Anti-prostatic cancer sulfonamide compound and application thereof

Abstract

The invention discloses an anti-prostatic cancer sulfonamide compound and application thereof, and belongs to the technical field of medicines. The structural formula of the sulfonamide compound is shown as a general formula (I), and the structure of the sulfonamide compound is different from that of traditional steroidal and non-steroidal androgen receptor antagonists. The compound has remarkable androgen receptor transcription inhibition activity, can be effectively combined with an androgen receptor ligand binding pocket in a competitive manner, blocks homologous dimerization of an androgen receptor, inhibits nuclear translocation of the androgen receptor, inhibits expression of downstream genes in an androgen receptor signal channel, and can be used for inhibiting the transcription of the androgen receptor. Further, the effects of antagonizing the transcriptional activity of an androgen receptor and inhibiting tumor cell proliferation are achieved; the compound can effectively inhibit the transcriptional activity of a mutant androgen receptor related to the clinical drug resistance of enzalutamide and the proliferation activity of drug-resistant cells, and shows better safety. The invention provides a new choice for prostatic cancer treatment and drug development.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a sulfonamide compound and its pharmaceutical applications, especially its application in the preparation of drugs for treating prostate cancer. Background Art

[0002] Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate gland and is the disease with the highest incidence rate and the second highest mortality rate among male malignant tumors globally. In the early stage, prostate cancer often has no symptoms. Approximately 70% of prostate cancer patients are in the middle and late stages at the time of initial diagnosis, and the five-year survival rate is only 53.8%.

[0003] The progression of prostate cancer is closely related to the persistent activation of the androgen receptor (AR) signaling pathway. The androgen receptor (AR) is a type of ligand-gated transcription factor and is also a member of the steroid hormone receptor superfamily. It is widely distributed in most organs and tissues in the human body and mediates the functions of androgens in promoting the development of male reproductive organs, stimulating the growth of bones and muscles, etc. AR contains four independent domains with different functions: the amino-terminal domain (NTD), the DNA-binding domain (DBD), the ligand-binding domain (LBD), and a flexible hinge region that connects DBD and LBD. Among them, LBD is composed of 11 α-helices (helix 1, helix 3 to helix 12) and two anti-parallel β-sheets, and contains a ligand-binding pocket (LBP), a co-activator binding site activation function region 2 (AF2), and a binding function region 3 (BF3). Among them, the LBP pocket formed by the amino termini of helix 3, helix 5, and helix 11 is the binding site for endogenous androgens (Sack J S, et al., 2001).

[0004] Currently, the main strategy for the development of prostate cancer treatment drugs is to target the AR protein and prevent the activation of the AR signaling pathway, thereby blocking a series of downstream biological responses that promote the development of the disease. Among them, as the first-line drug for prostate cancer treatment, the main principle of action of AR antagonists is to competitively bind to the AR ligand-binding pocket with endogenous androgens, prevent the activation of the AR signaling pathway and the transcription of downstream target genes, so that the proteins required for the growth of tumor cells cannot be expressed, thereby achieving the purpose of tumor regression. Unfortunately, however, both the first-generation and second-generation AR antagonists are very prone to acquired drug resistance. Point mutations within the LBP pocket, such as W742C, F877L, H875Y, and T878A, will reduce the requirement of the LBP pocket for ligand-binding specificity, resulting in the activation of AR by other steroid hormones or even antagonists (Penson D F, et al., 2016; Tan J, et al., 1997; Borgmann H, et al., 2018; Hara T, et al., 2003.).

[0005] Therefore, the development of AR antagonists with new structures to overcome the drug resistance of commercially available AR antagonists is currently a hot topic and a difficult point in the research and development of prostate cancer drugs at home and abroad. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of novel androgen receptor antagonists based on the sulfonamide nucleus and apply them to the preparation of drugs for treating androgen receptor-mediated diseases such as prostate cancer.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a sulfonamide compound, and the structural general formula of the sulfonamide compound is shown as formula (I),

[0009]

[0010] wherein, R1 is selected from hydrogen, fluorine, chlorine, bromine, cyano, nitro, methyl, methoxy, methyl ester group, trifluoromethyl, trifluoromethoxy, difluoromethoxy;

[0011] X is selected from O or N-R a , wherein R a is selected from hydrogen, methyl, ethyl;

[0012] R2 is selected from methyl, methylamino,

[0013] Preferably, the structural formula of the sulfonamide compound is shown as any one of formula (1) to (27), corresponding to the following compounds A1 to A6, B1 to B6, C1 to C3, D1 to D12,

[0014]

[0015]

[0016] The present invention also provides a pharmaceutically acceptable salt form of the sulfonamide compound, or its solvate, or its stereoisomer, or its prodrug molecule, or its deuterated compound.

[0017] The pharmaceutically acceptable salt can be, but is not limited to, organic acid salts, inorganic acid salts, organic base salts or inorganic base salts. The organic acid salts can be, but are not limited to, oxalate, lactate, p-toluenesulfonate, malate, citrate, fumarate, camphorsulfonate or methanesulfonate; the inorganic acid salts can be, but are not limited to, nitrate, sulfate, hydrohalide or phosphate; the organic base salts can be, but are not limited to, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine or tert-butylamine; the inorganic base salts can be, but are not limited to, sodium hydroxide, potassium hydroxide or lithium hydroxide.

[0018] The solvate refers to a variable-stoichiometry complex formed by a solute (the compound of formula (I) provided by the present invention) and a solvent. The solvent includes but is not limited to pharmaceutically acceptable solvents such as water, methanol, ethanol, and acetic acid.

[0019] The present invention has found through research that the sulfonamide compounds have significant antagonistic activity against androgen receptors and exhibit good biological activity in in vitro and in vivo biological evaluations. Therefore, such compounds can be applied to the treatment of tumors related to abnormal expression or mutation of androgen receptors.

[0020] Specifically, the present invention provides the use of the sulfonamide compound or its pharmaceutically acceptable salt or its solvate or its stereoisomer or its prodrug molecule or its deuterated compound in the preparation of an androgen receptor antagonist.

[0021] Furthermore, the androgen receptor antagonist is a competitive inhibitor of the androgen receptor ligand pocket, an androgen receptor nuclear translocation inhibitor, or an androgen receptor dimerization inhibitor. Mechanistic studies have shown that the sulfonamide compounds can play a role in antagonizing the transcriptional activity of androgen receptors by competitively binding to the androgen receptor ligand-binding pocket, blocking the homodimerization of androgen receptors, and inhibiting the entry of androgen receptors into the nucleus.

[0022] The present invention also provides the use of the sulfonamide compound or its pharmaceutically acceptable salt or its solvate or its stereoisomer or its prodrug molecule or its deuterated compound in the preparation of a drug for the treatment of prostate cancer, breast cancer, or ovarian cancer. The occurrence or progression of the prostate cancer, breast cancer, or ovarian cancer is related to the high expression of androgen receptors or the persistent activation of the androgen receptor signaling pathway.

[0023] The prostate cancer includes but is not limited to metastatic prostate cancer and castration-resistant prostate cancer. It should be noted that the pharmaceutical uses of the compounds are not limited to the preparation of drugs for the above-mentioned tumors.

[0024] Furthermore, the prostate cancer is enzalutamide-resistant prostate cancer. Research has shown that the sulfonamide compounds can significantly inhibit the proliferation of enzalutamide-resistant prostate cancer cells. The present invention provides a potential treatment strategy for overcoming clinically generated enzalutamide resistance.

[0025] Furthermore, the prostate cancer is prostate cancer with a point mutation in the ligand-binding pocket of the androgen receptor, and the point mutation is F877L, F877L / T878A, W742C, T878G, or H875Y / T878A. The point mutation is a mutation in the androgen receptor ligand-binding pocket caused by the long-term use of first-generation and second-generation AR antagonists. Research has shown that the sulfonamide compounds have antagonistic activity against the above mutant androgen receptors.

[0026] The present invention also provides a pharmaceutical composition, comprising an effective dose of the sulfonamide compound described above, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug molecule thereof, or a deuterated compound thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition is used for treating diseases related to abnormal expression or mutation of androgen receptor.

[0027] The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, including conventional diluents in the pharmaceutical field, excipients such as water, fillers such as starch, binders such as cellulose derivatives, gelatin, etc., wetting agents such as glycerol, disintegrants such as agar, calcium carbonate, etc., adsorbent carriers such as kaolin and saponite clay, surfactants such as cetyl alcohol, absorption promoters such as quaternary ammonium compounds, lubricants such as talc powder, etc. Flavoring agents, sweetening agents, etc. can be added when necessary.

[0028] The pharmaceutical preparation is suitable for administration by any appropriate route, such as nasal spray, oral spray (inhalant), oral administration (including buccal or sublingual administration), rectal administration, topical administration (including buccal, sublingual or transdermal administration), parenteral administration (including subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection) route. The preparation of the present invention can be prepared by any method known in pharmacy. For example, by mixing the active ingredient with a carrier or excipient.

[0029] Specifically, the dosage form of the pharmaceutical composition can be, but is not limited to, capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

[0030] The compound or its pharmaceutically acceptable salt provided by the present invention can be used in combination with existing anti-tumor drugs, and the anti-tumor drugs can be, but are not limited to: mitosis inhibitors (such as vinblastine); tubulin depolymerization inhibitors (such as taxol); alkylating agents (such as cisplatin); antimetabolites (such as 5-fluorouracil); intercalating antibiotics (such as actinomycin); enzymes (such as asparaginase); topoisomerase inhibitors (such as etoposide and camptothecin); biological response modifiers (such as interferon); proteasome inhibitors (such as bortezomib).

[0031] The beneficial effects of the present invention are as follows:

[0032] The present invention provides a novel class of androgen receptor antagonists based on the sulfonamide nucleus, whose structures are different from those of traditional steroid and non-steroid antagonists. This class of compounds has significant androgen receptor transcriptional inhibitory activity, can effectively compete for binding to the ligand-binding pocket of the androgen receptor, block the homodimerization of the androgen receptor, inhibit the nuclear translocation of the androgen receptor, and inhibit the expression of downstream genes in the androgen receptor signaling pathway, thereby playing a role in antagonizing the transcriptional activity of the androgen receptor and inhibiting the proliferation of tumor cells. In addition, the compounds provided by the present invention can effectively inhibit the transcriptional activity of mutant androgen receptors related to the clinical resistance of the second-generation androgen receptor antagonist enzalutamide and the proliferation activity of resistant cells, and show good safety. Therefore, this class of compounds can be used as androgen receptor antagonists in the treatment of diseases related to the androgen receptor. The present invention provides new options for the treatment and drug development of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the synthetic routes of Compounds A1 - A5, B1 - B6 and D1 - D12.

