Synthesis and application of AR protein degradation agent

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to synthesis and application of an AR protein degradation agent. The AR protein degradation agent modified by using norbornene, adamantanecarboxylic acid and adamantanacetic acid as hydrophobic labels can effectively inhibit proliferation of tumor cells and induce androgen protein degradation, and dose dependence and time dependence are shown; according to the compound disclosed by the invention, by changing the chain length, the combination of a target protein ligand and a receptor is not influenced, and the target protein can be recognized by ubiquitin ligase; according to the preferable protein degradation agent, norbornene is used as a hydrophobic label, the groups are smaller than those of adamantanecarboxylic acid and adamantanacetic acid which are used as hydrophobic label groups, and the druggability is good; the parent nucleus of the AR protein degradation agent compound is paired with AR protein, target protein is degraded through a hydrophobic tag, and the tumor inhibition effect is better than that of an existing AR inhibitor enzalutamide; the compound provided by the invention is few in preparation steps, simple in purification method and high in yield.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to the synthesis and application of an AR protein degrader. Background Technology

[0002] Prostate cancer (PCa) ranks among the leading causes of cancer-related illnesses in men worldwide, and its incidence is showing a year-on-year increasing trend. Abnormal activation of the androgen receptor (AR) is closely related to the development of PCa. The AR consists of four functional domains: the N-terminal domain (NTD), the DNA-binding domain (DBD), the hinge domain (HD), and the carboxyl-terminal ligand domain (LBD). The NTD is responsible for the transcriptional activation of the AR; mutations or amplifications of the NTD enhance the transcriptional activation capacity of the AR. The binding of the LBD to androgens promotes AR dimerization and entry into the cell nucleus, upregulating the expression of proliferative genes, inhibiting apoptosis, and leading to malignant cell proliferation. Existing therapeutic drugs inhibit tumor cell growth by blocking the binding of androgens to the AR. However, traditional antagonists suffer from drug resistance issues, and their blocking is incomplete, resulting in less than ideal efficacy. Protein degraders can degrade pathogenic proteins, fundamentally eliminating pathogenic factors and potentially overcoming drug resistance. They also offer solutions for some difficult-to-drug targets. Currently, the most commonly used protein degradation method is PROTAC (targeted protein degradation chimera), but it suffers from drawbacks such as large molecular weight and poor drug-likeness. Hydrophobic-tagged protein degraders are designed by linking hydrophobic tags (such as adamantane and norbornene) to a linker chain, and then to a target protein ligand. Hydrophobic tags generally have smaller molecular weights and better drug-likeness, potentially making them a superior protein degradation method compared to PROTAC. The design of hydrophobic-tagged protein degraders relies heavily on molecular synthesis and testing; even slight differences in the linker chain, hydrophobic tag, and linkage method can significantly impact protein degradation activity. Therefore, designing and synthesizing different types of hydrophobic-tagged protein degraders with different linker chains for screening holds promise for discovering protein degraders with superior activity and drug-likeness, providing a better treatment option for prostate cancer. Summary of the Invention

[0003] The purpose of this invention is to provide an AR protein degrader with excellent protein degradation ability and anti-tumor activity, achieved through a bifunctional hydrophobic tag. By degrading AR, the protein degrader of this invention can overcome the problems of drug resistance and incomplete blocking of antagonists, and is expected to become an effective means of treating prostate cancer. It also provides a new design idea for the development of prostate cancer drugs, thereby solving the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention provides an AR protein degrader and a pharmaceutically acceptable salt thereof, said AR protein degrader being composed of a hydrophobic tag molecule and a parent nucleus;

[0006] The parent core has the structure shown in Formula I, and the hydrophobic tag molecules include the structures shown in B1 (norbornene), B2 (adamantane carboxylic acid), and B3 (adamantane acetic acid);

[0007] Formula I;

[0008] ;

[0009] The general structural formula of the AR protein degrader is shown in Formula II below:

[0010] Formula II.

[0011] Where n = 1, 2, 3.

