Novel compounds inhibiting th17 cell differentiation and uses thereof

Abstract

This invention belongs to the pharmaceutical field, specifically relating to novel compounds that inhibit Th17 cell differentiation and their applications. Specifically, this invention provides various novel compounds, their tautomers, stereoisomers, isotope markers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs, which possess biological activity in inhibiting the differentiation of CD4+ T cells into Th17 cells and can be used for the prevention and / or treatment of Th17 cell-related autoimmune deficiency diseases (especially multiple sclerosis). The various compounds of this invention have novel structures and excellent biological activity, making them ideal candidate drug compounds for the treatment of MS.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to novel compounds that inhibit Th17 cell differentiation and their applications. Background Technology

[0002] Multiple sclerosis (MS) is a complex chronic disease of the central nervous system, belonging to the category of autoimmune diseases. Its characteristics include neurodegeneration, demyelination of the brain and spinal cord, and axonal damage. More than 2 million people worldwide are affected, typically in young adults aged 20-40, with a particularly severe impact on women (approximately 2:1 ratio). Its pathogenesis is multifactorial, involving genetic and environmental risk factors (latitude, vitamin D deficiency, smoking, obesity, Epstein-Barr virus infection) (Marcus R., JAMA. 2022;328(20):2078). MS is characterized by its temporal and spatial prevalence, clinically causing muscle weakness, sensory disturbances, cognitive impairment, and fatigue. It is the most common non-traumatic neurological disease among young people and has become a rapidly growing global health problem.

[0003] There is currently no cure for MS. Although various treatments have been developed and widely used, no new drugs with good safety and efficacy have been developed. First-line acute treatment mainly involves plasma exchange and high-dose glucocorticoids, usually methylprednisolone; during remission, disease-modifying therapies (DMTs) are the main treatments, including immunomodulators and immunosuppressants such as interferon beta, glamer acetate, dimethyl fumarate, fingolimod, and occraizumab. While existing treatments have some efficacy, they suffer from problems such as high drug prices, serious toxic side effects, limited administration methods, and inability to repair damaged neurons. Glamer acetate can only be administered subcutaneously, thus limiting its use; long-term injection can lead to dyspnea, chest tightness, palpitations, and anxiety. Fingolimod can cause macular edema, elevated transaminases, and an increased risk of infection (Imane Boutitah-Benyaich et al., Sig Transduct Target Ther 10, 324 (2025)). Progressive multifocal leukoencephalopathy (PML) has been reported in patients with severe chronic lymphocytopenia treated with dimethyl fumarate for MS (Gwendoline Montes Diaz et al., Autoimmunity Reviews, 2018, 17(12): p.1240-1250). Therefore, there is an urgent need to develop safe and effective drugs to alleviate the suffering of MS patients. The experimental autoimmune encephalomyelitis (EAE) mouse model, due to its similar clinical, pathological, and histological characteristics to multiple sclerosis, has been widely used in multiple sclerosis drug discovery.

[0004] T cells have long been considered the most important players in the pathogenesis of MS. Effector memory CD4 T cells can differentiate into Th1 and Th17 cells, which secrete pro-inflammatory cytokines, including interferon-γ (IFN-γ) or interleukin-17 (IL-17) and interleukin-22 (IL-22), respectively. Studies have found that the expression level of IL-17 is significantly increased in the cerebrospinal fluid and peripheral blood of MS patients, which can induce the production of reactive oxygen species that attack blood-brain barrier endothelial cells, causing inflammatory cell invasion and leading to continuous disease progression (Shi Y et al., Front. Immunol. 13:932152). Helper T cells (Th) expressing IL-17 play a key role in MS and are considered important therapeutic targets for neuroinflammatory diseases such as MS. Therefore, developing drugs that target Th17 differentiation-related signals is expected to provide new treatment strategies for MS.

[0005] JQ1 is a small thiazolidinedione molecule with strong affinity, capable of binding to and inhibiting target proteins of interest within bromine domain proteins. JQ1 is commonly used in cancer therapy, exhibiting activity against hematologic malignancies, solid tumors, lung adenocarcinoma, neuroblastoma, and medullary blastoma. While potent and highly selective, it faces limitations in drug development, such as undesirable pharmacokinetic properties (short half-life) and potential off-target effects and toxicity. Therefore, its primary value lies in providing crucial lead compound templates for subsequent drug design. Currently, JQ1, as an experimental compound still under investigation, has not yet been approved for the treatment of multiple sclerosis, but its target and mechanism of action have shown significant therapeutic potential in preclinical studies.

[0006] In conclusion, there is still a need to develop novel inhibitors of Th17 cell differentiation in order to provide more candidate drugs for the treatment of multiple sclerosis. Summary of the Invention

[0007] Based on existing technologies, this invention has conducted a series of optimization studies on the structure of the MS lead compound JQ1, aiming to obtain novel compound molecules with superior biological activity in inhibiting the differentiation of CD4+ T cells into Th17 cells, with the expectation of serving as candidate drug compounds for the treatment of MS. Specifically, through structural modification and in vitro activity screening experiments, JQ1 and 31 modified novel compounds were evaluated, thereby discovering several novel compounds (especially JQ24, JQ30, and JQ31), which exhibit significantly superior activity in inhibiting the differentiation of CD4+ T cells into Th17 cells.

[0008] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In one embodiment, the present invention provides compounds, their tautomers, stereoisomers, isotope labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs as shown below: , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0009] In one embodiment, the present invention specifically provides the following compounds, their tautomers, stereoisomers, isotope labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs: , , .

[0010] In one embodiment, the present invention more specifically provides the following compounds, their tautomers, stereoisomers, isotope labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs: .

[0011] In one embodiment, the compounds provided by the present invention are selected from: (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-(3-(4-methylpiperazin-1-yl)propyl)acetamide (JQ2); 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]aza-4-yl)acetic acid (JQ6); tert-butyl 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetate (JQ7); 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)tert-butyl acetate (JQ8); 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)acetamide (JQ9); (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methanesulfonyl)methyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine (JQ10); ((6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-6-yl)methyl(methyl)sulfonylimine (JQ11); 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetamide (JQ12); S-tert-butyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl) ethoxylate (JQ13); 4-((6S)-6-(2-(tert-butoxy)-2-oxoethyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)pyridine 1-oxide (JQ14); 2-((6S)-4-(4-chlorophenyl)-3-cyclopropyl-2,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (JQ15); 2-((6S)-2,3,9-trimethyl-4-(pyridin-4-yl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (JQ16); tert-butyl(S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoate (JQ17); (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enamide (JQ18); 2-((3S)-5-(4-chlorophenyl)-1-(1-cyanoethyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)tert-butyl acetate (JQ19); 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-1-(isoxazolidin-2-yl)ethane-1-one (JQ20); 2-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-5-ethyl-1,3,4-oxadiazole (JQ21); 5-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-3-ethyl-1,2,4-oxadiazole (JQ22); (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-dimethylbut-2-enamide (JQ23); (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-diethylbut-2-enamide (JQ24); (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-N-ethylbut-2-enamide (JQ25); (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-morpholinobut-2-en-1-one (JQ26); (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-(isoxazolidin-2-yl)but-2-en-1-one (JQ27); tert-butyl(S)-2-[5-(4-chlorophenyl)-6,7-dimethyl-2-(1-methyl-1H-pyrazol-5-yl)-3H-thieno[2,3-e][1,4]diaza-3-yl]acetate (JQ28); (S,E)-2-[3-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]propyl-1-en-1-yl]-5-ethyl-1,3,4-oxadiazole (JQ29); (E)-1-(3,3-difluoropyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (JQ30); (E)-1-(pyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (JQ31); or (E)-1-(azacyclobut-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (JQ32).

[0012] In one embodiment, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound described herein, its tautomer, stereoisomer, isotope label, hydrate, solvate, pharmaceutically acceptable salt, or prodrug.

[0013] In one embodiment, the pharmaceutical composition of the present invention is a Th17 cell differentiation inhibitor.

[0014] In one embodiment, the pharmaceutical composition of the present invention is a therapeutic or preventative agent for multiple sclerosis.

[0015] In another embodiment, the present invention provides a kit for inhibiting Th17 cell differentiation, comprising the compound described herein, its tautomers, stereoisomers, isotope markers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs.

[0016] In one embodiment, the Th17 cells of the present invention are derived from humans or rodents.

[0017] In another embodiment, the present invention provides the use of the compounds of the present invention, their tautomers, stereoisomers, isotope markers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, or pharmaceutical compositions of the present invention in the preparation of a medicament for inhibiting Th17 cell differentiation.

[0018] In yet another embodiment, the present invention provides the use of the compounds described herein, their tautomers, stereoisomers, isotope labels, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, or pharmaceutical compositions described herein in the preparation of medicaments for the treatment or prevention of multiple sclerosis.

[0019] The beneficial technical effects of the present invention include at least the following: This invention provides a variety of novel compounds, their tautomers, stereoisomers, isotope markers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs, which possess biological activity in inhibiting the differentiation of CD4+ T cells into Th17 cells and can be used for the prevention and / or treatment of Th17 cell-related autoimmune deficiency diseases (especially multiple sclerosis). The compounds of this invention have novel structures and excellent biological activity, making them ideal candidate drug compounds for the treatment of MS. Attached Figure Description

[0020] Figure 1 This shows a statistical graph of the relative differentiation values ​​of Th17 cells in the presence of each of the 32 compounds in Example 1.

[0021] Figure 2 The IC50 values ​​of compounds JQ1, JQ24, JQ30, and JQ31 are shown to inhibit Th17 differentiation in mice.

[0022] Figure 3 The IC50 values ​​of compounds JQ1, JQ24, JQ30, and JQ31 are shown to inhibit human Th17 differentiation. Detailed Implementation

[0023] Example 1: Preparation of the compound This application uses JQ1 as a lead compound template, and through continuous optimization of the drug structure, 32 candidate compounds were obtained in sequence.

