Benzimidazole compound and use thereof

Abstract

A benzimidazole compound and a use thereof. The compound is as shown in formula (I) or (II). The compound can inhibit inflammasomes, exhibits high activity targeting NLRP3 inflammasomes, and can be used in the preparation of a drug for treating diseases related to NLRP3 inflammasomes.

Description

Benzimidazole compounds and their applications Technical Field

[0001] This invention relates to a compound, its preparation method and application, and more particularly to a benzimidazole compound and its application, belonging to the field of pharmaceutical compounds. Background Technology

[0002] The NLRP3 inflammasome has been extensively studied in the field of inflammation, representing one of the most thoroughly researched areas. The NLRP3 inflammasome is a protein complex composed of NLRP3 protein, an apoptosis-associated speck-like protein (ASC) domain, and a cysteine-containing aspartate-specific proteinase-1 (Caspase-1). NLRP3 mainly includes an N-terminal pyrin domain (PYD), a central NACHT domain (nucleoside triphosphatase domain), and a C-terminal LRR (leucine-rich repeat) domain. The ASC contains two conserved domains: the PYD domain and the caspase-recruitment domain (CARD). The ASC domain acts as a bridge between NLRP3 and pro-caspase-1, binding to the upstream NLRP3 protein and pro-caspase-1 through PYD-PYD and CARD-CARD isotype interactions, respectively. Pro-caspase-1 consists of a CARD domain and a p20-p10 domain. The p20-p10 domain can cleave under activation conditions to generate p20 and p10, transforming into mature caspase-1. NLRP3 protein oligomerizes and recruits the ASC domain. The ASC domain then polymerizes, assembling into a single, abundant fibrous structure called an "ASC spot," which binds to caspase-1 and allows it to approach and induce activation. Once activated, caspase-1 cleaves pro-IL-1β and pro-IL-18 into homologous inflammatory cytokines and cleaves Gasdermin D (GSDMD). GSDMD cleavage releases the pore-forming N-terminal region from the autoinhibitory C-terminal domain, ultimately leading to pyroptosis.

[0003] Through a deeper understanding of the NLRP3 inflammasome activation pathway, the applicant focused on the process by which NEK7 assists in the conformational changes of NLRP3. NEK7 assists in opening the closed bicyclic cage conformation by bridging adjacent LRR domains in the NLRP3 oligomer, ultimately forming the disc-shaped active NLRP3 structure.

[0004] Some existing benzimidazole compounds can block the interaction between NLRP3 and NEK7 through their LRR region. However, the bioactivity of imidazopyridine core series compounds still needs further improvement. Summary of the Invention

[0005] Objective of the invention: The present invention aims to provide a benzimidazole compound with good biological activity; another objective of the present invention is to provide an application of the benzimidazole compound.

[0006] Technical solution: The compounds of the present invention as shown in general formula (I) or (II) or their pharmaceutically acceptable salts:

[0007] Among them, R 1 for R a C1-C6 alkylene or -SO2-

[0008] R b It is a halogen;

[0009] R 2 for R C H, C1-C6 alkyl, halogen, or -NO2; R d for P is 1-5; q is 0-3;

[0010] L is R e For -S-, -SO-, -SO2-, or -NH-; R f The following are not specified: C1-C6 alkylene, -SO2-, or -CO- (carbonyl); where Rf represents the absence of Rf and L represents the absence of Rf.

[0011] R 3 for R g It is a benzene ring, a 4-6 membered aliphatic ring, or a 4-5 membered heterocycle containing an oxygen atom;

[0012] R h It can be halogen, H, C1-C6 alkyl, C1-C6 alkoxy, -CN, trifluoromethyl or -O-CF3;

[0013] R 4 for R i It is a C1-C6 alkyl or C2-C4 alkenyl group.

[0014] Preferably, L is Among them, the one closest to R e The linker is attached to the imidazopyridine nucleus, near R. f The connection key and R 3 connect.

[0015] Furthermore, R 1 for R a It is a C1-C3 alkylene group or -SO2-.

[0016] R b It is a halogen;

[0017] R 2 for R C H, C1-C3 alkyl, halogen, or -NO2; R d for P is 1-5; q is 0-3;

[0018] Where L is R e For -S-, -SO-, -SO2-, or -NH-; R f It is absent, C1-C6 alkylene, -SO2- or -CO-;

[0019] R 3 for R g It is a benzene ring, a 4-6 membered aliphatic ring, or a 4-5 membered heterocycle containing an oxygen atom;

[0020] R h It can be a halogen, H, C1-C3 alkyl, C1-C3 alkoxy, -CN, trifluoromethyl or -O-CF3;

[0021] R 4 for R i It is a C1-C3 alkyl or C2-C4 alkenyl group.

[0022] Furthermore, R 1 for R a It is methylene or -SO2-.

[0023] R b It can be F, Cl, Br or I;

[0024] R 2 for R C H, F, Cl, Br or I, methyl, propyl, -NO2; R d for P is 1, 2, 3, 4 or 5; q is 0, 1, 2 or 3.

[0025] Preferably, R 1 Selected from:

[0026] R 2 Selected from:

[0027] Furthermore,

[0028] L is R e For -S-, -SO-, -SO2-, or -NH-; R f For non-existent, methylene (-CH2-), -SO2-, or -CO-

[0029] R 3 for R g It is a benzene ring, cyclopentyl, cyclohexyl, or a 4-membered heterocycle containing one oxygen atom and three carbon atoms;

[0030] R h The following are the possible values: F, Cl, Br, I, H, methyl, methoxy, -CN, trifluoromethyl, -O-CF3;

[0031] R 4 for R d for P is 1, 2, 3, 4, or 5; q is 0, 1, 2, or 3; R i It can be ethyl or vinyl.

[0032] Preferably,

[0033] L is selected from:

[0034] R 3 Selected from:

[0035] R 4 Selected from:

[0036] Preferably, L is selected from: In this group, the left-side connecting bonds of each group are connected to the imidazopyridine core, and the right-side connecting bonds are connected to R. 3 connect.

[0037] On the other hand, the present invention provides a compound or a pharmaceutically acceptable salt thereof, selected from any of the following structures:

[0038] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0039] On the other hand, the present invention provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating NLRP3 inflammasome-related diseases.

[0040] Preferably, the NLRP3 inflammasome-related diseases are selected from chronic inflammation, autoimmune diseases, metabolic diseases, neurodegenerative diseases, gout, pseudogout, atherosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type II diabetes, Alzheimer's disease, Parkinson's disease, multiple sclerosis, kidney injury, renal fibrosis, acute and chronic heart failure, liver fibrosis, liver injury, inflammatory bowel disease, rheumatoid arthritis, cardiovascular and cerebrovascular diseases, etc.

[0041] On the other hand, the present invention provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of an NLRP3 inflammasome inhibitor.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The compounds of the present invention have high biological activity and can exert inhibitory effects in both the initiation and activation stages of the NLRP3 inflammasome. In the initiation stage, it may inhibit IL-1β expression, thereby reducing the production of inflammatory mediators; in the activation stage, it significantly reduces the secretion of IL-1β in LPS and ATP-induced THP-M cells. Attached Figure Description

[0043] Figure 1 shows the IC50 of compound JM-31 in inhibiting the NLRP3 inflammasome. 50 THP-M cells were treated with different concentrations of compounds for 24 hours, followed by LPS for 3 hours and ATP for 1 hour. The IL-1β content in the cell supernatant was detected by ELISA, and the IC50 was calculated. 50 Values. Three independent experiments were conducted for each concentration, expressed as mean ± sem (n = 3).

[0044] Figure 2 illustrates the inhibitory effect of compound JM-31 on the NLRP3 inflammasome. Western blotting was used to analyze the effect of compound JM-31 on the levels of key proteins in the NLRP3 inflammasome pathway.

[0045] Figure 3 shows the effects of compound JM-31 on different stages of the NLRP3 inflammasome pathway. THP-M cells were treated with compound JM-31 for 24 h before (a) and after (b) LPS treatment for 3 h, followed by ATP treatment for 1 h. The IL-1β content in the cell supernatant was detected by ELISA. Each data point represents three independent experiments and is expressed as mean ± sem (n = 3), **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0046] Figure 4 shows the effect of compound JM-31 on the transcriptional levels of key proteins in the NLRP3 inflammasome pathway. THP-M cells were treated with compound JM-31 for 24 h, followed by LPS treatment for 4 h. NLRP3 and IL-β levels were detected by RT-qPCR, with GAPDH as an internal control. Each data point represents three independent experiments and is expressed as mean ± sem (n = 3). NS: not significant, *p < 5, **p < 0.01. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0048] Examples 1-32 below illustrate the synthesis of intermediates.

[0049] Synthesis of Intermediate 2 in Example 1

[0050] Synthesis of 3H-imidazo[4,5-b]pyridine-2-thiol (2)

[0051] 1.0 g (9.16 mmol) of 2,3-diaminopyridine was dissolved in a 12 mL mixture of ethanol and water. Potassium hydroxide (514.10 mg, 9.16 mmol) and carbon disulfide (1.40 g, 18.33 mmol) were added, and the mixture was stirred in a sand bath at 80 °C for 8 h until TLC analysis showed complete reaction of starting material 1. The reaction solution was cooled to room temperature, the pH was adjusted to slightly acidic, and the mixture was allowed to stand for 2 h to precipitate a black solid. The precipitate was filtered and dried under vacuum. 1.08 g of a black solid was obtained, with a yield of 78%.

[0052] Synthesis of Intermediate 3 in Example 2

[0053] Synthesis of 2-((cyclohexylmethyl)thio)-3H-imidazo[4,5-b]pyridine (3)

[0054] Intermediate 2 (0.5 g, 3.31 mmol) was weighed and dissolved in DMF (2 mL). Na₂CO₃ (701.04 mg, 6.61 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, bromomethylcyclohexane (708.78 mg, 3.97 mmol) was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 2. The reaction mixture was cooled to ambient temperature, and then diluted with a large amount of water. The pH of the solution was adjusted to near neutral, and the mixture was extracted three times with EA. The organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na₂SO₄. The mixture was purified by column chromatography using a DCM-MeOH system to give 0.45 g of a dark brown solid, with a yield of 55%. 1 H NMR(300MHz, DMSO-d6)δ(ppm):12.93(d,J=139.9Hz,1H),8.19(d,J=32.2Hz,1H),7.90–7.66(m,1H),7.13(t,J=6.5H z,1H),3.23(d,J=6.9Hz,2H),1.89–1.79(m,2H),1.73–1.55(m,4H),1.27–1.09(m,3H),1.02(qd,J=12.1,3.4Hz,2H).

[0055] Synthesis of Intermediate 5 in Example 3

[0056] Synthesis of 2-((4-fluorophenyl)thio)-1H-benzo[d]imidazole (5)

[0057] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 50 °C for 0.5 h. Then, 496.78 μL (3.99 mmol) of p-fluorobenzyl bromide was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. After filtration, the precipitate was purified by slurry mixing in a PE-EA system, yielding 0.80 g of a white crystalline solid, with a yield of 93%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.47 (m, 4H), 7.18–7.07 (m, 4H), 4.55 (s, 2H).

