Mechanism of Antifungal Triazoles and Related Drugs : Electron Transfer , Reactive Oxygen Species and Oxidative Stress

Aromatic triazoles are known to be effective antifungal agents. The generally accepted mode of action entails inhibition of ergosterol in the membrane. A hypothesis is presented herein based on Electron Transfer (ET), Reactive Oxygen Species (ROS) and Oxidative Stress (OS) which may be a unifying mechanism. Many substances in vivo are known to operate in a multifaceted manner. A prior article provides evidence for involvement of ET-ROS-OS for a variety of antifungal agents. In the present case, the active entity appears to be a highly conjugated imine. For drugs containing alcohol substituents, dehydration would provide the requisite conjugation. A few related aromatic nitrogen heterocycles are also addressed.


Introduction
In 1990, a review presented a unifying mechanism for antifungal agents based on ET-ROS-OS [1].Of particular interest involving the ET drugs are the iminium, e.g.triarylmethane dyes, such as gentian violet (Figure 1).The dyes are known to generate ROS via redox cycling.The one-electron reduction product interacts with oxygen to produce ROS which can damage DNA.
A prior integrating, mechanistic theme is as follows: A large portion of bioactive substances, including metabolites, utilize Electron Transfer (ET) processes which are believed to perform vital roles in physiological responses [2].Groups, such as quinones (or phenolic precursors), metal complexes, aromatic nitro compounds (or reduced hydroxylamine and nitroso derivatives), and conjugated imine or iminium species, are key players in ET.Redox cycling from such species is depicted in In some instances, like respiration or neurochemistry, normal electrical effects are brought about by ET involvement.Bioactive substances with ET groups broadly show reduction potential ranges within the physiologically responsive spectrum (more positive than about -0.5 V).Consequently, the production of ROS from ET in vivo, at low concentration, can be favorable in cell signaling, but at high concentration it can be toxic.Relatively stable radical cations are generated from electron donors, such as phenols, N-heterocycles or disulfides in proteins.The mechanisms of action of drugs and toxins (e.g., anti-infective agents [3], anticancer drugs [4], carcinogens [5], reproductive toxins [6], nephrotoxins [7], hepatotoxins [8], cardiovascular toxins [9], nerve toxins [10], mitochondrial toxins [8], abused drugs [11], pulmonary toxins [12], ototoxins [13] and various other categories [14] have increasingly been attributed to ET, ROS, and OS over the past decade.

The Hypothesis
The ET-ROS theoretical scheme has been receiving mounting acceptance through increasing experimental evidence.The evidence provides data on the involvement of the common ROS mentioned previously, e.g.lipid peroxidation, degradation products of oxidation, depletion of antioxidants (AOs), effect of exogenous AOs, and DNA oxidation and cleavage products, and electrochemical data, which supports the ET-ROS scheme.Frequent observations showing a myriad of ET substances present a diversity of activities (e.g.multiple-drug properties) and toxic effects, which is consistent with this comprehensive, unifying mechanism of action.
The triazole class, important antifungal agents, is usually considered to function by inhibition of ergosterol resulting in membrane insult.This article presents a unifying hypothesis based on ET-ROS-OS for mechanistic involvement in a multifaceted approach.Triazole is an aromatic heterocycle containing an imine group, one of the principal classes of ET agents.Extended conjugation is required for ET which is provided by benzenoid substituents.In some cases, conjugation arises via alcohol dehydration which generates a vinylogous system.The basic imine in the heterocycle readily undergoes protonation to an iminium which is generally more electron affinic in ET.The imine can also increase electron attraction by hydrogen bonding or N-oxidation.Redox by iminium is depicted in Scheme 3.

Evaluation of the Hypothesis Itraconazole
This triazole antifungal agent is also used for meningitis and systemic infections, such as for candidiasis (Figure 2).Several studies demonstrate activity as an anticancer agent [15,16,17].A review deals with the unifying theme of ET-ROS-OS for anticancer drugs, including conjugated imine [4].The agent is closely related structurally to posaconazole.
The literature addresses mode of action as being the same as other members of the class, namely inhibition of ergosterol synthesis.It is distinct in inhibiting the hedgehog signaling pathway [18,19].In relation to the ET-ROS-OS scheme, the aromatic triazole (Hückel's Rule, 4n + 2 π electrons, n = 1) is in conjugation with the benzene substituent, thus providing stabilization of the generated radical anion (Figure 3).Metabolism mainly entails hydroxylation of the triazole ring [20].Studies of ocular toxicity in rabbits indicates safety except for high doses which caused retinal necrosis [21].In one study, synthesized triazole fungicides were made and showed antifungal activity against Aspergillus niger and Aspergillus flavus [22].The conjugation with the triazole is realized by the bromophenyl group (Figure 4).Synthesized triazole-quinone/phenyl conjugates were tested for their antimicrobial activity and found to exhibit antifungal and antibacterial activity [23] (Figure 5).The quinolone portion attached to the triazole provides the requisite conjugation.

