents a hydrogen atom.

4. The compound of claim 3, a pharmaceutically acceptable salt thereof, a readily hydrolyzable ester thereof, or an isomer thereof:

wherein,

R¹and R²Each represents halogen or C_1-6An alkyl group;

q represents a saturated, partially saturated, unsaturated, 5-to 10-membered heterocyclic group having 1 to 2 members selected from N;

R³、R⁴and R⁵Each represents a hydrogen atom or a carbonyl group.

5. The compound of claim 4, a pharmaceutically acceptable salt thereof, a readily hydrolyzable ester thereof, or an isomer thereof:

wherein,

R¹represents halogen;

R²represents C_1-6An alkyl group;

q represents

6. The compound of claim 5, a pharmaceutically acceptable salt thereof, a readily hydrolyzable ester thereof, or an isomer thereof:

7. the invention further claims intermediates of the compounds shown in the general formula (I) in the preparation process, namely the compounds shown in the general formulas (II) and (III), pharmaceutically acceptable salts, easily hydrolysable esters or isomers thereof,

wherein R is¹、R²、R³、R⁴、R⁵A, B, C, D, Q, m and n are as defined in claim 1.

8. A process for the preparation of a compound of formula (I), a pharmaceutically acceptable salt, an easily hydrolysable ester or an isomer thereof, which process comprises reacting a compound of formula (IV), a pharmaceutically acceptable salt or an easily hydrolysable ester thereof, with a compound of formula (V), a pharmaceutically acceptable salt, an easily hydrolysable ester or an isomer thereof,

wherein R is¹、R²、R³、R⁴、R⁵A, B, C, D, Q, m and n are as defined in claim 1.

9. A pharmaceutical composition comprising a compound, a pharmaceutically acceptable salt thereof, an easily hydrolysable ester thereof or an isomer thereof according to any one of claims 1 to 6 and one or more pharmaceutically acceptable carriers and/or diluents, in any pharmaceutically acceptable dosage form.

10. The use of a compound, a pharmaceutically acceptable salt thereof, an easily hydrolysable ester thereof or an isomer thereof according to any one of claims 1-6 in the manufacture of a medicament for the treatment and/or prevention of a tumour.