Novel JOSD2 covalent inhibitor, preparation method and application thereof, and pharmaceutical composition

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel JOSD2 covalent inhibitor, a preparation method and application thereof, and a pharmaceutical composition. The invention provides a novel JOSD2 covalent inhibitor, a compound with a structure as shown in a formula (I), a stereoisomer thereof and a pharmaceutically acceptable salt form, the novel JOSD2 covalent inhibitor has certain inhibitory activity on JOSD2 and can effectively inhibit and kill colorectal cancer tumor cells, the preparation method is easy to operate, post-treatment is simple and convenient, reaction starting raw materials are cheap and easy to obtain, the reaction efficiency is high, and the novel JOSD2 covalent inhibitor is suitable for industrial production. The covalent JOSD2 inhibitor is expected to be used for developing the covalent JOSD2 inhibitor and treating related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a novel JOSD2 covalent inhibitor, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] JOSD2 (Josephin domain containing 2) is one of the members of the deubiquitinating enzyme MJDs family, and is a serine / threonine protein kinase. The MJD family also includes three members, namely JOSD1, Ataxin 3 and Ataxin 3-like. Their common feature is that they have a Josephin domain composed of about 180 amino acids. For a long time, the abnormality of MJDs has been considered to be closely related to neurodegenerative diseases. In recent years, studies have successively pointed out that MJDs also play an important role in the occurrence and development of malignant tumors. These include upregulating the protein level of mutant P53 and promoting the malignant progression of gastric cancer; deubiquitinating and stabilizing the transcription factor KLF5 to promote the proliferation and survival of breast cancer cells; regulating the protein level of the mutant JAK2-V617F of the protein kinase JAK2 and promoting the malignant progression of hematological malignancies.

[0003] JOSD2 can deubiquitinate and regulate the metabolic enzyme complex composed of aldolase A, phosphofructokinase-1 and phosphoglycerate dehydrogenase, and then regulate the glucose metabolism of tumor cells; it can also bind to β1-catenin (CTNNB1) and reduce its ubiquitination level, enhance the conduction of the Wnt signaling pathway, and play a role in promoting the survival and migration of hepatocellular carcinoma. In cholangiocarcinoma, JOSD2 deubiquitinates, stabilizes and activates the important pro-cancer transcription factor YAP / TAZ, inducing tumor progression. At the same time, JOSD2 can remove the K6-type ubiquitin chain of the key tumor suppressor protein LKB1 in non-small cell lung cancer, preventing it from forming a ternary complex to play a tumor suppressor role, thereby promoting the malignant proliferation of non-small cell lung cancer cells. JOSD2 is overexpressed in colorectal cancer cells. By stabilizing the KRAS protein level and activating the mTOR pathway, it promotes the proliferation and differentiation of cancer cells, and is expected to be a new target for the treatment of colorectal cancer.

[0004] Chinese Patent Publication No. CN111139299A discloses the application of JOSD2 protein in the preparation of drugs for treating malignant tumors. Experimental evidence in this invention shows that the survival period of malignant tumor patients with high JOSD2 expression is significantly lower than that of patients with low expression, and its expression in cancer tissues is significantly more specific than that in normal tissues. Knocking down JOSD2 protein in cancer cells can significantly inhibit the proliferation and colony formation ability of cancer cells. In addition, knocking down JOSD2 protein in cancer cells can completely inhibit the ability of cancer cells to form xenograft tumors in vivo. This invention can prepare products for diagnosing malignant tumors and predicting the prognosis of malignant tumors, as well as JOSD2 siRNA and JOSD2 inhibitors, which can be used as specific biomarker proteins for diagnosing malignant tumors and predicting the prognosis of malignant tumors, making the diagnosis of malignant tumors more accurate and rapid, and providing new target therapeutic drugs for the prevention and treatment of malignant tumors.

[0005] Another Chinese Patent Publication No. CN116077666A discloses the application of JOSD2 inhibitors in the preparation of anti-cancer therapeutic drugs. This invention discovers that the deubiquitinating enzyme JOSD2 plays an important role in the occurrence and development of esophageal squamous cell carcinoma. When the expression of JOSD2 is reduced, the activity and proliferation of esophageal squamous cancer cells are significantly inhibited, and the therapeutic effect of chemotherapy drugs is also significantly enhanced.

[0006] In view of the resolved crystal structure of JOSD2, it is of great significance to further develop new JOSD2 inhibitors and fully study their in vivo mechanism of action. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a vacuum rapid high-temperature hot-pressing sintering process for graphite brushes, its application, and graphite brushes.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A novel JOSD2 covalent inhibitor, a compound having the structure shown in formula (I), its stereoisomers, and pharmaceutically acceptable salt forms:

[0010]

[0011] In formula (I), R2 is selected from halogen, -CH3, -N(CH3)2, -CF3, or none;

[0012] Ring B is selected from any one of the following: Substituted or unsubstituted C6-C8 cycloalkyl, substituted or unsubstituted C6-C8 heterocycloalkyl, substituted or unsubstituted C6-C 10 bridged cycloalkyl, substituted or unsubstituted C6-C 10 bridged heterocycloalkyl, substituted or unsubstituted C7-C12 Spiroalkyl and substituted or unsubstituted C7-C 12 Spiroheteroalkyl;

[0013] L2 is selected from NH, or none;

[0014] X is selected from O, S, NH or NCH3;

[0015] L1 is selected from NH, one or more of;

[0016] Ring A is selected from any one of the following: C5-C6 heteroalkyl, C5-C 10 Bridged heteroalkyl and C7-C 12 Spiroheteroalkyl;

[0017] R1 is selected from and -CN, one or more of.

[0018] Preferably, the substitution is mono-substituted or di-substituted by R2.

[0019] Preferably, the carbon atoms of the C5-C6 heteroalkyl, C5-C 10 Bridged heteroalkyl and C7-C 12 Spiroheteroalkyl are mono-substituted or multi-substituted by N or O.

[0020] Preferably, ring B is selected from any one of the following:

[0021] Ring A is selected from any one of the following:

[0022] m are all 0 or 1, and n are all 1 or 2.

[0023] More preferably, ring B is selected from any one of the following:

[0024] Ring A is selected from any one of the following:

[0025] Further preferably, the novel JOSD2 covalent inhibitor is selected from one of the following compounds:

[0026]

[0027]

[0028] The present invention also provides a method for preparing the above novel JOSD2 covalent inhibitor, which comprises the following steps: first, reducing or hydrolyzing the compound shown in formula (II) to generate the compound shown in formula (III), and then performing acid deprotection of Boc and substitution reaction to obtain the product.

[0029]

[0030] In the above formula, R3 is -NH2 or -CH2OH; R4 is -NO2, -COOH or -COOCH3; the definitions of R2, L1, L2, X, A and B are as described above.

[0031] The present invention also provides a pharmaceutical composition, which comprises the above novel JOSD2 covalent inhibitor or the novel JOSD2 covalent inhibitor prepared by the above preparation method and a pharmaceutically acceptable salt or other drugs.

[0032] Preferably, the other drugs are camptothecin and its derivatives, paclitaxel and its derivatives, gemcitabine and its derivatives, platinum drugs, gefitinib, afatinib or osimertinib.

[0033] The present invention also provides the use of the above novel JOSD2 covalent inhibitor or the novel JOSD2 covalent inhibitor prepared by the above preparation method in the preparation of a drug for treating JOSD2-related diseases.

[0034] Preferably, the related diseases are colorectal cancer, non-small cell lung cancer, cholangiocarcinoma, hepatocellular carcinoma, thymoma, pancreatic cancer, colorectal cancer, melanoma, glioma, glioblastoma multiforme, ovarian cancer or bladder cancer.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a novel JOSD2 covalent inhibitor, which is a compound having the structure shown in formula (I), its stereoisomers and pharmaceutically acceptable salt forms. It has certain inhibitory activity against JOSD2, can effectively inhibit and kill colorectal cancer tumor cells, and its preparation method is easy to operate, the post-treatment is simple, the starting materials of the reaction are cheap and easy to obtain, the reaction efficiency is high, and it is expected to be used in the development of covalent JOSD2 inhibitors and the treatment of related diseases. Detailed embodiments

[0037] It should be noted that the raw materials used in the present invention are all ordinary commercially available products. Among them, the homologous recombination kit, catalog number CU201-03, is purchased from Beijing TransGen Biotech Co., Ltd.; BL21 Escherichia coli, model BL21(DE3), is purchased from TOLOBIO Co., Ltd.; HCT116 cells are purchased from the Cell Bank of the Chinese Academy of Sciences; trypsin is purchased from Hangzhou Keyi Biotechnology Co., Ltd.; the complete medium is Mcacoy’S 5A medium (containing 10% FBS), which is purchased from Zhejiang Senrui Biotechnology Co., Ltd.

[0038] Example 1: Synthesis of N-(6-(adamantane-1-carboxamido)benzothiazol-2-yl)-1-cyanopyrrolidine-3-carboxamide (A1)

[0039]

[0040] Step 1: Synthesis of intermediate 1-2

[0041] Weigh (R)-1-BOC-pyrrolidine-3-carboxylic acid (215 mg, 1 mmol) into a 30 mL dichloromethane (DCM) solution, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (570 mg, 1.5 mmol) and N,N-diisopropylethylamine (DIPEA) (387 mg, 3 mmol), stir and react for 30 min, then add 2-amino-6-nitrobenzothiazole (Compound 1-1) (195.01 mg, 1 mmol) and stir and react for 12 h. TLC detects that the reaction is complete, add pure water to quench, extract with DCM, spin-dry the solvent, and purify by silica gel column chromatography with an elution gradient of PE:EA = 2:1 to obtain intermediate 1-2 as a white solid. Yield: 78.2%; ESI-MS: m / z = 393 [M+H] + .

