N-heterocyclic carbene-gold (i)-phenylalkyne complex and its antibacterial application

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an N-heterocyclic carbene-gold (I)-phenyl alkyne complex and antibacterial application thereof. The N-heterocyclic carbene-gold (I)-phenyl alkyne complex has good bacteriostatic activity, and the minimum inhibitory concentration (MIC) is less than 5 muM. Meanwhile, it is found that the N-heterocyclic carbene-gold (I)-phenyl alkyne complex can enter cells, inhibit the growth and reproduction of intracellular bacteria by using the acidic environment in cells, and the cytotoxicity is significantly reduced compared with existing drugs, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a N-heterocyclic carbene-gold(I)-phenylalkyne complex and its antibacterial application. BACKGROUND

[0002] For more than 90 years since the discovery of penicillin in 1928, antibacterial drugs, especially antibiotics, have played a vital role in modern medicine. However, Gram-negative bacteria (G - -bacteria) are resistant to many antibiotics due to their double-membrane structure, complex carbohydrate network, charged outer membrane, and efflux pumps. According to reports, no new class of antibiotics has been approved for treating G - -bacterial infections in the past 50 years. The last class of anti-G - -bacterial antibiotics is artificially synthesized quinolones. Therefore, there is an urgent need to develop new drugs for treating G-bacterial infections.

[0003] More and more researches focus on the unique lifestyle of bacteria to avoid antibiotic killing and escape the host immune system. According to the difference of the infection site, bacteria can be divided into extracellular bacteria and intracellular bacteria. Extracellular bacteria mainly survive and multiply in the extracellular environment of the body, damaging the tissues and organs of the host; while intracellular bacteria refer to bacteria that invade the host cells through their own virulence system and survive and grow in them to avoid the adverse extracellular environment. Intracellular bacteria can be further divided into bacteria that reside in modified phagosomes (Salmonella, Mycobacterium, Francisella), inclusion bodies (Chlamydia), lysosomes (Legionella, Coxiella), or cytoplasm (Listeria, Shigella) according to the location of bacteria in the cell. Due to the characteristics of intracellular bacteria to escape the host immune system and avoid antibiotic killing, compared with extracellular bacteria, intracellular bacteria are a difficult problem faced today.

[0004] Salmonella enterica is a G - -bacterium that can invade cells to infect humans and animals, which belongs to the Enterobacteriaceae family and can cause fever, chills, abdominal pain, diarrhea, etc. after infection. Salmonellosis affects about 200 million people worldwide, causing more than 200,000 deaths each year; according to the data of 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 infection can cause persistent infection, reinfection, and long-term carriage. Fluoroquinolones, cephalosporins, or macrolide antibiotics are commonly used in clinical treatment of Salmonella-induced infections.

[0005] Salmonella enterica serovar Typhimurium (STm) is a typical intracellular pathogen. During the infection of macrophages and neutrophils, STm forms Salmonella-containing vacuoles (SCVs) intracellularly, which can escape extracellular host immune defenses (serum complement, antibodies, bile acids, antimicrobial peptides, etc.) and antibiotics (for example, the first-line drug of STm, ciprofloxacin, is negatively charged, making it difficult to penetrate the two layers of cell membranes inside the host cells to exert antibacterial action at SCVs). Importantly, intracellular STm can also form antibiotic-tolerant persisters (a type of bacteria with weak metabolic activity but not proliferation in the cell, similar to a dormant state, which are not easily killed by antibiotics), which can easily lead to chronic infection and disease recurrence. Therefore, new antibacterial drugs are needed to address this threat.

[0006] Prior art research has found that heterocyclic carbene metal complexes with specific structures have antibacterial activity. For example, Chinese patent application CN 118852216 A discloses that a large steric heterocyclic carbene gold(I) complex has antibacterial activity and inhibits 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, with an optimal MIC of 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 prior art does not disclose whether the complexes have the same killing or inhibiting effect on intracellular bacteria. As is known to those skilled in the art, in addition to the nature of the metal center and its reactivity characteristics, the overall chemical structure of the NHC-metal complex also affects the biological behavior of each NHC. Therefore, not all NHC-metal complex structural compounds have excellent antibacterial performance, and some NHC-metal complex structural compounds have high cytotoxicity.

