CN116102478A - Preparation method and anti-infection application of 1,4-dicarbonylthiosemicarbazide compound - Google Patents

Abstract

The invention belongs to the technical field of anti-infective drugs, and relates to a preparation method of a1, 4-dicarbonyl thiosemicarbazide compound and application of the compound as an anti-infective drug.

The compound is shown as a formula (I), wherein A is optionalFrom benzene rings, halogenated benzenes, pyridines and pyrimidines; wherein R is ₁ Selected from unsubstituted or halogenated C1-C6 alkyl, C3-C6 cycloalkyl; r is R ₂ Selected from the group consisting of C4-C8 alkylbenzene, C3-C8 cycloalkylbenzene, C3-C8 cycloalkyl, C1-C8 alkyl substituted C3-C8 cycloalkyl, R ₂ The substitution position of (c) is selected from ortho, meta or para. The 1, 4-dicarbonyl thiourea compound has strong inhibition effect on gram positive bacteria, especially methicillin-resistant staphylococcus aureus (MRSA), methicillin-resistant staphylococcus epidermidis (MRSE) and vancomycin-resistant enterococci (VRE), and can be used for preparing drug-resistant bacteria drugs.

Description

Preparation method of 1,4-dicarbonylthiosemicarbazide compounds and their anti-infection uses

Technical Field

The present invention belongs to the technical field of anti-infective drugs, and relates to 1,4-dicarbonylthiosemicarbazide compounds, a method for preparing the compounds, and their use as anti-infective drugs.

Background Art

At present, bacterial resistance to antibiotics has become a serious public health safety issue. In clinical bacterial infectious diseases, almost 70% of pathogens show varying degrees of resistance to one or more antibiotics. Especially in the "post-antibiotic era" of antibiotic drug research, the overall slowdown of antimicrobial drug research and development and the double attack of global drug resistance, clinical drugs for bacterial infections may soon face the severe challenge of "no drugs available". In clinical practice, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus (VRE) are the most common resistant Gram-positive bacteria. The development of antimicrobial drugs with new mechanisms of action and new structural types is of great clinical significance for the prevention and treatment of the above-mentioned drug-resistant bacterial infections.

1,4-diacylthiosemicarbazide compounds have a wide range of biological and physiological activities. This class of compounds shows certain activity in inhibiting the growth of microorganisms in the fields of antibacterial, antiparasitic, antiviral and antifungal. In particular, there are many reports on the anti-infection research of 1,4-diarylthiosemicarbazide compounds and. Based on the results of current literature reports, 1,4-diarylthiosemicarbazide compounds and 1-acyl-4-arylthiosemicarbazide compounds show certain inhibitory activity against bacteria, and their minimum inhibitory concentration (MIC) is high, which does not have the value of being developed as antibacterial drugs (Molecules, 2019, 24, 1490; Monatshefte fur Chemie, 2007, 138, 511-516; DE3033554A1; WO2016070021). In 2018, the patent (CN108456157) disclosed the antibacterial activity of 1-aroyl-4-acylthiosemicarbazide compounds against Gram-positive bacteria. The antibacterial activity range was 0.1 μg/mL–100 μg/mL. Its main structural feature was substituted aromatic groups such as alkoxy-substituted benzene and alkoxy-substituted biphenyl as its aromatic characteristic structure. The present invention discovers for the first time that when aromatic groups such as C4-C8 alkylbenzene, C3-C8 cycloalkylbenzene, C3-C8 cycloalkyl, and C1-C8 alkyl-substituted C3-C8 cycloalkyl are used as the characteristic structures of the aromatic acyl group, the in vitro antibacterial activity is significantly better than the currently reported thiosemicarbazide compounds, and the compound has significant inhibitory activity against clinically isolated drug-resistant Gram-positive bacteria (methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and vancomycin-resistant Enterococcus, etc.), with a MIC range of 0.06μg/mL-8.0μg/mL, and the inhibitory activity results against clinical drug-resistant strains show that they are significantly better than those of the positive control drug.

Summary of the invention

The purpose of the present invention is to provide a class of 1,4-dicarbonylthiosemicarbazide compounds with stronger antibacterial activity, a preparation method and the use of the compounds as antibacterial drugs.

The present invention provides the following technical solutions:

The present invention provides a class of 1,4-dicarbonylthiosemicarbazide compounds, which have a compound as shown in formula (I):

in:

A can be selected from a benzene ring, a halogenated benzene, pyridine and pyrimidine;

_R1 is selected from unsubstituted or halogenated C1-C6 alkyl, C3-C6 cycloalkyl;

R ₂ is selected from C4-C8 alkylbenzene, C3-C8 cycloalkylbenzene, C3-C8 cycloalkyl, C1-C8 alkyl substituted C3-C8 cycloalkyl, and the substitution position of R ₂ is selected from ortho, meta or para;

_R1 wherein the C1-C6 alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight-chain or side-chain pentyl, straight-chain or side-chain hexyl; _R1 wherein the C3-C6 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Preferably, when A is selected from a benzene ring, the C3-C8 cycloalkyl group described in _R2 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl; the C3-C8 cycloalkyl group substituted by C1-C8 alkyl group described in _R2 is selected from cyclopropyl substituted by C1-C8 alkyl group, cyclobutyl substituted by C1-C8 alkyl group, cyclopentyl substituted by C1-C8 alkyl group, cyclohexyl substituted by C1-C8 alkyl group, cycloheptyl substituted by C1-C8 alkyl group, and cyclooctyl substituted by C1-C8 alkyl group; the substitution position of _R2 is selected from ortho, meta or para, as shown in formula Ia, and n can be selected from 0, 1, 2, 3, 4, 5,

Preferably, when A is selected from a benzene ring and a halogenated benzene, _R1 is selected from an unsubstituted C1-C6 alkyl group and an unsubstituted C3-C6 cycloalkyl group, and _R2 is a C4-C8 alkyl substituted phenyl group selected from n-butylbenzene, isobutylbenzene, sec-butylbenzene, tert-butylbenzene, a linear or side-chain pentylbenzene, a linear or side-chain hexylphenyl, a linear or side-chain heptylphenyl, a linear or side-chain octylphenyl, wherein the C4-C8 alkyl group is selected from the ortho position, the meta position or the para position at the benzene substitution position; as shown in formula Ib, n can be selected from 0, 1, 2, 3, 4, 5, and X can be selected from F, Cl, Br,

Preferably, when A is selected from a benzene ring and a halogenated benzene, _R1 is selected from an unsubstituted C1-C6 alkyl group and an unsubstituted C3-C6 cycloalkyl group, and _R2 is a C3-C8 cycloalkylbenzene selected from cyclopropanebenzene, cyclobutylbenzene, cyclopentylbenzene, cyclohexylbenzene, cycloheptylbenzene and cyclooctylbenzene, wherein the substitution position of the C3-C8 cycloalkyl group on the benzene ring is selected from the ortho position, meta position or para position; the substitution position of _R2 is selected from the ortho position, meta position or para position, as shown in formula Ic, n can be selected from 0, 1, 2, 3, 4, 5, and X can be selected from F, Cl, Br,

Preferably, when A is selected from pyridine and pyrimidine, _R1 is an unsubstituted C1-C6 alkyl group or a C3-C6 cycloalkyl group, the C4-C8 alkyl substituted phenyl group of _R2 is selected from n-butylbenzene, isobutylbenzene, sec-butylbenzene, tert-butylbenzene, linear or side-chain pentylbenzene, linear or side-chain hexylphenyl, linear or side-chain heptylphenyl, linear or side-chain octylphenyl; the substitution position of _R2 is selected from ortho, meta or para.

