CN110054625B

CN110054625B - N-acylbenzenesulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor, and preparation method and application thereof

Info

Publication number: CN110054625B
Application number: CN201810047659.0A
Authority: CN
Inventors: 方浩; 李孝贤; 杨新颖; 梁涛; 刘仁帅; 周易
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-01-18
Filing date: 2018-01-18
Publication date: 2022-01-07
Anticipated expiration: 2038-01-18
Also published as: CN110054625A

Abstract

The invention relates to an N-acyl benzene sulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor, a preparation method and application thereof, wherein the compound has a structure shown as a general formula I. The compound has the activities of resisting histone deacetylase and inhibiting Bcl-2 protein, can be used for preparing medicaments for preventing or treating related mammal diseases caused by abnormal expression of histone deacetylase and abnormal expression of Bcl-2 protein, and also relates to the pharmaceutical application of the composition of the compound with the structure shown in the general formula I.

Description

N-acylbenzenesulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor, and preparation method and application thereof

Technical Field

The invention relates to a N-acyl benzene sulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor, a preparation method thereof, a pharmaceutical composition and medical application, and belongs to the technical field of medicines.

Background

Apoptosis is an important homeostatic mechanism of multicellular organisms, it actively eliminates unwanted or abnormal cells, and plays an important role in embryonic development, maintenance of homeostasis, and resistance to normal physiological processes such as pathogens. Disorders of this mechanism can induce a variety of diseases, such as cancer, autoimmune diseases, cardiovascular diseases, and neurodegenerative diseases. Research shows that the Bcl-2 protein family is closely related to the process of apoptosis and is also an important target for treating tumors. More than 20 Bcl-2 family proteins can be classified into two major classes, namely anti-apoptotic proteins and pro-apoptotic proteins according to functions. These proteins all possess 1-4 Bcl-2homology domains (Bcl-2homology domains, including BH1, BH2, BH3 and BH 4). Anti-apoptotic proteins Bcl-2, Bcl-x_LBcl-W, Mcl-1 and A1/Bfl-1 can be combined with pro-apoptotic proteins to enable tumor cells to escape from apoptosis, and the structure of the pro-apoptotic proteins generally comprises four domains of BH 1-4. The proapoptotic proteins are further divided into two classes of BH3-only proteins and multidomain proteins, wherein Bim, Bad, Bid, Puma, Noxa and the like belong to BH3-only proteins, while Bax and Bak belong toIn multidomain pro-apoptotic proteins (also known as pro-apoptotic effectors). Anti-apoptotic proteins bind to the helices of the BH3 domain of pro-apoptotic proteins through their hydrophobic pocket (BH3 binding pocket). Bcl-2 protein family members mutually regulate and control a mitochondrial apoptosis pathway through protein-protein interaction (PPI) to induce apoptosis of cells. Currently only one small molecule inhibitor of Venetocalax (ABT-199) is approved by the FDA in the United states for marketing.

Histone Deacetylases (HDACs) are one of the most important and most studied epigenetic targets. Histone deacetylases and Histone Acetyltransferases (HATs) in humans cooperate to maintain normal levels of histone acetylation. Wherein, histone deacetylase makes chromatin structure compact by removing acetyl group of histone epsilon-N-acetyl lysine, and inhibits transcription. In tumor cells, histone deacetylase is over-expressed, so that histone is over-deacetylated, and cell cycle inhibitor p21 is inhibited^WAF1/CIP1The expression of the tumor suppressor factor P53 is reduced, the stability and the activity of the tumor suppressor factor p53 are reduced, and the expression levels of the cell factors hypoxia inducible factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF) promoting the angiogenesis of the tumor are increased.

Human histone deacetylase has 18 subtypes, and is divided into Zn²⁺Dependent (Class I, II, IV) and NAD⁺Dependent (Class III): class I (HDAC1 ~ 3,8), Class II (II a, HDAC4 ~ 5,7, 9; II b, HDAC6,10), Class II (SIRT1 ~ 7) and Class IV (HDAC 11). The distribution of tissues and the role played by each subtype in the body are different.

