Design, Synthesis And Bioactivities Evaluation Of Novel Pleuromutilin Derivatives

Pleuromutilin was first isolated in a crystalline form cultures of two species of basidiomycetes,Pleurotus mutilus and P. passeckerianus by Kavanagh and his colleagues in1951. pleuromutilinderivatives interfered with bacterial protein synthesis via a specific interaction with the23S rRNA ofthe50S bacterial ribosome subunit. The domain V of23S rRNA at the peptidyl transferase center (PTC)is mutilins derivatives binding site, in which the tricyclic core of the pleuromutilin is positioned in apocket close to the A-tRNA binding site, whereas the C-14extension points toward the P-tRNA bindingsite. Thus these compounds prevent the correct positioning of the tRNAs for peptide transfer, andinhibit the peptidyl trasferase. Structure activity relationship (SAR) studies showed that the presence ofthioether group at C-22position of pleuromutilin and a basic group enhances antibacterial activity. Sothe molecular modifications of pleuromutilin were focused essentially on the modifications of C-14glycolic acid chain, by which could be the most promising approach to obtain optimum antibacterialactivity.Valnemulin as the second generation drug for animal using has higher antibioactivity than tiamulin.However related analogues of valnemulin are still scarce. A serial of new pleuromutilin derivatives (4a-i,5a-f,6a-b) were designed and synthesized based on the structure of valnemulin. Meanwhile. thiadiazdebased structural scaffolds form an essential constituent of many synthetic drugs with this moietyexhibiting a wide range of biological activities such as antibacterial, antitabercular, antidepressant andantimycobacterial activity. We have synthesized three types of pleuromutilin derivatives bearingthiadiazde moiety: compounds9a-f,11a-c, and13a-f. All the synthesized compounds were purified withcolumn chromatography, followed by characterized with means of IR (KBr),1H NMR and13C NMRspectral analysis.Some compound were cultivated their single crystal, and the cultivated single crytal4a,4b,4c,5a and9e were screen to the crystal structure studies after X-ray diffraction.All the synthesized pleuromutilin derivatives4a-i,5a-f,6a-b,8,12a-f,13a-c, and15a-f were screenedfor their in vitro minimum inhibitory concentration (MIC) test against MRSA, MRSE, E.coli, andS.agalactia by agar dilution method according to the National Committee for Clinical LaboratoryStandards (NCCLS). The MICs of34new pleuromutilin derivatives in vitro against MRSA, MRSE,E.coli, and S.agalactia ranged from8to0.25μg/mL,32to0.5μg/mL,64to1μg/mL, and32to0.25μg/mLrespectively.Also, Oxford cup assay was carried out to evaluate the antibacterial activity of allthe synthesized compounds against the above mentioned four bacterial strains. The results which werereported as diameters of growth inhibition (mm) were in agreement with that of MICs. Among all thepleuromutilin derivatives examined, five compounds5a,5c,8,11b,13c showed good antibacterialactivity, especially for the MRSA, MRSE and S.agalactia.Compound8has the similar construction with azamulin which had strongly inhibition of cytochromeP450, especially the CYP3A4. To further study azamulin and compound8inhibition activities againstCYP3A4, docking simulations were performed using CYP3A4and50S ribosome. In the docking mode, the azamulin and3NXU revealed the best confirmation and lowest free energy of binding affinity (ΔGb)which was up to-9.9kcal/mol. While the free energy of binding affinity for the compound8dockingwith3NXU reached to-9.2kcal/mol and was higher than that of azamulin docking with3NXU. FromMIC values and the zones of inhibition it was observed that some synthesised compounds with similarconstruction showed different antibacterial activity. To shed light on this question, compounds5a-c,9d-f,11a-c and13a-d were docked to1XBP to compare their binding mode. The results show thatcompounds5a,5c,9d,9f,11b and13a have a lower free energy of binding affinity than that of theiranalogues. The compound5c obtained the lowest free energy of binding affinity which reaches to-11.55kcal/mol. Those results agree with well the reults of inhibitory tests. In addition, the dockingmodels of all the compounds are similar with tiamulin which RMSD are less than2and lie in activitypocket of1XBP.