Synthesis and application of SecA targeted inhibitor SWHT124

Abstract

The invention provides synthesis and application of a SecA targeted inhibitor SWHT124, which comprises the following steps: mixing 2, 4, 6-trichloropyrimidine, a compound shown as a formula I and potassium carbonate, adding an organic solvent I, uniformly stirring, reacting at room temperature for 5-6 hours, and purifying to obtain the inhibitor. The invention also provides an application of the inhibitor SWHT124 in preparation of antibacterial drugs. Compared with a common pyrimidine compound in the prior art, the pyrimidine compound disclosed by the invention is easier to generate nucleophilic substitution reaction, and the obtained inhibitor is high in purity, few in byproducts and obvious in antibacterial effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical materials, and in particular relates to the synthesis and application of a SecA targeted inhibitor SWHT124. Background Art

[0002] Staphylococcus aureus (S. aureus) infection has always been a hot issue, and with the increase in drug-resistant bacteria, it is urgent to find new and effective anti-S. aureus inhibitors. SecA is a unique and essential protein in bacteria. It is the only known energy conversion enzyme in the Sec transport system and is involved in the secretion of S. aureus-related virulence factors. Therefore, it is an ideal target for the development of new antibacterial agents.

[0003] SecA is an important member of the AAAATPase family. As the only energy conversion enzyme in the Sec protein transport pathway, it is a "power pump" that drives polypeptides through the channel through the hydrolysis cycle of ATP. SecA is the key core of the Sec transporter and is essential for the survival of a wide spectrum of bacteria. About 95% of the envelope proteins and secretory proteins synthesized by bacteria must pass through the inner membrane (IM) through the Sec transport system. SecA plays a dual role in protein transport: it participates in the recruitment and transport of substrates to the Sec channel; it also acts as an ATP-dependent nanomotor that enables polypeptides to pass through the IM. The Sec pathway is unique to some pathogenic Gram-positive bacteria, which have two SecA proteins: SecA1 (the SecA homolog of all bacteria) and SecA2. Electron microscopy observations show that SecA exists with the membrane in the form of soluble protein, corresponding to a soluble globular structure and a lipid-bearing ring structure. Some researchers have also proposed possible relationships between structural domains and functional domains in this regard.

[0004] There is no large-scale, substantial screening work for SecA inhibitors in the prior art, and no inhibitors for S. aureus SecA have been found, and there are no SecA-related drugs on the market. The anti-S. aureus drugs in the prior art mainly inhibit protein synthesis by binding to the 50S and 30S subunits of bacterial ribosomes and blocking the translocation of peptidyl t-RNA, which is different from their antibacterial action targets. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides the synthesis and application of a SecA targeted inhibitor SWHT124, which is more susceptible to nucleophilic substitution reaction than the pyrimidine compounds commonly used in the prior art. The obtained inhibitor has high purity, few by-products, and obvious antibacterial effect.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The first object of the present invention is to provide a synthesis and application of a SecA targeted inhibitor SWHT124, comprising the following steps: mixing 2,4,6-trichloropyrimidine, a compound of formula I and potassium carbonate, adding an organic solvent I and stirring evenly, reacting at room temperature for 5 to 6 hours, and obtaining the inhibitor after purification;

[0008] The structural formula of the compound of formula I is

[0009] The structural formula of the inhibitor is

[0010] Furthermore, the molar ratio of the 2,4,6-trichloropyrimidine, the compound of formula I and potassium carbonate is 0.8-1.2:0.8-1.2:0.8-1.2.

[0011] Furthermore, the molar ratio of the 2,4,6-trichloropyrimidine, the compound of formula I and potassium carbonate is 1:1:1.

[0012] Furthermore, the organic solvent I is acetone.

[0013] Furthermore, the purification process is as follows: after the reaction is completed, the solution is extracted with ethyl acetate and saturated brine to obtain an organic layer; the extracted product is dried with anhydrous sodium sulfate to obtain a crude product; and then a mixed solution of dichloromethane and methanol is added to separate and obtain the inhibitor.