[0007] Based on the above technical problems to be solved urgently, how to make the antibacterial drugs more efficient to reach the intracellular pathogenic bacteria, and at the same time use the environmental characteristics of intracellular bacteria to kill intracellular bacteria, and have low cytotoxicity, so as to solve the long-term clinical challenges faced by the prior art, has become one of the key points of developing new antibacterial drugs. SUMMARY

[0008] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the prior art, and to provide the application of N-heterocyclic carbene-gold(I)-phenyl alkyne complex in bactericidal or preparation of antibacterial agents.

[0009] Another object of the present application is to provide the application of N-heterocyclic carbene-gold(I)-phenyl alkyne complex in the preparation of drugs for treating intracellular bacterial infection.

[0010] The above object of the present application is achieved by the following technical scheme:

[0011] The present application protects the application of N-heterocyclic carbene-gold(I)-phenyl alkyne complex in bactericidal or preparation of antibacterial agents, and the structure of the N-heterocyclic carbene-gold(I)-phenyl alkyne complex is shown in 1a:

[0012]

[0013] The bactericidal is under acidic conditions; the bactericide is used under acidic conditions.

[0014] The present application finds that compound 1a has excellent bactericidal effect under acidic conditions through a large number of researches.

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

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

[0017] Preferably, the gram-negative bacteria is Salmonella bacteria.

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

[0019] Preferably, the pharmaceutically acceptable excipient is one or more of dispersants, wetting agents, disintegrants, binders, antifoaming agents, antifreezing agents, thickening agents, fillers and solvents.

[0020] Preferably, the dosage form of the bacteriostatic agent can be aqueous agent, soluble powder, water-dispersible granules, soluble liquid.

[0021] Further, the application protects the use of N-heterocyclic carbene-gold(I)-phenyl alkyne complex in the preparation of a drug for treating intracellular bacterial infection, wherein the N-heterocyclic carbene-gold(I)-phenyl alkyne complex has the structure shown in 1a:

[0022]

[0023] The N-heterocyclic carbene-gold(I)-phenyl alkyne complex can effectively enter cells, and is activated by the acidic environment in the cells to produce an active complex, thereby inhibiting the proliferation of intracellular bacteria in the cells, and the cytotoxicity of the N-heterocyclic carbene-gold(I)-phenyl alkyne complex is significantly reduced compared with existing drugs.

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

[0025] Preferably, the drug further contains a pharmaceutically acceptable excipient.

[0026] The application has the following beneficial effects:

[0027] The N-heterocyclic carbene-gold(I)-phenyl alkyne complex has good bacteriostatic activity, and the minimum inhibitory concentration (MIC) is less than 5 μM. At the same time, it is found that the N-heterocyclic carbene-gold(I)-phenyl alkyne complex can enter cells, utilize the acidic environment in the cells to inhibit the growth and reproduction of intracellular bacteria, and the cytotoxicity is significantly reduced compared with existing drugs, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The chromatogram is for the stability (acidic activation) of the compound 1a of the application in the LPM culture medium with acid.

[0029] Figure 2 The fluorescence intensity contrast chart is for the generation of active gold of the compound 1a and the NHC-Au-L similar complex NHC-Au-Cl of the application in different pH environments.

[0030] Figure 3 The minimum inhibitory concentration (MIC) change chart is for the compound 1a and the NHC-Au-L similar complex NHC-Au-Cl of the application to the Salmonella typhimurium (STm) cultured in the LPM culture medium with different component contents.

[0031] Figure 4 The bactericidal ability determination chart is for the compound 1a and the antibiotic to the Salmonella typhimurium (STm) in a dormant state.

