Targeting hdac10 compounds, methods of making and using the same

Abstract

The application provides a targeted HDAC10 compound and a preparation method and application thereof. The targeted HDAC10 compound has the following structure general formula (I) or structure general formula (II) shown in the following structure general formula (I) or structure general formula (II) and pharmaceutically acceptable salts, hydrates or solvates thereof: the compound shown in the formula (I) of the application can be used as an HDAC10 subtype selective inhibitor and has high selective HDAC10 inhibitory activity; the compound shown in the formula (II) of the application can be used as an HDAC10 subtype selective degradation agent and shows a higher HDAC10 degradation effect.

Description

Technical Field

[0001] This invention relates to the field of organic compound synthesis and pharmaceutical application technology, and in particular to a HDAC10-targeting compound, its preparation method and application. Background Technology

[0002] Histone deacetylases (HDACs) are overexpressed in a variety of cancers and are closely associated with high tumor invasiveness and poor prognosis, thus making them an important target for cancer therapy (see: Falkenberg K J. et al., Nat. Rev.Drug Discov. 2014, 13(9): 673-91; Ho TC S. et al., J. Med. Chem. 2020, 63(21): 12460-84.). Currently, several HDAC inhibitors have been approved by drug regulatory authorities in various countries for the treatment of hematologic malignancies such as peripheral T-cell lymphoma and cutaneous T-cell lymphoma, and a large number of HDAC inhibitors are being used in clinical trials (see: Cao Y. et al., Pharmacol Ther. 2025, 265: 108749; Shah RR, Drug Saf.2019, 42(2): 235-45, Quinn DI. et al., Invest. New Drugs. 2021, 39(3): 812-20.). However, HDAC inhibitors currently used in clinical practice generally have serious toxic side effects, mainly due to their poor selectivity for HDAC family members. Therefore, there is an urgent need to develop HDAC inhibitors and degrading agents with high subtype selectivity.

[0003] HDAC10 belongs to the Class IIb HDAC subfamily. Similar to other HDAC isotypes, HDAC10 also has multiple functions, including epigenetic regulation and post-translational modification (see: Lambona C. et al., Curr Opin StructBiol. 2023, 82: 102668.). However, unlike other HDAC isotypes, HDAC10 is also a polyamine deacetylase. Under conditions of exogenous polyamine restriction or difluoromethylornithine (DFMO) inhibition of endogenous polyamine synthesis, HDAC10 can catalyze the conversion of N8-acetylsemine to spermine to regulate intracellular polyamine levels, thereby using N8-acetylsemine as a polyamine alternative source to maintain tumor growth. Correspondingly, knocking out or inhibiting HDAC10 can inhibit tumor cell growth by interfering with intracellular polyamine homeostasis (see: Hai Y. et al., Nat. Commun. 2017, 8: 15368.). A growing body of research confirms that autophagy helps tumor cells cope with environmental stresses such as hypoxia, nutrient deprivation, and drug therapy. It plays a crucial role in tumor maintenance and progression and is closely related to the development of tumor drug resistance. Therefore, targeting and inhibiting autophagy has become an important direction in the field of tumor therapy. For example, autophagy inhibitors can effectively overcome the adaptive resistance of tumor cells to RAF-MEK-ERK signaling pathway inhibitors (see: Jain V. et al., Trends Pharmacol. Sci. 2023, 44(5):290-302.). It is worth noting that HDAC10 is a novel autophagy intervention target; therefore, HDAC10 inhibitors are expected to inhibit autophagy and enhance the efficacy of RAF-MEK-ERK signaling pathway inhibitors and other anticancer drugs (see: Oehme I. et al., Proc Natl Acad Sci US A. 2013, 110(28): E2592-601.). With the continuous discovery of its physiological and pathological functions, HDAC10 is gradually being regarded as a potential therapeutic target for tumors and a variety of non-tumor diseases.

[0004] In recent years, targeted protein degradation technology has provided new research ideas for solving problems that traditional small molecule inhibitors cannot solve. Targeted protein degradation chimeras (PROTACs) are heterobifunctional molecules that bind to both target protein ligands and E3 ubiquitin ligase ligands via linkers (see: Békés M. et al., Nat. Rev. Drug Discov. 2022, 21(3):181-200.). Unlike classical inhibitors that block protein function by occupying the protein's active pocket, degradative agents can induce the overall degradation of the target protein through the ubiquitin-proteasome system, thereby eliminating its enzyme activity dependence and protein scaffold function, while also overcoming drug resistance (see: Nalawansha D A. et al., Cell Chem Biol. 2020, 27(8):998-1014.). Existing studies have developed various selective degraders targeting HDAC6, HDAC8, and other subtypes, demonstrating the degradability of HDAC family proteins. This strategy also suggests that it may be an important direction for overcoming the insufficient selectivity and significant toxicity of traditional HDAC inhibitors (see: Cao Z. et al., ACS Chem Biol. 2021, 16(12), 2746–2751; Xiao Y. et al., Cell Chem Biol. 2023, 30(11), 1421–1435.e12.). Therefore, the development of HDAC10-targeting degraders will not only help to elucidate the functions of HDAC10 in polyamine metabolism, autophagy regulation, and tumor drug resistance, but may also provide novel therapeutic strategies for the intervention of HDAC10-related diseases.

[0005] However, few HDAC10 subtype selective inhibitors have been reported so far. DKFZ-728 is a highly active and selective HDAC10 inhibitor, but its druggability is not ideal. Existing HDAC10 degraders are also scarce, and most act on both HDAC6 and HDAC10 simultaneously; further development is needed for degraders targeting a single HDAC10 subtype. Therefore, there is an urgent need to develop novel, druggable HDAC10 subtype selective inhibitors and degraders. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a target HDAC10 compound, its preparation method, and its applications. The compound represented by formula (I) of this invention can serve as a selective inhibitor of HDAC10 subtypes, exhibiting highly selective HDAC10 inhibitory activity; the compound represented by formula (II) of this invention can serve as a selective degrader of HDAC10 subtypes, demonstrating a high HDAC10 degradation effect.

[0007] The technical solution of the present invention is as follows:

[0008] 1. Targeting HDAC10 compounds

[0009] Compounds targeting HDAC10 are compounds having the structure shown in general formula (I) or general formula (II) as well as their pharmaceutically acceptable salts, hydrates, or solvates:

[0010]

[0011] Wherein, R is selected from phenyl, substituted phenyl, naphthyl, substituted naphthyl, quinolinyl, substituted quinolinyl, quinoxalinyl, substituted quinoxalinyl, indoleyl, substituted indoleyl, furan ring, substituted furan ring, thiophene ring, substituted thiophene ring, benzyl, substituted benzyl, phenethyl or substituted phenethyl;

[0012] L is selected from , , or , where m=4-9, n=0-2, o=0-2, p=0-1, X=C or N.

[0013] According to a preferred embodiment of the present invention, the HDAC10-targeting compound is selected from one of the following compounds, and their pharmaceutically acceptable salts, hydrates, or solvates:

[0014]

[0015]

[0016] 2. Preparation method of HDAC10-targeting compounds

[0017] The preparation method of the HDAC10-targeting compound is selected from one of the following:

[0018] (a) Compound 1 reacts with 1-Boc-4-aminopiperidine to give compound 2. Compound 2 is deprotected to give compound 3. Compound 3 reacts with methyl 4-bromobutyrate to give compound 4. Compound 4 reacts with potassium hydroxylamine to give compounds M1-M29.

[0019] The reaction formula is as follows:

[0020]

[0021] Wherein, the substituent R is the same as the substituent R in compound (I).

[0022] The reagents and conditions in the above reaction formula:

[0023] a. 1-Boc-4-aminopiperidine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, N,N-dimethylformamide, reacted in an ice bath to room temperature;

[0024] b. Trifluoroacetic acid, dichloromethane, reaction at room temperature;

[0025] c. Methyl 4-bromobutyrate, potassium carbonate, N,N-dimethylformamide, reaction at 70°C;

[0026] d. Potassium hydroxylamine, methanol, reaction at room temperature.

