CN111423379B

CN111423379B - Substituted 3-indazole Mcl-1 protein inhibitor, preparation method and application

Info

Publication number: CN111423379B
Application number: CN202010434371.6A
Authority: CN
Inventors: 万义超; 李园华; 闫春兴; 温佳俊
Original assignee: Hunan University of Science and Technology
Current assignee: Hunan University of Science and Technology
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2021-08-03
Anticipated expiration: 2040-05-21
Also published as: CN111423379A; AU2021100660A4

Abstract

The invention discloses a substituted 3-indazole Mcl-1 protein inhibitor, a preparation method and application thereof, wherein the compound has a structure shown as a general formula (I):

Description

Substituted 3-indazole Mcl-1 protein inhibitor, preparation method and application

Technical Field

The invention relates to a substituted 3-indazole Mcl-1 protein inhibitor, and preparation, a pharmaceutical composition and medical application thereof, belonging to the technical field of medicines.

Background

Apoptosis, also known as Programmed Cell Death (PCD), is a highly ordered cellular process. The process can eliminate damaged or redundant cells in vivo, and plays an important role in the aspects of normal embryonic development of organisms and maintenance of cellular homeostasis. Escape of apoptosis is an important marker of tumor cells and also one of the important reasons for tumor cell resistance to conventional chemotherapy and radiation therapy. Therefore, in the last 20 years, the effective restoration of normal apoptosis of tumor cells by targeting key regulatory factors of apoptosis has become an anticancer strategy with potential in the current machines. So far, there are two main mature apoptosis pathways, one is called death receptor mediated pathway, also called exogenous apoptosis pathway; the other is called the mitochondrially mediated pathway, also called the endogenous apoptotic pathway.

The B cell lymphoma/leukemia-2 (B-cell leukemia/lymphoma-2, Bcl-2) protein family is an important regulatory factor in the endogenous pathway of apoptosis and plays an important regulating role in the apoptosis pathway. This family of proteins can be classified into multidomain pro-apoptotic Bcl-2 proteins (e.g., Bax, Bak, and Bok), BH3-only proteins (e.g., Bad, Bid, and Noxa), and anti-apoptotic Bcl-2 proteins (e.g., Bcl-2, Bcl-xL, and Mcl-1), depending on their structure and function. When cells are stimulated by some intracellular or extracellular death signals (such as DNA damage, ultraviolet radiation and oncogene activation), oligomerization of pro-apoptotic Bcl-2 proteins (such as Bax and Bak) results in altered permeability of the outer membrane of mitochondria, which in turn releases some cytokines (such as cytochrome c and Smac proteins) into the cells, thereby inducing caspase cascade reactions, which ultimately lead to apoptosis. The high expression of anti-apoptotic Bcl-2 protein in various cancer cells is considered to be one of the important causes of escape of cancer cells from apoptosis and acquisition of drug resistance. Therefore, antagonizing the activity of anti-apoptotic Bcl-2 proteins would undoubtedly find a way for cancer therapy.

Over the past several decades, a number of small molecule inhibitors against the anti-apoptotic Bcl-2 proteins have been reported in succession and have shown good anti-cancer activity. In particular, the selective Bcl-2 inhibitor Venetocalax/ABT-199 is approved by the U.S. Food and Drug Administration (FDA) for marketing, and is greatly encouraging drug workers to treat chronic lymphocytic leukemia patients with 17p chromosome deletions. However, ABT-199 has poor binding capacity with Mcl-1, and has undesirable effects on tumors related to Mcl-1 overexpression, thereby greatly limiting the clinical application of the tumor. Furthermore, the up-regulation of Mcl-1 protein is also one of the mechanisms of cancer cell resistance to conventional chemotherapeutic drugs such as vincristine, paclitaxel, gemcitabine and cisplatin. Therefore, the development of novel Mcl-1 protein inhibitors is a research topic with great challenge and great application value in the research of antitumor drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a substituted 3-indole Mcl-1 protein inhibitor and a preparation method of the compound.

The invention further provides a pharmaceutical composition and medical application of the compound.

The technical scheme of the invention is as follows:

substituted 3-indazole Mcl-1 protein inhibitor

A substituted 3-indole Mcl-1 protein inhibitor is a compound with a structure shown in a general formula (I) or a pharmaceutically acceptable salt thereof.

In the general formula (I), R₁Is alkyl, aryl, heteroaryl; r₁Preferably optionally substituted C1-C10 alkyl, C3-C10 cycloalkyl, C5-C15 aryl, and mono-heterocyclic aryl containing 5 or 6 ring atoms, or bis-heterocyclic aryl having 8 to 15 ring atoms, the heterocyclic aryl containing 1-4 heteroatoms independently selected from O, S, N or oxidized S or N; the carbon atom or the nitrogen atom is a connecting point of a heteroaromatic ring structure, and a stable aromatic ring is kept;

in the general formula (I), R₂Is optionally substituted aryl, heteroaryl; r₂Preferably an optionally substituted C5-C15 aryl group, and a mono-heterocyclic aryl group containing 5 or 6 ring atoms, or a bis-heterocyclic aryl group having 8 to 15 ring atoms, the heterocyclic aryl group containing 1-4 heteroatoms independently selected from O, S, N or oxidized S or N; the carbon atom or the nitrogen atom is a connecting point of a heteroaromatic ring structure, and a stable aromatic ring is kept;

the group or substituent is selected from the group consisting of hydroxy, halogen, nitro, cyano, guanidino, carboxy, haloC 1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, aralkoxy, heteroaryl having 5-10 ring atoms containing 1-2 heteroatoms, 1-3 of the above groups or substituents being attached at any accessible position to produce a stable compound.