To date, the us FDA has approved four histone deacetylase pan-inhibitors for sale: vorinostat (SAHA), Romidepsin (FK-228), Belinostat (PXD-101) and Panobinostat (LBH-589). Of these, Vorinostat and 3 omidinsin are used for the treatment of cutaneous T-cell lymphoma, Belinostat for the treatment of peripheral T-cell lymphoma, Panobinostat in combination with other drugs for the treatment of multiple myeloma. However, the inhibitors that are available on the market still have many disadvantages, especially the ineffectiveness to solid tumors at normal dose and the cardiotoxicity, which results in the clinical application of the drugs being greatly limited. The partial structure of the Bcl-2 inhibitor is introduced into the prior HDACs inhibitor, and the defect of histone deacetylation inhibitor can be compensated. Therefore, the development of novel effective dual-target inhibitors is a challenging and research-worthy topic in the research of antitumor drugs.

Disclosure of Invention

The invention provides a N-acyl benzene sulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor, and also provides a preparation method and application of the compound.

The technical scheme of the invention is as follows:

mono, N-acyl benzene sulfonamide hydroxamic acid Bcl-2, HDAC double-target inhibitor

A double-target inhibitor of N-acyl benzene sulfonamide hydroxamic acids Bcl-2 and HDAC is a compound with a structure shown in a general formula I, a stereoisomer and a pharmaceutically acceptable salt thereof,

in the general formula I, R is substituted by hydrogen, halogen, nitro, cyano, trifluoromethyl, trifluoromethoxy and carboxyl group, C₁-C₁₂Alkyl, cycloalkyl, or C substituted by one or more of hydroxy, halogen, nitro, cyano, carboxyl groups₁-C₁₂Alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, which may be the same or different and may have one or more of the following substituents or may be unsubstituted: hydroxy, halogen, nitro, cyano, guanidino, carboxy, haloC₁-C₁₂Alkyl radical, C₁-C₁₂Alkoxy radical, C₁-C₁₂Alkyl radical, C₃-C₁₂Cycloalkyl, aryl, heteroaryl;

in the general formula I, X is carbon, nitrogen, oxygen or sulfur;

in the general formula I, Z is aryl, heteroaryl, cyclohexyl, heterocycle, methyl, ethyl, propyl, isopropyl, butyl and isobutyl;

in the general formula I, n_lIs 0 to 3, n₂Is 0 to 5.

According to a preferred embodiment of the invention, in formula I,

r is hydrogen, halogen, nitro, cyano, trifluoromethyl, trifluoromethoxy or carboxyl group;

x is carbon or nitrogen;

z is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

n₁is 0 to 3; n is₂Is 0 to 5.

According to the invention, it is further preferred that the compound of the above general formula I is one of the following:

2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((2- (hydroxyamino) -2-oxoethyl) amino) -3-nitrophenyl) sulfonyl) benzamide (4A),

2- ((1H-pyrrolo [2,3-B ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((4- (hydroxycarbamoyl) benzyl) amino) -3-nitrophenyl) sulfonyl) benzamide (4B),

1- (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl 3,4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) -N-hydroxy-4-carboxamide (4C),

1- (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl 3,4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) -N-hydroxy-3-carboxamide (4D),

(E) -2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5-, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- (3- (hydroxyamino) -3-oxoprop-1-en-1-yl) phenyl) sulfonyl) benzamide (4E).

The above compounds are preferred, and the following numbers in parentheses are numbers corresponding to the structures of the compounds in the following schemes and table 1.

Detailed Description

The terms and definitions used herein have the following meanings:

"aryl" means an aromatic hydrocarbon containing a ring system, such as phenyl or naphthyl, optionally fused with a cycloalkyl group, preferably having 5 to 7 ring atoms, more preferably having 5 to 6 ring atoms. Preferred aryl groups contain 5 to 15 carbon atoms.

"heteroaryl" is an aromatic heterocycle, which may be a monocyclic or bicyclic group. They contain one or more, preferably 1-4, more preferably 1-3, even more preferably 1-2 heteroatoms independently selected from O, S and N. Heteroaryl includes oxidized S or N, such as sulfinyl, sulfonyl, and N-oxide of a tricyclic nitrogen. The carbon or nitrogen atom is the point of attachment to the heteroaromatic ring structure, thereby maintaining a stable aromatic ring. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, benzo [ b ] thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furyl, benzofuryl, and indolyl.

"arylalkyl" means C₁-C₆An alkylene-linked aryl group.

"Heteroarylalkyl" means C₁-C₆An alkylene-linked heteroaryl group.