[0027] (ii) Compound 5 and compound 6 undergo a condensation reaction to generate compound 7. Compound 7 is hydrolyzed to give compound 8. Compound 9 is reduced to generate compound 10. Compound 8 and compound 10 undergo a condensation reaction to generate compound 11. Compound 11 reacts with potassium hydroxylamine to give compounds N1-N6.

[0028] The reaction formula is as follows:

[0029]

[0030] The reagents and conditions in the above reaction formula:

[0031] a. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, reaction at room temperature;

[0032] b. Lithium hydroxide monohydrate, methanol, tetrahydrofuran, water, reaction at room temperature;

[0033] c. Hydrogen, palladium on carbon, methanol, reaction at room temperature;

[0034] d. Potassium hydroxylamine, methanol, reaction at room temperature.

[0035] (iii) Compound 5 and Compound 12 undergo a condensation reaction to generate Compound 14. After deprotection under acidic conditions, Compound 14 undergoes a reductive amination reaction with Compound 13 to generate Compound 15. Compound 16 is oxidized to generate Compound 17. After deprotection under acidic conditions, Compound 15 undergoes a reductive amination reaction with Compound 17 to generate Compound 18. Compound 18 reacts with potassium hydroxylamine to generate Compound N7-N10.

[0036] The reaction formula is as follows:

[0037]

[0038] The reagents and conditions in the above reaction formula:

[0039] a. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, N,N-dimethylformamide, reacted in an ice bath to room temperature;

[0040] b. Trifluoroacetic acid, dichloromethane, reaction at room temperature; sodium triacetoxyborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, reaction at room temperature;

[0041] c. Manganese dioxide, dichloromethane, reaction at room temperature;

[0042] d. Trifluoroacetic acid, dichloromethane, reaction at room temperature; sodium cyanoborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, methanol, reaction at room temperature;

[0043] e. Potassium hydroxylamine, methanol, reaction at room temperature.

[0044] (iv) Compound 19 and Compound 20 undergo a coupling reaction to obtain Compound 21. Compound 21 is hydrolyzed under alkaline conditions and then undergoes a condensation reaction with Compound 5 to obtain Compound 22. Compound 22 is deprotected under acidic conditions and then undergoes a reductive amination reaction with Compound 17 to obtain Compound 23. Compound 23 reacts with potassium hydroxylamine to obtain Compound N11-N12.

[0045] The reaction formula is as follows:

[0046]

[0047] The reagents and conditions in the above reaction formula:

[0048] a. Cuprous iodide, triethylamine, bis(triphenylphosphine)palladium dichloride, N,N-dimethylformamide, reaction at 100°C;

[0049] b. Lithium hydroxide monohydrate, tetrahydrofuran, methanol, water, reaction at room temperature; 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, reaction at room temperature;

[0050] c. Ethyl hydrochloride solution, ethyl acetate, reaction at room temperature; sodium cyanoborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, methanol, reaction at room temperature;

[0051] d. Potassium hydroxylamine, methanol, reaction at room temperature.

[0052] (v) Compound 24 and Compound 25 undergo a coupling reaction to give Compound 26. Compound 26 is hydrolyzed under alkaline conditions and then undergoes a condensation reaction with Compound 5 to give Compound 27. Compound 27 is deprotected under acidic conditions and then undergoes a reductive amination reaction with Compound 17 to give Compound 28. Compound 28 reacts with potassium hydroxylamine to give Compound N13-N14.

[0053] The reaction formula is as follows:

[0054]

[0055] The reagents and conditions in the above reaction formula:

[0056] a. Cuprous iodide, triethylamine, bis(triphenylphosphine)palladium dichloride, N,N-dimethylformamide, reaction at 100°C;

[0057] b. Lithium hydroxide monohydrate, tetrahydrofuran, methanol, water, reaction at room temperature; 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, reaction at room temperature;

[0058] c. Ethyl hydrochloride solution, ethyl acetate, reaction at room temperature; sodium cyanoborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, methanol, reaction at room temperature;

[0059] d. Potassium hydroxylamine, methanol, reaction at room temperature.

[0060] 3. Applications of compounds targeting HDAC10

[0061] HDAC10-targeting compounds are used as selective inhibitors or degraders of HDAC10 subtypes in the preparation of drugs for the prevention or treatment of diseases associated with abnormal HDAC10 expression or activity.

[0062] According to a preferred embodiment of the present invention, the disease associated with abnormal HDAC10 expression or activity is cancer, inflammatory disease, or autoimmune disease.

[0063] Preferably, the cancer is colorectal cancer, bladder cancer, melanoma, cervical cancer, ovarian cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, neuroblastoma, lymphoma, or leukemia.

[0064] Preferably, the inflammatory disease or autoimmune disease is inflammatory bowel disease, temporomandibular joint osteoarthritis, or synovial inflammatory disease.

[0065] The beneficial effects of this invention are as follows:

[0066] To address the issues of limited existing HDAC10 subtype selective inhibitors, insufficient drug-likeness of reported representative HDAC10 inhibitors, and a relative lack of development of selective HDAC10 degrading agents, this invention provides a series of novel isohydroxamic acid-based HDAC10 subtype selective inhibitors and degrading agents, enriching the structural types of HDAC10-targeting drugs and providing new candidate compounds and technical solutions for the intervention and treatment of HDAC10-related diseases.

[0067] The compounds M1-M29 of this invention, as HDAC10 subtype selective inhibitors, possess novel structures, high activity, and high selectivity for HDAC10 inhibition. Compared with the existing inhibitor DKFZ-728, the inhibitors of this invention exhibit comparable HDAC10 inhibitory activity.

[0068] The HDAC10 subtype selective inhibitor described in this invention shows promising application potential at the cellular level. In vitro evaluation of its antitumor cell proliferation activity indicates that the representative compound M1, when used alone, exhibits low direct cytotoxicity to tumor cells. However, when combined with an inhibitor of the RAF-MEK-ERK signaling pathway, it significantly enhances the inhibitory effect of the relevant inhibitor on HCT116 colon cancer cells. This result demonstrates that the HDAC10 subtype selective inhibitor described in this invention can not only be used as an HDAC10-targeting inhibitor but also as a sensitizer in combination therapy to improve the antitumor efficacy of RAF-MEK-ERK signaling pathway inhibitors.

[0069] The HDAC10 subtype selective inhibitor described in this invention also exhibits good preliminary safety and drug-likeness. Normal hepatocyte toxicity evaluation results showed that the representative compound M1 had low toxicity to normal hepatocytes; mouse liver microsomal stability evaluation results showed that M1 had good metabolic stability, significantly better than the previously reported representative compound DKFZ-728, indicating that the HDAC10 subtype selective inhibitor obtained through structural optimization in this invention has the potential to be further developed into a drug candidate.

[0070] Furthermore, this invention designs and synthesizes a series of HDAC10-targeting degraders (compounds N1-N14) by changing the length and properties of the linkers in the compounds, and successfully screens effective HDAC10-targeting degraders through WB (Western Blot) experiments.

[0071] In summary, this invention provides two classes of compounds targeting HDAC10: HDAC10 subtype-selective inhibitors and HDAC10 subtype-selective degraders. The inhibitor portion exhibits definite HDAC10 enzyme activity inhibition, good subtype selectivity, low normocytotoxicity, and good metabolic stability, and can enhance the antitumor activity of RAF-MEK-ERK signaling pathway inhibitors. The degrader portion provides a novel strategy for intervening in HDAC10 function by degrading the HDAC10 protein. Therefore, this invention has significant application value for developing drugs to treat diseases related to abnormal HDAC10 expression or activity. Attached Figure Description

[0072] Figure 1This is a graph showing the degradation activity of the degrading agent in the target compound on intracellular HDAC10. Detailed Implementation

[0073] The present invention will be further described below with reference to embodiments, but is not limited thereto.