[0058] Synthesis of Intermediate 6 in Example 4

[0059] Synthesis of 2-((cyclohexylmethyl)thio)-1H-benzo[d]imidazole (6)

[0060] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of N,N-dimethylformamide. Anhydrous sodium carbonate (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 561.43 μL (3.99 mmol) of bromomethylcyclohexane was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by PE-EA column chromatography, yielding a white needle-like solid. 1 H NMR (300MHz, Chloroform-d) δ (ppm): 7.50 (s, 2H), 7.19 (dd, J = 6.0, 3.2Hz, 2H), 3.27 (d, J =6.9Hz,2H),1.88(d,J=12.5Hz,2H),1.68(m,4H),1.33–1.13(m,3H),1.13–0.94(m,2H).

[0061] Synthesis of Intermediate 8 in Example 5

[0062] Synthesis of 2-((cyclohexylmethyl)thio)-1H-imidazolium (8)

[0063] 0.5 g (4.99 mmol) of 2-mercaptoimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (1.06 mg, 9.99 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 1.33 g (7.49 mmol) of bromomethylcyclohexane was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 7. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. The solution was extracted three times with EA, and the organic phases were combined and washed with saturated brine. The solution was purified by PE-EA column chromatography to give 0.42 g of a white crystalline solid, with a yield of 43%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 12.15 (s, 1H), 7.11 (s, 1H), 6.90 (s, 1H), 2.88 (d, J = 6.8Hz, 2H ),1.82–1.72(m,2H),1.70–1.57(m,1H),1.43(m,1H),1.21–1.04(m,3H),1.01–0.83(m,2H).

[0064] Synthesis of Intermediate 9 in Example 6

[0065] Synthesis of 2-((4-fluorobenzyl)thio)-1H-imidazolium (9)

[0066] 0.5 g (4.99 mmol) of 2-mercaptoimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (1.06 mg, 9.99 mmol) was added, and the mixture was stirred in a sand bath at 50 °C for 1 h. Then, 1.50 g (5.99 mmol) of p-bromobenzyl bromide was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of the starting material. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. The solution was extracted three times with EA (extractant ether), and the organic phases were combined, washed with a saturated sodium chloride solution, and dried. The solution was purified by PE-EA column chromatography to give 0.83 g of a white crystalline solid, with a yield of 62%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 12.22 (s, 1H), 7.45 (m, 2H), 7.23–7.08 (m, 3H), 6.94 (s, 1H), 4.19 (s, 2H).

[0067] Synthesis of Intermediate 11 in Example 7

[0068] Synthesis of N-(4-fluorobenzyl)-2-nitroaniline (11)

[0069] 2-Mercaptoimidazole (2.0 g, 14.48 mmol) was dissolved in N,N-dimethylformamide (20 mL). K₂CO₃ (2.40 mg, 17.38 mmol) was added under ice bath conditions, followed by slow, dropwise addition of p-fluorobenzyl bromide (2.74 g, 14.48 mmol). The reaction mixture was heated to 60 °C until TLC analysis showed complete reaction of the starting material. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. The solution was extracted three times with EA (extractant extractant), and the organic phases were combined and washed with saturated brine. The solution was concentrated under reduced pressure and purified by PE-EA column chromatography to give 2.40 g of a yellow oil, with a yield of 67%.

[0070] Synthesis of Intermediate 12 in Example 8

[0071] Synthesis of 1-(4-fluorobenzyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (12)

[0072] Intermediate 11 (2.40 g, 9.75 mmol) was dissolved in anhydrous ethanol (20 mL), and iron powder (2.72 g, 48.73 mmol) and a saturated solution of ammonium chloride were added with stirring at room temperature. The mixture was refluxed at 85 °C for 3–4 h until the reaction was complete as detected by TLC. The solid in the reaction system was removed by diatomaceous earth filtration, the solvent was evaporated, diluted with an appropriate amount of water, and extracted three times with EA. The combined organic phases were washed with a saturated sodium chloride solution. The solvent was evaporated, dissolved in anhydrous tetrahydrofuran (10 mL), and N,N-carbonyldiimidazole (4.75 g, 29.27 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated, and the mixture was dried under vacuum to obtain a yellow solid.

[0073] Synthesis of Intermediate 13 in Example 9

[0074] Synthesis of 4-fluoro-N-(2-nitrophenyl)benzenesulfonamide (13)

[0075] 4-Fluorobenzenesulfonyl chloride (2.82 g, 14.48 mmol) was slowly added dropwise to a solution of o-nitroaniline (2.0 g, 14.48 mmol) in anhydrous pyridine (10 mL). The mixture was stirred in a sand bath at 125 °C for 6 h until all the reactants had reacted. After cooling, the reaction solution was diluted with a large amount of water, extracted with EA, and the organic phases were combined, washed with a saturated NaCl solution, and dried over anhydrous sodium sulfate. The solution was purified by column chromatography using a PE-EA system to give 4.0 g of a yellow solid, with a yield of 93%.

[0076] Synthesis of Intermediate 14 in Example 10

[0077] Synthesis of 1-((4-fluorophenyl)sulfonyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (14)

[0078] Intermediate 13 (4.0 g, 13.50 mmol) was dissolved in anhydrous ethanol (30 mL), and iron powder (3.77 g, 67.51 mmol) and a saturated solution of ammonium chloride were added with stirring at room temperature. The mixture was refluxed at 85 °C for 3–4 h until the reaction was complete as detected by TLC. The solid was filtered off with diatomaceous earth, the solvent was evaporated, and an appropriate amount of water was added. After extraction with EA, the organic phases were combined and washed with a saturated solution of NaCl. The mixture was dried over anhydrous sodium sulfate. The solvent was evaporated, dissolved in anhydrous tetrahydrofuran (10 mL), and N,N-carbonyldiimidazole (6.58 g, 40.56 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated, and the mixture was dried under vacuum to give a yellow solid.

[0079] Synthesis of Intermediate 15 in Example 11

[0080] Synthesis of 2-((4-chlorobenzyl)thio)-1H-benzo[d]imidazole (15)

[0081] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 50 min. 523 μL (4.00 mmol) of p-chlorobenzyl bromide was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.70 g of a white solid (77% yield). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 12.59 (s, 1H), 7.52–7.31 (m, 6H), 7.13 (m, 2H), 4.55 (s, 2H).

[0082] Synthesis of Intermediate 16 in Example 12

[0083] Synthesis of 2-((4-bromobenzyl)thio)-1H-benzo[d]imidazole (16)

[0084] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 ml of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 550 μl (4.02 mmol) of 4-bromobenzyl bromide was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 1.01 g of a white solid (94% yield). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.54–7.36 (m, 6H), 7.12 (m, 2H), 4.54 (s, 2H).

[0085] Synthesis of Intermediate 17 in Example 13

[0086] Synthesis of 2-(benzylthio)-1H-benzo[d]imidazole (17)

[0087] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, benzyl bromide (475 μL, 4.00 mmol) was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.69 g of a white solid, with a yield of 86%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.49–7.40 (m, 4H), 7.29 (m, 3H), 7.12 (m, 2H), 4.56 (s, 2H).

[0088] Synthesis of Intermediate 18 in Example 14

[0089] Synthesis of 2-((4-methylbenzyl)thio)-1H-benzo[d]imidazole (18)

[0090] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 560 μL (4.01 mmol) of 4-methylbenzyl bromide was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.61 g of a white solid (72% yield). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.44 (dd, J=5.9, 3.2Hz, 2H), 7.32 (d, J=7.7Hz, 2H), 7.18–7.03 (m, 4H), 2.25 (s, 3H).

[0091] Synthesis of Intermediate 19 in Example 15

[0092] Synthesis of 2-((4-methoxybenzyl)thio)-1H-benzo[d]imidazole (19)

[0093] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 583 μL (4.00 mmol) of 4-methoxybenzyl bromide was slowly added dropwise, and the reaction continued until reactant 4 was completely reacted. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.45 g of a white solid (50% yield). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 12.54 (s, 1H), 7.44 (s, 2H), 7.36 (d, J = 8.3Hz, 2H), 7.12 (m, 2H), 6.86 (d, J = 8.3Hz, 2H), 4.51 (s, 2H), 3.71 (s, 3H).

[0094] Synthesis of Intermediate 20 in Example 16

[0095] Synthesis of 2-((4-(trifluoromethyl)benzyl)thio)-1H-benzo[d]imidazole (20)

[0096] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 ml of N,N-dimethylformamide. Anhydrous sodium carbonate (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 618 μl (4.00 mmol) of 4-trifluoromethylbenzyl bromide was slowly added dropwise, and the reaction continued until reactant 4 was completely reacted. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.79 g of a white solid, with a yield of 76%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.67 (s, 4H), 7.45 (d, J = 5.3Hz, 2H), 7.17–7.09 (m, 2H), 4.65 (s, 2H).

[0097] Synthesis of Intermediate 21 in Example 17

[0098] Synthesis of 2-((4-methylbenzyl)thio)-1H-benzo[d]imidazole (21)

[0099] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 560 μL (4.01 mmol) of 4-methylbenzyl bromide was slowly added dropwise, and the reaction continued until TLC analysis showed complete reaction of starting material 4. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.81 g of a white solid (75% yield). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.64–7.53 (m, 2H), 7.44 (dt, J = 7.4, 3.7Hz, 2H), 7.30 (d, J = 8.1Hz, 2H), 7.12 (m, 2H), 4.59 (s, 2H).

[0100] Synthesis of Intermediate 22 in Example 18

[0101] Synthesis of 4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzonitrile (22)

[0102] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of N,N-dimethylformamide. Anhydrous sodium carbonate (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 784 mg (4.00 mmol) of 4-bromomethylbenzonitrile was slowly added dropwise, and stirring continued until reactant 4 was completely reacted. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.56 g of a white solid, with a yield of 63%.

[0103] Synthesis of intermediate 23 in Example 19

[0104] Synthesis of 2-((cyclopropylmethyl)thio)-1H-benzi[d]imidazole (23)

[0105] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of DMF. Na₂CO₃ (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 523 μl (4.00 mmol) of bromomethylcyclopropane was slowly added dropwise, and stirring continued until the reaction of reactant 4 was complete. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.45 g of a white solid (58% yield). 1H NMR (300MHz, DMSO-d6) δ (ppm): 12.48 (s, 1H), 7.42 (d, J = 41.4Hz, 2H), 7.10 (dd, J = 6.0, 3.1Hz ,2H),3.29(d,J=7.3Hz,2H),2.21(m,1H),1.79(m,2H),1.69–1.42(m,4H),1.36–1.21(m,2H).

