A piperidine quaternary ammonium compound, a preparation method and use thereof

Abstract

This invention belongs to the field of pharmaceutical chemistry technology, and discloses a piperidine quaternary ammonium compound with antibacterial activity and rapid bactericidal effect, its preparation method, and its uses. The piperidine quaternary ammonium compound is prepared via O-alkylation, Mannich reaction, and quaternization reaction. The structure of this compound is shown in Formulas I to IV. Antibacterial activity experiments show that this compound has clear antibacterial activity against a variety of cocci, with a minimum inhibitory concentration (MIC) of 0.5 μg / mL against Staphylococcus aureus, and 8 μg / mL against Enterococcus gargarizans; the MIC against Staphylococcus capsulatum and Staphylococcus epidermidis reaches 4 μg / mL, and the MIC against Streptococcus salivarius reaches 8 μg / mL.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and discloses a piperidine quaternary ammonium compound with antibacterial activity, its preparation method and uses. Background Technology

[0002] Since Alexander Fleming's discovery of penicillin in 1928, antibiotics have become an effective means of treating various bacterial infections. Subsequently, numerous antibiotic drugs have been discovered, including penicillins (such as amoxicillin), cephalosporins (such as cefadroxil), quinolones (such as levofloxacin), tetracyclines (such as tigecycline), macrolides (erythromycin), rifamycins (rifampin), aminoglycosides (such as streptomycin), and glycopeptides (such as vancomycin). These drugs have diverse chemical structures, different antibacterial spectra, and mechanisms of action, playing a crucial role in the treatment of microbial infectious diseases. Their structures are shown in Formula V.

[0003]

[0004]

[0005] Antibiotics are prone to bacterial resistance during use, and this problem poses a serious threat to human health and economic development worldwide. In 2019 alone, bacterial resistance caused approximately 5 million deaths globally (①Christopher JLM, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis, Lancet, 2022, 399:629.). Recently, the World Health Organization released a report on the Global Antimicrobial Resistance and Use Surveillance System (GLASS) report (②WHO. Global antimicrobial resistance and use surveillance system (GLASS) report: 2022. https: / / www.who.int / publications / i / item / 9789240062702). This report analyzed antimicrobial resistance trends since 2017 and found that among bloodstream infection pathogens reported in 76 countries and regions, the infection rates of "superbugs" resistant to third-generation cephalosporins, *Escherichia coli*, and methicillin-resistant *Staphylococcus aureus* were 42% and 35%, respectively. It is evident that the problem of bacterial resistance is becoming increasingly serious.

[0006] Antibiotic resistance mechanisms have also received widespread attention from scientists, but many unresolved issues remain regarding the exact mechanisms of bacterial resistance (③Alexander JAN, et al. Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus. Nature, 2023, 613, 375; ④Yang, KB, et al. High-resolution landscape of an antibiotic binding site. Nature, 2023, 622, 180; ⑤Lázár V, et al. Antibiotic combinations reduce Staphylococcus aureus clearance. Nature, 2022, 610, 540.). In conclusion, antibiotic resistance is an increasingly serious global problem, and the discovery of antibiotics with novel structural features will provide new options for overcoming antibiotic resistance.

[0007] In their previous research on TRPV3 (transformation receptor potential cation channel V member subtype 3) inhibitors, the applicant unexpectedly discovered that compounds of formula VI with phenylalkyl ether structural fragments have significant antibacterial activity (MIC of 32 μg / mL against Staphylococcus aureus). This structure is significantly different from the existing antibiotic structures. Further research revealed that compounds of formula VII have even better antibacterial activity, and an invention patent was applied for (⑥ Li Wen, et al. A phenylalkyl ether compound and its preparation method and use, CN117209456, 2023-09-13).

[0008]

[0009] Based on the above research, the applicant conducted extensive literature review and designed novel compounds. The newly designed compounds retain the structural features of the phenylalkyl ethers verified in the aforementioned patent CN117209456, and attempt to link aliphatic chains containing halogens or ether bonds to the N atom of the piperidine group, resulting in several series of piperidine quaternary ammonium compounds. Further synthesis and antibacterial activity evaluation yielded beneficial research results. Summary of the Invention

[0010] Therefore, the present invention aims to provide a class of compounds having a piperidine quaternary ammonium structure, which have effective antibacterial activity.