"Alkyl (Alkyl)", alone or in combination, refers to a group derived from an alkane, containing from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, if not specifically indicated. Which is a straight or branched alkyl group and includes straight or branched alkyl groups containing or interrupted by cycloalkyl moieties. Straight or branched alkyl groups are attached at any accessible site (available point) to produce stable compounds. Examples include, but are not limited to, 4- (isopropyl) -cyclohexylethyl or 2-methyl-cyclopropylpentyl. In many embodiments, alkyl is a straight or branched alkyl group containing 1 to 15 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and the like.

An "alkylene" is a divalent alkane-derived radical of carbon atoms, straight or branched, in which two hydrogen atoms are removed from the same carbon atom or different carbon atoms. Examples of alkylene groups include, but are not limited to, -CH₂-、-CH₂CH₂-and-CH₂CH(CH₃)-。

"cycloalkyl" is a substituted or unsubstituted, saturated or unsaturated cyclic group containing carbon atoms and/or one or more heteroatoms. The rings may be monocyclic or fused, bridged or spiro ring systems. The number of ring atoms in each ring is 3 to 8, more preferably 3 to 6, such as cyclopropyl, cyclopentyl, cyclohexyl, adamantyl and the like.

"alkoxy" means the group-O-alkyl.

"halogen", alone or in combination, means all halogens, i.e. chlorine (Cl), fluorine (F), bromine (Br) or iodine (I).

By "pharmaceutically acceptable salt" is meant a therapeutically effective and non-toxic salt form of the compound of formula I. It may form an anionic salt from any acidic group (e.g. carboxyl) or a cationic salt from any basic group (e.g. amino). Many such salts are known in the art. A cationic salt formed on any acidic group (e.g., a carboxyl group), or an anionic salt formed on any basic group (e.g., an amino group). Many of these salts are known in the art, such as cationic salts including salts of alkali metals (e.g., sodium and potassium) and alkaline earth metals (e.g., magnesium and calcium) and organic salts (e.g., ammonium salts). Anionic salts may also be conveniently obtained by treating the basic form of I with the corresponding acid, such acids including inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1, 2, 3-propanetriacid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, cyclohexylsulfinic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid and the like. These salts are well known to the skilled artisan and the skilled artisan can prepare any of the salts provided by the knowledge in the art. In addition, the skilled artisan may select one salt and select another salt depending on solubility, stability, ease of formulation, and the like. The determination and optimization of these salts is within the experience of the skilled artisan.

"stereoisomers" as used herein defines all possible stereoisomeric forms of the compounds of the invention or physiological derivatives thereof. Unless otherwise indicated, the chemical designation of the compounds of the invention includes mixtures of all possible stereochemical forms, which mixtures comprise all diastereomers and enantiomers of the basic structural molecule, as well as the substantially pure individual isomeric forms of the compounds of the invention, i.e. containing less than 10%, preferably less than 5%, in particular less than 2%, most preferably less than 1% of other isomers. Various stereoisomeric forms of the peptoid compounds of the present invention are expressly included within the scope of the present invention.

The compounds of formula I may also exist in other protected forms or derivatives which will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

The substituents described above may themselves be substituted by one or more substituents. Such substituents include those listed in C.Hansch and A.Leo, scientific Constants for Correlation Analysis in Chemistry and Biology (1979). Preferred substituents include alkyl, alkenyl, alkoxy, hydroxy, oxy, nitro, amino, aminoalkyl (e.g., aminomethyl, and the like), cyano, halogen, carboxy, carbonylalkoxy (e.g., carbonylethoxy, and the like), thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl (e.g., piperidinyl, morpholinyl, pyrrolyl, and the like), imino, hydroxyalkyl, aryloxy, arylalkyl, and combinations thereof.

"pharmaceutical composition" refers to a preparation containing a therapeutically significant amount of an active agent, which is prepared in a form suitable for administration to a patient. Thus, the preparation does not contain any component or components in such amounts that a properly cautious medical practitioner finds the preparation unsuitable for administration to an ordinary subject. In many cases, such pharmaceutical compositions are sterile preparations.

The specific temperature range of "room temperature" referred to in the present invention is 25-30 ℃.

Preparation method of double-target inhibitor of di, N-acyl benzene sulfonamide hydroxamic acid Bcl-2 and HDAC

A method for preparing a N-acyl benzene sulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor comprises the following steps:

the method comprises the following steps of carrying out nucleophilic substitution reaction on different substituted benzene sulfonamides 1A-1D and amino compounds under a reflux condition to obtain intermediates 2A-2D, carrying out Heck reaction on different substituted benzene sulfonamides 1E and benzene sulfonamides to obtain an intermediate 2E, carrying out amide condensation on the intermediates 2A-2E and acid at room temperature to obtain intermediates 3A-3E, and carrying out reaction on the intermediates 3A-3E in a methanol solution of hydroxylamine to obtain target compounds 4A-4E.

