Design,Synthesis And Biological Studies Of Novel Antifungal Compounds

The global incidence of life-threatening invasive fungal infections has increased significantly,causing more than one million deaths each year.The incidence of invasive fungal infections is also increasing in China.Currently available antifungal drugs have limited efficacy,lack broad-spectrum antimicrobial activity,have severe toxic side effects and are susceptible to drug resistance.There is an urgent need to develop new antifungal drugs to overcome this dilemma.This thesis consists of two parts.The first part focuses on the optimization research of triazole antifungal drugs.Three series of 88 novel triazole compounds containing methoxy side chains were designed and synthesized.Their antifungal activities were evaluated in vitro and in vivo.The aim was to obtain novel,efficient,broad-spectrum,anti-resistant fungi,and low-toxicity triazole lead compounds.The second part discusses drug combination therapy as an effective method to overcome drug resistance.We conducted a systematic structural modification of ENOblock,a triazines hit compound obtained from our screening.This resulted in the design and synthesis of 87 novel triazines in six series.We then screened these compounds for in vitro synergistic antifungal activity,summarized the structure-activity relationship,conducted a preliminary mechanism study,and evaluated the pharmacodynamics in vivo.Our goal was to discover new antifungal lead compounds with novel structure types and mechanisms of action.All 175 products synthesized in this thesis were confirmed using ¹H-NMR,¹³C-NMR,and LC-MS.I.Design,synthesis and evaluation of antifungal activity of novel triazole compoundsTriazoles are the most developed antifungal drugs among the existing antifungal arsenals.This chapter presents optimization studies on the structure of azoles side chains based on bioisosterism,scaffold hopping,and conformational adjustment strategies.Three series of novel triazoles were designed and synthesized:A（methoxy-1,2,3-triazolyl ring）,B（methoxy-pyrazolyl ring）,and C（methoxy-alkynyl group）.The compounds were tested for antifungal activity in vitro.The MIC values of the compounds were obtained by using seven pathogenic fungal strains for primary screening to summarize SAR.Then,the anti-resistant fungi activity of selected compounds was tested using five drug-resistant fungal strains.The compounds A2,A18,B4,B7,C14,and C16 were tested for their inhibitory effects on virulence factors.Hyphal and biofilm formation,as well as mature biofilm disruption experiments,were conducted.To determine the antifungal mechanism,gas chromatography was used to detect the effect of each compound on sterol content and composition in fungal cell membranes,verifying the inhibitory effect of the compounds on the target enzyme CYP51.The cytotoxicity assay demonstrated that the preferred compounds were not significantly toxic to HUVEC cells.Molecular docking was used to simulate the binding modes of A18,B7,and C16 with Candida albicans CYP51,and to analyze the potential amino acid residues for the formation of interactions between each compound and the target enzyme.Additionally,several genetically recombinant strains were utilized to study the anti-resistance mechanism.The study found that C14 and C16 are effective against azole-resistant strains that overexpress the Mdr1 efflux pump,Erg11,and the Erg11 amino acid mutation.In vivo evaluation of antifungal activity was conducted using two biological models.The G.mellonella larva-C.albicans infection model was used for preliminary screening of the preferred compounds A18,B7,and C14,and C14 was found to be the most effective.C14 was evaluated on a mouse model of C.albicans systemic infection.The results demonstrated that C14（5 mg/kg）significantly extended the survival time of mice.Through the above in vitro and in vivo activity studies,the overall SAR suggested that the C series,which was designed as a linear side chain,had better antifungal effects than the other two ring-containing structures.In summary,we obtained a novel triazole lead compound C14 with highly efficient,broad-spectrum,anti-resistant fungi,and low-toxicity properties.II.Discovery,structure optimisation and evaluation of antifungal activity of novel triazine compoundsSeveral small molecule compounds with no or weak antifungal activity can restore the susceptibility of drug-resistant fungi to drugs when combined with antifungal drugs;these small molecules are known as synergists.To discover novel compounds with synergistic activity,this chapter focuses on the structure optimisation studies of ENOblock,a previously obtained triazine hit compound.The molecule has a 1,3,5-triazine parent nucleus and contains three different amino substituent side chains.A total of six series of new triazines,D to I,were designed and synthesised by replacing the poor medicinal properties fragments in the side chains.Among them,the F series of compounds containing amino-thiourea fragments have synergistic activity against drug-resistant fungi.SARs show that:amino-thiourea is the key pharmacophore for synergistic effects.Once sulphur atoms are substituted by oxygen or ring cyclisation,it would lose antifungal activity;F series compounds with strong electron withdrawing group substitution in the para position of the terminal benzene ring have synergistic effects.In addition,compounds F12 and F13 with-CN and-NO₂substituents in the terminal benzene ring have single antifungal activity against resistant fungi.The optimisation of ENOblock by structural simplification strategy revealed that the hydroxyl-substituted triazine hydrazones H17-H19 exhibited good synergistic activities.In particular,when the hydroxyl group was in the ortho position,compound H17 also possessed a single anti-resistant fungi effect.Following the hydroxyl-substituted structures,the series of compounds H23-H32,obtained by introducing nitro groups,among which H25（2-OH-5NO₂）was the most effective with MIC=1.0μg/m L against drug-resistant Candida albicans901 and 904,respectively.To investigate whether the preferred compounds of the F-series and the H-series（F12,F13,H17,H23-H26）could be used as novel antifungal leads.These compounds were tested for in vitro antifungal activity against ten pathogenic fungi（sensitive and resistant）.It was found that only H23 and H25 exhibited broad-spectrum antifungal activity and could be used as single antifungal compounds,especially H25.The time-growth and fungicidal curves showed that H25 inhibited fungal growth in a concentration-dependent manner and had a fungicidal effect within 12 h.H25 was able to inhibit hyphal and biofilm formation at4μg/m L,but had limited effect on disruption of mature biofilm.A preliminary study of the antifungal mechanism showed that H25 could reduce the mannan content of the fungal cell wall,causing changes in cell wall components and damaging cell morphology.H25 also had low cytotoxicity,low haemolytic activity and stable in vitro metabolism in liver microsomes.In vivo efficacy experiments showed that 10 mg/kg H25 was able to significantly reduce the fungal burden in mouse kidneys.In conclusion,from the synergism strategy,we finally obtained a novel triazine hydrazone compound H25 through hit-to-lead optimisation.This promising compound with a novel structure and a novel antifungal mechanism is worthy of in-depth study.