[0011] The piperidine quaternary ammonium compound of the present invention has the structural formulas shown in Formulas I to IV:

[0012]

[0013] In Equation I, m is an integer between 2 and 5;

[0014] In Equation II, n is an integer between 8 and 10;

[0015] In Equation III, x is an integer from 1 to 4;

[0016] In equation IV, y is 1 or 2.

[0017] It may also be other pharmaceutically acceptable salts, or solvates, or hydrates, or prodrugs, or metabolites thereof.

[0018] The present invention also provides a method for preparing the aforementioned compound, comprising the following steps:

[0019] Step 1: Using p-hydroxyacetophenone and n-bromobutane as raw materials, compound a is obtained through an O-alkylation reaction;

[0020]

[0021] Step 2: Compound a reacts with piperidine and paraformaldehyde in a Mannich reaction to give compound b;

[0022]

[0023] Step 3: Compound b is mixed with saturated sodium carbonate, and a neutralization reaction occurs to obtain compound c;

[0024]

[0025] Step 4: Compound c is mixed with different brominated derivatives to undergo quaternization reaction, yielding target compounds 1-13;

[0026]

[0027] The applicant of this invention retained the structural features of the previously verified phenylalkyl ether and attached an aliphatic chain containing halogen or ether bonds to the N atom of the piperidine group, thus obtaining piperidine quaternary ammonium compounds 1-13 as shown in Formulas I to IV above. The structure of these compounds was confirmed, and their contents were all greater than 99.0% as determined by high performance liquid chromatography. Based on this, the activity of the obtained compounds was evaluated.

[0028] The piperidine quaternary ammonium compounds described in this invention exhibit excellent activity against a variety of cocci, indicating that these compounds possess superior antibacterial activity. In particular, compounds 4, 6, 10, and 13 show MIC values ​​between 0.5 and 1 μg / mL against Staphylococcus aureus, which are superior to the clinically used drugs amoxicillin and tigecycline; compounds 6, 10, and 13 have a MIC value of 16 μg / mL against Enterococcus faecalis, similar to the activity of the positive control erythromycin; and compounds 6 and 13 have a MIC value of 8 μg / mL against Enterococcus fowleri.

[0029] The activity of compounds 6, 10, and 13 against Staphylococcus aureus was similar to that of the positive control drug vancomycin. Compounds 6, 10, and 13 had a MIC of 4 μg / mL against Staphylococcus aureus, which was superior to the positive controls erythromycin and tigecycline. Compound 4 had a MIC of 8 μg / mL against Staphylococcus aureus, which was superior to the positive control erythromycin and similar to that of tigecycline. Compounds 4, 6, and 13 had a MIC of 4 μg / mL against Staphylococcus cephalosporins, which was superior to the positive controls vancomycin and tigecycline. Compounds 5 and 10 had a MIC of 8 μg / mL against Staphylococcus cephalosporins, which was superior to the activity of tigecycline. Compound 6 had a MIC of 4 μg / mL against Staphylococcus epidermidis, which was superior to the positive controls vancomycin and tigecycline. The MIC values ​​of the compounds against Streptococcus salivarius and Streptococcus aureus ranged from 16 to 128 μg / mL, and all showed activity values ​​superior to the positive control chlorhexidine.

[0030] The present invention has the following beneficial effects:

[0031] This invention discloses a piperidine quaternary ammonium compound with antibacterial activity, its preparation method, and its uses. This compound exhibits significant antibacterial activity in antibacterial activity tests, with a minimum inhibitory concentration (MIC) reaching 0.5 μg / mL. The compound prepared by this invention offers advantages such as novel structure and high antibacterial activity for antibacterial drug development, providing a new option for developing drugs with antibacterial activity.

[0032] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0033] Figure 1 This is a line graph showing the bactericidal kinetics of compound 6 of the present invention;

[0034] Figure 2 This is an example of plate coating for the bactericidal experiment of compound 6 and vancomycin of the present invention (both compound 6 and vancomycin were subjected to bactericidal experiments at 4 times the minimum inhibitory concentration); wherein, Figure a is compound 6; Figure b is vancomycin, and the samples are arranged from top to bottom according to the sampling time;

[0035] Figure 3 This is a bar chart of nucleic acid leakage experiments for compound 6 of the present invention;

[0036] Figure 4 This is a line graph showing the effect of membrane components PG, CL, and PE on the MIC value of compound 6 of the present invention. Detailed Implementation

[0037] The following detailed description of specific embodiments, presented in the form of experimental examples, further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0038] The chemical reagents used in the specific embodiments of the present invention are obtained by purchasing commercially available products.