[0014] Furthermore, the mass ratio of dichloromethane to methanol is 15-20:1.

[0015] Furthermore, the preparation method of the compound of formula I comprises the following steps: mixing 3,5-bis(trichloromethyl)benzoyl chloride and thiosemicarbazide under ice conditions, slowly adding an organic solvent II, and stirring overnight under nitrogen protection; then slowly adding saturated sodium carbonate to the mixture to terminate the reaction to obtain a compound of formula III; adding a sodium hydroxide solution to the compound of formula III, heating and refluxing, and then adding hydrochloric acid to adjust the pH value to neutral to obtain a compound of formula I;

[0016] The structural formula of the compound of formula III is

[0017] Furthermore, the molar ratio of the 3,5-bis(trichloromethyl)benzoyl chloride to the thiosemicarbazide is 0.8-1.2:0.8-1.2.

[0018] Furthermore, the molar ratio of the 3,5-bis(trichloromethyl)benzoyl chloride to the thiosemicarbazide is 1:1.

[0019] Furthermore, the organic solvent II is tetrahydrofuran.

[0020] Furthermore, the synthesis and application of a SecA targeted inhibitor SWHT124 comprises the following steps:

[0021] 1) Mixing 3,5-bis(trichloromethyl)benzoyl chloride and thiosemicarbazide under ice, slowly adding organic solvent II, and stirring overnight under nitrogen protection; then slowly adding saturated sodium carbonate to the mixture to terminate the reaction to obtain a compound of formula III;

[0022] 2) adding sodium hydroxide solution to the compound of formula III obtained in step 1) and heating to reflux, and then adding hydrochloric acid to adjust the pH value to neutral to obtain the compound of formula I;

[0023] 3) After mixing 2,4,6-trichloropyrimidine, the compound of formula I obtained in step 2) and potassium carbonate, adding organic solvent I and stirring evenly, reacting at room temperature for 5 to 6 hours, extracting the solution after the reaction with ethyl acetate and saturated brine, and taking the organic layer; drying the extracted product with anhydrous sodium sulfate to obtain a crude product; then adding a mixed solution of dichloromethane and methanol to separate and obtain the inhibitor;

[0024] The reaction path is as follows:

[0025]

[0026] Furthermore, the compound of formula III obtained in step 1) is first extracted with ethyl acetate and saturated brine, and the organic layer is taken; the extracted product is dried over anhydrous sodium sulfate and then used in the reaction in step 2).

[0027] The second object of the present invention is to provide the use of the inhibitor obtained by the above preparation method in the preparation of antibacterial drugs.

[0028] Furthermore, the inhibitor is used in combination with PMBN in the preparation of antibacterial drugs.

[0029] Compared with the prior art, the beneficial technical effects of the present invention are:

[0030] The preparation process of the invention is simple, the raw materials are easily available, and the operation is convenient. Using 2,4,6-trichloropyrimidine as the raw material makes it easier for nucleophilic substitution reaction to occur, which promotes the reaction, reduces the generation of by-products, and improves the purity and yield of the antibacterial agent. The inhibitor obtained by the invention is a targeted Seca inhibitor with broad-spectrum antibacterial activity, and has good antibacterial activity against multiple strains such as E. coli ATCC25922, B. subtilis 168, S. aureus ATCC 700699, E. coli NR698, E. coli 010004, P. aeruginosa 40, etc. When used in combination with PMBN, it not only increases the permeability of the inhibitor, but also does not affect the antibacterial effect of the inhibitor.

[0031] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the Sec pathway transport of the present invention.

[0033] Figure 2 The graph shows the antibacterial effect of the inhibitor of the present invention on four bacteria: E. coli ATCC 25922, B. subtilis 168, S. aureus ATCC 700699, and E. coli NR698.

