Aryl oxadiazole compound as well as synthesis and application thereof

Abstract

The invention discloses an aryl oxadiazole compound with a chemical structural formula as shown in the specification or pharmaceutically acceptable salt thereof. In-vitro experiment results show that the compound has a remarkable inhibition effect on DENV, the therapeutic index (TI) of the compound is far higher than that of a clinical reference substance ribavirin, the TI value of a preferred compound such as I-29 exceeds 1131.86, and the TI value of the preferred compound is far higher than that of the clinical reference substance ribavirin. The characteristics of high efficiency and low toxicity are highlighted. In addition, the preparation method is simple in process, high in yield, mild in reaction condition and suitable for large-scale production and clinical popularization.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drugs in biomedical technology, specifically relating to a class of aryl oxadiazole compounds and their pharmaceutically acceptable salts, their preparation methods, and their application in the preparation of drugs for the prevention and / or treatment of dengue virus (DENV) infection. Background Technology

[0002] Dengue fever, commonly known as "breakbone fever," is an acute vector-borne infectious disease caused by four closely related dengue virus serotypes (DENV-1 to DENV-4). It is one of the most serious public health problems worldwide. In recent years, with climate change, accelerated urbanization, and increased population mobility, the scope of dengue fever has continued to expand, and the threat has become increasingly severe.

[0003] Dengue virus belongs to the Flaviviridae family and has the following key characteristics that make treatment difficult: (1) Serotype diversity: there is no cross-protection between the four serotypes (DENV-1 to DENV-4). Antibodies produced after infection with a certain serotype cannot neutralize the heterotype virus and may instead aggravate the condition of secondary infection through antibody-dependent enhancement (ADE). (2) Complex viral structure: non-structural proteins encoded by DENV (such as NS3 and NS5) are key targets for viral replication, but existing inhibitors are prone to drug resistance. (3) Lack of ideal animal models: the virus has high host specificity, and preclinical research tools are limited, hindering the drug development process. These characteristics make the development of broad-spectrum and highly effective anti-DENV drugs an international challenge.

[0004] Currently, the clinical management of dengue fever mainly focuses on symptomatic and supportive treatment, lacking specific antiviral drugs. The main limitations of existing methods include: (1) Vaccine limitations: the only approved vaccine, CYD-TDV (Sanofi), is only applicable to people aged 9-45 years, and has low protective efficacy against serotype 2 (approximately 50%), with safety controversies (such as increased risk of severe illness). (2) Drug gap: the broad-spectrum antiviral drug ribavirin has weak inhibitory activity against DENV (EC5). 50 (2) High concentration (≥50 μM), low therapeutic index (TI≈4.37), and side effects such as hemolytic anemia, which cannot meet clinical needs. (3) Insufficient preventive measures: Relying on public health measures such as mosquito control has limited effect and is difficult to sustain. Therefore, it is urgent to develop new high-activity, low-toxicity anti-DENV compounds. Summary of the Invention

[0005] To address the problems of insufficient activity, low therapeutic index, and complex preparation processes in existing anti-dengue virus drugs, this invention provides a class of aryl oxadiazole compounds or pharmaceutically acceptable salts thereof. These compounds exhibit significant inhibitory effects on dengue virus (DENV), with a therapeutic index (TI) far exceeding that of the clinical control drug ribavirin, and also have low toxicity and are easy to prepare.

[0006] The chemical structural formulas of the aryl oxadiazole compounds of this invention are as follows: ; In the formula: R1 is selected from substituted or unsubstituted aromatic groups, aliphatic groups, heterocyclic groups, and substituents are selected from halogens, trifluoromethoxy, p-toluenesulfonyl, methanesulfonyl, alkyl, alkoxy, cyano, etc.; n is 0, 1, or 2; R1 includes, but is not limited to, phenyl, pyridyl, indole, and cyclohexyl. R2 is selected from halogens and C1-C4 alkoxy groups.

[0007] The aryl oxadiazole compounds of this invention include, but are not limited to, compounds of formulas I-1 to I-33 listed in the table below, which have been analyzed by proton nuclear magnetic resonance spectroscopy (NMR). 1 The structure was confirmed by 1H NMR and mass spectrometry (MS), and its specific properties, yield and activity data are detailed in the Examples section of the instruction manual; Table 1 ; ; .

[0008] Another objective of this invention is to provide a method for preparing the above-mentioned compound. This method is simple, has a high yield, is suitable for industrial production, and uses mild reaction conditions in each step with high yields (up to 87%), and the intermediates are easy to purify. The specific operation is as follows: (1) Esterification reaction: In the presence of solvent and chlorination reagent, substituted aromatic acetic acid reacts with alcohol to generate ester compounds as shown in Formula II; ; In the formula, R2 is selected from halogens and C1-C4 alkoxy groups, and R3 is a C1-C5 alkyl group; The alcohol is one of methanol, ethanol, propanol, butanol, and pentanol; the chlorinating agent is one or more of thionyl chloride, phosphorus oxychloride, phosphorus pentachloride, and oxalyl chloride; the molar ratio of substituted aromatic acetic acid to alcohol is 1:(5-40); and the reaction is carried out at 0-25℃. (2) Bromination reaction: In a solvent, ester compounds react with N-bromosuccinimide (NBS) and azobisisobutyronitrile (AIBN) to generate α-bromoesters as shown in Formula III; ; The molar ratio of the ester compound to N-bromosuccinimide is 1:(1-2), and the molar ratio of the ester compound to azobisisobutyronitrile is 1:(0.02-1); the solvent is carbon tetrachloride; the reaction is carried out under reflux at 65-80℃. (3) Nucleophilic substitution reaction: At room temperature and in the presence of solvent, α-bromoester reacts with 3,5-dimethoxyaniline under the catalysis of a basic reagent to generate the aryl ester shown in formula IV; ; The alkaline reagent is selected from one or more of potassium carbonate, sodium carbonate, sodium hydroxide, triethylamine, and diisopropylethylamine; the molar ratio of α-bromoester to alkaline reagent is 1:(1-3), and the molar ratio of α-bromoester to 3,5-dimethoxyaniline is 1:(1-1.5); the solvent is acetonitrile. (4) Hydrazolysis reaction: In a solvent, aryl esters react with hydrazine hydrate under reflux at 75-85℃ to generate aryl hydrazide as shown in formula V; ; The molar ratio of the aryl ester to hydrazine hydrate is 1:(3-10); the solvent is anhydrous ethanol. (5) Acyl chloride reaction: In a solvent, aromatic or fatty carboxylic acids react with a chlorinating agent to produce acyl chloride as shown in formula VII; ; In the formula, R1 is selected from substituted or unsubstituted aromatic groups, aliphatic groups, and heterocyclic groups; the substituents are selected from halogens, trifluoromethoxy, p-toluenesulfonyl, methanesulfonyl, alkyl, alkoxy, cyano, etc.; n is 0, 1, or 2. The chlorinating agent is one or more of thionyl chloride, phosphorus oxychloride, phosphorus pentachloride, and oxalyl chloride; the molar ratio of aromatic or aliphatic carboxylic acid to chlorinating agent is 1:(2-6); the solvent is anhydrous tetrahydrofuran or dichloromethane; the reaction is carried out at 0-25℃. (6) Condensation reaction: Under alkaline conditions, the aryl hydrazide shown in formula V reacts with the acyl chloride shown in formula VII to generate the aryl dihydrazide shown in formula VIII; ; The molar ratio of the aryl hydrazide to the acyl chloride is 1:(1-2); the basic reagent is triethylamine, and the molar ratio of the basic reagent to the aryl hydrazide is 1:(1-2); the reaction is carried out at 0-25℃; (7) Cyclization reaction: Aryl bishydrazide reacts with p-toluenesulfonyl chloride under alkaline conditions to undergo dehydration and cyclization, producing aryl oxadiazole compounds as shown in Formula I; ; The molar ratio of aryl diazid to p-toluenesulfonyl chloride is 1:(1.2-2); the basic reagent is diisopropylethylamine, and the molar ratio of the basic reagent to aryl diazid is 1:(2-5); the reaction is carried out at 25-45℃.