[0012] R is , or .

[0013] Furthermore, the molecular structure of the AR protein degrader includes the structures shown in J1, J2, J3, J4, J5, J6, J7, J8, and J9 below;

[0014]

[0015] .

[0016] Preferably, the molecular structure of the AR protein degrader is shown in J2;

[0017] .

[0018] The preparation route for an AR protein degrader as described above, and its pharmaceutically acceptable salt, is as follows:

[0019] ;

[0020] ;

[0021] ;

[0022] .

[0023] The use of an AR protein degrader as described above, and its pharmaceutically acceptable salt, in the preparation of remedies for diseases associated with abnormal AR protein expression.

[0024] Furthermore, diseases associated with abnormal AR protein expression include prostate cancer, kidney cancer, and breast cancer.

[0025] The use of an AR protein degrader as described above, and its pharmaceutically acceptable salt, in the preparation of a medicament for treating cancers associated with abnormal AR protein expression.

[0026] Furthermore, cancers associated with abnormal AR protein expression include prostate cancer, kidney cancer, and breast cancer.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This invention provides a protein degrading agent that degrades androgens. The protein degrading agent obtained by this invention can be used in the treatment of prostate cancer. The protein degrading agent obtained by this invention can effectively inhibit the proliferation of tumor cells and induce the degradation of androgen proteins in a dose- and time-dependent manner. By changing the chain length, the compounds of this invention can enable the target protein to be recognized by ubiquitin ligases without affecting the binding of the target protein ligand to the receptor.

[0029] The preferred protein degrading agent obtained in this invention uses norbornene as a hydrophobic tag, and the group is smaller than that of adamantane carboxylic acid and adamantane acetic acid as hydrophobic tag groups, exhibiting good drug-like properties. The core of the compound described in this invention pairs with AR proteins, degrading the target protein through a hydrophobic tag, and its tumor-inhibiting efficacy is superior to that of the existing AR inhibitor enzalutamide. The compound described in this invention requires fewer preparation steps, has a simple purification method, and achieves high yield. Attached Figure Description

[0030] Figure 1 The effects of different concentrations of J2 (10 mg / kg, 20 mg / kg, 40 mg / kg), the control group, and oral administration of the androgen receptor inhibitor enzalutamide on animal body weight were investigated.

[0031] Figure 2 The changes in tumor volume of J2 after administration of different concentrations of 10 mg / kg, 20 mg / kg, and 40 mg / kg, as well as the control group and oral androgen receptor inhibitor enzalutamide;

[0032] Figure 3To detect the degradation activity of different concentrations of compound J2 on AR protein in LNCaP cells by Western blotting;

[0033] Figure 4 To detect the degradation activity of 5 μM compound J2 on AR protein in LNCaP cells at different time points by Western blotting;

[0034] Figure 5 This is the general structural formula of the AR protein degrader of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] Taking a compound with a chain length of n=2 and a hydrophobic label of norbornene as an example, the specific implementation scheme is as follows.

[0038] A protein degrading agent with norbornene (B1) as a hydrophobic group is prepared by the following steps:

[0039] Preparation of compound 4-2: 2-(4-bromobutyl)isoindoline-1,3-dione (1 g, 3.54 mmol), methyl 2-amino-2-methylpropionate (1.09 g, 7.09 mmol), potassium carbonate (1.96 g, 14.18 mmol), and potassium iodide (588.38 mg, 3.54 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL) and stirred overnight at 80 °C. The mixture was then extracted three times with ethyl acetate, and the organic layers were collected, dried over anhydrous sodium sulfate, concentrated, and directly added to the next reaction step.

[0040] ;

[0041] Preparation of compound 4-3: Untreated compound 4-2 was dissolved in anhydrous tetrahydrofuran (20 mL), followed by the addition of triethylamine (79.35 μL, 569.3 μmol), and 4-isothiocyanate-2-(trifluoromethyl)benzonitrile (753.48 mg, 3.3 mmol) was added under ice bath conditions. The mixture was stirred at room temperature for 3 hours, the solvent was evaporated, and the mixture was extracted three times with ethyl acetate. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated, and then subjected to silica gel column chromatography to obtain the desired compound 4-3 (650 mg, two-step yield 55.48%).