[0034] Figure 3 This is the carbon spectrum of the inhibitor SWHT124 obtained in Example 1 of the present invention.

[0035] Figure 4 This is the hydrogen spectrum of the inhibitor SWHT124 obtained in Example 1 of the present invention.

[0036] Figure 5 This is the SM spectrum of the inhibitor SWHT124 obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection that the present invention is intended to protect.

[0038] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. PMBN is a polymyxin nonapeptide, which is a polymyxin derivative with the fatty acid tail and N-terminal diaminobutyryl group removed, and has no direct antibacterial activity against intestinal bacteria.

[0039] Example 1

[0040] A synthesis and application of SecA targeting inhibitor SWHT124, comprising the following steps:

[0041] 1) 1 mol of 3,5-bis(trichloromethyl)benzoyl chloride and 1 mol of thiosemicarbazide are mixed on ice, tetrahydrofuran is slowly added, and stirred overnight under nitrogen protection; on the second day, saturated sodium carbonate is slowly added to the mixture to terminate the reaction, and the compound of formula III is obtained by filtration; ethyl acetate and saturated saline are added to the compound of formula III to extract the organic layer, and then anhydrous sodium sulfate is added to the organic layer after extraction to dry it. The crude product after extraction is purified by rotary evaporation and silica gel column chromatography, and is eluted in a mixture of dichloromethane and methanol in a mass ratio of 30:1-15:1;

[0042] 2) adding 5% by mass of sodium hydroxide solution to the compound of formula III obtained in step 1) and heating and refluxing for 5 h, then adding hydrochloric acid to adjust the pH value to 6-7, filtering the precipitated white solid to obtain the compound of formula I;

[0043] 3) Mix 1 mol of 2,4,6-trichloropyrimidine, 1 mol of the compound of formula I obtained in step 2) and 1 mol of potassium carbonate, add acetone and stir evenly, react at room temperature for 5 to 6 hours, extract the solution after the reaction with ethyl acetate and saturated brine, and take the organic layer; dry the extracted product with anhydrous sodium sulfate to obtain a crude product; then add a mixture of dichloromethane and methanol in a mass ratio of 18:1 to separate and obtain the inhibitor SWHT124. Through detection, the total yield of the inhibitor SWHT124 was 59.7%. The carbon spectrum of the inhibitor SWHT124 obtained is as follows Figure 3 As shown, the hydrogen spectrum of the inhibitor SWHT124 is as follows Figure 4 As shown, the SM spectrum of the inhibitor SWHT124 is as follows Figure 5 shown.

[0044] The reaction path is as follows:

[0045]

[0046] Example 2

[0047] A synthesis and application of SecA targeting inhibitor SWHT124, comprising the following steps:

[0048] 1) 0.8 mol of 3,5-bis(trichloromethyl)benzoyl chloride and 1.2 mol of thiosemicarbazide were mixed on ice, tetrahydrofuran was slowly added, and the mixture was stirred overnight under nitrogen protection; on the second day, saturated sodium carbonate was slowly added to the mixture to terminate the reaction, and the compound of formula III was obtained by filtration; ethyl acetate and saturated saline were added to the compound of formula III to extract the organic layer, and then anhydrous sodium sulfate was added to the extracted organic layer to dry it for later use;

[0049] 2) adding 5% by mass of sodium hydroxide solution to the compound of formula III obtained in step 1) and heating and refluxing for 4 hours, then adding hydrochloric acid to adjust the pH value to 6-7, filtering the precipitated white solid to obtain the compound of formula I;

[0050] 3) 1 mol of 2,4,6-trichloropyrimidine, 1.2 mol of the compound of formula I obtained in step 2) and 0.8 mol of potassium carbonate were mixed, acetone was added and stirred evenly, and the mixture was reacted at room temperature for 5 to 6 hours. After the reaction was completed, the solution was extracted with ethyl acetate and saturated brine, and the organic layer was taken; the extracted product was dried with anhydrous sodium sulfate to obtain a crude product; then a mixed solution of dichloromethane and methanol in a mass ratio of 20:1 was added to separate the inhibitor SWHT124. Through detection, the total yield of the inhibitor SWHT124 was 56.6%.