Preferably, the 1,4-dicarbonylthiosemicarbazide compounds provided by the present invention are:

A1: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A2: N-(2-(4'-pentyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A3: N-(2-(4'-hexyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A4: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)propionamide

A5: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)isopropylamide

A6: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)cyclopropylcarboxamide

A7: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)cyclobutylcarboxamide

A8: N-(2-(4'-butyl-3-fluoro-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A9: N-(2-(4-cyclopropylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

A10: N-(2-(4-cyclopentylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

A11: N-(2-(4-cyclohexylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

A12: N-(2-(4-(4-ethylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A13: N-(2-(4-(4-propylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A14: N-(2-(4-(4-butylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A15: N-(2-(4-(4-pentylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A16: N-(2-(4'-cyclopropyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A17: N-(2-(4'-cyclobutyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A18: N-(2-(4'-cyclopentyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A19: N-(2-(4'-cyclohexyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

B1: N-(2-(2-(4-butyl)pyridine-5-carbonyl)hydrazine-1-thiocarbonyl)acetamide

B2: N-(2-(2-(4-butylphenyl)pyridine-5-carbonyl)hydrazine-1-thiocarbonyl)propionamide

C1: N-(2-(6-(4-butylphenyl)nicotinoyl)hydrazine-1-thiocarbonyl)acetyl

C2: N-(2-(6-(4-butylphenyl)nicotinoyl)hydrazine-1-thiocarbonyl)propionamide

Secondly, the present invention provides a method for preparing 1,4-dicarbonylthiosemicarbazide compounds, which is characterized by comprising the following reaction steps:

(1) Step 1: Mix a cycloalkyl boronic acid reagent/C4-C6 alkyl benzene boronic acid reagent/cycloalkyl benzene boronic acid reagent with 1-2 equivalents of ortho-, meta- or para-halogenated benzoic acid methyl ester/picolinate/pyrimidine carboxylate, add a catalyst Pb(PPh ₃ ) ₄ and 2 equivalents of Na ₂ CO ₃ , fill with argon gas for protection, heat and react in toluene: ethanol: water for 6-12 hours, directly add an appropriate amount of water after the reaction is completed, extract the reaction mixture with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, separate and purify by column chromatography to obtain the following compound (this is used as a representative structure for illustration, but is not limited to this):

(2) Step 2: The intermediate obtained in step 1 is mixed with hydrazine hydrate in an alcohol solution, and heated under reflux for reaction. After the reaction is completed, the crude product is directly concentrated under reduced pressure, and the solid is washed with water and dried to obtain the following compound (this is used as a representative structure for illustration, but is not limited thereto):

(3) Step 3: At room temperature, potassium isothiocyanate was dissolved in acetonitrile, and an equimolar amount of alkanoyl chloride/substituted alkanoyl chloride was added dropwise thereto. The mixture was stirred at room temperature for 30 min to 12 h to obtain the following compound:

The product does not require post-treatment and can be directly used in the next reaction after filtration;

(4) Step 4: Weigh an appropriate amount of hydrazide intermediate, dissolve it in an appropriate amount of acetonitrile, and add 1.5-2.0 times the equivalent of isothiocyanate acetonitrile solution to the reaction system at room temperature. The reaction can be carried out at room temperature or under heating conditions. TLC is used to monitor the progress of the reaction. After the reaction is completed, the crude product is concentrated under reduced pressure, washed with water, and recrystallized from an organic solvent to obtain a compound of formula I:

in:

A can be selected from a benzene ring, a halogenated benzene, pyridine and pyrimidine;

_R1 is selected from unsubstituted or halogenated C1-C6 alkyl, unsubstituted C3-C6 cycloalkyl;

R ₂ is selected from C4-C8 alkylbenzene, C3-C8 cycloalkylbenzene, C3-C8 cycloalkyl, C1-C8 alkyl substituted C3-C8 cycloalkyl, and the substitution position of R ₂ is selected from ortho, meta or para.

The synthetic route is as follows:

At the same time, the present invention provides the use of the 1,4-dicarbonylthiosemicarbazide compound as an antibacterial drug. Preferably, the antibacterial use is mainly against Gram-positive bacteria, wherein the Gram-positive bacteria include Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis or Enterococcus faecium.

The present invention proves through in vitro antibacterial experimental tests that the thiosemicarbazide compounds can significantly inhibit the growth of Staphylococcus aureus, Staphylococcus aureus epidermidis, and Bacillus subtilis. Some of the compounds show strong inhibitory effects on resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant enterococci (VER) and vancomycin-resistant Staphylococcus aureus in further in vitro antibacterial activity screening.

The present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a 1,4-dicarbonylthiourea compound as described in Formula I and a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is selected from injections, tablets, pills, capsules, suspensions or emulsions. The carrier comprises conventional excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

Beneficial technical effects: The present invention provides a class of 1,4-dicarbonylthiosemicarbazide compounds (such as formula I) with a completely new structure, whose structural characteristics have not been reported in the literature. Such compounds have significant inhibitory activity against clinically isolated drug-resistant Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus, etc. The antibacterial activity is significantly better than that of the listed drug vancomycin, and its MIC range is 0.06 μg/mL-8.0 μg/mL. Such 1,4-dicarbonylthiosemicarbazide compounds may provide a new solution for vancomycin-resistant Gram-positive bacteria.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific examples, but the examples are only used to illustrate the present invention, rather than to limit the present invention. All experimental methods in the examples are conventional methods unless otherwise specified; the materials and chemical reagents used are reagents and materials directly obtained from commercial channels unless otherwise specified.

1. Preparation method of A-series compounds

General synthesis route 1:

Operation steps: add two equivalents of cesium carbonate, 0.01 equivalents of bis(triphenylphosphine)palladium dichloride, one equivalent of methyl p-bromobenzoate (or methyl 2-fluoro-4-bromobenzoate) and 1.2 equivalents of boric acid compound to a reaction vessel protected by argon gas and filled with a mixed solvent (toluene: water: ethanol = 3:3:1) in sequence, and heat the reaction system to 80°C and stir for 4 hours. After the reaction, the reaction solution is washed with water several times until neutral, and the organic phase is concentrated by a rotary evaporator after extraction, and then separated by silica gel column chromatography to obtain a coupling product. The coupling product is dissolved in ethanol, 3 to 5 equivalents of hydrazine hydrate are added, and the reaction system is stirred and heated under reflux for 4 hours. After cooling, a solid is precipitated, the solid is filtered and washed, and the solid is dissolved in acetonitrile for standby use. Add 1.1 equivalents of potassium thiocyanate to another reaction vessel filled with acetonitrile solvent, stir to dissolve, then add 1.2 equivalents of acyl chloride, stir and react for 5 minutes, the system is milky white, pass the reaction solution through a microporous filter membrane and quickly add it to the acetonitrile system reserved in the previous step, stir and react at room temperature for 2 hours, add a small amount of water to the reaction system after the reaction is completed, and filter the solid to obtain the product.