[0042] Compound 4-3 was detected by ¹H NMR (500 MHz, Chloroform-d) as follows: δ 8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.82 (dd, J = 5.1, 3.1 Hz, 2H), 7.74 (dd, J = 5.1, 3.1 Hz, 2H), 7.61 (dd, J = 7.6, 2.3 Hz, 1H), 3.96 (dt, J = 12.6, 5.9 Hz, 1H), 3.75 (dt, J = 13.4, 5.7 Hz, 3H), 1.80 – 1.68 (m, 4H), 1.46 (s, 6H).

[0043] ;

[0044] Preparation of compound 4-4: Compound 4-3 (650 mg, 1.26 mmol) was dissolved in ethanol, followed by the addition of 80% hydrazine hydrate (614 μL, 12.63 mmol), and stirred at room temperature for 2 days. After the reaction was complete, the solvent was evaporated to dryness, dissolved in dichloromethane, and the insoluble matter was removed by filtration. The filtrate was then evaporated to dryness, concentrated, and subjected to silica gel column chromatography to obtain the desired compound 4-4 (452.2 mg, yield 93.11%).

[0045] Compound 4-4 was detected by ¹H NMR (500 MHz, Chloroform-d) with the following results: δ 8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3 Hz, 1H), 3.93 (dt, J = 12.7, 6.7 Hz, 1H), 3.71 (dt, J = 12.6, 6.7 Hz, 1H), 2.83 (tt, J = 6.5, 5.2 Hz, 2H), 1.81 (t, J = 6.5 Hz, 2H), 1.71 – 1.62 (m, 2H), 1.55 (ttd, J = 7.3, 5.3, 1.0 Hz). Hz, 2H), 1.46 (s, 6H).

[0046] ;

[0047] Preparation of J2: Norborneol (53.91 mg, 390.2 μmol) was dissolved in dichloromethane (6 ml), and diisopropylethylamine (50.43 mg, 390.2 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (148.37 mg, 390.2 μmol) were added separately. After stirring at room temperature for 30 minutes, 4-4 (100 mg, 260.1 μmol) was added, and the reaction was stirred at room temperature for 2 hours. The solvent was evaporated, and the mixture was extracted three times with ethyl acetate. The organic layer was collected, dried with anhydrous sodium sulfate, concentrated, and then subjected to silica gel column chromatography to obtain the desired compound J2 (126.3 mg, yield 96.22%).

[0048] Compound J2 was detected by ¹H NMR (500 MHz, Chloroform-d) as follows: δ 8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3 Hz, 1H), 6.81 (t, J = 4.9 Hz, 1H), 6.09 (ddt, J = 7.5, 5.7, 1.8 Hz, 1H), 5.87 (dd, J = 7.5, 4.5 Hz, 1H), 3.96 (dt, J = 12.6, 6.3 Hz, 1H), 3.77 (dt, J = 12.6, 6.3 Hz, 1H), 3.30 – 3.22 (m, 1H), 3.10 (tt, J = 5.9, 5.0 Hz, 2H), 2.85(qdd, J = 4.6, 1.8, 0.9 Hz, 1H), 2.62 (dtd, J = 6.9, 5.1, 1.8 Hz, 1H), 1.96(dt, J = 12.2, 4.9 Hz, 1H), 1.81 (dt, J = 12.2, 4.8 Hz, 1H), 1.74 – 1.63 (m,2H), 1.58 – 1.51 (m, 2H), 1.51 – 1.42 (m, 8H), 1.38 (dt, J = 12.5, 4.4 Hz,1H);13C NMR (125 MHz) δ 180.51, 178.12, 175.36, 138.52, 138.39, 136.10, 135.55, 132.15, 124.17, 123.36, 120.28, 116.35, 99.90, 67.80, 47.43, 47.24, 44.70, 42.52, 40.73, 40.51, 30.42, 26.70, 25.28, 22.19.