[0039] The instruments used for detection and analysis in this invention are as follows:

[0040] Nuclear magnetic resonance imaging (MRI) scanner, specifically the Bruker Advance Digital 400 MRI scanner from Switzerland.

[0041] High-resolution mass spectrometers: Bruker SolanX 70FT-MS (Switzerland); Agilent 6540TOF.

[0042] High-performance liquid chromatograph (HPLC) instrument: Agilent Technologies 1260 Infinity II HPLC instrument.

[0043] Sterilizer, Bingshan Songyang Biotechnology (Dalian) Co., Ltd., MVS-83 Vertical Pressure Steam Sterilizer.

[0044] 37℃ incubator, Shanghai Yiheng DHP-9602 (vertical) constant temperature incubator.

[0045] Shaker, Shanghai Jinghong Experimental Equipment Co., Ltd. THZ-412 benchtop constant temperature shaker.

[0046] Microplate reader, Synergy H1 full-function microplate reader from Bertek Instruments, Inc.

[0047] The NanoDrop One / OneC micro UV-Vis spectrophotometer, manufactured by Thermo Fisher Scientific, Inc.

[0048] Experiments 1-4 are experiments on the synthesis of compounds, and Experiments 5-9 are experiments on the activity evaluation of compounds.

[0049] Preparation of Compound 1 in Experimental Example 1

[0050]

[0051] 8.1 g (0.06 mol) of p-hydroxyacetophenone was weighed into a three-necked flask and dissolved in 30 mL of LMF. Then, anhydrous potassium carbonate (24.9 g, 0.18 mol), n-butane bromide (6.6 mL, 0.06 mol), and NaY zeolite (8.1 g) were added sequentially. The reaction was carried out at room temperature for 6 h, with TLC-PE:EA ratio of 3:1 to monitor the reaction progress. After the reaction was complete, the system was transferred to a separatory funnel, and 100 mL of water and 50 mL of ethyl acetate were added for extraction three times. The organic layers were combined, washed six times with water, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a transparent oily liquid compound a, yield: 95.3%. Compound a: 1 H NMR (400MHz, CDCl3) δ7.91(d,J=8.9Hz,2H),6.91(d,J=8.9Hz,2H),4.02(t,J=6.5Hz,2H),2. 54(s,3H),1.78(dq,J=8.6,6.6Hz,2H),1.50(dt,J=14.9,7.4Hz,2H),0.98(t,J=7.4Hz,3H).

[0052] Compound a (7.68 g, 0.04 mol) and piperidine (5.44 g, 0.064 mol) were weighed and dissolved in 25 mL of isopropanol. Paraformaldehyde (0.24 g, 0.04 mol) and 2 drops of trifluoroacetic acid were added with stirring. The mixture was microwaved and reacted for 7 h. After cooling to room temperature, the mixture was filtered, and the filter cake was dried to obtain crude compound b. The crude product was recrystallized from the crude product with 20 mL of water at 60 °C, yielding compound b (85.7%). Compound b: 1 H NMR (400MHz, CDCl3) δ7.81 (d, J = 8.7Hz, 2H), 6.78 (d, J = 8.7Hz, 2H), 3.87 (t, J = 6.5Hz, 2H), 3.02 (t, J = 7. 5Hz,2H),2.67(t,J=7.5Hz,2H),1.64(dt,J=8.4,6.6Hz,3H),1.53–1.27(m,7H),0.86(t,J=7.4Hz,4H).

[0053] In a 100 mL beaker, compound b (4.88 g, 0.015 mol) was taken, 30 mL of distilled water and 15 mL of saturated sodium carbonate solution were added, and the mixture was stirred for 30 min. The mixture was then transferred to a separatory funnel, 50 mL of water was added, and the mixture was washed three times with ethyl acetate. The organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain compound c. The yield was 82.1%.