In the general formula (I), X is carbonyl or sulfonyl or-CH₂-；

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

R₁is halogen C1-C6 alkyl, C₁-C₆Alkyl, C3-C8 cycloalkyl, substituted by 1-2 hydroxy, halogen, nitro, cyano groupsSubstituted or unsubstituted morpholine group, piperazine group-linked aromatic group Ar or aromatic group Ar, -NH-R₄(ii) a Ar is phenyl, naphthyl, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, thiazolyl, benzothiazolyl, thienyl, benzo [ b ] b]Thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furyl, benzofuryl and indolyl; r₄C1-C6 alkyl substituted or unsubstituted by 1-2 hydroxy, halogen, nitro, cyano substituents, and the above aromatic group Ar to which C1-C3 alkylene is attached; the substituent is hydroxyl, halogen, nitryl, cyano, guanidyl, carboxyl, halogen C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, and heteroaryl containing 1-2 heteroatoms and having 5-10 ring atoms;

R₂is an aromatic group Ar or an aromatic group Ar, -NH-R connected by a morpholine group or a piperazine group which is substituted or not substituted by 1 to 2 hydroxyl, halogen, nitro or cyano substituents₄(ii) a Ar is phenyl, naphthyl, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, thiazolyl, benzothiazolyl, thienyl, benzo [ b ] b]Thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furyl, benzofuryl and indolyl; r₄C1-C6 alkyl substituted or unsubstituted by 1-2 hydroxy, halogen, nitro, cyano substituents, and the above aromatic group Ar to which C1-C3 alkylene is attached; the substituent is hydroxyl, halogen, nitryl, cyano, guanidyl, carboxyl, halogen C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, and heteroaryl containing 1-2 heteroatoms and having 5-10 ring atoms;

x is carbonyl or sulfonyl or-CH₂-；

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

1-benzyl-N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 a);

1-benzyl-N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7 b);

1-benzyl-N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 c);

1- (4-bromobenzyl) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 d);

1- (4-bromobenzyl) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7 e);

1- (4-bromobenzyl) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 f);

1- (4-methylbenzyl) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 g);

1- (4-methylbenzyl) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7H);

1- (4-methylbenzyl) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 i);

1- (3, 4-dichlorobenzyl) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 j);

1- (3, 4-dichlorobenzyl) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 k);

1- (naphthyl-2-methylene) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 l);

1- (naphthyl-2-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 m);

1- ([1,1' -biphenyl ] -4-methylene) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7N);

1- ([1,1' -biphenyl ] -4-methylene) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7 o);

1- ([1,1' -biphenyl ] -4-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 p);

1- (4 '-methyl- [1,1' -biphenyl ] -4-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 q);

1- (4 '-chloro- [1,1' -biphenyl ] -4-methylene) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 r);

1- (4 '-chloro- [1,1' -biphenyl ] -4-methylene) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7 s);

1- (4 '-chloro- [1,1' -biphenyl ] -4-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7 t).

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" refers to an aryl group linked by a C1-C6 alkylene group.

"heteroarylalkyl" refers to a heteroaryl group linked by a C1-C6 alkylene group.

"arylalkenyl" refers to an aryl group attached to a C2-C6 alkenyl group.

"heteroarylalkenyl" refers to a heteroaryl group attached to a C2-C6 alkenyl 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)-。

"Alkenyl" means, alone or in combination, a straight or branched chain hydrocarbon containing from 2 to 6, preferably from 2 to 4, carbon atoms and containing from 1 to 2, preferably one, carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl.

"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.

The compounds of general formula (I) may also exist in other protected forms or derivatives, which are obvious to a person skilled in the art and which 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 room temperature is the environmental temperature of the experimental operation and is controlled within the range of 10-30 ℃.

Preparation method of bi-substituted 3-indazole Mcl-2 protein inhibitor

A preparation method of a substituted 3-indazole Mcl-2 protein inhibitor comprises the following steps: indazole-3-carboxylic acid is used as an initial raw material, methyl protection is carried out on carboxyl by using esterification reaction to generate an intermediate 5, and then nucleophilic substitution reaction is carried out on nitrogen atoms of an indazole ring and benzyl with different substitution to generate key intermediates 6a-6 g; and finally, condensing the intermediate 6a-6g with various substituted benzene sulfonamides under the action of a condensing agent 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) to obtain a target compound 7a-7 t.

The synthetic route is as follows:

wherein R is₁-R₂Is as defined above for formula I;

reagents and conditions: a) refluxing acetyl chloride, methanol and saturated sodium bicarbonate solution; b) various substituted bromobenzyls, potassium carbonate, N-Dimethylformamide (DMF), room temperature; c) 1mol/L sodium hydroxide solution, tetrahydrofuran, room temperature; ii.2- (7-Azobenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), N, N-Diisopropylethylamine (DIEA), dichloromethane, room temperature.

