Use of 5-fluoro-2'-deoxycytidine in the preparation of antibacterial drugs

Abstract

The present invention provides the use of 5-fluoro-2'-deoxycytidine (CDP-3) in the preparation of antibacterial drugs. The present invention discovers for the first time that CDP-3 can inhibit the synthesis of bacterial phosphatidylglycerol by targeting bacterial phosphatidylglycerol phosphate synthase, and has antibacterial and broad-spectrum synergistic activities. Its MICs against Staphylococcus aureus and Streptococcus are 4 μg / mL and 8 μg / mL respectively, and the MIC 90 against methicillin-resistant Staphylococcus aureus is 8 μg / mL, and the MIC against hypervirulent Klebsiella pneumoniae is 16 μg / mL. In addition, CDP-3 can significantly enhance the antibacterial efficacy of antibiotics such as clindamycin, tilmicosin, tetracycline, florfenicol, gentamicin, chloramphenicol, rifampicin and ofloxacin against multi-drug resistant Gram-negative bacteria, and has broad-spectrum synergistic activity. Sub-inhibitory concentrations of CDP-3 can not only inhibit bacterial growth, but also reduce bacterial biofilm formation, and inhibit virulence and pathogenicity. CDP-3 not only increases the survival rate of Galleria mellonella infected with MRSA, but also shows excellent therapeutic efficacy against animal models infected with multi-drug resistant Gram-negative bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of antibacterial agents, and particularly 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 pathogenic bacteria resistance has made the originally effective drugs gradually become less effective or even ineffective. In 2019, the number of deaths globally related to bacterial resistance reached 1.27 million, and the top-ranked pathogens in terms of lethality were Escherichia coli, Staphylococcus aureus, Streptococcus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Mycobacterium tuberculosis (Lancet, 2022). The current situation of bacterial resistance in China is also not optimistic. Research shows that the resistance rates of 51 strains of Staphylococcus aureus isolated from Xinjiang Uygur Autonomous Region to 11 commonly used antibiotics are as follows: ampicillin (51 / 51, 100%), azithromycin (44 / 51, 86.27%), sulfisoxazole (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 rates of 33 strains of porcine Streptococcus isolated from Jiangxi region to clindamycin, co-trimoxazole, and gentamicin reached 100%, and among them, 12 strains (12 / 33, 40%) were resistant to 9 drugs simultaneously. Resistance genes of tetracyclines, macrolides, aminoglycosides, and sulfonamides were detected in all 33 strains of Streptococcus, and the detection rates were tetO (78.8%), tetM (15.2%), ermB (90.9%), mefA (40%), ermA (9%), strA (97%), strB (93.93%), Aph3 (72.7%), sul1 (100%), sul2(21.2%) (Huang Yuting, 2023). As bacteria adapt to antibacterial drugs, they have evolved mechanisms to resist all existing antibiotics. Although the development of new antibacterial drugs is a rigid demand in medical and health care, developing drugs with the same mechanism of action as existing antibacterial drugs will inevitably lead to drug resistance quickly. Therefore, it is a question worthy of in-depth consideration what kind of antibacterial mechanism the drugs should have. On the other hand, since the treatment cycle of antibiotic drugs is shorter than that of drugs for hypertension, diabetes, leukemia, etc.; and due to the severity of bacterial drug resistance, newly developed antibiotics may be classified as reserve drugs rather than entering the market for circulation. Given the low expected economic return, pharmaceutical companies lack the motivation to develop new antibiotic drugs. In the past nearly 50 years, only daptomycin, a new antibacterial drug from natural sources, has been put into clinical use. It is reported that one of the main reasons why most antibacterial drugs are ineffective against Gram-negative bacteria is that the amount entering bacterial cells is insufficient (Richter & Hergenrother, 2019). If a compound that can increase the intracellular accumulation of antibacterial drugs in bacteria can be developed, then this compound will have broad-spectrum synergistic effects on existing antibiotics (such as polymyxin), will reverse the drug resistance of bacteria to existing antibacterial drugs, and will extend the service life of existing drugs, undoubtedly having great economic prospects.

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

[0004] 5-Fluoro-2'-deoxycytidine has been extensively studied in leukemia and anti-tumor fields. According to the inventor's search, there is no report on the antibacterial activity of 5-fluoro-2'-deoxycytidine. According to the reports of 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 have similar structures to 5-fluoro-2'-deoxycytidine, have certain antibacterial activities (doi:10.1016 / j.ijantimicag.2023.107076, see Table 4). In this invention, the antibacterial activities of gemcitabine and azacitidine were actually measured. 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 is 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, >128 μg / mL, respectively, which were significantly weaker than those of CDP-3 (Table 1, Table 2). Summary of the Invention

[0005] The present invention provides an antibacterial compound with a broad antibacterial spectrum, good antibacterial activity, strong broad-spectrum synergistic effect, and significant in vivo therapeutic effect for clinical use. The present invention discovers the targeting of 5-fluoro-2'-deoxycytidine (CDP-3) to bacterial PgsA and first proposes its new use in antibacterial. There is still no public report on the application of 5-fluoro-2'-deoxycytidine in antibacterial. The present invention first proposes 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 that of existing antibiotics due to its targeting of bacterial PgsA. Its discovery and further application in antibacterial will enrich the reservoir of antibacterial drug source molecules, alleviate the severe situation of drug resistance, and also provide a new option for the treatment of infections caused by drug-resistant pathogenic bacteria in clinical practice. Specifically, the present invention proposes the following technical solutions:

[0006] The first object of the present invention is to provide the use of 5-fluoro-2'-deoxycytidine in the preparation of antibacterial drugs.

[0007] Further, the pathogenic 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.

