Synthesis And Antibacterial Evaluation Of Platensimycin-Silver(Ⅰ) Salt And Its 2,4-Dinitrobenzyl Derivative

Staphylococcus aureus is one of the leading pathogens of hospital and community-acquired infections.These infections have posed a serious threat to human health.However,the development of antimicrobial drugs has lagged far behind the emergence and spread of drug-resistant bacteria,including methicillin-resistant staphylococcus aureus（MRSA）.Therefore,there is an urgent need to develop antibiotics with new mechanisms of action and discover new therapeutic approaches to address the challenge of multi-drug-resistant pathogens.Platensimycin（PTM）is a highly selective antibiotic that inhibits fatty acid synthesis by selectively competing for the active site of bacterial fatty acid synthase.It shows potent antibacterial activity against Gram-positive bacteria in vitro and in vivo.However,we previously found that PTM is a bacteriostatic antibiotic,and is particularly ineffective against persistent bacteria such as residue MRSA in the infected macrophages.In addition,the poor in vivo pharmacokinetic properties of PTM also limit its further development and clinical application.The antibacterial activity and the pharmacokinetic properties of PTM may be improved by chemical modification or nano-delivery strategies.In this Thesis,the synergistic antibacterial effect of different forms of Ag including Ag⁺and Ag nanoparticles（AgNPs）with PTM,and the antibacterial activity of PTM-Ag（Ⅰ）salt was studied.We showed that both Ag⁺and AgNPs can have a synergistic antibacterial effect with PTM against S.aureus,and the silver salt of PTM has better antibacterial activity than PTM.In addition,we also synthesized a 2,4-dinitrobenzyl derivative of PTM and investigated its antibacterial activity,supporting the adoption of the antibody-recruiting molecule（AMR）strategy for its in vivo antibacterial evaluation.The main research contents of this Thesis are as follows:Synthesis and evaluation of the antibacterial activity of silver salt of PTM.We first verified that PTM can synergize with AgNO₃ to exhibit excellent antibacterial activity against the tested S.aureus strains.Although low concentrations of PTM and AgNO₃ had no effect on the growth of the clinically isolated E.coli strain,their combination showed significant antibacterial effect on the E.coliΔtol C mutant strain with the deletion of the efflux pump gene tol C.We also synthesized AgNPs by chemical reduction and discovered that AgNPs could also increase the activity of PTM against Streptococcus aureus ATCC 29213.However,the rapid aggregation of AgNPs in bacterial culture medium inspired us to adopt other approaches for the combination of PTM and Ag⁺.Silver sulfadiazine,a complex of the antibiotic sulfadiazine and Ag（Ⅰ）,has been widely used clinically for the treatment of infections in burn wounds.Therefore,we hypothesized that PTM and Ag（Ⅰ）could also form silver salts,which may show excellent antibacterial effects due to their synergetic effects.We first synthesized the silver salt of PTM and found that PTM and Ag⁺in PTM-Ag（Ⅰ）were in a 1:4 ratio by thermogravimetric analysis.X-ray photoelectron spectroscopy revealed that the nitrogen atoms on the amide in PTM and PTM-Ag（Ⅰ）had different binding energies,which indicates the formation of PTM-Ag（Ⅰ）.There is a special symmetric stretching vibration peak of the carboxylate group of benzoic acid on PTM of the sliver salt at 1540.23 cm^-1 in Fourier transform infrared spectroscopy.This also supports the formation of the PTM-Ag（Ⅰ）complex and is consistent with its ¹H NMR spectrum in solution.PTM-Ag（Ⅰ）showed excellent antibacterial efficacy against a variety of S.aureus including MRSA.For example,the anti-staphylococcus activity of PTM-Ag（Ⅰ）（5μM）was about 50-fold better than that of PTM（5μM）against strains MRSA 2968,MRSA 2954,and MRSA 3161.Finally,we explored the antibacterial mechanism of PTM-Ag（Ⅰ）against MRSA2968 using the fluorescent probe pyridinium iodide dye（PI）2’,7’-dichlorodihydrofluorescein diacetate and found that its antibacterial activity was associated with increased bacterial cell membrane permeability and overproduction of reactive oxygen species.Synthesis and evaluation of the antimicrobial activity of 2,4-dinitrophenyl derivatives of platensimycin.Since about 1%of antibodies in human serum can bind nitroarene epitopes,2,4-dinitrobenzene derivatives have been used as antigens against anti-2,4-dinitrobenzene Ig G antibodies for the development of new small molecule drugs that exploit the function of the immune system.We hypothesized that a nitroarene-platensimycin conjugate may become an AMR to allow the association of PTM to anti-DNP Ig G antibodies,thereby prolonging its half-life in serum and improving the antimicrobial efficacy.We then designed and synthesized the PTM derivative LL-2 containing 2,4-dinitrobenzene in two steps and discovered that its minimum inhibitory concentration against S.aureus was 4μg/m L.In human serum,the half-life of LL-2 was approximately twice that of PTM,indicating its superior serum stability to PTM.In conclusion,we investigated the antibacterial effects of the combination of PTM with different forms of Ag,and further synthesized and biologically evaluated the silver salt of PTM and a 2,4-dinitrobenzene derivative of PTM,respectively.We discovered that（1）PTM combined with Ag⁺or AgNPs can exert synergistic anti-S.aureus activity,and the silver salt of PTM had superior anti-S.aureus activity than PTM alone.One of the reasons was that the synergistic effect of PTM and Ag（Ⅰ）increased the permeability of bacterial cell membrane and the intracellular reactive oxygen level;（2）2,4 dinitrobenzene derivative of PTM was more stable than PTM in human serum,while its in vitro antibacterial activity was reduced.These studies not only provide two options for the development of PTM-based antibiotics,but also are instrumental for the development of new generation antibiotics using the AMR strategy or drug combination in the form of silver salt.