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

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-substituted 3-indazole Mcl-1 protein inhibitor

The invention also provides application of the series of compounds in preparing medicaments for preventing or treating related mammal diseases caused by abnormal expression of Mcl-1 protein. The mammal diseases related to the abnormal expression of the Mcl-1 protein comprise cancer, neurodegenerative diseases, virus infection, inflammation, leukemia, malaria, diabetes and the like.

In addition, the present invention also includes a pharmaceutical composition suitable for oral administration to a mammal comprising any of the compounds of formula (I) above, 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 formulae (I) and II, and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

Assays for both inhibitory and cellular activity were performed to evaluate the biological activity of the compounds in vitro.

In vitro enzyme inhibition experiment, use is made ofA fluorescence polarization experiment determination method adopts 5-FAM labeled Bid-BH3 polypeptide as a fluorescence labeling molecule in a specific measurement system, the molecule can be specifically combined with Mcl-1 protein, and the dissociation constant (K) of the molecule_d) The combination of the two results in higher polarization values around 30-60 nM. If the tested target compound can be combined with the target protein, binding of Bid and the protein is competitively inhibited, so that the polarization value is reduced, a quantity-effect curve of competitive binding of the target compound is obtained, and finally, an inhibition constant K is calculated_i。

The MTT detection method is used for testing the cell activity of the compound, tumor cell suspensions (acute granulocyte leukemia cell line HL-60, prostate cancer cell line PC-3, breast cancer cell line MDA-MB-231 and chronic granulocyte leukemia cell line K562) and normal liver cell lines LO2 are respectively inoculated in a 96-well plate, culture media containing compounds with different concentrations are added into each well, after incubation, MTT staining is carried out, after continuous incubation, the absorbance OD value of each well is measured at 570nm on an enzyme labeling instrument, and the cell growth inhibition rate is calculated, so that the activity of the compound is determined.

In vitro enzyme inhibition experiments show that part of the compounds in the invention have stronger inhibitory activity on Mcl-1 protein, wherein the activities of the compounds 7m, 7q, 7t and the like are equivalent to that of a positive control drug AT-101, and the inhibitory activity of the compound 7k on the Mcl-1 protein is higher than that of the positive control drug AT-101. Meanwhile, in an in vitro anti-tumor cell proliferation test, the compounds 7K, 7m and 7q have good inhibitory activity on three tumor cells, namely PC-3, MDA-MB-231 and K562. The best compound 7k has obvious inhibition on the three cells, the activity of the compound is equivalent to that of a positive control drug AT-101, the compound has great development prospect, and the compound can be used for guiding the discovery of a novel Mcl-1 protein inhibitor.

Detailed Description

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

Example 1.1 Synthesis of methyl H-indazole-3-carboxylate (5)

Acetyl chloride (10mL, 100mmol) was slowly added to dry methanol (100mL) in an ice bath. After stirring for 30min, 1H-indazole-3-carboxylic acid 4(4.05g, 25.0 mm) was addedol). The reaction was then refluxed for 4h in an oil bath. After cooling to room temperature, saturated NaHCO was used₃And adjusting the pH value of the solution to 8-9. The precipitate was collected by filtration, washed with water and dried in vacuo to give compound 5 as a white solid in 94% yield. Compound 5 was used in the next step without further purification.

Synthesis of benzyl-1H-indazole-3-carboxylic acid methyl ester (6a)

Compound 5(0.88g, 5mmol) is dissolved in 20mL dry DMF solution and then K is slowly added₂CO₃(2.07g, 15 mmol). After stirring at room temperature for 30min, benzyl bromide (0.65mL, 5.5mmol) was added. Stirring was continued for 30min and quenched by addition of 100mL of 1mol/L hydrochloric acid. Then, the reaction was extracted 2 times with ethyl acetate. The ethyl acetate layers were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate. After filtration and spin-drying, separation and purification by silica gel column chromatography (petroleum ether: ethyl acetate: 20: 1) gave compound 6a as a white solid in 50% yield, mp:82-84 ℃.¹H NMR(500MHz,DMSO-d₆),δ8.12(d,J＝8.0Hz,1H),7.89(d,J＝8.5Hz,1H),7.52-7.49(m,1H),7.38-7.29(m,6H),5.81(s,2H),3.94(s,3H).

Synthesis of 1-benzyl-N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7a)

Intermediate 6a was dissolved in 10mL of a mixed solvent (THF: 1mol/L NaOH ═ 1: 1), and stirred at room temperature overnight. After the organic solvent was distilled off under reduced pressure, the mixture was acidified with 1mol/L hydrochloric acid until the pH was 2 to 3. The resulting white precipitate was collected, dried in vacuo and dissolved in 20mL dry DMF. After stirring for 10min, DIEA (0.35mL, 2mmol) and HATU (0.46g, 1.2mmol) were added sequentially. After stirring for 30min, 4-nitrobenzenesulfonamide (0.24g, 1.2mmol) was added. After stirring at room temperature for 6 hours, 100mL of a 1mol/L hydrochloric acid solution was added, followed by extraction with ethyl acetate 2 times. The ethyl acetate layers were combined, washed with saturated brine and dried over anhydrous magnesium sulfate. After filtration and spin-drying, separation and purification by silica gel column chromatography (petroleum ether: ethyl acetate: 10: 9) gave 0.06g of 7 as a yellow solid in 14% yield, mp: 173-.¹H NMR(500MHz,DMSO-d₆),δ12.88(s,1H),8.49(d,J＝8.5Hz,2H),8.33(d,J＝9.0Hz,2H),8.00(d,J＝8.0Hz,1H),7.85(d,J＝8.5Hz,1H),7.49-7.46(m,1H),7.35-7.26(m,6H),5.82(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.23,150.71,145.56,141.07,136.74,135.37,129.76,129.18,128.39,127.99,127.84,125.00,124.27,123.32,121.48,111.58,53.32.HRMS(ESI)m/zCalcd for C₂₁H₁₆N₄O₅S[M-H]^-:435.0769,Found:435.0765.