[0009] Another objective of this invention is to apply the above-mentioned aryl oxadiazole compounds in the preparation of drugs for the prevention and / or treatment of dengue virus infection. The compounds prepared by introducing specific aromatic or aliphatic groups can effectively inhibit viral replication, and the structural optimization significantly reduces cytotoxicity. In vitro experimental data show that the compounds of this invention exhibit significant inhibitory activity against DENV, especially compounds I-21, I-27, I-28, and I-29.

[0010] The drug of this invention comprises the compound of this invention as the active ingredient, and may also contain pharmaceutically acceptable excipients (such as diluents and binders) to formulate dosage forms such as capsules, tablets, and injections. Furthermore, this active ingredient can be used in combination with other antiviral drugs (such as NS5 inhibitors) to form combination therapies, thereby enhancing efficacy and reducing the risk of drug resistance.

[0011] The aryl oxadiazole compounds prepared by this invention have a significant inhibitory effect on DENV, and their anti-DENV therapeutic index is higher than that of the current clinical drug ribavirin. They can be used as candidate drugs for anti-DENV. Furthermore, the preparation method of this invention is simple, has a high yield, and is suitable for industrial production. Detailed Implementation

[0012] The following examples further illustrate the content of the present invention, but these examples do not limit the scope of protection of the present invention. Unless otherwise specified, the methods in the examples are conventional methods, and unless otherwise specified, the reagents used are conventional commercial reagents or reagents prepared according to conventional methods. Example 1: Preparation of compound I-1, the preparation route is as follows:

[0013] 1. Preparation of methyl p-fluorophenylacetate (intermediate 2-1) 1 g (6.49 mmol) of p-fluorophenylacetic acid was added to a 250 mL round-bottom flask and dissolved thoroughly in anhydrous tetrahydrofuran. After cooling to 0 °C in an ice bath, 0.7 mL (9.73 mmol) of thionyl chloride and 1-3 drops of N,N-dimethylformamide (DMF) were slowly added dropwise. The ice bath was removed, and the mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC until the starting material spot disappeared. The solvent was removed under reduced pressure, and the crude acyl chloride obtained was used directly in the next step without purification. The above acyl chloride was dissolved in anhydrous tetrahydrofuran, and anhydrous triethylamine (1.35 mL, 9.74 mmol) was added. 80 mL of methanol was slowly added dropwise in an ice bath. After the addition was complete, the mixture was stirred at room temperature for 30 min. The reaction was monitored by TLC until complete. The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (100 mL × 3). The organic phases were collected and combined, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain a colorless oily product, methyl p-fluorophenylacetate 2-1, with a yield of 85%.

[0014] 2. Preparation of methyl 2-bromo-2-(4-fluorophenyl)acetate (intermediate 3-1) Methyl p-fluorophenylacetate (1 g, 5.95 mmol) was placed in a 250 mL round-bottom flask and dissolved in carbon tetrachloride. Azobisisobutyronitrile (39 mg, 0.24 mmol) and N-bromosuccinimide (1.27 g, 7.14 mmol) were added sequentially. The mixture was heated in an oil bath to 80 °C and refluxed for 10–12 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature to quench the reaction. The solvent was removed under reduced pressure, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were collected and combined, washed with saturated saline solution, dried over anhydrous sodium sulfate, concentrated, and then purified by silica gel column chromatography to obtain a colorless oily intermediate 3–1, with a yield of 68%. 3. Preparation of methyl 2-((3,5-dimethoxyphenyl)amino)-2-(4-fluorophenyl)acetate (intermediate 4-1) Intermediate 3-1 (1 g, 4.05 mmol), 3,5-dimethoxyaniline (0.744 mg, 4.86 mmol), and anhydrous potassium carbonate (0.84 mg, 6.07 mmol) were placed in a 250 mL round-bottom flask, dissolved in acetonitrile, and stirred at room temperature for 24–48 h. The reaction was monitored by TLC until completion, the reaction was quenched, the solvent was removed by vacuum distillation, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were collected and combined, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 4-1 (white solid) in 53% yield. 4. Preparation of 2-((3,5-dimethoxyphenyl)amino)-2-(4-fluorophenyl)acetylhydrazine (intermediate 5-1) Compound 4-1 (1.00 g, 4.7 mmol) was weighed and dissolved in anhydrous ethanol. 1.5 mL of hydrazine hydrate (48.21 mmol) was added, and the mixture was refluxed at 80 °C for 10-15 h. The reaction was monitored by TLC until complete. The reaction was quenched, and the solvent was removed under reduced pressure. The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were collected and washed successively with 100 mL of water and saturated sodium chloride solution to remove excess hydrazine hydrate. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 5-1 (white solid) in 87% yield. 5. Preparation of 1H-indole-3-formyl chloride (intermediate 6-1) Indolecarboxylic acid (0.2 g, 1.24 mmol) was added to a 100 mL round-bottom flask, anhydrous tetrahydrofuran was added and stirred thoroughly to dissolve, the mixture was cooled in an ice bath, and thionyl chloride (0.135 mL, 1.86 mmol) and DMF (1-2 drops) were added sequentially. The mixture was heated to room temperature and stirred for about 3 h. The reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure to obtain intermediate 6-1 (oily crude product), which was used directly in the next step of the reaction. 6. Preparation of N'-(2-((3,5-dimethoxyphenyl)amino)-2-(4-fluorophenyl)acetyl)-1H-indole-3-carboxylhydrazine (intermediate 7-1) Intermediate 5-1 (0.2 g, 0.6 mmol) was dissolved in anhydrous tetrahydrofuran, and anhydrous triethylamine (0.13 mL, 0.94 mmol) was added. The solution of freshly prepared intermediate 6-1 (0.29 mg, 0.63 mmol) in anhydrous tetrahydrofuran was slowly added dropwise under ice bath conditions. After the addition was complete, the mixture was brought to room temperature and stirred for 30 min. The reaction was monitored by TLC to indicate completion. The reaction was quenched with methanol, concentrated, and extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain intermediate 7-1 (white solid), with a yield of 66%. 7. Preparation of N-((5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-1) Intermediate 7-1 (0.2 g, 0.4 mmol) was dissolved in anhydrous dichloromethane, and p-toluenesulfonyl chloride (0.22 g, 1.3 mmol) and diisopropylethylamine (0.3 mL, 1.73 mmol) were added. The mixture was heated under reflux in an oil bath for about 8-24 hours. The reaction was monitored by TLC until complete. The reaction was quenched, and the mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with 100 mL of saturated saline solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by column chromatography, and recrystallized to obtain the target compound I-1. ; The product was a white solid, with a yield of 23%. 1 H NMR (400 MHz, DMSO-d6) δ 12.04 (s, 1H, NH), 8.14 (s, 1H, ArH), 8.03 – 8.01 (m, 1H, ArH), 7.70 – 7.66 (m, 2H, ArH), 7.52 (dd, J =6.2, 3.7 Hz 1H, ArH), 7.26 (m, 4H, ArH), 6.87 (dd, J = 8.4, 3.0 Hz, 1H, NH), 6.22 (dd, J = 8.3, 3.0 Hz, 1H, CH), 6.01 – 6.00 (m, 2H, ArH), 5.81–5.80 (m, 1H, ArH), 3.63 (s, 6H, CH3); m / z [M+H] + : 444.1595. Example 2: Compound I-2 was prepared using the same method as compound I-1, except that step (5) used 1-p-toluenesulfonyl-1H-indole-3-carboxylic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(1-p-toluenesulfonyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (Formula I-2); the product is a white solid, yield 42%, mp: 210℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H, ArH), 8.09 (d, J = 7.8 Hz, 1H, ArH), 8.03 (t, J =6.8, 3H, ArH), 7.69 (t, J = 9.1, 2H, ArH), 7.51- 7.41 (m, 4H, ArH), 7.26 (t,J = 8.9, 2H, ArH), 6.87 (d, J = 8.4 Hz, 1H, NH), 6.27 (d, J = 8.3 Hz, 1H,CH), 6.01 (d, J = 1.9 Hz, 2H, ArH), 5.82 (t, J = 2.0 Hz, 1H, ArH), 3.63 (s,6H, CH3), 2.32 (s, 3H, CH3); m / z [M + +H] + : 598.1682. Compound I-3 was prepared using the same method as compound I-1, except that step (5) used m-fluoromethoxybenzoic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(3-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (Formula I-3); white solid, yield 73%, mp: 140℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.00(d, J = 1.9 Hz, 1H, ArH), 7.88 (s, 1H, ArH), 7.76 (t, J = 8.0 Hz, 1H, ArH),7.68- 7.64 (m, J = 8.8, 2.1 Hz, 3H, ArH), 7.26 (t, J = 8.9 Hz, 2H, ArH), 6.88 (d, J = 8.6 Hz, 1H, NH), 6.28 (d, J = 8.5, Hz, 1H, CH), 6.00 (d, J = 2.1 Hz,2H, ArH), 5.82 (d, J = 2.1 Hz, 1H, ArH), 3.64 (s, 6H, CH3); m / z [M + +H] + :489.1300. Compound I-4 was prepared using the same method as compound I-1, except that in step (5), p-trifluoromethoxybenzoic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (Formula I-4); white solid, yield 72%, mp: 130℃; 1H NMR (400 MHz, DMSO-d6) δ 8.11(d, J = 8.8, 2H, ArH), 7.67- 7.59 (m, 4H, ArH), 7.26 (t, J = 8.9 Hz, 2H,ArH), 6.87 (d, J = 8.4 Hz, 1H, NH), 6.24 (d, J = 8.3 Hz, 1H, CH), 5.99 (d, J= 2.0 Hz, 2H, ArH), 5.81 (t, J = 2.0 Hz, 1H, ArH), 3.64 (s, 6H, CH3);m / z [M + +H] + : 489.1300.

