1, 3, 4-oxadiazole compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine, and discloses a 1, 3, 4-oxadiazole compound as well as a preparation method and application thereof, the molecular structure of the 1, 3, 4-oxadiazole compound comprises structures as shown in formula (I) to formula (VI), and the structures as shown in formula (I) to formula (VI) are defined in the specification. The 1, 3, 4-oxadiazole compound provided by the invention has an inhibition effect on the activity of an ASADH enzyme and mycobacterium tuberculosis, and can be used for preparing a medicine for inhibiting the activity of the ASADH enzyme and a medicine for treating mycobacterium tuberculosis infection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a 1,3,4-oxadiazole compound, its preparation method, and its application. Background Technology

[0002] Treatment for tuberculosis caused by drug-sensitive Mycobacterium tuberculosis requires at least six months, while treatment for tuberculosis caused by multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis requires even longer periods of second-line anti-tuberculosis drugs. Due to the long treatment duration and the toxicity of second-line drugs, patients often fail to complete treatment or do not strictly adhere to the treatment regimen. Although drugs such as rifampin, isoniazid, and ethambutol are considered the ultimate cure for tuberculosis, drug resistance still limits their effectiveness. Therefore, in the face of the increasingly serious global tuberculosis crisis, there is an urgent need to develop anti-tuberculosis drugs with new structures and mechanisms to address the threat of drug-resistant bacteria in existing tuberculosis treatments.

[0003] The bacterial aspartate metabolic pathway is responsible for the biosynthesis of lysine, threonine, isoleucine, and methionine, and is crucial for the survival of Mycobacterium tuberculosis. Aspartate β-semialdehyde dehydrogenase (ASADH) is a core enzyme in this pathway, participating in bacterial aspartate metabolism. In prokaryotes, inhibiting ASADH activity leads to the loss of diaminobenzoic acid esters, resulting in insufficient cell wall synthesis and subsequent bacterial death. Therefore, ASADH, a core enzyme in the aspartate metabolic pathway, is considered a potential target for developing anti-tuberculosis drugs. For drug development, the aspartate metabolic pathway has a crucial characteristic: "It exists in microorganisms and plants, but is completely absent in the human body." This means that if we can safely inhibit ASADH activity, we can effectively "starve" bacteria without affecting human cells. Therefore, developing ASADH enzyme inhibitors has a high safety profile for long-term treatment in patients infected with multidrug-resistant Mycobacterium tuberculosis.

[0004] 1,3,4-Oxadiazoles are a crucial class of five-membered aromatic heterocyclic compounds, consisting of one oxygen atom, two nitrogen atoms, and two carbon atoms, with the heteroatoms located at positions 1, 3, and 4, respectively. Due to their unique electronic structure and excellent biological activity, they are frequently used in modern drug development as substitutes for amide bonds to address drug metabolic stability issues. As rigid and stable amide mimics, 1,3,4-Oxadiazoles possess excellent hydrogen bond recognition capabilities and moderate lipophilicity. Furthermore, their 2,5-disubstituted linear structure facilitates modular synthesis, allowing for efficient matching of the spatial and electronic requirements of various enzyme active sites. However, there is currently no research on the application of 1,3,4-Oxadiazoles in the preparation of ASADH enzyme inhibitors. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a 1,3,4-oxadiazole compound that inhibits both ASADH enzyme activity and Mycobacterium tuberculosis, along with its preparation method and applications.

[0006] The technical solution of the present invention is as follows: First, the present invention provides a 1,3,4-oxadiazole compound, the molecular structure of which includes the structures shown in formula (I) to (VII); (I), (II) (Ⅲ), (Ⅳ), (V) (VI) (VII); Wherein, R1 is at least one of ortho-, meta-, or para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, or nitro groups; R2 is at least one of the following: ortho-, meta-, or para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, or nitro. R3 is at least one of nitrogen-nitrogen dimethyl, nitrogen-nitrogen diethyl, piperidine, and tetrahydropyrrole.

