Design,Synthesis And Biological Evaluation Of Novel Anti-resistant Fungal Lead Compounds

Recently,invasive fungal infections(IFIs)are emerging as high-incidence infectious diseases with high mortality and threatening human health due to limited antifungal drugs and serious drug resistance in clinic.It is urgent to find novel antifungal drugs,especially anti-resistant drugs.With the clarification of fungal resistance mechanism and related signal pathway,several inhibitors of key proteins were discovered to possess antifungal activity,which lay the foundation for discovery of novel antifungal drugs.In this thesis,based on histone deacetylase(HDAC)and heat shock protein 90(Hsp90),a series of CYP51/HDAC dual inhibitors,Hsp90 inhibitors and Hsp90/HDAC dual inhibitors were designed and synthesized,and assayed for in vitro and in vivo antifungal activities.Also,the antifungal mechanisms of selected highly potent compounds were preliminarily studied.Taken together,the results provided a series of new lead compounds for the development of novel antifungal agents.1.Design,synthesis and antifungal biological evaluation of novel CYP51/HDAC dual inhibitors.Lanosterol 14?-demethylase(CYP51)is the target of azole antifungal drugs.In this part,twenty-nine CYP51/HDAC dual inhibitors were designed and synthesized via connecting the key pharmacophores of azoles and HDAC inhibitors.The results of antifungal activity in vitro revealed that most of the newly-synthesized compounds not only exhibited potent activity against sensitive fungi,but also showed strong inhibitory effects against azole-resistant fungi.Among them,compounds B8 and C6 exhibited excellent anti-resistant fungal activity with MIC80 in the range of 2-8 ?g/m L and 0.25-0.5 ?g/m L,respectively.Additionally,compounds B8 and C6 could block the biosynthesis of ergosterol and inhibit fungal cell HDAC catalytic activity.The investigation of resistant genes revealed that compounds B8 and C6 could down-regulate the expression of ERG11(encoding CYP51)and efflux pump genes(CDR1,CDR2,MDR1).And the influence of efflux pump genes was demonstrated by rhodamine 6G efflux assay.The further mechanism studies suggested that compound B8 and C6 could inhibit the formation of fungal hypha and biofilm,down-regulate the expression of related genes(EFG1,TEC1,CPH1,ALS3).Importantly,in vivo antifungal efficacy studies suggested that compounds B8 and C6 could alleviate the renal fungal burden of IFIs mice significantly by intraperitoneal and oral administration,and extend the survival time of infected mice by intraperitoneal injection.2.Design,synthesis of novel Hsp90 inhibitors for the integrated treatment of IFIs and tumorCancer patients are vulnerable to IFIs.Due to serious resistance of current antifungal agents,it is significant to discover new drugs with anti-resistant fungal and antitumor activities for improving the life quality of cancer patients.Ganetespib is an Hsp90 inhibitor undergoing clinical trials for the chemotherapy of tumor.Initially,Ganetespib was proven to exhibit potent synergistic antifungal activity,with FICI ranges from 0.023 to 0.039.Thus,it is a valuable lead compound to find novel anti-resistant fungal and antitumor compounds.Then,thirty Hsp90 inhibitors were designed and synthesized by introducing and replacing alkyl groups and bioisosteric replacement.In vitro biological evaluations showed that most compounds exhibited strong Hsp90? inhibition activities,good synergistic anti-resistant fungal activities with fluconazole and potent antitumor activities.Among them,compounds F3 and F5 exhibited excellent synergistic anti-resistant fungal activities(FICI = 0.047-0.063 and 0.039-0.063,respectively)and antimuor activities(IC50 = 0.025-0.15 ?M and 0.021-0.23 ?M,respectively),comparable to Ganetespib.Antifungal mechanisms research demonstrated that compounds F3 and F5 could significantly down-regulate the expression of resistant genes(ERG11,CDR1,CDR2,MDR1),ruduce rhodamine 6G efflux,inhibit the formation of fungal biofilm and influence the expression of biofilm related genes.Moreover,compounds F3 and F5 could effectively stimulate the cell cycle of HEL arrest in G0/G1 phase,according to the mechanism study of antitumor.3.Design,synthesis and synergistic anti-resistant fungal biological evaluation of novel Hsp90/HDAC dual inhibitors.In this part,seventeen Hsp90/HDAC dual inhibitors were designed and synthesized by combing the key pharmacophores of Ganetespib and HDAC inhibitors to dually block the calcium-calcineurin signaling pathway.According to the results of biological evaluation in vitro,the preliminary structure activity relationship was obtained for the target compounds.Among them,compounds J5 and J9 showed potent Hsp90? and HDAC inhibition,excellent synergistic effect with fluconazole against resistant fungi and weak cytotoxicity.The antifungal mechanism studies suggested that compounds J5 and J9 inhibited biofilm formation and influence the expression of related genes.Additionally,compounds J5 could down-regulated the expression of resistant genes(ERG11,CDR1).Importantly,the antifungal efficacy in vivo suggested that both compounds J5 and J9 could synergize fluconazole to reduce the renal fungal burden of IFIs mice.