The synthetic route is as follows:

wherein, R, X, Z, n_l、n₂Is as defined above for formula I;

reagents and conditions: a) various amines, acetonitrile, N-diisopropylethylamine, 82 ℃; b) 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, dichloromethane, room temperature; c) methanol solution of hydroxylamine, room temperature; d) triethylamine, palladium acetate, triphenylphosphine, N-dimethylformamide.

The above amines are glycine methyl ester, 4- (aminomethyl) benzoic acid methyl ester, 4-piperidine carboxylic acid methyl ester, and 3-piperidine carboxylic acid ethyl ester.

The structural formula of the target compound in the synthetic route is shown in the following table 1:

TABLE 1 structural formulas of the target compounds

According to a preferred embodiment of the present invention, the compounds of the invention are prepared by the following specific steps, for example 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((2- (hydroxyamino) -2-oxoethyl) amino) -3-nitrophenyl) sulfonyl) benzamide (4A):

(1) synthesis of (2-nitro-4-sulfamoylphenyl) glycine methyl ester (2A)

Dissolving 700mg of 4-chloro-3-nitrobenzenesulfonyl chloride in 20ml of acetonitrile, respectively adding 800mg of glycine methyl ester hydrochloride and 1.64g of DIEA, and reacting at 80 ℃ for 10 hours; after the reaction is finished, cooling to room temperature, diluting with ethyl acetate, washing the organic phase twice with 1M citric acid and saturated saline respectively, drying with anhydrous magnesium sulfate, and spin-drying; purifying with silica gel column to obtain yellow solid 920mg with yield 76.7%;

(2) synthesis of ethyl (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3-1, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) glycinate (3A)

260mg of 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoic acid was dissolved in 10ml of dichloromethane and 110mg of DMAP, 140mg of EDCI and 130mg of 2A, respectively, were added; reacting for 12 hours at room temperature, washing with 1M citric acid and saturated salt water twice after the reaction is finished, drying the organic phase with anhydrous magnesium sulfate, spin-drying, and purifying by a silica gel column to obtain 180mg of yellow solid with the yield of 47.4%;

(3) synthesis of 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((2- (hydroxyamino) -2-oxoethyl) amino) -3-nitrophenyl) sulfonyl) benzamide (4A)

Preparation of hydroxylamine methanol solution:

solution A: dissolving 4.67g of dried hydroxylamine hydrochloride in 24mL of methanol to obtain the solution;

solution B: dissolving 5.61g of potassium hydroxide in 14mL of methanol to obtain the potassium hydroxide solution;

dropwise adding the solution B into the solution A at 0 ℃, continuously stirring in the dropwise adding process, continuously reacting for 30min at 0 ℃ after the dropwise adding is finished, filtering to remove solids to obtain a colorless and transparent hydroxylamine methanol solution, and drying for later use;

adding 6mL of hydroxylamine methanol solution into 30mg of the compound 3A, and stirring at room temperature for 0.5 h; removing most of organic solvent, adding small amount of water, adjusting pH to 5-7 with 2M dilute hydrochloric acid, and filtering; the filter cake was washed with water and dried to give the desired compound 4A in 84.8% yield.

The specific procedures for the compounds will be described in detail in the examples.

The skilled person can vary the above steps to increase the yield, and can determine the synthetic route according to the basic knowledge in the art, such as choice of reactants, solvents and temperature, and can increase the yield by using various conventional protecting groups to avoid side reactions. These conventional protection methods can be found, for example, in T.Greene, Protecting Groups in Organic Synthesis.

