Synthesis of four quaternary ammonium salt derivatives and their application in treating Staphylococcus aureus infection

Abstract

The present invention discloses four quaternary ammonium salt derivatives, their synthesis methods, and applications. One quaternary ammonium salt derivative of the present invention has the chemical name (E)-N,N,N-trimethyl-1-(4-(phenyldiazenyl)phenyl)methylammonium bromide (Compound 1e). Compound 1e, synthesized using aniline as a starting material, exhibits excellent antibacterial activity against Staphylococcus aureus at 500 μg / mL.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, in particular to a synthesis method of four quaternary ammonium salt derivatives and applications thereof in resisting Staphylococcus aureus infection. Background Art

[0002] Cationic antimicrobials, as a type of antibacterial compound, lack specific targets in microorganisms. They generally interact with bacterial membranes through electrostatic forces, creating pores in the membranes and causing irreversible damage. Through this mode of action, cationic antimicrobials can completely destroy the membrane structure of microorganisms, thereby eliminating the possibility of microbial resistance to these antimicrobials at the source.

[0003] Common cationic antimicrobial agents generally include quaternary ammonium salts, quaternary phosphonium salts, halogenamines, etc. There are many explanations for the antibacterial mechanism of quaternary ammonium salts. One widely accepted theory is that it is through the N + It interacts with the negative charge on the surface of the bacterial cell membrane, absorbs part of the anionic bacterial cell membrane into its internal gaps, causing the microbial membrane to wrinkle and deform, and the intracellular substances to leak, thereby killing the bacteria; there are also reports that the N in the group + Can interact with intracellular Mg 2+ , Ca 2+ etc. to carry out ion exchange, causing electrolytic imbalance and death of bacteria. Among the existing antibacterial drug research strategies, the synthesis of cationic antibacterial agents has attracted much attention among researchers: Tsai et al. synthesized a cationic dipeptide lysine-cysteine ​​(KC) based on cysteine, which has inhibitory effects on both Gram-positive and Gram-negative bacteria. It combines the effects of dipeptide and bacterial cell wall and has excellent anti-resistance properties; in addition, many researchers have introduced cations into polymers. For example, Yan et al. quaternized brominated poly (butylene succinate), and the hydrophilicity of the resulting polyester was greatly improved. Cationic copolyesters showed excellent antibacterial properties against Staphylococcus aureus and Escherichia coli. However, new small molecule cationic antibacterial compounds have been rarely reported, and we hope to fill these gaps. Summary of the Invention

[0004] The purpose of the present invention is to provide a synthesis method of four quaternary ammonium salt derivatives and their application as anti-Staphylococcus aureus infection agents, so as to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for synthesizing a quaternary ammonium salt derivative (Compound 1d), comprising the following steps:

[0006] Step 1: 1.24 ml (10.74 mmol) of p-toluyl chloride was slowly added to a mixture of 0.98 ml (10.74 mmol) of aniline and 1.64 ml (11.81 mmol) of triethylamine in 50 ml of dichloromethane at 0°C, and the mixture was stirred at room temperature for 5 hours; the reaction mixture was extracted with DCM, the organic phases were combined and dried over Na2SO4, and the crude mixture was purified by silica gel column chromatography with PE / EtOAc 5:1 to 3:1 to obtain 4-methyl-N-phenylbenzamide (Compound 1b) as a white solid in an 89.5% yield;

[0007] Step 2: Under nitrogen atmosphere, 0.30 g (1.42 mmol) of compound 1b, 2.78 g (15.62 mmol) of NBS, and 0.47 g (2.84 mmol) of AIBN were dissolved in 30 ml of carbon tetrachloride. The reaction solution was heated to reflux for 24 h, and the solvent was removed by rotary evaporation. The mixture was extracted with DCM, and the organic phases were combined and dried over Na2SO4 to obtain a crude product of 4-(bromomethyl)-N-phenylbenzamide (compound 1c).

[0008] Step 3: 0.20 g (0.69 mmol) of compound 1c and 30% ethanol (in ethanol) were dissolved in 0.955 ml (3.63 mmol) of trimethylamine in 20 ml of ethanol, and the mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by neutral alumina column chromatography with DCM / MeOH (10:1 to 5:1) to obtain the product N,N,N-trimethyl-1-(4-(phenylcarbamoyl)phenyl)methylammonium bromide (compound 1d) as a pale yellow solid in a yield of 36.4%.

[0009] Preferably, the compound 1d is used for resisting Staphylococcus aureus infection.