[0054] Compound c (2.89 g, 0.01 mol) was dissolved in 10 mL of acetone. 1-Bromo-2-fluoroethane (6.35 g, 0.05 mol) was added with stirring, and the reaction was carried out at room temperature for 12 h. The reaction progress was monitored by TLC using dichloromethane:methanol = 10:1. After the reaction was complete, the system was transferred to a 100 mL beaker, 30 mL of ethyl acetate was added, and the mixture was stirred for 10 min. The mixture was then centrifuged, and the supernatant was discarded. The precipitate was then added back to 30 mL of ethyl acetate. This process was repeated twice to obtain compound 1, with a yield of 63.6%. Compound 1: 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=8.4Hz,2H),7.07(d,J=8.4Hz,2H),4.19–3.95(m,3H),3.54(dt,J=18.8,6.4Hz,3H),3.47(s,2H),2.99( s,3H),1.83(t,J=6.6Hz,1H),1.78(s,3H),1.75(s,1H),1.70(q,J=7.0Hz,2H),1.64(s,2H),1.43(h,J=7.4Hz,3H),0.93(t,J=7.4Hz,3H). 13 C10 NMR (101MHz, CDCl3) δ 194.39, 163.92, 130.73, 130.69, 128.32, 114.64, 114.29, 68.08, 53.75, 52.21, 32.84, 31.04, 22.56, 21.87, 19.12, 13.89, 13.61. HRMS Molecular weight: [C101MHz, CDCl3] 18 H 27 NO2] + :290.2115.

[0055] Following the preparation method of Example 1, compounds 2–4 were obtained:

[0056]

[0057]

[0058] Preparation of compound 5 in Experimental Example 2

[0059]

[0060] Compound c (2.89 g, 0.01 mol) was weighed and dissolved in 10 mL of acetone. 1,8-Dibromooctane (13.60 g, 0.05 mol) was added with stirring, and the reaction was carried out at room temperature for 12 h. The reaction progress was monitored by TLC using a dichloromethane:methanol ratio of 10:1. After the reaction was complete, the system was transferred to a 100 mL beaker, 30 mL of ethyl acetate was added, and the mixture was stirred for 10 min. The mixture was then centrifuged, and the supernatant was discarded. The precipitate was then added back to 30 mL of ethyl acetate. This process was repeated twice to obtain compound 5, with a yield of 71.8%. Compound 5: 1 H NMR (400MHz, MeOD) δ8.29(d,J=8.5Hz,2H),7.29(d,J=8.5Hz,2H),4.35(t,J=6.4Hz,2H),3.92–3.75(m,7H),3.57(s,2H),3.32(d t,J=13.2,6.5Hz,2H),2.10(ddt,J=44.0,22.4,14.9Hz,10H),1.77(tt,J=26.4,11.4Hz,6H),1.26(t,J=7.4Hz,4H).HRMS molecular weight: [C 18 H 27 NO2] + :290.2154.

[0061] Following the preparation method of Example 2, compounds 6 and 7 were obtained:

[0062]

[0063]

[0064] Preparation of compound 8 in Experimental Example 3

[0065]

[0066] Compound c (2.89 g, 0.01 mol) was dissolved in 10 mL of acetone. Bromomethyl methyl ether (6.25 g, 0.05 mol) was added with stirring, and the reaction was carried out at room temperature for 12 h. The reaction progress was monitored by TLC using dichloromethane:methanol = 10:1. After the reaction was complete, the system was transferred to a 100 mL beaker, 30 mL of ethyl acetate was added, and the mixture was stirred for 10 min. The mixture was then centrifuged, and the supernatant was discarded. The precipitate was then added back to 30 mL of ethyl acetate. This process was repeated twice to obtain compound 8, with a yield of 64.0%. Compound 8: 1H NMR (400MHz, DMSO-d6) δ7.97(d,J=8.9Hz,2H),7.06(d,J=8.9Hz,2H),4.86( d,J=9.7Hz,1H),4.06(t,J=6.5Hz,3H),3.73–3.64(m,2H),3.58(d,J=8.2Hz, 5H),3.55–3.39(m,2H),3.06(s,1H),3.05–2.91(m,2H),1.79(s,2H),1.74–1 .62(m,3H),1.41(dt,J=12.1,6.1Hz,2H),0.92(t,J=7.4Hz,5H).HRMS molecular weight: [C 18 H 27 NO2] + :290.2116.

[0067] Following the preparation method of Example 3, compounds 9–11 were obtained:

[0068]

[0069]

[0070] Preparation of compound 12 in Experimental Example 4

[0071]

