Dihydropyrazole derivatives, preparation method thereof and application of dihydropyrazole derivatives in preparation of antibacterial drugs

Abstract

The invention relates to the technical field of chemical synthesis, in particular to a dihydropyrazole derivative, a preparation method thereof and application of the dihydropyrazole derivative in preparation of antibacterial drugs. The dihydropyrazole derivative is prepared from corresponding raw materials through aldol condensation, Michael addition and acylation reaction, the reaction conditions are mild, the steps are simple, and the raw materials are low in cost and easy to obtain. The compound has excellent activity of resisting staphylococcus aureus, methicillin-resistant staphylococcus aureus, ampicillin-resistant staphylococcus aureus, pseudomonas aeruginosa and bacillus subtilis. The invention is of great significance to development and research of new antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, in particular to a class of dihydropyrazole derivatives, a preparation method thereof and an application thereof in the preparation of antibacterial drugs. Background Art

[0002] Antimicrobial drugs play a vital role in treating acute and chronic infections caused by various microorganisms. However, with the widespread use of antimicrobial drugs in clinical practice, bacteria and other microorganisms have evolved unique survival mechanisms to limit the effectiveness of drugs. The multiple resistance mechanisms of microorganisms and their combined effects will cause antimicrobial drugs to gradually lose their efficacy, reduce the adverse effects of drugs on microorganisms, thereby allowing microorganisms to survive and provide the possibility for subsequent antimicrobial treatment. Multidrug-resistant bacteria (such as ESBLs-positive Escherichia coli and carbapenem-resistant Klebsiella pneumoniae) hydrolyze drugs through β-lactamases or change the structure of PBPs, resulting in the ineffectiveness of β-lactam antibiotics. Therefore, the development of new antimicrobial drugs is very necessary and urgent for researchers.

[0003] Peptidoglycan is an important component of the cell wall of all bacteria (except mycobacteria), giving the bacterial cell wall its mechanical strength and determining its shape. Peptidoglycan polymers counteract the osmotic pressure generated by the cytoplasmic contents by forming a meshwork around the bacterial cytoplasmic membrane. Therefore, interfering with peptidoglycan synthesis leads to cell rupture. Inhibiting peptidoglycan synthesis is an effective bactericidal strategy with the additional advantage of being highly specific for bacteria because peptidoglycan and the enzymes required for its biosynthesis have no human counterparts. To date, antibiotic drugs actually only utilize a small part of the cell wall biosynthesis pathway, and most of them act in the bacterial periplasm, leaving the cytoplasmic steps of peptidoglycan biosynthesis underutilized as potential targets.

[0004] MurA (UDP-N-acetylglucosamine-enolpyruvyltransferase), attached with high affinity to UDP-MurNAc, catalyzes the first step in bacterial cell wall biosynthesis, exerting inhibitory or regulatory effects that are crucial for bacterial survival. MurA, a novel target and a key rate-limiting enzyme in peptidoglycan synthesis, is a key inhibitor of cell wall precursor synthesis. Its inhibitors exhibit no cross-resistance with PBPs and can circumvent β-lactamase-mediated resistance mechanisms. However, existing MurA inhibitors suffer from poor selectivity and low bioavailability (e.g., quinolone derivatives also exhibit DNA gyrase inhibitory activity). Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to design and synthesize a new type of highly selective MurA enzyme inhibitor to provide an effective alternative treatment for multidrug-resistant bacterial infections, especially pathogens resistant to β-lactam antibiotics.

[0006] The present invention provides novel 3,5-disubstituted dihydropyrazole MurA enzyme inhibitors, which have significant antibacterial effects and are generally stronger than fosfomycin, a MurA enzyme inhibitor currently used in clinical practice. The antibacterial effect on β-lactam antibiotic-resistant bacteria is significantly higher than that of conventional β-lactam antibiotics.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A class of dihydropyrazole derivatives, with the following structural formula:

[0009]

[0010] Ar1 is one of phenyl, nitro-substituted phenyl, alkyl-substituted phenyl, and halogen-substituted phenyl. The substitution position is not limited, and the number of carbon atoms of the alkyl group is 1 to 5.

[0011] Preferably, Ar1 is one of a phenyl group monosubstituted with a nitro group, a phenyl group monosubstituted with an alkyl group, and a phenyl group monosubstituted with a halogen group. The substitution position is not limited, and the number of carbon atoms of the alkyl group is 1 to 5.

[0012] Ar2 is one of furyl, thienyl and pyrrolyl.

[0013] Preferably, Ar2 is furyl or thienyl.

[0014] R is a halogen-monosubstituted alkyl group, and the number of carbon atoms of the alkyl group is 1 to 5.

[0015] Preferably, R is a methyl group monosubstituted with halogen.

