Thiohydantoin compound with dual inhibitory effects on androgen receptor and histone deacetylase 6 and use thereof

Abstract

The present invention discloses a thiohydantoin compound having dual inhibitory effects on the androgen receptor and histone deacetylase 6. The present invention also discloses a pharmaceutically acceptable salt of the thiohydantoin compound having dual inhibitory effects on the androgen receptor and histone deacetylase 6. The present invention also discloses the use of the compound in preparing a dual inhibitor of the androgen receptor and histone deacetylase 6. The present invention also discloses the use of the compound in preparing a drug for treating tumors associated with the androgen receptor and histone deacetylase 6. The drug prepared using the compound has an anti-prostate cancer effect, providing a new direction for the research of anti-prostate cancer drugs and having important significance for the development of anti-prostate cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a thiohydantoin compound, specifically a thiohydantoin compound with dual inhibitory effects on androgen receptor and histone deacetylase 6 and a use thereof. Background Art

[0002] Prostate cancer is one of the most common malignancies in men. Despite advances in early detection and treatment, PCa remains a leading cause of cancer-related morbidity and mortality, ranking second worldwide among malignant tumors affecting men. Prostate cancer is a malignant tumor caused by the abnormal proliferation of cells in prostate tissue. In the early stages of the disease, PCa can be identified by elevated prostate-specific antigen (PSA) levels, which may be accompanied by urinary symptoms. As the tumor progresses, the symptoms of prostate cancer become increasingly severe, with symptoms such as ureteral obstruction, systemic pain, and cancer cell metastasis.

[0003] In 1941, Huggins and Hodges demonstrated that reducing serum androgen concentrations by eliminating androgens (e.g., through orchiectomy) or administering exogenous estrogens could effectively induce tumor regression and alleviate clinical symptoms. This groundbreaking research first revealed the close relationship between prostate cancer and androgen activity in the body. Currently, clinical treatments for prostate cancer include surgery, chemotherapy, radiotherapy, immunotherapy, and androgen deprivation therapy (ADT), all aimed at prolonging patient survival. ADT, by suppressing androgen levels, can temporarily delay the progression of PCa. However, after 2-3 years of treatment, patients often develop drug resistance, leading to castration-resistant prostate cancer (CRPC), for which there is currently no effective treatment.

[0004] With the increasing prevalence of prostate cancer in recent years, the clinical demand for more effective drugs has become increasingly urgent. The progression of CRPC involves the interaction of multiple genes and pathways within the androgen receptor. Although the specific mechanisms are not fully understood, it has been revealed that inhibiting a single target or signaling pathway alone is not sufficient to effectively prevent CRPC progression. Therefore, the development of multi-target, multi-pathway anti-PCa drugs is a major research direction for the future.

[0005] AR (androgen receptor) and HDAC (histone deacetylase 6) play key roles in the development and progression of various tumors. A phase I / II clinical trial (NCT00878436) demonstrated that the HDAC inhibitor panobinostat combined with the AR antagonist bicalutamide had a synergistic therapeutic effect in the treatment of CRPC. AR antagonists, as single-target drugs, are unable to fully inhibit tumor cell signaling by blocking only a single receptor, resulting in limited efficacy and the development of drug resistance. Furthermore, combination therapy may affect pharmacokinetic properties due to drug interactions, increasing the risk of adverse reactions. Therefore, research on single-molecule, multi-target anti-tumor drugs is becoming a hot topic, aiming to overcome these limitations and improve therapeutic efficacy.

[0006] AR is an important target for the treatment of prostate cancer. After binding to androgens intracellularly, it dissociates from heat shock protein 90 (HSP90), undergoes conformational changes, forms a dimer, and enters the nucleus through the nuclear pore. Acting as a transcription factor, it regulates the transcription of target genes, thereby stimulating the growth of PCa cells. Histone deacetylases are a class of proteases that catalyze the deacetylation of histone lysine residues. Increased HDAC expression leads to histone deacetylation, causing DNA to wrap more tightly around the histones, thereby affecting the expression of tumor suppressor genes. HDACs have been found to increase during treatment with AR antagonists. In particular, HDACs are responsible for regulating DNA / histone interactions in chromatin in both anti-androgen-sensitive and resistant cell lines. HDACs have also been shown to be essential enzymes for AR function.

