Benz[b]oxepine compounds and uses thereof

Abstract

The application discloses a benzo[b]oxepane compound and application thereof, and belongs to the technical field of medicines.The structural general formula of the compound is shown as formula (I).The compound has high androgen receptor antagonistic activity, and can effectively inhibit androgen-induced downstream signal transcription of the androgen receptor.The compound has good biological activity at the protein level, the cell level and the animal level, and has good safety.Therefore, the compound can be applied to the preparation of medicines for treating diseases such as tumors caused by abnormal expression of the androgen receptor, including but not limited to prostate cancer, breast cancer and ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to benzo[b]oxetine heptane compounds and their pharmaceutical uses, particularly in the preparation of drugs for treating prostate cancer. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors occurring in men worldwide, posing a significant threat to human health and endangering human life. Because the proliferation of prostate cancer cells depends on androgens, androgen deprivation therapy has become a primary treatment method in clinical practice and the foundation of systemic treatment for prostate cancer patients. Lowering androgen levels through surgical or medical castration can significantly control tumor progression and prolong patient survival. However, long-term androgen deprivation therapy can lead to castration-resistant prostate cancer, which is more aggressive and deadly, severely reducing patient survival rates.

[0003] Given the importance of androgens in the development and progression of prostate cancer, androgen receptor (AR) antagonists are widely used clinically for the treatment of prostate cancer, including castration-resistant prostate cancer. As an important target in prostate cancer treatment, the structure and mechanism of action of AR have been extensively studied. AR belongs to the steroid receptor family within the nuclear receptor family and functions as a transcription factor. When AR binds to androgens such as testosterone (T) and dihydrotestosterone (DHT) and is activated, AR recruits coactivators, forms homodimers, and translocates to the nucleus. It then regulates the expression of downstream genes, such as prostate-specific antigen (PSA) and transmembrane serine protease 2 (TMPRSS2), by recognizing androgen response elements. Under normal circumstances, the androgen receptor signaling pathway promotes the differentiation of prostate epithelial cells, while abnormal signaling pathways regulate cell cycle, survival, and proliferation, thereby leading to tumor progression.

[0004] The androgen receptor (AR) consists of four main components: an N-terminal domain, a DNA-binding domain, a hinge region, and a ligand-binding domain (LBD). Androgens bind to the AR via the ligand-binding pocket (LBP) at the LBD. Currently used AR antagonists exert their activity by acting on this site and competing with androgens for the ligand-binding pocket. Examples include first-generation nonsteroidal AR antagonists flutamide and bicalutamide, and second-generation antagonists enzalutamide and dalolutamide. However, long-term use of first- and second-generation AR antagonists inevitably leads to drug resistance mutations, resulting in treatment failure. Specifically, the W742L / C or T878A / S mutation causes bicalutamide to change from an antagonist to a partial agonist or agonist. The F877L mutation causes enzalutamide to become an agonist, significantly reducing the drug's clinical efficacy.

[0005] Therefore, designing and discovering novel AR antagonists that act on other domains can help overcome drug resistance problems in the clinical treatment of prostate cancer, avoid treatment failure caused by cross-resistance, and increase patients' drug choices and treatment benefits in clinical treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a novel compound with androgen receptor antagonistic activity, which can be used in the preparation of drugs for treating androgen receptor-mediated diseases such as prostate cancer.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a benzo[b]oxacycloheptane compound, or a pharmaceutically acceptable salt thereof, said compound having a structure of the general formula shown in formula (I):

[0009]

[0010] Among them, R1 is selected from hydrogen, chlorine, and bromine;

[0011] R2 is selected from chlorine,

[0012]

[0013] Preferably, the structural formula of the compound is shown in any of the examples in Table 1.

[0014] Table 1

[0015]

[0016] In this invention, the pharmaceutically acceptable salts include, but are not limited to, organic acid salts, inorganic acid salts, organic base salts, and inorganic base salts; the organic acid salts include, but are not limited to, oxalate, lactate, p-toluenesulfonate, malate, citrate, fumarate, camphorsulfonate, and methanesulfonate; the inorganic acid salts include, but are not limited to, nitrates, sulfates, hydrohalates, and phosphates; the organic base salts include, but are not limited to, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, and tert-butylamine; and the inorganic base salts include, but are not limited to, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0017] This invention has revealed that the benzo[b]oxetine heptane compounds exhibit significant antagonistic activity against androgen receptors, stably binding to the androgen receptor ligand-binding domain, and demonstrating good biological activity in both in vivo and in vitro biological evaluations. Therefore, these compounds can be applied to the treatment of tumors associated with abnormal androgen receptor expression or mutations.

[0018] Specifically, the present invention provides the use of the aforementioned benzo[b]oxetine heptane compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or prodrug molecules thereof, or deuterated derivatives thereof, in the preparation of androgen receptor antagonists.

[0019] This invention also provides the use of the aforementioned benzo[b]oxetine heptane compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or prodrug molecules thereof, or deuterated derivatives thereof, in the preparation of androgen receptor nuclear translocation inhibitors or androgen receptor dimerization inhibitors. Studies have shown that these compounds can effectively inhibit the activation and translocation of androgen receptors to the cell nucleus; and can effectively inhibit androgen receptor dimerization.

