Synthesis,Structure Identification And Biological Activity Evaluation Of Novel Pleuromutilin Derivatives

Although more than 31 lead classes of antibiotics and 160 kinds of antibiotics have been found from the 1890s–1980s,six lead class antibiotics have been approved for use in the past forty years.Meanwhile,many antibiotics have been found to be serious drug resistant in clinical.The discovery of new antibiotics has gotten stuck in bottleneck.The diterpene antibiotic pleuromutilin,discovered and isolated from basidiomycetes,Pleurotus mutilus(Fr.)Sacc.Drosophila subatrata and P.Passeckerianus Pilat in 1951,showed good antibacterial activities to Gram-positive bacteria.Pleuromutilin and its derivatives inhibit protein synthesis by blocking the peptidyl transferase center(PTC)and intervening t RNA binding A-site and P-site,which are different from chloramphenicols,macrolides,tetracyclines and oxazoxanone antibiotics.The ABC transporters are the major cause of resistance mechanism of pleuromutilin derivatives according to a few reports.Pleuromutilin derivatives have attracted more attentions to be developed new antibiotics because of their specifically antibacterial mechanism and low frequency resistance.We first modified the tricyclic carbon skeleton of pleuromutilin and 3 series derivatives were designed and synthesized.Then,50 new derivatives were designed and synthesized by substitution their hydroxyl at C-22 with N and S atom according to structure-activity relationship.All the derivatives were characterized by Nuclear Magnetic Resonance(NMR),Infrared Spectroscopy(IR),and High Resolution Mass Spectrometer(HRMS).Some compounds were further confirmed by X-ray single crystal diffraction.The synthesized pleuromutilin derivatives and tiamulin fumarate used as reference drug were screened for their in vitro antibacterial activity,including minimum inhibitory concentrations(MICs).The results showed that 14 compounds(4d,4f,5a,5c,5d,6b,7a,7i,7f,8,9a,12,14 f and 14h)displayed higher or comparative antibacterial activities than tiamulin(MRSA and S.aureus).Compound 5a showed satisfactory antibacterial activity to Pasteurella multocida.The results of time-kill curve showed only only 39 CFU/m L bacteria counts by 1.5 μg/m L compound 5a in 12 h.Compound 12 and 14 h showed the best antibacterial activities with MIC of 0.125 and 0.0625 μg/m L against MRSA and MRSE.Compounds 7a,7b,7d and 7h displayed promising antibacterial activity against VRE(MIC were 0.0625,0.25,0.25,0.125 μg/m L,respectively).Compounds 9a and 12 were the most active antibacterial agents and therefore used to evaluate their in vitro time-kill assay and in vivo efficacy in mouse model.The median effective dose of compound 9a and 12 were 5.47 mg/kg and 4.22 mg/kg,while tiamulin fumarate was 5.95 mg/kg.Docking experiments for compounds 5a,5b,5f,7a-7i,12 and 14a-14 i carried out on PTC of 23 S r RNA provided the information about the binding model.The result of molecular docking showed the side chains of pleuromutilin derivatives affect their antibacterial activities.The steric hindrance of side chain obviously affected the binding modes between the ribosomes and pleuromutilin derivatives.Meanwhile pleuromutilin derivatives bearing the hydroxyl or amino groups could enhance its bacteriostatic activities.