[0051] Example 3

[0052] A synthesis and application of SecA targeting inhibitor SWHT124, comprising the following steps:

[0053] 1) 1.2 mol of 3,5-bis(trichloromethyl)benzoyl chloride and 0.8 mol of thiosemicarbazide were mixed on ice, tetrahydrofuran was slowly added, and the mixture was stirred overnight under nitrogen protection; on the second day, saturated sodium carbonate was slowly added to the mixture to terminate the reaction, and the compound of formula III was obtained by filtration; ethyl acetate and saturated saline were added to the compound of formula III to extract the organic layer, and then anhydrous sodium sulfate was added to the extracted organic layer to dry it for later use;

[0054] 2) adding 5% by mass of sodium hydroxide solution to the compound of formula III obtained in step 1) and heating and refluxing for 5 h, then adding hydrochloric acid to adjust the pH value to 6-7, filtering the precipitated white solid to obtain the compound of formula I;

[0055] 3) 1.2 mol of 2,4,6-trichloropyrimidine, 1 mol of the compound of formula I obtained in step 2) and 0.8 mol of potassium carbonate were mixed, acetone was added and stirred evenly, and the mixture was reacted at room temperature for 5 to 6 hours. After the reaction was completed, the solution was extracted with ethyl acetate and saturated brine, and the organic layer was taken; the extracted product was dried with anhydrous sodium sulfate to obtain a crude product; then a mixture of dichloromethane and methanol in a mass ratio of 15:1 was added to separate the inhibitor SWHT124. Through detection, the total yield of the inhibitor SWHT124 was 57.9%.

[0056] Experimental example: Antibacterial effect detection

[0057] Dilute the 0.5 McFarland concentration bacterial solution 20 times to make the bacterial content about 1*10 7 CFU / mL, take 10μL of diluted bacterial solution in a sterile 96-well culture plate; then add 5μL of different concentrations of drug solution and negative control sterile DMSO; finally add 185μL MH broth medium to form a 200μL system, mix and shake for 1min, culture at 37℃ for 24h, observe the turbidity of the liquid in the 96-well culture plate, and measure the OD600 value to determine whether the bacteria grow and its sensitivity to the inhibitor. See Appendix Figure 1 SWHT124 had inhibitory effects on four bacteria, namely E. coli ATCC 25922, B. subtilis 168, S. aureus ATCC 700699, and E. coli NR698, at the experimental concentrations. The growth inhibition effect on S. aureus ATCC 700699 and E. coli NR698 was particularly obvious.

[0058] The MTT method was used to determine the MIC of the inhibitor SWHT124 against several bacteria. The 0.5 McFarland concentration bacterial solution was diluted 1000 times, and 20 μL of the diluted bacterial solution was taken into a sterile 96-well culture plate. 80 μL of drug-containing culture medium with different concentrations of dilution was added. PMBN (polymyxin nonapeptide) was added to the drug-containing culture medium to form a 10 μL system. After culturing at 37°C for 8 hours, 10 μL of MTT solution (5 mg / ml) was added to each well and cultured for 2 hours. After 2 hours, 100 μL of DMSO was added to each well, and the 96-well plate was placed at 37°C for 10 minutes and OD570 was measured. The MIC of each bacterium was measured by comparing with the blank group without bacteria.

[0059] See Table 1, the MIC and inhibitor concentration of PMBN inhibitors against several bacteria. It can be seen that several common drug-resistant Gram-negative bacteria were selected and PMBN was added. PMBN itself has no direct antibacterial activity against intestinal bacteria, but PMBN can enhance the activity of Gram-negative bacteria that are usually unable to pass through Gram-negative bacteria. -PMBN at 25 μg / ml (3 μg / ml for Pseudomonas aeruginosa) can be used to enhance the permeability of the inhibitor without affecting G - growth, but made the inhibitors effective against Gram-negative bacteria. The combined use of PMBN and the inhibitors greatly reduced the MIC.

