Hydantoyl thiourea compound with dual inhibition effects of androgen receptor and histone deacetylase 6 and application of hydantoyl thiourea compound

Abstract

The invention discloses a hydantoin thiourea compound with dual inhibition effects of an androgen receptor and histone deacetylase 6, and further discloses a pharmaceutically acceptable salt of the hydantoin thiourea compound with the dual inhibition effects of the androgen receptor and the histone deacetylase 6. The invention further discloses application of the compound in preparation of a dual inhibitor serving as an androsinesin receptor and histone deacetylase 6. The invention also discloses application of the compound in preparation of drugs for treating tumors related to androgen receptors and histone deacetylase 6. The medicine prepared from the compound has an anti-prostatic cancer effect, provides a new direction for the research of the anti-prostatic cancer medicine, and is of great significance to the development of the anti-prostatic cancer medicine.

Description

Technical Field

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

[0002] Prostate cancer is one of the most common malignancies in men. Despite the progress in early detection and treatment, PCa remains a major factor leading to cancer-related morbidity and mortality, ranking second in the world among malignancies affecting men. Prostate cancer is a malignant tumor formed by abnormal proliferation of cells in the prostate tissue. In the early stage of the disease, patients can identify PCa by detecting an increase in the value of prostate-specific antigen (PSA), and at this time, urinary system symptoms may occur. As the tumor develops, the symptoms caused by prostate cancer will become more malignant, and symptoms such as ureteral obstruction, systemic pain, and cancer cell metastasis will appear.

[0003] In 1941, Huggins and Hodges were able to effectively induce tumor regression and relieve clinical symptoms by eliminating androgens (such as orchiectomy) or administering exogenous estrogen to reduce serum androgen concentration. This pioneering study first revealed the close relationship between prostate cancer and in vivo androgen activity. Currently, the clinical treatment methods for prostate cancer include surgical treatment, chemotherapy, radiotherapy, immunotherapy, and androgen deprivation therapy (ADT), etc., aiming to prolong the survival time of patients. Among them, ADT can stagewise delay the progression of PCa by inhibiting androgen levels. However, after 2-3 years of treatment, patients often develop into castration-resistant prostate cancer (CRPC) due to drug resistance, and there is currently no effective treatment method for CRPC.

[0004] In recent years, with the increasing prevalence of prostate cancer year by year, the clinical demand for more effective drugs has become increasingly urgent. The process of CRPC progression involves the result of multi-gene and multi-pathway interactions of androgen receptor. Although the specific mechanism is not fully understood, it has been revealed that inhibiting a single target or a single signaling pathway alone is not sufficient to effectively prevent the progression of CRPC. Therefore, developing multi-target and multi-pathway anti-PCa drugs is the main direction of future research.

[0005] Androgen receptor (AR) and histone deacetylase 6 (HDAC6) play key roles in the occurrence and development of various tumors. A phase I / II clinical trial (NCT00878436) showed that the HDAC inhibitor panobinostat combined with the AR antagonist bicalutamide has a synergistic therapeutic effect on CRPC. As a single-target drug, AR antagonist is difficult to comprehensively inhibit the signal transduction of tumor cells only by blocking a single receptor, with limited efficacy and easy to develop drug resistance. In addition, combination therapy may affect the pharmacokinetic properties due to drug interactions and increase the risk of adverse reactions. Therefore, the research on single-molecule multi-target anti-tumor drugs is becoming a current hotspot, aiming to overcome the above limitations and improve the therapeutic effect.

[0006] As an important target for the treatment of prostate cancer, after binding to androgens intracellularly, AR dissociates from heat shock protein 90 (HSP90), forms a dimer after conformational change, enters the nucleus through the nuclear pore, and as a transcription factor, 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. The increased expression of HDAC leads to histone deacetylation, making DNA more tightly wound around histones, thus affecting the expression of tumor suppressor genes. When using AR antagonists for treatment, an increase in HDACs was found. Among them, in androgen-sensitive and drug-resistant cell lines, HDAC is responsible for regulating the interaction between DNA / histones in chromatin, and HDAC has also been proven to be an enzyme necessary for AR function.