[0016] The dihydropyrazole derivatives can be prepared by the following synthetic route:

[0017]

[0018] (1) Aryl ethyl ketone (Ar1COCH3) and aryl formaldehyde (Ar2COH) undergo aldol condensation under the catalysis of a base (preferably 10% sodium hydroxide solution) to produce chalcone intermediate I. The reactants are dissolved in anhydrous ethanol and stirred at room temperature. The reaction endpoint is monitored by thin-layer chromatography.

[0019] (2) The chalcone intermediate I obtained in step (1) is dissolved in anhydrous ethanol, and then hydrazine hydrate is added, and the mixture is stirred under reflux to react. The reaction end point is monitored by thin layer chromatography to finally generate a chalcone intermediate II.

[0020] (3) The chalcone intermediate II obtained in step (2) is dissolved in dichloromethane. RCOCl is added dropwise to the dichloromethane solution of the chalcone intermediate II under an ice bath while stirring the reaction. The reaction is monitored by TLC until completion, and the product is separated and purified to obtain a dihydropyrazole derivative.

[0021] Preferably, in step (1), the molar ratio of aryl ethyl ketone (Ar1COCH3) to aryl formaldehyde (Ar2COH) is 1:1.

[0022] Preferably, in step (2), the molar ratio of chalcone intermediate I to hydrazine hydrate is 1:5.

[0023] Preferably, in step (3), the molar ratio of chalcone intermediate II to RCOCl is 1:1.

[0024] The new MurA enzyme inhibitor provided by the present invention has excellent inhibitory activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and ampicillin-resistant Staphylococcus aureus, and can also effectively fight other pathogens such as Pseudomonas aeruginosa and Bacillus subtilis.

[0025] The present invention also provides an antibacterial drug, which is prepared from the dihydropyrazole derivative alone or by mixing the dihydropyrazole derivative with a pharmaceutically acceptable carrier.

[0026] The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the field of pharmaceutical preparations, and is selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, colorants, flavoring agents, and surfactants.

[0027] The antibacterial drug can be in the form of tablets, injections, capsules, emulsions, granules, pills, suppositories, etc.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0029] 1. The dihydropyrazole MurA enzyme inhibitors of the present invention have stronger inhibitory effects on Gram-positive bacteria such as Staphylococcus aureus than the MurA enzyme inhibitor fosfomycin currently used in clinical practice; some compounds also have stronger inhibitory effects on Staphylococcus aureus than conventional penicillins and cephalosporins β-lactam antibiotics.

[0030] 2. For bacteria resistant to β-lactam antibiotics, such as methicillin-resistant Staphylococcus aureus, the dihydropyrazole MurA enzyme inhibitors of the present invention are not affected by the resistance mechanism and have a significantly stronger antibacterial effect than conventional β-lactam antibiotics.

[0031] 3. It also has a significant inhibitory effect on other pathogenic bacteria, such as Pseudomonas aeruginosa and Bacillus subtilis. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0034] Example 1: Synthesis of WA-1

[0035] Synthesis of WA-1 Chalcone Intermediate I

[0036]

[0037] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Then, 2.88 g (0.03 mol) of furfural and 4.95 g (0.03 mol) of p-nitroacetophenone were added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude yellow powder.

[0038] Synthesis of WA-1 Chalcone Intermediate Ⅱ

[0039]

[0040] 2.43 g (0.01 mol) of WA-1 chalcone intermediate I was added to a 250 mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30 mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5 g (0.05 mol) of 50% hydrazine hydrate solution was added dropwise while stirring and refluxing. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude, tan powder.

[0041] Synthesis of WA-1

[0042]

[0043] Dissolve 2.57 g (0.01 mol) of WA-1 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (3:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide solution. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry it to obtain a brown crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (4:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a yellow solid (1.23 g). The yield for this step is 32.54%.

[0044] WA-1 NMR spectrum data:

[0045] 1 H NMR (600MHz, DMSO) δ8.36-8.31(m,2H),8.11-8.07(m,2H),7.59(dd,J=1.8,0.9Hz,1H),6.44-6.39(m,2 H),5.78-5.73(m,1H),4.51-4.43(m,2H),3.85(dd,J=18.1,11.9Hz,1H),3.51(dd,J=18.1,4.9Hz,1H).

[0046] Example 2: Synthesis of WA-2

[0047] Synthesis of WA-2 Chalcone Intermediate I

[0048]

[0049] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Then, 2.88 g (0.03 mol) of furfural and 4.03 g (0.03 mol) of p-methylacetophenone were added sequentially under stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude muddy yellow powder.

[0050] Synthesis of WA-2 Chalcone Intermediate Ⅱ

[0051]

[0052] 2.12 g (0.01 mol) of WA-2 chalcone intermediate I was added to a 250 mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30 mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5 g (0.05 mol) of 50% hydrazine hydrate solution was added dropwise while continuing the stirring and reflux reaction. The reaction was monitored by TLC until completion. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude dark yellow powder.