[0034] Figure 2 It is the synthetic routes of Compounds C1 - C3.

[0035] Figure 3 It is the transcriptional inhibitory activities of enzalutamide, bicalutamide, darolutamide, and Compound B5 against AR mutants related to clinical resistance.

[0036] Figure 4 It is the effects of Compound B5 on prostate cancer tumor markers.

[0037] Figure 5 It is the effects of Compound B5 on the expression of downstream genes regulated by AR at the mRNA level.

[0038] Figure 6 It is the effects of Compound B5 on the expression of downstream genes regulated by AR at the protein level.

[0039] Figure 7 It is the proliferation inhibition activity curve of Compound B5 against enzalutamide-resistant cells.

[0040] Figure 8 It is the competitive activity of Compound B5 against the ligand-binding pocket of AR at a series of concentration gradients.

[0041] Figure 9 It is the effects of Compound B5 on the nuclear localization of AR at a concentration of 10 μM.

[0042] Figure 10 It is the effects of Compound B5 on the homodimerization of AR at concentration gradients of 10 μM, 1 μM, and 0.1 μM.

[0043] Figure 11 In vivo anti-tumor activity results of compound B5 in a nude mouse model transplanted with LNCaP cells, where A is the tumor volume; B is the tumor weight; C is the body weight of nude mice; D is the plasma PSA level.

[0044] Figure 12 In vivo anti-tumor activity results of compound B5 in a nude mouse model transplanted with enzalutamide-resistant LNCaP cells, where A is the tumor volume; B is the body weight of nude mice. Specific implementation manners

[0045] The present invention will be further described below in conjunction with specific embodiments. The specific embodiments are for illustrative purposes only and should not be construed as limiting the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0046] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0047] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0048] Example 1: Synthesis of target molecules A1 to A6

[0049] The synthetic routes of compounds A1 to A6 are as Figure 1 shown.

[0050] 1. Preparation of N′-[4-[(4-chlorophenyl)methoxy]phenyl]-N,N-dimethylsulfonamide (compound A1)

[0051] (a) Synthesis of intermediate 1-chloro-4-(4-nitrophenoxymethyl)benzene (compound 2a)

[0052] Dissolve 4-nitrophenol (10 mmol, 1.0 equivalent), 4-chlorobenzyl chloride (1a) (10 mmol, 1.0 equivalent) and potassium carbonate (K2CO3, 20 mmol, 2.0 equivalents) in N,N-dimethylformamide (DMF, 20 mL), and heat and stir at 80 °C for 12 hours. After the reaction is completed, add an appropriate amount of water (10 mL) to quench the reaction system, and extract three times with ethyl acetate (15 mL). Combine the organic phases, wash them successively with water and saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product 2a.

[0053] (b) Synthesis of intermediate 4-(4-chlorobenzyloxy)aniline (compound 3a)

[0054] The product 2a (5 mmol, 1.0 equiv), reduced iron powder (15 mmol, 3.0 equiv), and ammonium chloride (NH4Cl, 45 mmol, 9.0 equiv) obtained in the previous step were dissolved in 70% ethanol solution (20 mL), heated to reflux and maintained for 2 hours. After the reaction was completed, it was quickly filtered through a diatomaceous earth pad while it was hot to remove the iron powder and insoluble impurities. The ethanol was removed by concentration under reduced pressure. Water was added to the residue, and it was extracted three times with ethyl acetate. The organic phases were combined and washed successively with water and saturated sodium chloride. After the organic phase was dried over anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain the crude product, and the target compound 3a was obtained with ethyl acetate / petroleum ether (v / v, 1:5).

[0055] (c) N′-[4-[(4-chlorophenyl)methoxy]phenyl]-N,N-dimethylsulfamide (A1)

[0056] Compound 3a (1 mmol, 1.0 equiv), N,N-diisopropylethylamine (DIPEA, 5 mmol, 5.0 equiv), and 4-dimethylaminopyridine (DMAP, 0.1 mmol, 0.1 equiv) were dissolved in anhydrous tetrahydrofuran (THF, 3 mL). An appropriate amount of dimethylaminosulfonyl chloride (4a) (2 mmol, 2.0 equiv) was slowly added at room temperature. Subsequently, the reaction mixture was stirred and heated to reflux. After 8 hours of reaction, an appropriate amount of water (10 mL) was added to the reaction system to quench the reaction, and it was extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed successively with water and saturated sodium chloride. After drying over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure. The crude product was separated by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:10) as the eluent to obtain a pink solid A1 with a yield of 70% and a purity of 98.86%.

[0057] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, DMSO-d6): δ 9.60 (s, 1H), 7.45 (s, 4H), 7.17–7.09 (m, 2H), 6.99–6.91 (m, 2H), 5.05 (s, 2H), 2.65 (s, 6H). 13 C NMR (126 MHz, DMSO-d6): δ 155.30, 136.64, 132.89, 132.07, 130.04, 128.93, 122.79, 115.73, 69.05, 38.28. HRMS (ESI) m / z calcd for C 15 H 17 ClN2NaO3S [M+Na] + : 363.0541; found, 363.0539. The structural formula of compound A1 is shown in formula (1).

[0058] 2. Preparation of N′-[4-[(4-bromophenyl)methoxy]phenyl]-N,N-dimethylsulfonamide (Compound A2)

[0059] Referring to the synthesis of Compound 2a, replacing 4-chlorobenzyl chloride with 4-bromobenzyl chloride (1b), Compound 1-bromo-4-(4-nitrophenoxymethyl)benzene (2b) was obtained; referring to the synthesis of Compound 3a, replacing 2a with 2b, Compound 4-(4-bromobenzyloxy)aniline (3b) was obtained; referring to the synthesis of Compound A1, replacing 3a with 3b, brown solid A2 was obtained with a yield of 65% and a purity of 97.76%.

[0060] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR(500MHz,DMSO-d6):δ9.62(s,1H),7.61(d,J=8.0Hz,2H),7.42(d,J=8.0Hz,2H),7.16(d,J=8.0Hz,2H),6.97(d,J=8.0Hz,2H),5.06(s,2H),2.67(s,6H). 13 C NMR(126MHz,DMSO-d6):δ155.28,137.07,132.08,131.86,130.34,122.78,121.43,115.73,69.07,38.28.HRMS(ESI)m / z calcd for C 15 H 17 BrN2NaO3S[M+Na] + :407.0035; found,407.0031. The structural formula of Compound A2 is shown in Formula (2).

[0061] 3. Preparation of N′-[4-[(4-nitrophenyl)methoxy]phenyl]-N,N-dimethylsulfonamide (Compound A3)

[0062] Referring to the synthesis of Compound 2a, replacing 4-chlorobenzyl chloride with 4-nitrobenzyl chloride (1c), Compound 1-nitro-4-(4-nitrophenoxymethyl)benzene (2c) was obtained; referring to the synthesis of Compound 3a, replacing 2a with 2c, Compound 4-(4-nitrobenzyloxy)aniline (3c) was obtained; referring to the synthesis of Compound A1, replacing 3a with 3c, brown solid A3 was obtained with a yield of 68% and a purity of 99.56%.

[0063] The nuclear magnetic resonance and mass spectrometry data are as follows: 11H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.26 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 8.5 Hz, 2H), 7.18–7.11 (m, 2H), 7.02–6.94 (m, 2H), 5.23 (s, 2H), 2.65 (s, 6H). 13 13C NMR (126 MHz, DMSO-d6): δ 155.03, 147.49, 145.57, 132.31, 128.76, 124.12, 122.75, 115.78, 68.72, 38.27. HRMS (ESI) m / z calcd for C 15 H 17 N3NaO5S [M+Na] + : 374.0781; found, 374.0783. The structural formula of compound A3 is shown in formula (3).

[0064] 4. Preparation of N′-[4-[(4-methylphenyl)methoxy]phenyl]-N,N-dimethylsulfonamide (Compound A4)

[0065] Referring to the synthesis of compound 2a, 4-chlorobenzyl chloride was replaced with 4-methylbenzyl chloride (1d) to obtain compound 1-methyl-4-(4-nitrophenoxymethyl)benzene (2d); referring to the synthesis of compound 3a, 2a was replaced with 2d to obtain compound 4-(4-methylbenzyloxy)aniline (3d); referring to the synthesis of compound A1, 3a was replaced with 3d to obtain a brown solid A4 with a yield of 70% and a purity of 95.37%.

[0066] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 1H NMR (500 MHz, DMSO-d6): δ 9.57 (s, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.21–7.07 (m, 4H), 6.97–6.90 (m, 2H), 4.99 (s, 2H), 2.64 (s, 6H), 2.29 (s, 3H). 13 13C NMR (126 MHz, DMSO-d6): δ 155.55, 137.57, 134.52, 131.85, 129.47, 128.35, 122.84, 115.69, 69.78, 38.28, 21.29. HRMS (ESI) m / z calcd for C 16 H 20 N2NaO3S [M+Na] + : 343.1087; found, 343.1088. The structural formula of compound A4 is shown in formula (4).

[0067] 5. Preparation of N′-[4-[(4-methoxyphenyl)methoxy]phenyl]-N,N-dimethylsulfonamide (Compound A5)

[0068] Referring to the synthesis of Compound 2a, replacing 4-chlorobenzyl chloride with 4-methoxybenzyl chloride (1e) gave Compound 1-methoxy-4-(4-nitrophenoxymethyl)benzene (2e); referring to the synthesis of Compound 3a, replacing 2a with 2e gave Compound 4-(4-methoxybenzyloxy)aniline (3e); referring to the synthesis of Compound A1, replacing 3a with 3e gave yellow solid A5 in a yield of 65% and a purity of 95.93%.

[0069] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, DMSO-d6): δ 9.57 (s, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 6.97–6.89 (m, 4H), 4.95 (s, 2H), 3.75 (s, 3H), 2.65 (s, 6H). 13 C NMR (126 MHz, DMSO-d6): δ 159.48, 155.59, 131.81, 130.05, 129.40, 122.84, 115.68, 114.29, 69.65, 55.59, 38.28. HRMS (ESI) m / z calcd for C 16 H 20 N2NaO4S [M+Na] + : 359.1036; found, 359.1037. The structural formula of Compound A5 is shown in Formula (5).