[0008] The present invention investigated the antibacterial activity of 5-fluoro-2'-deoxycytidine (CDP-3) against common clinical pathogenic bacteria, its synergistic activity with existing antibiotics, and its effectiveness in an animal infection model. The results showed that the MICs of CDP-3 against Staphylococcus aureus and Streptococcus were 4 μg / mL and 8 μg / mL, respectively, the MIC against highly virulent Klebsiella pneumoniae was 16 μg / mL, the MIC against vancomycin-resistant Enterococcus faecium was 32 μg / mL, and the MIC 90 against MRSA (n = 43) was 8 μg / mL. CDP-3 can significantly enhance the antibacterial activity of antibiotics such as clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, gentamicin, chloramphenicol, rifampicin, and ofloxacin against multi-drug resistant Gram-negative bacteria. Sub-inhibitory concentrations of CDP-3 can not only inhibit bacterial growth but also inhibit bacterial biofilm formation and induce attenuation of virulence and pathogenicity. At the same time, CDP-3 not only increased the survival rate of Galleria mellonella infected with MRSA T144 (MIC = 4 μg / mL), but also showed excellent therapeutic efficacy against Galleria mellonella infected with multi-drug resistant Escherichia coli ( E. coli B2, MIC = 64 μg / mL), highly virulent Klebsiella pneumoniae (CRHVKP4, MIC = 16 μg / mL), and multi-drug resistant Salmonella ( S. enteritis 15E475, MIC = 64 μg / mL). When CDP-3 was used in combination with clindamycin, it significantly improved the healing effect of clindamycin on the skin wounds of mice infected with E. coli B2. The application of CDP-3 in the field of antibacterials 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.

[0009] The second object of the present invention is to provide an antibacterial agent, the active ingredient of which includes 5-fluoro-2'-deoxycytidine.

[0010] Further, the antibacterial agent further includes at least one of rifampicin, ofloxacin, cefepime, clindamycin, tilmicosin, roxithromycin, niclosamide, tetracycline, florfenicol, gentamicin, and chloramphenicol.

[0011] The inventors found that in addition to its excellent antibacterial activity, 5-fluoro-2'-deoxycytidine can be used in combination with other antibacterial drugs to enhance the antibacterial activity of other commonly used clinical antibacterial drugs, especially against strains that have developed sufficient drug resistance. Based on this, the third objective of the present invention is to provide the use of a composition of 5-fluoro-2'-deoxycytidine and other common antibacterial drugs in antibacterial. Specifically, the third aspect of the present invention proposes the following uses for the combined use of 5-fluoro-2'-deoxycytidine and other common antibacterial agents.

[0012] 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 antibacterial drug against Escherichia coli. The Escherichia coli is preferably multidrug-resistant Escherichia coli (such as E. coli B2), or a clinical strain of Escherichia coli (such as E. coli 1653).

[0013] The inventors found that 5-fluoro-2'-deoxycytidine at 32 μg / mL can increase the MIC of clindamycin, tilmicosin, and niclosamide against multidrug-resistant Escherichia coli ( E. coli B2) by 8 to 64 times; 5-fluoro-2'-deoxycytidine at 32 μg / mL can increase the MIC of tetracycline, florfenicol, and gentamicin against a clinical strain of Escherichia coli ( E. coli 1653) by 8 to 32 times.

[0014] The present invention also provides the use of a composition of 5-fluoro-2'-deoxycytidine and at least one of chloramphenicol, tilmicosin, and clindamycin in the preparation of an antibacterial drug against Salmonella. The Salmonella is preferably multidrug-resistant Salmonella ( S. enteritis 15E475).

[0015] The inventors found that 5-fluoro-2'-deoxycytidine at 8 μg / mL can increase the MIC of chloramphenicol, tilmicosin, and clindamycin against multidrug-resistant Salmonella ( S. enteritis 15E475) by 8 to 256 times.

[0016] The present invention also provides the use of a composition of 5-fluoro-2'-deoxycytidine and at least one of clindamycin, rifampicin, and ofloxacin in the preparation of an antibacterial drug against Klebsiella pneumoniae; the inventors found that 5-fluoro-2'-deoxycytidine at 8 μg / mL can increase the MIC of clindamycin against Klebsiella pneumoniae ( K. pneumoniae WNX-1) by 16 times; 5-fluoro-2'-deoxycytidine at 4 μg / mL can increase the MIC of rifampicin and ofloxacin against highly virulent Klebsiella pneumoniae ( K. pneumoniae CRHVKP4) by 16 times.

[0017] There is no particular limitation on the dosage form of the pharmaceutical composition provided by the present invention, such as tablets, capsules, granules, suspensions, injections or suspensions.

[0018] The pharmaceutical composition includes excipients, and the excipients are selected from at least one of fillers, binders, wetting agents, disintegrants, flavoring agents, preservatives, pH regulators, antioxidants.

[0019] The beneficial effects of the present invention are as follows:

[0020] This application discovers a new application and antibacterial mechanism of 5-fluoro-2'-deoxycytidine (CDP-3) in antibacterial. CDP-3 shows excellent antibacterial or synergistic activity against common pathogenic bacteria in clinic. Its MICs against Staphylococcus aureus and Streptococcus are 4 μg / mL and 8 μg / mL respectively, the MIC against Klebsiella pneumoniae with high drug resistance and high virulence (CRHVKP4) is 16 μg / mL, the MIC against Enterococcus faecium resistant to vancomycin is 32 μg / mL, and the MIC against MRSA (n = 43) 90 is 8 μg / mL. At 32 μg / mL, it can increase the MICs of clindamycin, tilmicosin, and niclosamide against multidrug-resistant Escherichia coli ( E. coli B2) by 8 to 64 times; at 32 μg / mL, it can increase the MICs of tetracycline, florfenicol, and gentamicin against clinical strains of Escherichia coli ( E. coli 1653) by 8 to 32 times; at 8 μg / mL, it can increase the MICs of chloramphenicol, tilmicosin, and clindamycin against multidrug-resistant Salmonella ( S. enteritis 15E475) by 8 to 256 times; at 8 μg / mL, it can increase the MIC of clindamycin against Klebsiella pneumoniae ( K. pneumoniae WNX-1) by 16 times, and at 4 μg / mL, it can increase the MICs of rifampicin and ofloxacin against Klebsiella pneumoniae ( K. pneumoniae CRHVKP4) by 16 times.

