CN107793371B

CN107793371B - Bromodomain recognition protein inhibitor and preparation method and application thereof

Info

Publication number: CN107793371B
Application number: CN201610802181.9A
Authority: CN
Inventors: 沈竞康; 缪泽鸿; 熊兵; 胡剑萍; 王迎庆; 宋姗姗
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2016-09-05
Filing date: 2016-09-05
Publication date: 2020-12-15
Anticipated expiration: 2036-09-05
Also published as: WO2018041260A1; CN107793371A

Abstract

The present invention relates to compounds of general formula (I), and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates, and crystalline forms thereof. The compound of the general formula (I) can inhibit bromodomain recognition protein, and is used for regulating and controlling the apparent state of cells and treating a series of diseases and symptoms mediated by the bromodomain recognition protein, particularly treating hematological malignant tumors, midline cancers, inflammations and the like.

Description

Bromodomain recognition protein inhibitor and preparation method and application thereof

Technical Field

The invention relates to a novel bromodomain recognition protein inhibitor and a preparation method and application thereof; the invention also relates to application of the compounds in preparing medicaments for treating diseases mediated by bromodomain recognition proteins, in particular to application in preparing medicaments for treating diseases such as hematological malignancy, midline cancer, inflammation and the like.

Background

In 1964, Allfey et al found lysine acetylation as a protein post-translational modification (PTM), and they simultaneously found acetylation and methylation of histones and proposed that these post-translational modifications regulate RNA synthesis. Histone acetylation neutralizes the positive charge on the-amino group of a histone-specific lysine, resulting in weaker binding of negatively charged DNA to histone and relaxation of chromatin structure, which allows access of polymerase, transcription factors, and other transcription-related complexes to DNA, resulting in transcriptional activation of genes. Acetylation of histones can thus activate the transcriptional expression of a particular gene, whereas deacetylation of histones is the opposite of acetylation of histones and can therefore result in silencing of expression of a particular gene. Bromodomain proteins (BRDs) recognize histone terminally acetylated lysine residues, which act by recruiting protein complexes to influence the transcription process. 61 bromodomains are found on 46 different proteins in the human genome and can be divided into eight major families (as shown in FIG. 1), of which the BET (Bromo-and Extra-terminal) family is currently being studied more.

The BET family includes broadly expressed BRD2, BRD3, BRD4, and BRDT specifically expressed in testis.

The BET family functions primarily to recruit transcription regulatory complexes to acetylated chromatin, thereby controlling specific gene networks involved in cell proliferation and cell cycle progression. For example, BRD4 and BRDT modulate transcriptional elongation, primarily by interacting with positive transcriptional elongation factor (P-TEFb), resulting in activation of RNA polymerase II. BET family protein disorders are associated with many diseases, such as cancer, inflammation, etc., which make BET proteins an attractive drug target.

In 2010, two BET family selective inhibitors, (+) -JQ1 (see Filipakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W.B.; Fedorov, O.; Morse, E.M.; Keats, T.; Hickman, T.T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M.R.; Wang, Y.; Chktie, A.L.; West, N.; Cameron, M.J.; Schwarz, B.; Heightman, T.D.; La.; Thanggu, N.; French, C.A.; O.; Kung, K.; Knanase, S.D.; Semlik Send.; Thinkray, K.; C.A.; Wilford K.; C.S.I.; C.S.S.I. Thi C.I.; C.S. Thinless, C.I.; C. Thinkkum. K.; C. K. K.; C. Thi C. K. K.; C. K. supplement, C. K. K.; C. K. K.; C. K. 3, C. K. and C. K. 3, C. K. K.; C. A. K. K.; C. A. A.; and C. A.; C. A. A. With the continuing efforts of researchers, more BET family selective inhibitors are being discovered and more inhibitors of the non-BET family of bromodomain proteins have been studied in recent years, which may help to better understand protein function and its associated diseases.

In recent years, dual inhibitors of kinase-bromodomain recognition proteins have been studied, and BRD4 has been shown to exhibit atypical kinase properties, i.e., phosphorylation of the serine 2 position at the C-terminus of RNA polymerase IIC. Wherein the PLK1 inhibitor BI2536 and JAK2 inhibitor TG101209 have good activity on BRD4, and IC₅₀25nM and 130nM, respectively. These early discoveries allow one to rationally design dual inhibitors of kinase-bromodomain recognition proteins or selective bromodomain recognition protein inhibitors from kinase inhibitors, where selective inhibitors can reduce off-target side effects.

Disclosure of Invention

The inventors of the present invention rationally designed, synthesized and studied selective bromodomain recognition protein inhibitors from dual inhibitors of kinase-bromodomain recognition proteins. The inventors obtained the following important information by detailed analysis of the crystal structure (PDB ID: 4O 74): wherein the lactam structure in domain 1 is a key hydrogen bond with Asn140 in the BRD4KAc pocket, and the two nitrogen atoms in domain 2 are key hydrogen bonds with the hinge region of the kinase (FIG. 2A); the phenylamide structure and its attached group extended out of the binding pocket and did not enter the WPF subconjunctival site of the BRD4 binding site (fig. 2B). We analyzed the structure, retaining the lactam structure that forms the key hydrogen bond with BRD4 and replacing the N atom that forms the key hydrogen bond with the hinge region of the kinase with a C atom. A series of compounds described herein were thus synthesized and tested for activity. The test result shows that the compound has very high BRD4 target binding activity and growth inhibition activity of BRD4 sensitive cells. The inventors also obtained a unique mode of action of the compounds in the present application through crystal experiments and complex structure analysis: the compound unexpectedly enters the WPF sub-binding site, has quite different binding modes, and the resolved crystal structure is shown in figure 2C.

Therefore, an object of the present invention is to provide a class of bromodomain recognition protein inhibitors having novel structures, i.e., compounds represented by the general formula (I), and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystal forms thereof, which are useful for treating, preventing and inhibiting related diseases mediated by bromodomain recognition proteins;

another object of the present invention is to provide a process for producing a compound represented by the general formula (I);

still another object of the present invention is to provide a pharmaceutical composition comprising a compound represented by the general formula (I), and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof;

the invention also aims to provide the compound represented by the general formula (I) and application of a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof; the compound represented by the general formula (I) and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof are selective inhibitors acting on bromodomain recognition protein, and can inhibit the acetylation of lysine recognized by the bromodomain recognition protein.

The invention also aims to provide the application of the compound shown in the general formula (I) and the stereoisomer, the pharmaceutically acceptable salt, the prodrug, the solvate, the hydrate or the crystal form thereof in preparing the medicines for treating the diseases such as hematological malignant tumor, midline cancer, inflammation and the like.

The invention provides a compound shown as a general formula (I), and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate and a crystal form thereof:

wherein:

x is C or N;

R₁is hydrogen atom, substituted or unsubstituted C1-C20 straight chain or branched chain alkyl, substituted or unsubstituted C1-C20 straight chain or branched chain alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, and the substituent is halogen, hydroxyl, amino, nitro or cyano;

R₂is substituted or unsubstituted C1-C20 straight-chain or branched-chain alkyl, substituted or unsubstituted C1-C20 straight-chain or branched-chain alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, the substituent is halogen, hydroxyl, amino, nitro or cyano, R is₂The configuration of (A) can be R type or S type or racemate;

R₃is substituted or unsubstituted C1-C20 linear or branched alkyl, substituted or unsubstituted C1-C20 linear or branched alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted benzyl, and the substituents are independently selected from the following groups: halogen, hydroxy, amino, nitro, cyano, a 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, a C1-C3 straight or branched chain alkyl group, or a C1-C3 straight or branched chain alkoxy group;

l is

or-CH₂-, wherein R₅Is a hydrogen atom, C1-C6 straight or branched chainA chain alkyl group;

R₄is C1-C20 straight chain or branched chain alkyl, halogen substituted C1-C20 straight chain or branched chain alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C3-C10 cycloalkyl and substituted or unsubstituted C6-C20 aromatic ring, substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C6-C12 aryl C1-C6 alkyl, substituted or unsubstituted 5-10 membered heterocyclic or heteroaryl group containing 1-3 heteroatoms selected from N, O and S, substituted or unsubstituted 5-10 membered heterocyclic or heteroaryl group containing 1-3 heteroatoms selected from N, O and S and substituted or unsubstituted C6-C12 aryl, the substituents are independently selected from 1-4 groups as follows: halogen, hydroxy, amino, nitro, cyano, C1-C6 linear or branched alkyl or C1-C6 linear or branched alkoxy, or

When L is

When R is₄And R₅May form, with the nitrogen atom to which they are attached, a substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl group containing a N atom and 0-2 heteroatoms of O, S and a substituted or unsubstituted C6-C20 aryl group, said substituents being independently selected from 1-2 of the following groups: halogen, hydroxyl, amino, nitro, cyano, C1-C6 straight chain or branched chain alkyl or C1-C6 straight chain or branched chain alkoxy.

Preferably:

x is C or N;

R₁is hydrogen atom, C1-C6 straight chain or branched chain alkyl or halogen substituted C1-C6 straight chain or branched chain alkyl;

R₂is C1-C6 straight chain or branched chain alkyl or halogen substituted C1-C6 straight chain or branched chain alkyl, R₂The configuration of (A) can be R type or S type or racemate;

R₃is a substituted or unsubstituted C1-C6 straight or branched chain alkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group, a substituted or unsubstituted benzyl group, and the substituents are independently selected from 1-5 groups as follows: halogen, hydroxy, 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, C1-C3 straight or branched alkyl, or C1-C3 straight or branched alkylAn oxy group;

l is

or-CH₂-, wherein R₅Is a hydrogen atom, a C1-C3 straight chain or branched chain alkyl group;

R₄is C1-C6 straight chain or branched chain alkyl, halogen substituted C1-C6 straight chain or branched chain alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 cycloalkyl and substituted or unsubstituted C6-C12 aromatic ring, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C6-C12 aryl C1-C4 alkyl, substituted or unsubstituted 5-10 membered heterocyclic or heteroaryl group containing 1-3 heteroatoms selected from N, O and S, substituted or unsubstituted 5-10 membered heterocyclic or heteroaryl group containing 1-3 heteroatoms selected from N, O and S and substituted or unsubstituted C6-C10 aryl, the substituents are independently selected from 1-4 groups as follows: halogen, hydroxy, C1-C3 straight or branched chain alkyl or C1-C3 straight or branched chain alkoxy, or

When L is

When R is₄And R₅May form, with the nitrogen atom to which they are attached, a substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl group containing a N atom and 0-2 heteroatoms of O, S and a substituted or unsubstituted C6-C12 aryl group, said substituents being independently selected from 1-2 of the following groups: halogen, hydroxyl, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy.

Preferably:

x is C or N;

R₁is a hydrogen atom or a C1-C3 straight or branched alkyl group;

R₂is C1-C6 straight chain or branched chain alkyl, R₂The configuration of (A) can be R type or S type or racemate;

R₃is a substituted or unsubstituted C1-C6 straight or branched chain alkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group, a substituted or unsubstituted benzyl group, the substituents are independently selected from 1-4 groups as follows: halogen, containing 1-2 substituents selected fromA 5-7 membered heterocyclyl of heteroatoms of N and O, a C1-C3 linear or branched alkyl group, or a C1-C3 linear or branched alkoxy group;

l is

R₄is substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkyl and substituted or unsubstituted C6-C12 aromatic ring, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C6-C12 aryl C1-C4 alkyl, substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl containing 1-3 heteroatoms selected from N, O and S, substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl containing 1-3 heteroatoms selected from N, O and S, and substituted or unsubstituted C6-C10 aryl, the substituents being independently selected from 1-3 groups as follows: halogen, hydroxy, C1-C3 straight or branched chain alkyl or C1-C3 straight or branched chain alkoxy, or

When L is

When R is₄And R₅May form, with the nitrogen atom to which they are attached, a substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl group containing a N atom and 0-2 heteroatoms of O, S and a substituted or unsubstituted C6-C10 aryl group, said substituents being independently selected from 1-2 of the following groups: halogen, hydroxyl, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy.

More preferably:

x is C or N;

R₁is a hydrogen atom, a methyl group or an ethyl group;

R₂is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, R₂The configuration of (A) can be R type or S type or racemate;

R₃is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxyethyl, a 5-to 6-membered hetero atom containing 1 to 2 hetero atoms selected from N and OCyclyl-substituted C1-C3 straight or branched chain alkyl (preferably morpholinoethyl, tetrahydrofurylmethyl), halogen or C1-C3 alkoxy-substituted benzyl (preferably Br-substituted benzyl or methoxybenzyl);

l is

or-CH₂-, wherein R₅Is a hydrogen atom, a methyl group or an ethyl group;

R₄is substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkyl and substituted or unsubstituted C6-C10 aromatic ring, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C6-C12 aryl C1-C3 alkyl, substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl containing 1-2 heteroatoms selected from N and O and substituted or unsubstituted C6-C10 aryl, the substituents being independently selected from 1-3 groups as follows: halogen, C1-C3 straight or branched chain alkyl or C1-C3 straight or branched chain alkoxy; preferably cyclopentyl, cyclohexyl, methylcyclohexyl, phenyl, methylphenyl, chlorophenyl, fluorophenyl, methoxyphenyl, isoxazolyl, 1,2, 4-trimethylpyrazol-3-yl, methylbenzyl, 2, 4-dimethylphenyl, 3-fluoro-4-methylphenyl, 2,4, 6-trimethylphenyl or pyrimidinyl, or

When L is

When R is₄And R₅And a substituted or unsubstituted C6-C10 aryl group containing a 5-6 membered heterocyclic or heteroaryl group containing a N atom and optionally an O atom and which is substituted or unsubstituted, said substituents being independently selected from 1 to 2 of the following groups: halogen, C1-C3 straight or branched chain alkyl or C1-C3 straight or branched chain alkoxy; preferably R₄And R₅May form with the nitrogen atom to which they are attached an indolinyl group, a methyl-substituted indolinyl group, a F-substituted indolinyl group or

Preferably, when L is-CH₂When is, R₄Containing an N atom.

Most preferably, the compound is selected from:

the compounds of formula (I) may contain asymmetric or chiral centers and may therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof (e.g., racemic mixtures), are included within the scope of the present invention.

The compounds of formula (I) may also exist in different tautomeric forms, all of which are included within the scope of the invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconverted via a low energy barrier.

The compounds of formula (I) may exist in unsolvated forms as well as solvated forms comprising pharmaceutically acceptable solvents such as water, ethanol and the like, and the compounds of the present invention include solvated as well as unsolvated forms.

The compound represented by the general formula (I) has a basic group and thus can form a pharmaceutically acceptable salt (i.e., a pharmaceutically acceptable salt) with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid or an organic acid such as ascorbic acid, nicotinic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, oxalic acid, malic acid, glycolic acid, succinic acid, propionic acid, acetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like, including a pharmaceutically acceptable acid addition salt, by treating a free base of the compound represented by the general formula (I) with the inorganic acid or the organic acid.

For purposes of illustration, the reaction schemes shown below provide possible routes for synthesizing the compounds of the present invention as well as key intermediates. For a more detailed description of the individual reaction steps, see the examples section below. The compounds of formula (I) of the present invention may be synthesized by methods including those well known in the chemical arts, particularly in light of the description of the present invention. The starting materials are generally available from commercial sources such as sigma aldrich or are readily prepared using methods well known to those skilled in the art.

The compounds in the reaction scheme include salts thereof, for example, salts as defined for the compounds having the general formula (I), etc., i.e., salts of the corresponding compounds are obtained by treating the free base form of the compounds with an organic or inorganic acid.