[0070] 6. Preparation of 4-((4-((N,N-dimethylsulfamoyl)amino)phenoxy)methyl)phenyl acetate (Compound A6)

[0071] Referring to the synthesis of Compound 2a, replacing 4-chlorobenzyl chloride with methyl 4-chloromethylbenzoate (1f) gave methyl 4-((4-nitrophenoxy)methyl)benzoate (2f); referring to the synthesis of Compound 3a, replacing 2a with 2f gave methyl 4-((4-aminophenoxy)methyl)benzoate (3f); referring to the synthesis of Compound A1, replacing 3a with 3f gave yellow solid A6 in a yield of 58% and a purity of 97.14%.

[0072] The nuclear magnetic resonance and mass spectrometry data are as follows: 11H NMR (500 MHz, DMSO-d6): δ 9.61 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 5.16 (s, 2H), 3.85 (s, 3H), 2.65 (s, 6H). 13 13C NMR (101 MHz, DMSO-d6): δ 166.52, 155.24, 143.18, 132.16, 129.83, 129.45, 128.03, 122.77, 115.74, 69.23, 52.67, 38.27. HRMS (ESI) m / z calcd for C 17 H 20 N2NaO5S [M+Na] + : 387.0985; found, 387.0990. The structural formula of compound A6 is shown in formula (6).

[0073] Example 2: Synthesis of target molecules B1 - B6

[0074] The synthetic routes of compounds B1 - B6 are as Figure 1 shown.

[0075] 1. Preparation of N-(4-benzyloxyphenyl)-4-hydroxybenzenesulfonamide (Compound B1)

[0076] (a) Synthesis of intermediate 1-benzyloxy-4-nitrobenzene (2 g)

[0077] Dissolve 4-nitrophenol (10 mmol, 1.0 equiv), benzyl chloride (1 g) (10 mmol, 1.0 equiv), and potassium carbonate (K2CO3, 20 mmol, 2.0 equiv) in N,N-dimethylformamide (DMF, 20 mL), and heat and stir at 80 °C for 12 h. After the reaction is completed, add an appropriate amount of water (10 mL) to quench the reaction system, and extract three times with ethyl acetate (15 mL). Combine the organic phases, wash successively with water and saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product 1-benzyloxy-4-nitrobenzene (2 g).

[0078] (b) Synthesis of intermediate 4-benzyloxyaniline (3 g)

[0079] The product obtained in the previous step, 1-benzyloxy-4-nitrobenzene (2 g) (5 mmol, 1.0 equiv), reduced iron powder (15 mmol, 3.0 equiv), and ammonium chloride (NH4Cl, 45 mmol, 9.0 equiv) were dissolved in 70% ethanol solution (20 mL), heated to reflux and maintained for 2 hours. After the reaction was completed, it was quickly filtered through a diatomaceous earth pad while it was hot to remove the iron powder and insoluble impurities. The ethanol was removed by concentration under reduced pressure. Water was added to the residue, and it was extracted three times with ethyl acetate. The organic phases were combined and washed successively with water and saturated sodium chloride. After the organic phase was dried over anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain the crude product, and the target compound 4-benzyloxyaniline (3 g) was obtained through ethyl acetate / petroleum ether (v / v, 1:5).

[0080] (c) N-(4-benzyloxyphenyl)-4-hydroxybenzenesulfonamide (B1)

[0081] 4-Benzyloxyaniline (3 g) (1 mmol, 1.0 equiv), N,N-diisopropylethylamine (DIPEA, 5 mmol, 5.0 equiv), and 4-dimethylaminopyridine (DMAP, 0.1 mmol, 0.1 equiv) were dissolved in anhydrous tetrahydrofuran (THF, 3 mL), and an appropriate amount of 4-hydroxybenzenesulfonyl chloride (4b) (2 mmol, 2.0 equiv) was slowly added at room temperature. Subsequently, the reaction mixture was stirred and heated to reflux, and after reacting for 8 hours, an appropriate amount of water (10 mL) was added to the reaction system to quench it, and it was extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed successively with water and saturated sodium chloride. After drying over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure. The crude product was separated by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:10) as the eluent to obtain a brown solid B1 with a yield of 55% and a purity of 96.25%.

[0082] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, DMSO-d6): δ 7.52–7.26 (m, 7H), 6.98–6.79 (m, 6H), 4.99 (s, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 161.63, 155.89, 137.46, 131.42, 131.29, 129.82, 129.55, 128.90, 128.35, 128.28, 123.58, 115.95, 115.60, 69.83. HRMS (ESI) m / z calcd for C 19 H 17 NNaO4S [M+Na] + : 378.0770; found, 378.0775. The structural formula of compound B1 is shown in formula (7).

[0083] 2. Preparation of 4-acetyl-N-(4-benzyloxyphenyl)benzenesulfonamide (Compound B2)

[0084] Referring to the synthesis of Compound B1, 4-hydroxybenzenesulfonyl chloride (4b) was replaced with 4-acetylbenzenesulfonyl chloride (4c) to obtain yellow solid B2 with a yield of 70% and a purity of 99.07%.

[0085] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, DMSO-d6): δ 10.14 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.50–7.26 (m, 5H), 7.05–6.84 (m, 4H), 4.99 (s, 2H), 2.59 (s, 3H). 13 C NMR (126 MHz, DMSO-d6): δ 197.78, 156.30, 143.61, 140.08, 137.37, 130.37, 129.45, 128.91, 128.37, 128.27, 127.57, 124.02, 115.78, 69.84, 27.52. HRMS (ESI) m / z calcd for C 21 H 19 NNaO4S [M+Na] + : 404.0932; found, 404.0938. The structural formula of Compound B2 is shown in Formula (8).

[0086] 3. Preparation of 4-(methoxyphenyl)-N-(methylsulfonamido)aniline (Compound B3)

[0087] Referring to the synthesis of Compound B1, 4-hydroxybenzenesulfonyl chloride (4b) was replaced with methylsulfonamido chloride (4d) to obtain light brown solid B3 with a yield of 65% and a purity of 95.07%.

[0088] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, DMSO-d6): δ 9.35 (s, 1H), 7.47–7.29 (m, 5H), 7.10 (t, J = 8.0 Hz, 3H), 6.98–6.92 (m, 2H), 5.05 (s, 2H), 2.45 (d, J = 5.0 Hz, 3H). 1313C NMR (126 MHz, DMSO-d6): δ 155.04, 137.69, 132.40, 128.93, 128.31, 128.19, 121.84, 115.67, 69.89, 28.77. HRMS (ESI) m / z calcd for C 14 H 16 N2NaO3S [M+Na] + : 315.0774; found, 315.0778. The structural formula of Compound B3 is shown in Formula (9).

[0089] 4. Preparation of N-(4-Benzyloxyphenyl)morpholine-4-sulfonamide (Compound B4)

[0090] Referring to the synthesis of Compound B1, 4-Hydroxybenzenesulfonyl chloride (4b) was replaced with Morpholine-4-sulfonyl chloride (4e) to obtain yellow solid B4 with a yield of 55% and a purity of 98.49%.

[0091] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 1H NMR (500 MHz, Chloroform-d): δ 7.50–7.32 (m, 5H), 7.23–7.14 (m, 2H), 7.00–6.91 (m, 2H), 6.19 (s, 1H), 5.07 (s, 2H), 3.71–3.63 (m, 4H), 3.26–3.20 (m, 4H). 13 13C NMR (126 MHz, Chloroform-d): δ 156.95, 136.63, 129.54, 128.65, 128.13, 127.51, 124.59, 115.63, 70.36, 66.24, 46.47. HRMS (ESI) m / z calcd for C 17 H 20 N2NaO4S [M+Na] + : 371.1036; found, 371.1044. The structural formula of Compound B4 is shown in Formula (10).

[0092] 5. Preparation of N-(4-Benzyloxyphenyl)piperidine-1-sulfonamide (Compound B5)

[0093] Referring to the synthesis of Compound B1, 4-Hydroxybenzenesulfonyl chloride (4b) was replaced with Piperidine-1-sulfonyl chloride (4f) to obtain yellow solid B5 with a yield of 70% and a purity of 99.73%.

[0094] The nuclear magnetic resonance and mass spectrometry data are as follows: 11H NMR (500 MHz, Chloroform-d): δ 7.44–7.36 (m, 4H), 7.35–7.31 (m, 1H), 7.18–7.12 (m, 2H), 6.95–6.90 (m, 2H), 6.26 (s, 1H), 5.04 (s, 2H), 3.24–3.14 (m, 4H), 1.57–1.51 (m, 4H), 1.48 (m, 2H). 13 13C NMR (126 MHz, Chloroform-d): δ 156.52, 136.76, 130.21, 128.62, 128.08, 127.51, 123.94, 115.51, 70.35, 47.23, 25.31, 23.65. HRMS (ESI) m / z calcd for C 18 H 22 N2NaO3S [M+Na] + : 369.1243; found, 369.1240. The structural formula of compound B5 is shown in formula (11).

[0095] 6. Preparation of N-(4-Benzyloxyphenyl)-4-methylpiperidine-1-sulfonamide (Compound B6)

[0096] Referring to the synthesis of compound B1, 4-Hydroxybenzenesulfonyl chloride (4b) was replaced with 4-Methylpiperidine-1-sulfonyl chloride (4g) to obtain yellow solid B6 with a yield of 50% and a purity of 99.12%.

[0097] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 1H NMR (500 MHz, Chloroform-d): δ 7.46–7.28 (m, 5H), 7.19–7.10 (m, 2H), 6.97–6.89 (m, 2H), 6.11 (s, 1H), 5.04 (s, 2H), 3.71 (d, J = 12.0 Hz, 2H), 2.70 (td, J = 12.0, 2.5 Hz, 2H), 1.62 (d, J = 13.0 Hz, 2H), 1.44–1.36 (m, 1H), 1.19–1.10 (m, 2H), 0.90 (d, J = 6.5 Hz, 3H). 13 13C NMR (126 MHz, Chloroform-d): δ 156.53, 136.76, 130.20, 128.63, 128.08, 127.50, 123.95, 115.51, 70.34, 46.69, 33.48, 30.25, 21.52. HRMS (ESI) m / z calcd for C 19 H 24N2NaO3S[M+Na] + : 383.1400; found, 383.1405. The structural formula of Compound B6 is shown in Formula (12).

[0098] Example 3: Synthesis of Target Molecules C1 - C3

[0099] The synthetic routes of Compounds C1 - C3 are as Figure 2 shown.