[0042] Step 2: Synthesis of intermediate 1-3

[0043] Add 5% Pd / C (20 mg, 10%) dissolved in 10 mL methanol (ETOH) to intermediate 1-2 (196 mg, 0.5 mmol). Bubble H2 into this mixture and stir at 40 °C for 12 h. After the reaction is complete, filter the Pd / C solid, spin-dry the filtrate to obtain intermediate 1-3 as a pale yellow solid. Yield: 76.2%; ESI-MS: m / z = 363 [M+H] + .

[0044] Step 3: Synthesis of intermediate 1-4

[0045] 180 mg (1 mmol) of adamantane carboxylic acid was weighed and dissolved in DCM. HATU (570 mg, 1.5 mmol) and DIPEA (387 mg, 3 mmol) were added. After stirring for 30 min, intermediate 1-3 (362 mg, 1 mmol) was added and the mixture was stirred for 12 h. The reaction was monitored by TLC until completion, then quenched with pure water. The mixture was extracted with DCM, the solvent was evaporated, and purification was performed by silica gel column chromatography with an elution gradient of PE:EA = 1:1 to obtain intermediate 1-4 as a white solid. Yield: 69.2%; ESI-MS: m / z = 525 [M+H] + 。

[0046] Step 4: Synthesis of intermediate 1-5

[0047] 262 mg (0.5 mmol) of intermediate 1-4 was weighed and 2 mL of 4 mol / L HCl(EA) was added. The mixture was stirred for 1 h, the solvent was evaporated to obtain intermediate 1-5 as a white solid. Yield: 92.1%; ESI-MS: m / z = 425 [M+H] + 。

[0048] Step 5: Synthesis of compound A1

[0049] 212 mg (0.5 mmol) of intermediate 1-5 was weighed and dissolved in 10 mL of DCM. Under a N2 atmosphere, DIPEA (129 mg, 1 mmol) was added, then cyanogen bromide (BrCN) (53 mg, 0.5 mmol) was added. The reaction was carried out in an ice bath for 30 min and then quenched with water. The mixture was extracted with DCM, the solvent was evaporated, and purification was performed by silica gel column chromatography with an elution gradient of PE:EA = 1:1 to obtain compound A1. Yield: 69.4%, ESI-MS: m / z = 450 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 7.72 - 7.54 (m, 2H), 3.64 - 3.38 (m, 5H), 2.23 - 1.96 (m, 6H), 1.88 (d, J = 3.1 Hz, 6H), 1.67 (t, J = 3.2 Hz, 7H).

[0050] The overall yield of compound A1 was 26.3% and the purity was 94.44%.

[0051] Example 2: Synthesis of (R)-N-(6-(adamantane-1-carboxamido)benzo[d]thiazol-2-yl)-1-(2-chloroacetyl)pyrrolidine-3-carboxamide (A2)

[0052]

[0053] Synthesis of Compound A2: Intermediate 1-5 (424 mg, 1 mmol) was added to 6 mL of dichloromethane. After purging with nitrogen for protection, it was placed in an ice-water bath, and TEA (1.2 mL, 7 mmol) was added. The reaction was carried out for 10 min. Chloroacetyl chloride (55 μL, 0.7 mmol) was dissolved in 1 mL of dichloromethane. After cooling to 0 °C, it was slowly added dropwise to the reaction system in the ice-water bath, and the reaction was carried out for 45 min. After the reaction was complete, the reaction system was added to 10 mL of water, extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation. The crude product was purified by column chromatography (PE:EA = 1:1 - 1:2) to obtain Compound A2 as a white solid. Yield: 52%, ESI-MS: m / z = 501 [M+H] + 。 1 H NMR (400 MHz, Chloroform-d) δ 8.24 (dd, J = 6.7, 2.1 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H), 7.57 (t, J = 9.1 Hz, 1H), 7.31 (ddd, J = 13.8, 8.7, 2.2 Hz, 1H), 4.03 (d, J = 5.0 Hz, 2H), 3.87 - 3.67 (m, 3H), 3.47 - 3.29 (m, 2H), 2.42 - 2.30 (m, 2H), 2.27 - 2.13 (m, 3H), 2.09 (t, J = 3.3 Hz, 3H), 1.98 (d, J = 2.9 Hz, 6H), 1.27 (d, J = 1.9 Hz, 1H), 1.24 (d, J = 5.9 Hz, 2H).

[0054] The total yield of Compound A1 was 19.8% and the purity was 98.13%.

[0055] Example 3: Synthesis of (R)-N-(adamantan-1-yl)-2-(1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazole-6-carboxamide (A3)

[0056]

[0057] Step 1: Synthesis of Intermediate 3-2

[0058] Weigh (R)-1-BOC-pyrrolidine-3-carboxylic acid (215 mg, 1 mmol) into 30 mL of dichloromethane solution, add HATU (570 mg, 1.5 mmol) and DIPEA (387 mg, 3 mmol), stir and react for 30 min, then add methyl 2-aminobenzothiazole-6-carboxylate (208.3 mg, 1 mmol) and stir and react for 12 h. After detecting the completion of the reaction by TLC, add pure water to quench the reaction, extract with DCM, rotary evaporate the solvent, and purify by silica gel column chromatography with an elution gradient of PE:EA = 2:1 to obtain intermediate 3-2 as a white solid. Yield: 78.2%; ESI-MS: m / z = 406 [M+H] + 。

[0059] Step 2: Synthesis of intermediate 3-3

[0060] Add 10 mL of 1 mol / L sodium hydroxide to intermediate 3-2 (203 mg, 0.5 mmol), stir at 40 °C for 2 h, then slowly add 2 M hydrochloric acid dropwise to the reaction solution, separate the solid and liquid using a suction funnel, and freeze-dry the solid to obtain intermediate 3-3. Yield: 76.2%; ESI-MS: m / z = 392 [M+H] + 。

[0061] Step 3: Synthesis of intermediate 3-4

[0062] Weigh intermediate 3-3 (392 mg, 1 mmol) and dissolve it in DCM, add HATU (570 mg, 1.5 mmol) and DIPEA (387 mg, 3 mmol), stir and react for 30 min, then add adamantylamine (151 mg, 1 mmol) and stir and react for 12 h. After detecting the completion of the reaction by TLC, add pure water to quench the reaction, extract with DCM, rotary evaporate the solvent, and purify by silica gel column chromatography with an elution gradient of PE:EA = 1:1 to obtain intermediate 3-4 as a white solid. Yield: 69.2%; ESI-MS: m / z = 525 [M+H] + 。

[0063] Step 4: Synthesis of intermediate 3-5

[0064] Weigh intermediate 3-4 (262 mg, 0.5 mmol) and add 2 mL of 4 mol / L HCl (EA), stir and react for 1 h, rotary evaporate the solvent to obtain intermediate 3-5 as a white solid. Yield: 92.1%; ESI-MS: m / z = 425 [M+H] + 。

[0065] Step 5: Synthesis of compound A3

[0066] Weigh the intermediate 3-5 (212 mg, 0.5 mmol), dissolve it in 10 mL of DCM, add DIPEA (129 mg, 1 mmol) and BrCN (53 mg, 0.5 mmol) under N2 atmosphere, then carry out the reaction in an ice bath for 30 min, and quench with water. After extraction with DCM and rotary evaporation, it is purified by silica gel column chromatography with an elution gradient of PE:EA = 1:1 to obtain compound A3, yield: 64.5%, ESI-MS: m / z = 450 [M+H] + 。

[0067] The total yield of compound A3 is 24.5%, and the purity is 95.15%.

[0068] Example 4: Synthesis of (R)-1-cyano-N-(6-(cyclohexanecarboxamido)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide (A4)

[0069]

[0070] Step 1: Synthesis of intermediate 4-1

[0071] Referring to Step 3 of Example 1, replace adamantaneformic acid with cyclohexanecarboxylic acid to obtain intermediate 4-1, ESI-MS: m / z = 473 [M+H] + 。

[0072] Step 2: Synthesis of intermediate 4-2

[0073] Referring to Step 4 of Example 1 to obtain intermediate 4-2, ESI-MS: m / z = 373 [M+H] + 。

[0074] Step 3: Synthesis of compound A4

[0075] Referring to Step 5 of Example 1, replace intermediate 1-5 with intermediate 4-2 to obtain compound A4. Yield: 72.1%, ESI-MS: m / z = 398 [M+H] + 。 1 H NMR (400 MHz, Methanol-d4) δ 8.20 (d, J = 2.2 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.46 - 7.36 (m, 1H), 3.66 (qd, J = 9.7, 7.0 Hz, 2H), 3.56 (dt, J = 9.2, 6.8 Hz, 1H), 3.46 (dt, J = 9.3, 7.1 Hz, 1H), 3.28 (t, J = 7.0 Hz, 1H), 2.40 - 2.13 (m, 3H), 1.48 (qd, J = 12.3, 3.2 Hz, 2H), 1.29 - 1.14 (m, 3H).

[0076] The total yield of Compound A4 is 17.1%, and the purity is 98.85%.

[0077] Example 5: Synthesis of (S)-1-(adamantane-1-carbonyl)-N-(2-((R)-1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazol-6-yl)pyrrolidine-2-carboxamide (A5)

[0078]

[0079] Step 1: Synthesis of Intermediate 5-1

[0080] Referring to Step 3 of Reference Example 1, replace adamantaneformic acid with (adamantane-1-carbonyl)-D-proline to obtain Intermediate 5-1, ESI-MS: m / z = 622 [M+H] + .

[0081] Step 2: Synthesis of Intermediate 5-2

[0082] Referring to Step 4 of Reference Example 1 to obtain Intermediate 5-2, ESI-MS: m / z = 522 [M+H] + .

[0083] Step 3: Synthesis of Compound A5

[0084] Referring to Step 5 of Reference Example 1, replace Intermediate 1-5 with Intermediate 5-2 to obtain Compound A5. Yield: 61.43%, ESI-MS: m / z = 547 [M+H] + . 1 H NMR (400 MHz, Chloroform-d) δ 8.65 (s, 1H), 7.38 (dd, J = 8.8, 4.1 Hz, 1H), 6.60 (s, 1H), 5.07 (t, J = 6.2 Hz, 1H), 3.74 - 3.52 (m, 5H), 3.41 (tt, J = 10.3, 7.5 Hz, 1H), 2.31 - 2.22 (m, 2H), 2.19 - 2.11 (m, 2H), 2.09 - 2.06 (m, 4H), 2.01 (d, J = 3.8 Hz, 8H), 1.25 (d, J = 2.5 Hz, 6H).