[0053] Synthesis of WA-2

[0054]

[0055] Dissolve 2.26 g (0.01 mol) of WA-2 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (4:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide solution. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry it to obtain a yellow-brown crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (8:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a white solid (1.19 g). The yield for this step is 34.29%.

[0056] WA-2 NMR spectrum data:

[0057] 1 H NMR (500MHz, DMSO) δ7.71(dd,J=8.2,3.6Hz,2H),7.56(dd,J=1.9,0.9Hz,1H),7.31-7.26(m,2H),6.38(ddd,J=18.4,3.3,1.2Hz,2 H),5.66(dd,J=11.8,4.6Hz,1H),4.41(d,J=1.2Hz,2H),3.72(dd,J=18.0,11.8Hz,1H),3.38(dd,J=18.0,4.7Hz,1H),2.35(s,3H).

[0058] Example 3: Synthesis of WA-3

[0059] Synthesis of WA-3 Chalcone Intermediate I

[0060]

[0061] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Then, 2.88 g (0.03 mol) of furfural and 4.14 g (0.03 mol) of p-fluoroacetophenone were added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude muddy yellow powder.

[0062] Synthesis of WA-3 Chalcone Intermediate Ⅱ

[0063]

[0064] 2.16g (0.01mol) of WA-3 chalcone intermediate I was added to a 250mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5g (0.05mol) of 50% hydrazine hydrate solution was added dropwise while stirring and refluxing. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude yellow powder.

[0065] Synthesis of WA-3

[0066]

[0067] Dissolve 2.30 g (0.01 mol) of WA-3 chalcone intermediate II in 50 mL of dichloromethane, and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (3:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide solution. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture, remove the solvent by rotary evaporation, cool to room temperature, and dry it to obtain a dark yellow crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (6:1, v / v). The separated liquid is rotary evaporated and dried to obtain a white solid (1.33 g). The yield for this step is 37.89%.

[0068] NMR spectrum data of WA-3:

[0069] 1H NMR (500MHz, CDCl3) δ7.73-7.68(m,2H),7.44-7.39(m,2H),7.31(dd,J=1.9,0.8Hz,1H),6.39(d,J=3.3Hz,1H),6.32(dd,J=3.4,1.8Hz,1H) ,5.70(dd,J=11.6,4.8Hz,1H),4.34(d,J=11.1Hz,1H),4.26(d,J=11.1Hz,1H),3.60(dd,J=17.6,11.6Hz,1H),3.48(dd,J=17.6,4.8Hz,1H).

[0070] Example 4: Synthesis of WA-4

[0071] Synthesis of WA-4 Chalcone Intermediate I

[0072]

[0073] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Then, 2.88 g (0.03 mol) of furfural and 4.64 g (0.03 mol) of p-chloroacetophenone were added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude dark yellow powder.

[0074] Synthesis of WA-4 Chalcone Intermediate Ⅱ

[0075]

[0076] 2.33 g (0.01 mol) of WA-4 chalcone intermediate I was added to a 250 mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30 mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5 g (0.05 mol) of 50% hydrazine hydrate solution was added dropwise while stirring and refluxing. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude orange-yellow powder.

[0077] Synthesis of WA-4

[0078]

[0079] Dissolve 2.47 g (0.01 mol) of WA-4 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (2:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide solution. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry to obtain a brown crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (4:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a 1.20 g yellow solid, with a yield of 32.61%.

[0080] WA-4 NMR spectrum data:

[0081] 1 H NMR (500MHz, DMSO) δ7.87-7.81(m,2H),7.59-7.52(m,3H),6.38(ddd,J=16.2,3.3,1.3Hz,2H),5.69( dd,J=11.8,4.7Hz,1H),4.42(d,J=1.7Hz,2H),3.76(dd,J=18.1,11.8Hz,1H),3.40(d,J=4.7Hz,1H).

[0082] Example 5: Synthesis of WA-5

[0083] Synthesis of WA-5 Chalcone Intermediate I

[0084]

[0085] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Then, 2.88 g (0.03 mol) of furfural and 5.97 g (0.03 mol) of p-bromoacetophenone were added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude yellow powder.

[0086] Synthesis of WA-5 Chalcone Intermediate Ⅱ

[0087]

[0088] 2.77 g (0.01 mol) of WA-5 chalcone intermediate I was added to a 250 mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30 mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5 g (0.05 mol) of 50% hydrazine hydrate solution was added dropwise while continuing the reaction under stirring and reflux. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude dark brown powder.