[0060] Table 1 Antibacterial effect of inhibitors on different bacteria

[0061]

[0062] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0063] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Synthesis and application of a SecA targeted inhibitor SWHT124, characterized in that: The following steps are involved: After mixing 2,4,6-trichloropyrimidine, the compound of formula I and potassium carbonate, adding organic solvent I and stirring evenly, reacting at room temperature for 5 to 6 hours, and obtaining the inhibitor after purification; The structural formula of the compound of formula I is The structural formula of the inhibitor is 2. The synthesis and application of a SecA targeted inhibitor SWHT124 as claimed in claim 1, characterized in that: The molar ratio of the 2,4,6-trichloropyrimidine, the compound of formula I and potassium carbonate is 0.8-1.2:0.8-1.2:0.8-1.

2.

3. The synthesis and application of a SecA targeted inhibitor SWHT124 as claimed in claim 2, characterized in that: The molar ratio of the 2,4,6-trichloropyrimidine, the compound of formula I and potassium carbonate is 1:1:

1.

4. The synthesis and application of a SecA targeted inhibitor SWHT124 as claimed in claim 1, characterized in that: The purification process is as follows: after the reaction is completed, the solution is extracted with ethyl acetate and saturated brine to obtain an organic layer; the extracted product is dried with anhydrous sodium sulfate to obtain a crude product; and then a mixed solution of dichloromethane and methanol is added to separate and obtain the inhibitor.

5. The synthesis and application of a SecA targeted inhibitor SWHT124 as claimed in claim 1, characterized in that: The preparation method of the compound of formula I comprises the following steps: mixing 3,5-bis(trichloromethyl)benzoyl chloride and thiosemicarbazide under ice conditions, slowly adding an organic solvent II, and stirring overnight under nitrogen protection; then slowly adding saturated sodium carbonate to the mixture to terminate the reaction to obtain a compound of formula III; adding a sodium hydroxide solution to the compound of formula III, heating and refluxing, and then adding hydrochloric acid to adjust the pH value to neutral to obtain a compound of formula I; The structural formula of the compound of formula III is 6. The synthesis and application of a SecA targeted inhibitor SWHT124 as claimed in claim 5, characterized in that: The molar ratio of the 3,5-bis(trichloromethyl)benzoyl chloride to the thiosemicarbazide is 0.8-1.2:0.8-1.

2.

7. The synthesis and use of a SecA targeted inhibitor SWHT124 according to any one of claims 1 to 6, characterized in that: The following steps are involved: 1) Mixing 3,5-bis(trichloromethyl)benzoyl chloride and thiosemicarbazide under ice, slowly adding organic solvent II, and stirring overnight under nitrogen protection; then slowly adding saturated sodium carbonate to the mixture to terminate the reaction to obtain a compound of formula III; 2) adding sodium hydroxide solution to the compound of formula III obtained in step 1) and heating to reflux, and then adding hydrochloric acid to adjust the pH value to neutral to obtain the compound of formula I; 3) After mixing 2,4,6-trichloropyrimidine, the compound of formula I obtained in step 2) and potassium carbonate, adding organic solvent I and stirring evenly, reacting at room temperature for 5 to 6 hours, extracting the solution after the reaction with ethyl acetate and saturated brine, and taking the organic layer; drying the extracted product with anhydrous sodium sulfate to obtain a crude product; then adding a mixed solution of dichloromethane and methanol to separate and obtain the inhibitor; The reaction path is as follows:

8. Use of the inhibitor obtained according to the preparation method according to any one of claims 1 to 7 in the preparation of antibacterial drugs.

9. The use according to claim 8, characterized in that: The inhibitor is used in combination with PMBN in the preparation of antibacterial drugs.

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