[0007] According to literature reports, AR and HDAC6 are closely related to the progression of prostate cancer. HSP90 is a substrate of HDAC6 and a molecular chaperone of AR. Inhibiting HDAC6 activity causes Hsp90 to be overacetylated, thereby weakening HSP90's ability to bind to ATP. The disruption of chaperone function leads to the degradation of the client protein (AR) by the proteasome. At the same time, the AR-HSP90 interaction allows AR to maintain high affinity for binding to androgen ligands, but inhibiting HDAC6 can effectively affect the formation of a complex between AR and the heat shock protein HSP90, thereby affecting AR activation, stability, and nuclear migration. Therefore, it is speculated that improving HDAC6 selectivity is beneficial for inhibiting the proliferation of prostate cancer cells. Therefore, dual-target inhibitors of AR and HDAC6 have potential anti-tumor activity and therapeutic potential.

[0008] Therefore, an object of the present invention is to provide a class of AR and HDAC6 dual-target inhibitors that improve the selective inhibitory activity of HDAC6 while maintaining AR inhibitory activity, which have potential anti-prostate cancer tumor activity and potential for treating prostate cancer. Summary of the Invention

[0009] In response to the above-mentioned technical problems in the prior art, the present invention provides a thiohydantoin compound with dual inhibitory effects on androgen receptors and histone deacetylase 6 and its use. The thiohydantoin compound with dual inhibitory effects on androgen receptors and histone deacetylase 6 and its use are intended to solve the technical problem of poor effect of drugs in the prior art in treating prostate problems.

[0010] The present invention also provides a thiohydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6, selected from any one of the following structural formulas:

[0011] .

[0012] The present invention also provides a pharmaceutically acceptable salt of the above-mentioned thiohydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6.

[0013] The present invention also provides a pharmaceutical composition comprising the above-mentioned compound and a pharmaceutically acceptable carrier and / or excipient.

[0014] The present invention also provides a pharmaceutical composition comprising the above-mentioned pharmaceutically acceptable salt, solvate, polymorph, isomer or prodrug and a pharmaceutically acceptable carrier and / or excipient.

[0015] Furthermore, the pharmaceutically acceptable adjuvant and / or excipient is a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler and / or a disintegrant.

[0016] Furthermore, the dosage form of the drug is an aqueous dispersion, liquid, gel, syrup, slurry, suspension, aerosol, controlled release agent, fast dissolving agent, effervescent agent, lyophilized agent, tablet, powder, pill, dragee, capsule, delayed release agent, extended release agent, pulse controlled release agent, multi-particulate or immediate release agent.

[0017] The present invention also provides the use of the above compound in the preparation of a dual inhibitor of androgen receptor and histone deacetylase 6.

[0018] The present invention also provides use of the pharmaceutically acceptable salt in the preparation of a dual inhibitor of androgen receptor and histone deacetylase 6.

[0019] The present invention also provides use of the above compound in preparing a drug for treating tumors related to androgen receptor and histone deacetylase 6.

[0020] The present invention also provides use of the pharmaceutically acceptable salt in preparing a drug for treating tumors associated with androgen receptor and histone deacetylase 6.

[0021] This invention utilizes structural modifications based on an AR / HDAC dual-target inhibitor to enhance HDAC6 selectivity. Using the AR / HDAC dual-target inhibitor developed by Meng Xiangguo et al. (CN 109796437B: Hydantoin and thiohydantoin compounds with dual AR and HDAC inhibitory activity and uses) as the lead compound, the original triazole benzene ring fragment and linker length (n=4-6) were retained. The inhibitors were modified using methods reported in the literature for enhancing HDAC6 selectivity, taking into account the structure-activity relationship of the parent nucleus and the substrate and electrical properties of the receptor / enzyme protein cavity. The goal is to enhance HDAC6 selectivity while maintaining AR inhibitory activity.

[0022] The compounds of the present invention competitively bind to AR, antagonizing androgens, thereby inhibiting AR and thus being effective against prostate cancer. On the other hand, the activity of HDAC is closely related to the development of tumors. The compounds of the present invention first bind to AR, selectively accumulate, and then release and bind to the second target HDAC, enhancing the anti-tumor activity of the compounds.