Example-1:

Compound A1: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction operation refers to the general synthetic route 1. 9.3mmol cesium carbonate, 0.05mmol bis(triphenylphosphine) palladium dichloride, 4.7mmol methyl p-bromobenzoate and 5.6mmol 4-n-butylphenylboronic acid are added to a reaction vessel protected by argon gas and filled with 40mL of a mixed solvent (toluene: water: ethanol = 3:3:1). The reaction system is heated to 80°C and stirred for 4 hours. After the reaction is completed, the reaction solution is washed with water (3×10mL) several times until neutral, extracted, and concentrated under reduced pressure to obtain a crude product. The crude product is redissolved in dichloromethane, 100-200 mesh silica gel is added in 3 times the amount of the crude product and mixed thoroughly, and after reduced pressure concentration, the crude product-silica gel powder mixture is purified by rapid column chromatography to obtain a coupling product. 2.2mmol of the coupling product is dissolved in 10mL of ethanol, 9mmol of hydrazine hydrate is added, and the reaction system is stirred and heated under reflux for 4 hours, and then the heating is stopped, and the solid is cooled to precipitate. The solid is filtered and washed, and the solid is dissolved in acetonitrile for standby use. Add 3.8mmol potassium thiocyanate to another reaction container and dissolve it in 10mL acetonitrile, stir and dissolve, then add 4mmol acetyl chloride, stir and react for 5 minutes, the system is milky white, pass the reaction solution through a microporous filter membrane and quickly add it to the acetonitrile system reserved in the previous step, stir and react at room temperature for 2 hours, add a small amount of water to the reaction system after the reaction is completed, and filter the solid to obtain the product. The yield is: 36%, off-white solid. . HRMS calcd.forC ₂₀ H ₂₃ N ₃ O ₂ S[M+Na] ⁺ 392.1403, found: 392.1411; ¹ H NMR (500MHz, DMSO-d ₆ ) δ12.1 (s, 1H), 11.6 (s, 1H), 11.1 (s, 1H), 8.0 (d, J = 7.8Hz, 2H), 7.8 (d ,J＝7.9Hz,2H),7.7(d,J＝7.7Hz,2H), 7.3(d,J＝7.7Hz,2H),2.6(t,J＝7.7Hz,2H),2.2(s,3H),1.6(m,2H),1.3(d,J＝7.5Hz,2H), 0.9(t,J＝7.4Hz,3H).

Example-2:

Compound A2: N-(2-(4'-pentyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction steps refer to Example-1, the starting material is 0.5 g of 4-pentylphenylboronic acid, and the acyl chloride is acetyl chloride. The yield is: 32%, light yellow solid. HRMS calcd for C ₂₁ H ₂₅ N ₃ O ₂ S[M+Na] ⁺ 406.1560, found: 404.1565; ¹ HNMR (500MHz, DMSO-d ₆ )δ12.2(s,1H),11.6(s,1H),11.08(s,1H),8.0(d,J＝7.8Hz,2H),7.8(d,J ＝7.9Hz,2H),7.7(d,J＝7.5Hz,2H),7.3(d,J＝7.5Hz,2H),3.3(s,2H),2.6(t,J＝7.8Hz,2H), 2.2(s,3H),1.6(t,J＝7.5Hz,2H),1.3(q,J＝7.3Hz,4H),0.9(t,J＝6.7Hz,3H).

Example-3:

Compound A3: N-(2-(4'-hexyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 0.75 g of 4-hexylphenylboronic acid, the acyl chloride is acetyl chloride, the yield is 37%, light yellow solid. HRMS calcd for C ₂₂ H ₂₇ N ₃ O ₂ S[M+Na] ⁺ 420.1824,found:420.1828; ¹ H NMR (500MHz, DMSO-d ₆ )δ12.2(s,1H),11.6(s,1H),11.1(s,1H),8.0(d,J＝7.9Hz,2H),7.8(d, J＝7.9Hz,2H),7.7(d,J＝7.4Hz,2H),7.3(d,J＝7.5Hz,2H),3.3(s,3H),2.6(d,J＝7.6Hz,2H), 2.2(s,3H),1.6(t,J＝7.5Hz,2H),1.3(s,7H),0.9(d,J＝6.4Hz ,3H).

Example-4:

Compound A4: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)propionamide

The reaction procedure is as in Example 1, the starting material is 1.0 g of 4-butylphenylboronic acid, the acyl chloride is propionyl chloride, the yield is 37%, light yellow solid. HRMS calcd for C ₁₉ H ₂₃ N ₃ O ₂ S[M+Na] ⁺ 408.1456,found:408.1472; ¹ HNMR (500MHz, DMSO-d ₆ )δ12.21(s,1H),11.64(s,1H),11.49(s,1H),9.29(s,2H),8.40(d,J＝ 8.0 Hz,2H),7.41(d,J＝8.0Hz,2H),2.69(t,J＝7.7Hz,2H),2.55–2.47(m,3H),1.62(p,J＝7.5Hz, 2H),1.35(m,2H),1.09(t,J＝7.5Hz,3H),0.93(t,J＝7.3Hz ,3H).

Example-5:

Compound A5: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)isopropylamide

The reaction procedure is as in Example 1, the starting material is 1.0 g of 4-n-butylphenylboronic acid, and the acyl chloride is isobutyryl chloride. The yield is 48%, light yellow powder. HRMS calcd for C ₂₂ H ₂₇ N ₃ O ₂ S[M+Na] ⁺ 420.1824, found: 408.1826; ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.20(s,1H),11.58(s,1H),11.07(s,1H),7.98(d,J＝8.0Hz,2H), 7.81(d,J＝8.1Hz,2H),7.66(d,J＝7.8Hz,2H),7.32(d,J＝7.7Hz,2H),2.81(m,1H),2.61(t,J ＝4.4Hz,2H),1.63(m,2H),1.31(m,2H),1.11(d,J＝5.5Hz,6H),0.92(s,3H).

Example-6:

Compound A6: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)cyclopropylcarboxamide

The reaction procedure is as in Example 1, the starting material is 1.0 g of 4-n-butylphenylboronic acid, the acyl chloride is cyclopropylcarbonyl chloride, the yield is 33%, light yellow powder. HRMS calcd for C ₂₂ H ₂₅ N ₃ O ₂ S[M+Na] ⁺ 418.1667, found: 418.1670; ¹ H NMR (500MHz, DMSO-d ₆ ) δ11.91 (d, J = 5.0Hz, 1H), 11.07 (d, J = 4.8Hz, 1H), 7.98 (d, J = 7.1Hz, 2H) ,7.80(d,J＝7.6Hz,2H),7.66(d,J＝7.6Hz,2H),7.31(d,J＝7.6Hz,2H),2.60(t,J＝7.7Hz,2H),2.09(d,J＝6.0Hz,1H),1.62(m,2H),1.32(m,2H),1.01-0.88( m,7H).