[0074] Meanwhile, unless otherwise specified, all reagents used in the embodiments are commercially available; and unless otherwise specified, the methods and equipment used can be based on existing technologies.

[0075] Example 1 below illustrates the preparation of the HDAC10 subtype selective inhibitor of the present invention; Examples 2-5 illustrate the preparation of the HDAC10 subtype selective degrader of the present invention.

[0076] Example 1. Preparation of compounds M1-M29, taking compound M1 as an example.

[0077] Synthesis route:

[0078]

[0079] The specific synthesis method and steps are as follows:

[0080] Compound 2a: Weigh 0.50 g (4.09 mmol) of compound 1a into a pear-shaped flask, slowly add 25 mL of DMF, and stir until the solution is clear. Under ice bath conditions, add EDCI (1.18 g, 6.14 mmol), HOBt (0.83 g, 6.14 mmol), and DIPEA (1.06 g, 8.18 mmol) sequentially. After stirring and activating for 30 min, add 1-Boc-4-aminopiperidine (0.98 g, 4.91 mmol). Remove from ice bath, allow to warm naturally to room temperature, and stir. After the starting material spot disappears under TLC monitoring, quench the reaction by pouring the reaction solution into 100 mL of water. Extract the aqueous phase three times with 60 mL of ethyl acetate. Combine the organic phases and wash twice with 80 mL of saturated sodium bicarbonate solution and once with 80 mL of saturated brine. Dry the product over anhydrous magnesium sulfate, filter, and concentrate to obtain the crude product. The crude product was slurried with 10 mL of petroleum ether / ethyl acetate (v / v = 1:1), stirred for 6 h, and then filtered to give 1.10 g of a white solid, with a yield of 89%. NMR data are as follows: 1¹H NMR (400 MHz, DMSO-d⁶) δ 8.27 (d, J = 7.8 Hz, 1H), 7.87 – 7.80 (m, 2H), 7.52 (dd, J = 8.3, 6.2 Hz, 1H), 7.45 (t, J = 7.3 Hz, 2H), 3.96 (m, 3H), 2.84 (s, 2H), 1.83 – 1.73 (m, 2H), 1.41 (m, 11H). Mass spectrometry data are as follows: ESI-MS, m / z = 327.38 [M+Na] + .

[0081] Compound 3a: Compound 2a (0.10 g, 0.33 mmol) was weighed and placed in a pear-shaped flask. 3 mL of dichloromethane was slowly added, and the mixture was stirred until the solution became clear. Trifluoroacetic acid (0.57 g, 4.95 mmol) was then slowly added dropwise under stirring. The reaction was stirred at room temperature for 2 h. After the reaction starter spot disappeared as detected by TLC, dichloromethane and trifluoroacetic acid were removed by vacuum distillation. 3 mL of methanol was added to the viscous residue, and evaporation was continued. This process was repeated until a solid precipitated. 6 mL of frozen diethyl ether was added and the mixture was stirred until a white solid precipitated. Stirring was continued for 4 h to promote crystal precipitation. The solid was filtered, dried under vacuum, and finally yielded 93 mg of trifluoroacetate 3a, with a yield of 89%. NMR data are as follows: 1 ¹H NMR (400 MHz, DMSO-d6) δ 8.81 – 8.29 (m, 3H), 7.93 – 7.74 (m, 2H), 7.61 – 7.36 (m, 3H), 4.06 (m, 1H), 3.33 (d, J = 12.1 Hz, 2H), 3.14 – 2.88 (m, 2H), 2.06 – 1.86 (m, 2H), 1.84 – 1.63 (m, 2H). Mass spectrometry data are as follows: ESI-MS, m / z = 205.33 [M+H] + .

[0082] Compound 4a: Compound 3a (0.32 g, 0.98 mmol) was weighed and placed in a pear-shaped flask. 25 mL of DMF was added, and the mixture was stirred until the solution was clear. Potassium carbonate (0.55 g, 3.92 mmol) and methyl 4-bromobutyrate (0.27 g, 1.47 mmol) were added sequentially, and the mixture was stirred at 70 °C for 4 h. After the reaction starting material spot disappeared as detected by TLC, the reaction solution was slowly poured into 100 mL of water for quenching. The aqueous phase was extracted three times with 60 mL of ethyl acetate. The combined organic phases were washed twice with 80 mL of saturated sodium bicarbonate solution and once with 80 mL of saturated saline solution. The crude product was obtained after drying with anhydrous magnesium sulfate and filtration concentration. The crude product was purified by column chromatography (MeOH / DCM, v / v = 1:100 – 1:25) to obtain 0.23 g of a white solid, with a yield of 77%. NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 7.7 Hz, 1H), 7.86 – 7.81 (m,2H), 7.51 (dd, J = 8.4, 6.1 Hz, 1H), 7.45 (t, J = 7.3 Hz, 2H), 3.74 (m, 1H),3.60 (s, 3H), 2.83 (d, J = 11.3 Hz, 2H), 2.35 – 2.24 (m, 4H), 1.94 (t, J =11.1 Hz, 2H), 1.76 (d, J = 10.2 Hz, 2H), 1.68(p, J =7.1 Hz,2H),1.54(qd,J =12.2, (3.5 Hz, 2H). Mass spectrometry data are as follows: ESI-MS, m / z = 305.50 [M+H] + .

[0083] Compound M1: Compound 4a (0.90 g, 2.94 mmol) was weighed and placed in a pear-shaped flask. 5 mL of a 2.25 M potassium hydroxylamine methanol solution was slowly added, and the mixture was stirred at room temperature for 1 h. After the reaction starter spot disappeared as detected by TLC, methanol was removed by vacuum distillation. 1 mL of water was added to the residue, and 3 M hydrochloric acid aqueous solution was slowly added dropwise until the pH of the solution reached 7-8. After the solid precipitated, stirring was continued for 4 h to promote crystal growth. The mixture was filtered and dried to obtain 0.83 g of a white solid, with a yield of 92%. Mp: 140-142 ℃. NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.70 (s, 1H), 8.26 (d, J = 7.7Hz, 1H), 7.84 (d, J = 7.5 Hz, 2H), 7.58 – 7.38 (m, 3H), 3.74 (m, 1H), 2.84(d, J = 11.1 Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.01 – 1.87 (m, 4H), 1.82 –1.70 (m, 2H), 1.70 – 1.50(m, 4H). 13 C10 NMR (101 MHz, DMSO-d6) δ 169.58, 166.09, 135.22, 131.47, 128.62, 127.75, 57.75, 52.86, 47.51, 31.99, 30.80, 23.25. Mass spectrometry data are as follows: ESI-MS, m / z = 306.33 [M+H] + Retention time: 17.1 min.

[0084] The preparation of compounds M2-M29 is the same as that of compound M1 above, except that the types of substituents R in compound 1 are varied.

[0085] Example 2. Preparation of compounds N1-N6, taking compound N2 as an example.

[0086] Synthesis route:

[0087]

[0088] Compound 7a: Compound 6a (0.11 g, 0.62 mmol) was weighed and placed in a pear-shaped flask. 8 mL of DMF was slowly added with continuous stirring. HATU (0.30 g, 0.78 mmol) and DIPEA (0.40 g, 3.12 mmol) were added, and the mixture was stirred and activated for 30 min. Then, compound 5 (0.25 g, 0.52 mmol) was added, and the reaction was stirred at room temperature for 2 h. After the reaction starter spots disappeared as detected by TLC, the reaction solution was slowly poured into 100 mL of water for quenching. The aqueous phase was extracted three times with 60 mL of ethyl acetate. The combined organic phases were washed once with 80 mL of saturated brine. The crude product was obtained after drying with anhydrous magnesium sulfate and filtration concentration. The crude product was purified by column chromatography (MeOH / DCM, v / v = 1:100 – 1:25) to obtain 0.21 g of a pink solid, with a yield of 68%. NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 9.3 Hz, 1H), 7.46 – 7.35 (m, 4H), 4.96 – 4.85 (m, 1H), 4.51 (d,J = 9.3 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 3.61 (s, 2H), 3.57(s, 3H), 2.46 (s, 3H), 2.28 (t, J = 7.5 Hz, 2H), 2.21 (q, J = 6.5 Hz, 1H),2.11 (dt, J = 14.1, 7.2 Hz, 1H), 2.05 – 1.95 (m, 1H), 1.79 (ddd, J = 13.0, 8.5, 4.7 Hz, 1H), 1.55 – 1.44 (m, 4H), 1.37 (d, J = 7.0 Hz, 3H), 1.26 – 1.22 (m, 2H), 0.93 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 600.96 [M+H] + .

