Design,Synthesis And Bioactivity Evaluation Of Selenium-containing Small Molecular CYP51 Inhibitors

The treatment of fungal infections,especially invasive fungal infections,faces great challenges recent years.The changes of fungal infection types and variation of fungi lead to an increase of drug-resistant strains.However,the limited types and drawbacks of antifungal drugs available for clinical are far from meeting the clinical needs for the treatment of fungal infections.Therefore,the discovery of novel lead antifungal compounds with high efficacy and low toxicity is significance for the development of drugs in treatment of invasive fungal infections.At present,this problem can be solved from three aspects:1.The discovery of new antifungal drug targets,and the study on relationship of between fungal-resistance mechanism and antifungal drugs.2.To develop new structure of compounds or introduce other antifungal mechanisms in traditional targets.3.The structural modification of traditional drugs.Obviously,the latter two are economical and practical drug research strategies with short development cycle.Lanosterol 14?-demethylase(CYP51)is the most active target of antifungal drugs in clinic.CYP51 is involved in the biosynthesis of ergosterol which was the main component of fungal cell membrane and fungal reproduction can be effectively influenceed by inhibiting CYP51.At present,all clinical application of the treatment of invasive fungal infection of CYP51 inhibitor are azole compounds.There is still great promise to develop new CYP51 inhibitors with more broad spectrum,high efficiency,low resistance and low toxicity,due to the advantage of broad-spectrum antifungal activity,exact curative effect and structural diversity of azoles.It was reported that organicselenium compounds possess variety of biological activities,and many reports on antifungal activities of small molecule compounds containing selenium have also been reported.One of the research directions of organicselenium pharmacochemistry is how to effectively introduce selenium-containing functional groups or selenium-containing structures into drug molecules so as to improve or change the biological activity.In our previous research work,a large number of selenium-containing compounds with excellent antifungal activity were found,which were proved that the introduction of selenium into the antifungal compounds could effectively enhance the antifungal activity and improve the related pharmacokinetic properties of the compounds.Therefore,organicselenium compounds pocess important research value and development prospect in the field of antifungal drugs.In this paper,based on the structure-activity relationships of the CYP51 inhibitors and the previous research results of selenium-containing antifungal compounds,a total of 132 target compounds of were designed and synthesized according to the relevant drug development strategies.Meanwhile,the pharmacological activity evaluation and mechanism of target compounds were systematically studied.All of target compounds had not been reported in the literature,and their structures were confirmed by 1H NMR,13C NMR and HRMS.24 series S target compounds(S01-S24)with 1,2,3-selenadiazole were designed and synthesized by introducing 1,2,3-selenadiazole fragment into the structural framework of classical CYP51 azole-inhibitors by scaffold hopping.The results of in vitro antifungal activity showed that compounds S01,S03,S07 and S11 pocess good fungistatic activities,fungicidal activities and anti-Candida albicans biofilm activities.In addition,some compounds also showed good fungistatic activities and fungicidal activities against azole-resistant strains,indicating that they have important research prospects in the treatment of multi-drug resistant fungal infections.Studies of antifungal mechanism showed that compound S07 exhibited moderate inhibitory activity of CYP51 and pocess antifungal activity of promoting the production of reactive oxygen species(ROS)in fungal cells.The interaction mode of compound S07 with CYP51(PDB ID:4UYM)was investigated by molecular docking method.And the cytotoxicity test showed that the target compounds had moderate cytotoxicity.It was found that the target compounds with the dual mechanism included CYP51 inhibitory activity and ROS production activity of fungal cells had outstanding antifungal activity,in the study of biological activity and SAR of series S compounds.Based on the results and molecular hybridization,18 series M compounds(M01-M18)with diselenium bond and 18 series N compounds(N01-N18)with selenium ether were designed and synthesized.The results of preliminary in vitro antifungal activity evaluation showed that some of the target compounds had good fungistatic activities and fungicidal activities,among which the representative compounds M01,M03,M05,N02,N03 and N05 not only had good antifungal activity against azole-resistant strains,but also had anti-Candida albicans biofilm activities.Studies of antifungal mechanism showed that compound M01 and N03 exhibited inhibitory activity of CYP51 and pocess antifungal activity of promoting the production of reactive oxygen species(ROS)in fungal cells.Cytotoxicity test,hemolysis test and in vitro liver microsomal stability test showed that series M compounds had lower toxicity,extremely low hemolysis and good metabolic stability compared with series N compounds.In vivo pharmacodynamics experiments showed that compound M01 had good antifungal activity in mouse and could significantly reduce the fungal load in mouse kidneys.The molecular docking method was used to study the interaction mode of compounds M01 and N03 with CYP51(PDB ID:5TZ1),which provided a basis for further structural optimization of the compounds.Based on the biological activity and structure-activity studies of series S,M and N compounds,we choose miconazole as lead compounds,which can promote fungi to produce ROS,to design and synthesis of 30 miconazole containing-selenium analogues(A01-A30)by using bioisosterism.In vitro antifungal activity tests showed that the target compounds showed good fungistatic activities against 8 common fungi strains and 5 azole-resistant strains.Compound A03 has better anifungal activity and anti-biofilm activity than miconazole.Further mechanism study showed that Compound A03 has better CYP51 inhibitory activity than miconazole and fluconazole and has the same antifungal activity of promoting the production of ROS in fungal cells as miconazole.However,cytotoxicity test,hemolysis test and in vitro liver microsomal stability test showed that A03 had moderate cytotoxicity,potential hemolysis,and poor stability of liver microsomes.Therefore,further structural optimization of A03 was required.In order to improve the potential hemolysis,cytotoxicity and poor metabolic stability of compound A03 by structural optimization,a total of 42 series B and C compounds(B01-B28 and C01-C14)were designed and synthesized.In vitro antifungal activity test showed that compound B17 pocessed satisfactory antifungal activity and processed lower metabolic rate,cytotoxicity and hemolysis than compound A03.Meanwhile,compound B17 also showed good fungicidal activity,anti-biofilm activity and fungistatic activity against azole-resistant strains.Further study on the antifungal mechanism showed that compound B17 pocessed good CYP51 inhibitory activity and ROS production activity.In vivo antifungal activity test showed that compound B17 could significantly reduce the fungal load of mouse kidney.The pharmacokinetic evaluation and molecular docking study of potent compound B17 further clarified its mechanism of action and its research potential as an antifungal lead compound,which provided a research basis for the follow-up research work.The research work in this paper has actively explored and expanded the application of organicselenium compounds in the field of antifungal drugs.Our work provided a new idea,which added dual antifungal mechanism of CYP51 inhibitory activity and ROS production activity to antifungal new drugs,and research basis for the research of antifungal new drugs.