[0007] According to the literature, AR and HDAC6 are closely related to the progression of prostate cancer. As a substrate of HDAC6 and a molecular chaperone of AR, inhibiting the activity of HDAC6 will cause Hsp90 to be hyperacetylated, thereby weakening the ability of HSP90 to bind to ATP. The disruption of the chaperone function leads to the degradation of the client protein (AR) by the proteasome. At the same time, the AR-HSP90 interaction enables AR to maintain a high affinity for binding to androgen ligands, but inhibiting HDAC6 can effectively affect the formation of a complex between AR and heat shock protein HSP90, thereby affecting the activation, stability and nuclear migration of AR. Therefore, it is speculated that enhancing the selectivity of HDAC6 is beneficial to 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 the AR inhibitory activity, which have potential anti-prostate cancer tumor activity and therapeutic potential for prostate cancer. Summary of the Invention

[0009] In view of the above technical problems in the prior art, the present invention provides a hydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6 and its uses, and the hydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6 and its uses are to solve the technical problem of poor therapeutic effect of drugs on prostate in the prior art.

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

[0011] The present invention also provides a pharmaceutically acceptable salt of the above-mentioned hydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6.

[0012] The present invention also provides a pharmaceutical composition, comprising the above compound and a pharmaceutically acceptable carrier and / or excipient.

[0013] 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.

[0014] Further, the pharmaceutically acceptable auxiliary and / or excipient is a diluent, binder, surfactant, wetting agent, adsorption carrier, lubricant, filler and / or disintegrant.

[0015] Further, the dosage form of the drug is an aqueous dispersant, liquid, gel, syrup, medicinal pulp, suspension, aerosol, controlled release agent, rapid solvent, effervescent agent, freeze-dried agent, tablet, powder, pill, sugar-coated pill, capsule, delayed release agent, extended release agent, pulsed controlled release agent, multi-particle agent or immediate release agent.

[0016] 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.

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

[0018] The present invention also provides the use of the above compound in the preparation of a drug for treating tumors related to androgen receptor and histone deacetylase 6.

[0019] The present invention also provides the use of the above-mentioned pharmaceutically acceptable salt in the preparation of a drug for treating tumors related to androgen receptor and histone deacetylase 6.

[0020] The present invention performs structural modification on the basis of an AR / HDAC dual-target inhibitor in order to achieve an improvement in the selectivity of HDAC6. Using the AR / HDAC dual-target inhibitor developed from the hydantoin and hydantoin-thiourea compounds with dual inhibition of AR and HDAC by Meng Xiangguo et al. (CN 109796437B) as the lead compound; retaining the original triazole benzene ring fragment and the linker chain length (n = 4 - 6), combining the parent nucleus structure-activity relationship and the requirements of the receptor / enzyme protein cavity for the substrate and electrical properties; and performing modification through the methods for improving the selectivity of HDAC6 reported in known literature. It is expected to improve the selectivity of HDAC6 while maintaining the AR inhibitory activity.

[0021] The compounds of the present invention competitively bind to AR, antagonize androgens, achieve the effect of inhibiting AR, and thus are 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 to bind to the second target HDAC, enhancing the anti-tumor activity of the compounds.

[0022] The present invention modifies the Zn 2+ binding group, hydrophobic linker chain substituent, and recognition group substituent, and investigates the action law of the structural modification of such compounds on the selectivity of HDAC6. The AR / HDAC6 dual-target inhibitor has a molecular pharmacophore that inhibits the activity of HDAC6 by locally aggregating AR, thereby reducing the poor selectivity and toxicity problems of single HDAC inhibitors. It solves the problem of low potency of the existing technology against castration-resistant prostate cancer caused by overexpression of AR and gene mutations of AR.

[0023] Compared with the existing technology, the technological progress of the present invention is significant. Compared with the AR / HDAC dual-target inhibitor developed from the hydantoin and hydantoin-thiourea compounds with dual inhibition of AR and HDAC by Meng Xiangguo et al. (CN 109796437B), while maintaining the inhibitory effect on AR, the inhibition of histone deacetylase 6 is significantly improved, and an AR degradation effect is produced. It provides a new direction for the research of anti-prostate cancer drugs and is of great significance for the development of anti-prostate cancer drugs.