[0020] This invention also provides the use of the aforementioned benzo[b]oxetine heptane compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or prodrug molecule thereof, or deuterated derivatives thereof, in medicaments for treating prostate cancer, breast cancer, and ovarian cancer. The prostate cancers include, but are not limited to, metastatic prostate cancer and castration-resistant prostate cancer. It is worth noting that the pharmaceutical use of these compounds is not limited to the preparation of drugs for the aforementioned tumors.

[0021] The therapeutic mechanism of the drug includes: its active ingredient, benzo[b]oxetine heptane compounds, inhibits tumor cell proliferation by antagonizing androgen receptor activity.

[0022] Furthermore, the prostate cancer includes AR F877L / T878A Mutant prostate cancer or AR W742C Mutant prostate cancer. Studies have shown that the benzo[b]oxetine heptane compounds provided in this invention also exhibit excellent antagonistic activity against drug-resistant prostate cancer with gene mutations in the ligand-binding domain (LBD) of the androgen receptor (AR), such as F877L, T878A, and W742C.

[0023] The present invention also provides a pharmaceutical composition for treating tumors associated with abnormal expression or mutation of androgen receptors, comprising an effective dose of the benzo[b]oxetine compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof, or a deuterated thereof, and a pharmaceutically acceptable carrier.

[0024] The carrier is a commonly used excipient, filler, disintegrant, diluent, surfactant, absorption enhancer, adsorbent, binder, lubricant, humectant, flavoring agent, sweetener, etc. in the pharmaceutical field. The pharmaceutical formulations of this invention can be prepared according to conventional methods in the pharmaceutical field, including but not limited to capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention provides a novel class of benzo[b]oxetine heptane compounds. These compounds exhibit excellent androgen receptor antagonistic activity, effectively inhibiting the AR signaling pathway, AR nuclear translocation, and dimerization to antagonize androgen receptors, thereby effectively inhibiting tumor cell proliferation. They maintain antagonistic activity against clinically identified AR drug-resistant mutants and have minimal toxic side effects on normal cells. Therefore, these compounds can be applied to the treatment of tumors related to abnormal androgen receptor expression or mutations, including but not limited to the treatment of prostate cancer, metastatic prostate cancer, castration-resistant prostate cancer, breast cancer, and ovarian cancer. Attached Figure Description

[0027] Figure 1 This is a flowchart of the preparation process for compounds A4 to A16.

[0028] Figure 2 The results show the affinity test results between compounds A2 and A8 and AR LBD.

[0029] Figure 3 The results show the transcriptional repression activity of compound A8 against clinically common AR drug-resistant mutants.

[0030] Figure 4 The results show the effect of compound A8 on AR nuclear translocation.

[0031] Figure 5 The results show the inhibitory effect of compound A8 on AR dimerization.

[0032] Figure 6 The results show the inhibitory effect of compound A8 on the proliferation of LNCaP xenografts. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Compounds A1-A3 are from the Chemdiv compound library, with their numbers D443-0124, D443-0279, and D443-0425.

[0037] Example 1: Synthesis of target molecule A4

[0038] The process flow diagram for the preparation of 7-chloro-N-(3-(chloromethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (compound A4) is shown below. Figure 1 As shown, the specific method is as follows:

[0039] (a) Synthesis of 3-(chloromethyl)-1,2,4-thiadiazole-5-amine (compound I-2)

[0040] 2-Chloroacetamidine hydrochloride (I-1, 640 mg, 5.0 mmol) was dissolved in anhydrous methanol (10 mL), cooled to 0 °C, and triethylamine (17.5 mL, 12.5 mmol) and liquid bromine (719 mg, 4.5 mmol) were added dropwise. A methanol solution of potassium thiocyanate (534 mg, 5.5 mmol) was slowly added dropwise to the system, and the mixture was stirred at 0 °C for 2 h. The reaction mixture was quenched with an appropriate amount of water, extracted with ethyl acetate, the organic layer was washed with saturated saline solution, dried over anhydrous Na₂SO₄, concentrated, and the residue was subjected to column chromatography to give a white solid with a yield of 75%. 1 H NMR (500MHz, DMSO-d6) δ8.05 (s, 2H), 4.53 (s, 2H); ESI-MS: m / z=150.0[M+H] + .

[0041] (b) Synthesis of methyl (E)-4-(4-chloro-2-formylphenoxy)but-2-enoate (compound I-5)

[0042] 5-Chloro-2-hydroxybenzaldehyde (I-3, 939 mg, 6.0 mmol) and (E)-4-bromobut-2-enoate methyl ester (I-4, 1.3 g, 7.2 mmol) were dissolved in DMF (10 mL), and then potassium carbonate (1.6 g, 12.0 mmol) was added to the system and stirred at room temperature for 4 h. After the reaction was complete, an appropriate amount of aqueous solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, concentrated, and the residue was subjected to column chromatography to give a white solid with a yield of 84%. 1H NMR(500MHz,Chloroform-d)δ10.47(s,1H),7.81(d,J=2.7Hz,1H),7.49(dd,J=8.9,2.8Hz,1H),7.09(dt,J=15.8,4.1Hz,1 H), 6.90 (d, J=8.9Hz, 1H), 6.20 (dt, J=15.8, 2.0Hz, 1H), 4.83 (dd, J=4.1, 2.1Hz, 2H), 3.78 (s, 3H); ESI-MS: m / z=255.0[M+H] + .

