Benzene methanesulfonyl compound taking AR / AR-V7 double protein as target spot as well as preparation method and application of benzene methanesulfonyl compound

Abstract

The invention relates to a benzenesulfonyl compound taking AR / AR-V7 double protein as a target spot and a preparation method and application thereof, the molecular structure of the benzenesulfonyl compound is shown as a formula I: # imgabs0 #, and R is selected from any one of hydrogen atom, halogen, alkyl, hydroxyl, methoxyl, ester group, cyano and nitro. Comprising the following preparation steps: taking p-aminobenzene sulfonyl chloride as an initial raw material, and performing boc protection to obtain a compound 1; performing condensation reaction to obtain a compound 2; carrying out boc removal reaction to obtain a compound 3; and reacting the compound 3 with each substituted phenylboronic acid to obtain the target compound. Compared with the prior art, the benzenesulfonyl compound prepared by the invention has excellent prostate cancer cell inhibitory activity, has AR / AR-V7 double protein targets, and can be used for preparing drugs for treating prostate cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a phenylmethylsulfonyl compound targeting AR / AR-V7 double proteins, and a preparation method and application thereof. Background Art

[0002] According to a 2020 survey by the World Health Organization, cancer is the leading cause of death in most countries around the world. [1] According to the survey statistics of the International Agency for Research on Cancer, the number of cases of prostate cancer among male cancer patients in 2020 is second only to lung cancer. [2] In recent years, the incidence of prostate cancer has shown a clear and sustained growth trend. [3] .

[0003] Malignant transformation of prostate cancer demonstrates a progressive, multistep process, progressing from prostatic intraepithelial neoplasia to localized prostate cancer, locally invasive prostate adenocarcinoma, and finally metastatic prostate cancer. [4] This complex biological process involves the interaction of multiple factors and changes in driver genes, including but not limited to the activation of inflammatory signaling pathways, DNA damage, aging, activation of the MYC gene, and TMPRSS-ERG gene fusion. [5] . Because androgens play a very important role in the occurrence of prostate cancer, androgen deprivation therapy (ADT) has become the main treatment for advanced prostate cancer. However, although ADT can significantly reduce the concentration of prostate-specific antigen (PSA) in serum in the early stage, almost all patients will develop resistance during the treatment and transform into castration-resistant prostate cancer (CRPC). [6] Despite this, subsequent studies have found that the condition of CRPC patients has not improved under low androgen secretion, the AR (androgen receptor) signaling pathway is still activated, and some AR proteins have variants. These AR variants can still promote the proliferation of cancer cells through the AR pathway in the absence of androgens. [7] Current research further reveals that the AR variant most closely associated with prostate cancer, especially CRPC tumors, is AR-V7 [8] AR-V7, as a variant of androgen receptor (AR), plays a key role in the progression of prostate cancer due to its structural characteristics. AR-V7 has a complete NTD domain and DBD domain, but lacks the LBD domain and hinge region. Instead, it is encoded by a short peptide sequence whose function is not yet clear, which is encoded by exon 3 (CE3). [9] .

[0004] CYP17A1 is the most important enzyme in androgen production

[10] , and abiraterone is an inhibitor of this enzyme. When the CYP17A1 enzyme is inhibited, the production of androgens will be blocked. However, since the protein transcribed by the AR-V7 splice variant can continue to activate the AR gene downstream pathway in the absence of androgens, abiraterone cannot be used as a targeted drug for AR-V7. The LBD binding domain of AR-FL (AR full-chain protein) is the target of enzalutamide, and the production of AR-V7 mutants directly prevents enzalutamide from binding to AR, resulting in CRPC patients becoming resistant to enzalutamide.

[11] Therefore, neither paclitaxel, abiraterone nor enzalutamide can be used as drugs targeting AR-V7. There is an urgent need to develop targeted drugs targeting AR-V7 to solve the problem that there are no targeted drugs available for castration-resistant prostate cancer.

[0005] The references involved in the above background technology are:

[0006] [1]SUNG H, FERLAY J, SIEGEL RL, et al.Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36Cancers in185Countries[J].CA Cancer J Clin, 2021,71(3):209-49.3

[0007] [2]JEMAL A, BRAY F, CENTER M, et al.Global cancer statistics[J].CACancer J Clin, 2011, 61(2):69-90.

[0008] [3]Zheng R, Zhang S, Zeng H, et al. Cancer incidence and mortality in China, 2016[J]. Journal of the National Cancer Center, 2022, 2(1)1-9.

[0009] [4]Huggins C,Hodges C V.Studies on prostate cancer:The effect ofcastration,of estrogen and of androgen injection on serum phophatases inmetastatic carcinoma of the prostate[J].The Journal of Urology,2002,168(1):9-12.

[0010] [5]Rouse W B,Tompkins V S,O'Leary C A,et al.The RNA secondarystructure of androgen receptor-FL and V7 transcripts reveals novel regulatoryregions[J].Nucleic Acids Research,2024,654:563-579.