Mechanism of Antifungal Triazoles and Related Drugs: Electron Transfer, Reactive Oxygen Species and Oxidative Stress
Copyright: © 2017 Peter K, et al.

Posaconazole
This antifungal agent is quite similar in structure to itraconazole (Figure 6).It is also used to counter Candida species and is very effective against Chagas disease and lupus [24,25].
The mechanism is reported to entail blockage of ergosterol synthesis and impairing enzymes, such as ATPase, lanosterol demethylase and ET enzymes.Interaction with the ET enzyme adds credence to ET action by the drug.Rationale for participation in the unifying ET-ROS-OS scheme is provided in the itraconazole section, involving conjugation of the aromatic triazole with the benzenoid group (C 6 H 4 ).

Efinaconazole
This drug, also called Jublia, is used to treat fungal infections, mainly of the nail (Figure 7).The drug inhibits ergosterol biosynthesis in the membrane, thereby adversely affecting fungal cell growth [26].Evidence is presented in the prior sections pointing to involvement of conjugation by triazole substituents.
Although Jublia itself does not fit into the scheme, dehydration of the alcohol would yield a conjugated vinylogous triazole metabolite shown in Figure 8.The acid catalyst depicted in the pseudo-5-membered ring is derived from the protonated triazole (Figure 7).Hence this entity should be capable of participating in the unifying ET-ROS-OS mechanistic scheme.

Fluconazole
This triazole, which bears close structural resemblance to Jublia, is marketed for treatment of fungal infections (Figure 9).It plays a role where other drugs have failed.
Although there are reports of various adverse effects, such as nausea, rash, fatigue, anorexia, headache and seizures, the drug appears to be relatively safe [27].It is similar to other triazoles by inhibiting ergosterol synthesis in the membrane.In relation to

Mechanism of Antifungal Triazoles and Related Drugs: Electron Transfer, Reactive Oxygen Species and Oxidative Stress
Copyright: © 2017 Peter K, et al.
the ET-ROS-OS mechanism, the drug can be compared with Jublia in potentially undergoing metabolism to the conjugated species illustrated in Figure 10.It is interesting that the same vinylog is formed from dehydration involving the other triazole ring.

Voriconazole
This antifungal triazole agent belongs to the alcohol category (Figure 11).It is similar to amphotericin B and fluconazole in efficacy [28,29].Side effects are similar to others of this class except for blurred vision or increased sensitivity to light.Metabolically, hydroxylation appears to be more important than N-oxidation [30].
There are prior studies on phenazine and quinoxaline N-oxides [31,32].The imine-N-oxide is related to iminium in exhibiting enhanced electron affinity.Protonation would increase cationic character.As for the other alcohols, dehydration would generate a conjugated vinylogous metabolite (Figure 12).A similar structure is derived from dehydration involving the other heterocycle.

Isavuconazole
Antifungal activity is also exhibited by this triazole (Figure 13).The potency was compared in vivo versus those of voriconazole and fluconazole [33].Isavuconazole also belongs to the alcoholcontaining class.In vivo dehydration can conceivably generate the conjugated, vinylogous metabolite illustrated in Figure 14.
The compound should be capable of redox cycling in accord with the ET-ROS-OS mode of action.Alternatively, dehydration might occur to produce the vinylogous system incorporating the thiazole heterocycle.

Mechanism of Antifungal Triazoles and Related Drugs: Electron Transfer, Reactive Oxygen Species and Oxidative Stress
Copyright: © 2017 Peter K, et al.

Pramiconazole
This triazole antifungal agent has been less investigated in comparison to its closely related structural analogs, namely itraconazole and posaconazole (Figure 15).A significant portion of the prior discussion for the similar two drugs should also be applicable in this case.Close inspection of the structure suggests that the drug may function as a precursor of the triazole alcohol class.The acetal segment could readily undergo hydrolysis to a keto group capable of reduction to an alcohol.Subsequent dehydration, would yield the vinylogous, conjugated system which would be capable of ET.

Propiconazole
A study was made of modulation of glutathione-related antioxidant defense system of fish treated by the triazole fungicide propiconazole (Figure 16) [34].OS in rainbow trout was measured, as well as oxidative damage.It interferes with esgosterol.Mechanistic aspects are discussed under pramiconazole.In rats, hepatotoxicity was displayed by propiconazole, triadimefon, and myclobutanil [35].A prior review applies the ET-ROS-OS scheme to hepato toxins [8].

Suvorexant
This drug, not an antifungal agent, belongs to the triazole class, and is structurally related to itraconazole and posaconazole in possessing a benzenoid groups in conjugation with the heterocycle (Figure 17).Suvorexant, mainly used for treatment of insomnia, is an orexin receptor antagonist [36].The amidesubstituent on the benzene ring should aid in delocalization of the radicals in the ET process.The triazole in this drug is related to the 1,2-diimine class which is known to possess ET properties as a simple functionality [37].