[0049] .

[0050] Example 2

[0051] J1 was prepared in the same way as in Example 1, except that raw material 4-4 was replaced with 2-(2-bromoethyl)isoindoline-1,3-dione.

[0052] The results of the detection of compound J1 are as follows: 1H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 1.9 Hz, 1H), 7.61 (dd, J = 7.6, 1.9Hz, 1H), 6.98 (t, J = 3.8 Hz, 1H), 6.09 (ddd, J = 7.5, 5.7, 1.8 Hz, 1H), 5.87 (dd, J = 7.5, 4.6 Hz, 1H), 4.11 – 3.97 (m, 2H), 3.55 – 3.42 (m, 2H), 3.30 –3.22 (m, 1H), 2.85 (qdd, J = 4.6, 1.8, 0.9 Hz, 1H), 2.64 (dt, J = 6.4, 5.1Hz, 1H), 1.95 (dt, J = 12.5, 5.0 Hz, 1H), 1.81 (dt, J = 12.4, 5.0 Hz, 1H), 1.48 (s, 3H), 1.43 (s, 4H), 1.38 (dt, J = 12.5, 4.4 Hz, 1H).

[0053] 13 C NMR (125 MHz) δ 180.30, 177.73, 174.84, 138.39, 138.33, 136.10,135.55, 132.15, 124.17, 123.36, 120.28, 116.35, 99.90, 67.94, 47.41, 47.24,44.96, 43.07, 40.73, 38.84, 30.41, 22.19.

[0054] Example 3

[0055] J3 was prepared in the same manner as in Example 1, except that raw material 4-4 was replaced with 2-(6-bromohexyl)isoindoline-1,3-dione.

[0056] The results of the detection of compound J3 are as follows: 1H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 6.81 (t, J = 4.9 Hz, 1H), 6.09 (ddd, J = 7.5, 5.7, 1.8 Hz, 1H), 5.87 (dd, J = 7.5, 4.6 Hz, 1H), 3.99 – 3.90 (m, 1H), 3.72 (dt, J = 12.9, 6.6 Hz,1H), 3.30 – 3.22 (m, 1H), 3.16 – 3.03 (m, 2H), 2.85 (pdd, J = 4.7, 1.8, 1.0Hz, 1H), 2.62 (dt, J = 6.4, 5.1 Hz, 1H), 1.95 (dt, J = 12.5, 5.0 Hz, 1H), 1.81 (dt, J = 12.5, 4.8 Hz, 1H), 1.65 (pd, J = 6.5, 1.1 Hz, 2H), 1.53 – 1.27(m, 14H).

[0057] 13 C NMR (125 MHz) δ 180.51, 178.12, 175.36, 138.52, 138.39, 136.10,135.55, 132.15, 124.17, 123.36, 120.28, 116.35, 99.90, 67.81, 47.43, 47.24,44.70, 42.64, 40.77, 40.73, 30.41, 28.64, 27.43, 26.78, 26.70, 22.19.

[0058] Example 4

[0059] The preparation of J4 is the same as in Example 2, except that raw material B1 is replaced with B2.

[0060] The results of the detection of compound J4 are as follows: 1H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 6.62 (t, J = 4.0 Hz, 1H), 4.11 – 3.96 (m, 2H), 3.50 (td, J = 4.8,4.0 Hz, 2H), 2.03 (dt, J = 11.1, 5.5 Hz, 3H), 1.86 (dd, J = 13.0, 5.1 Hz,3H), 1.80 (dd, J = 13.0, 5.3 Hz, 3H), 1.71 (t, J = 5.7 Hz, 6H).

[0061] 13 C NMR (125 MHz) δ 180.30, 179.42, 174.84, 138.33, 136.10, 132.15,124.17, 123.36, 120.28, 116.35, 99.90, 67.94, 43.56, 42.74, 40.03, 39.70,36.93, 27.53, 22.19.