[0024] (1) Compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (JQ1, molecular formula C 23 H 25 Exemplary preparation of ClN4O2S (molecular weight 456.99)

[0025] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (1.0 g, 2.49 mmol) and 1,1-di-tert-butoxy-N,N-dimethylmethylamine (2.53 g, 12.45 mmol) were mixed in toluene (20 mL) and stirred at 110 °C for 5 hours. The reaction mixture was extracted with ethyl acetate (100 mL × 3), washed with water (100 mL), and the solvent was removed under reduced pressure. The solution was purified by column chromatography to obtain (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (0.50 g, 1.10 mmol, yield: 44.8%). LC-MS (ESI): m / z 457.1 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6)δ 7.51 (d, J = 8.7 Hz, 2 H), 7.44 (d, J = 8.5 Hz, 2 H), 4.43 (dd, J = 8.1, 6.4 Hz, 1H), 3.40 – 3.34 (m, 1 H), 3.30-3.27 (m, 1 H), 2.61 (s, 3 H), 2.42 (s, 3 H), 1.64 (s, 3 H), 1.43 (s, 9 H). (2) Compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-(3-(4-methylpiperazin-1-yl)propyl)acetamide (JQ2, molecular formula C 27 H 34 Exemplary preparation of ClN7OS (molecular weight 540.13)

[0026] HATU (30 mg, 0.08 mmol) and DIEA (32 mg, 0.25 mmol) were added to a DMF solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (20 mg, 0.05 mmol) and 3-(4-methylpiperazin-1-yl)propyl-1-amine (16 mg, 0.10 mmol), and the mixture was stirred overnight at room temperature. (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-(3-(4-methylpiperazin-1-yl)propyl)acetamide (10 mg, 0.02 mmol, yield: 40.0%, white solid) was purified by preparative liquid chromatography. LC-MS (ESI): m / z 540.2 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 8.20 (t, J = 5.5 Hz, 1 H), 7.50 (d, J = 8.7 Hz, 2 H), 7.43 (d, J =8.5 Hz, 2H), 4.50 (t, J = 7.1 Hz, 1 H), 3.22 (dd, J = 7.0, 4.3 Hz, 2 H), 3.12 (dd, J =11.6, 6.0 Hz, 2 H), 2.69 – 2.66 (m, 1 H), 2.60 (s, 3 H), 2.48 – 2.31 (m, 12 H), 2.23 (s, 3 H), 1.63 (s, 3 H), 1.62 – 1.55 (m, 2 H). (3) Compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiophene[3,2-F][1,2,4]triazol[4,3-A][1,4]diaza-6-yl)acetamide (JQ3, CAS No.: 1446144-04-2)

[0027] Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., specification: 25mg / bottle.

[0028] (4) Compound (R)-(-)2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-F][1,2,4]triazolo[4,3-A][1,4]diazacycloheptane-6-yl)tert-butyl acetate (JQ4, CAS No.: 1268524-71-5)

[0029] Purchased from Shanghai BIDE Pharmaceutical Technology Co., Ltd., specification: 50mg / bottle.

[0030] (5) Compound (S)-2-(6-(4-chlorophenyl)-1-methyl-4H-benzo[C]isoxazolo[4,5-E]azacycloheptane-4-yl)acetamide (JQ5, CAS No.: 1380087-89-7)

[0031] Purchased from Shanghai BIDE Pharmaceutical Technology Co., Ltd., specification: 50mg / bottle.

[0032] (6) Compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]aza-4-yl)acetic acid (JQ6, molecular formula C 24 H 23 Exemplary preparation of ClN2O3 (molecular weight 422.91)

[0033] Step 1: Synthesis of compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]aza-4-yl)acetic acid (intermediate product) 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]aza-4-yl)acetamide (30 mg, 0.08 mmol) was dissolved in HCl (0.5 mL, 3.00 mmol), H2O (0.5 mL), and 1,4-dioxane (3 mL), and the mixture was stirred at 70 °C for 3 hours. H2O and DCM were added to the reaction mixture, the organic phase was separated, the organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The solution was purified by silica gel column chromatography to obtain 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]aza-4-yl)acetic acid (10 mg, 0.03 mmol, yield: 33.24%, white solid). LC-MS (ESI): m / z 367.1 [M + H] + Step 2: Synthesis of tert-butyl acetate of compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]aza-4-yl)acetate 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]azaphosphon-4-yl)acetic acid (8 mg, 0.02 mmol) and 1,1-di-tert-butoxy-N,N-dimethylmethylamine (30 mg, 0.15 mmol) were dissolved in toluene (2 mL) and stirred for 3 hours. Ethyl acetate and H₂O were added to the reaction mixture, the organic phase was separated, the organic layer was washed with saturated brine, dried over anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The product was purified by preparative liquid chromatography to give tert-butyl 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazo[4,5-e]azaphosphon-4-yl)acetic acid (2.5 mg, 0.006 mmol, yield: 30%, white solid). LC-MS (ESI): m / z 423.1 [M + H] + . 1 H NMR (400 MHz, CDCl3) δ 7.66 –7.59 (m, 2 H), 7.43 (d, J = 7.7 Hz, 1 H), 7.39 – 7.29 (m, 5 H), 4.46 (s, 1 H), 3.51 – 3.30 (m, 2 H), 2.58 (s, 3 H), 1.53 (s, 9 H). (7) Compound tert-butyl 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetate (JQ7, molecular formula C 25 H 27 Exemplary preparation of ClN4O2S (molecular weight 483.03)

[0034] Step 1: Synthesis of compound 2-cyclopropyl-N-methoxy-N-methylacetamide Cyclopropionic acid (10 g, 99.9 mmol) and SOCl2 (23.76 g, 199.70 mmol) were added to anhydrous DCM (50 mL), followed by a catalytic amount of DMF (100 μL). The mixture was stirred for 2 hours, and N,O-dimethylhydroxylamine hydrochloride (10.72 g, 109.80 mmol) was added, followed by dropwise addition of DIEA (38.73 g, 299.60 mmol). The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with H2O (200 mL), 10% NaOH aqueous solution (3 × 150 mL), saturated NaHCO3 aqueous solution (200 mL), H2O (200 mL), and saturated brine (200 mL). The organic layer was separated and dried over anhydrous MgSO4. The solvent was removed by filtration and concentration under reduced pressure to obtain 2-cyclopropyl-N-methoxy-N-methylacetamide (10.00 g, yield 69.9%, yellow oil). LC-MS (ESI): m / z 144.2 [M + H] + . Step 2: Synthesis of compound 1-cyclopropylacetone 2-Cyclopropyl-N-methoxy-N-methylacetamide (2.00 g, 13.97 mmol) was dissolved in THF (25 mL). Then, with stirring at 0 °C, methyl magnesium bromide (13.9 mL, 27.93 mmol, 2 M in THF) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with NH4Cl (aq, 100 mL), extracted with ethyl acetate (2 × 100 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 1-cyclopropylacetone (0.60 g, 43.8% yield, as a yellow oil), which was used directly in the next reaction without further purification. LC-MS (ESI): m / z 99.1 [M + H] + . Step 3: Synthesis of compound (2-amino-5-cyclopropyl-4-methylthiophene-3-yl)(4-chlorophenyl) methyl ketone A mixture of sulfur (62 mg, 1.95 mmol), 3-(4-chlorobenzene)-3-oxypropionitrile (350 mg, 1.95 mmol), 1-cyclopropylprop-2-one (191 mg, 1.95 mmol), and morpholine (170 mg, 1.95 mmol) in EtOH (10 mL) was heated to 70 °C and reacted for 12 hours. The reaction mixture was then cooled to room temperature and poured into brine (100 mL). The aqueous layer was extracted with ethyl acetate (3 × 50 mL), and the combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was performed by column chromatography (combflash RF system, 40 g silica gel, gradient 0-100% ethyl acetate / n-hexane) to give (2-amino-5-cyclopropyl-4-methylthiophene-3-yl)(4-chlorophenyl) methyl ketone (300 mg, 52.76% yield, yellow solid). LC-MS (ESI): m / z 292.1 [M + H] + . Step 4: Synthesis of tert-butyl(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-5-cyclopropyl-4-methylthiophene-2-yl)amino)-4-oxobutyrate HCTU (851 mg, 2.06 mmol) and DIEA (532 mg, 4.11 mmol) were added sequentially to a DMF (10 mL) solution of (R)-2-(((9H-fluorene-9-methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutyric acid (846 mg, 2.06 mmol). The mixture was then stirred at room temperature for 5 minutes. Solid (2-amino-5-cyclopropyl-4-methylthiophene-3-yl)(4-chlorophenyl) ketone (300 mg, 1.03 mmol) was added. After stirring at room temperature for 16 hours, ethyl acetate (100 mL) and brine (100 mL) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purified by column chromatography (Combiflash RF, 40 g silica gel, gradient 0-100% ethyl acetate / n-hexane) to give tert-butyl(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-5-cyclopropyl-4-methylthiophene-2-yl)amino)-4-oxobutyrate (0.50 g, yield 70.9%). LC-MS (ESI): m / z 629.1 [M + H - 56] + . Step 5: Synthesis of compound (S)-3-amino-4-((3-(4-chlorobenzoyl)-5-cyclopropyl-4-methylthiophen-2-yl)amino)-4-oxobutyrate Piperidine (2 mL) was added to a 10 mL DMF solution of tert-butyl(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-5-cyclopropyl-4-methylthiophen-2-yl)amino)-4-oxobutyrate (200 mg, 0.29 mmol), and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system, the organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain tert-butyl(S)-3-amino-4-((3-(4-chlorobenzoyl)-5-cyclopropyl-4-methylthiophen-2-yl)amino)-4-oxobutyrate (110 mg, yield 81.4%). LC-MS (ESI): m / z 463.3 [M + H] + . Step Six: Synthesis of tert-butyl(R)-2-(5-(4-chlorophenyl)-7-cyclopropyl-6-methyl-2-oxo-2,3-dihydro-1H-thiopheno[2,3-e][1,4]diaza-3-yl)acetate To a solution of tert-butyl(S)-3-amino-4-((3-(4-chlorobenzoyl)-5-cyclopropyl-4-methylthiophene-2-yl)amino)-4-oxobutyrate (110 mg, 0.24 mmol) in 10 mL of EtOH, 2 mL of AcOH was added, and the mixture was stirred at 80 °C for 16 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction mixture, the organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give tert-butyl(R)-2-(5-(4-chlorophenyl)-7-cyclopropyl-6-methyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate (100 mg, yield 94.6%). LC-MS (ESI): m / z 445.2 [M + H] + . Step 7: Synthesis of tert-butyl 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetate tBuOK (38 mg, 0.34 mmol) was added to a THF (15 mL) solution of tert-butyl(R)-2-(5-(4-chlorophenyl)-7-cyclopropyl-6-methyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate (100 mg, 0.23 mmol). The temperature was raised to -10 °C, and the mixture was stirred for 30 minutes. The mixture was then cooled to -78 °C, and diethylphosphoryl chloride (91 mg, 0.34 mmol) was added. The temperature was raised to -10 °C, and the mixture was stirred for 45 minutes. Acetylhydrazine (42 mg, 0.56 mmol) was added to the reaction mixture, and the mixture was stirred at 23 °C for 1 hour. Butanol (2.25 mL) was added, and the mixture was heated to 90 °C. After reacting for 1 hour, the solvent was removed under reduced pressure. Purification by column chromatography (40 g silica gel gradient 0-100% ea-hexane) yielded tert-butyl 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetate (90 mg, 82.9% yield, white solid). LC-MS (ESI): m / z 427.1 [M + H - 56] + . 1 HNMR (400 MHz, DMSO- d 6) δ: 7.54 - 7.50 (m, 2 H), 7.46 - 7.43 (m, 2 H), 4.43 (d, J = 8.2, 6.3 Hz, 1 H), 3.41 - 3.24 (m, 2 H), 2.59 (s, 3 H), 2.14 (t, J = 8.3 Hz, 1 H), 1.75 (s, 3 H), 1.43 (s, 9 H), 1.14 - 1.06 (m, 2 H), 0.87 - 0.73 (m, 1 H), 0.70 - 0.66 (m, 1 H). (8) Compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)tert-butyl acetate (JQ8, molecular formula C 24 H 25 Exemplary preparation of ClN4O3S (molecular weight 485.00)