[0021] CDP-3 not only increases the survival rate of Galleria mellonella infected with MRSA T144 (MIC = 4 μg / mL), but also against E. coli B2 (MIC = 64 μg / mL), K. pneumoniae CRHVKP4 (MIC = 16 μg / mL) and S. enteritisGreater wax moths infected with multi-drug resistant negative bacteria such as 15E475 (MIC = 64 μg / mL) also showed excellent therapeutic effects. Although CDP-3 has low antibacterial activity against negative bacteria, it has good in vivo therapeutic effects on negative bacteria infections, which undoubtedly provides a new treatment strategy for clinical treatment of negative bacteria infections. The discovery of the new use of CDP-3 provides a compound with a completely new antibacterial mechanism, enriching the antibacterial drug source molecule library. Its further application will provide a new therapeutic drug for the prevention and control of clinical related bacterial diseases; the revelation of its antibacterial mechanism proves the druggability of bacterial PgsA protein, and will also provide a theoretical reference for the development of more drugs targeting bacterial PgsA. The submission of this application will provide a new option for extending the service life of existing antibiotics and alleviating the severe situation of bacterial drug resistance, promote the research and development of drugs with new antibacterial mechanisms, and contribute to China's independent new drug innovation strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the changes in the phospholipid fatty acid chains of Staphylococcus aureus after treatment with CDP-3;

[0023] Figure 2 shows the changes in the phospholipid abundance of Staphylococcus aureus after treatment with CDP-3;

[0024] Figure 3 is the bactericidal curve of CDP-3 against MRSA T144;

[0025] Figure 4 is the growth curve of MRSA T144 under different compound conditions;

[0026] Figure 5 is the inhibitory effect of CDP-3 on bacterial biofilm formation;

[0027] Figure 6 is the inhibitory effect of CDP-3 on bacterial virulence;

[0028] Figure 7 is the toxicity display of CDP-3 on mice;

[0029] Figure 8 is the therapeutic effect of CDP-3 on the greater wax moth infection model;

[0030] Figure 9 is the therapeutic effect of CDP-3 combined with clindamycin on the multi-drug resistant Escherichia coli skin infection model. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present application will be further described in detail in conjunction with specific embodiments. The provided embodiments are only for clarifying the present application and not for limiting the scope of the present application. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present application in any way.

[0032] Example 1 Antibacterial Activity of Cytidine Derivatives against Common Clinical Pathogens

[0033] Pick a single colony of the test strain into BHI broth and culture it on a shaker at 37 °C until the logarithmic growth phase of the bacteria. Use a McFarland turbidimeter to adjust the turbidity of the bacteria to a McFarland turbidity of 0.5, and dilute it 100-fold with MHB broth medium to about 10 6 CFU / mL. Take 100 μL of the above bacterial solution and add it to a 96-well U-bottom plate. Dissolve the cytidine series derivatives in phosphate buffer (PBS). Add 100 μL of MHB broth medium to each column of the 96-well U-bottom plate. Take 100 μL of a certain concentration of the compound and add it to the first column of the 96-well U-bottom plate, and serially dilute it 2-fold to the tenth column. Add an additional 100 μL of the test bacterial solution to the twelfth column as a positive control, and the eleventh column as a negative control. Place the 96-well U-bottom plate in an incubator at 37 °C for 18 - 20 h, read the experimental results, and take the lowest drug concentration that visibly inhibits bacterial growth as the MIC value of the compound.

[0034] The present invention tested the antibacterial properties of various cytidine derivatives, and the general formula structure is shown as formula (I) below. The MIC results of various compounds against various types of bacteria are shown in Tables 1 and 2.

[0035] The MICs of cefoxitin, clindamycin, erythromycin, chloramphenicol, florfenicol, linezolid, and vancomycin against MRSA T144 are 8 μg / mL, >128 μg / mL, >128 μg / mL, 32 μg / mL, 64 μg / mL, 1 μg / mL, and 1 μg / mL, respectively. From the data in Table 1, it can be seen that the antibacterial activity of CDP-3 against MRSA T144 is significantly stronger than that of commonly used clinical antibiotics such as cefoxitin, clindamycin, erythromycin, chloramphenicol, and florfenicol (Table 1).

[0036] From the data in Table 2, it can be seen that 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), vancomycin-resistant Enterococcus faecium (VRE 10), etc., and the MICs are 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL respectively. The MIC of CDP-3 against MRSA (n = 43)90 is 8 μg / mL.

[0037] (I)

[0038] Table 1. Bacteriostatic efficacy of cytidine derivatives against MRSA T144

[0039] .

[0040] According to the reports of Jordheim LP et al. (2012) and Zheng et al. (2024), gemcitabine (CDP-8) and azacitidine (CDP-9) with similar structures to CDP-3 (doi:10.1016 / j.ijantimicag.2023.107076, see Appendix 4) have certain antibacterial activities. In this invention, the antibacterial activities of gemcitabine, azacitidine and CDP-3 were compared. The results showed that the antibacterial activities of CDP-3 against positive bacteria such as MRSA T144, MRSA T50, ATCC29213, RN4220, and streptococcus were significantly stronger than those of gemcitabine and azacitidine (Table 1, Table 2); the antibacterial activities against negative bacteria such as Klebsiella pneumoniae CRHVKP4 and Escherichia coli ATCC 25922 were also higher than those of gemcitabine (CDP-8) and azacitidine (CDP-9) (Table 1, Table 2). Although the above comparative documents publicly mentioned that gemcitabine and azacitidine have antibacterial activities, it was found through actual measurement that the antibacterial activity of azacitidine (CDP-9) was weak (MIC≥128 μg / mL), and the antibacterial activity of gemcitabine (CDP-8) was also significantly lower than that of CDP-3. Based on this, the applicant proposed that compared with the publicly disclosed similar structures, the activity of CDP-3 proposed in this application was significantly better, and this application had certain creativity.