[0043] (c) Synthesis of methyl 7-chlorobenzo[b]oxepine-4-carboxylate (compound I-6)

[0044] Compound I-5 (1.1 g, 4.2 mmol) was dissolved in dichloromethane (8 mL), and then DBU (1.2 mL, 8.2 mmol) was added to the system and stirred at room temperature for 6 h. After the reaction was complete, an appropriate amount of water was added to the system, followed by extraction with an appropriate amount of ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, concentrated, and the residue was subjected to column chromatography to give a white solid with a yield of 50%. 1 H NMR(500MHz,Chloroform-d)δ7.57–7.49(m,2H),7.40(dt,J=8.7,0.7Hz,1H),7.31(dd,J=8 .7,2.1Hz,1H),6.87(s,1H),6.58(d,J=15.7Hz,1H),3.82(s,3H); ESI-MS:m / z=237.0[M+H] + .

[0045] (d) Synthesis of 7-chlorobenzo[b]oxepine-4-carboxylic acid (compound I-7)

[0046] Compound I-6 (850 mg, 3.6 mmol) was dissolved in tetrahydrofuran and water (3:1, 20 mL), and sodium hydroxide (1.2 g, 30.0 mmol) was added. The mixture was reacted overnight at room temperature. Part of the solvent was removed under reduced pressure, and the pH was adjusted to 3-4 with 2 mol / L dilute hydrochloric acid. The mixture was then filtered to give a white solid I-7, in 93% yield; ESI-MS: m / z = 223.0 [M+H]. + .

[0047] (e) Synthesis of 7-chloro-N-(3-(chloromethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxepine-4-carboxamide (compound A4)

[0048] Compound I-7 (222 mg, 1.0 mmol), compound I-2 (149 mg, 1.0 mmol), EDCI (382 mg, 2.0 mmol), and 4-PPY (15 mg, 0.1 mmol) were dissolved in DCM (10 mL) and refluxed with stirring overnight. The solvent was removed under reduced pressure, an appropriate amount of water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline solution, dried over anhydrous Na2SO4, concentrated under reduced pressure, and the residue was subjected to column chromatography to give a white solid A4 in 42% yield. 1 H NMR(500MHz,Chloroform-d)δ9.73(s,1H),7.70(d,J=15.1Hz,1H),7.53(d,J=2.2Hz,1H),7.39–7.36(m,1H ),7.30(dd,J=8.8,2.1Hz,1H),6.96(s,1H),6.70(d,J=15.2Hz,1H),4.62(s,2H).ESI-MS:m / z=353.9[M+H] + .

[0049] Example 2: Synthesis of target molecules A5-A16

[0050] 1. Preparation of 7-chloro-N-(3-(pyrrolidone-1-ylmethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A5)

[0051] Compound A4 (1.4 g, 4.0 mmol) and tetrahydropyrrole (500 μL, 6.0 mmol) were dissolved in acetonitrile (20 mL), and then triethylamine (1.11 mL, 8.0 mmol) was added. The mixture was reacted overnight at room temperature. The solvent was removed under reduced pressure, and an appropriate amount of water was added. The mixture was extracted with ethyl acetate, and the organic layer was washed with a saturated saline solution. The solution was then dried over anhydrous Na2SO4, concentrated under reduced pressure, and the residue was subjected to column chromatography to give a white solid A5 in 65% yield. 1 H NMR(500MHz,DMSO-d6)δ12.91(s,1H),7.86–7.77(m,2H),7.71(d,J=8.8Hz,1H),7.46(dd,J=8.8,2.2Hz,1H),7 .41(s,1H),6.94(d,J=15.7Hz,1H),3.90(s,2H),2.68(s,4H),1.73(p,J=3.3Hz,4H).ESI-MS:m / z=398.1[M+H] + .

[0052] 2. Preparation of 7-chloro-N-(3-(piperidin-1-ylmethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A6)

[0053] Following the synthesis of compound A5, tetrahydropyrrole was replaced with piperidine, yielding a white solid in 52% yield; 1 H NMR(500MHz,DMSO-d6)δ13.26(s,1H),7.87–7.77(m,2H),7.71(d,J=8.8Hz,1H),7.46(dd,J=8.8,2.2Hz,1H),7.42(s,1H), 6.93(d,J=15.6Hz,1H),3.66(s,2H),2.46(s,4H),1.49(q,J=5.7Hz,4H),1.35(d,J=16.0Hz,2H).ESI-MS:m / z=403.1[M+H] + .

