Design,Synthesis,Structure-Activity Relationship And Antifungal Activity Of Triazole Derivatives

During the past three decades,the morbidity of life-threatening invasive fungal infections?IFIs?has been increasing among the patients with immunosuppression or immunodeficiency.Generally,Candida albicans,Cryptococus neoformans and Aspergillus fumigatus are the major pathogenic agents for systemic fungal infections.It is estimated that invasive fungal infections collectively kills over 1.6 million people worldwide each year.Currently,the antifungal drugs treating IFIs mainly include azoles,polyenes,flucytosine,and echinocandins.Azoles are widely used as first-line drugs in the clinic,which act by inhibiting fungal lanosterol 14?-demethylase?CYP51?to block the biosynthesis of ergosterol,thus affecting the integrity of fungal cell membrane,resulting in the death of fungi.However,azole antifungal agents are also facing challenges in the problems of antifungal spectrum,drug safety,and drug resistance.For examples,fluconazole is mainly used for treating candidiasis,but useless to Aspergillosis,while voriconazole is mainly used for the treatment of Aspergillus fumigatus lung infection,but demonstrates lower efficacy in Cryptococus neoformans.Furthermore,the strong inhibition of human CYP3A4 and poor oral bioavailability limit the clinical application of itraconazole and posaconazole.Besides,fungal drug resistance to azoles has become the most common and severe ones among antifungal drugs.Thus,it is desperately needed to develop novel antifungal drugs with broad spectrum,high efficacy,low toxicity and anti-resistance.Aimed at searching for the novel azole antifungal agent,this research has experienced four stages.The research is focused on the drug design,synthesis,and in vitro antifungal evaluation at the first three stages,and then the research features efficacy evaluation on animal models,preliminary toxicity and pharmacokinetics at the fourth stage.The drug design and synthesis employed in the stages are mainly based on:the SAR studies on azoles,the enzyme structure information,novel strategy on the synthetic routes of compounds,and newly obtained SAR at the former stages.As an exploration at the first stage,the category A compounds(6A_1-12)with a side chain containing 1,2,3-triazole were designed and synthesized from alknyl intermediate reacting with aryl azides via click reaction.This type of compounds is a racemate with one chiral center which performed well in the in vitro antifungal activity against the tested iolates of Candida albicans,Cryptococus neoformans,and Aspergillus fumigatus.The importance of the side chain containing 1,2,3-triazole was preliminarily explored at this stage.At the second stage,one more chiral center was introduced to the category A compounds to yield category B compounds(12B_1-33 and 14B_1-6)which bear a chiral chain moiety composed of a chiral tertiary alcohol and a chiral carbon linking to a methyl group.These compounds exhibited in vitro antifungal activity superior to category A compounds.A series of active compounds were obtained with highly potent activity and broad antifungal spectrum.Subsequently,the 1,2,3-triazole in category B and A compounds were respectively replaced by isoxazole and pyrrazole,to obtain category D(22D_1-21)and category F(30F,31F,28F_1-5 and 33F_1-22)with longer side chain.SAR studies indicated that the contribution of the moieties to antifungal activity is generally ranked as 1,2,3-triazole>isoxazole>pyrrazole,suggesting that 1,2,3-triazole linking to chiral chain moiety deserves further investigation.At the third stage,2,4-diflourophenyl group of some compounds in category B and D was replaced by 2,5-diflourophenyl group to produce corresponding compounds 12B_34-36 and 22D_22-29,respectively.The in vitro antifungal activity screening results indicated that 2,4-diflourophenyl group performed better than 2,5-diflourophenyl group.The replacement of the chiral tertiary alcohol moiety with tetrahydrogenfuran ring generated compounds of category C(18C_1-11)and E?23E?.SAR studies demonstrated that the compounds with chiral methyl tertiary alcohol moiety displayed higher antifungal activity than the corresponding racemates in category A and F,as well as the compounds with tetrahydrogenfuran ring in category C and E.The results confirmed that the methyl tertiary alcohol plays a critically essential role in the antifungal activity of these compounds.Summing up studies at the first three stages,124 compounds in 6 categories were designed,synthesized,and evaluated based on the previous study and newly reported information of two CYP51 structures,as well as the new synthetic route of the key chiral intermediate?2R,3S?