Cephalosporin derivative containing 1, 3, 4-thiadiazole as well as preparation method and application of cephalosporin derivative

Abstract

The invention aims to provide a cephalosporin derivative containing 1, 3, 4-thiadiazole as well as a preparation method and application thereof, the structure of the cephalosporin derivative is shown as a general formula I. In the formula I, R1 and R2 are independently selected from H or C1-6 alkyl; r3 is selected from H or C1-6 alkyl; r4 is selected from C1-6 alkyl groups or C3-6 cycloalkyl groups; r5 is selected from H, C1-6 alkyl groups, C3-6 cycloalkyl groups, substituted 4-6-membered heterocyclic rings and substituents thereof, or aromatic rings and substituents thereof; x is selected from N or CH. The invention also provides a preparation method and application of the compound. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of new compounds and their application, and in particular to a cephalosporin derivative containing 1,3,4-thiadiazole and a preparation method and application thereof. Background Art

[0002] Bacterial infection is a major threat to global public health, and its pathogenic mechanism has evolved from local lesions to multi-system invasion. The evolution of drug resistance caused by the misuse of antibiotics has accelerated the spread of multi-drug resistant strains represented by methicillin-resistant Staphylococcus aureus (MRSA), causing traditional treatment options to face the risk of failure. The current development of anti-infective drugs faces dual challenges: insufficient antibacterial activity of new compounds and the continued rise in resistance rates of traditional antibiotics. Taking β-lactam antibiotics as an example, their clinical efficacy decline is due to the structural alienation of PBP2a, a key target of drug-resistant bacteria. This protein hinders drug binding through a hydrophobic active cavity, resulting in a significant decrease in the efficiency of transpeptidase inhibition. In view of this molecular mechanism, computer-aided drug design based on target structure has become a core strategy to break through the drug resistance barrier. By optimizing the functional group configuration of cephalosporin derivatives and enhancing the binding efficiency with allosteric sites, it provides a new path for the treatment of drug-resistant bacterial infections.

[0003] In view of the increasingly severe situation of methicillin-resistant Staphylococcus aureus (MRSA) resistance, scientists are committed to developing a variety of strategies to overcome the problem of bacterial resistance. Among them, one of the most promising research directions focuses on designing new antibacterial drug molecules - by constructing a spatial structure that is completely different from traditional antibiotics, targeting and enhancing the specific binding ability with penicillin-binding protein 2a (PBP2a), thereby effectively overcoming the resistance mechanism and significantly improving the antibacterial efficacy. Summary of the invention

[0004] The present invention aims to provide a cephalosporin derivative containing 1,3,4-thiadiazole and a preparation method and use thereof.

[0005] The technical solution of the present invention is as follows:

[0006] Cephalosporin derivatives containing 1,3,4-thiadiazole and pharmaceutically acceptable salts thereof, the structure of which is shown in general formula I:

[0007]

[0008] in:

[0009] R1 and R2 are each independently selected from H or C 1-6 alkyl;

[0010] R3 is selected from H or C 1-6 alkyl;

[0011] R4 is selected from C1-6 Alkyl or C 3-6 Cycloalkyl;

[0012] R5 is selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, substituted 4-6 membered heterocycle and its substituents, or aromatic ring and its substituents;

[0013] X is selected from N or CH.

[0014] Preferably, R5 is selected from C 4-6 Cycloalkyl, or aromatic ring and its substituents.

[0015] Preferably, R1, R2, R3, R4 are each independently selected from C 1-3 Alkyl.

[0016] Preferably, the cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt are selected from the following compounds:

[0017]

[0018] Preferably, the synthesis method of the cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt is carried out according to the following reaction formula:

[0019]

[0020] In the above reaction formula,

[0021] The process of preparing Intermediate 1 in Step 1 includes the following steps: Add Raw Material 1, Raw Material 2 and Solvent a into a dry reaction vessel, stir to dissolve, cool to 0 - 5°C, slowly dropwise add Base 1, then keep the temperature for reaction for 3 - 6 h, and obtain Intermediate 1 by column chromatography of the reaction product;

[0022] The process of preparing Intermediate 2 in Step 2 includes the following steps: In an atmosphere of dichloromethane, at a temperature of -5 - 0°C, slowly add phosphorus pentachloride and pyridine into a dry reaction vessel successively, keep the temperature and stir for 1 - 2 h, then add Intermediate 1 and react for 2 - 5 h, filter out the insoluble substances, cool the filtrate to 0 - 5°C, add trifluoroacetic acid, naturally warm up to room temperature and react for 2 - 5 h, then add Solvent b to precipitate and collect the solid to obtain Intermediate 2;

[0023] The process of preparing the compound of general formula (I) in Step 3 includes the following steps: Add Intermediate 2, Raw Material 3 and Solvent c into a dry reaction vessel, cool to 0 - 5°C, slowly dropwise add Base 2, then naturally warm up to room temperature and react for 5 - 10 h, precipitate and collect the solid to obtain the finished product of the compound of general formula (I).