[0010] A method for synthesizing a quaternary ammonium salt derivative (Compound 2d) specifically comprises the following steps:

[0011] Step 1: 1.24 ml (10.74 mmol) of p-toluyl chloride was slowly added to a mixture of 0.98 ml (10.74 mmol) of aniline and 1.64 ml (11.81 mmol) of triethylamine in 50 ml of dichloromethane at 0°C, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was extracted with DCM, the organic phases were combined and dried over Na2SO4, and the crude mixture was purified by silica gel column chromatography with PE / EtOAc 5:1 to 3:1 to obtain 4-methyl-N-phenylbenzamide (Compound 2b) as a white solid in a yield of 93.5%.

[0012] Step 2: Under nitrogen atmosphere, 0.30 g (1.42 mmol) of compound 1b, 2.78 g (15.62 mmol) of NBS, and 0.47 g (2.84 mmol) of AIBN were dissolved in 30 ml of carbon tetrachloride. The reaction solution was heated to reflux for 24 h, and the solvent was removed by rotary evaporation. The mixture was extracted with DCM, and the organic phases were combined and dried over Na2SO4 to obtain a crude product of 4-(bromomethyl)-N-phenylbenzamide (compound 2c).

[0013] Step 3: 0.20 g (0.69 mmol) of compound 1c and 30% ethanol (in ethanol) were dissolved in 0.955 ml (3.63 mmol) of trimethylamine in 20 ml of ethanol, and the mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by neutral alumina column chromatography with DCM / MeOH (10:1 to 5:1) to obtain the product N,N,N-trimethyl-1-(4-(phenylcarbamoyl)phenyl)methylammonium bromide (compound 2d) as a pale yellow solid in a yield of 29.3%.

[0014] Preferably, the compound 2d is used for resisting Staphylococcus aureus infection.

[0015] A method for synthesizing a quaternary ammonium salt derivative (compound 3e) specifically comprises the following steps:

[0016] Step 1: 1 g, 9.33 mmol of p-methylaniline was dissolved in 10 ml of DCM. To this solution was added 11.47 g, 18.66 mmol of a 50 ml aqueous solution of potassium peroxodisulfate complex. The solution was stirred at room temperature under a nitrogen atmosphere until TLC monitoring showed complete consumption of the starting material. After separation, the aqueous phase was extracted twice with DCM. The combined organic layers were washed with 0.1 M HCl, saturated sodium bicarbonate solution, water, and brine, and dried over anhydrous sodium sulfate. After removal of the solvent, the residue was vacuum distilled at ambient temperature to give a green liquid product (compound 3b) in a yield of 50.4%.

[0017] Step 2: Under a nitrogen atmosphere, 0.41 ml (4.54 mmol) of aniline was slowly added to a solution of 0.55 g (4.54 mmol) of compound 3b in 5 ml of acetic acid. The mixture was stirred at room temperature for 9 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel flash column chromatography (hexane) to give an orange solid product (compound 3c) in a yield of 54.6%.

[0018] Step 3: Under a nitrogen atmosphere, 0.1 g, 0.51 mmol of compound 3c, 0.09 g, 0.51 mmol of NBS, and 0.005 g, 0.03 mmol of AIBN were dissolved in 10 ml of carbon tetrachloride. The reaction mixture was refluxed for 24 hours, the solvent was evaporated, and the residue was purified by silica gel flash column chromatography (hexane) to obtain the product (compound 3d) as an orange-red solid in a yield of 41.6%.

[0019] Step 4: To a solution of 0.1 g (0.36 mmol) of compound 3d in 10 ml of ethanol was added 0.955 ml (3.63 mmol) of 30% trimethylamine in ethanol. The mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by preparative liquid chromatography to afford an orange solid product (compound 3e) in a yield of 19.5%.

[0020] Preferably, the compound 3e is used for resisting Staphylococcus aureus infection.

[0021] A method for synthesizing a quaternary ammonium salt derivative (compound 4e) specifically comprises the following steps:

[0022] Step 1: 1 g, 9.33 mmol of p-methylaniline was dissolved in 10 ml of DCM. To the solution was added 11.47 g, 18.66 mmol of a 50 ml aqueous solution of potassium peroxodisulfate complex. The solution was stirred at room temperature under a nitrogen atmosphere until TLC monitoring showed complete consumption of the starting material. After separation, the aqueous phase was extracted twice with DCM. The combined organic layers were washed with 0.1 M HCl, saturated sodium bicarbonate solution, water, and brine, and dried over anhydrous sodium sulfate. After removal of the solvent, the residue was vacuum distilled at ambient temperature to give a green liquid product (compound 4b) in a yield of 50.4%.