[0015] Compound I-5 was prepared using the same method as compound I-1, except that step (5) used coumarin-3-carboxylic acid as a raw material to synthesize the corresponding acyl chloride; ; 3-(5-(((3,5-dimethoxyphenyl)amino)(4-fluorophenyl)methyl)-1,3,4-oxadiazol-2-yl)-2H-benzopyran-2-one (Formula I-5): white solid, yield 77%, mp: 180℃; 1 H NMR (400 MHz, DMSO-d6) δ8.87 (s, 1H, ArH), 7.96 (dd, J = 7.9, 1.6 Hz, 1H, ArH), 7.76 (ddd, J = 8.7,7.3, 1.6 Hz, 1H, ArH), 7.67- 7.64 (m, 2H, ArH), 7.49(d, J = 2.2 Hz, 1H, ArH), 7.44 (d, J = 1.8 Hz, 1H, ArH), 7.26(t, J = 2.2 Hz, 2H, ArH), 6.88 (d, J = 8.3Hz, 1H, NH), 6.26 (d, J = 8.3 Hz, 1H, CH), 5.98 (d, J = 2.1 Hz, 2H, ArH), 5.81 (t, J = 2.1 Hz, 1H, ArH), 3.63 (s, 6H, CH3); m / z [M + +H] + : 473.1382. Compound I-6 was prepared using the same method as compound I-1, except that in step (5), cinnamic acid was used as a raw material to synthesize the corresponding acyl chloride; ; (E)-N-((4-fluorophenyl)(5-styryl-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (Formula I-6): white solid, yield 42%, mp: 143℃; 1 H NMR (400 MHz, DMSO-d6) δ 6.91(d, J =7.0 Hz, 2H, ArH), 6.78 (dd, J = 8.4, 5.4 Hz, 2H, ArH), 6.71 (d, J = 16.5 Hz,1H, CH=CH), 6.60- 6.56 (m, 3H, (m, 2H, ArH), 4.95(m, 1H, ArH), 3.64 (s, 6H, CH3); m / z [M + +H] + : 431.1647.

[0016] Compound I-7 was prepared using the same method as compound I-1, except that step (5) used 4-(methanesulfonyl)benzoic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(4-(methanesulfonyl)phenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-7): white solid, yield 60%, mp: 110℃; 1H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J= 8.9Hz, 2H, ArH), 8.15 (d, J = 8.4Hz, 2H, ArH), 7.67 (t, J = 7.2 Hz, 2H,ArH), 7.25 (d, J = 8.9Hz, 2H, ArH), 6.91 (d, J = 8.5 Hz, 1H, NH), 6.29 (d, J= 8.4 Hz, 1H, CH), 5.99 (d, J = 1.8 Hz, 2H, ArH), 5.81 (t, J = 2.6 Hz, 1H,ArH), 3.63 (s, 6H, CH3), 3.33 (s, 3H, CH3); m / z [M + +H] + : 486.1260.

[0017] Compound I-8 was prepared using the same method as compound I-1, except that step (5) used 3-indoleacetic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-8): white solid, 19% yield, mp: 90℃; 1 H NMR (400 MHz, DMSO-d6) δ 11.01 (s,1H, NH), 7.54- 7.51 (m, 2H, ArH), 7.41 (d, J = 7.9 Hz, 1H, ArH), 7.35 (d, J =8.1 Hz, 1H, ArH), 7.26 (d, J = 2.4 Hz, 1H, ArH), 7.17 (t, J = 8.8 Hz, 2H,ArH), 7.12- 7.02 (m, 1H, ArH), 6.95 (t, J = 7.2 Hz, 1H, ArH), 6.75 (d, J =8.5 Hz, 1H, NH), 6.10 (d, J = 8.5 Hz, 1H, CH), 5.92 (d, J = 2.1 Hz, 2H, ArH), 5.77 (t, J = 2.1 Hz, 1H, ArH), 4.32 (s, 2H, CH2), 3.59 (s, 6H, CH3); m / z [M + +H] +: 458.1750.