[0085] The total yield of Compound A5 is 33.4%, and the purity is 96.29%.

[0086] Example 6: Synthesis of (R)-N-(adamantane-1-yl)-2-(1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazole-7-carboxamide (A6)

[0087]

[0088] Step 1: Synthesis of Intermediate 6-2

[0089] Referring to Step 1 of Reference Example 3, Intermediate 6-2 was obtained by using Compound 6-1 instead of Compound 3-1. ESI-MS: m / z = 406 [M+H] + .

[0090] Step 2: Synthesis of Intermediate 6-3

[0091] Referring to Step 2 of Reference Example 3, Intermediate 6-3 was obtained by using Intermediate 6-2 instead of Intermediate 3-2. ESI-MS: m / z = 392 [M+H] + .

[0092] Step 3: Synthesis of Intermediate 6-4

[0093] Referring to Step 3 of Reference Example 3, Intermediate 6-4 was obtained by using Intermediate 6-3 instead of Intermediate 3-3. ESI-MS: m / z = 525 [M+H] + .

[0094] Step 4: Synthesis of Intermediate 6-5

[0095] Referring to Step 4 of Reference Example 3, Intermediate 6-5 was obtained by using Intermediate 6-4 instead of Intermediate 3-4. ESI-MS: m / z = 425 [M+H] + .

[0096] Step 5: Synthesis of Compound A6

[0097] Referring to Step 5 of Reference Example 3, Compound A6 was obtained by using Intermediate 6-5 instead of Intermediate 3-5. Yield: 86.75%, ESI-MS: m / z = 450 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.90 - 7.83 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H), 3.64 (dd, J = 9.6, 7.7 Hz, 1H), 3.56 (dd, J = 9.6, 6.1 Hz, 1H), 3.49 - 3.35 (m, 3H), 2.22 (dtd, J = 13.2, 7.4, 5.8 Hz, 1H), 2.11 (d, J = 3.1 Hz, 6H), 2.09 - 2.03 (m, 4H), 1.67 (d, J = 3.2 Hz, 6H).

[0098] The overall yield of Compound A6 was 5.5% and the purity was 99.93%.

[0099] Example 7: Synthesis of (R)-N-(4-(adamantane-1-carboxamido)benzothiazol-2-yl)-1-cyanopyrrolidine-3-carboxamide (A7)

[0100]

[0101] Step 1: Synthesis of Intermediate 7-2

[0102] Compound 7-1 (0.975 g, 5 mmol) was added to 10 mL of pyridine, and (R)-1-Boc-3-carboxypyrrolidine (1.07 g, 5 mmol) was added. The mixture was stirred at -20 °C for 30 min, and phosphorus oxychloride (0.5 mL, 5.5 mmol) was slowly added dropwise. After reacting at -20 °C for 1 h, the reaction system was transferred to room temperature and continued to react for 2 h. After the reaction was complete, pyridine was removed by rotary evaporation. The reaction system was added to 15 mL of water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation to obtain Intermediate 7-2. ESI-MS: m / z = 393 [M+H] + 。

[0103] Step 2: Synthesis of Intermediate 7-3

[0104] Referring to Step 2 of Example 1, Intermediate 7-2 was used instead of Intermediate 1-2 to obtain Intermediate 7-3. ESI-MS: m / z = 363 [M+H] + 。

[0105] Step 3: Synthesis of Intermediate 7-4

[0106] Referring to Step 3 of Example 1, Intermediate 7-3 was used instead of Intermediate 1-3 to obtain Intermediate 7-4. ESI-MS: m / z = 525 [M+H] + 。

[0107] Step 4: Synthesis of Intermediate 7-5

[0108] Referring to Step 4 of Example 1, Intermediate 7-4 was used instead of Intermediate 1-4 to obtain Intermediate 7-5. ESI-MS: m / z = 425 [M+H] + 。

[0109] Step 5: Synthesis of Compound A7

[0110] Referring to Step 5 of Example 1, Intermediate 7-5 was used instead of Intermediate 1-5 to obtain Compound A7. ESI-MS: m / z = 450 [M+H] + 。 11H NMR (400 MHz, Chloroform-d) δ 8.71 (s, 1H), 8.45 (dd, J = 8.0, 0.9 Hz, 1H), 7.49 (dd, J = 8.1, 1.0 Hz, 1H), 3.78 - 3.60 (m, 3H), 3.47 (dtd, J = 8.8, 7.3, 1.2 Hz, 1H), 3.28 (p, J = 7.4 Hz, 1H), 2.36 - 2.25 (m, 2H), 2.10 - 2.06 (m, 3H), 1.99 (d, J = 2.9 Hz, 6H), 1.78 - 1.66 (m, 6H).

[0111] The overall yield of compound A7 was 17.6%, and the purity was 98.57%.

[0112] Example 8: Synthesis of (R)-N-(6-(Adamantan-1-ylcarbamoyl)benzo[d]thiazol-2-yl)-1-(ethenylsulfonyl)piperidine-3-carboxamide (A8)

[0113]

[0114] Referring to Step 5 of Reference Example 1, using chloroethylsulfonyl chloride instead of cyanogen bromide, compound A8 was obtained. ESI-MS: m / z = 515 [M + H] + 。

[0115] The overall yield of compound A8 was 16.7%, and the purity was 99.65%.

[0116] Example 9: Synthesis of (R)-N-(6-(Bicyclo[2.2.1]heptane-1-carboxamido)benzo[d]thiazol-2-yl)-1-cyanopyrrolidine-3-carboxamide (A9)

[0117]

[0118] Step 1: Synthesis of Intermediate 9-1

[0119] Referring to Step 3 of Reference Example 1, using norbornane-1-carboxylic acid instead of adamantane-1-carboxylic acid, Intermediate 9-1 was obtained. ESI-MS: m / z = 485 [M + H] + 。

[0120] Step 2: Synthesis of Intermediate 9-2

[0121] Referring to Step 4 of Reference Example 1, using Intermediate 9-1 instead of Intermediate 1-4, Intermediate 9-2 was obtained. ESI-MS: m / z =

[0122] 385 [M + H] + 。

[0123] Step 3: Synthesis of Compound A9

[0124] Refer to Step 5 of Example 1. Replace intermediate 1-5 with intermediate 9-2 to obtain compound A9. ESI-MS: m / z = 410 [M+H] + .

[0125] The total yield of compound A9 is 28.7%, and the purity is 99.45%.

[0126] Example 10: Synthesis of (S)-1-(adamantane-1-carbonyl)-N-(2-((R)-1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazol-7-yl)pyrrolidine-3-acetamide (A10)

[0127]

[0128] Step 1: Synthesis of intermediate 10-2

[0129] Refer to Step 1 of Example 1. Replace compound 1-1 with compound 10-1 to obtain intermediate 10-2. ESI-MS: m / z = 393 [M+H] + .

[0130] Step 2: Synthesis of intermediate 10-3

[0131] Refer to Step 2 of Example 1. Replace intermediate 1-2 with intermediate 10-2 to obtain intermediate 10-3. ESI-MS: m / z = 363 [M+H] + .

[0132] Step 3: Synthesis of intermediate 10-4

[0133] Refer to Step 3 of Example 1. Replace intermediate 1-3 with intermediate 10-3 to obtain intermediate 10-4. ESI-MS: m / z = 622 [M+H] + .

[0134] Step 4: Synthesis of intermediate 10-5

[0135] Refer to Step 4 of Example 1. Replace intermediate 1-4 with intermediate 10-4 to obtain intermediate 10-5. ESI-MS: m / z = 522 [M+H] + .

[0136] Step 5: Synthesis of compound A10

[0137] Refer to Step 5 of Example 1. Replace intermediate 1-5 with intermediate 10-5 to obtain compound A10. ESI-MS: m / z = 547 [M+H] + . 11H NMR (400 MHz, Chloroform-d) δ 7.53 (dd, J = 18.2, 7.8 Hz, 1H), 7.45 (dd, J = 8.0, 3.3 Hz, 1H), 7.37 - 7.27 (m, 1H), 3.90 (q, J = 5.9, 5.1 Hz, 2H), 3.71 - 3.49 (m, 3H), 3.44 - 3.22 (m, 2H), 2.16 (t, J = 6.8 Hz, 4H), 2.09 (s, 12H), 1.75 (s, 7H).

[0138] The total yield of compound A10 was 20.3%, and the purity was 97.76%.

[0139] Example 11: Synthesis of (S)-N-(adamantan-1-yl)-2-(1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazole-7-carboxamide (A11)

[0140]

[0141] Step 1: Synthesis of Intermediate 11-1

[0142] Referring to Step 1 of Reference Example 3, Intermediate 11-1 was obtained by using (S)-1-BOC-pyrrolidine-3-carboxylic acid instead of (R)-1-BOC-pyrrolidine-3-carboxylic acid. ESI-MS: m / z = 406 [M+H] + .

[0143] Step 2: Synthesis of Intermediate 11-2

[0144] Referring to Step 2 of Reference Example 3, Intermediate 11-2 was obtained by using Intermediate 11-1 instead of Intermediate 3-2. ESI-MS: m / z = 392 [M+H] + .

[0145] Step 3: Synthesis of Intermediate 11-3

[0146] Referring to Step 3 of Reference Example 3, Intermediate 11-3 was obtained by using Intermediate 11-2 instead of Intermediate 3-3. ESI-MS: m / z = 525 [M+H] + .

[0147] Step 4: Synthesis of Intermediate 11-4

[0148] Referring to Step 4 of Reference Example 3, Intermediate 11-4 was obtained by using Intermediate 11-3 instead of Intermediate 3-4. ESI-MS: m / z = 425 [M+H] + .