Example-7:

Compound A7: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)cyclobutylcarboxamide

The reaction procedure is as in Example 1, the starting material is 1.0 g of 4-n-butylphenylboronic acid, the acyl chloride is cyclobutylcarbonyl chloride, the yield is 48%, light yellow powder. HRMS calcd for C ₂₃ H ₂₇ N ₃ O ₂ S[M+Na] ⁺ 432.1824, found: 432.1827; ¹ H NMR (500MHz, DMSO-d ₆ ) δ12.34 (s, 1H), 11.81 (s, 1H), 11.17 (s, 1H), 8.01 (d, J = 8.2Hz, 2H), 7 .82(d,J＝6.1Hz,2H),7.72-7.64(m,2H),7.33(t,J＝8.6Hz,2H),2.62(t,J＝6.1Hz,2H), 2.10(m,1H),1.63(m,2H),1.33-0.92(m,13H).

Example-8:

Compound A8: N-(2-(4'-butyl-3-fluoro-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction steps refer to Example-1, the starting material is 1.0 g of 4-n-butylphenylboronic acid, and the acyl chloride is acetyl chloride. The yield is: 40%, off-white powder. HRMS calcd for C ₂₀ H _22F N ₃ O ₂ S[M+Na] ⁺ 410.1417, found:410.1420; ¹ H NMR (500MHz, DMSO-d ₆ )δ12.47(s,1H),11.65(s,1H),10.97(d,J＝3.3Hz,1H),7.84(t,J＝7.9Hz, 1H),7.68(t,J＝8.0Hz,3H),7.66(d,J＝4.0Hz,1H),7.32(d,J＝7.9Hz,2H),2.63(t,J＝7.7Hz, 2H), 2.15 (s, 3H), 1.58 (p, J = 7.6 Hz, 2H), 1.32 (h, J = 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). Example-9:

Compound A9: N-(2-(4-cyclopropylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 1 g of cyclopropylboronic acid, and the acyl chloride is acetyl chloride. The yield is 29%, light yellow powder. HRMS calcd for C ₁₃ H ₁₅ N ₃ O ₂ S[M+Na] ⁺ 300.0885, found: 300.0889; ¹ H NMR (500 MHz, DMSO-d ₆ )δ12.11(s,1H),11.56(s,1H),10.92(s,1H),7.78(d,J＝7.8Hz,2H),7.20(d,J ＝7.8Hz,2H),2.14(s,3H),2.04-1.93(m,1H),1.02(d,J＝7.9Hz,2H),0.75(d,J＝5.2Hz,2H). Example 10:

Compound A10: N-(2-(4-cyclopentylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 1 g of cyclopentylboronic acid, the acyl chloride is acetyl chloride, the yield is 45%, light yellow powder. HRMS calcd for C ₁₅ H ₁₉ N ₃ O ₂ S[M+Na] ⁺ 328.1198, found: 328.1202; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ12.14 (s, 1H), 11.60 (s, 1H), 10.96 (s, 1H), 7.85 (d, J＝7.8Hz, 2H), 7 .41(d,J＝7.6Hz,2H),3.07(p,J＝8.7Hz,1H),2.18(s,3H),2.07(q,J＝9.7,8.9Hz,2H),1.81(d,J＝7.4Hz,2H),1.69(p,J＝7.6,5.8Hz,2H),1.58(p,J＝9.1Hz, 2H).

Example-11:

Compound A11: N-(2-(4-cyclohexylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 1 g of cyclohexylboronic acid, the acyl chloride is acetyl chloride, the yield is 48%, light yellow powder. HRMS calcd for C ₁₆ H ₂₁ N ₃ O ₂ S[M+Na] ⁺ 342.1247, found: 342.1242; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ12.10 (s, 1H), 11.56 (s, 1H), 10.92 (s, 1H), 7.81 (d, J = 8.0Hz, 2H), 7 .36(d,J＝8.0Hz,2H),2.62–2.53(m,1H),2.14(s,3H),1.80(dd,J＝11.9,4.7Hz,4H),1.71(d,J＝13.0Hz,1H),1.48–1.34(m,4H),1.31–1.18(m,1H).

Example-12:

Compound A12: N-(2-(4-(4-ethylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

The reaction procedure is as in Example 1, the starting material is 1 g of 4-ethylcyclohexylboronic acid, and the acyl chloride is acetyl chloride. The yield is 44%, light yellow powder. HRMS calcd for C ₁₈ H ₂₅ N ₃ O ₂ S[M+Na] ⁺ 370.1667, found: 370.1671; ¹ H NMR (500MHz, DMSO-d ₆ )δ12.14(s,1H),11.60(s,1H),10.96(s,1H),7.85(d,J＝7.9Hz,2H), 7.39(d,J＝7.9Hz,2H),2.58(d,J＝12.2Hz,1H),2.18(s,3H),1.86(t,J＝12.6Hz,4H),1.49 (qd,J＝12.9,12.3,3.5Hz,2H), 1.26(dt,J＝20.2,10.0Hz,3H), 1.07(q,J＝11.0,10.3Hz,2H), 0.92(t,J＝7.2Hz,3H).

Example-13:

Compound A13: N-(2-(4-(4-propylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

The reaction procedure is as in Example 1, the starting material is 1 g of 4-n-propylcyclohexylboronic acid, and the acyl chloride is acetyl chloride. The yield is 33%, light yellow powder. HRMS calcd for C ₁₉ H ₂₇ N ₅ O ₂ S[M+Na] ⁺ 383.1624, found: 383.1628; ¹ H NMR (500MHz, DMSO-d ₆ )δ11.59(s,1H),10.95(s,1H),7.84(d,J＝8.0Hz,2H),7.38(d,J＝ 7.9Hz,2H),2.60–2.54(m,1H),2.17(s,3H),1.85(td,J＝11.1,10.1,5.0Hz,4H),1.49(qd,J＝ 13.1,3.6Hz,2H),1.34(dq,J＝16.5,8.8,8.1Hz,3H),1.22(q,J＝7.2,6.7Hz,2H),1.07(qd,J＝13.1,3.4Hz,2H),0.91(t,J＝7.3Hz,3H).

Example-14:

Compound A14: N-(2-(4-(4-butylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

The reaction procedure is as in Example 1, the starting material is 1 g of 4-n-butylcyclohexylboronic acid, and the acyl chloride is acetyl chloride. The yield is 42%, light yellow powder. HRMS calcd for C ₂₀ H ₂₉ N ₃ O ₂ S[M+Na] ⁺ 398.1980, found: 398.1984; ¹ H NMR (500MHz, DMSO-d ₆ )δ12.12(s,1H),11.57(s,1H),10.92(s,1H),7.81(d,J＝7.9Hz,2H), 7.35(d,J＝7.9Hz,2H),2.54(d,J＝12.3Hz,1H),2.14(s,3H),1.80(d,J＝11.5Hz,4H),1.45(q, J＝12.5Hz,2H),1.33–1.25(m,5H),1.21(d,J＝7.7Hz,2H),1.03(q,J＝12.3Hz,2H),0.88(d, J＝6.7Hz,3H).