[0024] Preferably, in step 1, solvent a is selected from tetrahydrofuran, methyltetrahydrofuran, dichloromethane or acetone; base 1 is triethylamine, diisopropylethylamine or n-butylamine.

[0025] Preferably, in step 2, solvent b is selected from acetone, isopropanol, isobutanol or propanol.

[0026] Preferably, in step 3, solvent c is selected from tetrahydrofuran, methyltetrahydrofuran, dichloromethane or acetone; base 2 is triethylamine, diisopropylethylamine or n-butylamine.

[0027] The present invention also discloses the use of the above-mentioned cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salts in the preparation of anti-infective drugs.

[0028] The present invention also discloses the use of the above-mentioned cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salts in the preparation of antibacterial drugs.

[0029] The compound of general formula I in the present invention is a class of cephalosporin antibiotics with good chemical stability, high PBP2a affinity and strong antibacterial activity.

[0030] The cephalosporin derivative containing 1,3,4-thiadiazole of the present invention has been confirmed to have good antibacterial activity through pharmacological experiments, and thus it can be expected to be developed into a pharmaceutical use for preventing and treating antibacterial drugs, and can also be developed into a pharmaceutical use for preventing and treating anti-infective drugs, with potential huge social and economic benefits. Detailed Embodiments

[0031] To better understand the essence of the present invention, the preparation process of the compound is first described in the form of examples below. The examples give some physical, chemical and spectroscopic data of the compound. It must be noted that the examples of the present invention are used to illustrate the present invention rather than to limit the present invention. Simple improvements made to the present invention according to the essence of the present invention all fall within the scope of protection required by the present invention.

[0032] Example 1

[0033] Preparation of (7R)-7-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido]-3-[[(3S,5S)-5-[(5-cyclopropyl-1,3,4-thiadiazol-2-yl)carbamoyl]-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 1) (R1 and R2 are H, R3 and R4 are methyl, R5 is cyclopropyl, X is N):

[0034]

[0035] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (3.1 g, 11.0 mmol) in dichloromethane (50 mL), cool to 0 - 5 °C and stir for 5 minutes. Slowly add diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the temperature for reaction for 5 h. Remove the solvent under reduced pressure. The residue is purified by column chromatography (ethyl acetate: petroleum ether = 5:1) to obtain 5.4 g of a light yellow solid, with a yield of 71.1%.

[0036] Intermediate 2: Add dichloromethane (30 mL) to a reaction flask, cool to -5 - 0 °C, and successively and slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol). Keep the temperature and stir for 1 h, then add Intermediate 1 (3.8 g, 5.0 mmol) and react for 3 h. Filter off the insoluble substances. Cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h. Then add isopropanol (50 mL) to precipitate a light yellow solid. After vacuum drying, 1.3 g of Intermediate 2 is obtained, with a yield of 57.0%.

[0037] Compound 1: Add Intermediate 2 (0.9 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and dichloromethane (10 mL) to a dry reaction flask, cool to 0 - 5 °C, slowly add triethylamine (0.4 mL, 3.0 mmol), then raise the temperature to room temperature and react for 10 h. A light yellow solid precipitates. After vacuum drying, 1.1 g of the finished product of the compound of general formula (I) is obtained, with a yield of 82.5%.

[0038] HRMS Calcd for C 23 H 26 N 10 O6S4: [M + H] + 667.6823, found 667.6314. 1 H NMR (400 MHz, DMSO-d6) δ: 9.61 (1H, s), 9.17 (1H, d), 7.79 (2H, brs), 5.75 (1H, d), 5.03 (1H, d), 3.97 (3H, s), 3.62 (1H, d), 3.48 (1H, d), 3.19 (1H, d), 2.98 (1H, d), 2.75 - 2.83 (1H, m), 2.43 - 2.59 (3H, m), 2.36 (3H, s), 2.20 - 2.27 (1H, m), 1.04 - 1.35 (4H, m).

[0039] Example 2

[0040] Preparation of (7R)-7-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido]-3-[[(3S,5S)-5-((5-ethyl-1,3,4-thiadiazol-2-yl)carbonyl)-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 2) (R1 and R2 are H, R3 and R4 are methyl, R5 is isopropyl, X is N):

[0041]

[0042] Intermediate 1: Dissolve Raw Material 1 (5.8 g, 10.0 mmol) and Raw Material 2 (3.0 g, 11.0 mmol) in tetrahydrofuran (50 mL), cool to 0 - 5 °C and stir for 5 minutes. Slowly add diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the reaction at a constant temperature for 5 h. Remove the solvent under reduced pressure. The residue is purified by column chromatography (ethyl acetate:petroleum ether = 10:1) to obtain 6.0 g of a light yellow solid with a yield of 80.6%.

[0043] Intermediate 2: Add dichloromethane (30 mL) to the reaction flask, cool to -5 - 0 °C, and successively add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) slowly. Keep stirring at a constant temperature for 1 h, then add Intermediate 1 (3.8 g, 5.0 mmol) and react for 4 h. Filter out the insoluble substances. Cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (2.0 mL, 27.0 mmol), raise the temperature to room temperature and react for 3 h. Then add isopropanol (50 mL) to precipitate a light yellow solid. After vacuum drying, 1.4 g of Intermediate 2 is obtained with a yield of 59.6%.