[0106] Synthesis of Intermediate 24 in Example 20

[0107] Synthesis of 2-((oxetane-3-ylmethyl)thio)-1H-benzo[d]imidazole (24)

[0108] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of N,N-dimethylformamide. Anhydrous sodium carbonate (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 385 μL (4.00 mmol) of 3-bromomethyloxetane was slowly added dropwise, and stirring continued until reactant 4 was completely reacted. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by slurry extraction using a PE-EA system, yielding 0.32 g of a white solid, with a yield of 44%.

[0109] Synthesis of Intermediate 26 in Example 21

[0110] Synthesis of 4-(bromomethyl)-N-(3-methoxypropyl)benzamide (26)

[0111] 1.0 g (9.30 mmol) of 4-bromomethylbenzoic acid was dissolved in 10 mL of anhydrous dichloromethane. 1.0 g (11.16 mmol) of 3-methoxypropylamine was added, and the mixture was stirred in a sand bath at 0 °C for 20–30 min. Then, 4.24 g (4.01 mmol) of DIPEA was slowly added dropwise, and the reaction was continued for 1 h. The mixture was concentrated under reduced pressure and purified by column chromatography using a PE / EA system to give 2.26 g of a white solid, with a yield of 85%.

[0112] Synthesis of intermediate 27 in Example 22

[0113] Synthesis of 2-((4-fluorobenzyl)sulfinyl)-1H-benzo[d]imidazole (27)

[0114] Intermediate 5 (0.5 g, 1.94 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). After stirring in an ice bath for 30 min, OXONE (0.38 g, 1.26 mmol) aqueous solution was added in portions, and stirring was maintained for 1 h. After the starting material reacted completely, it was diluted with a large amount of water, and the pH was adjusted to 6-7 with saturated NaHCO3 solution. Extraction was performed using EA, and the organic phases were combined and washed with saturated NaCl solution. The mixture was concentrated under reduced pressure, purified by column chromatography in a PE / EA system, and dried under vacuum to give 0.28 g of white solid, with a yield of 52%. 1 H NMR (300MHz, Chloroform-d) δ (ppm): 10.80 (s, 1H), 7.84 (s, 1H), 7.46 (s, 1H), 7.37 (p, J = 4.8, 3.8Hz, 2H), 7.04 (m, 2H), 6.90 (m, 2H), 4.58–4.26 (m, 2H).

[0115] Synthesis of intermediate 28 in Example 23

[0116] Synthesis of 2-((4-fluorobenzyl)sulfonyl)-1H-benzo[d]imidazole (28)

[0117] Intermediate 5 (0.5 g, 1.94 mmol) was weighed and dissolved in anhydrous tetrahydrofuran (5 mL). After stirring at room temperature for 30 min, an aqueous solution of OXONE (0.76 g, 2.52 mmol) was added, and the mixture was stirred overnight at room temperature. After the starting material reacted completely, it was diluted with a large amount of water, and the pH was adjusted to 6-7 with saturated NaHCO3 solution. The mixture was extracted with EA, and the organic phases were combined and washed with saturated NaCl solution. The solution was concentrated under reduced pressure, purified by column chromatography in a PE / EA system, and dried under vacuum to give 0.41 g of a white solid, with a yield of 73%. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 13.79 (s, 1H), 7.70 (s, 2H), 7.39 (m, 2H), 7.27 (dd, J = 8.5, 5.6Hz, 2H), 7.14 (t, J = 8.7Hz, 2H), 5.00 (s, 2H).

[0118] Synthesis of intermediate 29 in Example 24

[0119] Synthesis of 2-((cyclohexylmethyl)thio)-1H-benzo[d]imidazole (29)

[0120] 0.5 g (3.33 mmol) of 2-mercaptobenzimidazole was dissolved in 2 mL of N,N-dimethylformamide. Anhydrous sodium carbonate (705.65 mg, 6.66 mmol) was added, and the mixture was stirred in a sand bath at 45 °C for 1 h. Then, 561.43 μL (3.99 mmol) of bromocyclohexane was slowly added dropwise, and stirring continued until reactant 4 was completely reacted. After cooling, the reaction solution was diluted with a large amount of water to adjust the pH to near neutral. At this point, a large amount of white flocculent precipitate formed. The precipitate was purified by PE-EA column chromatography, yielding 0.401 g of a white needle-like solid, with a yield of 49%.

[0121] Synthesis of intermediate 31 in Example 25

[0122] Synthesis of 4-((2-chloro-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (31)

[0123] 2-Chlorobenzimidazole (2 g, 13.31 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL). Sodium hydroxide (1.05 g, 7.51 mmol) was rapidly added with stirring at room temperature, followed by intermediate 15 (3.00 g, 10.49 mmol). The mixture was stirred at room temperature for 24 h. After the starting material reacted completely, it was diluted with a large amount of water and extracted three times with EA. The organic phases were combined and washed with a saturated NaCl solution. The solution was concentrated under reduced pressure, purified by column chromatography in a PE / EA system, and dried under vacuum to give 0.78 g of a pale yellow solid, with a yield of 81%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.41 (t, J = 5.7Hz, 1H), 7.78 (d, J = 8.2Hz, 2H), 7.69–7.60 (m, 1H), 7.61–7.55 (m, 1H), 7.3 0–7.19(m,4H),5.58(s,2H),3.36(s,1H),3.31(d,J=6.5Hz,1H),3.28–3.22(m,2H),3.21(s,3H),1.72(p,J=6.6Hz,2H).

[0124] Synthesis of intermediate 33 in Example 26

[0125] Synthesis of 2-((4-fluorobenzyl)sulfonyl)-1H-benzo[d]imidazole (33)

[0126] 0.5 g (3.76 mmol) of 2-aminobenzimidazole was dissolved in 5 mL of anhydrous DMF. Triethylamine (0.76 g, 7.51 mmol) was added with stirring at room temperature, followed by the slow dropwise addition of 1.19 g (7.51 mmol) of 4-fluorobenzoyl chloride, while maintaining stirring at room temperature. After the starting material reacted completely, the mixture was diluted with a large amount of water and extracted three times with ethyl acetate. The organic phases were combined, washed with a saturated NaCl solution, and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure and dried under vacuum to give 0.78 g of a off-white solid, with a yield of 81%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.26–8.17 (m, 2H), 8.05–7.96 (m, 1H), 7.45 (m, 2H), 7.32 (m, 3H), 7.14 (dd, J = 5.9, 3.2Hz, 2H).

[0127] Synthesis of intermediate 34 in Example 27

[0128] Synthesis of N-(1H-benzo[d]imidazol-2-yl)-4-fluorobenzenesulfonamide (34)

[0129] 0.5 g (3.76 mmol) of 2-aminobenzimidazole was dissolved in 5 mL of anhydrous dichloromethane. 4-fluorobenzenesulfonyl chloride (0.876 g, 4.51 mmol) was added with stirring in an ice bath, followed by stirring for 10 min. The mixture was then stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure, purified by PE / EA column chromatography, and dried under vacuum to give 0.63 g of a white solid (57% yield). 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.04–7.97(m,2H),7.71(dd,J=8.3,1.1Hz,1H),7.30(dd,J=7.9,1.3Hz,1H),7.25–7.04(m,5H),6.13(s,2H).

[0130] Synthesis of Intermediate 35 in Example 28

[0131] Synthesis of tert-butyl 4-(4-(bromomethyl)benzoyl)piperazine-1-carboxylic acid ester (35)

[0132] 4-Bromomethylbenzoic acid (1.0 g, 4.65 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (15 mL). 1-tert-butyloxycarbonylpiperazine (0.87 g, 4.65 mmol), HOBT (0.75 g, 5.58 mmol), and EDCI (1.07 g, 5.58 mmol) were added, and the mixture was stirred at room temperature for 4-5 h. After the reaction mixture was completely reacted (TLC monitoring), stirring was stopped. The reaction solution was diluted with a large amount of water and extracted 2-3 times with EA. The organic phases were combined and washed with saturated NaCl solution. The solution was concentrated under reduced pressure and purified by column chromatography using a PE / EA system to give 1.66 g of a white solid (93% yield).

[0133] Synthesis of intermediate 37 in Example 29

[0134] Synthesis of N-(4-(hydroxymethyl)phenyl)acrylamide (37)

[0135] 1.0 g (8.12 mol) of 4-aminobenzyl alcohol was dissolved in 5 mL of anhydrous tetrahydrofuran. Under ice bath conditions, 3.15 g (24.36 mmol) of N,N-diisopropylethylamine was added, followed by slow dropwise addition of acryloyl chloride (808.38 mg, 8.93 mmol). After N2 replacement, the mixture was kept in an ice bath for 20 min, then stirred at room temperature for 4–5 h. TLC was used to monitor the reaction until complete, after which stirring was stopped. The mixture was extracted four times with DCM. The combined organic phases were washed with 1 M NaOH solution and saturated NaCl solution, and dried over anhydrous magnesium sulfate. The solution was concentrated under reduced pressure and purified by PE / EA column chromatography to give 0.71 g of a white solid, with a yield of 49.34%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 10.12 (s, 1H), 7.63 (d, J = 8.3Hz, 2H), 7.28 (d, J = 8.2Hz, 2H), 6.45 (dd, J = 17.0, 10.0 Hz,1H),6.26(dd,J=16.9,2.2Hz,1H),5.76(dd,J=10.0,2.2Hz,1H),5.14(t,J=5.7Hz,1H),4.46(d,J=5.7Hz,2H).

[0136] Synthesis of intermediate 38 in Example 30

[0137] Synthesis of N-(4-(bromomethyl)phenyl)acrylamide (38)

[0138] Intermediate 37 (0.5 g, 2.82 mmol) was weighed and dissolved in anhydrous dichloromethane (3 mL). Triethylamine (0.43 g, 4.23 mmol) was added under ice bath conditions, followed by slow dropwise addition of thionyl chloride (402.79 mg, 3.39 mmol). The mixture was kept on ice for 20 min, then stirred at room temperature for 24 h. After the reaction was complete as monitored by TLC, stirring was stopped, and the mixture was concentrated under reduced pressure and purified by PE / EA column chromatography to give 0.34 g of a white solid, with a yield of 50%.

[0139] Synthesis of intermediate 39 in Example 31

[0140] Synthesis of N-(4-(hydroxymethyl)phenyl)propionamide (39)

[0141] 4-Aminobenzyl alcohol (1.0 g, 8.12 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). Under ice bath conditions, N,N-diisopropylethylamine (3.15 g, 24.36 mmol) was added, followed by slow dropwise addition of propionyl chloride (826.39 mg, 8.93 mmol). After N2 replacement, the mixture was kept in an ice bath for 20 min, then stirred at room temperature for 4–5 h. TLC was used to monitor the reaction until complete, after which stirring was stopped. The mixture was extracted four times with DCM, and the combined organic phases were washed successively with 1 M NaOH solution and saturated brine, and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure and purified by PE / EA column chromatography to give 0.65 g of a white solid, with a yield of 45%. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 9.80 (s, 1H), 7.53 (d, J = 8.4Hz, 2H), 7.21 (d, J = 8.3Hz, 2H), 5.08 (t, J =5.7Hz,1H),4.41(d,J=5.7Hz,2H),2.30(q,J=7.5Hz,2H),1.38–1.20(m,1H),1.07(t,J=7.5Hz,3H).