Application of tri, N-acylbenzenesulfonamide hydroxamic acid Bcl-2 and HDAC double-target inhibitor

The activity screening experiment shows that the compound has the activities of resisting histone deacetylase and inhibiting Bcl-2 protein, so the invention also provides the application of the series of compounds in preparing medicaments for preventing or treating related mammal diseases caused by abnormal expression of histone deacetylase and Bcl-2 protein. In addition, the present invention also includes a pharmaceutical composition suitable for oral administration to a mammal comprising a compound of any of the above general formulas I, and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

In addition, the present invention also includes a pharmaceutical composition suitable for parenteral administration to a mammal comprising a compound of any of the above general formula I, and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

In the test experiment of the Bcl-2 family protein small molecule inhibitor by applying the fluorescence polarization technology, in the specific measurement system adopted, the fluorescence labeling molecule is 5-FAM labeled Bid-BH3 polypeptide and can be matched with Bcl-2 family protein (Bcl-x)_LBcl-2 and Mcl-1) and their dissociation constant (K)_d) The combination of these two results in higher polarization values around 30-60 nM. If the test compound is also capable of specifically binding to these proteins, it will compete with the fluorescently labeled polypeptide and inhibit its binding to the proteins, resulting in a decrease in polarization. Comparing the polarization value generated by adding the test compound with the negative control and the positive control, if the reduction level of the polarization value relative to the negative control reaches about half of the difference between the negative value and the positive value, the test compound is considered to have obvious activity, and the IC can be further accurately measured₅₀Value, and then derive its competitive inhibition constant K_i。

In vitro enzyme inhibition experiment, oligopeptide substrate containing acetylated lysine side chain (Color de Lys)^TMSubstrate) under the action of HDAC, deacetylation takes place. Increased sensitivity of deacetylated product in acetylation reagent (Color de Lys)^TMDeveloper), produces an absorbance value at 405nm that is proportional to the HDACs inhibition of the test compound. By measuring the absorbance at 405nm of the control group and the test compound group, the inhibition rate of the test compound can be calculated and the IC can be obtained₅₀The value is obtained.

In vitro enzyme inhibition experiments show that the compound has stronger inhibitory activity and selectivity on Bcl-2, and the activity of all the compounds is equivalent to that of positive control ABT-263. The compounds have great development prospect and can be used for guiding the discovery of novel efficient Bcl-2 and HDAC double-target inhibitors.

Detailed Description

The present invention will be further described with reference to the following examples, but is not limited thereto.

Example 1.2 Synthesis of- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((2- (hydroxyamino) -2-oxoethyl) amino) -3-nitrophenyl) sulfonyl) benzamide (4A)

Synthesis of (2-nitro-4-sulfamoylphenyl) glycine methyl ester (2A)

Dissolving 4 mg of chlorine-3-nitrobenzene sulfonyl chloride in 20ml of acetonitrile, adding 800mg of glycine methyl ester hydrochloride and 1.64g of DIEA respectively, and reacting at 82 ℃ for 10 hours; after the reaction, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, and the organic phase was washed twice with 1M citric acid and saturated brine, dried over anhydrous magnesium sulfate, and spin-dried. Purify by silica gel column to obtain 920mg of yellow solid with 76.7% yield.¹H NMR(400MHz,DMSO)δ7.93(d,J＝8.4Hz,2H),7.84(d,J＝8.4Hz,2H),7.72(d,J＝16.1Hz,1H),7.46(s,2H),6.79(d,J＝16.1Hz,1H),3.75(s,3H).

Synthesis of Compounds 2B-2D reference 2A.

Synthesis of ethyl (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3-1, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) glycinate (3A)

2- ((1H-pyrrolo [2, 3-b)]Pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl)]-2-yl) methyl) piperazin-1-yl) benzoic acid 260mg dissolved in 10ml dichloromethane, DMAP110mg, EDCI140mg and 2a130mg were added respectively. The reaction was carried out at room temperature for 12 hours. After the reaction, the mixture was washed twice with 1M citric acid and saturated brine, respectively. The organic phase was dried over anhydrous magnesium sulfate and spin dried. Purification on a silica gel column gave 180mg of a yellow solid in 47.4% yield.¹H NMR(400MHz,DMSO)δ11.68(s,1H),11.40(s,1H),8.78(t,J＝5.0Hz,1H),8.59(d,J＝1.9Hz,1H),8.04(d,J＝2.4Hz,1H),7.87–7.81(m,1H),7.57–7.53(m,1H),7.49(dd,J＝5.8,2.8Hz,2H),7.34(d,J＝8.3Hz,2H),7.04(d,J＝8.3Hz,2H),6.99(d,J＝9.2Hz,1H),6.67(dd,J＝8.9,1.5Hz,1H),6.43–6.35(m,1H),6.18(d,J＝1.5Hz,1H),4.33(d,J＝5.8Hz,2H),3.72(s,3H),3.12–3.02(m,4H),2.76(s,2H),2.19(m,4H),2.16–2.11(m,2H)1.95(s,2H),1.38(t,J＝6.1Hz,2H),0.92(s,6H).