[0062] Example 5

[0063] J5 was prepared in the same manner as in Example 1, except that raw material B1 was replaced with B2.

[0064] The results of the detection of compound J5 are as follows: 1H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 6.28 (t, J = 5.2 Hz, 1H), 3.96 (dt, J = 12.6, 6.3 Hz, 1H), 3.77 (dt,J = 12.6, 6.3 Hz, 1H), 3.18 (q, J = 4.7 Hz, 2H), 2.03 (hept, J = 5.5 Hz, 3H), 1.89 – 1.76 (m, 6H), 1.74 – 1.69 (m, 7H), 1.69 – 1.65 (m, 1H), 1.57 – 1.48 (m, 2H), 1.46 (s, 6H).

[0065] 13 C NMR (125 MHz) δ 180.51, 179.83, 175.36, 138.52, 136.10, 132.15,124.17, 123.36, 120.28, 116.35, 99.90, 67.80, 43.52, 42.52, 41.65, 39.70,36.93, 27.53, 26.49, 25.28, 22.19.

[0066] Example 6

[0067] The preparation of J6 is the same as in Example 3, except that the raw material B1 is replaced with B2.

[0068] The results of the detection of compound J6 are as follows: 1H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 6.34 (t, J = 5.2 Hz, 1H), 3.99 – 3.90 (m, 1H), 3.77 – 3.68 (m, 1H), 3.13 (q, J = 4.8 Hz, 2H), 2.03 (hept, J = 5.5 Hz, 3H), 1.86 (dd, J = 13.0,5.1 Hz, 3H), 1.80 (dd, J = 13.0, 5.3 Hz, 3H), 1.71 (t, J = 5.7 Hz, 6H), 1.65(p, J = 6.4 Hz, 2H), 1.54 – 1.45 (m, 8H), 1.38 – 1.28 (m, 4H).

[0069] 13 C NMR (125 MHz) δ 180.51, 179.83, 175.36, 138.52, 136.10, 132.15,124.17, 123.36, 120.28, 116.35, 99.90, 67.81, 43.52, 42.64, 41.94, 39.70,36.93, 28.40, 27.53, 27.43, 26.78, 26.70, 22.19.

[0070] Example 7

[0071] The preparation of J7 is the same as in Example 2, except that the raw material B1 is replaced with B3.

[0072] The results of the detection of compound J7 are as follows: 1H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 7.11 (t, J = 3.2 Hz, 1H), 4.04 (t, J = 5.7 Hz, 2H), 3.49 (td, J =5.7, 3.2 Hz, 2H), 2.05 – 1.95 (m, 5H), 1.66 (t, J = 5.7 Hz, 6H), 1.62 (d, J =4.9 Hz, 6H), 1.46 (s, 6H).

[0073] 13 C NMR (125 MHz) δ 180.30, 174.84, 174.10, 138.33, 136.10, 132.15,124.17, 123.36, 120.28, 116.35, 99.90, 67.94, 47.28, 42.92, 42.64, 38.09,37.67, 34.82, 29.77, 22.19.

[0074] Example 8

[0075] The preparation of J8 is the same as in Example 1, except that the raw material B1 is replaced with B3.

[0076] The results of the detection of compound J8 are as follows: 1 H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 6.42 (t, J = 4.4 Hz, 1H), 3.96 (dt, J = 12.6, 6.3 Hz, 1H), 3.77 (dt,J = 12.6, 6.3 Hz, 1H), 3.14 (td, J = 5.6, 4.4 Hz, 2H), 2.05 – 1.95 (m, 5H),1.74 – 1.60 (m, 14H), 1.56 – 1.48 (m, 2H), 1.46 (s, 6H).

[0077] 13C NMR (125 MHz) δ 180.51, 175.36, 174.57, 138.52, 136.10, 132.15,124.17, 123.36, 120.28, 116.35, 99.90, 67.80, 47.31, 42.64, 42.52, 39.75,37.67, 34.82, 29.77, 26.69, 25.28, 22.19.

