CN110862358B

CN110862358B - Aryl-substituted oxadiazinone compound and preparation method and application thereof

Info

Publication number: CN110862358B
Application number: CN201911144299.7A
Authority: CN
Inventors: 胡文浩; 蔡游燕; 袁燕秋; 王佳; 钱宇
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2021-09-10
Anticipated expiration: 2039-11-20
Also published as: CN110862358A

Abstract

The invention discloses an aryl-substituted oxadiazinone compound and a preparation method and application thereof. The structure of the compound is shown as a formula I; the compound disclosed by the invention is novel in structure, simple in preparation process, mild in reaction condition, few in steps, fast in reaction, low in cost, less in generated waste, simple and safe in operation, high in atom economy, high in selectivity and high in yield, and cheap and easily-obtained compounds are used as raw materials; the compound has a mother nucleus with good druggability, achieves obvious activity inhibition through covalent modification effect on target FabH, has high selectivity and strong inhibition effect on the FabH target, can well inhibit bacteria such as staphylococcus aureus and even drug-resistant bacteria such as methicillin-resistant staphylococcus aureus, and can be prepared into an antibacterial drug which is selective to bacteria and nontoxic to human body.

Description

Aryl-substituted oxadiazinone compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical chemistry and pharmacotherapeutics, in particular to an aryl-substituted oxadiazinone compound and a preparation method and application thereof.

Background

Antibiotics have been used for over 70 years in the treatment of various pathogenic microbial infections and have evolved into several broad families of drugs including beta-lactams, tetracyclines, aminoglycosides, macrolides and polypeptides, polyenes, and the like. As a basic drug, antibiotics are one of the largest categories in clinical medication in China, and are closely related to the national life and the stable development of the pharmaceutical industry in China. However, it is worth to be reminded that China is also a serious disaster area for abusing antibiotics, and the increasingly wide drug resistance of pathogenic bacteria caused by overuse of antibiotics becomes a serious medical problem concerned by China and even the world. Wherein, the detection rate of methicillin-resistant staphylococcus aureus (MRSA), one of the super bacteria, in domestic clinical treatment reaches 40-70%, and the detection rate has a tendency of increasing year by year, thereby greatly threatening the life health of people in China. Therefore, the next generation of antibiotics that can overcome the resistance of pathogenic bacteria is urgently needed in clinical treatment, and the development of antibacterial drugs aiming at new mechanisms and new targets has become a key way for overcoming the resistance of bacteria.

The problem of antibiotic resistance is forcing us to renew old antibiotics in order to seek new targets to cope with emerging threats. With the development of molecular biology technology, scientists have identified many targets of antibacterial drugs and have conducted more intensive research. In recent years, key enzymes involved in the bacterial fatty acid biosynthesis pathway have attracted a great deal of attention from scientists. It has been found that de novo synthesis of fatty acids is critical to the survival and pathogenicity of bacteria. Also, fatty acid synthases of higher animals and microorganisms vary greatly: in eukaryotic cells, fatty acid synthesis is performed by a type I synthetase (FASI) composed of multiple subunits, whereas most bacteria use a type II fatty acid synthetase system (FASI) in which each step is catalyzed by a separate, single-function enzyme. These findings illustrate that: key enzymes in the bacterial Fatty Acid Synthesis (FAS) pathway are highly potential novel antibacterial drug targets. Among them, β -ketoacyl-ACP synthase (FabH) controls the initial step of bacterial fatty acid biosynthesis, is ubiquitous in pathogens and does not have its homologous proteins in humans, and has become a hotspot in the target study of novel antibacterial drugs. Small molecule inhibitors that inhibit the activity of FabH enzymes associated with the FASII pathway are becoming increasingly attractive as a strategy to address drug-resistant bacteria such as methicillin-resistant staphylococcus aureus; is expected to become an antibacterial drug which is selective to bacteria and nontoxic to human bodies.

Disclosure of Invention

The invention aims to provide an aryl-substituted oxadiazinone compound. The compound has a novel structure, has high-efficiency bacteriostatic action, particularly shows good inhibitory action on strains with drug resistance, has a bacteriostatic mechanism that fatty acid synthesis is influenced by inhibiting the catalytic activity of FabH enzyme covalently bound with bacterial fatty acid biosynthesis; the compound has high selectivity to a target and has a mother nucleus with good druggability.

The invention also aims to provide a preparation method of the aryl-substituted oxadiazinone compound.

The invention further aims to provide application of the aryl-substituted oxadiazinone compound.

The above object of the present invention is achieved by the following scheme:

an aryl-substituted oxadiazinone compound is shown in a formula I:

wherein R is₁Is C_1～6Alkyl radical, C_1～6Cycloalkyl, benzyl, substituted benzyl, phenethyl or substituted phenethyl;

R₂is hydrogen, halogen, cyano, nitro, hydroxy, substituted or unsubstituted C_1～4Alkyl, substituted or unsubstituted C_1～4Alkoxy, substituted or unsubstituted C_1～4Cycloalkyl, substituted or unsubstituted C_1～4Cycloalkoxy, substituted or unsubstituted phenyl, substituted or unsubstituted phenoxy, substituted or unsubstituted benzyl, substituted or unsubstituted pentaheterocyclyl;

the substitution means that at least 1 site is substituted with the following substituent: halogen, cyano, nitro, amino, hydroxy, carboxyl, C_1～4Alkyl radical, C_1～4Haloalkyl, C_1～4Alkoxy radical, C_1～4Haloalkoxy, phenyl, benzyl, phenoxy or methylthio;

the heterocyclic radical is oxygen, sulfur orC of nitrogen atom_5～6Aryl or cycloalkyl groups of (a).

Preferably, said R is₁Is C_1～6Straight or branched alkyl, C_1～6Cycloalkyl, phenyl, benzyl or phenethyl.

More preferably, said R₁Is methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, methylcyclopropane, n-pentyl, isopentyl, tert-pentyl, cyclopentyl, methylcyclobutyl, n-hexyl, isohexyl, tert-hexyl, cyclohexyl, phenyl or benzyl.

More preferably, said R₁Is isopropyl, isoamyl, methylcyclopropane or methylcyclobutyl.

Preferably, said R is₂Is hydrogen, halogen, cyano, nitro, hydroxyl, substituted or unsubstituted phenyl, substituted or unsubstituted phenoxy, substituted or unsubstituted benzyl, substituted or unsubstituted pyridyl, substituted or unsubstituted 4-tetrahydropyridinyl;

the substitution means that at least 1 site is substituted with the following substituent: halogen, cyano, nitro, amino, hydroxyl, carboxyl, methyl, ethyl, propyl, butyl, methoxy, ethoxy, phenyl or methylthio.