[0044] Compound 2: Add Intermediate 2 (0.9 g, 2.0 mmol), Raw Material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) to a dry reaction flask, cool to 0 - 5 °C, slowly add n-butylamine (0.3 mL, 3.0 mmol), then raise the temperature to room temperature and react for 5 h. A light yellow solid precipitates. After vacuum drying, 1.0 g of the finished product of the compound of general formula (I) is obtained with a yield of 76.3%.

[0045] HRMS Calcd for C 22 H 26 N 10 O6S4: [M + H] + 654.7439, found 654.7321. 11H NMR (400 MHz, DMSO-d6) δ: 9.73 (1H, s), 9.28 (1H, d), 7.81 (2H, brs), 5.69 (1H, d), 5.11 (1H, d), 3.91 (3H, s), 3.66 (1H, d), 3.52 (1H, d), 3.17 (1H, d), 2.95 (1H, d), 2.58 - 2.75 (1H, m), 2.38 - 2.48 (3H, m), 2.31 (3H, s), 2.15 - 2.25 (1H, m), 1.11 - 1.29 (4H, m).

[0046] Example 3

[0047] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido]-3-[[(3S,5S)-1-methyl-5-[(5-phenyl-1,3,4-thiadiazol-2-yl)carbamoyl]-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 3) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is phenyl, X is N):

[0048]

[0049] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (3.5 g, 11.0 mmol) in tetrahydrofuran (50 mL), cool to 0 - 5 °C and stir for 5 minutes, slowly drip in diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the reaction at a constant temperature for 3 h, remove the solvent under reduced pressure, and subject the residue to column chromatography (ethyl acetate: petroleum ether = 8:1) to obtain 5.6 g of a light yellow solid with a yield of 69.7%.

[0050] Intermediate 2: Add dichloromethane (30 mL) to the reaction flask, cool to -5 - 0 °C, slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) successively, stir at a constant temperature for 2 h, then add Intermediate 1 (4.0 g, 5.0 mmol) and react for 3 h, filter off the insoluble matter, cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h, then add isobutanol (50 mL) to precipitate a light yellow solid, and obtain 1.6 g of Intermediate 2 after vacuum drying with a yield of 62.5%.

[0051] Compound 1: In a dry reaction flask, add intermediate 2 (1.0 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL). Cool to 0 - 5 °C, slowly add triethylamine (0.4 mL, 3.0 mmol) dropwise, then raise the temperature to room temperature and react for 5 h. A light yellow solid precipitates. After vacuum drying, 1.1 g of the finished product of the compound of general formula (I) is obtained, with a yield of 78.2%.

[0052] HRMS Calcd for C 26 H 26 N 10 O6S4: [M + H] + 703.7716, found 703.7633. 1 H NMR (400 MHz, DMSO-d6) δ: 9.81 (1H, s), 9.43 (1H, d), 7.86 (2H, brs), 7.01 - 7.55 (5H, m), 5.57 (1H, d), 5.15 (1H, d), 3.88 (3H, s), 3.61 (1H, d), 3.21 (1H, d), 2.99 (1H, d), 2.59 - 2.71 (1H, m),

[0053] 2.36 - 2.55 (3H, m), 2.27 (3H, s), 2.01 - 2.18 (1H, m).

[0054] Example 4

[0055] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-8-oxo-3-[[(3S,5S)-5-(5-p-tolyl-1,3,4-thiadiazole-2-carboxamido)-1-methylpyrrolidin-3-yl]thio]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 4) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is p-tolyl, X is N):

[0056]

[0057] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (3.7 g, 11.0 mmol) in tetrahydrofuran (50 mL). Cool to 0 - 5 °C and stir for 5 minutes, slowly add triethylamine (2.0 mL, 14.0 mmol) dropwise, then keep the temperature and react for 4 h. Remove the solvent under reduced pressure. The residue is subjected to column chromatography (ethyl acetate: petroleum ether = 8:1) to obtain 5.5 g of a light yellow solid, with a yield of 67.3%.

[0058] Intermediate 2: Add dichloromethane (30 mL) to a reaction flask, cool to -5 - 0 °C, slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) successively, stir at the same temperature for 2 h, then add Intermediate 1 (4.1 g, 5.0 mmol) and react for 3 h. Filter off the insoluble matter, cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h. Then add isobutanol (50 mL) to precipitate a light yellow solid. After vacuum drying, 1.5 g of Intermediate 2 is obtained, with a yield of 56.3%.

[0059] Compound 1: Add Intermediate 2 (1.1 g, 2.0 mmol), starting material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) to a dry reaction flask, cool to 0 - 5 °C, slowly dropwise add triethylamine (0.4 mL, 3.0 mmol), then raise the temperature to room temperature and react for 5 h. A light yellow solid precipitates. After vacuum drying, 1.1 g of the finished product of the compound of general formula (I) is obtained, with a yield of 76.7%.