[0078] Example 9

[0079] The preparation of J9 is the same as in Example 3, except that the raw material B1 is replaced with B3.

[0080] The results of the detection of compound J9 are as follows: 1 H NMR (500 MHz, Chloroform-d) δ8.06 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 7.6, 2.3Hz, 1H), 6.44 (t, J = 4.4 Hz, 1H), 3.99 – 3.90 (m, 1H), 3.72 (dt, J = 12.8,6.6 Hz, 1H), 3.11 (td, J = 5.6, 4.4 Hz, 2H), 2.05 – 1.95 (m, 5H), 1.69 – 1.60(m, 14H), 1.55 – 1.46 (m, 2H), 1.46 (s, 6H), 1.38 – 1.28 (m, 4H).

[0081] 13 C NMR (125 MHz) δ 180.51, 175.36, 174.62, 138.52, 136.10, 132.15,124.17, 123.36, 120.28, 116.35, 99.90, 67.81, 47.31, 42.64, 40.03, 37.67,34.82, 30.07, 29.77, 27.43, 26.78, 26.70, 22.19.

[0082] Example 10

[0083] This embodiment investigates the antitumor proliferation effects of the J1-J9 compounds synthesized in Examples 1-9, using enzalutamide as a positive control, to examine their inhibitory effects on LNCaP, VCaP, and 22Rv1 cells, and also examines the degradation of AR in LNCaP by the J1-J9 compounds.

[0084] First, LNCaP, VCaP, and 22Rv1 cells in logarithmic growth phase were digested with 0.25% trypsin. After digestion, the cell suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in fresh complete culture medium (prepared by Xavier Biotech + 10% PBS) for cell counting. Subsequently, cells were seeded into 96-well plates at a density of 5000 cells / well, with 100 μL of cell suspension per well. The 96-well plates were incubated at 37°C and 5% CO2 for 24 hours to allow complete cell adhesion.

[0085] Then, in the drug-treated group: the test compounds J1-J9 were diluted to the required concentrations using complete culture medium to obtain drug-containing medium. 100 μL of drug-containing medium was added to each well of the 96-well plate after the above culture. In the blank group: 100 μL of complete culture medium was added to each well of the 96-well plate after the above culture. In the zeroing group: no cells were present; only 100 μL of complete culture medium was added to each well. Each group had three replicates. Drugs were administered at concentration gradients of 50, 100, 200, 400, 800, and 1000 μM. After drug administration, the 96-well plates were incubated at 37°C and 5% CO2 for 72 h. Under light-protected conditions, 20 μL of MTT solution (5 mg / mL, dissolved in PBS) was added to each well. After incubating in the dark for 4 hours, remove the 96-well plate, remove the culture medium, add 150 μL of DMSO to each well, and shake for 10-15 minutes to completely dissolve the formazan in the DMSO. Then, measure the absorbance at 570 nm using a microplate reader. The proliferation inhibition rate is calculated using the following formula:

[0086] Proliferation inhibition rate PI (%) = 100% - (A treatment group - A zeroing group) / (A blank group - A zeroing group) * 100%;

[0087] Wherein, A is the absorbance of the drug administration group, A is the absorbance of the zeroing group, and A is the absorbance of the blank group; the above absorbances are the average values ​​of each group.

[0088] Western blotting was used to determine the degradation activity of the test compounds on the target proteins and to calculate the DC50 value. Log-phase LNCaP cells were seeded in culture plates and cultured at 37°C and 5% CO2 until cell adhesion. 100 μL of the test compounds at concentration gradients of 50, 100, 200, 400, 800, and 1000 μM were added, with a DMSO control group included. Each group was incubated at least three times, and incubation continued for 18–24 hours. After incubation, the culture medium was discarded, and the cells were washed with pre-cooled PBS. Lysis was performed on ice with RIPA lysis buffer containing protease inhibitors. The supernatant was collected by centrifugation, and protein concentration was determined using the BCA method (manufacturer: Beyotime). Protein samples were denatured and then subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with skim milk powder, and incubated sequentially with the target protein primary antibody, internal control antibody, and corresponding secondary antibody. Protein band images were acquired after ECL chemiluminescence imaging. ImageJ software was used for quantitative analysis of band grayscale values. The grayscale ratio of the target protein to the internal control was normalized, and the protein expression level of the solvent control group was set to 100%. The remaining percentage of the target protein at each concentration was calculated. A four-parameter logistic fitting model was used to plot the dose-response curve. The compound concentration corresponding to a 50% target protein degradation rate was obtained from the curve, which is the DC50 value.

