AU2021100660A4 - Substituted 3-Indazole MCL-1 Inhibitor, Preparation Method and Use Thereof - Google Patents

Abstract

The present disclosure provides a substituted 3-indazole Mcl-i inhibitor, a preparation method and use thereof. The substituted 3-indazole Mcl-i inhibitor is a compound having the 0 O / 'R2 NH N / N structure of general formula (I): (1) . The compound disclosed by the present disclosure has relatively strong inhibitory activity against Mcl-i protein and may prevent or treat related mammalian diseases caused by aberrant expression of Mcl-i protein. The present disclosure further relates to pharmaceutical use of a composition containing the compound represented by the general formula (I).

Description

SUBSTITUTED 3-INDAZOLE MCL-1 INHIBITOR, PREPARATION METHOD AND USETHEREOF TECHNICAL FIELD The present disclosure relates to a substituted 3-indazole Mcl-i inhibitor and preparation thereof, a pharmaceutical composition, and medical use, and belongs to the technical field of medicine. BACKGROUND Apoptosis, also known as programmed cell death (PCD), is a highly ordered cellular process. This process can remove damaged or redundant cells from the body and plays an important role in the normal embryonic development of organisms and the maintenance of cell homeostasis. Escaping apoptosis is not only an important hallmark of tumor cells, but also one of the important reasons why tumor cells are resistant to conventional chemotherapy and radiotherapy. Therefore, over the past 20 years, it has become a potential anti-cancer strategy to effectively restore the normal apoptosis of tumor cells by targeting key regulators of apoptosis. So far, there are two major well-studied apoptotic pathways: one is called death receptor-mediated pathway, also called extrinsic apoptotic pathway; the other is called mitochondrial-mediated pathway, also called intrinsic apoptotic pathway. B-cell leukemia/lymphoma-2 (Bcl-2) protein family is a class of important regulatory factors in the intrinsic apoptotic pathway and plays an important regulatory role in the apoptotic pathway. According to differences in structure and function, this family can be divided into pro-apoptotic multi-domain Bcl-2 proteins (such as Bax, Bak, and Bok), BH3-only proteins (such as Bad, Bid, and Noxa), and anti-apoptotic Bcl-2 proteins (such as Bcl-2, Bcl-xL, and Mcl-i). When cells are stimulated by some intracellular or extracellular death signals (such as DNA damage, ultraviolet radiation, and oncogene activation), the oligomerization of pro-apoptotic Bcl-2 proteins (such as Bax and Bak) may lead to a change in mitochondrial outer membrane permeabilization, and further, some cytokines (such as cytochrome C and Smac protein) are released into the cell, and further induce a cascade of caspases, ultimately leading to cell apoptosis. The anti-apoptotic Bcl-2 proteins are highly expressed in a plurality of cancer cells and are regarded as one of the important reasons for cancer cells to escape apoptosis and acquire drug resistance. Therefore, antagonizing the activity of the anti-apoptotic Bcl-2 proteins undoubtedly finds a way for us to treat cancers. Over the past several decades, a plurality of small molecule inhibitors targeting anti-apoptotic Bcl-2 proteins have been reported successively and shown good anti-cancer activity. In particular, selective Bcl-2 inhibitor Venetoclax/ABT-199 was approved by the Food and Drug Administration (FDA) for the treatment of patients with chronic lymphocytic leukemia with 17p chromosome deletion, which substantially encouraged pharmaceutical workers. However, ABT-199 poorly binds to Mcl-1, and has less desirable effect on cancer cells with Mel-1 overexpression, which substantially limits clinical application thereof In addition, the up-regulation of Mel-1 protein is one of the mechanisms by which cancer cells develop resistance to conventional chemotherapeutic drugs, such as vincristine, paclitaxel, gemcitabine, and cisplatin. Therefore, it is a very challenging research topic with great application value to develop novel Mcl- Iinhibitors in studying anti-tumor drugs. SUMMARY In view of the shortcomings of the prior art, the present disclosure provides a substituted 3-indazole Mel- Iinhibitor, and further provides a preparation method thereof. The present disclosure further provides pharmaceutical compositions and medical use of the substituted 3-indazole Mcl- Iinhibitor. The technical solutions of the present disclosure are as follows: I. Substituted 3-indazole Mci-i inhibitor A substituted 3-indazole Ml-i inhibitor is provided, where the substituted 3-indazole Mcl-i inhibitor is a compound having the structure of general formula (I) and a pharmaceutically acceptable salt thereof. 0 0'z// 0 R2 NH

N / N XR (I)