[0007] Preferably, the compound of formula (I) is N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-cyanobenzamide; the compound of formula (II) is N-(5-(benzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-yl)-3-methylbenzamide; the compound of formula (III) is N-(5-(benzofuran-2-yl)-1,3,4-oxadiazol-2-yl)-4-nitrobenzamide; and the compound of formula (IV) is N-(5-(1H-indole) -2-yl)-1,3,4-oxadiazol-2-yl)-4-cyanobenzamide; the compound with formula (V) is N-(5-(benzothiophen-2-yl)-1,3,4-oxadiazol-2-yl)-3-fluorobenzamide; the compound with formula (VI) is 3-bromo-N-(5-(isoquinoline-3-yl)-1,3,4-oxadiazol-2-yl)benzamide; the compound with formula (VII) is N-(5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-yl)-3-fluorobenzamide.

[0008] Secondly, the present invention provides a method for preparing 1,3,4-oxadiazole compounds with the structure shown in formula (I), wherein the 1,3,4-oxadiazole ring of the compound of formula (I) is formed by an oxidative cyclization reaction of a urea compound. Specifically, the preparation method includes the following steps: Step 1: The aminourea hydrochloride is condensed with R1-substituted benzaldehyde to obtain an aminourea compound; Step 2: The acetal urea compounds prepared in Step 1 undergo an oxidative cyclization reaction to yield 2-amino-1,3,4-oxadiazole compounds. Step 3: The 2-amino-1,3,4-oxadiazole compound prepared in step 2 undergoes an amide condensation reaction with R2-substituted benzoic acid to obtain compound (Ⅰ); Wherein, the general chemical formula of R1-substituted benzaldehyde is R1-C6H4-CHO, and the general chemical formula of R2-substituted benzoic acid is R2-C6H4-COOH. R1 is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups. R2 is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups.

[0009] Preferably, in some embodiments of the present invention, the R1 substituted aldehyde is selected from 4-chlorobenzaldehyde, and the R2 substituted benzoic acid is selected from p-cyanobenzoic acid.

[0010] Thirdly, the present invention provides a method for preparing 1,3,4-oxadiazole compounds with structures shown in formulas (II) to (VII), wherein the 1,3,4-oxadiazole ring of the compounds of formulas (II) to (VIII) is obtained by cyclization and desulfurization of N-acylthioaminourea formed by the reaction of benzoyl isothiocyanate with aromatic acyl hydrazide. Specifically, the preparation method includes the following steps: Step 1: Preparation of benzoyl isothiocyanate: Potassium thiocyanate is added to acetonitrile solvent, and benzoyl chloride with an R2 group on the benzene ring is added to react and obtain benzoyl isothiocyanate; the R2 group is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups; preferably, the benzoyl chloride with an R2 group on the benzene ring includes 3-methylbenzoyl chloride, 4-nitrobenzoyl chloride, 4-cyanobenzoyl chloride, 3-fluorobenzoyl chloride, and 3-bromobenzoyl chloride; Step 2, Preparation of aromatic acylhydrazides: Ethyl aromatic carboxylate is reacted with hydrazine hydrate via a hydrazinolysis reaction to obtain the aromatic acylhydrazides; the ethyl aromatic carboxylate includes benzothiazole-2-carboxylate, benzofuran-2-carboxylate, indole-2-carboxylate, benzothiophene-2-carboxylate, quinoline-2-carboxylate, and 4-(dimethylamino)benzoate; the aromatic acylhydrazides include benzothiazole-2-acylhydrazides, benzofuran-2-acylhydrazides, indole-2-acylhydrazides, benzothiophene-2-acylhydrazides, quinoline-2-acylhydrazides, and 4-(dimethylamino)benzoate. Step 3: The aromatic hydrazide prepared in step 2 is reacted with the benzoyl isothiocyanate prepared in step 1 and then cyclized and desulfurized to obtain compounds of formula (II) to (VII).

