5α-Reductase Degradation Molecules, Their Preparation Methods, and Applications

Abstract

This invention discloses 5α-reductase degrading molecules, their preparation methods, and applications. Four 5α-reductase degrading molecules can induce proteasome-dependent degradation of 5α-reductase, achieving complete clearance of the enzyme protein rather than merely inhibiting its activity, thus obtaining a more durable therapeutic effect. Under equal-dose conditions, the 5α-reductase degrading molecules are more effective than the traditional inhibitor finasteride in treating benign prostatic hyperplasia (BPH). These four 5α-reductase degrading molecules can effectively degrade 5α-reductase protein and significantly inhibit the development of BPH, exhibiting good biocompatibility. This provides a new strategy and candidate drugs for the treatment of BPH, prostate cancer, androgenetic alopecia, acne, and other diseases related to 5α-reductase.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to 5α-reductase degradation molecules, their preparation methods, and applications. Background Technology

[0002] 5α-reductase is a key steroid metabolic enzyme that primarily catalyzes the conversion of testosterone into the more potent dihydrotestosterone (DHT). DHT is a potent androgen with an affinity approximately 2-5 times that of testosterone, playing important physiological functions in tissues such as the prostate, sebaceous glands, and hair follicles. However, abnormal 5α-reductase activity and elevated DHT levels are closely associated with a variety of diseases, including benign prostatic hyperplasia (BPH), prostate cancer, androgenetic alopecia, and acne.

[0003] Currently, 5α-reductase inhibitors, such as finasteride and dutasteride, are mainly used in clinical practice to reduce DHT levels by inhibiting the activity of 5α-reductase, thereby treating related diseases. Although these inhibitors have achieved certain clinical efficacy, they still have several limitations: (1) Inhibitors can only temporarily inhibit enzyme activity and cannot clear the enzyme protein itself, resulting in the need for long-term medication; (2) Some patients do not respond well to inhibitors or gradually develop drug resistance; (3) Long-term use may cause adverse reactions such as sexual dysfunction and breast hyperplasia.

[0004] The discovery of new compounds that can effectively degrade 5α-reductase has significant medical value. Summary of the Invention

[0005] This invention provides 5α-reductase degrading molecules, their preparation methods, and applications. The four 5α-reductase degrading molecules provided by this invention can effectively degrade 5α-reductase protein and significantly inhibit the development of benign prostatic hyperplasia (BPH), and have good biocompatibility. They provide new strategies and candidate drugs for the treatment of diseases related to 5α-reductase, such as BPH, prostate cancer, androgenic alopecia, and acne.

[0006] To achieve this objective, the present invention provides the following technical solution:

[0007] In a first aspect, this invention provides the use of a 5α-reductase degrading molecule in the preparation of a medicament for the prevention / treatment of 5α-reductase-related diseases, wherein the structural formula of the 5α-reductase degrading molecule is:

[0008]

[0009]

[0010] Preferably, the 5α-reductase-related diseases include benign prostatic hyperplasia (BPH), prostate cancer, androgenetic alopecia, or acne. Preferably, the BPH is testosterone-induced BPH.

[0011] A second aspect of the present invention provides a medicament for the prevention / treatment of 5α-reductase-related diseases, comprising 5α-reductase degrading molecules, said 5α-reductase degrading molecules including VHL-01, VHL-02, VHL-03 and VHL-04;

[0012] The structural formula of VHL-01 is:

[0013]

[0014] The structural formula of VHL-02 is:

[0015]

[0016] The structural formula of VHL-03 is:

[0017]

[0018] The structural formula of VHL-04 is:

[0019]

[0020] Preferably, the effective dose of the 5α-reductase degrading molecule is not less than 5 mg / kg.

[0021] Preferably, the effective dose of the 5α-reductase degrading molecule is 60-80 mg / kg.

[0022] Preferably, it also includes pharmaceutically acceptable excipients, including excipients, diluents, lubricants, disintegrants, binders, and coating materials.

