Novel compounds for the treatment of multiple sclerosis

Abstract

The present application belongs to the field of medicine, and particularly relates to a novel compound for treating multiple sclerosis. Specifically, the present application provides a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, wherein the compound or the salt or the pharmaceutical composition comprising the same has the biological activity of inhibiting CD4 + The biological activity of T cells differentiating into Th17 cells can be used for preventing and / or treating autoimmune deficiency diseases related to Th17 cells, for example, multiple sclerosis (MS). The compound of the present application is novel in structure, excellent in biological activity, and can be used as an ideal candidate drug compound for treating MS.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to novel compounds for the treatment of multiple sclerosis. Background Technology

[0002] Th17 cells are CD4 + Helper T cells are a distinct subset that primarily drive inflammatory responses by secreting effector cytokines such as IL-17A, IL-17F, IL-21, and IL-22 (E Park et al., Exp Mol Med, 2025, 57(9): p.1913-1927). The immune imbalance resulting from overactivation of Th17 cells and dysfunction of regulatory T cells (Tregs) has been shown to be involved in the pathogenesis of various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, psoriasis, type 1 diabetes, Hashimoto's thyroiditis, and inflammatory bowel disease.

[0003] Currently, there are numerous reports on intervention strategies targeting Th17 cells to improve autoimmune diseases. In rheumatoid arthritis, upregulating the expression of core transcription factors STAT3 and RORC, which are related to Th17 differentiation, can promote Th17 cell differentiation and thus accelerate disease progression (G Yan et al., Mol Med, 2024, 30(1): p.237). Carnosol, a naturally occurring polyphenol diterpenoid compound, can significantly alleviate the severity of collagen-induced arthritis and reduce inflammatory responses by inhibiting Th17 cell differentiation by limiting IL-6R (CD126) expression (J Chen et al., Biomed ResInt, 2023, 2023: p.1179973). Tripterygium wilfordii and ZnSO4 inhibit Th17 differentiation by regulating glycolysis and improving mitochondrial function, respectively, thereby inhibiting the occurrence of arthritis in mouse models (MY Shen et al., Immunopharmacol Immunotoxicol, 2022, 44(6): p.838-849; SY Lee et al., Exp Mol Med, 2025, 57(1): p.221-234). In type 1 diabetes, interventions such as metformin, neutralizing anti-IL-17 antibodies, and recombinant IL-25 can inhibit Th17 cell differentiation and promote Treg cell development, reduce inflammatory infiltration of immune cells around the islets of Langerhans, and lower the level of glutamate decarboxylase 65 (GAD65) autoantibodies, thereby significantly alleviating the autoimmune response in NOD mice (W Chen et al., PLoS One, 2022, 17(11): p.e0277061; JA Emamaullee et al., Diabetes, 2009, 58(6):p.1302-1311). In multiple sclerosis, it has been reported that natural compounds, small molecule inhibitors, and biologics targeting specific membrane proteins can effectively alleviate neuroinflammation and clinical symptoms by inhibiting the differentiation or function of Th17 cells. Examples include ivy saponins, blocking rejection-directing molecule A (RGMa), and NF-κB inhibitors (D Guan et al., IUBMBLife, 2024, 76(10): p.845-857; F Yang et al., CNS Neurosci Ther, 2025, 31(8): p.e70555). Therefore, developing drugs targeting Th17 cell differentiation holds promise as an effective strategy for treating autoimmune diseases.

[0004] 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.

[0005] Currently, there are no drugs that can cure MS in clinical practice. Although various treatment methods 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. Although 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 injected subcutaneously, thus limiting its use. Long-term injection can cause dyspnea, chest tightness, palpitations, and anxiety. Fingolimod can cause macular edema, elevated transaminase levels, and an increased risk of infection (ImaneBoutitah-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.

[0006] 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 less-than-ideal 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 (particularly the inhibition of effector memory CD4+ T cell differentiation into Th17 cells) have shown significant therapeutic potential in preclinical studies.

[0007] In summary, there is still a need to develop novel inhibitors of Th17 cell differentiation in order to provide more drug candidates for the treatment of autoimmune diseases. Summary of the Invention

[0008] Based on existing technologies, this invention has conducted a series of optimization studies on the structure of the JQ1 compound, 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 autoimmune diseases (e.g., MS). Specifically, through structural modification and in vitro activity screening experiments, JQ1 and 31 modified novel compounds were evaluated, thereby discovering a number of novel compounds (e.g., compounds reported in Chinese invention patent application CN202610249180X, particularly JQ24, JQ30, and JQ31), which exhibit significantly superior activity in inhibiting the differentiation of CD4+ T cells into Th17 cells.