[0054] 3. Preparation of 7-chloro-N-(3-(thiomorpholinemethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A7)

[0055] Following the synthesis of compound A5, tetrahydropyrrole was replaced with thiomorpholine to give a white solid in 85% yield; 1 HNMR(500MHz,DMSO-d6)δ13.31(s,1H),7.85–7.78(m,2H),7.71(d,J=8.8Hz,1H),7.46(dd,J=8.8,2.2Hz,1H),7.42( s,1H),6.93(d,J=15.6Hz,1H),3.72(s,2H),2.76(dd,J=6.4,3.6Hz,4H),2.64–2.58(m,4H); ESI-MS: m / z=421.1[M+H] + .

[0056] 4. Preparation of 7-chloro-N-(3-(morpholinomethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A8)

[0057] Following the synthesis of compound A5, tetrahydropyrrole was replaced with morpholine to give a white solid in 80% yield; 1H NMR (500MHz, CDCl3-d6) δ10.33(s,1H),7.75(d,J=15.2Hz,1H),7.59(dd,J=2.1,0.6Hz,1H),7.43(dt,J=8.8,0.8Hz,1H),7.36(dd,J=8.8 ,2.1Hz,1H),7.00(s,1H),6.79(dd,J=15.2,0.6Hz,1H),3.83(s,2H),3.79–3.73(m,4H),2.62(t,J=4.7Hz,4H); ESI-MS:m / z=405.1[M+H] + .

[0058] 5. Preparation of 7-chloro-N-(3-((1,1-thiomorpholino)methyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A9)

[0059] Following the synthesis of compound A5, tetrahydropyrrole was replaced with thiomorpholine-1,1-dioxide to give a white solid in 75% yield. 1 H NMR(500MHz,DMSO-d6)δ13.32(s,1H),7.86–7.79(m,2H),7.73–7.70(m,1H),7.47(dd,J=8.8,2.2Hz,1H),7.43(s, 1H), 6.95 (d, J=15.6Hz, 1H), 3.92 (s, 2H), 3.12 (dd, J=7.2, 3.2Hz, 4H), 3.07–3.02 (m, 4H); ESI-MS: m / z=453.0[M+H] + .

[0060] Preparation of 6,7-chloro-N-(3-(piperazin-1-ylmethyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (compound A10)

[0061] Following the synthesis of compound A5, tetrahydropyrrole was replaced with piperazine to give a white solid in 47% yield; 1 H NMR (500MHz, DMSO-d6) δ13.56(s,1H),9.49(s,2H),7.85(dd,J=8.9,6.7Hz,2H),7.72(dt,J=8.9,0.7H z,1H),7.50–7.44(m,2H),7.04(d,J=15.6Hz,1H),4.42(s,8H),3.17(s,2H); ESI-MS:m / z=404.1[M+H] + .

[0062] 7. Preparation of 7-chloro-N-(3-(4-methylpiperazin-1-yl)methyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A11)

[0063] Following the synthesis of compound A5, tetrahydropyrrole was replaced with 4-methylpiperazine to give a white solid in 47% yield; 1 H NMR(500MHz,DMSO-d6)δ12.96(s,1H),7.87–7.79(m,2H),7.71(d,J=8.8Hz,1H),7.47(dd,J=8.8,2.3Hz,1 H),7.42(s,1H),6.94(d,J=15.6Hz,1H),3.67(s,2H),2.40(s,8H),2.20(s,3H); ESI-MS:m / z=418.1[M+H] + .

[0064] 8. 7-Chloro-N-(3-((4-hydroxypiperidin-1-yl)methyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (compound A12)

[0065] Following the synthesis of compound A5, tetrahydropyrrole was replaced with 4-hydroxypiperidine to give a white solid in 62% yield; 1 H NMR (500MHz, DMSO-d6) δ13.26(s,1H),7.86–7.77(m,2H),7.71(d,J=8.8Hz,1H),7.46(dd,J=8.8,2.2Hz,1H),7.42(s,1H),6.93(d,J=15.6Hz,1H),4. 58–4.50(m,1H),3.66(s,2H),3.43(s,1H),2.76(d,J=10.2Hz,2H),2.20(s ,2H),1.74–1.64(m,2H),1.39(d,J=10.7Hz,2H); ESI-MS:m / z=419.1[M+H] + .

[0066] 9. Preparation of 7-chloro-N-(3-((4-(methanesulfonyl)piperazin-1-yl)methyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A13)

[0067] Following the synthesis of compound A5, tetrahydropyrrole was replaced with 1-methanesulfonylpiperazine to give a white solid in 68% yield; 1H NMR (500MHz, DMSO-d6) δ13.30(s,1H),7.86–7.79(m,2H),7.71(d,J=8.8Hz,1H),7.48–7.42(m,2H),6.94(d,J= 15.6Hz,1H),3.76(s,2H),3.11(t,J=4.9Hz,4H),2.87(s,3H),2.61(t,J=5.0Hz,4H); ESI-MS:m / z=482.1[M+H] + .