[0072] Compound c (2.89 g, 0.01 mol) was dissolved in 10 mL of acetone. Bromomethyl ether (4.73 g, 0.05 mol) was added with stirring, and the reaction was carried out at room temperature for 12 h. The reaction progress was monitored by TLC using dichloromethane:methanol = 10:1. After the reaction was complete, the system was transferred to a 100 mL beaker, 30 mL of ethyl acetate was added, and the mixture was stirred for 10 min. The mixture was then centrifuged, and the supernatant was discarded. The precipitate was then added back to 30 mL of ethyl acetate. This process was repeated twice to obtain compound 12, with a yield of 49.6%. Compound 12: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.98 (d, J = 8.9Hz, 2H), 7.07 (d, J = 8.9Hz, 2H), 4.08 (t, J = 6.5Hz, 4H), 3.62 (t, J = 7.4Hz, 3H), 3.48 (s, 1H), 3.34 (td, J = 7.3, 4.9Hz, 3H), 3.00–2.85 (m, 4H), 1.94–1.62 (m, 8H), 1.51–1.29 (m, 3H), 0.94 (t, J = 7.4Hz, 5H). HRMS molecular weight: [C 18 H 27 NO2] + :290.2114.

[0073] Following the preparation method of Example 4, compound 13 was obtained:

[0074]

[0075] Example 5: Evaluation of antibacterial activity against Escherichia coli and Staphylococcus aureus

[0076] The activity of the compounds synthesized in this invention against *Escherichia coli* and *Staphylococcus aureus*, both facultative anaerobes, was evaluated using a micro-broth dilution method. The culture medium used was MHB broth. The specific operating steps are as follows:

[0077] (1) Prepare an antibacterial compound solution with a concentration of 25600 μg / mL and store it in a refrigerator at 4℃ for later use.

[0078] (2) For the test bacteria Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), single colonies of 0.5–1 mm were picked up with an inoculation loop and inoculated into MHB broth medium. The medium was incubated on a shaker at 37°C and 220 rpm for 4 hours, and then diluted to a bacterial count of approximately 1 × 10⁻⁶. 5 Prepare a bacterial culture at CFU / mL for later use.

[0079] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL into a 1.5 mL sterile centrifuge tube, and add 990 μL of sterile ultrapure water to dilute it to a compound solution with a concentration of 256 μg / mL.

[0080] (4) Take a 96-well plate and add 100 μL of MHB broth to each well. Then add 100 μL of the compound solution to the first well. Dilute stepwise using the two-fold dilution method to obtain the antibacterial compound solution. The concentrations of the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Finally, add 100 μL of the prepared bacterial suspension to make the total volume of each well 200 μL. The eleventh well contains 200 μL of MHB broth, and the twelfth well contains 100 μL of MHB broth and 100 μL of bacterial suspension as a blank control. Place the inoculated 96-well plate in a 37°C incubator and observe the bacterial growth for 16 hours. Vancomycin, meropenem, tigecycline, and amoxicillin are used as positive controls. Perform at least three parallel tests for each compound. The MIC value of the compound is the first well that does not become cloudy (indicating no bacterial growth) as observed by the naked eye.

[0081] Table 1. MIC results of the compounds of this invention against Escherichia coli and Staphylococcus aureus.

[0082]

[0083] As shown in Table 1, compounds 1 through 13 of this invention all exhibit a certain degree of antibacterial activity, which can be summarized as follows:

[0084] (1) Compounds 4, 6 and 13 had a MIC of 0.5 μg / mL against Staphylococcus aureus, which was superior to the antibacterial activity of the positive control drugs vancomycin, tigecycline and amoxicillin.

[0085] (2) The MIC of compound 10 against Staphylococcus aureus is 1 μg / mL, which is similar to the activity of the positive control drug vancomycin;

[0086] (3) Other compounds have MICs of 4 to 32 μg / mL against Staphylococcus aureus, and have a certain degree of antibacterial activity;

[0087] (4) The MIC of the synthesized compounds 1-13 against Escherichia coli was 32-128 μg / mL, which was weaker than that of the positive control drug meropenem (0.25 μg / mL).

[0088] Example 6 Evaluation of antibacterial activity against various enterococci, staphylococci, and streptococci

[0089] The activity of the compounds synthesized in this invention against various enterococci, staphylococci, and streptococci was evaluated using a micro-broth dilution method. Since the various enterococci, staphylococci, and streptococci used can grow rapidly under aerobic conditions, cationic-regulated MHB broth (CAMHB broth) was employed as the culture medium. The specific operating steps are as follows:

[0090] (1) Prepare an antibacterial compound solution with a concentration of 25600 μg / mL and store it in a refrigerator at 4℃ for later use.