[0009] In this regard, embodiments of the present invention include, but are not limited to, the following: In some embodiments, the present invention provides a pharmaceutical composition comprising a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient. Formula (I) Wherein, R1 and R2 are independently selected from C2-C6 straight-chain alkyl groups, or R1 and R2 together with the N atom attached to them. Piperidinyl (i.e., ), aziridine heptyl (i.e., ), or aziridine octyl (i.e., ); where R 1a and R 1b Each of the following is independently selected from H, halogen, methyl, or halogen-substituted methyl groups; R3, R4, and R5 are each independently selected from H, halogens, C1-3 alkyl groups, or halogen-substituted methyl groups; and The R6 is selected from halogens; and The n is selected from 0 to 6.

[0010] In this invention, the selection of pharmaceutical excipients varies depending on the route of administration and characteristics of action, and can generally be fillers, diluents, binders, wetting agents, disintegrants, lubricants, emulsifiers, suspending agents, etc., which are conventional in the art.

[0011] In some embodiments, the present invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof: Formula (I) Wherein, R1 and R2 are independently selected from C2-6 straight-chain alkyl groups, or R1 and R2 together with the N atom attached to them. Piperidinyl, aziridine-heptyl, or aziridine-octyl; R 1a and R 1b Each of the following is independently selected from H, halogen, methyl, or halogen-substituted methyl groups; R3, R4, and R5 are each independently selected from H, halogens, C1-3 alkyl groups, or halogen-substituted methyl groups; and The R6 is selected from halogens; and The n is selected from 0-6; Wherein, the compound represented by formula (I) is not any of the following: , , Or, or its pharmaceutically acceptable salt, stereoisomer, or tautomer.

[0012] In some embodiments, n in this invention is selected from 0, 1, 2, 3, 4, 5 or 6, preferably 0, 1, 2 or 3, more preferably 1, 2 or 3, and most preferably 1.

[0013] In some embodiments, R1 and R2 of the present invention are each independently selected from C2 or C3 straight-chain alkyl groups, or R1 and R2 are formed together with the N atom attached to them. Or piperidinyl; and The R 1a and R 1b They are selected independently from H and halogens, respectively.

[0014] In some embodiments, n is selected from 0-3; and / or R3, R4, and R5 are each independently selected from C1-3 straight-chain alkyl groups.

[0015] In some embodiments, R1 and R2 of the present invention are each independently selected from C2 alkyl groups, or R1 and R2 are formed together with the N atom attached to them. , wherein, the R 1a and R 1b Each is independently selected from H or halogen; The n is 1; and R3, R4, and R5 are each independently selected from C1 alkyl groups.

[0016] In some embodiments, the C3-6 alkyl group of the present invention is a straight-chain alkyl group, such as n-propyl, n-butyl, n-pentyl, or n-hexyl.

[0017] Unless otherwise stated, the halogens described in this invention are F, Cl, Br, and I.

[0018] In some implementations, the halogen in R6 is Cl.

[0019] In some implementation schemes, R 1a and R 1b They are F respectively.

[0020] In some embodiments, the compound represented by formula (I) in the pharmaceutical composition of the present invention is selected from any of the following: , , Or, or its pharmaceutically acceptable salt, stereoisomer, or tautomer.

[0021] In some embodiments, the compound represented by formula (I) in the pharmaceutical composition of the present invention is selected from: Or, or its pharmaceutically acceptable salt, stereoisomer, or tautomer.

[0022] In this invention, the term "pharmaceutically acceptable salt" refers to a salt formed from a suitable nontoxic organic acid, inorganic acid, organic base, or inorganic base with a compound of Formula I, which retains the biological activity of the compound of Formula I. The organic acid may be one or more of the conventional salt-forming organic acids in the art, preferably methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, hydroxyethylsulfonic acid, naphthalenesulfonic acid, and salicylic acid. The inorganic acid may be one or more of the conventional salt-forming inorganic acids in the art, preferably hydrochloric acid, sulfuric acid, and phosphoric acid. The organic base may be one or more of the conventional salt-forming organic bases in the art, preferably pyridines, imidazoles, pyrazines, indoles, purines, tertiary amines, and anilines. The tertiary amine organic base is preferably triethylamine and / or N,N-diisopropylethylamine. The aniline-based organic base is preferably N,N-dimethylaniline. The pyridine-based organic base is preferably one or more of pyridine, methylpyridine, 4-dimethylaminopyridine, and 2-methyl-5-ethylpyridine. The inorganic base can be any conventional salt-forming inorganic base in the art, preferably one or more of alkali metal hydrides, alkali metal hydroxides, alkali metal alkoxides, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium bicarbonate, and sodium bicarbonate. The alkali metal hydrides are preferably sodium hydride and / or potassium hydride. The alkali metal hydroxides are preferably one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide. The alkali metal alkoxides are preferably one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide, and sodium tert-butoxide.

[0023] In some embodiments, the present invention also provides the use of the pharmaceutical compositions described herein, compounds of formula (I), or pharmaceutically acceptable salts thereof in the preparation of a medicament / reagent for inhibiting Th17 cell differentiation.

[0024] In some implementations, the reagent is used for non-diagnostic / therapeutic purposes.

[0025] In some embodiments, the Th17 cells of the present invention are derived from humans or mammals. Preferably, the mammal is, for example, a rodent.