[0149] Step 5: Synthesis of Compound A11

[0150] Referring to Step 5 of Example 3, replace intermediate 3-5 with intermediate 11-4 to obtain compound A11, ESI-MS: m / z = 450 [M+H] + 。 1 H NMR (400 MHz, Chloroform-d) δ 7.81 (dd, J = 6.4, 2.5 Hz, 1H), 7.50 - 7.43 (m, 2H), 6.09 (s, 1H), 3.77 - 3.54 (m, 4H), 3.41 (dt, J = 9.3, 7.3 Hz, 1H), 2.80 (s, 1H), 2.32 - 2.13 (m, 12H), 1.73 (s, 7H).

[0151] The total yield of compound A11 was 27.4%, and the purity was 96.05%.

[0152] Example 12: Synthesis of (3R)-1-cyano-N-(7-(3,5-dimethyladamantane-1-carboxamido)benzothiazol-2-yl)pyrrolidine-3-carboxamide (A12)

[0153]

[0154] Step 1: Synthesis of intermediate 12-1

[0155] Referring to Step 3 of Example 1, replace intermediate 1-3 with intermediate 10-3 to obtain intermediate 12-1, ESI-MS: m / z = 553 [M+H] + 。

[0156] Step 2: Synthesis of intermediate 12-2

[0157] Referring to Step 4 of Example 1, replace intermediate 1-4 with intermediate 12-1 to obtain intermediate 12-2, ESI-MS: m / z = 453 [M+H] + 。

[0158] Step 5: Synthesis of compound A12

[0159] Referring to Step 5 of Example 1, replace intermediate 1-5 with intermediate 12-2 to obtain compound A12, ESI-MS: m / z = 478 [M+H] + 。 11H NMR (400 MHz, Chloroform-d) δ 7.72 (s, 1H), 7.46 (dd, J = 5.6, 3.4 Hz, 1H), 7.36 - 7.31 (m, 2H), 3.63 (dd, J = 9.7, 6.6 Hz, 1H), 3.54 (s, 1H), 3.41 - 3.33 (m, 1H), 3.25 (p, J = 7.1 Hz, 1H), 3.03 (d, J = 7.3 Hz, 1H), 2.23 - 2.11 (m, 3H), 1.85 (s, 2H), 1.63 (q, J = 12.2 Hz, 4H), 1.45 - 1.36 (m, 4H), 1.35 - 1.24 (m, 2H), 0.89 (s, 6H).

[0160] The total yield of compound A12 was 13.3%, and the purity was 99.63%.

[0161] Example 13: Synthesis of (R)-N-(2-(N-(1-cyanopyrrolidin-3-yl)sulfamoyl)amino)benzo[d]thiazol-7-yl)adamantane-1-carboxamide (A13)

[0162]

[0163] Step 1: Synthesis of intermediate 13-2

[0164] In a 1st glass bottle, at 0 °C, chlorosulfonyl isocyanate (600 mg, 4.24 mmol) and 2-bromoethanol (0.3 mL, 4.24 mmol) were added to DCM (6 mL), and the mixture was stirred for 1 h. Meanwhile, in a 2nd glass bottle, at 0 °C, (S)-1-Boc-3-aminopyrrolidine (947.64 mg, 5.08 mmol) and triethylamine (TEA)

[0165] (1.19 mL, 8.48 mmol) were added to DCM (6 mL), and the mixture was stirred for 1 h. After 1 h, at 0 °C, the reaction mixture in the 2nd glass bottle was added to the 1st glass bottle. The resulting reaction mixture was stirred at room temperature for 2 h, poured into water (30 mL), and extracted with DCM. The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain intermediate 13-2, a yellow oil, with a yield of 98.3%, ESI-MS: m / z = 336 [M + H] + .

[0166] Step 2: Synthesis of intermediate 13-3

[0167] 2-Amino-7-nitrobenzothiazole (350 mg, 1.79 mmol) was added to a round-bottom flask, followed by acetonitrile (15 mL) and TEA (0.65 mL, 4.68 mmol). The reaction mixture was stirred at room temperature for 15 min, then intermediate 13-2 (1.43 g, 4.48 mmol) was added thereto and the temperature was raised to 70 °C for stirring reaction. After the raw materials were reacted completely, it was cooled to room temperature, 30 mL of water was added to the reaction solution, and then it was extracted three times with ethyl acetate. The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH = 20:1) to obtain intermediate 13-3, a yellow solid, with a yield of 82.1%, ESI-MS: m / z = 444 [M+H] + 。

[0168] Step 3: Synthesis of intermediate 13-4

[0169] Intermediate 13-3 (650 mg, 1.43 mmol) was added to a two-necked round-bottom flask, followed by 10 mL of methanol, and then palladium on carbon (195 mg) was added. After changing the gas, the reaction was stirred at room temperature under a H2 atmosphere. After the reaction was completed, it was filtered through diatomaceous earth and concentrated under reduced pressure to obtain intermediate 13-4, a white solid, with a yield of 92.3%, ESI-MS: m / z = 414 [M+H] + 。

[0170] Step 4: Synthesis of intermediate 13-5

[0171] Intermediate 13-4 (515 mg, 1.25 mmol) was added to a two-necked round-bottom flask, followed by 8 mL of DCM, and then TEA (435.5 μL, 2.5 mmol) was added. After changing the gas, the solution was stirred under N2 protection and cooled to 0 °C in an ice bath. A DCM solution (1 mL) of 1-adamantanecarbonyl chloride (198.7 mg, 1.0 mmol) was added dropwise, and after the addition was complete, it was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with saturated NaHCO3 solution, 1 M HCl solution and saturated NaCl solution. The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure to obtain intermediate 13-5, a white solid, with a yield of 96.1%, ESI-MS: m / z = 576 [M+H] + 。

[0172] Step 5: Synthesis of intermediate 13-6

[0173] Add intermediate 13-5 (120 mg, 0.21 mmol) to a round-bottom flask, then add 12 mL of DCM, cool to 0 °C in an ice bath, and then add 1.2 mL of trifluoroacetic acid. After the addition is complete, stir the reaction at room temperature. After the reaction is completed, concentrate under reduced pressure to obtain intermediate 13-6, a white solid, with a yield of 93.4%, ESI-MS: m / z = 476 [M+H] + 。

[0174] Step 6: Synthesis of compound A13

[0175] Add intermediate 13-6 (100 mg, 0.2 mmol) to a round-bottom flask, then add 3 mL of THF and K2CO3 (110.5 mg, 0.8 mmol), and stir at room temperature for 15 min. Cool to 0 °C in an ice bath, then add BrCN (30 mg, 0.24 mmol), and then stir the reaction at room temperature. After the reaction is completed, add 6 mL of water to the reaction solution, extract three times with ethyl acetate, combine the organic phases, dry over Na2SO4, filter and concentrate under reduced pressure to obtain the crude product. The crude product is purified by thin-layer chromatography (DCM:MeOH = 15:1) to obtain compound A13, a white solid, yield: 20%, ESI-MS: m / z = 501 [M+H] + 。 1 1H NMR (400 MHz, Chloroform-d) δ 7.94 (s, 1H), 7.23 - 7.18 (m, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 5.88 (d, J = 17.1 Hz, 1H), 3.97 (d, J = 7.7 Hz, 1H), 3.52 - 3.28 (m, 5H), 2.11 - 2.07 (m, 3H), 2.00 - 1.95 (m, 7H), 1.82 - 1.68 (m, 7H).

[0176] The total yield of compound A13 is 13.4% and the purity is 97.73%.

[0177] Example 14: Synthesis of (R)-N-(6-(adamantane-1-carboxamido)benzo[d]oxazol-2-yl)-1-cyanopyrrolidine-3-carboxamide (A14)

[0178]

[0179] Step 1: Synthesis of intermediate 14-2

[0180] Refer to Step 1 of Example 1, use compound 14-1 instead of compound 1-1 to obtain intermediate 14-2, ESI-MS: m / z = 377 [M+H] + 。

[0181] Step 2: Synthesis of Intermediate 14-3

[0182] Referring to Step 2 of Reference Example 1, Intermediate 14-3 was obtained by using Intermediate 14-2 instead of Intermediate 1-2. ESI-MS: m / z = 347 [M+H] + .

[0183] Step 3: Synthesis of Intermediate 14-4

[0184] Referring to Step 3 of Reference Example 1, Intermediate 14-4 was obtained by using Intermediate 14-3 instead of Intermediate 1-3. ESI-MS: m / z = 509 [M+H] + .

[0185] Step 4: Synthesis of Intermediate 14-5

[0186] Referring to Step 4 of Reference Example 1, Intermediate 14-5 was obtained by using Intermediate 14-4 instead of Intermediate 1-4. ESI-MS: m / z = 409 [M+H] + .

[0187] Step 5: Synthesis of Compound A14

[0188] Referring to Step 5 of Reference Example 1, Compound A14 was obtained by using Intermediate 14-5 instead of Intermediate 1-5. ESI-MS: m / z = 434 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 9.93 (s, 1H), 8.31 (d, J = 2.2 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.47 (dd, J = 8.8, 2.0 Hz, 1H), 3.67 - 3.47 (m, 2H), 3.45 -

[0189] 3.37 (m, 2H), 2.30 (td, J = 9.8, 8.1, 5.8 Hz, 1H), 2.24 - 2.11 (m, 1H), 2.11 - 1.94 (m, 1H), 1.84 - 1.67 (m, 4H), 1.61 (d, J = 11.5 Hz, 1H), 1.39 (qd, J = 12.3, 2.9 Hz, 2H), 1.29 - 1.06 (m, 4H).

[0190] The total yield of Compound A14 was 31.8%, and the purity was 97.92%.

[0191] Example 15: Synthesis of (3R)-1-Cyano-N-(6-(3,5-Dimethyladamantane-1-carboxamido)-1H-benzo[d]imidazol-2-yl)pyrrolidine-3-carboxamide (A15)

[0192]

[0193] Step 1: Synthesis of Intermediate 15-2

[0194] Referring to Step 1 of Reference Example 1, Intermediate 15-2 was obtained by using Compound 15-1 instead of Compound 1-1. ESI-MS: m / z = 376 [M+H] + .