[0089] Table 1. Inhibition of prostate cancer cells (IC50) and degradation of androgen proteins (DC50) by compounds J1-J9.

[0090]

[0091] As shown in Table 1, the half-maximal inhibitory concentrations (WMCs) of compounds J1-J9 against the three types of tumor cells were all lower than those of enzalutamide, with compound J2 exhibiting a higher tumor proliferation inhibitory capacity.

[0092] As shown in Table 1, compounds J1-J9 all require a half-degradation concentration of less than 30 μM for AR proteins in LNCaP, with compound J2 exhibiting a higher AR protein degradation capacity.

[0093] Example 11

[0094] Compound J2 was selected for further investigation into its ability to degrade AR proteins. The concentration-dependent degradation activity of the test compounds J1-J9 on the target proteins was determined using Western blotting. Logarithmically growing LNCaP cells were seeded in culture plates and cultured at 37°C and 5% CO2 until cell adhesion. 100 μL of the test compounds J1-J9 at concentration gradients of 1, 2, 5, 10, and 50 μM were added, with a DMSO control group included. Each group was incubated at least three times, and incubation continued for 18-24 hours. After incubation, the culture medium was discarded, and the cells were washed with pre-cooled PBS, lysed on ice with RIPA lysis buffer containing protease inhibitors, and the supernatant was collected by centrifugation. Protein concentration was determined using the BCA method, and the loading volume was standardized. After denaturation, the protein samples were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with skim milk powder, and incubated sequentially with the primary antibody, internal control antibody, and corresponding secondary antibody for the target protein AR. Protein band images were acquired after ECL chemiluminescence imaging. ImageJ software was used to perform quantitative analysis on the grayscale values ​​of the strips.

[0095] The results are as follows Figure 3 As shown, Western blotting revealed that as the concentration of compound J2 increased, the protein immunoblot became shallower and the protein degradation rate increased, indicating that the degradation of AR protein by the compound was dose-dependent.

[0096] Example 12

[0097] Compound J2 was dissolved in DMSO to prepare a 5 μM concentration. LNCaP cells in the logarithmic growth phase were digested, centrifuged, resuspended, and counted. The cells were then subjected to a 1x10⁻⁶ solution. 6 Cells were seeded into 6-well plates at a density of cells per well. After 24 hours of complete cell adhesion, 100 μL of 5 μM J2 was added to each well. Cells were seeded at 2, 4, 6, 8, 16, 24, and 36 hours according to the designated groups. The solution was then washed off with PBS, and the 6-well plates were removed. The method for detecting protein content was the same as in Example 11.

[0098] The results are as follows Figure 4 As shown, by Western blotting, it was found that as the treatment time of compound J2 increased, the protein immunoblot in the imaging became shallower and the protein degradation rate became higher, indicating that the degradation of AR protein by the compound is time-dependent.

[0099] Example 13

[0100] Evaluation of the in vivo antitumor activity of J2 in LNCaP tumor-bearing mouse model

[0101] A human prostate cancer LNCaP cell-bearing model was established using male nude mice. LNCaP cells in logarithmic growth phase were subcutaneously instilled into the right axilla of the mice, allowing tumors to form and grow continuously. The tumors were targeted when their average volume reached approximately 150 mm². 3 At that time, 35 tumor-bearing mice with good tumor growth status were selected to enter the drug administration experiment.