[0089] Synthesis of WA-5

[0090]

[0091] Dissolve 2.91 g (0.01 mol) of WA-5 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (2:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry it to obtain a brown crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (3:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a white solid (1.49 g). The yield for this step is 36.16%.

[0092] WA-5 NMR spectrum data:

[0093] 1 H NMR (500MHz, CDCl3) δ7.66-7.60(m,2H),7.60-7.55(m,2H),7.31(dd,J=1.8,0.9Hz,1H),6.39(d,J=3.3Hz,1H),6.32(dd,J=3.3,1.8Hz,1H) ,5.70(dd,J=11.6,4.8Hz,1H),4.34(d,J=11.0Hz,1H),4.26(d,J=11.1Hz,1H),3.60(dd,J=17.6,11.6Hz,1H),3.48(dd,J=17.6,4.8Hz,1H).

[0094] Example 6: Synthesis of WB-1

[0095] Synthesis of WB-1 Chalcone Intermediate I

[0096]

[0097] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Thiophene-2-carboxaldehyde (3.36 g, 0.03 mol) and p-nitroacetophenone (4.95 g, 0.03 mol) were then added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude muddy yellow powder.

[0098] Synthesis of WB-1 Chalcone Intermediate Ⅱ

[0099]

[0100] 2.59g (0.01mol) of WB-1 chalcone intermediate I was added to a 250mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30mL of anhydrous ethanol was added, stirred, and heated to reflux (70°C). Once completely dissolved, 5g (0.05mol) of 50% hydrazine hydrate solution was added dropwise while stirring and refluxing. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude, tan powder.

[0101] Synthesis of WB-1

[0102]

[0103] Dissolve 2.73 g (0.01 mol) of WB-1 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (2:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry to obtain an orange-yellow crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (4:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a yellow solid (1.09 g). The yield for this step is 27.65%.

[0104] NMR spectrum data of WB-1:

[0105] 1H NMR (500MHz, DMSO) δ8.36-8.30(m,2H),8.13-8.07(m,2H),7.45(dt,J=5.1,1.7Hz,1H),7.11-7.07(m,1H),6.97(ddd,J=5.1,3.6,1.8Hz,1H) ,5.96(dd,J=11.5,4.3Hz,1H),4.51(d,J=11.4Hz,1H),4.42(d,J=11.4Hz,1H),3.95(dd,J=18.3,11.6Hz,1H),3.50(dd,J=18.3,4.3Hz,1H).

[0106] Example 7: Synthesis of WB-2

[0107] Synthesis of WB-2 Chalcone Intermediate I

[0108]

[0109] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Thiophene-2-carboxaldehyde (3.36 g, 0.03 mol) and p-methylacetophenone (4.10 g, 0.03 mol) were then added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude yellow powder.

[0110] Synthesis of WB-2 Chalcone Intermediate Ⅱ

[0111]

[0112] 2.28g (0.01mol) of WB-2 chalcone intermediate I was added to a 250mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30mL of anhydrous ethanol was added, stirred, and heated to reflux (70°C). Once completely dissolved, 5g (0.05mol) of 50% hydrazine hydrate solution was added dropwise while continuing the stirring and reflux reaction. The reaction was monitored by TLC until completion. After completion, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude white powder.

[0113] Synthesis of WB-2

[0114]

[0115] Dissolve 2.42 g (0.01 mol) of WB-2 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (2:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry to obtain a yellow crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (4:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a white solid (1.51 g). The yield for this step is 41.60%.

[0116] NMR spectrum data of WB-2:

[0117] 1 H NMR (500MHz, CDCl3) δ7.69-7.64(m,2H),7.25(d,J=8.0Hz,2H),7.18(dd,J=5.2,1.2Hz,1H),7.08-7.03(m,1H),6.92(dd,J =5.1,3.5Hz,1H),5.89(d,J=11.3Hz,1H),4.37-4.27(m,2H),3.72(dd,J=17.7,11.3Hz,1H),3.37(dd,J=17.7,4.1Hz,1H).

[0118] Example 8: Synthesis of WB-3

[0119] Synthesis of WB-3 Chalcone Intermediate I

[0120]

[0121] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Thiophene-2-carboxaldehyde (3.36 g, 0.03 mol) and p-fluoroacetophenone (4.14 g, 0.03 mol) were then added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude yellow-gray powder.

[0122] Synthesis of WB-3 Chalcone Intermediate Ⅱ

[0123]

[0124] 2.32g (0.01mol) of WB-3 chalcone intermediate I was added to a 250mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5g (0.05mol) of 50% hydrazine hydrate solution was added dropwise while continuing the stirring and reflux reaction. The reaction was monitored by TLC until completion. After completion, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude yellow powder.

[0125] Synthesis of WB-3

[0126]

[0127] Dissolve 2.46 g (0.01 mol) of WB-3 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (4:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry to obtain a yellow crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (8:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a white solid (1.22 g). The yield for this step is 33.24%.