[0100] 1. Preparation of N-(4-benzylaminophenyl)piperidine-1-sulfonamide (Compound C1)

[0101] (a) Preparation of Intermediate N-(4-nitrophenyl)benzylamine (Compound 5a)

[0102] Dissolve 4-nitroaniline (10 mmol, 1.0 equivalent), benzyl chloride (1 g) (10 mmol, 1.0 equivalent), and potassium carbonate (K2CO3, 20 mmol, 2.0 equivalents) in N,N-dimethylformamide (DMF, 20 mL), and heat and stir at 80 °C for 12 hours. After the reaction is completed, add an appropriate amount of water (10 mL) to quench the reaction system, and extract three times with ethyl acetate (15 mL). Combine the organic phases, wash them successively with water and saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product N-(4-nitrophenyl)benzylamine (5a).

[0103] (b) Preparation of N-(4-benzylaminophenyl)piperidine-1-sulfonamide (Compound 6a)

[0104] Dissolve the product N-(4-nitrophenyl)benzylamine (5a) (5 mmol, 1.0 equivalent), reduced iron powder (15 mmol, 3.0 equivalents), and ammonium chloride (NH4Cl, 45 mmol, 9.0 equivalents) obtained in the previous step in 70% ethanol solution (20 mL), heat to reflux and maintain for 2 hours. After the reaction is completed, quickly filter through a diatomaceous earth pad while it is hot to remove the iron powder and insoluble impurities, concentrate under reduced pressure to remove ethanol, add water to the residue, and extract three times with ethyl acetate. Combine the organic phases, wash them successively with water and saturated sodium chloride. After drying the organic phase over anhydrous sodium sulfate, concentrate under reduced pressure to obtain a crude product, and obtain the target compound N-benzyl-1,4-phenylenediamine (6a) through ethyl acetate / petroleum ether (v / v, 1:5).

[0105] (c) N-(4-benzylaminophenyl)piperidine-1-sulfonamide (Compound C1)

[0106] Dissolve compound N-benzyl-1,4-phenylenediamine (6a) (1 mmol, 1.0 equiv), N,N-diisopropylethylamine (DIPEA, 5 mmol, 5.0 equiv), and 4-dimethylaminopyridine (DMAP, 0.1 mmol, 0.1 equiv) in anhydrous tetrahydrofuran (THF, 3 mL), and slowly add an appropriate amount of piperidine-1-sulfonyl chloride (4e) (2 mmol, 2.0 equiv) at room temperature. Subsequently, stir the reaction mixture and heat it to reflux. After reacting for 8 hours, add an appropriate amount of water (10 mL) to quench the reaction, and extract it three times with ethyl acetate (15 mL). Combine the organic phases, wash them successively with water and saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent. The crude product is separated by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:10) as the eluent to obtain a yellow solid C1 with a yield of 68% and a purity of 96.75%.

[0107] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, Chloroform-d): δ 7.38–7.32 (m, 4H), 7.31–7.27 (m, 1H), 7.13–7.00 (m, 2H), 6.63–6.51 (m, 2H), 5.94 (s, 1H), 4.31 (s, 2H), 4.06 (s, 1H), 3.24–3.11 (m, 4H), 1.54–1.52 (m, 4H), 1.51–1.45 (m, 2H). 13 C NMR (126 MHz, Chloroform-d): δ 138.87, 128.70, 127.58, 127.42, 125.41, 113.40, 77.23, 48.62, 47.25, 25.36, 23.70. HRMS (ESI) m / z calcd for C 18 H 23 N3NaO2S [M + H] + : 346.1584; found, 346.1585. The structural formula of compound C1 is shown in formula (13).

[0108] 2. Preparation of N-(4-(benzyl(methyl)amino)phenyl)piperidine-1-sulfonamide (Compound C2)

[0109] (a) Preparation of intermediate N-methyl-N-(4-nitrophenyl)benzylamine (Compound 5b)

[0110] Under the condition of an ice bath at 0 °C, compound N-benzyl-4-nitrobenzene (1 mmol, 1.0 equivalent) was slowly added to a DMF mixture of sodium hydride (NaH, 1.2 mmol, 1.2 equivalents) and methyl iodide (1.1 mmol, 1.1 equivalents). Subsequently, the reaction system was gradually warmed to room temperature and stirred for 1 hour. After the reaction was completed, an appropriate amount of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed successively with deionized water and saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product.

[0111] (b) Referring to the synthesis of compound 6a, 5a was replaced with 5b to obtain compound 6b. Referring to the synthesis of compound C1, compound 6a was replaced with 6b to obtain compound C2. The yield was 59%, and the purity was 98.52%.

[0112] The nuclear magnetic resonance and mass spectrometry data were as follows: 1 H NMR (500 MHz, Chloroform-d): δ 7.33–7.31 (2H), 7.24 (s, 1H), 7.20 (d, J = 7.5 Hz, 2H), 7.15–6.99 (m, 2H), 6.67 (d, J = 9.0 Hz, 2H), 5.93 (s, 1H), 4.52 (s, 2H), 3.19 (t, J = 5.5 Hz, 4H), 3.01 (s, 3H), 1.56–1.54 (m, 4H), 1.49–1.48 (m, 2H). 13 C NMR (126 MHz, Chloroform-d): δ 148.15, 138.66, 128.64, 127.03, 126.76, 125.84, 125.42, 112.77, 56.83, 47.28, 38.79, 25.41, 23.76. HRMS (ESI) m / z calcd for C 19 H 25 N3NaO2S [M+Na] + : 360.1740; found, 360.1748. The structural formula of compound C2 is shown in formula (14).

[0113] 3. Preparation of N-(4-(benzyl(ethyl)amino)phenyl)piperidine-1-sulfonamide (Compound C3)

[0114] Referring to the synthesis of compound 5b, methyl iodide was replaced with ethyl iodide to obtain compound 5c; referring to the synthesis of compound 6a, 5a was replaced with 5c to obtain compound 6c; referring to the synthesis of compound C1, compound 6a was replaced with 6c to obtain the yellow solid compound C3. The yield was 53%, and the purity was 96.71%.

[0115] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, Chloroform-d): δ 7.35 (t, J = 7.5 Hz, 2H), 7.29–7.23 (m, 3H), 7.12–7.07 (m, 2H), 6.72–6.59 (m, 2H), 5.95 (s, 1H), 4.54 (s, 2H), 3.50 (q, J = 7.0 Hz, 2H), 3.23 (t, J = 5.5 Hz, 4H), 1.62–1.57 (m, 4H), 1.55–1.52 (m, 2H), 1.24 (t, J = 7.0 Hz, 3H). 13 C NMR (126 MHz, Chloroform-d): δ 128.63, 126.91, 126.56, 125.71, 112.57, 54.12, 47.26, 45.47, 25.41, 23.77, 12.15. HRMS (ESI) m / z calcd for C 20 H 27 N3NaO2S [M+Na] + : 374.1897; found, 374.1903. The structural formula of Compound C3 is shown in Formula (15).

[0116] Example 4: Synthesis of Target Molecules D1 - D12

[0117] The synthetic routes of Compounds D1 - D12 are as Figure 1 shown.

[0118] 1. Preparation of N-(4-((4-Fluorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D1)

[0119] (a) Synthesis of Intermediate 1-Fluoro-4-((4-nitrophenoxy)methyl)benzene (Compound 2h)

[0120] Dissolve 4-nitrophenol (10 mmol, 1.0 equivalent), 1-chloromethyl-4-fluorobenzene (1h) (10 mmol, 1.0 equivalent), and potassium carbonate (K2CO3, 20 mmol, 2.0 equivalents) in N,N-dimethylformamide (DMF, 20 mL), and heat and stir at 80 °C for 12 hours. After the reaction is completed, add an appropriate amount of water (10 mL) to quench the reaction system, and extract three times with ethyl acetate (15 mL). Combine the organic phases, wash them successively with water and saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product 1-fluoro-4-((4-nitrophenoxy)methyl)benzene (2h).

[0121] (b) Synthesis of Intermediate 4-((4-Fluorobenzyl)oxy)aniline (Compound 3h)

[0122] The product obtained in the previous step, 1-fluoro-4-((4-nitrophenoxy)methyl)benzene (2h) (5 mmol, 1.0 equivalent), reduced iron powder (15 mmol, 3.0 equivalents), and ammonium chloride (NH4Cl, 45 mmol, 9.0 equivalents) were dissolved in 70% ethanol solution (20 mL), heated to reflux and maintained for 2 hours. After the reaction was completed, it was quickly filtered through a diatomaceous earth pad while it was hot to remove the iron powder and insoluble impurities. The ethanol was removed by concentration under reduced pressure. Water was added to the residue, and it was extracted three times with ethyl acetate. The organic phases were combined and washed successively with water and saturated sodium chloride. After drying over anhydrous sodium sulfate, the organic phase was concentrated under reduced pressure to obtain a crude product, and the target compound 4-((4-fluorobenzyl)oxy)aniline (3h) was obtained through ethyl acetate / petroleum ether (v / v, 1:5).

[0123] (c) N-(4-((4-fluorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D1)

[0124] Compound 4-((4-fluorobenzyl)oxy)aniline (2h) (1 mmol, 1.0 equivalent), N,N-diisopropylethylamine (DIPEA, 5 mmol, 5.0 equivalents), and 4-dimethylaminopyridine (DMAP, 0.1 mmol, 0.1 equivalent) were dissolved in anhydrous tetrahydrofuran (THF, 3 mL). An appropriate amount of piperidine-1-sulfonyl chloride (2 mmol, 2.0 equivalents) was slowly added at room temperature. Subsequently, the reaction mixture was stirred and heated to reflux. After 8 hours of reaction, an appropriate amount of water (10 mL) was added to quench the reaction, and it was extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed successively with water and saturated sodium chloride. After drying over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure. The crude product was separated by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:10) as the eluent to obtain a yellow solid D1 with a yield of 62% and a purity of 97.60%.

[0125] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, Chloroform-d): δ 7.42 (dd, J = 8.5, 5.5 Hz, 2H), 7.20–7.15 (m, 2H), 7.10 (t, J = 8.5 Hz, 2H), 6.96–6.90 (m, 2H), 6.12 (s, 1H), 5.02 (s, 2H), 3.26–3.17 (m, 4H), 1.60–1.56 (m, 4H), 1.54–1.47 (m, 2H). 13¹³C NMR (126 MHz, Chloroform-d): δ 161.59, 156.35, 132.50, 130.33, 129.37 (q, J = 8.3 Hz), 124.01, 115.54 (q, J = 21.3 Hz), 115.51, 69.68, 47.24, 25.32, 23.65. HRMS (ESI) m / z calcd for C 18 H 21 FN₂NaO₃S [M+Na] + : 387.1149; found, 387.1155. The structural formula of compound D1 is shown in formula (16).