Example-15:

Compound A15: N-(2-(4-(4-pentylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

The reaction procedure is as in Example 1, the starting material is 1 g of 4-ethylcyclopentylboronic acid, and the acyl chloride is acetyl chloride. The yield is 39%, light yellow powder. HRMS calcd for C ₂₁ H ₃₁ N ₅ O ₂ S[M+Na] ⁺ 412.2137, found: 412.2141; ¹ H NMR (500MHz, DMSO-d ₆ )δ12.14(s,1H),11.59(s,1H),10.95(s,1H),7.84(d,J＝7.9Hz,2H), 7.38(d,J＝7.9Hz,2H),2.56(s,1H),2.17(s,3H),1.89–1.80(m,4H),1.48(dt,J＝13.9,7.0Hz, 2H), 1.32 (ddd, J＝16.7, 11.3, 5.4Hz, 7H), 1.24 (p, J＝7.3, 6.7Hz, 3H), 1.12–1.01 (m, 2H), 0.90 (t, J＝6.9Hz, 3H).

Example-16:

Compound A16: N-(2-(4'-cyclopropyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 1 g of 4-cyclopropylphenylboronic acid, and the acyl chloride is acetyl chloride. The yield is 38%, light yellow powder. HRMS calcd for C ₁₉ H ₁₉ N ₃ O ₂ S[M+Na] ⁺ 373.1090,found:373.1100; ¹ HNMR (500MHz, DMSO-d ₆ )δ12.16(s,1H),11.62(s,1H),11.10(s,1H),8.00(d,J＝8.2Hz,2H),7. 82 (d,J＝8.3Hz,2H),7.66(d,J＝7.9Hz,2H),7.23(d,J＝7.8Hz,2H),2.19(s,3H),2.00(dt,J＝8.8,5.2Hz,1H),1.02(d,J＝7.9Hz,2H),0.75(d,J＝5.2Hz,2H).

Example-17:

Compound A17: N-(2-(4'-cyclobutyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 1 g of 4-cyclobutylphenylboronic acid, and the acyl chloride is acetyl chloride. The yield is 42%, light yellow powder. HRMS calcd for C ₂₀ H ₂₁ N ₃ O ₂ S[M+Na] ⁺ 390.1247, found:390.1255; ¹ HNMR (500MHz, DMSO-d ₆ )δ12.17(s,1H),11.62(s,1H),11.10(s,1H),8.00(d,J＝8.0Hz,2H),7.83(d,J＝8.0Hz,2H),7.71(d,J＝7.9Hz,2H),7.38(d,J＝7.8Hz,2H),3.60(t,J＝8.8Hz,1H), 2.35(q,J＝9.0,8.5Hz,2H),2.19(s,4H),2.14(dd,J＝9.5,2.6Hz,2H),2.08–1.97(m,1H), 1.86(d,J＝9.6Hz,1H).

Example-18:

Compound A18: N-(2-(4'-cyclopentyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction operation steps refer to Example-1, the starting material is 1g of 4-cyclopentylphenylboronic acid, and the acyl chloride is acetyl chloride. The yield is: 46%, light yellow powder. HRMS calcd for C ₂₁ H ₂₃ N ₃ O ₂ S[M+Na] ⁺ 404.1403., found: 404.1408; ¹ HNMR (500MHz, DMSO-d ₆ )δ12.15(s,1H),11.62(s,1H),11.10(s,1H),8.00(d,J＝8.1Hz,2H),7.83 (d,J＝8.1Hz,2H),7.69(d,J＝7.8Hz,2H),7.40(d,J＝8.2Hz,2H),3.10–3.01(m,1H),2.19(s, 3H), 2.07 (p, J＝7.4Hz, 2H), 1.81 (q, J＝7.4Hz, 2H), 1.69 (q, J＝6.6Hz, 2H), 1.60 (q, J＝9.4 Hz, 2H).

Example-19:

Compound A19: N-(2-(4'-cyclohexyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction procedure is as in Example 1, the starting material is 1 g of 4-cyclohexylphenylboronic acid, and the acyl chloride is acetyl chloride. The yield is 41%, light yellow powder. HRMS calcd for C ₂₂ H ₂₅ N ₃ O ₂ S[M+Na] ⁺ 418.1667, found:418.1671; ¹ HNMR (500MHz, DMSO-d ₆ )δ12.16(d, J＝2.5Hz,1H),11.62(s,1H),11.10(d, J＝2.5Hz,1H),8.00(d, J＝8.2Hz,2H),7.83(d, J＝8.2Hz,2H),7.69(d, J＝7.9Hz,2H),7.38(d, J＝7.9Hz,2H), 2.64–2.56(m,1H),2.19(s,3H),1.85(d,J＝10.0Hz,4H),1.75(d,J＝12.9Hz,1H),1.49–1.39 (m,4H),1.30(t,J＝12.4Hz,1H).

2. Preparation method of B-series compounds:

General synthesis route:

Operation steps: add two equivalents of cesium carbonate, 0.01 equivalents of bis(triphenylphosphine)palladium dichloride, one equivalent of 2-chloropyrimidine-5-carboxylic acid ethyl ester and 1.2 equivalents of boric acid compound to a reaction vessel protected by argon gas and filled with a mixed solvent (toluene: water: ethanol = 3:3:1) in sequence, and heat the reaction system to 80°C and stir for 4 hours. After the reaction is completed, the reaction solution is washed with water several times until neutral, and the organic phase is concentrated by a rotary evaporator after extraction, and then separated by silica gel column chromatography to obtain the coupling product. The coupling product is dissolved in ethanol, and 3 to 5 equivalents of hydrazine hydrate are added. The reaction system is stirred and heated under reflux for 4 hours. After cooling, a solid is precipitated, and the solid is filtered and washed, and the solid is dissolved in acetonitrile for standby use. To another reaction vessel filled with acetonitrile solvent, add 1.1 equivalents of potassium thiocyanate, stir to dissolve, then add 1.2 equivalents of acyl chloride, stir to react for 5 minutes, the system becomes milky white, the reaction solution is passed through a microporous filter membrane and quickly added to the acetonitrile system reserved in the previous step, stir and react at room temperature for 2 hours, after the reaction is completed, add a small amount of water to the reaction system, and filter out the solid to obtain the product.