Synthesis of Compounds 3B-3E reference is made to 3A.

Synthesis of 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((2- (hydroxyamino) -2-oxoethyl) amino) -3-nitrophenyl) sulfonyl) benzamide (4A)

Preparation of hydroxylamine methanol solution:

adding 6mL of hydroxylamine methanol solution into 30mg of the compound 3A, and stirring at room temperature for 0.5 h; removing most of organic solvent by rotary evaporation, adding a small amount of water, adjusting pH to 5-7 with 2M dilute hydrochloric acid, and filtering; the filter cake was washed with water and dried to give the desired compound 4A in 84.8% yield.¹H NMR(400MHz,DMSO)δ11.73(s,1H),10.86(s,1H),10.06(s,1H),9.02(s,1H),8.81(t,J＝4.9Hz,1H),8.59(s,1H),8.05(d,J＝2.1Hz,1H),7.88–7.81(m,1H),7.56(s,1H),7.50(d,J＝8.7Hz,2H),7.36(d,J＝8.0Hz,2H),7.06(d,J＝7.9Hz,2H),6.87(d,J＝9.2Hz,1H),6.69(d,J＝8.5Hz,1H),6.40(s,1H),6.21(s,1H),4.05(d,J＝5.2Hz,2H),3.19(m,4H),2.98(s,2H),2.42(m,4H),2.21(s,2H),1.97(s,2H),1.40(s,2H),0.93(s,6H).

Synthesis of Compounds 4B-4E reference was made to 4A.

Example 2- ((1H-pyrrolo [2,3-B ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((4- (hydroxycarbamoyl) benzyl) amino) -3-nitrophenyl) sulfonyl) benzoyl (4B)

Intermediates and target compounds were prepared as in example 1. Yield: 82.2 percent.¹H NMR(400MHz,DMSO)δ10.63(s,1H),10.07(s,1H),9.20(dd,J＝12.5,6.5Hz,1H),8.60(s,1H),8.04(d,J＝2.0Hz,1H),7.91(d,J＝7.9Hz,1H),7.72(d,J＝7.1Hz,2H),7.53(d,J＝6.1Hz,2H),7.48(dd,J＝8.2,3.3Hz,2H),7.43(d,J＝7.8Hz,1H),7.36(d,J＝8.0Hz,2H),7.06(d,J＝7.9Hz,2H),6.91–6.80(m,1H),6.72–6.63(m,1H),6.43–6.35(m,1H),6.19(s,1H),4.83–4.66(m,2H),3.25–3.12(m,4H),2.99(s,2H),2.40(dd,J＝28.8,5.5Hz,3H),2.21(s,2H),1.97(s,2H),1.40(t,J＝4.6Hz,2H),0.93(s,6H).

Example 3.1- (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl 3,4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) -N-hydroxy-4-carboxamide (4C)

Intermediates and target compounds were prepared as in example 1. Yield: 81.5 percent.¹H NMR(400MHz,DMSO)δ11.76(s,1H),10.56(s,2H),10.10(s,2H),8.77(s,1H),8.27(d,J＝1.6Hz,1H),8.06(d,J＝2.1Hz,1H),7.84(dd,J＝8.8,1.5Hz,1H),7.57(d,J＝1.9Hz,1H),7.55–7.49(m,2H),7.37(d,J＝8.1Hz,2H),7.26(d,J＝9.0Hz,1H),7.07(d,J＝8.1Hz,2H),6.69(d,J＝9.1Hz,1H),6.42(s,1H),6.21(s,1H),3.27(s,4H),3.14(s,2H),2.98(dd,J＝8.1,5.9Hz,2H),2.68–2.54(m,3H),2.26(d,J＝0.6Hz,3H),1.98(s,2H),1.70(d,J＝0.8Hz,4H),1.41(t,J＝5.7Hz,2H),0.93(s,6H).