[0023] Step 2: Under a nitrogen atmosphere, 0.41 ml (4.54 mmol) of aniline was slowly added to a solution of 0.55 g (4.54 mmol) of compound 4b in 5 ml of acetic acid. The mixture was stirred at room temperature for 9 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel flash column chromatography (hexane) to give an orange solid product (compound 4c) in a yield of 43.1%.

[0024] Step 3: Under a nitrogen atmosphere, 0.1 g, 0.51 mmol of compound 3c, 0.09 g, 0.51 mmol of NBS, and 0.005 g, 0.03 mmol of AIBN were dissolved in 10 ml of carbon tetrachloride. The reaction mixture was refluxed for 24 hours, the solvent was evaporated, and the residue was purified by silica gel flash column chromatography (hexane) to obtain the product (compound 4d) as an orange-red solid in a yield of 55.4%.

[0025] Step 4: To a solution of 0.1 g (0.36 mmol) of compound 4d in 10 ml of ethanol (30% in ethanol) was added 0.955 ml (3.63 mmol) of trimethylamine. The mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by preparative liquid chromatography to afford an orange solid product (compound 4e) in a yield of 49.6%.

[0026] Preferably, the compound 4e is used for resisting Staphylococcus aureus infection.

[0027] Compared with the prior art, the beneficial effects of the present invention are: the compound 1e synthesized using aniline as the starting material has excellent antibacterial activity against Pseudomonas aeruginosa, and the minimum inhibitory concentration (MIC) can reach. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagrams of the structural formulas of four quaternary ammonium salt derivatives of the present invention;

[0029] Figure 2 Schematic diagram of the reaction synthesis routes of four quaternary ammonium salt derivatives of the present invention;

[0030] Figure 3 Schematic diagram of the CFU test of Staphylococcus aureus with different concentrations of compounds 3e, 4e, 1d, and 2d. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] The technical solutions of the present invention are as follows:

[0033] Four quaternary ammonium salt derivatives (3aR, 4R, 5R, 7S, 8S, 9R, 9aS, 12R)-8-hydroxy-4, 7, 9, 12-tetramethyl-3-oxo-7-vinyldecahydro-4, 9a-propoxycyclopenta[8]annulene-5-yl 2-(4-((E)-2-((2-naphthoyl)oxy)imino)-2-(R1-yl)ethyl)piperazin-1-yl)acetate, having Figure 1 The structural formula shown is:

[0034] A method for synthesizing the above-mentioned quaternary ammonium salt derivative is characterized by comprising the following steps:

[0035] Step 1. at 0 ℃, p-methylbenzoyl chloride (1.24ml, 10.74mmol) is slowly added to a mixture of aniline (0.98ml, 10.74mmol) and triethylamine (1.64ml, 11.81mmol) in dichloromethane (50ml), and the mixture is stirred at room temperature for 5 hours. The reaction mixture is extracted with DCM, the organic phase is merged and dried with Na2SO4. The crude mixture is purified by silica gel column chromatography (PE / EtOAc 5:1 to 3:1) to obtain 4-methyl-N-phenylbenzamide (compound 1b) as a white solid.

[0036] Step 2. Under a nitrogen atmosphere, compound 1b (0.30 g, 1.42 mmol), NBS (2.78 g, 15.62 mmol), and AIBN (0.47 g, 2.84 mmol) were dissolved in carbon tetrachloride (30 ml). The reaction solution was heated at reflux for 24 hours, the solvent was removed by rotary evaporation, and the mixture was extracted with dichloromethane. The organic phases were combined and dried over Na2SO4 to obtain crude 4-(bromomethyl)-N-phenylbenzamide (compound 1c), which was directly used in the next reaction. Compound 1c (0.20 g, 0.69 mmol) and trimethylamine (30% in ethanol, 0.955 ml, 3.63 mmol) were dissolved in ethanol (20 ml) and stirred at 80°C for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by neutral alumina column chromatography (DCM / MeOH 10:1 to 5:1) to give the product N,N,N-trimethyl-1-(4-(phenylcarbamoyl)phenyl)methanium bromide (Compound 1d) as a pale yellow solid.

[0037] The reaction synthesis route is shown in 2:

[0038] The present invention is further described in detail by the following examples, but the scope of the present invention is not limited by these examples.

[0039] Example 1. Synthesis and antibacterial activity study of compound 1d.

[0040] 1. Synthesis of compound 1b.

[0041] The reaction process is the same as step 1, with a yield of 89.5%. 1 H NMR(500MHz,Chloroform-d)δ7.72(s,1H),7.71–7.69(m,2H),7.59–7.55(m,2H),7 .31–7.28(m,2H),7.21(d,J=8.0Hz,2H),7.07(tt,J=7.3,1.2Hz,1H),2.35(s,3H).