[0195] Step 2: Synthesis of Intermediate 15-3

[0196] Referring to Step 2 of Reference Example 1, Intermediate 15-3 was obtained by using Intermediate 15-2 instead of Intermediate 1-2. ESI-MS: m / z = 346 [M+H] + .

[0197] Step 3: Synthesis of Intermediate 15-4

[0198] Referring to Step 3 of Reference Example 1, Intermediate 15-4 was obtained by using Intermediate 15-3 instead of Intermediate 1-3. ESI-MS: m / z = 536 [M+H] + .

[0199] Step 4: Synthesis of Intermediate 15-5

[0200] Referring to Step 4 of Reference Example 1, Intermediate 15-5 was obtained by using Intermediate 15-4 instead of Intermediate 1-4. ESI-MS: m / z = 436 [M+H] + .

[0201] Step 5: Synthesis of Compound A15

[0202] Referring to Step 5 of Reference Example 1, Intermediate 15-5 was used instead of Intermediate 1-5 to obtain a light pink solid (A15). Yield: 59.3%, ESI-MS: m / z = 461 [M+H] + . 1 H NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.55 (s, 1H), 7.34 (d, J = 8.5 Hz, 1H), 6.98 (d, J = 8.6 Hz, 1H), 3.57 (q, J = 5.0, 4.2 Hz, 2H), 3.54 - 3.49 (m, 1H), 3.43 (dd, J = 9.1, 7.2 Hz, 1H), 3.32 (p, J = 7.2 Hz, 1H), 2.25 - 2.14 (m, 3H), 1.83 (d, J = 3.2 Hz, 2H), 1.62 (q, J = 12.1 Hz, 4H), 1.45 - 1.36 (m, 4H), 1.22 (s, 2H), 0.90 (s, 7H).

[0203] The total yield of compound A15 was 22.9%, and the purity was 97.57%.

[0204] Example 16: Synthesis of (R)-N-(adamantan-1-yl)-2-(1-cyanopyrrolidine-3-carboxamido)-1H-benzo[d]imidazole-7-carboxamide (A16)

[0205]

[0206] Step 1: Synthesis of intermediate 16-2

[0207] Referring to Step 1 of Reference Example 3, intermediate 16-2 was obtained by using compound 16-1 instead of compound 3-1. ESI-MS: m / z = 389 [M+H] + 。

[0208] Step 2: Synthesis of intermediate 16-3

[0209] Referring to Step 2 of Reference Example 3, intermediate 16-3 was obtained by using intermediate 16-2 instead of intermediate 3-2. ESI-MS: m / z = 375 [M+H] + 。

[0210] Step 3: Synthesis of intermediate 16-4

[0211] Referring to Step 3 of Reference Example 3, intermediate 16-4 was obtained by using intermediate 16-3 instead of intermediate 3-3 and 3,5-dimethyladamantanamine instead of adamantanamine. ESI-MS: m / z = 536 [M+H] + 。

[0212] Step 4: Synthesis of intermediate 16-5

[0213] Referring to Step 4 of Reference Example 3, intermediate 16-5 was obtained by using intermediate 16-4 instead of intermediate 3-4. ESI-MS: m / z = 436 [M+H] + 。

[0214] Step 5: Synthesis of compound A16

[0215] Referring to Step 5 of Reference Example 2, compound A16 was obtained by using intermediate 16-5 instead of intermediate 3-5. ESI-MS: m / z = 461 [M+H] + 。 11H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 3.74 - 3.18 (m, 8H), 2.16 (dt, J = 33.5, 6.2 Hz, 3H), 1.95 (s, 2H), 1.76 (q, J = 11.7 Hz, 4H), 1.42 - 1.23 (m, 4H), 1.14 (q, J = 12.6, 12.1 Hz, 2H), 0.84 (s, 6H).

[0216] The total yield of compound A16 was 20.5%, and the purity was 96.8%.

[0217] Example 17: Synthesis of (R)-N-(6-(Adamantan-1-carboxamido)-1H-benzo[d]imidazol-2-yl)-1-cyanopyrrolidine-3-carboxamide (A17)

[0218]

[0219] Step 1: Synthesis of Intermediate 17-1

[0220] Referring to Step 3 of Example 1, Intermediate 15-3 was used instead of Intermediate 1-3 to obtain Intermediate 17-1, ESI-MS: m / z = 508 [M+H] + .

[0221] Step 2: Synthesis of Intermediate 17-2

[0222] Referring to Step 4 of Example 1, Intermediate 17-1 was used instead of Intermediate 1-4 to obtain Intermediate 17-2, ESI-MS: m / z = 408 [M+H] + .

[0223] Step 3: Synthesis of Compound A17

[0224] Referring to Step 5 of Example 1, Intermediate 17-2 was used instead of Intermediate 1-5 to obtain Compound A17, ESI-MS: m / z = 433 [M+H] + . 1 1H NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.56 (s, 1H), 7.35 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 3.56 (d, J = 7.1 Hz, 2H), 3.44 (d, J = 8.3 Hz, 1H), 3.30 (d, J = 7.3 Hz, 1H), 2.12 (s, 4H), 2.01 (s, 6H), 1.78 (s, 6H).

[0225] The total yield of Compound A17 is 31.7%, and the purity is 99.31%.

[0226] Example 18: Synthesis of (3R)-1-cyano-N-(6-(3,5-dimethyladamantane-1-carboxamido)-1H-benzo[d]imidazol-2-yl)pyrrolidine-3-carboxamide (A18)

[0227]

[0228] Step 1: Synthesis of Intermediate 18-1

[0229] Referring to Step 1 of Reference Example 1, Intermediate 18-1 was obtained by using Compound 15-1 instead of Compound 1-1. ESI-MS: m / z = 389 [M+H] + .

[0230] Step 2: Synthesis of Intermediate 18-2

[0231] Referring to Step 2 of Reference Example 1, Intermediate 18-2 was obtained by using Intermediate 18-1 instead of Intermediate 1-2. ESI-MS: m / z = 360 [M+H] + .

[0232] Step 3: Synthesis of Intermediate 18-3

[0233] Referring to Step 3 of Reference Example 1, Intermediate 18-3 was obtained by using Intermediate 18-2 instead of Intermediate 1-3. ESI-MS: m / z = 550 [M+H] + .

[0234] Step 4: Synthesis of Intermediate 18-4

[0235] Referring to Step 4 of Reference Example 1, Intermediate 18-4 was obtained by using Intermediate 18-3 instead of Intermediate 1-4. ESI-MS: m / z = 450 [M+H] + .

[0236] Step 5: Synthesis of Compound A18

[0237] Referring to Step 5 of Reference Example 1, Compound A18 was obtained by using Intermediate 18-4 instead of Intermediate 1-5. ESI-MS: m / z = 475 [M+H] + .

[0238] The total yield of Compound A18 is 23.8%, and the purity is 97.58%.

[0239] Example 19: Synthesis of N-(2-(5-cyanooctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)-1H-benzo[d]imidazol-6-yl)-3,5-dimethyladamantane-1-carboxamide (A19)

[0240]

[0241] Step 1: Synthesis of Intermediate 19-2

[0242] Compound 19-1 (450 mg, 2.71 mmol) and glycolic acid (310 mg, 4.07 mmol) were added to a round-bottom flask, followed by 10 mL of HCl (5.5 M). The temperature was raised to 100 °C and stirred overnight. After the reaction was completed, it was cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was washed with ethanol, and the residue was recovered to obtain Intermediate 19-2, a brownish-black solid. ESI-MS: m / z = 194 [M+H] + .

[0243] Step 2: Synthesis of Intermediate 19-3

[0244] Referring to Step 2 of Example 1, Intermediate 19-2 was used instead of Intermediate 1-2 to obtain Intermediate 19-3. ESI-MS: m / z = 164 [M+H] + .

[0245] Step 3: Synthesis of Intermediate 19-4

[0246] Intermediate 19-3 (427 mg, 2.62 mmol) was added to a round-bottom flask, followed by DMF (6 mL). Then 3,5-dimethyladamantane carboxylic acid (594.5 mg, 3.93 mmol), HATU (1.49 g, 3.93 mmol) and DIEA (1.37 mL, 7.86 mmol) were added, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, water was added to the reaction solution, and it was extracted three times with ethyl acetate. The organic phases were combined, dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography (DCM:MeOH = 50:1) gave Intermediate 19-4, a yellow solid. ESI-MS: m / z = 354 [M+H] + .

[0247] Step 4: Synthesis of Intermediate 19-5

[0248] Intermediate 19-4 (579 mg, 1.64 mmol) was added to a round-bottom flask, followed by acetone (10 mL). Then, an aqueous solution (5 mL) of potassium permanganate (286.2 mg, 1.81 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH = 50:1) to obtain Intermediate 19-5 as a white solid. ESI-MS: m / z = 368 [M+H] + 。

[0249] Step 5: Synthesis of Intermediate 19-6

[0250] Intermediate 19-5 (161 mg, 0.42 mmol) was added to a round-bottom flask, followed by DMF (2 mL). Then, tert-butyl hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (133.4 mg, 0.63 mmol), HATU (239.6 mg, 0.63 mmol), and DIEA (220 μL, 1.26 mmol) were added, and the mixture was stirred at room temperature for 1 h. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH = 40:1) to obtain Intermediate 19-6 as a yellow solid. ESI-MS: m / z =

[0251] 562 [M+H] + 。

[0252] Step 6: Synthesis of Intermediate 19-7

[0253] Intermediate 19-6 (179 mg, 0.32 mmol) was added to a round-bottom flask, followed by DCM (2 mL). Then, EA / HCl (2 mL) was added, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain Intermediate 19-7 as a white solid. ESI-MS: m / z = 462 [M+H] + 。

[0254] Step 7: Synthesis of Compound A19

[0255] Intermediate 19-7 (147 mg, 0.32 mmol) was added to a round-bottom flask, followed by DCM (2 mL). TEA (135 μL, 0.97 mmol) and cyanogen bromide (41.4 mg, 0.39 mmol) were added at about 0 °C, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by thin-layer chromatography (DCM:MeOH = 30:1) to obtain Compound A19 as a white solid. ESI-MS: m / z = 487 [M+H] +。

[0256] The total yield of compound A19 was 24.8%, and the purity was 98.4%.