[0091] (2) For the test bacteria, *Enterococcus faecalis*, *Enterococcus facium*, *Enterococcus casseliflavus*, *Enterococcus gallinarum*, *S. lentus*, *S. simulans*, *S. capitis*, *S. epidermidis*, *S. salivarius*, and *S. sobrinus* were selected using an inoculation loop. Single colonies of 0.5–1 mm were picked up and inoculated into CAMHB broth medium. The medium was incubated on a shaker at 37°C and 220 rpm for 4 hours, and then diluted to approximately 1 × 10⁻⁶ colonies. 5 Prepare a bacterial culture at CFU / mL for later use.

[0092] (3) Take 10 μL of the antibacterial compound solution with a concentration of 25600 μg / mL into a 1.5 mL sterile centrifuge tube, and add 990 μL of sterile ultrapure water to dilute it to a compound solution with a concentration of 256 μg / mL.

[0093] (4) Take a 96-well plate and add 100 μL of CAMHB broth to each well. Then add 100 μL of the compound solution to the first well. Dilute stepwise using the two-fold dilution method to obtain the antibacterial compound solution. The concentrations of the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Finally, add 100 μL of the prepared bacterial suspension to make the total volume of each well 200 μL. The eleventh well contains 200 μL of CAMHB broth, and the twelfth well contains 100 μL of CAMHB broth and 100 μL of bacterial suspension as a blank control. Place the inoculated 96-well plate in a 37°C incubator and observe the bacterial growth for 16 hours. Vancomycin, erythromycin, tigecycline, and chlorhexidine are used as positive controls. Perform at least three parallel tests for each compound. The MIC value of the compound is the first well that does not become cloudy (indicating no bacterial growth) as observed by the naked eye.

[0094] Table 2. MIC results of the compounds of this invention against Enterococci.

[0095]

[0096] As shown in Table 2, the compounds of this invention exhibit certain anti-enterococcal activity, which can be summarized as follows:

[0097] (1) The MIC values ​​of the compounds of the present invention against Enterococcus faecalis are 16 to 128 μg / mL. Among them, compounds 10 and 13 have an MIC value of 16 μg / mL against Enterococcus faecalis, showing certain antibacterial activity.

[0098] (2) The MIC values ​​of the compounds of the present invention against Enterococcus faecalis are 16–128 μg / mL. Among them, the MIC values ​​of compounds 6, 10 and 13 against Enterococcus faecalis are 16 μg / mL, which is similar to the activity of the positive control erythromycin.

[0099] (3) The MIC values ​​of the compounds of the present invention against Enterococcus faecalis are 16 to 64 μg / mL. Among them, compounds 3, 4, 6, and 10 to 13 have an MIC value of 16 μg / mL against Enterococcus faecalis, showing certain antibacterial activity.

[0100] (4) The MIC values ​​of the compounds of the present invention against Enterococcus fowleri range from 8 to 128 μg / mL. Among them, the MIC values ​​of compounds 6 and 13 against Enterococcus fowleri are 8 μg / mL, which is similar to the activity of the positive control drug vancomycin; the MIC values ​​of compounds 4 and 10 against Enterococcus fowleri are 16 μg / mL, which shows certain antibacterial activity.

[0101] Table 3. MIC results of the compounds of this invention against Staphylococcus aureus.

[0102]

[0103] As shown in Table 3, the compounds of this invention exhibit strong anti-staphylococcal activity, which can be summarized as follows:

[0104] (1) The MIC values ​​of the compounds of the present invention against Staphylococcus aureus are 16-64 μg / mL. Among them, compounds 4 and 6 have an MIC value of 16 μg / mL against Staphylococcus aureus, showing certain antibacterial activity.

[0105] (2) The MIC values ​​of the compounds of the present invention against Staphylococcus aureus range from 4 to 64 μg / mL. Among them, compounds 6, 10 and 13 have an MIC value of 4 μg / mL against Staphylococcus aureus, which is superior to the positive control erythromycin and tigecycline; compound 4 has an MIC value of 8 μg / mL against Staphylococcus aureus, which is superior to the positive control erythromycin and has similar activity to tigecycline; compound 7 has an MIC value of 16 μg / mL against Staphylococcus aureus, which is similar to the activity of erythromycin.

[0106] (3) The MIC values ​​of the compounds of the present invention against Staphylococcus aureus are 4–64 μg / mL. Among them, the MIC values ​​of compounds 4, 6 and 13 against Staphylococcus aureus are 4 μg / mL, which is similar to the activity of the positive control drug vancomycin and superior to the activity of the positive control drug tigecycline; the MIC values ​​of compounds 5 and 10 against Staphylococcus aureus are 8 μg / mL, which is superior to the activity of tigecycline; the MIC values ​​of compounds 3, 7, 11 and 12 against Staphylococcus aureus are 16 μg / mL, which is similar to the activity of tigecycline.