[0023] The present invention is to Zn 2+ By modifying the binding group, hydrophobic linker substituent, and recognition group substituent, and examining the effects of structural modifications on HDAC6 selectivity, the AR / HDAC6 dual-target inhibitors accumulate molecular pharmacophores that inhibit HDAC6 activity locally at the AR, thereby reducing the poor selectivity and toxicity of single-agent HDAC inhibitors. This addresses the low efficacy of existing technologies against castration-resistant prostate cancer caused by AR overexpression and AR gene mutations.

[0024] Compared with existing technologies, the present invention represents a significant technological advancement. Compared with the AR / HDAC dual-target inhibitor developed by Meng Xiangguo et al. ("Hydantoin and thiohydantoin compounds with dual AR and HDAC inhibitory effects and uses thereof" (CN 109796437B)), the present invention significantly enhances histone deacetylase 6 inhibition while maintaining AR inhibition, and also produces AR degradation. This provides a new direction for the research of anti-prostate cancer drugs and is of great significance for the development of such drugs.

[0025] It should be understood that within the scope of the present invention, the above-mentioned technical features and the technical features described in detail below (e.g., in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they are not described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1: The effects of compound I-3 and lead compound B4 on the expression of LNCaP prostate cancer cell-related proteins were studied by Western-Blot. DETAILED DESCRIPTION

[0027] Through extensive and in-depth research, the inventors have modified the structure of the AR / HDAC dual-target inhibitor B4 (shown below), achieving improved HDAC6 selectivity while maintaining AR inhibitory activity, and completed the present invention on this basis.

[0028]

[0029] The compounds of formula (I) described above can be synthesized using standard synthetic techniques or well-known techniques in combination with the methods described herein. In addition, the solvents, temperatures and other reaction conditions mentioned herein may vary.

[0030] The starting materials used in the synthesis of compounds of formula (I) can be synthesized or obtained from commercial sources. The compounds described herein and other related compounds having different substituents can be synthesized using known techniques and raw materials. The general method of preparing the compounds can be modified by using appropriate reagents and conditions to introduce different groups into the molecular formula provided herein.

[0031] The compounds of Formula (I) described herein are synthesized using the synthetic routes shown in the following schemes. In some embodiments, the compounds described herein can be prepared by the following methods. The following methods and examples are intended to illustrate these methods. These schemes and examples should not be construed as limiting the present invention in any way. The compounds described herein can also be synthesized using standard synthetic techniques known to those skilled in the art, or a combination of methods known in the art and the methods described herein.

[0032] The synthesis method of compound (I) may include the following steps:

[0033]

[0034] Example 1 Preparation of Compound I:

[0035] Step A: Synthesis of intermediate S16

[0036] Under nitrogen protection and controlled at 0°C in an ice bath, 3-fluoro-4-iodotoluene S15 (10.00 g, 42.37 mmol) and trimethylsilyl acetylene (4.58 g, 46.60 mmol) were dissolved in triethylamine (50.00 mL, 5 V). Tetrakis(triphenylphosphine)palladium (489.60 mg, 423.68 μmol) and CuI (1.21 g, 6.36 mmol) were then added in sequence. The mixture was stirred at 0°C for 1 h and then at room temperature for 1 h. A solid precipitated in the reaction solution, which was filtered. The filtrate was added to 20 mL of a saturated solution of ammonia and ammonium chloride (1:4), extracted with ethyl acetate, washed three times with water, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (n-heptane) to obtain 7.50 g of an oily product, S16, in a yield of 85.79%. 1 H NMR (400 MHz, DMSO-d6) δ7.39 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 10.8 Hz, 1H), 7.02 (d, J = 7.8 Hz,1H), 2.33 (s, 3H), 0.23 (s, 9H).