[0060] HRMS Calcd for C 27 H 28 N 10 O6S4: [M + H] + 717.8045, found 717.6952. 1 H NMR (400 MHz, DMSO-d6) δ: 9.77 (1H, s), 9.52 (1H, d), 7.78 (2H, brs), 7.21 - 7.56 (4H, m), 5.63 (1H, d), 5.09 (1H, d), 3.83 (3H, s), 3.63 (1H, d), 3.22 (1H, d), 3.02 (1H, d), 2.63 - 2.74 (1H, m),

[0061] 2.41 - 2.55 (3H, m), 2.35 (3H, s), 2.23 (3H, s), 1.97 - 2.06 (1H, m).

[0062] Example 5

[0063] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido]-3-[[(3S,5S)-1-methyl-5-[(5-methyl-1,3,4-thiadiazol-2-yl)carbamoyl]-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 5) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is cyclopentyl, X is N):

[0064]

[0065] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (2.8 g, 11.0 mmol) in tetrahydrofuran (50 mL), cool to 0 - 5 °C, stir for 5 minutes, slowly dropwise add triethylamine (2.0 mL, 14.0 mmol), then keep the temperature for reaction for 5 h, remove the solvent under reduced pressure, and subject the residue to column chromatography (ethyl acetate: petroleum ether = 3:1) to obtain 5.0 g of a light yellow solid with a yield of 67.0%.

[0066] Intermediate 2: Add dichloromethane (30 mL) to the reaction flask, cool to -5 - 0 °C, slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) successively, keep the temperature and stir for 2 h, then add Intermediate 1 (3.7 g, 5.0 mmol) and react for 3 h, filter off the insoluble matter, cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h, then add isopropanol (50 mL) to precipitate a light yellow solid, and obtain 1.2 g of Intermediate 2 after vacuum drying with a yield of 52.5%.

[0067] Compound 1: Add Intermediate 2 (0.9 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) to a dry reaction flask, cool to 0 - 5 °C, slowly dropwise add n-butylamine (0.3 mL, 3.0 mmol), then raise the temperature to room temperature and react for 10 h to precipitate a light yellow solid. After vacuum drying, 1.1 g of the finished product of the compound of general formula (I) is obtained with a yield of 78.0%.

[0068] HRMS Calcd for C 21 H 24 N 10 O6S4: [M + H] + 641.6856, found 641.6714. 1 H NMR (400 MHz, DMSO-d6) δ: 9.85 (1H, s), 9.31 (1H, d), 7.92 (2H, brs), 5.69 (1H, d), 5.17 (1H, d), 3.85 (3H, s), 3.69 (1H, d), 3.55 (1H, d), 3.24 (1H, d), 2.93 (1H, d), 2.55 - 2.75 (1H, m), 2.51 (3H, s), 2.28 - 2.44 (3H, m), 2.16 (3H, s).

[0069] Example 6

[0070] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-8-oxo-3-[[(3S,5S)-5-(5-p-tolyl-1,3,4-thiadiazole-2-carboxamido)-1-methylpyrrolidin-3-yl]thio]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 6) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is p-chlorophenyl, X is N):

[0071]

[0072] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (3.9 g, 11.0 mmol) in tetrahydrofuran (50 mL), cool to 0 - 5 °C and stir for 5 minutes. Slowly add diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the reaction at a constant temperature for 5 h. Remove the solvent under reduced pressure. The residue is purified by column chromatography (ethyl acetate: petroleum ether = 5:1) to obtain 6.7 g of a light yellow solid with a yield of 80.3%.

[0073] Intermediate 2: Add dichloromethane (30 mL) to the reaction flask, cool to -5 - 0 °C, and slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) successively. Keep stirring at a constant temperature for 1 h, then add Intermediate 1 (4.2 g, 5.0 mmol) and react for 5 h. Filter out the insoluble matter. Cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (2.0 mL, 27.0 mmol), raise the temperature to room temperature and react for 3 h. Then add isopropanol (50 mL) to precipitate a light yellow solid. After vacuum drying, 1.3 g of Intermediate 2 is obtained with a yield of 58.8%.

[0074] Compound 2: Add Intermediate 2 (1.1 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) to a dry reaction flask, cool to 0 - 5 °C, slowly add triethylamine (0.4 mL, 3.0 mmol), then raise the temperature to room temperature and react for 5 h. A light yellow solid precipitates. After vacuum drying, 1.0 g of the compound of general formula (I) is obtained with a yield of 67.8%.

[0075] HRMS Calcd for C 26 H 25 ClN 10 O6S4: [M + H] + 738.1013, found 738.0631. 11H NMR (400 MHz, DMSO-d6) δ: 9.76 (1H, s), 9.47 (1H, d), 7.88 (2H, brs), 7.52 - 7.76 (4H, m), 5.61 (1H, d), 5.14 (1H, d), 3.77 (3H, s), 3.61 (1H, d), 3.21 (1H, d), 2.95 (1H, d), 2.60 - 2.75 (1H, m),

[0076] 2.38 - 2.51 (3H, m), 2.20 (3H, s), 1.91 - 2.03 (1H, m).