[0018] Compound I-9 was prepared using the same method as compound I-1, except that in step (5) indole-6-carboxylic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(1H-indol-6-yl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-9): white solid, yield 33%, mp: 90℃; 1 H NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H,NH), 8.21 (d, J = 1.6 Hz, 1H, ArH), 7.72- 7.65 (m, 3H, ArH), 7.54 (d, J = 8.5Hz, 1H, ArH), 7.50 (t, J = 2.8 Hz, 1H, ArH), 7.26 (t, J = 8.8 Hz, 2H, ArH), 6.89 (d, J = 8.3 Hz, 1H, NH), 6.61 (s, 1H, ArH), 6.22 (d, J = 8.3 Hz, 1H,CH), 6.00 (d, J = 2.1 Hz, 2H, ArH), 5.80 (t, J = 2.1 Hz, 1H, ArH), 3.64 (s,6H, CH3); m / z [M + +H] + : 444.1592.

[0019] Compound I-10 was prepared using the same method as compound I-1, except that step (5) used 3,4-dimethoxybenzoic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-10): white solid, 30% yield, mp: 88℃; 1H NMR (400 MHz, DMSO-d6) δ 7.66- 7.62(m, 2H, ArH), 7.52 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 7.44 (d, J = 2.1 Hz, 1H,ArH), 7.25 (t, J = 8.7 Hz, 2H, ArH), 7.15 (d, J = 8.5 Hz, 1H, ArH), 6.86 (d,J = 8.5 Hz, 1H, NH), 6.21 (d, J = 8.4 Hz, 1H, CH), 5.98 (d, J = 2.1 Hz, 2H,ArH), 5.80 (t, J = 2.2 Hz, 1H, ArH), 3.84 (s, 6H, CH3), 3.63 (s, 6H, CH3); m / z[M + +H] + : 465.1695.

[0020] Compound I-11 was prepared using the same method as compound I-1, except that step (5) used 3,4-dimethoxyphenylacetic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(3,4-dimethoxybenzyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-11): white solid, 17% yield, mp: 83℃; 1 H NMR (400 MHz, DMSO-d6) δ 7.58 -7.54 (m, 2H, ArH), 7.22 (t, J = 8.8 Hz, 2H, ArH), 6.86 (d, J = 8.2 Hz, 1H,NH), 6.82 (d, J = 2.1 Hz, 1H, ArH), 6.76- 6.72 (m, 2H, ArH), 6.12 (d, J = 8.5Hz, 1H, CH), 5.93 (d, J = 2.1 Hz, 2H, ArH), 5.78 (t, J = 2.1 Hz, 1H, ArH), 4.17 (s, 2H, CH2), 3.71 (s, 3H, CH3), 3.66 (s, 3H, CH3), 3.60 (s, 6H, CH3); m / z [M + +H] + : 479.1856.

[0021] Compound I-12 was prepared using the same method as compound I-1, except that step (5) used 4-(methanesulfonyl)phenylacetic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(4-(methanesulfonyl)benzyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-12): white solid, yield 23%, mp: 90℃; 1 H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J= 8.4 Hz, 2H, ArH), 7.58- 7.52 (m, 4H, ArH), 7.22 (d, J = 8.9 Hz, 2H, ArH), 6.76 (d, J = 8.6 Hz, 1H, NH), 6.14 (d, J = 8.5 Hz, 1H, CH), 5.93 (d, J = 2.1Hz, 2H, ArH), 5.80 (t, J = 2.2 Hz, 1H, ArH), 4.42 (s, 2H, CH2), 3.61 (s, 6H,CH3), 3.22 (s, 3H, CH3); m / z [M + +H] + : 497.1421.

[0022] Compound I-13 was prepared using the same method as compound I-1, except that in step (5), piperic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(benzo[d][1,3]dioxono-5-yl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-13): white solid, yield 46%, mp: 115℃; 1H NMR (400 MHz, DMSO-d6) δ7.67- 7.63 (m, 2H, ArH), 7.51 (dd, J = 8.1, 1.7 Hz, 1H, ArH), 7.46 (d, J =1.7 Hz, 1H, ArH), 7.27- 7.23 (m, 2H, ArH), 7.13 (d, J = 8.2 Hz, 1H, ArH), 6.85 (d, J = 8.4 Hz, 1H, NH), 6.21 (d, J = 8.4 Hz, 1H, CH), 6.17 (s, 2H,CH2), 5.98 (d, J = 2.2 Hz, 2H, ArH), 5.81 (t, J = 2.1 Hz, 1H, ArH), 3.63 (s, 6H, CH3); m / z [M + +H] + : 449.1387.

[0023] Compound I-14 was prepared using the same method as compound I-1, except that in step (5), piperidine was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(benzo[d][1,3]dioxono-5-ylmethyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-14): white solid, yield 21%, mp: 90℃; 1 H NMR (400 MHz, DMSO-d6) δ 7.58- 7.55 (m, 2H, ArH), 7.24- 7.19 (m, 2H, ArH), 6.84(m, 2H, ArH), 6.77- 6.70 (m, 2H, ArH), 6.13 (d, J = 8.6 Hz, m / z [M + +H] + : 463.1543.

[0024] Compound I-15 was prepared using the same method as compound I-1, except that in step (5), nicotinic acid was used as a raw material to synthesize the corresponding acyl chloride;

[0025] ;

[0026] N-((4-fluorophenyl)(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-15): white solid, 35% yield, mp: 90℃; 1 H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J =2.3, 1H, ArH), 8.81 (dd, J = 4.9, 1.7 Hz, 1H, ArH), 8.34 (ddd, J = 8.0, 2.2,1.6 Hz, 1H, ArH), 7.768- 7.62 (m, 3H, ArH), 7.25 (d, J = 8.9 Hz, 2H, J = 4.9,1.7 Hz), 6.87 (d, J = 8.5 Hz, 1H, NH), 6.27 (d, J = 8.4 Hz, 1H, CH), 5.99 (d,J = 2.2 Hz, 2H, ArH), 5.81 (t, J = 2.1 Hz, 1H, ArH), 3.63 (s, 6H, CH3); m / z [M + +H] + : 406.1441.

[0027] Compound I-16 was prepared using the same method as compound I-1, except that step (5) used 2,2-difluoro-1,3-benzodioxo-5-carboxylic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(2,2-difluorobenzo[d][1,3]dioxotropine-5-yl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-16): white solid, 55% yield, mp: 119℃; 1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 1.7 Hz, 1H, ArH), 7.85 (dd, J = 8.4, 1.7 Hz, 1H), 7.68- 7.64 (m, 3H, ArH), 7.28 – 7.22 (m, 2H, ArH), m / z [M + +H] + : 485.1199.