[0037] Step B: Synthesis of intermediate S17

[0038] Under nitrogen, intermediate S16 (8.70 g, 42.16 mmol) was dissolved in acetonitrile (87.00 ml, 10 V). Azobisisobutyronitrile (1.04 g, 6.32 mmol) was then added, followed by N-bromosuccinimide (8.26 g, 46.38 mmol) in two portions. The reaction temperature was raised to reflux and allowed to react for 4 h. After monitoring the reaction by UV-visible spot-plate TLC, the remaining N-bromosuccinimide was added and the reaction continued for 8 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate / water, washed three times with water, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (n-heptane) to obtain 5.21 g of oily product S17 in a yield of 43.32%. 1 H NMR (400 MHz, DMSO- d 6)δ 7.51 (t, J = 7.7 Hz, 1H), 7.40 (dd, J = 10.4, 1.5 Hz, 1H), 7.29 (dd, J=8.0, 1.6 Hz, 1H), 4.71 (s, 2H), 0.24 (s, 9H). δ 7.51 (t, J = 7.7 Hz, 1H), 7.40(dd, J = 10.4, 1.5 Hz, 1H), 7.29 (dd, J = 8.0, 1.6 Hz, 1H), 4.71 (s, 2H), 0.24 (s, 9H).

[0039] Step C: Synthesis of intermediate S19

[0040] Under nitrogen protection, intermediate S17 (5.00 g, 17.53 mmol) and raw material 1-aminocyclobutane-1-carboxylic acid methyl ester S18 (2.72 g, 21.04 mmol) were dissolved in dimethylacetamide (50.00 ml, 10 V). Potassium acetate (4.30 g, 43.82 mmol) as a base and cuprous chloride (694.15 mg, 7.01 mmol) as a catalyst were added in sequence. 2-ethylcyclohexanone (1.04 g, 6.32 mmol) was added as a ligand. The reaction temperature was raised to 100 °C. After reacting for 2 h, the reaction was monitored by spot plate TLC under ultraviolet light. The reaction was stopped after the reaction of the raw materials was complete. After cooling to room temperature, ammonia:ammonium chloride saturated solution (1:4) was added for 20 min. mL, extracted with ethyl acetate / water, washed three times with water, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (n-heptane) to obtain 2.36 g of oily product S19 with a yield of 51.52%. 1 H NMR (400 MHz, DMSO-d6) δ 7.46 (t, J = 7.7 Hz, 1H), 7.27 (d, J = 10.9 Hz, 1H),7.18 (d, J = 9.2 Hz, 1H), 4.41 (s, 1H), 3.61 (s, 3H), 3.56 (d, J = 6.1 Hz,2H), 2.94 (d, J = 6.1 Hz, 1H), 2.39 – 2.20 (m, 2H), 2.02 – 1.88 (m, 3H), 1.77(tdt, J = 9.3, 4.4, 2.3 Hz, 1H).

[0041] Step D: Synthesis of intermediate S20

[0042] Under nitrogen protection, 5-amino-3-(trifluoromethyl)picolinonitrile S1 (5.00 g, 26.72 mmol) was dissolved in acetone (50.00 mL, 5 V) in an ice bath at 0°C. Thiophosgene (9.22 g, 80.16 mmol) was slowly added dropwise and stirred for 1 h. After the reaction was complete, ice water was added, the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at low temperature to afford a dark brown oil, S20. The crude product was refrigerated and used directly in the next step without further purification.

[0043] Step E: Synthesis of intermediate S21

[0044] Under nitrogen, intermediate S19 (0.64 g, 2.45 mmol) and intermediate S20 (1.12 g, 4.90 mmol) were dissolved in dimethylacetamide (6.40 ml, 10 V) and triethylamine (495.71 mg, 4.90 mmol) was added. The mixture was heated to 100°C and stirred for 2 h. After completion of the reaction, it was cooled to room temperature. The mixture was extracted with ethyl acetate, washed three times with water, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (n-heptane) to obtain 0.84 g of yellow oil S21 in a yield of 74.81%. MS: 459.1 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 9.23 (d, J = 1.8Hz, 1H), 8.78 (d, J = 1.9 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.39 (d, J =10.7 Hz, 1H), 7.33 – 7.25 (m, 1H), 5.31 (s, 2H), 4.49 (s, 1H), 2.67 – 2.57(m, 2H), 2.53 (s, 1H), 2.11 – 1.93 (m, 1H), 1.81 (dtd, J = 14.4, 10.0, 4.7Hz, 1H), 1.25 (d, J = 9.4 Hz, 1H).