[0077] Example 7

[0078] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido]-3-[[(3S,5S)-5-[(5-Cyclobutyl-1,3,4-thiadiazol-2-yl)carbamoyl]-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 7) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is cyclobutyl, X is N):

[0079]

[0080] Intermediate 1: Dissolve Raw Material 1 (5.8 g, 10.0 mmol) and Raw Material 2 (3.3 g, 11.0 mmol) in tetrahydrofuran (50 mL), cool to 0 - 5 °C and stir for 5 minutes, slowly dropwise add diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the reaction at a constant temperature for 5 h, remove the solvent under reduced pressure, and subject the residue to column chromatography (ethyl acetate: petroleum ether = 5:1) to obtain 5.4 g of a light yellow solid with a yield of 71.1%.

[0081] Intermediate 2: Add dichloromethane (30 mL) to the reaction flask, cool to -5 - 0 °C, successively and slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol), keep stirring at a constant temperature for 2 h, then add Intermediate 1 (3.8 g, 5.0 mmol) and react for 3 h, filter off the insoluble matter, cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h, then add isopropanol (50 mL) to precipitate a light yellow solid, and obtain 1.3 g of Intermediate 2 after vacuum drying with a yield of 57.0%.

[0082] Compound 1: In a dry reaction flask, add intermediate 2 (1.0 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL). Cool to 0 - 5 °C, slowly add n-butylamine (0.3 mL, 3.0 mmol) dropwise, then raise the temperature to room temperature and react for 8 h. A light yellow solid precipitates. After vacuum drying, 1.0 g of the finished product of the compound of general formula (I) is obtained, and the yield is 73.5%.

[0083] HRMS Calcd for C 24 H 28 N 10 O6S4: [M+H] + 681.6801, found 681.6648. 1 H NMR (400 MHz, DMSO-d6) δ: 9.69 (1H, s), 9.23 (1H, d), 7.74 (2H, brs), 5.66 (1H, d), 5.14 (1H, d), 3.86 (3H, s), 3.69 (1H, d), 3.54 (1H, d), 3.21 (1H, d), 2.99 (1H, d), 2.59 - 2.71 (1H, m), 2.37 - 2.49 (3H, m), 2.33 (3H, s), 2.14 - 2.25 (1H, m), 1.85 - 2.01 (4H, m), 1.51 - 1.63 (2H, m).

[0084] Example 8

[0085] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[[(3S,5S)-5-[(5-Cyclopentyl-1,3,4-thiadiazol-2-yl)carbamoyl]-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 8) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is cyclopentyl, X is N):

[0086]

[0087] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (3.4 g, 11.0 mmol) in tetrahydrofuran (50 mL). Cool to 0 - 5 °C and stir for 5 minutes. Slowly add diisopropylethylamine (2.5 mL, 14.0 mmol) dropwise, then keep the temperature and react for 5 h. Remove the solvent under reduced pressure. The residue is purified by column chromatography (ethyl acetate: petroleum ether = 5:1) to obtain 5.2 g of a light yellow solid, and the yield is 65.2%.

[0088] Intermediate 2: Add dichloromethane (30 mL) into a reaction flask, cool it to -5 - 0 °C, slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) successively, stir at a constant temperature for 1 h, then add Intermediate 1 (4.0 g, 5.0 mmol) and react for 3 h. Filter out the insoluble substances, cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h, then add isopropanol (50 mL) to precipitate a light yellow solid. After vacuum drying, 1.5 g of Intermediate 2 is obtained, and the yield is 58.8%.

[0089] Compound 1: Add Intermediate 2 (1.0 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) into a dry reaction flask, cool it to 0 - 5 °C, slowly dropwise add triethylamine (0.4 mL, 3.0 mmol), then raise the temperature to room temperature and react for 5 h. A light yellow solid precipitates. After vacuum drying, 1.1 g of the finished product of the compound of general formula (I) is obtained, and the yield is 79.2%.

[0090] HRMS Calcd for C 25 H 30 N 10 O6S4: [M + H] + 695.6437, found 695.6119. 1 H NMR (400 MHz, DMSO - d6) δ: 9.81(1H, s), 9.35(1H, d), 7.77(2H, brs), 5.66(1H, d), 5.14(1H, d), 3.91(3H, s), 3.67(1H, d), 3.50(1H, d), 3.25(1H, d), 2.96(1H, d), 2.54 - 2.68(1H, m), 2.31 - 2.43(3H, m), 2.21(3H, s), 2.01 - 2.13(1H, m), 1.65 - 1.91(8H, m).

[0091] Example 9

[0092] (7R)-7-((Z)-2-(5-Amino-1,2,4-thiadiazolyl)-2-(methoxyimino)acetamido)-3-(((3S,5S)-5-((5-(3,4-Dimethoxyphenyl)-1,3,4-thiadiazol-2-yl)carbonyl)pyrrolidin-3-yl)thio)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 9) Preparation (R1 and R2 are H, R3 and R4 are methyl, R5 is 3-methoxyphenyl, X is N):

[0093]

[0094] Intermediate 1: Dissolve raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (4.2 g, 11.0 mmol) in tetrahydrofuran (20 mL), cool to 0 - 5 °C and stir for 5 minutes. Slowly drop in diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the temperature for reaction for 5 h. Remove the solvent under reduced pressure. The residue is subjected to column chromatography (ethyl acetate: petroleum ether = 10:1) to obtain 7.1 g of a light yellow solid with a yield of 77.3%.

