Application of 5-fluoro-2 '-deoxycytidine in preparation of antibacterial drugs

Abstract

The invention provides an application of 5-fluoro-2 '-deoxycytidine (CDP-3) in the aspect of preparing an antibacterial agent. It is found for the first time that CDP-3 can inhibit synthesis of bacterial phosphatidylglycerol by targeting bacterial phosphatidylglycerol phosphate synthase, and the CDP-3 has antibacterial and broad-spectrum synergistic activity. The MIC of the compound on staphylococcus aureus and streptococcus is 4 mu g / mL and 8 mu g / mL respectively, the MIC 90 of the compound on methicillin-resistant staphylococcus aureus is 8 mu g / mL, and the MIC of the compound on high-toxicity klebsiella pneumoniae is 16 mu g / mL. In addition, the CDP-3 can significantly enhance the antibacterial efficacy of antibiotics such as clindamycin, tilmicosin, tetracycline, florfenicol, gentamicin, chloramphenicol, rifampicin and ofloxacin on multiple drug-resistant negative bacteria, and has broad-spectrum synergistic activity. CDP-3 with sub-inhibitory concentration not only can inhibit the growth of bacteria, but also can reduce the formation of bacterial biofilms and inhibit toxicity and pathogenicity. The CDP-3 not only improves the survival rate of MRSA infected greater wax moths, but also shows excellent treatment efficacy on animal models infected by multiple drug-resistant negative bacteria.

Description

Technical Field

[0001] The invention belongs to the field of antibacterial preparations, and specifically relates to the use of 5-fluoro-2'-deoxycytidine in the preparation of antibacterial drugs. Background Art

[0002] With the widespread use of antibiotics, the development of pathogen resistance has made the originally effective drugs gradually inefficient or even ineffective. In 2019, the number of deaths related to bacterial resistance reached 1.27 million worldwide, with the top mortality rates being Escherichia coli, Staphylococcus aureus, Streptococcus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Mycobacterium tuberculosis (Lancet, 2022). The current situation of bacterial resistance in my country is not optimistic either. Studies have shown that the resistance rates of 51 strains of Staphylococcus aureus isolated from Xinjiang to 11 commonly used antibiotics are as follows: ampicillin (51 / 51, 100%), azithromycin (44 / 51, 86.27%), sulfamethoxazole (43 / 51, 84.31%), ciprofloxacin (15 / 51, 29.41%), amoxicillin (8 / 51, 15.69%), cefoxitin (6 / 51, 11.76%) and chloramphenicol (3 / 51, 5.88%) (He Tengfei, 2022). The resistance rate of 33 strains of porcine streptococci isolated from Jiangxi to clindamycin, cotrimoxazole and gentamicin reached 100%, of which 12 strains were resistant to 9 drugs at the same time (12 / 33, 40%). Resistance genes to tetracyclines, macrolides, aminoglycosides, and sulfonamides were detected in 33 strains of streptococci, with detection rates of tetO (78.8%), tetM (15.2%), ermB (90.9%), oeLh (40%), ermA (9%), sr (97%), strB (93.93%), Aph3 (72.7%), sul1 (100%), sul2(21.2%) (Huang Yuting, 2023). As bacteria adapt to antimicrobial drugs, they have evolved mechanisms to resist all existing antibiotics. Although the development of new antimicrobial drugs is a rigid demand for medicine and health, if drugs with the same mechanism of action as existing antimicrobial drugs are developed, they will inevitably lead to drug resistance soon. Therefore, it is worth thinking deeply about what kind of antimicrobial mechanism to develop drugs. On the other hand, since the treatment cycle of antibiotics is shorter than that of drugs for hypertension, diabetes, leukemia, etc., and due to the severity of bacterial resistance, newly developed antibiotics may be classified as reserve drugs instead of entering the market. Given the low economic return expectations, pharmaceutical companies lack the motivation to develop new antibiotic drugs. In the past 50 years, only daptomycin, a new antimicrobial drug of natural origin, has been put into clinical use. It is reported that one of the main reasons why most antimicrobial drugs are not effective against negative bacteria is that the amount entering bacterial cells is insufficient (Richter & Hergenrother, 2019). If a compound that can increase the intracellular accumulation of antibiotics in bacteria can be developed, this compound will have a broad-spectrum synergistic effect on existing antibiotics (such as polymyxin), will reverse bacterial resistance to existing antibiotics, and extend the service life of existing drugs, which undoubtedly has huge economic prospects.

[0003] Bacterial phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL) are not only the basic skeletons of the phospholipid bilayer of bacterial cell membranes, but also participate in individual signal transduction, virulence, invasion, resistance to external pressure and various other important life activities. They are important components for maintaining normal physiological activities of bacteria. Therefore, phospholipid-related synthases, such as phosphatidylglycerol phosphate synthase (PgsA) and transporters, are considered to be attractive new antibacterial targets. Specifically, Staphylococcus aureus phospholipids are composed of PG, CL and lysylphosphatidylglycerol (L-PG), among which PG is the most abundant phospholipid in Staphylococcus aureus, accounting for about 60% of the total phospholipids. PE, PG and CL are the main phospholipids in Escherichia coli, of which PG accounts for about 15%. It can be seen that PG is a common phospholipid in Gram-positive and Gram-negative bacteria, and PG is rare in most mammals and only serves as a surfactant on the surface of alveolar cells. Therefore, PG and its synthase have high specificity between bacteria and mammalian cells and are candidate antibacterial targets with great research value.

[0004] 5-Fluoro-2′-deoxycytidine has been studied mainly in leukemia and anti-tumor. According to the inventor's search, there are no reports on the antibacterial effect of 5-Fluoro-2′-deoxycytidine. According to Jordheim LP et al. (2012) and Zheng et al. (2024), gemcitabine (doi:10.1016 / j.ijantimicag.2012.01.019, see the results section) and azacitidine, which are similar structures to 5-Fluoro-2′-deoxycytidine, have certain antibacterial activity (doi:10.1016 / j.ijantimicag.2023.107076, see Appendix 4). The present invention actually measured the antibacterial activity of gemcitabine and azacitidine, and the results showed that the MIC values ​​of azacitidine against MRSA T144, MRSA T50, ATCC29213, RN4220, Streptococcus, and Klebsiella pneumoniae (CRHVKP4) were all greater than 128 μg / mL (Table 1, Table 2), indicating that the actual antibacterial activity of azacitidine was very weak. The MIC values ​​of gemcitabine against MRSA T144, MRSA T50, ATCC29213, RN4220, Streptococcus, and Klebsiella pneumoniae (CRHVKP4) were 16, 16, 8, 8, >128, and >128 μg / mL, respectively, which were significantly weaker than CDP-3 (Table 1, Table 2). Summary of the invention