[0142] Synthesis of intermediate 40 in Example 32

[0143] Synthesis of N-(4-(bromomethyl)phenyl)propionamide (40)

[0144] Intermediate 39 (0.5 g, 2.82 mmol) was weighed and dissolved in anhydrous dichloromethane (3 mL). Triethylamine (0.43 g, 4.23 mmol) was added under ice bath conditions, followed by slow dropwise addition of thionyl chloride (402.79 mg, 3.39 mmol). The mixture was kept on ice for 20 min, then stirred at room temperature for 24 h. After the reaction was complete as monitored by TLC, stirring was stopped, and the mixture was concentrated under reduced pressure and purified by PE / EA column chromatography to give 0.41 g of a white solid, with a yield of 61%.

[0145] Examples 33-62 below describe the synthesis of the final product.

[0146] Example 33 Synthesis of final product JM-1

[0147] Synthesis of 4-((2-((cyclohexylmethyl)thio)-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-1)

[0148] Intermediate 3 (0.2 g, 0.81 mmol) was weighed and dissolved in N,N-dimethylformamide (2 mL). NaOH (64.68 mg, 1.62 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (277.65 mg, 0.97 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 3 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.25 g of a white solid was obtained, with a yield of 67%. mp 166.6–167.1 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.44(t,J=5.6Hz,1H),8.21–8.14(m,1H),7.99–7.92(m,1H),7.79(d,J=8.1H z,2H),7.48(d,J=8.0Hz,2H),7.12(dd,J=7.6,6.4Hz,1H),5.79(s,2H),3.33(t,J=6.3Hz,2H),3.26(q,J=8.4 ,7.6Hz,2H),3.21(s,3H),3.17(d,J=6.8Hz,2H),1.83(d,J=12.3Hz,2H),1.74–1.69(m,2H),1.68–1.63(m,2H ),1.61–1.56(m,1H),1.26–1.17(m,1H),1.14(d,J=9.4Hz,2H),1.00(t,J=12.0Hz,2H).HRMS(ESI)calcd.for C 26 H 26 FN3O3[M+H] + 452.2246, found 453.2320.

[0149] Example 34 Synthesis of the final product JM-2

[0150] Synthesis of 4-((2-((4-fluorobenzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-2)

[0151] Intermediate 5 (0.2 g, 0.77 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 ml). NaOH (61.94 mg, 1.55 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (265.88 mg, 0.93 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 5 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.25 g of a white solid was obtained, with a yield of 67%. mp 158.6–159.6 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.40(t,J=5.6Hz,1H),7.72(d,J=8.0Hz,2H),7.65–7.58(m,1H),7.48(m,3H),7.18(d,J=6.2Hz,2H),7.15(d,J= 2.0Hz,2H),7.11(d,J=8.9Hz,2H),5.41(s,2H),4.62(s,2H),3.33(d,J=6.2Hz,2H),3.25(t,J=6.5Hz,2H),3.21(s,3H),1.71(p,J=6.7Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ(ppm):166.22,161.94(d, 1 J C-F =244.4Hz),151.50,143.42,139.68,136.62,134.39(d, 4 J C-F =3.0Hz), 131.47(d, 3 J C-F =9.1Hz),128.00,127.21,122.36,118.34,115.74(d, 2 J C-F =21.3Hz),110.36,70.14,58.36,46.84,36.96,35.42,29.66.HRMS(ESI)calcd.for C 26 H 32 N4O2S[M+H] + 463.1730, found 464.1800.

[0152] Example 35 Synthesis of the final product JM-3

[0153] Synthesis of 4-((2-((cyclohexylmethyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-3)

[0154] Intermediate 6 (0.2 g, 0.81 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 ml). NaOH (64.94 mg, 1.62 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (278.77 mg, 0.97 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 6 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.18 g of a white solid was obtained, with a yield of 49%. mp 143.2–144.7 °C. 1 H NMR(300MHz,DMSO-d6)δ(ppm):8.41(t,J=5.7Hz,1H),7.77(d,J=8.2Hz,2H),7.63–7.53(m,1H),7.51– 7.40(m,1H),7.22(d,J=8.1Hz,2H),7.18–7.13(m,2H),5.45(s,2H),3.32(d,J=6.3Hz,2H),3.26(d,J=6 .7Hz,4H),3.21(s,3H),1.80(s,1H),1.78–1.73(m,2H),1.71(s,1H),1.70–1.63(m,2H),1.62(d,J=7.9 Hz,1H),1.57(dd,J=6.9,3.4Hz,1H),1.31–1.19(m,1H),1.13(d,J=9.6Hz,2H),0.98(q,J=11.7Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ(ppm):166.20,152.42,143.47,139.82,136.67,134.50,128.03,127.20,122.1 6,118.17,110.14,70.15,58.36,46.79,37.62,36.97,32.29,29.68,26.23,25.88.HRMS(ESI)calcd.for C 26 H 33 N3O2S[M+H] +451.2293, found 452.2372.

[0155] Example 36 Synthesis of the final product JM-4

[0156] Synthesis of 4-((2-((cyclohexylmethyl)thio)-1H-imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-4)

[0157] Intermediate 8 (0.2 g, 1.02 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (81.50 mg, 2.04 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (437.32 mg, 1.53 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 8 was completely reacted. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.18 g of a white solid was obtained, with a yield of 44%. mp 123.5–125.1 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.47 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.36 (d, J=1.4Hz,1H),7.19(d,J=8.1Hz,2H),7.00(d,J=1.3Hz,1H),5.22(s,2H),3.34(t,J=6. 3Hz,2H),3.28(d,J=6.3Hz,2H),3.22(s,3H),2.84(d,J=6.8Hz,2H),1.73(p,J=6.3Hz, 3H),1.68–1.55(m,4H),1.37(m,1H),1.19–1.00(m,3H),0.85(q,J=11.6,10.8Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.18,141.28,140.69,134.35,129.40,127.95,127.27,122.8 4,70.18,58.36,49.25,41.03,37.64,36.99,32.20,29.70,26.27,25.88.HRMS(ESI)calcd.for C 22 H 32 N3O2S[M+H] + 401.2137, found 402.2219.

[0158] Example 37 Synthesis of the final product JM-5

[0159] Synthesis of (4-((2-((cyclohexylmethyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)phenyl)(piperazin-1-yl)methyl ketone (JM-5)

[0160] Intermediate 9 (0.2 g, 0.74 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (59.44 mg, 1.49 mmol) was added, and the mixture was stirred at room temperature for 10 min. Intermediate 35 (284.80 mg, 0.74 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediates 3 and 9 were completely reacted. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. Then, HCl / EA (15 mL) was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated, dissolved in an appropriate amount of water, and the solution was adjusted with saturated sodium bicarbonate solution until a large amount of white solid precipitated. 0.13 g of white solid was obtained, with a yield of 38%. mp 129.1–129.7 °C. 1 H NMR(300MHz,DMSO-d6)δ(ppm):7.48–7.39(m,2H),7.37–7.24(m,3H),7.20–7.11(m,2H),7.07–6.99 (m,3H),5.05(s,2H),4.16(s,2H),3.50(s,2H),3.16(s,2H),2.76–2.53(m,4H),1.35–1.18(m,1H).

[0161] Example 38 Synthesis of the final product JM-6

[0162] Synthesis of 4-((3-(4-fluorobenzyl)-2-carbonyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-6)

[0163] Intermediate 12 (0.2 g, 0.83 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (49.35 mg, 1.24 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (236.26 mg, 0.83 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 12 was completely reacted. After stopping stirring, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.21 g of a white solid was obtained, with a yield of 57%. mp 147.5–149.5 °C. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.41 (t, J = 5.7Hz, 1H), 7.81–7.75 (m, 2H), 7.44–7.35 (m, 4H), 7.22–7.14 (m, 3H), 7.1 2–7.07(m,1H),7.04–6.95(m,2H),5.14(s,2H),5.10(s,2H),3.30–3.24(m,2H),3.22(s,3H),1.72(p,J=6.6Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.44,162.09(d, 1 J C-F =242.2Hz),154.32,140.47,134.55,133.80(d, 4 J C-F =3.3Hz), 130.08(d, 3 J C-F =8.3Hz),129.28,128.12,127.72,121.85,116.04(d, 2 J C-F =21.0Hz),108.97,70.29,58.45,44.16,43.72,37.11,29.79.HRMS(ESI)calcd.for C 26 H 26 FN3O2[M+H] + 447.1958, found 448.2041.

[0164] Example 39 Synthesis of the final product JM-7

[0165] Synthesis of 1-(4-fluorobenzyl)-3-(4-(piperazin-1-carbonyl)benzyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (JM-7)

[0166] Intermediate 12 (0.2 g, 0.83 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (66.04 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 35 (316.43 mg, 0.83 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 12 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined. After washing with saturated NaCl solution, the mixture was dried over anhydrous sodium sulfate. The solution was purified by PE-EA column chromatography. 0.15 g of a white solid was obtained, with a yield of 42%. 1 H(300MHz,DMSO-d6)δ(ppm):7.40(dd,J=8.3,5.4Hz,3H),7.35(d,J=1.8Hz,3H),7.18(d,J=8.7Hz,2H),7.16–7.11(m,2H ),7.01(dd,J=5.8,3.2Hz,2H),5.13(s,2H),5.09(s,2H),3.50(s,2H),3.20(s,2H),2.62(s,4H),1.24(d,J=6.9Hz,1H).

[0167] Example 40 Synthesis of the final product JM-8

[0168] Synthesis of 1-((4-fluorophenyl)sulfonyl)-3-(4-nitrobenzyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (JM-8)

[0169] Intermediate 14 (0.2 g, 0.68 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (54.74 mg, 1.37 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, 4-nitrobenzyl bromide (177.39 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 14 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA (extractant extract), and the organic phases were combined. After washing with saturated brine, the solution was dried over anhydrous sodium sulfate. The solution was purified by PE-EA column chromatography. 0.21 g of a white solid was obtained, with a yield of 72%. mp 133.4–133.2 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.20–8.07 (m, 4H), 7.88–7.81 (m, 1H), 7.58–7.48 (m, 2H), 7.47–7.41 (m, 2H), 7.22 (dd, J = 4.3, 2.3Hz, 3H), 5.14 (s, 2H). 13C NMR(101MHz,DMSO-d6)δ(ppm):166.27(d, 4 J C-F =255.5Hz),143.82,134.86,133.65(d, 4 J C-F =3.0Hz), 131.36(d, 3 J C-F =11.1Hz),128.81,128.32,125.65,125.36,124.33,123.50,119.81,117.65(d, 2 J C-F =9.1Hz),114.96,114.75,113.15,110.02,43.90.HRMS(ESI)calcd.for C 20 H 14 N3O5S[M+H] + 427.0638, found 428.0718.