[0089] Compound 8a: Compound 7a (0.21 g, 0.35 mmol) was weighed and placed in a pear-shaped flask. 9 mL of a mixed solvent of tetrahydrofuran / methanol / water (v / v / v = 1:1:1) was added and stirred continuously. Lithium hydroxide monohydrate (0.14 g, 3.50 mmol) was added, and the mixture was stirred at room temperature for 2 h. After the reaction starter spot disappeared as detected by TLC, the solution was removed by vacuum distillation. 1 mL of water was added to the residue, and 3 M hydrochloric acid aqueous solution was slowly added dropwise until the pH of the solution reached approximately 5. A solid precipitated. The solid was filtered and dried under vacuum to obtain 0.19 g of a white solid, with a yield of 93%. The obtained compound did not require further purification. Mass spectrometry data are as follows: ESI-MS, m / z = 587.05 [M+H] + .

[0090] Compound 10: Compound 9 (0.5 g, 1.43 mmol) was weighed and placed in a pear-shaped flask. 10 mL of anhydrous methanol was added, and the solid was dissolved by sonication. Palladium on carbon (50 mg), representing 10% of the weight of Compound 9, was added. The entire system was purged three times with hydrogen gas, and the reaction was carried out at room temperature for 6 h with stirring. After the reaction starter spot disappeared as detected by TLC, the palladium on carbon was removed by diatomaceous earth filtration. The filtrate was then distilled under reduced pressure to remove the solvent, yielding 0.38 g of a dark green solid (84% yield). No further purification was required. Mass spectrometry data are as follows: ESI-MS, m / z = 320.48 [M+H] + .

[0091] Compound 11a was prepared using the same method as compound 7a. Compound 11a was prepared from compounds 10 and 8a, yielding 120 mg of a white solid in 43% yield. NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.10(s, 1H), 8.98 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.14 (s, 1H), 7.79 (d, J =8.7 Hz, 3H), 7.65 (d, J = 8.3 Hz, 2H), 7.46 – 7.35 (m, 4H), 5.11 (d, J = 3.6Hz, 1H), 4.98 – 4.83 (m, 1H), 4.51 (d, J = 9.3 Hz, 1H), 4.42 (t, J = 8.0 Hz,1H), 4.31 – 4.24 (s, 1H), 3.79 (s, 2H), 3.60 (s, 3H), 3.10 – 2.82 (m, 6H), 2.45 (s, 3H), 2.36 – 2.26 (m, 5H), 2.16 – 2.10 (m, 1H), 2.04 – 1.98 (m, 1H), 1.84 – 1.70 (m, 5H), 1.64 – 1.54 (t, J = 7.6 Hz, 4H), 1.53 – 1.45 (m, 2H), 1.37 (d, J = 7.0 Hz, 3H), 1.32 – 1.25 (m, 2H), 0.93 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 888.58 [M+H] + .

[0092] Compound N2: Compound 11a (0.12 g, 0.14 mmol) was weighed and placed in a pear-shaped flask. 4 mL of a 2.25 M potassium hydroxylamine methanol solution was slowly added, and the mixture was stirred at room temperature for 1 h. After the reaction starter spot disappeared as detected by TLC, methanol was removed by vacuum distillation. 1 mL of water was added to the residue, and the pH was adjusted to 7-8 by slowly adding 3 M hydrochloric acid aqueous solution. After solid precipitation, stirring was continued for 4 h to promote crystal growth. The resulting crude product was filtered and vacuum dried. Purification was performed using water (containing 0.1% formic acid) and acetonitrile as the mobile phase via a preparative-grade HPLC column, finally yielding 28 mg of a white solid as the formate of compound N2, with a yield of 23%. NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 10.00 (s, 1H), 8.92 (s,1H), 8.57 (s, 1H), 8.31 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.73(dd, J = 8.9, 6.3 Hz, 3H), 7.58 (d, J = 8.4 Hz, 2H), 7.40 – 7.27 (m, 4H), 5.03 (s, 1H), 4.97 – 4.85 (m, 1H), 4.44 (d, J = 9.3 Hz, 1H), 4.35 (t, J = 8.0Hz, 1H), 4.21 (s, 1H), 3.68 (s, 1H), 3.54 (d, J = 3.5 Hz, 2H), 2.82 (d, J =11.0 Hz, 2H), 2.39 (s, 3H), 2.31 – 2.18 (m, 5H), 2.10 – 1.88 (m, 6H), 1.72(ddd, J = 13.2, 8.9, 4.6 Hz, 3H), 1.51 (tdd, J = 26.9, 15.2, 7.8 Hz, 9H), 1.30 (d, J = 7.0 Hz, 3H), 1.21 (p, J = 8.7 Hz, 3H), 0.85 (s, 9H). The mass spectrometry data are as follows: ESI-MS, m / z = 889.95 [M+H] + .

[0093] The preparation of compounds N1, N3-N6 is the same as that of compound N2, except that the value of m in compound 6 is changed.

[0094] Example 3. Preparation of compounds N7-N10, taking compound N10 as an example.

[0095] Synthesis route:

[0096]

[0097] Compound 14a: Weigh compound 12a (0.13 g, 0.50 mmol) into a pear-shaped flask, slowly pour in 10 mL of DMF, stir until the solution is clear, add EDCI (0.12 g, 0.63 mmol), HOBt (85 mg, 0.63 mmol) and DIPEA (0.22 g, 1.68 mmol) in sequence under ice bath, stir and activate for 30 min, then add compound 5 (0.2 g, 0.42 mmol). Remove from the ice bath and allow to naturally warm to room temperature with stirring for 5 h. After the starting material spot disappears as monitored by TLC, the reaction solution is quenched in 100 mL of water. The aqueous phase is extracted three times with 60 mL of ethyl acetate. The organic phases are combined and washed once with 80 mL of saturated brine. After drying with anhydrous magnesium sulfate and filtration and concentration, the crude product is obtained. The crude product is purified by column chromatography (MeOH / DCM, v / v = 1:100–1:20) to finally obtain 0.13 g of white solid, with a yield of 48%. NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.33 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 9.2 Hz, 1H), 7.46 – 7.35 (m, 4H), 5.05 (d, J = 3.6 Hz, 1H), 4.97 – 4.87 (m, 1H), 4.51 (d,J = 9.2 Hz, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 3.87 (d, J = 12.9Hz, 2H), 3.61 (d, J = 3.5 Hz, 2H), 2.61 (s, 2H), 2.45 (s, 3H), 2.20 (dd, J = 13.7, 7.5 Hz, 1H), 2.08 (dd, J = 13.7, 6.7 Hz, 1H), 2.04 – 1.96 (m, 1H), 1.88– 1.75 (m, 2H), 1.57 (d, J = 13.0 Hz, 2H), 1.39 (s, 12H), 1.07 – 0.97 (m, 2H), 0.94 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 670.15 [M+H] + .