[0005] The present invention provides a clinically effective antibacterial compound with a broad antibacterial spectrum, good antibacterial activity, strong broad-spectrum synergistic effect, and significant in vivo therapeutic effect. The present invention discovers the targeting of 5-fluoro-2′-deoxycytidine (CDP-3) to bacterial PgsA, and proposes its new antibacterial use for the first time. At present, there is still no public report proposing the application of 5-fluoro-2′-deoxycytidine in antibacterial aspects. The present invention proposes for the first time that 5-fluoro-2′-deoxycytidine (CDP-3) can exert antibacterial / synergistic activity by targeting bacterial phosphatidylglycerol phosphate synthase (PgsA) and interfering with the synthesis of bacterial phosphatidylglycerol (PG). CDP-3 has an antibacterial mechanism different from existing antibiotics because it targets bacterial PgsA. Its discovery and further application in antibacterial aspects will enrich the molecular reservoir of antibacterial drug sources, alleviate the severe situation of drug resistance, and provide a new option for the treatment of infections caused by drug-resistant pathogens in clinical practice. Specifically, the present invention proposes the following technical solutions: The first object of the present invention is to provide the use of 5-fluoro-2′-deoxycytidine in the preparation of antibacterial drugs.

[0006] Furthermore, the pathogen in the antibacterial drug is selected from one of Staphylococcus aureus, Streptococcus, Enterococcus, Clostridium perfringens, Bacillus anthracis, Escherichia coli, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Bordetella pertussis, and Mycobacterium tuberculosis.

[0007] The present invention investigates the antibacterial activity of 5-fluoro-2′-deoxycytidine (CDP-3) against common clinical pathogenic bacteria, the synergistic activity against existing antibiotics, and the effectiveness in animal infection models. The results show that the MICs of CDP-3 against Staphylococcus aureus and Streptococcus are 4μg / mL and 8μg / mL, respectively, the MIC against highly virulent Klebsiella pneumoniae is 16μg / mL, the MIC against vancomycin-resistant Enterococcus faecium is 32μg / mL, and the MIC against MRSA (n=43) is 16μg / mL. 90 8μg / mL. CDP-3 can significantly enhance the antibacterial activity of antibiotics such as clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, gentamicin, chloramphenicol, rifampicin and ofloxacin against multidrug-resistant negative bacteria. Subinhibitory concentrations of CDP-3 can not only inhibit bacterial growth, but also inhibit bacterial biofilm formation, induce virulence and weaken pathogenicity. At the same time, CDP-3 not only improves the survival rate of MRSA T144 (MIC = 4μg / mL) infected with greater wax moth, but also has a significant effect on multidrug-resistant Escherichia coli ( E. coli B2, MIC = 64 μg / mL), highly virulent Klebsiella pneumoniae (CRHVKP4, MIC = 16 μg / mL), and multidrug-resistant Salmonella ( S. enteritis 15E475, MIC = 64μg / mL) also showed excellent therapeutic efficacy. When CDP-3 is used in combination with clindamycin, it can significantly improve the efficacy of clindamycin against E. coli The healing effect of skin wounds of mice infected with B2. The application of CDP-3 in the field of antibacterial will provide a new means for the prevention and treatment of clinical drug-resistant bacterial infections, and has great clinical application prospects and economic value.

[0008] A second object of the present invention is to provide an antibacterial agent wherein the active ingredient comprises 5-fluoro-2'-deoxycytidine.

[0009] Furthermore, the antibacterial agent also includes at least one of rifampicin, ofloxacin, cefepime, clindamycin, tilmicosin, roxithromycin, niclosamide, tetracycline, florfenicol, gentamicin, and chloramphenicol.

[0010] The inventors have found that in addition to having excellent antibacterial activity, 5-fluoro-2′-deoxycytidine can also be used in combination with other antibacterial drugs to improve the antibacterial activity of other common clinical antibacterial drugs, especially for strains that have developed sufficient drug resistance. Based on this, the third object of the present invention is to provide the use of a combination of 5-fluoro-2′-deoxycytidine and other common antibacterial drugs in antibacterial treatment. Specifically, the third aspect of the present invention proposes the following use of 5-fluoro-2′-deoxycytidine in combination with other common antibacterial agents.

[0011] The present invention also provides the use of a composition of 5-fluoro-2′-deoxycytidine and at least one of clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, and gentamicin in the preparation of an anti-Escherichia coli drug. The Escherichia coli is preferably a multidrug-resistant Escherichia coli (e.g. E. coli B2), clinical strains of E. coli (e.g. E. coli 1653).

[0012] The inventors found that 5-fluoro-2′-deoxycytidine at 32 μg / mL could inhibit the effects of clindamycin, tilmicosin, and niclosamide on multidrug-resistant Escherichia coli ( E. coli B2) by 8 to 64 times; 5-fluoro-2′-deoxycytidine at 32 μg / mL can inhibit the effects of tetracycline, florfenicol, and gentamicin on clinical strains of Escherichia coli ( E. coli 1653) increased the MIC by 8 to 32 times.

[0013] The present invention also provides a use of a composition of 5-fluoro-2′-deoxycytidine and at least one of chloramphenicol, tilmicosin, and clindamycin in the preparation of an anti-Salmonella drug. The Salmonella is preferably a multidrug-resistant Salmonella ( S. enteritis 15E475).

[0014] The inventors found that 5-fluoro-2′-deoxycytidine at 8 μg / mL could effectively inhibit the effects of chloramphenicol, tilmicosin, and clindamycin on multidrug-resistant Salmonella ( S. enteritis 15E475) has a MIC that is increased by 8 to 256 times.

[0015] The present invention also provides a use of a composition of 5-fluoro-2′-deoxycytidine and at least one of clindamycin, rifampicin and ofloxacin in the preparation of an anti-Klebsiella pneumoniae drug; the inventors found that 5-fluoro-2′-deoxycytidine at 8 μg / mL can reduce the effect of clindamycin on Klebsiella pneumoniae ( K. pneumoniae WNX-1) by 16 times; 5-fluoro-2′-deoxycytidine at 4 μg / mL could inhibit the activity of rifampicin and ofloxacin against highly virulent Klebsiella pneumoniae ( K. pneumoniae The MIC of CRHVKP4 was increased by 16 times.