[0095] Intermediate 2: Add dichloromethane (20 mL) to the reaction flask, cool to -5 - 0 °C, and slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) successively. Keep the temperature and stir for 2 h, then add Intermediate 1 (4.3 g, 5.0 mmol) and react for 4 h. Filter out the insoluble substances. Cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (2.0 mL, 27.0 mmol), raise the temperature to room temperature and react for 5 h. Then add isobutanol (50 mL) to precipitate a light yellow solid. After vacuum drying, 1.8 g of Intermediate 2 is obtained with a yield of 67.3%.

[0096] Compound 2: Add Intermediate 2 (1.2 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) to a dry reaction flask, cool to 0 - 5 °C, slowly dropwise add n-butylamine (0.3 mL, 3.0 mmol), then raise the temperature to room temperature and react for 10 h. A light yellow solid precipitates. After vacuum drying, 1.3 g of the finished product of the compound of general formula (I) is obtained with a yield of 85.3%.

[0097] HRMS Calcd for C 28 H 30 N 10 O8S4: [M + H] + 763.6636, found 763.5873. 1 H NMR (400 MHz, DMSO-d6) δ: 9.81 (1H, s), 9.35 (1H, d), 7.77 (2H, brs), 7.06 - 7.33 (3H, m), 5.66 (1H, d), 5.14 (1H, d), 4.10 (3H, s), 3.91 (3H, s), 3.80 (3H, s), 3.67 (1H, d), 3.50 (1H, d), 3.25 (1H, d), 2.54 - 2.68 (1H, m), 2.31 - 2.43 (3H, m), 2.21 (3H, s), 2.01 - 2.13 (1H, m).

[0098] Pharmacodynamic Experimental Study of Example 10

[0099] I. Preparation of the comparative drugs:

[0100] 1. Comparative Example 1:

[0101] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[[(3S,5S)-5-(3-Methylcyclopentylcarbamoyl)-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 10) was prepared as follows:

[0102]

[0103] Intermediate 1: Raw material 1 (5.8 g, 10.0 mmol) and raw material 2 (3.6 g, 11.0 mmol) were dissolved in tetrahydrofuran (50 mL), cooled to 0 - 5 °C and stirred for 5 minutes. Diisopropylethylamine (2.5 mL, 14.0 mmol) was slowly added dropwise, and then the reaction was carried out at a constant temperature for 5 h. The solvent was removed under reduced pressure. The residue was purified by column chromatography (ethyl acetate: petroleum ether = 10:1) to obtain 6.2 g of a light yellow solid with a yield of 76.4%.

[0104] Intermediate 2: Dichloromethane (30 mL) was added to the reaction flask, cooled to -5 - 0 °C, phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol) were slowly added successively, and the mixture was stirred at a constant temperature for 2 h. Then Intermediate 1 (4.1 g, 5.0 mmol) was added and the reaction was carried out for 4 h. The insoluble substances were filtered off. The filtrate was cooled to 0 - 5 °C, trifluoroacetic acid (2.0 mL, 27.0 mmol) was added, and the reaction was carried out at room temperature for 3 h. Then isopropanol (50 mL) was added to precipitate a light yellow solid. After vacuum drying, 1.3 g of Intermediate 2 was obtained with a yield of 59.6%.

[0105] Compound 2: In a dry reaction flask, Intermediate 2 (0.9 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL) were added, cooled to 0 - 5 °C, and n-butylamine (0.3 mL, 3.0 mmol) was slowly added dropwise. Then the reaction was carried out at room temperature for 5 h. A light yellow solid was precipitated. After vacuum drying, 0.9 g of the finished product of the compound of general formula (I) was obtained with a yield of 73.8%.

[0106] HRMS Calcd for C 24 H 32 N8O6S3: [M + H] + 625.6639, found 625.6528. 11H NMR (400 MHz, DMSO-d6) δ: 9.69 (1H, s), 9.33 (1H, d), 7.85 (2H, brs), 5.66 (1H, d), 5.17 (1H, d), 3.94 (3H, s), 3.69 (1H, d), 3.55 (1H, d), 3.29 (1H, d), 3.17 (1H, d), 2.95 (1H, d), 2.58 - 2.75 (1H, m), 2.38 - 2.48 (3H, m), 2.33 (3H, s), 1.75 - 1.89 (3H, m), 1.55 - 1.69 (4H, m), 0.85 - 0.99 (3H, m).