[0045] Step F: Synthesis of Intermediate S22-a

[0046] Under nitrogen, intermediate S21 (0.30 g, 654.4 μmol) and intermediate S13-a (0.52 g, 1.31 mmol) were dissolved in dimethyl sulfoxide (3 mL, 10 V) and the catalyst, cuprous iodide (124.63 mg, 654.40 μmol), was added. The temperature was raised to 45°C and stirred for 2 h. After completion of the reaction, the mixture was cooled to room temperature. 3 mL of a saturated ammonia:ammonium chloride solution (1:4) was added. The mixture was extracted with ethyl acetate, washed three times with water, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate / petroleum ether) to afford 0.12 g of S22-a as a white solid in a yield of 21.35%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.23 (s, 1H), 9.25 (d, J = 2.0 Hz, 1H), 8.80 (d, J = 2.0 Hz,1H), 8.36 (d, J = 3.6 Hz, 1H), 8.11 (t, J = 7.9 Hz, 1H), 7.49 – 7.38 (m, 2H), 7.33 – 7.23 (m, 15H), 5.34 (s, 2H), 4.30 (t, J = 6.7 Hz, 2H), 2.75 – 2.60 (m,2H), 2.54 (s, 2H), 2.09 – 1.96 (m, 1H), 1.82 (dt, J = 10.2, 5.4 Hz, 3H), 1.57– 1.50 (m, 2H), 1.21 – 1.12 (m, 2H).

[0047] Step G: Synthesis of intermediate S22-b

[0048] Compound S22-b was synthesized by a method similar to that described in step S22-a in Example 1, except that S13-a was replaced by S13-b, with a yield of 26.26%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.16 (s, 1H),9.25 (s, 1H), 8.80 (d, J = 2.0 Hz, 1H), 8.40 (d, J = 3.6 Hz, 1H), 8.10 (t, J= 7.9 Hz, 1H), 7.41 (dd, J = 17.0, 10.1 Hz, 2H), 7.34 – 7.27 (m, 15H), 5.34(s, 2H), 4.33 (t, J = 7.0 Hz, 2H), 2.66 (q, J = 10.2 Hz, 2H), 2.54 (s, 2H), 2.07 – 1.97 (m, 1H), 1.78 (ddp, J = 26.7, 20.9, 6.7 Hz, 5H), 1.24 (t, J = 7.4Hz, 2H), 0.97 (dt, J = 13.5, 7.3 Hz, 2H).

[0049] Step H: Synthesis of Intermediate S22-c

[0050] Compound S22-c was synthesized by a method similar to that described in step S22-a in Example 1, except that S13-a was replaced by S13-c, with a yield of 29.07%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.16 (s, 1H),9.25 (d, J = 1.9 Hz, 1H), 8.80 (d, J = 2.0 Hz, 1H), 8.43 (d, J = 3.6 Hz, 1H),8.10 (t, J = 7.9 Hz, 1H), 7.41 (dd, J = 16.8, 10.0 Hz, 2H), 7.30 (dd, J =13.2, 4.8 Hz, 15H), 5.34 (s, 2H), 4.38 (t, J = 7.0 Hz, 2H), 2.71 – 2.61 (m,2H), 2.54 (s, 2H), 2.09 – 1.95 (m, 1H), 1.78 (dp, J = 21.7, 7.0 Hz, 5H), 1.19– 1.06 (m, 4H), 0.99 (q, J = 7.4, 6.8 Hz, 2H).