In the general formula (I), Ri may be alkyl, aryl, or heteroaryl; R may preferably be optionally substituted Cl-C10 alkyl, C3-C1 cycloalkyl, C5-C15 aryl, and heteromonocyclic aryl having 5 or 6 ring atoms, or bicyclic heterocyclic aryl having 8-15 ring atoms, where a heterocyclic aryl may contain 1-4 heteroatoms, and the heteroatoms may be independently selected from 0, S, N, or oxidized S or N; a carbon atom or nitrogen atom may be an attachment point of heteroaromatic ring structure, maintaining a stable aromatic ring; in the general formula (I), R2 may optionally be substituted aryl or heteroaryl; R2 may preferably be optionally substituted C5-C15 aryl, heteromonocyclic aryl having 5 or 6 ring atoms, or bicyclic heterocyclic aryl having 8-15 ring atoms, where a heterocyclic aryl may contain 1-4 heteroatoms, and the heteroatoms may be independently selected from 0, S, N, or oxidized S or N; a carbon atom or nitrogen atom may be an attachment point of heteroaromatic ring structure, maintaining a stable aromatic ring; a group or substituent may be selected from hydroxyl, halogen, nitro, cyano, guanidino, carboxyl, halo C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C6 -C10 aryl, aralkyloxy, and heteroaryl having 5-10 ring atoms and 1-2 heteroatoms; 1-3 of the above groups or substituents may be connected at any available point to produce a stable compound; and in the general formula (I), X may be selected from carbonyl, sulfonyl, or -CH2-. According to the present disclosure, preferably, in the general formula (I), Ri may be halo C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, aromatic group Ar or aromatic group Ar attached to a morpholinyl or piperazinyl substituted or unsubstituted by 1-2 hydroxyl, halogen, nitro, or cyano substituents, or -NH-R4; Ar may phenyl, naphthyl, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, quinazolinyl, purinyl, quinolyl, pyrimidinyl, pyrrolyl, pyrazolyl, thiazolyl, benzothiazolyl, thienyl, benzo[b]thienyl, isoxazolyl, oxthiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furyl, benzofuryl, and indyl having 0 or 1 substituent; R4may be Cl-C6 alkyl substituted or unsubstituted by 1-2 hydroxyl, halogen, nitro, or cyano substituents, and the above aromatic group Ar attached to C1-C3 alkylene; the substituent may be hydroxy, halogen, nitro, cyano, guanidino, carboxyl, halo C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, and heteroaryl having 5-10 ring atoms and 1-2 heteroatoms; R2may be aromatic group Ar or aromatic group Ar attached to a morpholinyl or piperazinyl substituted or unsubstituted by 1-2 hydroxyl, halogen, nitro, or cyano substituents, or -NH-R4; Ar may phenyl, naphthyl, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, quinazolinyl, purinyl, quinolyl, pyrimidinyl, pyrrolyl, pyrazolyl, thiazolyl, benzothiazolyl, thienyl, benzo[b]thienyl, isoxazolyl, oxthiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furyl, benzofuryl, and indyl having or 1 substituent; R4may be Cl-C6 alkyl substituted or unsubstituted by 1-2 hydroxyl, halogen, nitro, or cyano substituents, and the above aromatic group Ar attached to C1-C3 alkylene; the substituent may be hydroxy, halogen, nitro, cyano, guanidino, carboxyl, halo C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C5-C1O aryl, and heteroaryl having 5-10 ring atoms and 1-2 heteroatoms; and X may be selected from carbonyl, sulfonyl, or -CH2-. According to the present disclosure, further preferably, the compound represented by the general formula (I) may be one of the following: 1-benzyl-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7a); 1-benzyl-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7b); 1-benzyl-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7c); 1-(4-bromophenyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7d); 1-(4-bromobenzyl)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7e); 1-(4-bromobenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7f);

1-(4-methylbenzyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7g); 1-(4-methylbenzyl)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7h); 1-(4-methylbenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7i); 1-(3,4-dichlorobenzyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7j); 1-(3,4-dichlorobenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7k); 1-(naphthyl-2-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (71); 1-(naphthyl-2-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7m); 1-([1,1'-biphenyl]-4-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7n); 1-([1,1'-biphenyl]-4-methylene)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7o); 1-([1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxa mide (7p); 1-(4'-methyl-[1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole 3-carboxamide (7q); 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxa mide (7r); 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carbox amide (7s); and 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3 -carboxamide (7t). For the above preferred compounds, numbers in parentheses are those corresponding to the following reaction routes and the compound structures in Table 1. Brief description of the disclosure The terms and definitions used herein have the following meanings: "Aryl" refers to an aromatic hydrocarbon containing a ring system, such as phenyl or naphthyl, which may be optionally fused with cycloalkyl; the cycloalkyl may preferably have -7 ring atoms, and more preferably 5-6 ring atoms. Preferably, the aryl may contain 5-15 carbon atoms. "Heteroaryl" is an aromatic heterocyclic ring and may be a monocyclic or bicyclic group. The heteroaryl may contain one or more, preferably 1-4, more preferably 1-3, and even more preferably 1-2 heteroatoms, the heteroatoms may be independently selected from 0, S and N. The heteroaryl may include oxidized S or N, such as sulfinyl, sulfonyl, and tricyclic oxynitride.

A carbon atom or nitrogen atom may be an attachment point of heteroaromatic ring structure, thereby maintaining a stable aromatic ring. Examples of the heteroaryl mat include, but not be limited to, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, quinolyl, pyrimidinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxthiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazinyl, furyl, benzofuryl, and indyl. "Aralkyl" refers to an aryl group to which a C1-C6 alkylene group is attached. "Heteroaralkyl" refers to a heteroaryl group to which a C1-C6 alkylene group is attached. "Arylalkenyl" refers to an aryl group to which a C2-C6 alkenyl group is attached. "Heteroarylalkenyl" refers to a heteroaryl group to which a C2-C6 alkenyl group is attached. "Alkyl", alone or in combination, refers to a group derived from an alkane, containing 1-20 and preferably 1-12 (unless otherwise specified) carbon atoms. The alkyl may be a linear or branched alkyl group, and may include a linear or branched alkyl group containing or interrupted by a cycloalkyl portion. The linear or branched alkyl group may be attached at any available point to produce a stable compound. Examples thereof may include, but not be limited to, 4-(isopropyl)-cyclohexylethyl or 2-methyl-cyclopropylpentyl. In many examples, the alkyl group may be a linear or branched alkyl group containing 1-15, 1-8, 1-6, 1- 4, or 1-2 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl and similar alkyl groups. "Alkylene" is a carbon atom group derived from a bivalent alkane, linear or branched. Herein, two hydrogen atoms may be removed from the same carbon atom or different carbon atoms. Examples of the alkylene may include, but not be limited to, -CH2-, -CH2CH2-, and -CH2CH(CH3)-. "Alkenyl", alone or in combination, herein refers to a linear or branched hydrocarbon, which contains 2-6 and preferably 2-4 carbon atoms, and has 1-2 and preferably a carbon-carbon double bond. Examples of the alkenyl may include, but not be limited to, vinyl, propenyl, isopropenyl, and butenyl. "Cycloalkyl" is a substituted or unsubstituted, saturated or unsaturated cyclic group containing carbon atoms and/or one or more heteroatoms. The ring may be a single, fused, bridged, or spirocyclic ring system. There may be 3-8 and more preferably 3-6 ring atoms in each ring, such as cyclopropyl, cyclopentyl, cyclohexyl, adamantyl, and similar groups. "Alkoxy" indicates a group-O-alkyl. "Halogen", alone or in combination, refers to all halogens, namely chlorine (Cl), fluorine (F), bromine (Br), or iodine (I). "Pharmaceutically acceptable salt" refers to a therapeutic and non-toxic salt form of the compound represented by the general formula (I). The pharmaceutically acceptable salt may form an anionic salt from any acidic group (such as carboxyl), or form a cationic salt from any basic group (such as amino). A plurality of such salts have been known in the art. There are cationic salts formed on any acidic group (such as carboxyl) or anionic salts formed on any basic group (such as amino). A plurality of such salts have been known in the art. For example, cationic salts may include alkali metal (such as sodium and potassium) and alkaline earth metal (such as magnesium and calcium) salts and organic salts (such as ammonium salts). The anionic salts may further be conveniently obtained by treating the basic form of the general formula (I) with the corresponding acid. Such acids may include inorganic acids such as sulfuric acid, nitric acid, and phosphoric acid; 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-tricarballylic acid, methanesulfonic acid, ethanesulfonic acid, benzene methanesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfinic acid, salicylic acid, and 4-aminosalicylic acid. These salts are well known to those skilled in the art, and those skilled in the art may prepare any salt provided by knowledge in the art. In addition, those skilled in the art may choose one salt instead of another based on factors such as solubility, stability, and ease of formulation. The determination and optimization of these salts are within the experience of those skilled in the art. The compound represented by the general formula (I) may further exist in other protected forms or derivative forms, and these forms are obvious to those skilled in the art and should be included in the scope of the present disclosure. The foregoing substituents per se may be substituted by one or more substituents. Such substituents may include those listed in C. Hansch and A. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology (1979). Preferred substituents may include alkyl, alkenyl, alkoxy, hydroxyl, oxy, nitro, amino, aminoalkyl (such as aminomethyl), cyano, halogen, carboxyl, calkoxycarbonyl (such as ethoxycarbonyl), thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl (such as piperidyl, morpholinyl, and pyrrolyl.), imino, hydroxyalkyl, aryloxy, aralkyl, and combination 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. Therefore, the preparation does not contain any one component or a plurality of components in such an amount that a properly prudent medical practitioner finds that the preparation is not suitable for administration to ordinary subjects. In many cases, this pharmaceutical composition is a sterile preparation. Room temperature refers to an ambient temperature where an experiment is operated, being controlled within the range of 10-30°C. II. Method for preparing the substituted 3-indazole Mcl-i inhibitor