The preparation method of the compound represented by the structural general formula (I) comprises the following steps,

the reaction scheme I:

step a: compound 1A with amino acid NH₂R₂COOH to obtain a compound 1B;

step b: reacting the compound 1B under the condition of tin dichloride dihydrate to obtain a compound 1C;

step c: compounds 1C and R₁I reacts under the condition of sodium hydride to obtain a compound 1D;

step d: compounds 1D and sulfonamides R₄SO₂NH₂Obtaining a compound 1E through a coupling reaction under the conditions of allyl palladium chloride dimer, 2-di-tert-butyl phosphino-2 ',4',6' -triisopropyl biphenyl and potassium carbonate;

step e: compounds 1E and R₅I reacting under sodium hydride to obtain the compoundObject 1F.

Reaction scheme two:

step a: compound 2A with Primary amine R₃NH₂Reacting to obtain a compound 2B;

step b: reacting the compound 2B under the condition of iron powder and ammonium chloride to obtain a compound 2C;

step c: 1) compound 2C with different 2-bromoalkanoyl bromides

Reacting to obtain an intermediate, and 2) carrying out intramolecular nucleophilic reaction on the intermediate under the condition of N, N-diisopropylethylamine to obtain a compound 2D;

step d: compounds 2D and R₁I, reacting to obtain a compound 2E;

step e: compounds 2E and sulfonamides R₄SO₂NH₂Obtaining a compound 2F in a general formula through a coupling reaction under the conditions of allyl palladium chloride dimer, 2-di-tert-butyl phosphino-2 ',4',6' -triisopropyl biphenyl and potassium carbonate;

step f: compounds 2F and R₅I reacts under the condition of sodium hydride to obtain a compound 2G;

alternatively, after steps a-d, instead of performing steps e and f, the following steps are performed:

step g: 1) compound 2E with tert-butyl carbamate in Pd (OAc)₂2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl and cesium carbonate through a coupling reaction to obtain an intermediate, and 2) deprotecting the intermediate under the action of trifluoroacetic acid to obtain a compound 2H;

step h: compound 2H with different acid chlorides R₄COCl reaction to obtain a compound 2I;

step i: compounds 2I and R₅Reaction of I under sodium hydride conditions affords compound 2J.

The reaction route is three:

step a: reacting the compound 3A with thionyl chloride and methanol to obtain a compound 3B;

step b: compounds 3B with different primary amines R₃NH₂Reacting to obtain a compound 3C;

step c: reacting the compound 3C under the conditions of iron powder and ammonium chloride to obtain a compound 3D;

step d: 1) compound 3D with different 2-bromoalkanoyl bromides

Reacting to obtain an intermediate, and 2) carrying out intramolecular nucleophilic reaction on the intermediate under the condition of N, N-diisopropylethylamine to obtain a compound 3E;

step e: compounds 3E and R₁I reacts under the condition of sodium hydride to obtain a compound 3F;

step f: hydrolyzing the compound 3F under the condition of lithium hydroxide to obtain a compound 3G;

step g: compound 3G with amine R₄R₅The NH gives compound 3H by condensation reaction.

The reaction route is four:

step a: compound 4A with 2-bromoalkanoyl bromide

Reacting to obtain a compound 4B;

step b: compound 4B with amine R₃NH₂Reacting to obtain a compound 4C;

step c: carrying out intramolecular nucleophilic reaction on the compound 4C under the condition of N, N-diisopropylethylamine to obtain a compound 4D;

step d: compounds 4D and R₁I reacts under the condition of sodium hydride to obtain a compound 4E;

step e: compounds 4E and sulfonamides R₄SO₂NH₂Performing coupling reaction on allyl palladium chloride dimer, 2-di-tert-butyl phosphino-2 ',4',6' -triisopropyl biphenyl and potassium carbonate to obtain a compound 4F;

step f: compounds 4F and R₅I reacts under the condition of sodium hydride to obtain a compound 4G;

step g: compound 4E with amine R₄R₅NH in triethylamine, 4-dimethylaminopyridine, molybdenum hexacarbonyl, [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride and 1, 8-diazabicycloundecen-7-ene react under the condition to obtain a compound 4H;

alternatively, after steps a-d, instead of performing steps e and f or g, the following steps are performed:

step h: 1) compound 4E with tert-butyl carbamate in Pd (OAc)₂2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl and cesium carbonate under the condition of coupling reaction to obtain an intermediate, and 2) deprotecting the intermediate under the action of trifluoroacetic acid to obtain a compound 4I;

step i: compound 4I and acid chloride R₄COCl reaction to obtain a compound 4J;

step j: compounds 4J and R₅I reacts under the condition of sodium hydride to obtain a compound 4K,

in schemes one to four, R₁-R₄L and X are as defined above.

Reaction scheme five:

step a: compound 5A with amine R₄H, carrying out reduction ammoniation reaction to obtain a compound 5B;

step b: compound 5B with different amines R₃NH₂Reacting to obtain a compound 5C;

step c: reacting the compound 5C under the conditions of tin dichloride dihydrate and concentrated hydrochloric acid to obtain a compound 5D;

step d: 1) compound 5D with different 2-bromoalkanoyl bromides

Reacting to obtain an intermediate, and 2) carrying out intramolecular nucleophilic reaction on the intermediate under the condition of N, N-diisopropylethylamine to obtain a compound 5E;

step e: compounds 5E and R₁Reaction of I under sodium hydride conditions affords compound 5F.

In scheme five, R₁-R₄And X is as described above, and R₄Containing an N atom.

Preliminary studies have shown that the following diseases, conditions and/or disorders are mediated by inhibitors of bromodomain recognition proteins: hematologic malignancies, midline cancer, and inflammation.

Accordingly, the compounds of the present invention represented by general formula (I), and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystal forms thereof are useful for treating diseases, conditions and/or disorders mediated by bromodomain recognition protein inhibitors.

The present invention provides a method of treating diseases, conditions and/or disorders mediated by bromodomain recognition protein inhibitors, comprising administering to a patient an effective amount of a compound of formula (I), and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystal forms thereof.

The invention also provides a pharmaceutical composition, which comprises a therapeutically effective amount of the compound represented by the general formula (I), and one or more of stereoisomer, pharmaceutically acceptable salt, prodrug, solvate, hydrate and crystal form thereof, and at least one excipient, diluent or carrier.

Further, the compounds represented by the general formula (I), and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates or crystal forms thereof can be used in monotherapy or in combination therapy. When used in combination therapy, the compounds of the present invention represented by general formula (I), and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystal forms thereof, are generally used in conjunction with small molecule compound, radiation, antibody-based therapies (e.g. herceptin and rituximab), anticancer vaccination, gene therapy, cell therapy, hormone therapy or cytokine therapy.

Typical formulations are prepared by mixing a compound of formula (I) of the invention with a carrier, diluent or excipient. Suitable carriers, diluents or excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient employed will depend upon the mode and purpose for which the compounds of the invention are to be administered. Solvents are generally selected based on the solvent that one skilled in the art would consider safe (GRAS) for administration to a mammal. Generally, safe solvents are non-toxic aqueous solvents such as water, as well as other non-toxic solvents that are soluble or miscible with water. Suitable aqueous solvents include mixtures of one or more of water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), and the like. The formulation may also include one or more buffering agents, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifying agents, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents, or other known additives to provide an elegant presentation of the drug (i.e., the compound of the present invention or pharmaceutical composition thereof) or to aid in the manufacture of the pharmaceutical product (i.e., the drug).

The formulation may be prepared using conventional solution mixing procedures. For example, the drug substance in bulk form (i.e., the compound of formula (I) of the present invention or a stabilized form of the compound (e.g., a complex with a cyclodextrin derivative or other known complexing agent) is dissolved in a suitable solvent in the presence of one or more of the excipients described above.

According to the methods of the present invention, a compound of the present invention or a combination of a compound of the present invention and at least one other agent (referred to herein as a "combination"), is preferably administered in the form of a pharmaceutical composition. Thus, the compounds or combinations of the present invention can be administered to a patient separately or together in any known oral, rectal, transdermal, parenteral (e.g., intravenous, intramuscular, or subcutaneous) intracisternal, intravaginal, intraperitoneal, intravesical, topical (e.g., powder, ointment, or liquid drop), buccal, or nasal dosage form.

Compositions suitable for parenteral injection generally include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers or diluents (including solvents and carriers) include mixtures of one or more of water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like); vegetable oils (such as olive oil); and injectable organic esters such as ethyl oleate. The desired particle size may be maintained, for example, by the use of a coating such as lecithin, in the case of a dispersion, or by the use of surfactants to maintain suitable fluidity.

These compositions may also contain excipients such as preservatives, wetting agents, emulsifying agents and dispersing agents. Microbial contamination of the composition can be avoided by various bactericides and fungicides, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. These compositions may also include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical composition can also be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

Solid dosage forms for oral administration may include capsules, tablets, powders, and granules. In solid dosage forms, the compounds or combinations of the present invention are mixed with at least one inert excipient, diluent or carrier. Suitable excipients, diluents or carriers include materials such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders (e.g., starches, lactose, sucrose, mannitol, silicic acid, and the like); (b) binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, etc.); (c) humectants (such as glycerin, etc.); (d) disintegrating agents (such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, specific complex silicate, sodium carbonate, etc.); (e) solution retarding agents (e.g., paraffin, etc.); (f) accelerated absorbents (such as quaternary ammonium compounds and the like); (g) wetting agents (e.g., acetyl alcohol, glyceryl monostearate, etc.); (h) adsorbents (such as kaolin, bentonite, etc.); and/or i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, etc.). In the case of capsules and tablets, the dosage form may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose and high molecular weight polyethylene glycols and the like as excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the compounds of the present invention or compositions thereof, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide; oils (e.g., cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, etc.); glycerol; tetrahydrofurfuryl alcohol; fatty acid esters of polyethylene glycol and sorbitan; or mixtures of several of these substances, and the like.

In addition to these inert diluents, the compositions can also include excipients such as one or more of wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, and perfuming agents.

For suspensions, in addition to the compounds or combinations of the present invention, carriers such as suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, or mixtures of several of these, may be included.

Compositions for rectal or vaginal administration preferably include suppositories which can be prepared by mixing the compounds or combinations of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ordinary room temperature and liquid at body temperature and therefore melt in the rectum or vagina to release the active compound.

The compounds of the invention and combinations of the compounds of the invention with hematological cancer or inflammatory drugs in dosage forms for topical administration may include ointments, powders, sprays, and inhalants. The medicament may be mixed under sterile conditions with a pharmaceutically acceptable excipient, diluent or carrier, and any preservatives, buffers or propellants which may be required. Ophthalmic formulations, ophthalmic ointments, powders and solutions are also intended to be within the scope of the present invention.

It is known that the compounds (or compositions) of the invention can be placed in the drinking water, whereby a therapeutic dose of the compound is taken along with the daily drinking water supply. The compound can be metered directly into the drinking water, preferably in the form of a liquid water-soluble concentrate (such as an aqueous solution of a water-soluble salt).

Paste formulations may be prepared by dispersing the drug in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil and the like.

Pills containing an effective amount of a compound, pharmaceutical composition or combination of the present invention may be prepared by mixing a compound or composition of the present invention with a diluent such as carbowax, carnauba wax or the like; a lubricant such as magnesium stearate or calcium stearate may also be added to enhance the pelleting process.

The invention also provides the application of the compound represented by the general formula (I) and the stereoisomer, the pharmaceutically acceptable salt, the prodrug, the solvate, the hydrate or the crystal form thereof as a selective inhibitor of the bromodomain recognition protein, and the application in preparing medicaments for treating related diseases mediated by the bromodomain recognition protein. The relevant diseases mediated by the bromodomain recognition protein include, but are not limited to, hematological malignancies, midline cancer, and inflammation.

The invention also provides a compound represented by the general formula (I), and application of a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form of the compound to preparation of a medicament for treating diseases such as hematological malignant tumor, midline cancer, inflammation and the like.

The invention also provides a compound represented by the general formula (I), and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof, which is used for treating related diseases mediated by the bromodomain recognition protein.

The invention also encompasses isotopically-labeled compounds of the invention, which are identical to those recited herein, except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as:²hydrogen, hydrogen,³Hydrogen, hydrogen,¹¹Carbon, carbon,¹³Carbon, carbon,¹⁴Carbon, carbon,¹³Nitrogen, nitrogen,¹⁵Nitrogen, nitrogen,¹⁵Oxygen, oxygen,¹⁷Oxygen, oxygen,¹⁸Oxygen, oxygen,³¹Phosphorus, phosphorus,³²Phosphorus, phosphorus,³⁵Sulfur, sulfur,¹⁸Fluorine,¹²³Iodine,¹²⁵Iodine and³⁶chlorine.

Certain isotopically-labelled compounds of the invention (e.g. with³H and¹⁴c-labeled those) for compound and/or substrate tissue distribution assays. Tritiation (i.e., tritiation) is particularly preferred³H) And carbon-14 (i.e.¹⁴C) Isotopes because they are easy to prepare and detect. Also, heavier isotopes such as deuterium (i.e., deuterium)²H) Substitution may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may be preferred in certain circumstances. Positron emitting isotopes, e.g.¹⁵O、¹³N、¹¹C and¹⁸f was used in Positron Emission Tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the present invention can generally be followed by methods analogous to those disclosed in the schemes and/or in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagentTo prepare the compound.

Drawings

FIG. 1 is a schematic representation of a gene tree for human bromodomain proteins.

FIG. 2A is a design of a selective BDR4 inhibitor from a PLK-BRD4 dual inhibitor; FIG. 2B shows the binding pattern of BI2536 and BRD 4; figure 2C shows the binding pattern of compound 8 to BRD 4.

Detailed Description

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, the examples provided below are merely intended to further illustrate the invention and are not intended to limit the scope of the invention in any way.

The starting materials may be obtained from commercial sources or prepared by methods known in the art or according to the methods described herein.

The structure of the compound is determined by nuclear magnetic resonance¹H-NMR) and/or Mass Spectrometry (MS). NMR was measured using a Mercury-400 nuclear magnetic resonance apparatus manufactured by Varian corporation, and deuterated chloroform (CDCl) was used as a solvent₃) Deuterated methanol (CD)₃OD), deuterated dimethyl sulfoxide (DMSO-d)₆) Or deuterated acetonitrile (CD)₃CN), TMS as internal standard. MS was measured using a Thermo Finnigan LCQ-Deca XP model (ESI) liquid chromatography-mass spectrometer. ISCO is used for separating and purifying product by column chromatography

Rf 75 rapid preparation chromatograph, and the carrier adopts 200-mesh and 300-mesh silica gel of Qingdao ocean chemical plant.