[0011] [6]Dransfield D T,Namdev N,Jacoby D B,et al.Correlation ofgaleterone-induced degradation of the androgen receptor with inhibition of adeubiquitinating enzyme[J].Journal of Clinical Oncology,2016,34(2_suppl):345-345.

[0012] [7]Brooks N,Pegg N,Worthington J,et al.Therapeutic targeting of thep300 / CBP bromodomain for the treatment of castration-resistant prostatecancer[J].Cancer Research,2018,78(16Supplement):A091-A091.

[0013] [8] Dransfield DT, Jacoby DB, Namdev N, et al. Galeterone to target proteasomal degradation of the androgen receptor in prostate tumor cells: Anovel mechanism of action for treatment of AR-V7+CRPC[J]. Journal of Clinical Oncology, 2016, 34(15_suppl):e14092-e14092.

[0014] [9] Antonarakis ES, Lu C, Chen Y, et al. AR splice variant 7 (AR-V7) and response to taxanes in men with metastatic castration-resistant prostatecancer (mCRPC) [J]. Journal of Clinical Oncology, 2015.

[0015]

[10] Mediwala SN, Sun H, Szafran AT, et al. The activity of the androgenreceptor variant AR-V7 is regulated by FOXO1 in a PTEN-PI3K-AKT-dependent way[J]. The Prostate, 2012.

[0016]

[11] Antonarakis ES, Lu C, Wang H, et al. AR-V7andresistance toenzalutamide and abiraterone in prostate cancer[J].N EnglJ Med.2014,371(11):1028-1038. Summary of the invention

[0017] Currently available drugs cannot target AR-V7 mutants, so there is no targeted drug available for castration-resistant prostate cancer. The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein and its preparation method and application.

[0018] The purpose of the present invention can be achieved by the following technical solutions:

[0019] One of the technical solutions of the present invention is to provide a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein, wherein the molecular structure of the phenylmethylsulfonyl compound is as shown in Formula I:

[0020]

[0021] Wherein, R is selected from any one of a hydrogen atom, a halogen, an alkyl group, a hydroxyl group, a methoxy group, an ester group, a cyano group, and a nitro group.

[0022] Preferably, the R is a hydroxyl group, a cyano group, or an ester group.

[0023] The second technical solution of the present invention is to provide a method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein as described in the first technical solution, comprising the following steps:

[0024] S1, dissolving p-aminobenzenesulfonyl chloride and di-tert-butyl dicarbonate in a solvent, reacting under alkaline conditions, and washing and drying the product to obtain compound 1;

[0025] S2, dissolving benzamide in DMF, adding sodium hydride, cooling to 0°C, adding the compound 1 obtained in step S1 dropwise, stirring and reacting, washing and drying the reaction product to obtain compound 2;

[0026] S3, dissolving the compound 2 obtained in step S2 in a solvent, adding trifluoroacetic acid dropwise at low temperature, and reacting at room temperature after the addition is complete, and extracting and drying the reaction product to obtain compound 3;

[0027] S4, dissolving the compound 3 obtained in step S3 and a phenylboronic acid derivative in a solvent, adding a catalyst, reacting under alkaline conditions and a gas atmosphere to obtain a product, and washing and purifying the product to obtain a final product, i.e., a phenylmethylsulfonyl compound.

[0028] Furthermore, the structural formulas of compound 1, compound 2 and compound 3 are respectively as shown in the formula:

[0029]

[0030] Further, in step S1, the p-aminobenzenesulfonyl chloride is used as a starting material; the di-tert-butyl dicarbonate is used as a protecting agent for the amino group to provide protection for the amino group on the p-aminobenzenesulfonyl chloride; the solvent is dichloromethane; the alkaline condition is achieved by adding triethylamine (TEA); the amount of the p-aminobenzenesulfonyl chloride, di-tert-butyl dicarbonate, triethylamine and solvent is in the relationship of 1mmol:1.2mmol:2mmol:(1.5-2)mL, and the amount of the solvent refers to the total amount used to dissolve the aminobenzenesulfonyl chloride, di-tert-butyl dicarbonate and triethylamine, that is, it is sufficient to ensure that all substances are fully dissolved.

[0031] Furthermore, in step S1, the process conditions of the reaction are: reaction temperature is 0-25°C, and reaction time is 6-12h.

[0032] Furthermore, in step S1, the washing and drying operation after the reaction is: the product is first washed 2-4 times with a phosphorous acid solution, the concentration of the phosphorous acid solution is 1-3M; after washing, the liquid is separated, and the organic phase is washed 2-4 times with a saturated sodium bicarbonate solution; after washing, the dichloromethane is dried to obtain compound 1.

[0033] In some specific embodiments of the present application, TLC is used to monitor whether the reaction of the raw material aminobenzenesulfonyl chloride is complete. After the reaction is complete, N,N-dimethylethylenediamine is added and stirring is continued for 1 hour to remove excess BOC anhydride (di-tert-butyl dicarbonate) to avoid its interference with subsequent reactions.