[0102] Thirty-five tumor-bearing mice were sorted according to tumor volume and then randomly divided into 5 groups of 7 mice each, as follows:

[0103] The solvent control group, the low-dose J2 group (10 mg / kg), the medium-dose J2 group (20 mg / kg), the high-dose J2 group (40 mg / kg), and the positive control group (enzalutamide, 10 mg / kg) were included.

[0104] In the experiment, 100% polyethylene glycol 200 (PEG200) was used as the solvent. The J2 dose groups were administered at doses of 10 mg / kg, 20 mg / kg, and 40 mg / kg, respectively; the positive control group received enzalutamide at 10 mg / kg; and the solvent control group received an equal volume of PEG200 solvent. Mice in each group were administered the above doses for 40 consecutive days.

[0105] During the administration period, the long diameter (L) and short diameter (W) of the tumor were measured using calipers 2-3 times per week, and the changes in mouse body weight were recorded simultaneously.

[0106] Tumor volume is calculated using the following formula:

[0107] Tumor volume (mm) 3 ) = (L × W 2 ) / 2;

[0108] To evaluate the antitumor effect of the drug, the tumor growth inhibition rate (TGI) was calculated:

[0109] TGI(%)=(Vc-Vt) / (Vc-Vo)×100;

[0110] in:

[0111] Vc represents the median tumor volume in the control group at the end of treatment;

[0112] Vt is the median tumor volume in the treatment group at the end of treatment;

[0113] Vo is the median tumor volume at the start of treatment.

[0114] At the end of the experiment, the tumor volume of each group was statistically analyzed using a two-tailed unpaired t-test (GraphPadPrism 8.0 software).

[0115] The body weight change curve of J2 in LNCaP tumor-bearing mice is as follows: Figure 1 As shown, the effect of compound J2 on animal body weight during the dosing period was evaluated. The tumor growth inhibitory effect of J2 in LNCaP tumor-bearing mice is shown in the figure. Figure 2 As shown, the effects of different doses of J2 on tumor volume changes were compared with those of the solvent control group and the positive control group enzalutamide (10 mg / kg).

[0116] Experimental results showed that compound J2 could inhibit tumor growth in LNCaP-bearing mice to varying degrees at doses of 10 mg / kg, 20 mg / kg, and 40 mg / kg, demonstrating good in vivo antitumor activity. Meanwhile, mouse body weight was monitored throughout the 40-day dosing period, and the results showed no significant decrease in body weight in any of the treatment groups, indicating that compound J2 did not exhibit significant toxicity within the aforementioned dose range.

[0117] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0118] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. An AR protein degrading agent, a pharmaceutically acceptable salt thereof, characterized in that, The AR protein degrader consists of a hydrophobic tag molecule and a parent nucleus; The parent core has the structure shown in Formula I, and the hydrophobic tag molecule includes the structures shown in B1, B2, and B3; Formula I; ； Where n = 1, 2, 3.

2. The AR protein degrading agent according to claim 1, and its pharmaceutically acceptable salt, characterized in that, The molecular structure of the AR protein degrader includes the structures shown in J1, J2, J3, J4, J5, J6, J7, J8, and J9 below; 。 3. The AR protein degrading agent according to claim 2, and its pharmaceutically acceptable salt, characterized in that, The molecular structure of the AR protein degrader is shown in J2; 。 4. A method for preparing an AR protein degrading agent as described in any one of claims 1-3, and a pharmaceutically acceptable salt thereof, characterized in that, The preparation route is shown below: ； ； ； 。 5. The use of an AR protein degrader as described in any one of claims 1-3, and a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating diseases related to abnormal AR protein expression.

6. The application according to claim 5, characterized in that, Diseases associated with abnormal AR protein expression include prostate cancer, kidney cancer, and breast cancer.

7. The use of an AR protein degrader as described in any one of claims 1-3, and a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating cancers associated with abnormal AR protein expression.

8. The application according to claim 7, characterized in that, Cancers associated with abnormal AR protein expression include prostate cancer, kidney cancer, and breast cancer.

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