More preferably, said R₂Is hydrogen, fluorine, chlorine, bromine, iodine, phenyl, benzyl, phenoxy, benzyloxy, nitrophenyl, methoxyphenyl, ethoxyphenyl, aminophenyl, hydroxyphenyl, pyridyl, 4-tetrahydropyridinyl or N-tert-butyl-4-tetrahydropyridinyl.

More preferably, said R₂Is hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, methoxy, ethoxy, 2-methoxyphenyl, 2-nitrophenyl, 2-aminophenyl, 3-pyridyl or N-tert-butyl-4-tetrahydropyridinyl.

More preferably, the aryl-substituted oxadiazinone compound is represented by one of the following structural formulas:

the invention also provides a preparation method of the aryl-substituted oxadiazinone compound, which comprises the following steps:

s1, performing substitution reaction on amide compounds shown in a formula 1 on amino to prepare compounds shown in a formula 2;

s2, mixing the compound of the formula 2 with triethylamine and methylsulfonyl chloride for reaction to prepare a compound of a formula 3;

s3, reacting the compound of the formula 3 with the compound of the formula 4 under an alkaline condition to prepare a compound of the formula 5;

and S4, mixing the compound shown in the formula 5 with triphosgene for reaction, then adding pyridine, and carrying out heating reflux reaction to prepare the target compound shown in the formula I.

Preferably, the reaction temperature in the step S2 is-30 to 50 ℃; the reaction temperature in the step S3 is-50 ℃;

in the step S4, the reaction temperature of the compound shown in the formula 5 and triphosgene is-30-50 ℃; the temperature of the heating reflux condition in the step S4 is 40-130 ℃.

More preferably, the reaction temperature in the step S2 is-30 to 0 ℃; the reaction temperature in the step S3 is-50-0 ℃;

in the step S4, the reaction temperature of the compound shown in the formula 5 and triphosgene is-10-0 ℃; the temperature of the heating reflux condition in the step S4 is 60-80 ℃.

More preferably, the reaction temperature in the step S2 is-10 to 0 ℃; the reaction temperature in step S3 is-30 to-10 ℃.

Preferably, the specific process of step S1 is: the amide compound shown in the formula 1 is mixed with a formaldehyde aqueous solution under an alkaline condition, and the mixture is heated to 40-100 ℃ for reaction, so that the compound shown in the formula 2 can be prepared.

More preferably, the temperature heated in step S1 is 60 ℃.

Preferably, the solvent for the reaction of step S1 is ethanol; the alkaline condition is the addition of potassium carbonate.

Preferably, the post-processing procedure in step S1 is: after the reaction is finished, concentrating to remove the solvent, extracting with dichloromethane and water, combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, spin-drying the solvent, and separating by column chromatography to obtain the compound shown in the formula 2.

More preferably, the mobile phase of the column chromatography separation process is dichloromethane and methanol in a volume ratio of 20: 1.

Preferably, the post-processing procedure of step S2 is: after the reaction is finished, removing the solvent, and performing column chromatography separation to obtain a compound shown in the formula 3; more preferably, the mobile phase of the column chromatography separation process is dichloromethane and methanol in a volume ratio of 10: 1.

Preferably, the solvent for the reaction described in step S2 is anhydrous THF.

Preferably, the solvent for the reaction of step S3 is dichloromethane; the alkaline condition is the addition of potassium carbonate.

Preferably, the post-processing procedure of step S3 is: after the reaction is finished, removing the solvent, extracting with dichloromethane and water, combining organic phases, washing with saturated saline water, drying with anhydrous sodium sulfate, and then separating by column chromatography to obtain a compound of formula 4; more preferably, the mobile phase of the column chromatography separation process is dichloromethane and methanol in a volume ratio of 30: 1.

Preferably, the solvent for the reaction described in step S4 is anhydrous THF.

Preferably, the post-processing procedure of step S4 is: after the reaction is finished, cooling to room temperature, then filtering, removing the solvent, and separating by column chromatography to obtain the compound of formula 4.

The application of the aryl-substituted oxadiazinone compound, the isomer thereof or the pharmaceutically acceptable salt thereof in preparing the antibacterial drugs is also within the protection scope of the invention.

Preferably, the bacteriostatic drug is a drug for inhibiting staphylococcus aureus, escherichia coli or methicillin-resistant staphylococcus aureus infection.

The invention also protects the application of the aryl-substituted oxadiazinone compound, the isomer thereof or the pharmaceutically acceptable salt thereof in preparing the bacterial FabH enzyme inhibitor.

Compared with the prior art, the invention has the following beneficial effects:

the compound has a novel structure, has a mother nucleus with good druggability, can be covalently combined with FabH enzyme biosynthesized by bacterial fatty acid, has strong inhibition effect and good selectivity, can well inhibit bacteria such as staphylococcus aureus, even has good inhibition effect on drug-resistant bacteria such as methicillin-resistant staphylococcus aureus, and can be prepared into an antibacterial agent which has selectivity on bacteria and is nontoxic to human bodies.

In addition, the preparation process of the compound is simple, the compound which is cheap and easy to obtain is used as a raw material, the reaction condition is mild, the steps are few, the reaction is fast, the cost is low, the generated waste is few, the operation is simple and safe, the atom economy is high, the selectivity is high, the yield is high, and the large-scale production in factories is facilitated.

Drawings

FIG. 1 is a hydrogen spectrum of the compound of example 1.

FIG. 2 is a carbon spectrum of the compound of example 1.

FIG. 3 is a hydrogen spectrum of the compound of example 9.

FIG. 4 is a carbon spectrum of the compound of example 9.

FIG. 5 is a hydrogen spectrum of the compound of example 10.

FIG. 6 is a carbon spectrum of the compound of example 10.

FIG. 7 is a hydrogen spectrum of the compound of example 14.

FIG. 8 is a carbon spectrum of the compound of example 14.

FIG. 9 is a hydrogen spectrum of the compound of example 15.

FIG. 10 is a carbon spectrum of the compound of example 15.

FIG. 11 is the IC of Compound Oxa1 for inhibition of FabH₅₀Graph is shown.

FIG. 12 IC of Compound Oxa4 for inhibition of FabH₅₀Graph is shown.

FIG. 13 is MALDI-TOF mass spectrometry analysis of the co-incubation of compound Oxa1 with FabH.

FIG. 14 is a LC-MS characterization of protein degradation peptide fragments incubated with compound Oxa1 and FabH.

FIG. 15 is a MALDI-TOF mass spectrometry analysis of co-incubation of compound Oxa1 with the FabH C112A mutant.