[0068] 10. Preparation of 7-chloro-N-(3-((4-(cyclopropanecarbonyl)piperazin-1-yl)methyl)-1,2,4-thiadiazol-5-yl)benzo[b]oxetane-4-carboxamide (Compound A14)

[0069] Following the synthesis of compound A5, tetrahydropyrrole was replaced with 1-cyclopropylformylpiperazine to give a white solid in 88% yield; 1 H NMR (500MHz, DMSO-d6) δ13.31(s,1H),7.86–7.79(m,2H),7.71(d,J=8.8Hz,1H),7.47(dd,J=8.8,2.2Hz,1H),7.43(s,1H),6.94(d,J=15.6Hz, 1H),3.70(d,J=35.4Hz,4H),3.46(s,2H),2.54(s,2H),2.46(s,2H),1.95(tt,J=7.9,4.9Hz,1H),0.73–0.66(m,4H); ESI-MS: m / z=472.1[M+H] + .

[0070] 11. Preparation of ethyl piperazine-1-carboxylate (compound A15) of 4-((5-(7-chlorobenzo[b]oxetane-4-carboxamido)-1,2,4-thiadiazol-3-yl)methyl)piperazine-1-carboxylate

[0071] Following the synthesis of compound A5, tetrahydropyrrole was replaced with ethyl N-piperazine carboxylate to give a white solid in 82% yield; 1H NMR (500MHz, DMSO-d6) δ13.30(s,1H),7.85–7.79(m,2H),7.71(d,J=8.8Hz,1H),7.47(dd,J=8.8,2.2Hz,1H),7.43(s,1H),6.93(d,J=15.6 Hz,1H),4.02(q,J=7.1Hz,2H),3.72(s,2H),3.36(t,J=4.7Hz,4H),2.47(t,J=5.1Hz,4H),1.17(t,J=7.1Hz,3H); ESI-MS: m / z=476.1[M+H] + .

[0072] 12. Preparation of tert-butyl piperazine-1-carboxylate (compound A16) of 4-((5-(7-chlorobenzo[b]oxetane-4-carboxamido)-1,2,4-thiadiazol-3-yl)methyl)piperazine-1-carboxylate

[0073] Following the synthesis of compound A5, tetrahydropyrrole was replaced with N-BOC-piperazine to give a white solid in 77% yield; 1 H NMR(500MHz,DMSO-d6)δ13.30(s,1H),7.86–7.78(m,2H),7.73–7.69(m,1H),7.47(dd,J=8.8,2.2Hz,1H),7.43(s,1 H),6.93(d,J=15.6Hz,1H),3.71(s,2H),3.31(s,4H),2.45(t,J=5.1Hz,4H),1.38(s,9H); ESI-MS: m / z=504.1[M+H] + .

[0074] Biological Experiment Example 1: Test of the transcriptional repressive activity of compounds A1-A16 on androgen receptors

[0075] Detection Principle: Cells were derived from the Cell Bank of the Chinese Academy of Sciences. The plasmid pLJM1-ARR2PB-eGFP, containing the androgen receptor (AR)-responsive promoter ARR2PB, was stably transfected into androgen-dependent prostate cancer cells (LNCaP). An enhanced green fluorescent protein (eGFP) expression fragment was linked downstream of ARR2PB; therefore, eGFP expression is positively correlated with AR transcriptional activity. The LNCaP-ARR2PB-eGFP cell line, constructed through stable transfection, allows for the quantitative detection of the compound's antagonistic activity against AR by detecting eGFP expression.

[0076] Detection Procedure: LNCaP-ARR2PB-eGFP cells were starved for 4-5 days in RPMI-1640 medium containing 5% hormone-free fetal bovine serum treated with activated charcoal and dextran to consume the original hormones and eliminate interference. Once the cells reached a suitable density, they were digested and centrifuged to collect the cells. The cell suspension density was adjusted to 3.5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 10 cells / well into 96-well plates. After cell attachment the following day, each well was treated with 5 nM dihydrotestosterone (DHT) and different concentration gradients of the test compound. A blank control group was also included, receiving only blank culture medium. After 72 hours of incubation, fluorescence intensity was measured using a microplate reader (excitation wavelength: 485 nm; emission wavelength: 535 nm). The half-maximal inhibitory concentration (IC50) of the compound antagonizing AR was then calculated. 50 ).

[0077] Detection results: As shown in Table 2, A1–A16 all exhibited good AR transcriptional repression activity, IC50 50 The activity ranges from 0.04 μM to 6.7 μM. Among them, the activities of A2, A3, and A6 are similar to those of enzalutamide, while the activities of A7, A8, A9, A13, A14, and A15 are superior to those of enzalutamide, showing good application prospects.