Example 2.1 Synthesis of benzyl-N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7b)

Intermediates and target compounds were prepared as in example 1. The yield was 18%, mp: 166-.¹H NMR(500MHz,DMSO-d₆),δ12.64(s,1H),8.09(d,J＝8.5Hz,2H),8.02(d,J＝8.5Hz,1H),7.84(d,J＝9.0Hz,1H),7.76(d,J＝9.0Hz,2H),7.49-7.46(m,1H),7.35-7.25(m,6H),5.81(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.08,141.03,139.12,139.05,136.76,135.47,130.11,129.79,129.16,128.38,128.00,127.79,124.19,123.29,121.52,111.54,53.29.HRMS(ESI)m/z Calcd for C₂₁H₁₆ClN₃O₃S[M-H]^-:424.0528,Found:424.0528.

Example 3.1 Synthesis of benzyl-N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7c)

Intermediates and target compounds were prepared as in example 1. The yield was 22%, mp: 181-.¹H NMR(500MHz,DMSO-d₆),δ12.87(s,1H),8.69(d,J＝1.5Hz,1H),8.33(dd,J＝8.5,2.0Hz,1H),8.10(d,J＝8.5Hz,1H),8.03(d,J＝8.0Hz,1H),7.85(d,J＝8.5Hz,1H),7.50-7.47(m,1H),7.34-7.27(m,6H),5.82(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.47,147.60,141.06,140.35,136.76,135.49,133.56,132.95,131.16,129.17,128.38,127.97,127.82,125.69,124.25,123.31,121.58,111.57,53.28.HRMS(ESI)m/z Calcd for C₂₁H₁₅ClN₄O₅S[M-H]^-:469.0379,Found:469.0381.

Example 4.1 Synthesis of- (4-bromobenzyl) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7d)

Intermediates and target compounds were prepared as in example 1. The yield was 27%, mp: 184-.¹H NMR(500MHz,DMSO-d₆),δ12.85(s,1H),8.48(d,J＝8.5Hz,2H),8.31(d,J＝9.0Hz,2H),8.00(d,J＝8.0Hz,1H),7.84(d,J＝8.5Hz,1H),7.55(d,J＝8.5Hz,2H),7.50-7.47(m,1H),7.33-7.29(m,3H),5.80(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ160.68,150.22,144.99,140.55,135.67,135.04,131.60,129.75,129.27,127.44,124.51,123.85,122.78,121.14,121.01,111.00,52.05.HRMS(ESI)m/z Calcd for C₂₁H₁₅BrN₄O₅S[M-H]^-:514.9853,Found:514.9851.

Example 5.1 Synthesis of- (4-bromobenzyl) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7e)

Intermediates and target compounds were prepared as in example 1. The yield was 15%, mp: 170-.¹H NMR(500MHz,DMSO-d₆),δ12.64(s,1H),8.08(d,J＝8.5Hz,2H),8.01(d,J＝8.5Hz,1H),7.85(d,J＝8.5Hz,1H),7.76(d,J＝8.5Hz,2H),7.55(d,J＝8.0Hz,2H),7.50-7.47(m,1H),7.33-7.30(m,3H),5.79(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ160.55,140.52,138.60,138.55,135.70,135.17,131.59,129.76,129.61,129.30,127.39,123.75,122.75,121.12,121.06,110.95,52.01.HRMS(ESI)m/z Calcd for C₂₁H₁₅BrClN₃O₃S[M-H]^-:501.9633,Found:501.9634.

Example 6.1 Synthesis of- (4-bromobenzyl) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7f)

Intermediates and target compounds were prepared as in example 1. The yield is 10%, mp: 198-.¹H NMR(500MHz,DMSO-d₆),δ12.92(s,1H),8.69(d,J＝1.5Hz,1H),8.33-8.31(m,1H),8.10(d,J＝8.5Hz,1H),8.03(d,J＝8.0Hz,1H),7.85(d,J＝8.5Hz,1H),7.55-7.48(m,3H),7.35-7.29(m,3H),5.81(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.32,147.63,141.06,140.33,136.18,135.73,133.57,132.97,132.09,131.19,130.23,129.75,127.93,125.69,124.32,123.31,121.63,111.48,52.56.HRMS(ESI)m/z Calcd for C₂₁H₁₄BrClN₄O₅S[M-H]^-:546.9484,Found:546.9487.