[0126] 2. Preparation of N-(4-((3-fluorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D2)

[0127] Referring to the synthesis of compound 2h, replacing 1-chloromethyl-4-fluorobenzene with 1-chloromethyl-3-fluorobenzene (1i), compound 1-fluoro-3-((4-nitrophenoxy)methyl)benzene (2i) was obtained; referring to the synthesis of compound 3h, replacing 2h with 2i, compound 4-(3-fluorobenzyloxy)aniline (3i) was obtained; referring to the synthesis of compound D1, replacing 3h with 3i, brown solid D2 was obtained with a yield of 50% and a purity of 98.97%.

[0128] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 ¹H NMR (500 MHz, Chloroform-d): δ 7.35 (td, J = 8.0, 6.0 Hz, 1H), 7.21–7.12 (m, 4H), 7.02 (td, J = 8.5, 2.0 Hz, 1H), 6.94–6.88 (m, 2H), 6.14 (s, 1H), 5.04 (s, 2H), 3.26–3.14 (m, 4H), 1.54 (m, 4H), 1.49 m, 2H). 13 ¹³C NMR (126 MHz, Chloroform-d): δ 163.01 (q, J = 246.4 Hz), 156.16, 139.39 (d, J = 7.4 Hz), 130.48, 130.17 (d, J = 8.2 Hz), 123.87, 122.73 (d, J = 2.8 Hz), 115.51, 114.91 (d, J = 21.2 Hz), 114.24 (d, J = 22.2 Hz), 69.50 (d, J = 2.0 Hz), 47.23, 25.31, 23.64. HRMS (ESI) m / z calcd for C 18 H 21 FN₂NaO₃S [M+Na]+ : 387.1149; found, 387.1154. The structural formula of compound D2 is shown in formula (17).

[0129] 3. Preparation of N-(4-((2-fluorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D3)

[0130] Referring to the synthesis of reference compound 2h, replacing 1-chloromethyl-4-fluorobenzene with 1-chloromethyl-2-fluorobenzene (1j), compound 1-fluoro-2-((4-nitrophenoxy)methyl)benzene (2j) was obtained; referring to the synthesis of reference compound 3h, replacing 2h with 2j, compound 4-(2-fluorobenzyloxy)aniline (3j) was obtained; referring to the synthesis of compound D1, replacing 3h with 3j, yellow solid D3 was obtained with a yield of 55% and a purity of 98.15%.

[0131] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, Chloroform-d): δ 7.49 (m, 1H), 7.32 (m, 1H), 7.19–7.13 (m, 3H), 7.12–7.06 (m, 1H), 6.97–6.91 (m, 2H), 6.08 (s, 1H), 5.11 (s, 2H), 3.25–3.12 (m, 4H), 1.53 (m, 4H), 1.51–1.45 (m, 2H). 13 C NMR (126 MHz, Chloroform-d): δ 160.49 (q, J = 247.0 Hz), 156.29, 130.39, 129.84 (q, J = 8.2 Hz), 129.75 (q, J = 3.7 Hz), 124.29 (q, J = 3.7 Hz), 123.97, 115.52, 115.40 (q, J = 21.2 Hz), 64.05 (q, J = 4.4 Hz), 47.25, 25.32, 23.64 HRMS (ESI) m / z calcd for C 18 H 21 FN2NaO3S [M+Na] + : 387.1149; found, 387.1154. The structural formula of compound D3 is shown in formula (18).

[0132] 4. Preparation of N-(4-((4-chloro-3-fluorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D4)

[0133] Referring to the synthesis of reference compound 2h, 1-chloromethyl-4-fluorobenzene was replaced with 1-chloro-4-chloromethyl-2-fluorobenzene (1k) to obtain compound 1-chloro-2-fluoro-4-((4-nitrophenoxy)methyl)benzene (2k); referring to the synthesis of reference compound 3h, 2h was replaced with 2k to obtain compound 4-((4-chloro-3-fluorobenzyl)oxy)aniline (2k); referring to the synthesis of reference compound D1, 3h was replaced with 3k to obtain a yellow solid D4 with a yield of 60% and a purity of 97.86%.

[0134] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR(500MHz,Chloroform-d):δ7.42(t,J=8.0Hz,1H),7.25(dd,J=9.0,2.0Hz,1H),7.16(m,3H),6.97–6.84(m,2H),6.20(s,1H),5.03(s,2H),3.22(t,J=5.5Hz,4H),1.56(m,4H),1.53–1.46(m,2H). 13 C NMR(126MHz,Chloroform-d):δ158.21(q,J=250.7Hz),155.90,137.75,130.71(q,J=13.5Hz),123.85,123.43(q,J=4.0Hz),115.51,115.47(q,J=21.8Hz),68.94,47.23,25.30,23.63.HRMS(ESI)m / zcalcd for C 18 H 20 ClFN2NaO3S[M+Na] + :421.0759;found,421.0764。The structural formula of compound D4 is shown in formula (19).

[0135] 5. Preparation of N-(4-((2-chloro-3-fluorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D5)

[0136] Referring to the synthesis of reference compound 2h, 1-chloromethyl-4-fluorobenzene was replaced with 2-chloro-1-chloromethyl-3-fluorobenzene (1l) to obtain compound 2-chloro-1-fluoro-3-((4-nitrophenoxy)methyl)benzene (2l); referring to the synthesis of reference compound 3h, 2h was replaced with 2l to obtain compound 4-((2-chloro-3-fluorobenzyl)oxy)aniline (3l); referring to the synthesis of reference compound D1, 3h was replaced with 3l to obtain a yellow solid D5 with a yield of 55% and a purity of 98.68%.

[0137] The nuclear magnetic resonance and mass spectrometry data are as follows: 11H NMR (500 MHz, Chloroform-d): δ 7.37 (d, J = 8.0 Hz, 1H), 7.32–7.28 (m, 1H), 7.21–7.17 (m, 2H), 7.14 (dd, J = 8.0, 2.0 Hz, 1H), 7.00–6.88 (m, 2H), 6.15 (s, 1H), 5.17 (s, 2H), 3.31–3.16 (m, 4H), 1.55 - 1.57 (m, 4H), 1.54–1.47 (m, 2H). 13 13C NMR (126 MHz, Chloroform-d): δ 158.22 (q, J = 248.5 Hz), 155.94, 136.95, 130.72, 127.74 (q, J = 7.9 Hz), 123.80 (q, J = 5.1 Hz), 119.60 (q, J = 18.4 Hz), 115.79 (q, J = 21.1 Hz), 115.54, 67.07, 47.23, 25.31, 23.64. HRMS (ESI) m / z calcd for C 18 H 20 ClFN2NaO3S [M + Na] + : 421.0759; found, 421.0763. The structural formula of compound D5 is shown in formula (20).

[0138] 6. Preparation of N-(4-((3-chlorobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D6)

[0139] Referring to the synthesis of compound 2h, replacing 1-chloromethyl-4-fluorobenzene with 1-chloro-3-chloromethylbenzene (1m), compound 1-chloro-3-((4-nitrophenoxy)methyl)benzene (2m) was obtained; referring to the synthesis of compound 3h, replacing 2h with 2m, compound 4-((3-chlorobenzyl)oxy)aniline (3m) was obtained; referring to the synthesis of compound D1, replacing 3h with 3m, yellow solid D6 was obtained with a yield of 62% and a purity of 99.17%.

[0140] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 1H NMR (500 MHz, Chloroform-d): δ 7.45 (d, J = 2.0 Hz, 1H), 7.35–7.29 (m, 3H), 7.20–7.14 (m, 2H), 6.96–6.89 (m, 2H), 6.22 (s, 1H), 5.04 (s, 2H), 3.21 (t, J = 5.5 Hz, 4H), 1.59–1.54 (m, 4H), 1.50 (m, 2H). 1313C NMR (126 MHz, Chloroform-d): δ 156.13, 138.85, 134.57, 130.51, 129.90, 128.18, 127.45, 125.37, 123.85, 115.51, 69.48, 47.23, 25.31, 23.64. HRMS (ESI) m / z calcd for C 18 H 21 ClN2NaO3S [M+Na] + : 403.0854; found, 403.0859. The structural formula of compound D6 is shown in formula (21).

[0141] 7. Preparation of N-(4-((3-(trifluoromethyl)benzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D7)

[0142] Referring to the synthesis of compound 2h, replacing 1-chloromethyl-4-fluorobenzene with 1-chloro-3-trifluoromethylbenzene (1n) gives compound 1-[(4-nitrophenoxy)methyl]-3-(trifluoromethyl)benzene (2n); referring to the synthesis of compound 3h, replacing 2h with 2n gives compound 4-[3-(trifluoromethyl)benzyloxy]aniline (3n); referring to the synthesis of compound D1, replacing 3h with 3n gives yellow solid D7 with a yield of 68% and a purity of 98.96%.

[0143] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 1H NMR (500 MHz, Chloroform-d): δ 7.71 (s, 1H), 7.62 (t, J = 7.0 Hz, 2H), 7.53 (t, J = 8.0 Hz, 1H), 7.22–7.13 (m, 2H), 6.97–6.87 (m, 2H), 6.22 (s, 1H), 5.11 (s, 2H), 3.22 (t, J = 5.3 Hz, 4H), 1.58–1.55 (m, 4H), 1.54–1.49 (m, 2H). 13 13C NMR (126 MHz, Chloroform-d): δ 156.08, 137.81, 131.03 (q, J = 31.8 Hz), 130.60, 129.10, 124.87 (q, J = 3.6 Hz), 124.01 (q, J = 272.4 Hz), 124.09 (q, J = 4.0 Hz), 123.89, 115.50, 69.51, 47.23, 25.31, 23.63. HRMS (ESI) m / z calcd for C 19 H 21 F3N2NaO3S [M+Na] +: 437.1117; found, 437.1120. The structural formula of compound D7 is shown in formula (22).

[0144] 8. Preparation of N-(4-((3-methoxybenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D8)

[0145] Referring to the synthesis of reference compound 2h, 1-chloromethyl-4-fluorobenzene was replaced with 1-chloro-3-methoxybenzene (1o) to obtain compound 3-[(4-nitrophenoxy)methyl]-1-methoxybenzene (2o); referring to the synthesis of reference compound 3h, 2h was replaced with 2o to obtain compound 4-[(3-methoxybenzyl)oxy]aniline (3o); referring to the synthesis of compound D1, 3h was replaced with 3o to obtain yellow solid D8 with a yield of 49% and a purity of 97.46%.