[0011] Fourthly, the present invention provides a pharmaceutical composition comprising at least one of the above-described 1,3,4-oxadiazole compounds, their isomers, pharmaceutically acceptable salts thereof, solvates, and prodrugs. Fifthly, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a drug for inhibiting ASADH enzyme activity.

[0012] In a sixth aspect, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating Mycobacterium tuberculosis infection.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The 1,3,4-oxadiazole compounds with structures shown in formulas (I) to (VII) provided by this invention exhibit inhibitory effects on both ASADH enzyme and Mycobacterium tuberculosis. In the antibacterial activity experiment, compound N-(5-(4-chlorophenyl)-1,3,4-oxadiazole-2-yl)-4-cyanobenzamide (compound No. 1) with structure (I) showed antibacterial activity against various bacilli, while compound N-(5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazole-2-yl)-3-fluorobenzamide (compound No. 7) with structure (VII) showed a significant inhibitory effect on Mycobacterium tuberculosis Ra. In the ASADH enzyme activity inhibition experiment, the half-maximal inhibitory concentration (IC50) of compound No. 1 was significantly lower than that of Mycobacterium tuberculosis Ra. 50 The half-maximum inhibitory concentration (IC50) of compound 7 was 1.365 μM. 50 The concentration was 0.7486 μM, indicating that they could effectively inhibit half of the ASADH enzyme activity at a very low concentration. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the steps for preparing compound No. 1 in Example 1 of the present invention; Figure 2 This is a schematic diagram of the steps for preparing compound No. 2 in Example 2 of the present invention; Figure 3 This is a schematic diagram illustrating the steps involved in preparing compound No. 7 in Example 7 of the present invention; Figure 4 This is a schematic diagram illustrating the principle of the ASADH enzyme inhibitory activity assay. Figure 5 The graph shows the inhibitory activity of compound 1 against ASADH enzyme. Figure 6 The graph shows the inhibitory activity of compound 7 against ASADH enzyme. Specific implementation methods The present invention will be further described in detail below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] The structures and names of the compounds prepared in the following examples are shown in Table 1 below.

[0016] Table 1. Structures and names of 1,3,4-oxadiazole compounds prepared in each example

[0017] Example 1: Preparation of Compound No. 1 See Figure 1 As shown, the preparation method of compound No. 1 includes the following steps: Step 1: Synthesize compound 1-2, “2-(4-chlorobenzylmethyl)hydrazine-1-formamide”: 10 mmol of 4-chlorobenzaldehyde was added to 10 mL of methanol, and 11 mmol of aminourea hydrochloride and 15 mmol of sodium acetate were added to 10 mL of water. The mixture was stirred at room temperature and reacted for 30 min. After the reaction was completed by TLC, the solvent was evaporated under reduced pressure and the white solid was collected to obtain compound 1-2, “2-(4-chlorobenzylmethyl)hydrazine-1-carboxamide”.

[0018] Step 2: Synthesize compounds 1-3 "(5-(4-chlorophenyl)-1,3,4-oxadiazole-2-amine): Compounds 1-2 (2 mmol), potassium carbonate (2.8 mmol), and iodine (6 mmol) from the previous step were added to solvent 1,4-dioxane (5 mL). After heating at 60 °C for 5 h, the reaction was detected by TLC. After the reaction was completed, 5% sodium thiosulfate solution (10 mL) was added, and the aqueous phase was extracted three times with dichloromethane (50 mL). The organic phases were combined, the solvent was evaporated under reduced pressure, and the solution was purified by column chromatography to obtain compound 1-3, "(5-(4-chlorophenyl)-1,3,4-oxadiazole-2-amine).