[0028] Compound I-17 was prepared using the same method as compound I-1, except that step (5) used cyclohexylcarboxylic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((5-cyclohexyl-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-17): pale yellow liquid, yield 19%; 1 H NMR (400 MHz, DMSO-d6) δ 7.58-7.54 (m, 2H, ArH),7.25-7.20 (m, 2H, ArH), 6.72 (d, J = 8.4 Hz, 1H, NH), 6.10 (d, J = 8.4 Hz,1H, CH), 5.94 (d, J = 2.1 Hz, 2H, ArH), 5.79-5.78 (m, 1H, ArH), 3.61 (s, 6H,CH3), 1.61-1.51 (s, 6H, CH,CH2), 1.10-1.0 (m, 3H, CH2), 0.94-0.85 (m, 2H,CH2); m / z [M + +H] + : 411.1958.

[0029] Compound I-18 was prepared using the same method as compound I-1, except that step (5) used cyclohexylacetic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(cyclohexylmethyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-18): colorless liquid, yield 19%; 1 H NMR (400 MHz, DMSO-d6) δ 7.58-7.55 (m, 2H, ArH),7.25-7.20 (m, 2H, ArH), 6.75 (d, J = 8.5 Hz, 1H, NH), 6.10(d, J = 8.4 Hz, 1H,CH), 5.94 (d, J = 2.1 Hz, 2H, ArH), 5.79 (t, J = 2.2 Hz, 1H, ArH), 3.60 (s,6H, CH3), 2.90(s, 1H, CH), 1.94 (d, J = 6.9 Hz, 2H, CH2),1.71 – 1.62 (m, 3H,CH2, CH), 1.51 – 1.42 (m, 2H, CH2), 1.38-1.21 (m, 5H, CH2); m / z [M + +H] + :425.2113.

[0030] Compound I-19 was prepared using the same method as compound I-1, except that step (5) used 4-benzopyranone-2-carboxylic acid as a raw material to synthesize the corresponding acyl chloride; ; 2-(5-(((3,5-dimethoxyphenyl)amino)(4-fluorophenyl)methyl)-1,3,4-oxadiazol-2-yl)-4H-benzopyran-4-one (I-19): white solid, yield 51%, mp: 214℃; 1H NMR (400 MHz, DMSO-d6) δ8.09 (dd, J = 7.9, 1.7 Hz, 1H, ArH), 7.92- 7.87 (m, 1H, ArH), 7.78 (d, J = 8.6Hz, 1H, ArH), 7.70- 7.66 (m, 2H, ArH), 7.59- 7.55 (m, 1H, ArH), 7.30- 7.25(m, 2H, ArH), 7.06 (s, 1H, ArH), 6.91 (d, J = 8.6 Hz, 1H, NH), 6.36 (d, J =8.6 Hz, 1H, CH), 6.01 (d, J = 2.1 Hz, 2H, ArH), 5.83 (t, J = 2.1 Hz, 1H,ArH), 3.64 (s, 6H, CH3); m / z [M + +H] + : 473.4604.

[0031] Compound I-20 was prepared using the same method as compound I-1, except that in step (5), p-chlorobenzoic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-20): white solid, 79% yield, mp: 214℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.00- 7.97 (m,2H, ArH), 7.70- 7.64 (m, 4H, ArH), 7.28- 7.23 (m, 2H, ArH), 6.88 (d, J = 8.4Hz, 1H, NH), 6.25 (d, J = 8.4 Hz, 1H, CH), 5.98 (d, J = 2.2 Hz, 2H, ArH), 5.81 (t, J = 2.1 Hz, 1H, ArH), 3.63 (s, 6H, CH3); m / z [M + +H] + : 439.1099.

[0032] Compound I-21 was prepared using the same method as compound I-1, except that step (5) used quinoline-4-carboxylic acid as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(quinolin-4-yl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-21): white solid, yield 38%, mp: 154℃; 1 H NMR (400 MHz, DMSO-d6) δ 9.14 (d, J =4.5 Hz, 1H, ArH), 8.98 (dd, J = 8.6, 1.4 Hz, 1H, ArH), 8.18 (dd, J = 8.4, 1.3Hz, 1H, ArH), 8.08 (d, J = 4.5 Hz, 1H, ArH), 7.94- 7.90(m, 1H, ArH), 7.81(ddd, J = 8.4, 6.9, 1.4 Hz, 1H, ArH), 7.74- 7.70 (m, 2H, ArH), 7.28(t, J =8.9 Hz, 2H, ArH), 6.97 (d, J = 8.6 Hz, 1H, m / z [M + +H] + : 456.1592.

[0033] Compound I-22 was prepared using the same method as compound I-1, except that in step (5), p-methoxybenzoic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((4-fluorophenyl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-22): white solid, 65% yield, mp: 165℃; 1H NMR (400 MHz, DMSO-d6) δ 7.93- 7.90(m, 2H, ArH), 7.67- 7.63 (m, 2H, ArH), 7.27- 7.23 (m, 2H, ArH), 7.15- 7.13(m, 2H, ArH), 6.87 (d, J = 8.4 Hz, 1H, NH), 6.21 (d, J = 8.3 Hz, 1H, ArH), 5.98 (d, J = 2.1 Hz, 2H, ArH), 5.81 (t, J = 2.1 Hz, 1H, ArH), 3.84 (s, 3H,CH3), 3.63 (s, 6H, CH3); m / z [M + +H] + : 435.1590.

[0034] Compound I-23 was prepared using the same method as compound I-1, except that in step (5), p-bromobenzoic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(4-bromophenyl)-1,3,4-oxadiazol-2-yl)(4-fluorophenyl)methyl)-3,5-dimethoxyaniline (I-23): white solid, 73% yield, mp: 151℃; 1 H NMR (400 MHz, DMSO-d6) δ 7.92- 7.89 (m,2H, ArH), 7.84- 7.81 (m, 2H, ArH), 7.67- 7.64 (m, 2H, ArH), 7.28- 7.23 (m,2H, ArH), 6.88 (d, J = m / z [M + +H] + : 483.0590.

[0035] Compound I-24 was prepared using the same method as compound I-1, except that in step (5), p-fluorobenzoic acid was used as a raw material to synthesize the corresponding acyl chloride; ;

[0036] N-((4-fluorophenyl)(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-24): white solid, 75% yield, mp: 90℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.05 – 8.02 (m,2H, ArH), 7.68- 7.64 (m, 2H, ArH), 7.48- 7.44 (m, 2H, ArH), 7.28- 7.23 (m,2H, ArH), 6.88 (d, J = m / z [M + +H] + : 423.1390.

[0037] Compound I-25 was prepared using the same method as compound I-1, except that step (1) used 4-chloro-2-methoxyphenylacetic acid as the starting material to synthesize the corresponding methyl ester 2-2;

[0038] N-((5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)(4-chloro-2-methoxyphenyl)methyl)-3,5-dimethoxyaniline (I-25): white solid, 20% yield, mp: 150℃; 1 H NMR (400 MHz, DMSO-d6) δ12.03 (s, 1H, NH), 8.15 (d, J = 2.9 Hz, 1H, ArH), 8.02 (d, J = 7.1 Hz, 1H,ArH), 7.60- 7.49 (m, 2H, ArH), 7.30- 7.16 (m, 3H, ArH), 7.10 (dd, J = 8.2,2.0 Hz, 1H, ArH), 6.86 (d, J = 8.3 Hz, 1H, CH), 6.18 (d, J = 8.1 Hz, 1H, NH), 5.89 (d, J = 2.2 Hz, 2H, ArH), 5.82 (t, J = 2.1 Hz, 1H, ArH), 3.88 (s, 3H, CH3), 3.63 (s, 6H, CH3); m / z [M+ +H] + : 490.1406.