Detailed Description

The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

The structure of 6- (4-chlorophenyl) -3- (cyclopropylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa1) is shown below:

the preparation process comprises the following steps:

step 1: preparation of 4-chloro-N- (hydroxymethyl) benzamide

P-chlorobenzamide (1.00g, 6.42mmol, 1.00eq) was dissolved in an aqueous ethanol solution (ethanol: water ═ 20 mL: 10mL, V/V), and 37% aqueous formaldehyde solution (0.7mL, 9.63mmol, 1.50eq) and potassium carbonate (0.89g, 6.42mmol, 1.00eq) were added, followed by stirring at 60 ℃ overnight. After stirring with heating stopped, ethanol was concentrated and dried by spinning, and extracted three times with dichloromethane and water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was spun and column chromatographed (dichloromethane: methanol ═ 20:1, V/V) to give 1.12g of a white solid with a yield of 95%.

Step 2: preparation of methyl (4-chlorobenzoylamino) methanesulfonate

4-chloro-N- (hydroxymethyl) benzamide (1.12g, 6.10mmol, 1.00eq) was dissolved in anhydrous THF (10mL) and triethylamine (1.70mL, 12.2mmol, 2.00eq) and methanesulfonyl chloride (0.7mL, 9.15mmol, 1.50eq) were added slowly under ice bath. The reaction was carried out for 30 minutes, and column chromatography (dichloromethane: methanol 10:1, V/V) was carried out after spin-drying the solvent to obtain 1.20g of a viscous liquid with a yield of 85%.

And step 3: 4-chloro-N- (((cyclopropylmethyl) amino) methyl) benzamide

Cyclopropylmethylamine (0.65g, 9.18mmol, 2.00eq) was dissolved in dichloromethane, potassium carbonate 0.95g, 6.88mmol, 1.50eq was added, methyl (4-chlorobenzoylamino) methanesulfonate (1.20g, 4.59mmol, 1.00eq) was added slowly with stirring at-30 ℃ and stirring was continued for 30 min. After the solvent was dried by spinning, extraction was performed three times with dichloromethane and water, and organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying and column chromatography (dichloromethane: methanol ═ 30:1, V/V) to obtain 0.22g of a white solid in 80% yield.

And 4, step 4: preparation of 6- (4-chlorophenyl) -3- (cyclopropylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one

4-chloro-N- (((cyclopropylmethyl) amino) methyl) benzamide (0.22g, 0.93mmol, 1.00eq) was dissolved in THF (5mL), triphosgene (0.11g, 0.37mmol, 0.40eq) and THF (5mL) were dissolved in an ice bath, slowly dropped into the reaction solution, and stirred at room temperature. After 2 hours, pyridine (0.15mL, 1.86mmol, 2.00eq) was added dropwise to the reaction solution in an ice bath, refluxed at 70 ℃ for 3 hours, cooled to room temperature, and filtered. The solvent was spin-dried and column chromatography was carried out to give 0.18g of a white solid product in 84% yield.

¹H NMR(400MHz,CDCl₃)δ7.96(d,J＝7.7Hz,2H),7.41(d,J＝7.7Hz,2H),5.05(s,2H),3.32(d,J＝7.1Hz,2H),1.20–0.93(m,1H),0.61(d,J＝8.0Hz,2H),0.31(d,J＝4.8Hz,2H).¹³C NMR(126MHz,CDCl₃)δ152.57,147.85,138.33,128.99,128.76,128.31,62.78,50.74,8.58,3.59.HRMS(ESI)m/z calculated for C₁₃H₁₃N₂O₂Cl[M+Na]⁺,287.0558；found 287.0568.

Example 2

The structure of 3-benzyl-6- (4-chlorophenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa2) is shown below:

the preparation process comprises the following steps: the cyclopropylmethylamine was changed to benzylamine and the remaining required starting materials, reagents and preparation were the same as in example 1. A white solid was obtained in 88% yield.

¹H NMR(500MHz,CDCl₃)δ7.94(d,J＝7.6Hz,2H),7.40(d,J＝7.7Hz,2H),7.38–7.31(m,5H),4.86(s,2H),4.62(s,2H).¹³C NMR(126MHz,CDCl₃)δ152.39,148.14,138.43,134.72,129.04,129.00,128.79,128.33,128.22,62.32,49.63.HRMS(ESI)m/z calculated for C₁₆H₁₃N₂O₂Cl[M+Na]⁺,323.0558；found 323.0547.

Example 3

The structure of 6- (4-fluorophenyl) -3- (cyclopropylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa3) is shown below:

the preparation process comprises the following steps: p-chlorobenzamide was replaced with p-fluorobenzamide, and the remaining required starting materials, reagents and preparation were the same as those in example 1. A white solid was obtained in 93% yield.

¹H NMR(500MHz,CDCl₃)δ8.13–7.93(m,2H),7.11(t,J＝8.2Hz,2H),5.05(s,2H),3.32(d,J＝7.1Hz,2H),1.14–0.97(m,1H),0.61(d,J＝7.4Hz,2H),0.31(d,J＝4.7Hz,2H).¹³C NMR(126MHz,CDCl₃)δ166.17,164.16,150.26,130.01,126.00,115.62,62.75,50.75,8.61,3.60.HRMS(ESI)m/z calculated for C₁₃H₁₃N₂O₂F[M+Na]⁺,271.0853；found 271.0860.

Example 4

The structure of 3-butyl-6- (4-chlorophenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa4) is shown below:

the preparation process comprises the following steps: the cyclopropylmethylamine was replaced with n-butylamine, and the remaining required starting materials, reagents and preparation were the same as in example 1. A white solid was obtained in 90% yield.

¹H NMR(500MHz,CDCl₃)δ7.95(d,J＝7.5Hz,2H),7.41(d,J＝7.5Hz,2H),4.94(s,2H),3.40(t,J＝7.4Hz,2H),1.69–1.59(m,2H),1.39(dd,J＝14.9,7.3Hz,2H),0.97(t,J＝7.3Hz,3H).¹³C NMR(126MHz,CDCl₃)δ151.79,146.73,137.36,128.00,127.76,127.26,61.90,45.09,27.77,18.92,12.71.HRMS(ESI)m/z calculated for C₁₃H₁₅N₂O₂Cl[M+Na]⁺,289.0714；found 289.0697.

Example 5

The structure of 6- (4-chlorophenyl) -3-isopropyl-3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa5) is shown below:

the preparation process comprises the following steps: the cyclopropylmethylamine was changed to isopropylamine and the remaining required starting materials, reagents and preparation were the same as in example 1. A white solid was obtained in 91% yield.

¹H NMR(500MHz,CDCl₃)δ7.95(d,J＝8.3Hz,2H),7.40(d,J＝8.4Hz,2H),4.89(s,2H),4.57–4.31(m,1H),1.29(d,J＝6.9Hz,6H).¹³C NMR(126MHz,CDCl₃)δ152.98,147.44,138.35,129.01,128.77,128.28,57.41,46.86,19.01.HRMS(ESI)m/z calculated for C₁₂H₁₃N₂O₂Cl[M+Na]⁺,275.0558；found 275.0583.