[0098] Compound 15a: Compound 14a (0.13 g, 0.19 mmol) was weighed into a pear-shaped flask, and 2 mL of dichloromethane was added. The mixture was stirred until the solution was clear, and then trifluoroacetic acid (0.33 g, 2.91 mmol) was slowly added dropwise under stirring. The mixture was stirred at room temperature for 2 h. After the reaction starter spot disappeared as detected by TLC, dichloromethane and trifluoroacetic acid were removed by vacuum distillation. 3 mL of methanol was added to the viscous residue and the evaporation was continued. The above operation was repeated until a solid precipitated. After vacuum drying, the solid was used directly in the next reaction. The dried compound was dissolved in 5 mL of tetrahydrofuran, and triethylamine (115 mg, 1.14 mmol) was added and stirred at room temperature for 10 min. Compound 13a (60 mg, 0.29 mmol) and glacial acetic acid (114 mg, 1.90 mmol) were added and stirred at room temperature for 30 min. Sodium triacetoxyborohydride (100 mg, 0.48 mmol) was added and the mixture was stirred at room temperature for 12 h. After the reaction starting material spot disappeared as detected by TLC, the solvent was removed by vacuum distillation. The compound was diluted with 100 mL of dichloromethane, washed twice with 80 mL of saturated sodium bicarbonate aqueous solution, and once with 80 mL of saturated brine. The crude product was obtained after drying with anhydrous magnesium sulfate and filtration concentration. The crude product was purified by column chromatography (MeOH / DCM, v / v = 1:100 – 1:12) to finally obtain 0.11 g of a white solid, with a two-step yield of 72%. NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.41 (d, J = 7.8Hz, 1H), 7.85 (s, 1H), 7.48 – 7.34 (m, 4H), 5.12 (d, J = 3.5 Hz, 1H), 4.95 –4.85 (m, 1H), 4.51 (d, J = 9.3 Hz, 1H), 4.41 (t, J = 8.1 Hz, 1H), 4.27 (s,1H), 3.90 (s, 2H), 3.60 (d, J = 3.4 Hz, 2H), 2.75 (s, 4H), 2.46 (s, 3H), 2.23– 1.91 (m, 6H), 1.83 – 1.52 (m, 9H), 1.38 (s, 12H), 1.23 (s, 2H), 1.16 (t, J = 7.2 Hz, 1H), 0.93 (d, J = 6.6 Hz, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 767.99 [M+H] + .

[0099] Compound 17: Compound 16 (0.25 g, 0.75 mmol) was weighed and placed in a pear-shaped flask. 20 mL of dichloromethane was added, followed by manganese dioxide (0.98 g, 11.22 mmol). The mixture was stirred at room temperature for 24 h. After the starting material spot disappeared as detected by TLC, the mixture was filtered through diatomaceous earth to remove the residue. The filtrate was then distilled under reduced pressure to remove the solvent, yielding 0.18 g of an orange solid (72% yield). No further purification was required. Mass spectrometry data are as follows: ESI-MS, m / z = 334.17 [M+H] + .

[0100] Compound 18a: Weigh 0.30 g (0.39 mmol) of compound 15a into a pear-shaped flask, add 4 mL of dichloromethane, and stir until the solution is clear. Then, slowly add 0.89 g (7.82 mmol) of trifluoroacetic acid under stirring, and stir the reaction at room temperature for 4 h. After the reaction starter spot disappeared as detected by TLC, remove dichloromethane and trifluoroacetic acid by vacuum distillation. Add 3 mL of methanol to the viscous residue and continue evaporation. Repeat the above operation until a solid precipitates. After vacuum drying, use directly for the next reaction. The dried compound was dissolved in 20 mL of a tetrahydrofuran / methanol (v / v = 4:1) mixture, and triethylamine (79 mg, 0.78 mmol) was added. The mixture was stirred at room temperature for 10 min, then compound 17 (0.16 g, 0.47 mmol) and glacial acetic acid (280 mg, 4.86 mmol) were added. The mixture was stirred at room temperature for 30 min, then sodium cyanoborohydride (62 mg, 0.98 mmol) was added. The mixture was stirred at room temperature for 12 h. After the starting material spot disappeared as detected by TLC, the solvent was removed by vacuum distillation. The compound was diluted with 100 mL of dichloromethane, washed twice with 80 mL of saturated sodium bicarbonate aqueous solution, and once with 80 mL of saturated saline solution. The crude product was obtained after drying and filtration with anhydrous magnesium sulfate. The crude product was purified by column chromatography (MeOH / DCM, v / v = 1:100 – 1:12, eluent containing 1% triethylamine) to finally obtain 0.15 g of a white solid, with a two-step yield of 39%. Mass spectrometry data are as follows: ESI-MS, m / z = 983.99 [M+H] + .

[0101] The preparation method of compound N10 is the same as that of compound N2. Compound N10 was prepared using compound 18a as a starting material, yielding 50 mg of a white solid as the formate of compound N10, with a yield of 33%. The NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ10.39 (s, 1H), 9.00 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.22 (s, 4H), 7.84 (d,J = 9.0 Hz, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.47 – 7.33 (m, 6H), 4.95 – 4.85(m, 1H), 4.51 (d, J = 9.2 Hz, 1H), 4.41 (t, J = 8.1 Hz, 1H), 4.28 (s, 1H), 3.79 (s, 1H), 3.60 (s, 2H), 3.50 (s, 2H), 2.93 (d, J = 10.9 Hz, 2H), 2.84 (s, 2H), 2.77 (d, J = 10.6 Hz, 2H), 2.46 (s, 3H), 2.36 (d, J = 7.6 Hz, 2H), 2.22– 1.88 (m, 14H), 1.84 – 1.76 (m, 3H), 1.68 – 1.55 (m, 8H), 1.37 (d, J = 7.0Hz, 3H), 1.24 – 1.02 (m, 5H), 0.93 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 985.19 [M+H] + .

[0102] The preparation of compounds N7-N9 is the same as that of compound N10, except that the value of n in compound 12 and the value of o in compound 13 are changed.

[0103] Example 4. Preparation of compounds N11-N12, taking compound N11 as an example.

[0104] Synthesis route:

[0105]

[0106] Compound 21a: Compound 19 (0.48 g, 2.29 mmol), compound 20a (0.50 g, 1.91 mmol), Pd(PPh3)2Cl2 (0.27 g, 0.38 mmol), and CuI (72 mg, 0.38 mmol) were placed in a double-necked flask and dissolved in 6 mL of DMF. The system was evacuated and purged three times with argon. During stirring, 2 mL of TEA was added using a disposable syringe. The mixture was stirred at 100 °C for 12 h. After the reaction starting material spot disappeared as detected by TLC, the reaction solution was poured into 100 mL of water and extracted three times with 80 mL of ethyl acetate. The combined organic phases were washed once with saturated brine. The crude product was obtained by drying with anhydrous magnesium sulfate and filtration and concentration. The crude product was purified by column chromatography (PE / EA, v / v = 100:0 – 25:1) to obtain 0.60 g of a yellow oil, with a yield of 92%. The NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 7.92 (dd, J =8.0, 1.0 Hz, 2H), 7.54 (dd, J = 8.4, 1.9 Hz, 2H), 3.85 (s, 3H), 3.64 (dd, J =12.5, 6.2 Hz, 2H), 3.15 (d, J = 13.9 Hz, 2H), 2.90 (dt, J = 9.2, 5.1 Hz, 1H), 1.88 – 1.74 (m, 2H), 1.59 – 1.46 (m, 2H), 1.40 (s, 9H).