[0078] Table 2. Antagonistic activity of compounds against AR transcriptional levels

[0079] serial number <![CDATA[IC 50 (μM)]]> serial number <![CDATA[IC 50 (μM)]]> serial number <![CDATA[IC 50 (μM)]]> A1 1.58 A2 0.14 A3 0.17 A4 6.70 A5 0.37 A6 0.17 A7 0.05 A8 0.04 A9 0.06 A10 0.27 A11 0.68 A12 0.52 A13 0.07 A14 0.04 A15 0.09 A16 0.34 Enzalutamide 0.14

[0080] Biological Experiment Example 2: Assay of the Proliferative Activity of Compounds A1-A16 on AR-Positive Prostate Cancer Cells LNCaP

[0081] Detection Principle: The MTT assay is a commonly used method for detecting cell proliferation inhibition. MTT can be reduced to formazan, a water-insoluble blue-purple crystalline compound, by succinate dehydrogenase and cytochrome C in the mitochondria of living cells. The amount of formazan produced is linearly positively correlated with the number of living cells within a certain range. LNCaP cells are AR-positive androgen-dependent cell lines. AR antagonists can inhibit the proliferation of LNCaP cells by antagonizing the effects of androgens. A certain amount of MTT solution is added to drug-treated LNCaP cells to form formazan, which is then dissolved and crystallized using DMSO or a triplet solution. The optical density at 570 nm wavelength is measured using an ELISA reader to reflect the number of living cells, thus indicating the effect of the drug on LNCaP proliferation.

[0082] Detection Procedure: LNCaP cells grown to the logarithmic growth phase were digested and collected. The cell suspension concentration was adjusted, and the cells were seeded into 96-well clear plates at a density of 3000 cells / well. After 24 hours, the cells adhered. 10 μL of a concentration gradient compound solution diluted with culture medium was added to each well. A blank control group (cell-free) and a solvent control group (culture medium only) were also included. Incubation continued for 72 hours. 10 μL of 5 mg / mL MTT solution was added to each well, and incubation was performed for 2–4 hours. Then, 100 μL of a triple solution was added to the well, and incubation was performed overnight to dissolve formazan. OD values ​​at 570 nM and 620 nM were measured using a microplate reader, and the increments were calculated.

[0083] Results: As shown in Table 3, all compounds exhibited good inhibitory activity against LNCaP proliferation. Except for compound A1, the proliferation inhibitory activities of the other compounds were superior to enzalutamide, and most compounds had IC50 < 2 μM, demonstrating excellent therapeutic potential at the cellular level.

[0084] Table 3. Inhibitory activity of compounds against LNCaP proliferation

[0085] serial number <![CDATA[IC 50 (μM)]]> serial number <![CDATA[IC 50 (μM)]]> serial number <![CDATA[IC 50 (μM)]]> A1 28.04 A2 2.81 A3 7.91 A4 5.56 A5 1.67 A6 2.05 A7 1.74 A8 1.52 A9 1.18 A10 1.07 A11 0.69 A12 1.70 A13 1.41 A14 1.10 A15 1.94 A16 2.59 Enzalutamide 19.10

[0086] Biological Experiment Example 3: Protein Thermal Migration Assay for Detecting the Binding of Compounds A2 and A8 with AR LBD

[0087] Detection Principle: Protein thermal migration technology is one of the methods for detecting molecule-protein binding forces based on protein stability. The principle is that during gradual heating, the secondary structure of a protein gradually unfolds, exposing autofluorescent groups (tryptophan, tyrosine, etc.). The unfolding process of the protein is detected by detecting changes in its autofluorescence, and the change in the protein's Tm value reflects its stability. Upon binding to small molecule compounds, the protein is induced to undergo conformational changes, making it more stable or unstable, resulting in an increase or decrease in the Tm value, thus verifying the affinity between the compound and the protein.

[0088] Detection Procedure: Dilute the purified AR LBD protein to 0.8 mg / mL with binding buffer. Dilute the analyte compound to twice its final concentration with binding buffer. Mix the protein and compound solutions 1:1 and incubate for 30 minutes. Transfer the sample to a high-precision capillary pipette via capillary action and heat it from 25°C to 90°C at a rate of 2°C / min. Collect the fluorescence signal and calculate the first derivative and Tm value.

[0089] Test results: such as Figure 2As shown, through incubation with different concentrations of A8, the Tm value of AR LBD continuously shifts towards increasing temperature, indicating that A8 can form a stable binding with AR LBD. Calculations of the ΔTm value reveal that the addition of 100 μM A2 also stabilizes AR LBD, but its activity is slightly lower than that of A8.

[0090] Biological Experiment Example 4: Dual-luciferase reporter gene assay for the effect of compound A8 on AR F877L / T878A AR W742C activity of mutants

[0091] Detection Principle: PC3 is an AR-negative prostate cancer cell line. Transfecting PC3 cells with pCMV-AR, ARR2PB-Luc, and Renilla plasmids allows for the quantitative detection of the activity of compounds against mutant AR. Luc is a firefly luciferase; linking it downstream of the ARR2PB promoter allows for the regulation of Luc expression through AR transcriptional activity. The Renilla plasmid expresses Renjani luciferase and can be used to correct transfection efficiency in the transfection system. Detecting the expression ratio of firefly luciferase to Renjani luciferase allows for the quantitative detection of the interaction between transcription factors and target promoters.