Example 4.1- (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl 3,4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) -N-hydroxy-3-carboxamide (4D)

Intermediates and target compounds were prepared as in example 1. Yield: 80.1 percent.¹H NMR(400MHz,DMSO)δ11.74(s,1H),10.55(s,1H),10.04(s,1H),8.81(s,1H),8.26(d,J＝1.8Hz,1H),8.06(d,J＝2.3Hz,1H),7.84(dd,J＝8.9,1.7Hz,1H),7.57(d,J＝2.1Hz,1H),7.52(d,J＝8.6Hz,2H),7.36(d,J＝8.2Hz,2H),7.29(d,J＝9.0Hz,1H),7.05(d,J＝8.2Hz,2H),6.74–6.63(m,1H),6.42(d,J＝1.0Hz,1H),6.19(s,1H),3.27(d,J＝12.5Hz,2H),3.17(s,4H),3.12–3.01(m,2H),2.95(dd,J＝14.7,6.8Hz,2H),2.47–2.32(m,4H),2.25–2.14(m,2H),1.97(s,2H),1.81(d,J＝6.8Hz,1H),1.76–1.66(m,1H),1.59(ddd,J＝35.1,18.1,6.9Hz,2H),1.40(t,J＝5.9Hz,2H),0.93(s,6H).

EXAMPLE 5 (E) -2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- (3- (hydroxyamino) -3-oxoprop-1-en-1-yl) phenyl) sulfonyl) benzamide (4E)

Intermediates and target compounds were prepared as in example 1. Yield: 84.1 percent.¹H NMR(400MHz,DMSO)δ11.81(s,1H),11.06(s,1H),10.88(s,1H),10.34(s,5H),10.12(d,J＝1.2Hz,1H),8.07(d,J＝0.5Hz,1H),7.86(d,J＝7.9Hz,2H),7.66(d,J＝7.8Hz,1H),7.61–7.44(m,4H),7.39(d,J＝7.9Hz,2H),7.10(d,J＝7.9Hz,2H),6.72(d,J＝8.3Hz,1H),6.64(s,1H),6.43(s,1H),6.27(s,1H),3.64(dd,J＝14.2,11.6Hz,4H),3.37–3.16(m,4H),2.83–2.59(m,2H),2.37(s,2H),2.01(s,2H),1.50–1.37(m,2H),0.94(s,6H).

Experimental example 1 evaluation test of Activity of target Compound on Bcl-2 protein

Experimental reagent:

Bid-BH3 polypeptide with N-terminal fluorescently labeled by 5-FAM

(5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG) in 1 XPBS;

test buffer: 1 × PBS;

correcting the solution: 1nM fluoroescein, 10mM NaOH.

An experimental instrument: the multifunctional microplate reader of TECAN Infinite F200 PRO.

The experimental steps are as follows:

1) and adding the target protein and the small molecular compound to be detected into the test buffer solution, uniformly mixing, and incubating for 30min at room temperature in a dark place. Then adding fluorescence labeled Bid BH3 polypeptide to ensure that the total volume of each solution is 200 mu L, mixing uniformly, and incubating for 20min at room temperature in a dark place.

2) 60 μ L of each of the above-mentioned solution and the calibration solution was transferred to a black 384-well plate (three sets in parallel), and fluorescence polarization was immediately detected on a microplate reader with 485nm as an excitation wavelength and 535nm as an emission wavelength, and the fluorescence polarization value of the calibration solution was set to 20 mP.

3) All compounds were first screened initially at three typical concentrations (1. mu.M, 10. mu.M, 50. mu.M), each compound was assayed in duplicate in 3 wells on the same plate, and the results of the polarization values were averaged. And (4) calculating the inhibition rate according to the measurement results of the negative control, the positive control and the polarization value of the tested compound. Targets commonly used in assaysThe protein concentration is 300-500 nM, the fluorescence labeling polypeptide adopts 5-FAM-Bid-BH3 polypeptide, and the positive compound adopts Gossypol or ABT-263. If the test result shows that the compound has an inhibition rate of more than 50% at a concentration of 50 mu M and the inhibition rate shows obvious dose-dependent relationship at three concentrations tested, the compound is considered to have specific binding with the target protein, and a more accurate IC (integrated circuit) needs to be further determined₅₀Numerical values.

4) The complete binding curve was determined at 7 different concentrations (1nM,10nM,100nM, 1. mu.M, 10. mu.M, 50. mu.M, 100. mu.M) for compounds showing significant activity in the primary screen. Each compound was subjected to 3 replicate wells on the same plate and the results of the polarization measurements were averaged. Data were processed and plotted using GraphPad Prism software to obtain the IC of the compound₅₀The value is obtained.

Based on the total concentration of protein, total concentration of fluorescent polypeptide, dissociation constant of protein-polypeptide complex, and IC of detection compound used in the measurement₅₀Values, competitive inhibition constant K for test compounds, using the calculation methods described in the literature_i。

The results are shown in Table 2.