[0026] In some embodiments, the present invention also provides the use of the pharmaceutical composition of the present invention or a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating an autoimmune disease, said autoimmune disease being a Th17-mediated autoimmune disease.

[0027] In some embodiments, the autoimmune diseases described in this invention include: psoriasis, vitiligo, dermatitis, autoimmune encephalomyelitis, asthma, Arthur's reaction, rheumatoid arthritis, multiple sclerosis, autoimmune myocarditis, autoimmune uveitis, type 1 diabetes, systemic lupus erythematosus, and inflammatory bowel disease.

[0028] In some embodiments, the autoimmune disease described in this invention is autoimmune encephalomyelitis or multiple sclerosis.

[0029] In one embodiment, the compounds provided by the present invention are selected from: (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)-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); or (S,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).

[0030] The beneficial technical effects of the present invention include at least the following: This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising thereof, which have the biological activity of 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, such as multiple sclerosis. Many of 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

[0031] Figure 1 The graph shows the relative values ​​of Th17 cell differentiation in the presence of each of the 32 compounds in Example 1, with the significance level set as: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; ns indicates no statistical difference ( p≥0.05).

[0032] Figure 2 The IC50 values ​​of compounds JQ1, JQ24, JQ30, and JQ31 are shown to inhibit mouse Th17 differentiation.

[0033] Figure 3 The IC50 values ​​of compounds JQ1, JQ24, JQ30, and JQ31 are shown to inhibit human Th17 differentiation.

[0034] Figure 4 The pharmacokinetic curve is shown in the figure after oral administration of 10 mg / kg JQ24 to rats.

[0035] Figure 5 The pharmacokinetic curve of rats after oral administration of 10 mg / kg JQ30 is shown.

[0036] Figure 6 The pharmacokinetic curve of rats after oral administration of 10 mg / kg JQ31 is shown.

[0037] Figure 7 This shows the pharmacokinetic (PK) comparison of JQ24, JQ30, and JQ31 in SD rats.

[0038] Figure 8 This shows the changes in body weight of mice in each group during the experiment.

[0039] Figure 9 Clinical scores of EAE mice during JQ31 treatment are displayed, with the significance level set at: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; ns indicates no statistical difference ( p ≥0.05).

[0040] Figure 10 This shows the relative weight changes in EAE mice during JQ31 treatment.

[0041] Figure 11 The effect of JQ31 treatment on spinal cord inflammatory infiltration was shown, with the significance level set at: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; ns indicates no statistical difference ( p ≥0.05). Detailed Implementation

[0042] Example 1: Preparation of the compound This application uses JQ1 as a lead compound template. Through continuous structural optimization, 31 candidate compounds were obtained in sequence. After screening and cell experiments, it was demonstrated that, compared with compound JQ1, compounds JQ24, JQ30 and JQ31 have significantly better functions in inhibiting the differentiation of CD4+ cells into Th17 cells.

[0043] (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)

[0044] (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, CAS No.: 202592-23-2; manufacturer: Bid Pharmaceutical) 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, 2H), 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, 3H), 2.42 (s, 3 H), 1.64 (s, 3 H), 1.43 (s, 9 H).

[0045] (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)

[0046] 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).

[0047] (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)

[0048] Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., specification: 25mg / bottle.

[0049] (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)

[0050] Purchased from Shanghai BIDE Pharmaceutical Technology Co., Ltd., specification: 50mg / bottle.

[0051] (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)

[0052] Purchased from Shanghai BIDE Pharmaceutical Technology Co., Ltd., specification: 50mg / bottle.

[0053] (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)

[0054] 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 NaCl solution, 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] + .

[0055] 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]aza-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 NaCl solution, 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]aza-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).

[0056] (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)

[0057]

[0058]

[0059] 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 NaCl solution (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] + .

[0060] 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 Na2SO4, 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] + .

[0061] 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 a saturated NaCl solution (100 mL). The aqueous layer was extracted with ethyl acetate (3 × 50 mL), and the combined organic phases were washed with a saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, 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] + .

[0062] 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 for 5 minutes at room temperature. Solid (2-amino-5-cyclopropyl-4-methylthiophene-3-yl)(4-chlorophenyl) ketone (300 mg, 1.03 mmol) was added. After stirring for 16 hours at room temperature, 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 saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, 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] + .

[0063] 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 anhydrous 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] + .

[0064] 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] + .

[0065] 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 Under conditions of -78℃ 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 and reacted for 1 hour. The solvent was then 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).

[0066] (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)

[0067] 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, 11.14 mmol) was added to a solution of 3-(4-chlorobenzene)-3-oxopropionitrile (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 a saturated NaCl solution (100 mL). The aqueous layer was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with a saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Purification by column chromatography (Combi flash RF system, 40 g silica gel, gradient 0-100% ethyl acetate-n-hexane) 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] + .

[0068] 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 NaCl solution (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. (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, 71.2%) was purified by column chromatography (Combi flash RF, 40 g silica gel, gradient 0-100% ethyl acetate-hexane). LC-MS (ESI): m / z 687.1 [M + H] + .