[0016] The dosage form of the pharmaceutical composition provided by the present invention is not particularly limited, such as tablets, capsules, granules, suspensions, injections or suspensions.

[0017] The pharmaceutical composition comprises excipients, and the excipients are selected from at least one of fillers, binders, wetting agents, disintegrants, flavoring agents, preservatives, pH regulators, and antioxidants.

[0018] The beneficial effects of the present invention are as follows: This application discovered a new antibacterial application and antibacterial mechanism of 5-fluoro-2′-deoxycytidine (CDP-3). CDP-3 showed excellent antibacterial or synergistic activity against common clinical pathogenic bacteria. Its MICs against Staphylococcus aureus and Streptococcus were 4μg / mL and 8μg / mL, respectively, and its MIC against highly resistant and highly virulent Klebsiella pneumoniae (CRHVKP4) was 16μg / mL, and its MIC against vancomycin-resistant Enterococcus faecium was 32μg / mL. Its MIC against MRSA (n=43) was 90 At 32 μg / mL, clindamycin, tilmicosin, and niclosamide could be used to treat multidrug-resistant Escherichia coli ( E. coli B2) increased MIC by 8 to 64 times; at 32 μg / mL, tetracycline, florfenicol, and gentamicin could inhibit clinical strains of Escherichia coli ( E. coli 1653) by 8 to 32 times; at 8 μg / mL, chloramphenicol, tilmicosin, and clindamycin could be used to inhibit multidrug-resistant Salmonella ( S. enteritis 15E475) increased by 8 to 256 times; at 8 μg / mL, clindamycin could inhibit Klebsiella pneumoniae ( K. pneumoniae WNX-1) increased the MIC by 16 times, and at 4 μg / mL, it could inhibit the effects of rifampicin and ofloxacin on Klebsiella pneumoniae ( K. pneumoniae The MIC of CRHVKP4 was increased by 16 times.

[0019] CDP-3 not only improved the survival rate of G. mellonella infected with MRSA T144 (MIC = 4 μg / mL), but also E. coli B2 (MIC = 64 μg / mL), K. pneumoniae CRHVKP4 (MIC = 16 μg / mL) and S. enteritisIt also showed excellent therapeutic effects on wax moths infected with multidrug-resistant negative bacteria such as 15E475 (MIC=64μg / mL). Although CDP-3 has low antibacterial activity against negative bacteria, it has good in vivo therapeutic effects on infections caused by negative bacteria, which undoubtedly provides a new therapeutic strategy for the treatment of clinical negative bacterial infections. The discovery of new uses of CDP-3 provides a compound with a new antibacterial mechanism and enriches the molecular library of antibacterial drug sources. Its further application will provide a new therapeutic drug for the prevention and control of clinically related bacterial diseases; the revelation of its antibacterial mechanism proves the targetability of bacterial PgsA protein, and will also provide a theoretical reference for the development of more drugs that can target bacterial PgsA. The proposal of this application will provide a new option for extending the service life of existing antibiotics and alleviating the severe situation of bacterial resistance. It will promote the research and development of drugs with new antibacterial mechanisms and help my country's independent strategy for new drug innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the change in the fatty acid chain of phospholipids of Staphylococcus aureus after CDP-3 treatment; Figure 2 is the change in phospholipid abundance of S. aureus after CDP-3 treatment; Figure 3 is the bactericidal curve of CDP-3 against MRSA T144; Figure 4 is the growth curve of MRSA T144 under different compound conditions; Figure 5 It is the inhibitory effect of CDP-3 on bacterial biofilm formation; Figure 6 It is the inhibitory effect of CDP-3 on bacterial virulence; Figure 7 It is the display of toxicity of CDP-3 to mice; Figure 8 is the therapeutic effect of CDP-3 on the G. mellonella infection model; Fig. 9 The therapeutic effect of CDP-3 combined with clindamycin on the multidrug-resistant Escherichia coli skin infection model. DETAILED DESCRIPTION

[0021] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0022] Example 1 Antibacterial activity of cytidine derivatives against common clinical pathogens Pick a single colony of the test strain and place it in BHI broth and culture it on a shaker at 37°C until the bacterial logarithmic growth phase. Use a McFarland turbidimeter to adjust the bacterial turbidity to 0.5, and dilute it 100 times with MHB broth to about 10 6 CFU / mL, take 100 μL of the above bacterial solution and add it to a 96-well U-shaped plate. Dissolve the cytidine derivatives in phosphate buffered saline (PBS), add 100 μL of MHB broth medium to each column of the 96-well U-shaped plate, take 100 μL of a certain concentration of compound and add it to the first column of the 96-well U-shaped plate, dilute it 2-fold to the 10th column, add an additional 100 μL of the test bacterial solution to the 12th column as a positive control, and the 11th column as a negative control. Place the 96-well U-shaped plate in a 37°C constant temperature incubator and culture it for 18-20 h, read the experimental results, and take the lowest drug concentration that can inhibit bacterial growth visible to the naked eye as the MIC value of the compound.

[0023] The present invention tests the antibacterial performance of various cytidine derivatives, and the general structure is shown in the following formula (I). The MIC results of various compounds against various types of bacteria are shown in Tables 1 and 2.

[0024] The MICs of cefoxitin, clindamycin, erythromycin, chloramphenicol, florfenicol, linezolid and vancomycin against MRSA T144 were 8 μg / mL, >128 μg / mL, >128 μg / mL, 32 μg / mL, 64 μg / mL, 1 μg / mL and 1 μg / mL, respectively. As can be seen from the data in Table 1, the antibacterial activity of CDP-3 against MRSA T144 was significantly stronger than that of cefoxitin, clindamycin, erythromycin, chloramphenicol, florfenicol and other commonly used clinical antibiotics (Table 1).

[0025] As can be seen from the data in Table 2 , CDP-3 has good antibacterial activity against bacteria such as Staphylococcus aureus (ATCC 29213), Staphylococcus aureus (RN4220), Streptococcus (ATCC 35246), highly virulent Klebsiella pneumoniae (CRHVKP4), and vancomycin-resistant Enterococcus faecium (VRE 10), with MICs of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL, respectively, and MICs against MRSA (n=43). 90 8 μg / mL.

[0026] (I) Table 1. Antibacterial efficacy of cytidine derivatives against MRSA T144 .