[0024] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1:Study the effects of Compound I-3 and lead compound B4 on the expression levels of proteins related to LNCaP prostate cancer cells by Western-Blot. Detailed implementation mode

[0026] Based on extensive and in-depth research, the present inventors carried out structural modification on the AR / HDAC dual-target inhibitor B4 (shown below). On the basis of maintaining the AR inhibitory activity, the selectivity of HDAC6 was improved, and on this basis, the present invention was completed.

[0027]

[0028] The compounds of formula (I) described above can be synthesized using standard synthetic techniques or well-known techniques in combination with the methods described in the text. In addition, the solvents, temperatures and other reaction conditions mentioned herein can be changed.

[0029] The starting materials for the synthesis of the compounds of formula (I) can be synthesized or obtained from commercial sources. The compounds described herein and other related compounds with different substituents can be synthesized using well-known techniques and raw materials. The general methods for the preparation of the compounds can be changed by using appropriate reagents and conditions for introducing different groups in the molecular formulas provided herein.

[0030] The compounds of formula (I) described herein are synthesized according to the synthetic route shown in the following scheme. In some embodiments, the compounds described herein can be prepared by the following methods. The following methods and examples are for illustrative purposes only. These procedures 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 by combining known methods in the art and the methods described herein.

[0031] The synthetic method of Compound (I) may include the following steps:

[0032] Example 1 Preparation of Compound I: Step A: Synthesis of Intermediate S16 Under nitrogen protection, the temperature was controlled at 0 °C in an ice bath. 3-Fluoro-4-iodotoluene S15 (10.00 g, 42.37 mmol) and trimethylsilylacetylene (4.58 g, 46.60 mmol) were dissolved in triethylamine (50.00 mL, 5 V). Then, catalyst tetrakis(triphenylphosphine)palladium (489.60 mg, 423.68 μmol) and CuI (1.21 g, 6.36 mmol) were added successively. The mixture was stirred at 0 °C for 1 h and then at room temperature for 1 h. Solids precipitated in the reaction solution, and filtration was carried out by suction. 20 mL of ammonia water:saturated ammonium chloride solution (1:4) was added to the filtrate, and it was extracted with ethyl acetate, washed with water three times, then washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure in vacuo, and the crude product was purified by column chromatography (n-heptane) to obtain 7.50 g of an oily substance S16 with 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).

[0033] Step B: Synthesis of intermediate S17 Under nitrogen protection, intermediate S16 (8.70 g, 42.16 mmol) was dissolved in acetonitrile (87.00 ml, 10V). Then, azobisisobutyronitrile (1.04 g, 6.32 mmol) was added. N-Bromosuccinimide (8.26 g, 46.38 mmol) was added in two portions. The reaction temperature was raised to reflux, and after reacting for 4 h, when it was monitored by TLC under UV light and the reaction could not proceed further, the remaining N-bromosuccinimide was added and the reaction continued for 8 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate / water, washed with water three times, then washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure in vacuo, and the crude product was purified by column chromatography (n-heptane) to obtain 5.21 g of an oily substance S17 with 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).

[0034] Step C: Synthesis of Intermediate S19 Under nitrogen protection, dissolve Intermediate S17 (5.00 g, 17.53 mmol) and raw material methyl 1-aminocyclobutane-1-carboxylate S18 (2.72 g, 21.04 mmol) in dimethylacetamide (50.00 ml, 10 V), then successively add potassium acetate (4.30 g, 43.82 mmol) as the base, copper(I) chloride (694.15 mg, 7.01 mmol) as the catalyst, and then add 2-ethylcyclohexanone (1.04 g, 6.32 mmol) as the ligand. Heat the reaction temperature to 100 °C. After reacting for 2 h, monitor the reaction by TLC visualization under ultraviolet light. Stop the reaction after the reaction raw materials are completely reacted. After cooling to room temperature, add 20 mL of ammonia: saturated ammonium chloride solution (1:4), extract with ethyl acetate / water, wash three times with water, then wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The crude product is purified by column chromatography (n-heptane) to obtain 2.36 g of oily substance S19, with a yield of 51.52%. 1 1H 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).