[0107] Compound 22a: Weigh 0.30 g (0.87 mmol) of compound 21a into a pear-shaped flask, add 9 mL of a mixed solvent of tetrahydrofuran / methanol / water (v / v / v = 1:1:1) and stir continuously. Add lithium hydroxide monohydrate (0.35 g, 8.70 mmol) and stir at room temperature for 2 h. After the reaction starting material spot disappears as detected by TLC, remove the solution by vacuum distillation. Add 1 mL of water to the residue and slowly add 3 M hydrochloric acid aqueous solution until the pH of the solution reaches about 5. A solid precipitates out. Filter the solid and dry it under vacuum. The obtained compound does not require further purification. Place the dried compound into a dry pear-shaped flask, add 10 mL of DMF to dissolve the compound, add HATU (0.39 g, 1.03 mmol) and DIPEA (0.53 g, 4.13 mmol) while stirring, and activate at room temperature for 30 min. Add compound 5 (0.33 g, 0.69 mmol) and stir at room temperature for 2 h. After the reaction starting material spots disappeared as detected by TLC, the reaction solution was poured into 100 mL of water and extracted three times with 80 mL of ethyl acetate. The combined organic phases were washed once with saturated brine. The crude product was obtained after drying with anhydrous magnesium sulfate and filtration concentration. The crude product was purified by column chromatography (MeOH / DCM, v / v = 0:100 – 3:100) to finally obtain 0.42 g of a pale yellow solid, with a two-step yield of 65%. NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.40 (d, J = 7.8 Hz,1H), 8.01 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.3 Hz,2H), 7.45 – 7.36 (m, 4H), 5.11 (s, 1H), 4.99 – 4.85 (m, 1H), 4.75 (d, J = 9.0Hz, 1H), 4.45 (t, J = 8.1 Hz, 1H), 4.31 (s, 1H), 3.75 – 3.58 (m, 4H), 3.19 –3.07 (m, 2H), 2.89 (tt, J = 8.3, 3.9 Hz, 1H), 2.46 (s, 3H), 2.02 (qd, J = 8.1, 5.6 Hz, 1H), 1.88 – 1.77 (m, 3H), 1.57 – 1.48 (m, 2H), 1.40 (s, 9H), 1.38 (d, J = 7.0 Hz, 3H), 1.03 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 756.22 [M+H] + .

[0108] Compound 23a: Weigh 0.30 g (0.40 mmol) of compound 22a into a dry pear-shaped flask, add 3 mL of ethyl acetate to dissolve the compound, and add 4 mL of 4 M ethyl acetate hydrochloride solution while stirring. Stir at room temperature for 12 h, and a solid gradually forms in the system. After the reaction starting material spot disappears as detected by TLC, filter the solid and dry it under vacuum. The obtained compound does not require further purification. Place the dried compound into a dry pear-shaped flask, add 12 mL of a mixed solvent of tetrahydrofuran and methanol (v / v = 4:1), add triethylamine (73 mg, 0.72 mmol), and stir at room temperature for 10 min. Add compound 17 (0.18 g, 0.54 mmol) and glacial acetic acid (259 mg, 4.32 mmol), and stir at room temperature for 30 min. Add sodium cyanoborohydride (113 mg, 1.80 mmol), and stir at room temperature for 12 h. After the reaction starting material spot disappeared as detected by TLC, the solvent was removed by vacuum distillation. The compound was diluted with 100 mL of dichloromethane, washed twice with 80 mL of saturated sodium bicarbonate aqueous solution, and once with 80 mL of saturated brine. The crude product was obtained after drying with anhydrous magnesium sulfate and filtration concentration. The crude product was purified by column chromatography (MeOH / DCM, v / v = 1:100 – 1:12, eluent containing 0.2% triethylamine), finally yielding 0.17 g of a pale yellow solid, with a two-step yield of 44%. NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.42 (d, J = 7.8Hz, 1H), 8.23 ​​(s, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.90 – 7.75 (m, 4H), 7.48 –7.32 (m, 8H), 5.13 (d, J = 3.6 Hz, 1H), 4.98 – 4.87 (m, 1H), 4.75 (d, J = 9.0Hz, 1H), 4.45 (t, J = 8.1 Hz, 1H), 4.30 (d, J = 4.9 Hz, 1H), 3.67 (s, 2H),3.60 (s, 3H), 3.52 (s, 2H), 3.03 (s, 3H), 2.89 (s, 2H), 2.68 (s, 3H), 2.46 (s, 3H), 2.33 (t, J = 7.8 Hz, 2H), 2.18 (s, 2H), 2.06 – 1.99 (m, 1H), 1.94 –1.57 (m, 12H), 1.38 (d, J = 3.7 Hz, 3H), 1.03 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 973.13 [M+H] + .

[0109] The preparation method of compound N11 is the same as that of compound N2. Compound N11 was prepared using compound 23a as a starting material, yielding 80 mg of a white solid as the formate of compound N11, with a yield of 47%. The NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ10.37 (s, 1H), 8.98 (s, 1H), 8.40 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 5.8 Hz, 2H), 7.99 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 7.9 Hz,2H), 7.47 – 7.33 (m, 8H), 4.98 – 4.88 (m, 1H), 4.75 (d, J = 9.0 Hz, 1H), 4.45(t, J = 8.1 Hz, 1H), 4.33 – 4.28 (m, 1H), 3.76 (s, 1H), 3.67 (d, J = 3.0 Hz, 2H), 3.52 (s, 2H), 2.88 (d, J = 11.0 Hz, 2H), 2.67 (s, 3H), 2.46 (s, 3H), 2.30 (t, J = 7.3 Hz, 2H), 2.18 (s, 2H), 2.08 – 1.93 (m, 5H), 1.92 – 1.73 (m, 5H), 1.71 – 1.54 (m, 6H), 1.38 (d, J = 7.0 Hz, 3H), 1.03 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 973.47 [M+H] + .

[0110] Compound N12 is prepared in the same manner as compound N11, except that the value of p in compound 20 is changed.

[0111] Example 5. Preparation of compounds N13-N14, taking compound N13 as an example.

[0112]

[0113] Compound 26a: Compound 24a (1.02 g, 2.98 mmol), compound 25 (0.62 g, 3.87 mmol), Pd(PPh3)2Cl2 (0.42 g, 0.60 mmol), and CuI (0.11 g, 0.60 mmol) were placed in a double-necked flask and dissolved in 8 mL of LDM. The system was purged with argon three times under vacuum. During stirring, TEA (2 mL) was added using a disposable syringe. The mixture was stirred at 100 °C for 12 h. After the reaction starting material spot disappeared as detected by TLC, the reaction solution was poured into 100 mL of water and extracted three times with 80 mL of ethyl acetate. The combined organic phases were washed once with saturated brine. The crude product was obtained by drying with anhydrous magnesium sulfate and filtration and concentration. The crude product was slurryed with 15 mL of anhydrous methanol to obtain 0.35 g of white solid, yield 28%. NMR data are as follows: 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 8.35 (d, J = 2.3 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.75 – 7.70 (m, 1H), 7.65 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 9.1 Hz, 1H), 3.87 (s, 3H), 3.63 – 3.56 (m, 4H), 3.45 – 3.41 (m, 4H), 1.43 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 421.98 [M+H] + .

[0114] Compound 27a was prepared using the same method as compound 22a. Compound 27a was prepared from compounds 26a and 5, yielding 0.31 g of a white solid, with a two-step yield of 51%. The NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 7.94 – 7.88 (m, 2H), 7.72 (dd, J = 8.9, 2.4 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.47 – 7.37 (m, 4H), 6.89 (d, J = 9.0 Hz, 1H), 5.01 – 4.88 (m, 1H), 4.76 (d, J = 9.1 Hz, 1H), 4.46 (t, J = 8.1 Hz, 1H), 4.31 (d, J = 4.3 Hz, 1H), 3.68 (d, J = 3.1 Hz, 2H), 3.61 – 3.56 (m, 4H), 3.45 – 3.40 (m, 4H), 2.46 (s, 3H), 2.08 – 1.98 (m, 1H), 1.86 – 1.76 (m, 1H), 1.43 (s, 9H), 1.38 (d, J = 7.0 Hz, 3H), 1.04 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 834.61 [M+H] + .