[0027] According to Jordheim LP et al. (2012) and Zheng et al. (2024), CDP-3-similar structures gemcitabine (CDP-8) and azacitidine (CDP-9) (doi:10.1016 / j.ijantimicag.2023.107076, see Appendix 4) have certain antibacterial activity. The present invention compares the antibacterial activity of gemcitabine, azacitidine and CDP-3. The results show that CDP-3 has significantly stronger antibacterial activity against positive bacteria such as MRSA T144, MRSA T50, ATCC29213, RN4220, and Streptococcus than gemcitabine and azacitidine (Table 1, Table 2); the antibacterial activity against negative bacteria such as Klebsiella pneumoniae CRHVKP4 and Escherichia coli ATCC 25922 is also higher than gemcitabine (CDP-8) and azacitidine (CDP-9) (Table 1, Table 2). Although the above comparative documents publicly mention that gemcitabine and azacitidine have antibacterial activity, it has been found through actual measurements that the antibacterial activity of azacitidine (CDP-9) is weak (MIC ≥ 128 μg / mL), and the antibacterial activity of gemcitabine (CDP-8) is also significantly lower than that of CDP-3. Based on this, the applicant proposes that compared with similar structures that have been disclosed, the CDP-3 proposed in this application has significantly better activity, and this application has certain creativity.

[0028] Table 2. Antibacterial efficacy of cytidine derivatives against common clinical pathogens ; Note: Escherichia coli B2 is a multidrug-resistant bacterium that carries 25 multidrug resistance genes, including blaNDM-5 , mcr-1 , tetA , mxDV , oeLh , arr-2 , aph(4) , aac(3)-IV and fosA3 The MICs of cefepime, polymyxin, ofloxacin, rifampicin, and tetracycline against E. coli B2 were 32, 2, 32, 128, and 128 μg / mL, respectively. Enterococcus faecium VRE10 is a vancomycin-resistant strain, and CRHVKP4 is a highly resistant and highly virulent Klebsiella pneumoniae. 43 MRSA strains were isolated from clinical sites. “ / ” indicates not tested.

[0029] CDP-3 is 5-F, CDP-4 is 5-CH3, CDP-5 is 5-Cl, CDP-6 is 5-Br, and CDP-7 is 5-I. From CDP-3 to CDP-7, only the 5-substituent is different, and the properties of the 5-substituent are very similar. In theory, the activity should show a gradient change. However, as shown in Table 2, the MIC values ​​of CDP-4, CDP-5, CDP-6 and CDP-7 against MRSA T144, Staphylococcus aureus ATCC 29213 and Staphylococcus aureus RN4220 are all greater than 128 μg / mL, and only CDP-3 (5-F) shows good antibacterial activity (MIC=4 μg / mL). The above data prove that the discovery of CDP-3 was obtained after rigorous screening and research.

[0030] Example 2: CDP-3 enhances the efficacy of common antibiotics The chessboard broth dilution method was used to determine the synergistic efficacy (FIC) of CDP-3 and common antibiotics against multidrug-resistant Escherichia coli, Salmonella, and Klebsiella pneumoniae. Single bacterial colonies were picked from the agar plate and placed in MHB broth. The bacteria were cultured in a shaking incubator at 37°C until the logarithmic growth phase. The bacterial turbidity was adjusted to a McFarland turbidity of 0.5 using a McFarland turbidimeter and diluted 100 times (~1.0×10 6 CFU / mL) and set aside. Use a pipette to take 100 μL of MHB and add it to each well of a 96-well U-shaped plate. Dilute the prepared antibiotic with PBS to about 8 times the MIC, take 100 μL and add it to the first column of the 96-well U-shaped plate, dilute it to the seventh column with a pipette, and discard the pipette tip; at the same time, dilute the CDP-3 stock solution to about 512 μg / mL with PBS, take 100 μL and add it to wells 1 to 8 of the eighth row of the 96-well U-shaped plate, and dilute it to the second row with a pipette. Then add 100 μL of the diluted test bacterial solution to each well. The 11th and 12th columns are negative and positive controls containing only MHB culture medium and only test bacterial solution, respectively. After incubation at 37°C for 18-20 h, read the FIC value. Among them, FIC = MIC (A drug combination) / MIC (A drug alone) + MIC (B drug combination) / MIC (B drug alone).

[0031] The results showed that the FIC index of CDP-3 and commonly used antibiotics was within 0.5 (Table 3), indicating that the CDP-3 provided by the present invention has a strong synergistic effect with commonly used antibiotics. CDP-3 can significantly improve the antibacterial activity of antibiotics such as clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, gentamicin, chloramphenicol, tilmicosin, roxithromycin, rifampicin and ofloxacin against negative bacteria such as Escherichia coli, Salmonella, Klebsiella pneumoniae, and Acinetobacter baumannii. In particular, the antibacterial activity of niclosamide against drug-resistant Escherichia coli (B2) can be increased by at least 64 times, and the MIC value of clindamycin against drug-resistant Salmonella enteritidis (15E475) can be increased by 128 times or more. CDP-3 makes originally insensitive antibacterial drugs sensitive, significantly reversing the bacterial resistance to existing antibiotics.

[0032] When gemcitabine (CDP-8) with similar structure is used in combination with roxithromycin, gentamicin and clindamycin, the synergistic effect is significantly lower than that of CDP-3 proposed in this application (Table 4). The above results show that CDP-3 not only has excellent antibacterial activity itself, but can also be used in combination with existing antibiotics to enhance the antibacterial activity of old drugs, which is undoubtedly conducive to extending the service life of old drugs and giving new vitality to existing antibiotics.

[0033] Table 3. Synergistic effect of CDP-3 on commonly used antibiotics ; Note: a, b: MIC of antibiotics in the presence or absence of CDP-3; the MICs of ampicillin, chloramphenicol, florfenicol, cefotaxime, ceftazidime and doxycycline against Salmonella Enteritidis (15E475) were 8 μg / mL, >256 μg / mL, >128 μg / mL, >0.56 μg / mL, 64 μg / mL and 32 μg / mL, respectively.

[0034] Table 4. Comparison of the synergistic effects of CDP-3 and gemcitabine in combination with antibiotics ; Note: The test strain is Acinetobacter baumannii (Ab 17-38); a, b: MIC of antibiotics in the presence or absence of CDP-3 and gemcitabine (CDP-8).