[0107] (4) The MIC values ​​of the compounds of the present invention against Staphylococcus epidermidis range from 4 to 128 μg / mL. Among them, compound 6 has an MIC value of 4 μg / mL against Staphylococcus epidermidis, which is superior to the positive controls vancomycin and tigecycline; compound 4 has an MIC value of 8 μg / mL against Staphylococcus epidermidis, which is similar to the activities of vancomycin and tigecycline; and compounds 10, 12 and 13 have an MIC value of 16 μg / mL against Staphylococcus epidermidis, showing certain antibacterial effects.

[0108] Table 4. MIC results of the compounds of this invention against Streptococcus.

[0109]

[0110]

[0111] As shown in Table 4, the compounds of this invention exhibit strong anti-streptococcal activity, which can be summarized as follows:

[0112] (1) The compounds of the present invention have MIC values ​​of 8–64 μg / mL against Streptococcus salivarius, and their activity values ​​are all superior to the positive control chlorhexidine. Among them, compounds 3, 4, and 6 have MIC values ​​of up to 8 μg / mL against Streptococcus salivarius, and have strong antibacterial activity. Compounds 7 and 10–13 have MIC values ​​of 16 μg / mL against Streptococcus salivarius, and have certain antibacterial activity.

[0113] (2) The MIC values ​​of the compounds of the present invention against distant streptococci range from 16 to 128 μg / mL, and their activity values ​​are all superior to the positive control chlorhexidine. Among them, compound 13 has an MIC value of up to 16 μg / mL against Streptococcus salivarius, exhibiting strong antibacterial activity, while compounds 4, 6, 10, and 12 have an MIC value of 32 μg / mL against Streptococcus salivarius, exhibiting certain antibacterial activity.

[0114] As shown in Tables 1-4, these compounds exhibit good antibacterial activity against Enterococcus, Staphylococcus, and Streptococcus. Therefore, Examples 7-9 use compound 6 as an example to explore the antibacterial mechanism of this class of compounds.

[0115] Experiment Example 7: Bactericidal Kinetics Experiment

[0116] (1) Pick a single colony of Staphylococcus aureus (0.5–1 mm) with an inoculation loop and inoculate it into MHB broth medium. Incubate on a shaker at 37°C and 220 rpm for 16–18 h. Then dilute with MHB broth medium at a concentration of 1 × 10⁻⁶. 4 The mixture was divided into 7 separate ep ep tubes and cultured for 2 hours. Then, 4×MIC and 8×MIC compounds, vancomycin (control drug), and PBS (blank control) were added, and the mixture was cultured for another 2 hours.

[0117] (2) At each specified time point of 0, 1, 2, 3, 4, and 6 h after adding the compound, 100 μL of bacterial culture was aspirated and centrifuged at 3500 rpm for 2 min. The supernatant was discarded, the culture was resuspended in PBS, and then diluted 10-fold. 100 μL of the diluted bacterial culture was evenly spread on MHA solid agar plates and incubated in a constant temperature incubator at 37 °C.

[0118] (3) After incubation for 16 hours, calculate the total number of bacteria on the agar plate. Then, calculate the number of colonies per milliliter based on the dilution factor.

[0119] The broken line was obtained by calculating the number of colonies per milliliter. Figure 1 The plate coating results for compound 6 and the positive control vancomycin at 4 times the MIC concentration at each time point are shown in the figure. Figure 2 a in middle, b in middle 2. (Passed) Figure 1 It can be seen that for compound 6, when Staphylococcus aureus was administered 4×MIC, the bacteria were completely killed after 3 hours of treatment, and when administered 8×MIC, the bacteria were completely killed after 2 hours of treatment. In contrast, the control drug vancomycin did not kill all the bacteria after 6 hours. Therefore, it can be seen that the bactericidal speed of compound 6 is much stronger than that of vancomycin.

[0120] Experiment Example 8: Nucleic Acid Leakage Experiment

[0121] Pick single colonies of Staphylococcus aureus (0.5–1 mm) using an inoculation loop and inoculate them into MHB broth. Incubate on a shaker at 37°C and 220 rpm for 4–6 hours. Centrifuge the bacterial suspension at 3500 rpm and 4°C for 5 minutes. Resuspend the suspension in PBS, centrifuge again, and repeat twice to achieve a final bacterial concentration of 1 × 10⁻⁶. 8 CFU / mL. Different concentrations of compound 6, melitin (positive control), polymyxin B (negative control), and blank control PBS were added to EP tubes to achieve final concentrations of 8, 16, and 32 in the bacterial suspension.