[0107] 2. Comparative Example 2:

[0108] (7R)-7-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(methoxyimino)acetamido]-3-[[(3S,5S)-5-((3,4-Dimethoxyphenyl)carbonyl)-1-methylpyrrolidin-3-yl]thio]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (Compound 11) Preparation:

[0109]

[0110] Intermediate 1: Dissolve Raw Material 1 (5.8 g, 10.0 mmol) and Raw Material 2 (3.3 g, 11.0 mmol) in tetrahydrofuran (50 mL), cool to 0 - 5 °C and stir for 5 minutes, slowly add diisopropylethylamine (2.5 mL, 14.0 mmol), then keep the reaction at a constant temperature for 5 h, remove the solvent under reduced pressure, and the residue is purified by column chromatography (ethyl acetate: petroleum ether = 5:1) to obtain 4.7 g of a light yellow solid with a yield of 60.4%.

[0111] Intermediate 2: Add dichloromethane (30 mL) to the reaction flask, cool to -5 - 0 °C, successively and slowly add phosphorus pentachloride (3.4 g, 16.5 mmol) and pyridine (1.4 mL, 17.3 mmol), keep stirring at a constant temperature for 1 h, then add Intermediate 1 (3.9 g, 5.0 mmol) and react for 5 h, filter off the insoluble matter, cool the filtrate to 0 - 5 °C, add trifluoroacetic acid (1.5 mL, 20.0 mmol), raise the temperature to room temperature and react for 2 h, then add isopropanol (50 mL) to precipitate a light yellow solid, and obtain 1.3 g of Intermediate 2 after vacuum drying with a yield of 52.6%.

[0112] Compound 1: In a dry reaction flask, add intermediate 2 (1.0 g, 2.0 mmol), raw material 3 (0.7 g, 2.0 mmol) and tetrahydrofuran (10 mL). Cool to 0 - 5 °C, slowly add n-butylamine (0.3 mL, 3.0 mmol) dropwise, then raise the temperature to room temperature and react for 10 h. A light yellow solid precipitates. After vacuum drying, 1.0 g of the finished product of the compound of general formula (I) is obtained, and the yield is 73.7%.

[0113] HRMS Calcd for C 26 H 30 N8O8S3: [M + H] + 679.5843, found 679.5441. 1 H NMR (400 MHz, DMSO-d6) δ: 9.67 (1H, s), 9.31 (1H, d), 7.77 (2H, brs), 7.08 - 7.35 (4H, m), 5.66 (1H, d), 5.16 (1H, d), 4.05 (3H, s), 3.85 (3H, s), 3.75 (3H, s), 3.65 (1H, d), 3.48 (1H, d), 3.19 (1H, d), 2.94 (1H, d), 2.56 - 2.76 (1H, m), 2.33 - 2.45 (3H, m), 2.31 (3H, s).

[0114] II. Pharmacodynamic experiments

[0115] 1. Antibacterial activity test

[0116] In this study, the antibacterial activity of the compound was evaluated by the microbroth dilution method according to the CLSI guidelines. Five clinically common pathogenic bacteria, namely Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae, were selected as test strains, and cefpirome was used as the positive control. The experimental procedures were carried out in a standardized manner: First, the test compound was prepared into a 50 μg / mL stock solution with sterile water, and a concentration series was established by two-fold serial dilution. The positive control group was subjected to equivalent dilution treatment synchronously to ensure the reliability of the method. The strain inoculation step was strictly followed. A bacterial suspension containing 5 × 105 CFU was quantitatively inoculated onto the agar plate using a multi-point inoculator. This inoculum size was verified by preliminary experiments to ensure uniform colony distribution and result reproducibility. The inoculated plates were incubated in a 37 °C constant temperature incubator for 18 hours to simulate the bacterial proliferation conditions under human infection. Finally, the minimum inhibitory concentration (MIC) was determined by visual reading.

[0117] The experimental results are shown in Table 1:

[0118] Table 1 Minimum inhibitory concentration (MIC) of the compounds of the present invention against common bacteria

[0119]

[0120] Experiments show that compounds 1-9 of the present invention have significant antibacterial activities against both Gram-positive and Gram-negative bacteria. Among them, the inhibitory effects of compounds 4, 7, and 8 on Staphylococcus aureus are better than those of cefpirome, and their antibacterial efficacies against the remaining strains are comparable. The activity of the comparative example is weak, confirming that the introduction of the 1,3,4-thiadiazole heterocycle can significantly improve the antibacterial performance.

[0121] 2. Molecular docking experiment with PBP2a

[0122] Computer-aided drug design method was used to carry out the molecular docking study of 1,3,4-thiadiazole cephalosporin derivatives. First, the two-dimensional structure of the target compound was constructed and converted to the PDBQT format by Open Babel to complete the ligand preparation. Subsequently, the crystal structure of PBP2a (3ZG0) was obtained from the RCSB database. After removing the crystallization water and the original ligand by PyMOL, the receptor protein was converted to the PDBQT format. During the docking process of AutoDock Vina, the docking parameters were set for the active pocket of this protein: 20.8×26.7×90.1, grid Except for adjusting the exhaustive parameter to 20, the remaining were kept as the software default settings for the molecular docking simulation.