[0032] Figure 5Figure 1 shows the anti-intracellular bacterial activity of compound 1a of the present application against S. Typhimurium (STm)-infected macrophages. DETAILED DESCRIPTION

[0033] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. Unless otherwise specified, the reagents, methods and apparatus used in the following examples are those conventional in the art.

[0034] The CAS number of NHC-Au-Cl described in the embodiments of the present application is 847755-59-3; Auranofin (Goldin) is a clinically approved drug, which is used as a control group for antibacterial efficacy.

[0035] The LPM medium (formula: 5mM KCl, 7.5mM (NH4)2SO4, 0.5mM K2SO4, 10mM Glucose, 49μM MgCl2, 337μM KH2PO4, 0.05% Casamino acids, 80mM MES (2-morpholinoethanesulfonic acid), pH 5.8) described in the embodiments of the present application is a medium that simulates the environment of Salmonella-containing vacuoles (SCVs) containing S. Typhimurium, i.e. simulates the intracellular environment of macrophages, which has the characteristics of low magnesium, low phosphate and low pH.

[0036] M9 medium: minimal 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 examples of the present application, the S. 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 1-4

[0040] The synthesis route of the compounds is as follows:

[0041]

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

[0043] S1. Take 0.2 g of chlorinated 1,3-dimethyl imidazole dissolved 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 filter under reduced pressure, concentrate the solvent, precipitate by adding n-pentane, wash, and dry, 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 dissolved in 15 mL of ethanol, and react at 80°C for 2 h; then spin dry the solvent; dissolve the solid in dichloromethane (DCM), filter under reduced pressure, wash with n-pentane, and dry, to obtain the target product 1a.

[0045] Comparative Example 1 Compound 1c, the difference between the specific synthesis method and that of compound 1a is that chlorinated 1,3-dimethyl imidazole is replaced by 1,3-dibutyl imidazole chloride salt.

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

[0047] Comparative Example 3 Compound 1b, the specific synthesis method is as follows:

[0048] S1. Take 0.22 g of 2,6-diisopropylphenyl-1,3-imidazole chloride salt and 0.15 g of dimethyl sulfide gold chloride dissolved in acetone, and stir-react at 60°C for 10 min; then add 0.05 g of sodium acetate, and continue to react at 60°C for 50 min; spin dry the solvent. Dissolve the solid in dichloromethane (DCM), filter under reduced pressure with diatomite, wash with n-pentane, and dry, to 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 phenylacetylene, and 0.04 g of sodium acetate dissolved in ethanol, and react at room temperature for 6 h; then spin dry the solvent; dissolve the solid in dichloromethane (DCM), filter under reduced pressure, wash with n-pentane, and dry, to obtain the target product compound 1b.

[0050] Comparative Example 4 Compound 1m, the synthesis method is as follows: take 0.05 g of 2,6-diisopropylphenyl-1,3-imidazole gold chloride obtained in the S1 step of compound 1b, 0.01 g of phenylboronic acid, and 0.033 g of potassium carbonate dissolved in ethanol, and react at 35°C for 16 h; after the reaction is complete, spin dry the solvent, dissolve the solid in dichloromethane (DCM), filter under reduced pressure, wash with n-pentane, 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 1-4, and the specific process is as follows:

[0056] Salmonella typhimurium (STm) monoclonal was picked from LB solid medium and cultured in M9 medium overnight, the next day it was expanded to the logarithmic growth phase OD 600 600=0.5, then inoculated into M9 medium containing 1 or 5 μM of the compound at a ratio of 1:1000 and cultured for 24 h, and finally the effect of different concentrations of different compounds on the growth of bacteria in the medium was observed. Bacterial growth, the bacterial solution was turbid; no bacterial growth, the bacterial solution was clear.

[0057] The results show that at a concentration of 5 μM, the bacterial solution containing compound 1a is clear, i.e. the bacteria do not grow at a concentration of 5 μM of compound 1a; while compounds 1b, 1c, 1i and 1m still grow at 1 or 5 μM, the bacterial solution is turbid, showing no antibacterial activity.