Example 6

The structure of 6- (4-chlorophenyl) -3- (cyclobutylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa6) is shown below:

the preparation process comprises the following steps: the cyclopropylmethylamine was changed to cyclobutylmethylamine, and the remaining required starting materials, reagents and preparation were the same as in example 1. A white solid was obtained in 89% yield.

¹H NMR(400MHz,CDCl₃)δ7.94(d,J＝8.7Hz,2H),7.40(d,J＝8.7Hz,2H),4.91(s,2H),3.45(d,J＝7.5Hz,2H),2.78–2.61(m,1H),2.21–2.04(m,2H),1.99–1.88(m,2H),1.86–1.73(m,2H).¹³C NMR(126MHz,CDCl₃)δ152.76,147.98,138.35,129.01,128.76,128.28,63.18,51.48,33.43,26.45,18.45.HRMS(ESI)m/z calculated for C₁₄H₁₅N₂O₂Cl[M+Na]⁺,301.0714；found 301.0721.

Example 7

The structure of 6- (4-bromophenyl) -3- (cyclopropylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa7) is shown below:

the preparation process comprises the following steps: the p-chlorobenzamide was replaced with p-bromobenzamide, and the remaining required starting materials, reagents and preparation were the same as in example 1. A white solid was obtained in 87% yield.

¹H NMR(400MHz,CDCl₃)δ8.29–7.67(m,2H),7.67–7.39(m,2H),5.04(s,2H),3.32(d,J＝7.2Hz,2H),1.08(m,1H),0.75–0.49(m,1H),0.31(q,J＝4.9Hz,1H).¹³C NMR(101MHz,CDCl₃)δ152.69,147.83,131.79,129.17,128.78,126.90,62.80,50.76,8.58,3.60.HRMS(ESI)m/z calculated for C₁₃H₁₃N₂O₂Br[M+Na]⁺,331.0053；found331.0057.

Example 8

The structure of 6- (4-chlorophenyl) -3-isopentyl-3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa8) is shown below:

the preparation process comprises the following steps: the cyclopropylmethylamine was changed to isoamylamine and the remaining required starting materials, reagents and preparation were the same as in example 1. A white solid was obtained in 89% yield.

¹H NMR(400MHz,Acetone)δ8.09–7.88(m,2H),7.65–7.33(m,2H),5.04(s,2H),3.63–3.29(m,2H),1.65(tt,J＝15.2,7.7Hz,1H),1.56(dd,J＝14.8,7.2Hz,2H),0.95(d,J＝6.5Hz,6H).¹³C NMR(101MHz,CDCl₃)δ152.81,147.66,138.37,129.02,128.76,128.30,62.79,44.74,35.40,25.87,22.41.HRMS(ESI)m/z calculated for C₁₄H₁₇N₂O₂Cl[M+Na]⁺,303.0871；found 303.0870.

Example 9

The structure of 3- (cyclopropylmethyl) -6- (4- (N-BOC-1,2,5, 6-tetrahydropyridine) phenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa9) is shown below:

the preparation process comprises the following steps: mixing 6- (4-bromophenyl) -3- (cyclopropylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (100mg, 0.32mmol, 1.00eq), N-BOC-1,2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester (117mg, 0.38mmol, 1.20eq), [1,1' -bis (diphenylphosphine)) Ferrocene]Palladium dichloride complex (26mg, 0.03mmol, 0.10eq), sodium carbonate (102mg, 0.96mmol, 3.00eq) was placed in a dry flask. The flask was evacuated and charged with N₂And (5) ventilating for three times. Anhydrous 1, 4-dioxane (3mL) was added. Heated to 110 ℃ and stirred under reflux for 18 hours. After cooling to room temperature, the reaction was diluted with EtOAc and passed through SiO₂And (5) filtering. After the filtrate was spin-dried, extracted three times with EtOAc and water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was spin-dried and column chromatographed to give 105mg of a white solid in 80% yield.

¹H NMR(500MHz,CDCl₃)δ7.99(d,J＝8.5Hz,2H),7.45(d,J＝8.4Hz,2H),6.18(s,1H),5.07(s,2H),4.12(s,2H),3.66(t,J＝5.4Hz,2H),3.33(d,J＝7.1Hz,2H),2.56(s,2H),1.51(s,9H),1.23–0.97(m,1H),0.62(q,J＝5.4Hz,2H),0.44–0.18(q,2H).¹³C NMR(101MHz,CDCl₃)δ154.78,153.25,148.16,144.10,128.47,127.80,124.83,79.78,62.80,50.72,28.48,27.20,8.61,3.57.HRMS(ESI)m/z calculated for C₂₃H₂₉N₃O₄Cl[M+Na]⁺,434.2050；found 434.2059.

Example 10

The structure of 3- (cyclopropylmethyl) -6- (4- (pyridin-3-yl) phenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa10) is shown below:

step 1: preparation of 3- (cyclopropylmethyl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one

The preparation process comprises the following steps: mixing 6- (4-bromophenyl) -3- (cyclopropylmethyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (1.40g, 4.50mmol, 1.00eq), pinacol diboron (1.70g, 6.70mmol, 1.50eq), [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride complex (329mg, 0.45mmol, 0.10eq), potassium carbonate (1.3g, 13.50mmol, 3.00eq) were placed in a dry flask. The flask was evacuated and charged with N₂And (5) ventilating for three times. Anhydrous DMF (18mL) was added. Heating to 80 deg.C and stirring overnight. After cooling to room temperature, the reaction was diluted with EtOAc and passed through SiO₂And (5) filtering. After the filtrate was spin-dried, extracted three times with EtOAc and water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was spin-dried and column chromatographed to give 1.4g of a white solid in 87% yield.

Step 2: preparation of 3- (cyclopropylmethyl) -6- (4- (pyridin-3-yl) phenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one

The preparation process comprises the following steps: 3- (cyclopropylmethyl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (50mg, 0.14mmol, 1.00eq), 3-iodopyridine (43mg, 0.21mmol, 1.50eq), [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride complex (10mg, 0.014mmol, 0.10eq), potassium carbonate (41mg, 0.42mmol, 3.00eq) was placed in a dry flask. The flask was evacuated and charged with N₂And (5) ventilating for three times. Anhydrous DMF (1mL) was added. Heat to 80 ℃ and stir overnight. After cooling to room temperature, the reaction was diluted with EtOAc and passed through SiO₂And (5) filtering. After the filtrate was spin-dried, extracted three times with EtOAc and water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was spin-dried and column chromatographed to give 39mg of a white solid in 93% yield.