[0257] Example 20: Synthesis of N-(2-(5-cyanooctahydropyrrolo[3,4-c]pyrrole-2-carbonyl)-1H-benzo[d]imidazol-6-yl)-3,5-dimethyladamantane-1-carboxamide (A20)

[0258]

[0259] Step 1: Synthesis of intermediate 20-1

[0260] Referring to Step 5 of Reference Example 19, tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate was used instead of tert-butyl hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate to obtain intermediate 20-1, ESI-MS: m / z = 562 [M+H] + 。

[0261] Step 2: Synthesis of intermediate 20-2

[0262] Referring to Step 6 of Reference Example 19, intermediate 20-1 was used instead of intermediate 19-6 to obtain intermediate 20-2, ESI-MS: m / z = 462 [M+H] + 。

[0263] Step 3: Synthesis of compound A20

[0264] Referring to Step 7 of Reference Example 19, intermediate 20-2 was used instead of intermediate 19-7 to obtain compound A20, ESI-MS: m / z = 487 [M+H] + 。 11H NMR (400 MHz, Chloroform-d) δ 11.63 (s, 1H), 8.42 (d, J = 2.1 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.51 - 7.45 (m, 1H), 6.99 (dd, J = 8.8, 2.1 Hz, 1H), 4.61 (ddd, J = 10.6, 7.5, 3.0 Hz, 1H), 4.37 (dd, J = 12.9, 5.0 Hz, 1H), 4.15 (d, J = 5.3 Hz, 1H), 3.83 (dd, J = 13.3, 4.8 Hz, 1H), 3.72 (qd, J = 7.1, 3.3 Hz, 2H), 3.45 (ddd, J = 18.3, 10.0, 4.8 Hz, 2H), 3.09 (ddtd, J = 36.2, 12.3, 7.6, 4.9 Hz, 2H), 2.20 (p, J = 3.2 Hz, 1H), 1.81 (d, J = 3.4 Hz, 2H), 1.75 (s, 3H), 1.65 - 1.58 (m, 3H), 1.46 - 1.35 (m, J = 2.9 Hz, 4H), 0.90 (s, 6H).

[0265] The total yield of compound A20 was 31.2% and the purity was 96.8%.

[0266] Example 21: Synthesis of 2-(6-cyano-2,6-diazaspiro[3.3]heptane-2-carbonyl)-N-(3,5-dimethyladamantan-1-yl)-1H-benzo[d]imidazole-7-carboxamide (A21)

[0267]

[0268] Step 1: Synthesis of intermediate 21-2

[0269] Referring to Step 1 of Reference Example 19, using compound 21-1 instead of compound 19-1 to obtain intermediate 21-2, ESI-MS: m / z = 193 [M+H] + .

[0270] Step 2: Synthesis of intermediate 21-3

[0271] Referring to Step 3 of Reference Example 3, using intermediate 21-2 instead of intermediate 3-3 (i.e., 3,5-dimethyladamantanamine instead of adamantanamine) to obtain intermediate 21-3, ESI-MS: m / z = 354 [M+H] + .

[0272] Step 3: Synthesis of intermediate 21-4

[0273] Referring to Step 4 of Reference Example 19, Intermediate 21-4 was obtained by using Intermediate 21-3 in place of Intermediate 19-4. ESI-MS: m / z = 368 [M+H] + .

[0274] Step 4: Synthesis of Intermediate 21-5

[0275] Referring to Step 5 of Reference Example 19, Intermediate 21-5 was obtained by using Intermediate 21-4 in place of Intermediate 19-5 (i.e., tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate in place of tert-butyl hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate). ESI-MS: m / z = 548 [M+H] + .

[0276] Step 5: Synthesis of Intermediate 21-6

[0277] Referring to Step 6 of Reference Example 19, Intermediate 21-6 was obtained by using Intermediate 21-5 in place of Intermediate 19-6. ESI-MS: m / z = 448 [M+H] + .

[0278] Step 6: Synthesis of Compound A21

[0279] Referring to Step 7 of Reference Example 19, Compound A21 was obtained by using Intermediate 21-6 in place of Intermediate 19-7. ESI-MS: m / z = 473 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.43 (s, 1H), 5.28 (d, J = 13.5 Hz, 1H), 5.01 (d, J = 14.7 Hz, 2H), 4.38 (d, J = 14.3 Hz, 1H), 4.27 (t, J = 11.6 Hz, 2H), 3.85 (s, 1H), 3.76 (d, J = 16.0 Hz, 1H), 3.46 (s, 1H), 2.13 (s, 1H), 1.96 (s, 2H), 1.83 (d, J = 11.6 Hz, 2H), 1.64 (dd, J = 25.2, 11.8 Hz, 3H), 1.36 (s, 3H), 1.28 (d, J = 12.4 Hz, 3H), 1.21 (s, 3H), 1.15 (s, 2H), 0.83 (d, J = 10.0 Hz, 6H).

[0280] The overall yield of Compound A21 was 9.1% and the purity was 98.3%.

[0281] Example 22: Synthesis of N-(adamantan-1-yl)-2-(7-cyano-2,7-diazaspiro[4.4]nonane-2-carbonyl)-6-fluoro-1H-benzo[d]imidazole-7-carboxamide (A22)

[0282]

[0283] Step 1: Synthesis of Intermediate 22-1

[0284] Referring to Step 3 of Reference Example 3, Intermediate 22-1 was obtained by using Intermediate 21-2 instead of Intermediate 3-3. ESI-MS: m / z = 326 [M+H] + .

[0285] Step 2: Synthesis of Intermediate 22-2

[0286] Referring to Step 4 of Reference Example 19, Intermediate 22-2 was obtained by using Intermediate 22-1 instead of Intermediate 19-4. ESI-MS: m / z = 340 [M+H] + .

[0287] Step 3: Synthesis of Intermediate 22-3

[0288] Referring to Step 5 of Reference Example 19, Intermediate 22-3 was obtained by using Intermediate 22-2 instead of Intermediate 19-5 (i.e., 2-BOC-2,7-diazaspiro[4.4]nonane instead of tert-butyl hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate). ESI-MS: m / z =

[0289] 548 [M+H] + .

[0290] Step 4: Synthesis of Intermediate 22-4

[0291] Referring to Step 6 of Reference Example 19, Intermediate 22-4 was obtained by using Intermediate 22-3 instead of Intermediate 19-6. ESI-MS: m / z = 448 [M+H] + .

[0292] Step 5: Synthesis of Compound A22

[0293] Referring to Step 7 of Reference Example 19, Compound A22 was obtained by using Intermediate 22-4 instead of Intermediate 19-7. ESI-MS: m / z = 473 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.43 (s, 1H), 5.28 (d, J = 13.5 Hz, 1H), 5.01 (d, J = 14.7 Hz, 2H), 4.38 (d, J = 14.3 Hz, 1H), 4.27 (t, J = 11.6 Hz, 2H), 3.85 (s, 1H), 3.76 (d, J = 16.0 Hz, 1H), 3.46 (s, 1H), 2.13 (s, 1H), 1.96 (s, 2H), 1.83 (d, J = 11.6 Hz, 2H), 1.64 (dd, J = 25.2, 11.8 Hz, 3H), 1.36 (s, 3H), 1.28 (d, J = 12.4 Hz, 3H), 1.21 (s, 3H), 1.15 (s, 2H), 0.83 (d, J = 10.0 Hz, 6H).

[0294] The total yield of compound A22 was 19.6% and the purity was 97.1%.

[0295] Example 23: Synthesis of N-((1R,5S)-3-cyano-3-azabicyclo[3.1.0]hexan-6-yl)-6-(3,5-dimethyladamantan-1-carboxamido)-1H-benzo[d]imidazole-2-carboxamide (A23)

[0296]

[0297] Step 1: Synthesis of intermediate 23-1

[0298] Referring to Step 5 of Reference Example 19, tert-butyl (1R,5S)-6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylate was used instead of tert-butyl hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate to obtain intermediate 23-1, ESI-MS: m / z = 548 [M+H] + 。

[0299] Step 2: Synthesis of intermediate 23-2

[0300] Referring to Step 6 of Reference Example 19, intermediate 23-1 was used instead of intermediate 19-6 to obtain intermediate 23-2, ESI-MS: m / z = 448 [M+H] + 。

[0301] Step 3: Synthesis of compound A23

[0302] Referring to Step 7 of Reference Example 19, intermediate 23-2 was used instead of intermediate 19-7 to obtain compound A23, ESI-MS: m / z = 473 [M+H] + 。1 1H NMR (400 MHz, Chloroform-d) δ 12.13 (s, 1H), 8.25 (s, 1H), 7.99 (s, 1H), 7.65 - 7.59 (m, 1H), 7.44 - 7.30 (m, 1H), 7.12 (dd, J=8.8, 2.0 Hz, 1H), 3.68 - 3.56 (m, 4H), 2.86 - 2.77 (m, 1H), 2.21 - 2.15 (m, 1H), 2.08 - 1.95 (m, 4H), 1.80 (d, J=3.2 Hz, 2H), 1.59 (q, J=12.1 Hz, 4H), 1.44 - 1.34 (m, 4H), 0.87 (s, 6H).

[0303] The total yield of compound A23 was 26.1% and the purity was 98.47%.

[0304] Example 24: Synthesis of N-(adamantan-1-yl)-2-((1R,5S)-3-cyano-3-azabicyclo[3.1.0]hexane-6-carboxamido)-1H-benzo[d]imidazole-7-carboxamide (A24)

[0305]

[0306] Step 1: Synthesis of intermediate 24-1

[0307] Referring to Step 1 of Reference Example 3, using compound 16-1 instead of compound 3-1 to obtain intermediate 24-1, ESI-MS: m / z = 401 [M+H] + .