[0034] Furthermore, in step S2, the amount of compound 1, benzamide, sodium hydride and DMF used is in the following relationship: 1 mmol: (1.1-1.3) mmol: (5-10) mmol: (1.5-2) mL.

[0035] Furthermore, in step S2, the process conditions for the stirring reaction are: the reaction temperature is 0-25°C, and the reaction time is 12-24h.

[0036] Furthermore, in step S2, the sodium hydride and compound 1 should be added / dropped slowly.

[0037] Furthermore, in step S2, the washing and drying operation after the reaction is: first use a mixed solvent of ether and water to quench the reaction; separate the liquids, wash the organic phase with saturated sodium chloride for 2-4 times, then dry it with anhydrous sodium sulfate, and evaporate the ether under reduced pressure to obtain a white solid compound 2.

[0038] Furthermore, in step S3, the solvent is dichloromethane, and the amount of the compound 2, trifluoroacetic acid, and the solvent is in the ratio of 1 mmol:(2-8) mmol:3 mL.

[0039] Furthermore, in step S3, the reaction temperature is 0-25°C, and the reaction time is 6-12h.

[0040] Furthermore, in step S3, the extraction and drying operation after the reaction is: first use a saturated sodium bicarbonate solution to adjust the pH to 8-9; add dichloromethane for extraction, wash the organic phase with a saturated sodium chloride solution 2-4 times, dry over anhydrous sodium sulfate, and dry the dichloromethane under reduced pressure to obtain a white solid compound 3.

[0041] Further, in step S4, the phenylboronic acid derivative is a compound of the following structural formula:

[0042]

[0043] Wherein R is selected from any one of a hydrogen atom, a halogen, an alkyl group, a hydroxyl group, a methoxy group, an ester group, a cyano group, and a nitro group.

[0044] Further, in step S4, the solvent is dichloromethane, the catalyst is copper acetate, and the gas atmosphere is oxygen or air atmosphere; the alkaline condition is achieved by adding triethylamine (TEA);

[0045] Further, in step S4, the amount of the compound 3, the phenylboronic acid derivative, triethylamine, copper acetate and the solvent is in the following relationship: 1 mmol: (1.1-1.3) mmol: 3 mmol: (0.05-1) mmol: (5-10) mL;

[0046] Furthermore, in step S4, the reaction temperature is 35-50°C, the reaction time is 12-48h, and reflux is required during the reaction.

[0047] Furthermore, in step S4, the washing and purification operation of the product after the reaction is: first wash the organic phase three times with a saturated sodium chloride solution; then purify it by column chromatography, wherein the eluent for the column chromatography purification is ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is 1:20-1:5.

[0048] The third technical solution of the present invention is to provide an application of a phenylmethylsulfonyl compound targeting the AR / AR-V7 dual protein as described in one of the above technical solutions, wherein the phenylmethylsulfonyl compound targeting the AR / AR-V7 dual protein is used to prepare a drug that inhibits the activity of prostate cancer cells and inhibits the malignant progression of prostate cancer.

[0049] In some specific embodiments, the drug is a phenylmethylsulfonyl compound targeting AR / AR-V7 dual proteins and treating prostate cancer mice, and can effectively inhibit the malignant progression of prostate cancer in mice.

[0050] Compared with the prior art, this application has at least the following advantages:

[0051] (1) The present invention is based on a computer-aided drug design method, and the phenylmethylsulfonyl compounds synthesized by structural design targeting the prostate cancer cell target AR / AR-V7 dual protein have better binding mode with the protein pocket, which can significantly improve the inhibitory activity and selectivity of the compounds.

[0052] (2) Compared with the prostate cancer cell inhibitors targeting AR protein reported in the prior art, such as enzalutamide (IC 50 =36nM) activity, the phenylmethylsulfonyl compounds prepared by the present invention have excellent prostate cancer cell inhibition activity, especially the three preferred compounds 4-1, 4-4 and 4-7, whose half inhibition concentration IC 50 They are 6.94 μM, 1.27 μM, and 0.12 μM respectively; when mice are used as experimental subjects, the phenylmethylsulfonyl compounds provided by the present invention have excellent therapeutic effects on prostate cancer mice.

[0053] (3) The phenylmethylsulfonyl compounds provided in the present application can target both AR and AR-V7 simultaneously, and have better applicability than the drugs in the prior art that only target one of AR or AR-V7, thus providing a basis for the future development of drugs with multiple targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0055] Figure 1 A synthetic route for a phenylmethylsulfonyl compound targeting AR / AR-V7 dual proteins provided in this application;