[0035] Step D: Synthesis of Intermediate S20 Under nitrogen protection, the temperature was controlled at 0 °C in an ice bath. 5-Amino-3-(trifluoromethyl)picolyl nitrile S1 (5.00 g, 26.72 mmol) was dissolved in acetone (50.00 mL, 5 V), and thiophosgene (9.22 g, 80.16 mmol) was slowly added dropwise. The mixture was stirred and reacted for 1 h. After the raw materials reacted completely, ice water was added, and the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at low temperature to obtain a black-brown oily substance S20. Without purification, the crude product was stored refrigerated and directly used in the next step.

[0036] Step E: Synthesis of intermediate S21 Under nitrogen protection, 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 then triethylamine (495.71 mg, 4.90 mmol) was added. The temperature was raised to 100 °C and the mixture was stirred for 2 h. After the reaction was completed, it was cooled to room temperature. It 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 a yellow oily substance S21, with a yield of 74.81%. MS: 459.1 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 9.23 (d, J J = 1.8Hz, 1H), 8.78 (d, J J = 1.9 Hz, 1H), 7.56 (t, J J = 7.7 Hz, 1H), 7.39 (d, J 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 J = 14.4, 10.0, 4.7Hz, 1H), 1.25 (d, J J = 9.4 Hz, 1H).

[0037] Step F: Synthesis of intermediate S22-a Under nitrogen protection, 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 then copper iodide catalyst (124.63 mg, 654.40 μmol) was added. The temperature was raised to 45 °C and stirred for 2 h. After the reaction was completed, it was cooled to room temperature. 3 mL of ammonia: saturated ammonium chloride solution (1:4) was added. It was extracted with ethyl acetate, washed with water three times, then washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure in vacuo, and the crude product was purified by column chromatography (ethyl acetate / petroleum ether) to obtain 0.12 g of white solid S22-a, with a yield of 21.35%. 1 H NMR (400MHz, 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).

[0038] Step G: Synthesis of intermediate S22-b The synthesis of compound S22-b was carried out by a procedure similar to that for the synthesis of S22-a described in Example 1, except that S13-a was replaced with S13-b, and the yield was 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).

[0039] Step H: Synthesis of Intermediate S22-c The synthesis of Compound S22-c was carried out by a procedure similar to that of Step S22-a described in Example 1, except that S13-a was replaced with S13-c, and the yield was 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).

[0040] Step I: Synthesis of Compound I-1 Under nitrogen protection and with the temperature 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, obtaining Compound I-1. After the reaction, the trifluoroformic acid in the reaction solution was diluted with water, and then saturated sodium bicarbonate solution was slowly added to quench the reaction, with bubbles generated simultaneously. The pH value of the reaction system was adjusted to 6 - 7, extracted with methanol / dichloromethane, washed with water three times, then washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure in vacuo, and the crude product was purified by column chromatography (methanol / dichloromethane) to obtain 32.00 mg of white solid I-1 with 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).

[0041] Step J: Synthesis of Compound I-2 The synthesis of Compound I-2 was carried out by a procedure similar to that of Step I-1 described in Example 1, except that S22-a was replaced with S22-b, and 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).

[0042] Step K: Synthesis of Compound I-3 The synthesis of Compound I-3 was carried out by a procedure similar to that of Step I-1 described in Example 1, except that S22-a was replaced with S22-c, and 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

[0043] The experiment used apalutamide and vorinostat as positive control compounds. All test samples were prepared at an initial concentration of 10 -2 M, dissolved using cell-grade dimethyl sulfoxide as a solvent, aliquoted, and stored frozen at -20 °C for later use.

[0044] Step A: Testing the growth inhibitory activity of AR / HDAC6 dual-target compounds against human prostate cancer cell line LNCaP Experimental method 1×10 4 cells were seeded in 96-well plates. After the cells adhered for 24 h, they were treated with blank control (medium), different concentrations of the compound (120, 100, 80, 40, 20, 10, 5, 2.5, 1.25 μmol / L, and the final concentration of DMSO was controlled below 0.1%) in the corresponding medium for 48 h. After the treatment, 10 μL of CCK-8 solution was added to each well and incubated in an incubator at 37 °C for 3 h. After incubation, the absorbance (450 nm) was measured using a microplate reader.