[0051] Step I: Synthesis of Compound I-1

[0052] Under nitrogen protection and controlled at 0°C in an ice bath, intermediate S22-a (0.12 g, 139.71 μmol) was dissolved in dichloromethane. Trifluoroacetic acid (0.12 ml, 1 V) and triisopropylsilane (0.60 ml, 5 V) were added to remove the trityl protecting group, yielding compound I-1. After the reaction, the trifluoroacetic acid in the reaction solution was diluted with water, and then saturated sodium bicarbonate solution was slowly added to quench the reaction, generating bubbles. The pH of the reaction system was adjusted to 6-7, and the mixture was extracted with methanol / dichloromethane, washed three times with water, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (methanol / dichloromethane) to yield 32.00 mg of I-1 as a white solid in a yield of 37.15%. MS: 617.2 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 10.43 (s, 1H), 9.25 (s, 1H), 8.80 (s, 1H), 8.71 (s, 1H), 8.45 (d, J = 3.1 Hz, 1H), 8.10 (t, J = 7.8 Hz, 1H), 7.41 (dd, J = 28.0, 9.9 Hz, 2H), 5.34 (s, 2H), 4.44 (t, J = 6.8 Hz, 2H), 2.66 (q, J =10.6 Hz, 2H), 2.56 – 2.51 (m, 2H), 2.02 (d, J = 8.9 Hz, 3H), 1.89 – 1.78 (m, 3H), 1.52 – 1.43 (m, 2H).

[0053] Step J: Synthesis of Compound I-2

[0054] Compound I-2 was synthesized similarly to step I-1 described in Example 1, except that S22-a was replaced with S22-b. The yield was 29.53%. MS: 631.2 [M+H] + . 1 H NMR (400 MHz, DMSO- d6) δ10.34 (s, 1H), 9.26 (s, 1H), 8.81 (s, 1H), 8.67 (s, 1H), 8.45 (s, 1H), 8.11(t, J = 7.9 Hz, 1H), 7.42 (dd, J = 27.0, 10.0 Hz, 2H), 5.34 (s, 2H), 4.43 (t, J = 7.0 Hz, 2H), 2.67 (q, J = 10.5 Hz, 2H), 2.53 (d, J = 9.5 Hz, 2H), 2.03(q, J = 9.3 Hz, 1H), 1.94 (t, J = 7.3 Hz, 2H), 1.85 (tt, J = 14.6, 6.9 Hz,3H), 1.53 (p, J = 7.4 Hz, 2H), 1.24 (q, J = 7.3 Hz, 3H).

[0055] Step K: Synthesis of Compound I-3

[0056] Compound I-3 was synthesized similarly to step I-1 described in Example 1, except that S22-a was replaced with S22-c. The yield was 36.69%. MS: 645.2 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ10.34 (s, 1H), 9.25 (s, 1H), 8.80 (s, 1H), 8.66 (s, 1H), 8.45 (s, 1H), 8.10(t, J = 7.9 Hz, 1H), 7.41 (dd, J = 27.3, 9.9 Hz, 2H), 5.34 (s, 2H), 4.42 (t, J = 7.0 Hz, 2H), 2.66 (q, J = 10.9, 10.2 Hz, 2H), 2.52 (d, J = 10.1 Hz, 2H),2.02 (q, J= 9.5 Hz, 1H), 1.92 (t, J = 7.3 Hz, 2H), 1.84 (dq, J = 18.2, 10.8,8.6 Hz, 3H), 1.47 (p, J = 7.3 Hz, 2H), 1.34 – 1.18 (m, 6H). Example 2

[0057] Apalutamide and vorinostat were used as positive control compounds, and all test samples were -2 The initial concentration of M was prepared, and cell-grade dimethyl sulfoxide was used as the solvent for dissolution, followed by aliquoting and storage at -20 °C.

[0058] Step A: Anti-growth activity test of AR / HDAC6 dual-target compounds against human prostate cancer cell LNCaP

[0059] Experimental methods

[0060] 1×10 4 Cells were seeded in 96-well plates and allowed to adhere for 24 hours before treatment with blank control (culture medium) or various concentrations of compound (120, 100, 80, 40, 20, 10, 5, 2.5, and 1.25 μmol / L, with the final DMSO concentration kept below 0.1%) in the corresponding culture medium for 48 hours. Following treatment, 10 μL of CCK-8 solution was added to each well and incubated in a 37°C incubator for 3 hours. After incubation, absorbance was measured at 450 nm using a microplate reader.

[0061] Data Analysis

[0062] Cell growth survival rate = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 ) × 100%

[0063] Statistical analysis was performed based on the absorbance data to evaluate the inhibitory effect of the compound on cell growth. The dose-effect relationship and half-maximal inhibitory concentration (IC) of the compound were further analyzed by calculating the cell survival rate. 50 ).