[0023] Preferably, the dosage form of the drug includes: oral solid dosage form, oral liquid dosage form, injection, or topical dosage form.

[0024] Preferably, the 5α-reductase-related diseases include benign prostatic hyperplasia, prostate cancer, androgenetic alopecia, or acne.

[0025] A third aspect of the present invention provides a method for preparing 5α-reductase degradation molecules, wherein the 5α-reductase degradation molecules include VHL-01, VHL-02, VHL-03 or VHL-04;

[0026] The preparation method of VHL-01 is as follows:

[0027] S1. Synthesis of compound 1-A: Compound 1-A was prepared as a solid powder using compound 2-[2-(2-aminoethoxy)ethoxy]ethanol, 1-androsten-3-one-4-aza-17b-carboxylic acid and DIPEA;

[0028] S2, Synthesis of Compound 1-B: Compound 1-A was dissolved and Dys-Martin oxidant was added to prepare solid powder compound 1-B;

[0029] Synthesis of S3 and VHL-01: Compound E3 ligase ligand 1A, HATU and DIPEA were dissolved and compound 1-B was added to prepare solid powder compound VHL-01;

[0030] The preparation method of VHL-02 is as follows:

[0031] S1, Synthesis of Compound 2-A: Solid powder compound 2-A was prepared using compounds amino-polyethylene glycol-tert-butyl propionate, 1-androsten-3-one-4-aza-17b-carboxylic acid and DIPEA;

[0032] S2, Synthesis of Compound 2-B: Compound 2-A was dissolved and TFA was added to prepare solid powder compound 2-B;

[0033] Synthesis of S3 and VHL-02: Compound E3 ligase ligand 1A, HATU and DIPEA were dissolved and compound 2-B was added to prepare solid powder compound VHL-02;

[0034] The preparation method of VHL-03 is as follows:

[0035] S1, Synthesis of Compound 3-A: Solid powder compound 3-A was prepared using compound tert-butyl 9-amino-4,7-dioxanonanoate, 1-androsten-3-one-4-aza-17b-carboxylic acid and DIPEA;

[0036] S2, Synthesis of Compound 3-B: Compound 3-A was dissolved and TFA was added to prepare solid powder compound 3-B;

[0037] Synthesis of S3 and VHL-03: Compound E3 ligase ligand 1A, HATU and DIPEA were dissolved, and compound 3-B was added to prepare solid powder compound VHL-03;

[0038] The preparation method of VHL-04 is as follows:

[0039] S1, Synthesis of Compound 4-A: Solid powder compound 4-A was prepared using compound 15-amino-4,7,10,13-tetraoxapentadecanoate tert-butyl ester and DIPEA;

[0040] S2, Synthesis of compound 4-B: Compound 4-A was dissolved and TFA was added to prepare solid powder compound 4-B;

[0041] Synthesis of S3 and VHL-04: Compound E3 ligase ligand 1A, HATU and DIPEA were dissolved and compound 4-B was added to prepare solid powder compound VHL-04.

[0042] Compared with the prior art, the beneficial effects and significant progress of the present invention are as follows:

[0043] 1. This invention provides four 5α-reductase degradation molecules. These 5α-reductase degradation molecules can achieve complete clearance of the enzyme protein by inducing proteasome-dependent degradation of 5α-reductase, rather than just inhibiting its activity, thereby obtaining a more lasting therapeutic effect.

[0044] 2. Under equal dosage conditions, the 5α-reductase degradation molecule of the present invention is more effective than the traditional inhibitor finasteride in treating benign prostatic hyperplasia;

[0045] 3. The 5α-reductase degradation molecule of the present invention has good biocompatibility, providing a safety guarantee for clinical application. Attached Figure Description

[0046] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0047] Obviously, the accompanying drawings described below are only some of the drawings of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort, but these other drawings are also within the scope of the drawings required for the embodiments of the present invention.

[0048] Figure 1 The NMR and RPHPLC chromatograms of VHL-01, VHL-02, VHL-03 and VHL-04 in Example 1 of the present invention are shown.