[0035] Example 3 Affinity of cytidine derivatives for bacterial phosphatidylglycerol phosphate synthase (PgsA) In order to determine the antibacterial mechanism and structure-activity relationship of CDP-3, PgsA protein was expressed, and the affinity of cytidine derivatives to bacterial phosphatidylglycerol phosphate synthase (PgsA) was measured by surface plasmon resonance (SPR). The affinity constant of cytidine, the endogenous ligand of PgsA, to PgsA is 1.37×10 -9The affinity constants of CDP-3 (5-F) to CDP-7 (5-I) and PgsA were 1.76×10 -9 M, 14.9×10 -9 M. The above results show that the affinity of CDP-3 to PgsA is close to that of its endogenous ligand, confirming that CDP-3 can compete with endogenous ligands to bind to PgsA, resulting in a decrease in the efficiency of bacterial synthesis of phosphatidylglycerol and achieving an antibacterial effect. At the same time, when the 5th position of cytidine is -I (CDP-7), the binding affinity of CDP-7 to PgsA is 14.9×10 -9 M, which is 1 / 10 of the CDP-3 binding affinity. Based on this, it is proposed that fluorine substitution at the 5-position of cytidine is the optimal choice for exerting antibacterial activity.

[0036] In order to reveal the bacterial phospholipid reprogramming induced by CDP-3 targeting PgsA, the changes in bacterial phospholipid abundance and phospholipid fatty acid chain composition under the action of CDP-3 were studied. Staphylococcus aureus RN4220 cultured for 6-8 h was diluted 4 times for later use. 5 mL of 320 μg / mL CDP-3 was added to 795 mL of sterile MHB medium to a final concentration of 2 μg / mL (1 / 2MIC). 1 mL of the reserved bacterial solution was added to make the final concentration of the bacterial solution about 10 6 cfu / mL, this group is the experimental group. Another 400 mL of sterile MHB medium was added with only bacterial solution without CDP-3, which was set as the blank control group. The above culture solution was placed on a shaker at 37°C and 200 rpm for incubation. The blank control group was incubated for 12 h, and the experimental group was incubated for 24 h. After the incubation, the bacteria were collected by centrifugation at 4°C and 8000 rpm for 10 min. The collected bacterial precipitate was washed with PBS, and the wet weight of the bacteria was weighed after removing the supernatant. After adding 5 mL of PBS to resuspend the bacteria, 15 mL of extraction solution (chloroform: methanol = 1:2, V: V) was added. After appropriate vortexing, it was placed on a shaker at 37°C and 200 rpm for 1.5 h to completely extract bacterial phospholipids. The shaken liquid was placed in a -20°C refrigerator for 15 min to facilitate liquid stratification. After the liquid was stratified, about 5 mL of the bottom chloroform layer was carefully aspirated, and the chloroform was removed by nitrogen blowing to obtain the total bacterial phospholipids. Finally, the total bacterial phospholipids were analyzed by gas chromatography-mass spectrometry.

[0037] The results are as follows Figure 1As shown in the figure, compared with the untreated blank group, the abundance of odd-numbered chains in the total fatty acid chains of Staphylococcus aureus significantly decreased and the abundance of even-numbered chains significantly increased after CDP-3 treatment. The abundances of PG and CL in the blank group were 17058.95 ± 399.93 μg / mL and 1866.34 ± 42.30 μg / mL, respectively, and the abundances of PG and CL in the experimental group were 9021.18 ± 50.36 μg / mL and 363.76 ± 11.19 μg / mL, respectively. Figure 2 It can be seen that compared with the blank group, the abundance of PG and CL of Staphylococcus aureus decreased by 1.89 and 5.13 times respectively after treatment with 1 / 2 MIC CDP-3. The above results show that the abundance of PG and CL of Staphylococcus aureus decreased significantly in the presence of CDP-3, which further proves that the antibacterial mechanism of CDP-3 is to inhibit the synthesis of bacterial PG. And the decrease in the abundance of CL of Staphylococcus aureus is significantly greater than that of PG. This may be because when facing huge survival pressure, bacteria adjust their own phospholipid metabolism state to ensure the abundance of PG first in order to survive, and reduce the further conversion of PG to CL. It can be seen that for the survival of Staphylococcus aureus (positive bacteria), PG is more important than CL and is the basis for maintaining basic life activities. On the other hand, the content of even-chain fatty acids increased significantly under the action of 1 / 2 MIC CDP-3, which suggests that even-chain fatty acid chains are more important than odd-chain fatty acids for survival.

[0038] Example 4 Killing curve: Pick a single MRSA T144 clone in 1 mL BHI broth medium and culture it at 37°C and 200 rpm until the bacteria reach the logarithmic growth phase. Then, adjust the concentration of the MRSA T144 culture medium to a McFarland turbidity of 0.5. Add 5 mL MHB medium to each well of a 6-well plate, take 0.05 μL of the diluted bacterial solution and add it to MHB to obtain a concentration of about 10 6 CFU / mL of bacterial solution. Different concentrations of CDP-3 (from 1 / 8×MIC to 8×MIC) were added to the bacterial MHB, and an equal volume of PBS was added as a negative control group. Three replicates were set for each treatment and cultured in a 37°C incubator. 100 μL of culture solution was taken out at 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h, diluted in PBS and then plated for counting. The number of colonies was calculated after the MHA agar plate was inverted and cultured at 37°C for 24 h.

[0039] Figure 3This is the bactericidal curve of CDP-3 against MRSA T144. The results show that CDP-3 is a time-dependent bactericidal agent and it still has the effect of inhibiting bacterial growth at sub-inhibitory concentrations, suggesting that during clinical treatment, even if the concentration of CDP-3 in the body is reduced to below the MIC value, it can still effectively inhibit bacterial growth. Growth curve: Pick a single MRSA T144 clone in 1 mL BHI broth and culture it at 37°C, 200 rpm until the bacteria reach the logarithmic growth phase. Then, adjust the concentration of the MRSA T144 culture medium to ~10 6 cfu / mL. Use a gun to take 100 μL of the diluted culture medium to the 1st to 6th columns of a 96-well flat-bottom plate, and then use a gun to take 1 / 4×MIC of vancomycin (VAN), ofloxacin (OFL), cefquinome (CEFQ), roxithromycin (ROX) and CDP-3 and add them to the 1st to 6th rows, as the VAN group, OFL group, CEFQ group, ROX group and CDP-3 group, respectively. MHB was added to the 6th row as a blank control group. The 96-well plate was placed in a 37°C incubator for incubation, and the plates were taken out at 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h, respectively, and the OD was measured using an enzyme reader. 600nm , and plot the growth curves under different antimicrobial compounds.