[0019] Step 3: Add HOBT (1.2 mmol), EDCI (1.2 mmol), p-cyanobenzoic acid (1.2 mmol) and DMAP (2 mmol) to solvent DCM (2 mL), stir for 30 min, then add compounds 1-3 obtained in the previous step. After reacting for 4-5 h, the reaction is detected by TLC to indicate that the reaction is complete. The solution is then purified by column chromatography to obtain a pale yellow solid, which is compound 1.

[0020] The NMR data for compound No. 1 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 12.48 (s,1H), 8.15 (d, J = 7.2 Hz, 2H), 8.01(d, J = 6.9 Hz, 2H), 7.93 (d, J = 7.0 Hz,2H), 7.62 – 7.54 (m,3H). 13 C NMR (151MHz, d 6-DMSO) δ 176.30, 170.66, 165.13, 137.05, 133.13, 130.16, 129.66,128.34, 122.59, 118.65, 115.41. Example 2: Preparation of compound 2 See Figure 2 As shown, the preparation method of compound No. 2 includes the following steps: Step 1: Synthesis of compound 2-2, "benzo[d]thiazole-2-carboxylic acid ethyl ester": Benzo[d]thiazole-2-carboxylic acid (2 mmol) was added to solvent ethanol (3 mL), followed by concentrated sulfuric acid (5 mmol). The mixture was heated at 60 °C for 10 h. After the reaction was complete as detected by TLC, the mixture was further processed by adding saturated sodium bicarbonate solution and extracting three times with ethyl acetate. The organic phases were combined, the solvent was evaporated under reduced pressure, and the mixture was purified by column chromatography with hexane:ethyl acetate = 10:1 as the eluent to obtain compound 2-2, "benzo[d]thiazole-2-carboxylic acid ethyl ester".

[0021] Step 2: Synthesize compound 2-3, “benzo[d]thiazol-2-acylhydrazide”: The 2-2 compound (2 mmol) and hydrazine hydrate (2 mL) from the previous step were added to the solvent water (2 mL). After reacting at room temperature for 10 h, TLC was performed. The white solid obtained by filtration was the 2-3 compound "benzo[d]thiazol-2-acylhydrazine".

[0022] Step 3: Synthesize compound 2-5, "3-methylbenzoyl isothiocyanate": Potassium thiocyanate (1.2 mmol) was added to acetonitrile (5 mL) and 3-methylbenzoyl chloride (1 mmol) was added dropwise. After reacting for 30 min, the solvent was evaporated under reduced pressure and purified by rapid column chromatography to obtain a yellow oily liquid, which was compound 2-5, "3-methylbenzoyl isothiocyanate".

[0023] Step 4: Synthesize compound 2-6, “N-(2-(benzo[d]thiazol-2-carbonyl)hydrazine-1-thiocarbonyl)-3-methylbenzamide”: Compounds 2-3 (1 mmol) and 2-5 (1 mmol) were added to the solvent acetonitrile (5 mL), and the reaction was carried out for 3-4 h. The reaction was detected by TLC. The solvent was evaporated under reduced pressure, water was added, and the mixture was filtered to obtain a pale yellow solid compound. The compound was recrystallized from methanol to obtain compound 2-6, “N-(2-(benzo[d]thiazol-2-carbonyl)hydrazine-1-thiocarbonyl)-3-methylbenzamide”.

[0024] Step 5: Add the synthesized compounds 2-6 (1 mmol) and potassium iodate (2 mmol) from the previous step to the solvent water (10 mL). After reacting for 4-5 h, the reaction was detected by TLC. After the reaction was complete, the mixture was filtered, the residue was collected, and the residue was recrystallized from ethyl acetate to obtain compound 2.