[0042] 2. Synthesis of compound 1d.

[0043] The reaction process is the same as step 2, with a yield of 36.4%. 1 H NMR(500MHz,DMSO-d6)δ10.54(s,1H),8.17–8.14(m,2H),7.86–7.82(m,2H),7.72(d, J=8.1Hz,2H),7.36(t,J=7.9Hz,2H),7.12(t,J=7.4Hz,1H),4.71(s,2H),3.10(s,9H).

[0044] 3. Anti-Staphylococcus aureus activity of compound 1d.

[0045] CFU analysis: The antibacterial activity of each single substance extract against Staphylococcus aureus was determined by the CFU count method on agar plates. The resulting colonies were counted to determine the CFU and growth inhibitory activity of the drug. The bacterial suspension was incubated with different concentrations of the extract for 3 hours. Subsequently, 30 μl of 10-fold serial dilutions of bacteria at different concentrations were dropped onto TSB and LB agar plates and incubated at 37°C for 24 hours, with triplicates per group. After incubation at 37°C for 24 hours, the colonies were counted. Representative images of BHI agar plates were acquired using an iPhone 12. The results are shown in the figure below.

[0046] Example 2. Synthesis and antibacterial activity study of compound 2d.

[0047] 1. Synthesis of compound 2b.

[0048] The reaction process is the same as step 1, with a yield of 93.5%. 1 H NMR(500MHz,Chloroform-d)δ7.91(s,1H),7.81–7.77(m,2H),7.69–7.64(m,2 H),7.40–7.35(m,2H),7.29(d,J=8.3Hz,2H),7.19–7.14(m,1H),2.44(s,3H).

[0049] 2. Synthesis of compound 2d.

[0050] The reaction process is the same as step 2, with a yield of 29.3%. 1 H NMR(500MHz,DMSO-d6)δ10.56(s,1H),8.01–7.98(m,2H),7.98–7.95(m,2H),7.6 4–7.59(m,1H),7.57–7.53(m,2H),7.53–7.50(m,2H),4.53(s,2H),3.04(s,9H).

[0051] 3. Anti-Staphylococcus aureus activity of compound 2d.

[0052] The test process is the same as above, and the results are shown in the figure below.

[0053] Example 3. Synthesis and antibacterial activity study of compound 3e.

[0054] 1. Synthesis of compound 3b.

[0055] Toluidine (1g, 9.33mmol) is dissolved among the DCM (10ml), in this solution, add the aqueous solution (50ml) of potassium peroxydisulfate complex salt (11.47g, 18.66mmol), under nitrogen atmosphere, stirring at room temperature this solution, until TLC monitoring shows that starting raw material is consumed completely, after layering, aqueous phase is extracted twice with DCM, with 0.1M HCl, saturated sodium bicarbonate solution, water, salt water washing and merging organic layer, and use anhydrous sodium sulfate drying. After removing solvent, resistates is vacuum distilled at ambient temperature, obtains green liquid product. Productive rate: 50.4%. 1 H NMR (500MHz, Chloroform-d) δ8.05–8.01(m,1H),7.72(d,J=8.0Hz,1H),7.31(d,J=8.0Hz,1H),7.23(d,J=8.5Hz,1H),2.37(d,J=9.6Hz,3H).

[0056] 2. Synthesis of compound 3c.

[0057] Under nitrogen, aniline (0.41 ml, 4.54 mmol) was slowly added to a solution of compound 13'b (0.55 g, 4.54 mmol) in acetic acid (5 ml). The mixture was stirred at room temperature for 9 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel flash column chromatography (hexane) to afford the product as an orange solid. Yield: 54.6%. 1 H NMR (500MHz, Chloroform-d) δ7.96–7.92(m,2H),7.89–7.85(m,2H),7.54(dd,J=8.3,6.6Hz,2H),7.51–7.47(m,1H),7.35(d,J=8.2Hz,2H),2.47(s,3H).

[0058] 3. Synthesis of compound 3d.

[0059] Under a nitrogen atmosphere, compound 13'c (0.1 g, 0.51 mmol), NBS (0.09 g, 0.51 mmol), and AIBN (0.005 g, 0.03 mmol) were dissolved in carbon tetrachloride (10 ml). The reaction mixture was refluxed for 24 hours. The solvent was evaporated, and the residue was purified by silica gel flash column chromatography (hexane) to obtain the product as an orange-red solid. Yield: 41.6%. 1 H NMR(500MHz,Chloroform-d)δ7.95(dd,J=7.1,1.8Hz,2H),7.93–7.91(m,2H),7.58–7.56(m,2H),7.56–7.53(m,2H),7.53–7.49(m,1H),4.58(s,2H).