¹H NMR(500MHz,CDCl₃)δ8.89(s,1H),8.64(d,J＝4.6Hz,1H),8.13(d,J＝7.7Hz,2H),7.92(d,J＝7.3Hz,1H),7.66(d,J＝7.6Hz,2H),7.42–7.38(m,1H),5.09(s,2H),3.34(d,J＝7.1Hz,2H),1.16–1.03(m,1H),0.62(d,J＝7.6Hz,2H),0.33(d,J＝4.7Hz,2H).¹³C NMR(126MHz,CDCl₃)δ153.04,149.16,148.29,148.04,141.30,135.55,134.45,129.54,128.42,127.13,123.66,62.87,50.77,8.62,3.61.HRMS(ESI)m/z calculated for C₁₈H₁₇N₃O₂[M+H]⁺,308.1394,found 308.1395.

Example 11

The structure of 3- (cyclopropylmethyl) -6- (2 '-nitro- [1,1' -biphenyl ] -4-yl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa11) is shown below:

the preparation process comprises the following steps: the 3-iodopyridine was replaced with 2-iodonitrobenzene, and the remaining required raw materials, reagents and preparation methods were the same as in step 2 of example 10. A yellow solid was obtained in 85% yield.

¹H NMR(500MHz,CDCl₃)δ8.08(d,J＝7.9Hz,2H),7.92(d,J＝8.0Hz,1H),7.65(t,J＝7.4Hz,1H),7.53(t,J＝7.7Hz,1H),7.45(d,J＝7.5Hz,1H),7.39(d,J＝7.9Hz,2H),5.08(s,2H),3.33(d,J＝7.0Hz,2H),1.15–1.03(m,1H),0.61(d,J＝7.3Hz,2H),0.32(d,J＝4.3Hz,2H).¹³C NMR(126MHz,CDCl₃)δ152.98,148.96,148.04,141.25,135.58,132.58,131.83,129.67,128.79,128.08,127.99,124.38,62.85,50.75,8.62,3.60.HRMS(ESI)m/z calculated for C₁₉H₁₇N₃O₄[M+Na]⁺,374.1111,found 374.1105.

Example 12

The structure of 3- (cyclopropylmethyl) -6- (2 '-methoxy- [1,1' -biphenyl ] -4-yl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa12) is shown below:

the preparation process comprises the following steps: the 3-iodopyridine was replaced with 2-iodoanisole, and the remaining required raw materials, reagents and preparation methods were the same as in step 2 of example 10. A white solid was obtained in 92% yield.

¹H NMR(500MHz,CDCl₃)δ8.05(d,J＝7.8Hz,2H),7.61(d,J＝7.8Hz,2H),7.34(d,J＝7.9Hz,2H),7.04(t,J＝7.4Hz,1H),7.00(d,J＝8.1Hz,1H),5.07(s,2H),3.82(s,3H),3.33(d,J＝7.0Hz,2H),1.17–1.02(m,1H),0.61(d,J＝7.4Hz,2H),0.32(d,J＝4.4Hz,2H).¹³C NMR(126MHz,CDCl₃)δ156.49,153.49,148.29,142.46,130.76,129.61,129.32,128.17,127.34,120.95,111.36,62.84,55.59,50.74,8.65,3.60.HRMS(ESI)m/z calculated for C₂₀H₂₀N₂O₃[M+Na]⁺,359.1366,found 359.1365.

Example 13

The structure of 3- (cyclopropylmethyl) -6- (2 '-amino- [1,1' -biphenyl ] -4-yl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa13) is shown below:

the preparation process comprises the following steps: the 3-iodopyridine was replaced with 2-iodoaniline, and the remaining required raw materials, reagents and preparation methods were the same as in step 2 of example 10.

¹H NMR(500MHz,CDCl₃)δ8.09(d,J＝7.6Hz,2H),7.55(d,J＝7.6Hz,2H),7.18(t,J＝7.6Hz,1H),7.14(d,J＝7.5Hz,1H),6.84(t,J＝7.4Hz,1H),6.78(d,J＝8.0Hz,1H),5.08(s,2H),3.77(s,2H),3.34(d,J＝7.0Hz,2H),1.16–1.03(m,1H),0.62(d,J＝7.4Hz,2H),0.33(d,J＝4.5Hz,2H).¹³C NMR(126MHz,CDCl₃)δ153.26,148.13,143.51,143.43,130.31,129.13,129.08,128.54,128.15,126.37,118.78,115.84,62.83,50.75,8.63,3.60.HRMS(ESI)m/z calculated for C₁₉H₁₉N₃O₂[M+Na]⁺344.1369,found344.1354.

Example 14

The structure of 3- (cyclopropylmethyl) -6- (4-hydroxyphenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa14) is shown below:

the preparation process comprises the following steps: 3- (cyclopropylmethyl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (500mg, 1.37mmol, 1.00eq), sodium perborate tetrahydrate (630mg, 4.09mmol, 3.00eq) were dissolved in THF (5.0mL)/H₂O (5.0mL), reacted at room temperature for 1 hour, and the reaction mixture was saturated with NH₄The Cl solution was diluted, extracted twice with EtOAc and the combined organic phases were washed with saturated NaHCO₃Washing the solution with saturated saline, drying with anhydrous sodium sulfate, spin-drying the solvent, and performing column chromatography to obtain 286mg of white solidThe rate was 85%.

¹H NMR(500MHz,CDCl₃)δ7.87(d,J＝7.8Hz,2H),7.09(s,1H),6.89(d,J＝7.9Hz,2H),5.03(s,2H),3.32(d,J＝6.6Hz,2H),1.07(s,1H),0.60(d,J＝6.7Hz,2H),0.31(d,J＝3.7Hz,2H).¹³C NMR(126MHz,CDCl₃)δ159.85,153.30,148.87,129.73,121.55,115.56,62.62,50.79,8.62,3.61.HRMS(ESI)m/z calculated for C₁₃H₁₄N₂O₃[M+Na]⁺269.0897,found 269.0909.

Example 15

The structure of 3- (cyclopropylmethyl) -6- (4-methoxyphenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa15) is shown below:

the preparation process comprises the following steps: 3- (cyclopropylmethyl) -6- (4-hydroxyphenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (30mg, 0.12mmol, 1.00eq), iodomethane (52mg, 0.36mmol, 3.00eq), potassium carbonate (66mg, 0.48mmol, 4.00eq), the flask was evacuated and charged with N₂And (5) ventilating for three times. Anhydrous DMF (1mL) was added. After stirring at room temperature for 0.5 h, the reaction was diluted with EtOAc and passed through SiO₂And (5) filtering. After the filtrate was spin-dried, extracted three times with EtOAc and water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was spin-dried and column chromatographed to give 25mg of a white solid in 80% yield.