[0170] Example 41 Synthesis of the final product JM-9

[0171] Synthesis of 1-((4-fluorophenyl)sulfonyl)-3-(4-methylbenzyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (JM-9)

[0172] Intermediate 14 (0.2 g, 0.68 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (54.74 mg, 1.37 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, 4-methylbenzyl bromide (151.96 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 14 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.15 g of a white solid was obtained, with a yield of 55%. mp 127.5–128.5 °C. 1 H NMR(300MHz,DMSO-d6)δ(ppm):8.16–8.08(m,2H),7.86–7.79(m,1H),7.59–7.46 (m,2H),7.19(dq,J=4.8,2.9,2.4Hz,3H),7.06(s,4H),4.92(s,2H),2.22(s,3H).

[0173] Example 41 Synthesis of the final product JM-10

[0174] Synthesis of 1-benzyl-3-((4-fluorophenyl)sulfonyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (JM-10)

[0175] Intermediate 14 (0.2 g, 0.68 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (54.74 mg, 1.37 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, benzyl bromide (140.44 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 14 was completely reacted. After stopping stirring, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.18 g of a white solid was obtained, with a yield of 69%. mp 158.6–159.6 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.13 (m, 2H), 7.83 (m, 1H), 7.58–7.46 (m, 2H), 7.28–7.19 (m, 6H), 7.18–7.12 (m, 2H), 4.98 (s, 2H). 13 C NMR(101MHz,DMSO-d6)δ(ppm):164.98,150.62,136.11,133.66(d, 4 J C-F =3.0Hz), 131.34(d, 3 J C-F =10.3Hz),129.77,129.16,128.23,127.65,125.55,125.31,123.31,117.62(d, 2 J C-F =23.3Hz),113.10,110.15,44.34.HRMS(ESI)calcd.for C 20 H 15 N₂O₃S[M+H] + 382.0787, found 383.0858.

[0176] Example 42 Synthesis of the final product JM-11

[0177] Synthesis of 1-(4-fluorobenzyl)-3-((4-fluorophenyl)sulfonyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (JM-11)

[0178] Intermediate 14 (0.2 g, 0.68 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (54.74 mg, 1.37 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, 4-methylbenzyl bromide (155.21 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 14 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.15 g of a white solid was obtained, with a yield of 54%. mp 137.1–137.5 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.16–8.09 (m, 2H), 7.82 (dd, J = 6.1, 2.4Hz, 1H) ,7.53(t,J=8.8Hz,2H),7.29–7.19(m,5H),7.12(t,J=8.8Hz,2H),4.96(s,2H). 13 C NMR(101MHz,DMSO-d6)δ(ppm):167.27(d, 1 J C-F =255.5Hz), 143.82, 133.65(d, 4 J C-F =3.1Hz), 131.36(d, 3 J C-F =10.2Hz), 128.81, 128.32(d, 3 J C-F =8.5Hz),125.65,125.36,124.33,123.50,119.81,117.65(d, 2 J C-F =23.2Hz), 114.96, 114.85(d, 2 J C-F =21.5Hz),113.15,110.02,43.90.HRMS(ESI)calcd.for C 20 H 14 F2N2O3S[M+H] + 400.0693, found 401.0758.

[0179] Example 43 Synthesis of the final product JM-12

[0180] Synthesis of 4-((3-((4-fluorophenyl)sulfonyl)-2-carbonyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-12)

[0181] Intermediate 14 (0.2 g, 0.68 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (54.74 mg, 1.37 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (234.98 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 14 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA (extractant ether), and the organic phases were combined. After washing with saturated brine, the mixture was dried over anhydrous sodium sulfate. The solution was purified by column chromatography using a PE-EA system. 0.1 g of a white solid was obtained, with a yield of 30%. mp 160.3–161.2 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.40(d,J=6.0Hz,1H),8.18–8.09(m,2H),7.88–7.79(m,1H),7.72(d,J=8.3Hz,2H),7.53(t,J =8.8Hz,2H),7.21(m,5H),5.03(s,2H),3.36(s,1H),3.32(s,1H),3.27(t,J=6.5Hz,2H),3.21(s,3H),1.71(p,J=6.6Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):165.86,165.59(d, 1 J C-F =252.8Hz),150.26,138.67,134.29,133.36(d, 4 J C-F =2.3Hz), 131.02(d, 3 J C-F =9.8Hz),129.35,127.68,127.16,125.10,123.02,117.30(d, 2 J C-F =23.3Hz),112.76,109.77,69.82,58.02,43.79,36.64,29.32.HRMS(ESI)calcd.for C 25 H 24 FN3O5S[M+H] + 497.1421, found 498.1499.

[0182] Example 44 Synthesis of the final product JM-13

[0183] Synthesis of N-(3-methoxypropyl)-4-((2-((4-methylbenzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)benzamide (JM-13)

[0184] Intermediate 15 (0.2 g, 0.73 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (58.23 mg, 1.46 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (249.96 mg, 0.87 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 15 was completely reacted. After stopping stirring, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.27 g of a white solid was obtained, with a yield of 77%. mp 155.3–156.0 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.41(t,J=5.6Hz,1H),7.71(d,J=8.0Hz,2H),7.64–7.59(m,1H),7.49–7.43(m,3H),7.35(d,J=8.3Hz ,2H),7.20–7.11(m,4H),5.41(s,2H),4.61(s,2H),3.32(d,J=6.2Hz,2H),3.25(d,J=6.4Hz,2H),3.21(s,3H),1.71(p,J=6.6Hz,2H). 13 CNMR(75MHz,DMSO-d6)δ(ppm):165.35,150.78,142.59,138.79,136.28,135.67,133.69 128.54,127.10,126.32,121.42,117.43,109.43,69.26,57.47,45.94,36.07,35.35,28.79,20.27.HRMS(ESI)calcd.for C 26 H 26 ClN3O2S[M+H] + 479.1434, found 480.1514.

[0185] Example 45 Synthesis of the final product JM-14

[0186] Synthesis of 4-((2-((4-bromophenylmethyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-14)

[0187] Intermediate 16 (0.2 g, 0.63 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (50.12 mg, 1.25 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (215 mg, 0.75 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 16 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.26 g of a white solid was obtained, with a yield of 78%. mp 147.7–148.9 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.39 (s, 1H), 7.72 (m, 2H), 7.61 (d, J = 7.2Hz, 1H), 7.48 (m, 3H), 7.43 –7.35(m,2H),7.22–7.09(m,4H),5.41(s,2H),4.60(s,2H),3.35–3.17(m,7H),1.77–1.65(m,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.17,151.30,143.35,139.58,137.10,136.57,134.52,132.46,131.21,128. 82,127.95,127.13,122.32,118.29,110.30,70.08,58.30,46.79,36.90,35.35,29.61.HRMS(ESI)calcd.for C 26 H 26 BrN3O2S[M+H] + 523.0929, found 524.1003.

[0188] Example 46 Synthesis of the final product JM-15

[0189] Synthesis of 4-((2-(benzylthio)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-15)

[0190] Intermediate 17 (0.2 g, 0.83 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (66.57 mg, 1.66 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (285.78 mg, 0.99 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 17 was completely reacted. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by PE-EA column chromatography. 0.31 g of a white solid was obtained, with a yield of 84%. mp 143.1–145.0 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.41 (t, J = 5.7Hz, 1H), 7.72 (d, J = 8.3Hz, 2H), 7.65–7.58 (m, 1H), 7.45 (m, 3H), 7.34 –7.24(m,3H),7.21–7.11(m,4H),5.41(s,2H),4.62(s,2H),3.33–3.22(m,4H),3.21(s,3H),1.71(p,J=6.6Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.32,152.44,143.62,128.14,127.29,122.28,118.28 ,110.27,70.26,58.47,46.90,38.10,37.08,32.23,29.79,25.35.HRMS(ESI)calcd.for C 26 H 27 N3O2S[M+H] + 445.1824, found 446.1905.

[0191] Example 47 Synthesis of the final product JM-16

[0192] Synthesis of N-(3-methoxypropyl)-4-((2-((4-methylbenzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)benzamide (JM-16)

[0193] Intermediate 18 (0.2 g, 0.79 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (62.90 mg, 1.57 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (270 mg, 0.94 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 18 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.30 g of a white solid was obtained, with a yield of 83%. mp 147.5–148.8 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.38(t,J=5.6Hz,1H),7.70(d,J=8.0Hz,2H),7.64–7.58(m,1H),7.48–7.41(m,1H),7.31(d,J=7.8Hz,2H ),7.21–7.14(m,3H),7.14–7.05(m,3H),5.40(s,2H),4.57(s,2H),3.32-3.22(m,4H),3.21(s,3H),2.26(s,3H),1.71(p,J=6.6Hz,2H). 13 CNMR(75MHz,DMSO-d6)δ(ppm):166.09,151.49,143.32,139.55,137.63,136.48,134.42,129.32,128.84, 127.85,127.10,122.21,118.20,110.21,70.03,58.24,46.73,36.85,36.23,29.55.HRMS(ESI)calcd.for C 27 H 29 N3O2S[M+H] + 459.1980, found 460.2049.

[0194] Example 48 Synthesis of the final product JM-17

[0195] Synthesis of 4-((2-((4-methoxybenzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-17)

[0196] Intermediate 19 (0.2 g, 0.74 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (59.18 mg, 1.48 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (254.04 mg, 0.89 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 19 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.26 g of a white solid was obtained, with a yield of 74%. mp 151.1–152.5 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.39(t,J=5.6Hz,1H),7.72(d,J=8.0Hz,2H),7.66–7.59(m,1H),7.45(m,1H),7.40–7.32(m,2H),7.23–7.10(m,4 H),6.91–6.81(m,2H),5.40(s,2H),4.57(s,2H),3.71(s,3H),3.33(d,J =6.3Hz,2H),3.26(t,J=6.6Hz,2H),3.21(s,3H),1.72(p,J=6.6Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.35,164.78,161.51,147.18,141.11,139.48,135.74,134.78,134.28,128.08,127.11, 126.75,124.94,123.83,121.82,116.30,116.02,112.97,70.28,60.09,58.50,48.15,37.10,29.80.HRMS(ESI)calcd.for C 27 H 29 N3O3S[M+H] + 475.1930, found 476.2008.

[0197] Example 49 Synthesis of the final product JM-18

[0198] Synthesis of N-(3-methoxypropyl)-4-((2-((4-(trifluoromethyl)benzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)benzamide (JM-18)

[0199] Intermediate 20 (0.2 g, 0.86 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (68.86 mg, 1.72 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (295.60 mg, 1.03 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 20 was completely reacted. After stopping stirring, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.20 g of a white solid was obtained, with a yield of 60%. mp 172.3–172.9 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.43(t,J=5.6Hz,1H),7.72(d,J=8.3Hz,2H),7.66(s,4H),7.64–7.59(m,1H),7.52–7.43(m,1H) ,7.24–7.11(m,4H),5.43(s,2H),4.71(s,2H),3.33(t,J=6.3Hz,2H),3.26(q,J=6.7Hz,2H),3.21(s,3H),1.71(p,J=6.6Hz,2H). 13 C NMR (75MHz, DMSO) δ (ppm): 166.92, 151.89, 144.05, 143.82, 140.31, 137.38, 135.28, 130.87, 128.70, 127.88, 126.47 (q, 4 J C-F =3.75Hz), 123.10(q, 3 J C-F =10.5Hz),119.05,111.07,70.81,59.04,47.54,41.44,41.17,40.89,40.61,40.33,40.05,39.78,37.64,36.07,30.35.HRMS(ESI)calcd.for C 27 H 26 F3N3O2S[M+H] + 513.1698, found 514.1771.

