N-heterocyclic carbene-gold (I)-benzyne complex and antibacterial application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an N-heterocyclic carbene-gold (I)-benzyne complex and antibacterial application thereof. The N-heterocyclic carbene-gold (I)-benzyne complex disclosed by the invention has relatively good antibacterial activity, and the minimum inhibitory concentration (MIC) of the N-heterocyclic carbene-gold (I)-benzyne complex is less than 5 mu M. Meanwhile, research finds that the N-heterocyclic carbene-gold (I)-benzyne complex can enter cells and inhibit growth and reproduction of intracellular bacteria by utilizing an intracellular acid environment, the cytotoxicity is remarkably reduced compared with that of an existing drug, and the N-heterocyclic carbene-gold (I)-benzyne complex has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to an N-heterocyclic carbene-gold (I)-benzyne complex and its antibacterial application. Background Art

[0002] In the more than 90 years since the discovery of penicillin in 1928, antimicrobial drugs, especially antibiotics, have played a vital role in modern medicine. - Many antibiotics are ineffective against G. - Infection caused by bacteria, the last type of anti-G - The antibiotics against gram-negative bacteria are synthetic antibiotics called quinolones. Therefore, there is an urgent need to develop new drugs to treat infections caused by gram-negative bacteria.

[0003] More and more studies have focused on the unique lifestyle of bacteria to avoid antibiotic killing and evade the host immune system. According to the different sites of bacterial infection, they can be divided into extracellular bacteria and intracellular bacteria. Extracellular bacteria mainly survive and reproduce in the extracellular body environment, damaging the host's tissues and organs; while intracellular bacteria refer to bacteria that invade host cells through their own virulence system and survive and grow in them to avoid the adverse extracellular environment. Intracellular bacteria can live in modified phagosomes (Salmonella, Mycobacterium, Francisella), inclusion bodies (Chlamydia), lysosomes (Legionella, Coxiella) or cytoplasm (Listeria, Shigella) according to the different localization of bacteria in the cell. Because intracellular bacteria have the characteristics of evading host immunity and avoiding antibiotic killing, compared with extracellular bacteria, intracellular bacteria are a thorny problem facing us today.

[0004] Salmonella enterica is a G-type bacterium that can invade cells and infect humans and animals. - Bacteria, which belong to the family Enterobacteriaceae, can cause fever, chills, abdominal pain, diarrhea, etc. after infection. Salmonellosis affects about 200 million people worldwide and causes more than 200,000 deaths each year; according to the United States Department of Agriculture Economic Research Service, the economic impact of Salmonella on the European Union exceeds 3 billion euros per year. At the same time, both typhoid and non-typhoid Salmonella infections can cause persistent infection, reinfection and long-term carriage. Fluoroquinolones, cephalosporins or macrolide antibiotics are usually used clinically to treat infections caused by Salmonella.

[0005] Salmonella enterica serovar Typhimurium (STm) is a typical intracellular pathogen. During the infection of macrophages and neutrophils, STm forms Salmonella-containing vacuoles (SCVs) inside the cells. These intracellular vesicles containing Salmonella can be protected from extracellular host immune defense (serum complement, antibodies, bile acid, antimicrobial peptides, etc.) and antibiotic killing (for example, ciprofloxacin, the first-line drug for STm, is negatively charged, making it difficult to penetrate the two cell membranes in the host cell to reach SCVs and exert antibacterial effects). Importantly, STm intracellular bacteria can also form antibiotic-resistant persisters (persisters, a type of bacteria that have weak metabolic activity in the bacteria but do not proliferate, similar to a dormant state, which is not easily killed by antibiotics), which can easily lead to chronic infection and disease recurrence. Therefore, new antimicrobial drugs are needed to address this threat.