The method for preparing the substituted 3-indazole Mcl-i inhibitor includes the following steps: using indazole-3-carboxylic acid as a starting material, esterfying carboxyl to produce an intermediate 5; conducting a nucleophilic substitution reaction on a nitrogen atom of an indazole ring with differently substituted benzyl groups to produce key intermediates 6a to 6g; finally, condensing the intermediates 6a to 6g with various substituted benzene sulfonamides to obtain target compounds 7a to 7t in the presence of a condensing agent 2-(7-azabenzotriazol-1-yl)-NN,N',N'-tetramethyluronium hexafluorophosphate (HATU). A synthetic route is as follows: 0 0 OH -0 0 NH oH a b c N C N N 0 N N ~ N N ~ N' H H I I

4 5 6a-6g 7a-7t

where, definitions of Ri-R2 are the same as those described in the general formula I; reagents and conditions are as follows: in step a), reagents used are acetyl chloride, methanol, and saturated sodium bicarbonate solution, and the condition is reflux; in step b), reagents used are differently substituted benzyl bromides, potassium carbonate, and N,N-dimethylformamide (DMF), and the condition is room temperature; in step c): i. reagents used are 1 mol/L sodium hydroxide solution and tetrahydrofuran (THF), and the condition is room temperature; and ii. reagents used are 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA), and dichloromethane, and the condition is room temperature. Structural formulas of the target compounds in the synthetic route are shown in Table 1 below:

o NH NH R2

N N XR1

7a-7t

Table 1 Structural formulas of the target compounds

Compd R1 R2 X

7a Ph 4-NO2-Ph -CH2

7b Ph 4-Cl-Ph -CH2

7c Ph 3-NO2-4-Cl-Ph -CH2-

7d 4-Br-Ph 4-NO2-Ph -CH2 7e 4-Br-Ph-CH2- 4-Cl-Ph -CH2 7f 4-Br-Ph-CH2- 3-NO2-4-Cl-Ph -CH2 7g 4-CH3-Ph-CH2- 4-NO2-Ph -CH2 7h 4-CH3-Ph-CH2- 4-Cl-Ph -CH2 7i 4-CH3-Ph-CH2- 3-NO2-4-Cl-Ph -CH2 7j 3,4-di-Cl-Ph-CH2- 4-NO2-Ph -CH2 7k 3,4-di-Cl-Ph-CH2- 3-NO2-4-Cl-Ph -CH2 71 Naph-2-CH2- 4-NO2-Ph -CH2 7m Naph-2-CH2- 3-NO2-4-Cl-Ph -CH2 7n Biph-4-CH2- 4-NO2-Ph -CH2 7o Biph-4-CH2- 4-Cl-Ph -CH2 7p Biph-4-CH2- 3-NO2-4-Cl-Ph -CH2 7q 4-CH3-Biph-4'-CH2- 3-NO2-4-Cl-Ph -CH2 7r 4-Cl-Biph-4'-CH2- 4-NO2-Ph -CH2 7s 4-Cl-Biph-4'-CH2- 4-Cl-Ph -CH2 7t 4-Cl-Biph-4'-CH2- 3-NO2-4-Cl-Ph -CH2

The specific operation steps of the compounds will be described in detail in the examples. Those skilled in the art may modify the foregoing steps to improve the yield. They may determine the synthetic route based on the basic knowledge in the art, such as selection of reactants, solvents and temperature, and may avoid the occurrence of side reactions to increase yield by using various conventional protecting groups. These conventional protection methods may be found in, for example, T. Greene, Protecting Groups in Organic Synthesis. III. Use of the substituted 3-indazole Mc-1 inhibitor The present disclosure further provides use of the compounds in the preparation of medicaments for preventing or treating related mammalian diseases caused by aberrant expression of Mcl-1 protein. The related mammalian diseases caused by aberrant expression of Mcl-i protein include cancers, neurodegenerative diseases, viral infections, inflammation, leukemia, malaria, and diabetes. In addition, the present disclosure further includes a pharmaceutical composition suitable for oral administration to mammals, including any one of the compounds represented by the foregoing general formula (I) and a pharmaceutically acceptable carrier, and optionally including one or more of pharmaceutically acceptable excipients.