[0045] Data analysis Cell growth survival rate = (OD 实验组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) × 100% Based on the absorbance data, statistical analysis was performed to evaluate the inhibitory effect of the compound on cell growth. By calculating the cell survival rate, the dose-effect relationship and the half-maximal inhibitory concentration (IC 50 ) of the compound were further analyzed.

[0046] Experimental results: The results of the growth inhibitory effect test on LNCaP prostate cancer cells are shown in Table 1. The IC 50 of the positive drug apalutamide was 106.10 μM, and the inhibitory activities of compound class I and the lead compound B4 were comparable. As the linking chain of compound class I increased from 4 carbons in I-1 to 6 carbons in I-3, the growth inhibitory activity against LNCaP prostate cancer cells gradually increased.

[0047] Table 1 Growth Inhibitory Effect of Compounds on LNCaP Prostate Cancer Cells

[0048] Step B: Selective Inhibitory Activity of AR / HDAC6 Dual-Target Compounds against HDAC Experimental Method (1) The test compounds were serially diluted 3-fold from an initial concentration of 10 μM, and the positive control (SAHA) was serially diluted 3-fold from an initial concentration of 3 μM, with a total of 8 concentration points. Each concentration was assayed in duplicate. Control wells were set up as follows: Both the Max well (containing DMSO and enzyme) and the Min well (enzyme-free well) were added with 250 nL of 100% DMSO.

[0049] (2) Prepare 1.67× enzyme solution with 1× reaction solution. Add 15 μL of 1.67× enzyme solution to each well, and add 15 μL of 1.6× reaction buffer (instead of enzyme solution) to the Min well. Incubate at room temperature for 15 min to allow the enzyme to fully bind to the compound.

[0050] (3) Prepare 2.5× substrate mixture with 1× reaction buffer. Add 10 μL of 2.5× substrate mixture to each well and start the detection immediately.

[0051] (4) Use a Synergy multi-functional microplate reader to dynamically monitor the change in fluorescence signal and record the data for subsequent analysis.

[0052] Data Analysis Kinetic data in the linear phase of the enzymatic reaction were used. The slope was obtained by linear regression analysis, and then the percentage inhibition rate was calculated using the following formula: %Inhibition = 100×[Mean(Max)−Sample Signal] / [Mean(Max)−Mean(Min)] Where: Mean(Max) is the mean of the slope values of each Max well; Mean(Min) is the mean of the slope values of each Min well; Sample Signal is the slope value of the compound well.

[0053] Fitting the dose-effect curve: Using the log value of the compound concentration as the X-axis and the corresponding percentage inhibition rate as the Y-axis, the log(inhibitor) vs. response - Variable slope function of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve, and thus the IC 50 value of each compound inhibiting enzyme activity was obtained.

[0054] The experimental results are shown in Table 2: The inhibitory activities of SAHA against the two targets are almost similar. It is a typical broad-spectrum HDAC inhibitor without the effect of selective inhibition. Among the class I compounds, when the number of carbons in I-1 increases from 4 to 6 in I-3, the selective inhibitory activity against HDAC1 / HDAC6 decreases, and the selective inhibitory activity of I-1 (HDAC6 IC 50 = 32.84 nM, HDAC1 IC 50 = 861.95 nM) against HDAC6 is increased by 26 times, which is better than the lead compound B4 (HDAC6 IC 50 = 13.60 nM, HDAC1IC 50 = 28.94 nM). Therefore, when the recognition group is benzyl, introducing a branched chain or increasing the chain length on the hydrophobic linker will affect its selective inhibitory activity against HDAC6.

[0055] Table 2 IC 50 values of the compounds against HDAC1 and HDAC6

[0056] Step C: Effects of AR / HDAC6 dual-target compounds on the expression levels of related proteins Experimental method Using B4 as the positive control, LNCaP cells (Cell Bank of the Chinese Academy of Sciences) were seeded in 6-well plates at a density of 2×10 5 cells / well and treated with three concentrations (1.25, 5, and 40 μmol / L) of the test compound I-3 for 24 h.