Preparation examples:

example 1

Reagents and conditions: a) (R) -2-aminobutyric acid, potassium carbonate, ethanol, water, 80 ℃,3 hours; b) tin dichloride dihydrate, ethanol and concentrated hydrochloric acid at 80 ℃ for 3 hours; c) sodium hydride at 0 ℃, 30 minutes, methyl iodide, room temperature, 2 hours; d) p-toluenesulfonamide, allyl palladium chloride dimer, tBuXPhos, potassium carbonate, 2-methyltetrahydrofuran, 80 ℃ for 24 hours.

a) Dissolving the compound A (2g,9.09mmol) and (R) -2-aminobutyric acid (0.937g,9.09mmol) in 10mL of ethanol, adding 6N potassium carbonate aqueous solution (2.51g,18.18mmol), refluxing at 80 ℃ for 3 hours, monitoring the reaction by TLC, adjusting the pH to 7-8 with dilute hydrochloric acid after the reaction is finished, evaporating the solvent, extracting with ethyl acetate (20mL x 2) and 20mL of water, combining organic layers, back-extracting with saturated saline solution 40mL once, drying the organic phase with anhydrous sodium sulfate, and evaporating the organic phase to obtain 2.7g of white solid B, wherein the yield is 98.18%.¹H NMR(400MHz,CDCl₃)8.41-8.32(m,1H),8.07(dd,J＝9.2,0..9Hz,1H),6.93-6.89(m,1H),6.84(ddd,J＝9.1,1.9,0.9Hz,1H),4.24(q,J＝6.5Hz,1H),2.20-1.97(m,2H),1.09(t,J＝7.4Hz,3H)。

b) Compound B (1.5g,4.95mmol) was dissolved in 15mL ethanol, then stannous chloride dihydrate (4.47g,19.79mmol) was added to the above solution in 5mL ethanol and 1.0mL concentrated hydrochloric acid, the mixture was refluxed at 80 ℃ for 3 hours, the reaction was monitored by TLC, after the reaction was completed, the pH was adjusted to 7-8 with 2N sodium hydroxide, the filtrate was filtered, the filtrate was evaporated to dryness, extracted with ethyl acetate (20mL x 2) and 20mL water, the organic layers were combined, back-extracted once with 40mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was evaporated to dryness to give 1.24g white solid C, yield 98.41%.¹H NMR(400MHz,CDCl₃)8.78(s,1H),6.90-6.78(m,2H),6.60(d,J＝8.1Hz,1H),4.06-3.97(m,1H),3.87(dd,J＝7.3,5.1Hz,1H),1.91-1.73(m,2H),1.03(dd,J＝8.1,6.7Hz,3H).

c) Compound C (1.2g,4.70mmol) was dissolved in 5mL N, N-Dimethylformamide (DMF), cooled to 0 ℃, sodium hydride (0.452g,18.82mmol) was added, stirred at 0 ℃ for half an hour, methyl iodide (1.17mL,18.8mmol) was added at 0 ℃, then reacted at room temperature for 2 hours, monitored by TLC, after the reaction was completed, the pH was adjusted to 7-8 with dilute hydrochloric acid, extracted with ethyl acetate (20mL × 2) and 100mL water, the organic layers were combined, back-extracted once with 40mL saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 1.12g of light yellow oily liquid D, 84.21% yield.

d) Compound D (0.3g,1.06mmol) and p-methylbenzeneSulfonamide (0.272g,1.59mmol) and potassium carbonate (0.293g,2.12mmol) were dissolved in 2mL 2-methyltetrahydrofuran, allylpalladium chloride dimer (8mg,0.021mmol), tBuXPhos (9mg,0.021mmol) were added with nitrogen purging, followed by reaction at 80 ℃ for 24 hours, monitoring the reaction by TLC, after completion of the reaction, extraction with ethyl acetate (10mL x 2) and 10mL water, combining the organic layers, back-extraction with 20mL saturated brine, drying the organic phase over anhydrous sodium sulfate, purification of the organic phase by flash chromatography column while stirring with ethyl acetate/petroleum ether gradient elution to give 0.2g white solid E, compound 1, 50.63% yield. MS (ESI) [ M + H ]]⁺：374.18；¹H NMR(400MHz,CDCl₃)7.67(d,J＝8.3Hz,2H),7.37(s,1H),7.20(d,J＝8.1Hz,2H),6.68(d,J＝8.5Hz,1H),6.49(dd,J＝8.4,2.3Hz,1H),6.40(d,J＝2.3Hz,1H),3.83(dd,J＝7.5,5.1Hz,1H),3.29(s,3H),2.81(s,3H),2.35(s,3H),1.68–1.56(m,1H),1.53–1.43(m,1H),0.78(t,J＝7.5Hz,3H).

Example 2

The compound of example 2 was prepared by the same method as in example 1, except that (S) -2-aminopropionic acid was used instead of (R) -2-aminobutyric acid in step a.

MS(ESI)[M+H]⁺：360.16。¹H NMR(400MHz,CDCl₃)7.70(d,J＝8.3Hz,2H),7.48(s,1H),7.23(d,J＝8.1Hz,2H),6.74(d,J＝8.5Hz,1H),6.56(dd,J＝8.5,2.2Hz,1H),6.45(d,J＝2.2Hz,1H),3.98(q,J＝6.8Hz,1H),3.31(s,3H),2.76(s,3H),2.38(s,3H),1.07(d,J＝6.8Hz,3H)。

Example 3

Except that (S) -2-aminopropionic acid is used instead of (R) -2-aminobutyric acid in step a; the compound of example 3 was prepared in the same manner as in example 1 except that o-chlorobenzenesulfonamide was used instead of p-methylbenzenesulfonamide in step d.

MS(ESI)[M+H]⁺：380.13。¹H NMR(400MHz,DMSO-d₆)10.39(s,1H),8.03(d,J＝7.7Hz,1H),7.60(d,J＝5.9Hz,2H),7.49(t,J＝6.3Hz,1H),6.84(d,J＝8.5Hz,1H),6.53(d,J＝8.0Hz,1H),6.43(s,1H),3.93(d,J＝6.6Hz,1H),3.15(s,3H),2.66(s,3H),0.88(d,J＝6.5Hz,3H)。

Example 4

The compound of example 4 was prepared by the same method as in example 1, except that (S) -2-aminobutyric acid was used instead of (R) -2-aminobutyric acid in step a.

MS(ESI)[M+H]⁺：374.16。¹H NMR(400MHz,CDCl₃)7.66(d,J＝8.2Hz,2H),7.20(d,J＝8.2Hz,3H),6.69(d,J＝8.4Hz,1H),6.48(dd,J＝8.4,2.1Hz,1H),6.39(d,J＝2.0Hz,1H),3.83(dd,J＝7.5,5.2Hz,1H),3.30(s,3H),2.81(s,3H),2.36(s,3H),1.69–1.56(m,1H),1.47(dt,J＝21.6,7.4Hz,1H),0.79(t,J＝7.5Hz,3H)。

Example 5

The compound of example 5 was prepared in the same manner as in example 1, except that p-fluorobenzenesulfonamide was replaced with p-methylbenzenesulfonamide in step d.

MS(ESI)[M+H]⁺：378.43。¹H NMR(400MHz,CDCl₃)7.81-7.75(m,2H),7.14-7.07(m,2H),6.96(s,1H),6.71(d,J＝8.4Hz,1H),6.45(dd,J＝8.4,2.3Hz,1H),6.39(d,J＝2.3Hz,1H),3.86(dd,J＝7.5,5.1Hz,1H),3.32(s,3H),2.84(s,3H),1.72–1.59(m,1H),1.51(dt,J＝13.9,7.4Hz,1H),0.81(t,J＝7.5Hz,3H)。

Example 6

Except that (S) -2-aminobutyric acid is used instead of (R) -2-aminobutyric acid in step a; the compound of example 6 was prepared in the same manner as in example 1 except that p-fluorobenzenesulfonamide was replaced with p-methylbenzenesulfonamide in step d.

MS(ESI)[M+H]⁺：378.14。¹H NMR(400MHz,CDCl₃)7.81-7.75(m,2H),7.14-7.06(m,2H),7.04(s,1H),6.71(d,J＝8.4Hz,1H),6.45(dd,J＝8.4,2.3Hz,1H),6.39(d,J＝2.3Hz,1H),3.86(dd,J＝7.5,5.1Hz,1H),3.32(s,3H),2.84(s,3H),1.66(ddd,J＝14.0,7.5,5.1Hz,1H),1.51(dt,J＝14.0,7.4Hz,1H),0.81(t,J＝7.5Hz,3H)。

Example 7

The compound of example 7 was prepared by the same method as in example 1, except that D-leucine was used instead of (R) -2-aminobutyric acid in step a.

MS(ESI)[M+H]⁺：402.53。¹H NMR(400MHz,CDCl₃)7.66(d,J＝7.9Hz,2H),7.18(t,J＝6.4Hz,2H),6.70(d,J＝6.6Hz,1H),6.56–6.48(m,1H),6.40(s,1H),3.87(t,J＝7.1Hz,1H),3.30–3.22(m,3H),2.78(d,J＝2.8Hz,3H),2.34(d,J＝6.1Hz,3H),2.06–1.99(m,2H),1.64–1.51(m,1H),0.86(ddd,J＝25.6,8.0,2.9Hz,6H)。

Examples 8 and 9

Reagents and conditions: a) cyclopropylamine, 1, 2-dichloroethane, at 80 ℃ under reflux for 12 hours; b) reacting iron powder, ammonium chloride solution and ethanol at 80 ℃ for 1 hour; c) 1.2-Bromopropionyl bromide, N, N-Diisopropylethylamine (DIPEA), bisChloromethane, and reacting for 2 hours at room temperature; 2. acetonitrile, DIPEA, 80 ℃ reaction overnight; d) sodium hydride, N, N-Dimethylformamide (DMF) and methyl iodide react for 1 hour at room temperature; e) p-methylbenzenesulfonamide, allylpalladium chloride dimer, 2-di-tert-butylphosphino-2 ',4',6' -triisopropylbiphenyl (tBuXPhos), potassium carbonate and 2-methyltetrahydrofuran, and reacting at 80 ℃ for 24 hours; f) cesium carbonate, tert-butyl carbamate, Pd (OAc)₂2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl (Xphos) and dioxane, and reacting at 100 ℃ overnight; g) p-methylbenzoyl chloride, triethylamine and dichloromethane are reacted at room temperature overnight.

Preparation of the compound of example 8:

a) compound a (10g,45.45mmol) was dissolved in 30mL1, 2-dichloroethane, cyclopropylamine (6.29mL,90.91mmol) was added, followed by reflux at 80 ℃ for 12 hours, the reaction was monitored by TLC, after completion of the reaction the solvent was evaporated, extracted with dichloromethane (40mL x 2) and 40mL water, the organic layers were combined, back-extracted once with 80mL saturated brine, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give 10.2g of red solid B, yield 87.33%.¹H NMR(400MHz,CDCl₃)8.05(s,1H),7.96(d,J＝9.1Hz,1H),7.45(d,J＝2.0Hz,1H),6.77(dd,J＝9.1,2.0Hz,1H),2.60–2.50(m,1H),0.97–0.88(m,2H),0.69–0.61(m,2H).

b) Compound B (10g,38.90mmol) was dissolved in 25mL ethanol, ammonium chloride (10.41g,194.55mmol) in water (10mL) was added, iron powder (10.89g,194.55mmol) was added, the reaction was carried out at 80 ℃ for 1 hour, monitored by TLC, after completion of the reaction the iron powder was filtered off with celite, extracted with ethyl acetate (40mL x 2) and 40mL water, the organic layers were combined, back-extracted once with 80mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was evaporated to dryness to give 5.2g of red oily liquid C, 58.89% yield.¹H NMR(400MHz,CDCl₃)7.13(d,J＝2.1Hz,1H),6.79(dd,J＝8.1,2.1Hz,1H),6.53(d,J＝8.1Hz,1H),3.59–3.03(m,2H),2.45–2.32(m,1H),0.76(q,J＝6.4Hz,2H),0.57–0.44(m,2H).

c) Compound C (5.2g,22.90mmol) was dissolved in dry 15mL of dichloromethane, cooled to 0 deg.C, DIPEA (7.98mL,45.80mmol) and 2-bromopropionyl bromide (3.12mL,29.77mmol) were added, and the reaction was carried out at room temperature for 2 hours using TMonitoring the reaction by LC, extracting with dichloromethane (40mL x 2) and 40mL of water after the reaction is finished, combining organic layers, back-extracting once with saturated saline water 80mL, drying the organic phase with anhydrous sodium sulfate, evaporating the organic phase to obtain an intermediate, dissolving the intermediate in 20mL of acetonitrile, adding 8mL of DIPEA, reacting overnight at 80 ℃, monitoring the reaction by TLC, evaporating the solvent after the reaction is finished, extracting with dichloromethane (40mL x 2) and 40mL of water, combining the organic layers, back-extracting once with saturated saline water 80mL, drying the organic phase with anhydrous sodium sulfate, stirring the organic phase with silica gel, purifying by a flash chromatography column, and eluting with ethyl acetate/petroleum ether at a gradient of 0-30% to obtain 2g of white solid D with the yield of 31.06%. MS (ESI) [ M + H ]]⁺：281.17；¹H NMR(400MHz,CDCl₃)9.10(s,1H),7.18(d,J＝1.8Hz,1H),6.91(dd,J＝8.2,1.9Hz,1H),6.64(d,J＝8.2Hz,1H),4.04(q,J＝6.8Hz,1H),2.45–2.35(m,1H),1.24(d,J＝6.9Hz,3H),1.00(td,J＝10.5,6.2Hz,1H),0.84–0.75(m,1H),0.63(dt,J＝10.2,5.1Hz,1H),0.56(ddd,J＝14.2,8.1,4.1Hz,1H).

d) Compound D (2g,7.11mmol) was dissolved in 8mL DMF, cooled to 0 ℃, sodium hydride (0.512g,21.34mmol) was added, iodomethane (0.67mL,10.67mmol) was added after reaction for half an hour at 0 ℃, then reacted for 1 hour at room temperature, monitored by TLC, cooled to 0 ℃ after reaction was completed, diluted hydrochloric acid was added to adjust pH to 7-8, then extracted with dichloromethane (20mL × 2) and 20mL water, the organic layers were combined, back extracted once with 40mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography column while stirred with silica gel, eluted with a gradient of 0-25% ethyl acetate/petroleum ether to give 1.8g white solid E, yield 85.71%. MS (ESI) [ M + H ]]⁺：295.15；¹H NMR(400MHz,CDCl₃)7.14(d,J＝2.2Hz,1H),6.91(dd,J＝8.5,2.1Hz,1H),6.70(d,J＝8.5Hz,1H),4.04(p,J＝7.0Hz,1H),3.24(s,3H),2.36–2.27(m,1H),1.10(d,J＝6.9Hz,3H),0.91(dtd,J＝10.8,6.3,4.7Hz,1H),0.79–0.68(m,1H),0.60–0.51(m,1H),0.46(ddt,J＝10.4,6.5,4.1Hz,1H).

e) Compound E (0.2g,0.68mmol), p-methylbenzenesulfonamide (0.174g,1.02mmol) and potassium carbonate (0.187g,1.36mmol) were dissolved in 2mL of 2-methyltetrahydrofuran and dimerized by addition of allylpalladium chloride with nitrogen purgeBody (6mg,0.014mmol), tBuXPhos (6mg,0.014mmol), purged with nitrogen, then reacted at 80 ℃ for 24 hours, monitored by TLC, extracted with ethyl acetate (10mL x 2) and 10mL water after the reaction was complete, combined organic layers, back extracted once with saturated brine 20mL, dried over anhydrous sodium sulfate, and purified by flash chromatography on silica gel with a 0-30% gradient of ethyl acetate/petroleum ether to give 0.15g of F as a white solid, compound 8, 57.47% yield. MS (ESI) [ M + H ]]⁺：386.22；¹H NMR(400MHz,CDCl₃)7.85(s,1H),7.71(d,J＝7.5Hz,2H),7.21(d,J＝7.4Hz,2H),6.91(s,1H),6.74(d,J＝8.3Hz,1H),6.65(d,J＝7.8Hz,1H),4.08(d,J＝6.2Hz,1H),3.27(s,3H),2.36(s,3H),2.27(s,1H),1.10(d,J＝6.2Hz,3H),0.83(s,1H),0.73(s,1H),0.55(s,1H),0.30(s,1H).