[0049] Figure 2 This is a Western blot result of the degradation of 5α-reductase by VHL-01, VHL-02, VHL-03 and VHL-04 in BPH-1 cells according to Example 2 of the present invention.

[0050] Figure 3 Immunofluorescence detection and quantitative analysis of 5α-reductase expression in organoids of benign prostatic hyperplasia using VHL-01, VHL-02, VHL-03 and VHL-04 in Example 3 of the present invention;

[0051] Figure 4This is a dynamic observation of the inhibitory effect of VHL-01 and finasteride on the growth of organoids in benign prostatic hyperplasia in Example 4 of the present invention.

[0052] Figure 5 This is a comparison of the therapeutic effects of VHL-01 and finasteride on a rat model of benign prostatic hyperplasia in Example 5 of the present invention.

[0053] Figure 6 The effects of different doses of VHL-01, finasteride, and PBS on the heart, liver, spleen, lungs, and kidneys of a rat model of testosterone-induced benign prostatic hyperplasia are shown in Example 5 of this invention.

[0054] Figure 7 This study compares the effects of different doses of VHL-01, finasteride, and PBS on blood routine and liver and kidney function in a rat model of benign prostatic hyperplasia, as described in Example 5 of this invention. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0056] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0057] It should be noted that the terms "first," "second," and "third" (if present), etc., in the specification, claims, and accompanying drawings of the embodiments of this invention, are only used to distinguish different objects and not to describe a specific order. Furthermore, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or devices.

[0058] In this invention, DIPEA: N,N-Diisopropylethylamine is N,N-diisopropylethylamine.

[0059] In this invention, DCM: Dichloromethane is dichloromethane.

[0060] In this invention, HATU:2-(7-AZOBENZOTRIAZOLYL-1-OXY)-N,N,N',N'-TETRAMETHYLURONIUM is N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate.

[0061] In this invention, EA: Ethyl acetate is ethyl acetate.

[0062] In this invention, DMF: N,N-Dimethylformamide is N,N-dimethylformamide.

[0063] In this invention, TFA: Trifluoroacetic acid is trifluoroacetic acid.

[0064] In this invention, Dess-Martin periodinane is a Dess-Martin oxidant.

[0065] In this invention, 2-[2-(2-AMINOETHOXY)ETHOXY]ETHANOL is 2-[2-(2-aminoethoxy)ethoxy]ethanol.

[0066] In this invention, 4-Aza-5a-androstan-1-ene-3-one-17b-carboxylic acid is 1-androsten-3-one-4-aza-17b-carboxylic acid.

[0067] In this invention, L-Prolinamide,3-methyl-L-valyl-4-hydroxy-N-[(1S)-1-[4-(4-methyl-5-thiazolyl)phenyl]ethyl]-,(4R)- is E3 ligand 1A.

[0068] In this invention, Amino-PEG1-t-Butyl ester is amino-polyethylene glycol-tert-butyl propionate.

[0069] In this invention, 3-[2-(2-Aminoethoxy)ethoxy]propanoic acid 1,1-dimethylethyl ester is tert-butyl 9-amino-4,7-dioxanoic acid.

[0070] In this invention, tert-Butyl 15-aMino-4,7,10,13-tetraoxapentadecanoate is tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate.

[0071] The technical solution of the present invention will now be described in detail with reference to specific embodiments.

[0072] Example 1: Synthesis of 5α-reductase degradation molecules

[0073] In this embodiment, a total of four 5α– reductase degradation molecules were synthesized, namely VHL-01, VHL-02, VHL-03 and VHL-04.