[0064] Experimental results: The results of the growth inhibition test on LNCaP prostate cancer cells are shown in Table 1. The positive drug apalutamide IC 50=106.10 μM, indicating that the inhibitory activity of Class I compounds was comparable to that of lead compound B4. The growth inhibitory activity of Class I compounds against LNCaP prostate cancer cells gradually increased as the length of the linker chain increased from 4 carbon atoms in I-1 to 6 carbon atoms in I-3.

[0065] Table 1 Inhibitory effects of compounds on the growth of LNCaP prostate cancer cells

[0066]

[0067] Step B: Selective inhibitory activity of AR / HDAC6 dual-target compounds against HDAC

[0068] Experimental methods

[0069] (1) The test compound was diluted to a starting concentration of 10 μM and the positive control (SAHA) was diluted to a starting concentration of 3 μM. A total of 8 concentration points were tested in duplicate. Control wells were set up: 250 nL of 100% DMSO was added to the Max well (containing DMSO and enzyme) and the Min well (no enzyme well).

[0070] (2) Prepare a 1.67× enzyme solution using the 1× reaction solution. Add 15 μL of the 1.67× enzyme solution to each well. For the Min well, add 15 μL of the 1.6× reaction buffer (instead of the enzyme solution). Incubate at room temperature for 15 min to allow the enzyme to fully bind to the compound.

[0071] (3) Prepare 2.5× substrate mixture with 1× reaction buffer, add 10 μL of 2.5× substrate mixture to each well, and start the assay immediately.

[0072] (4) Use the Synergy multifunctional microplate reader to dynamically monitor changes in fluorescence signals and record data for subsequent analysis.

[0073] Data Analysis

[0074] The kinetic data of the linear phase of the enzymatic reaction were used to obtain the slope through linear regression analysis, and then the percentage inhibition rate was calculated using the following formula:

[0075] %Inhibition=100×[Mean(Max)−Sample Signal] / [Mean(Max)−Mean(Min)]

[0076] Where: Mean(Max) is the mean of the slope values of the Max wells; Mean(Min) is the mean of the slope values of the Min wells; Sample Signal is the slope value of the compound wells.

[0077] Fitting the dose-effect curve: The log value of the compound concentration is used as the X-axis and the corresponding percentage inhibition rate is used as the Y-axis. The log (inhibitor) vs. response -Variable slope analysis software GraphPad Prism 5 is used to fit the dose-effect curve to obtain the IC value of each compound for enzyme inhibition. 50 value.

[0078] The experimental results are shown in Table 2: SAHA has almost similar inhibitory activity against the two targets, and is a typical broad-spectrum HDAC inhibitor with no selective inhibitory effect. Among the class I compounds, when the 4 carbon atoms of I-1 are increased to 6 carbon atoms of I-3, the selective inhibitory activity against HDAC1 / HDAC6 decreases, and I-1 (HDAC6 IC 50 = 32.84 nM, HDAC1 IC 50 = 861.95 nM) showed a 26-fold increase in the selective inhibitory activity against HDAC6, which was superior to the lead compound B4 (HDAC6 IC 50 = 13.60 nM, HDAC1IC 50 = 28.94 nM). Therefore, when the recognition group is benzyl, the introduction of a branch chain on the hydrophobic linker or the increase of the branch chain length will affect its selective inhibitory activity against HDAC6.

[0079] Table 2 IC values of compounds against HDAC1 and HDAC6 50 value

[0080]

[0081] Step C: Effects of AR / HDAC6 dual-target compounds on related protein expression

[0082] Experimental methods

[0083] B4 was used as a positive control, and LNCaP cells (Cell Bank, Chinese Academy of Sciences) were cultured at 2×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate, and the test compound I-3 was added at three concentrations of 1.25, 5, and 40 μmol / L for 24 h.