[0035] Step 1: Synthesis of compound (2-amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-yl)(4-chlorophenyl) methyl ketone Sulfur (0.36 g, 11.136 mmol) was added to a solution of 3-(4-chlorobenzene)-3-oxypropionitrile (2 g, 11.13 mmol), tetrahydropyran-4-one (1.11 g, 11.13 mmol), and morpholine (0.97 g, 11.13 mmol) in EtOH (20 mL). The mixture was heated to 70 °C at room temperature and reacted for 12 hours. The reaction mixture was then cooled to room temperature and poured into brine (100 mL). The aqueous layer was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography (Combi flash RF system, 40 g silica gel, gradient 0-100% EA-PE) yielded (2-amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-yl)(4-chlorophenyl) methyl ketone (2.2 g, 67.2% yield, yellow solid). LC-MS (ESI): m / z 294.2 [M + H] + . Step 2: Synthesis of compound (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)amino)-4-oxobutyrate HCTU (845 mg, 2.04 mmol) and DIEA (528 mg, 4.08 mmol) were added sequentially to a DMF (15 mL) solution of (N-(((9H-fluorene-9-yl)methoxy)carbonyl)-D-aspartic acid-4-tert-butyl ester (840 mg, 2.04 mmol). The reaction was then stirred at room temperature for 5 minutes, and compound (2-amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-yl)(4-chlorophenyl) methyl ketone (300 mg, 1.02 mmol) was added. The reaction was stirred at room temperature for 16 hours, and ethyl acetate (50 mL) and brine (50 mL) were added. The two phases were separated, and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The solution was then subjected to column chromatography (Combi flash). RF, 40 g silica gel, gradient 0-100% ethyl acetate-hexane) purification yielded (S)-tert-butyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)amino)-4-oxobutyrate (0.50 g, yield 71.2%). LC-MS (ESI): m / z 687.1 [M + H] + . Step 3: Synthesis of compound (S)-tert-butyl-3-amino-4-((3-(4-chlorobenzoyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)amino)-4-oxobutyrate Piperidine (2 mL) was added to a DMF (10 mL) solution of compound (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)amino)-4-oxobutyrate (500 mg, 0.72 mmol), and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system to separate the organic phase. The organic phase was washed with saturated NaCl solution (50 mL × 3), dried over Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (Combi flash RF, 40 g silica gel, gradient 0-100% ethyl acetate / n-hexane) to give (S)-tert-butyl-3-amino-4-((3-(4-chlorobenzoyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)amino)-4-oxobutyrate (200 mg, yield 59.1%, yellow solid). LC-MS (ESI): m / z 465.1 [M + H] + . Step 4: Synthesis of compound (S)-tert-butyl 2-(5-(4-chlorophenyl)-2-oxo-1,2,3,6,7,9-hexahydropyrano[4',3':4,5]thiopheno[2,3-e][1,4]diaza-3-yl)acetate Add 5 mL of AcOH to a solution of (S)-tert-butyl-3-amino-4-((3-(4-chlorobenzoyl)-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)amino)-4-oxobutyrate (200 mg, 0.43 mmol) in EtOH (20 mL). Stir at 80 °C for 3 hours. Concentrate the reaction mixture under reduced pressure to remove the solvent. Dilute with water (100 mL) and extract with ethyl acetate (5 mL × 2). The combined organic phases were washed with saturated NaCl solution (50 mL), dried over Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (Combiflash RF, 40 g silica gel, gradient 0-100% ethyl acetate-hexane) to give compound (S)-tert-butyl-2-(5-(4-chlorophenyl)-2-oxo-1,2,3,6,7,9-hexahydropyrano[4',3':4,5]thieno[2,3-e][1,4]diaza-3-yl)acetate (150 mg, yield: 78.0%). LC-MS (ESI): m / z 447.2 [M + H] + . Step 5: Synthesis of tert-butyl 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)acetate At -78°C, tBuOK (36.15 mg, 0.32 mmol) was added to a THF (15 mL) solution of (S)-tert-butyl-2-(5-(4-chlorophenyl)-2-oxo-1,2,3,6,7,9-hexahydropyrano[4',3':4,5]thieno[2,3-e][1,4]diazepine-3-yl)acetate (120 mg, 0.26 mmol). The reaction temperature was raised to -10°C, and the mixture was stirred for 30 minutes. Then, it was cooled to -78°C, and diethylphosphoryl chloride (87 mg, 0.32 mmol) was added to the reaction system. The reaction system was then heated to -10°C and reacted for 45 minutes. Acetylhydrazine (30 mg, 0.40 mmol) was added to the reaction system. After stirring the reaction system at 23°C for 1 hour, butanol (2.25 mL) was added, and the mixture was heated to 90°C and reacted for 1 hour. The solvent was then removed under reduced pressure. Dilute with H₂O (100 mL) and extract with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Purified by column chromatography (Combiflash system, 40 g silica gel, gradient 0-100% ethyl acetate-hexane) to give 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-4-yl)tert-butyl acetate (100 mg, yield 76.8%). LC-MS (ESI): m / z 429.1 [M + H -56] + . 1 HNMR (400 MHz, DMSO-d6) δ: 7.53 (d, J = 8.7 Hz, 2 H), 7.46 (d, J = 8.6 Hz, 2 H), 4.97 – 4.70 (m, 2 H), 4.47 (d, J = 8.1 Hz, 1 H), 3.90 – 3.87 (m, 1 H), 3.60 – 3.57 (m, 1 H), 3.45 – 3.36 (m, 1 H), 3.29 (d, J = 8.1 Hz, 1 H), 2.62 (s, 3H), 2.07 – 1.86 (m, 2 H), 1.43 (s, 9 H). (9) Compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)acetamide (JQ9, molecular formula C 20 H 18 Exemplary preparation of ClN5O2S (molecular weight 427.91)

[0036] Step 1: Synthesis of compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)acetic acid TFA (5 mL) was added to a DCM (2 mL) solution of tert-butyl acetate (60 mg, 0.12 mmol) containing 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-4-yl)acetate, and the mixture was stirred at room temperature for 6 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system, the organic phase was separated, and the solution was rinsed with saturated NaCl solution (50 mL). 3) Wash, dry with Na2SO4, filter, and concentrate under reduced pressure to obtain 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazamine-4-yl)acetic acid (50 mg, yield 94.2%). LC-MS (ESI): m / z 429.0 [M + H] + . Step 2: Synthesis of compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-4-yl)acetamide To a DMF (5 mL) solution of compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazoxy-4-yl)acetic acid (50 mg, 0.12 mmol), EDCI (67 mg, 0.35 mmol), HOBt (47 mg, 0.35 mmol), DIEA (75 mg, 0.58 mmol), and ammonium carbonate (112 mg, 1.17 mmol) were added, and the mixture was stirred at 40 °C for 18 h. The reaction mixture was diluted with ethyl acetate and water. The organic phase was separated, washed with saturated NaCl solution, and concentrated under reduced pressure. The crude product was purified by high-performance preparative liquid chromatography (0.1% TFA conditions), and after drying, yielded compound 2-((4S)-6-(4-chlorophenyl)-1-methyl-7,10-dihydro-4H,8H-pyrano[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-4-yl)acetamide (28 mg, yield 56.1%, white solid). LC-MS (ESI): m / z 428.1 [M + H] + . 1 HNMR (400 MHz, DMSO-d6) δ: 7.65 (s, 1 H), 7.60 -7.50 (m, 2 H), 7.50 - 7.41 (m, 2 H), 6.98 (s, 1 H), 4.96 - 4.70 (m, 2 H), 4.54 - 4.50 (m, 1 H), 3.90 - 3.87 (m, 1 H), 3.61- 3.58 (m, 1 H), 3.24 (d, J = 7.0 Hz, 2 H), 2.61 (s, 3 H), 2.10 - 1.84 (m, 2 H). (10) Compound (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methanesulfonyl)methyl)-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazolium (JQ10, molecular formula C 19 H 19 Exemplary preparation of ClN4O2S2 (molecular weight 434.96)