[0057] (1) Protein preparation: The prostate cancer cells were digested with trypsin, centrifuged, and the supernatant was discarded. After centrifuging and washing with PBS and discarding the supernatant, the centrifuge tube containing the cells was placed on ice; it was mixed with RIPA buffer (Servicebio) containing a protease inhibitor mixture and then transferred to a 1.5 mL EP tube and lysed on ice for 30 min. Centrifuge at 12,000 rpm at 4 °C for 30 min to collect the supernatant. The protein sample was mixed with 6×Loading buffer (Beyotime) at a ratio of 1:5, denatured in a metal bath at 100 °C for 10 min, and then cooled on ice for standby.

[0058] (2) Electrophoresis and membrane transfer Select gels with different concentrations according to molecular weight. After setting up the apparatus, fill it with electrophoresis buffer. Pull out the comb teeth with both hands simultaneously, and use a pipette to add the diluted sample into the comb teeth holes. Electrophoresis is first carried out at a voltage of (80 V, 30 min). After the sample moves from the stacking gel to the separating gel, the voltage is (120 V, 1 h). When the Marker reaches the bottom of the gel, stop electrophoresis and pour out the buffer. After electrophoresis, place the peeled separating gel into the pre-prepared transfer buffer. Activate the PVDF membrane in methanol for 1 min, take it out and place it in the transfer buffer for later use. Adopt the wet transfer method (100 A, 60 min).

[0059] (3) Blocking and incubation After the transfer is completed, take out the PVDF membrane and incubate it on a shaker at room temperature for 1 h with a 5% skim milk powder solution (Beyotime). After that, wash the PVDF membrane three times with TBST for 5 min each time.

[0060] 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), and incubate it on a shaker in a 4°C refrigerator overnight. The next day, wash the PVDF membrane three times with TBST for 5 min each time.

[0061] Place it in an incubation box containing the secondary antibody dilution solution (HRP-labeled goat anti-mouse IgG Beyotime, HRP-labeled goat anti-rabbit IgG (H+L) Servicebio), and incubate it at room temperature for 1 h. After that, wash the PVDF membrane three times with TBST for 5 min each time.

[0062] (4) Development Prepare the ECL developing solution, evenly distribute the developing solution on the surface of the membrane, and use a fully automatic chemiluminescence imaging instrument for exposure and development.

[0063] Result analysis: Study the effects of compound I-3 and lead compound B4 on the expression levels of LNCaP prostate cancer cell-related proteins by Western-Blot (such as Figure 1 ), among which, the target protein AR and the downstream protein PSA gradually decrease in expression levels with the increase of compound concentration; AC-α-Tublin gradually increases in acetylation expression levels with the increase of compound concentration. The experiment shows that with the increase of concentration, the inhibitory activity of compound I-3 on HDAC6 also gradually increases, and the inhibitory and degradation effects on AR also gradually increase.

Claims

1. A hydantoin compound with dual inhibitory effects on androgen receptor and histone deacetylase 6, characterized in that, Selected from any of the following structural formulas: 。 2. A pharmaceutically acceptable salt of the hydantoin compound having dual inhibitory effects on androgen receptor and histone deacetylase 6 as claimed in claim 1.

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

4. A pharmaceutical composition, characterized in that: Comprising the pharmaceutically acceptable salt, solvate, polymorph, isomer or prodrug as claimed in 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 adjuvant and / or excipient is a diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler and / or disintegrant.

6. A pharmaceutical composition according to claim 3 or 4, characterized in that, The dosage form of the drug is an aqueous dispersant, liquid, gel, syrup, medicated pulp, suspension, aerosol, controlled release agent, rapid solvent, effervescent agent, lyophilized agent, tablet, powder, pill, sugar-coated pill, capsule, delayed release agent, extended release agent, pulsed controlled release agent, multi-particle agent or immediate release agent.

7. Use of the compound as claimed in claim 1 in the preparation of a dual inhibitor of androgen receptor and histone deacetylase 6.

8. Use of the pharmaceutically acceptable salt as claimed in claim 2 in the preparation of a dual inhibitor of androgen receptor and histone deacetylase 6.

9. Use of the compound as claimed in claim 1 in the preparation of a drug for treating tumors related to androgen receptor and histone deacetylase 6.

10. Use of the pharmaceutically acceptable salt as claimed in claim 2 in the preparation of a drug for treating tumors related to androgen receptor and histone deacetylase 6.

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