[0039] Compound I-26 was prepared using the same method as compound I-1, except that in step (1), 4-chloro-2-methoxyphenylacetic acid was used as a raw material to synthesize the corresponding methyl ester 2-2, and in step (5), 1-p-toluenesulfonyl-1H-indole-3-carboxylic acid was used as a raw material to synthesize the corresponding acyl chloride. ; N-((4-chloro-2-methoxyphenyl)(5-(1-p-toluenesulfonyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-26): white solid, yield 41%, mp: 134℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H, ArH), 8.10 (d, J = 7.6 Hz, 1H, ArH), 8.05- 8.02 (m,3H, ArH), 7.58 (d, J = 8.2 Hz, 1H, ArH), 7.51- 7.39 (m, 4H, ArH), 7.19 (d, J= 2.0 Hz, 1H, ArH), 7.10 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 6.87 (d, J = 8.3 Hz,1H, NH), 6.24 (d, J = 8.3 Hz, 1H, CH), 5.90 (d, J = 2.1 Hz, 2H, ArH), 5.83(t, J = 2.1 Hz, 1H, ArH), 3.87 (s, 3H, CH3), 3.64 (s, 6H, CH3), 2.31 (s, 3H, CH3); m / z [M + +H] + : 644.1495. Compound I-27 was prepared using the same method as compound I-1, except that in step (1) 4-chloro-2-methoxyphenylacetic acid was used as a raw material to synthesize the corresponding methyl ester 2-2, and in step (5) piperic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(benzo[d][1,3]dioxono-5-ylmethyl)-1,3,4-oxadiazol-2-yl)(4-chloro-2-methoxyphenyl)methyl)-3,5-dimethoxyaniline (I-27): white solid, 14% yield, mp: 147℃; 1H NMR (400 MHz, DMSO-d6) δ 7.44 (d, J = 8.3 Hz, 1H, ArH), 7.14 (d, J = 2.0 Hz, 1H, ArH), 7.05 (dd, J = 8.2, 2.0 Hz, 1H, ArH), 6.87- 6.83 (m, 2H, ArH, NH), 6.74- 6.70 (m,2H, ArH), 6.06 (d, J = 8.5 Hz, 1H, CH), 6.00 (s, 2H, CH2), 5.83 (d, J = 2.1Hz, 2H, ArH), 5.79 (t, J = 2.1 Hz, 1H, ArH), 4.16 (s, 2H, CH2), 3.79 (s, 3H,CH3), 3.61 (s, 6H, CH3); m / z [M + +H] + : 509.1353. Compound I-28 was prepared using the same method as compound I-1, except that in step (1) 4-chloro-2-methoxyphenylacetic acid was used as a raw material to synthesize the corresponding methyl ester 2-2, and in step (5) piperic acid was used as a raw material to synthesize the corresponding acyl chloride; ; N-((5-(benzo[d][1,3]dioxono-5-yl)-1,3,4-oxadiazol-2-yl)(4-chloro-2-methoxyphenyl)methyl)-3,5-dimethoxyaniline (I-28): white solid, yield 38%, mp: 172℃; 1 H NMR (400 MHz, DMSO-d6) δ 7.54 – 7.49 (m, 2H, ArH), 7.45 (d, J = 1.7 Hz, 1H, ArH), 7.17 (d,J = 2.0 Hz, 1H, ArH), 7.10 – 7.07 (m, 2H, ArH), 6.83 (d, J = 8.2 Hz, 1H. NH), 6.18 (d, J = 9.3 Hz, 3H, ArH,, CH), 5.86 (d, 2.1 Hz, 2H, ArH), 5.81(t, 2.2Hz, 1H, ArH), 3.86 (s, 3H, CH3), 3.62 (s, 6H, CH3); m / z [M + +H] + : 495.1195.

[0040] Compound I-29 was prepared using the same method as compound I-1, except that in step (1), 4-chloro-2-methoxyphenylacetic acid was used as the starting material to synthesize the corresponding methyl ester 2-2, and in step (5), formic acid was used as the starting material to synthesize the corresponding acyl chloride. ; 3-(5-((4-chloro-2-methoxyphenyl)((3,5-dimethoxyphenyl)amino)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (I-29): white solid, yield 66%, mp: 193℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.41(t, J = 1.7 Hz, 1H, ArH), 8.29 (d, J = 8.1 Hz, 1H, ArH), 8.10 (d, J = 7.9 Hz,1H, ArH), 7.81 (t, J = 7.9 Hz, 1H, ArH), 7.56 (d, J = 8.2 Hz, 1H, ArH), 7.19 (d, J = 2.0 Hz, 1H, ArH), 7.10 (dd, J = 8.2, 2.0 Hz, 1H, ArH), 6.84 (d, J =8.3 Hz, 1H, NH), 6.23 (d, J = 8.2 Hz, 1H, CH), 5.89 (d, J = 2.1 Hz, 2H, ArH), 5.83 (t, J = 2.1 Hz, 1H, ArH), 3.88 (s, 3H, CH3), 3.64 (s, 6H, CH3); m / z [M + +H] + : 476.1250.

[0041] Compound I-30 was prepared using the same method as compound I-1, except that in step (1), 4-chloro-2-methoxyphenylacetic acid was used as the starting material to synthesize the corresponding methyl ester 2-2, and in step (5), p-cyanobenzoic acid was used as the starting material to synthesize the corresponding acyl chloride.

[0042]

[0043] 4-(5-((4-chloro-2-methoxyphenyl)((3,5-dimethoxyphenyl)amino)methyl)-1,3,4-oxadiazol-2-yl)benzonitrile (I-30): white solid, yield 73%, mp: 158℃; 1H NMR (400 MHz, DMSO-d6) δ 8.15(d, J = 8.3 Hz, 2H, ArH), 8.07 (d, J = 8.1 Hz, 2H, ArH), 7.55 (d, J = 8.3 Hz,1H, ArH), 7.19 (d, J = 2.0 Hz, 1H, ArH), 7.10 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 6.85 (d, J = 8.2 Hz, 1H, NH), 6.24 (d, J = 8.2 Hz, 1H, CH), 5.89 (d, J = 2.1Hz, 2H, ArH), 5.83 (t, J = 2.1 Hz, 1H, ArH), 3.86 (s, 3H, CH3), 3.63 (s, 6H,CH3); m / z [M + +H] + : 476.1250.

[0044] Compound I-31 was prepared using the same method as compound I-1, except that in step (1), 4-chloro-2-methoxyphenylacetic acid was used as the starting material to synthesize the corresponding methyl ester 2-2, and in step (5), m-trifluoromethoxybenzoic acid was used as the starting material to synthesize the corresponding acyl chloride. ; N-((4-chloro-2-methoxyphenyl)(5-(3-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-31): white solid, yield 62%, mp: 135℃; 1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 7.8Hz, 1H, ArH), 7.93- 7.88 (m, 1H, ArH), 7.76 (t, J = 8.0Hz, 1H, ArH), 7.71- 7.63 (m, 1H, ArH), 7.55 (d, J = 8.2 Hz, 1H, ArH), 7.19 (d, J = 2.0 Hz, 1H, ArH), 7.10 (dd, J = 8.2, 2.0 Hz, 1H, ArH), 6.84 (d, J =8.3 Hz, 1H, NH), 6.23 (d, J = 8.3 Hz, 1H, CH), 5.89 (d, J = 2.1 Hz, 2H, ArH), 5.83 (t, J = 2.1 Hz, 1H, ArH), 3.87 (s, 3H, CH3), 3.64 (s, 6H, CH3); m / z [M + +H] + : 535.1115.