Preparation of the compound of example 9:

the procedure for the preparation of steps a) to d) was the same as that used for the preparation of example 8,

f) compound E (0.2g,0.68mmol), cesium carbonate (0.314g,0.96mmol) and tert-butyl carbamate (0.12g,1.03mmol) were dissolved in 2mL dioxane, purged with nitrogen, after which Pd (OAc) was added₂(0.046g,0.068mmol) and XPhos (0.052g,0.11mmol), purging with nitrogen, reacting at 100 ℃ overnight after purging, monitoring with TLC plate, extracting with ethyl acetate (10 mL. about.2) and 10mL water after reaction, combining organic layers, back-extracting with 20mL saturated saline solution once, drying the organic phase with anhydrous sodium sulfate, purifying the organic phase silica gel sample by flash chromatography, eluting with a gradient of 0-30% ethyl acetate/petroleum ether to obtain 0.16g yellow solid, dissolving the yellow solid in 3mL dry dichloromethane, cooling to 0 ℃, adding 1mL trifluoroacetic acid, reacting at room temperature for 2 hours, monitoring with TLC plate, cooling to 0 ℃ after reaction, and using saturated NaHCO₃The pH was adjusted to 7-8, then extracted with ethyl acetate (10mL × 2) and 10mL water, the organic layers were combined, back extracted once with 20mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography with silica gel, eluting with a gradient of 0-35% ethyl acetate/petroleum ether to give 0.1G of G as a white solid with a total yield of 63.69%.¹H NMR(400MHz,CDCl₃)7.18(d,J＝2.2Hz,1H),6.97(dd,J＝8.5,2.2Hz,1H),6.74(d,J＝8.5Hz,1H),4.08(q,J＝6.8Hz,1H),3.29(s,3H),2.42–2.32(m,1H),1.15(d,J＝6.9Hz,3H),1.00–0.92(m,1H),0.81–0.74(m,1H),0.65–0.57(m,1H),0.55–0.47(m,1H).

g) Compound G (0.1G,0.43mmol) was dissolved in 2mL of dichloromethane and p-methylbenzoyl chloride (0.075mL,0.56mmol) and triethylamine (0.18mL,1.29mmol) were added and the reaction was allowed to proceed overnight at room temperature, monitored on TLC plates, extracted with dichloromethane (10mL x 2) and 10mL water after completion of the reaction, the organic layers were combined, back-extracted once with 20mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash silica gel column chromatography eluting with a gradient of 0-30% ethyl acetate/petroleum ether to give 0.05G of H as a white solid, compound 9, 33.12% yield. MS (ESI) [ M + H ]]⁺：350.23；¹H NMR(400MHz,CDCl₃)7.97(s,1H),7.79(s,1H),7.77(s,1H),7.60(s,1H),7.28(s,1H),7.26(s,1H),7.08(dd,J＝8.5,2.2Hz,1H),6.87(d,J＝8.6Hz,1H),4.10(q,J＝6.8Hz,1H),3.33(s,3H),2.45–2.37(m,4H),1.16(d,J＝6.8Hz,3H),1.04–0.96(m,1H),0.81–0.72(m,1H),0.64–0.51(m,2H).

Example 10

Except that in step a cyclopentylamine is used in place of cyclopropylamine; the compound of example 10 was prepared by the same method as in example 8, except that step d was omitted.

MS(ESI)[M+H]⁺：400.18。¹H NMR(400MHz,DMSO-d₆)10.29(s,1H),9.77(s,1H),7.55(d,J＝8.3Hz,2H),7.31(d,J＝8.1Hz,2H),6.63(d,J＝8.2Hz,1H),6.45(dd,J＝8.2,2.1Hz,1H),6.43(s,1H),3.83(q,J＝6.7Hz,1H),3.50(p,J＝7.4Hz,1H),2.31(s,3H),1.88–1.72(m,2H),1.70–1.59(m,2H),1.57–1.45(m,3H),1.39–1.27(m,1H),0.88(d,J＝6.7Hz,3H)。

Example 11

The compound of example 11 was prepared by the same procedure as in example 8, except that cyclopentylamine was used instead of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：414.17。¹H NMR(400MHz,CDCl₃)7.64(d,J＝8.3Hz,2H),7.21(d,J＝8.0Hz,2H),7.08(s,1H),6.75(d,J＝8.4Hz,1H),6.57(d,J＝2.2Hz,1H),6.54(dd,J＝8.4,2.3Hz,1H),4.15(q,J＝6.8Hz,1H),3.59(p,J＝7.3Hz,1H),3.29(s,3H),2.36(s,3H),1.96–1.84(m,2H),1.72(dd,J＝11.4,6.1Hz,1H),1.66–1.50(m,4H),1.46–1.34(m,1H),0.97(d,J＝6.8Hz,3H)。

Example 12

Except that in step a cyclopentylamine is used in place of cyclopropylamine; the compound of example 12 was prepared in the same manner as in example 8, except that ethyl bromide was used instead of methyl iodide in step d.

MS(ESI)[M+H]⁺：428.19。¹H NMR(400MHz,CDCl₃)7.64(d,J＝8.3Hz,2H),7.22(d,J＝8.4Hz,2H),6.78(d,J＝8.5Hz,1H),6.76(s,1H),6.54(d,J＝2.3Hz,1H),6.51(dd,J＝8.4,2.3Hz,1H),4.10(q,J＝6.7Hz,1H),4.02(dq,J＝14.4,7.3Hz,1H),3.78(dq,J＝14.1,7.0Hz,1H),3.59(p,J＝7.3Hz,1H),2.37(s,3H),1.95–1.83(m,2H),1.78–1.69(m,1H),1.69–1.63(m,2H),1.61–1.55(m,2H),1.42(dt,J＝16.1,6.9Hz,1H),1.21(t,J＝7.1Hz,3H),0.96(d,J＝6.8Hz,3H)。

Example 13

The compound of example 13 was prepared by the same procedure as in example 8, except that isopropylamine was used instead of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：388.10。¹H NMR(400MHz,CDCl₃)7.66(d,J＝8.3Hz,2H),7.51(s,1H),7.20(d,J＝8.0Hz,2H),6.74(d,J＝8.5Hz,1H),6.60(d,J＝2.2Hz,1H),6.55(dd,J＝8.5,2.2Hz,1H),4.18–4.13(m,1H),3.69(dq,J＝13.3,6.6Hz,1H),3.28(s,3H),2.35(s,3H),1.17(dd,J＝8.0,6.8Hz,6H),1.01(d,J＝6.8Hz,3H)。

Example 14

The compound of example 14 was prepared by the same procedure as in example 8, except that 2-methylpropan-1-amine was used instead of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：402.22。¹H NMR(400MHz,CDCl₃)7.67(d,J＝8.2Hz,2H),7.48(s,1H),7.21(d,J＝8.1Hz,2H),6.73(d,J＝8.5Hz,1H),6.53(dd,J＝8.5,2.1Hz,1H),6.39(d,J＝2.0Hz,1H),3.95(q,J＝6.8Hz,1H),3.30(s,3H),3.07(dd,J＝13.7,5.5Hz,1H),2.49(dd,J＝13.7,8.8Hz,1H),2.36(s,3H),1.81–1.65(m,1H),1.01(d,J＝6.8Hz,3H),0.86(dd,J＝8.3,6.8Hz,6H)。

Example 15

The compound of example 15 was prepared by the same method as in example 8, except that 2-methoxyethylamine was used instead of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：404.50。¹H NMR(400MHz,CDCl₃)8.18(s,1H),7.62(d,J＝8.0Hz,2H),7.13(d,J＝7.9Hz,2H),6.68(d,J＝8.5Hz,1H),6.56(d,J＝8.5Hz,1H),6.51(s,1H),4.05(q,J＝6.5Hz,1H),3.47–3.40(m,2H),3.40–3.32(m,1H),3.24(s,3H),3.20(d,J＝6.1Hz,3H),3.10(dd,J＝13.2,5.7Hz,1H),2.28(s,3H),0.99(d,J＝6.7Hz,3H)。

Example 16

The compound of example 16 was prepared by the same procedure as in example 8, except that 2-morpholinoethane-1-amine was used in place of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：459.26。¹H NMR(400MHz,CDCl₃)7.63(d,J＝8.3Hz,2H),7.22(d,J＝8.0Hz,2H),6.78(d,J＝8.5Hz,1H),6.69(s,1H),6.54(d,J＝2.3Hz,1H),6.50(dd,J＝8.4,2.3Hz,1H),4.10(q,J＝6.7Hz,1H),4.02(dq,J＝14.3,7.1Hz,1H),3.78(dq,J＝14.3,7.2Hz,1H),3.58(p,J＝7.3Hz,1H),2.38(s,3H),1.95–1.83(m,2H),1.78–1.69(m,1H),1.66–1.50(m,5H),1.42(ddd,J＝16.0,13.4,6.9Hz,1H),1.21(t,J＝7.1Hz,3H),0.96(d,J＝6.8Hz,3H)。

Example 17

The compound of example 17 was prepared by the same procedure as in example 8, except that (tetrahydrofuran-2-yl) methylamine was used instead of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：430.23。¹H NMR(400MHz,CDCl₃)7.64(dd,J＝8.3,6.6Hz,2H),7.29(d,J＝3.0Hz,1H),7.20(d,J＝8.1Hz,2H),6.72(t,J＝8.1Hz,1H),6.57–6.50(m,1H),6.47(dd,J＝8.4,2.2Hz,1H),4.16(q,J＝6.8Hz,1H),4.03–3.95(m,1H),3.85(ddd,J＝15.2,11.0,6.9Hz,1H),3.75–3.68(m,1H),3.34(dd,J＝14.2,3.3Hz,1H),3.27(d,J＝3.7Hz,3H),2.99–2.90(m,1H),2.36(s,3H),2.00(ddd,J＝10.3,8.2,5.1Hz,1H),1.91–1.79(m,2H),1.60–1.46(m,1H),1.05(dd,J＝6.8,2.6Hz,3H)。

Example 18

The compound of example 18 was prepared by the same procedure as in example 8, except that (2-methoxyphenyl) methylamine was used instead of cyclopropylamine in step a.

MS(ESI)[M+H]⁺：466.07。¹H NMR(400MHz,CDCl₃)7.47(d,J＝8.3Hz,2H),7.39(s,1H),7.26–7.20(m,1H),7.18(d,J＝7.5Hz,1H),7.11(d,J＝8.1Hz,2H),6.88(d,J＝8.1Hz,1H),6.84(t,J＝7.5Hz,1H),6.70(d,J＝8.5Hz,1H),6.55(d,J＝2.2Hz,1H),6.46(dd,J＝8.5,2.2Hz,1H),4.45(d,J＝15.4Hz,1H),4.10–4.01(m,2H),3.86(s,3H),3.28(s,3H),2.33(s,3H),1.11(d,J＝6.8Hz,3H)。

Example 19

The compound of example 19 was prepared by the same procedure as in example 8, except that p-methoxybenzenesulfonamide was used instead of p-methylbenzenesulfonamide in step e.

MS(ESI)[M+H]⁺：402.09。¹H NMR(400MHz,CDCl₃)7.74–7.67(m,2H),6.92–6.87(m,2H),6.84(d,J＝2.3Hz,1H),6.74(d,J＝8.5Hz,1H),6.66(s,1H),6.53(dd,J＝8.5,2.4Hz,1H),4.06(q,J＝6.9Hz,1H),3.83(s,3H),3.27(s,3H),2.28(td,J＝6.6,3.3Hz,1H),1.12(d,J＝6.8Hz,3H),0.86(dt,J＝10.9,6.3Hz,1H),0.80–0.70(m,1H),0.58(dt,J＝10.3,4.5Hz,1H),0.36(dt,J＝10.3,5.2Hz,1H)。

Example 20

Reagents and conditions: a) thionyl chloride (SOCl)₂) Refluxing with methanol at 60 deg.C for 12 hr; b) cyclopropylamine, 1, 2-dichloroethane, at 80 ℃ under reflux for 12 hours; c) reacting iron powder, ammonium chloride solution and ethanol at 80 ℃ for 1 hour; d) reacting 2-bromopropionyl bromide, N, N-Diisopropylethylamine (DIPEA) and dichloromethane at room temperature for 2 hours; 2. acetonitrile, DIPEA, 80 ℃ reaction overnight; e) sodium hydride, N, N-Dimethylformamide (DMF) and methyl iodide react for 1 hour at room temperature; f) lithium hydroxide, THF/H₂O, reacting at room temperature overnight; g)2- (7-Azobenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), N, N-Diisopropylethylamine (DIPEA), N, N-bis (p-diisopropylethylamine)Methyl Formamide (DMF) was reacted at room temperature overnight.

a) Compound A (10g,54.02mmol) is dissolved in 26mL of methanol, cooled to 0 ℃ and SOCl is added₂(19.62mL,270.11mmol), reflux at 60 ℃ for 12 h, monitored by TLC plate, after completion of the reaction the solvent was evaporated, cooled to 0 ℃ and washed with saturated NaHCO₃The pH was adjusted to 7-8, followed by extraction with dichloromethane (40 mL. times.2) and 40mL water, and the combined organic layers were back-extracted once with 80mL of saturated brine, dried over anhydrous sodium sulfate and evaporated to dryness to give 10.2g of pale yellow solid B in 94.88% yield.¹H NMR(400MHz,CDCl₃)8.11(t,J＝7.8Hz,1H),7.97(s,1H),7.94(d,J＝3.0Hz,1H),3.98(s,3H).

b) Compound B (10g,50.25mmol) was dissolved in 50mL 1, 2-dichloroethane, cyclopropylamine (6.95mL,100.50mmol) was added, followed by reflux at 80 ℃ for 12 hours, monitored by TLC plates, after the reaction was complete the solvent was evaporated, extracted with dichloromethane (50mL x 2) and 50mL water, the organic layers were combined, back extracted once with 100mL saturated brine, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give 10.2g red solid C, yield 85.93%.¹H NMR(400MHz,DMSO-d₆)8.15(d,J＝8.8Hz,1H),8.03(s,1H),7.92(d,J＝1.7Hz,1H),7.22(dd,J＝8.8,1.8Hz,1H),3.89(s,3H),2.68(td,J＝6.6,3.0Hz,1H),0.94–0.84(m,2H),0.70–0.60(m,2H).

c) Compound C (10g,42.33mmol) was dissolved in 30mL ethanol, ammonium chloride (11.32g,211.65) in water (10mL) was added, iron powder (11.86g,211.67mmol) was added, the reaction was carried out at 80 ℃ for 1 hour, monitored by TLC plate, after the reaction was completed, the iron powder was filtered off with celite, extracted with ethyl acetate (40mL × 2) and 40mL water, the organic layers were combined, back-extracted once with 80mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was evaporated to dryness to give 5.2g of yellow solid D, yield 59.56%. MS (ESI) [ M + H ]]⁺：207.67。

d) Compound D (5.2g,25.21mmol) was dissolved in dry 20mL of dichloromethane, cooled to 0 deg.C, DIPEA (8.78mL,50.42mmol) and 2-bromopropionyl bromide (3.96mL,37.82mmol) were added and the reaction was allowed to proceed at room temperature for 2 hours, monitored by TLC plates, extracted with dichloromethane (40 mL. multidot.2) and 40mL water after completion of the reaction, the organic layers were combined and back-extracted once with 80mL of saturated brine and the organic phase was sulfuric acid anhydrousSodium drying, evaporating an organic phase to obtain an intermediate, dissolving the intermediate in 20mL of acetonitrile, adding 9mL of LDIPEA, reacting at 80 ℃ overnight, monitoring by a TLC plate, evaporating the solvent after the reaction is finished, extracting with dichloromethane (40mL of 2) and 40mL of water, combining organic layers, back-extracting with 80mL of saturated saline solution once, drying the organic phase with anhydrous sodium sulfate, stirring the organic phase with silica gel, purifying by a flash chromatography column, and eluting with a gradient of 0-40% of ethyl acetate/petroleum ether to obtain 3g of white solid E, wherein the yield is 45.73%. MS (ESI) [ M + H ]]⁺：261.20；¹H NMR(400MHz,CDCl₃)8.61(s,1H),7.75(d,J＝1.6Hz,1H),7.53(dd,J＝8.1,1.8Hz,1H),6.78(d,J＝8.1Hz,1H),4.13–4.05(m,1H),3.90(s,3H),2.52–2.45(m,1H),1.24(d,J＝6.9Hz,3H),1.10–1.02(m,1H),0.86–0.78(m,1H),0.68–0.60(m,1H),0.60–0.51(m,1H).

