Design,synthesis And Antibacterial Activity Against MRSA Of Pleuromutilin Derivatives Possessing A 1,2,4-Triazole Linker

With the large-scale use or even abuse of antibacterial drugs in veterinary clinics,the problem of bacterial resistance in animal sources is becoming more and more serious and it is urgent to develop new animal-specific antibacterial drugs.Pleuromutilin is a diterpenoid natural product with a 5-6-8 tricyclic ring produced by Pleurotus mutilus（Clitopilus scyphoides）and Pleurotus passeckerianus（Clitopilus passeckerianus）,which have strong bacteriostatic activity against some gram-negative bacteria,most positive bacteria and mycoplasma.Pleuromutilin is not easily cross-resistant with other antibacterial agents and has broad development prospects.The researchers performed a large number of transformations on pleuromutilin.By introducing a thiol-containing reactive group into the side chain at the C14 position of pleuromutilin,three listed drugs of tiamulin,valnemulin and ritomeline were obtained,among which tiamulin and valnemulin are special antibacterial agents for animals.1,2,4-triazole is an important pharmaceutical intermediate with biological activities such as antibacterial,anti-inflammatory,analgesic and anti-cancer,and is widely used in the research and development of new drugs.In this study,derivatives 13a～13c,14a～14c,15a～15c,16a～16c,17a～17c and 18a～18c were designed and synthesized using 1,2,4-triazole ring as the connecting arm.It involves an acylation reaction,a self-condensation ring,a sulfonylation reaction,and a nucleophilic substitution reaction.These derivatives were purified by recrystallization and column chromatography,and confirmed by IR,HR-MS,¹H NMR and ¹³C NMR.In vitro minimum inhibitory concentration（MIC）test of all the derivatives against（S.aureus）ATCC29213,clinical isolation of Staphylococcus aureus AD₃,MRSA,was carried out and MIC results showed 0.0625～2μg/m L,0.03～0.25μg/m L,0.0625～1μg/m L respectively.Derivatives 13a,14a,15b and 16a have the best in vitro anti-MRSA activity and are superior to tiamulin,so the in vitro bactericidal curves of the four derivatives were studied.The results showed that the derivative 13a had a bacteriostatic effect on MRSA at 4×MIC,but failed to achieve complete elimination,was a time-dependent bacteriostatic agent;Derivative 14a,15b and 16a achieve complete elimination of MRSA at 4×MIC and are time-dependent bactericides.In this paper,the in vivo anti-MRSA activity of single-dose derivatives 14a,16a was studied in a model of neutrophil-reducing thigh infection in mice.The results showed that intramuscular injection of 20 mg/kg dose of 14a,16a can significantly reduce the number of bacteria in the thigh muscle of mice.Both of them had better antibacterial efficacy than tiamulin,and the activity of derivative 14a was better than 16a.The in vivo anti-MRSA activity of single dose 14a was further studied in a model of neutrophil reduction in mice.The results showed that the survival rate of mice treated with intravenous injection of 30mg/kg 14a was 50%,which was higher than that of the same dose of tiamulin treatment group.This study established a method for the determination of derivative 14a in mouse plasma by LC-MS/MS.Healthy mice were intravenously injected with 14a at a single dose（10mg/kg）,plasma samples were collected,and the pharmacokinetic parameters were analyzed using the non-atrioventricular model of Win Nonlin 5.2 software.The results showed that derivative 14a had a T_1/2 of 0.40±0.08 h,CL of 5.73±0.38 L/h·kg,MRT of 0.48±0.05 h,and AUC of 1.75±0.11 h·μg/m L.According to the comprehensive analysis,the derivative 14a belongs to a rapid elimination drug,and the degree of absorption in the body is low.