[0069] 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 anhydrous 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] + .

[0070] 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 (50 mL × 2). The combined organic phases were washed with saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (Combiflash RF, 40 g silica gel, gradient 0-100% ethyl acetate-n-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] + .

[0071] 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℃, 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 min. 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 min. 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 h. 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-n-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).

[0072] (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)

[0073] 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 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 (60 mg, 0.12 mmol), 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 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]diaza-4-yl)acetic acid (50 mg, yield 94.2%). LC-MS (ESI): m / z 429.0 [M + H] + .

[0074] 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).

[0075] (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)

[0076]

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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 Add to a 2 mL solution of (R)-5-(4-chlorophenyl)-6,7-dimethyl-3-((methylthio)methyl)-1,3-dihydro-2H-thiopheno[2,3-e][1,4]diaza-2-one (170 mg, 0.46 mmol) in THF. t Buok (57 mg, 0.51 mmol) 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, acetylhydrazine (51 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 1 h. 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 solution was purified by preparative liquid chromatography-mass spectrometry (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.

[0081] 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- d 6) 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, 3 H), 1.69 – 1.58 (m, 3H).

[0082] (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)

[0083] 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).

[0084] (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)

[0085] 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 anhydrous 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] + .

[0086] 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- d6) 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).

[0087] (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

[0088] 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.

[0089] 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).

[0090] (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)

[0091] 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 anhydrous 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) δ8.39 – 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, 3 H), 2.44 (s, 3 H), 1.77 (s, 3 H), 1.42 (s, 9 H).

[0092] (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)

[0093]

[0094] 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] + .

[0095] 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 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 NaCl solution (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 Na₂SO₄, 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] + .

[0096] 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 anhydrous 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] + .

[0097] 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 anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The solution was purified by column chromatography (gradient 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]diazyl-3-yl)acetate (150 mg, yield 78.0%). LC-MS (ESI): m / z 445.1 [M + H] + .

[0098] 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 Under conditions of -78℃ 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, 2H), 0.91 – 0.64 (m, 2H).

[0099] (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)

[0100]

[0101] 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 saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, 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] + .

[0102] 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 a saturated NaCl solution (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 a saturated NaCl solution (50 mL), dried over anhydrous Na₂SO₄, 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] + .

[0103] 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] + .

[0104] 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 NaCl solution. Extract the aqueous layer with ethyl acetate (3 × 50 mL). Wash the combined organic layers with saturated NaCl solution (50 mL), dry to anhydrous Na₂SO₄, 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] + .

[0105] 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℃,t BuOK (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 min, 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), and the reaction was heated to 90 °C for 1 hour. All solvents were 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).

[0106] (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)

[0107] 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 Add 2-methylpropyl-2-yl(triphenyl-λ) to a solution of (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 (89 mg, 0.23 mmol) and toluene (2 mL). 5 -phosphine subunit) acetate (78 mg, 0.21 mmol, purchased from Bidepharm) was reacted with stirring at 110 °C for 4 h. 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 purified by high-performance liquid 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 (400 MHz, 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, 1 H), 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).

[0108] (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)

[0109] 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 high performance liquid 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).

[0110] (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)

[0111] 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 t Buok (40 mg, 0.36 mmol) was added to a microwave-safe tube containing 3 mL of THF. The sealed tube was microwave-safe 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 anhydrous 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]diaza-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).

[0112] (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)

[0113] 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] + .

[0114] 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).

[0115] (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)

[0116] 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).

[0117] (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)

[0118] 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-hydroxypropanediol (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 at 150 °C for 4 hours in a microwave reactor. Ethyl acetate (50 mL) and H₂O (50 mL) were then added to the reaction mixture. The organic phase was separated, washed with saturated NaCl 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).

[0119] (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)

[0120]

[0121]

[0122] 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] + .

[0123] 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 HATU (1.14 g, 3.00 mmol), DIEA (772 mg, 6.00 mmol), and N,O-dimethylhydroxylamine hydrochloride (389 mg, 4.00 mmol, CAS No.: 6638-79-5; manufacturer: Bied Pharmaceuticals) were added 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) and 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] + .

[0124] 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. After the reaction was complete, ethyl acetate (100 mL) and water (100 mL) were added to dilute the mixture. The organic phase was separated, further washed with saturated NaCl solution, dried with anhydrous Na₂SO₄, 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] + .

[0125] 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 tert-butyl acetate (220 mg, 0.59 mmol, CAS No.: 35000-38-5; manufacturer: BIDE Pharmaceuticals) was reacted at 100 °C for 3 hours with stirring. 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, yield 69%, yellow solid). LC-MS (ESI): m / z 484.1 [M+ H] + .

[0126] 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] + .

[0127] 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] + . 1 HNMR (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).

[0128] (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 28Exemplary preparation of ClN5OS (molecular weight 482.04)

[0129] 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. (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) was purified by high performance liquid chromatography (ACN in water, 0%–45%). 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).