[0006] Prior art studies have found that heterocyclic carbene metal complexes with specific structures have antibacterial activity. For example, Chinese patent application CN 118852216 A discloses a large sterically hindered heterocyclic carbene gold (I) complex with antibacterial activity, and an inhibitory effect on Salmonella typhimurium; Claudia Schmidt et al. disclosed that heterocyclic carbene gold (I) complexes have excellent antibacterial activity against Gram-positive bacteria, but weak antibacterial activity against Gram-negative bacteria, and the best MIC is 37-42 μM (Schmidt, Claudia; Karge, Bianka; Misgeld, Rainer; et al. Gold (I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-Positive Directed Antibacterial Effects. [J] Chemistry-A European Journal, 2017). However, the above-mentioned prior art does not disclose whether it also has a killing or inhibitory effect on intracellular bacteria, and it is well known to those skilled in the art that in addition to the properties of the metal center and its reactive characteristics, the overall chemical structure of the NHC-metal complex will also affect the biological behavior of each NHC. Therefore, not all NHC-metal complex structure compounds have excellent antibacterial properties, and some NHC-metal complex structure compounds are more toxic to cells.

[0007] Based on the above technical problems that need to be solved urgently, how to make antibiotics reach the location of intracellular pathogens more efficiently, and at the same time utilize the environmental characteristics of intracellular bacteria to kill intracellular bacteria with low cytotoxicity, so as to solve the clinical challenges that existing technologies have long faced, has become one of the key points in the research and development of new antibiotics. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art and provide the application of N-heterocyclic carbene-gold (I)-benzyne complex in sterilization or preparation of antibacterial agent.

[0009] Another object of the present invention is to provide the use of N-heterocyclic carbene-gold (I)-benzyne complex in the preparation of drugs for treating intracellular bacterial infections.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention protects the use of N-heterocyclic carbene-gold (I)-benzyne complexes in sterilization or preparation of antibacterial agents. The structure of the N-heterocyclic carbene-gold (I)-benzyne complex is shown in 1a:

[0012]

[0013] The sterilization is carried out under acidic conditions; the bactericide is used under acidic conditions.

[0014] The present invention has found through a large number of studies that compound 1a has excellent bactericidal effect under acidic conditions.

[0015] Preferably, the acidic condition is pH ≤ 5.8, under which compound 1a can be activated by acid, while pH > 5.8 may result in failure of acid activation of compound 1a.

[0016] Preferably, the antibacterial spectrum of the bactericidal or bacteriostatic agent includes Gram-negative bacteria.

[0017] Preferably, the Gram-negative bacteria are Salmonella bacteria.

[0018] Preferably, the antibacterial agent further comprises a pharmaceutically acceptable excipient.

[0019] Preferably, the pharmaceutically acceptable excipient is one or more of a dispersant, a wetting agent, a disintegrant, a binder, a defoaming agent, an antifreeze agent, a thickener, a filler and a solvent.

[0020] Preferably, the dosage form of the antibacterial agent can be an aqueous solution, a soluble powder, a water-dispersible granule, or a soluble liquid.

[0021] Furthermore, the present invention protects the use of an N-heterocyclic carbene-gold (I)-benzyne complex in the preparation of a drug for treating intracellular bacterial infection. The structure of the N-heterocyclic carbene-gold (I)-benzyne complex is shown in 1a:

[0022]

[0023] N-heterocyclic carbene-gold (I)-benzyne complexes can effectively enter cells and be activated by the acidic environment within cells to produce active complexes, thereby inhibiting the proliferation of intracellular bacteria within cells. At the same time, compared with existing drugs, their cytotoxicity is significantly reduced.

[0024] Preferably, the intracellular bacteria are Gram-negative bacteria.

[0025] Preferably, the medicine further contains pharmaceutically acceptable excipients.

[0026] The present invention has the following beneficial effects:

[0027] The N-heterocyclic carbene-gold (I)-benzyne complex of the present invention has good antibacterial activity, and its minimum inhibitory concentration (MIC) is less than 5 μM. At the same time, the study found that the N-heterocyclic carbene-gold (I)-benzyne complex of the present invention can enter the cell, use the acidic environment in the cell to inhibit the growth and reproduction of intracellular bacteria, and the cytotoxicity is significantly reduced compared with existing drugs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a chromatogram of the stability study of the compound 1a of the present invention in acidic LPM medium (acid activation).