-2-?2,4-diflourohenyl?-3-methl-1-?1H-1,2,4-triazole-1-yl?-4-pentyne-2-ol?11a?.All target compounds are reported for the first time,and their structures were confirmed by ¹H NMR,LC-MS,and ¹³C NMR?for part of the compounds?.A series of compounds featuring medium-length side chains were obtained to have potent in vitro antifungal activities against the tested three fungi.Moreover,a new SAR was obtained:1)Introduction of electron-withdrawing groups and hydrophilic groups to the aromatic ring at the end of the side chain is not preferable for activity,while halogen substitution is beneficial to activity,especially when the ortho fluorine atom at phenyl ring may increase the activity against Aspergillus fumigatus.Molecular docking studies suggested that ortho fluorine atom might form H bond with residue Tyr122 of CYP51;2)H bond donors or receptors at the end of the side chain containing pyrazole are preferable for antifungal activity.Among them the compound 28F₁ bearing 4-cyanobenzyl group exhibited the highest in vitro antifungal activity.Molecular docking suggested that the cyano group may promote antifungal activity due to the H bond of the 4-CN with Lys90.At the fourth stage,evaluation of efficacy on animal model,preliminary toxicity,preliminary pharmacokinetics and mechanism of action study were carried out.First of all,compound 22D₂₀,which performed well in vitro activity,was submitted to animal infection models.However,22D₂₀ did not show protective effect on the model infected by Candida albicans SC5314,at a dose of 2 mg/kg.The failure was presumably attributed to the possibility of its low metabolic stability.Subsequently,four compounds(22D₁,22D₂,22D₉ and 22D₂₀)were subjected to metabolic stability screening in mouse liver microsomes,and the results demonstrated that the metabolic stability of the substituents has the following order:-OCF₃>-Cl>-H>-F.According to the metabolic stability screening results,seven compounds(12B₄,12B₅,12B₂₇,12B₂₈,22D₁,22D₉ and 28F₁)were selected and subjected to murine models of disseminated Candida albicans SC5314 infection.All the tested compounds but 12B₄ displayed antifungal efficacies superior to or equivalent to fluconazole.Compounds 12B₅ and 22D₉ were extensively subjected to murine models of disseminated Cryptococus neoformans H99,and Aspergillus fumigatus 7544 infection.Compound 12B₅ showed antifungal efficacy outperforming fluconazole against Cryptococus neoformans H99 infection,and equivalent to voriconaole against Aspergillus fumigatus 7544 infection.Compound 22D₉ showed antifungal efficacy significantly superior to fluconazole against Cryptococus neoformans H99 and Candida albicans SC5314 infections,but no efficacy against Aspergillus fumigatus7544 infection.To deal with the challenging problem of fungal drug-resistance,the efficacy of compound 12B₅ was evaluated in the murine model of disseminated drug-resistant Candida albicans infection.Compound 12B₅ could significantly prolong the survival of the tested mice at a dose of 0.5 mg/kg,while fluconazole and voriconazole exhibited no protective efficacy.Finally,studies on the mechanism of action showed that 12B₅,like other azole drugs,blocks the biosynthesis of fungal ergosterol by acting on CYP51.The data of subacute toxicity of compound 12B₅ is more outstanding than that of voricoanzole.The pharmacokinetic characteristics of 12B₅ were assessed on rats,and the results indicated that 12B₅ has acceptable half-life and excellent oral bioavailability.To summarize,after four stages of research,a novel orally bioavailable triazole compound 12B₅ with broad spectrum antifungal activity and higher safety was obtained,especially featuring antifungal efficacy superior to fluconazole and voriconazole in treating the murine model of disseminated Candida albicans,Cryptococus neoformans,and drug-resistant Candida albicans infections,which renders compound 12B₅ deserving further investigation as a drug candidate.