Example 7.1 Synthesis of- (4-methylbenzyl) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7g)

Intermediates and target compounds were prepared as in example 1. The yield was 25%, mp: 192-.¹H NMR(500MHz,DMSO-d₆),δ12.94(s,1H),8.49(dd,J＝7.0,2.0Hz,2H),8.32(dd,J＝7.0,2.0Hz,2H),7.99(d,J＝8.0Hz,1H),7.83(d,J＝8.5Hz,1H),7.48-7.45(m,1H),7.32(t,J＝7.5Hz,1H),7.26(d,J＝8.0Hz,2H),7.15(d,J＝8.0Hz,2H),5.76(s,2H),2.25(s,3H).¹³C NMR(125MHz,DMSO-d₆),δ161.27,150.69,145.59,140.95,137.69,135.27,133.71,129.75,129.69,128.03,127.76,124.99,124.22,123.34,121.47,111.62,53.16,21.13.HRMS(ESI)m/z Calcd for C₂₂H₁₈N₄O₅S[M-H]^-:449.0925,Found:449.0926.

Example 8.1 Synthesis of- (4-methylbenzyl) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7H)

Intermediates and target compounds were prepared as in example 1. The yield was 25%, mp: 165-.¹H NMR(500MHz,DMSO-d₆),δ12.61(s,1H),8.08-8.05(m,2H),8.00(d,J＝8.0Hz,1H),7.82(d,J＝9.0Hz,1H),7.76-7.74(m,2H),7.48(t,J＝8.0Hz,1H),7.32(t,J＝7.5Hz,1H),7.26(d,J＝8.0Hz,2H),7.14(d,J＝8.0Hz,2H),5.75(s,2H),2.25(s,3H).¹³C NMR(125MHz,DMSO-d₆),δ161.07,140.92,139.11,139.05,137.67,135.42,133.72,130.11,129.80,129.69,128.05,127.72,124.16,123.30,121.48,111.59,53.14,21.14.HRMS(ESI)m/z Calcd for C₂₂H₁₈ClN₃O₃S[M-H]^-:438.0685,Found:438.0684.

Example 9.1 Synthesis of- (4-methylbenzyl) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7i)

Intermediates and target compounds were prepared as in example 1. The yield was 25%, mp: 202-.¹H NMR(500MHz,DMSO-d₆),δ12.87(s,1H),8.69(d,J＝2.0Hz,1H),8.33(dd,J＝8.5,2.0Hz,1H),8.10(d,J＝8.5Hz,1H),8.02(d,J＝8.0Hz,1H),7.83(d,J＝8.5Hz,1H),7.49-7.46(m,1H),7.33(t,J＝7.5Hz,1H),7.25(d,J＝8.0Hz,2H),7.15(d,J＝8.0Hz,2H),5.76(s,2H),2.25(s,3H).¹³C NMR(125MHz,DMSO-d₆),δ161.41,147.62,140.96,140.35,137.69,135.35,133.71,133.57,132.96,131.18,129.69,128.02,127.76,125.69,124.22,123.35,121.55,111.61,53.17,21.13.HRMS(ESI)m/z Calcd for C₂₂H₁₇ClN₄O₅S[M-H]^-:483.0535,Found:483.0538.

Example 10.1 Synthesis of- (3, 4-dichlorobenzyl) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7j)

Intermediates and target compounds were prepared as in example 1. The yield was 28%, mp: 204-.¹H NMR(500MHz,DMSO-d₆),δ12.93(s,1H),8.51(d,J＝8.5Hz,2H),8.35(d,J＝8.5Hz,2H),8.02(d,J＝8.0Hz,1H),7.91(d,J＝8.5Hz,1H),7.73(s,1H),7.62(d,J＝8.5Hz,1H),7.53-7.50(m,1H),7.36-7.30(m,2H),5.85(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.13,150.83,145.45,141.08,137.87,135.66,131.75,131.49,131.17,130.16,129.78,128.39,128.07,125.05,124.41,123.25,121.55,111.42,51.90.HRMS(ESI)m/z Calcd for C₂₁H₁₄Cl₂N₄O₅S[M-H]^-:502.9989,Found:502.9988.

Example 11.1 Synthesis of- (3, 4-dichlorobenzyl) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7k) intermediates and the title compound were prepared as in example 1. yield 28%, mp: 179-.¹H NMR(500MHz,DMSO-d₆),δ12.81(s,1H),8.69(d,J＝2.0Hz,1H),8.33(dd,J＝8.5,2.0Hz,1H),8.10(d,J＝8.5Hz,1H),8.04(d,J＝8.5Hz,1H),7.89(d,J＝8.5Hz,1H),7.70(s,1H),7.62(d,J＝8.5Hz,1H),7.53-7.50(m,1H),7.36(t,J＝7.5Hz,1H),7.29(dd,J＝8.5,1.5Hz,1H),5.84(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.30,149.87,147.63,141.08,137.86,133.57,132.97,131.76,131.44,131.15,130.17,128.38,128.06,126.69,126.19,125.70,124.38,123.27,121.67,111.40,51.89.HRMS(ESI)m/z Calcd for C₂₁H₁₃Cl₃N₄O₅S[M-H]^-:538.9570,Found:538.9571.