[0146] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR (500 MHz, Chloroform-d): δ 7.32 (t, J = 8.0 Hz, 1H), 7.19–7.13 (m, 2H), 7.04–6.97 (m, 2H), 6.97–6.91 (m, 2H), 6.89 (dd, J = 8.5, 2.5 Hz, 1H), 6.21 (s, 1H), 5.04 (s, 2H), 3.84 (s, 3H), 3.21 (t, J = 5.5 Hz, 4H), 1.58–1.53 (m, 4H), 1.52–1.49 (m, 2H). 13 C NMR (126 MHz, Chloroform-d): δ 159.87, 156.47, 138.37, 130.23, 129.68, 123.93, 119.64, 115.52, 113.52, 112.96, 70.21, 55.27, 47.23, 25.31, 23.64. HRMS (ESI) m / z calcd for C 19 H 24 N2NaO4S [M+Na] + : 399.1349; found, 399.1355. The structural formula of compound D8 is shown in formula (23).

[0147] 9. Preparation of N-(4-((3-(difluoromethoxy)benzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D9)

[0148] For the synthesis of reference compound 2h, 1-chloromethyl-4-fluorobenzene was replaced with 1-chloro-3-difluoromethoxybenzene (1p) to obtain compound 1-difluoromethoxy-3-[(4-nitrophenoxy)methyl]benzene (2p); for the synthesis of reference compound 3h, 2h was replaced with 2p to obtain compound 4-[(3-difluoromethoxybenzyl)oxy]aniline (3p); for the synthesis of reference compound D1, 3h was replaced with 3p to obtain a yellow solid D9 with a yield of 71% and a purity of 97.82%.

[0149] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR(500MHz,Chloroform-d):δ7.38(t,J=8.0Hz,1H),7.28–7.24(m,1H),7.20(t,J=2.0Hz,1H),7.18–7.13(m,2H),7.08(dd,J=8.5,2.5Hz,1H),6.94–6.89(m,2H),6.38-6.67(t,J=75Hz,1H),6.47(s,1H),5.04(s,2H),3.26–3.11(m,4H),1.57–1.50(m,4H),1.50–1.48(m,2H). 13 C NMR(126MHz,Chloroform-d):δ156.08,151.47,139.13,130.58,130.05,124.18,123.71,118.96,118.41,117.95,115.88(q,J=259.8Hz),115.50,113.82,69.56,47.22,25.30,23.63.HRMS(ESI)m / z calcd forC 19 H 22 F2N2NaO4S[M+Na] + :435.1161;found,435.1165. The structural formula of compound D9 is shown in formula (24).

[0150] 10. Preparation of N-(4-((3-(trifluoromethoxy)benzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D10)

[0151] For the synthesis of reference compound 2h, 1-chloromethyl-4-fluorobenzene was replaced with 1-chloromethyl-3-trifluoromethoxybenzene (1q) to obtain compound 1-[(4-nitrophenoxy)methyl]-3-trifluoromethoxybenzene (2q); for the synthesis of reference compound 3h, 2h was replaced with 2q to obtain compound 4-[(3-trifluoromethoxybenzyl)oxy]aniline (3q); for the synthesis of reference compound D1, 3h was replaced with 3q to obtain yellow solid D10 with a yield of 58% and a purity of 99.14%.

[0152] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 H NMR(500MHz,Chloroform-d):δ7.43(t,J=8.0Hz,1H),7.36(d,J=8.0Hz,1H),7.31(s,1H),7.23–7.15(m,3H),6.96–6.91(m,2H),6.15(s,1H),5.08(s,2H),3.21(m,4H),1.56–1.54(m,4H),1.52–1.50(m,2H). 13 C NMR(126MHz,Chloroform-d):δ156.09,149.51,139.18,130.54,130.01,125.47,123.91,120.38,119.79,115.51,69.38,47.24,25.31,23.63.HRMS(ESI)m / z calcd for C 19 H 21 F3N2NaO4S[M+Na] + :453.1066; found, 453.1065. The structural formula of compound D10 is shown in formula (25).

[0153] 11. Preparation of N-(4-((3-cyanobenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D11)

[0154] For the synthesis of reference compound 2h, 1-chloromethyl-4-fluorobenzene was replaced with 3-(chloromethyl)benzonitrile (1r) to obtain compound 3-[(4-nitrophenoxy)methyl]benzonitrile (2r); for the synthesis of reference compound 3h, 2h was replaced with 2r to obtain compound 3-[(4-aminophenoxy)methyl]benzonitrile (3r); for the synthesis of reference compound D1, 3h was replaced with 3r to obtain yellow solid D11 with a yield of 55% and a purity of 99.03%.

[0155] The nuclear magnetic resonance and mass spectrometry data are as follows: 11H NMR (500 MHz, Chloroform-d): δ 7.78 (s, 1H), 7.72–7.64 (m, 2H), 7.54 (t, J = 8.0 Hz, 1H), 7.23–7.18 (m, 2H), 6.98–6.92 (m, 2H), 6.23 (s, 1H), 5.11 (s, 2H), 3.24 (t, J = 5.5 Hz, 4H), 1.59–1.56 (m, 4H), 1.55–1.51 (m, 2H). 13 13C NMR (126 MHz, Chloroform-d): δ 155.76, 138.46, 131.66, 131.49, 130.86, 130.73, 129.45, 123.72, 118.61, 115.47, 112.82, 69.00, 47.22, 25.31, 23.64. HRMS (ESI) m / z calcd for C 19 H 21 N3NaO3S [M+Na] + : 394.1196; found, 394.1202. The structural formula of compound D11 is shown in formula (26).

[0156] 12. Preparation of N-(4-((3-methylbenzyl)oxy)phenyl)piperidine-1-sulfonamide (Compound D12)

[0157] Referring to the synthesis of compound 2h, replacing 1-chloromethyl-4-fluorobenzene with 1-chloromethyl-3-methylbenzene (1s) gives compound 1-methyl-3-[(4-nitrophenoxy)methyl]benzene (2s); referring to the synthesis of compound 3h, replacing 2h with 2s gives compound 4-[(3-methylphenyl)oxy]aniline (3s); referring to the synthesis of compound D1, replacing 3h with 3s gives yellow solid D11 with a yield of 61% and a purity of 98.56%.

[0158] The nuclear magnetic resonance and mass spectrometry data are as follows: 1 1H NMR (500 MHz, Chloroform-d): δ 7.32 (d, J = 7.5 Hz, 1H), 7.30–7.23 (m, 2H), 7.21–7.15 (m, 3H), 6.99–6.93 (m, 2H), 6.12 (s, 1H), 5.04 (s, 2H), 3.23 (t, J = 5.5 Hz, 4H), 2.41 (s, 3H), 1.57–1.56 (m, 4H), 1.55–1.48 (m, 2H). 1313C NMR(126MHz,Chloroform-d):δ156.62,138.34,136.63,130.12,128.85,128.53,128.27,124.62,124.01,115.48,70.41,47.24,25.32,23.65,21.42.HRMS(ESI)m / z calcd forC 19 H 24 N2NaO3S[M+Na] + :383.1400; found, 383.1406. The structural formula of Compound D12 is shown in Formula (27).

[0159] Biological Test Example 1: Evaluation Experiment of Androgen Receptor Antagonistic Ability

[0160] Detection Principle: Androgen receptor (AR), as a transcription factor, needs to bind to a specific sequence to exert its transcriptional activity. In the detection method of the present invention, a reporter gene enhanced green fluorescent protein (eGFP) controlled by the ARR2PB promoter is introduced into androgen receptor-dependent prostate cancer cells (LNCaP), so that LNCaP cells express eGFP regulated by androgen receptor, and a prostate cancer cell line (LNCaP-ARR2PB-eGFP) is constructed. After treatment with test compounds at different concentration gradients, the level of eGFP expression in LNCaP-ARR2PB-eGFP cells is detected, and the strength of the androgen receptor antagonistic ability of the compound can be measured.

[0161] Detection Steps: The LNCaP-ARR2PB-eGFP cells are starved in a complete medium without androgen for 3 - 5 days to reduce the background fluorescence value to a lower level. Select starved cells in the logarithmic growth phase. After trypsin digestion is terminated, centrifuge, discard the supernatant, resuspend the cell pellet with a complete medium without androgen, count the cells and adjust the cell concentration with the medium, and then inoculate them into a 96-well plate at a density of 3.5×10 4 cells / well, with a volume of 90 μL per well. Set up a group without dihydrotestosterone (DHT) and a group without the compound. Place the culture plate in an incubator with saturated humidity at 37 °C and 5% CO2 and continue to culture for 24 hours until the cells are basically adherent. Add 10 μL of a medium containing both DHT and the test compound. Usually, the final concentration of DHT is 5 nM, and the initial screening concentration of the compound is 10 μM. Determine the half-maximal inhibitory concentration (IC 50) When the final concentrations of the test compounds were 50, 16.667, 5.556, 1.852, 0.617, 0.206, 0.069, and 0.023 μM in sequence, 3 replicate wells were set. They were placed in an incubator and cultured for another 72 hours. The fluorescence intensity values near the wavelength of 530 nm were detected with a Synergy H1 multimode microplate reader under the excitation light of 485 nm wavelength, and the AR antagonistic activity of the test compounds at the cellular level was calculated.

[0162] Detection results: As shown in Table 1, the compounds of the present invention exhibited AR antagonistic activity, among which the antagonistic activities of compounds A1, B5, D2, D7, and D8 were particularly prominent (IC 50 <1 μM).

[0163] Table 1. Inhibitory activities of compounds on the transcriptional level and proliferation of LNCaP cells

[0164]

[0165] Biological test example 2: Evaluation of the inhibitory activity of compounds on the proliferation of LNCaP cells by cell proliferation inhibition experiment

[0166] Detection principle: LNCaP cells are a classic cell line derived from lymph node metastases of human prostate cancer. They are from prostate cancer patients with lymph node metastases, have metastatic characteristics, and are an AR-positive and androgen-dependent prostate cancer cell line. Usually, the thiazolyl blue (MTT) method is one of the commonly used methods for detecting cell proliferation inhibition. Its principle is: MTT is a yellow dye that can be reduced to a water-insoluble blue-violet crystalline formazan under the action of succinate dehydrogenase and cytochrome C in the mitochondria of living cells. If the cells die, MTT cannot be reduced. Therefore, the amount of formazan produced is linearly positively correlated with the number of living cells within a certain range. The formazan crystals can be dissolved with triple buffer, and the optical density at 570 nm wavelength measured with a microplate reader can reflect the number of living cells.