[0040] Figure 4 The growth curve of MRSA T144 under different compound conditions. Unlike commonly used antibiotics, CDP-3 showed a significant inhibitory effect on bacterial growth at 1 / 8 MIC, while common antibacterial drugs such as vancomycin (VAN), ofloxacin (OFL), cefquinome (CEFQ), and roxithromycin (ROX) had no inhibitory effect on bacterial growth at sub-inhibitory concentrations ( Figure 4 ). It can be seen that compared with commonly used antibiotics, CDP-3 has significant antibacterial advantages.

[0041] Example 5 Effect of CDP-3 on bacterial biofilm formation Crystal violet staining was used to elucidate the inhibitory effect of CDP-3 on S. aureus and E. coli biofilms. S. aureus RN4220 or E. coli B2 cultures were first grown for 8 h and diluted to ~10 6cfu / mL. 0.1 mL of the dilution was added to each well of a 6-well plate. Then, 1.9 mL of MHB containing 1 / 16~1 / 4 × MIC CDP-3 or 1 / 8 × MIC concentrations of ROX, Kana, OFL, VAN, and CEFQ was added, respectively, and fresh MHB without antibiotics was used as a control, with 3 replicates per group. The plates were incubated at 37°C for 48 h to form biofilms. The floating cells in each well were removed by washing three times with 4 mL of PBS. The biofilm formed on the bottom surface of the microplate wells was fixed at 65°C for 1 h. Then, the biofilm was stained with 400 µL of 0.25% (W / V) crystal violet solution at room temperature for 5 min. The excess crystal violet was removed by washing three times with 4 mL of PBS. After drying under a clean bench for 15 min, the stained biofilms of different groups were observed and recorded using an optical microscope (10×10). Then, the bound crystal violet was extracted with 400 μL of 95% ethanol, and the OD values ​​of different groups were detected at 490 nm by a microplate reader.

[0042] The results of each treatment group are as follows Figure 5 The results showed that CDP-3 could significantly reduce the formation of RN4220 biofilm at 1 / 4~1 / 16 MIC concentrations ( P ≤ 0.0005) ( Figure 5 A), and its inhibitory effect on biofilm formation is significantly stronger than existing antibiotics such as roxithromycin (ROX), kanamycin (KANA), ofloxacin (OFL), vancomycin (VAN), and cefquinome (CEFQ) ( P ≤ 0.0054)( Figure 5 B). It is worth emphasizing that compared with the control group without drug addition, the biofilm formation level after treatment with 1 / 8×MIC roxithromycin and kanamycin was significantly higher than that of the untreated group ( P ≤ 0.0023) ( Figure 5 B), indicating that roxithromycin and kanamycin promote biofilm formation at sub-inhibitory concentrations. As part of bacterial non-genetic drug resistance, biofilm plays an important role in resisting disinfectants, antibiotics, recurrent infections, asymptomatic infections, and drug resistance evolution. Therefore, interfering with or removing biofilms has become an increasingly important part of antibacterial infection treatment. Compared with existing antibiotics, CDP-3 showed excellent ability to inhibit biofilm formation, once again demonstrating the advantages of CDP-3 provided by the present invention in antibacterial treatment, and also demonstrating the advantages of targeted phospholipid antibacterial.

[0043] Example 6 The reduction of bacterial biofilm formation induced by CDP-3 suggests its potential in inhibiting bacterial virulence. Therefore, further studies were conducted on the role of CDP-3 in inhibiting staphyloxanthin, motility, and endocytosis.

[0044] 1. Effect of CDP-3 on staphyloxanthin a) Pick a single MRSA T144 clone and place it in 1 mL BHI broth medium. Cultivate at 37°C, 200 rpm until the logarithmic growth phase. Then, adjust the concentration of the MRSA T144 culture medium to a McFarland turbidity of 0.5. Add 5 mL MHB medium to each well of a 6-well plate, take 0.05 μL of the diluted bacterial solution and add it to MHB to obtain a concentration of about 10 6 CFU / mL of bacterial solution. CDP-3 and vancomycin at 8×MIC concentrations were added to the MHB with bacteria, and an equal volume of PBS was added as a negative control group. Three replicates were set for each treatment and cultured in a 37°C incubator. 100 μL of culture solution was taken out at 8 h, diluted in PBS in multiples, and then plated for counting. The MHA agar plate was inverted and cultured at 37°C for 24 h before observing the colony morphology.

[0045] b) MIC of 43 MRSA clinical isolates was determined by agar dilution method 90 After the test results are read, use an agar plate image acquisition instrument to compare 1 / 2×MIC 90 、1 / 4×MIC 90 The agar plates with CDP-3 and vancomycin were photographed to observe the morphology of 43 MRSA clinical isolates under CDP-3 and vancomycin treatment.

[0046] 2. Effect of CDP-3 on the motility of Staphylococcus aureus According to the label, weigh an appropriate amount of soy broth medium into 200 mL of distilled water, add 0.8 g of agar powder, mix well, sterilize, and cool appropriately, add an appropriate amount of CDP-3 solution and pour into a 90 mm plate to make a CDP-3 soft agar plate containing 1 / 8~1 / 2×MIC. Soft agar without CDP-3 was used as a blank control. E. coli ATCC 25922 monoclonal clone was cultured in 50 mL BHI broth at 37°C and 200 rpm for 8 h, and then centrifuged at 4°C and 9000 rpm for 10 min to collect the bacteria. The precipitate was resuspended in 1 mL PBS and 5 μL of about 10 μL was added to the center surface of the agar plate using a 10 μL pipette tip. 9 cfu / mL, and after being dried by air blowing for 15 min in a clean bench, the bacteria were cultured in a 37°C incubator for 3 days, and the movement range of the colonies in different treatment groups was observed.

[0047] 3. Effect of CDP-3 on the cellular entry ability of Staphylococcus aureus The RAW264.3 cells were revived and cultured until they were completely attached. The cells were collected by centrifugation, resuspended in fresh culture medium, and 1.8 mL was pipetted into a 6-well plate. 100 μL of Staphylococcus aureus RN4220 (~10 6 cfu / mL) of bacterial solution was added, and then 100 μL of CDP-3 was added to the 6-well plate, so that the concentration of CDP-3 was 1 / 8~1 / 2×MIC. The group without CDP-3 was used as a blank control, and each group was repeated 3 times. The 6-well plate was placed in a 37°C incubator for incubation, and the cells of each group were collected after 4 h and 8 h, respectively. After breaking, the S. aureus RN4220 was plated and counted, and the number of intracellular bacteria in different treatment groups was counted to analyze the effect of CDP-3 on the ability of S. aureus to enter the cell.