[0074] 1.1 Synthesis of VHL-01

[0075] (1) Synthesis of compound 1-A

[0076] In a 50 mL round-bottom flask, compounds 2-[2-(2-AMINOETHOXY)ETHOXY]ETHANOL (1.0 eq), 4-Aza-5a-androstan-1-ene-3-one-17b-carboxylic acid (1.3 eq), and DIPEA (2.4 eq) were dissolved sequentially in 5 mL of anhydrous DCM. After stirring the reaction mixture at room temperature, HATU (1.2 eq) was added, and the mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction mixture, followed by extraction with EA (3 × 50 mL). The organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound 1-A as a solid powder.

[0077] (2) Synthesis of compound 1-B

[0078] In a 50 mL round-bottom flask, compound 1-A (1.0 eq) was dissolved in 5 mL of anhydrous DCM, followed by the addition of Dess-Martinperiodinane (2.5 eq). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed by TLC monitoring, a saturated NaHCO3-Na2S2O4 solution was added to the reaction mixture and stirred for 20 min. Then, DCM (3 × 50 mL) was added for extraction, and the organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound 1-B as a solid powder.

[0079] (3) Synthesis of VHL-01

[0080] In a 50 mL round-bottom flask, compounds L-Prolinamide, 3-methyl-L-valyl-4-hydroxy-N-[(1S)-1-[4-(4-methyl-5-thiazolyl)phenyl]ethyl]-,(4R)-(1.3 eq), HATU (1.1 eq), and DIPEA (2.6 eq) were dissolved in 7 mL of anhydrous DMF. After stirring the reaction system at room temperature for 0.5 h, compound 1-B (1 eq) dissolved in 3 mL of anhydrous DMF was added to the reaction system, and the reaction system was stirred overnight at room temperature. After the reaction progress was monitored by TLC, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction system, followed by extraction with EA (3 × 50 mL), and the organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a solid powder compound VHL-01.

[0081] 1.2 Synthesis of VHL-02

[0082] (1) Synthesis of compound 2-A

[0083] In a 50 mL round-bottom flask, compounds Amino-PEG1-t-Butyl ester (1.0 eq), 4-Aza-5a-androstan-1-ene-3-one-17b-carboxylic acid (1.3 eq), and DIPEA (2.4 eq) were dissolved sequentially in 5 mL of anhydrous DMF. After stirring the reaction mixture at room temperature, HATU (1.2 eq) was added, and the mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction mixture, followed by extraction with EA (3 × 50 mL). The organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound 2-A as a solid powder.

[0084] (2) Synthesis of compound 2-B

[0085] In a 50 mL round-bottom flask, compound 2-A (1 eq) was added and dissolved in 5 mL of 80% TFA solution (TFA / DCM = 4:1, v / v). The reaction system was stirred at room temperature for 1.5 h, and the reaction progress was monitored by TLC until completion. The reaction system was concentrated under reduced pressure, washed with diethyl ether, and the residual hydrochloric acid was removed by vacuum filtration to obtain compound 2-B.

[0086] (3) Synthesis of VHL-02

[0087] In a 50 mL round-bottom flask, compounds L-Prolinamide, 3-methyl-L-valyl-4-hydroxy-N-[(1S)-1-[4-(4-methyl-5-thiazolyl)phenyl]ethyl]-,(4R)-(1.3 eq), HATU (1.1 eq), and DIPEA (2.6 eq) were dissolved in 7 mL of anhydrous DMF. After stirring the reaction system at room temperature for 0.5 h, compound 2-B (1 eq) dissolved in 3 mL of anhydrous DMF was added to the reaction system, and the reaction system was stirred overnight at room temperature. After the reaction progress was monitored by TLC, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction system, followed by extraction with EA (3 × 50 mL), and the organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a solid powder compound VHL-02.

[0088] 1.3 Synthesis of VHL-03

[0089] (1) Synthesis of compound 3-A

[0090] In a 50 mL round-bottom flask, compounds 3-[2-(2-Aminoethoxy)ethoxy]propanoicacid 1,1-dimethylethyl ester (1.0 eq), 4-Aza-5a-androstan-1-ene-3-one-17b-carboxylic acid (1.3 eq), and DIPEA (2.4 eq) were dissolved sequentially in 5 mL of anhydrous DMF. After stirring the reaction mixture at room temperature, HATU (1.2 eq) was added, and the mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction mixture, followed by extraction with EA (3 × 50 mL). The organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound 3-A as a solid powder.