[0200] Example 50 Synthesis of the final product JM-19

[0201] Synthesis of N-(3-methoxypropyl)-4-((2-((4-(trifluoromethoxy)benzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)benzamide (JM-19)

[0202] Intermediate 21 (0.2 g, 0.62 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (49.33 mg, 1.23 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (211.77 mg, 0.74 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 21 was completely reacted. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.23 g of a white solid was obtained, with a yield of 70%. mp 173.9–175.5 °C. 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.41(t,J=5.6Hz,1H),7.73(d,J=8.1Hz,2H),7.66–7.60(m,1H),7.60–7.55(m,2H),7.51–7.44(m,1H),7 .33–7.25(m,2H),7.18(m,4H),5.42(s,2H),4.66(s,2H),3.34(t,J=6.3Hz,2H),3.31–3.23(m,2H),3.21(s,3H),1.72(p,J=6.6Hz,2H). 13 CNMR(75MHz,DMSO-d6)δ(ppm):165.59,150.74,147.36,142.74,138.97,136.96,136.03,133.94,130.67,127 .35,126.54,121.72,120.83,117.68,109.68,69.48,57.67,46.19,36.30,34.54,29.00.HRMS(ESI)calcd.for C 27 H 26 F3N3O3S[M+H] + 529.1647, found 530.1716.

[0203] Example 51 Synthesis of the final product JM-20

[0204] Synthesis of 4-((2-((4-cyanophenylmethyl)thio)-1H-benzimidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-20)

[0205] Intermediate 22 (0.2 g, 0.75 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (60.30 mg, 1.72 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (258.85 mg, 0.90 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 22 was completely reacted. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.12 g of a white solid was obtained, with a yield of 34%. mp 157.1–157.7 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.41 (t, J = 5.6 Hz, 1H), 7.75 (t, J = 7.8 Hz, 4H), 7.63 (m, 3H), 7.47 (m, 1H), 7.17 (m, 4H), 5.42 (s, 2H), 4.70 (s, 2H), 3.34 (t, J = 6.3Hz, 2H), 3.27 (q, J = 6.8Hz, 2H), 3.21 (s, 3H), 1.72 (p, J = 6.6Hz, 2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):165.79,150.65,143.73,139.18,136.29,134.16,132.38,129.93,127.59, 126.76,122.01,118.74,117.95,110.05,69.72,57.93,46.44,36.55,35.11,29.24.HRMS(ESI)calcd.for C 27 H 26 N4O2S[M+H] + 470.1776, found 471.1851.

[0206] Example 52 Synthesis of the final product JM-21

[0207] Synthesis of 4-((2-((cyclopentylmethyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-21)

[0208] Intermediate 23 (0.2 g, 0.86 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (68.86 mg, 1.72 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (295.60 mg, 1.03 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 23 was completely reacted. After stopping stirring, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.19 g of a white solid was obtained, with a yield of 50%. mp 159.1–140.3 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.45 (d, J = 6.1 Hz, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 6 .8Hz,1H),7.45(d,J=7.4Hz,1H),7.22(d,J=7.9Hz,2H),7.18–7.09(m,2H),5.45(s,2H), 3.31(s,2H),3.26(d,J=6.5Hz,2H),3.21(d,J=1.4Hz,3H),2.22(p,J=7.6Hz,1H),1.73(s ,2H),1.70(d,J=6.5Hz,2H),1.64–1.54(m,2H),1.50(d,J=8.0Hz,2H),1.32–1.21(m,2H). 13 C NMR (75MHz, DMSO-d6) δ (ppm): 166.44, 154.32, 140.47, 134.55, 133.82, 130.08, 129.28, 128.12, 127. 72,121.85,116.18,115.90,108.97,70.29,58.45,44.16,43.72,37.11,29.79.HRMS(ESI)calcd.for C 25 H 31 N3O2S[M+H] + 437.2137, found 438.2207.

[0209] Example 53 Synthesis of the final product JM-22

[0210] Synthesis of N-(3-methoxypropyl)-4-((2-((4-(trifluoromethoxy)benzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)benzamide (JM-22)

[0211] Intermediate 24 (0.2 g, 0.67 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (53.98 mg, 1.35 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 26 (231.73 mg, 0.81 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 24 was completely reacted. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.19 g of a white solid was obtained, with a yield of 66%. mp 138.2–140.1 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.42 (t, J = 5.6 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7. 63–7.55(m,1H),7.47(m,1H),7.25–7.12(m,4H),5.44(s,2H),4.62(dd,J=7.8, 6.0Hz,2H),4.34(t,J=6.1Hz,2H),3.67(d,J=7.5Hz,2H),3.41-3.38(m,1H),3. 33(d,J=6.1Hz,2H),3.27(t,J=6.5Hz,2H),3.21(s,3H),1.71(p,J=6.6Hz,2H).

[0212] Example 53 Synthesis of the final product JM-23

[0213] 4-((2-(phenylmethylsulfinyl)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide

[0214] Intermediate 27 (0.2 g, 0.78 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (62.42 mg, 1.56 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (267.95 mg, 0.94 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until the reaction was complete. After stirring was stopped, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na₂SO₄. The mixture was purified by PE-EA column chromatography. 0.14 g of a white solid was obtained, with a yield of 39%. mp 143.5–145.0 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.41 (d, J = 6.2 Hz, 1H), 7.85 (dd, J = 7.1, 4.0 Hz, 1H),7.69(d,J=8.4Hz,2H),7.54(m,1H),7.34(m,2H),7.26(d,J=6.0Hz,2H),7. 12(d,J=8.2Hz,4H),5.74–5.51(m,2H),4.86–4.63(m,2H),3.29(s,2H),3.25(d ,J=6.9Hz,2H),3.20(d,J=3.2Hz,3H),1.77–1.65(m,2H).HRMS(ESI)calcd.for C 26 H 26 FN3O4S[M+H] + 479.1697, found 480.1760.

[0215] Example 54 Synthesis of the final product JM-24

[0216] Synthesis of 4-((2-(phenylmethylsulfinyl)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-24)

[0217] Intermediate 28 (0.2 g, 0.73 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (58.75 mg, 1.47 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (252.20 mg, 0.88 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until the reaction was complete. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.15 g of a white solid was obtained, with a yield of 43%. mp 151.1–152.0 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.40 (t, J = 5.7Hz, 1H), 7.92 (m, 1H), 7.69 (dd, J = 8 .4,2.3Hz,2H),7.62–7.55(m,1H),7.47–7.39(m,2H),7.34(m,2H),7.19(m,2H),7 .11–7.01(m,2H),5.71–5.62(m,2H),5.13(d,J=2.2Hz,2H),3.32(dd,J=6.3,2.3H z, 2H), 3.25 (d, J = 6.8Hz, 2H), 3.20 (d, J = 2.4Hz, 3H), 1.69 (td, J = 7.8, 5.7Hz, 2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.26,147.08,141.00,139.37,135.62,134.67,134.17(q, 3 J C-F =8.3Hz),127.97,127.00,126.65,124.83,123.72,121.70,116.04(d, 2 J C-F =21.8Hz),112.85,70.17,59.99,58.38,48.06,37.00,29.68.HRMS(ESI)calcd.for C 26 H 26 FN3O4S[M+H] + 495.1628, found 496.1707.

[0218] Example 55 Synthesis of the final product JM-25

[0219] Synthesis of 4-((2-(cyclohexylamino)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-25)

[0220] Weigh intermediate 30 (0.2 g, 0.86 mmol) and dissolve anhydrous N,N-dimethylformamide (2 mL). Add NaOH (68.86 mg, 1.72 mmol) and stir at room temperature for 10 min. Then add intermediate 26 (295.60 mg, 1.03 mmol) and stir at room temperature for 2-3 h until the starting material reacts completely. After stopping stirring, dilute the reaction solution with plenty of water, extract three times with EA, combine the organic phases, wash with saturated NaCl solution, and dry with anhydrous sodium sulfate. Purify by column chromatography using a PE-EA system. 0.16 g of white solid was obtained, yield 43%. mp 143.0-145.0℃. 1 H NMR(300MHz,DMSO-d6)δ(ppm):8.43(t,J=5.6Hz,1H),7.83–7.74(m,2H),7.66–7.57(m,1 H),7.51–7.42(m,1H),7.27–7.11(m,4H),5.47(s,2H),3.99–3.86(m,1H),3.35(d,J=6.4 Hz,2H),3.28(d,J=6.4Hz,2H),3.23(d,J=1.2Hz,3H),2.11(d,J=12.2Hz,2H),1.74(p,J= 7.8,7.4Hz,4H),1.62–1.44(m,4H),1.40(s,1H),1.37–1.21(m,2H).HRMS(ESI)calcd.for C 25 H 31 N3O2S[M+H] + 437.2137, found 438.2214.

[0221] Example 56 Synthesis of the final product JM-26

[0222] Synthesis of 4-((2-((4-fluorophenyl)amino)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-26)

[0223] Intermediate 31 (0.2 g, 0.56 mmol) was weighed, and 4-fluoroaniline (186.32 mg, 1.68 mmol) was added under N2 protection. The mixture was heated and stirred at 160 °C for 16 h until the starting material was completely reacted. After cooling the reaction solution, an appropriate amount of EA was added, and a large amount of pale blue solid precipitated. The solid was filtered off, dissolved in DCM, and purified by DCM-MeOH column chromatography. 0.12 g of blue solid was obtained, with a yield of 52%. 1H NMR (300MHz, DMSO-d6) δ (ppm): 9.13 (d, J = 3.1 Hz, 1H), 8.36 (s, 1H), 7.90 (p, J =3.9Hz,2H),7.76(m,2H),7.39(d,J=7.7Hz,1H),7.22(d,J=10.4Hz,2H),7.16 (m,2H),7.04(t,J=5.0Hz,1H),6.98(d,J=7.2Hz,1H),5.58(d,J=3.1Hz,2H), 3.32(t,2H),3.25(d,J=6.2Hz,2H),3.21(d,J=3.3Hz,3H),1.73–1.67(m,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):166.61,151.07,142.61,140.67,137.92,134.64,134.23,128.27,127.20,121.91,120.35(d, 3 J C-F =6.8Hz), 117.04, 115.85(q, 2 J C-F =21.8Hz),109.33,70.43,58.60,45.43,37.25,29.93.HRMS(ESI)calcd.for C 25 H 25 FN4O2[M+H] + 432.1962, found 433.2038.