[0029] Figure 2 This is a comparison chart of the fluorescence intensity of the coumarin precursor probe of the present invention for detecting the generation of active gold in compound 1a and NHC-Au-L similar complex NHC-Au-Cl in different pH environments.

[0030] Figure 3 The graph shows the changes in the minimum inhibitory concentration (MIC) of compound 1a of the present invention and its NHC-Au-L analog complex NHC-Au-Cl against Salmonella typhimurium (STm) cultured in LPM medium with different component contents.

[0031] Figure 4 The graph is a determination of the bactericidal ability of the compound 1a of the present invention and antibiotics against dormant-like Salmonella typhimurium (STm).

[0032] Figure 5The graph shows the anti-intracellular bacterial activity of compound 1a of the present invention against macrophages infected with Salmonella typhimurium (STm). DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] The CAS number of NHC-Au-Cl described in the examples of the present invention is 847755-59-3; Auranofin is a clinically approved drug for marketing, and is used as a control group for antibacterial efficacy.

[0035] The LPM medium described in the embodiment of the present invention (formula: 5mM KCl, 7.5mM (NH4)2SO4, 0.5mM K2SO4, 10mM Glucose, 49μM MgCl2, 337μM KH2PO4, 0.05% Casamino acids, 80mM MES (2-morpholineethanesulfonic acid), pH 5.8) is a culture medium that simulates the environment of vesicles (SCVs) containing Salmonella typhimurium, that is, simulates the intracellular environment of macrophages, and has the characteristics of low magnesium, low phosphate and low pH.

[0036] M9 medium: minimum nutrient medium, formula: 6.78g / L Na2HPO4, 3g / L KH2PO4, 0.5g / L NaCl, 4g / L Glucose, 1g / L NH4Cl, 0.493g / L MgSO4·7H2O, 0.011g / L CaCl2.

[0037] In the application example of the present invention, the Salmonella typhimurium is S. Typhimurium ATCC 14028 (STm).

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Synthesis of compounds of Example 1 and Comparative Examples 1 to 4

[0040] The synthetic route of the compound is as follows:

[0041]

[0042] The specific synthesis method of compound 1a in Example 1 is as follows:

[0043] S1. Take 0.2 g of 1,3-dimethylimidazole chloride and dissolve it in dichloromethane (DCM), add 0.1 g of silver oxide, and react at room temperature in the dark for 3 h; then gradually add 0.26 g of dimethyl sulfide gold chloride solution dissolved in dichloromethane, and continue to react at room temperature for 3 h; then reduce the pressure and filter, concentrate the solvent, add n-pentane to precipitate it, wash it, and drain it to obtain the intermediate product compound I;

[0044] S2. Take 66 mg of compound I, 25 mg of phenylacetylene and 54 mg of sodium methoxide and dissolve them in 15 mL of ethanol. React at 80°C for 2 h. Then spin dry the solvent. Add dichloromethane (DCM) to dissolve the solid, filter under reduced pressure, wash with n-pentane and dry it to obtain the target product 1a.

[0045] The specific synthesis method of compound 1c in comparative example 1 is different from that of compound 1a in that 1,3-dimethylimidazole chloride is replaced by 1,3-dibutylimidazole chloride.

[0046] Comparative Example 2 Compound 1i, the specific synthesis method is different from that of Compound 1a in that phenylacetylene is replaced by 4-(4-ethynylphenyl)pyridine.

[0047] The specific synthesis method of compound 1b in comparative example 3 is:

[0048] S1. Take 0.22g of 2,6-diisopropylphenyl-1,3-imidazole chloride and 0.15g of dimethyl sulfide gold chloride and dissolve them in acetone, stir and react at 60°C for 10min, then add 0.05g of sodium acetate, continue to react at 60°C for 50min, and spin dry the solvent. Add dichloromethane (DCM) to dissolve the solid, filter under reduced pressure with diatomaceous earth, wash with n-pentane and dry, and obtain 2,6-diisopropylphenyl-1,3-imidazole gold chloride;

[0049] S2. Take 0.11 g of 2,6-diisopropylphenyl-1,3-imidazole gold chloride, 0.04 g of benzylene and 0.04 g of sodium acetate, dissolve them in ethanol, react at room temperature for 6 hours, and then spin-dry the solvent; add dichloromethane (DCM) to dissolve the solid, filter under reduced pressure, wash with n-pentane and dry it to obtain the target product compound 1b.