[0128] NMR spectrum data of WB-3:

[0129] 1 H NMR (500MHz, DMSO) δ7.91(ddd,J=8.8,5.4,2.7Hz,2H),7.43(dd,J=5.1,1.3Hz,1H),7.34(dd,J=9.7,7.9Hz,2H),7.07(dt,J=3.6,1.0Hz,1H),6.96(dd,J=5 .0,3.5Hz,1H),5.90(dd,J=11.4,4.0Hz,1H),4.46(d,J=11.3Hz,1H),4.38(d ,J=11.2Hz,1H),3.88(dd,J=18.2,11.4Hz,1H),3.43(dd,J=18.2,4.1Hz,1H).

[0130] Example 9: Synthesis of WB-4

[0131] Synthesis of WB-4 Chalcone Intermediate I

[0132]

[0133] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Thiophene-2-carboxaldehyde (3.36 g, 0.03 mol) and p-chloroacetophenone (4.64 g, 0.03 mol) were then added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude, pale yellow powder.

[0134] Synthesis of WB-4 Chalcone Intermediate Ⅱ

[0135]

[0136] 2.49 g (0.01 mol) of WB-4 chalcone intermediate I was added to a 250 mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30 mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5 g (0.05 mol) of 50% hydrazine hydrate solution was added dropwise while stirring and refluxing. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude yellow powder.

[0137] Synthesis of WB-4

[0138]

[0139] Dissolve 2.63 g (0.01 mol) of WB-4 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (3:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry to obtain a crude yellow product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (5:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a white solid (1.17 g). The yield for this step is 30.49%.

[0140] NMR spectrum data of WB-4:

[0141] 1H NMR (500MHz, CDCl3) δ7.48 (dd, J=5.1, 1.1Hz, 1H), 7.33-7.28 (m, 2H), 7.27 (dd, J=3.6, 1.2Hz, 2H), 7.22-7.16 (m, 2H), 7.09 (dd, J=5.1,3.6Hz,1H),4.33(d,J=10.9Hz,1H),4.27(d,J=10.9Hz,1H),3.80(dd,J=17.6,11.6Hz,1H),3.17(dd,J=17.6,4.7Hz,1H).

[0142] Example 10: Synthesis of WB-5

[0143] Synthesis of WB-5 Chalcone Intermediate I

[0144]

[0145] A 250 mL three-necked flask equipped with an electric stirrer and a reflux condenser was added with 30 mL of anhydrous ethanol. Thiophene-2-carboxaldehyde (3.36 g, 0.03 mol) and p-bromoacetophenone (5.97 g, 0.03 mol) were then added sequentially with stirring at room temperature. After stirring, 50 mL of 10% sodium hydroxide was added dropwise. The reaction was allowed to react at room temperature and monitored by TLC until completion. After completion of the reaction, the solid was filtered, washed three times with anhydrous ethanol, and dried to obtain a crude yellow powder.

[0146] Synthesis of WB-5 Chalcone Intermediate Ⅱ

[0147]

[0148] 2.91 g (0.01 mol) of WB-5 chalcone intermediate I was added to a 250 mL three-necked flask equipped with an electric stirrer, reflux condenser, and a thermostatic oil bath. 30 mL of anhydrous ethanol was then added, stirred, and heated to reflux (70°C). Once completely dissolved, 5 g (0.05 mol) of 50% hydrazine hydrate solution was added dropwise while stirring and refluxing. The reaction was monitored by TLC until complete. After completion of the reaction, the cooled reaction solution was concentrated under reduced pressure to remove excess solvent, yielding a crude pale yellow powder.

[0149] Synthesis of WB-5

[0150]

[0151] Dissolve 3.07 g (0.01 mol) of WB-5 chalcone intermediate II in 50 mL of dichloromethane and add the resulting solution to a 250 mL three-necked flask. Add 1.57 g (0.01 mol) of bromoacetyl chloride dropwise to the three-necked flask under an ice bath, stirring the reaction. Monitor the reaction by TLC until completion. The developing solvent is petroleum ether:ethyl acetate (4:1, v / v). After completion, adjust the pH of the reaction solution to neutral with 10% sodium hydroxide. Extract the organic layer with dichloromethane and dry it over anhydrous sodium sulfate. Filter the mixture and remove the solvent by rotary evaporation. Cool to room temperature and dry it to obtain a brown crude product. Purify the crude product using a 200-300 mesh silica gel column with a mobile phase of petroleum ether:ethyl acetate (8:1, v / v). Rotary evaporate the separated liquid and dry it to obtain a yellow solid (1.17 g). The yield for this step is 27.35%.