[0037] Step 1: Synthesis of compound (9H-fluorene-9-yl)methyl(R)-(1-((3-(4-chlorobenzoyl)-4,5-dimethylthiophene-2-yl)amino)-3-(methylthio)-1-oxopropane-2-yl)carbamate To a DMF (20 mL) solution of compound (2-amino-4,5-dimethylthiophene-3-yl)(4-chlorophenyl) ketone (1.00 g, 3.76 mmol, purchased from Leyan), N-(((9H-fluorene-9-yl)methoxy)carbonyl)-S-methyl-L-cysteine ​​(2.60 g, 7.52 mmol), HCTU (3.10 g, 7.52 mmol), and DIEA (1.91 g, 15.05 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (100 mL). The organic layer was separated, washed with saturated NaCl solution (100 mL), and concentrated under reduced pressure. Purified by silica gel column chromatography (n-hexane / ethyl acetate = 3:1) to (9H-fluorene-9-yl)methyl(R)-(1-((3-(4-chlorobenzoyl)-4,5-dimethylthiophene-2-yl)amino)-3-(methylthio)-1-oxopropane-2-yl)carbamate (1.50 g, 2.47 mmol, 65%, yellow oil). LC-MS: [M + H] + 606.2 Step 2: Synthesis of compound (R)-2-amino-N-(3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)-3-(methylthio)propionamide Diethylamine (0.3 g, 4.79 mmol) and (9H-fluorene-9-yl)methyl(R)-(1-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-3-(methylthio)-1-oxopropane-2-yl)carbamate (1.40 g, 2.39 mmol) were added to DCM (20 mL) and stirred at room temperature for 3 hours. The solvent was removed under reduced pressure, and the solution was purified by preparative liquid chromatography-mass spectrometry (150 g SepaFlash® C18 flash column, eluent 0–100% FA in H2O / CAN @ 60 mL / min) to obtain (R)-2-amino-N-(3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)-3-(methylthio)propionamide (500 mg, 1.30 mmol, 54%, yellow solid). LC-MS: [M + H] + 383.1 Step 3: Synthesis of compound (R)-5-(4-chlorophenyl)-6,7-dimethyl-3-((methylthio)methyl)-1,3-dihydro-2H-thiopheno[2,3-e][1,4]diazamine-2-one Acetic acid (75 mg, 1.25 mmol) was added to a solution of (R)-2-amino-N-(3-(4-chlorobenzoyl)-4,5-dimethylthiophene-2-yl)-3-(methylthio)propionamide (480 mg, 1.25 mmol) in 20 mL of EtOH. The mixture was stirred at 85 °C for 30 min and concentrated under reduced pressure. Purification by silica gel column chromatography (n-hexane / ethyl acetate = 1:1) yielded compound (R)-5-(4-chlorophenyl)-6,7-dimethyl-3-((methylthio)methyl)-1,3-dihydro-2H-thieno[2,3-e][1,4]diazide-2-one (400 mg, 1.09 mmol, 87%, yellow solid). LC-MS: [M + H] + 365.0 Step 4: Synthesis of compound (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methylthio)methyl)-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazolium To a THF (2 mL) solution of compound (R)-5-(4-chlorophenyl)-6,7-dimethyl-3-((methylthio)methyl)-1,3-dihydro-2H-thieno[2,3-e][1,4]diazepine-2-one (170 mg, 0.46 mmol), tBuOK (57 mg, 0.51 mmol) was added, and the mixture was stirred at -78 °C for 30 min. Diethylphosphoryl chloride (150 mg, 0.55 mmol) was added, and the mixture was stirred at -78 °C for 1 h. Then, hydrazine acetate (51 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 1 h. Finally, n-butanol (2 mL) was added to the reaction mixture, and the mixture was stirred at 90 °C for 1 h and concentrated under reduced pressure. The residues were purified by preparative liquid chromatography-mass spectrometry (C18 (ISCO®) purification column; 60 g SepaFlash® C18 flash column, eluent 0–100% FA in H2O / ACN @ 60 mL / min) to obtain (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methylthio)methyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazolium (60 mg, 0.14 mmol, 31%, yellow solid). LC-MS: [M + H] + 403.0 Step 5: Synthesis of compound (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methanesulfonyl)methyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazolium To a DCM (1 mL) solution of (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methylthio)methyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine (35 mg, 87 μmol, batch number: N24076-012-P), m-chloroperoxybenzoic acid (35 mg, 0.17 mmol) was added and stirred at room temperature for 2 hours. (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methanesulfonyl)methyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazolium (25 mg, 57 μmol, 65%, white solid) was purified by preparative liquid chromatography-mass spectrometry (C18 (ISCO®); 60 g SepaFlash® C18 flash column, eluent 0–100% FA in H2O / ACN @ 60 mL / min). LC-MS: [M + H] + : 435.0. 1 H NMR (400 MHz, DMSO-d6) δ 7.55 – 7.45 (m, 4 H), 4.69 – 4.60 (m, 1 H), 4.48 – 4.39 (m, 1 H), 4.12 – 4.02 (m, 1 H), 3.25 (s, 3 H), 2.62 (s, 3 H), 2.45 – 2.38 (m, 3H), 1.69 – 1.58 (m, 3H). (11) Compound ((6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-6-yl)methyl(methyl)sulfonylimine (JQ11, molecular formula C 19 H 20 Exemplary preparation of ClN5OS2 (molecular weight 433.97)

[0038] Step 1: Synthesis of compound ((6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-6-yl)methyl(methyl)sulfonylimide To a solution of compound (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-((methylthio)methyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine (15 mg, 37 μmol, batch number: N24076-012-P) in EtOH (1 mL), (diacetoxy)iodobenzene (36 mg, 0.11 mmol) and ammonium acetate (11 mg, 0.14 mmol) were added, and the mixture was stirred at room temperature for 2 hours. (6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-6-yl)methyl(methyl)sulfonylimine (12 mg, 29 μmol, 77%, white solid) was purified by preparative liquid chromatography (C18 (ISCO®); 60 g SepaFlash® C18, eluent 0–100% FA in H2O / ACN @ 60 mL / min) using LC-MS: [M + H] + : 434.1. 1 H NMR (400 MHz, DMSO- d 6) δ 7.55 – 7.44 (m, 4 H), 4.72 – 4.56 (m, 1 H), 4.42 – 4.22 (m, 1 H), 4.16 – 3.97 (m, 1 H), 3.97 – 3.77 (m, 1 H), 3.19 – 3.10 (m, 3 H), 2.66 – 2.59 (m, 3 H), 2.45 – 2.37 (m, 3 H), 1.63 (s, 3 H). (12) Compound 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetamide (JQ12, molecular formula C 21 H 20 Exemplary preparation of ClN5OS (molecular weight 425.94)

[0039] Step 1: Synthesis of compound 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid TFA (2 mL) was added to a 2 mL solution of 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl)acetate tert-butyl acetate (45 mg, 0.09 mmol), and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system. The organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (39 mg, yield 98.1%). LC-MS (ESI): m / z 427.2 [M + H] + . Step 2: Synthesis of compound 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl)acetamide EDCl (52.54 mg, 0.27 mmol), HOBt (37.03 mg, 0.27 mmol), DIEA (59.04 mg, 0.46 mmol), and ammonium carbonate (87.78 mg, 0.91 mmol) were added to a DMF (5 mL) solution of 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl)acetic acid. The mixture was stirred at 40 °C for 18 hours. The reaction mixture was diluted with ethyl acetate and water. The organic phase was separated, washed with saturated NaCl solution, combined, and concentrated under reduced pressure. The crude product was purified by reversed-phase high-performance liquid chromatography (0.1% TFA conditions) and dried to give 2-((6S)-4-(4-chlorophenyl)-2-cyclopropyl-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-6-yl)acetamide (25 mg, yield 64.2%, white solid). LC-MS (ESI): m / z 426.1 [M +H] + . 1 HNMR (400 MHz, DMSO- d 6) δ: 7.66 (s, 1 H), 7.55 - 7.49 (m, 2 H), 7.48 - 7.41 (m, 2 H), 6.98 (s, 1 H), 4.48 (t,J = 7.0 Hz, 1 H), 3.25 - 3.20 (m, 2 H), 2.58 (s, 3 H), 2.18 - 2.07 (m, 1 H), 1.75 (s, 3 H), 1.17- 1.03 (m, 2 H), 0.88 - 0.75 (m, 1 H), 0.70– 0.67 (m, 1 H). (13) Compound S-tert-butyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl) ethoxylate (JQ13, molecular formula C 23 H 25 Exemplary preparation of ClN4OS2 (molecular weight 473.05) Step 1: Synthesis of compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid

[0040] TFA (4 mL, 0.65 mmol) was added to a DCM solution of tert-butyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazolo-6-yl)acetate (300 mg, 0.65 mmol), and the mixture was stirred at 0 °C for 30 min. The reaction mixture was then stirred overnight at room temperature. The mixture was concentrated under reduced pressure to give 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazolo-6-yl)acetic acid (260 mg, 0.64 mmol, 98%, yellow solid). LC-MS: [M + H] + 401.0 Step 2: Synthesis of compound S-tert-butyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl) ethoxylate HATU (128 mg, 0.33 mmol), DIEA (72 mg, 0.56 mmol), and tert-butyl mercaptan (22 mg, 0.24 mmol, purchased from Aladdin) were added to a DMF (2 mL) solution of 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl)acetic acid (90 mg, 0.22 mmol), and stirred at room temperature for 2 hours. S-tert-butyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl) ethoxylate (40 mg, 85 μmol, 37%, white solid) was purified by preparative liquid chromatography (C18 (ISCO®) 60 g SepaFlash® C18, eluent 0–100% FA in H2O / ACN @ 60 mL / min) using H2O / ACN. LC-MS: [M + H] + : 473.1. 1 H NMR (400 MHz, DMSO- d 6) δ 7.53 – 7.40 (m, 4 H), 4.57 – 4.51 (m, 1 H), 3.65 – 3.56 (m, 2 H), 2.60 (s, 3 H), 2.42 (s, 3 H), 1.63 (s, 3 H), 1.46 (s, 9 H). (14) Compound 4-((6S)-6-(2-(tert-butoxy)-2-oxoethyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)pyridine 1-oxide (JQ14, molecular formula C 22 H 25 Exemplary preparation of N5O3S (molecular weight 439.53)

[0041] Step 1: Synthesis of compound 4-((6S)-6-(2-(tert-butoxy)-2-oxoethyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-4-yl)pyridine 1-oxide Add m-chloroperoxybenzoic acid (31 mg, 0.18 mmol) to a DCM solution of 2-((6S)-2,3,9-trimethyl-4-(pyridin-4-yl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl)tert-butyl acetate (50 mg, 0.12 mmol) in 3 mL. Stir at room temperature for 3 hours. Add ethyl acetate (50 mL) and H2O (50 mL) to the reaction system. Separate the organic phase, wash with saturated NaCl solution (50 mL × 3), dry with Na2SO4, filter, and concentrate under reduced pressure. 4-((6S)-6-(2-(tert-butoxy)-2-oxoethyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-4-yl)pyridine 1-oxide (20 mg, 38.0%) was purified by high performance liquid chromatography (TFA conditions). LC-MS (ESI): m / z 440.1 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 8.39 –8.04 (m, 2 H), 7.45 – 7.19 (m, 2 H), 4.58 – 4.25 (m, 1 H), 3.36 (s, 2 H), 2.59 (s, 3H), 2.44 (s, 3 H), 1.77 (s, 3 H), 1.42 (s, 9 H). (15) Compound 2-((6S)-4-(4-chlorophenyl)-3-cyclopropyl-2,9-dimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (JQ15, molecular formula C 25 H 27 Exemplary preparation of ClN4O2S (molecular weight 483.03)