[0025] The NMR data for compound No. 2 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 12.42 (s, 1H), 8.22 (dd, J= 44.7, 8.1 Hz,2H), 7.93 – 7.79 (m, 2H), 7.62 (ddd, J = 24.7, 10.4, 4.6 Hz, 2H), 7.45 (dt, J =14.0, 6.9 Hz, 2H), 2.38 (s, 3H). 13 C NMR (151 MHz, d 6-DMSO) δ 159.58, 153.27,151.28, 138.65, 135.15, 134.22, 129.40, 129.13, 127.98, 127.91, 126.10,124.59, 123.45, 21.43. Example 3: Preparation of Compound No. 3 The preparation method is the same as in Example 2, except that benzo[d]thiazole-2-carboxylic acid is used instead of benzo[d]thiazole-2-carboxylic acid, and 4-nitrobenzoyl chloride is used instead of 3-methylbenzoyl chloride, to obtain compound No. 3.

[0026] The NMR data for compound No. 3 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 12.77 (s, 1H), 8.32 (d, J = 8.5 Hz, 2H), 8.21 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.7 Hz,1H), 7.74 – 7.65 (m, 2H), 7.46(t, J = 7.7 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H). 13 C NMR (151 MHz, d 6-DMSO) δ155.42, 150.33, 140.40, 130.45, 127.72, 127.52, 124.74, 124.19, 123.18,112.35, 110.40. Example 4: Preparation of compound 4 The preparation method is the same as in Example 2, except that indole-2-carboxylic acid is used instead of benzo[d]thiazole-2-carboxylic acid, and 4-cyanobenzoyl chloride is used instead of 3-methylbenzoyl chloride, to obtain compound No. 4.

[0027] The NMR data for compound No. 4 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 8.17 (d, J = 7.5 Hz, 2H), 8.02 (d, J = 7.6 Hz, 2H), 7.47 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H). 13 C NMR (151 MHz, d 6-DMSO) δ 137.86, 133.19, 131.32,129.73, 125.88, 122.99, 122.24, 121.83, 118.70, 115.46, 113.20. Example 5: Preparation of compound 5 The preparation method is the same as in Example 2, except that benzo[d]thiazole-2-carboxylic acid is used instead of benzo[d]thiazole-2-carboxylic acid, and 3-fluorobenzoyl chloride is used instead of 3-methylbenzoyl chloride, to obtain compound No. 5.

[0028] The NMR data for compound No. 5 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 12.46 (s,1H), 8.14 – 8.05 (m,2H), 8.01 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.59 (dd, J =13.8, 7.9 Hz,1H), 7.55 – 7.41 (m,3H). 13 C NMR (151 MHz, d6-DMSO) δ 163.24,161.61, 140.30, 139.39, 131.45, 131.40, 127.40, 127.03, 125.98, 125.85,125.15, 124.67, 123.43, 120.48, 120.34, 115.77, 115.62. Example 6: Preparation of compound 6 The preparation method is the same as in Example 2, except that isoquinoline-3-carboxylic acid is used instead of benzo[d]thiazole-2-carboxylic acid, and 3-bromobenzoyl chloride is used instead of 3-methylbenzoyl chloride, to obtain compound 6.

[0029] The NMR data for compound 6 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 12.41 (s,1H), 8.19 (s,1H), 8.14 – 8.05 (m,2H), 8.01 (d, J = 6.7 Hz, 2H), 7.83 (d, J = 7.5 Hz, 1H), 7.48 (dt, J = 15.5, 7.4Hz, 3H). 13 C NMR (151 MHz, d 6-DMSO) δ 140.31, 139.39, 136.11, 131.52, 131.40,128.03, 127.40, 127.01, 125.98, 125.85, 124.66, 123.43, 122.35. Example 7: Preparation of compound 7 See Figure 3 As shown, the preparation method of compound No. 7 includes the following steps: Step 1: Synthesis of compound 7-2 "4-(dimethylamino)benzoate ethyl ester": Nitrogen dimethyl, ethyl p-bromobenzoate and potassium carbonate were added to the solvent DMF and reacted to obtain compound 7-2 "4-(dimethylamino)benzoate ethyl ester".