e) Compound E (3g,11.53mmol) was dissolved in 8mL DMF, cooled to 0 ℃, sodium hydride (0.83g,34.58mmol) was added, iodomethane (1.08mL,17.30mmol) was added after reaction for half an hour at 0 ℃, then reacted for 1 hour at room temperature, monitored by TLC plates, cooled to 0 ℃ after completion of the reaction, diluted hydrochloric acid was added to adjust pH to 7-8, then extracted with dichloromethane (20mL × 2) and 20mL water, the organic layers were combined, back extracted once with 40mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography column with silica gel, eluted with a gradient of 0-25% ethyl acetate/petroleum ether to give 2.8g colorless transparent liquid F in 88.61% yield. MS (ESI) [ M + H ]]⁺：275.18；¹H NMR(400MHz,CDCl₃)7.75(d,J＝1.9Hz,1H),7.59(dd,J＝8.3,1.9Hz,1H),6.93(d,J＝8.4Hz,1H),4.12(q,J＝6.9Hz,1H),3.89(s,3H),3.35(s,3H),2.48–2.42(m,1H),1.15(d,J＝6.9Hz,3H),1.03(dtd,J＝9.7,6.3,4.6Hz,1H),0.84–0.76(m,1H),0.66–0.58(m,1H),0.56–0.48(m,1H).

f) Dissolving compound F (2.8G,10.22mmol) in 15mL tetrahydrofuran and 5mL water, adding lithium hydroxide monohydrate (1.72G,40.88mmol), reacting overnight at room temperature, monitoring by TLC plate, adjusting pH to 6-7 with dilute hydrochloric acid after reaction, extracting with ethyl acetate (20mL 2) and 20mL water, combining organic layers, back-extracting once with saturated saline water 40mL, drying the organic phase with anhydrous sodium sulfate, evaporating the organic phase to dryness to obtain 2.4G white solid G, and obtaining the productThe rate was 90.23%. MS (ESI) [ M + H ]]⁺：261.08；¹H NMR(400MHz,CDCl₃)7.83(d,J＝1.9Hz,1H),7.70(dd,J＝8.3,1.9Hz,1H),6.99(d,J＝8.4Hz,1H),4.17(q,J＝6.9Hz,1H),3.39(s,3H),2.55–2.43(m,1H),1.19(d,J＝6.9Hz,3H),1.06(tt,J＝11.1,5.4Hz,1H),0.88–0.77(m,1H),0.66(dt,J＝10.0,4.7Hz,1H),0.60–0.51(m,1H).

g) Compound G (0.2G,0.77mmol) was dissolved in 2mL DMF, HATU (0.29G,0.77mmol) was added, after reaction for half an hour at room temperature p-methylaniline (0.1G,0.92mmol) and DIPEA (0.14mL,0.77mmol) were added, reaction was overnight at room temperature, monitored on TLC plates, after completion of the reaction extracted with ethyl acetate (20mL × 2) and 40mL water, the organic layers were combined, back extracted once with 40mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography with silica gel stirred and eluted with a gradient of 0-35% ethyl acetate/petroleum ether to give 0.1G white solid H, compound 20, 37.31% yield. MS (ESI) [ M + H ]]⁺：350.16；¹H NMR(400MHz,CDCl₃)8.03(s,1H),7.67(d,J＝1.7Hz,1H),7.54(d,J＝8.3Hz,2H),7.33(dd,J＝8.2,1.8Hz,1H),7.15(d,J＝8.3Hz,2H),6.91(d,J＝8.3Hz,1H),4.12(q,J＝6.8Hz,1H),3.34(s,3H),2.43(ddd,J＝9.9,6.6,3.7Hz,1H),2.33(s,3H),1.15(d,J＝6.8Hz,3H),0.99(td,J＝10.6,6.2Hz,1H),0.78(td,J＝11.2,6.4Hz,1H),0.60(dt,J＝10.0,4.5Hz,1H),0.51(dt,J＝14.3,7.1Hz,1H).

Example 21

The compound of example 21 was prepared by the same method as in example 20, except that N-methylcyclohexylamine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：356.24。¹H NMR(400MHz,CDCl₃)7.10(s,1H),6.89(s,2H),4.09(q,J＝6.8Hz,1H),3.62–3.46(m,1H),3.33(s,3H),3.02–2.75(m,3H),2.43–2.33(m,1H),1.70(d,J＝29.8Hz,4H),1.47(d,J＝58.7Hz,4H),1.13(d,J＝6.8Hz,3H),1.05(s,2H),0.97–0.85(m,1H),0.77(td,J＝11.3,6.4Hz,1H),0.60(td,J＝10.2,4.6Hz,1H),0.49(td,J＝10.2,4.1Hz,1H)。

Example 22

The compound of example 22 was prepared by the same method as in example 20, except that N-ethylcyclohexylamine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：370.26。¹H NMR(400MHz,CDCl₃)7.07(s,1H),6.90(d,J＝8.0Hz,1H),6.87(d,J＝8.0Hz,1H),4.10(q,J＝6.8Hz,1H),3.52(d,J＝8.4Hz,1H),3.43(d,J＝12.8Hz,2H),3.34(s,3H),2.44–2.33(m,1H),1.75(s,4H),1.52(dd,J＝17.9,8.1Hz,3H),1.25(d,J＝12.2Hz,3H),1.14(d,J＝6.8Hz,3H),1.06(s,3H),0.92(dt,J＝10.7,6.3Hz,1H),0.78(dt,J＝11.4,6.3Hz,1H),0.62(td,J＝10.1,4.8Hz,1H),0.50(td,J＝10.2,4.1Hz,1H)。

Example 23

The compound of example 23 was prepared by the same method as in example 20 except that isoxazolyl amine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：327.35。¹H NMR(400MHz,CDCl₃)8.74(dd,J＝4.5,1.4Hz,1H),8.47(dd,J＝8.4,1.4Hz,1H),7.94(d,J＝1.9Hz,1H),7.88(dd,J＝8.4,2.0Hz,1H),7.47(dd,J＝8.4,4.5Hz,1H),7.07(d,J＝8.4Hz,1H),4.19(q,J＝6.9Hz,1H),3.41(s,3H),2.49(ddd,J＝10.0,6.6,3.6Hz,1H),1.21(d,J＝6.9Hz,3H),1.07–1.00(m,1H),0.86–0.81(m,1H),0.69–0.62(m,1H),0.56(ddd,J＝10.3,5.1,3.2Hz,1H)。

Example 24

The compound of example 24 was prepared by the same procedure as in example 20 except that 1,3, 5-trimethyl-1H-pyrazolyl-4-amine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：368.25。¹H NMR(400MHz,CDCl₃)7.67(d,J＝1.9Hz,1H),7.63(s,1H),7.37(dd,J＝8.3,1.9Hz,1H),6.92(d,J＝8.3Hz,1H),4.10(q,J＝6.8Hz,1H),3.67(s,3H),3.33(s,3H),2.47–2.39(m,1H),2.13(s,3H),2.10(s,3H),1.13(d,J＝6.9Hz,3H),1.03–0.93(m,1H),0.77(ddd,J＝11.0,10.3,6.4Hz,1H),0.59(dt,J＝10.4,4.6Hz,1H),0.55–0.46(m,1H)。

Example 25

The compound of example 25 was prepared in the same manner as in example 20, except that p-methylbenzylamine was used in place of p-methylaniline in step g.

MS(ESI)[M+H]⁺：364.17。¹H NMR(400MHz,CDCl₃)7.64(s,1H),7.23(d,J＝7.8Hz,3H),7.12(d,J＝7.8Hz,2H),6.86(d,J＝8.3Hz,1H),6.75(d,J＝4.9Hz,1H),4.57(d,J＝5.3Hz,2H),4.10(q,J＝6.8Hz,1H),3.31(s,3H),2.45–2.36(m,1H),2.32(s,3H),1.12(d,J＝6.8Hz,3H),0.97(td,J＝10.7,6.0Hz,1H),0.76(td,J＝11.1,6.1Hz,1H),0.57(dd,J＝9.9,4.6Hz,1H),0.50(dd,J＝10.0,4.1Hz,1H)。

Example 26

The compound of example 26 was prepared by the same method as in example 20, except that o-anisidine was used instead of p-anisidine in step g.

MS(ESI)[M+H]⁺：366.11。¹H NMR(400MHz,CDCl₃)8.57(s,1H),8.53(dd,J＝7.9,1.6Hz,1H),7.70(d,J＝1.9Hz,1H),7.36(dd,J＝8.3,2.0Hz,1H),7.07(td,J＝7.7,1.7Hz,1H),7.02(dd,J＝7.8,1.4Hz,1H),6.98(d,J＝8.3Hz,1H),6.91(dd,J＝8.0,1.3Hz,1H),4.15(q,J＝6.8Hz,1H),3.92(s,3H),3.37(s,3H),2.53–2.45(m,1H),1.18(d,J＝6.9Hz,3H),1.09–0.98(m,1H),0.84(ddd,J＝9.4,7.6,4.8Hz,1H),0.65(dt,J＝10.1,4.4Hz,1H),0.60–0.52(m,1H)。

Example 27

The compound of example 27 was prepared by the same method as in example 20, except that N-methyl-p-methylaniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：364.21。¹H NMR(400MHz,CDCl₃)7.03(d,J＝7.9Hz,2H),6.99(d,J＝8.4Hz,1H),6.94(s,2H),6.92(s,1H),6.75(d,J＝8.2Hz,1H),4.00(q,J＝6.7Hz,1H),3.48(s,3H),3.27(s,3H),2.28(s,3H),2.12(s,1H),1.02(d,J＝6.8Hz,3H),0.68(d,J＝6.4Hz,2H),0.49(d,J＝8.2Hz,1H),0.11(d,J＝9.0Hz,1H)。

Example 28

The compound of example 28 was prepared by the same method as in example 20, except that 2, 4-dimethylaniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：364.23。¹H NMR(400MHz,CDCl₃)7.81(d,J＝7.8Hz,1H),7.68(s,1H),7.60(s,1H),7.35(dd,J＝8.2,1.8Hz,1H),7.07(d,J＝9.0Hz,2H),6.98(d,J＝8.3Hz,1H),4.16(q,J＝6.8Hz,1H),3.39(s,3H),2.53–2.45(m,1H),2.32(d,J＝4.6Hz,6H),1.19(d,J＝6.8Hz,3H),1.02(d,J＝4.4Hz,1H),0.83(dt,J＝11.2,6.2Hz,1H),0.64(dd,J＝10.3,4.3Hz,1H),0.57(d,J＝3.9Hz,1H)。

Example 29

The compound of example 29 was prepared by the same method as in example 20, except that aniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：336.17。¹H NMR(400MHz,DMSO-d₆)10.14(s,1H),7.77(d,J＝8.0Hz,2H),7.66(s,1H),7.57(d,J＝8.3Hz,1H),7.35(t,J＝7.8Hz,2H),7.17(d,J＝8.3Hz,1H),7.09(t,J＝6.9Hz,1H),4.05(q,J＝6.7Hz,1H),3.31(s,3H),2.55(d,J＝3.1Hz,1H),1.09(d,J＝6.8Hz,3H),1.04–0.95(m,1H),0.80(d,J＝5.5Hz,1H),0.64(d,J＝4.1Hz,1H),0.43(d,J＝3.8Hz,1H)。

Example 30

The compound of example 30 was prepared in the same manner as in example 20, except that N-methylaniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：350.19。¹H NMR(400MHz,CDCl₃)7.21(t,J＝7.6Hz,2H),7.12(t,J＝7.4Hz,1H),7.03(d,J＝7.5Hz,2H),6.98(dd,J＝8.2,1.8Hz,1H),6.91(d,J＝1.7Hz,1H),6.73(d,J＝8.3Hz,1H),3.97(q,J＝6.8Hz,1H),3.49(s,3H),3.24(s,3H),2.13–2.03(m,1H),0.99(d,J＝6.8Hz,3H),0.70–0.60(m,2H),0.49–0.41(m,1H),0.13–0.04(m,1H)。

Example 31

The compound of example 31 was prepared by the same method as in example 20, except that p-anisidine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：366.24。¹H NMR(500MHz,CDCl₃)7.81(s,1H),7.67(s,1H),7.55(d,J＝8.6Hz,2H),7.32(dd,J＝8.2,1.4Hz,1H),6.94(d,J＝8.2Hz,1H),6.90(d,J＝8.9Hz,2H),4.14(q,J＝6.8Hz,1H),3.81(s,3H),3.37(s,3H),2.53–2.41(m,1H),1.17(d,J＝6.8Hz,3H),1.07–0.96(m,1H),0.81(td,J＝11.4,6.3Hz,1H),0.63(dt,J＝10.1,4.7Hz,1H),0.55(dd,J＝10.1,4.2Hz,1H)。

Example 32

The compound of example 32 was prepared by the same method as in example 20, except that p-methylcyclohexylamine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：356.27。¹H NMR(400MHz,CDCl₃)7.59(t,J＝2.1Hz,1H),7.17(td,J＝8.2,1.9Hz,1H),6.90(dd,J＝10.8,8.3Hz,1H),6.07(dd,J＝103.5,8.1Hz,1H),4.20(s,1H),4.12(qd,J＝6.8,3.1Hz,1H),3.34(d,J＝4.0Hz,3H),2.47(tt,J＝6.3,3.3Hz,1H),2.13–2.02(m,1H),1.90–1.52(m,8H),1.16–1.12(m,3H),1.01(dt,J＝10.7,6.1Hz,1H),0.92(dd,J＝15.2,6.5Hz,3H),0.84–0.75(m,1H),0.65–0.58(m,1H),0.54(dt,J＝10.2,4.3Hz,1H)。

Example 33

The compound of example 33 was prepared by the same method as in example 20, except that cyclohexylamine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：342.25。¹H NMR(400MHz,CDCl₃)7.59(d,J＝1.9Hz,1H),7.17(dd,J＝8.2,1.9Hz,1H),6.89(d,J＝8.3Hz,1H),5.97(d,J＝8.0Hz,1H),4.12(q,J＝6.9Hz,1H),4.04–3.89(m,1H),3.35(s,3H),2.52–2.42(m,1H),2.03(d,J＝12.0Hz,2H),1.76(d,J＝4.4Hz,1H),1.65(d,J＝13.2Hz,1H),1.43(dd,J＝24.3,12.2Hz,2H),1.29–1.18(m,4H),1.15(d,J＝6.9Hz,3H),1.02(dt,J＝10.9,6.2Hz,1H),0.79(dt,J＝11.1,6.5Hz,1H),0.62(dt,J＝10.6,4.2Hz,1H),0.53(ddd,J＝14.5,8.4,4.3Hz,1H)。

Example 34

The compound of example 34 was prepared by the same method as in example 20 except that N-methyl-p-methylcyclohexylamine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：370.29。¹H NMR(400MHz,CDCl₃)7.09(s,1H),6.88(s,2H),4.08(q,J＝6.8Hz,1H),3.49(s,1H),3.32(s,3H),2.92(s,3H),2.89–2.74(m,1H),2.37(s,1H),1.74(s,4H),1.51(dd,J＝29.9,21.7Hz,4H),1.13(d,J＝6.8Hz,3H),1.00–0.87(m,3H),0.83–0.71(m,2H),0.60(d,J＝4.5Hz,1H),0.48(d,J＝4.1Hz,1H)。

Example 35

The compound of example 35 was prepared by the same method as in example 20, except that p-methylaniline was replaced with N-ethyl-p-methylaniline in step g.