[0115] Compound 28a was prepared using the same method as compound 23a. Compound 28a was prepared from compounds 27a and 17, yielding 110 mg of a yellow solid as compound 28a, with a two-step yield of 30%. The NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.42 (d, J = 7.8 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 7.91 (dd, J = 8.4, 4.1Hz, 2H), 7.81 (dd, J = 8.2, 3.8 Hz, 2H), 7.69 (dd, J = 8.9, 2.5 Hz, 1H), 7.59– 7.55 (m, 2H), 7.45 – 7.38 (m, 6H), 6.87 (d, J = 9.1 Hz, 1H), 5.14 (d, J =3.5 Hz, 1H), 4.98 – 4.87 (m, 1H), 4.76 (d, J = 9.0 Hz, 1H), 4.46 (t, J = 8.1Hz, 1H), 4.31 (s, 1H), 3.87 – 3.74 (m, 2H), 3.68 (s, 2H), 3.60 (s, 3H), 3.59– 3.56 (m, 4H), 3.13 – 2.94 (m, 8H), 2.46 (s, 3H), 2.19 – 1.97 (m, 3H), 1.85– 1.71 (m, 6H), 1.70 – 1.56 (m, 3H), 1.38 (d, J = 6.9 Hz, 3H), 1.04 (s, 9H). Mass spectrometry data are as follows: ESI-MS, m / z = 1051.27 [M+H] + .

[0116] The preparation method of compound N13 is the same as that of compound N2. Compound N13 was prepared using compound 28a as a starting material, yielding 50 mg of a white solid as the formate of compound N13, with a yield of 45%. The NMR data are as follows: 1H NMR (400 MHz, DMSO-d6) δ10.45 (s, 1H), 9.88 (s, 1H), 8.92 (s, 1H), 8.72 (s, 1H), 8.34 (d, J = 7.8 Hz,2H), 8.25 (d, J = 2.3 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.62 (dd, J = 8.8, 2.5 Hz, 1H), 7.51 (d, J =8.0 Hz, 2H), 7.41 – 7.26 (m, 6H), 6.81 (d, J = 9.1 Hz, 1H), 5.06 (d, J = 3.6Hz, 1H), 4.92 – 4.80 (m, 1H), 4.69 (d, J = 9.1 Hz, 1H), 4.39 (t, J = 8.1 Hz,1H), 4.25 (s, 1H), 3.93 (s, 1H), 3.61 (s, 2H), 3.52 (s, 6H), 2.83 (s, 4H), 2.42 – 2.34 (m, 7H), 2.05 – 1.69 (m, 10H), 1.31 (d, J = 7.0 Hz, 3H), 1.17 (s, 2H), 0.97 (s, 9H). The mass spectrometry data are as follows: ESI-MS, m / z = 1051.64 [M+H] + .

[0117] Compound N14 is prepared in the same manner as compound N13, except that the type of substituent X in compound 24 is changed.

[0118] Experimental Example 1. Evaluation of the in vitro HDAC inhibitory activity and subtype selectivity of the target compound's inhibitor.

[0119] This invention utilizes fluorescence analysis to determine the inhibitory activity of the target compound for HDAC4, HDAC6, HDAC8, HDAC10, and HDAC11.

[0120] The experimental results (Table 1) show that all the inhibitors of the present invention have an inhibition rate of more than 50% against HDAC10 at a concentration of 1 μM, but the inhibition rates against HDAC4, HDAC6, HDAC8 and HDAC11 are less than 20%. This preliminarily proves that the inhibitors of the present invention have good selective inhibitory activity against HDAC10 subtypes.

[0121] Table 1. Results of in vitro HDAC inhibition rate tests for each subtype of the compound

[0122]

[0123] Next, the in vitro inhibitory activity of the compound M1, which exhibited superior activity, against HDAC4, HDAC6, HDAC8, and HDAC11 was determined (Table 2). The results showed that at a concentration of 10 μM, M1 inhibited HDAC4, HDAC6, HDAC8, and HDAC11 by less than 40%, confirming that M1 has excellent selectivity for the HDAC10 isotype.

[0124] Table 2. Results of in vitro HDAC inhibition rate tests for each HDAC isoform of compound M1

[0125]

[0126] Subsequently, this invention employed a TR-FRET experiment with DKFZ-728 as a control compound to more accurately determine the HDAC10 inhibitory activity of the representative compound M1, and the IC50 of compound M1 was determined. 50 The value (7.2 ± 0.5 nM) is comparable to that of DKFZ-728 (9.0 ± 2.3 nM) (Table 3), indicating that compound M1 has excellent HDAC10 inhibitory activity.

[0127] Table 3. In vitro inhibition of HDAC10 activity by compound M1 and DKFZ-728

[0128]

[0129] Experimental Example 2. In vitro antitumor cell proliferation activity and normal cytotoxicity of compound M1

[0130] This invention employs the CCK-8 method to conduct a series of experiments on compound M1, including its in vitro antitumor cell proliferation activity and normal cell toxicity.

[0131] HCT116 colon cancer cells possess a mutant G13D and a wild-type KRAS gene. Since KRAS is located upstream of the RAF-MEK-ERK signaling pathway, HCT116 is a RAF-MEK-ERK signaling pathway-dependent tumor cell and can be used to evaluate the antitumor activity of RAF-MEK-ERK signaling pathway inhibitors alone and in combination with HDAC10 subtype selective inhibitors.

[0132] In vitro experimental results showed that the HDAC10 subtype selective inhibitor M1 did not inhibit the proliferation of HCT116 colon cancer cells by more than 50% at concentrations of 3.33 μM and 6.67 μM (Table 4), indicating that the HDAC10 subtype selective inhibitor alone has low cytotoxicity.

[0133] Table 4. In vitro anti-cancer cell proliferation activity of compound M1

[0134]

[0135] To investigate the anticancer potential of combining an HDAC10 subtype selective inhibitor with a RAF-MEK-ERK signaling pathway inhibitor, this invention evaluated the inhibitory effects of the MEK inhibitor trametinib alone and in combination with the HDAC10 subtype selective inhibitor M1 on HCT116 cells, using the traditional autophagy inhibitor chloroquine as a control. Specifically, the inhibitory rates of trametinib at concentrations of 4.6 nM and 13.7 nM, as well as the IC50 of trametinib at these concentrations, were tested. 50 As shown in Table 5, the results indicate that both the HDAC10 subtype selective inhibitor M1 and the traditional autophagy inhibitor chloroquine significantly reduced the IC50 of Trametinib against HCT116. 50 The inhibitor M1 of this invention, when used in combination with Trametinib, showed superior efficacy.

[0136] Table 5. Anti-cancer cell proliferation activity of Trametinib alone and in combination

[0137]

[0138] Subsequently, this invention further evaluated the antitumor activity of the ERK inhibitor Ulixertinib alone and in combination with M1 or chloroquine; specifically, the inhibition rate of Ulixertinib at concentrations of 0.12 μM and 1.11 μM, as well as the IC50 of Ulixertinib in single and combined use, were tested. 50 As shown in Table 6, both the HDAC10 subtype selective inhibitor M1 and the traditional autophagy inhibitor chloroquine significantly enhanced the inhibitory activity of Ulixertinib against HCT116 cells. The combination of the inhibitor M1 and Ulixertinib in this invention showed even better results.

[0139] Table 6. Anti-cancer cell proliferation activity of ulixertinib alone and in combination

[0140]

[0141] As the core organ for drug metabolism, the liver's toxicity assessment is a key focus in drug safety studies. This study used L02 normal hepatocytes to evaluate the hepatotoxicity of compound M1. As shown in Table 7, M1 inhibited L02 cells by 8% and 11% at concentrations of 3.33 μM and 30 μM, respectively, indicating very low toxicity to normal hepatocytes. This preliminarily demonstrates the safety of HDAC10 subtype selective inhibitors.