[0045] Compound I-32 was prepared using the same method as compound I-1, except that in step (1), 4-chloro-2-methoxyphenylacetic acid was used as the starting material to synthesize the corresponding methyl ester 2-2, and in step (5), p-trifluoromethoxybenzoic acid was used as the starting material to synthesize the corresponding acyl chloride. ; N-((4-chloro-2-methoxyphenyl)(5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-32): white solid, yield 60%, mp: 135℃; 1H NMR (400 MHz, DMSO-d6) δ 8.15- 8.07 (m, 2H, ArH), 7.60- 7.58 (m, 2H, ArH), 7.53 (d, J = 8.2 Hz,1H, ArH), 7.17 (d, J = 2.0 Hz, 1H, ArH), 7.10 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 6.84 (d, J = 8.2 Hz, 1H, NH), 6.21 (d, J = 8.2 Hz, 1H, CH), 5.89 (d, J = 2.1Hz, 2H, ArH), 5.83 (t, J = 2.1 Hz, 1H, ArH), 3.87 (s, 3H, CH3), 3.64 (s, 6H,CH3); m / z [M + +H] + : 535.1115.

[0046] Compound I-33 was prepared using the same method as compound I-1, except that in step (1), 4-chloro-2-methoxyphenylacetic acid was used as the starting material to synthesize the corresponding methyl ester 2-2, and in step (5), 5-fluoro-1-p-toluenesulfonyl-1H-indole-3-carboxylic acid was used as the starting material to synthesize the corresponding acyl chloride. ; N-((4-chloro-2-methoxyphenyl)(5-(5-fluoro-1-p-toluenesulfonyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)methyl)-3,5-dimethoxyaniline (I-33): white solid, yield 32%, mp: 225℃; 1H NMR (400MHz, DMSO-d6) δ 8.67 (s, 1H, CH), 8.06- 8.01 (m, 4H, ArH), 7.57- 7.48 (m, 2H, ArH), 7.42 (d, J = 8.2 Hz, 2H, ArH), 7.19 (d, J = 2.0 Hz, 1H, ArH), 7.10 (dd,J = 8.2, 2.0 Hz, 1H, ArH), 6.86 (d, J = 8.2 Hz, 1H, NH), 6.23 (d, J = 8.2 Hz,1H, CH), 5.89 (d, J = 2.1 Hz, 2H, ArH), 5.82 (t, J = 2.1 Hz, 1H, ArH), 3.87(s, 3H, CH3), 3.63 (s, 6H, CH3), 2.32 (s, 3H, CH3); m / z [M++H] + : 662.1400.

[0047] Example 3: In vitro anti-DENV activity experiments of compounds I-1 to I-33 1. The in vitro cellular level anti-DENV activity was evaluated using Vero cells and the DENV-II D01090 (GenBank: KY882458) virus strain, as follows: Drugs and samples: Compounds I-1 to I-33 were dissolved in DMSO to prepare 50 mM stock solutions and stored at 4°C. The positive control compound ribavirin was purchased from Guangzhou Angfei Biotechnology Co., Ltd., dissolved in DMSO, and stored at 4°C.

[0048] 2. MTT assay for drug cytotoxicity: Prepare Vero cells in good growth condition, seed them in 96-well plates, and adjust the cell density to 1×10⁶ cells / well. 5Cells / mL, volume 100 μL / well, surrounded by 200 μL / well of PBS to prevent evaporation, cultured overnight until cell density reaches above 90%; a blank control group (Mock) and a solvent control group (DMSO) were set up, and the compound was serially diluted to a total of 6 concentrations (200, 100, 50, 25, 12.5, 6.25 μmol / L), with 3 replicates for each compound concentration to reduce experimental error. The diluted compound was added to the wells in descending order of concentration to make each well 100 μL, and cultured at 37°C in a 5% CO2 cell incubator for 3 days. Three days later, the culture plates were removed, and 20 μL of MTT (5 mg / mL) was added to each well. The plates were then incubated at 37°C with 5% CO2 for 4 hours to ensure thorough mixing of live cells and formazan. After 4 hours, the supernatant was aspirated, and 100 μL of 12% SDS-50% DMF solution was added. The plates were then incubated at 37°C with 5% CO2 for 12 hours until the formazan was fully dissolved. Once the formazan was fully dissolved, the OD value was measured using a microplate reader (detection wavelength 570 nm, reference wavelength 630 nm). A dose-response curve was plotted based on the OD value and the compound concentration, and the cytotoxic half-maximal concentration (CMC) of the compound was calculated. 50 ).

[0049] 3. Plaque assay to determine the inhibitory activity of the compound against DENV: Vero cells were seeded in 12-well plates at 3 × 10⁶ wells. 5 Cells / well. After 24 hours of culture, DENV-II was added at 500 μL / well (serum-free DMEM medium diluted with virus, MOI=0.5). After adsorption for 2-4 hours, maintenance solid medium was prepared at a 1:1 ratio. 4% FBS DMEM medium containing the compound was mixed with 2% low-melting-point agarose and added at 1 mL / well to the cell culture plate to achieve a final concentration of 2% FBS DMEM solid maintenance medium, ensuring basic cell viability. After solidification, the plate was inverted and placed in a 37℃, 5% CO2 cell culture incubator. Cell pathogenesis was observed daily, generally for 5-6 days. Implantation was performed with 4% paraformaldehyde for 30 min, followed by washing three times and staining with 0.8% crystal violet. Images were acquired and plaques were counted using an ELISA reader (CTL, Immunospot S6 Universal), and the half-maximal effective drug concentration (EC50) was calculated. 50 (50% effective concentration).

[0050] 4. qPCR method for detecting the inhibitory activity of the compound against DENV: Vero cells were seeded in 24-well plates at 3 × 10⁶ cells / well. 5Count / well. After 24 hours of incubation, 200 μL of DENV-II (serum-free DMEM medium diluted with virus, MOI=0.5) was added to each well. After adsorption for 2-4 hours, serially diluted compounds were added, and the mixture was incubated at 37°C for another 72 hours. The supernatant was collected for viral RNA extraction and quantification. Viral RNA yield was calculated using a serial dilution standard curve. A dose-response curve was plotted based on viral RNA copy number and compound concentration, and the half-maximal effective concentration (EC50) of the compound was calculated. 50 ).

[0051] Based on the experimental results, a dose-response curve was plotted using GraphPad Prism 9.1.0, and the 50% effective concentration (EC50) of the sample inhibiting the virus was calculated using the Reed & Muench method. 50 ), 50% cell growth inhibitory concentration (CC) 50 ) and the Therapeutic Index (TI). The Therapeutic Index (TI = CC) 50 / EC 50 () represents the half-maximal inhibitory concentration (Cg) of the drug on cells. 50 and the half-maximal effective concentration (EC50) against the virus 50 The ratio of the drug's safety to its safety index represents the safety of the drug; the higher the value, the safer the drug.