In addition, the present disclosure further includes a pharmaceutical composition suitable for parenteral administration to mammals, including any one of the compounds represented by the foregoing general formulas (I) and (II) and a pharmaceutically acceptable carrier, and optionally including one or more of pharmaceutically acceptable excipients. The in vitro bioactivity of the compounds is tested and evaluated by inhibiting enzyme activity and cell viability. Fluorescence polarization assay is used in the in vitro enzyme inhibition assay. In a specific measurement system, a 5-FAM-labeled Bid-BH3 peptide is used as a fluorescence marker molecule, and the molecule may specifically bind to a Mcl-i protein; with dissociation constant (Kd) thereof being about 30-60 nM, binding both produces a higher polarization value. On the condition that a target compound to be tested is able to bind to a target protein, binding of Bid to the protein will be competitively inhibited, resulting in a decrease in the polarization value; further, a dose-response curve of competitive binding of the target compound is obtained, and an inhibition constant Ki is finally calculated. The cell viability of the compounds is tested by the MTT assay; tumor cell suspensions (acute myeloid leukemia cell line HL-60, prostate cancer cell line PC-3, breast cancer cell line MDA-MB-231, and chronic myeloid leukemia cell line K562) and normal hepatocyte cell line L02 are inoculated into a 96-well plate, respectively, each well is added with a medium supplemented with different concentrations of compounds; after incubation, the cell lines are stained with MTT; after continued incubation, for each well, absorbance (OD value) at 570 nm is measured on a microplate reader, and cell growth inhibition rate is calculated to determine the activity of the compounds. The in vitro enzyme inhibition assay shows that some of the compounds in the present disclosure have strong inhibitory activity against the Mcl-i protein, among which compounds 7m, 7q, and 7t have comparable activity to positive control drug AT-101, and compound 7k has stronger inhibitory activity against the Mcl-1 protein than the positive control drug AT-101. Meanwhile, in the in vitro anti-tumor cell proliferation assay, compounds 7k, 7m, and 7q have better inhibitory activity against three kinds of tumor cells: PC-3, MDA-MB-231, and K562. Herein, the best compound, 7k, significantly inhibits these three kinds of cells, has comparable activity to the positive control drug AT-101, possesses great development prospects, and may be used to guide the discovery of novel Mcl-Iinhibitors. DETAILED DESCRIPTION The present disclosure will be further described below in conjunction with examples, but is not limited thereto. Example 1. Synthesis of methyl 1H-indazole-3-carboxylate (5)

Acetyl chloride (10 mL, 100 mmol) was slowly added to absolute methanol (100 mL) in an ice bath. After stirring for 30 min, 1H-indazole-3-carboxylic acid 4 (4.05 g, 25.0 mmol) was added. Subsequently, reflux reaction was conducted in an oil bath for 4 h. After cooling to room temperature, pH was adjusted to 8-9 with saturated NaHCO3 solution. Precipitates were collected by filtration, washed with water, and dried in vacuum to obtain white solid compound 5, with a yield of 94%. Compound 5 was used directly in the next step without purification. Synthesis of methyl 1-benzyl-1H-indazole-3-carboxylate (6a) Compound 5 (0.88 g, 5 mmol) was dissolved in 20 mL of dry DMF solution, and K2CO3 (2.07 g, 15 mmol) was slowly added. After stirring for 30 min at room temperature, benzyl bromide (0.65 mL, 5.5 mmol) was added. Stirring was continued for 30 min, and the reaction was quenched with 100 mL of 1 mol/L hydrochloric acid was added. Subsequently, the mixture was extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate. After filtration and spin-drying, the residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain white solid compound 6a with a yield of 50%, mp: 82-84C. 1 H NMR (500 MHz, DMSO-d), 8.12 (d, J= 8.0 Hz, 1H), 7.89 (d, J= 8.5 Hz,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-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7a) Intermediate 6a was dissolved in 10 mL of mixed solvent (THF: 1 mol/L NaOH = 1:1), and stirred at room temperature overnight. After the organic solvent was evaporated under reduced pressure, the mixture was acidified with 1 mol/L hydrochloric acid until the pH was 2-3. The resulting white precipitate was collected, vacuum dried, and dissolved in 20 mL of dried DMF. After stirring for 10 min, DIEA (0.35 mL, 2 mmol) and HATU (0.46 g, 1.2 mmol) were added sequentially. After stirring for 30 min, 4-nitrobenzenesulfonamide (0.24 g, 1.2 mmol) was added. After stirring for 6 h at room temperature, 100 mL of1 mol/L hydrochloric acid solution was added, and the mixture was extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate. After filtration and spin-drying, the residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 9) to obtain 0.06 g of yellow solid 7 with a yield of 14%, mp: 173-174 0C. 1H NMR (500 MHz, DMSO-d), 12.88 (s, 1H), 8.49 (d, J= 8.5 Hz, 2H), 8.33 (d, J=9.0 Hz, 2H), 8.00 (d, J= 8.0 Hz, 1H), 7.85 (d, J= 8.5 Hz, 1H), 7.49-7.46 (m, 1H), 7.35-7.26 (m, 6H), 5.82 (s, 2H). 13 C NMR (125 MHz, DMSO-d), 161.23, 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/z Calcd for C21HI6N405S [M-H]-: 435.0769, Found: 435.0765. Example 2. Synthesis of