[0058] Structure-activity analysis of the N-heterocyclic carbene-gold (I)-phenyl alkyne complex and its derivatives screened above: if the N substituent on the N-heterocyclic carbene (NHC) is too large or the conjugated ring of the NHC is destroyed, the antibacterial activity (MIC>5 μM) will be lost, and when the substituent on the NHC is small, the antibacterial activity is strong. In addition, 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 in Example 1, NHC-Au-Cl (CAS No.: 847755-59-3) and the clinically marketed drug Auranofin, and simultaneously determines the minimum inhibitory concentration (MIC), and the specific process is as follows:

[0061] Cytotoxicity to THP-1 (human monocytic leukemia cells) suspension cells: CCK-8 colorimetric method was used. 2×10 4 THP-1 cells were evenly plated in a 96-well plate at 50 μL per well, and the test compounds were diluted to the required concentration in advance, then 50 μL of the test compound at different concentrations was added to each well; 48 h after administration, 10 μL of CCK-8 solution was added to each well, incubated at 37°C for 2-3 h, shaken for 1 min, and the absorbance of each well was measured at 450 nm, and the IC 50.

[0062] Cytotoxicity on PMA (Phorbol Myristate Acetate) induced THP-1 cells to differentiate into M0 type macrophages: MTT colorimetric method was used. THP-1 cells were evenly spread in 96-well plates at 2 x 10 4 cells per well, 100 nM PMA was added to induce THP-1 cells to differentiate into M0 type macrophages for 24 h, then the PMA-containing cell culture medium was removed and new culture medium was added for relaxation culture for 24 h; different gold (I) complexes were added to the corresponding wells of the 96-well plate after half-gradient dilution; 20 μL of MTT (5 mg / mL MTT was dissolved in PBS) was added to each well after 24 h of administration, and incubation was carried out at 37 °C for 4 h, then 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, and the IC 50 .

[0063] Cytotoxicity on PBMC (human peripheral blood mononuclear cells): was determined by using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Cat# G3580). PBMC cells were evenly spread in 96-well plates at 1 x 10 5 cells per well, and 50 μL of each test compound was added to each well after half-gradient dilution to the required concentration; 72 h after administration, detection was carried out according to the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Cat# G3580) instruction manual, and finally the IC of different compounds on cells was calculated. 50 .

[0064] The experimental results are shown in Table 1, and the IC 50 value of compound 1a is higher than that of NHC-Au-Cl (CAS No.: 847755-59-3) and Auranofin, a clinically marketed drug, i.e., the cytotoxicity of compound 1a is lower.

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

[0066]

[0067] MIC determination: STm (S. Typhimurium ATCC 14028) monoclonal was picked from LB solid culture medium and cultured in M9 or LPM culture medium overnight, and then expanded to the logarithmic growth phase with an OD 600 value of 0.5 at a ratio of 1:100, inoculated into M9 culture medium containing half-gradient dilution concentrations of test compounds at a ratio of 1:1000, and cultured for 24 h, and finally the OD 600, observe the growth of bacteria, the bacterial solution is clear or OD 600 The lowest concentration with OD value of 0 is the minimum inhibitory concentration (MIC) of the compound. As can be seen from the results in Table 2, the compound 1a of the present application has strong antibacterial activity, and its MIC is much lower than that of the existing drug Auranofin, and its antibacterial activity is basically the same as that of the 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 on bacteria

[0069]

[0070] Application Example 3

[0071] In this application example, the stability (acid activation) change of compound 1a in LPM medium with acid was studied, and the specific process was as follows:

[0072] 20 μM of compound 1a was added to LPM medium, and after mixing, 1 mL was taken at 0, 2 h, respectively, and then filtered with 0.22 μm membrane filter, and then detected by ultra performance liquid chromatography (UPLC) instrument. The mobile phase was CH3CN: H2O = 90%: 10%, the detection wavelength was 254 nm, the chromatographic column was carbon octadecyl bonded silica gel column (Cosmosil 5C18-MS-II chromatographic column, 5 μm, 4.6x250 mm), the flow rate was 1 mL / min, and the column temperature was room temperature. The control group was DMSO (LPM), and the concentration of NHC-Au-Cl was 20, 200 μM. As shown in Figure 1 After incubation of 1a in LPM medium for 2 h, the peak of compound 1a completely disappeared at 254 nm, and a new peak with a retention time of 7.73 min was generated. However, after UPLC of NHC-Au-Cl with different concentrations, a peak of its own at 7.77 min was also generated, indicating that compound 1a was activated in LPM medium with acid, and generated NHC-Au-L similar complex NHC-Au-Cl.