Example-20:

Compound B1: N-(2-(2-(4-butyl)pyridine-5-carbonyl)hydrazine-1-thiocarbonyl)acetamide

The reaction operation refers to the general synthetic route 2. 9.3mmol cesium carbonate, 0.05mmol bis(triphenylphosphine) palladium dichloride, 4.7mmol 2-chloropyrimidine-5-carboxylic acid ethyl ester and 5.6mmol 4-n-butylphenylboronic acid are added to the reaction vessel with 40mL mixed solvent (toluene: water: ethanol = 3: 3: 1) protected by argon gas. The reaction system is heated to 80°C and stirred for 4 hours. After the reaction is completed, the reaction solution is washed with water (3×10mL) several times until neutral, extracted, and concentrated under reduced pressure to obtain a crude product. The crude product is redissolved in dichloromethane, 100-200 mesh silica gel is added 3 times the amount of the crude product and fully mixed. After concentrating under reduced pressure, the crude product-silica gel powder mixture is purified by rapid column chromatography to obtain a coupling product. 2.2mmol of the coupling product is dissolved in 10mL of ethanol, 9mmol of hydrazine hydrate is added, and the reaction system is stirred and heated under reflux for 4 hours, and then the heating is stopped, and the solid is cooled to precipitate. The solid is filtered and washed, and the solid is dissolved in acetonitrile for standby use. Add 3.8mmol potassium thiocyanate to another reaction container and dissolve it in 10mL acetonitrile, stir and dissolve, then add 4mmol acetyl chloride, stir and react for 5 minutes, the system is milky white, the reaction solution is passed through a microporous filter membrane and quickly added to the acetonitrile system reserved in the previous step, stir and react at room temperature for 2 hours, after the reaction is completed, add a small amount of water to the reaction system, solid precipitation is filtered to obtain the product. The yield is: 29%, light yellow powder. HRMS calcd forC ₁₈ H ₂₁ N ₅ O ₂ S[M+Na] ⁺ 394.1308,found:394.1322; ¹ H NMR(400MHz,DMSO-d ₆ )δ12.14(s, 1H),11.66(s,1H),11.46(s,1H),9.25(s,2H),8.36(d,J =7.7Hz,2H),7.36(d,J＝7.9Hz,2H),2.64(d,J＝8.2Hz,2H),2.16(s,3H),1.57(d,J＝8.0Hz,2H),1.37–1.24(m,2H),0.90(d,J＝8.0Hz,3H).

Example-21:

Compound B2: N-(2-(2-(4-butylphenyl)pyridine-5-carbonyl)hydrazine-1-thiocarbonyl)propionamide

The reaction procedure is as in Example 20, the starting material is 1 g of 4-n-butylphenylboronic acid, and the acyl chloride is propionyl chloride. The yield is: 35%, light yellow solid. HRMS calcd for C ₁₉ H ₂₃ N ₅ O ₂ S[M+Na] ⁺ 408.1456, found: 408.1472; ¹ H NMR (500MHz, DMSO-d ₆ )δ12.21(s, 1H), 11.64(s, 1H), 11.49(s, 1H), 9.29(s, 2H), 8.40(d, J＝8.0 Hz, 2H), 7.41(d, J＝8.0Hz, 2H), 2.69(t, J＝7.7Hz, 2H), 2.55–2.47(m, 3H), 1.62(p, J＝7.5Hz, 2H), 1.35 (h, J = 7.4Hz, 2H), 1.09 (t, J = 7.5Hz, 3H), 0.93 (t, J = 7.3Hz, 3H).

3. Preparation method of C-series compounds:

General synthesis route:

Operation steps: add two equivalents of cesium carbonate, 0.01 equivalents of bis(triphenylphosphine)palladium dichloride, one equivalent of 6-chloronicotinate methyl ester and 1.2 equivalents of boric acid compound to a reaction vessel protected by argon gas and filled with a mixed solvent (toluene: water: ethanol = 3:3:1) in sequence, heat the reaction system to 80°C, and stir for 4 hours. After the reaction is completed, the reaction solution is washed with water several times until neutral, and the organic phase is concentrated by a rotary evaporator after extraction, and then separated by silica gel column chromatography to obtain a coupling product. The coupling product is dissolved in ethanol, 3 to 5 equivalents of hydrazine hydrate are added, the reaction system is stirred and heated under reflux for 4 hours, and a solid is precipitated after cooling, the solid is filtered and washed, and the solid is dissolved in acetonitrile for standby use. Add 1.1 equivalents of potassium thiocyanate to another reaction vessel filled with acetonitrile solvent, stir to dissolve, then add 1.2 equivalents of acyl chloride, stir to react for 5 minutes, the system is milky white, pass the reaction solution through a microporous filter membrane and quickly add it to the acetonitrile system reserved in the previous step, stir and react at room temperature for 2 hours, add a small amount of water to the reaction system after the reaction is completed, and filter the solid to obtain the product.

Example-22:

Compound C1: N-(2-(6-(4-butylphenyl)nicotinoyl)hydrazine-1-thiocarbonyl)acetyl

The reaction operation refers to the general synthetic route 3. 9.3mmol cesium carbonate, 0.05mmol bis(triphenylphosphine) palladium dichloride, 4.7mmol 6-chloronicotinate methyl ester and 5.6mmol 4-n-butylphenylboronic acid are added to the reaction vessel with 40mL mixed solvent (toluene: water: ethanol = 3:3:1) protected by argon gas. The reaction system is heated to 80°C and stirred for 4 hours. After the reaction is completed, the reaction solution is washed with water (3×10mL) several times until neutral, extracted, and concentrated under reduced pressure to obtain a crude product. The crude product is redissolved in dichloromethane, 100-200 mesh silica gel is added in 3 times the amount of the crude product and fully mixed. After reduced pressure concentration, the crude product-silica gel powder mixture is purified by rapid column chromatography to obtain a coupling product. 2.2mmol of the coupling product is dissolved in 10mL of ethanol, 9mmol of hydrazine hydrate is added, and the reaction system is stirred and heated under reflux for 4 hours, and then the heating is stopped, and the solid is cooled to precipitate. The solid is filtered and washed, and the solid is dissolved in acetonitrile for standby use. Add 3.8mmol potassium thiocyanate to another reaction container and dissolve it in 10mL acetonitrile, stir and dissolve, then add 4mmol acetyl chloride, stir and react for 5 minutes, the system is milky white, pass the reaction solution through a microporous filter membrane and quickly add it to the acetonitrile system reserved in the previous step, stir and react at room temperature for 2 hours, add a small amount of water to the reaction system after the reaction is completed, filter and obtain the product after the solid precipitates. The yield is 32%, light yellow powder. HRMS calcd for C ₁₉ H ₂₂ N ₄ O ₂ S[M+Na] ⁺ 393.1356, found: 393.1269; ¹ H NMR (500MHz, DMSO-d ₆ ) δ12.13 (s, 1H), 11.62 (s, 1H), 11.28 (s, 1H), 9.10 (d, J = 2.3Hz, 1H), 8 .29(dd,J＝8.3,2.3Hz,1H),8.09(t,J＝8.0Hz,3H),7.34(d,J＝7.8Hz,2H),2.64(t,J＝7.7Hz,2H),2.16(s,3H),1.59(p,J＝7.6Hz, 2H), 1.32 (q, J = 7.5Hz, 2H), 0.90 (t, J = 7.4Hz, 3H).