MS(ESI)[M+H]⁺：378.31。¹H NMR(400MHz,CDCl₃)7.03(s,1H),7.01(s,1H),6.97(dd,J＝8.2,1.9Hz,1H),6.92(d,J＝2.4Hz,2H),6.90(s,1H),6.73(d,J＝8.3Hz,1H),4.04–3.86(m,3H),3.25(s,3H),2.27(s,3H),2.17–2.06(m,1H),1.20(t,J＝7.1Hz,3H),1.00(d,J＝6.8Hz,3H),0.75–0.61(m,2H),0.52–0.41(m,1H),0.18–0.07(m,1H)。

Example 36

The compound of example 36 was prepared by the same method as in example 20, except that indoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：362.26。¹H NMR(400MHz,CDCl₃)7.32(s,1H),7.21(d,J＝7.5Hz,1H),7.11(d,J＝1.8Hz,1H),7.09(d,J＝1.8Hz,1H),7.01(s,1H),6.96(s,1H),6.94(s,1H),4.13(q,J＝6.9Hz,3H),3.37(s,3H),3.13(t,J＝8.2Hz,2H),2.40(d,J＝3.4Hz,1H),1.18(d,J＝6.8Hz,3H),0.87(s,1H),0.79(dd,J＝12.9,7.8Hz,1H),0.61(dd,J＝10.0,4.7Hz,1H),0.50(s,1H)。

Example 37

The compound of example 37 was prepared by the same procedures as in example 20, except that 5-methylindoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：376.26。¹H NMR(400MHz,CDCl₃)7.32(s,1H),7.10(s,1H),7.08(s,1H),7.03(s,1H),6.95(s,1H),6.93(s,1H),4.13(dd,J＝13.5,6.7Hz,3H),3.37(s,3H),3.09(t,J＝8.3Hz,2H),2.40(s,1H),2.30(s,3H),1.18(d,J＝6.8Hz,3H),0.89(s,1H),0.79(d,J＝4.9Hz,1H),0.62(d,J＝4.5Hz,1H),0.51(s,1H)。

Example 38

The compound of example 38 was prepared by the same procedures as in example 20 except that 5-fluoroindoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：380.24。¹H NMR(400MHz,DMSO)7.28(s,1H),7.16(s,1H),7.13(s,3H),7.00(s,1H),4.11(t,J＝8.3Hz,2H),4.03(q,J＝6.8Hz,1H),3.29(s,3H),3.10(t,J＝8.2Hz,2H),2.47(s,1H),1.09(d,J＝6.8Hz,3H),0.95(s,1H),0.77(d,J＝5.0Hz,1H),0.61(d,J＝4.4Hz,1H),0.39(s,1H)。

Example 39

The compound of example 39 was prepared by the same method as in example 20 except that 4-methylindoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：376.25。¹H NMR(400MHz,CDCl₃)7.31(s,1H),7.10(d,J＝1.8Hz,1H),7.08(d,J＝1.8Hz,1H),6.96(s,1H),6.93(s,1H),6.85(s,1H),4.13(dd,J＝13.6,6.7Hz,3H),3.37(s,3H),3.03(t,J＝8.2Hz,2H),2.40(d,J＝3.1Hz,1H),2.25(s,3H),1.18(d,J＝6.8Hz,3H),0.87(s,1H),0.78(d,J＝5.2Hz,1H),0.61(dd,J＝9.5,5.1Hz,1H),0.50(s,1H)。

Example 40

Except that in step b cyclopentylamine is used in place of cyclopropylamine; the compound of example 40 was prepared in the same manner as in example 20, except that 2, 4-dimethylaniline was used in place of the p-methylaniline in step g.

MS(ESI)[M+H]⁺：392.12。¹H NMR(400MHz,CDCl₃)7.77(d,J＝7.6Hz,1H),7.62(s,1H),7.45(s,1H),7.30(dd,J＝8.3,1.8Hz,1H),7.06(d,J＝7.9Hz,2H),6.99(d,J＝8.3Hz,1H),4.23(q,J＝6.8Hz,1H),3.96–3.84(m,1H),3.40(s,3H),2.31(d,J＝6.7Hz,6H),2.13–1.99(m,2H),1.84–1.74(m,1H),1.74–1.70(m,1H),1.68–1.55(m,4H),1.07(d,J＝6.8Hz,3H)。

EXAMPLE 41

Except that in step b cyclopentylamine is used in place of cyclopropylamine; the compound of example 41 was prepared in the same manner as in example 20 except that indoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：390.14。¹H NMR(400MHz,CDCl₃)7.21(d,J＝7.4Hz,1H),7.09(s,1H),7.07(s,1H),7.04(s,1H),7.01(s,1H),6.98(s,1H),6.96(s,1H),4.22(q,J＝6.8Hz,1H),4.14(t,J＝7.9Hz,2H),3.85–3.71(m,1H),3.40(s,3H),3.13(t,J＝8.2Hz,2H),1.98(d,J＝6.0Hz,2H),1.73(d,J＝12.4Hz,1H),1.68–1.51(m,5H),1.06(d,J＝6.8Hz,3H)。

Example 42

The compound of example 42 was prepared by the same procedure as in example 20, except that cyclopentylamine was used instead of cyclopropylamine in step b.

MS(ESI)[M+H]⁺：378.10。¹H NMR(400MHz,CDCl₃)8.18(s,1H),7.54(d,J＝8.1Hz,2H),7.44(s,1H),7.31(d,J＝7.1Hz,1H),7.13(d,J＝8.1Hz,2H),6.90(d,J＝8.3Hz,1H),4.18(q,J＝6.8Hz,1H),3.87–3.76(m,1H),3.35(s,3H),2.31(s,3H),2.05–1.95(m,2H),1.74(d,J＝6.1Hz,1H),1.66–1.52(m,5H),1.01(d,J＝6.8Hz,3H)。

Example 43

Except that in step b cyclopentylamine is used in place of cyclopropylamine; replacing 2-bromopropionyl bromide with 2-bromobutyryl bromide in step d; the compound of example 43 was prepared in the same manner as in example 20 except that 2, 4-dimethylaniline was used in place of the p-methylaniline in step g.

MS(ESI)[M+H]⁺：406.27。¹H NMR(400MHz,CDCl₃)7.75(d,J＝7.4Hz,1H),7.63(s,1H),7.48(s,1H),7.31(dd,J＝8.3,1.9Hz,1H),7.06(s,1H),7.04(s,1H),6.97(d,J＝8.3Hz,1H),3.95(q,J＝7.2Hz,2H),3.40(s,3H),2.31(s,3H),2.29(s,3H),2.07–1.96(m,2H),1.82–1.72(m,2H),1.70–1.49(m,6H),0.89(t,J＝7.5Hz,3H)。

Example 44

Except that in step b cyclopentylamine is used in place of cyclopropylamine; replacing 2-bromopropionyl bromide with 2-bromobutyryl bromide in step d; the compound of example 44 was prepared by the same procedure as in example 20, except that indoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：404.27。¹H NMR(400MHz,DMSO)7.27(s,1H),7.25(s,1H),7.12(t,J＝9.2Hz,3H),7.08(s,1H),7.01(t,J＝7.3Hz,1H),4.05(td,J＝8.8,3.9Hz,2H),3.91–3.83(m,2H),3.32(s,3H),3.08(t,J＝8.3Hz,2H),1.93(d,J＝6.4Hz,2H),1.70–1.51(m,5H),1.47–1.28(m,3H),0.80(t,J＝7.4Hz,3H)。

Example 45

The compound of example 45 was prepared by the same method as in example 20 except that pyrimidin-4-amine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：338.38。¹H NMR(400MHz,CDCl₃)8.74(dd,J＝4.5,1.4Hz,1H),8.47(dd,J＝8.4,1.4Hz,1H),7.94(d,J＝1.9Hz,1H),7.89(dd,J＝8.4,2.0Hz,1H),7.47(dd,J＝8.4,4.5Hz,1H),7.07(d,J＝8.4Hz,1H),4.19(q,J＝6.8Hz,1H),3.42(s,3H),2.53–2.46(m,1H),1.18(s,3H),1.07–0.99(m,1H),0.87–0.81(m,1H),0.67(dt,J＝10.1,4.8Hz,1H),0.61–0.53(m,1H)。

Example 46

The compound of example 46 was prepared by the same method as in example 20, except that 2-fluoroaniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：354.18。¹H NMR(400MHz,CDCl₃)8.47(t,J＝8.0Hz,1H),8.08(s,1H),7.68(d,J＝1.9Hz,1H),7.36(dd,J＝8.2,1.9Hz,1H),7.18(dd,J＝13.7,5.5Hz,1H),7.15–7.04(m,2H),6.99(d,J＝8.3Hz,1H),4.16(q,J＝6.8Hz,1H),3.38(s,3H),2.56–2.45(m,1H),1.19(d,J＝6.8Hz,3H),1.05(dt,J＝10.9,5.2Hz,1H),0.88–0.80(m,1H),0.65(dt,J＝10.1,4.4Hz,1H),0.56(dt,J＝10.2,4.1Hz,1H)。

Example 47

The compound of example 47 was prepared by the same method as in example 20, except that 3-fluoro-4-methylaniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：368.18。¹H NMR(400MHz,CDCl₃)7.96(d,J＝12.5Hz,1H),7.66(d,J＝1.9Hz,1H),7.56(dd,J＝11.5,1.9Hz,1H),7.31(dd,J＝8.3,1.9Hz,1H),7.21–7.17(m,1H),7.13(t,J＝8.2Hz,1H),6.93(d,J＝8.3Hz,1H),4.14(q,J＝6.8Hz,1H),3.36(s,3H),2.52–2.39(m,1H),2.25(s,3H),1.16(d,J＝6.9Hz,3H),1.00(dd,J＝12.2,7.4Hz,1H),0.86–0.76(m,1H),0.67–0.58(m,1H),0.58–0.49(m,1H)。

Example 48

The compound of example 48 was prepared by the same method as in example 20, except that cyclopentylamine was used instead of p-tolylamine in step g.

MS(ESI)[M+H]⁺：328.25。¹H NMR(400MHz,CDCl₃)7.59(d,J＝1.9Hz,1H),7.16(dd,J＝8.2,2.0Hz,1H),6.89(d,J＝8.3Hz,1H),6.06(d,J＝7.1Hz,1H),4.44–4.32(m,1H),4.12(q,J＝6.9 Hz,1H),3.34(s,3H),2.52–2.40(m,1H),2.17–2.03(m,2H),1.76–1.68(m,2H),1.68–1.62(m,2H),1.56–1.43(m,2H),1.15(d,J＝6.9 Hz,3H),1.02(ddd,J＝10.9,8.5,5.5 Hz,1H),0.84–0.73(m,1H),0.67–0.56(m,1H),0.57–0.47(m,1H)。

Example 49

The compound of example 49 was prepared by the same method as in example 20 except that N-methylcyclopentylamine was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：342.24。¹H NMR(400 MHz,CDCl₃)7.15(s,1H),6.91(d,J＝1.4 Hz,2H),4.19(s,1H),4.11(q,J＝6.8 Hz,1H),3.34(s,3H),2.93(s,3H),2.44–2.37(m,1H),1.68(s,7H),1.52(d,J＝6.9 Hz,1H),1.15(d,J＝6.9Hz,3H),0.99–0.90(m,1H),0.78(ddd,J＝11.2,10.4,6.5 Hz,1H),0.68–0.57(m,1H),0.51(ddt,J＝10.2,6.3,4.1 Hz,1H)。

Example 50

The compound of example 50 was prepared by the same method as in example 20, except that 2,4, 6-trimethylaniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：378.48。¹H NMR(400 MHz,CDCl₃)7.71(d,J＝1.9 Hz,1H),7.40(dd,J＝8.2,1.9 Hz,1H),7.32(s,1H),6.98(d,J＝8.2 Hz,1H),6.94(s,2H),4.16(q,J＝6.9 Hz,1H),3.39(s,3H),2.54–2.46(m,1H),2.29(s,3H),2.26(s,6H),1.19(d,J＝6.9 Hz,3H),1.06–0.99(m,1H),0.82(dd,J＝12.9,7.9 Hz,1H),0.64(dd,J＝10.3,4.4 Hz,1H),0.58(dd,J＝10.2,4.1 Hz,1H)。

Example 51

The compound of example 51 was prepared by the same procedures as in example 20 except that 2, 5-dimethylindoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：390.21。¹H NMR(400 MHz,CDCl₃)7.27(s,1H),7.22(s,1H),7.07(d,J＝7.8 Hz,1H),7.03(s,1H),6.97–6.89(m,1H),6.83(s,1H),4.78(s,1H),4.19–4.06(m,1H),3.44–3.32(m,4H),2.59(d,J＝15.5Hz,1H),2.37(s,1H),2.28(s,3H),1.33–1.22(m,3H),1.17(d,J＝6.8Hz,3H),0.98–0.86(m,1H),0.81–0.69(m,1H),0.61(dd,J＝10.4,4.2Hz,1H),0.55(dd,J＝10.3,4.0Hz,1H)。

Example 52

The compound of example 52 was prepared by the same procedures as in example 20, except that 2-methylindoline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：376.26。¹H NMR(400MHz,CDCl₃)7.21(s,1H),7.19(s,1H),7.09(d,J＝8.7Hz,1H),7.02(s,1H),6.99(d,J＝7.2Hz,1H),6.95(s,1H),6.94–6.89(m,1H),4.77(s,1H),4.17–4.06(m,1H),3.43(dd,J＝17.3,8.1Hz,1H),3.36(d,J＝3.4Hz,3H),2.63(dd,J＝15.8,2.9Hz,1H),2.35(s,1H),1.25(d,J＝12.0Hz,3H),1.16(dd,J＝6.8,2.2Hz,3H),0.96–0.83(m,1H),0.79–0.69(m,1H),0.65–0.57(m,1H),0.57–0.48(m,1H)。

Example 53

Except that 3-bromobenzylamine is used in step b instead of cyclopropylamine; the compound of example 53 was prepared in the same manner as in example 20, except that aniline was used instead of p-methylaniline in step g.

MS(ESI)[M+H]⁺：464.11。¹H NMR(400MHz,CDCl₃)7.69(s,1H),7.60(s,1H),7.58(s,1H),7.49(s,1H),7.42(d,J＝8.2Hz,1H),7.36(t,J＝6.4Hz,2H),7.33(dd,J＝6.7,1.5Hz,1H),7.29(d,J＝7.7Hz,1H),7.23(s,1H),7.21(d,J＝7.8Hz,1H),7.14(t,J＝7.4Hz,1H),7.01(d,J＝8.3Hz,1H),4.62(d,J＝14.8Hz,1H),4.15(d,J＝14.9Hz,1H),4.02(q,J＝6.8Hz,1H),3.44(s,3H),1.14(d,J＝6.8Hz,3H)。

Examples 54 and 55

Reagents and conditions: a) reacting 2-bromopropionyl bromide, sodium carbonate and dichloromethane at room temperature for 2 hours; b) cyclopropylamine, acetonitrile, N, N-Diisopropylethylamine (DIPEA), at 80 ℃ under reflux for 12 hours; c) n, N-Diisopropylethylamine (DIPEA) and N, N-Dimethylformamide (DMF) react at 150 ℃ overnight; d) sodium hydride, N, N-Dimethylformamide (DMF) and methyl iodide react for 1 hour at room temperature; e) p-methylbenzenesulfonamide, allylpalladium chloride dimer, 2-di-tert-butylphosphino-2 ',4',6' -triisopropylbiphenyl (tBuXPhos), potassium carbonate and 2-methyltetrahydrofuran, and reacting at 80 ℃ for 24 hours; f)2, 4-dimethylaniline, 4-dimethylaminopyridine, molybdenum hexacarbonyl, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, 1, 8-diazabicycloundecen-7-ene (DBU), triethylamine, dioxane, at 85 ℃ under reflux for 24 hours.