[0152] NMR spectrum data of WB-5:

[0153] 1 H NMR (500MHz, CDCl3) δ7.66–7.59(m,2H),7.57(d,J=8.4Hz,2H),7.20(dd,J=5.1,1.3Hz,1H),7.05(td,J=3.6,1.2Hz,1H),6.93(dd,J=5. 1,3.6Hz,1H),5.91(dd,J=11.4,4.4Hz,1H),4.54(d,J=3.1Hz,1H),4.34–4.26(m,1H),3.74(dd,J=17.7,11.4Hz,1H),3.40–3.31(m,1H).

[0154] Example 11: Antibacterial Activity Detection - Minimum Inhibitory Concentration (MIC) Determination

[0155] Experimental Preparation 1. Preparation of Reagents

[0156] (1) Preparation of 9% normal saline: Weigh 0.9 g of sodium chloride and pour it into a 200 mL conical flask. Add 100 mL of ultrapure water and seal the flask with a sterile sealing film. Sterilize the flask in an autoclave (121°C, 20 min).

[0157] (2) Preparation of ceftriaxone sodium solution: dissolve ceftriaxone sodium powder in an appropriate amount of 9% saline to prepare a 10 mg / mL stock solution, and then use sterile liquid culture medium to prepare the required concentration.

[0158] (3) Preparation of fosfomycin disodium solution: Weigh a certain amount of fosfomycin disodium according to the potency conversion and dissolve it in sterile liquid culture medium to prepare a 10 mg / mL stock solution for later use.

[0159] (4) Preparation of ampicillin sodium solution: Weigh a certain amount of ampicillin sodium according to the potency conversion and dissolve it in sterile liquid culture medium to prepare a 10 mg / mL stock solution for later use.

[0160] (5) Preparation of test product solutions: Accurately weigh the compounds obtained in Examples 1-10 using an electronic balance, dissolve them in DMSO to obtain 10 mg / mL stock solutions for later use, and then use DMSO to prepare the desired concentrations.

[0161] Experimental Preparation II. Preparation of Culture Medium

[0162] (1) Preparation of LB liquid medium: Weigh 2.00 g of sodium chloride, 1.00 g of yeast extract, and 2.00 g of peptone, pour into a conical flask, dissolve in 200 mL of ultrapure water, seal, and sterilize in an autoclave (121°C, 20 min). After completion, store in a refrigerator at 4°C until use.

[0163] (2) Preparation of LB solid medium: Weigh 2.00 g of sodium chloride, 1.00 g of yeast extract, and 2.00 g of peptone into a conical flask. Add 3.00 g of agar, dissolve in 200 mL of ultrapure water, seal, and sterilize in an autoclave (121°C, 20 min). After sterilization, wait until the temperature drops to 50°C, pour the liquid into a plate, cool until solidified, seal, and place upside down overnight.

[0164] (3) Preparation of NB solid culture medium: Add 1.85 g of NB nutrient broth powder and 2.00 g of agar to a conical flask, dissolve with 100 mL of ultrapure water, seal the flask, and sterilize in an autoclave (121°C, 20 min). After sterilization, wait until the temperature drops to 50°C, pour the liquid into a plate, cool until solidified, seal the flask, and place it upside down overnight.

[0165] (4) Preparation of NB liquid culture medium: Dissolve 1.85 g of NB nutrient broth powder in 100 mL of ultrapure water in a conical flask, seal the flask, and sterilize it in an autoclave (121°C, 20 min). After sterilization, store in a refrigerator at 4°C until ready for use.

[0166] Experimental Preparation III: Cultivation of Ampicillin-Resistant Staphylococcus aureus

[0167] Using the nine-square grid method, a series of gradient concentrations were marked on the back of the NB solid culture medium dish, and then 100 μL of 1×10 5A Staphylococcus aureus suspension containing 100 CFU / mL of S. aureus was plated onto a solid culture medium and evenly spread using a sterile inoculating loop. Sterile filter paper was then placed at the corresponding positions on the solid culture medium. 20 μL of ampicillin solution prepared in NB liquid medium at 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 μg / mL was added to the filter paper. After this procedure, the culture was incubated in a 37°C incubator for 24 hours. The appropriate strain was then selected using an inoculating loop. After several generations of culture, stable, drug-resistant strains were obtained.

[0168] Determination of minimum inhibitory concentration (MIC):

[0169] Bacterial Activation: Select the appropriate culture medium based on the bacterial species to be activated (LB medium for E. coli and Bacillus subtilis, NB medium for Staphylococcus aureus and Pseudomonas aeruginosa). Remove the frozen bacterial strain and quickly thaw the cryovial in a 37°C water bath, gently shaking to allow the vial to thaw. Immediately after thawing, wipe the surface of the cryovial with alcohol for disinfection. Then, add the bacterial suspension to the appropriate liquid culture medium, gently shake, and incubate at 30-37°C for 18-24 hours.