[0048] After VAN treatment, MRSA T144 has a typical golden appearance ( Figure 6 Ac and Ad), while after CDP-3 treatment, MRSA T144 showed a transparent appearance ( Figure 6 The results showed that CDP-3 can significantly inhibit the staphyloxanthin of Staphylococcus aureus. Staphyloxanthin is an important virulence factor of Staphylococcus aureus, which plays an important role in invasion, infection, and resistance to body immunity. The inhibitory effect of CDP-3 on staphyloxanthin will be beneficial to the treatment of clinically relevant infections. After treatment with CDP-3, the range of motion of Staphylococcus aureus RN4220 and Escherichia coli ATCC 25922 was significantly reduced ( Figure 6 B), indicating that CDP-3 can significantly inhibit the motility of Staphylococcus aureus. At the same time, after treatment with CDP-3, the number of Staphylococcus aureus RN4220 entering RAW264.7 cells was significantly reduced ( Figure 6 C), suggesting that CDP-3 plays a role in inhibiting the virulence of Staphylococcus aureus.

[0049] Example 7 Toxicity study of CDP-3 on mice To determine the dosage and safety of CDP-3 in the treatment experiment, a toxicity study of CDP-3 on mice was conducted. 24 Babl / C mice (25-30 g) were randomly divided into 4 groups, with 6 mice in each group. The mice were intraperitoneally injected with 10 mg / kg bw, 20 mg / kgb.w. and 40 mg / kg bw, respectively, for 5 consecutive days, twice a day. On the 5th day, half of the mice in each group were dissected, and the liver, kidney, lung, heart, spleen, stomach, intestine and other organs were removed to observe the pathological conditions of the mouse organs in each dose group. The remaining mice were discontinued and observed for 7 days, during which the survival rate of the mice was recorded. On the 12th day, the remaining mice were dissected, and the liver, kidney, lung, heart, spleen, stomach, intestine and other organs were removed to observe the recovery of organ damage.

[0050] The results showed that after continuous administration of three doses of high, medium and low for 5 days, mice in each dose group developed diarrhea, accompanied by gastric distension and intestinal fluid accumulation ( Figure 7 A. Figure 7 B), the spleen of mice in the 20 mg / kg bw and 40 mg / kg bw groups was significantly smaller than that in the PBS group ( Figure 7 A), the spleen of mice in the 10 mg / kg bw group was less reduced. After drug withdrawal, all the remaining mice in the 20 mg / kg b.w. and 40 mg / kg bw groups died, while all the mice in the 10 mg / kg bw group survived, and the lesions in the stomach, intestines and spleen of mice in the 10 mg / kg bw group were significantly improved ( Figure 7 The above results indicate that CDP-3 is safe for mice when injected intraperitoneally at a dose of 10 mg / kg bw, twice a day, for 5 consecutive days. In subsequent treatment experiments, it can be administered at a dose of 10 mg / kg bw or lower.

[0051] Example 10: Therapeutic effect of CDP-3 in animal infection models 1. MRSA infection model of Galleria mellonella Fifty G. mellonella were randomly divided into five groups, each with 10 animals, including control group, negative group, vancomycin (VAN) group, CDP-3 low-dose group (10 mg / kg bw) and high-dose group (20 mg / kg bw). 5 cfu / mL of MRSA T144 was injected into the right posterior gastropod of G. mellonella. At 1 h after infection, G. mellonella was injected with 10 μL of PBS, VAN (20 mg / kg bw), and CDP-3 (10 mg / kg bw or 20 mg / kg bw) at the left posterior gastropod, and the survival rate of G. mellonella was recorded within 6 days.

[0052] 2. Multidrug-resistant Negative Bacteria Infection Model of G. mellonella Fifty wax moths were randomly divided into five groups, each with 10 animals, including a control group, a negative group, a polymyxin sulfate (Coli) group, a CDP-3 low-dose group (10 mg / kg bw), and a high-dose group (20 mg / kg bw). E. coli B2 (10 7 cfu / mL), Klebsiella pneumoniae CRHVKP4 (10 5 cfu / mL), Salmonella 15E475 (10 5 cfu / mL) were injected into the right posterior gastropod of G. mellonella. At 0.5-1 h after infection, G. mellonella was injected with 10 μL PBS, Coli (20 mg / kg bw), and CDP-3 (10 mg / kg bw or 20 mg / kg bw) at the left posterior gastropod, and the survival rate of G. mellonella was recorded within 5 days.

[0053] 3. Multidrug-resistant Escherichia coli in mice ( E. coli B2) Skin infection model Twenty BABL / C mice were anesthetized with 200 μL 10% urethane intraperitoneally and randomly divided into five groups (n=4), including control group, negative group, polymyxin (Coli) group, clindamycin (CLI) group, CDP-3 group and +CDP-3 group. The hair on the back of the mice was carefully removed, and an 8 mm skin wound was made with a skin punch. Subsequently, 40 μL ~10 9 cfu / mL of Escherichia coli B2 culture fluid was dripped onto the wound. 24 h after infection, 40 μL PBS, Coli (8 μg / mL), CLI (50 μg / mL), CDP-3 (50 μg / mL) and CLI+CDP-3 (50 µg / mL+50 μg / mL) were dripped onto the wound of mice in different groups, once a day. Top views of the local infected skin of mice under different treatments were recorded at 0, 2, 4, 6 and 10 days.

[0054] In the MRSA infection model of G. wax moth, CDP-3 protected G. wax moth by 80% within 6 days of infection, which was slightly better than the 70% of vancomycin. Compared with the group without drug administration, CDP-3 increased the survival rate of G. wax moth infected with MRSA by 2 times ( Figure 8 A), which suggests that CDP-3 has a good therapeutic effect on systemic MRSA infection. E. coliIn the B2 infection model, within 5 days of infection, the protection rate of 20 mg / kg bw CDP-3 against G. mellonella was 90%, which was better than the protective efficacy of the positive drug Coli (50%) ( Figure 8 B). In the infection model of multidrug-resistant Klebsiella pneumoniae CRHVKP4 in G. mellonella, the protection rate of the 20 mg / kg bwCDP-3 group against G. mellonella was 50% within 5 days of infection, which was better than the protective efficacy of the positive drug Coli ( Figure 8 In the infection model of multidrug-resistant Salmonella 15E475 in G. mellonella, the protection rates of high and low CDP-3 groups against G. mellonella were 90% and 80%, respectively, within 5 days of infection, which was better than the 50% of positive drug Coli ( Figure 8 D).