[0122] 64 μg / mL. After culturing for another 2 hours, the bacterial culture was centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was collected, and the optical density (OD) of the solution at 260 nm was measured using an ultra-micro spectrophotometer. 260nm )value.

[0123] The measured OD 260nm Values ​​are recorded and processed to obtain a bar chart. Figure 3 .pass Figure 3 It can be seen that both melitoxin and compound 6 affect OD. 260nm The value increased significantly, indicating that compound 6, like the positive control melittin, can induce intracellular nucleic acid leakage in bacteria. Furthermore, the amount of nucleic acid leakage from compound 6 was higher than that from melittin, suggesting that compound 6 has a more significant membrane-disrupting effect than melittin. This strong membrane-disrupting ability promotes the leakage of intracellular substances, thereby accelerating the bacterial death process.

[0124] Experimental Example 9: Exploration of Potential Membrane Targets

[0125] (1) Pick a single colony of Staphylococcus aureus (0.5-1 mm) with an inoculation loop and inoculate it into MHB broth medium. Incubate on a shaker at 37°C and 220 rpm for 4-6 hours. Then dilute to a bacterial count of approximately 1×10⁻⁶. 5 A bacterial suspension of CFU / mL is prepared for later use.

[0126] (2) Take 80 μL of the antibacterial compound solution with a concentration of 25600 μg / mL into a 5 mL sterile centrifuge tube, add 3920 μL of sterile ultrapure water to dilute it to a compound solution with a concentration of 512 μg / mL, and set aside.

[0127] (3) Take a 96-well plate, add 50 μL of MHB broth medium to each well, and then add 100 μL of compound solution to the first well. Dilute stepwise using the two-fold dilution method to obtain antibacterial compound solution. The concentrations of the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Then add 50 μL of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) with concentrations of 0, 1, 2, 4, 8, 16, 32, and 64 μg / mL, respectively.

[0128] (4) Add 100 μL of the prepared bacterial suspension to make the total volume of each well 200 μL. Well 11 contains 200 μL of MHB broth, and well 12 contains 100 μL of MHB broth and 100 μL of bacterial suspension as a blank control. Place the inoculated 96-well plate in a 37°C incubator and observe bacterial growth for 16 h. Perform three replicates for each membrane component concentration. The first well that does not become cloudy (indicating no bacterial growth) is the MIC value of compound 6 at that membrane component concentration, as observed visually.

[0129] from Figure 4 It was found that PG and CL significantly affected the antibacterial activity of compound 6, while PE had little effect. When the concentrations of PG and CL were 32 μg / mL, the MIC of compound 6 increased 8-fold, while that of PE increased only 2-fold. When the concentrations of PG and CL were 64 μg / mL, the MIC of compound 6 increased 16-fold, while that of PE remained the same as at the 32 μg / mL concentration, increasing only 2-fold. These results indicate that compound 6 mainly acts on the cell membrane, and primarily on PG and CL.

Claims

1. A piperidine quaternary ammonium compound, characterized in that, Quaternary ammonium salts with molecular formulas shown in Formulas I to IV: In Equation I, m is an integer from 2 to 5; in Equation II, n is an integer from 8 to 10; in Equation III, x is an integer from 1 to 4. In equation IV, y is 1 or 2.

2. The piperidine quaternary ammonium compound according to claim 1, characterized in that, The compound is one of the following compounds: 。 3. The method for preparing the piperidine quaternary ammonium compound according to claim 1, characterized in that, This can be achieved through the following steps: (1) Using p-hydroxyacetophenone and n-bromobutane as raw materials, intermediate a is obtained by O-alkylation reaction; (2) Intermediate a undergoes a Mannich reaction with piperidine and paraformaldehyde to obtain intermediate b; (3) Intermediate b is mixed with saturated sodium carbonate and a neutralization reaction occurs to obtain intermediate c; (4) Intermediate c is mixed with different brominated products to undergo quaternization reaction, yielding target compounds 1-13; 。 4. The use of the piperidine quaternary ammonium compound according to claim 1 or 2 in the preparation of a drug, characterized in that, As an active ingredient, it is prepared into an anti-Staphylococcus aureus drug.

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