[0042] Step 1: Synthesis of (2-amino-4-cyclopropyl-5-methylthiophen-3-yl)(4-chlorophenyl) ketone Sulfur (1.43 g, 5.57 mmol) was added to a solution of 3-(4-chlorophenyl)-3-oxopropionitrile (1.00 g, 5.57 mmol), 1-cyclopropylprop-1-one (546 mg, 5.57 mmol), and TEA (0.77 mL, 5.57 mmol) in EtOH (20 mL). The mixture was heated to 95 °C and reacted for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The solution was diluted with H₂O, extracted with ethyl acetate, washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The solution was purified by column chromatography (0-100% ethyl acetate / n-hexane) to give (2-amino-4-cyclopropyl-5-methylthiophene-3-yl)(4-chlorophenyl) methyl ketone (300 mg, 18% yield, yellow oil). LC-MS (ESI): m / z 292.2 [M + H] + . Step 2: Synthesis of compound (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4-cyclopropyl-5-methylthiophene-2-yl)amino)-4-oxobutyrate (N240763-023-P1) HCTU (851 mg, 2.06 mmol) and DIEA (532 mg, 4.11 mmol) were added sequentially to a DMF (10 mL) solution of Fmoc-D-Asp(O-tBu)-OH (846 mg, 2.06 mmol). The mixture was stirred at room temperature for 5 minutes, and then (2-amino-4-cyclopropyl-5-methylthiophene-3-yl)(4-chlorophenyl) methyl ketone (300 mg, 1.03 mmol) was added. The mixture was stirred at room temperature for 18 hours, and then ethyl acetate (50 mL) and saturated brine (50 mL) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purified by column chromatography (gradient 0-70% ethyl acetate / n-hexane) to (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4-cyclopropyl-5-methylthiophen-2-yl)amino)-4-oxobutyrate (400 mg, yield 56.0%, brown oil). LC-MS (ESI): m / z 685.1 [M + H] + . Step 3: Synthesis of compound (S)-tert-butyl-3-amino-4-((3-(4-chlorobenzoyl)-4-cyclopropyl-5-methylthiophen-2-yl)amino)-4-oxobutyrate To a DMF (10 mL) solution of (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-(4-chlorobenzoyl)-4-cyclopropyl-5-methylthiophen-2-yl)amino)-4-oxobutyrate (350 mg, 0.51 mmol), piperidine (2 mL) was added, and the mixture was stirred at room temperature for 18 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction mixture. The organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain (S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4-cyclopropyl-5-methylthiophen-2-yl)amino)-4-oxobutyrate (200 mg, yield 84.0%, yellow solid). LC-MS (ESI): m / z 462.2 [M + H] + . Step 4: Synthesis of compound (S)-tert-butyl 2-(5-(4-chlorophenyl)-6-cyclopropyl-7-methyl-2-oxo-2,3-dihydro-1H-thiopheno[2,3-e][1,4]diaza-3-yl)acetate The compound (S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4-cyclopropyl-5-methylthiophene-2-yl)amino)-4-oxobutyrate was dissolved in EtOH (8 mL), followed by the addition of AcOH (2 mL). The reaction mixture was stirred at 80 °C for 3 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system, the organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The solution was purified by column chromatography (0–80% ethyl acetate / n-hexane) to give (S)-tert-butyl 2-(5-(4-chlorophenyl)-6-cyclopropyl-7-methyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate (150 mg, yield 78.0%). LC-MS (ESI): m / z 445.1 [M + H] + . Step 5: Synthesis of tert-butyl acetate of compound 2-((6S)-4-(4-chlorophenyl)-3-cyclopropyl-2,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazyl-6-yl)acetate tBuOK (30 mg, 0.27 mmol) was added to a THF (15 mL) solution of (S)-tert-butyl-2-(5-(4-chlorophenyl)-6-cyclopropyl-7-methyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepine-3-yl)acetate (80 mg, 0.18 mmol). The reaction mixture was heated to -10 °C and stirred for 30 min, then cooled to -78 °C. Diphenyl chlorophosphate (73 mg, 0.27 mmol) was added to the reaction mixture. The mixture was heated to -10 °C and stirred for 45 min. Acetylhydrazine (33 mg, 0.45 mmol) was added to the reaction mixture. After stirring the reaction mixture at 23 °C for 1 hour, butanol (2.25 mL) was added, and the mixture was heated to 90 °C and reacted for 1 hour. The solvent was then removed under reduced pressure. Purified by column chromatography (0-100% ethyl acetate / / n-hexane) to give tert-butyl 2-((6S)-4-(4-chlorophenyl)-3-cyclopropyl-2,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazan-6-yl)acetate (15 mg, yield 17.0%). LC-MS (ESI): m / z 483.2 [M + H] + . 1 HNMR (400MHz, DMSO-) d 6) δ: 7.64 – 7.35 (m, 4 H), 4.49 – 4.34 (m, 1 H), 2.60 (d, J = 8.2 Hz, 3H), 2.51 (s, 3H), 2.14 (t, J = 8.3, 1 H), 1.75 (s, 2 H), 1.44 (d, J = 8.8 Hz, 9 H), 1.10 (d, J = 8.2Hz, 2 H), 0.91 – 0.64 (m, 2 H). (16) Compound 2-((6S)-2,3,9-trimethyl-4-(pyridin-4-yl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (JQ16, molecular formula C 22 H 25 Exemplary preparation of N5O2S (molecular weight 423.54)

[0043] Step 1: Synthesis of compound 3-(4-pyridinyl)-4,5-dimethylthiophene-2-amine Sulfur (175 mg, 5.47 mmol) was added to a solution of 3-oxo-3-(pyridin-4-yl)propionitrile (800 mg, 5.47 mmol, purchased from Bidepharm), butanone (0.49 mL, 5.47 mmol), and morpholine (952 mg, 10.95 mmol) in EtOH (20 mL). The mixture was heated to 85 °C and reacted for 12 hours. The reaction mixture was then cooled to room temperature and poured into brine (100 mL). The aqueous phase was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The solution was purified by column chromatography (Combiflash RF system, 40 g silica gel, gradient 0-100% ethyl acetate-hexane) to give 3-(4-pyridinyl)-4,5-dimethylthiophene-2-amine (700 mg, 55% yield, yellow solid). LC-MS (ESI): m / z 233.2 [M + H] + . Step 2: Synthesis of compound (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-isonicotinyl-4,5-dimethylthiophene-2-yl)amino)-4-oxobutyrate HCTU (1.07 g, 2.58 mmol) and DIEA (668 mg, 5.17 mmol) were added sequentially to a 10 mL solution of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-D-aspartic acid-4-tert-butyl ester (1.06 g, 2.58 mmol, purchased from Bidepharm) in DMF. The mixture was then stirred at room temperature for 5 minutes, followed by the addition of 3-(4-pyridinyl)-4,5-dimethylthiophene-2-amine (300 mg, 1.29 mmol). After stirring at room temperature for 16 hours, ethyl acetate (50 mL) and saturated brine (50 mL) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purified by column chromatography (gradient 0-100% ethyl acetate / n-hexane) to give (S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-isonicotinyl-4,5-dimethylthiophene-2-yl)amino)-4-oxobutyrate (500 mg, yield 61%). LC-MS (ESI): m / z 626.1 [M + H] + . Step 3: Synthesis of tert-butyl(S)-3-amino-4-((3-isonicotinyl-4,5-dimethylthiophen-2-yl)amino)-4-oxobutyrate Piperidine (2 mL) was added to a DMF (10 mL) solution of compound (S)-tert-butyl-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((3-isonicotinyl-4,5-dimethylthiophen-2-yl)amino)-4-oxobutyrate (400 mg, 0.64 mmol). The mixture was stirred at room temperature for 16 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the system. The organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give tert-butyl(S)-3-amino-4-((3-isonicotinyl-4,5-dimethylthiophen-2-yl)amino)-4-oxobutyrate (180 mg, 69% yield, yellow solid). LC-MS (ESI): m / z 404.2 [M + H] + . Step 4: Synthesis of tert-butyl(S)-2-(6,7-dimethyl-2-oxo-5-(pyridin-4-yl)-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate Add 2 mL of AcOH to an 8 mL EtOH solution of tert-butyl(S)-3-amino-4-((3-isonicotinyl-4,5-dimethylthiophene-2-yl)amino)-4-oxobutyrate (300 mg, 0.74 mmol). Stir at 80 °C for 6 hours. Cool the reaction mixture to room temperature and pour it into 100 mL of saturated brine. Extract the aqueous layer with ethyl acetate (3 × 50 mL). Wash the combined organic layers with saturated brine (50 mL), dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purified by column chromatography (gradient 0-100% ethyl acetate / n-hexane) to give tert-butyl(S)-2-(6,7-dimethyl-2-oxo-5-(pyridin-4-yl)-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate (200 mg, yield: 69%, yellow solid). LC-MS (ESI): m / z 386.1 [M + H] + . Step 5: Synthesis of tert-butyl acetate of compound 2-((6S)-2,3,9-trimethyl-4-(pyridin-4-yl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl) At -78°C, tBuOK (98 mg, 0.87 mmol) was added to a THF (1 mL) solution of tert-butyl(S)-2-(6,7-dimethyl-2-oxo-5-(pyridin-4-yl)-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate (280 mg, 0.73 mmol). The reaction was heated to -10°C and stirred for 30 minutes, then cooled to -78°C. Diphenyl chlorophosphate (234 mg, 0.87 mmol) was added to the reaction system. The temperature was raised to -10°C, and acetylhydrazine (81 mg, 1.09 mmol) was added. The system was then stirred at 23°C for 1 hour, followed by the addition of butanol (2.25 mL), followed by heating to 90°C and reacting for 1 hour. All solvents were then removed under reduced pressure. Purification by column chromatography (gradient 0-100% ethyl acetate / n-hexane) yielded 2-((6S)-2,3,9-trimethyl-4-(pyridin-4-yl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)tert-butyl acetate (200 mg, 65% yield, white solid). LC-MS (ESI): m / z 424.2 [M + H] + . 1 HNMR (400 MHz, DMSO- d 6) δ: 8.74 – 8.53 (m, 2 H), 7.40 – 7.27 (m, 2 H), 4.56 – 4.40 (m, 1 H), 3.39-3.34 (m, 2 H), 2.63 (s, 3 H), 2.43 (s, 3 H), 1.64 (s, 3 H), 1.44 (s, 9 H). (17) Compound tert-butyl(S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoate (JQ17, molecular formula C 25 H 27 Exemplary preparation of ClN4O2S (molecular weight 483.03)

[0044] Step 1: Synthesis of tert-butyl(S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoate 2-methylpropyl-2-yl(triphenyl-λ5-phosphoryl) acetate (78 mg, 0.21 mmol, purchased from Bidepharm) was added to a solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetaldehyde (89 mg, 0.23 mmol) and toluene (2 mL), and the mixture was stirred at 110 °C for 4 hours. The crude product was purified by column chromatography (dichloromethane: methanol, 100 / 1 to 50 / 1) to give tert-butyl(S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoate (73 mg, yellow liquid). The product was further purified by C18 chromatography (ACN in H2O, 0%–100%, FA) to give tert-butyl(S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoate (6.43 mg, 5%, white solid). LC-MS (ESI): m / z 484.6 [M + H] + . 1 H NMR (400MHz, DMSO-) d 6) δ 7.51 (d, J = 8.8 Hz, 2 H), 7.46 (d, J = 8.6 Hz, 2 H), 7.08 – 7.04 (m, 1H), 5.99 (d, J = 15.7 Hz, 1 H), 4.30 (t, J = 7.0 Hz, 1 H), 3.31 – 3.20 (m, 2 H), 2.60 (s, 3 H), 2.41 (s, 3 H), 1.64 (s, 3 H), 1.43 (s, 9 H). (18) Compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enamide (JQ18, molecular formula C 21 H 20 Exemplary preparation of ClN5OS (molecular weight 425.94)