[0167] Detection steps: Cells in the logarithmic growth phase were selected. After trypsin digestion was terminated, they were centrifuged, the supernatant was removed, the cell pellet was resuspended with complete medium, and after cell counting and adjusting the cell concentration with the medium, at 5×10 3Inoculate at a density of / holes into a 96-well plate, with a volume of 90 μL per well, and set up zero-adjustment wells and control wells. Place the culture plate in an incubator with saturated humidity at 37 °C and 5% CO2. After culturing for 24 hours, add 10 μL of the culture medium containing the compound to be tested. The final concentrations of the compound to be tested are 50, 16.667, 5.556, 1.852, 0.617, 0.206, 0.069, and 0.023 μM in sequence. Set up 3 replicates and continue culturing in the incubator for 72 hours. Add 10 μL of MTT solution (5 mg / mL) to each well, culture for 3 - 5 hours until crystals form. Add 100 μL of triple solution and incubate overnight to completely dissolve the formazan crystals. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at 570 nm.

[0168] Detection results: As shown in Table 1, the compounds of the present invention have good proliferation inhibitory activity against prostate cancer cell line LNCaP. The IC 50 values of most compounds are close to enzalutamide, and among them, compounds A1, A2, A5, B2, B5, D2, D6, D7, D8, D9, and D10 all exhibit better proliferation inhibitory activity than enzalutamide.

[0169] In summary, the antagonistic activity of B5 is close to that of enzalutamide, and its proliferation inhibitory activity is significantly better than that of enzalutamide. Subsequently, further functional tests on B5 were carried out.

[0170] Biological test example 3: Detect the inhibitory activity of the compound against clinically drug-resistant related AR mutants.

[0171] Detection principle: Amino acid mutations in the ligand-binding pocket of the androgen receptor can lead to abnormal activation of the AR signaling pathway by AR antagonists or non-specific ligands, thereby promoting tumor progression. Among them, the somatic mutation H875Y derived from human CWR22 cells can enhance the sensitivity of AR to estradiol and progesterone and achieve abnormal activation by changing its interaction with the p160 co-activator. The T877A mutation is related to the development of resistance to the first-generation anti-androgen hydroxyflutamide, and it has been reported that this mutation can promote the growth of prostate cancer cells. The W742C mutation may cause bicalutamide to produce an agonist effect, while the T878G mutation can transform enzalutamide into a partial agonist. In addition, the F877L mutation was detected in the plasma DNA of male CRPC patients receiving treatment with the second-generation antagonists enzalutamide and apalutamide. Therefore, in the present invention, a dual-luciferase reporter gene assay system was constructed to detect the inhibitory effect of compound B5 on drug-resistant AR mutants.

[0172] Detection steps: After starving PC3 cells with a medium containing 5% charcoal-stripped serum for 3 days, seed them at a density of 1×10 4Cells were seeded into a 96-well white plate at a density of 96 wells per cell. To ensure a final volume of 100 μL per well after subsequent transfection, 75 μL of cells were seeded into each well during plating. After the cells adhered for 24 hours, 50 ng of AR mutant plasmid (such as AR F877L 、AR F877L / T878A 、AR H875Y / T878A 、AR W742C or AR T878G plasmid), 20 ng of pGL4-ARR2PB-Luc plasmid, and 5 ng of Renilla plasmid were added to each well, along with 0.25 μL of HG-Transgene transfection reagent (GeneCopoeia), and 25 μL of serum-free medium. The cells were transfected for 24 hours. The medium containing the transfection reagent in the wells was removed, and 90 μL of pre-warmed RPMI-1640 medium containing 5% charcoal-stripped serum was added. Then, 10 μL of medium containing the compound was added. The final concentrations of the test compounds were 50, 16.667, 5.556, 1.852, 0.617, 0.206, 0.069, and 0.023 μM, respectively. Three replicate wells were set up and incubated in the incubator for an additional 24 hours.

[0173] Test results: As Figure 3 shown, B5 exhibited dose-dependent transcriptional inhibitory effects on all tested mutants. For the F877L, F877L / T878A, W742C, T878G, and H875Y / T878A mutants, the IC 50 values of B5 were 0.20 μM, 0.26 μM, 0.23 μM, 5.55 μM, and 0.20 μM, respectively, showing a 3- to 12-fold activity advantage compared to darolutamide, indicating that B5 can antagonize the activity of mutant ARs caused by the representative first- and second-generation AR antagonists used in long-term clinical practice. Biological Test Example 4: Detection of the effect of compounds on prostate cancer tumor markers

[0174] Detection principle: Prostate-Specific Antigen (PSA) is a serine protease secreted by prostate epithelial cells. Its serum level is usually less than 4 ng / mL in healthy men, while prostate cancer (especially advanced or metastatic cases) can cause a significant increase in PSA, making it a core biomarker for prostate cancer screening, diagnosis, and efficacy monitoring. The expression of PSA is strictly regulated by the AR signaling pathway. Using the AR-positive prostate cancer cell line LNCaP, the activity of candidate AR antagonists was rapidly evaluated by detecting the total prostate-specific antigen (tPSA) content in cells after drug treatment.

[0175] Detection procedure: After the detection of LNCaP-ARR2PB-eGFP cells was completed, 300 μL of the supernatant was taken and used with Detection of the 2000XPi system. Subsequently, 300 μL of the supernatant of each sample was collected and sent to the Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital, Hangzhou) for detection of tPSA level using the e 801 electrochemiluminescence immunoassay analyzer (Roche Diagnostics, Switzerland).

[0176] Detection results: As Figure 4 shown, compound B5 can significantly inhibit the expression of PSA (IC 50 = 0.16 μM).

[0177] Biological test example 5: Detection of the effect of the compound on the expression of genes regulating the AR signaling pathway

[0178] Detection principle: As a nuclear receptor transcription factor, the core function of AR is to form a dimer after binding to androgens (such as DHT), transfer to the nucleus to bind to androgen response elements (ARE), and regulate the transcription of downstream target genes. Detecting the changes in the mRNA and protein levels of these marker genes can directly reflect the degree of inhibition of AR transcriptional activity, which is the gold standard for verifying whether a compound truly blocks the AR signaling pathway.

[0179] Detection steps: In the present invention, fluorescence quantitative PCR (qPCR) was used to quantitatively analyze the mRNA expression of AR and AR-regulated genes (KLK3, TMPRSS2, FKBP5): LNCaP cells were cultured in phenol red-free RPMI-1640 medium containing 5% charcoal-stripped serum and seeded in 6-well plates at a density of 4 × 10 5 cells / well. The culture plates were placed in an incubator with saturated humidity at 37 °C and 5% CO2. After the cells adhered for 24 hours, enzalutamide and compound B5 were added respectively at concentrations of 0.1 μM, 1 μM, and 10 μM. The negative control group was added with an equal volume of DMSO, and the positive control group and the drug administration group were both added with DHT at a final concentration of 10 nM. After treatment for 48 hours, mRNA was extracted using the EZ-10 DNAaway RNA Mini-Preps kit and reverse transcribed into cDNA. Using the 1st cDNA SuperMix for qPCR, and finally amplified using the qPCR SYBR Green Master Mix.

[0180] Western blotting (WB) was used to analyze the protein expression of AR and AR-regulated genes (PSA, TMRPSS2, FKBP5) after drug administration: LNCaP cells were cultured in phenol red-free RPMI-1640 medium containing 5% charcoal-stripped serum and seeded in 6-well plates at a density of 4 × 10 5Cells were seeded in 6-well plates at a density of [number of cells / holes], and the culture plates were placed in an incubator with saturated humidity at 37°C and 5% CO₂. After the cells adhered for 24 hours, enzalutamide and compound B5 were added respectively, with concentrations set at 0.1 μM, 1 μM, and 10 μM. The negative control group was added with an equal volume of DMSO, and the positive control group and the drug administration group were both added with DHT at a final concentration of 10 nM. After 48 hours of drug administration, the supernatant was removed. The cells were lysed and centrifuged, and the protein supernatant was collected. Proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane; non-specific sites were blocked with 5% skim milk, and then incubated with primary antibodies (including AR, TMRPSS2, FKBP5, GAPDH, and PSA, overnight at 4°C) and HRP-labeled secondary antibodies (for one hour at room temperature) in sequence; finally, signals were detected by an ECL chemiluminescence imaging system, and gray-scale analysis was performed using the internal reference protein GAPDH as a reference.

[0181] Detection results: As Figure 5 shown, similar to enzalutamide, compound B5 had a dose-dependent inhibitory effect on the mRNA expression of AR-regulated genes KLK3, TMPRSS2, and FKBP5, but had no effect on the mRNA expression of AR itself. As Figure 6 shown, the WB experiment confirmed that compound B5 had the same inhibitory effect on the protein expression of AR target genes. This experiment further demonstrated that the compound of the present invention has an antagonistic effect on AR transcription.

[0182] Biological test example 6: Detection of the proliferation inhibitory activity of the compound against enzalutamide-resistant cells

[0183] Detection principle: Enzalutamide is a second-generation AR antagonist used to treat castration-resistant prostate cancer. However, long-term use is prone to cause drug resistance, manifested as abnormal activation of the AR signaling pathway (such as AR mutation, upregulation of the AR splice variant AR-V7 expression) or bypass signal compensation (such as glucocorticoid receptor activation). In this experiment, based on the enzalutamide-resistant model LNCaP-enzR induced by the LNCaP cell line, it can simulate clinical drug resistance progression and is an important tool for screening new AR antagonists against drug resistance. Therefore, the MTT method was used to test whether compound B5 could inhibit the proliferation inhibitory activity of LNCaP-enzR cells.

[0184] Detection steps: Cells in the logarithmic growth phase were selected. After trypsin digestion was terminated, the cells were centrifuged, the supernatant was removed, and the cell pellet was resuspended with complete medium. After cell counting and adjusting the cell concentration with the medium, the cells were seeded at a density of 5×10 3Inoculate at a density of / holes into a 96-well plate, with a volume of 90 μL per well, and set up zero-adjustment wells and control wells. Place the culture plate in an incubator with saturated humidity, at 37 °C and 5% CO2. After culturing for 24 hours, add 10 μL of the culture medium containing the compound to be tested. The final concentrations of the compound to be tested are 50, 16.667, 5.556, 1.852, 0.617, 0.206, 0.069, and 0.023 μM in sequence. Set up 3 replicates and continue culturing in the incubator for 72 hours. Add 10 μL of MTT solution (5 mg / mL) to each well, and culture for 3 - 5 hours until crystals form. Add 100 μL of the triple solution and incubate overnight to completely dissolve the formazan crystals. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at 570 nm.

[0185] Detection results: As Figure 7 shown, compound B5 can significantly inhibit the proliferation of LNCaP-enzR cells in vitro, and its half-maximal inhibitory concentration (IC 50 ) is 5.40 μM, indicating that the compounds in the present invention may become a potential strategy to overcome clinically generated enzalutamide resistance.