[0055] As mentioned above, CDP-3 E. coli The MIC value of B2 and Salmonella 15E475 was 64 μg / mL. When CDP-3 was administered at 20 mg / kg (20 μg / g), the concentration in the body of G. mellonella could not reach 64 μg / mL, which means that the dose of 20 mg / kg bw had no significant effect on E. coli B2 and Salmonella 15E475 theoretically have no bactericidal activity, but 20 mg / kg bw of CDP-3 did show excellent protective efficacy. Combined with the above-mentioned inhibition of bacterial phosphatidylglycerol synthesis by CDP-3, which is an important component required for bacterial membrane composition, signal exchange, and virulence expression, it is speculated that this phenomenon is due to the fact that sub-inhibitory concentrations of CDP-3 inhibit the virulence of bacteria, maintaining the bacteria in a low-vitality state and thus increasing the survival rate of G. mellonella. This ability to maintain bacteria in a low-vitality state will undoubtedly help the recovery of bacterial infections and will provide a new strategy for the treatment of clinical multidrug-resistant negative bacterial infections.

[0056] Fig. 9 The therapeutic effect of CDP-3 combined with clindamycin on the multidrug-resistant Escherichia coli skin infection model. In the mouse skin infection model, the CLI+CDP-3 group showed the best wound healing effect, which was stronger than the positive control Coli group ( Fig. 9 A). When CLI was used alone, scabs still formed on the wound on the 10th day. However, when CLI was used in combination with CDP-3, the wound healing effect was significantly improved. CDP-3 also showed excellent wound healing effect when used alone, and its effect was even slightly stronger than that of the Coli group ( Fig. 9B). CDP-3 has no bactericidal activity against E. coli B2 at 50 μg / mL. Combined with the above-mentioned inhibitory effect of CDP-3 on bacterial virulence, it is speculated that the superior effect of CDP-3 comes from its inhibition of bacterial virulence. After the bacterial virulence is inhibited, wound inflammation is reduced. Although CDP cannot directly kill E. coli B2, compared with Coli, which has a killing effect, CDP-3 still shows a stronger wound healing effect, which indicates the importance of inhibiting bacterial virulence in the treatment of bacterial infections, proves the great potential of CDP-3 targeting bacterial phospholipids in antibacterial treatment applications, and also indicates the huge economic benefits of this application.

[0057] In summary, this application discovered a new application of CDP-3 in antibacterial applications: it can target bacterial phosphatidylglycerol phosphate synthase (PgsA) to exert antibacterial and synergistic activities, and is the first antibacterial compound discovered so far that targets PgsA to act on bacterial phosphatidylglycerol (PG) synthesis. Due to its new mechanism of action that is different from existing antibiotics, CDP-3 has good antibacterial activity against sensitive and resistant Staphylococcus aureus (MRSA), Streptococcus, Enterococcus faecium and other positive bacteria and highly virulent Klebsiella pneumoniae (CRHVKP4) (Table 1, Table 2). The inhibition of the synthesis of PG, a phospholipid component of bacterial cell membrane, leads to increased bacterial permeability, so that CDP-3 can significantly improve the antibacterial activity of antibiotics such as clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, gentamicin, chloramphenicol, tilmicosin, roxithromycin, rifampicin and ofloxacin against drug-resistant Escherichia coli, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii and other negative bacteria (Table 3). Due to the restriction of the synthesis of phosphatidylglycerol, a basic component of bacterial cell membrane, bacteria are restricted in biofilm formation, movement, virulence, and pathogenicity, so that CDP-3 has a better protective effect than bactericides (such as polymyxins) against infections caused by multidrug-resistant negative bacteria under the premise of low antibacterial activity ( Figure 8 , Fig. 9 ), which suggests that CDP-3 will also have good potential in the treatment of clinical multi-drug resistant negative bacterial infections. CDP-3 was administered to mice at a dose of 10 mg / kg bw, twice a day for 5 consecutive days, which only caused mild diarrhea in mice, and the symptoms recovered after drug withdrawal, indicating that the toxicity of CDP-3 is within an acceptable range. At present, the current situation of bacterial resistance is severe and new drug development is insufficient. The new application of CDP-3 in antibacterial aspects proposed in this invention undoubtedly brings hope to the treatment of clinical drug-resistant pathogenic bacteria infections.

Claims

1. Use of 5-fluoro-2′-deoxycytidine in the preparation of antibacterial drugs; the bacteria in the antibacterial drugs are selected from one of Staphylococcus aureus, Streptococcus, Enterococcus, Clostridium perfringens, Bacillus anthracis, Escherichia coli, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Bordetella pertussis, and Mycobacterium tuberculosis.

2. An antibacterial agent, characterized in that the active ingredient includes 5-fluoro-2′-deoxycytidine; the bacteria in the antibacterial agent are selected from one of Staphylococcus aureus, Streptococcus, Enterococcus, Clostridium perfringens, Bacillus anthracis, Escherichia coli, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Bordetella pertussis, and Mycobacterium tuberculosis.

3. The antibacterial agent according to claim 2, characterized in that The antibacterial agent also includes at least one of rifampicin, ofloxacin, cefepime, clindamycin, tilmicosin, roxithromycin, niclosamide, tetracycline, florfenicol, gentamicin and chloramphenicol.

4. Use of a composition of 5-fluoro-2′-deoxycytidine and at least one of clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol and gentamicin in the preparation of an anti-Escherichia coli drug.

5. Use of a combination of 5-fluoro-2′-deoxycytidine and at least one of chloramphenicol, tilmicosin and clindamycin in the preparation of an anti-Salmonella drug.

6. Use of a combination of 5-fluoro-2′-deoxycytidine and at least one of clindamycin, rifampicin and ofloxacin in the preparation of an anti-Klebsiella pneumoniae drug.

7. The use according to any one of claims 1, 4 to 6, characterized in that: The dosage of 5-fluoro-2′-deoxycytidine is 5 mg / kg / d.

8. The use according to any one of claims 1, 4 to 6, characterized in that: The dosage form of the medicine is tablet, capsule, granule, suspension, injection or suspension.

9. The use according to any one of claims 1, 4 to 6, characterized in that: The medicine further comprises excipients, which include fillers, binders, wetting agents, disintegrants, flavoring agents, preservatives, pH regulators or antioxidants.

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