[0030] Step 2 is the same as Step 2-4 of Example 2, except that: Compound 7-2 is used instead of Compound 2-2 "benzo[d]thiazole-2-carboxylic acid ethyl ester", and 3-fluorobenzoyl chloride is used instead of 3-methylbenzoyl chloride, to obtain Compound 7-6 "N-(2-(4-(dimethylamino)benzoyl)hydrazine-1-thiocarboxyl)-3-fluorobenzoamide".

[0031] Step 3: Add the 7-6 compound (0.5 mmol) synthesized in the previous step and EDCI (0.6 mmol) to the solvent dichloromethane (5 mL), react at room temperature for 3-4 h, and after the reaction is complete as detected by TLC, extract with water and extract three times with dichloromethane. Combine the organic phases, evaporate the solvent under reduced pressure, and recrystallize the obtained solid ethyl acetate to obtain compound 7.

[0032] The NMR data for compound 7 prepared above are as follows: 1 H NMR (600 MHz, d 6-DMSO) δ 12.19 (s,1H), 7.86 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 9.6 Hz, 1H), 7.71 (d, J = 8.5 Hz,2H), 7.61 – 7.52 (m,1H), 7.48 (t, J = 7.9Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 2.97 (s, 6H). 13 C NMR (151 MHz, d 6-DMSO) δ 163.25,163.25, 161.63, 161.63, 152.73, 152.73, 131.38, 131.33, 127.88, 127.88,125.07, 125.07, 120.25, 120.11, 115.68, 115.68, 115.52, 115.52, 112.33,112.33, 110.10, 110.10, 39.88. The following tests were conducted on the minimum inhibitory concentration of compounds 1-7 prepared in the above examples to test their ability to inhibit Mycobacterium tuberculosis and ASADH enzyme activity.

[0033] I. Minimum Inhibitory Concentration Test for Mycobacterium tuberculosis The minimum inhibitory concentration (MIC) values ​​of compounds 1-7 were determined using the broth microdilution method as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0034] The specific testing steps are as follows: 1. Preparation of 7H9 broth basal medium: Weigh 2.35g of commercially available Middlebrook into a 500 mL Erlenmeyer flask, add 450 mL of ultrapure water, add 0.2 mL of glycerol and 0.05 g of Tween 80 per 90 mL, autoclave for 30 min, autoclave at 121℃ for 30 min, cool to room temperature, and add 10% enrichment broth ADC.

[0035] 2. Preparation of bacterial suspension: Dilute the test strain with 7H9 broth basal medium and incubate for 24 hours to activate the strain. Prepare a bacterial suspension with a concentration equivalent to 1 McFarland standard using the activated bacterial suspension, and then dilute it 1:1000 with 7H9 broth basal medium to a concentration equivalent to 1×10⁻⁶. 5 Prepare a bacterial culture of CFu / mL for later use.

[0036] 3. Preparation of concentration gradient of the test compound: The test compound was dissolved in 3% DMSO solution and then prepared into an initial solution with a concentration of 256 μg / mL using 7H9 broth basal medium. Then, the concentration gradient was prepared by the two-fold dilution method.

[0037] Specifically, first, add 100 μL of 7H9 broth basal medium to each well in rows B, C, and D of a 96-well plate. Then, add 100 μL of the initial solution of the test compound to the first well of each row (i.e., B1, C1, D1). After thorough mixing, transfer 100 μL of the mixture from B1, C1, and D1 to the corresponding wells in rows B, C, and D2 (i.e., B2, C2, D2). Mix again, and then transfer 100 μL of the mixture from B2, C2, and D2 to the corresponding wells in rows B3 and C3. D3, repeat the above operation to the twelfth well (B12, C12, D12) of the three rows BCD. Take 100 μL of the mixture from B12, C12, and D12 and discard it, so that the drug concentration of the first well to the twelfth well of the three rows BCD decreases by a factor of two, that is, the drug concentration (μg / mL) of the first well to the twelfth well are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625, respectively.