[0056] Figure 2Indicates that compound 4-7 (E7) targets AR and AR-V7 to inhibit cell proliferation; the numbers in the figure indicate: (A) (B) CWR22RV1 and C4-2B prostate cancer cells were treated with AR / AR-V7 small molecule inhibitors of different sequences 4-2 (E2), 4-4 (E7), 4-7 (E1), 4-6 (E3), 4-1 (E4), 4-9 (E5) and 4-10 (E22) to analyze cell clone formation ability, and NC was the control group; (C) (D) CWR22RV1 and C4-2B were treated with AR small molecule inhibitors 4-4 (E7) and 4-7 (E1), and BrdU staining was used to analyze cell proliferation ability; (E) (F) CWR22RV1 and C4-2B cells were treated with AR small molecule inhibitors 4-4 (E7) and 4-7 (E1) for 48 hours, and proteins were extracted, and Western blot was used to analyze the cell proliferation ability. Blot was used to detect the protein expression of AR and AR-V7; (G) CWR22RV1 was treated with the AR / AR-V7 small molecule inhibitor 4-7 (E1), mRNA was extracted, and qRT-PCR was used to detect the changes in AR target genes;

[0057] Figure 3 The AR / AR-V7 small molecule inhibitor 4-7(E1) inhibits the ability of prostate cancer cells to form tumors in situ in mice; the numbers in the figure indicate: (A) Mice were intraperitoneally injected with different concentrations of the AR small molecule inhibitor 4-7(E1), and the changes in the size of the prostate in situ tumors in mice were analyzed by in vivo imaging at intervals of 5 days; (B) Statistical analysis of the volume of mouse prostate tumors on the 25th day; (C) Statistical analysis of the weight of mouse prostate tumors on the 25th day; (E) Immunohistochemical staining analysis of AR protein in mouse prostate cancer tissue (F) Immunohistochemical staining analysis of AR-V7 protein in mouse prostate cancer tissue. DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0059] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0060] Those skilled in the art should know that the "4-aminosulfonyl chloride" and "p-aminosulfonyl chloride" described in the following examples are the same concept.

[0061] The embodiments provided in this application first use the computer-aided drug design method to virtually screen the receptor target protein with the AR variant AR-V7 (PDB ID: 6J1L). The molecular docking uses the Surflex-Dock module in SYBYL-X2.1 (Tripos, Inc., USA). First, some small molecules were screened out in the ZINC15 database according to the five principles of Lipinskid drugs and other conditions, and then about 20 lead compounds were obtained through virtual screening. Through molecular dynamics simulation, a sulfonamide lead compound was selected, and then a series of new prostate cancer cell inhibitors with AR and AR-V7 dual protein targets were designed with the lead compound as the parent for the NTD domains of AR and AR-V7. The details are as follows:

[0062] Embodiment 1:

[0063] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-1) targeting AR / AR-V7 dual proteins (see the specific synthesis route). Figure 1 ), comprising the following steps:

[0064] (1) In a 100 mL round-bottom flask, 4-aminosulfonyl chloride (3.82 g, 20 mmol) and di-tert-butyl dicarbonate (5.232 g, 24 mmol) were added to 30 mL of dichloromethane, and triethylamine (4.04 g, 40 mmol) was slowly added dropwise at 0°C. After the addition was complete, the mixture was warmed to room temperature and stirred for 6 hours. After the reaction of the raw materials was complete as monitored by TLC (which is a routine operation in the art), N,N-dimethylethylenediamine (0.44 g, 5 mmol) was added and stirring was continued for 1 hour. After stirring was completed, the mixture was washed three times with a 1 M phosphorous acid solution. The organic layer was separated and then washed three times with a saturated sodium bicarbonate solution. After separation, the dichloromethane was dried under reduced pressure to obtain compound 1.

[0065] (2) In a 100 mL round-bottom flask, benzamide (1.33 g, 11 mmol) was dissolved in DMF, the temperature was lowered to 0°C, a turbid DMF solution of sodium hydride (2.33 g, 100 mmol) was slowly added, and a DMF solution of compound 1 (2.91 g, 10 mmol) was subsequently added dropwise. After the addition was complete, the temperature was raised to room temperature and stirred for 12 hours. In the above steps, the total volume of DMF was about 30-40 mL. After the reaction was completed by TLC monitoring, a mixed solvent of ether and water was added to quench the reaction. The DMF was dissolved in the aqueous phase and removed, and the organic layer was washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, and the ether was dried under reduced pressure to obtain a white solid compound 2.

[0066] (3) In a 100 mL round-bottom flask, compound 2 (3.76 g, 10 mmol) was dissolved in 30 mL of dichloromethane, trifluoroacetic acid (6 ml) was added at 0°C, the temperature was raised to room temperature after the addition was complete, and the mixture was stirred for 6 hours. After the reaction of the raw material was completed by TLC monitoring, a saturated sodium bicarbonate solution was added to adjust the pH to 8-9, and dichloromethane was added for extraction. The organic phase was washed three times with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the dichloromethane was evaporated under reduced pressure to obtain a white solid compound 3.

[0067] (4) In a 25 mL round-bottom flask, compound 3 (276 mg, 1 mmol), p-hydroxyphenylboronic acid (179 mg, 1.3 mmol), copper acetate (20 mg, 0.1 mmol) and triethylamine (303 mg, 3 mmol) were dissolved in 10 mL of dichloromethane and refluxed at 45° C. for 12 hours. After the reaction was completed, the organic phase was washed three times with a saturated sodium chloride solution, and the product was purified by column chromatography with petroleum ether: ethyl acetate = 10:1 to obtain the target compound 4-1 with a yield of 76.5%.