1-benzyl-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7b) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 18%, mp: 166-167°C. 1H NMR (500 MHz, DMSO-d), 12.64 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 8.02 (d, J =8.5 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 9.0 Hz, 2H), 7.49-7.46 (m, 1H), 7.35-7.25 (m, 6H), 5.81 (s, 2H). 1 3 C NMR (125 MHz, DMSO-d), 8 161.08, 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 C21HI6ClN303S [M-H]-: 424.0528, Found: 424.0528. Example 3. Synthesis of 1-benzyl-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7c) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 22%, mp: 181-183°C. 1H NMR (500 MHz, DMSO-d), 12.87 (s, 1H), 8.69 (d, J = 1.5 Hz, 1H), 8.33 (dd, J= 8.5, 2.0 Hz, 1H), 8.10 (d, J= 8.5 Hz, 1H), 8.03 (d, J= 8.0 Hz, 1H), 7.85 (d, J= 8.5 Hz, 1H), 7.50-7.47 (m, 1H), 7.34-7.27 (m, 6H), 5.82 (s, 2H). 13 C NMR (125 MHz, DMSO-d), 8 161.47, 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 C21Hi5ClN405S [M-H]-: 469.0379, Found: 469.0381. Example 4. Synthesis of 1-(4-bromophenyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7d) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 27%, mp: 184-186°C. 1H NMR (500 MHz, DMSO-d), 12.85 (s, 1H), 8.48 (d, J = 8.5 Hz, 2H), 8.31 (d, J=9.0 Hz, 2H), 8.00 (d, J= 8.0 Hz, 1H), 7.84 (d, J= 8.5 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.50-7.47 (m, 1H), 7.33-7.29 (m, 3H), 5.80 (s, 2H). 13C NMR (125 MHz, DMSO-d), 8 160.68, 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) mlz Calcd for C21Hi5BrN405S [M-H]-: 514.9853, Found: 514.9851. Example 5. Synthesis of 1-(4-bromobenzyl)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7e) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 15%, mp: 170-172°C. 1H NMR (500 MHz, DMSO-d), 12.64 (s, 1H), 8.08 (d, J = 8.5 Hz, 2H), 8.01 (d, J=8.5 Hz, 1H), 7.85 (d, J= 8.5 Hz, 1H), 7.76 (d, J= 8.5 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.50-7.47 (m, 1H), 7.33-7.30 (m, 3H), 5.79 (s, 2H). 13C NMR (125 MHz, DMSO-d), 8 160.55, 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) mlz Calcd for C21Hl5BrCN303S [M-H]-: 501.9633, Found: 501.9634.

Example 6. Synthesis of 1-(4-bromobenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7f) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 10%, mp: 198-199°C. 1H NMR (500 MHz, DMSO-d), 12.92 (s, 1H), 8.69 (d, J = 1.5 Hz, 1H), 8.33-8.31 (m, 1H), 8.10 (d, J= 8.5 Hz, 1H), 8.03 (d, J= 8.0 Hz, 1H), 7.85 (d, J= 8.5 Hz, 1H), 7.55-7.48 (m, 3H), 7.35-7.29 (m, 3H), 5.81 (s, 2H). 13 C NMR (125 MHz, DMSO-d), 8 161.32, 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 C21H14BrClN40S [M-H]-: 546.9484, Found: 546.9487. Example 7. Synthesis of 1-(4-methylbenzyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7g) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 25%, mp: 192-193°C. 1H NMR (500 MHz, DMSO-d), 12.94 (s, 1H), 8.49 (dd, J = 7.0, 2.0 Hz, 2H), 8.32 (dd, J =7.0, 2.0 Hz, 2H), 7.99 (d, J= 8.0 Hz, 1H), 7.83 (d, J= 8.5 Hz, 1H), 7.48-7.45 (m, 1H), 7.32 (t, J= 7.5 Hz, 1H), 7.26 (d, J= 8.0 Hz, 2H), 7.15 (d, J= 8.0 Hz, 2H), 5.76 (s, 2H), 2.25 (s, 3H). 1 3 C NMR (125 MHz, DMSO-d), 8 161.27, 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 C22H18N40S [M-H]-: 449.0925, Found: 449.0926. Example 8. Synthesis of 1-(4-methylbenzyl)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7h) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 25%, mp: 165-166°C. 1H NMR (500 MHz, DMSO-d6), 8 12.61 (s, 1H), 8.08-8.05 (m, 2H), 8.00 (d, J= 8.0 Hz, 1H), 7.82 (d, J= 9.0 Hz, 1H), 7.76-7.74 (m, 2H), 7.48 (t, J= 8.0 Hz, 1H), 7.32 (t, J= 7.5 Hz, 1H), 7.26 (d, J= 8.0 Hz, 2H), 7.14 (d, J= 8.0 Hz, 2H), 5.75 (s, 2H), 2.25 (s, 3H). 1 3 C NMR (125 MHz, DMSO-d6), 8 161.07, 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 C22Hi8ClN303S [M-H]-: 438.0685, Found: 438.0684. Example 9. Synthesis of 1-(4-methylbenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7i) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 25%, mp: 202-203°C. 1H NMR (500 MHz, DMSO-d), 12.87 (s, 1H), 8.69 (d, J = 2.0 Hz, 1H), 8.33 (dd, J= 8.5, 2.0 Hz, 1H), 8.10 (d, J= 8.5 Hz, 1H), 8.02 (d, J= 8.0 Hz, 1H), 7.83 (d, J= 8.5 Hz, 1H), 7.49-7.46 (m, 1H), 7.33 (t, J= 7.5 Hz, 1H), 7.25 (d, J= 8.0 Hz, 2H), 7.15 (d, J= 8.0 Hz, 2H), 5.76 (s, 2H), 2.25 (s, 3H). 13C NMR (125 MHz, DMSO-d),8 161.41,