[0038] 4. Preparation of control: Using E2 from a 96-well plate Well E4 (2 μL DMSO + 98 μL 7H9 broth basal medium) served as the solvent control, and wells F2-F4 served as the blank control (200 μL 7H9 broth basal medium). Well G2... G4 is a positive control (100 μL of control drug, with rifampin, clarithromycin and ampicillin as the control drugs).

[0039] 5. Inoculation and culture: Add 100 μL of the prepared bacterial solution to each well (no bacterial solution is added to the blank growth control), place the 96-well plate in a 37°C incubator for 24 h, and observe the bacterial growth in each well. The MIC value is the concentration of the test compound in the well that completely inhibits bacterial growth.

[0040] The test results are shown in Table 2 below. Compounds 1-7 all exhibited some degree of antibacterial activity. Compound 1 showed antibacterial activity against multiple bacilli, while compound 7 showed a significant inhibitory effect on Ra. Table 2. Results of Minimum Inhibitory Concentration (μg / mL) Test

[0041] II. Binding free energy of the test compound docking with the ASADH enzyme molecule Download the PDB crystal structure of the Mtb-ASADH enzyme (PDB ID: 3TZ6), and use AutoDock 4.2 software to perform dehydration and hydrogenation operations on it. Prepare the mol2 file of the ligand of the test compound and minimize its energy. Perform hydrogenation operations on it as well. Use AutoDock 4.2 software to calculate the binding free energy of the docking between the test compound and the Mtb-ASADH enzyme molecule. The results are shown in Table 3 below.

[0042] Table 3. Binding free energy of the test compounds

[0043] As can be seen from Table 3 above, compounds 1-7 all have a certain binding capacity for ASADH enzyme.

[0044] III. AsADH enzyme inhibitory activity experiment See the experimental detection principle. Figure 4 As shown, aspartic acid is converted to aspartic phosphate by aspartate kinase. In the presence of NADPH, aspartic phosphate is catalyzed by ASADH enzyme to produce aspartic semialdehyde. NADPH then transfers hydrogen to NADP+. NADDH has an absorption at 340 nm, while NADP+ does not. The inhibition rate of this compound on the enzyme is determined by the change in NADPH.

[0045] The specific steps are as follows: Preparation of Tris-HCl buffer and NADPH system: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM dithiothreitol, 5 mM ASP, 2 mM ATP, 1 mM NADPH. A total volume of 100 μL was added to a 96-well plate, along with 100 μL of the dissolved compound in 3% DMSO, 3 U of aspartate kinase, and 0.3 U of aspartate semialdehyde dehydrogenase. The MtASADH heat-inactivated group served as a positive control, while the negative control group contained only 3% DMSO. Compounds 1 and 7, with good anti-tuberculosis activity, were selected to verify ASADH enzyme inhibitory activity, with concentration gradients of 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM. The IC50 of the test compounds against ASADH enzyme was calculated using GraphPad Prism software. 50 value.

[0046] See results Figure 5 , Figure 6 As shown, the IC50 of compound 1 50 =1.365 μM, IC50 of compound 7 50 =0.7486μM, all showed good ASADH enzyme inhibitory activity, which preliminarily indicates that this type of compound may exert its anti-tuberculosis effect by inhibiting ASADH.

[0047] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, any improvements and modifications made based on the core ideas of the present invention without departing from the spirit and scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A 1,3,4-oxadiazole compound, characterized in that: The molecular structure of the compound includes the structures shown in formulas (I) to (VII); （Ⅰ）， （Ⅱ）， （Ⅲ）， （Ⅳ）， （Ⅴ）， （Ⅵ）， （Ⅶ）； Wherein, R1 is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups; R2 is selected from at least one of the following: ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro. R3 is selected from at least one of nitrogen-nitrogen dimethyl, nitrogen-nitrogen diethyl, piperidine, and tetrahydropyrrole.