[0091] (2) Synthesis of the compound 3-B

[0092] In a 50 mL round-bottom flask, compound 3A (1 eq) was added and dissolved in 5 mL of 80% TFA solution (TFA / DCM = 4:1, v / v). The reaction mixture was stirred at room temperature for 1.5 h, and the reaction was monitored by TLC until completion. The reaction mixture was concentrated under reduced pressure, washed with diethyl ether, and filtered under vacuum to remove residual hydrochloric acid, yielding compound 3-B.

[0093] (3) Synthesis of VHL-03

[0094] In a 50 mL round-bottom flask, compounds L-Prolinamide, 3-methyl-L-valyl-4-hydroxy-N-[(1S)-1-[4-(4-methyl-5-thiazolyl)phenyl]ethyl]-,(4R)-(1.3 eq), HATU (1.1 eq), and DIPEA (2.6 eq) were dissolved in 7 mL of anhydrous DMF. After stirring the reaction system at room temperature for 0.5 h, compound 3-B (1 eq) dissolved in 3 mL of anhydrous DMF was added to the reaction system, and the reaction system was stirred overnight at room temperature. After the reaction progress was monitored by TLC, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction system, followed by extraction with EA (3 × 50 mL), and the organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a solid powder compound VHL-03.

[0095] 1.4 Synthesis of VHL-04

[0096] (1) Synthesis of compound 4-A

[0097] In a 50 mL round-bottom flask, compounds tert-Butyl 15-aMino-4,7,10,13-tetraoxapentadecanoate (1.0 eq), 4-Aza-5a-androstan-1-ene-3-one-17b-carboxylicacid (1.3 eq), and DIPEA (2.4 eq) were dissolved sequentially in 5 mL of anhydrous DMF. After stirring the reaction mixture at room temperature, HATU (1.2 eq) was added, and the mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction mixture, followed by extraction with EA (3 × 50 mL). The organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a solid powder, compound 4-A.

[0098] (2) Synthesis of compound 4-B

[0099] In a 50 mL round-bottom flask, compound I (1 eq) was added and dissolved in 5 mL of 80% TFA solution (TFA / DCM = 4:1, v / v). The reaction system was stirred at room temperature for 1.5 h, and the reaction progress was monitored by TLC until completion. The reaction system was concentrated under reduced pressure, washed with diethyl ether, and the residual hydrochloric acid was removed by vacuum filtration to obtain compound 4-B.

[0100] (3) Synthesis of VHL-04

[0101] In a 50 mL round-bottom flask, compounds L-Prolinamide, 3-methyl-L-valyl-4-hydroxy-N-[(1S)-1-[4-(4-methyl-5-thiazolyl)phenyl]ethyl]-,(4R)-(1.3 eq), HATU (1.1 eq), and DIPEA (2.6 eq) were dissolved in 7 mL of anhydrous DMF. After stirring the reaction system at room temperature for 0.5 h, compound 4-B (1 eq) dissolved in 3 mL of anhydrous DMF was added to the reaction system, and the reaction system was stirred overnight at room temperature. After the reaction progress was monitored by TLC, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction system, followed by extraction with EA (3 × 50 mL), and the organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a solid powder compound VHL-04.

[0102] The NMR and RPHPLC chromatograms of the four 5α-reductase degradation molecules VHL-01, VHL-02, VHL-03, and VHL-04 prepared in this embodiment are shown below. Figure 1 As shown in Figure 1, the chemical structures of VHL-01, VHL-02, VHL-03, and VHL-04 are presented. The structures were confirmed and their purity analyzed by 1H NMR and reversed-phase high-performance liquid chromatography (RPHPLC). The results show that the characteristic peaks of each molecule in the 1H NMR spectrum are clear and consistent with the target structures. RPHPLC analysis indicates that the purity of all molecules reaches over 95%, meeting the standards for subsequent pharmacodynamic evaluation and providing a stable and reliable material basis for further bioactivity experiments.