¹H NMR(400MHz,CDCl₃)δ7.96(d,J＝9.0Hz,2H),6.92(d,J＝9.0Hz,2H),5.02(s,2H),3.85(s,3H),3.31(d,J＝7.1Hz,2H),1.20–0.89(m,1H),0.76–0.37(m,2H),0.38–0.23(m,2H).¹³C NMR(126MHz,CDCl₃)δ162.66,153.23,148.37,129.47,122.15,113.79,62.71,55.42,50.69,8.65,3.57.HRMS(ESI)m/z calculated for C₁₄H₁₆N₂O₃[M+Na]⁺,283.1053,found 283.1057.

Example 16

The structure of 3- (cyclopropylmethyl) -6- (4-ethoxyphenyl) -3, 4-dihydro-2H-1, 3, 5-oxadiazin-2-one (Oxa16) is shown below:

the preparation process comprises the following steps: methyl iodide was replaced with ethyl iodide, and the remaining required raw materials, reagents and preparation method were the same as those in example 15. A white solid was obtained in 94% yield.

¹H NMR(500MHz,CDCl₃)δ7.95(d,J＝7.7Hz,2H),6.91(d,J＝7.8Hz,2H),5.02(s,2H),4.08(q,J＝6.8Hz,2H),3.31(d,J＝7.0Hz,2H),1.43(t,J＝6.8Hz,3H),1.14–0.94(m,1H),0.59(d,J＝7.0Hz,2H),0.31(d,J＝3.5Hz,2H).¹³C NMR(126MHz,CDCl₃)δ162.08,153.28,148.39,129.45,121.94,114.23,63.66,62.71,50.68,14.70,8.66,3.57.HRMS(ESI)m/z calculated for C₁₅H₁₈N₂O₃[M+Na]⁺,297.1210,found 297.1201.

Example 17 bioactive fractions

Firstly, determining the inhibition effect of the compound on a target FabH.

1. Experimental methods

Compound inhibition was determined by an enzyme-to-substrate catalytic activity assay. The test enzyme FabH is expressed and purified from Escherichia coli; the substrate Malonyl-ACP is obtained by catalyzing holoACP and manonyl-CoA by FabD;

the experimental method is as follows:

1.1 the staphylococcus aureus FabH with His6 mark at the N-terminal is expressed and purified from escherichia coli by the following specific method:

FabH (SAOUHSC _00920, GenBank. TM. accession No. 3920807) was amplified from Staphylococcus aureus ATCC29213 using Phusion high-fidelity PCR and used to express N-terminal 6 XHis-tagged FabH. Obtaining staphylococcus aureus FabH DNA by a colony PCR method: selecting single clone from LB agar plate, adding 20 μ L0.2% SDS lysate, vortexing for 10s, boiling at 98 deg.C for 10min, centrifuging, collecting supernatant, and constructing NheI/HindIII (FabH) -pET28b (+) recombinant plasmid (forward primer: 5' -AAAAAAGCTA)GCATGAACGTGGGTATTAAAGGTT-3 ' reverse primer: 5'-AAAAAAAAGCTTCTATTTTCCCCATTTTATTGTCA-3'). The PCR procedure was: (1) pre-denaturation: at 95 ℃ for 3 min; (2) denaturation: 30s at 94 ℃; (3) annealing: 58.7 ℃ for 30 s; (4) primer extension: 72 ℃ for 2 min. Circulating the steps (2) to (4) for 30 times; (5) extension: 72 ℃ for 5 min. The PCR product is cut by NheI/HindIII enzyme and then is connected by T4DNA ligase to obtain a recombinant plasmid pET28b (+) -S.a FabH, the plasmid is introduced into DH5 alpha, and a positive monoclonal is selected for sequencing, extracted and purified. The pET28b (+) -S.a FabH recombinant plasmid was introduced into E.coli BL21(DE3), and the monoclonal was picked up and cultured overnight at 37 ℃ in 5mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and the overnight-cultured bacterial solution was poured into 1L of LB liquid medium containing 50. mu.g/mL of kanamycin and cultured to OD₆₀₀0.8, IPT G was added to a final concentration of 1.0mM, induced at 37 ℃ for 3h, centrifuged at 15000rpm for 30min, the supernatant was discarded, the pellet was resuspended in Buffer A (20mM Tris-HCl, pH8.0, 500mM NaCl), protease inhibitor, DnaseI was added, and then sonicated by cell sonicator for 15min (amplitude 50%, 2s on, 2s off). The cell lysate was centrifuged at 15000rpm at 4 ℃ for 30 min. A1 mL nickel column was pre-equilibrated with Binding Buffer (Buffer A, 10mM imidazole), and the supernatant was filtered through a 0.45 μm filter and loaded. Wash with 5mL Wash Buffer (Buffer A, 30mM imidazole, 1mM DTT) and finally elute protein with 3mL Elution Buffer (Buffer A, 200mM imidazole, 1mM DTT). Protein purity was analyzed by SDS-PAGE, and purified S.a FabH was concentrated and replaced with a buffer containing 20mM Tris-HCl, pH8.0, 100mM NaCl, 1mM TCEP, 15% (v/v) glycerol, snap frozen with liquid nitrogen, and stored at-80 ℃.

1.2 expression and purification of FabD: FabD (NC-000913.3, GenBank TM accession No. 945766) was amplified from BL21-pLySs using Phusion high-fidelity PCR for expression of N-terminal His 6-tagged FabD. 20uL BL21-pLySs with ddH₂O washing twice, adding 25mM NaOH and 0.2mM EDTA mixed solution, boiling for 1h at 98 ℃, cooling on an ice box, adding 70uL 40mM Tris-HCl, centrifuging, collecting supernatant, and storing at-20 ℃. A NdeI/BamHI (FabD) -pET28b (+) recombinant plasmid was constructed, and a forward primer 5'-AAAAAACATATGATGACGCAATTTGCATTTGTG-3' (Nde I) and a reverse primer 5'-AAAAAAGGATCCTTAAAGCTCGAGCGCCGC-3' (BamH I) were designed. PCR procedureComprises the following steps: (1) pre-denaturation: at 95 ℃ for 3 min; (2) denaturation: 30s at 94 ℃; (3) annealing: at 54.3 ℃ for 30 s; (4) primer extension: circulating the steps (2) to (4) for 30 times at 72 ℃ for 2 min; (5) extension: 72 ℃ for 5 min. After the PCR product is cut by NdeI/BamHI, T4DNA ligase is connected to obtain a recombinant plasmid pET28b (+) -FabD, the plasmid is introduced into DH5 alpha, and a positive monoclonal is selected for sequencing, extracted and purified.