Preparation of the compound of example 54:

a) compound a (1g,3.97mmol) was dissolved in dry 10mL dichloromethane, cooled to 0 ℃, sodium carbonate (0.42g,3.97mmol) and 2-bromopropionyl bromide (0.5mL,4.77mmol) were added, then reacted at room temperature for 2 hours, monitored by TLC, extracted with dichloromethane (20mL x 2) and 20mL water after the reaction was complete, the organic layers were combined, back extracted once with saturated brine 40mL, the organic phase was dried over anhydrous sodium sulfate, evaporated to dryness to give 1.2g white solid B, 78.43% yield.¹H NMR(400MHz,CDCl₃)8.70(s,1H),8.54(d,J＝8.5Hz,1H),7.46(d,J＝8.5Hz,1H),4.59(q,J＝7.1Hz,1H),1.99(d,J＝7.1Hz,3H).

b) Compound B (1g,2.59mmol) was dissolved in 8mL acetonitrile, DIPEA (1.13mL,6.48mmol) and cyclopropylamine (0.18mL,2.59mmol) were added, followed by reflux at 80 ℃ for 12 hours, monitoring the reaction by TLC, after the reaction was completed evaporating the solvent, extracting with dichloromethane (20mL x 2) and 20mL water, combining the organic layers, back-extracting once with saturated brine 40mL, drying the organic phase over anhydrous sodium sulfate, purifying the organic phase silica gel sample by flash chromatography eluting with a gradient of 0-10% ethyl acetate/petroleum ether to give 0.8g white solid C in 85.11% yield. MS (ESI) [ M + H ]]⁺：362.02；¹H NMR(400MHz,CDCl₃)10.03(s,1H),8.67(dd,J＝9.3,8.5Hz,1H),7.39(t,J＝8.8Hz,1H),3.43(p,J＝7.2Hz,1H),2.39–2.26(m,1H),1.43(dd,J＝9.3,7.2Hz,3H),0.54(ddd,J＝12.2,4.9,3.3Hz,1H),0.47(tdd,J＝9.2,7.4,4.0Hz,3H).

c) Compound C (0.8g,2.22mmol) was dissolved in 8mL DMF, DIPEA (0.77mL,4.44mmol) was added, then refluxed at 150 ℃ for 12 hours, the reaction was monitored by TLC, after the reaction was completed, the solvent was evaporated to dryness, extracted with ethyl acetate (20mL × 2) and 20mL water, the organic layers were combined, back-extracted once with saturated brine 40mL, dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography with silica gel, eluting with a gradient of ethyl acetate/petroleum ether of 0-20% to give 0.5g white solid D with a yield of 79.37%.¹H NMR(400MHz,DMSO-d₆)10.61(s,1H),6.93(d,J＝7.9Hz,1H),6.89(d,J＝7.8Hz,1H),4.00(q,J＝6.9Hz,1H),2.65–2.58(m,1H),1.21(d,J＝6.9Hz,3H),0.94(dt,J＝11.5,6.6Hz,1H),0.68(dt,J＝9.3,6.7Hz,1H),0.61(dd,J＝10.6,5.5Hz,1H),0.46(dd,J＝10.3,4.6Hz,1H).

d) Compound D (0.5g,1.77mmol) was dissolved in 4mL DMF, cooled to 0 ℃, sodium hydride (0.13g,5.31mmol) was added, iodomethane (0.22mL,3.54mmol) was added after reaction for half an hour at 0 ℃, then reacted for 1 hour at room temperature, monitored by TLC, cooled to 0 ℃ after completion of the reaction, diluted hydrochloric acid was added to adjust pH to 7-8, then extracted with dichloromethane (15mL × 2) and 15mL water, the organic layers were combined, back extracted once with 30mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography column while stirred with silica gel and eluted with a gradient of 0-25% ethyl acetate/petroleum ether to give 0.4g colorless transparent liquid E in 76.19% yield. MS (ESI) [ M + H ]]⁺：296.18；¹H NMR(400MHz,CDCl3)6.81(d,J＝8.1Hz,1H),6.76(d,J＝8.0Hz,1H),4.08(q,J＝6.9Hz,1H),3.17(s,3H),2.61–2.50(m,1H),1.16(d,J＝6.9Hz,3H),0.98–0.89(m,1H),0.68–0.57(m,1H),0.50–0.36(m,2H).

e) Compound E (0.2g,0.68mmol), p-methylbenzenesulfonamide (0.175g,1.02mmol) and potassium carbonate (0.19g,1.36mmol) were dissolved in 2-methyltetrahydrofuran, and allylpalladium chloride dimer (6mg,0.014mmol), tBuXPhos (6mg,0.014mmol), again purged with nitrogen, and then 80% purged with nitrogenAfter 24 hours of reaction, the reaction was monitored by TLC, after the reaction was complete, extracted with ethyl acetate (10mL × 2) and 10mL water, the organic layers were combined, back-extracted once with 20mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was purified by flash chromatography column with silica gel, eluting with a gradient of 0-30% ethyl acetate/petroleum ether, to give 0.15g of F as a white solid, compound 54, in 57.25% yield. MS (ESI) [ M + H ]]⁺：387.19；¹H NMR(400MHz,CDCl₃)7.79(d,J＝8.3Hz,2H),7.28(s,2H),7.04(s,1H),6.98(d,J＝8.3Hz,1H),6.75(d,J＝8.3Hz,1H),4.14(q,J＝6.8Hz,1H),3.25(s,3H),2.47(dd,J＝7.0,3.3Hz,1H),2.40(s,3H),1.21(d,J＝6.9Hz,3H),0.87(d,J＝5.3Hz,1H),0.75–0.64(m,1H),0.49(d,J＝5.1Hz,1H),0.36(d,J＝4.9Hz,1H).

Preparation of the compound of example 55:

the procedure for the preparation of steps a) to d) was the same as that used for the preparation of example 54,

f) compound E (0.2g,0.68mmol), 2, 4-dimethylaniline (0.165g,1.36mmol), triethylamine (0.189mL,1.36mmol) and 4-dimethylaminopyridine (0.083g,0.68mmol) were dissolved in dioxane, molybdenum hexacarbonyl (0.09g,0.34mmol) was added after purging with nitrogen and [1,1' -bis (diphenylphosphino) ferrocene ] was added after purging with nitrogen]Palladium dichloride (55mg,0.068mmol), 1, 8-diazabicycloundec-7-ene (0.153mL,1.02mmol) was added after purging with nitrogen, then purging with nitrogen, followed by reaction at 85 ℃ for 24 hours, the reaction was monitored by TLC, after completion of the reaction, ethyl acetate (10mL × 2) and 10mL water were extracted, the organic layers were combined, back-extracted once with 20mL of saturated brine, dried over anhydrous sodium sulfate, stirred on silica gel and purified by flash chromatography, eluting with a gradient of 0-30% ethyl acetate/petroleum ether to give 0.15G of red solid G, compound 55, 56.82% yield. MS (ESI) [ M + H ]]⁺：365.22；¹H NMR(400MHz,CDCl₃)9.94(s,1H),8.23(d,J＝8.2Hz,1H),7.82(d,J＝8.0Hz,1H),7.21(d,J＝8.1Hz,1H),7.09(d,J＝8.6Hz,1H),7.03(s,1H),4.29(q,J＝6.8Hz,1H),3.36(s,3H),2.82–2.69(m,1H),2.37(s,3H),2.31(s,3H),1.33(d,J＝6.9Hz,3H),1.11–1.03(m,1H),0.87–0.85(m,2H),0.67(d,J＝3.7Hz,1H).

Example 56

The compound of example 56 was prepared by the same method as in example 55, except that 5-methylindoline was used instead of 2, 4-dimethylaniline in step f.

MS(ESI)[M+H]⁺：377.22。¹H NMR(400MHz,CDCl₃)8.19(d,J＝7.8Hz,1H),7.55(d,J＝8.1Hz,1H),7.17(d,J＝8.1Hz,1H),7.07(s,1H),7.05(s,1H),4.80(dd,J＝20.2,9.3Hz,1H),4.37–4.30(m,1H),4.25(q,J＝6.9Hz,1H),3.35(s,3H),3.11(t,J＝8.2Hz,2H),2.69(s,1H),2.33(s,3H),1.27(d,J＝6.9Hz,3H),0.98(s,1H),0.78(s,1H),0.61(s,2H)。

Example 57

The compound of example 57 was prepared by the same procedures as in example 55 except that indoline was used instead of 2, 4-dimethylaniline in step f.

MS(ESI)[M+H]⁺：363.13。¹H NMR(400MHz,CDCl₃)8.33(s,1H),7.57(s,1H),7.27(s,1H),7.25(s,1H),7.20(d,J＝8.0Hz,1H),7.09(s,1H),4.82(s,1H),4.41–4.32(m,1H),4.28(d,J＝6.7Hz,1H),3.38(s,3H),3.18(t,J＝8.1Hz,2H),2.72(s,1H),1.30(d,J＝6.9Hz,3H),1.01(s,1H),0.80(s,1H),0.63(s,2H)。

Example 58

The compound of example 58 was prepared by the same method as in example 55, except that 4-methylindoline was used instead of 2, 4-dimethylaniline in step f.

MS(ESI)[M+H]⁺：377.22。¹H NMR(400MHz,CDCl₃)8.16(d,J＝7.3Hz,1H),7.53(d,J＝7.9Hz,1H),7.17(d,J＝8.0Hz,2H),6.89(d,J＝7.3Hz,1H),4.82(d,J＝10.5Hz,1H),4.34(dt,J＝11.8,7.9Hz,1H),4.25(q,J＝6.8Hz,1H),3.34(s,3H),3.05(t,J＝8.3Hz,2H),2.69(s,1H),2.26(s,3H),1.27(d,J＝6.9Hz,3H),0.97(d,J＝11.4Hz,1H),0.78(s,1H),0.61(s,2H)。

Example 59

The compound of example 59 was prepared by the same method as in example 55, except that 2, 5-dimethylindoline was used instead of 2, 4-dimethylaniline in step f.

MS(ESI)[M+H]⁺：391.17。¹H NMR(400MHz,CDCl₃)8.26(s,1H),7.58(s,1H),7.17(d,J＝8.0Hz,1H),7.05(s,1H),5.79(s,1H),4.25(s,1H),3.42(d,J＝8.7Hz,1H),3.39(s,1H),3.35(s,3H),2.64(s,2H),2.33(s,3H),1.29(s,6H),1.05–0.90(m,1H),0.77(s,1H),0.60(s,2H)。

Example 60

Reagents and conditions: a) indoline, sodium triacetoxyborohydride, 1, 2-dichloroethane, 0 ℃,2 hours; b) cyclopropylamine, 1, 2-dichloroethane, at 80 ℃ under reflux for 12 hours; c) tin dichloride dihydrate and concentrated hydrochloric acid at 0-room temperature for 3 hours; d) reacting 2-bromopropionyl bromide, N, N-Diisopropylethylamine (DIPEA) and dichloromethane at room temperature for 2 hours; 2. acetonitrile, DIPEA, 80 ℃ reaction overnight; e) sodium hydride, N, N-Dimethylformamide (DMF), methyl iodide were reacted at room temperature for 1 hour.

a) Compound A (1.7g,10.07mmol) was dissolved in 10mL of 1, 2-dichloroethane, cooled to 0 deg.C, indoline (1g,8.39mmol) was added, after reaction at 0 deg.C for half an hour sodium triacetoxyborohydride (2.14g,10.07mmol) was added, followed by reaction at 0 deg.C for 2 hours, monitored by TLC plates, after the reaction was complete extracted with dichloromethane (20 mL. about.2) and 20mL of saturated sodium bicarbonate, and the mixture was combinedAnd the organic layer was purified by flash column chromatography with silica gel and eluted with a gradient of 0-3% ethyl acetate/petroleum ether to give 1.7g of red oily liquid B in 74.43% yield.¹H NMR(400MHz,CDCl₃)8.06(t,J＝8.0Hz,1H),7.39–7.29(m,2H),7.16(d,J＝7.2Hz,1H),7.07(t,J＝7.7Hz,1H),6.75(t,J＝7.4Hz,1H),6.40(d,J＝7.8Hz,1H),4.32(s,2H),3.41(t,J＝8.2Hz,2H),3.06(t,J＝8.3Hz,2H).

b) Compound B (1.7g,6.24mmol) was dissolved in 10mL 1, 2-dichloroethane, cyclopropylamine (0.86mL,12.48mmol) was added and the reaction was carried out at 80 ℃ for 12 h, monitored by TLC plates, after completion of the reaction the solvent was evaporated, extracted with dichloromethane (20mL x 2) and 20mL water, the organic layers were combined, dried over anhydrous sodium sulphate and evaporated to dryness to give 1.8g of red solid C in 93.26% yield.¹H NMR(400MHz,CDCl₃)8.18–8.08(m,2H),7.30(s,1H),7.13(d,J＝7.3Hz,1H),7.07(t,J＝7.7Hz,1H),6.74–6.67(m,2H),6.49(d,J＝7.8Hz,1H),4.27(s,2H),3.40(t,J＝8.3Hz,2H),3.03(t,J＝8.3Hz,2H),2.59–2.47(m,1H),0.84(q,J＝6.7Hz,2H),0.65–0.57(m,2H).

c) Compound C (1.8g,5.82mmol) was dissolved in 4mL of concentrated hydrochloric acid, tin dichloride dihydrate (6.24g,27.65mmol) in concentrated hydrochloric acid (4mL) was added and the reaction was allowed to proceed at room temperature for 3 hours, monitored by TLC plates, after completion of the reaction the pH was neutralized to 7-8 with saturated sodium bicarbonate, extracted with ethyl acetate (20mL x 2) and 20mL of water, the organic layers were combined and back-extracted once with 40mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to give 1.4g of red oily liquid D in 85.89% yield.

d) Compound D (1.4g,5.01mmol) was dissolved in dry 8mL dichloromethane, cooled to 0 ℃, DIPEA (1.75mL,10.02mmol) and 2-bromopropionyl bromide (0.63mL,6.01mmol) were added, then reacted at room temperature for 2 hours, monitored by TLC plates, extracted with dichloromethane (20mL x 2) and 20mL water after the reaction was complete, the organic layers were combined, back-extracted once with saturated brine 40mL, the organic phase was dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography column with silica gel, eluted with a gradient of ethyl acetate/petroleum ether of 0-15% to give 200mg yellow solid intermediate,¹H NMR(400MHz,CDCl₃)7.72(s,1H),7.20(d,J＝8.0Hz,1H),7.15(s,1H) 7.10(d, J ═ 7.1Hz,1H),7.05(d, J ═ 7.6Hz,1H),6.80(d, J ═ 7.0Hz,1H),6.67(t, J ═ 7.4Hz,1H),6.55(d, J ═ 7.8Hz,1H),4.58(q, J ═ 7.0Hz,1H),4.24(s,2H),3.33(t, J ═ 8.3Hz,2H),2.98(t, J ═ 8.3Hz,2H),2.43(dt, J ═ 10.1,3.4Hz,1H),1.97(d, J ═ 7.0Hz,3H),0.71(q, J ═ 6.5Hz,2H), 0.51-0.45H,; the intermediate was dissolved in 2mL acetonitrile, 1mL ipiea was added, followed by 80 ℃ overnight reaction, monitored by TLC plates, after completion of the reaction the solvent was evaporated, extracted with dichloromethane (10mL x 2) and 10mL water, the organic layers were combined, back extracted once with 20mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was evaporated to dryness without purification to give 0.15g pale yellow powder E, total yield 8.98%.

e) Compound E (0.1g,0.30mmol) was dissolved in 2mL dmf, cooled to 0 ℃, sodium hydride (22mg,0.90mmol) was added, reacted at 0 ℃ for half an hour, iodomethane (37uL,0.60mmol) was added at 0 ℃, then reacted at room temperature for 1 hour, monitored by TLC plates, after the reaction was completed, the pH was neutralized to 7-8 with saturated sodium bicarbonate, then extracted with ethyl acetate (10mL × 2) and 20mL water, the organic layers were combined, back extracted once with saturated brine, dried over anhydrous sodium sulfate, the organic phase was purified by flash chromatography column while stirring with ethyl acetate/petroleum ether gradient 0-25% to give 40mg white powder F, compound 60, 38.38% yield. MS (ESI) [ M + H ]]⁺：347.99。¹H NMR(400MHz,CDCl₃)7.10(s,2H),7.06(d,J＝7.8Hz,1H),6.87(s,2H),6.67(t,J＝7.3Hz,1H),6.56(d,J＝7.8Hz,1H),4.24(q,J＝14.8Hz,2H),4.10(q,J＝6.8Hz,1H),3.39–3.28(m,5H),2.98(t,J＝8.4Hz,2H),2.35(s,1H),1.17(d,J＝6.8Hz,3H),0.86(dd,J＝12.1,6.4Hz,1H),0.79–0.71(m,1H),0.65–0.57(m,1H),0.48(d,J＝4.4Hz,1H).