[0084] (1) Protein preparation: Prostate cancer cells were digested with trypsin, centrifuged, and the supernatant discarded. The cells were washed with PBS by centrifugation, the supernatant discarded, and the centrifuge tube containing the cells was placed on ice. The cells were mixed with RIPA buffer containing a protease inhibitor cocktail (Servicebio) and transferred to a 1.5 mL EP tube. The tube was lysed on ice for 30 min. The supernatant was collected by centrifugation at 12,000 rpm for 30 min at 4°C. The protein sample was mixed with 6× Loading buffer (Biyuntian) at a ratio of 1:5, denatured in a metal bath at 100°C for 10 min, and cooled on ice for later use.

[0085] (2) Electrophoresis transfer

[0086] Choose gels of different concentrations based on molecular weight. After setting up the apparatus, fill it with electrophoresis buffer. Simultaneously pull out the comb teeth with both hands and use a pipette to add the diluted sample to the comb holes. Electrophoresis begins at 80 V for 30 minutes. Once the sample has passed from the stacking gel to the separating gel, increase the voltage to 120 V for 1 hour. When the marker reaches the bottom of the gel, stop the electrophoresis and discard the buffer. After the electrophoresis is complete, place the stripped separating gel in the pre-prepared transfer buffer. Activate the PVDF membrane in methanol for 1 minute, remove it, and place it in the transfer buffer. Wet transfer is performed at 100 A for 60 minutes.

[0087] (3) Closed incubation

[0088] After the transfer was completed, the PVDF membrane was removed and incubated with 5% skim milk powder solution (Beyotime) on a shaker at room temperature for 1 h. After the transfer was completed, the PVDF was washed three times with TBST for 5 min each time.

[0089] Add the prepared primary antibody dilution solution (Anti-Androgen Receptor Recombinant antibody Abcam, Anti-alpha Tubulin (acetyl K40) Recombinant antibody Abcam, KLK3 / PSA Recombinant antibody Proteintech), incubate on a shaker in a 4°C refrigerator overnight, and wash the PVDF membrane three times with TBST for 5 minutes each the next day.

[0090] Place the plate in an incubator containing secondary antibody dilution (HRP-conjugated goat anti-mouse IgG Beyotime, HRP-conjugated goat anti-rabbit IgG (H+L) Servicebio) and incubate at room temperature for 1 hour. Wash the PVDF three times with TBST for 5 minutes each.

[0091] (4) Development

[0092] Prepare ECL color developing solution, evenly distribute the color developing solution on the membrane surface, and use a fully automatic chemiluminescence imaging instrument for exposure and development.

[0093] Results analysis: Western-Blot was used to study the effects of compound I-3 and lead compound B4 on the expression of LNCaP prostate cancer cell-related proteins (e.g. Figure 1 ), the target protein AR and the downstream protein PSA expression levels gradually decreased with increasing compound concentration; the acetylated expression level of AC-α-Tublin gradually increased with increasing compound concentration. Experiments showed that compound I-3's inhibitory activity against HDAC6 gradually increased with increasing concentration, and its inhibitory and degradation effects on AR also gradually increased.

Claims

1. A thiohydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6, characterized in that: Select any one of the following structural formulas: 。 2. A pharmaceutically acceptable salt or prodrug of the thiohydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6 according to claim 1.

3. A pharmaceutical composition, characterized in that: Comprising the compound according to claim 1 and a pharmaceutically acceptable carrier and / or excipient.

4. A pharmaceutical composition, characterized in that: Comprising the pharmaceutically acceptable salt or prodrug according to claim 2 and a pharmaceutically acceptable carrier and / or excipient.

5. A pharmaceutical composition according to claim 3 or 4, characterized in that: The pharmaceutically acceptable carrier and / or excipient is a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler and / or a disintegrant.

6. A pharmaceutical composition according to claim 3 or 4, characterized in that: The dosage form of the drug is an aqueous dispersion, liquid, gel, syrup, slurry, suspension, aerosol, controlled release agent, fast dissolution agent, effervescent agent, lyophilized agent, tablet, powder, pill, dragee, capsule, delayed release agent, extended release agent, pulse controlled release agent, multiparticulate or immediate release agent.

7. Use of the compound according to claim 1 in the preparation of a medicament for treating prostate cancer.

8. Use of the pharmaceutically acceptable salt or prodrug according to claim 2 in the preparation of a medicament for treating prostate cancer.

Images