[0041] Table 2. Bacteriostatic efficacy of cytidine derivatives against common clinical pathogenic bacteria

[0042] ;

[0043] Note: Escherichia coli B2 is a multi-drug resistant bacterium carrying 25 multi-drug resistance genes, including blaNDM-5 , mcr-1 , tetA , mdfA , oqxAB , arr-2 , aph(4) , aac(3)-IV and fosA3。The MIC values of cefepime, polymyxin, ofloxacin, rifampicin, and tetracycline against Escherichia coli B2 were 32, 2, 32, 128, and 128 μg / mL, respectively. Enterococcus faecium VRE10 is a vancomycin-resistant bacterium, and CRHVKP4 is a highly drug-resistant and highly virulent Klebsiella pneumoniae. 43 strains of MRSA were isolated from the clinic. " / " indicates not tested.

[0044] 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 substituents at the 5-position are different, and the properties of the substituents at the 5-position are very similar. In theory, the activities 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 were all greater than 128 μg / mL. Only CDP-3 (5-F) showed good antibacterial activity (MIC = 4 μg / mL). The above data prove that the discovery of CDP-3 was obtained through strict screening and research.

[0045] Example 2: Synergistic efficacy of CDP-3 against common antibiotics

[0046] The checkerboard broth dilution method was used to determine the synergistic efficacy (FIC) of the combination of CDP-3 and common antibiotics against multi-drug resistant Escherichia coli, Salmonella, and Klebsiella pneumoniae. Single bacterial colonies on the agar plate were picked and cultured in MHB broth at 37 °C in a shaker until the logarithmic growth phase. The turbidity of the bacteria was adjusted to a McFarland turbidity of 0.5 using a McFarland turbidimeter and diluted 100-fold (~1.0×10 6 CFU / mL) with MHB medium and then used for later experiments. 100 μL of MHB was taken with a multi-channel pipette and added to each well of a 96-well U-shaped plate. The prepared antibiotics were diluted to approximately 8 times the MIC with PBS, and 100 μL was taken and added to the first column of the 96-well U-shaped plate, and then serially diluted to the seventh column with a multi-channel pipette, and the pipette tips were discarded; at the same time, the CDP-3 stock solution was diluted to approximately 512 μg / mL with PBS, and 100 μL was taken and added to wells 1-8 in the eighth row of the 96-well U-shaped plate, and then serially diluted to the second row with a multi-channel pipette. Subsequently, 100 μL of the diluted test bacterial solution was added to each well. The eleventh column and the twelfth column were negative and positive controls containing only MHB medium and only the test bacterial solution, respectively. After incubation at 37 °C for 18 - 20 h, the FIC value was read. Among them, FIC = MIC (drug A in combination) / MIC (drug A alone) + MIC (drug B in combination) / MIC (drug B alone).

[0047] The results showed that the FIC indices of CDP-3 and common antibiotics were all within 0.5 (Table 3), indicating that CDP-3 provided by the present invention and common antibiotics all had strong synergistic effects. CDP-3 could significantly improve the antibacterial activities of antibiotics such as clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, gentamicin, chloramphenicol, tilmicosin, roxithromycin, rifampicin, and ofloxacin against Gram-negative bacteria such as Escherichia coli, Salmonella, Klebsiella pneumoniae, and Acinetobacter baumannii. In particular, it could increase the antibacterial activity of niclosamide against drug-resistant Escherichia coli (B2) by at least 64 times, and increase the MIC value of clindamycin against drug-resistant Salmonella enteritidis (15E475) by 128 times or more. CDP-3 made the originally insensitive antibacterial drugs become sensitive, significantly reversing the drug resistance of bacteria to existing antibiotics.

[0048] When gemcitabine with a similar structure (CDP-8) was used in combination with roxithromycin, gentamicin, and clindamycin, the synergistic effect was significantly lower than that of CDP-3 proposed in this application (Table 4). The above results indicated that CDP-3 not only had excellent antibacterial activity itself, but also could be used in combination with existing antibiotics to enhance the antibacterial activity of old drugs, which undoubtedly helped to extend the service life of old drugs and gave new vitality to existing antibiotics.

[0049] Table 3. Synergistic effect of CDP-3 on common antibiotics

[0050] ;

[0051] 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.

[0052] Table 4. Comparison of synergistic effects of CDP-3 and gemcitabine when combined with antibiotics

[0053] ;

[0054] Note: The test strain was Acinetobacter baumannii (Ab 17-38); a, b: MIC of antibiotics in the presence or absence of CDP-3 and gemcitabine (CDP-8).

[0055] Example 3 Affinity between cytidine derivatives and bacterial phosphatidylglycerol phosphate synthase (PgsA)

[0056] To determine the antibacterial mechanism and structure-activity relationship of CDP-3, the PgsA protein was expressed, and the affinity of cytidine derivatives for bacterial phosphatidylglycerol phosphate synthase (PgsA) was measured using surface plasmon resonance (SPR) technology. The affinity constant of the endogenous ligand cytidine of PgsA for PgsA was 1.37×10 -9 M, and the affinity constants of CDP-3 (5-F) and CDP-7 (5-I) for PgsA were 1.76×10 -9 M and 14.9×10 -9 M, respectively. The above results indicate that the affinity of CDP-3 for PgsA is close to that of its endogenous ligand, confirming that CDP-3 can competitively bind to PgsA with the endogenous ligand, resulting in a decrease in the efficiency of bacterial phosphatidylglycerol synthesis and achieving an antibacterial effect. At the same time, when the 5-position of cytidine is -I (CDP-7), the binding affinity of CDP-7 for PgsA is 14.9×10 -9 M, which is 1 / 10 of the binding affinity of CDP-3. Based on this, it is proposed that fluorine substitution at the 5-position of cytidine is the optimal choice for exerting antibacterial activity.