[0308] Step 2: Synthesis of intermediate 24-2

[0309] Referring to Step 2 of Reference Example 3, using intermediate 24-1 instead of intermediate 3-2 to obtain intermediate 24-2, ESI-MS: m / z = 387 [M+H] + .

[0310] Step 3: Synthesis of intermediate 24-3

[0311] Referring to Step 3 of Reference Example 3, using intermediate 24-2 instead of intermediate 3-3 to obtain intermediate 24-3, ESI-MS: m / z = 520 [M+H] + .

[0312] Step 4: Synthesis of intermediate 24-4

[0313] Referring to Step 4 of Reference Example 3, using intermediate 24-3 instead of intermediate 3-4 to obtain intermediate 24-4, ESI-MS: m / z = 420 [M+H] + .

[0314] Step 5: Synthesis of Compound A24

[0315] Referring to Step 5 of Reference Example 3, Compound A24 was obtained by using Intermediate 24-4 instead of Intermediate 3-5. ESI-MS: m / z = 445 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 7.72 (dd, J = 7.7, 1.2 Hz, 1H), 7.59 (dd, J = 7.9, 1.2 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 3.66 - 3.52 (m, 4H), 2.18 (t, J = 2.1 Hz, 2H), 2.15 (d, J = 2.8 Hz, 6H), 2.11 - 2.07 (m, 4H), 1.70 (d, J = 3.1 Hz, 6H).

[0316] The total yield of Compound A24 was 25.1%, and the purity was 93.57%.

[0317] Example 25: Synthesis of 2-((1R,5S)-3-cyano-3-azabicyclo[3.1.0]hexane-6-carboxamido)-N-(3,5-dimethyladamantan-1-yl)-1H-benzo[d]imidazole-7-carboxamide (A25)

[0318]

[0319] Step 1: Synthesis of Intermediate 25-1

[0320] Referring to Step 3 of Reference Example 3, Intermediate 25-1 was obtained by using Intermediate 24-2 instead of Intermediate 3-3 (i.e., 3,5-dimethyladamantanamine instead of adamantanamine). ESI-MS: m / z = 548 [M+H] + .

[0321] Step 2: Synthesis of Intermediate 25-2

[0322] Referring to Step 4 of Reference Example 3, Intermediate 25-2 was obtained by using Intermediate 25-1 instead of Intermediate 3-4. ESI-MS: m / z = 448 [M+H] + .

[0323] Step 3: Synthesis of Compound A25

[0324] Referring to Step 5 of Reference Example 3, Compound A25 was obtained by using Intermediate 25-2 instead of Intermediate 3-5. ESI-MS: m / z = 473 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 7.72 (dd, J = 7.7, 1.2 Hz, 1H), 7.59 (dd, J = 7.9, 1.2 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 3.66 - 3.53 (m, 5H), 2.22 - 2.10 (m, 5H), 1.99 (d, J = 3.1 Hz, 2H), 1.84 (d, J = 11.8 Hz, 2H), 1.75 (d, J = 11.9 Hz, 2H), 1.41 (d, J = 11.8 Hz, 2H), 1.32 (d, J = 12.4 Hz, 2H), 1.18 (d, J = 6.6 Hz, 2H), 0.88 (s, 6H).

[0325] The total yield of compound A25 was 14.9%, and the purity was 98.62%.

[0326] Example 26: Synthesis of 2 - ((1R,5S,6r)-3 - cyano - 3 - azabicyclo[3.1.0]hexane - 6 - carboxamido)-N-(3,5 - dimethyladamantan - 1 - yl)-1 - methyl - 1H - benzo[d]imidazole - 7 - carboxamide (A26)

[0327]

[0328] Step 1: Synthesis of intermediate 26 - 1

[0329] Intermediate 26 - 1 (548 mg) and potassium carbonate (207 mg) were added to a flask, dissolved in 10 mL of THF, then methyl iodide (142 mg) was added, and the mixture was heated at 40 °C for 4 h. Subsequently, the compound was separated by column chromatography with a separation polarity of DCM:MeOH = 40:1. Intermediate 26 - 1 (43 mg) was obtained with a yield of 7.6%, and ESI - MS m / z = 561 [M + H] + 。

[0330] Step 2: Synthesis of intermediate 26 - 2

[0331] Referring to Step 4 of Example 3, intermediate 26 - 1 was used instead of intermediate 3 - 4 to obtain intermediate 26 - 2, ESI - MS: m / z = 462 [M + H] + 。

[0332] Step 3: Synthesis of compound A26

[0333] Referring to Step 5 of Example 3, intermediate 26 - 2 was used instead of intermediate 3 - 5 to obtain compound A26, ESI - MS: m / z = 487 [M + H] + 。

[0334] The overall yield of compound A26 was 37.1%, and the purity was 95.15%.

[0335] Example 27: Synthesis of (1R,5S)-3-cyano-N-(6-(3,5-dimethyladamantane-1-carboxamido)-1H-benzo[d]imidazol-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxamide (A27)

[0336]

[0337] Step 1: Synthesis of intermediate 27-1

[0338] Referring to Step 1 of Reference Example 1, using compound 25-1 instead of compound 1-1, i.e., (1R,5S,6S)-3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid instead of (R)-1-BOC-pyrrolidine-3-carboxylic acid, intermediate 27-1 was obtained. ESI-MS: m / z = 388 [M+H] + .

[0339] Step 2: Synthesis of intermediate 27-2

[0340] Referring to Step 2 of Reference Example 1, using intermediate 27-1 instead of intermediate 1-2, intermediate 27-2 was obtained. ESI-MS: m / z = 358 [M+H] + .

[0341] Step 3: Synthesis of intermediate 27-3

[0342] Referring to Step 3 of Reference Example 1, using intermediate 27-2 instead of intermediate 1-3 and using 3,5-dimethyladamantane carboxylic acid instead of adamantane carboxylic acid, intermediate 27-3 was obtained. ESI-MS: m / z = 548 [M+H] + .

[0343] Step 4: Synthesis of intermediate 27-4

[0344] Referring to Step 4 of Reference Example 1, using intermediate 27-3 instead of intermediate 1-4, intermediate 27-4 was obtained. ESI-MS: m / z = 448 [M+H] + .

[0345] Step 5: Synthesis of compound A27

[0346] Referring to Step 5 of Reference Example 1, using intermediate 27-4 instead of intermediate 1-5, compound A27 was obtained. ESI-MS: m / z = 473 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 11.86 (s, 2H), 9.01 (s, 1H), 7.78 (s, 1H), 7.29 -

[0347] 7.21 (m, 2H), 3.59 (d, J = 9.8 Hz, 2H), 3.52 (d, J = 9.9 Hz, 2H), 2.12 (s, 2H), 2.08 (p, J = 3.1 Hz, 1H), 1.96 (t, J = 3.1 Hz, 1H), 1.72 (d, J = 3.6 Hz, 2H), 1.52 (q, J = 12.3 Hz, 5H), 1.40 - 1.26 (m, 5H), 0.83 (s, 6H).

[0348] The total yield of compound A27 was 13.2% and the purity was 95.4%.

[0349] Comparative Example 1: Synthesis of N-(5-acrylamidobenzothiazol-2-yl)-1-methylpiperidine-4-carboxamide (B1)

[0350]

[0351] Step 1: Synthesis of intermediate B1-2

[0352] Weigh 1-methylpiperidine-4-carboxylic acid (143 mg, 1 mmol), add HATU (570 mg, 1.5 mmol) and DIPEA (387 mg, 3 mmol), stir and react for 30 min, then add 2-amino-5-nitrobenzothiazole (195 mg, 1 mmol), stir and react for 12 h. Detect the completion of the reaction by TLC, add pure water to quench, extract with DCM, and rotary evaporate the solvent. Purify by silica gel column chromatography, and the elution gradient is PE:EA = 5:1. Obtain intermediate B1-2 as a white solid. Yield: 61.3%; ESI-MS: m / z = 321 [M + H] + .

[0353] Step 2: Synthesis of intermediate B1-3

[0354] Add 5% Pd / C (32 mg, 10%) to intermediate B1-2 (320 mg, 1 mmol), dissolve it in 10 mL of methanol. Pass H2 into this mixture and stir at 40 °C for 12 h. After the reaction is complete, filter the Pd / C solid, and rotary evaporate the filtrate to obtain intermediate B1-3 as a pale yellow solid. Yield: 86.5%; ESI-MS: m / z = 291 [M + H] + .

[0355] Step 3: Synthesis of compound B1

[0356] Weigh the intermediate B1-3 (145 mg, 0.5 mmol), add it to 10 mL of DCM, add DIPEA (129 mg, 1 mmol) under N2 atmosphere, then add acryloyl chloride (45 mg, 0.5 mmol), and then carry out an ice bath reaction for 30 min. Quench with water. After extraction with DCM and rotary evaporation, purify it by silica gel column chromatography, and the elution gradient is PE:EA = 2:1 to obtain compound B1. Yield: 71.2%; ESI-MS: m / z = 345 [M+H] + 。

[0357] The total yield of compound B1 is 37.7%, and the purity is 97.6%.