[0073] Application Example 4

[0074] In this application example, the fluorescence intensity of compound 1a and NHC-Au-Cl in different pH environments was determined and compared by using coumarin precursor probe (probe-1), and the specific process was as follows:

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

[0076] The coumarin precursor probe (probe-1) responds to active gold to produce fluorescence, and the gold in compound 1a is not active gold, and the fluorescence intensity produced by the probe in response is weak; although the active gold in NHC-Au-Cl, the chloride ion needs to be removed to expose Au (I) to make probe-1 produce obvious fluorescence. Therefore, in the LPM medium with low chloride ion concentration, the chloride ion is easy to remove, and the fluorescence intensity of probe-1 in the LPM medium is high. In the M9 medium, the chloride ion concentration is high, which leads to weak fluorescence intensity of NHC-Au-Cl even in acidic M9 medium. As shown in Table 1, in acidic LPM medium (pH = 5.8), 1a and NHC-Au-Cl can both activate probe-1, and the fluorescence intensity is similar, but when the pH of LPM medium is adjusted to neutral (pH = 7.10), the fluorescence intensity produced by 1a and NHC-Au-Cl to activate probe-1 is reduced, indicating that 1a can be activated under acidic conditions in LPM medium to produce high fluorescence intensity. The same results are obtained in M9 medium, but because in M9 medium there may be other ions and other reasons, the fluorescence intensity of 1a and NHC-Au-Cl is weak, especially when the pH is 7, the fluorescence intensity of compound 1a is extremely weak, which is not reflected in Table 1, and the fluorescence intensity of NHC-Au-Cl is 4248. Figure 2 Figure 2

[0077] Application Example 5

[0078] In this application example, the MIC changes of compound 1a and NHC-Au-Cl to STm cultured in LPM medium with different component contents were studied, and the specific process was as follows:

[0079] STm monoclonal was picked from LB solid medium and inoculated into LPM medium supplemented with different medium components (containing 10 mM Mg 2+ LPM medium containing 10 mM KH2PO4 3- ​​LPM medium (pH 7.1) overnight, and then subcultured in the same medium to the logarithmic growth phase (OD 600 0.5, and then inoculated in the same medium containing half-gradient dilution concentrations of the test compound at a ratio of 1:1000 for 24 h. Finally, the OD 600 value of each sample was determined, and the MIC of the test compound was calculated. As Figure 3 increased in the LPM medium at pH 7.1, but this phenomenon did not occur in the group supplemented with magnesium ions and phosphates, indicating that the antibacterial activity of 1a is closely related to the pH of 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 on the quasi-dormant Salmonella typhimurium (STm), and the specific process is as follows:

[0082] STm monoclonal was picked from LB solid medium and cultured overnight in MHB medium. The next day, it was subcultured at a ratio of 1:100 to OD 600 0.4-0.5, centrifuged to discard the supernatant, and the bacterial pellet was washed twice with PBS. Then, it was resuspended with PBS and starved for 24 h at 37°C with shaking, which was quasi-dormant STm. The PBS containing quasi-dormant STm was divided and 20 μM of 1a, NHC-Au-Cl, Auranofin, Ampicillin, Kanamycin, and Ciprofloxacin were added, respectively, and incubated at 37°C with shaking. Every other time (i.e., 0, 2, 6, 18, and 24 h), the bacterial solution was taken, centrifuged to discard the supernatant, and the bacterial pellet was washed three times with PBS and then resuspended with PBS. After gradient dilution, 5 μL of the bacterial solution was spotted on a plate to observe the colony-forming units (CFU) (each group was repeated three times at each time point).