[0103] Example 2: Protein Degradation Experiment at the Molecular and Cellular Level

[0104] This example is a molecular-cellular level protein degradation experiment targeting four molecules that degrade 5α-reductase.

[0105] (1) Cell Culture

[0106] The human prostate cancer cell line LNCaP was purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0107] (2) Western blot analysis of the protein degradation effect of 5α-reductase on degrading molecules

[0108] BPH-1 cells were loaded at 2 × 10 5Cells were seeded at a density of [number] cells / well in 6-well plates and cultured for 24 hours. Then, different concentrations (0, 0.03, 0.1, 0.3, 1, 3, 10 μM) of 5α-reductase degradation molecules (VHL-01, VHL-02, VHL-03, and VHL-04) were added for 48 hours, with an equal volume of DMSO as a control (NC group). Cells were collected and lysed with RIPA lysis buffer containing protease inhibitors at 4°C for 30 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. The supernatant was collected. Protein concentration was determined using a BCA protein quantification kit.

[0109] An equal volume of protein (30 μg) was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk powder at room temperature for 1 hour, rabbit anti-human 5α-reductase type 2 (SRD5A2) antibody (1:1000 dilution) was added and incubated overnight at 4°C. After washing three times with TBST, horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 hour. After washing three times with TBST, the protein was developed using an ECL kit. Protein expression was normalized using Tubulin as an internal control.

[0110] The results are as follows Figure 2 As shown, compared with the DMSO control group, all 5α-reductase degrading molecules were able to degrade 5α-reductase protein in BPH-1 cells in a dose-dependent manner. Among them, VHL-01 showed the strongest protein degradation activity, almost completely degrading the target protein at concentrations of 3 μM and 10 μM; VHL-02 and VHL-03 showed the next strongest degradation effects, while VHL-04 showed a relatively weaker degradation effect, but it also significantly inhibited protein expression at a high concentration (10 μM).

[0111] Example 3: Dose-dependent experiment

[0112] Human prostatic hyperplasia organoids were treated with 3 μM and 10 μM concentrations of VHL-01, VHL-02, VHL-03, and VHL-04, as well as a DMSO negative control (NC), for 48 hours. Immunofluorescence staining was then performed to detect 5α-reductase protein expression. 5α-reductase was labeled with Alexa Fluor 488 (green fluorescence), F-actin with Alexa Fluor 594 (red fluorescence), and cell nuclei were stained with DAPI (blue fluorescence).

[0113] The results are as follows Figure 3As shown, representative images for each group display the protein fluorescence signal (top row: three-color composite image; bottom row: green channel displayed separately). The right side shows the quantitative statistics of the relative fluorescence intensity of 5α-reductase in each group, with data expressed as mean ± standard deviation (SD). Statistical analysis was performed using the t-test, with *P < 0.05 considered statistically significant. It is evident that VHL-01 significantly reduced the expression of 5α-reductase in organoids at both concentrations, with fluorescence signal intensity significantly lower than the NC group and other 5α-reductase degradation groups. VHL-02, VHL-03, and VHL-04 showed moderate degradation effects, with the degradation effects decreasing sequentially from VHL-02, VHL-03, to VHL-04. Furthermore, all four groups exhibited a dose-dependent trend.

[0114] Example 4: Dynamic observation of the inhibitory effect of VHL-01 and finasteride on the growth of organoids in benign prostatic hyperplasia.

[0115] Human prostatic hyperplasia organoids were randomly divided into a negative control group (NC), a finasteride (3μM) group, and a VHL-01 (3μM) group. They were cultured and observed for 7 consecutive days, and the changes in organoid size were recorded on days 0, 3, 5, and 7.