[0045] Step 1: Synthesis of compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enamide (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoic acid (30 mg, 0.07 mmol, the preparation process can refer to the preparation process of compound JQ23) was added to a DMF (3 mL) solution of EDCI (20 mg, 0.10 mmol), HOBt (14 mg, 0.10 mmol), DIEA (45 mg, 0.35 mmol) and ammonium carbonate (101 mg, 1.05 mmol), and the reaction was stirred at 40 °C for 18 hours. The crude product was purified by dichloromethane:methanol (from 100 / 1 to 50 / 1) column chromatography to (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enamide (10 mg, yellow liquid). The product (10 mg) was further purified by C18 chromatography (ACN in H2O, 0%-100%, FA) to (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enamide (3.07 mg, 0.01 mmol, 10%, white solid). LC-MS (ESI): m / z 426.10[M + H] + . 1 H NMR (400MHz, DMSO-) d 6) δ 7.51 (d, J = 8.8 Hz, 2 H), 7.46 (d, J = 8.6 Hz, 2 H), 7.42 (s, 1 H), 6.93 (s, 1 H), 6.88 – 6.78 (m, 1 H), 6.09 (d, J = 15.6 Hz, 1 H), 4.22 (t, J = 7.0 Hz, 1H), 3.26 – 3.20 (m, 2 H), 2.60 (s, 3 H), 2.41 (s, 3 H), 1.64 (s, 3 H). (19) Compound 2-((3S)-5-(4-chlorophenyl)-1-(1-cyanoethyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thiopheno[2,3-e][1,4]diaza-3-yl)tert-butyl acetate (JQ19, molecular formula C 24 H 26 Exemplary preparation of ClN3O3S (molecular weight 472.00)

[0046] Step 1: Synthesis of tert-butyl acetate of compound 2-((3S)-5-(4-chlorophenyl)-1-(1-cyanoethyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thiopheno[2,3-e][1,4]diaza-3-yl) tert-butyl(S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl)acetate (50 mg, 0.12 mmol), 2-bromopropionitrile (32 mg, 0.24 mmol), and tBuOK (40 mg, 0.36 mmol) were added to a microwave-safe tube containing 3 mL of THF. The tube was sealed and microwaved at 110 °C for 120 minutes. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction mixture. The organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The compound 2-((3S)-5-(4-chlorophenyl)-1-(1-cyanoethyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazathio-3-yl)tert-butyl acetate (35 mg, 62% yield) was purified by high performance liquid chromatography (TFA conditions). LC-MS (ESI): m / z 472.1 [M + H] + . 1 H NMR (400MHz, DMSO-) d 6) δ 7.64 -7.54 (m, 2 H), 7.50 - 7.45 (m, 2 H), 5.85 - 5.71 (m, 1 H), 4.29 - 4.01 (m, 1 H), 3.18 - 3.09 (m, 1 H), 3.01 - 2.95 (m, 1 H), 2.45 - 2.34 (m, 3H), 1.64 (d, J = 7.1 Hz, 1 H), 1.62 (s, 3 H), 1.47 (d, J = 7.2 Hz, 2 H), 1.38 (s, 9 H). (20) Compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-1-(isoxazoline-2-yl)ethane-1-one (JQ20, molecular formula C 22 H 22 Exemplary preparation of ClN5O2S (molecular weight 455.96)

[0047] Step 1: Synthesis of compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid TFA (4 mL) was added to a DCM (4 mL) solution of tert-butyl acetate (300 mg, 0.66 mmol, purchased from Bidepharm). The mixture was stirred at room temperature for 3 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system. The organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (260 mg, yield 98%). LC-MS (ESI): m / z 401.2 [M + H] + . Step 2: Synthesis of compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-1-(isoxazolidin-2-yl)ethane-1-one To a DMF (5 mL) solution of compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (50 mg, 0.13 mmol, batch number: N240763-071-P), EDCI (48 mg, 0.25 mmol), HOBt (34 mg, 0.25 mmol), DIEA (81 mg, 0.62 mmol), and tetrahydroisoxazole (18 mg, 0.25 mmol, purchased from Leyan) were added, and the mixture was stirred at room temperature for 18 hours. The reaction system was diluted with ethyl acetate and water. The organic phase was separated, further washed with saturated NaCl solution, collected, and concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (0.1% TFA conditions) and dried to give compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-1-(isoxazolidin-2-yl)ethane-1-one (30 mg, yield 52%, white solid). LC-MS (ESI): m / z 455.8 [M + H] + . 1 HNMR (400 MHz, DMSO- d 6) δ: 7.50 (d, J = 8.7 Hz, 2 H), 7.47 – 7.28 (m, 2 H), 4.53 (t, J = 7.0 Hz, 1 H), 4.03 (t, J =7.0 Hz, 2 H), 3.74 – 3.44 (m, 4 H), 2.60 (s, 3 H), 2.46 – 2.38 (m, 3 H), 2.34 – 2.26 (m, 2 H), 1.75 – 1.54 (m, 3 H). (21) Compound 2-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-5-ethyl-1,3,4-oxadiazole (JQ21, molecular formula C 22 H 21 Exemplary preparation of OClN6S (molecular weight 452.96)

[0048] Step 1: Synthesis of compound 2-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-5-ethyl-1,3,4-oxadiazole Compound 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (263 mg, 0.66 mmol) and propionyl hydrazine (347 mg, 3.94 mmol, purchased from Leyan) were added to POCl3 (5 mL). The mixture was stirred at 100 °C for 4 hours. The reaction mixture was concentrated under reduced pressure and purified by high performance liquid chromatography to give 2-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-5-ethyl-1,3,4-oxadiazole (14 mg, 0.031 mmol, 4.74%). LC-MS (ESI): m / z 401.0 [M + H] + . 1 H NMR (400 MHz, Chloroform-d) δ 7.41 – 7.33 (m, 4 H), 4.80 (dd, J = 7.9, 6.4 Hz, 1 H), 4.12 (dd, J = 7.1, 2.4 Hz, 2 H), 2.93 (q, J =7.6 Hz, 2 H), 2.71 (s, 3 H), 2.44 (s, 3 H), 1.70 (s, 3 H), 1.43 (t, J = 7.6 Hz, 3 H). (22) Compound 5-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-3-ethyl-1,2,4-oxadiazole (JQ22, molecular formula C 22 H 21 Exemplary preparation of OClN6S (molecular weight 452.96)

[0049] Step 1: Synthesis of compound 5-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-3-ethyl-1,2,4-oxadiazole N-(1-azolylallyl)hydroxylamine (127 mg, 1.45 mmol) and K₂CO₃ (200 mg, 1.45 mmol) were added to a solution of 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (100 mg, 0.24 mmol) in toluene (3 mL). The reaction mixture was reacted in a microwave reactor at 150 °C for 4 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were then added to the reaction mixture. The organic phase was separated, washed with saturated sodium chloride solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and the crude product was purified by reversed-phase high-performance liquid chromatography (0.1% TFA conditions). After drying, the compound 5-(((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)methyl)-3-ethyl-1,2,4-oxadiazole (7.51 mg, 0.017 mmol, 6.88%) was obtained. LC-MS (ESI): m / z 452.8 [M + H] + . 1 H NMR (400 MHz, Chloroform-d)δ 7.44 – 7.34 (m, 4 H), 4.74 (dd, J = 8.9, 5.5 Hz, 1 H), 4.28 – 4.07 (m, 2 H), 2.79 (q, J = 7.6 Hz, 2 H), 2.71 (s, 3 H), 2.48 – 2.41 (m, 3 H), 1.75 – 1.68 (m, 3 H), 1.35 (t, J = 7.6 Hz, 3 H). (23) Compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-dimethylbut-2-enamide (JQ23, molecular formula C 23 H 24 Exemplary preparation of ClN5OS (molecular weight 453.99)

[0050] Step 1: Synthesis of compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid TFA (10 mL) was added to a 10 mL solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphos-6-yl)acetic acid tert-butyl ester (800 mg, 1.66 mmol, purchased from Bidepharm) in DCM, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding residue (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphos-6-yl)acetic acid (890 mg, 99% yield). The crude product was used directly in the next step. LC-MS (ESI): m / z 401.2 [M + H] + . Step 2: Synthesis of compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-methoxy-N-methylacetamide To a DMF (10 mL) solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (800 mg, 2.00 mmol), HATU (1.14 g, 3.00 mmol), DIEA (772 mg, 6.00 mmol), and N,O-dimethylhydroxylamine hydrochloride (389 mg, 4.00 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (100 mL). The organic phase was separated, washed further with saturated NaCl solution, and concentrated under reduced pressure. Purification by dichloromethane:methanol (from 100 / 1 to 50 / 1) column chromatography yielded (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazathio-6-yl)-N-methoxy-N-methylacetamide (600 mg, 67% yield, as a yellow solid). LC-MS (ESI): m / z 444.1 [M+H] + . Step 3: Synthesis of compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetaldehyde Compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-methoxy-N-methylacetamide (500 mg, 1.13 mmol) and dried tetrahydrofuran (15 mL) were added to a round-bottom flask dried under argon atmosphere. The mixture was cooled to 0 °C, and then LiAlH4 (86 mg, 2.25 mmol) was added, and the mixture was stirred at 0 °C for 30 minutes. The reaction mixture was then diluted with ethyl acetate (100 mL) and water (100 mL) at the settling point. The organic phase was separated, further washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to give compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetaldehyde (200 mg, yield 46%, yellow solid). LC-MS (ESI): m / z 385.1 [M + H] + . Step 4: Synthesis of tert-butyl (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoic acid ester Add compound (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetaldehyde (150 mg, 0.39 mmol) to toluene (5 mL), followed by 2-(triphenyl-λ) 5 220 mg (0.59 mmol) of tert-butyl butyl acetate (phosphane) was reacted with stirring at 100 °C for 3 h. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1~5 / 1) to give (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoic acid tert-butyl ester (130 mg, 69% yield, yellow solid). LC-MS (ESI): m / z 484.1 [M + H] + . Step 5: Synthesis of compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoic acid TFA (5 mL) was added to a 5 mL solution of tert-butyl (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphos-6-yl)but-2-enoic acid (130 mg, 0.27 mmol) in DCM, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure to give (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphos-6-yl)but-2-enoic acid (110 mg, 95.7% yield). The crude product was used directly in the next step. LC-MS (ESI): m / z 427.3 [M + H] + . Step Six: Synthesis of compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-dimethylbut-2-enamide To a DMF (1 mL) solution of compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoic acid (30 mg, 0.07 mmol), HATU (40 mg, 0.11 mmol), DIEA (27 mg, 0.21 mmol), and dimethylamine (0.11 mL, 0.21 mmol) were added, and the mixture was stirred at room temperature for 6 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction system, the organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazo-6-yl)-N,N-dimethylbut-2-enamide (18 mg, 56% yield, white solid) was purified by high performance liquid chromatography (TFA conditions). LC-MS (ESI): m / z 453.9 [M + H] + . 1HNMR (400 MHz, DMSO-d6) δ 7.52 – 7.50 (m, 2 H), 7.47 – 7.45 (m, 2 H), 6.93 – 6.79 (m, 1 H), 6.74 – 6.62 (m, 1 H), 4.26 (t, J = 7.0 Hz, 1 H), 3.28 (t, J = 7.4 Hz, 2 H), 3.06 (s, 3 H), 2.87 (s, 3 H), 2.61 (s, 3 H), 2.41 (d, J = 0.9 Hz, 3 H), 1.69 – 1.59 (m, 3 H). (24) Compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-diethylbut-2-enamide (JQ24, molecular formula C 25 H 28 Exemplary preparation of ClN5OS (molecular weight 482.04)