[0186] Biological test example 7: Verification of the affinity between the compound and the target by AR competition experiment

[0187] Detection principle: Based on the test principle of the Thermo Fisher AR competition detection kit, the AR LBD protein and Fluormone TM Tracer form an AR LBD / Fluormone TM Tracer complex with a relatively high fluorescence polarization value. Compounds that compete with Fluormone TM Tracer for binding to the LBP site will cause the Tracer to turn into a free state, and the fluorescence polarization value of the system will decrease. The competitive affinity between the test compound and AR LBP can be quantitatively evaluated by the change in the fluorescence polarization value.

[0188] Detection steps: Sequentially add 2× ligand dilution solution and 2× AR LBD / Fluormone TM Tracer complex to a 384-well plate. Gently mix the 384-well plate on a shaker and incubate at room temperature in the dark for 4 hours. Use an ELISA reader to measure the fluorescence polarization value of each well and calculate the competitive affinity between the compound and LBP.

[0189] Detection results: As Figure 8 shown, both compound B5 and enzalutamide play an antagonistic role by targeting the AR ligand-binding pocket, and the competitive binding IC 50 of B5 is 3.49 μM, slightly better than that of enzalutamide (IC 50 = 3.70 μM).

[0190] Biological Test Example 8: Detecting the Effect of a Compound on the Nuclear Entry of Androgen Receptor

[0191] Detection Principle: An important mechanism of action of second-generation antagonists represented by enzalutamide is that it can block the entry of AR into the cell nucleus. In this experiment, in order to detect whether a compound can affect the translocation of DHT-induced AR from the cytoplasm to the nucleus, the LNCaP cells treated with the compound were subjected to cytoplasmic / nuclear separation, and immunoblotting was used to quantitatively analyze the effect of the compound on the intracellular localization of endogenous AR.

[0192] Detection Procedure: LNCaP cells were cultured in phenol red-free RPMI-1640 medium containing 5% charcoal-stripped serum. After three days of starvation treatment, they were seeded into 10 cm 6 culture dishes at a density of 1 × 10 2 cells / well. A DMSO blank control group, a DHT positive control group, an enzalutamide negative control group, and a test compound group were set up. The culture dishes were placed in an incubator with saturated humidity, at 37 °C, and 5% CO2. After the cells adhered for 24 hours, 10 μM enzalutamide and compound B5 were added for pretreatment for 16 hours, and then DHT with a final concentration of 10 nM was added, and incubation was continued for 2 hours.

[0193] Detection Results: As Figure 9 shown, compound B5, similar to the positive drug enzalutamide, can significantly prevent the nuclear entry of androgen receptor, indicating that compound B5 is a second-generation androgen receptor antagonist.

[0194] Biological Test Example 9: Detecting the Effect of a Compound on AR Homodimerization

[0195] Detection Principle: The homodimerization of AR is a key step in its ligand-dependent activation. It forms a dimer interface through the intramolecular interaction between the N-terminal domain and the ligand-binding domain, promoting the stable binding of AR to the androgen response element on DNA and recruiting coactivators (such as SRC, p300) to initiate the transcription of target genes (such as PSA, TMPRSS2). In this experiment, the NanoBiT luciferase technique was used to detect the effect of the compound on AR dimers. The principle is composed of two small-sized NanoLuc luciferase subunits (Large BiT and Small BiT). When the target proteins interact, the two subunits bind and reconstruct the active luciferase, catalyzing the substrate to emit light, thereby detecting the changes in protein interactions in cells in real time and with high sensitivity.

[0196] Detection Procedure: Based on Promega Corporation's System, construct the AR carboxyl-terminal fusion SmBiT plasmid AR::SmBiT and the AR amino acid fusion LgBiT plasmid LgBiT::AR. Culture 293T cells in phenol red-free DMEM medium containing 5% charcoal-absorbed serum, and after 2 days of starvation treatment, seed them into a 96-well white culture plate at a density of 8×10 3 cells / well. After the cells adhere for 12 hours, transfect 50 ng of AR::SmBiT and 50 ng of LgBiT::AR plasmid into each well. Place the culture plate in an incubator with saturated humidity at 37 °C and 5% CO2 for 24 hours, then remove the medium. Add 90 μL of Opti-MEM low-serum medium and 10 μL of the test compound to each well and incubate for 4 hours. Subsequently, add 25 μL live cell detection reagent, measure the baseline luciferase activity using a Synergy H1 multimode microplate reader, and then add 1 nM DHT to each well and continuously record the real-time fluorescence signal for 30 minutes to quantify the ligand-dependent dimerization dynamics of AR.

[0197] Detection results: As Figure 10 shown, both enzalutamide and compound B5 showed significant concentration-dependent AR dimerization inhibitory activity. This further indicates that the compounds in the present invention can not only inhibit the nuclear entry of AR, but also disrupt the assembly of the dimerization-dependent transcription complex, thereby achieving the inhibitory effect on the AR signaling pathway.

[0198] Biological test example 10: Evaluation of the in vivo antitumor effect of the compound in a nude mouse model transplanted with LNCaP cells

[0199] Detection principle: Construct a tumor model by subcutaneous transplantation of human prostate cancer cells (LNCaP) into nude mice, and observe the effects of the compound on tumor volume, weight, and the plasma PSA level of mice to evaluate its in vivo antitumor activity and safety.

[0200] Detection steps: Select male Balb / c nude mice at 4-6 weeks of age. Mix 100 μL of LNCaP cells (1×10 7 cells) with an equal volume of Matrigel and inject it subcutaneously into the right groin of the mice. Measure the tumor volume every 3 days (calculation formula: length × width 2 / 2). When the tumor volume reaches 100 mm 3 , randomly divide them into 3 groups: vehicle control group, enzalutamide group (50 mg / kg, administered by gavage), and B5 group (50 mg / kg, administered by gavage). Administer the drug once a day, and measure the body weight change during the experiment to evaluate the toxicity. After the treatment is completed, collect blood from the orbital venous plexus, centrifuge at 5000 rpm, and take the supernatant to detect the tPSA level. After sacrificing the mice, completely dissect the tumors and weigh them.

[0201] Detection results: AsFigure 11 As shown, the tumor growth inhibition rate of the B5 administration group was 75.63%, the tumor volume and weight were significantly lower than those of the vehicle control group (*p < 0.05), and the plasma tPSA level in the B5 group decreased to 11.08 ng / mL, indicating that B5 significantly inhibited the PSA expression in LNCaP xenograft tumor-bearing mice.

[0202] Biological Test Example 11: Evaluation of the in vivo anti-tumor effect of a compound in a nude mouse model transplanted with enzalutamide-resistant LNCaP cells (LNCaP-EnzR)

[0203] Detection principle: A tumor model was constructed by subcutaneous transplantation of LNCaP-EnzR into nude mice, and the in vivo anti-tumor activity and safety of the compound were evaluated by observing the effects of the compound on tumor volume and mouse body weight.

[0204] Detection steps: Male Balb / c nude mice at 4 - 6 weeks of age were selected. After mixing 100 μL of LNCaP-EnzR (1×10 7 cells) with an equal volume of Matrigel, it was subcutaneously injected into the right groin of the mice. The tumor volume was measured every 3 days (calculation formula: length × width 2 / 2). When the tumor volume reached 100 mm 3 , the mice were randomly divided into two groups: the vehicle control group and the B5 group (50 mg / kg, administered by gavage), and the drug was administered once a day. The body weight changes were detected during the experiment to evaluate the toxicity.

[0205] Detection results: As Figure 12 shown, in the oral administration of compound B5 at 50 mg / kg, the tumor volume was significantly lower than that of the vehicle control group, indicating that B5 significantly inhibited the in vivo proliferation of enzalutamide-resistant LNCaP cell xenograft tumors. At the same time, its body weight did not show a significant decrease during the entire dosing period, indicating its high safety.

Claims

1. A sulfonamide compound or a pharmaceutically acceptable salt thereof or a solvate thereof or a stereoisomer thereof or a prodrug molecule thereof or a deuterated product thereof, characterized in that: The general structural formula of the sulfonamide compound is shown in formula (I), wherein R1 is selected from hydrogen, fluorine, chlorine, bromine, cyano, nitro, methyl, methoxy, methylcarbomethoxy, trifluoromethyl, trifluoromethoxy, difluoromethoxy; X is selected from O or NR a , where R a is selected from hydrogen, methyl, ethyl; R2 is selected from methyl, methylamino, 2. The sulfonamide compound or its pharmaceutically acceptable salt or solvate or stereoisomer or prodrug molecule or deuterated product thereof according to claim 1, characterized in that: The structural formula of the sulfonamide compound is shown in any one of formulas (1) to (27), 3. The sulfonamide compound or a pharmaceutically acceptable salt thereof or a solvate thereof or a stereoisomer thereof or a prodrug molecule thereof or a deuterated product thereof according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salt is an organic acid salt, an inorganic acid salt, an organic base salt or an inorganic base salt, the organic acid salt is oxalate, lactate, p-toluenesulfonate, malate, citrate, fumarate, camphorsulfonate or methanesulfonate; the inorganic acid salt is nitrate, sulfate, hydrohalide or phosphate; the organic base salt is sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine or tert-butylamine; the inorganic base salt is sodium hydroxide, potassium hydroxide or lithium hydroxide.

4. Use of the sulfonamide compound or a pharmaceutically acceptable salt or a solvate or a stereoisomer or a prodrug molecule or a deuterated product thereof as claimed in any one of claims 1 to 3 in the preparation of an androgen receptor antagonist.

5. The use according to claim 4, characterized in that The androgen receptor antagonist is an androgen receptor ligand pocket competitive inhibitor, an androgen receptor nuclear translocation inhibitor or an androgen receptor dimerization inhibitor.

6. Use of the sulfonamide compound or a pharmaceutically acceptable salt or a solvate or a stereoisomer or a prodrug molecule or a deuterated product thereof as claimed in any one of claims 1 to 3 in the preparation of a drug for treating prostate cancer, breast cancer or ovarian cancer.

7. The use according to claim 6, characterized in that The prostate cancer is enzalutamide-resistant prostate cancer.

8. The use according to claim 6, characterized in that The prostate cancer is a prostate cancer in which a point mutation occurs in the ligand binding pocket of the androgen receptor, and the point mutation is F877L, F877L / T878A, W742C, T878G or H875Y / T878A.

9. A pharmaceutical composition, characterized in that The invention comprises an effective dose of a sulfonamide compound as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a solvate thereof or a stereoisomer thereof or a prodrug molecule thereof or a deuterated product thereof, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

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