[0068] The obtained target compound 4-1 is a light yellow powder with the following structure:

[0069]

[0070] Its NMR data are as follows:

[0071] 1 H NMR(400MHz,Chloroform-d)δ8.60(s,1H),8.07–7.96(m,4H),7.82(d,J=8.4Hz,1H),7.68(t ,J=8.2Hz,3H),7.64–7.55(m,3H),7.48(d,J=7.5Hz,1H),7.41(t,J=7.5Hz,2H),7.20(s,1H).

[0072] 13 C NMR(101MHz,DMSO-d6)δ165.70,150.92,136.34,133.83,133.51,130.59,129.05,128.79, 126.63,123.93,115.20,113.44,112.77,40.61,40.40,40.19,39.99,39.78,39.57,39.36.

[0073] HRMS(ESI)calcd for C19H16N2O4S[M+H]+: 369.0864; Found: 369.08638.

[0074] Embodiment 2:

[0075] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-2) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equal molar amount of "p-methoxyphenylboronic acid".

[0076] The obtained target compound 4-2 is an off-white solid with a yield of 78%. The structure is shown below:

[0077]

[0078] Its NMR data are as follows:

[0079] 1 H NMR(400MHz,DMSO-d6)δ8.68(s,1H),7.88(d,J=8.3Hz,3H),7.81–7.72(m,3H),7.62(t, J=7.4Hz,1H),7.49(t,J=7.6Hz,2H),7.17–7.14(m,2H),6.97–6.94(m,3H),3.76(s,3H).

[0080] 13 C NMR(101MHz,DMSO-d6)δ165.70,150.92,136.34,133.83,133.51,130.59,129.05,128.79,126 .63,123.93,115.20,113.44,112.77,55.75,40.61,40.40,40.19,39.99,39.78,39.57,39.36.

[0081] HRMS(ESI)calcd for C20H18N2O4S[M+H]+: 383.1021; Found: 383.10224.

[0082] Embodiment 3:

[0083] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-3) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equimolar amount of "phenylboronic acid".

[0084] The obtained target compound 4-3 is a white powder with a yield of 70%. The structure is shown below:

[0085]

[0086] Its NMR data are as follows:

[0087] 1 H NMR (400MHz, DMSO-d6) δ8.93(s,1H),7.83(d,J=2.1Hz,4H),7.81(d,J=1.6Hz,3H),7.41–7.38(m,3H),7.34(d,J=1.6Hz,4H),7.15(s,1H).

[0088] 13 C NMR(101MHz,DMSO-d6)δ165.71,149.46,141.31,134.58,133.55,132.29,130.55,130.51,129.89, 129.06,128.82,127.85,122.98,120.46,114.05,40.63,40.42,40.21,40.00,39.79,39.58,39.38.

[0089] HRMS(ESI)calcd for C19H16N2O3S[M+Na]+: 375.0776; Found: 375.07768.

[0090] Embodiment 4:

[0091] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-4) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the methods are the same, except that the "p-hydroxyphenylboronic acid 179 mg (1.3 mmol)" in step (4) is replaced by an equimolar amount of "p-cyanophenylboronic acid".

[0092] The obtained target compound 4-4 is an off-white solid with a yield of 58%. The structure is shown below:

[0093]

[0094] Its NMR data are as follows:

[0095] 1H NMR (400MHz, Chloroform-d) δ8.70 (s, 1H), 8.27 (dd, J = 8.2, 1.4Hz, 1H), 8.14–8.09 (m, 1H), 7.99–7. 95(m,1H),7.88(dt,J=6.6,1.6Hz,2H),7.75–7.62(m,5H),7.54–7.51(m,2H),7.28(d,J=8.1Hz,2H).

[0096] 13 C NMR(101MHz,DMSO-d6)δ165.71,149.46,141.31,134.58,133.55,132.29,130.55,130.51,129.89, 129.06,128.82,127.85,122.98,120.46,114.05,40.63,40.42,40.21,40.00,39.79,39.58,39.38.

[0097] HRMS(ESI)calcd for C20H15N3O3S[M+H]+: 378.0868; Found: 378.08693.

[0098] Embodiment 5:

[0099] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-5) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equal molar amount of "3,4-methylenephenylboronic acid".

[0100] The obtained target compound 4-5 is an off-white solid with a yield of 78%. The structure is shown below:

[0101]

[0102] Its NMR data are as follows:

[0103] 1H NMR (400MHz, DMSO-d6) δ8.71(s,1H),7.87(d,J=7.6Hz,2H),7.80–7.77(m,2H),7.61(d,J=7.4Hz,1H),7.49(t,J=7.7H z,2H),6.97(d,J=8.8Hz,2H),6.90(d,J=8.2Hz,1H),6.82(d,J=2.0Hz,1H),6.67(dd,J=8.2,2.2Hz,1H),6.02(s,2H).