Pharmacological test examples

Method for testing enzymatic activity of bromodomain recognition protein BRD4 inhibitor

The binding activity of compounds to BRD4(I) was tested using the Fluorescence Anisotropy assay (FA). The principle of FA test is to calculate the fluorescence polarization values in the horizontal direction and the vertical direction for correlation analysis by detecting the molecular weight change before and after the interaction between the fluorescein labeled small molecule and other molecules. If the binding equilibrium between the fluorescently labeled small molecules and the large molecules is established, the fluorescence is excited and moves slowly, and the measured fluorescence polarization value is increased. If the combination between the fluorescence labeling micromolecule and the macromolecule is replaced by other ligands, the rotation or overturning speed of the fluorescence labeling micromolecule in a free state is increased, the emitted light is depolarized relative to an excitation light plane, the measured polarized light value is reduced, and the fluorescence anisotropy of the sample is calculated.

Fluorogenic substrate 1 was (+) -JQ1 conjugated to a fluorescent molecule, working at 5 nM. BRD4(I) protein working concentration 40nM, total reaction system 40. mu.L, buffer 50mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) pH 7.4, 150mM NaCl, 0.5mM 3- [3- (cholamidopropyl) dimethylamino]Propane sulfonic acid inner salt (CHAPS). The compound concentration of the primary screening is 1 mu M, and the IC of the compound with the inhibition rate of more than 60 percent under the condition is determined₅₀. Considering the solubility of the compounds and the effect of DMSO on the assay, a final DMSO concentration of 5% was chosen. All measurements were performed under these conditions. All the components are mixed and then are reacted for 4 hours in the dark at room temperature or are reacted overnight at 4 ℃ to determine the anisotropy value. The test was carried out using a 384-well microplate (cat # CLS3575) with a black, low-edge, NBS surface from Corning, Inc., the test apparatus being a BioTek synergy2 detector, excitation (excitation) being 485nM and emission (emission) being 530 nM. And reading blank values by taking the buffer as a system.

Numerical value processing: inhibition rate ═ C-F)/(C-B) × 100% (formula 1)

Wherein: c: anisotropy value for complete binding of fluorescent substrate to protein

B: fluorogenic substrate anisotropy background value

F: anisotropy value at the corresponding concentration of the Compound

The concentration of the compound and the corresponding inhibition rate were plotted as an S-curve. To give IC of the corresponding compound₅₀。

Fluorogenic substrate 1 for use in method for detecting BRD4 enzymatic Activity FA

Pharmacological data: the results of pharmacological testing of the compounds of the invention are disclosed in table 1 below, the control used in the test being the bromodomain protein recognizing BRD4 inhibitor (+) -JQ 1.

TABLE 1 Bromomomain protein BRD4 inhibitor enzyme Activity test results

It can be seen from Table 1 that many of the compounds have better molecular activity, especially compounds 27, 30, 35, 37 and 41 have slightly stronger molecular activity than the positive (+) -JQ 1. Summarizing the structure-activity relationship, it can be found that R₁The substituent group is methyl, so that the activity is better, and the activity of hydrogen atoms and ethyl is reduced; r₂The substituent group is better in methyl activity, the ethyl and isobutyl activities are reduced, and the configuration of the substituent group has little influence on the activity; r₃The site substituent is better in cyclopropyl activity, the isopropyl and cyclopentyl substitution activities are slightly reduced, and the activities of other larger substituents such as isobutyl, 2-methoxyethyl and the like are reduced; r₄The amide has better activity when the parent nucleus benzene ring is connected with carboxylic acid and the carboxylic acid is connected with different amino groups to form amide, wherein the activity is further improved when the nitrogen atom of the amide is substituted by methyl or ethyl compared with the activity without substitution.

Method for testing cell activity of BRD4 inhibitor of bromodomain recognition protein

Cell viability assay three cell lines HT-29, MM.1S and TY-82 were tested in total, and the test methods were (1) HT-29 cells: adopting human colon cancer HT-29 cells, respectively adding the compound for treating for 72h, and detecting the proliferation and growth inhibition effect and degree of the compound by an SRB method; (2) mm.1s cells: treating with human myeloma cell MM.1S for 72h, and detecting proliferation and growth inhibition effect and degree of the compound by ATP consumption method; (3) TY-82 cells: treating the linear cancer TY-82 cells with NUT-BRD4 for 72h, and detecting the proliferation inhibition effect of the compound by SRB method.

Pharmacological data: the results of pharmacological testing of some of the compounds of the invention are disclosed in table 2 below, with the control employed in the test being the bromodomain protein recognizing BRD4 inhibitor (+) -JQ 1.

It can be seen from Table 2 that the sensitivity of the compounds to MM.1S and TY-82 cell lines is higher than that of HT-29 cell line, and that the activity of compounds 37 and 39 to TY-82 cell line is very good, i.e.when R is₄The carboxylic acid is connected to the mother nucleus benzene ring, and the carboxylic acid is connected to 4-methyl or 5-methyl indoline, so that the activity is very good.

Method for testing metabolic stability and enzyme inhibition property of compound in liver microsome

3.1 metabolic stability test method

Metabolic stable incubation was performed with 150. mu.l liver microsomes (final concentration 0.5mg/ml), containing reduced coenzyme II (NADPH) (final concentration 1mM) and 1. mu.M compound, positive control or negative control, and the reaction was stopped with acetonitrile containing (imipramine, lot # 3221; tinidazole, lot # T3021) at 0min, 5min, 10min and 30min, vortexed for 10min, centrifuged at 15000rmp for 10min, and 50. mu.l of the supernatant was injected into a 96-well plate. The metabolic stability of the compounds was calculated by determining the relative decrease of the bulk drug.

3.2 direct inhibition assay (DI assay) test method

Direct inhibition of incubation was performed with 100. mu.l human liver microsomes (final concentration 0.2mg/ml) containing NADPH (final concentration 1mM), 10. mu.M compound, cocontail (Ketoconazole Ketoconazole 10. mu.M, Quinidine Quinidine 10. mu.M, Sulfaphenazole Sulfaphenazole 100. mu.M, naphazoline 10. mu.M, tranylcyclopromine 1000. mu.M), 10. mu.M DMSO as a negative control, and a mixed probe substrate (Midazolam 10. mu.M, Testosterone Testosterone 100. mu.M, dextromethorphan Dextromethon 10. mu.M, Diclofofenac 20. mu.M, Phenacetin Phocetin 100. mu.M, Mefenytoin 100. mu.M), and the reaction was terminated after 20min of incubation. The relative activity of the enzyme was calculated by measuring the relative production of the metabolite.

3.3 method for testing mechanistic inhibition test (TDI test)

The incubation was mechanically inhibited with 200. mu.l of human liver microsomes (final concentration 0.2mg/ml), 10. mu.M of compound, mixed positive inhibitor (oleandomycin acetate 10. mu.M, Paroxetine pareoxetine 10. mu.M, Tineilic Acid 10. mu.M, furalathin Furafylline 10. mu.M) or 10. mu.M negative control PRO, NADPH (final concentration 1mM) and mixed probe substrate (Midazolan Midazolam 5. mu.M, Testosterone Testosterone 50. mu.M, dextromethorphan Dextromethan 5. mu.M, dichlofophenolic Acid Diclofenac 10. mu.M, Phenacetin Phocetin 50. mu. M, S- (+) -Meytophenytoin S- (+) -meytoin 50. mu.M) were added after addition of NADPH (final concentration 1mM) or PBS, and the reaction was terminated after incubation for 0min, 5min, 10min and 30 min. The positive inhibitor CYP2C19 is independently prepared, and the inhibitor S- (+) -fluoxetine S- (+) -fluooxetine is 100 mu M. The enzyme activity was calculated by measuring the relative production of metabolites. Calculating k_obs。

Pharmacological data: the results of pharmacological testing of some of the compounds of the invention are set forth in table 3 below.

TABLE 3 metabolic stability and enzyme inhibition Properties of liver microsomes test results

As can be seen from Table 3, R₄The in vitro metabolic stability is better when the parent nucleus benzene ring is connected with carboxylic acid and the carboxylic acid is connected with different amino groups to form amide, especially the amide formed by p-toluidine, 2, 4-dimethylaniline and 3-fluoro-4-methylaniline.

Claims

1. A compound represented by the general formula (I):

wherein:

x is C or N;

R₂is C1-C6 straight chain or branched chain alkyl or halogen substituted C1-C6 straight chain or branched chain alkyl, R₂The configuration of (A) is R type or S type or racemate;

R₃is a substituted or unsubstituted C1-C6 straight or branched chain alkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group, a substituted or unsubstituted benzyl group, and the substituents are independently selected from 1-5 groups as follows: halogen, hydroxy, 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, C1-C3 straight or branched alkyl, or C1-C3 straight or branched alkoxy;

l is

R₄is C1-C6 straight chain or branched chain alkyl, halogen substituted C1-C6 straight chain or branched chain alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 cycloalkyl and substituted or unsubstituted C6-C12 aromatic ring, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C6-C12 aryl C1-C4 alkyl, substituted or unsubstituted 5-10 membered heterocyclic or heteroaryl group containing 1-3 heteroatoms selected from N, O and S, substituted or unsubstituted 5-10 membered heterocyclic or heteroaryl group containing 1-3 heteroatoms selected from N, O and S and substituted or unsubstituted C6-C10 aryl, the substituents are independently selected from 1-4 groups as follows: halogen, hydroxyl, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy,

or

When L is

When R is₄And R₅And to which they are connectedThe nitrogen atom forms a substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl group containing a N atom and 0-2 heteroatoms of O, S and a substituted or unsubstituted C6-C12 aryl group, the substituents being independently selected from 1-2 of the following groups: halogen, hydroxyl, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein,

x is C or N;

R₁is a hydrogen atom or a C1-C3 straight or branched alkyl group;

R₂is C1-C6 straight chain or branched chain alkyl, R₂The configuration of (A) is R type or S type or racemate;

R₃is a substituted or unsubstituted C1-C6 straight or branched chain alkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group, a substituted or unsubstituted benzyl group, the substituents are independently selected from 1-4 groups as follows: halogen, 5-7 membered heterocyclyl containing 1-2 heteroatoms selected from N and O, C1-C3 straight or branched alkyl or C1-C3 straight or branched alkoxy;

l is

R₄is substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkyl and substituted or unsubstituted C6-C12 aromatic ring, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C6-C12 aryl C1-C4 alkyl, substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl containing 1-3 heteroatoms selected from N, O and S, substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl containing 1-3 heteroatoms selected from N, O and S, and substituted or unsubstituted C6-C10 aryl, the substituents being independently selected from 1-4 groups as follows: halogen, hydroxyl, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy,

or

When L is

When R is₄And R₅And the nitrogen atom to which they are attached form a substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl group containing a N atom and 0-2 heteroatoms of O, S and a substituted or unsubstituted C6-C10 aryl group, said substituents being independently selected from 1-2 groups as follows: halogen, hydroxyl, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein,

x is C or N;

R₁is a hydrogen atom, a methyl group or an ethyl group;

R₂is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, R₂The configuration of (A) is R type or S type or racemate;

R₃is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxyethyl, a 5-6 membered heterocyclyl substituted C1-C3 straight or branched alkyl group containing 1-2 heteroatoms selected from N and O, halogen or a benzyl group substituted by C1-C3 alkoxy;

l is

or-CH₂-, wherein R₅Is a hydrogen atom, a methyl group or an ethyl group;

R₄is substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkyl and substituted or unsubstituted C6-C10 aromatic ring, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C6-C12 aryl C1-C3 alkyl, substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl containing 1-2 heteroatoms selected from N and O and substituted or unsubstituted C6-C10 aryl, the substituents being independently selected from 1-3 groups as follows: halogen, C1-C3 straight or branched chain alkyl or C1-C3 straight or branched chain alkylA branched alkoxy group;

or

When L is

When R is₄And R₅And the nitrogen atom to which they are attached form a substituted or unsubstituted 5-6 membered heterocyclyl or heteroaryl group containing a N atom and optionally an O atom and a substituted or unsubstituted C6-C10 aryl group, said substituents being independently selected from 1-2 of the following groups: halogen, C1-C3 straight chain or branched chain alkyl or C1-C3 straight chain or branched chain alkoxy.

4. The compound of the general formula (I) or a pharmaceutically acceptable salt thereof according to claim 3,

at R₃In the definition of (1) or (2) said 5-6 membered heterocyclyl substituted C1-C3 linear or branched alkyl group containing 1-2 heteroatoms selected from N and O is morpholinoethyl, tetrahydrofurylmethyl, said halogen or C1-C3 alkoxy substituted benzyl is Br substituted benzyl or methoxybenzyl.

5. The compound of the general formula (I) or a pharmaceutically acceptable salt thereof according to claim 3,

R₄is cyclopentyl, cyclohexyl, methylcyclohexyl, phenyl, methylphenyl, chlorophenyl, fluorophenyl, methoxyphenyl, isoxazolyl, 1,2, 4-trimethylpyrazol-3-yl, methylbenzyl, 2, 4-dimethylphenyl, 3-fluoro-4-methylphenyl, 2,4, 6-trimethylphenyl or pyrimidinyl,

or

When L is

When R is₄And R₅May form with the nitrogen atom to which they are attached an indolinyl group, a methyl-substituted indolinyl group, a F-substituted indolinyl group or

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein,

the compound is selected from:

7. a process for the preparation of a compound of formula (I) according to claim 1, which process comprises,

the reaction scheme I:

step a: compound 1A with amino acid NH₂R₂COOH to obtain a compound 1B;

step e: compounds 1E and R₅I reacts under the condition of sodium hydride to obtain a compound 1F,

or

Reaction scheme two:

step c: 1) compound 2C with different 2-bromoalkanoyl bromides

step d: compounds 2D and R₁I, reacting to obtain a compound 2E;

step i: compounds 2I and R₅Reacting the I under the condition of sodium hydride to obtain a compound 2J,

or

The reaction route is three:

step d: 1) compound 3D with different 2-bromoalkanoyl bromides

step g: compound 3G with amine R₄R₅NH is subjected to condensation reaction to obtain a compound 3H,

or

The reaction route is four:

step a: compound 4A with 2-bromoalkanoyl bromide

Reacting to obtain a compound 4B;

step b: compound 4B with amine R₃NH₂Reacting to obtain a compound 4C;

in schemes one to four, R₁-R₄L and X are as defined in claim 1,

or

Reaction scheme five:

step (ii) ofb: compound 5B with different amines R₃NH₂Reacting to obtain a compound 5C;

step d: 1) compound 5D with different 2-bromoalkanoyl bromides

step e: compounds 5E and R₁Reacting the compound I under the condition of sodium hydride to obtain a compound 5F,

in scheme five, R₁-R₃L and X are as defined in claim 1, R₄Is a substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl group containing 1-3 heteroatoms selected from N, O and S, a substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl group containing 1-3 heteroatoms selected from N, O and S, and a substituted or unsubstituted C6-C10 aryl group, said substituents being independently selected from 1-4 of the following groups: halogen, hydroxy, C1-C3 straight or branched chain alkyl or C1-C3 straight or branched chain alkoxy, and R₄Containing an N atom.

8. A pharmaceutical composition comprising a therapeutically effective amount of one or more of a compound of general formula (I) according to claim 1 and pharmaceutically acceptable salts thereof, and at least one excipient, diluent or carrier.

9. Use of a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a selective inhibitor of bromodomain recognition proteins.

10. Use of a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a disease associated with a bromodomain recognition protein mediated condition.

11. The use of claim 10, wherein the relevant diseases mediated by bromodomain recognition proteins include hematological malignancies, midline cancer, and inflammatory diseases.