Miconazole
This antifungal agent is quite akin to the triazole class except for imidazole in place of triazole (Figure 18).In addition, there is similarity in mode of action entailing inhibition of ergosterol synthesis in the membrane.The drug may function as a precursor of the alcohol class via ether cleavage followed by dehydration to the conjugated vinylogous system.Ether scission can occur by hydrolysis or oxidative dealkylation.

Clotrimazole
This antifungal medication is on the WHO list of important drugs.It also functions by inhibiting ergosterol biosynthesis in the membrane (Figure 19).
Although there are similarities to the triazole class, there is difference in the presence of the imidazole heterocycle.Another difference is the obvious absence of extended conjugation.However, there is precedent for radical delocalization during ET.Computational studies were performed on ET by the iminium metabolite of phencyclidine (PCP) [38].Although vinyl delocalization is not possible, the process can occur as depicted in Figure 20 via a pseudo-3-membered ring.A similar situation can be visualized for delocalization after electron uptake by PCP iminium as illustrated in Figure 21.
Similar delocalization is conceivable for the protonated (iminium) form of imidazole.

Mechanism of Antifungal Triazoles and Related Drugs: Electron Transfer, Reactive Oxygen Species and Oxidative Stress
Copyright: © 2017 Peter K, et al.

Echinocandins
Echinocandins comprise a recent class of antifungal drugs, including anidulafungin (ALF) and caspofungin (CPF) in addition to micafungin.The natural antifungal agent micafungin inhibits an essential polysaccharide in fungal cell walls [39,40,41].A result is osmotic instability and lysis of the cell (Figure 22).
The heteroaromatic oxazole portion incorporates the heterocycle in conjugation with two benzenoid rings which should make possible redox cycling in accord with the ET-ROS-OS theme (Figure 23).The heterocycle contains a conjugated imine which fits into the ET category.
There is additional significant literature.The most common adverse effects are nausea and enhanced levels of transamine [42].Rats developed liver tumors at high dose.Safety appears to be good in humans.The drug generates cell stress involving interference with the redox state that is counteracted by AOs [43].GSH levels are reduced.This behavior is in accord with participation of ET-ROS-OS.
All three are quite safe.ALF and CPF belong to the phenolic group of drugs which may function via the quinone metabolites in accord with ET-ROS-OS [44].ALF also incorporates a phenolic ether that has the potential for dealkylation to a quinone via a phenol conjugated with a bisphenyl.In animals, CPF can exhibit embryotoxic properties which may involve ROS [45,46].

Alprazolam
The drug, also called Xanax, is a member of the triazole family used mainly in treatment of anxiety (Figure 24) [47].It is related to some antifungal agents in being in conjugation with a benzenzoid.It is interesting in being incorporated with a benzodiazepine unit which also fits into the ET-ROS-OS scheme [48].Xanax became a blockbuster drug in the U.S. and alprazolam is the most prescribed.The potential for misuse is controversial.

Mechanism of Antifungal Triazoles and Related Drugs: Electron Transfer, Reactive Oxygen Species and Oxidative Stress
Copyright: © 2017 Peter K, et al.

Evaluation of hypothesis
There are various methods by which the hypothesis can be tested involving the parent compound or the active metabolite.Reduction potentials, based on polarography or cyclic voltammetry, provide information concerning ability to accomplish ET in vivo.The conjugated alkenes from alcohol dehydration could be synthesized for examination.Analytical methods can be used to detect the presence of ROS which are postulated to arise from ET. Addition of AOs would decrease ROS.

Other Support Material
A Quantitative Structure Activity Relationships (QSAR) study was performed on toxicity by triazole fungicides [49].Findings demonstrate that electron exchange may occur between the drugs and the target.The data adds credibility to the ET portion of the ET-ROS-OS unifying theme.In vitro experiments revealed hyperinduction of H2O2 production by the fungicide prothioconazole (Figure 25) [50].This important finding concerning ROS formation by the fungicide prothioconazole supports the ET-ROS-OS unifying theme.In another study, the physiological and biochemical responses in different tissues of fish from exposure to fungicide propiconazole [FC2] indicated that PCZ-induced the stressful conditions (Figure 16) [51].The stress is in in accord with OS arising from generation of ROS in the unifying mechanism.
The unifying mechanism involving ET-ROS-OS, which has received substantial support, is applied to anti-fungul triazoles based on the ET imine-iminium portion, as part of a multifaceted scheme.

Summary
The ET imine-iminium segment of antifungal triazoles is often part of a conjugated system.In some cases, conjugation may be achieved via metabolism, such as alcohol dehydration.Suggestions for future work relative to the unifying mechanism are presented.