[0057] To reveal the bacterial phospholipid reprogramming induced by CDP-3 targeting PgsA, changes in bacterial phospholipid abundance, phospholipid fatty acid chain composition, etc. under the action of CDP-3 were studied. Staphylococcus aureus RN4220 cultured for 6 - 8 h was diluted 4-fold and reserved. 5 mL of 320 μg / mL CDP-3 was added to 795 mL of sterile MHB medium to make the final concentration 2 μg / mL (1 / 2MIC). 1 mL of the reserved bacterial solution was aspirated and added to make the final concentration of the bacterial solution approximately 10 6 cfu / mL, and this group was the experimental group. Another 400 mL of sterile MHB medium was added with only the bacterial solution without CDP-3, which was set as the blank control group. The above culture solutions were placed in a shaker and cultured at 37°C and 200 rpm. The blank control group was cultured for 12 h, and the experimental group was cultured for 24 h. After the culture was completed, the bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 10 min. The collected bacterial cell precipitate was washed with PBS, and the supernatant was removed and the wet weight of the bacterial cells was weighed. After adding 5 mL of PBS to resuspend the bacterial cells, 15 mL of extraction solution (chloroform:methanol = 1:2, V:V) was added. After proper vortexing, it was placed in a shaker and shaken at 37°C and 200 rpm for 1.5 h to completely extract the bacterial phospholipids. The shaken liquid was placed in a -20°C refrigerator for 15 min to facilitate liquid stratification. After the liquid was stratified, approximately 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 using a gas chromatography-mass spectrometry instrument.

[0058] The results are as Figure 1As shown, compared with the untreated blank group, after treatment with CDP-3, the abundance of odd-chain fatty acids in the total fatty acid chain of Staphylococcus aureus decreased significantly, while the abundance of even-chain fatty acids increased significantly. 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, after treatment with 1 / 2 MIC of CDP-3, the abundances of PG and CL in Staphylococcus aureus decreased by 1.89 and 5.13 times respectively. The above results indicate that in the presence of CDP-3, the abundances of PG and CL in Staphylococcus aureus decreased significantly, which further proves that the antibacterial mechanism of CDP-3 is to inhibit the synthesis of bacterial PG. Moreover, the decrease in the abundance of CL in Staphylococcus aureus was significantly greater than that of PG, which may be due to the fact that when facing great survival pressure, bacteria adjusted their own phospholipid metabolism state to first ensure the abundance of PG and reduce the 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, under the action of 1 / 2 MIC CDP-3, the content of even-chain fatty acids increased significantly, which indicates that for survival, even-chain fatty acid chains are more important than odd-chain fatty acid chains.

[0059] Example 4

[0060] Bactericidal curve: Pick a single colony of MRSA T144 into 1 mL of BHI broth medium and culture the bacteria at 37°C and 200 rpm until the logarithmic growth phase. Subsequently, adjust the concentration of the MRSA T144 culture solution to a McFarland turbidity of 0.5. Add 5 mL of MHB medium to each well of a 6-well plate, and take 0.05 μL of the diluted bacterial solution and add it to the MHB to obtain a bacterial solution with a concentration of about 10 6 CFU / mL. Add different concentrations of CDP-3 (from 1 / 8×MIC to 8×MIC) to the bacteria-containing MHB, and add an equal volume of PBS as a negative control group. Set 3 replicates for each treatment and place them in an incubator at 37°C for culture. Take out 100 μL of the culture solution at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, dilute it serially in PBS and spread it on plates for counting. After the MHA agar plates are inverted and cultured at 37°C for 24 h, calculate the number of colonies.

[0061] Figure 3This is the bactericidal curve of CDP-3 against MRSA T144. The results show that CDP-3 is a time-dependent bactericide, and it still has the effect of inhibiting bacterial growth at sub-inhibitory concentrations, suggesting that even when the concentration of CDP-3 in vivo decreases below the MIC value during clinical treatment, it can still effectively inhibit bacterial growth.

[0062] Growth curve: Pick a single colony of MRSA T144 and inoculate it into 1 mL of BHI broth medium. Culture the bacteria at 37 °C and 200 rpm until they reach the logarithmic growth phase. Subsequently, adjust the concentration of the MRSA T144 culture solution to ~10 6 cfu / mL. Use a multichannel pipette to transfer 100 μL of the diluted culture solution into the first to sixth columns of a 96-well flat-bottom plate. Then, use a multichannel pipette to add 1 / 4×MIC of vancomycin (VAN), ofloxacin (OFL), cefquinome (CEFQ), roxithromycin (ROX), and CDP-3 into the first to sixth rows, respectively, as the VAN group, OFL group, CEFQ group, ROX group, and CDP-3 group. Add MHB to the sixth row of the well plate as a blank control group. Place the 96-well plate in an incubator at 37 °C and incubate. Take it out at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, respectively, and measure the OD 600nm value with an enzyme-linked immunosorbent assay (ELISA) reader to plot the growth curves under different antibacterial compounds.

[0063] Figure 4 This is the growth curve of MRSA T144 under different compound conditions. Different from common antibiotics, at 1 / 8 MIC, CDP-3 showed a significant effect of inhibiting bacterial growth, while common antibacterial drugs such as vancomycin (VAN), ofloxacin (OFL), cefquinome (CEFQ), and roxithromycin (ROX) did not have the effect of inhibiting bacterial growth at sub-inhibitory concentrations ( Figure 4 ). It can be seen that compared with common antibiotics, CDP-3 has significant antibacterial advantages.