Example 12.1 Synthesis of- (naphthyl-2-methylene) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7l)

Intermediates and target compounds were prepared as in example 1. The yield was 21%, mp: 200-.¹H NMR(500MHz,DMSO-d₆),δ12.99(s,1H),8.45(d,J＝8.0Hz,2H),8.29(d,J＝8.0Hz,2H),8.08(d,J＝8.0Hz,1H),7.88-7.83(m,5H),7.51-7.44(m,4H),7.30-7.27(m,1H),5.96(s,2H).¹³C NMR(126MHz,DMSO-d₆),δ161.92,150.30,148.23,141.06,134.50,133.19,132.87,130.79,129.58,128.90,128.22,128.06,127.55,126.96,126.73,125.90,124.69,123.73,123.43,122.07,122.03,111.33,53.42.HRMS(ESI)m/z Calcd for C₂₅H₁₈N₄O₅S[M-H]^-:485.0925,Found:485.0922.

Example 13.1 Synthesis intermediates of- (naphthyl-2-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7m) and the title compound can be prepared as in example 1. yield 30%, mp: 199-.¹H NMR(500MHz,DMSO-d₆),δ13.04(s,1H),8.71(d,J＝2.0Hz,1H),8.34(dd,J＝8.5,2.0Hz,1H),8.10(d,J＝8.5Hz,1H),8.05(d,J＝8.5Hz,1H),7.90(d,J＝10.0Hz,5H),7.52-7.46(m,4H),7.34(t,J＝7.5Hz,1H),6.00(s,2H).¹³C NMR(126MHz,DMSO)δ161.40,151.62,147.62,141.13,135.54,134.27,133.58,133.19,132.98,132.90,131.21,129.35,128.94,128.23,128.07,127.87,126.99,126.80,125.89,125.72,124.29,123.39,121.61,111.61,53.59.HRMS(ESI)m/z Calcd for C₂₅H₁₇ClN₄O₅S[M-H]^-:519.0535,Found:519.0539.

Example 14.1- ([1,1' -Biphenyl)]Synthetic intermediates and the title compound of (4-methylene) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7N) were prepared as in example 1. The yield was 24%, mp:191-192 ℃.¹H NMR(500MHz,DMSO-d₆),δ12.96(s,1H),8.49(d,J＝9.0Hz,2H),8.33(d,J＝9.0Hz,2H),8.02(d,J＝8.5Hz,1H),7.90(d,J＝8.5Hz,1H),7.64-7.61(m,4H),7.51-7.48(m,1H),7.46-7.43(m,4H),7.37-7.31(m,2H),5.87(s,2H).¹³C NMR(125MHz,DMSO),δ161.30,150.69,145.61,141.09,140.29,140.06,135.93,135.50,129.76,129.39,128.62,128.03,127.88,127.49,127.12,125.00,124.29,123.33,121.53,111.60,52.97.HRMS(ESI)m/z Calcd for C₂₇H₂₀N₄O₅S[M-H]^-:511.1082,Found:511.1080.

Example 15.1- ([1,1' -Biphenyl)]Synthetic intermediates of (e) -4-methylene) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7o) and the title compound were prepared as in example 1. The yield was 24%, mp: 222-.¹H NMR(500MHz,DMSO-d₆),δ12.68(s,1H),8.10(d,J＝9.0Hz,2H),8.03(d,J＝8.5Hz,1H),7.90(d,J＝8.5Hz,1H),7.77(d,J＝8.5Hz,2H),7.64-7.61(m,4H),7.51-7.48(m,1H),7.46-7.43(m,4H),7.37-7.31(m,2H),5.86(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.10,141.06,140.28,140.07,139.12,139.06,135.94,135.54,130.12,129.80,129.39,128.63,128.02,127.84,127.48,127.12,124.23,123.31,121.55,111.57,52.95.HRMS(ESI)m/z Calcd for C₂₇H₂₀ClN₃O₃S[M-H]^-:500.0841,Found:500.0848.

Example 16.1 Synthesis of- ([1,1' -biphenyl ] -4-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7p)

Intermediates and target compounds were prepared as in example 1. The yield was 11%, mp: 174-.¹H NMR(500MHz,DMSO-d₆),δ12.85(s,1H),8.70(s,1H),8.34(d,J＝8.5Hz,1H),8.10(d,J＝8.5Hz,1H),8.05(d,J＝8.0Hz,1H),7.90(d,J＝8.5Hz,1H),7.64-7.61(m,4H),7.52(t,J＝8.5Hz,1H),7.46(t,J＝8.5Hz,4H),7.37(q,J＝7.5Hz,2H),5.87(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.35,147.63,141.11,140.30,140.06,135.93,135.54,133.58,132.97,131.20,129.39,128.60,128.02,127.88,127.88,127.49,127.12,125.70,124.29,123.36,121.60,111.60,52.99.HRMS(ESI)m/z Calcd for C₂₇H₁₉ClN₄O₅S[M-H]^-:545.0692,Found:545.0689.