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 Caled for C22H17ClN405S [M-H]-: 483.0535, Found: 483.0538. Example 10. Synthesis of 1-(3,4-dichlorobenzyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7j) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 28%, mp: 204-205°C. 1H NMR (500 MHz, DMSO-d), ( 12.93 (s, 1H), 8.51 (d, J = 8.5 Hz, 2H), 8.35 (d, J= 8.5 Hz, 2H), 8.02 (d, J= 8.0 Hz, 1H), 7.91 (d, J= 8.5 Hz, 1H), 7.73 (s, 1H), 7.62 (d, J= 8.5 Hz,1H), 7.53-7.50 (m,1H), 7.36-7.30 (m, 2H), 5.85 (s, 2H). 13C NMR (125 MHz, DMSO-d), ( 161.13, 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 Called for C21H14Cl2N40S [M-H]-: 502.9989, Found: 502.9988. Example 11. Synthesis of 1-(3,4-dichlorobenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7k) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 28%, mp: 179-180°C. 1H NMR (500 MHz, DMSO-d), ( 12.81 (s, 1H), 8.69 (d, J = 2.0 Hz, 1H), 8.33 (dd, J= 8.5, 2.0 Hz, 1H), 8.10 (d, J= 8.5 Hz, 1H), 8.04 (d, J= 8.5 Hz,1H), 7.89 (d, J= 8.5 Hz,1H), 7.70 (s,1H), 7.62 (d, J= 8.5 Hz, 1H), 7.53-7.50 (m, 1H), 7.36 (t, J= 13 7.5 Hz, 1H), 7.29 (dd, J= 8.5, 1.5 Hz, 1H), 5.84 (s, 2H). C NMR (125 MHz, DMSO-d), 161.30,149.87, 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 Caled for C21H13Cl3N40S [M-H]-: 538.9570, Found: 538.9571. Example 12. Synthesis of 1-(naphthyl-2-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (71) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 21%, mp: 200-201°C. 1H NMR (500 MHz, DMSO-d), ( 12.99 (s, 1H), 8.45 (d, J = 8.0 Hz, 2H), 8.29 (d, J= 8.0 Hz, 2H), 8.08 (d, J= 8.0 Hz, 1H), 7.88-7.83 (m, 5H), 7.51-7.44 (m, 4H), 7.30-7.27 (m, 1H), 5.96 (s, 2H). 3 C NMR (126 MHz, DMSO-d), 161.92, 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 Caled for C25H18N40S [M-H]-: 485.0925, Found: 485.0922. Example 13. Synthesis of 1-(naphthyl-2-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7m) Preparation methods of the intermediate and the target compound are shown in Example 1.

The yield was 30%, mp: 199-200°C. 1H NMR (500 MHz, DMSO-d), 13.04 (s, 1H), 8.71 (d, J = 2.0Hz, 1H), 8.34 (dd,J= 8.5, 2.0Hz, 1H), 8.10 (d,J= 8.5 Hz, 1H), 8.05 (d,J= 8.5 Hz, 1H), 7.90 (d, J= 10.0 Hz, 5H), 7.52-7.46 (m, 4H), 7.34 (t, J= 7.5 Hz, 1H), 6.00 (s, 2H). 1 3 C NMR (126 MHz, DMSO) 8 161.40, 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 C2H17ClN405S [M-H]-: 519.0535, Found: 519.0539. Example 14. Synthesis of 1-([1,1'-biphenyl]-4-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide (7n) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 24%, mp: 191-192°C. 1H NMR (500 MHz, DMSO-d), 12.96 (s, 1H), 8.49 (d, J = 9.0 Hz, 2H), 8.33 (d, J =9.0 Hz, 2H), 8.02 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.5 Hz, 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 (125 MHz, DMSO), 8 161.30, 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 C27H2oN405S [M-H]-: 511.1082, Found: 511.1080. Example 15. Synthesis of 1-([1,1'-biphenyl]-4-methylene)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide (7o) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 24%, mp: 222-223°C. 1H NMR (500 MHz, DMSO-d), 12.68 (s, 1H), 8.10 (d, J = 9.0 Hz, 2H), 8.03 (d, J =8.5 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 8.5 Hz, 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 (125 MHz, DMSO-d6), 8 161.10, 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 C27H2aClN303S [M-H]-: 500.0841, Found: 500.0848. Example 16. Synthesis of 1-([1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxa mide (7p) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 11%, mp: 174-175°C. 1 H NMR (500 MHz, DMSO-d), 8 12.85 (s, 1H), 8.70 (s, 1H), 8.34 (d, J= 8.5 Hz, 1H), 8.10 (d, J=8.5 Hz, 1H), 8.05 (d, J= 8.0 Hz, 1H), 7.90 (d, J= 8.5 Hz, 1H), 7.64-7.61 (m, 4H), 7.52 (t, J= 8.5 Hz, 1H), 7.46 (t, J= 8.5 Hz, 4H), 7.37 (q, J= 7.5 Hz, 2H), 5.87 (s, 2H). 13C NMR (125 MHz, DMSO-d), 8 161.35, 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 C27H19ClN405S

[M-H]-: 545.0692, Found: 545.0689. Example 17. Synthesis of 1-(4'-methyl-[1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole 3-carboxamide (7q) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 27%, mp: 198-199°C. 1H NMR (500 MHz, DMSO-d), 8 12.91 (s, 1H), 8.70 (s, 1H), 8.34 (d, J= 8.5 Hz, 1H), 8.10 (d, J=8.5 Hz, 1H), 8.04 (d, J= 8.0 Hz, 1H), 7.90 (d, J= 8.5 Hz, 1H), 7.61 (d, J= 8.0 Hz, 2H), 7.52-7.49 (m, 3H), 7.42 (d, J= 8.0 Hz, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.25 (d, J= 8.0 Hz, 2H), 5.86 (s, 2H), 2.32 (s, 3H). 1 3 C NMR (125 MHz, DMSO-d6), 8 161.38, 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 C2H21ClN40S [M-H]-: 559.0848, Found: 559.0841. Example 18. Synthesis of 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxa mide (7r) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 24%, mp: 167-169°C. 1H NMR (500 MHz, DMSO-d), 12.94 (s, 1H), 8.49 (d, J = 9.0 Hz, 2H), 8.33 (d, J= 9.0 Hz, 2H), 8.01 (d, J=8.0 Hz, 1H), 7.90 (d, J= 8.5 Hz, 1H), 7.66-7.63 (m, 4H), 7.51-7.48 (m, 3H), 7.45 (d, J= 8.5 Hz, 2H), 7.34 (t, J=7.5 Hz, 1H), 5.87 (s, 2H). 1 3C NMR (125 MHz, DMSO-d), 8 161.26, 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 C27H19ClN405S [M-H]-: 545.0692, Found: 545.0694. Example 19. Synthesis of 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carbox amide (7s) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 15%, mp: 205-206°C. 1H NMR (500 MHz, DMSO-d), 12.65 (s, 1H), 8.09 (d, J = 8.0 Hz, 2H), 8.02 (d, J= 8.0 Hz, 1H), 7.89 (d, J =8.5 Hz, 1H), 7.76 (d, J= 8.5 Hz, 2H), 7.66-7.63 (m, 4H), 7.50-7.44 (m, 5H), 7.34 (t, J=7.5 Hz, 1H), 5.86 (s, 2H).13 C NMR (125 MHz, DMSO-d), 8 161.06, 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 C27H19Cl2N303S [M-H]-: 534.0451, Found: 534.0454. Example 20. Synthesis of 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3

