Modification Of C-14 Side Chain Of Pleuromutilin And Their Antibacterial Activities

As the wide-spreading bacterial resistance,most the currently used antibiotics are losing their efficiency or ineffective.Furthermore,only few new antibiotics are proved to be used in the clinic.Therefore,it is urgent to find and develop novel antimicrobial agents that are effective against drug-resistant bacteria and not prone to drug resistance.Recently,pleuromutilin derivatives have become a research hot topic in the world because of their high antibacterial activity and low cross-resistance.The modification of pleuromutilin side chain has led to four drugs:tiamulin,valnemulin,retapamulin and lefamulin.Tiamulin and valnemulin are veterinary drugs used for clinical infections of a variety of pathogens.Retapamulin is used for local skin infections or postoperative anti-infection and Lefamulin for the community acquired bacterial pneumonia（CABP）caused by microorganisms.Pleuromutilin compounds have completely realized the leap from veterinary to human use,displaying superior clinical prospects.In this study,pleuromutilin was activated by p-toluenesulfonyl chloride at C-22 hydroxyl group,followed by nucleophilic reaction with 4-hydroxy-2-mercapto-6-methylpyrimidine under alkaline conditions to produce 14-O-[（4-Hydroxy-6-methylpyrimidin-2-yl）mercaptoacetyl]mitilin（3）.The modifications of compound 3 with substituted nitrogen and oxygen atoms were lead to 19 novel derivatives with pyrimidine moieties.All synthesized compounds were characterized by infrared spectroscopy（IR）,nuclear magnetic resonance（NMR）and high resolution mass spectrometry（HRMS）.In addition,the obtained single crystals of compounds 5d and 5i were further used to confirm their chemical structures by X-ray diffraction.We measured the minimum inhibitory concentration（MIC）of all synthetic pleuromutilin derivatives.The results showed almost all compounds displayed potent antibacterial activities.Compounds 5a-5m with substituted nitrogen showed higher activities than compounds 6a-6e with substituted with oxygen.Especially compound 5c and 5g showed the best antibacterial activities against MRSA and other Gram-positive bacteria.Bactericidal curve of 5c and 5g was performed against MRSA based on the results of the MIC.Compound 5c showed relatively bacteriostatic kinetics against MRSA with time-concentration dependence.For further study the binding mode to the peptidyl transferase center（PTC）of the ribosome,we performed the molecular docking simulations on compound 5c with better antibacterial activity and compound 6d with lower activity,respectively.The results showed that hydrogen bond plays the most important role in the binding model.The free binding energy of compound 5c and 6d were-8.52 and-5.16 kcal/mol,respectively,which were consistent with their antibacterial activities.Mouse model of systemic infection of MRSA was established to study the in vivo efficacy of compound 5c.Compared with tiamulin,5c significantly increased the survival of infected mice(ED₅₀=18.02 mg/kg),reduced bacterial burden in lung,and attenuates lung tissue damage caused by MRSA.The genetic toxicity of compound 5c was evaluated by Ames test,Salmonella strains TA97a,TA98,TA100,TA102 and TA1535 were treated by five concentrations of 5c with or without the metabolic activation（S9）.The results showed revertant colonies in positive control groups were much more in double than that induced by five concentrations of 5c and DMSO,and there was no difference between the negative control group and the 5 dose groups with no dose-dependent effect,indicated that 5c should not be considered as a mutagen based on this test.