[0130] (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)

[0131] 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).

[0132] (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 C26 H 28 Exemplary preparation of OClN5S (molecular weight 494.05)

[0133] 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).

[0134] (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)

[0135] 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).

[0136] (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 H 27 Exemplary preparation of ClN4O2S (molecular weight 482.15)

[0137] 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] + .

[0138] 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] + .

[0139] (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)

[0140] 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).

[0141] (30) Compound (S,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)

[0142] 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, CAS No.: 163457-23-6; purchased from: Bid Pharmaceuticals) 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 (S,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, 2H), 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, 2 H), 2.71 – 2.65 (m, 3 H), 2.51 – 2.30 (m, 5 H), 1.69 (s, 3H).

[0143] (31) Compound (S,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)

[0144] 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 (S,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).

[0145] (32) Compound (S,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)

[0146] Step 1: Synthesis of (S,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 (S,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).

[0147] The correspondences between reagents and abbreviations involved in the synthesis of important candidate compounds are as follows: TFA: Trifluoroacetic acid DCM: Dichloromethane HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate DIEA: N,N-Diisopropylethylamine DMF: N,N-dimethylformamide THF: Tetrahydrofuran Example 2: Effect of the compound on inhibiting Th17 cell differentiation 1. The compounds synthesized in Example 1 were initially screened based on their inhibitory effect on mouse Th17 differentiation. The specific steps are as follows: (1) Primary mouse naïve CD4+ T cells were isolated. 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).

[0148] (2) In vitro differentiation of primary naïve CD4+ T cells 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 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).

[0149] (3) The candidate compounds in Example 1 were added respectively, and the drug’s toxicity to T cells and whether it inhibited the differentiation of naïve CD4+ T cells toward Th17 were analyzed.

[0150] 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.

[0151] 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 is set as: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001; ns indicates no statistical difference (p≥0.05).

[0152] Based on the results of flow cytometry analysis, JQ1, JQ24, JQ30, and JQ31 showed strong inhibitory effects. Therefore, these four candidate compounds were screened and their IC50 values ​​in the differentiation of mouse and human Th17 cells were determined.

[0153] 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.

[0154] 2. Verification of the inhibitory effect of the compound 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℃ 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℃ 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.

[0155] (2) In vitro differentiation of human primary CD4 T cells.

[0156] 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).

[0157] 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.

[0158] like Figure 3 As 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 drug activity ranking in inhibiting human Th17 cell differentiation is: JQ31 > JQ30 > JQ24 > JQ1. Compared to JQ1's activity in inhibiting mouse Th17 cell differentiation, JQ31 is more advantageous in inhibiting human Th17 cell differentiation and has greater translational potential in clinical applications.

[0159] Example 3: Study of JQ24, JQ30, and JQ31 in rat pharmacokinetic (PK) 1. Experimental Materials and Methods (1) Laboratory animals Eleven 7-8 week old male SD rats (Sprague-Dawley) were purchased from Sprague (Suzhou) Biotechnology Co., Ltd., and divided into three groups of three rats each, with two rats remaining for future use.

[0160] (2) Experimental methods i) Table 1 Grouping and Dosing Regimens:

[0161] Note: * Fasting is required for 12 hours before oral administration, and feeding can resume 4 hours after administration.

[0162] ii) Sample collection and processing Blood collection time points: 0.083 h (5 min), 0.25 h (15 min), 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after drug administration. Blood volume: Approximately 100 μL of whole blood was collected at each time point. Processing procedure: Whole blood was placed in anticoagulant tubes containing K2-EDTA and stored on ice. Centrifuged (6800 × g, 6 min, 2-8°C) within 1 hour to separate plasma. Plasma was aliquoted and stored at -80°C for analysis.

[0163] iii) Data Analysis Methods Analytical Validation: Quality control (QC) samples were used to assess analytical accuracy, requiring >66.7% of QC samples to have an accuracy within the range of 80-120%. Pharmacokinetic Parameter Calculation: Non-compartmental model analysis was performed using Phoenix WinNonlin 8.3.5 (Pharsight, USA, FDA certified). Calculated parameters included: AUC(0-t): Area under the drug-time curve from 0 to the last measurable time point.

[0164] AUC(0-∞): The area under the drug-time curve extrapolated to infinity.

[0165] T 1 / 2 Eliminate half-life.

[0166] C max Maximum blood drug concentration.

[0167] T max Peak time.

[0168] (3) Experimental results (i) Results of PK study of JQ24 in SD rats According to the experimental design, after rats were orally administered 10 mg / kg JQ24, blood samples were collected at different time points. Plasma was collected by centrifugation, and the blood drug concentration was analyzed by LC-MS / MS. Based on the blood drug concentration data at each time point, a drug-time curve was fitted, as shown below. Figure 4 As shown.

[0169] The pharmacokinetic parameters of JQ24 were calculated based on the blood drug concentration (see Table 2 below). According to the test results, the time to peak concentration (Tmax) of JQ24 after oral administration was 0.67 ± 0.29 h, the peak concentration (Cmax) was 193.33 ± 126.37 ng / mL, and the half-life was 1.00 ± 0.42 h.