-carboxamide (7t) Preparation methods of the intermediate and the target compound are shown in Example 1. The yield was 26%, mp: 194-195°C. 1H NMR (500 MHz, DMSO-d), 12.84 (s, 1H), 8.70 (d, J = 1.5 Hz, 1H), 8.34 (dd, J= 8.5, 2.0 Hz, 1H), 8.10 (d, J= 8.5 Hz, 1H), 8.04 (d, J= 8.5 Hz, 1H), 7.90 (d, J=8.5 Hz, 1H), 7.66-7.63 (m, 4H), 7.52-7.49 (m, 3H), 7.44 (d, J= 8.0 Hz, 2H), 7.35 (d, J= 7.5 Hz,1H), 5.87 (s, 2H). 13C NMR (125 MHz, DMSO-d), 161.40, 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) mlz Calcd for C27HiCl2N40S [M-H]-: 579.0302, Found: 579.0302. Target compound activity evaluation Experimental Example 1. Inhibition assay of target compounds against Mc-i protein (in vitro) Experimental reagents: Bid-BH3 polypeptide fluorescently labeled with 5-FAM on the N-terminus (5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG), dissolved in 1x PBS; Test buffer: 1x PBS; Calibration buffer: 1 nM fluorescein, 10 mM NaOH; Laboratory instrument: TECAN GENios Pro Microplate Reader. Experimental procedure: (1) A target protein and a small molecule compound to be tested were added into the test buffer, mixed well and incubated for 30 min at room temperature in the dark. Fluorescently labeled Bid BH3 peptide was added to make up to total volume of each solution, i.e. 200 [L, mixed well and incubated for 20 min at room temperature in the dark. (2) 60 L each of the above solution and calibration solution was transferred to a black 384-well plate (three parallel groups), and fluorescence polarization was immediately measured on a microplate reader. Using 485 nm as an excitation wavelength and 535 nm as an emission wavelength, the fluorescence polarization value of the calibration buffer was set at 20 mP. (3) All compounds were first screened at three typical concentrations (1 PM, 10 PM, and 50 pM), and each compound was measured in 3 replicates in parallel on the same plate, and polarization measurements were averaged. The inhibitory rate was calculated based on the polarization measurements of the negative control, the positive control and the test compound. The target protein concentration usually used in the assay is 300-500 nM, 5-FAM-Bid-BH3 peptide is used as a fluorescently labeled peptide, and AT-101 is used as a positive compound. On the condition that the assay results show that the compound has an inhibitory rate of greater than 50% at a concentration of 50 M, and the inhibitory rate shows a significant dose-dependent relationship at the three concentrations tested, it may be appreciated that the compound specifically binds to the target protein, and a relatively accurate IC5o value may needed to be further determined. (4) For compounds that showed significant activity in the preliminary screening, a complete binding curve was determined at 7 different concentrations (1 nM, 10 nM, 100 nM, 1 PM, 10 pM, 50 pM, 100 pM). Each compound was measured in 3 replicates in parallel on the same plate, and polarization measurements were averaged. GraphPad Prism software was used for data processing and plotting to obtain the IC5o values of the compounds. (5) According to the total concentration of the protein used in the measurement, the total concentration of the fluorescently labeled peptide, the dissociation constant of the protein-peptide complex, and the IC5o value of the test compound, the competitive inhibition constant Ki was obtained by using the calculation method in the following literature (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 experimental results are shown in Table 2. Table 2. The results of the in vitro inhibition assay of target compounds against Mcl-i protein No. Ki (M)a No. IC5o(paM)a No. IC5o(paM)a 7a >10 7h >10 7o >10 7b >10 7i 1.2 7p 1.1 7c >10 7j >10 7q 0.72 7d >10 7k 0.43 7r >10 7e >10 71 1.3 7s >10 7f 1.1 7m 0.52 7t 0.88 7g >10 7n >10 AT-101 0.45 aThe value in the table is an average of three test results

As can be seen from the table, when the indazole ring has a larger substituent at position 1, the compound has better inhibitory activity against Mcl-i protein, such as compounds 7k, 71, 7m, 7p, 7q, and 7t. When the sulfonamide moiety is 4-chloro-3-nitrobenzenesulfonamide, the activity is the best, for example, 7f is significantly better than 7d and 7e, and 7i is significantly better than 7g and 7h. The activity of compound 7k is higher than that of the positive control AT-101, which has important guiding significance for further development of Mcl-1 inhibitors with higher activity. Experimental Example 2. Anti-proliferative activities assay of target compound (in vitro) Three compounds with better enzyme activity were selected from 20 target compounds for in vitro anti-proliferative activities assay. The results are shown in Table 3. Explanation of Terms: Acute myeloid leukemia cell line HL-60, prostate cancer cell line PC-3, breast cancer cell line MDA-MB-231, chronic myeloid leukemia cell line K562, and normal hepatocyte cell line L02. IC5o: median inhibitory concentration. Materials: HL-60, PC-3, MDA-MB-231, K562, L02, methyl thiazolyl tetrazolium (MTT), 10% fetal bovine serum (FBS), and 96-well plate. Method: Cell culture: The cell lines are subjected to routine culture. Logarithmic growth phase cells were used in the experiment. For cell growth assay (MTT method), cell suspensions were adjusted to 5 x 104 cells/mL (suspended cells adjusted to 105 cells/mL) and plated into a 96-well plate (100 [L/well), respectively, with 2,000-5,000 cells per well. At 4 h after plating, 100 L of medium containing different concentrations of compounds was added to each well, and three replicates were set for each concentration; cell-free wells were used as blanks, wells with cells but without compound were used as blank wells of a compound, and gossypol was used as a positive control. The plate was incubated for 48 h at 37°C and 5%CO2, 10 L of 0.5% MTT staining solution was added to each well, and the incubation was continued. After 4 h, the plate was centrifuged for 30 min at 2,500 rpm, the media in wells were discarded, 150 L of DMSO was added to each well, and the plate was shaken for 5-10 min at 37°C. The absorbance (OD value) of each well was measured on a microplate reader at 570 nm, and the cell growth inhibition rate was calculated as follows: Inhibition rate %)_Mean OD value of the control wells-Mean OD value of the experimental wells X I00% Mean OD value of the control wells