Example 17.1- (4 '-methyl- [1,1' -biphenyl)]Synthetic intermediates and title compound of (e) -4-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7q) were prepared as in example 1. The yield was 27%, mp: 198-.¹H NMR(500MHz,DMSO-d₆),δ12.91(s,1H),8.70(s,1H),8.34(d,J＝8.5Hz,1H),8.10(d,J＝8.5Hz,1H),8.04(d,J＝8.0Hz,1H),7.90(d,J＝8.5Hz,1H),7.61(d,J＝8.0Hz,2H),7.52-7.49(m,3H),7.42(d,J＝8.0Hz,2H),7.35(t,J＝7.5Hz,1H),7.25(d,J＝8.0Hz,2H),5.86(s,2H),2.32(s,3H).¹³C NMR(125MHz,DMSO-d₆),δ161.38,147.62,141.08,140.35,140.20,137.37,137.14,135.58,135.52,133.57,132.97,131.19,129.98,128.59,127.87,127.19,126.93,125.70,124.29,123.35,121.60,111.60,53.00,21.11.HRMS(ESI)m/z Calcd for C₂₈H₂₁ClN₄O₅S[M-H]^-:559.0848,Found:559.0841.

EXAMPLE 18 Synthesis of 1- (4 '-chloro- [1,1' -biphenyl ] -4-methylene) -N- ((4-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7r)

Intermediates and target compounds were prepared as in example 1. The yield was 24%, mp: 167-.¹H NMR(500MHz,DMSO-d₆),δ12.94(s,1H),8.49(d,J＝9.0Hz,2H),8.33(d,J＝9.0Hz,2H),8.01(d,J＝8.0Hz,1H),7.90(d,J＝8.5Hz,1H),7.66-7.63(m,4H),7.51-7.48(m,3H),7.45(d,J＝8.5Hz,2H),7.34(t,J＝7.5Hz,1H),5.87(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.26,150.71,145.57,141.09,138.92,138.85,136.33,135.54,132.93,129.76,129.35,128.89,128.70,127.90,127.46,125.00,124.31,123.32,121.52,111.59,52.94.HRMS(ESI)m/z Calcd for C₂₇H₁₉ClN₄O₅S[M-H]^-:545.0692,Found:545.0694.

Example 19.1- (4 '-chloro- [1,1' -biphenyl)]Synthetic intermediates of (e) -4-methylene) -N- ((4-chlorobenzenesulfonyl) -1H-indazole-3-carboxamide (7s) and the title compound were prepared as in example 1. The yield was 15%, mp: 205-.¹H NMR(500MHz,DMSO-d₆),δ12.65(s,1H),8.09(d,J＝8.0Hz,2H),8.02(d,J＝8.0Hz,1H),7.89(d,J＝8.5Hz,1H),7.76(d,J＝8.5Hz,2H),7.66-7.63(m,4H),7.50-7.44(m,5H),7.34(t,J＝7.5Hz,1H),5.86(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.06,150.24,141.06,139.08,138.91,138.86,136.34,135.53,132.93,130.12,129.81,129.35,128.89,128.71,127.86,127.45,124.25,123.29,121.54,111.56,52.91.HRMS(ESI)m/z Calcd for C₂₇H₁₉Cl₂N₃O₃S[M-H]^-:534.0451,Found:534.0454.

Example 20.1- (4 '-chloro- [1,1' -biphenyl)]Synthetic intermediates and title compound of (4-methylene) -N- ((4-chloro-3-nitrobenzenesulfonyl) -1H-indazole-3-carboxamide (7t) were prepared as in example 1. The yield was 26%, mp: 194-.¹H NMR(500MHz,DMSO-d₆),δ12.84(s,1H),8.70(d,J＝1.5Hz,1H),8.34(dd,J＝8.5,2.0Hz,1H),8.10(d,J＝8.5Hz,1H),8.04(d,J＝8.5Hz,1H),7.90(d,J＝8.5Hz,1H),7.66-7.63(m,4H),7.52-7.49(m,3H),7.44(d,J＝8.0Hz,2H),7.35(d,J＝7.5Hz,1H),5.87(s,2H).¹³C NMR(125MHz,DMSO-d₆),δ161.40,147.62,141.11,140.35,138.92,138.85,136.33,135.60,133.57,132.97,132.93,131.19,129.35,128.89,128.68,127.89,127.45,125.70,124.30,123.34,121.61,111.58,52.94.HRMS(ESI)m/z Calcd for C₂₇H₁₈Cl₂N₄O₅S[M-H]^-:579.0302,Found:579.0302.

Evaluation of Activity of target Compound

EXAMPLE 1 test for Mcl-1 protein inhibition by the Compound of interest (In vitro)

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:

TECAN Genios Pro type multifunctional microplate reader.

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 (1mM, 10mM,50 mM), and each compound was assayed in 3 replicates on the same plate, and the results of the polarization measurements 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. The concentration of target protein usually adopted in the determination is 300-500 nM, the fluorescence labeling polypeptide adopts 5-FAM-Bid-BH3 polypeptide, and the positive compound adopts AT-101. If the test result shows that the compound has an inhibition rate of more than 50% at a concentration of 50mM 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 curves were determined at 7 different concentrations (1nM,10nM,100nM,1mM,10mM,50mM,100mM) 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.

(5) 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(Nikolovska-Coleska,Z.；et al.Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization.Anal Biochem.2004,332,261-273).

The results are shown in Table 2.

TABLE 2 in vitro inhibition test results of target compound on Mcl-1 protein

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

As can be seen from the table, the compounds have better inhibitory activity against Mcl-1 protein when the indazole ring has a larger substituent at position 1, such as compounds 7k, 7l, 7m, 7p, 7q and 7 t. When the sulfonamide moiety is 4-chloro-3-nitrobenzenesulfonamide, the activity is best, e.g., 7f is significantly better than 7d and 7e, 7i is significantly better than 7g and 7h, etc. The activity of the compound 7k is higher than that of a positive control AT-101, and the compound has important guiding significance for further developing an Mcl-1 protein inhibitor with higher activity.