[0170] Table 2. Pharmacokinetic (PK) parameters of JQ24 administered orally to rats at a dose of 10 mg / kg

[0171] Note: G1-1, G1-2, and G1-3 refer to the numbers of the three mice that were orally administered JQ24.

[0172] (ii) Results of PK study of JQ30 in SD rats According to the experimental design, after rats were orally administered 10 mg / kg JQ30, blood samples were collected at different time points, plasma was collected by centrifugation, and blood drug concentrations were analyzed by LC-MS / MS. Based on the blood drug concentration data at each time point, a drug-time curve was fitted, as shown below. Figure 5 As shown.

[0173] The pharmacokinetic parameters of JQ30 were calculated based on the blood drug concentration (as shown in Table 3). According to the test results, the time to peak concentration (Tmax) of JQ30 after oral administration was 0.83 ± 0.29 h, the peak concentration (Cmax) was 350.67 ± 132.46 ng / mL, and the half-life was 1.60 ± 0.62 h.

[0174] Table 3. Pharmacokinetic (PK) parameters of oral administration of 10 mg / kg JQ30 to rats

[0175] Note: G2-4, G2-5, and G2-6 refer to the numbers of the three mice that were orally administered JQ30.

[0176] (iii) Results of PK study of JQ31 in SD rats According to the experimental design, after rats were orally administered 10 mg / kg JQ31, blood samples were collected at different time points, plasma was collected by centrifugation, and the blood drug concentration was analyzed by LC-MS / MS. Based on the blood drug concentration data at each time point, a drug-time curve was obtained as shown in the figure. Figure 6 As shown.

[0177] The pharmacokinetic parameters of JQ31 were calculated based on the blood drug concentration (as shown in Table 4). According to the test results, the time to peak concentration (Tmax) of JQ31 after oral administration was 1.00 ± 0.00 h, the peak concentration (Cmax) was 486.00 ± 145.51 ng / mL, and the half-life was 2.40 ± 0.63 h.

[0178] Table 4. Pharmacokinetic (PK) parameters of JQ31 administered orally to rats at a dose of 10 mg / kg

[0179] Note: G3-7, G3-8, and G3-9 refer to the serial numbers of the three mice that were orally administered JQ31.

[0180] Based on the combined blood drug concentrations of the above compounds, a drug-time curve was plotted using the mean of the three sample data in each group. Figure 7 The curve shows that JQ31 has a higher exposure level.

[0181] The above results indicate that JQ31 exhibits the highest exposure, with significantly better area under the curve (AUCinf) and peak concentration (Cmax) compared to other compounds, suggesting superior oral bioavailability. It also demonstrates relatively optimal metabolic stability, with the lowest clearance (7,706 mL / h / kg) and the longest half-life (2.40 h), indicating a slower metabolic process in vivo and a more prolonged therapeutic effect.

[0182] Example 4: Pharmacodynamic study of JQ31 in a mouse EAE model As can be seen from Examples 1-3, considering factors such as in vitro activity, metabolic stability, solubility, and pharmacokinetic performance, specifically, JQ31 exhibits good in vitro activity at IC50. 50 The concentration reached 71.82 nM, representing the best level among the series of compounds screened in this application. Furthermore, the pharmacokinetic performance of JQ31 in rats, including peak concentration (Cmax), area under the curve (AUC), and elimination half-life (t), was also evaluated. 1 / 2 JQ31 has the highest clearance rate and the lowest clearance rate, making it significantly superior to other compounds in terms of drug potential and the core advantage molecule in this series of compounds.

[0183] The EAE model is a classic model for studying autoimmune diseases. Validating the efficacy of JQ31 in this model can further clarify its therapeutic potential and mechanism of action in vivo, providing crucial pharmacodynamic data support for subsequent drug development. Specific experimental methods and results are as follows: (1) Preparation of reagents and candidate drugs (i) PTX (pertussis toxin, purchased from List Biological Lab, catalog number: 180): Dissolve 50 µg of PTX in 0.5 mL of ultrapure water to prepare a PTX stock solution of 100 ng / µL. Store at 4 °C and dilute with sterile PBS to 1 ng / µL before use.

[0184] (ii) MOG emulsion a. MOG (myelin oligodendrocyte glycoprotein, purchased from Shanghai Jier Biotechnology, catalog number: 051716): Dissolve 10 mg of MOG35-55 protein in 4 mL of sterile PBS to a concentration of 2.5 mg / mL, and store at -20 ℃ for later use. b. Emulsification of MOG with CFA (purchased from Chondrex, catalog number: 7023): Add 2.5 mg / mL MOG dropwise to CFA at a 1:1 volume ratio and emulsify thoroughly.