[0104] 13 C NMR (101MHz, DMSO-d6) δ165.71,150.61,148.35,143.87,135.25,133.52,132.35,130.56,129.05,128. 79,127.01,115.13,113.14,109.11,104.18,101.65,40.61,40.40,40.19,39.98,39.77,39.56,39.35.

[0105] HRMS(ESI)calcd for C20H16N2O5S[M+Na]+: 419.0673; Found: 419.06734.

[0106] Embodiment 6:

[0107] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-6) targeting AR-V7 protein. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equimolar amount of "p-chlorophenylboronic acid".

[0108] The obtained target compound 4-6 is an off-white solid with a yield of 69%. The structure is shown below:

[0109]

[0110] Its NMR data are as follows:

[0111] 1 H NMR (400MHz, Methanol-d4) δ8.60(s,1H),8.19(d,J=8.6Hz,3H),8.09–8.04(m,6H),7.90(d,J=7.8Hz,2H),7.62(d,J=8.0Hz,3H).

[0112] 13C NMR(101MHz,Chloroform-d)δ165.16,157.28,149.12,143.99,137.63,136.48,135.85,131 .37,130.52,129.26,128.89,128.29,128.22,124.60,123.03,112.77,77.39,77.07,76.76.

[0113] HRMS(ESI)calcd for C19H15ClN2O3S[M+2H]+: 388.0462; Found: 388.04623.

[0114] Embodiment 7:

[0115] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-7) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equal molar amount of "methyl p-formate phenylboronic acid".

[0116] The obtained target compound 4-7 is an off-white solid with a yield of 79%. The structure is shown below:

[0117]

[0118] Its NMR data are as follows:

[0119] 1 H NMR (400MHz, DMSO-d6) δ8.71(s,1H),7.87(d,J=7.7Hz,2H),7.81–7.76(m,2H),7.61(d,J=7.4Hz,1H),7.49(t,J=7.7H z,2H),6.97(d,J=8.8Hz,2H),6.90(d,J=8.2Hz,1H),6.82(d,J=2.0Hz,1H),6.67(dd,J=8.2,2.2Hz,1H),3.42(s,3H).

[0120] 13C NMR (101MHz, DMSO-d6) δ165.71,150.61,148.35,143.87,135.25,133.52,132.35,130.56,129.05,128. 79,127.01,115.13,113.14,109.11,104.18,101.65,40.61,40.40,40.19,39.98,39.77,39.56,39.35.

[0121] HRMS(ESI)calcd for C21H18N2O5S[M+H]+: 411.0970; Found: 411.09703.

[0122] Embodiment 8:

[0123] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-8) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equimolar amount of "p-bromophenylboronic acid".

[0124] The obtained target compound 4-8 is an off-white solid with a yield of 71%. The structure is shown below:

[0125]

[0126] Its NMR data are as follows:

[0127] 1 H NMR (400MHz, Chloroform-d) δ8.70 (s, 1H), 7.85 (s, 2H), 7.76 (d, J = 7.4Hz, 3H), 7.64 (d, J = 7.5Hz, 6H), 7.40 (d, J = 5.4Hz, 3H).

[0128] 13 C NMR(101MHz,Chloroform-d)δ170.32,165.43,154.79,141.96,141.49,137.75,135.12,133.31,127.8 3,127.82,120.98,120.76,119.21,119.02,118.03,45.49,45.29,45.08,44.87,44.66,44.45,44.24.

[0129] HRMS(ESI)calcd for C19H15BrN2O3S[M+H]+: 430.9960; Found: 430.99611.

[0130] Embodiment 9:

[0131] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-9) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equal molar amount of "p-fluorophenylboronic acid".

[0132] The obtained target compound 4-9 is an off-white solid with a yield of 64%. The structure is shown below:

[0133]

[0134] Its NMR data are as follows:

[0135] 1 H NMR (400MHz, DMSO-d6) δ8.70(s,1H),8.42–8.32(m,4H),8.22(s,1H),7.94(d,J=5.1Hz,1H),7.86–7.77(m,4H),7.58–7.42(m,4H).

[0136] 13 C NMR(101MHz,DMSO-d6)δ169.20,158.28,144.20,143.94,137.17,136.57,133.01,130.42,130.39,127.24, 127.06,125.68,122.32,120.30,119.10,118.26,115.72,40.61,40.41,40.20,39.99,39.78,39.57,39.36.

[0137] HRMS(ESI)calcd for C19H15FN2O3S[M+H]+: 371.0821; Found: 371.08203.

[0138] Embodiment 10:

[0139] This example provides a method for preparing a phenylmethylsulfonyl compound (numbered as compound 4-10) targeting AR / AR-V7 dual proteins. Compared with Example 1, most of the operation steps are the same, except that the "179 mg (1.3 mmol) of p-hydroxyphenylboronic acid" in step (4) is replaced by an equimolar amount of "p-nitrophenylboronic acid".