[0170] The following experiments were all performed in a sterile operating table. In a 96-well plate, the stock solutions of the test product and the positive control drug (including: fosfomycin, ampicillin, cefotaxime) were diluted by the two-fold dilution method. 50 μL of the test product solution and the positive control drug solution with decreasing concentrations were added from left to right. There were three groups in total. The first group contained 50 μL of the antibacterial solution and 50 μL of 1×10 6 CFU / mL bacterial solution, the second group contained 50 μL blank medium and 50 μL 1×10 6The third group contained only 100 μL of blank culture medium. LB liquid medium was used for Escherichia coli and Bacillus subtilis, and NB liquid medium was used for Staphylococcus aureus and Pseudomonas aeruginosa. The bacterial strains were the following six: Escherichia coli quality control strain (purchased from China Center for Type Culture Collection, strain collection number: CCTCC AB 93154), Bacillus subtilis quality control strain (purchased from China Center for Type Culture Collection, strain collection number: CCTCC AB 90008), Staphylococcus aureus quality control strain (purchased from China Center for Type Culture Collection, strain collection number: CCTCC AB 99002), methicillin-resistant Staphylococcus aureus quality control strain (purchased from China Center for Type Culture Collection, strain collection number: CCTCC AB 2015107), ampicillin-resistant Staphylococcus aureus (self-cultured), and Pseudomonas aeruginosa quality control strain (purchased from China Center for Type Culture Collection, strain collection number: CCTCCAB 2013184). All cultures were incubated at 37°C for 24 hours in a constant-temperature incubator. Then, 10 μL of CCK-8 solution was added to each well of a 96-well plate. The plates were incubated at 37°C for 30 minutes, then removed and assayed for bacteriostasis. Wells showing bacterial growth will display an orange-yellow color with CCK-8. The minimum inhibitory concentration (MIC) was defined as the drug concentration at which no bacterial growth was visible to the naked eye. The results are shown in Table 1 below.

[0171] Table 1: Minimum inhibitory concentration of compounds (μmol / L)

[0172]

[0173] Example 12: Detection of enzyme inhibitory activity - half inhibitory concentration IC 50 Determination

[0174] Experimental Preparation 1. Extraction of MurA Enzyme

[0175] (1) Inoculation: 1×10 5 100 μL of CFU / mL Escherichia coli was added to 200 μL of LB liquid culture medium, and then 200 μL of 50 mg / mL kanamycin was added. The culture was placed in a 37°C constant temperature shaker at 250 rpm for 12 h.

[0176] (2) Expansion: Add 100 μL of 50 mg / mL kanamycin to 200 mL of LB liquid medium, then add 6 mL of the cultured bacterial solution in step (1), and place in a 37°C constant temperature shaker at 250 rpm for 2 h.

[0177] (3) Induction: After the expansion is completed, add 200 μL of 1 mol / L IPTG solution and place in a 37°C constant temperature shaker at 250 rpm for 3 h.

[0178] (4) Extraction: After the culture is completed, the bacterial liquid is poured into a centrifuge box and balanced, and placed in a low-temperature high-speed centrifuge, and the centrifugation conditions are set to -4°C, 4000 rpm, and 20 min. After the centrifugation is completed, the precipitate in the box is left and rinsed with PBS. The liquid is collected in a 50 mL centrifuge tube, placed on ice and disrupted with a cell disruptor for 12 min. After the end, the cell is placed in a low-temperature high-speed centrifuge, and the centrifugation conditions are set to -4°C, 8000 rpm, and 20 min. After the completion, the supernatant in the centrifuge tube is collected and column chromatography is performed using a nickel column.

[0179] (5) Collection: Before collecting the liquid, turn on the peristaltic pump and balance the nickel column with a balance solution (50 mL 0.2 M PB, 14.625 g NaCl, constant volume to 500 mL, pH = 7.4) for 15 min (speed of 7 rpm), and then load the liquid collected in step (4) onto the column (speed of 4 rpm). After the loading is completed, wash with a wash solution (50 mL 0.2 M PB, 14.625 g NaCl, 2.723 g imidazole, constant volume to 500 mL) for 7 min (speed of 8 rpm), and then start eluting the protein with an eluent (50 mL 0.2 M PB, 14.625 g NaCl, 17.020 g imidazole, constant volume to 500 mL) (speed of 3 rpm). The collected eluted protein liquid is placed in an ultrafiltration tube and then placed in a high-speed centrifuge, which is set to -4°C, 4000 rpm, and 20 min.

[0180] (6) Drying: After centrifugation, collect the upper liquid in the ultrafiltration tube, freeze it in a -80°C refrigerator overnight, and then perform vacuum freeze drying. The resulting protein powder is stored in a -20°C environment.