[0224] Example 57 Synthesis of the final product JM-27

[0225] Synthesis of 4-((2-(cyclohexylamino)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-27)

[0226] Intermediate 31 (0.2 g, 0.56 mmol) was weighed and cyclohexylamine (277.16 mg, 2.79 mmol) was added under N2 protection. The mixture was heated and stirred at 160 °C for 16 h until the starting material reacted completely. After stirring was stopped, the reaction solution was cooled to room temperature, diluted with a large amount of water, washed with 1N hydrochloric acid, and extracted three times with EA. The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by DCM-MeOH column chromatography. 0.1 g of a brown solid was obtained, with a yield of 42%. 1HNMR(300MHz,DMSO-d6)δ(ppm):8.38(t,J=5.7Hz,1H),7.75(d,J=8.1Hz,2H),7.23–7.15(m,3H),6.98(d,J=7.7Hz,1H),6.95(s,1H),6.80(t,J =7.5Hz,1H),6.58(d,J=7.8Hz,1H),5.33(s,2H),3.34–3.23(m,6H),3.2 1(s,3H),1.99(d,J=10.0Hz,2H),1.78–1.57(m,7H),1.38–1.20(m,6H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):165.73,154.01,142.74,140.13,134.18,133.55,127.23,126.44,120.34,11 8.08,114.75,107.55,69.56,57.73,51.37,43.94,36.36,32.72,29.06,25.30,24.83.HRMS(ESI)calcd.for C 25 H 32 ClN4O2[M+H] + 420.2525, found 421.2603.

[0227] Example 58 Synthesis of the final product JM-28

[0228] Synthesis of 4-((2-(phenylmethylsulfinyl)-1H-benzo[d]imidazol-1-yl)methyl)-N-(3-methoxypropyl)benzamide (JM-28)

[0229] Intermediate 33 (0.2 g, 0.84 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (67.43 mg, 1.69 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (289.47 mg, 1.01 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until the reactants reacted completely. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography using a PE-EA system. 0.19 g of a white solid was obtained, with a yield of 51%. mp 184.2–185.3 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm): 12.84 (s, 1H), 8.38 (t, J = 5.6Hz, 1H), 8.30 (m, 2H), 7.78 (d, J = 8.0Hz, 2H), 7.58–7.53 (m, 1H), 7.49 (d, J = 8.0Hz, 2H), 7.45–7.39(m,1H),7.26(t,J=8.8Hz,2H),7.20(m,2H),5.56(s,2H),3.32 (d,J=6.3Hz,2H),3.28–3.23(m,2H),3.20(s,3H),1.71(p,J=6.7Hz,2H). 13 C NMR (75MHz, DMSO-d6) δ (ppm): 173.18, 166.51, 163.13, 153.10, 140.26, 135.29, 134.75, 131.98 (d, J = 9.2Hz), 129.66, 128, 123.48, 115.48 112.85,110.48,70.34,58.52,49.26,45.17,37.16,29.84.HRMS(ESI)calcd.for C 26 H 25 FN4O3[M+H] + 460.1911, found 461.1983.

[0230] Example 59 Synthesis of the final product JM-29

[0231] Synthesis of N-(3-methoxypropyl)-4-((2-(benzenesulfonamide)-1H-benzo[d]imidazol-1-yl)methyl)benzamide (JM-29)

[0232] Intermediate 24 (0.2 g, 0.73 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (58.54 mg, 1.46 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 26 (251.29 mg, 0.878 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until the reaction was complete. After stirring was stopped, the reaction mixture was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na₂SO₄. The mixture was purified by PE-EA column chromatography. 0.15 g of a white solid was obtained, with a yield of 43%. mp 180.1–181.3 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.38 (t, J = 5.7Hz, 1H), 8.08–8.00 (m, 2H), 7.71–7.64 (m, 3H), 7.56–7.46 (m, 2H), 7.17–7.12 (m, 2H), 7 .10(m,1H),7.02(m,1H),6.92(m,1H),5.02(s,2H),3.36(s,1H),3.32(s,1H),3.29–3.23(m,2H),3.22(s,3H),1.71(p,J=6.6Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ(ppm):HRMS(ESI)calcd.for C 26 H 26 FN3O4S[M+H] + 495.1628, found 496.1707.

[0233] Example 60 Synthesis of the final product JM-30

[0234] Synthesis of (4-((2-((cyclohexylmethyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)phenyl)(piperazin-1-yl)methyl ketone (JM-30)

[0235] Intermediate 5 (0.2 g, 0.81 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (64.94 mg, 1.62 mmol) was added. The mixture was stirred at room temperature for 10 min. Intermediate 35 (311.14 mg, 0.81 mmol) was added, and the mixture was stirred at room temperature for 2-3 h until the starting material reacted completely. After stirring was stopped, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na2SO4. The mixture was purified by PE-EA column chromatography. Subsequently, HCl / EA (15 mL) was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated, dissolved in an appropriate amount of water, and the solution was adjusted with saturated sodium bicarbonate solution until a large amount of white solid precipitated. 0.23 g of white solid was obtained, with a yield of 63%. mp 146.3-147.1℃ 1H NMR(300MHz, DMSO-d6)δ(ppm):7.61–7.54(m,1H),7.53–7.45(m,1H),7.34(d,J=8.1Hz,2H),7.20(d,J=8.0Hz,2H),7.18–7.11(m,2H),5.44( s,2H),3.51(s,2H),3.31–3.12(m,4H),2.66(d,J=26.1Hz,4H),1.78(d,J=12.4Hz,2H),1.61(m,4H),1.27–0.89(m,6H).HRMS(ESI)calcd.for C 26 H 32 N4OS[M+H] + 448.2297, found 449.2010.

[0236] Example 61 Synthesis of the final product JM-31

[0237] Synthesis of N-(4-((2-((4-fluorobenzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)phenyl)acrylamide (JM-31)

[0238] Intermediate 5 (0.2 g, 0.77 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and NaOH (61.94 mg, 1.55 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, intermediate 37 (185.90 mg, 0.77 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 5 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na₂SO₄. The mixture was purified by PE-EA column chromatography. 0.10 g of a white solid was obtained, with a yield of 31%. mp 150.5–151.2℃ 1 H NMR(300MHz, DMSO-d6)δ(ppm):10.22(s,1H),8.90(d,J=1.6Hz,1H),8.15(m,1H),7.67(d,J=8.5Hz,2H),7.57–7.45(m,4H), 7.41(d,J=8.5Hz,2H),7.11(t,J=8.8Hz,2H),6.41(m,1H),6.29–6.16(m,1H),5.80–5.71(m,1H),5.55(s,2H),4.53(s,2H). 13C NMR(75MHz,DMSO-d6)δ(ppm):173.23,163.95,156.62,145.89,140.00,135.95(d, 4 J C-F =3.0Hz),132.42,132.07,131.77,131.41(d, 3 J C-F =8.25Hz),129.41,129.33,127.87,120.35,115.78(d, 2 J C-F =21.0Hz),110.95,61.47,34.51.HRMS(ESI)calcd.for C 24 H 20 FN3OS[M+H] + 417.1311, found 419.1340.

[0239] Example 62 Synthesis of the final product JM-32

[0240] Synthesis of N-(4-((2-((4-fluorobenzyl)thio)-1H-benzo[d]imidazol-1-yl)methyl)phenyl)propionamide (JM-32)

[0241] Intermediate 5 (0.2 g, 0.77 mmol) was weighed and dissolved in anhydrous N,N-dimethylformamide (2 mL). NaOH (61.94 mg, 1.55 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, intermediate 40 (185.90 mg, 0.77 mmol) was added, and the mixture was stirred at room temperature for 2–3 h until intermediate 5 was completely reacted. After stopping stirring, the reaction solution was diluted with a large amount of water, extracted three times with EA, and the organic phases were combined, washed with saturated NaCl solution, and dried over anhydrous Na₂SO₄. The mixture was purified by PE-EA column chromatography. 0.17 g of a white solid was obtained, with a yield of 52%. mp 141.1–142.5 °C. 1 H NMR(300MHz,DMSO-d6)δ(ppm):9.86(s,1H),7.60(m,1H),7.54–7.41(m,5H),7.21–7 .02(m,6H),5.27(s,2H),4.61(s,2H),2.27(q,J=7.5Hz,2H),1.04(t,J=7.6Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ172.53,161.99(d, 1 J C-F=242.3Hz),151.43,143.51,139.40,136.62,134.27(d, 4 J C-F =3.0Hz), 131.49(d, 3 J C-F =8.25Hz),131.05,128.07,122.29,119.68,118.31,115.74(d, 2 J C-F =21.8Hz),110.43,46.92,35.51,29.97,10.13.HRMS(ESI)calcd.for C 24 H 22 FN3OS[M+H] + 419.1468, found 420.1537.

[0242] Example 63 Biological Evaluation

[0243] (1) Culture of THP-1 cells

[0244] (1) Complete culture medium: 90% RPMI-1640 medium, 10% fetal bovine serum.

[0245] (2) Incubator environment: 37℃, air 95%, carbon dioxide 5%.

[0246] (3) Cell resuscitation: Take out THP-1 cell line (about 1 ml) from liquid nitrogen, place it in a 37℃ water bath, and after complete thawing (about 2 min), take out the cryovial, wipe off the moisture, spray with 70% ethanol for disinfection, centrifuge (800 rpm, 5 min), gently aspirate the supernatant, add 1 ml of freshly prepared complete culture medium, mix well, transfer to a culture flask containing 4 ml of complete culture medium, mix well, and place in an incubator for culture.

[0247] (4) Cell passage: Gently pipette the cells evenly and count them with a counting chamber. When the cell density reaches 800,000 to 1,000,000 / ml, passage the cells by half medium change and place them in an incubator for culture. Passage the cells every 2 to 3 days.

[0248] (5) Cell cryopreservation: Take 5 ml of cell suspension in good condition, transfer it to a centrifuge tube and centrifuge it (800 rpm, 5 min). Remove the supernatant, add 1 ml of cell cryopreservation solution, mix well, transfer it to a cryopreservation tube, seal it, and immediately store it in a liquid nitrogen tank.

[0249] (2) Cell experiments

[0250] Take healthy THP-1 cells, transfer them to centrifuge tubes and centrifuge (800 rpm, 5 min). Discard the supernatant, add freshly prepared complete culture medium, mix well, count the cells, and add more culture medium to bring the cell concentration to approximately 500,000 / ml. Add PMA to a final concentration of 100 ng / ml and mix well. Take a 96-well plate and add 100 μl of cell suspension to each well, so that the number of cells in each well is approximately 50,000. After adding cells, mix well by pipetting every 10 wells to ensure a uniform cell concentration in each well (for other sizes of plates, scale up proportionally to ensure consistent cell concentration). After adding cells, gently shake the 96-well plate to disperse the cells evenly and incubate for 12 h. Gently aspirate the supernatant with a pipette, add the pre-prepared drug solution along the wall, and add equal volumes of complete culture medium to the control and model groups. Incubate for 24 h. Prepare a 4 μg / ml LPS stock solution using complete culture medium, add an appropriate amount to each well (except for the blank group) to make the final LPS concentration 100 ng / ml, and continue incubation for 3 h. Prepare a 100 mg / ml ATP stock solution using PBS, add an appropriate amount to each well to make the final ATP concentration 5 mM, and continue incubation for 1 h.