[0083] The results are shown in Table 1. Figure 4 Compound 1a and NHC-Au-Cl can effectively kill quasi-dormant STm, and the antibacterial activity is significantly better than that of commercially available antibiotic drugs. In addition, NHC-Au-Cl exerts bactericidal effect faster than 1a because 1a needs to undergo an acid activation process to exert bactericidal effect. The above results show that 1a has potential against quasi-dormant intracellular bacteria.

[0084] Application Example 7

[0085] This application example studies the anti-intracellular bacterial activity of compounds 1a, NHC-Au-Cl, and auranofin against STm-infected macrophages. The specific procedures are as follows:

[0086] THP-1 cells were introduced at a rate of 2.4 × 10⁶ cells per well. 5 THP-1 cells were seeded into 24-well plates and induced to differentiate into macrophages by adding 100 nM PMA (phorbol) for 24 h. The PMA-containing culture medium was then removed, and fresh culture medium was added for relaxation and culture for 24 h. STm monoclonal antibodies were picked from LB solid medium and cultured overnight in RPMI 1640 medium containing 10% fetal bovine serum (FBS). The differentiation was then carried out according to OD... 600 Calculate the number of STm cells per mL of bacterial culture, dilute to the desired bacterial count, and infect macrophages at an infection ratio (MOI) of 50:1. Take the required volume of the above-mentioned STm-containing RPMI 1640 medium with 10% FBS, and then dilute it with RPMI 1640 medium containing 10% FBS. Remove the RPMI 1640 medium containing 10% FBS from the macrophages, wash once with PBS, and then remove it again. Add 400 μL of the above bacterial culture to each well, ensuring maximum contact with the macrophages, and infect for 30 min. After infection, discard all supernatant and add RPMI 1640 medium containing 100 μg / mL gentamicin with 10% FBS. After culturing in 1640 medium for 30 min, the cells were divided into two groups: ① One group was directly lysed to obtain intracellular bacteria, diluted and plated to obtain CFU / mL, denoted as T0 (i.e., the number of bacteria invading the macrophages after infection); ② The other group had its supernatant discarded, washed once with PBS, and then cultured in 1640 medium containing 10% FBS at concentrations of 50 μM 1a, 50 μM NHC-Au-Cl, 2.5 μM Auranofin, or an equal volume of DMSO for 7 h. Intracellular bacteria were then lysed and diluted and plated to obtain CFU / mL, denoted as T7. To determine the number of Salmonella in the cells, the cell culture medium was discarded, the cells were washed once with cold PBS, and 1 mL of 0.1% Triton [agent] was added to each well. The X-100 was vigorously pipetted into cold PBS and incubated at 37°C for 10 min to lyse the cells. The resulting solution was then serially diluted with PBS, and 200 μL was evenly spread onto LB solid medium. After incubation at 37°C overnight, the number of colonies on the plate was counted, and the colony forming units (CFU) per mL were 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 marketed drug Auranofin has poor anti-intracellular bacterial activity.

[0088] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. Use of N-heterocyclic carbene-gold (I)-phenylalkyne complexes for the preparation of antibacterial agents, characterized in that, The structure of the N-heterocyclic carbene-gold (I)-phenyl alkyne complex is shown in 1a: The antibacterial agent is used under acidic conditions.

2. Use according to claim 1, characterized in that, The acidic conditions are pH≤5.

8.

3. Use according to claim 1, characterized in that, The antibacterial spectrum of the antibacterial agent includes gram-negative bacteria.

4. Use according to claim 3, characterized in that, The gram-negative bacteria are Salmonella bacteria.

5. The use according to claim 1, characterized in that, The antibacterial agent further includes pharmaceutically acceptable excipients.

6. The use according to claim 1, characterized in that, The dosage form of the antibacterial agent can be aqueous agent, soluble powder, water dispersible granules.

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