EXAMPLE 2 Activity test for inhibition of cell proliferation by target Compound (In vitro)

From these, 3 compounds with better enzymatic activity were selected for the activity test of inhibiting cancer cell proliferation in vitro, and the results are shown in table 3.

Description of terms:

acute granulocytic leukemia cell line HL-60, prostate cancer cell line PC-3, breast cancer cell line MDA-MB-231, chronic granulocytic leukemia cell line K562 and normal liver cell line LO 2.

IC₅₀: half inhibitory concentration.

Materials:

HL-60, PC-3, MDA-MB-231, K562, LO2, MTT, 10% fetal bovine serum, 96-well plate

The method comprises the following steps:

the cell culture cell strain is cultured by a conventional method. Cells in logarithmic growth phase were used for all experiments.

Cell growth assay (MTT method) cell suspension was adjusted to 5X 10⁴/mL (suspension cells adjusted to 10)⁵mL), respectively inoculated in 96-well plates (100. mu.L/well), 2000-5000 cells/well. After plating for 4h, 100 μ L of medium containing compounds at different concentrations was added to each well, triplicate wells were set for each concentration, wells without cells were read as blanks, wells without cells with compounds were added as compound blanks, and gossypol was used as compound positive control. At 37 ℃ with 5% CO₂The culture medium is incubated for 48h, 10. mu.L of 0.5% MTT staining solution is added to each well, and the incubation is continued. After 4h, centrifuge at 2500rpm for 30min, discard the medium in the plate well, add 150. mu.L DMSO per well, shake at 37 ℃ for 5-10 min. Measuring the absorbance OD value of each hole at 570nm on an enzyme-labeling instrument, and obtaining the cellsThe growth inhibition rate was calculated as follows:

TABLE 3 results of antitumor cell proliferation assay of Compounds 7k, 7m and 7q

^aThe values in the table are the average of three tests, and the values after "+ -" indicate the standard deviation

In-vitro antiproliferative activity experiments of four tumor cells of HL-60, PC-3, MDA-MB-231 and K562 and normal cells of HUEVC are carried out on 3 compounds with better enzyme activity. Test data show that 3 tested target compounds have inhibition effects on three tumor cells, namely PC-3, MDA-MB-231 and K562, and particularly on K562 cells. Meanwhile, these three compounds had no effect on the growth of normal cell LO 2. The inhibition activity of the compound on three tumor cells of PC-3, MDA-MB-231 and K562 is equivalent to that of a positive control AT-101, which shows that the substituted 3-indazole Mcl-1 protein inhibitor has good development prospect, can be used for carrying out deep activity research, and developing a more active compound for preparing a medicament for preventing and treating related mammal diseases caused by abnormal expression of Mcl-1 protein.

Claims

1. A substituted 3-indazole Mcl-1 protein inhibitor is a compound with a structure shown in a general formula (I) and pharmaceutically acceptable salts thereof;

in the general formula (I),

R₁is 4-Br-Ph, 4-CH₃-Ph, 3,4-di-Cl-Ph, Naph-2, Biph-4 or 4-Cl-Biph-4';

R₂is 3-NO₂-4-Cl-Ph；

X is-CH₂-。

2. The preparation method of the substituted 3-indazole Mcl-1 protein inhibitor according to claim 1, which comprises the following steps:

the method comprises the following steps: indazole-3-carboxylic acid is used as a starting raw material, and methyl protection is carried out on carboxyl by using esterification reaction to generate an intermediate 5;

step two: nucleophilic substitution reaction is carried out on nitrogen atoms of indazole rings in the intermediate 5 and different substituted benzyl bromide, acyl chloride or sulfonyl chloride to generate key intermediates 6a-6 g;

step three: condensing the intermediate 6a-6g with various substituted benzene sulfonamides under the action of a condensing agent to obtain a target compound 7a-7t, namely a substituted 3-indazole Mcl-1 protein inhibitor;

wherein R is₁-R₂The definition of (A) is as described in the above general formula (I).

3. The process for preparing a substituted 3-indazole Mcl-1 protein inhibitor according to claim 2, wherein: reagents and conditions a: acetyl chloride, methanol, saturated sodium bicarbonate solution and refluxing.

4. The process for preparing a substituted 3-indazole Mcl-1 protein inhibitor according to claim 2, wherein: reagents and conditions b: various substituted bromobenzyls, potassium carbonate, N-dimethylformamide, room temperature.

5. The process for preparing a substituted 3-indazole Mcl-1 protein inhibitor according to claim 2, wherein: reagents and conditions c: 1mol/L sodium hydroxide solution, tetrahydrofuran, room temperature; ii.2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, dichloromethane, room temperature.

6. The use of a compound of claim 1 for the manufacture of a medicament for the prophylaxis or treatment of a disease in a mammal associated with aberrant expression of Mcl-1 protein.

7. The use according to claim 6, wherein the related mammal diseases caused by abnormal expression of Mcl-1 protein include cancer, neurodegenerative diseases, viral infection, inflammation, leukemia, malaria and diabetes.

8. 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.

9. 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.