[0092] Detection procedure: PC3 prostate cancer cell lines that do not express endogenous AR were starved with hormone-free serum for 2–3 days. Cells were collected, the cell suspension density was adjusted, and the cells were seeded into 96-well plates at 1 × 10⁶ cells per well. 4 Cells. The following day, 72 ng pCMV-AR, 24 ng ARR2PB-Luc, and 5 ng Renilla plasmid were mixed with serum-reduced medium (opti-MEM) and transfection reagent and incubated at room temperature for 30 min. After 24 h of co-transfection, the medium was replaced with fresh medium and serially diluted compounds and 2 nM DHT were added. After 24 h, the medium was discarded, and the cells were washed with 100 μL PBS. 20 μL of 1×PLB was added to each well, and the cells were shaken to fully lyse them. Then, 30 μL of firefly luciferase substrate was added to each well, and the first fluorescence value was measured. After the measurement, 30 μL of Renilla luciferase substrate was added to each well, mixed, and the second fluorescence value was measured. The ratio of the two sets of data was calculated, and the transcriptional activity of AR was analyzed.

[0093] Test results: such as Figure 3 As shown, AR transfection F877L / T878A Subsequently, enzalutamide (Enz) transforms from an antagonist to a partial agonist, exhibiting some antagonistic activity at low concentrations and agonistic activity at high concentrations. Transfected AR W742CSubsequently, bicalutamide (Bic) was converted into a full agonist, exhibiting agonistic activity at all tested concentrations. However, compounds A8 and dalolutamide both demonstrated excellent antagonistic activity in both mutant models, showing potential for treating drug-resistant prostate cancer.

[0094] Biological Experiment Example 5: The Effect of Compound A8 on AR Nuclear Translocation

[0095] Detection principle: As a transcription factor, AR translocates to the cell nucleus after activation by androgens, where it exerts its activity. By extracting and separating total protein from the cell nucleus and cytoplasm, and using Western blotting to detect the AR content in the nucleus and cytoplasm, the effect of the compound on AR nuclear translocation was tested.

[0096] Detection Procedure: LNCaP cells were starved with hormone-free serum for 2–3 days, and the cells were collected and seeded into 6-well plates. The next day, after cell adhesion, a compound with a final concentration of 10 μM was added to the wells, and the cells were incubated for 4–6 hours, followed by incubation with DHT for another 2 hours. Cells were collected by centrifugation, and nuclear and cytoplasmic proteins were isolated and extracted using a kit. The localization of the angiocytes (AR) was identified by Western blot. GAPDH was used as an endogenous control for cytoplasmic proteins, and Lamin B1 was used as an endogenous control for nuclear proteins. The separation efficiency of the nucleus and cytoplasm was assessed.

[0097] Test results: such as Figure 4 As shown, in the DMSO group without DHT, AR was localized in the cytoplasm, with only a very small amount of AR in the nucleus. In contrast, in the group with only DHT, AR was abundant in the nucleus, indicating that AR translocates to the nucleus after binding with DHT. The positive control drugs enzalutamide and compound A8 effectively inhibited nuclear translocation, and almost no AR was observed in the nucleus.

[0098] Biological Experiment Example 6: The Effect of Compound A8 on AR Dimerization

[0099] Detection principle: NanoLuc is an enzyme with luciferase activity, consisting of two inactive moieties, LgBiT and SmBiT, which are fused and expressed at the N-terminus and C-terminus of AR, respectively. When AR dimers, LgBiT and SmBiT bind together to form an active luciferase. When a substrate for this luciferase is added, NanoLuc catalyzes the cleavage of the substrate, emitting fluorescence. The degree of AR dimerization in cells can be determined by detecting the intensity of the fluorescence signal.

[0100] Detection procedure: 293T cell line was starved with hormone-free serum for 2 days, cells were collected, cell suspension density was adjusted, and cells were seeded into 96-well plates at 1×10⁶ cells per well. 4Cells. The following day, 50 ng LgBiT-AR and 50 ng AR-SmBiT plasmids were mixed thoroughly with serum-reduced medium (opti-MEM) and transfection reagent and incubated at room temperature for 30 min. After co-transfection for 24 h, the medium was replaced with fresh medium and serially diluted compounds were added. After incubation for 2–4 hours, luciferase substrate was added to the wells, and fluorescence values ​​were detected using a microplate reader for 10 minutes to obtain a baseline. DHT was added to the wells to a final concentration of 5 nM, and fluorescence values ​​were continuously detected for 1 hour. Data were recorded and AR dimerization was analyzed.

[0101] Test results: such as Figure 5 As shown, the DMSO group showed no hormone stimulation, therefore AR did not dimerize, and the fluorescence value remained at the baseline level. In the DHT group, the fluorescence signal rapidly increased after the addition of DHT, indicating that AR quickly dimerizes and exerts its activity after binding with DHT. Treatment with 2 μM and 0.2 μM A8 resulted in significantly lower fluorescence values ​​than the DHT group, indicating that A8 effectively inhibits AR dimerization. Biological Experiment Example 7: Detection of the effect of orally administered compound A8 on the proliferation of LNCaP cell xenograft models.

[0102] Detection procedure: After acclimatizing male BALB / c nude mice aged 4-6 weeks for one week, LNCaP tumor cells mixed with matrix gel at a 1:1 ratio were inoculated into their right back. The inoculation continued until the average tumor volume reached 100 mm². 3 Mice were randomly divided into two groups based on tumor volume: an enzalutamide group (40 mg / kg, once daily) and a compound A8 group (40 mg / kg, twice daily). Administered the drugs by gavage. During administration, mouse body weight and tumor volume were monitored, and tumor length and width were measured. Tumor volume (mm³) was calculated using the formula... 3 = Length (mm) × Width (mm) 2 / 2. On the last day of drug administration, the tumor was collected and weighed.