[0064] Example 5 Effect of CDP-3 on bacterial biofilm formation

[0065] The inhibitory effect of CDP-3 on the biofilms of Staphylococcus aureus and Escherichia coli was elucidated by the crystal violet staining method. First, culture the Staphylococcus aureus RN4220 or Escherichia coli B2 culture for 8 h and dilute it to ~10 6cfu / mL. Add 0.1 mL of the dilution to each well of a 6-well plate. Then, add 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 respectively. Use fresh MHB without antibiotics as a control, with 3 replicates for each group. Incubate the plates at 37 °C for 48 h to form biofilms. Wash 3 times with 4 mL of PBS to remove planktonic cells from each well. Fix the biofilms formed on the bottom surface of the microplate wells at 65 °C for 1 h. Then, stain the biofilms with 400 µL of 0.25% (W / V) crystal violet solution at room temperature for 5 min. Wash 3 times with 4 mL of PBS to remove the excess crystal violet. After drying with a blower in a laminar flow hood for 15 min, observe and record the stained biofilms of different groups using an optical microscope (10×10). Then, extract the bound crystal violet with 400 µL of 95% ethanol and detect the OD values of different groups at 490 nm using a microplate reader.

[0066] The results of each treatment group are as Figure 5 shown. The results indicate that CDP-3 can significantly reduce the formation of RN4220 biofilms at concentrations of 1 / 4 - 1 / 16 MIC ( P ≤ 0.0005) ( Figure 5 A), and its inhibitory effect on biofilm formation is significantly stronger than that of 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 drugs, the biofilm formation levels after treatment with 1 / 8×MIC roxithromycin and kanamycin are significantly higher than those of the untreated group ( P ≤ 0.0023) ( Figure 5 B), suggesting that roxithromycin and kanamycin can promote biofilm formation at sub-inhibitory concentrations. Biofilms, as part of bacterial non-genetic drug resistance, play important roles in resisting disinfectants, antibiotics, recurrent infections, asymptomatic infections, and drug resistance evolution. Therefore, interfering with or removing biofilms has increasingly become an important part of antibacterial infection treatment. Compared with existing antibiotics, CDP-3 exhibits excellent biofilm formation inhibitory ability, once again demonstrating the advantages of CDP-3 provided by the present invention in antibacterial treatment and also indicating the advantages of phospholipid-targeted antibacterial.

[0067] Example 6

[0068] The reduction in CDP-3-induced bacterial biofilm formation suggests its potential in inhibiting bacterial virulence. Therefore, further studies were conducted on the effects of CDP-3 on staphyloxanthin, motility, and invasion ability.

[0069] 1. Effect of CDP-3 on staphyloxanthin

[0070] a) Pick a single colony of MRSA T144 into 1 mL of BHI broth medium and culture it at 37 °C and 200 rpm until the logarithmic growth phase. Subsequently, adjust the concentration of the MRSA T144 culture solution to a McFarland turbidity of 0.5. Add 5 mL of MHB medium to each well of a 6-well plate, and take 0.05 μL of the diluted bacterial solution and add it to the MHB to obtain a bacterial solution with a concentration of approximately 10 6 CFU / mL. Add CDP-3 and vancomycin at 8×MIC concentration to the MHB containing bacteria, and add an equal volume of PBS as a negative control group. Set 3 replicates for each treatment and incubate them in a 37 °C incubator. Take out 100 μL of the culture solution at 8 h, serially dilute it in PBS and spread it on plates for counting. Observe the colony morphology after the MHA agar plates are incubated upside down at 37 °C for 24 h.

[0071] b) Use the agar dilution method to perform MIC 90 testing on 43 clinical isolates of MRSA. After reading the results, use an agar plate image acquisition instrument to take pictures of the agar plates of CDP-3 and vancomycin at 1 / 2×MIC 90 and 1 / 4×MIC 90 to observe the morphology of 43 clinical isolates of MRSA under the treatment of CDP-3 and vancomycin.

[0072] 2. Effect of CDP-3 on the motility of Staphylococcus aureus

[0073] Weigh an appropriate amount of soybean broth culture medium according to the label instructions and add it to 200 mL of distilled water. Then add 0.8 g of agar powder, mix well, sterilize, and cool appropriately. Add an appropriate amount of CDP-3 solution and pour it into a 90 mm petri dish to make a CDP-3 soft agar plate containing 1 / 8 - 1 / 2×MIC. The soft agar without CDP-3 is used as a blank control. Pick single colonies of MRSA T144 and E. coli ATCC 25922 into 50 mL of BHI broth medium and culture them at 37 °C and 200 rpm for 8 h. Then centrifuge at 9000 rpm for 10 min at 4 °C to collect the bacterial cells. After resuspending the pellet with 1 mL of PBS, use a 10 μL pipette tip to drop 5 μL of approximately 10 9The resuspended bacterial solution at cfu / mL was blown dry in a laminar flow hood for 15 min and then placed in an incubator at 37 °C for 3 d to observe the movement range of colonies in different treatment groups.

[0074] 3. Effect of CDP-3 on the ability of Staphylococcus aureus to enter cells

[0075] RAW264.3 cells were resuscitated, cultured until completely adherent, centrifuged to collect the cells, resuspended with fresh medium, and 1.8 mL was aspirated and added to a 6-well plate. Then, 100 μL of the bacterial solution of Staphylococcus aureus RN4220 (~10 6 cfu / mL) was added, and then 100 μL of CDP-3 was aspirated and added to the 6-well plate to make the concentration of CDP-3 1 / 8 - 1 / 2×MIC. The group without adding CDP-3 was used as a blank control, with 3 replicates in each group. The 6-well plate was placed in an incubator at 37 °C for culture. The cells of each group were collected at 4 h and 8 h respectively, broken, and the plates were coated with Staphylococcus aureus RN4220 for counting. The number of intracellular bacteria in different treatment groups was counted, and the effect of CDP-3 on the ability of Staphylococcus aureus to enter cells was analyzed.