[0050] Comparative Example 4 Synthesis method of compound 1m: Take 0.05 g of the product 2,6-diisopropylphenyl-1,3-imidazole gold chloride obtained in step S1 of compound 1b, 0.01 g of phenylboric acid and 0.033 g of potassium carbonate, dissolve them in ethanol, and react at 35°C for 16 hours; after the reaction is completed, spin-dry the solvent, add dichloromethane (DCM) to dissolve the solid, filter under reduced pressure, add n-pentane to wash, and finally spin-dry to obtain the target product compound 1m.

[0051] Table 1

[0052]

[0053]

[0054] Application Example 1

[0055] This application example studies the antibacterial activity of the compounds of Example 1 and Comparative Examples 1 to 4, and the specific process is as follows:

[0056] A single colony of Salmonella typhimurium (STm) was selected from LB solid medium and cultured in M9 medium overnight. The next day, the culture was expanded at a ratio of 1:100 to the logarithmic growth phase OD 600 After the value reached 0.5, the mixture was inoculated into M9 medium containing 1 or 5 μM compound at a ratio of 1:1000 and cultured for 24 hours. Finally, the effects of different compounds on bacterial growth in the culture medium were observed at different concentrations. If bacteria grow, the bacterial solution is turbid; if bacteria do not grow, the bacterial solution is clear.

[0057] The results showed that when the concentration of compound 1a was 5 μM, the bacterial solution containing bacteria was clear, which means that the bacteria did not grow at a concentration of 5 μM compound 1a; while the bacteria could still grow at 1 or 5 μM of compounds 1b, 1c, 1i, and 1m, and the bacterial solution was turbid, showing ineffective antibacterial activity.

[0058] The structure-activity analysis of the N-heterocyclic carbene-gold (I)-benzyne complexes and their derivatives screened above showed that if the N substituent on N-heterocyclic carbene (NHC) is too large or the conjugated ring of NHC is destroyed, the antibacterial activity will be lost (MIC>5μM), while when the substituent on NHC is small, the antibacterial activity is strong. In addition, when the complex structure is planar, the antibacterial activity is strong; when it is spherical or irregular, the antibacterial activity is weak.

[0059] Application Example 2

[0060] This application example studies the cytotoxicity of compound 1a, NHC-Au-Cl (CAS No.: 847755-59-3) and the clinically marketed drug Auranofin in Example 1, and simultaneously determines the minimum inhibitory concentration (MIC). The specific process is as follows:

[0061] Cytotoxicity to THP-1 (human monocytic leukemia cell) suspension cells: determined by CCK-8 colorimetry. 4 THP-1 cells were evenly plated in a 96-well plate with 50 μL per well. Each test compound was diluted in half to the required concentration in advance, and then 50 μL of the test compound of different concentrations was added to each well. After 48 hours of administration, 10 μL of CCK-8 solution was added to each well, incubated at 37°C for 2-3 hours, and the absorbance of each well was measured at 450 nm after shaking for 1 minute to calculate the IC of different compounds on cells. 50.

[0062] Cytotoxicity to M0 macrophages differentiated from THP-1 cells induced by PMA (phorbol) was determined by MTT colorimetry. THP-1 cells were plated at 2×10 4 100 nM PMA was added to induce 24 h to differentiate THP-1 cells into M0 macrophages. Then, the cell culture medium containing PMA was removed and new culture medium was added for 24 h of relaxation culture. Different gold (I) complexes were diluted in half and added to the corresponding wells of the 96-well plate. After 24 h of administration, 20 μL of MTT (MTT concentration was 5 mg / mL dissolved in PBS) was added to each well. After incubation at 37 °C for 4 h, the MTT mixture was discarded, 130 μL of DMSO was added to each well, and the absorbance of each well was measured at 490 nm after shaking for 1 min to calculate the IC of different compounds on cells. 50 .