[0185] (iv) JQ31: Accurately weigh JQ31 and dissolve it thoroughly in DMSO. Based on the required daily dosage, first prepare a high-concentration drug (100 mg / kg dose group): Transfer an appropriate amount of JQ31 (DMSO solution) to a new 15 mL centrifuge tube, add solubilol at a volume ratio of 2.5%, and add physiological saline at a volume ratio of 92.5%, to a final concentration of 3 mg / mL. Then dilute with 5% DMSO + 2.5% solubilol + 92.5% physiological saline to 1 mg / mL and 0.3 mg / mL, respectively. Prepare once daily.

[0186] (2) Establishment of mouse EAE model C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were anesthetized, and 0.2 mL of MOG emulsion was injected subcutaneously into the back of each mouse, followed by 0.2 mL of PTX injected via the tail vein. The same dose of PTX was injected a second time after 48 h.

[0187] (3) Animal grouping and administration Eighty adult male C57BL / 6 mice were randomly divided into a sham-operated group (normal control mice, n = 10) and a model group (70 mice). On the third day after the first immunization, 60 mice in the model group were selected based on body weight and health status and divided into six groups: a solvent control group (n = 10) and three treatment groups with different doses of JQ31 (3, 10, and 30 mg / kg, n = 10). Subsequently, mice were administered the solvent (5% DMSO + 2.5% solutol + 92.5% saline) or the corresponding dose of JQ31 (3, 10, and 30 mg / kg) by gavage according to body weight. Administering was performed daily according to the experimental protocol in Table 5, and clinical scores and body weight changes were observed and recorded.

[0188] Table 5 Dosing Regimen

[0189] a: Solvent control: 5% DMSO + 2.5% solutol + 92.5% physiological saline.

[0190] b: BID: Twice a day.

[0191] (4) Clinical observation and clinical scoring criteria Routine monitoring included the effects of treatment on the animals' normal behavior, such as daily activities, food intake, water consumption, weight gain or loss, and other abnormalities in the eyes or coat. Any deaths or clinical symptoms observed during the trial were recorded in the raw data.

[0192] Animals are scored based on their symptoms after becoming ill: 0 points: No disease observed; 1 point: Weak tail; 2 points: Weak or partial paralysis of one hind limb; 3 points: Paralysis of both hind limbs; 4 points: Paralysis of both hind limbs, and weakness or partial paralysis of the forelimbs; 5 points: Death.

[0193] (5) Weight measurement Weigh yourself and record your weight every day.

[0194] (6) Sample collection At the peak of disease incidence in EAE mice, the experiment was terminated, and the following tissue samples were collected for subsequent testing (Table 6). Mice were euthanized, and spinal cord tissue samples were collected from each group. Lumbar spinal cord tissue samples were taken and fixed in 4% paraformaldehyde solution for histopathological examination.

[0195] Table 6. Tissue Sample Collection

[0196] (7) Inflammatory cell infiltration (H&E) in spinal cord tissue Spinal cord tissue was first fixed with 4% paraformaldehyde, then dehydrated using a gradient ethanol process, embedded, and prepared into 3 μm thick paraffin sections. After baking, dewaxing, and rehydration, the sections were stained with hematoxylin and eosin (H&E) according to the H&E staining kit manual and mounted. The tissue sections were observed using an Olympus BX53 microscope, and images were acquired under 20x objective lens. Finally, quantitative histopathological assessment was performed according to the following criteria: 0 points: No inflammation; 1 point: Cell infiltration is limited to the perivascular area and meninges; 2 points: Mild cell infiltration (less than one-third of the white matter area is infiltrated by inflammatory cells); 3 points: Moderate cell infiltration (more than one-third of the white matter area is infiltrated by inflammatory cells); 4 points: Inflammatory cell infiltration can be observed throughout the entire white matter area.

[0197] (8) Statistical analysis All experimental results are expressed as mean ± standard error, and statistical analysis was performed using GraphPad Prism 8.0 software. The significance level was set at * p <0.05,** p<0.01, *** p <0.001, **** p <0.0001; ns indicates no statistical difference (p≥0.05).

[0198] (9) Experimental results During the experiment, the sham-operated group mice maintained normal weight gain and exhibited no abnormal behavior. Before the onset of the disease, the weight of mice in all treatment groups was within the normal range. In the high-dose JQ31 groups (10, 30 mg / kg), a few mice experienced a brief weight loss (<10%) in the initial treatment period (days 3-5), but did not show signs of toxicity such as reduced food intake or abnormal activity; their weight gradually returned to normal within the following week. From day 12 onwards, except for the sham-operated group, mice in all groups successively developed disease symptoms accompanied by weight loss, reaching near their lowest weight on days 15-16, and then slowly recovered. Figure 8 Mice in the JQ31-treated groups (3 mg / kg, 10 mg / kg, 30 mg / kg) were generally in better condition than the solvent control group, with a dose-dependent improvement in body weight loss. No drug-related deaths occurred in any of the JQ31 dose groups, and the degree of body weight loss was within an acceptable range, indicating that JQ31 is safe at the experimental doses.