[0251] To investigate the effect of the compound on the NLRP3 activation phase, THP-1 cells were seeded into wells of a plate as described above. LPS was added to a final concentration of 100 ng / ml, and the cells were incubated for 3 h. After the supernatant was removed, a pre-prepared 5 μM compound solution was added along the cell wall for 24 h of treatment. The cells were then treated with LPS for 3 h and ATP for 1 h. The IL-1β level in the culture supernatant was measured by ELISA, and the inhibition rate was calculated accordingly. The data obtained are the mean ± sem of three independent experiments (n = 3).

[0252] The blank group and the model group were given the same amount of complete culture medium and incubated in an incubator for 24 hours. ATP was added to make the final concentration 5 mM (or Nigericin was added to make the final concentration 10 μM), and the incubator was continued for another hour.

[0253] (3) Enzyme-linked immunosorbent assay (ELISA)

[0254] Taking IL-1β as an example, the detection of other proteins is similar:

[0255] (1) Seven IL-1β antibody standards with different concentrations were prepared using sample diluent: 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, 15.6 pg / mL, 7.8 pg / mL, 3.9 pg / mL, and 0 pg / mL for zero wells. Cell supernatant was diluted with sample diluent. 100 μl of standard or sample was added to each well, sealed with a sealing film, and incubated on a shaker (37℃, 90 rpm) for 1.5 h.

[0256] (2) Remove the microplate, shake it dry, add 100 μL of biotin-labeled anti-Human IL-1β antibody working solution (1×) to each well, cover with sealing film, and incubate on a shaker (37℃, 90 rpm) for 1 h.

[0257] (3) Remove the microplate, shake off the excess water, wash 3 times with 1× buffer, add at least 300 μL to each well, each time for about 1 min. Add 100 μl of the prepared ABC working solution (1×) to each well, cover with the sealing film, and incubate in a shaker (37℃, 90 rpm) for 0.5 h.

[0258] (4) Remove the microplate, shake off the excess water, wash 5 times with 1× buffer, add at least 300 μL to each well, each time for about 1-2 min. Add 90 μL of TMB chromogenic solution preheated to 37℃ to each well, cover with sealing film, wrap with aluminum foil, and incubate on a shaker (37℃, 90 rpm) for 15-20 min.

[0259] (5) Remove the microplate, add 100 μL of TMB stop solution to each well, and measure the OD value (450 nm) using a microplate reader.

[0260] (6) Using the zero-well as a control, a standard curve was established and the concentration was calculated. Inflammatory factor inhibition rate % = [1 - (sample absorbance - blank absorbance) / (model absorbance - blank absorbance)] × 100%.

[0261] (4) CCK-8 experiment

[0262] Differentiated THP-M macrophages were harvested. Six gradients were set up: a control group, a model group, and a drug group (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM). The supernatant was discarded. 100 μL of complete culture medium was added to the control and model groups, and an equal volume of pre-prepared drug solution was added to the drug group. After 48 h of culture, 10 μL of CCK-8 solution was added to each well, and culture was continued for another 4 h. The absorbance was measured at 450 nm using a microplate reader. Cell proliferation inhibition rate % = [(Absorbance of control wells - Absorbance of experimental wells) / (Absorbance of control wells - Absorbance of control wells)] × 100%.

[0263] Table 1 Note: a. In THP-M cells, after treatment with a 5 μM compound for 24 hours, followed by LPS treatment for 3 hours and ATP treatment for 1 hour, the IL-1β level in the culture supernatant was measured by ELISA, and the inhibition rate was calculated accordingly. The data obtained are mean ± sem (n = 3) from three independent experiments; b. The cytotoxicity of the target compound was detected using the CCK-8 assay. TC 50This indicates the compound concentration at which half of the cells survive; c.NA: not available.

[0264] (5) Inhibitory activity of JM-31 against NLRP3 inflammasome in THP-M cells

[0265] To evaluate the anti-inflammatory activity of JM-31 against the NLRP3 inflammasome, the half-maximal inhibitory concentration (IC50) of JM-31 against the NLRP3 inflammasome was measured using an ELISA assay. As shown in Figure 1, the compound was prepared into solutions at concentration gradients of 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, and 20 μM. THP-M cells were treated with the compound for 24 hours, followed by LPS treatment for 3 hours and ATP treatment for 1 hour. Cell supernatants were then collected, and the concentration of cleaved IL-1β in the supernatant was detected using an ELISA kit. Finally, the IC50 of JM-31 against the NLRP3 inflammasome was calculated. 50 The results showed that JM-31 had a half-maximal inhibitory concentration (IC50) of 1.28 μM against the NLRP3 inflammasome, and its activity was significantly better than that of I-19 (IC50). 50 =17.40μM) and II-8 (IC 50 =3.33μM).

[0266] (6) Inhibitory effect of compound JM-31 on NLRP3 inflammasome

[0267] To gain a deeper understanding of the potential inhibitory effect of compound JM-31 on the activation of the NLRP3 inflammasome, this application used Western blotting to evaluate the regulatory effect of JM-31 on the expression levels of core proteins in the NLRP3 inflammasome signaling pathway. THP-1 cells were administered different concentrations (0.5 μM, 1 μM, 10 μM) of JM-31 for 24 h, and the expression levels of key proteins in the pathway were measured. The results, as shown in Figure 2, indicate that JM-31 showed an inhibitory effect on the production of both IL-1β and caspase-1, particularly a significant inhibitory effect on IL-1β secretion. These findings suggest that JM-31 may exert its anti-inflammatory biological activity by intervening in the activation of the NLRP3 inflammasome. Further analysis showed that although JM-31 had no effect on the levels of NLRP3 and ASC proteins, it had a slight inhibitory trend on the expression levels of pro-IL-1 and βpro-caspase-1. This indicates that the inhibitory effect of JM-31 on the NLRP3 inflammasome is not limited to the inflammasome pathway itself, but may also involve the regulation of upstream inflammatory cytokine formation. Therefore, JM-31 may exert its anti-inflammatory effects through multiple mechanisms, which provides valuable information for further research on its potential as an anti-inflammatory therapeutic agent.

[0268] (7) Inhibitory effect of compound JM-31 on the activation phase of NLRP3 inflammasome.

[0269] To investigate the role of JM-31 in the activation phase of the NLRP3 inflammasome, different concentrations (0.5 μM, 1 μM, and 10 μM) of JM-31 were administered to THP-M cells before and after LPS treatment, with an administration time of 24 h. The activation of the NLRP3 inflammasome was simulated by using LPS and ATP to activate signaling pathways in the inflammatory response. Subsequently, the IL-1β level in the supernatant was quantitatively analyzed using ELISA. As shown in Figure 3, in both experimental groups, compared with the control group, the administered groups showed a significant concentration-dependent reduction in IL-1β levels. These results indicate that JM-31 exhibits significant anti-inflammatory activity in both the initiation and activation phases of the NLRP3 inflammasome.

[0270] (8) The effect of compound JM-31 on the initiation phase of NLRP3 inflammasome

[0271] The inflammatory response triggered by the NLRP3 inflammasome depends on the presence of specific proteins, with key components including NLRP3 and pro-IL-1β. Ensuring the synthesis of these proteins is a crucial step in the early activation of the NLRP3 inflammasome. To investigate the effect of compound JM-31 on the initiation phase of the NLRP3 inflammasome, we detected the mRNA levels of NLRP3 and IL-1β using real-time quantitative PCR (RT-qPCR). THP-M cells were treated with different concentrations (1 μM, 5 μM, 10 μM) of JM-31 for 24 h, followed by LPS stimulation for 3 h. Finally, using GAPDH as an internal control, the mRNA levels of NLRP3 and IL-1β were detected. The results are shown in Figure 4. Compared with the control group, the mRNA level of the inflammatory factor IL-1β was significantly reduced in the experimental group after treatment with different concentrations of the drug, and this reduction was concentration-dependent, while no significant change was observed in the mRNA level of NLRP3. These results suggest that compound JM-31 may target IL-1β during the initiation phase, inhibiting its expression and thus reducing the production of inflammatory mediators.

Claims

1. A compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof: in, R 1 for R a It is a C1-C6 alkylene group or -SO2-. R b It is a halogen; R 2 for R C H, C1-C6 alkyl, halogen, or -NO2; R d for P is 1-5; q is 0-3; Where L is R e For -S-, -SO-, -SO2-, or -NH-; R f It is absent, C1-C6 alkylene, -SO2- or -CO-; R 3 for R g It is a benzene ring, a 4-6 membered aliphatic ring, or a 4-5 membered heterocycle containing an oxygen atom; R h It can be halogen, H, C1-C6 alkyl, C1-C6 alkoxy, -CN, trifluoromethyl or -O-CF3; R 4 for R i It is a C1-C6 alkyl or C2-C4 alkenyl group.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, R 1 for R a It is methylene or -SO2-. R b It can be F, Cl, Br or I; R 2 for R C H, F, Cl, Br or I, methyl, propyl, -NO2; R d for P is 1, 2, 3, 4 or 5; q is 0, 1, 2 or 3.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, R 1 Selected from: R 2 Selected from:

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, L is R e For -S-, -SO-, -SO2-, or -NH-; R f The following are not present: methylene, -SO2-, or -CO-. R 3 for R g It is a benzene ring, cyclopentyl, cyclohexyl, or a 4-membered heterocycle containing one oxygen atom and three carbon atoms; R h The following are the possible values: F, Cl, Br, I, H, methyl, methoxy, -CN, trifluoromethyl, -O-CF3; R 4 for R d for P is 1, 2, 3, 4, or 5; q is 0, 1, 2, or 3; R i It can be ethyl or vinyl.

5. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, L is selected from: R 3 Selected from: R 4 Selected from:

6. A compound or a pharmaceutically acceptable salt thereof, selected from any of the following structures:

7. A pharmaceutical composition comprising the compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

8. Use of a compound according to any one of claims 1-6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating NLRP3 inflammasome-related diseases.

9. The application according to claim 8, characterized in that, The NLRP3 inflammasome-related diseases are selected from chronic inflammation, autoimmune diseases, metabolic diseases, neurodegenerative diseases, gout, pseudogout, atherosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type II diabetes, Alzheimer's disease, Parkinson's disease, multiple sclerosis, kidney injury, renal fibrosis, acute and chronic heart failure, liver fibrosis, liver injury, inflammatory bowel disease, rheumatoid arthritis, and cardiovascular and cerebrovascular diseases.

10. Use of a compound according to any one of claims 1-6 or a pharmaceutically acceptable salt thereof in the preparation of an NLRP3 inflammasome inhibitor.

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