[0063] Cytotoxicity to PBMC (human peripheral blood mononuclear cells): Kit (ThermoFisher, Cat. No. C35011). 5 PBMC cells were evenly plated in a 96-well plate with 50 μL per well. Each test compound was diluted in half to the required concentration in advance, and then 50 μL of the test compound of different concentrations was added to each well. After 72 hours of administration, the test compounds were divided into two groups according to the following formula: The test was performed according to the kit instructions, and the IC of different compounds on cells was calculated. 50 .

[0064] The experimental results are shown in Table 1. Compound 1a has a higher IC than NHC-Au-Cl (CAS No.: 847755-59-3) and the clinically listed drug Auranofin. 50 This indicates that compound 1a has lower toxicity to cells.

[0065] Table 1 IC of different compounds on cells 50 (μM) value

[0066]

[0067] MIC determination: STm (S. Typhimurium ATCC 14028) monoclones were picked from LB solid medium and cultured in M9 or LPM medium overnight, and then expanded at 1:100 to the logarithmic growth phase OD 600 After the value was 0.5, it was inoculated into M9 medium containing half-gradient dilution concentrations of the test compound at a ratio of 1:1000 and cultured for 24 h. Finally, the OD of each sample was measured. 600Observe the growth of bacteria, the clarification of bacterial solution or OD 600 The lowest concentration with a value of 0 is the minimum inhibitory concentration (MIC) of the compound. As shown in Table 2, compound 1a of the present invention has strong antibacterial activity, and its MIC is much lower than that of the existing drug Auranofin, which is basically consistent with the antibacterial activity of NHC-Au-Cl compound, and its cytotoxicity is much lower than that of NHC-Au-Cl and Auranofin.

[0068] Table 2 MIC (μM) values ​​of different compounds against bacteria

[0069]

[0070] Application Example 3

[0071] This application example studies the stability (acid activation) changes of compound 1a in acidic LPM medium. The specific process is as follows:

[0072] Add 20 μM compound 1a to LPM medium, mix well and incubate with shaking at 37°C. Pipette 1 mL at 0 and 2 h, filter with 0.22 μm filter membrane and use ultra-high performance liquid chromatography (UPLC) for detection. The mobile phase used CH3CN:H2O=90%:10%; the detection wavelength was 254 nm, the chromatographic column was octadecyl bonded silica gel column (Cosmosil 5C18-MS-II chromatographic column, 5 μm, 4.6x250 mm); flow rate: 1 mL / min; column temperature: room temperature. The control group was DMSO (LPM) and NHC-Au-Cl at concentrations of 20 and 200 μM. Figure 1 As shown in the figure, after 1a was incubated in LPM medium for 2 hours, the peak of compound 1a completely disappeared at 254nm, and a new peak with a retention time of 7.73min was generated. After NHC-Au-Cl with different concentrations was subjected to UPLC, a peak of its own was also generated at 7.77min, indicating that compound 1a was activated in the acidic LPM medium and produced NHC-Au-L similar complex NHC-Au-Cl.

[0073] Application Example 4

[0074] This application example uses a coumarin precursor probe (probe-1) to measure and compare the fluorescence intensity of compound 1a and NHC-Au-Cl in different pH environments. The specific process is as follows:

[0075] 20 μM 1a or NHC-Au-Cl was added to different culture media [LPM culture medium (pH=5.8), LPM culture medium (pH=7.10); M9 culture medium (pH=7.1), M9 culture medium (pH=5.88, the solution for adjusting pH was HCl), M9 culture medium (pH=5.87, the solution for adjusting pH was H2SO4)], and incubated at 37°C for 2 h; 500 μL of the above culture medium was respectively aspirated, 500 μL of CH3CN was added at a ratio of 1:1, and then 100 μM probe-1 was added, and the culture media were incubated with shaking at 37°C for 16 h, and the fluorescence intensity was measured at excitation wavelength / emission wavelength = 415 nm / 530 nm.