[0199] Mice were scored according to the clinical scoring criteria in (4), such as Figure 9 As shown, mice in the solvent control group began to show disease symptoms on day 12, reaching a peak on day 18 with a clinical score as high as 2.6 and an incidence rate of 100%. All of these indicators were within the normal range for our laboratory, indicating a normal EAE mouse model and controllable disease. The clinical scores of the 3 mg / kg, 10 mg / kg, and 30 mg / kg JQ31 treatment groups on day 18 were 2.3, 2.0, and 1.6, respectively, all lower than those of the solvent control group. During administration, JQ31 demonstrated a dose-dependent inhibitory effect on the disease score of EAE mice.

[0200] Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. It causes somatic sensory or motor dysfunction, accompanied by muscle malnutrition and atrophy, leading to weight loss. EAE mice also exhibit weight loss during disease progression, reflecting the severity of the disease. Similar to the disease progression in conventional EAE mouse models, the weight of these mice gradually decreased from day 10, reaching its lowest point around day 16. Figure 10 Different doses of JQ31 also showed different levels of weight loss, with 3, 10, and 30 mg / kg of JQ31 showing dose-dependent protection against weight loss in mice.

[0201] The H&E staining results of the spinal cord tissue were consistent with the clinical score. Figure 11 The Vehicle group of mice showed extensive immune cell infiltration in the spinal cord tissue, with an inflammation score as high as 2.6. Compared with the solvent control group, inflammatory cell infiltration was reduced in spinal cord samples from mice in all treatment groups. Specifically, the inflammation scores in the 3 mg / kg, 10 mg / kg, and 30 mg / kg JQ31 groups were 2.3, 1.9, and 1.6, respectively, showing a dose-dependent relationship. These results further clarify the therapeutic effect of JQ31 in the EAE mouse model.

[0202] The results showed that JQ31 dose-dependently reduced the clinical score of EAE mice, protected against weight loss, and reduced spinal cord inflammatory cell infiltration.

Claims

1. A pharmaceutical composition, characterized in that, It comprises a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient. Equation (I) Wherein, R1 and R2 are independently selected from C2-C6 straight-chain alkyl groups, or R1 and R2 together with the N atom attached to them. Piperidinyl, aziridine-heptyl, or aziridine-octyl; R 1a and R 1b Each of the following is independently selected from H, halogen, methyl, or halogen-substituted methyl groups; R3, R4, and R5 are each independently selected from H, halogens, C1-3 alkyl groups, or halogen-substituted methyl groups; The R6 is selected from halogens; and The n is selected from 0 to 6.

2. The pharmaceutical composition according to claim 1, characterized in that, R1 and R2 are each independently selected from C2 or C3 straight-chain alkyl groups, or R1 and R2 together with the N atom attached to them. Or piperidinyl; The R 1a and R 1b They are selected independently from H and halogens, respectively.

3. The pharmaceutical composition according to claim 1, characterized in that, The n is selected from 0 to 3; And / or, R3, R4, and R5 are each independently selected from C1-3 straight-chain alkyl groups.

4. The pharmaceutical composition according to claim 1, characterized in that, R1 and R2 are each independently selected from C2 alkyl groups, or R1 and R2 together with the N atom attached to them. The R 1a and R 1b Each is independently selected from H or halogen; The n is 1; and R3, R4, and R5 are each independently selected from C1 alkyl groups.

5. The pharmaceutical composition according to claim 1, characterized in that, The compound represented by formula (I) is selected from any of the following: , , Or, or its pharmaceutically acceptable salt, stereoisomer, or tautomer.

6. A compound of formula (I) or a pharmaceutically acceptable salt thereof: Equation (I) in, R1 and R2 are each independently selected from C2-C6 straight-chain alkyl groups, or R1 and R2 together with the N atom attached to them. Piperidinyl, aziridine-heptyl, or aziridine-octyl; R 1a and R 1b Each of the following is independently selected from H, halogen, methyl, or halogen-substituted methyl groups; R3, R4, and R5 are each independently selected from H, halogens, C1-3 alkyl groups, or halogen-substituted methyl groups; and The R6 is selected from halogens; and The n is selected from 0-6; Wherein, the compound represented by formula (I) is not any of the following: , , Or, or its pharmaceutically acceptable salt, stereoisomer, or tautomer.

7. Use of the pharmaceutical composition according to any one of claims 1-5 or the compound according to claim 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament / reagent for inhibiting Th17 cell differentiation.

8. The use according to claim 7, characterized in that, The Th17 cells are derived from humans or mammals.

9. Use of the pharmaceutical composition according to any one of claims 1-5 or the compound according to claim 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating an autoimmune disease, wherein the autoimmune disease is a Th17-mediated autoimmune disease.

10. The use according to claim 9, characterized in that, The autoimmune diseases mentioned include: psoriasis, vitiligo, dermatitis, autoimmune encephalomyelitis, asthma, Arthur's reaction, rheumatoid arthritis, multiple sclerosis, autoimmune myocarditis, autoimmune uveitis, type 1 diabetes, systemic lupus erythematosus, and inflammatory bowel disease.

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