[0181] Determination of enzyme inhibitory activity of the compounds obtained in Examples 1-10:

[0182] Before the experiment began, the enzyme was activated, and the substrate required for the enzyme reaction was prepared to the corresponding concentration. In a 96-well plate, 10 μL of ultrapure water, 10 μL of 6.5 mg / mL UNAG (urine N-acetyl-β-D-glucosaminidase), and 20 μL of 26.25 mg / mL MurA enzyme were added to each well. The target compound stock solution (prepared as in Example 11) was diluted to 11 concentrations by a two-fold dilution method. 10 μL of the target compound solution was added to each well in descending order from right to left. Two control groups were set up: a positive control group (10 μL of fosfomycin disodium solution at the same concentration) and a blank control group (10 μL of ultrapure water without the target compound solution). The plates were placed in a constant temperature incubator and incubated at 37°C for 5 minutes. After incubation, 10 μL of 16.80 mg / mL potassium phosphoenolpyruvate (PEP) was added to each well and incubated at 37°C for 25 minutes. After the incubation, add 50 μL of malachite green colorimetric reagent to each well and cover with tin foil, then place in a constant temperature incubator and set the incubation conditions to 37°C for 20 minutes. After the incubation is completed, the OD value is measured at a wavelength of 620 nm using a microplate reader to determine the inhibitory effect of the target compound on the enzyme activity, IC 50 If the concentration is less than 1000 μmol / L, the compound has inhibitory activity on the enzyme.

[0183] Table 2: Results of half-inhibitory concentration determination of compounds

[0184]

Claims

1. A class of dihydropyrazole derivatives, the structural formula is as follows: Ar1 is one of phenyl, nitro-substituted phenyl, alkyl-substituted phenyl, and halogen-substituted phenyl, the substitution position is not limited, and the number of carbon atoms of the alkyl group is 1 to 5; Ar2 is one of furyl, thienyl, and pyrrolyl; R is a halogen-monosubstituted alkyl group, and the number of carbon atoms of the alkyl group is 1 to 5.

2. The dihydropyrazole derivative according to claim 1, characterized in that Ar1 is one of a phenyl group monosubstituted with nitro, a phenyl group monosubstituted with alkyl, and a phenyl group monosubstituted with halogen, the substitution position is not limited, and the number of carbon atoms of the alkyl group is 1 to 5; and / or Ar2 is furyl or thienyl; and / or R is a methyl group monosubstituted with halogen.

3. A method for preparing the dihydropyrazole derivative according to claim 1 or 2, characterized in that: The following steps are involved: (1) Aryl ethyl ketone Ar1COCH3 and aromatic formaldehyde Ar2COH react under base catalysis through aldol condensation to generate chalcone intermediate I. The reactants are dissolved in anhydrous ethanol and stirred at room temperature. The reaction endpoint is monitored by thin layer chromatography. (2) dissolving the chalcone intermediate I obtained in step (1) in anhydrous ethanol, then adding hydrazine hydrate, stirring and reacting under reflux, and monitoring the reaction end point by thin layer chromatography to finally generate a chalcone intermediate II; (3) The chalcone intermediate II obtained in step (2) is dissolved in dichloromethane. RCOCl is added dropwise to the dichloromethane solution of the chalcone intermediate II under an ice bath while stirring the reaction. The reaction is monitored by TLC until completion, and the product is separated and purified to obtain a dihydropyrazole derivative.

4. The preparation method according to claim 3, characterized in that In step (1), the molar ratio of aryl ethyl ketone Ar1COCH3 and aryl formaldehyde Ar2COH is 1:1; In step (2), the molar ratio of chalcone intermediate I to hydrazine hydrate is 1:5; In step (3), the molar ratio of chalcone intermediate II to RCOCl is 1:

1.

5. Use of the dihydropyrazole derivative according to claim 1 or 2 in the preparation of MurA enzyme inhibitors.

6. Use of the dihydropyrazole derivative according to claim 1 or 2 in the preparation of antibacterial drugs.

7. Use of the dihydropyrazole derivative according to claim 1 or 2 in the preparation of a medicament for use against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, ampicillin-resistant Staphylococcus aureus, Bacillus subtilis and / or Pseudomonas aeruginosa.

8. An antibacterial drug, characterized in that The dihydropyrazole derivative according to claim 1 or 2 is prepared alone, or is prepared by mixing the dihydropyrazole derivative according to claim 1 or 2 with a pharmaceutically acceptable carrier.

9. The antibacterial drug according to claim 8, characterized in that The antibacterial drug is one of tablets, injections, capsules, emulsions, granules, pills, and suppositories.

10. The antibacterial drug according to claim 8 or 9, characterized in that: The antibacterial drug is a drug that is effective against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, ampicillin-resistant Staphylococcus aureus, Bacillus subtilis and / or Pseudomonas aeruginosa.

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