[0140] The target compound 4-10 was obtained as a yellow solid with a yield of 60%. The structure is shown below:

[0141]

[0142] Its NMR data are as follows:

[0143] 1 H NMR(400MHz,Chloroform-d)δ8.70(s,1H),7.93(d,J=8.5Hz,1H),7.65–7.58(m,2H),7.4 7(q,J=7.4,6.7Hz,5H),7.34(d,J=7.8Hz,2H),7.29–7.18(m,3H),7.13(d,J=7.8Hz,1H).

[0144] 13 C NMR(101MHz,Chloroform-d)δ163.49,154.44,153.76,145.28,144.73,139.02,136.01,128.50,127.6 7,127.09,124.18,121.19,119.07,114.81,112.68,45.39,45.18,44.97,44.76,44.55,44.35,44.14.

[0145] HRMS(ESI)calcd for C19H15N3O5S[M+H]+: 398.0766; Found: 398.07658.

[0146] Example 11 Inhibition of the activity of prostate cancer cells by phenylmethylsulfonyl compounds

[0147] In this example, the target compounds synthesized in Examples 1-10 were tested for prostate cancer cell inhibition activity, and the test for prostate cancer cell inhibition activity included the following experiments and operation steps:

[0148] (1) Cell lines and cell culture

[0149] 22RV1 and C4-2B cells were cultured in RPMI1640, 10% FBS, 100 units / ml penicillin and 0.1 mg / ml streptomycin. All cells were purchased from the Chinese Academy of Sciences Type Culture Collection, identified by short tandem repeat fingerprinting, and tested negative for mycoplasma.

[0150] (2) Real-Time Quantitative PCR

[0151] Total RNA was extracted from cell samples using TRIzol reagent (YEASEN, China). RNA concentration and purity were assessed using NanoDrop 2000 (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. qPCR reactions were performed on a QuantStudio 6Flex PCR instrument (Applied Biosystems, USA) using SYBR Green Master Mix (Servicebio, China). ACTIN was used as an internal control for normalization. Each sample was set up in triplicate, and the data were analyzed using the 2^-ΔΔCt method.

[0152] (3) Western Blot

[0153] Cell samples were lysed using RIPA lysis buffer (containing protease inhibitors) and then centrifuged to remove cell debris. Protein concentration was determined using the BCA method. Equal amounts of protein samples were separated by 8% SDS-PAGE. The separated proteins were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated overnight with a specific primary antibody (e.g., anti-β-ACTIN or an antibody against the target protein, diluted 1:1000). Subsequently, the corresponding secondary antibody (HRP-conjugated) was incubated and finally visualized using ECL reagent (YEASEN, China), and the signal was detected using a darkroom technique.

[0154] (4) EdU cell proliferation assay

[0155] Cells were treated according to the manufacturer's instructions (RIBOBIO, China). EdU (5-ethynyl-2'-deoxyuridine) was added to the cell culture medium and incubated for the specified time. Cells were fixed with 4% paraformaldehyde and stained with Click-iT reaction reagent. Nuclear staining was performed according to the kit instructions, and images were observed and captured using a fluorescence microscope (Zeiss, Germany).

[0156] The experimental results are shown in Table 1 and Figure 2 , Figure 2Among them, ** indicates 0.001 < p < 0.01, and * indicates 0.01 < p < 0.05. It can be seen that when compared with the activities of reported prostate cancer cell inhibitors targeting the AR protein target such as enzalutamide (IC 50 = 36 nM), it was found that multiple synthesized compounds all had good inhibitory activities. In particular, three preferred compounds among them, 4-1, 4-4, and 4-7, their half-maximal inhibitory concentration IC 50 were 6.94 μM, 1.27 μM, and 0.12 μM respectively.

[0157] Table 1

[0158]

[0159]

[0160] Example 12: Inhibitory effect of the AR / AR-V7 small molecule inhibitor 4-7 (E7) on the in situ tumorigenesis ability of prostate cancer in mice

[0161] (1) BALB / c nude mice model

[0162] All BALB / c nude male mice (6 - 8 weeks old) were purchased from Charles River Laboratories (Beijing, China). All animals used in this study received humane care in accordance with applicable animal-related regulations, policies, and guidelines. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Nanchang University.

[0163] After anesthetizing the nude mice, the abdominal skin was disinfected with an alcohol pad under sterile conditions. A longitudinal incision of about 1 cm was made in the lower middle abdomen, and the abdominal wall was opened layer by layer to locate the bladder. The bladder was gently lifted with instruments, and the prostate was found behind the bladder. Using a sterile micro adjustable syringe, 600,000 22RV1 cells were injected into the posterior lobe of the prostate of three groups of nude mice. Then the prostate was put back into the body, and the muscle and skin were sutured. The wound was disinfected, and the nude mice were sent back to the cage for feeding.