[0076] The coumarin precursor probe (probe-1) responds to active gold to produce fluorescence, but the gold in compound 1a is not active gold, and the fluorescence intensity produced by the probe response is weak; although NHC-Au-Cl is active gold, its chloride ions need to leave to expose Au (I) in order for probe-1 to produce obvious fluorescence. Therefore, in LPM medium with low chloride ion concentration, chloride ions are easy to leave, and the fluorescence intensity of probe-1 in LPM medium is high. However, the chloride ion concentration in M9 medium is relatively high, resulting in weak fluorescence intensity of NHC-Au-Cl even in acidic M9 medium. Figure 2 It can be seen that in the acidic LPM medium (pH = 5.8), 1a and NHC-Au-Cl can both activate probe-1, and the fluorescence intensity is similar. After the pH of the LPM medium is adjusted to neutral (pH = 7.10), the fluorescence intensity of 1a and NHC-Au-Cl activated probe-1 decreases, indicating that 1a can be activated under the acidic conditions in the LPM medium and produces high fluorescence intensity. The same results were obtained in the M9 medium, but because of the influence of more other ions in the M9 medium, the fluorescence intensity of 1a and NHC-Au-Cl was weak, especially at pH 7, the fluorescence intensity of compound 1a was extremely weak, specifically 588, at Figure 2 It is not reflected in the figure, NHC-Au-Cl is 4248.

[0077] Application Example 5

[0078] This application example studies the MIC changes of compound 1a and NHC-Au-Cl on STm cultured in LPM medium with different component contents. The specific process is as follows:

[0079] STm single clones were picked from LB solid medium and transferred to LPM medium supplemented with different medium components (containing 10 mM Mg 2+ LPM medium; containing 10mM KH2PO4 3-The cells were cultured overnight in LPM medium with pH 7.1, LPM medium with pH 5.8, and expanded in the same medium at a ratio of 1:100 to the logarithmic growth phase OD 600 After the value reached 0.5, the samples were inoculated into the same medium containing half-gradient dilutions of the test compound at a ratio of 1:1000 and cultured for 24 h. Finally, the OD of each sample was measured. 600 The MIC of the compound to be tested is calculated. Figure 3 The MIC of 1a against STm cultured in LPM medium at pH 7.1 increased, while the group supplemented with magnesium ions and phosphate did not have this phenomenon, indicating that the antibacterial activity of 1a is closely related to the pH in the environment.

[0080] Application Example 6

[0081] This application example studies the bactericidal ability of compound 1a, NHC-Au-Cl and different commercially available antibiotics against dormant Salmonella typhimurium (STm). The specific process is as follows:

[0082] Pick a single STm clone from the LB solid medium and culture it in MHB medium overnight. The next day, expand the culture at a ratio of 1:100 until the OD 600 The pH value was 0.4-0.5, the supernatant was discarded after centrifugation, and the bacterial precipitate was washed twice with PBS; then it was resuspended with PBS, and the bacteria were starved at 37°C for 24 hours, which was the quasi-dormant STm; the PBS containing the quasi-dormant STm was evenly divided, and 20 μM 1a, NHC-Au-Cl, Auranofin, Ampicillin, Kanamycin and Ciprofloxacin were added respectively, and incubated at 37°C with shaking; the bacterial solution was aspirated at regular intervals (i.e. 0, 2, 6, 18 and 24 hours), the supernatant was discarded after centrifugation, the bacterial precipitate was washed 3 times with PBS and then resuspended with PBS, and after gradient dilution, 5 μL of the bacterial solution was aspirated for spot plate observation of colony forming units (CFU) (each group and each time period were repeated 3 times in parallel).

[0083] The results are as follows Figure 4 Compound 1a and NHC-Au-Cl can effectively kill dormant STm, and their antibacterial activity is significantly better than that of commercially available antibiotics. In addition, NHC-Au-Cl exerts its bactericidal effect faster than 1a because 1a needs to undergo an acidic activation process before it can exert its bactericidal effect. The above results indicate that 1a has the potential to fight dormant intracellular bacteria.