[0116] The results are as follows Figure 4 As shown, the organoids in the NC group were significantly enlarged and showed significant fusion. Although the finasteride group showed some inhibition, the effect was limited. The number and size of organoids in the VHL-01 treatment group were significantly lower than those in the other two groups, indicating that its inhibitory effect on organoid growth and proliferation was significantly better than that of finasteride.

[0117] Example 5: Study on the therapeutic effect of VHL-01 in a C57 mouse model of benign prostatic hyperplasia.

[0118] 5.1 Establishment of an animal model of benign prostatic hyperplasia and experimental grouping

[0119] This experiment selected 8-week-old male C57BL / 6 mice, weighing approximately 20–22 g, and administered subcutaneous injections of testosterone propionate (10 mg / kg) daily for 14 consecutive days to successfully establish a mouse model of benign prostatic hyperplasia (BPH).

[0120] After the model was established, the mice were randomly divided into the following 7 experimental groups, with 6 to 7 mice in each group: (1) Model control group (PBS group); (2) Finasteride treatment group (5 mg / kg); (3) VHL-01 low-dose treatment group (5 mg / kg); (4) VHL-01 medium-low-dose treatment group (15 mg / kg); (5) VHL-01 medium-dose treatment group (30 mg / kg); (6) VHL-01 medium-high-dose treatment group (60 mg / kg); (7) VHL-01 high-dose treatment group (80 mg / kg).

[0121] From day 1 of modeling, mice in each drug treatment group (including the finasteride group and each dose group of VHL-01) were given the corresponding drugs via intraperitoneal injection daily for 14 consecutive days. The PBS group was given an equal volume of PBS via intraperitoneal injection daily.

[0122] 5.2 Prostate weight and prostate index detection and prostate histopathological analysis (HE staining)

[0123] After treatment, mice in each group were anesthetized and euthanized. The prostate tissue was completely removed, the surface moisture was dried, and the wet weight of the prostate was measured. The prostate index (prostate weight / mouse body weight × 100%) was calculated.

[0124] Prostate tissues from mice in the finasteride (5 mg / kg) group and the VHL-01 (5 mg / kg) group were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE) to observe the morphological and pathological changes of the prostate tissues.

[0125] Prostate weight and prostate index test results are as follows Figure 5 As shown in a and 5b, compared to the PBS model group, both the finasteride treatment group and the VHL-01 treatment groups at all doses significantly reduced prostate weight and prostate index. VHL-01 showed a significant effect at a dose of 5 mg / kg, with further enhanced efficacy at doses of 15, 30, and 60 mg / kg. The effect at the 80 mg / kg dose group was comparable to that at 60 mg / kg, suggesting that 60 mg / kg may be the upper limit of the effective dose for VHL-01 treatment. Overall, the therapeutic effect of VHL-01 was significantly superior to that of finasteride.

[0126] The results of prostate tissue pathological analysis (HE staining) are as follows: Figure 5 As shown in c, HE staining results showed that the degree of glandular dilation and epithelial hyperplasia in the prostate tissue of the finasteride group was reduced, but the disordered cell arrangement still existed; the histopathological improvement in the VHL-01 group was more obvious, with a significant reduction in glandular dilation, more regular epithelial cell arrangement, and a prostate tissue structure closer to normal.

[0127] 5.3 Safety assessment of major organs (HE staining)

[0128] After the experiment, HE staining was performed on the heart, liver, spleen, lung, and kidney tissues of mice in the PBS group, finasteride group, and different doses of VHL-01 group to assess the safety of the drug on major organs.