Example-23:

Compound C2: N-(2-(6-(4-butylphenyl)nicotinoyl)hydrazine-1-thiocarbonyl)propionamide

The reaction procedure is as in Example 22, the starting material is 1 g of 4-n-butylphenylboronic acid, and the acyl chloride is propionyl chloride. The yield is 32%, light yellow powder. HRMS calcd for C ₂₀ H ₂₄ N ₄ O ₂ S[M+H] ⁺ 385.1693, found:385.1701; ¹ HNMR (500MHz, DMSO-d ₆ )δ11.63(s,1H),11.32(s,1H),9.16(d,J＝2.3Hz,1H),8.34(dd,J＝8.4, 2.4Hz,1H),8.13(t,J＝7.5Hz,3H),7.38(d,J＝8.0Hz,2H),2.68(t,J＝7.7Hz,2H),2.52(q,J ＝7.4Hz,2H),1.62(q,J＝7.5Hz,2H),1.36(q,J＝7.4Hz,2H),1.11(t,J＝7.4Hz,3H),0.94(t,J＝7.3Hz,3H).

IV. In vitro antibacterial test method Experimental Example 1: Determination of the activity of compounds against Bacillus subtilis and non-resistant common clinical pathogens (Staphylococcus aureus, Staphylococcus epidermidis)

1. Preparation of test bacterial solution

Take 10g of sodium chloride, 10g of peptone, and 5g of yeast extract, add deionized water to 1000mL, adjust the pH to 7.0, and prepare LB medium after sterilization. Inoculate Bacillus subtilis (Bacillus subtilis CMCC 63501) into 20mL of LB medium, and culture it in a 37℃ incubator at a speed of 200 rpm for 12 hours. When the clarified LB medium becomes turbid, it indicates that the bacteria proliferate significantly and grow vigorously. At this time, dilute the bacterial solution with new LB liquid medium to make its OD600 value between 0.3 and 0.5, and dilute it 105 times with new LB medium as the test bacterial solution.

2. Determine the minimum inhibitory concentration

Take a clean and sterile 96-well cell culture plate, add 200μL of the prepared test bacterial solution to each well in the first column, and add 100μL of the test bacterial solution to each well in the second to twelfth columns. Take 4μL of the pre-prepared 5mg/ml DMSO solution of the sample to be tested and add it to each well in the first column (each sample is repeated three times), and set up a positive control group (i.e. 4μL of levofloxacin at the same concentration) and a blank control (i.e. no drug added). Starting from the first column of wells, 100 μL of sample was sucked from the previous column by an 8-channel micropipette and added to the next column of wells for 2-fold gradient dilution. 12 different concentrations of compound solutions were set, including 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.13 μg/ml, 1.56 μg/ml, 0.78 μg/ml, 0.39 μg/ml, 0.20 μg/ml, 0.10 μg/ml, and 0.05 μg/ml. The 96-well cell culture plate was placed in a 37°C incubator. After 18 hours of incubation, the growth of Bacillus subtilis in each well of the 96-well plate was observed. For each compound, the compound concentration corresponding to the well where no bacterial growth was observed was the minimum inhibitory concentration of the compound.

3. The activity of the compound against Staphylococcus aureus, Staphylococcus aureus and Enterococcus faecalis can also be determined by this method. The difference lies in the culture medium. The culture medium for Staphylococcus aureus is tryptone soy broth, the culture medium for Staphylococcus aureus is nutrient broth, and the culture medium for Enterococcus faecalis is brain heart infusion.

4. The anti-activities of the compounds against Bacillus subtilis (CMCC 63501), Staphylococcus aureus (ATCC29213) and Staphylococcus epidermidis (ATCC12228) are shown in Table 1.

Table-1 Antibacterial activity test results of compounds

Label: MIC: minimum inhibitory concentration.

Experimental Example 2: Antibacterial activity test of compounds against multiple standard or clinically isolated pathogenic bacteria

1. Type of bacteria

From the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and the Institute of Materia Medica, Chinese Academy of Medical Sciences, including 7 strains of Staphylococcus aureus, numbered ATCC25923 (MRSA), ATCC700699, ATCC700788, MRSA-1 (resistant clinical isolate), MRSA-2 (resistant clinical isolate), MRSA-3 (resistant clinical isolate), MSSA-1 (sensitive clinical isolate), MSSA-2 (sensitive clinical isolate); 5 strains of Staphylococcus epidermidis, numbered MRSE-1 (resistant clinical isolate), MRSE-2 (resistant clinical isolate), MRSE-3 (resistant clinical isolate), MSSE-1 and MRSE-2; 6 strains of Enterococcus faecalis, numbered ATCC 29212 (VSE); ATCC 51299 (VRE); ATCC 51575 (VSE); TCC700802 (VRE), EFA-1 and EFA-2; 2 strains of Enterococcus faecium, numbered EFM-1 and EFM-2.

2. Test methods

The MIC values of each test sample against the tested strains were determined by the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Succeptibility Test Procedure [Methods for Dilution Antimicrobial Succeptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Eleventh Edition, M07-A11, 2018]. In the test, 100 μl of the test sample solution of different concentrations was added to the 1st to 12th wells of a sterilized 96-well polystyrene plate, respectively, so that the final drug concentrations were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg/L, respectively (due to the poor water solubility of the compound, the test concentration of the drug was reduced to 32-0.015 mg/L). Then add 100μl of the test bacterial solution to each well (200μL per well), and the final concentration of the bacterial solution is about 10 ⁵ CFU/mL. After sealing, place it in a 35-37℃ incubator for 18-24h and judge the results. The minimum drug concentration that completely inhibits bacterial growth in the small well is its minimum inhibitory concentration (MIC). The sensitivity and resistance judgment criteria of vancomycin to bacteria (CLSI 2019) are shown in Table 2.

Table-2 Criteria for judging the sensitivity and resistance of vancomycin and other drugs to bacteria (CLSI 2019)

3. The inhibitory activity of representative compounds against the above-mentioned standard or clinically isolated clinical pathogenic bacteria strains is shown in Table-3.

Table-3 Antibacterial activity test (MIC) of compounds against various standard or clinically isolated pathogenic bacteria

Note: Methicillin-resistant Staphylococcus aureus: MRSA-1, MRSA-2, MRSA-3; Methicillin-sensitive Staphylococcus aureus: MSSA-1, MSSA-2; Vancomycin-intermediately sensitive Staphylococcus aureus: ATCC25923; Staphylococcus aureus: ATCC700699 (MRSA, VISA); Staphylococcus aureus: ATCC700788 (MRSA, VISA); Methicillin-resistant Staphylococcus epidermidis: MRSE-1, MRSE-2, MRSE-3; Methicillin-sensitive Staphylococcus epidermidis: MSSE-1, MSSE-2; Enterococcus faecalis: EFA-1, EFA-2; Vancomycin-sensitive Enterococcus faecalis: ATCC29212; Highly resistant Enterococcus faecalis: ATCC51299 (VRE); Enterococcus faecalis: ATCC700802 (VRE); Vancomycin-resistant Enterococcus faecalis: ATCC51575 (VRE); Enterococcus faecium: EFM-1, EFM-2.