2. The compound according to claim 1, characterized in that: The compounds of formula (I) include N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-cyanobenzamide; The compounds of formula (II) include N-(5-(benzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-yl)-3-methylbenzamide; The compounds of formula (III) include N-(5-(benzofuran-2-yl)-1,3,4-oxadiazol-2-yl)-4-nitrobenzamide; The compounds of formula (IV) include N-(5-(1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)-4-cyanobenzamide; The compounds of formula (V) include N-(5-(benzothiophene-2-yl)-1,3,4-oxadiazol-2-yl)-3-fluorobenzamide; The compounds of formula (VI) include 3-bromo-N-(5-(isoquinolin-3-yl)-1,3,4-oxadiazol-2-yl)benzamide; The compounds of formula (VII) include N-(5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-yl)-3-fluorobenzamide.

3. A method for preparing a 1,3,4-oxadiazole compound with the structure shown in formula (Ⅰ), characterized in that: The preparation method includes the following steps: Step 1: The aminourea hydrochloride is condensed with R1-substituted benzaldehyde to obtain an aminourea compound; Step 2: The acetal urea compounds prepared in Step 1 undergo an oxidative cyclization reaction to yield 2-amino-1,3,4-oxadiazole compounds. Step 3: The 2-amino-1,3,4-oxadiazole compound prepared in step 2 undergoes an amide condensation reaction with R2-substituted benzoic acid to obtain compound (Ⅰ); Wherein, the general chemical formula of R1-substituted benzaldehyde is R1-C6H4-CHO, and the general chemical formula of R2-substituted benzoic acid is R2-C6H4-COOH. R1 is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups. R2 is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups.

4. The preparation method according to claim 3, characterized in that: The R1 substituted aldehyde is selected from 4-chlorobenzaldehyde, and the R2 substituted benzoic acid is selected from p-cyanobenzoic acid.

5. A method for preparing a 1,3,4-oxadiazole compound with the structure shown in formulas (II) to (VII), characterized in that: The preparation method includes the following steps: Step 1: Preparation of benzoyl isothiocyanate: Potassium thiocyanate is added to acetonitrile solvent, and benzoyl chloride with an R2 group on the benzene ring is added to react and obtain benzoyl isothiocyanate; the R2 group is selected from at least one of ortho-, meta-, and para-substituted methyl, methoxy, fluorine, chlorine, bromine, phenoxy, cyano, and nitro groups. Step 2, Preparation of aromatic acylhydrazides: Ethyl aromatic carboxylate is reacted with hydrazine hydrate via a hydrazinolysis reaction to obtain the aromatic acylhydrazides; the ethyl aromatic carboxylate includes benzothiazole-2-carboxylate, benzofuran-2-carboxylate, indole-2-carboxylate, benzothiophene-2-carboxylate, quinoline-2-carboxylate, and 4-(dimethylamino)benzoate; the aromatic acylhydrazides include benzothiazole-2-acylhydrazides, benzofuran-2-acylhydrazides, indole-2-acylhydrazides, benzothiophene-2-acylhydrazides, quinoline-2-acylhydrazides, and 4-(dimethylamino)benzoate. Step 3: The aromatic hydrazide prepared in step 2 is reacted with the benzoyl isothiocyanate prepared in step 1 and then cyclized and desulfurized to obtain compounds of formula (II) to (VII).

6. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises at least one of the 1,3,4-oxadiazole compounds of claim 1 or 2, their isomers, their pharmaceutically acceptable salts, solvates, and prodrugs.

7. Use of the pharmaceutical composition of claim 6 in the preparation of a drug for inhibiting ASADH enzyme activity.

8. Use of the pharmaceutical composition of claim 6 in the preparation of a medicament for treating Mycobacterium tuberculosis infection.

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