[0129] HE staining results are as follows Figure 6As shown, no significant pathological changes were observed in the heart, liver, spleen, lung, and kidney tissues of mice in the finasteride group and different doses of VHL-01 groups. Specifically, the myocardial fiber structure of the heart was intact and neatly arranged, without degeneration, necrosis, or inflammatory cell infiltration; the hepatocytes were arranged in cords, the lobular structure was clear, and there was no fatty degeneration or inflammatory reaction; the red and white pulp of the spleen were clearly demarcated, the tissue structure was normal, and there was no hemorrhage, necrosis, or abnormal proliferation; the alveolar structure of the lungs was intact, and no edema, hemorrhage, or fibrosis was observed; the glomeruli and tubules of the kidneys were normal, without necrosis, inflammation, or other pathological damage. Overall, there were no significant differences in the histological morphology of the major organs between the different doses of VHL-01 and the finasteride group and the PBS control group, and no drug-related acute or chronic toxic reactions were observed. This indicates that VHL-01 has good histological safety in the major organs of experimental animals at effective doses, with no obvious side effects, providing important safety evidence for its further clinical development and application.

[0130] 5.4 Analysis of complete blood count and liver and kidney function indicators

[0131] A rat model of benign prostatic hyperplasia (BPH) was established using testosterone propionate induction. Rats were randomly divided into a PBS control group, a finasteride group, and different doses of VHL-01. After 28 days of continuous gavage administration, peripheral blood was collected from each group of rats to measure complete blood count (RBC), white blood cell count (WBC), hemoglobin (HGB), platelet count (PLT), and other routine blood indicators, as well as serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) and other liver and kidney function biochemical indicators. These studies were used to evaluate the safety effects of the drug on the blood system and liver and kidney function.

[0132] The results are as follows Figure 7 As shown, all blood routine and liver and kidney function indicators in all treatment groups were within the normal physiological range except for platelet count, and there were no statistically significant differences between the groups. Notably, the platelet (PLT) count in the VHL-01 group (5 mg / kg) was significantly higher than that in the finasteride group (5 mg / kg) (P<0.05), suggesting that VHL-01 may have a protective effect on platelets or have a smaller negative impact.

[0133] In summary, VHL-01 demonstrated excellent therapeutic efficacy and good safety in the C57 mouse model of benign prostatic hyperplasia, with particularly minimal impact on platelet count, indicating its promising clinical application as a treatment for benign prostatic hyperplasia.

[0134] In the description process of the above instruction manual:

[0135] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0136] Finally, it should be noted that:

[0137] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0138] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

The application of 1,5α-reductase degradation molecule VHL-01 in the preparation of drugs for treating benign prostatic hyperplasia, characterized in that, The structural formula of the 5α-reductase degradation molecule VHL-01 is: 。 2. The application as described in claim 1, characterized in that, The benign prostatic hyperplasia mentioned is testosterone-induced benign prostatic hyperplasia.

3. A drug for treating benign prostatic hyperplasia, characterized in that, This includes the 5α-reductase degradation molecule VHL-01, the structural formula of which is: 。 4. The drug as described in claim 3, characterized in that, The effective dose of the 5α-reductase-degrading molecule VHL-01 is not less than 5 mg / kg.

5. The drug as described in claim 4, characterized in that, The effective dose of the 5α-reductase-degraded molecule VHL-01 is 60-80 mg / kg.

6. The drug as described in claim 5, characterized in that, It also includes pharmaceutically acceptable excipients, including excipients, diluents, lubricants, disintegrants, binders, and coating materials.

7. The drug as described in claim 5, characterized in that, The dosage forms of the drug include: oral solid dosage forms, oral liquid dosage forms, injections, or topical preparations. A method for preparing 8.5α-reductase degradation molecules, characterized in that, The 5α-reductase degradation molecule is VHL-01 as described in claim 1; The preparation method of VHL-01 is as follows: S1. Synthesis of compound 1-A: Compound 1-A was prepared as a solid powder using compound 2-[2-(2-aminoethoxy)ethoxy]ethanol, 1-androsten-3-one-4-aza-17b-carboxylic acid and DIPEA; S2, Synthesis of Compound 1-B: Compound 1-A was dissolved and Dys-Martin oxidant was added to prepare solid powder compound 1-B; Synthesis of S3 and VHL-01: Compound E3 ligase ligand 1A, HATU and DIPEA were dissolved and compound 1-B was added to prepare solid powder compound VHL-01.

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