[0164] An appropriate amount of antibiotic was applied to the wound to prevent infection and abscess formation, and the activity level and food intake of the nude mice were observed daily. On the 15th, 20th, and 25th days, in vivo imaging equipment (MILABS, HOLAND) was used to capture images of the nude mice to obtain data; on the 25th day, the mice were sacrificed, and the volume and weight of the prostate tumors of the mice were measured and statistically analyzed.

[0165] (2) Immunohistochemistry (IHC)

[0166] The tissue samples will be paraffin-embedded and cut into 4 μm thick sections, and then placed on slides. Antigen retrieval is performed using an antigen retrieval solution (such as citrate buffer), followed by blocking with PBS containing 5% bovine serum albumin (BSA). The sections are incubated overnight with a specific primary antibody (such as an antibody against the target protein, diluted 1:200), and then incubated with the corresponding secondary antibody (such as HRP-conjugated). Staining is performed using the DAB color development system (Vector Labs, USA) and counterstained with hematoxylin. Use a microscope (such as Olympus BX53) to observe the staining results and take images.

[0167] The experimental results are shown in Figure 3 Compound 4-7 (E7) has a relatively good inhibitory effect on the size and volume of mouse tumors. It can be seen that compound 4-7 (E7) with the best cell inhibitory activity also shows excellent therapeutic effect on prostate cancer mice.

[0168] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein, characterized in that: The molecular structure of the phenylmethylsulfonyl compound is shown in Formula I: Wherein, R is selected from any one of a hydrogen atom, a halogen, an alkyl group, a hydroxyl group, a methoxy group, an ester group, a cyano group or a nitro group.

2. A method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein as claimed in claim 1, characterized in that: The steps include: S1, dissolving p-aminobenzenesulfonyl chloride and di-tert-butyl dicarbonate in a solvent, reacting under alkaline conditions, and washing and drying the product to obtain compound 1; S2, dissolving benzamide in DMF, adding sodium hydride, cooling to 0°C, adding the compound 1 obtained in step S1 dropwise, stirring for reaction, and washing and drying the reaction product to obtain compound 2; S3, dissolving the compound 2 obtained in step S2 in a solvent, adding trifluoroacetic acid dropwise at low temperature, and reacting at room temperature after the addition is complete, and extracting and drying the reaction product to obtain compound 3; S4, dissolving the compound 3 obtained in step S3 and a phenylboronic acid derivative in a solvent, adding a catalyst, reacting under alkaline conditions and a gas atmosphere to obtain a product, and washing and purifying the product to obtain a final product, i.e., a phenylmethylsulfonyl compound; The structural formulas of compound 1, compound 2 and compound 3 are shown as follows:

3. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 double protein according to claim 2, characterized in that: In step S1, alkaline conditions are achieved by adding triethylamine. The amount of aminobenzenesulfonyl chloride, di-tert-butyl dicarbonate, triethylamine and solvent is in the relationship of 1mmol:1.2mmol:2mmol:(1.5-2)mL, and the amount of the solvent is the total amount used to dissolve aminobenzenesulfonyl chloride, di-tert-butyl dicarbonate and triethylamine, ensuring that all substances are fully dissolved. The process conditions of the reaction are: the reaction temperature is 0-25°C, and the reaction time is 6-12h.

4. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein according to claim 2, characterized in that: In step S2, the dosage of compound 1, benzamide, sodium hydride and DMF is 1 mmol: (1.1-1.3) mmol: (5-10) mmol: (1.5-2) mL; the process conditions of the stirring reaction are: the reaction temperature is 0-25° C., and the reaction time is 12-24 h.

5. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 double protein according to claim 2, characterized in that: In step S3, the solvent is dichloromethane, and the amount of the compound 2, trifluoroacetic acid, and the solvent is in the following relationship: 1 mmol:(2-8) mmol:3 mL.

6. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein according to claim 2, characterized in that: In step S3, the reaction temperature is 0-25°C, and the reaction time is 6-12h.

7. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein according to claim 2, characterized in that: In step S4, the phenylboronic acid derivative is a compound of the following general structural formula: Wherein R is selected from any one of a hydrogen atom, a halogen, an alkyl group, a hydroxyl group, a methoxy group, an ester group, a cyano group, and a nitro group.

8. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein according to claim 2, characterized in that: In step S4, the solvent is dichloromethane, the catalyst is copper acetate, and the gas atmosphere is oxygen or air atmosphere; the alkaline condition is achieved by adding triethylamine; the dosage of the compound 3, phenylboronic acid derivative, triethylamine, copper acetate and solvent is in the following relationship: 1 mmol: (1.1-1.3) mmol: 3 mmol: (0.05-1) mmol: (5-10) mL.

9. The method for preparing a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein according to claim 2, characterized in that: In step S4, the reaction temperature is 35-50°C, and the reaction time is 12-48h.

10. A use of a phenylmethylsulfonyl compound targeting AR / AR-V7 dual protein as claimed in claim 1, characterized in that: The phenylmethylsulfonyl compound targeting the AR / AR-V7 dual protein is used to prepare a drug for inhibiting the activity of prostate cancer cells and inhibiting the malignant progression of prostate cancer.

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