[0123] The docking results are shown in Table 2:

[0124] Table 2 Simulation docking results of the compounds of the present invention with the active site of PBP2a

[0125]

[0126] The above experimental results show that the compounds of the present invention show significant binding ability to the active site of PBP2a, and the order of their affinity is positively correlated with the antibacterial activity. Specifically, compound 8 substituted with a cyclopentyl group shows the strongest binding strength, and its IC 50 value against Staphylococcus aureus is reduced by about 50% compared with the positive control; the binding potencies of compound 4 substituted with a p-tolyl group and compound 7 substituted with a cyclobutyl group are second, and their comprehensive antibacterial activities are lower than those of compound 8. Molecular docking reveals that 1,3,4-thiadiazole cephalosporin derivatives can enhance the antibacterial mechanism by strengthening the affinity with the PBP2a target protein.

[0127] Experiments show that the binding ability of the compounds of the present invention to the active site of PBP2a is positively correlated with their antibacterial activity. Among them, compound 8 substituted with a cyclopentyl group has the highest binding strength, and its IC 50 value against Staphylococcus aureus is reduced by 50% compared with the positive control; the binding potencies of compound 4 substituted with a p-tolyl group and compound 7 substituted with a cyclobutyl group are second, and their comprehensive antibacterial activities are relatively weak.

[0128] The experimental results show that the compounds synthesized in this study have significant binding ability to the active site of PBP2a, and their affinity is positively correlated with antibacterial activity. Among them, the compound 8 substituted with cyclopentyl has the strongest binding force, and the IC 50 value against Staphylococcus aureus decreased by about 50% compared with the positive control, showing the best antibacterial effect. The binding efficacy and antibacterial activity of the compound 4 substituted with p-tolyl and the compound 7 substituted with cyclobutyl are weaker than those of the compound 8. Molecular docking simulation shows that the cephalosporin derivatives modified with 1,3,4-thiadiazole effectively strengthen the molecular mechanism of inhibiting bacterial growth by enhancing the interaction with the PBP2a target protein.

[0129] Therefore, the cephalosporin compounds of the present invention have the characteristics of good chemical stability, high PBP2a affinity and strong antibacterial activity. In particular, the compounds 4, 7, and 8 in the examples have outstanding advantages and are new compounds with great potential for clinical application.

[0130] The above description is a detailed description of the preferred and feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. A cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt, the structure of which is shown in general formula I: Wherein: R1 and R2 are each independently selected from H or C 1-6 alkyl group; R3 is selected from H or C 1-6 alkyl; R4 is selected from C 1-6 alkyl or C 3-6 cycloalkyl; R5 is selected from H, C 1-6 alkyl, C 3-6 cycloalkyl, a substituted 4- to 6-membered heterocycle and its substituents, or an aromatic ring and its substituents; X is selected from N or CH.

2. The cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to claim 1, characterized in that: R5 is selected from C 4-6 cycloalkyl, or an aromatic ring and its substituents.

3. The cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to claim 1, characterized in that: R1, R2, R3, and R4 are each independently selected from C 1-3 alkyl groups.

4. The cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to claim 1, characterized in that: The cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt are selected from the following compounds:

5. The cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to claim 1, characterized in that, The synthesis method is carried out according to the following reaction formula: In the above reaction formula, The process of preparing intermediate 1 in step 1 Includes the following steps: Add raw material 1, raw material 2 and solvent a into a dry reaction vessel, stir and dissolve, cool to 0-5 °C, slowly dropwise add base 1, then keep the temperature for reaction for 3-6 h, and the reaction product is obtained by column chromatography to obtain intermediate 1; The process of preparing intermediate 2 in step 2 includes the following steps: In an atmosphere of dichloromethane, at a temperature of -5-0 °C, phosphorus pentachloride and pyridine are successively and slowly added into a dry reaction vessel, keep the temperature and stir for 1-2 h, then add intermediate 1 and react for 2-5 h, filter out the insoluble substances, cool the filtrate to 0-5 °C, add trifluoroacetic acid, naturally warm up to room temperature and react for 2-5 h, then add solvent b to precipitate and collect the solid to obtain intermediate 2; The process of preparing the compound of general formula (I) in step 3 includes the following steps: Add intermediate 2, raw material 3 and solvent c into a dry reaction vessel, cool to 0-5 °C, slowly dropwise add base 2, then naturally warm up to room temperature and react for 5-10 h, precipitate and collect the solid to obtain the finished product of the compound of general formula (I).

6. The cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to claim 5, characterized in that: In the said step 1, the solvent a is selected from tetrahydrofuran, methyltetrahydrofuran, dichloromethane or acetone; the base 1 is triethylamine, diisopropylethylamine or n-butylamine. In the said step 2, the solvent b is selected from acetone, isopropanol, isobutanol or propanol. In the said step 3, the solvent c is selected from tetrahydrofuran, methyltetrahydrofuran, dichloromethane or acetone; the base 2 is triethylamine, diisopropylethylamine or n-butylamine.

7. Use of the cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to any one of claims 1-6 in the preparation of antibacterial drugs.

8. Use of the cephalosporin derivative containing 1,3,4-thiadiazole and its pharmaceutically acceptable salt according to any one of claims 1-6 in the preparation of anti-infective drugs.

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