Claims (12)

1. A 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof as shown in formula (I),

in: A is selected from phenyl, halophenyl, pyridyl or pyrimidinyl; _R1 is selected from unsubstituted or halogenated C1-C6 alkyl, C3-C6 cycloalkyl; R ₂ is selected from C4-C8 alkylphenyl, C3-C8 cycloalkylphenyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl substituted by C1-C8 alkyl, and the substitution position of R ₂ is selected from ortho, meta or para. 2. The 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: _R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl; _R2 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl; C1-C8 alkyl-substituted cyclopropyl, C1-C8 alkyl-substituted cyclobutyl, C1-C8 alkyl-substituted cyclopentyl, C1-C8 alkyl-substituted cyclohexyl, C1-C8 alkyl-substituted cycloheptyl, C1-C8 alkyl-substituted cyclooctyl; the substitution position of _R2 is selected from ortho, meta or para, as shown in Ia, n can be selected from 0, 1, 2, 3, 4, 5,

3. The 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl; _R2 is selected from n-butylbenzene, isobutylbenzene, sec-butylbenzene, tert-butylbenzene, linear or side-chain pentylbenzene, linear or side-chain hexylphenyl, linear or side-chain heptylphenyl, linear or side-chain octylphenyl, wherein the C4-C8 alkyl group is selected from the ortho, meta or para position at the substitution position of the benzene ring; _R2 is selected from cyclopropanebenzene, cyclobutylbenzene, cyclopentylbenzene, cyclohexylbenzene, cycloheptylbenzene and cyclooctylbenzene, wherein the C3-C8 cycloalkyl group is selected from the ortho, meta or para position at the substitution position of the benzene ring; the substitution position of _R2 is selected from the ortho, meta or para position, as shown in Formula Ib and Formula Ic, n can be selected from 0, 1, 2, 3, 4, 5, X can be selected from F, Cl, Br,

4. The 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when A is selected from pyridine or pyrimidine, _R1 is an unsubstituted C1-C6 alkyl group or a C3-C6 cycloalkyl group, and _R2 is selected from n-butylbenzene, isobutylbenzene, sec-butylbenzene, tert-butylbenzene, a linear or side-chain pentylbenzene, a linear or side-chain hexylphenyl, a linear or side-chain heptylphenyl, a linear or side-chain octylphenyl; and the substitution position of _R2 is selected from the ortho position, the meta position or the para position. 5. The 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that the compound is selected from the following group: A1: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A2: N-(2-(4'-pentyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A3: N-(2-(4'-hexyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A4: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)propionamide

A5: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)isopropylamide

A6: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)cyclopropylcarboxamide

A7: N-(2-(4'-butyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)cyclobutylcarboxamide

A8: N-(2-(4'-butyl-3-fluoro-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A9: N-(2-(4-cyclopropylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

A10: N-(2-(4-cyclopentylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

A11: N-(2-(4-cyclohexylbenzoyl)hydrazine-1-thiocarbonyl)acetamide

A12: N-(2-(4-(4-ethylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A13: N-(2-(4-(4-propylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A14: N-(2-(4-(4-butylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A15: N-(2-(4-(4-ethylcyclohexyl)benzoyl)hydrazine-1-thiocarbonyl)acetyl

A16: N-(2-(4'-cyclopropyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A17: N-(2-(4'-cyclobutyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A18: N-(2-(4'-cyclopentyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

A19: N-(2-(4'-cyclohexyl-[1,1'-biphenyl]-4-carbonyl)hydrazine-1-thiocarbonyl)acetamide

B1: N-(2-(2-(4-butyl)pyridine-5-carbonyl)hydrazine-1-thiocarbonyl)acetamide

B2: N-(2-(2-(4-butylphenyl)pyridine-5-carbonyl)hydrazine-1-thiocarbonyl)propionamide

C1: N-(2-(6-(4-butylphenyl)nicotinoyl)hydrazine-1-thiocarbonyl)acetyl

C2: N-(2-(6-(4-butylphenyl)nicotinoyl)hydrazine-1-thiocarbonyl)propionamide

6. The method for preparing the 1,4-dicarbonylthiourea compound according to any one of claims 1 to 5, characterized in that it comprises the following reaction steps: 1) Mix a cycloalkyl boronic acid reagent/C4-C6 alkyl benzene boronic acid reagent/cycloalkyl benzene boronic acid reagent with 1-2 equivalents of ortho-, meta- or para-halogenated benzoic acid methyl ester/picolinate/pyrimidine carboxylate, add a catalyst Pb(PPh ₃ ) ₄ and 2 equivalents of Na ₂ CO ₃ , fill with argon gas for protection, heat in toluene: ethanol: water for reaction for 6-12 hours, directly add an appropriate amount of water after the reaction is completed, extract the reaction mixture with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, separate and purify by column chromatography to obtain the following compound:

2) The intermediate obtained in step 1 is mixed with hydrazine hydrate in an alcohol solution, heated to reflux for reaction, and after the reaction is completed, the crude product is directly concentrated under reduced pressure, the solid is washed with water, and dried to obtain the following compound:

3) At room temperature, potassium isothiocyanate was dissolved in acetonitrile, and an equimolar amount of alkanoyl chloride/substituted alkanoyl chloride was added dropwise thereto, and stirred at room temperature for 30 min to 12 h to obtain the following compound:

The product does not require post-treatment and can be directly used in the next reaction after filtration; 4) Weigh an appropriate amount of hydrazide intermediate, dissolve it in an appropriate amount of acetonitrile, and add 1.5-2.0 times the equivalent of isothiocyanate acetonitrile solution to the reaction system at room temperature. The reaction can be carried out at room temperature or under heating conditions. TLC is used to monitor the progress of the reaction. After the reaction is completed, the crude product is concentrated under reduced pressure, washed with water, and recrystallized from an organic solvent to obtain a compound of formula I:

in: A can be selected from a benzene ring, a halogenated benzene, pyridine and pyrimidine; _R1 is selected from unsubstituted or halogenated C1-C6 alkyl, unsubstituted C3-C6 cycloalkyl; _R2 is selected from C4-C8 alkylbenzene, C3-C8 cycloalkylbenzene, C3-C8 cycloalkyl, C1-C8 alkyl substituted C3-C8 cycloalkyl, _R2 substitution position is selected from ortho, meta or para, The synthetic route is as follows:

The definitions of R ₁ and R ₂ are the same as those in any one of claims 1-5. 7. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient. 8. The pharmaceutical composition according to claims 1-5, characterized in that the pharmaceutical composition is selected from injection, tablet, pill, capsule, suspension or emulsion. 9. Use of the 1,4-dicarbonylthiourea compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of anti-infective drugs. 10. The use according to claim 9, characterized in that the anti-infection agent is selected from Gram-positive bacteria. 11. The use according to claim 10, characterized in that the Gram-positive bacteria include Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis or Enterococcus faecium. 12. The use according to claim 11, characterized in that the Staphylococcus aureus includes methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Staphylococcus aureus; the Staphylococcus epidermidis includes methicillin-sensitive Staphylococcus epidermidis and methicillin-resistant Staphylococcus epidermidis; the Enterococcus faecalis includes vancomycin-sensitive Enterococcus faecalis and vancomycin-resistant Enterococcus faecalis; the Enterococcus faecium includes vancomycin-sensitive Enterococcus faecium and vancomycin-resistant Enterococcus faecium.