[0052] Using ribavirin, a clinical treatment drug, as a positive control, the compounds synthesized by the above method were subjected to anti-DENV activity experiments and cytotoxicity experiments. (1) Evaluation of anti-DENV activity: The compounds were first screened for antiviral activity at a concentration of 50 μM using the plaque method. After identifying the active compounds, the experimental concentration was reduced for secondary screening. Compounds that were only effective at high concentrations but had a narrow therapeutic window were eliminated. Finally, compounds that showed significant activity in the 10 μM range were selected. Their inhibition rate against viral replication was detected by qPCR, and the half-maximal effective concentration (EC50) was evaluated by concentration gradient test. 50 (2) Drug cytotoxicity: MTT assay was performed on compounds with antiviral activity, and the half-maximal toxic concentration (CMC) of the compounds was determined by concentration gradient. 50 ).

[0053] The detection results are shown in Tables 2 and 3. As can be seen from Table 2, the EC values ​​of 18 compounds... 50At concentrations below 50 μM, six compounds (I-2, I-13, I-21, I-27, I-28, and I-29) still exhibited antiviral effects when the experimental concentration was reduced to 10 μM. Furthermore, compounds I-21, I-27, I-28, and I-29 all showed inhibition rates exceeding 70% against DENV at 10 μM. Table 3 shows the EC50 values ​​of compounds I-21, I-27, I-28, and I-29. 50 The effective concentrations were 1.89±0.269 μM, 2.948±0.640 μM, 0.6415±0.109 μM, and 0.1767±0.0166 μM, respectively. Furthermore, in the MTT assay at the highest concentration of 200 μM, these compounds (I-21, I-27, I-28, and I-29) did not exhibit significant cytotoxicity, with therapeutic indices (TIs) of 105.82, 67.84, 311.77, and 1131.862, respectively. The therapeutic index (TI > 1131.862) of I-29 was approximately 259 times that of the positive control Ribavirin (TI > 4.37), indicating that this compound possesses excellent selective antiviral activity and could serve as a lead compound for further development of anti-DENV drugs.

[0054] Table 2. Initial screening results of anti-DENV activity ;

[0055] In the table, NA indicates no activity, and ND indicates not detected; Table 3. Results of the anti-DENV activity of the preferred compounds

[0056] The applicant declares that the present invention illustrates the aryl oxadiazole compounds, their preparation methods, and uses through the above embodiments. However, the present invention is not limited to the above embodiments, i.e., it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials for the products of the present invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An aryl oxadiazole compound with the following chemical structural formula, or a pharmaceutically acceptable salt thereof: ； In the formula: R1 is selected from substituted or unsubstituted aromatic groups, aliphatic groups, heterocyclic groups, and substituents are selected from halogens, trifluoromethoxy, p-toluenesulfonyl, methanesulfonyl, alkyl, alkoxy, cyano, etc.; n is 0, 1, or 2. R2 is selected from halogens and C1-C4 alkoxy groups.

2. The aryloxadiazole compound according to claim 1, characterized in that, The compound is selected from one of the following structures: ； ； 。 3. The method for preparing the aryloxadiazole compound according to claim 1 or 2, characterized in that, The steps are as follows: (1) Esterification reaction: In the presence of solvent and chlorination reagent, substituted aromatic acetic acid reacts with alcohol to generate ester compounds as shown in Formula II; ； In the formula, R2 is selected from halogens and C1-C4 alkoxy groups, and R3 is a C1-C5 alkyl group; (2) Bromination reaction: In a solvent, ester compounds react with N-bromosuccinimide and azobisisobutyronitrile to generate α-bromoesters as shown in Formula III; ； (3) Nucleophilic substitution reaction: At room temperature and in the presence of solvent, α-bromoester reacts with 3,5-dimethoxyaniline under the catalysis of a basic reagent to generate the aryl ester shown in formula IV; ； (4) Hydrazolysis reaction: In a solvent, aryl esters react with hydrazine hydrate under reflux at 75-85℃ to generate aryl hydrazide as shown in formula V; ； (5) Acyl chloride reaction: In a solvent, aromatic or fatty carboxylic acids react with a chlorinating agent to produce acyl chloride as shown in formula VII; ； In the formula, R1 is selected from substituted or unsubstituted aromatic groups, aliphatic groups, and heterocyclic groups; the substituents are selected from halogens, trifluoromethoxy, p-toluenesulfonyl, methanesulfonyl, alkyl, alkoxy, and cyano; n is 0, 1, or 2. (6) Condensation reaction: Under alkaline conditions, the aryl hydrazide shown in formula V reacts with the acyl chloride shown in formula VII to generate the aryl dihydrazide shown in formula VIII; ； (7) Cyclization reaction: Aryl bishydrazide reacts with p-toluenesulfonyl chloride under alkaline conditions to undergo dehydration and cyclization, producing aryl oxadiazole compounds as shown in Formula I; 。 4. The preparation method according to claim 3, characterized in that, In step (1), the alcohol is selected from methanol, ethanol, propanol, butanol, and pentanol, and the chlorination reagent is selected from thionyl chloride, phosphorus oxychloride, phosphorus pentachloride, and oxalyl chloride or more. The molar ratio of substituted aromatic acetic acid to alcohol is 1:(5-40), and the reaction is carried out at 0-25℃.

5. The preparation method according to claim 3, characterized in that, In step (2), the molar ratio of the ester compound to N-bromosuccinimide is 1:(1-2), and the molar ratio of the ester compound to azobisisobutyronitrile is 1:(0.02-1); the solvent is carbon tetrachloride; the reaction is carried out under reflux at 65-80℃.

6. The preparation method according to claim 3, characterized in that, In step (3), the alkaline reagent is selected from one or more of potassium carbonate, sodium carbonate, sodium hydroxide, triethylamine, and diisopropylethylamine; the molar ratio of α-bromoester to alkaline reagent is 1:(1-3), and the molar ratio of α-bromoester to 3,5-dimethoxyaniline is 1:(1-1.5); the solvent is acetonitrile.

7. The preparation method according to claim 3, characterized in that, In step (4), the molar ratio of aryl ester to hydrazine hydrate is 1:(3-10); the solvent is anhydrous ethanol.

8. The preparation method according to claim 3, characterized in that, In step (5), the chlorinating agent is one or more of thionyl chloride, phosphorus oxychloride, phosphorus pentachloride, and oxalyl chloride; the molar ratio of aromatic or aliphatic carboxylic acid to chlorinating agent is 1:(2-6); the solvent is anhydrous tetrahydrofuran or dichloromethane; the reaction is carried out at 0-25℃.

9. The preparation method according to claim 3, characterized in that, In step (6), the molar ratio of aryl hydrazide to acyl chloride is 1:(1-2); the basic reagent is triethylamine, and the molar ratio of the basic reagent to aryl hydrazide is 1:(1-2); the reaction is carried out at 0-25℃; in step (7), the molar ratio of aryl dihydrazide to p-toluenesulfonyl chloride is 1:(1.2-2); the basic reagent is diisopropylethylamine, and the molar ratio of the basic reagent to aryl dihydrazide is 1:(2-5); the reaction is carried out at 25-45℃.

10. The use of the aryl oxadiazole compound of claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of dengue virus infection.

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