[0076] After treatment with VAN, MRSA T144 had a typical golden appearance ( Figure 6 Ac and Ad), while after treatment with CDP-3, MRSA T144 showed a transparent appearance ( Figure 6 Aa and Ab), indicating that CDP-3 could significantly inhibit staphyloxanthin of Staphylococcus aureus. As an important virulence factor of Staphylococcus aureus, staphyloxanthin plays an important role in invasion, infection, and resistance to the body's immunity. The inhibitory effect of CDP-3 on staphyloxanthin will be beneficial to the treatment of clinical related infections. After treatment with CDP-3, the movement range of Staphylococcus aureus RN4220 and Escherichia coli ATCC 25922 was significantly reduced ( Figure 6 B), indicating that CDP-3 could 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 the role of CDP-3 in inhibiting the virulence of Staphylococcus aureus.

[0077] Example 7 Toxicity study of CDP-3 on mice

[0078] To determine the dosage and safety of CDP-3 during treatment trials, a toxicity study of CDP-3 on mice was conducted. Twenty-four Babl / C mice (25 - 30 g) were randomly divided into 4 groups, with 6 mice in each group. The mice were intraperitoneally injected with doses of 10 mg / kg b.w., 20 mg / kg b.w., and 40 mg / kg b.w. respectively, twice a day for 5 consecutive days. On the 5th day, half of the mice in each group were dissected, and organs such as the liver, kidney, lung, heart, spleen, stomach, and intestine were taken out to observe the pathological changes of the organs in the mice of each dosage group. The remaining mice were taken off the drug and observed for another 7 days, during which the survival rate of the mice was recorded. On the 12th day, the remaining mice were dissected, and organs such as the liver, kidney, lung, heart, spleen, stomach, and intestine were taken out to observe the recovery of organ damage.

[0079] The results showed that after continuous administration of the high, medium, and low doses for 5 days, diarrhea occurred in the mice of each dosage group, accompanied by gastric distension and intestinal fluid accumulation ( Figure 7 A, Figure 7 B). Compared with the PBS group, the spleens of the mice in the 20 mg / kg b.w. and 40 mg / kg b.w. groups were significantly smaller ( Figure 7 A), and the degree of spleen reduction in the 10 mg / kg b.w. group was less. After discontinuing the drug, all the remaining mice in the 20 mg / kg b.w. and 40 mg / kg b.w. groups died, while all the mice in the 10 mg / kg b.w. group survived, and the pathological changes of the stomach, intestine, and spleen in the 10 mg / kg b.w. group were significantly improved ( Figure 7 C). The above results indicate that intraperitoneal injection of CDP-3 at a dose of 10 mg / kg b.w., twice a day for 5 consecutive days, is relatively safe for mice, and a dose of 10 mg / kg b.w. or lower can be used for administration in subsequent treatment trials.

[0080] Example 10: Therapeutic effect of CDP-3 in an animal infection model

[0081] 1. MRSA infection model of Galleria mellonella

[0082] Fifty Galleria mellonella were randomly divided into 5 groups, with 10 in each group, including a control group, a negative group, a vancomycin (VAN) group, a low-dose CDP-3 group (10 mg / kg b.w.), and a high-dose group (20 mg / kg b.w.). 10 μL of ~2×10 5MRSA T144 at cfu / mL was injected into the right posterior abdominal foot of Galleria mellonella. At 1 h post-infection, 10 μL of PBS, VAN (20 mg / kg b.w.), and CDP-3 (10 mg / kg b.w. or 20 mg / kg b.w.) were injected into Galleria mellonella at the left posterior abdominal foot, and the survival rate of Galleria mellonella within 6 d was recorded.

[0083] 2. Multidrug-resistant negative bacteria infection model of Galleria mellonella

[0084] Fifty Galleria mellonella were randomly divided into 5 groups with 10 in each group, including a control group, a negative group, a polymyxin sulfate (Coli) group, a low-dose CDP-3 group (10 mg / kg b.w.), and a high-dose group (20 mg / kg b.w.). 10 μL of E. coli B2 (10 7 cfu / mL), Klebsiella pneumoniae CRHVKP4 (10 5 cfu / mL), and Salmonella 15E475 (10 5 cfu / mL) were injected into the right posterior abdominal foot of Galleria mellonella. At 0.5 - 1 h post-infection, 10 μL of PBS, Coli (20 mg / kg b.w.), and CDP-3 (10 mg / kg b.w. or 20 mg / kg b.w.) were injected into Galleria mellonella at the left posterior abdominal foot, and the survival rate of Galleria mellonella within 5 d was recorded.

[0085] 3. Multidrug-resistant Escherichia coli ( E. coli B2) skin infection model in mice

[0086] Twenty mice (BABL / C) were anesthetized by intraperitoneal injection of 200 μL of 10% ethyl carbamate, and then randomly divided into five groups (n = 4), including a control group, a negative group, a polymyxin (Coli) group, a clindamycin (CLI) group, a CDP-3 group, and a +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 of Escherichia coli B2 culture solution at ~10 9 cfu / mL was dropped on the wound. At 24 h post-infection, 40 μL of 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 dropped on the wounds of the mice in different groups once a day. The top views of the locally infected skin of the mice under different treatments were recorded at 0, 2, 4, 6, and 10 d respectively.

