Design,Synthesis And Biological Activity Of Antibacterial Conjugates Based On The Active Transport Strategy

As bacterial resistance is getting worse,it is urgent to develop new antibiotics.Based on the active transport mechanisms of transporters in the bacterial cell wall,three types of antibacterial conjugates are designed and biologically evaluated.The first part is on the design,synthesis and antibacterial studies of ferrichrome-methotrexate conjugates.As the siderophore of albomycin,ferrichrome plays a vital role for its antibacterial activity.It employs the transport proteins of bacterial iron transport system to efficiently carry antibacterial substances into bacteria.Methotrexate is clinically used to treat tumor and arthritis.However,due to its strong cytotoxicity and poor cell penetration,methotrexate can not be used directly as an antibacterial agent.Based on the selective uptake of siderophore between human cells and bacteria,ferrichrome-methotrexate conjugates were designed to reduce the cytotoxicity of methotrexate,enhance its antibacterial activity and therapeutic window.During the studies,the synthesis of ferrichrome with protective groups(tripeptide,siderophore)was optimized first,and the siderophore was used to build conjugates with methotrexate through suitable linkers.21 Ferrichrome-methotrexate conjugates and one fluorescent probe for mechanism verification were designed and synthesized.In vitro antibacterial activity studies suggested,that these conjugates have strong inhibitory effects against Streptococcus pneumoniae.By studying the structural composition of the linker,the Structure-Activity Relationship(SAR)of the linker was established.Finally,a compound with high bacteriostatic activity and low cytotoxicity was discovered(S.pneumoniae ATCC 49619,MIC 1.96 ng/m L).Its antibacterial activity is more than 1000times higher than that of methotrexate and 64 times higher than that of ciprofloxacin.Subsequently,fluorescent probe uptake experiment was carried out,and the results supported that the conjugate functioned within the bacteria cell.The activity of the siderophore-methotrexate conjugate to dihydrofolate reductase was examined,which exhibited the same level of IC₅₀ as methotrexate did,suggesting that the conjugation did not alter its affinity for dihydrofolate reductase.Finally,a preliminary animal experiment model was established using Balb/c mice.The mice were infected with a lethal dose of bacteria,and the effect of the drug on the survival rate was evaluated.Conjugate 2-4k exhibited a therapeutic effect similar to that of ampicillin,while all mice in the methotrexate group died.The second part is on the design,synthesis and antibacterial studies of vitamin B₁₂-antibiotic conjugates.Vitamin B₁₂plays an important role in the life of eukaryotes and prokaryotes.The transport system for bacterial absorption of vitamin B₁₂ and ferrichrome share a set of transporters.Since there has been no report on the conjugation of vitamin B₁₂with small molecule antibiotics,we designed and synthesized 11 vitamin B₁₂-antibiotic conjugates,and checked their antibacterial activities.In vitro antibacterial activity showed that the conjugates formed from vitamin B₁₂ and ampicillin or amoxicillin can effectively improve the antibacterial activity comparing with the parent drugs.In particular,conjugate 3-11a,exhibit a 520-fold increased in its inhibitory activity against E.coli(TACC 25922),and a 520-fold increased in the inhibitory activity against E.coli(ATCC 35150).It showed good selectivity for Gram-negative bacteria.Our studies sugges that vitamin B₁₂ as a transport carrier can significantly improve the effect of antibiotics.The third part is on the design,synthesis and antibacterial evaluation studies of2-chloro-3,4-dihydroxybenzoic acid-methotrexate conjugates.Small molecule siderphore mimics have been used to develop novel antibiotics.Based on small molecule siderphore mimics,siderophore(2-chloro-3,4-dihydroxybenzoic acid)-methotrexate conjugates were designed and synthesized in order to selectively treat Gram-negative bacterial infection.The study is to address the infection of Gram-negative bacteria through the differential absorption of siderphore by different bacteria.The in vitro antibacterial activity of the synthesized conjugate was studied,and the results showed that 4-5a had inhibitory activities against two Gram-negative bacteria(E.coli and P.aeruginosa).