[0103] Test results: such as Figure 6 As shown, compound A8, when administered orally at 40 mg / kg, effectively inhibited the proliferation of prostate cancer xenograft tumors, with effects similar to enzalutamide. Furthermore, no significant weight loss was observed in mice during the administration period. This indicates that compound A8 possesses both oral efficacy and high safety at this dosage.

Claims

1. A benz[b]oxepine compound of the general structure of formula (I) or a pharmaceutically acceptable salt thereof, ###0001### (I) wherein R1 is selected from hydrogen, chloro, bromo; R2 is selected from hydrogen, chloro, bromo; R3 is selected from hydrogen, chloro, bromo; R4 is selected from hydrogen, chloro, bromo; R5 is selected from hydrogen, chloro, bromo; R6 is selected from hydrogen, chloro, bromo; R7 is selected from hydrogen, chloro, bromo; R8 is selected from hydrogen, chloro, bromo; R9 is selected from hydrogen, chloro, bromo; R10 is selected from hydrogen, chloro, bromo; R11 is selected from hydrogen, chloro, bromo; R12 is selected from hydrogen, chloro, bromo; R13 is selected from hydrogen, chloro, bromo; R14 is selected from hydrogen, chloro, bromo; R15 is selected from hydrogen, chloro, bromo; R16 is selected from hydrogen, chloro, bromo; R17 is selected from hydrogen, chloro, bromo; R18 is selected from hydrogen, chloro, bromo; R19 is selected from hydrogen, chloro, bromo; R20 is selected from hydrogen, chloro, bromo; R21 is selected from hydrogen, chloro, bromo; R22 is selected from hydrogen, chloro, bromo; R23 is selected from hydrogen, chloro, bromo; R24 is selected from hydrogen, chloro, bromo; R25 is selected from hydrogen, chloro, bromo; R26 is selected from hydrogen, chloro, bromo; R27 is selected from hydrogen, chloro, bromo; R28 is selected from hydrogen, chloro, bromo; R29 is selected from hydrogen, chloro, bromo; R30 is selected from hydrogen, chloro, bromo; R31 is selected from hydrogen, chloro, bromo; R32 is selected from hydrogen, chloro, bromo; R33 is selected from hydrogen, chloro, bromo; R34 is selected from hydrogen, chloro, bromo; R35 is selected from hydrogen, chloro, bromo; R36 is selected from hydrogen, chloro, bromo; R37 is selected from hydrogen, chloro, bromo; R38 is selected from hydrogen, chloro, bromo; R39 is selected from hydrogen, chloro, bromo; R40 is selected from hydrogen, chloro, bromo; R41 is selected from hydrogen, chloro, bromo; R42 is selected from hydrogen, chloro, bromo; R43 is selected from hydrogen, chloro, bromo; R44 is selected from hydrogen, chloro, bromo; R45 is selected from hydrogen, chloro, bromo; R46 is selected from hydrogen, chloro, bromo; R47 is selected from hydrogen, chloro, bromo; R48 is selected from hydrogen, chloro, bromo; R49 is selected from hydrogen, chloro, bromo; R50 is selected from hydrogen, chloro, bromo; R51 is selected from hydrogen, chloro, bromo; R52 is selected from hydrogen, chloro, bromo; R53 is selected from hydrogen, chloro, bromo; R54 is selected from hydrogen, chloro, bromo; R55 is selected from hydrogen, chloro, bromo; R56 is selected from hydrogen, chloro, bromo; R57 is selected from hydrogen, chloro, bromo; R58 is selected from hydrogen, chloro, bromo; R59 is selected from hydrogen, chloro, bromo; R60 is selected from hydrogen, chloro, bromo; R61 is selected from hydrogen, chloro, bromo; R62 is selected from hydrogen, chloro, bromo; R63 is selected from hydrogen, chloro, bromo; R64 is selected from hydrogen, chloro, bromo; R65 is selected from hydrogen, chloro, bromo; R66 is selected from hydrogen, chloro, bromo; R67 is selected from hydrogen, chloro, bromo; R68 is selected from hydrogen, chloro, bromo; R69 is selected from hydrogen, chloro, bromo; R70 is selected from hydrogen, chloro, bromo; R71 is selected from hydrogen, chloro, bromo; R72 is selected from hydrogen, chloro, bromo; R73 is selected from hydrogen, chloro, bromo; R74 is selected from hydrogen, wherein ​ R2is selected from chloro, 2. The benzo[b]oxepine compound or pharmaceutically acceptable salt thereof according to claim 1, wherein ​ 3. The benzo[b]oxepane compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein ​ ​ ​ ​ 7. Use according to claim 6, wherein the prostate cancer is AR F877L / T878A mutant prostate cancer or AR W742C mutant prostate cancer.

8. A pharmaceutical composition, characterized by, ​ 9. The pharmaceutical composition of claim 8, wherein ​

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