The pET28b (+) -FabD recombinant plasmid was introduced into E.coli BL21-pLySs, and the single clones were picked up and cultured overnight at 37 ℃ in 5mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and the overnight-cultured bacterial solution was poured into 1L of LB liquid medium containing 50. mu.g/mL of kanamycin and cultured to OD₆₀₀0.8, IPT G was added to a final concentration of 1.0mM, induced at 37 ℃ for 4h, centrifuged at 15000rpm for 30min, the supernatant was discarded, the pellet was resuspended in Buffer A (50mM Tris-HCl, pH8.0, 500mM NaCl), protease inhibitor, DnaseI was added, and then sonicated by cell sonicator for 15min (amplitude 50%, 2s on, 2s off). The cell lysate was centrifuged at 15000rpm at 4 ℃ for 30 min. A1 mL nickel column was pre-equilibrated with Binding Buffer (Buffer A, 10mM imidazole), and the supernatant was filtered through a 0.45 μm filter and loaded. Wash with 5mL Wash Buffer (Buffer A, 30mM imidazole, 1mM DTT) and finally elute protein with 3mL Elution Buffer (Buffer A, 60mM imidazole, 1mM DTT). Protein purity was analyzed by SDS-PAGE, and the purified FabD was concentrated and replaced in a buffer containing 20mM HEPES, pH7.0, 100mM NaCl, 0.5mM TCEP, 50% (v/v) glycerol, snap frozen with liquid nitrogen, and stored at-80 ℃.

1.3 expression and purification of Holo-ACP: ACP (NC-000913.3, Gene ID:944805) and ACPs (NC-000913.3, Gene ID:947037) were amplified from BL21-pLySs using Phusion high-fidelity PCR for expression of N-terminal His 6-tagged holo-ACP. 20uL BL21-pLySs with ddH₂O washing twice, adding 25mM NaOH and 0.2mM EDTA mixed solution, boiling for 1h at 98 ℃, cooling on an ice box, adding 70uL 40mM Tris-HCl, centrifuging, collecting supernatant, and storing at-20 ℃. Constructing an ACP-ACPs-pETDuet-1 recombinant plasmid, and respectively designing an ACP forward primer 5 '-AAAAAAGGATCCATGAGCACTATCGAAGAACG(BamHI) reverse primer 5' -AAAAAAGAATTCTTACGCCTGGTGGCCGTT(EcoRI); ACPs Forward primer 5' -AAAAAACATATGGCAATATTAGGTTTAGGCA(NdeI) reverse primer 5' -AAAAAACTCGAGTTAACTTTCAATAA TTACCGTGG (XhoI). The PCR procedures were all as follows: (1) pre-denaturation: at 95 ℃ for 3 min; (2) denaturation: 30s at 94 ℃; (3) annealing: at 58.5 ℃ for 30 s; (4) primer extension: circulating the steps (2) to (4) for 30 times at 72 ℃ for 2 min; (5) extension: 72 ℃ for 5 min. And (3) sequentially carrying out corresponding enzyme digestion on the PCR product, then connecting the PCR product by using T4DNA ligase to obtain a recombinant plasmid ACP-ACPs-pETDuet-1, introducing the plasmid into DH5 alpha, selecting positive monoclonal for sequencing, extracting the plasmid and purifying. Introducing the ACP-ACPs-pETDuet-1 recombinant plasmid into Escherichia coli BL21-pLySs, selecting a monoclonal to be cultured in 5mL LB liquid culture medium containing 75 mu g/mL ampicillin at 37 ℃ overnight, pouring the overnight cultured bacterial liquid into 1L LB liquid culture medium containing 75 mu g/mL ampicillin to be cultured to OD₆₀₀0.5, IPT G was added to a final concentration of 0.5mM, induced at 37 ℃ for 4h, centrifuged at 15000rpm for 30min, the supernatant was discarded, the pellet was resuspended in BufferA (20mM Tris-HCl, pH8.0, 500mM NaCl), protease inhibitor, DnaseI was added, and then sonicated by a cell sonicator for 15min (amplitude 50%, 5s on, 5s off). The cell lysate was centrifuged at 15000rpm and 4 ℃ for 30 min. The supernatant was filtered through a 0.45 μm filter and applied to a 1mL nickel column to prepare Buffer A:20mM Tris-HCl, pH8.0, 500mM NaCl, 1mM DTT; buffer B: 20mM Tris-HCl, pH8.0, 500mM NaCl, 1mM DTT, 500mM imidazole. Setting the flow rate of a protein purifier at 0.5mL/min, washing with 10 times of column volume, 2% Buffer B and 98% Buffer A, performing gradient elution with 2% -22% Buffer B in 10 times of column volume, performing isocratic elution with 22% Buffer B in 5 times of column volume, collecting eluate, performing SDS-PAGE to determine protein purity, diluting the collected target protein with 20mM Tris-HCL (pH 8.0) for 20 times, and preparing Buffer A, namely 20mM HEPES, 1mM DTT and pH 7.0; buffer B: 1M KCl, 1mM DTT, 20mM HEPES, pH7.0, using 5mL ion exchange column, Hip Trap FF to separate holo-ACP-ACPs dimers, the purification steps were as follows: using MilliQ H in sequence₂Washing Hip Trap FF column with O, 100% Buffer B (1M KCl) and 2% Buffer B, loading, gradient eluting with 200mM KCl-600mM KCl and 15CV, isocratic eluting with 100% Buffer B (1M KCl) and 5CV, and sequentially eluting with 5-10CV, 100% Buffer A, 5-10CV and MilliQ H₂O, 5-10CV, 20% ethanol washing column. Purifying the obtained productThe holo-ACP was concentrated and replaced into a buffer containing 20mM HEPES (pH7.0), 100mM NaCl, the molecular weight of the concentrated holo-ACP was measured by MALDI-TOF-MS, and then the thiol content of the holo-ACP was measured to determine the holo-ACP concentration by the following method: an experimental group of 50uL holo-ACP +950uL 200uM DTNB and a blank group of 50uL (20mM HEPES +100mM NaCl) +950uL 200uM DTNB were set, respectively, and reacted at room temperature for 30min, and the absorbance A was measured at 412 nm. Determination of the SH concentration: -sh (um) ═ 73.53 (Asample-a blank) × dilution, after the holo-ACP concentration was determined, TCEP was added to a final concentration of 1mM, and the mixture was stored at-80 ℃ after dispensing.