Table 3 The results of the anti-tumor cell proliferation assay of compounds 7k, 7m and 7q IC5 0 (pM)a No. HL-60 PC-3 MDA-MB-231 K562 L02 7k 33.2 12.3 10.6 6.62 >50 7m >50 13.6 13.3 7.85 >50 7q >50 14.5 12.3 7.06 >50 AT-101 16.3 7.71 6.27 6.02 25.3 aThe value in the table is an average of three test results, and the value after "±" represents the standard deviation Three compounds with better enzyme activity were subjected to the in vitro anti-proliferative activities assay against HL-60, PC-3, MDA-MB-231, K562 cancer cells and normal cells HUEVC. The assay data showed that all of the three target compounds assayed had inhibitory effects on PC-3, MDA-MB-231, and K562 tumor cells, especially K562 cells. Meanwhile, these three compounds had no effect on the growth of normal cells L02. Herein, the compounds had comparable inhibitory activity against PC-3, MDA-MB-231, and K562 tumor cells to the positive control AT-101. This indicates that the substituted 3-indazole Mcl-i inhibitor has a good development prospect and in-depth activity studies can be carried out to develop more active compounds for the preparation of medicaments for preventing and treating related mammalian diseases caused by aberrant expression of Mcl-1 protein. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in Australia or any other country. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application, or patent cited in this text is not repeated in this text is merely for reasons for conciseness. Throughout the specification and claims, unless the context requires otherwise, the word ''comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims (5)

1. What is claimed is: 1. A substituted 3-indazole Mcl-i inhibitor, wherein the substituted 3-indazole Mcl-i inhibitor is a compound having the structure of general formula (I) and a pharmaceutically acceptable salt thereof; 0 0// O R2 NH

   N N

   (I)

   wherein, Ri is selected from the group consisting of an aromatic group Ar or an aromatic group Ar attached to a morpholinyl or piperazinyl substituted or unsubstituted by 1-2 hydroxyl, halogen, or nitro groups; Ar is selected from phenyl, naphthyl or indyl; R2 is selected from the group consisting of an aromatic group Ar or an aromatic group Ar attached to a morpholinyl or piperazinyl substituted or unsubstituted by 1-2 hydroxyl, halogen, or nitro groups; Ar is selected from phenyl, naphthyl or indyl; and X is selected from carbonyl, sulfonyl, or -CH2-.
2. 2. The substituted 3-indazole Mel-1 inhibitor according to claim 1, wherein the substituted 3-indazole Mel-i inhibitor is one of the following compounds: 1-benzyl-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-benzyl-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-benzyl-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(4-bromophenyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(4-bromobenzyl)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(4-bromobenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(4-methylbenzyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(4-methylbenzyl)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(4-methylbenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(3,4-dichlorobenzyl)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(3,4-dichlorobenzyl)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(naphthyl-2-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-(naphthyl-2-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-([1,1'-biphenyl]-4-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxamide;

   1-([1,1'-biphenyl]-4-methylene)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carboxamide; 1-([1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3-carboxa mide; 1-(4'-methyl-[1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole 3-carboxamide; 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-nitrophenyl)sulfonyl-1H-indazole-3-carboxa mide; 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-chlorophenyl)sulfonyl-1H-indazole-3-carbox amide; and 1-(4'-chloro-[1,1'-biphenyl]-4-methylene)-N-(4-chloro-3-nitrophenyl)sulfonyl-1H-indazole-3 -carboxamide.
3. 3. A method for preparing the substituted 3-indazole Mcl-i inhibitor according to claim 1 or 2, comprising the following steps:

   OH 0 0 NH a b

   C N C~ N A- N -~N

   H H XR1 X'R 4 5 6a-6g 7a-7t

   step 1: with indazole-3-carboxylic acid as a starting material, esterfying carboxyl to produce an intermediate 5; step 2: conducting a nucleophilic substitution reaction on a nitrogen atom of an indazole ring in the intermediate 5 with differently substituted benzyl bromides, acyl chlorides, or sulfonyl chlorides to produce key intermediates 6a to 6g; and step 3: condensing the intermediates 6a to 6g with various substituted benzene sulfonamides to obtain target compounds 7a to 7t, namely, substituted 3-indazole Mcl-i inhibitors, in the presence of a condensing agent; wherein, definitions of Ri-R2 are the same as those described in the above general formula

   (I); wherein reagents and condition a are as follows: acetyl chloride, methanol, saturated sodium bicarbonate solution, and reflux; or, wherein reagents and condition b are as follows: differently substituted benzyl bromides, potassium carbonate, N,N-dimethylformamide, and room temperature; or, wherein reagents and condition c are as follows: i. 1 mol/L sodium hydroxide solution, tetrahydrofuran (THF), and room temperature; and ii. 2-(7-azabenzotriazol-1-yl)-NN,N,N'-tetramethyluronium hexafluorophosphate,

   N,N-diisopropylethylamine, dichloromethane, and room temperature.
4. 4. Use of the substituted 3-indazole Mcl-i inhibitor according to claim 1 or 2 in the preparation of medicaments for preventing or treating related mammalian diseases caused by aberrant expression of Mcl-i protein; wherein the related mammalian diseases caused by aberrant expression of Mcl-i protein comprise cancers, neurodegenerative diseases, viral infections, inflammation, leukemia, malaria, and diabetes.
5. 5. A pharmaceutical composition suitable for oral administration to mammals or a pharmaceutical composition suitable for parenteral administration to mammals, comprising the substituted 3-indazole Mcl-i inhibitor according to any one of claims 1 to 2 and one or more of pharmaceutically acceptable carriers or excipients.