TABLE 2 results of Bcl-2 in vitro inhibition experiments by target compounds

Numbering	IC₅₀(nM)^a
		ABT-263	<10
4A	<10
		4B	<10
4C	<10
		4D	<10
4E	<10

^aThe values in the table are the average of the results of three tests

And (4) conclusion: as can be seen from the table, the inhibitory effect of the series of 5 compounds on Bcl-2 protein was comparable to that of the positive control drug. The test result has important guiding significance for further developing Bcl-2 small molecule inhibitors with higher activity.

EXAMPLE 2 test for inhibition of Histone deacetylase by the Compound of interest (In vitro)

Description of terms:

SAHA: vorinostat.

And (mM): millimoles per liter.

HeLa: cervical cancer cells.

Color de Lys^TMSubstrate: an oligopeptide substrate containing acetylated lysine side chains.

Color de Lys Dedeveloper: and (3) detecting a reagent by deacetylation reaction.

HDAC Assay Buffer: buffer, pH 8.0, containing 50mM Tris-HCl,137mM NaCl,2.7mM KCl,1mM MgCl₂。

Trichostatin A: an HDAC inhibitor.

IC₅₀: half inhibitory concentration.

[ Material]Stock solutions of target compound and positive control SAHA (50mM in dmso); enzymes (HeLa cell extract, major components HDAC1 and HDAC 2); color de Lys^TM Substrate；Color de Lys Developer(Developer)(ii) a HDAC Assay Buffer (Buffer); trichostatin A (TSA, 0.2mM in dimethyl sulfoxide); a 96-well plate; thermo Varioskan Flash full wavelength multifunctional microplate reader.

[ method ] preparation of the experiment according to the instructions for use of the kit:

1) diluting enzyme: diluting the Hela cell extract with Buffer at a volume ratio of 1: 2;

2) compound dilution: compounds (test compound and positive control SAHA) were diluted to 5x final concentration with Buffer;

3)Color de Lys^TMsubstrate: substrate was diluted 50-fold with Buffer (1mM, 2x final concentration);

4)Color de Lys^TMdeveloper: the detection reagent was prepared within 30min of use. First, Color de Lys was performed with precooler^TMDeveloper dilution 20 fold (e.g., 50. mu.L plus 950. mu.L Buffer); TSA was then diluted 100-fold with freshly prepared Developer solution (e.g., 10. mu.L to 1mL, where TSA concentration was 2. mu.M, 2 Xfinal concentration, which means 1. mu.M final concentration after addition to the reaction system).

In a 96-well plate, 15. mu.L of diluted enzyme and 10. mu.L of test compound were added to each well, and after incubation at 37 ℃ for 5min, 25. mu.L of substrate was added (blank wells were not added with enzyme and compound, but with Buffer instead, and control wells were replaced with Buffer). The 96-well plate was incubated for 30min in a shaker at 37 ℃. Then 50. mu.L of the currently prepared Color de Lys was added to each well^TMDeveloper, continue incubation. After 30min, measuring ultraviolet absorption at 405nm on enzyme labeling instrument, measuring 405nm absorbance of control group and target compound group, calculating target compound inhibition rate and obtaining IC₅₀The value is obtained. The results are shown in Table 3.

TABLE 3 results of HDAC in vitro inhibition assay by target compounds

Numbering	IC₅₀(μM)^a
		SAHA	0.319
4A	>1
		4B	>1
4C	>1
		4D	>1
4E	>1

^aThe values in the table are the average of the results of three tests

And (4) conclusion: as can be seen from the table, the activity of the target compound is not ideal, but the test result has good guiding significance for further developing Bcl-2 and HDACs dual-target inhibitors with higher activity.

Claims

1. The following compounds and pharmaceutically acceptable salts thereof:

1- (4- (N- (2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((4 '-chloro-5, 5-dimethyl 3,4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoyl) sulfamoyl) -2-nitrophenyl) -N-hydroxy-3-carboxamide (4D).

2. The use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of disorders in a mammal associated with abnormal expression of histone deacetylase and Bcl-2 protein.

3. A pharmaceutical composition suitable for oral administration to a mammal comprising a compound of claim 1 and one or more pharmaceutically acceptable carriers or excipients.

4. A pharmaceutical composition suitable for parenteral administration to a mammal comprising a compound of claim 1 and one or more pharmaceutically acceptable carriers or excipients.