[0051] Step 1: Synthesis of compound (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-diethylbut-2-enamide Diethylamine (0.87 mL, 8.43 mmol), DIEA (1.09 g, 8.43 mmol), and HATU (2.40 g, 6.32 mmol) were added to a DMF (2 mL) solution of (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)but-2-enoic acid (900 mg, 2.11 mmol), and the mixture was stirred at room temperature for 2 hours. Purified by C18 column chromatography (ACN in water, 0%–45%) to (S,E)-4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N,N-diethylbut-2-enamide (600 mg, 1.25 mmol, 59%, white solid). LC-MS: [M + H] + : 482.2. 1 H NMR (400 MHz, Chloroform-d ) δ 7.43 (m, 2 H), 7.32 (m, 2 H), 7.09 (m, 1 H), 6.54 (m, 1 H), 4.13 (m, 1H), 3.58 – 3.38 (m, 6 H), 2.67 (s, 3 H), 2.41 (s, 3 H), 1.70 (s, 3 H), 1.23 (m, 3 H), 1.15 (m, 3 H). (25) Compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-N-ethylbut-2-enamide (JQ25, molecular formula C 23 H 24 Exemplary preparation of OClN5S (molecular weight 453.99)

[0052] Step 1: Synthesis of compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-N-ethylbut-2-enamide DIEA (27.25 mg, 0.21 mmol) and HATU (32.06 mg, 0.084 mmol) were added to a DMF (2 mL) solution of (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid (30 mg, 0.070 mmol). The mixture was stirred at room temperature for 30 minutes, and then ethylamine (0.005 mL, 0.077 mmol) was added, followed by stirring at room temperature for another 6 hours. Ethyl acetate (50 mL) and H₂O (50 mL) were added to the reaction mixture, the organic phase was separated, washed with saturated NaCl aqueous solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-N-ethylbut-2-enamide (10.85 mg, 0.024 mmol, yield 34.01%, white solid) was purified by high performance liquid chromatography (TFA conditions). LC-MS (ESI): m / z 453.9 [M + H] + . 1 H NMR (400 MHz, Chloroform-d ) δ 7.49 – 7.35 (m, 4 H), 6.99 (t, J = 15.0, 7.4 Hz, 1H), 6.22(d, J = 15.3 Hz, 1 H), 6.09 (s, 1 H), 4.22 (t, J = 7.1 Hz, 1 H), 3.58 – 3.34 (m, 4 H), 2.83 (s, 3 H), 2.47 (s, 3 H), 1.75 (s, 3 H), 1.22 (t, J = 7.2 Hz, 3 H). (26) Compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-morpholinobut-2-en-1-one (JQ26, molecular formula C 26 H 28 Exemplary preparation of OClN5S (molecular weight 494.05)

[0053] Step 1: Synthesis of compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-morpholinobut-2-en-1-one Add (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid (30 mg, 0.070 mmol), HATU (29.39 mg, 0.077 mmol), and DIEA (27 mg, 0.21 mmol) to DMF (1 mL). Stir at room temperature for 1 hour, then add morpholine (0.018 mL, 0.21 mmol). Stir at room temperature for 5 hours. Add ethyl acetate (50 mL) and H2O (50 mL) to the reaction system, separate the organic phase, wash with saturated NaCl solution (50 mL × 3), dry with anhydrous Na2SO4, filter, and concentrate under reduced pressure. The compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-morpholinobut-2-en-1-one (16.40 mg, 0.033 mmol, yield 47.05%, white solid) was purified by high performance liquid chromatography (TFA conditions). LC-MS (ESI): m / z 495.08 [M + H] + . 1 H NMR (400MHz, Chloroform- d ) δ 7.45 (d, J = 8.5 Hz, 2 H), 7.37 (d, J = 8.4 Hz, 2 H), 7.04 (t, J =14.8, 7.3 Hz, 1 H), 6.67 – 6.57 (m, 1 H), 4.21 (m, J = 7.6, 6.5 Hz, 1 H), 3.69 (d, J =28.0 Hz, 8 H), 3.50 (m, J = 7.3, 6.8 Hz, 2 H), 2.80 (s, 3 H), 2.46 (s, 3 H), 1.74 (s, 3H). (27) Compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-(isoxazoline-2-yl)but-2-en-1-one (JQ27, molecular formula C 24 H 24 Exemplary preparation of ClN5O2S (molecular weight 482.00)

[0054] Step 1: Synthesis of (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]-1-(isoxazolidin-2-yl)but-2-en-1-one To a DMF (1 mL) solution of compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid (20 mg, 0.047 mmol), HATU (27 mg, 0.07 mmol), DIEA (18 mg, 0.14 mmol), and tetrahydroisoxazole (10 mg, 0.14 mmol) were added. The mixture was stirred at room temperature for 6 hours. 50 mL of H₂O and 50 mL of HCl were added to the reaction mixture. The organic phase was separated, washed with saturated NaCl solution (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazazo-6-yl]-1-(isoxazolidin-2-yl)but-2-en-1-one (10 mg, yield 44.0%, white solid) was purified by high-performance preparative liquid chromatography (TFA conditions). LC-MS (ESI): m / z 481.9 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.55 – 7.41 (m, 4H), 7.07 – 6.93 (m, 1 H), 6.63 (d, J = 15.6 Hz, 1 H), 4.30 (s, 1H), 3.91 (d, J = 6.8 Hz, 2 H), 3.64 (dd, J = 8.2, 6.6 Hz, 2 H), 3.35 – 3.26 (m, 2 H), 2.61 (s, 3 H), 2.41 (s, 3H), 2.30 – 2.20 (m, 2 H), 1.64 (s, 3 H). (28) Compound tert-butyl(S)-2-[5-(4-chlorophenyl)-6,7-dimethyl-2-(1-methyl-1H-pyrazol-5-yl)-3H-thiopheno[2,3-e][1,4]diaza-3-yl]acetate (JQ28, molecular formula C 25 H27 Exemplary preparation of ClN4O2S (molecular weight 482.15)

[0055] Step 1: Synthesis of tert-butyl(S)-2-[2-chloro-5-(4-chlorophenyl)-6,7-dimethyl-3H-thiopheno[2,3-e][1,4]diaza-3-yl]acetate To a DCM solution of tert-butyl(S)-2-[5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diaza-3-yl]acetate (300 mg, 0.716 mmol), 2-chloro-2-oxoacetyl chloride (363.55 mg, 2.864 mmol) and N,N-dimethylformamide (0.006 mL, 0.072 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The reaction system was washed with saturated NaHCO3 solution (10 mL). The organic phase was separated, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (n-hexane:ethyl acetate (97:3)) to give tert-butyl(S)-2-[2-chloro-5-(4-chlorophenyl)-6,7-dimethyl-3H-thieno[2,3-e][1,4]diazathio-3-yl]acetate (45 mg, 0.103 mmol, 14.37%, white solid). LC-MS (ESI): m / z 436.8 [M + H] + . Step 2: Synthesis of tert-butyl(S)-2-[5-(4-chlorophenyl)-6,7-dimethyl-2-(1-methyl-1H-pyrazol-5-yl)-3H-thieno[2,3-e][1,4]diaza-3-yl]acetate K₂CO₃ (18.96 mg, 0.137 mmol), (1-methyl-1H-pyrazol-5-yl)boronic acid (17 mg, 0.14 mmol), and Pd(dppf)Cl₂ (6 mg, 0.034 mmol) were added to tert-butyl(S)-2-[2-chloro-5-(4-chlorophenyl)-6,7-dimethyl-3H-thieno[2,3-e][1,4]diaza-3-yl]acetate (30 mg, 0.069 mmol) in water (0.5 mL) and dioxane (2 mL), respectively. The mixture was stirred at 65 °C for 3 hours. The reaction solution was directly purified by preparative high-performance liquid chromatography (TFA conditions) to give tert-butyl(S)-2-[5-(4-chlorophenyl)-6,7-dimethyl-2-(1-methyl-1H-pyrazol-5-yl)-3H-thieno[2,3-e][1,4]diazathio-3-yl]acetate (0.96 mg, yield 2.9%). LC-MS (ESI): m / z 482.9 [M + H] + . (29) Compound (S,E)-2-[3-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]propyl-1-en-1-yl]-5-ethyl-1,3,4-oxadiazole (JQ29, molecular formula C 24 H 24 Exemplary preparation of OClN6S (molecular weight 479.00)

[0056] Step 1: Synthesis of (S,E)-2-[3-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]propyl-1-en-1-yl]-5-ethyl-1,3,4-oxadiazole] (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid (30 mg, 0.070 mmol) was dissolved in POCl3 (1 mL), and propionyl hydrazine (12 mg, 0.14 mmol) was added at room temperature. The mixture was stirred at 100 °C for 8 hours. The reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography (TFA conditions) to give compound (S,E)-2-[3-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]propyl-1-en-1-yl]-5-ethyl-1,3,4-oxadiazole (1.2 mg, 0.003 mmol, 3.57%, white solid). LC-MS (ESI): m / z 479.1 [M + H] + . 1 H NMR (400 MHz, Chloroform- d ) δ 7.47 (d, J = 8.5 Hz, 2 H), 7.37 (d, J = 8.7 Hz, 2 H), 7.16 –7.09 (m, 1 H), 6.65 (dd, J = 16.2, 1.6 Hz, 1 H), 4.20 (t, J = 7.0 Hz, 1 H), 3.62 – 3.51 (m, 2 H), 2.89 (q, J = 7.6 Hz, 2 H), 2.72 (s, 3 H), 2.44 (d, J = 0.8 Hz, 3 H), 1.74 (d, J =0.9 Hz, 3 H), 1.40 (t, J = 7.6 Hz, 3 H). (30) Compound (E)-1-(3,3-difluoropyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (JQ30, molecular formula C 25 H 24 Exemplary preparation of ClF2N5OS (molecular weight 516.01)

[0057] Step 1: Synthesis of (E)-1-(3,3-difluoropyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one Compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid (880 mg, 2.06 mmol), HATU (1.18 g, 3.09 mmol), and DIEA (799 mg, 6.18 mmol) were added to a 15 mL DMF solution and stirred at room temperature for 30 minutes. Then, 3,3-difluoropyrrolidine hydrochloride (294 mg, 2.06 mmol, purchased from Bidepharm) was added to the reaction mixture and stirred at room temperature for 2 hours. The reaction was purified by preparative high-performance liquid chromatography (FA conditions) to give compound (E)-1-(3,3-difluoropyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (872 mg, 1.69 mmol, 81%, yellow solid). LC-MS: [M + H]+: 516.1. 1 H NMR (400 MHz, Chloroform- d ) δ 7.45 – 7.39 (m, 2 H), 7.36 – 7.30 (m, 2 H), 7.21 – 7.08 (m, 1 H), 6.48 – 6.27 (m, 1 H), 4.16 – 4.10 (m, 1 H), 3.97 – 3.74 (m, 4 H), 3.58 – 3.45 (m, 2H), 2.71 – 2.65 (m, 3 H), 2.51 – 2.30 (m, 5 H), 1.69 (s, 3 H). (31) Compound (E)-1-(pyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (JQ31, molecular formula C 25 H 26 Exemplary preparation of ClN5OS (molecular weight 480.03)