[0087] In the Galleria mellonella MRSA infection model, the protection rate of CDP-3 against G. mellonella within 6 days of infection was 80%, slightly better than the 70% of vancomycin. Compared with the non-drug group, CDP-3 doubled the survival rate of G. mellonella infected with MRSA ( Figure 8 of A), suggesting that CDP-3 has good therapeutic effects on systemic MRSA infections. In the multi-drug resistant E. coli B2 infection model of G. mellonella, within 5 days of infection, the protection rate of CDP-3 at 20 mg / kg b.w. against G. mellonella was 90%, superior to the protective efficacy of the positive drug Coli (50%) ( Figure 8 of B). In the multi-drug resistant Klebsiella pneumoniae CRHVKP4 infection model of G. mellonella, within 5 days of infection, the protection rate of the 20 mg / kg b.w. CDP-3 group against G. mellonella was 50%, superior to the protective efficacy of the positive drug Coli ( Figure 8 of C). In the multi-drug resistant Salmonella 15E475 infection model of G. mellonella, within 5 days of infection, the protection rates of the high and low CDP-3 groups against G. mellonella were 90% and 80% respectively, superior to the 50% of the positive drug Coli ( Figure 8 of D).

[0088] As mentioned above, the MIC values of CDP-3 against E. coli B2 and Salmonella 15E475 were 64 μg / mL. When CDP-3 was administered at 20 mg / kg (20 μg / g), the concentration in G. mellonella could not reach 64 μg / mL. That is, the dose of 20 mg / kg b.w. theoretically had no bactericidal activity against E. coli B2 and Salmonella 15E475, while 20 mg / kg b.w. of CDP-3 did show excellent protective efficacy. Combining the above inhibition of CDP-3 on bacterial phosphatidylglycerol synthesis, and phosphatidylglycerol is an important component required for bacterial membrane composition, signal communication, and virulence expression, it is speculated that this phenomenon is due to sub-inhibitory concentrations of CDP-3 inhibiting bacterial virulence, maintaining bacteria in a low-vitality state, thereby increasing the survival rate of G. mellonella. This ability to maintain bacteria in a low-vitality state undoubtedly contributes to the prognosis of bacterial infections and will provide new strategies for the treatment of clinical multi-drug resistant Gram-negative bacterial infections.

[0089] Figure 9 shows the therapeutic effect of CDP-3 in combination with clindamycin on the multi-drug 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 ( Figure 9A). When CLI was used alone, there was still scabbing at the wound on the 10th day, while when CLI was used in combination with CDP-3, the wound healing effect was significantly improved, and CDP-3 also showed excellent wound healing effect when used alone, and its effect was even slightly stronger than that of the Coli group ( Figure 9 B). CDP-3 had no bactericidal activity against Escherichia coli B2 at 50 μg / mL. Combining with the inhibitory effect of CDP-3 on bacterial virulence above, it was speculated that the excellent effect of CDP-3 was due to its inhibition of bacterial virulence. After the bacterial virulence was inhibited, the wound inflammation decreased. Although CDP could not directly kill Escherichia coli B2, compared with Coli with bactericidal effect, CDP-3 still showed a stronger wound healing effect, which indicated the importance of inhibiting bacterial virulence during the treatment of bacterial infections, proved the great potential of CDP-3 targeting bacterial phospholipids in antibacterial therapy applications, and also indicated the great economic benefits of this application.

[0090] In summary, this application discovered a new application of CDP-3 in antibacterial: it can target bacterial phosphatidylglycerol phosphate synthase (PgsA) to exert antibacterial and synergistic activities, and it is the first antibacterial compound discovered so far that targets PgsA to act on the synthesis of bacterial phosphatidylglycerol (PG). Due to having a brand-new mechanism of action different from existing antibiotics, CDP-3 has good antibacterial activities against positive bacteria such as sensitive and drug-resistant Staphylococcus aureus (MRSA), Streptococcus, Enterococcus faecium, etc. and highly virulent Klebsiella pneumoniae (CRHVKP4) (Table 1, Table 2). The inhibition of the synthesis of the bacterial cell membrane phospholipid component PG leads to an increase in the permeability of the bacterial cells, making CDP-3 significantly improve the antibacterial activities of antibiotics such as clindamycin, tilmicosin, niclosamide, tetracycline, florfenicol, gentamicin, chloramphenicol, tilmicosin, roxithromycin, rifampicin, and ofloxacin against negative bacteria such as drug-resistant Escherichia coli, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii, etc. (Table 3). Due to the limitation of the synthesis of phosphatidylglycerol, the basic component of the bacterial cell membrane, bacteria are restricted in aspects such as biofilm formation, motility, virulence, and pathogenicity, making CDP-3 have a better protective effect against infections caused by multi-drug resistant negative bacteria than bactericides (such as polymyxin) under the premise of low antibacterial activity ( Figure 8 、 Figure 9 ), which indicates 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 continuously at a dose of 10 mg / kg b.w. twice a day for 5 days, only causing slight diarrhea in mice, and the symptoms could recover after drug withdrawal, indicating that the toxicity of CDP-3 was within an acceptable range. In the current situation of severe bacterial drug resistance and insufficient new drug development, the new application of CDP-3 in antibacterial proposed by this invention undoubtedly brings hope for the treatment of clinical drug-resistant pathogenic bacterial infections.

Claims

Use of 5-fluorodeoxycytidine in the preparation of antibacterial drugs; the bacteria in the antibacterial drugs are Klebsiella pneumoniae CRHVKP4. Use of a composition of 5-fluorodeoxycytidine and at least one of rifampicin and ofloxacin in the preparation of a drug against Klebsiella pneumoniae; the Klebsiella pneumoniae is Klebsiella pneumoniae CRHVKP4.

3. The use according to claim 1 or 2, characterized in that, The dosage of 5-fluorodeoxycytidine is 5 mg / kg / d.

4. The use according to claim 1 or 2, characterized in that, The dosage form of the drug is tablet, capsule, granule, suspension, injection or suspension.

5. The use according to claim 2, characterized in that, The drug further comprises excipients, and the excipients include fillers, binders, wetting agents, disintegrants, flavoring agents, preservatives, pH regulators or antioxidants.

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