1.4Malonyl-ACP is obtained by FabD catalysis of holoACP and manonyl-CoA, and the specific method is as follows:

synthesis and purification of Malonyl-ACP: 125uM holo-ACP, 2.0mM malonyl-CoA, 6uM FabD, 20mM HEPES (pH7.0), 100mM NaCl, 1mM TCEP were prepared, reacted at 0 ℃ for 16 to 20 hours, and subjected to molecular weight measurement by MALDI-TOF-MS. Malonyl-ACP was purified by molecular sieves, buffer was prepared: 100mM NaCl, 20mM HEPES, pH7.0, purity by SDS-PAGE determination after concentration, using BCA method to determine the malony-ACP concentration, adding glycerol to the final concentration of 15%, subpackaging, at-80 ℃ storage.

1.5FabH IC₅₀Measurement of

The reaction components were mixed together in a batch-wise manner and then equally distributed according to the number of analyses performed. All experiments were performed in 6 replicates. The amounts correspond to a single reaction. 20 μ M isobutryyl-CoA, 0.05 μ g FabH, different concentrations of Oxa1, 100mM phosphate buffer (pH7.0) were mixed well, added to a black 384 well plate for reaction for 5min, 8 μ M malonyl-ACP was added, the mixture was mixed well, 40 μ L CPM (dissolved in 100% DMSO) was aspirated to terminate the reaction, and fluorescence at 384nm, 470nm was measured.

2. Results of the experiment

As shown in FIGS. 11 and 12, the IC of the compound Oxa1 acting on FabH was found to be₅₀Value of 2.6. mu.M, IC of Compound Oxa4 acting on FabH₅₀The value is 1.16 mu M, which shows that the series of compounds have good inhibition effect on the FabH enzyme synthesized by the bacterial fatty acid.

II, determining the inhibition mechanism of the compound on FabH

1. Experimental methods

Peptide modifications were characterized by co-incubation and MALDI-TOF mass spectrometry of the compounds with FabH protein and peptide profiling by trypsin digestion.

The experimental method is as follows:

the 100. mu.M series of compounds was reacted with 50. mu.M FabH and incubated for 4 hours at room temperature. A stock solution of 0.25. mu.g/. mu.L trypsin was prepared using a10 mM acetic acid solution and stored at-80 ℃. 7.5. mu.g of protein, 50mM NH were prepared₄HCO₃MS grade purified water (added to 50. mu.L) was added to the mixture, and then 0.15. mu.g trypsin (1: 50 by mass) was added to the mixture and incubated overnight at 37 ℃ using 2-layer stage-tips (C)₁₈/C₁₈) The purification was carried out according to the following steps: (A) wetting the suction head: 100 μ L of 100% methanol was added and centrifuged 3 times at 1200g for 10min, then 100 μ L of 0.1% TFA in methanol (50% MeOH) was added and centrifuged 3 times at 4000g for 4 min. (B) Balancing: add 100. mu.L of 0.1% TFA and centrifuge at 6000g for 4 min, 3 times. (C) The samples were loaded and centrifuged 2 times at 2000g for 12 min. (D) And (3) washing to remove salt: add 100. mu.L of 0.1% TFA and centrifuge at 6000g for 4 min. (E) TFA was washed off: 100 μ L of 0.1% formic acid solution was added and centrifuged at 6000g for 4 min 2 times. (F) And (3) elution: 50 μ L of 50% MeOH in 0.1% formic acid was added and centrifuged at 2000g for 4 min 2 times. (G) Dried in speed-vac for 20 minutes and then lyophilized, and the dried solution was dissolved in 40. mu.L of 0.1% formic acid solution for LC-MS analysis.

2. Results of the experiment

MALDI-TOF mass spectrometry of co-incubated fabhs showed +268 adducts, suggesting that the compound molecules may have performed 1:1 covalent modifications to fabhs (see fig. 13). To determine the modification site, compound-treated FabH was subjected to tryptic digestion and subsequent LC-MS analysis. Molecular weight peptides, MS, corresponding to adducts of 1 series of compounds were identified²The fragment suggested that its modification site was cysteine 112 (FIG. 14). to further define the covalent modification site, we mutated cysteine 112 to alanine by site-directed mutagenesis(ii) an amino acid. Under the same conditions as wild-type FabH, FabH C112A mutant failed to form the series of compound adducts at the same modification site as Cys112 (fig. 15). Taken together, the compounds inhibit FabH by covalently modifying cysteine 112 thereof.

And thirdly, determining the influence of the bacterial level of the compound on the FabH target bacteria.

1. Experimental methods

The antibacterial activity of the compounds was determined by the method of their MIC test against sensitive staphylococcus aureus ATCC29213, methicillin-resistant staphylococcus aureus (MRSA) and escherichia coli (e.

The experimental method is as follows:

the minimum inhibitory concentration MIC of Oxa1-16 was determined by LB broth microdilution. Picking 3-5 colonies to be detected with similar morphology on LB agar plate cultured overnight at 37 deg.C with inoculating loop, inoculating in 5mL LB broth culture medium, culturing at 37 deg.C and 225rpm for 4h, measuring OD₆₀₀The original bacterial liquid was diluted several times to OD₆₀₀The value is about 0.1, the bacterial liquid concentration is 10⁸cfu/mL, and then 10⁸Diluting the cfu/mL bacterial solution by 100 times to obtain 10⁶cfu/mL of bacterial solution for later use. Adding 50uL of compounds with different concentrations and a reference compound into a 96-well plate, taking 50uL of diluted bacterial liquid, culturing at 37 ℃ for 16-20h, and reading the result.

2. Results of the experiment

TABLE 1 MIC values for Oxa series of compounds

TABLE 2 MIC values of Oxa4 and each antibiotic for clinically isolated methicillin-resistant Staphylococcus aureus

The test results are shown in tables 1 and 2, and the series of compounds have good antibacterial activity in the MIC (minimal inhibitory concentration) measurement results of staphylococcus aureus. Moreover, Oxa4 showed better inhibitory activity against the MIC values of clinically isolated methicillin-resistant Staphylococcus aureus, compared to methicillin, vancomycin and linezolid antibiotics. From the data measured above, the series of compounds, especially the compounds Oxa1, Oxa3, Oxa4, Oxa6 and Oxa7 in the series have significant staphylococcus aureus inhibiting activity, and the parent nucleus has the advantages of good drug forming property, proper physical properties and the like, so that the structure-activity relationship can be studied deeply and improved.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An aryl-substituted oxadiazinone compound is characterized in that the aryl-substituted oxadiazinone compound is shown in one of the following structural formulas:

。

2. the use of an aryl-substituted oxadiazinone compound or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a bacteriostatic medicament for inhibiting infection by staphylococcus aureus or escherichia coli.

3. The use of an aryl-substituted oxadiazinone compound or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a bacteriostatic agent, wherein the bacteriostatic agent is an agent that inhibits methicillin-resistant staphylococcus aureus infection.

4. Use of the aryl-substituted oxadiazinones of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a bacterial FabH enzyme inhibitor.