[0058] Step 1: Synthesis of (E)-1-(pyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one Compounds (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid (600 mg, 1.40 mmol), HATU (801 mg, 2.10 mmol), and DIEA (544 mg, 4.21 mmol) were added to DMF (10 mL), and the mixture was stirred at room temperature for 30 minutes. Tetrahydropyrrole (99 mg, 1.40 mmol, purchased from Aladdin) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The reaction was purified by preparative high-performance liquid chromatography (FA conditions) to give compound (E)-1-(pyrrolidone-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (551.13 mg, 1.14 mmol, 81%, yellow solid). LC-MS: [M + H] + : 480.1. 1 H NMR (400 MHz, Chloroform- d ) δ 7.46 –7.40 (m, 2 H), 7.35 – 7.29 (m, 2 H), 7.13 – 7.03 (m, 1 H), 6.48 – 6.41 (m, 1 H), 4.15 – 4.09 (m, 1 H), 3.61 – 3.42 (m, 6 H), 2.71 – 2.62 (m, 3 H), 2.40 (s, 3 H), 2.01 –1.83 (m, 4 H), 1.69 (s, 3 H). (32) Compound (E)-1-(azacyclobut-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (JQ32, molecular formula C 24 H 24 Exemplary preparation of OClN5S (molecular weight 466.00)

[0059] Step 1: Synthesis of (E)-1-(azacyclobut-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiopheno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one In a DMF (1 mL) solution of compound (S,E)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-enoic acid, DIEA (45 mg, 0.35 mmol) and HATU (53 mg, 0.14 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Then, aziridine (8 mg, 0.14 mmol) was added, and the mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with H2O, extracted with ethyl acetate, washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The desired compound (E)-1-(azacyclobut-1-yl)-4-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl]but-2-en-1-one (2 mg, 0.004 mmol, 3.66%, white solid) was purified by preparative high-performance liquid chromatography (TFA conditions). LC-MS (ESI): m / z 466.1 [M + H] + . 1 H NMR (400 MHz, Chloroform- d ) δ 7.46 (d, J = 8.3 Hz, 2 H), 7.41 – 7.33 (m, 2 H), 7.08 – 7.02 (m, 1 H), 6.22 (d, J = 15.3 Hz, 1H), 4.28 (s, 2 H), 4.16 (dd, J = 17.6, 10.3 Hz, 2 H), 3.50 (q, J = 8.0 Hz, 2 H), 2.75 (s, 3 H), 2.45 (s, 3 H), 2.33 (q, J = 7.8 Hz, 2 H), 1.73 (s, 3 H). Example 2: Effect of the compound on inhibiting Th17 cell differentiation 1. The 32 compounds synthesized in Example 1 were initially screened for their inhibitory effects on mouse Th17 differentiation. Details are as follows: (1) Primary mouse naïve CD4+ T cells were isolated.

[0060] Single-cell suspensions were prepared from peripheral lymph nodes and spleens of 6-8 week old female C57BL / 6 mice (provided by the Experimental Animal Center of Zhejiang University, all housed in an SPF-grade environment). Naïve CD4+ T cells in the lymph nodes were sorted using a naïve CD4+ T sorting kit (purchased from Biolegend).

[0061] (2) In vitro differentiation of primary naïve CD4+ T cells.

[0062] After erythropoiesis, spleen cells were irradiated at 160 kV, 25 mA, and 30 Gy to obtain antigen-presenting cells (APC cells). After sorting, the cells and irradiated antigen-presenting cells were seeded at a 1:4 ratio on 96-well round-bottom plates. The T cell culture medium was RPMI 1640 medium (200 μL per well), supplemented with 50 mL of 10% fetal bovine serum, 5 mL of sodium pyruvate, 5 mL of 100× penicillin-streptomycin, and 2.5 mL of β-mercaptoethanol. To construct culture conditions for the differentiation of naïve CD4+ T cells into non-pathogenic Th17 cells, the following antibodies and cytokines were added for differentiation: anti-CD3 antibody (2 μg / ml, purchased from eBioscience), anti-CD28 antibody (3 μg / ml, purchased from eBioscience), IL2 (50 U / ml, purchased from biolegend), anti-IL4 antibody (10 μg / ml, purchased from biolegend), anti-IFNγ antibody (10 μg / ml, purchased from biolegend), anti-IL12 antibody (10 μg / ml, purchased from biolegend), TGFβ (5 ng / ml, purchased from biolegend), and IL6 (10 ng / ml, purchased from biolegend).

[0063] (3) Add the 32 compounds obtained from the screening to analyze the drug’s toxicity to T cells and whether it inhibits the differentiation of naïve CD4+ T cells toward Th17.

[0064] After culturing primary Naïve CD4+ T cells for 24 hours, drugs were added (final concentrations of 500 nM and 640 nM). Cells were cultured for another 24 hours; since the nutrients in the culture medium were almost completely depleted at this point, 200 μL of medium was added. Cells were then cultured for another 24 hours before flow cytometry staining analysis.

[0065] Flow cytometry (ACEA NovoCyte flow cytometer) was used to analyze CD4, IL-17, and Foxp3 levels to determine the percentage of differentiation into Th17 cells (CD4+ IL-17+) (number of Th17 cells / total number of cells); and the relative value of differentiation into Th17 cells was calculated (percentage of Th17 cells in the experimental group / percentage of Th17 cells in the control group). All experimental results are expressed as mean ± standard error and were statistically analyzed using GraphPad Prism 9.0 software (see [link to relevant documentation]). Figure 1 The significance level was set as follows: p <0.05, p <0.01, p <0.001, p <0.0001; ns indicates no statistical difference (p≥0.05).

[0066] Based on the results of flow cytometry analysis, 32 test compounds were initially screened. Among them, JQ2, JQ4, JQ6, JQ28, and JQ29 had little to no inhibitory effect on Th17 cell differentiation, while JQ1, JQ24, JQ30, and JQ31 had strong inhibitory effects. Therefore, the above four candidate compounds were screened and their IC50 values ​​on mouse and human Th17 cell differentiation were determined, and the remaining compounds were excluded.

[0067] like Figure 2 As shown, the IC50 values ​​of four compounds were determined by flow cytometry. The results showed that compound JQ31 effectively inhibited the differentiation of mouse CD4+ T cells into pathogenic Th17 cells, with the lowest IC50 value (71.82 nM), exhibiting superior activity compared to other candidate molecules. The drug activity ranking in inhibiting mouse Th17 cell differentiation was: JQ31 > JQ30 > JQ1 > JQ24.

[0068] 2. Verification of the inhibitory effect of the 32 compounds synthesized in Example 1 on human Th17 differentiation. Details are as follows: (1) Thawing of cryopreserved primary human CD4+ T cells (purchased from Shanghai Junxing Biotechnology Co., Ltd.): Preheat the 37°C water bath and T cell culture medium. Irradiate the cell stage with UV light, ensuring aseptic technique throughout the process. After picking up the cryovial with forceps, wrap it with PE gloves and place it in a 37°C water bath for 1 min 30s-1 min 50s, continuously shaking the cryovial and observing its color. A change from white to off-white or light yellow indicates complete thawing. Slowly transfer the thawed cells to a 15ml centrifuge tube using a Pasteur pipette. Add 1-2ml of preheated culture medium to the cryovial, bringing the total volume to 5ml. Mix gently by pipetting. Centrifuge the cells at 400g for 6-8 minutes, discard the supernatant, add an appropriate amount of culture medium, resuspend, and count.

[0069] (2) In vitro differentiation of human primary CD4 T cells.

[0070] Adjust the cell concentration to 15w cells per well in a 96-well plate, and add the following cytokines to induce Th17 cell differentiation: anti-CD3 antibody (5μg / ml, purchased from Biolegend), anti-CD28 antibody (2.5μg / ml, purchased from Biolegend), IL2 (100U / ml, purchased from Proteintech), anti-IL4 antibody (5μg / ml, purchased from Biolegend), anti-IFNγ antibody (5μg / ml, purchased from Biolegend), TGFβ (5ng / ml, purchased from Proteintech), IL6 (30ng / ml, purchased from Proteintech), IL1β (50ng / ml, purchased from Proteintech), and IL23 (50ng / ml, purchased from Proteintech).

[0071] After primary CD4 T cells were cultured for 48 hours, drug compounds were added (final concentrations were in gradients of 40 nM, 80 nM, 160 nM, 320 nM, 640 nM, and 1 μM), and the cells were cultured for another 6-7 days. The supernatant was then collected for ELISA detection.

[0072] like Figure 3As shown, the results of ELISA testing of IL-17A, a characteristic cytokine of Th17 cells, using a kit purchased from ProteinTech, indicate that JQ31 is the candidate molecule with the strongest inhibitory effect on human Th17 cell differentiation among the four compounds tested. Its IC50 value (48.13 nM) is significantly lower than the other three compounds, confirming the universality of JQ31's inhibitory effect. The order of drug activity in inhibiting human Th17 cell differentiation is: JQ31 > JQ30 > JQ24 > JQ1. Compared to JQ1's activity in inhibiting human Th17 cell differentiation, JQ31 has a greater advantage in inhibiting human Th17 cell differentiation and has greater translational potential in clinical applications.

Claims

1. The following compounds, their tautomers, stereoisomers, isotopic labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 2. The following compounds, their tautomers, stereoisomers, isotopic labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs: 、 、 。 3. The following compounds, their tautomers, stereoisomers, isotopic labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs: 。 4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a compound according to any one of claims 1-3, its tautomer, stereoisomer, isotope label, hydrate, solvate, pharmaceutically acceptable salt, or prodrug.

5. The pharmaceutical composition according to claim 4, characterized in that, The drug composition is a Th17 cell differentiation inhibitor.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is a therapeutic or preventative agent for multiple sclerosis.

7. A kit for inhibiting Th17 cell differentiation, characterized in that, The compound comprising any one of claims 1-3, its tautomers, stereoisomers, isotope labels, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs.

8. The reagent kit according to claim 7, characterized in that, The Th17 cells are derived from humans or rodents.

9. Use of the compound, tautomer, stereoisomer, isotope label, hydrate, solvate, pharmaceutically acceptable salt or prodrug, or pharmaceutical composition according to any one of claims 1-3, in the preparation of a medicament for inhibiting Th17 cell differentiation.

10. Use of the compound, tautomer, stereoisomer, isotope label, hydrate, solvate, pharmaceutically acceptable salt or prodrug, or pharmaceutical composition according to any one of claims 1-3 in the preparation of a medicament for the treatment or prevention of multiple sclerosis.

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