[0060] 4. Synthesis of compound 3e.

[0061] Trimethylamine (30% in ethanol, 0.955 ml, 3.63 mmol) was added to a solution of compound 13'd (0.1 g, 0.36 mmol) in ethanol (10 ml). The mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by preparative liquid chromatography (methanol:H2O, C18) to afford the product as an orange solid. Yield: 19.5%. 1 H NMR (500MHz, DMSO-d6) δ8.03–7.99(m,2H),7.93(ddt,J=6.6,2.5,1.3Hz,2H),7.78(d,J=8.1Hz,2H),7.67–7.60(m,3H),4.66(s,2H),3.09(s,9H).

[0062] 5. Anti-Staphylococcus aureus activity of compound 3e.

[0063] The test process is the same as above, and the results are shown in the figure below.

[0064] Example 4. Synthesis and antibacterial activity study of compound 4e.

[0065] 1. Synthesis of compound 4b.

[0066] The reaction process and results are the same as those of compound 3b above.

[0067] 2. Synthesis of compound 4c.

[0068] The reaction process was the same as that for the synthesis of compound 3c in Example 3. Yield: 43.1%. 1H NMR (500MHz, Chloroform-d) δ7.96–7.93(m,2H),7.86–7.80(m,2H),7.32(d,J=8.0Hz,2H),7.06–7.01(m,2H),3.92(s,3H),2.45(s,3H).

[0069] 3. Synthesis of compound 4d.

[0070] The reaction process was the same as that for the synthesis of compound 3d in Example 3. Yield: 55.4%. 1 H NMR (500MHz, Chloroform-d) δ7.97–7.93(m,2H),7.89–7.86(m,2H),7.57–7.53(m,2H),7.07–7.02(m,2H),4.58(s,2H),3.92(s,3H).

[0071] 4. Synthesis of compound 4e.

[0072] The reaction process was the same as that for the synthesis of compound 3e in Example 3. Yield: 49.6%. 1 H NMR (500MHz, DMSO-d6) δ7.94(t,J=8.9Hz,4H),7.75(d,J=8.0Hz,2H),7.17(d,J=8.6Hz,2H),4.66(s,2H),3.89(s,3H),3.09(s,9H).

[0073] 5. Anti-Staphylococcus aureus activity of compound 4e.

[0074] The test process is the same as above, and the results are shown in Figure 3 .

[0075] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for synthesizing a quaternary ammonium salt derivative, characterized in that: The specific steps include: Step 1: 1.24 ml (10.74 mmol) of p-toluyl chloride was slowly added to a mixture of 0.98 ml (10.74 mmol) of aniline and 1.64 ml (11.81 mmol) of triethylamine in 50 ml of dichloromethane at 0°C, and the mixture was stirred at room temperature for 5 hours; the reaction mixture was extracted with DCM, the organic phases were combined and dried over Na2SO4, and the crude mixture was purified by silica gel column chromatography with PE / EtOAc 5:1 to 3:1 to obtain 4-methyl-N-phenylbenzamide, i.e., compound 1b, as a white solid in a yield of 89.5%; Step 2: Under nitrogen atmosphere, 0.30 g (1.42 mmol) of compound 1b, 2.78 g (15.62 mmol) of NBS, and 0.47 g (2.84 mmol) of AIBN were dissolved in 30 ml of carbon tetrachloride. The reaction solution was heated to reflux for 24 h, and the solvent was removed by rotary evaporation. The mixture was extracted with DCM, and the organic phases were combined and dried over Na2SO4 to obtain 4-(bromomethyl)-N-phenylbenzamide, i.e., the crude product of compound 1c. Step 3: Dissolve 0.20 g (0.69 mmol) of compound 1c in 30% ethanol and 0.955 ml (3.63 mmol) of trimethylamine in 20 ml of ethanol. Stir at 80°C for 16 hours. Remove the solvent under reduced pressure, and purify the residue on a neutral alumina column with a DCM / MeOH ratio of 10:1 to 5:1 to obtain N,N,N-trimethyl-1-(4-(phenylcarbamoyl)phenyl)methylammonium bromide (compound 1d) as a pale yellow solid in a 36.4% yield.

2. Use of a quaternary ammonium salt derivative 1d, 2d, 3e, or 4e in the preparation of a drug for resisting Staphylococcus aureus infection, characterized in that: The structural formulas are: 。

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