[0142] Table 7. Inhibition rate of M1 alone on normal cells

[0143]

[0144] Experimental Example 3. In vitro stability experiment of compound M1 in mouse liver microsomes

[0145] Using diclofenac, whose metabolic pathway is well-defined, as a reference control, this invention preliminarily evaluated the in vitro liver microsomal stability of compound M1. The results are shown in Table 8. The half-life of compound M1 in mouse liver microsomes was 410.2 min, significantly higher than the reported half-life of DKFZ-728 in mouse liver microsomes (74 min) (see: Steimbach R R. etal., J Am Chem Soc. 2022, 144(41): 18861-75.). This indicates that obtaining the target compound M1 using a conformational constraint strategy yields better druggability than the lead compound DKFZ-728.

[0146] Table 8. Results of in vitro mouse liver microsomal stability evaluation of M1

[0147]

[0148] Experimental Example 4. Cellular HDAC10 Degradation Activity of the Degrading Agent in the Target Compound

[0149] This invention uses Western blotting (WB) to evaluate the degradation activity of the synthesized HDAC10-targeting degrader. Representative compounds were used to stimulate HCT116 cells at a concentration of 5 μM for 48 h, samples were collected, proteins were extracted, and WB experiments were performed. Experimental results ( Figure 1 The results showed that, compared with the blank control group, the selected compounds were able to reduce the protein level of HDAC10 to varying degrees, indicating that the degrading agents of the present invention have a good degradation effect on HDAC10 protein.

Claims

1. A compound targeting HDAC10, characterized in that, Compounds having the structure shown in general formula (I) or general formula (II) and their pharmaceutically acceptable salts, hydrates or solvates: ； Wherein, R is selected from phenyl, substituted phenyl, naphthyl, substituted naphthyl, quinolinyl, substituted quinolinyl, quinoxalinyl, substituted quinoxalinyl, indoleyl, substituted indoleyl, furan ring, substituted furan ring, thiophene ring, substituted thiophene ring, benzyl, substituted benzyl, phenethyl or substituted phenethyl; L is selected from , , or , where m=4-9, n=0-2, o=0-2, p=0-1, X=C or N.

2. The HDAC10-targeting compound according to claim 1, characterized in that, The HDAC10-targeting compound is selected from one of the following compounds, and its pharmaceutically acceptable salt, hydrate, or solvate: ； ； ； 。 3. The method for preparing the HDAC10-targeting compound as described in claim 1 or 2, characterized in that, Choose one of the following methods: (a) Compound 1 reacts with 1-Boc-4-aminopiperidine to give compound 2, compound 2 is deprotected to give compound 3, compound 3 reacts with methyl 4-bromobutyrate to give compound 4, and compound 4 reacts with potassium hydroxylamine to give compounds M1-M29. The reaction formula is as follows: ； Wherein, the substituent R is the same as the substituent R in compound (I); (ii) Compound 5 and compound 6 undergo a condensation reaction to generate compound 7. Compound 7 is hydrolyzed to give compound 8. Compound 9 is reduced to generate compound 10. Compound 8 and compound 10 undergo a condensation reaction to generate compound 11. Compound 11 reacts with potassium hydroxylamine to give compounds N1-N6. The reaction formula is as follows: ； (iii) Compound 5 and Compound 12 undergo a condensation reaction to generate Compound 14. After deprotection under acidic conditions, Compound 14 undergoes a reductive amination reaction with Compound 13 to generate Compound 15. Compound 16 is oxidized to generate Compound 17. After deprotection under acidic conditions, Compound 15 undergoes a reductive amination reaction with Compound 17 to generate Compound 18. Compound 18 reacts with potassium hydroxylamine to generate Compound N7-N10. The reaction formula is as follows: ； (iv) Compound 19 and Compound 20 undergo a coupling reaction to obtain Compound 21. Compound 21 is hydrolyzed under alkaline conditions and then undergoes a condensation reaction with Compound 5 to obtain Compound 22. Compound 22 is deprotected under acidic conditions and then undergoes a reductive amination reaction with Compound 17 to obtain Compound 23. Compound 23 reacts with potassium hydroxylamine to obtain Compound N11-N12. The reaction formula is as follows: ； (v) Compound 24 and Compound 25 undergo a coupling reaction to give Compound 26. Compound 26 is hydrolyzed under alkaline conditions and then undergoes a condensation reaction with Compound 5 to give Compound 27. Compound 27 is deprotected under acidic conditions and then undergoes a reductive amination reaction with Compound 17 to give Compound 28. Compound 28 reacts with potassium hydroxylamine to give Compound N13-N14. The reaction formula is as follows: 。 4. The method for preparing the HDAC10-targeting compound according to claim 3, characterized in that, Method (1) Reagents and conditions in the reaction formula: a. 1-Boc-4-aminopiperidine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, N,N-dimethylformamide, reacted in an ice bath to room temperature; b. Trifluoroacetic acid, dichloromethane, reaction at room temperature; c. Methyl 4-bromobutyrate, potassium carbonate, N,N-dimethylformamide, reaction at 70°C; d. Potassium hydroxylamine, methanol, reaction at room temperature.

5. The method for preparing the HDAC10-targeting compound according to claim 3, characterized in that, Method (II) Reagents and conditions in the reaction formula: a. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, reaction at room temperature; b. Lithium hydroxide monohydrate, methanol, tetrahydrofuran, water, reaction at room temperature; c. Hydrogen, palladium on carbon, methanol, reaction at room temperature; d. Potassium hydroxylamine, methanol, reaction at room temperature.

6. The method for preparing the HDAC10-targeting compound according to claim 3, characterized in that, Method (III) Reagents and conditions in the reaction formula: a. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, N,N-dimethylformamide, reacted in an ice bath to room temperature; b. Trifluoroacetic acid, dichloromethane, reaction at room temperature; sodium triacetoxyborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, reaction at room temperature; c. Manganese dioxide, dichloromethane, reaction at room temperature; d. Trifluoroacetic acid, dichloromethane, reaction at room temperature; sodium cyanoborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, methanol, reaction at room temperature; e. Potassium hydroxylamine, methanol, reaction at room temperature.

7. The method for preparing the HDAC10-targeting compound according to claim 3, characterized in that, Method (IV) Reagents and conditions in the reaction formula: a. Cuprous iodide, triethylamine, bis(triphenylphosphine)palladium dichloride, N,N-dimethylformamide, reaction at 100°C; b. Lithium hydroxide monohydrate, tetrahydrofuran, methanol, water, reaction at room temperature; 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, reaction at room temperature; c. Ethyl hydrochloride solution, ethyl acetate, reaction at room temperature; sodium cyanoborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, methanol, reaction at room temperature; d. Potassium hydroxylamine, methanol, reaction at room temperature.

8. The method for preparing the HDAC10-targeting compound according to claim 3, characterized in that, Method (5) Reagents and conditions in the reaction formula: a. Cuprous iodide, triethylamine, bis(triphenylphosphine)palladium dichloride, N,N-dimethylformamide, reaction at 100°C; b. Lithium hydroxide monohydrate, tetrahydrofuran, methanol, water, reaction at room temperature; 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, reaction at room temperature; c. Ethyl hydrochloride solution, ethyl acetate, reaction at room temperature; sodium cyanoborohydride, triethylamine, glacial acetic acid, tetrahydrofuran, methanol, reaction at room temperature; d. Potassium hydroxylamine, methanol, reaction at room temperature.

9. The application of the HDAC10-targeting compound as described in claim 1 or 2, characterized in that, It can be used as a selective inhibitor or degrader of HDAC10 subtypes in the preparation of drugs for the prevention or treatment of diseases associated with abnormal HDAC10 expression or activity.

10. The application according to claim 9, characterized in that, The diseases associated with abnormal HDAC10 expression or activity are cancer, inflammatory diseases, or autoimmune diseases; Preferably, the cancer is colorectal cancer, bladder cancer, melanoma, cervical cancer, ovarian cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, neuroblastoma, lymphoma, or leukemia; the inflammatory disease or autoimmune disease is inflammatory bowel disease, temporomandibular joint osteoarthritis, or synovial inflammatory disease.

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