[0084] Application Example 7

[0085] This application example studies the anti-intracellular bacterial activity of compound 1a, NHC-Au-Cl, and Auranofin against STm-infected macrophages. The specific process is as follows:

[0086] THP-1 cells were grown at 2.4 × 10 5 The cells were seeded into 24-well plates, 100 nM PMA (phorbol) was added for induction for 24 h to differentiate THP-1 cells into macrophages, and then the cell culture medium containing PMA was removed and a new culture medium was added for relaxation culture for 24 h; STm monoclonal clones were picked from LB solid culture medium and cultured overnight in RPMI 1640 culture medium containing 10% fetal bovine serum (FBS). 600 Calculate the number of STm in each mL of bacterial solution, dilute to the required number of bacteria, infect macrophages at an infection ratio (MOI) of 50:1, pipette the above-mentioned RPMI 1640 medium containing 10% FBS containing STm according to the required volume, and then dilute it with 1640 medium containing 10% FBS; remove the RPMI 1640 medium containing 10% FBS in the macrophages, add PBS to wash once, then remove it again, add 400 μL of the above bacterial solution to each well to make it contact with the macrophages as much as possible, and then infect for 30 minutes; after infection, discard all the supernatant, add RPMI 10% FBS containing 100 μg / mL gentamicin, After culturing in 1640 medium for 30 minutes, the cells were divided into two groups: ① one group directly lysed the cells to obtain intracellular bacteria, which were diluted and plated to obtain CFU / mL, recorded as T0 (i.e., the number of bacteria invading macrophages after infection); ② the other group discarded the supernatant, washed once with PBS, and replaced with 50μM 1a, 50μM NHC-Au-Cl, 2.5μM Auranofin or an equal volume of DMSO containing 10% FBS. After culturing for 7 hours, the cells were lysed to obtain intracellular bacteria, which were diluted and plated to obtain CFU / mL, recorded as T7; in order to determine the number of intracellular Salmonella, the cell culture medium was discarded, washed once with cold PBS, and 1mL of 0.1% Triton was added to each well. X-100 was vigorously pipetted with cold PBS and incubated at 37°C for 10 min to lyse the cells. The resulting solution was gradiently diluted with PBS, and 200 μL was evenly applied on LB solid culture medium. After incubation at 37°C overnight, the number of colonies on the plate was counted, and the colony forming unit (CFU) per mL was calculated.

[0087] like Figure 5 As shown, 1a and NHC-Au-Cl can significantly inhibit the growth of intracellular bacteria and have strong anti-intracellular bacterial activity, while the clinically available drug Auranofin has poor anti-intracellular bacterial activity.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of N-heterocyclic carbene-gold (I)-benzyne complex in sterilization, characterized in that: The structure of the N-heterocyclic carbene-gold (I)-benzyne complex is shown in 1a: The sterilization is carried out under acidic conditions.

2. Application of N-heterocyclic carbene-gold (I)-benzyne complex in the preparation of antibacterial agent, characterized in that: The structure of the N-heterocyclic carbene-gold (I)-benzyne complex is shown in 1a: The bactericide is used under acidic conditions.

3. The use according to claim 1 or 2, characterized in that: The acidic condition is pH ≤ 5.

8.

4. The use according to claim 1 or 2, characterized in that: The antibacterial spectrum of the bactericidal or bacteriostatic agent includes Gram-negative bacteria.

5. The application according to claim 4, characterized in that: The Gram-negative bacteria are Salmonella bacteria.

6. The use according to claim 2, characterized in that: The antibacterial agent also includes pharmaceutically acceptable excipients.

7. The use according to claim 2, characterized in that: The dosage form of the antibacterial agent can be aqueous solution, soluble powder, water dispersible granules, or soluble liquid.

8. Use of N-heterocyclic carbene-gold (I)-benzyne complexes in the preparation of drugs for treating intracellular bacterial infections, characterized in that: The structure of the N-heterocyclic carbene-gold (I)-benzyne complex is shown in 1a:

9. The use according to claim 8, characterized in that: The intracellular bacteria are Gram-negative bacteria.

10. The use according to claim 8, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

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