[0358] Experimental Example 1: In vitro JOSD2 enzyme activity inhibition experiment

[0359] 1. Experimental method:

[0360] (1) Prepare the GST-JOSD2 solution: Use PCR to amplify the JOSD2 gene, double-digest the PGEX-4T-1 empty vector, run agarose gel electrophoresis, and cut and recover the double-digested PGEX-4T-1; then use a homologous recombination kit to ligate JOSD2 and the double-digested PGEX-4T-1 together to obtain the JOSD2 expression vector: PGEX-4T-1-JOSD2, which has a GST tag. Transform the plasmid PGEX-4T-1-GST-JOSD2 into BL21 Escherichia coli, and add IPTG to induce its overnight low-temperature (16 °C) expression of GST-JOSD2 protein during the logarithmic growth phase. After overnight incubation, collect the bacterial cells, centrifuge at 12000 rpm for 2 min, and discard the supernatant; add 1×PBS to resuspend the bacterial cells, use a high-pressure cell disruptor to break Escherichia coli to release the protein at low temperature (4 °C), centrifuge at 12000 rpm for 30 min, and aspirate the supernatant through a 0.45 μM filter membrane. Connect the affinity column containing glutathione-agarose resin to the protein purifier, and the supernatant after passing through the membrane flows through the affinity column by the protein purifier, and GST-JOSD2 will bind to the affinity column. After the binding is completed, elute the GST-JOSD2 protein with reduced glutathione with a final concentration of 10 mM to obtain a solution containing GST-JOSD2, and add glycerol with a final volume concentration of 10-20% for long-term storage at -80 °C.

[0361] (2) Preparation of enzyme activity growth curve: Prepare UB-AMC-buffer containing NaVO3, 10 μM Leupeptin, 1 mM DTT, and 0.1 mg / mL Ovalbumin, and repeat the reaction in a black 96-well plate with a final reaction volume of 100 μL. Dilute GST-JOSD2 in UB-AMC-buffer, set the final concentration gradients to 1 nM, 2 nM, 4 nM, 8 nM, and 16 nM, and at the same time, each well contains the reaction substrate UB-AMC with a final concentration of 800 nM. The reaction is incubated at 37 °C and read every 2 min for 120 min. A substrate group and a UB-AMC-buffer group should be set for each detection. The readings are taken on a microplate reader with an excitation wavelength of 345 nm and an emission wavelength of 445 nm to obtain the enzyme activity growth curve.

[0362] (3) IC 50 (3) IC50 Test: Prepare the stock solutions of compounds A1 - A27 and B1 at 50-fold the final concentration respectively. Then dilute them to working concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.078125 μM respectively. Incubate compounds A1 - A27, B1, and the enzyme in a black 96-well plate with a reaction volume of 50 μL. Add 1 μL of DMSO or the diluted compound to the plate respectively and incubate at 37 °C for 30 min. Repeat the reaction in a black 96-well plate with a final reaction volume of 100 μL. After the incubation, prepare a substrate mixture with a concentration of 1600 nM using UB-AMC-buffer, take 50 μL of the substrate mixture and add it to the 96-well plate, and the final substrate concentration is 800 nM. The reaction is incubated at 37 °C and read every 2 min for 120 min. A substrate group, a buffer group, and a compound fluorescence group should be set for each detection. The readings are taken on a microplate reader with an excitation wavelength of 345 nm and an emission wavelength of 445 nm.

[0363] 2. Data processing: (1) Use the fluorescence value generated by the enzyme cleaving the substrate and the reaction time at each detection point to make an enzyme activity growth curve, and take the slope of the linear growth section as the enzyme activity value.

[0364] (2) The inhibition rate is calculated as follows:

[0365] Inhibition rate (%) = (1 - slope of the compound group ÷ slope of the DMSO group) × 100%

[0366] 3. Data analysis: Use the logarithm value of the concentration as the X-axis and the percentage inhibition rate as the Y-axis, and use the [Inhibitor] vs. normalized response - Variable slope of the analysis software GraphPad Prism 9 to fit the dose - effect curve, so as to obtain the IC50 of each compound on the enzyme activity. 50Value, and the inhibition rate results are shown in Table 1.

[0367] Table 1 Inhibition rate of the compounds of the present invention on enzyme activity (UB-AMC)

[0368]

[0369] As can be seen from Table 1, the compounds of the present invention can achieve potent inhibition of JOSD2 enzyme activity. And further determine the IC 50 value of the compounds of the present invention on the inhibition of JOSD2 enzyme activity in vitro, as shown in Table 2.

[0370] Table 2 IC 50 value of the compounds of the present invention on the inhibition of JOSD2 enzyme activity in vitro

[0371] Compound number <![CDATA[JOSD2(IC 50 , μM)]]> Compound number <![CDATA[JOSD2(IC 50 , μM)]]> A1 0.82 A2 2.87 A3 1.60 A4 8.04 A5 1.09 A6 0.36 A7 0.97 A10 4.77 A11 1.15 A12 1.00 A13 1.38 A15 0.24 A16 0.14 A24 2.22 A25 2.17 A27 0.07

[0372] Experimental Example 2: Proliferation inhibitory activity of the compounds of the present invention against colorectal cancer cell HCT116

[0373] 1. Experimental method: Select HCT116 cells in good growth state. After digestion with trypsin, collect the cells into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the waste liquid and resuspend the cells thoroughly with 1 mL of complete medium. Take 10 μL and use a cell counter to count. Calculate the amount of cell suspension needed according to 2500 cells / 100 μL per well. After thoroughly mixing the suspension with the complete medium, add it to a 96-well plate using a multi-channel pipette and incubate overnight in a 37 °C 5% CO2 incubator.

[0374] After 24 h, administer compounds A1-A3, A5-A7, A11, A15, A24, A25, and A27 respectively. Set 3 wells for each compound concentration. Prepare the compound concentration according to 2 times the highest concentration (final concentration) using the complete medium. Take 100 μL of the compound mixture at the highest concentration into the 96-well plate seeded with cells. Similarly, calculate the amount of compound mixture needed for each compound concentration, and add complete medium to make up to 100 μL. Finally, each well in the 96-well plate contains 200 μL. Incubate in a 37 °C 5% CO2 incubator for 72 h. Discard the culture solution in the 96-well plate, add 100 μL of 10% trichloroacetic acid (TCA) to each well and place it in a 4 °C refrigerator for fixation for 1 h. Discard the TCA, wash it with water, and dry the well plate; add 100 μL of 0.4% SRB staining solution to each well and stain at room temperature for 1 h. Discard the SRB, wash the 96-well plate with 1% glacial acetic acid until there is no red color, and dry the well plate; add 100 μL of 10 mM Tris to each well and shake on a shaker for 20 min; finally, measure the absorbance at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0375] 2. Data processing: The cell survival rate is calculated as follows:

[0376] Cell viability = (Absorbance value of treatment group - Absorbance value of blank group) ÷ (Absorbance value of control group - Absorbance value of blank group)

[0377] × 100%.

[0378] 3. Data analysis: Using the lg value of the concentration as the X-axis and the percentage viability as the Y-axis, a dose-response curve was plotted using the analysis software GraphPad Prism 9. The curve was fitted using the "Inhibitor vs. normalized response--Variable slope" model to obtain the IC 50 value, and the results are shown in Table 3.

[0379] Table 3 Proliferation inhibitory activity of the compound against colorectal cancer in vitro

[0380]

[0381] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A novel covalent inhibitor of JOSD2, characterized in that, Compounds having the structure shown in formula (I), their stereoisomers and pharmaceutically acceptable salt forms: In formula (I), R2 is selected from halogen, -CH3, -N(CH3)2, -CF3, or none; Ring B is selected from any one of the following: Substituted or unsubstituted C6-C8 cycloalkyl, substituted or unsubstituted C6-C8 heterocycloalkyl, substituted or unsubstituted C6-C 10 Bridged cycloalkyl, substituted or unsubstituted C6-C 10 Bridged heterocycloalkyl, substituted or unsubstituted C7-C 12 Spirocycloalkyl and substituted or unsubstituted C7-C 12 Spiroheterocycloalkyl; L2 is selected from NH, or none; X is selected from O, S, NH or NCH3; L1 is selected from one or more of NH, and the like; Ring A is selected from any one of the following: C5-C6 heteroalkyl, C5-C 10 bridged heteroalkyl and C7-C 12 spiro heteroalkyl; R1 is selected from one or more of -CN.

2. The novel JOSD2 covalent inhibitor according to claim 1, wherein The substitution is monosubstituted or disubstituted by R2; the carbon atoms of the C5-C6 heterocycloalkyl, C5-C 10 bridged heterocycloalkyl and C7-C 12 spiro heterocycloalkyl are monosubstituted or polysubstituted by N or O.

3. The novel JOSD2 covalent inhibitor according to claim 1, wherein The ring B is selected from any one of the following: The ring A is selected from any one of the following: m is 0 or 1, and n is 1 or 2.

4. The novel JOSD2 covalent inhibitor according to claim 3, wherein The ring B is selected from any one of the following: The ring A is selected from any one of the following:

5. The novel JOSD2 covalent inhibitor according to claim 3, wherein The novel JOSD2 covalent inhibitor is selected from one of the following compounds:

6. A preparation method of a novel JOSD2 covalent inhibitor according to any one of claims 1-5, characterized in that, It includes the following steps: first, the compound shown in formula (II) is subjected to a reduction or hydrolysis reaction to generate the compound shown in formula (III), and then acid deprotection of Boc and substitution reaction are carried out to obtain it. In the above formula, R3 is -NH2 or -CH2OH; R4 is -NO2, -COOH or -COOCH3; the definitions of R2, L1, L2, X, A and B are as described in claim 1.

7. A pharmaceutical composition, characterized in that, It includes the novel JOSD2 covalent inhibitor described in any one of claims 1-5 or the novel JOSD2 covalent inhibitor prepared by the preparation method described in claim 6 and pharmaceutically acceptable salts or other drugs.

8. The pharmaceutical composition according to claim 7, wherein The other drugs are camptothecin and its derivatives, paclitaxel and its derivatives, gemcitabine and its derivatives, platinum drugs, gefitinib, afatinib or osimertinib.

9. Use of a novel JOSD2 covalent inhibitor described in any one of claims 1-5 or a novel JOSD2 covalent inhibitor prepared by the preparation method described in claim 6 in the preparation of a drug for treating JOSD2-related diseases.

10. The application according to claim 9, wherein The related diseases are colorectal cancer, non-small cell lung cancer, cholangiocarcinoma, hepatocellular carcinoma, thymoma, pancreatic cancer, colorectal cancer, melanoma, glioma, glioblastoma multiforme, ovarian cancer or bladder cancer.

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