Yan Lin

Harley Betts

Sarah Keller

Kevin Cariou

Gilles Gasser

Recent Developments of Metal-based Compounds against Fungal Pathogens

Keywords: bioorganometallic chemistry, fungal pathogens, medicinal inorganic chemistry, medicinal organometallic chemistry, metal complexes, antimicrobials, antimycotic activity. Biographies

come

Sarah Keller did her PhD in Chemistry at the University of Basel under the supervision of C. E. Housecroft and E. C. Constable. For her two-year postdoctoral research project in the Gasser Group at Chimie ParisTech PSL University in Paris, she was awarded a Feoder Lynen Postdoctoral Fellowship from the Alexander von Humboldt Foundation and an Early Postdoc Mobility Fellowship from the Swiss National Science Foundation. She pursued several projects in the fields of inorganic and medicinal chemistry, such as the design and synthesis of organometallic drugs, or luminophores for sustainable light-emitting devices. She recently started working at hemotune AG, a spin-off company originating out of the Functional Materials Lab at ETH Zurich, where she is responsible for the development of surface-functionalized magnetic nanoparticles for medical applications. Gilles Gasser obtained his PhD in 2004 at the University of Neuchâtel (Switzerland) under the guidance of Helen Stoeckli-Evans. He then undertook two post-docs, first with the late Leone Spiccia (Monash University, Australia) in bioinorganic chemistry and then as an Alexander von Humboldt fellow with Nils Metzler Nolte (Ruhr-University Bochum, Germany) in bioorganometallic chemistry. Gilles started his independent scientific career at the University of Zurich (Switzerland) in 2010 before moving to Chimie ParisTech, PSL University (Paris, France) in 2016 to take a PSL Chair of Excellence. Gilles was the recipient of several fellowships and awards including the Alfred Werner Award from the Swiss Chemical Society, an ERC Consolidator Grant, the Jucker Award for his contribution to cancer research, the European BioInorganic Chemistry (EuroBIC) medal and the Pierre Fabre Award for therapeutic innovation from the French Société de Chimie Thérapeutique. Gilles' research interests lay in the use of metal complexes in different areas of medicinal and biological chemistry.

Introduction 1.1 Fungal infections and antifungal drugs

The fungal kingdom comprises an estimated 1.5 million species with more than 200 species associated with humans. [START_REF] Buckley | [END_REF] Fungi are ubiquitous in the environment and exhibit many strategies to facilitate their survival in changing surroundings. They can interact with plants, animals or humans in multiple ways, including symbiotic, commensal, latent or pathogenic relationships. 2 Humans can be infected by various routes, which include inhalation of spores or small yeast cells. Superficial infections are the most common fungal diseases in humans and affect 1.7 billion people in the world and are primarily caused by dermatophytes such as Epidermophyton, Microsporum, and Trichophyton. 3 These pathogenic fungi specialize in the infection of keratinous substances -such as skin, hair and nails -where they utilize the keratin for nutrition. Expansion of infections in immunocompromised patients, e.g. those with HIV/AIDS, may lead to systemic fungal infections that become life-threatening. High mortality rates, which can exceed 50%, have been attributed to the difficulty of diagnosis, and shortcomings in the current antifungal arsenal. 4 The most significant fungal species that cause human infections, allergies and toxicosis belong to one of the five main genera: Candida (C. moniliforme and F. solani) and Pneumocystis. 5 Additionally, it is predicted that the prevalence of fungal diseases will also increase with the progression of global warming. 6 Although fungi were recognized as pathogens prior to bacteria, progress in the development of effective antifungals has lagged behind antibacterial research. At least two reasons can potentially explain this oversight. First, before the HIV-era, the occurrence and severity of fungal infections was believed to be too low to warrant aggressive research by the pharmaceutical industry. Second, the eukaryotic nature of fungi makes them metabolically and physiologically similar to mammalian cells (aside from the presence of a fungal cell wall) and therefore offer limited pathogen-specific targets. Numerous tests have confirmed that antibiotics have poor activities against fungi. One reason for the difference in sensitivity between fungi and bacteria can be found in different cell wall permeabilities. The fungal cell wall is a complex intertwined layer of saccharides (Fig. 1) and significantly differs from mammal cells, this creates several target opportunities for the design of antifungal drugs. [START_REF] Gow | The Fungal Kingdom[END_REF] Fig. 1: Schematic representation of a fungal cell wall and cell membrane. Adapted from Mycoses Study Group Education and Research Consortium. [START_REF]Antifungal Pharmacology[END_REF] There are five main antifungal agent classes approved for human consumption: [START_REF] Howard | [END_REF] 1) The azoles (e.g., Fluconazole (FCZ, Fig. 2)) are the most commonly used antifungal drugs and target the cytochrome P-450-dependent enzyme, 14α-sterol demethylase (P-450 DM ), preventing the synthesis of ergosterol (Fig. 1, bottom left), a major component of fungal plasma membranes. 10 Cholesterol is an analogue of ergosterol in mammals and its synthesis can also be impacted by this enzyme. However, when azole agents are used at therapeutically relevant concentrations, their affinities for fungal P-450 DM are greater than for the mammalian enzyme. Exposure of fungi to azole agents results in ergosterol depletion, accumulation of lanosterol and other 14-methylated sterols and interferes with the "bulk" functions of ergosterol in fungal membranes, which alters the fluidity and integrity of the membrane. Disruption of the plasma membrane makes the fungal organism more vulnerable to further damage associated with nutrient transport and chitin synthesis. 11 As azole drugs are orally administered and have few side effects, they have been used widely, however, not long after their discovery, reports of clinical failure attributed to drug resistance appeared. 12 2) Polyenes, such as amphotericin B (AmB, Fig. 2), are naturally occurring macrolides produced by Grampositive bacteria and are widely used as a "gold standard" for treatment of systemic infections. They bind to ergosterol, allowing holes in the fungal cell membrane to form, disruption of chemiosmotic gradients and intracellular constituent leakage. Polyenes display potent antifungal activity but high mammalian toxicity and intravenous administration limit their application.

3) Echinocandins, such as caspofungin (CFG, Fig. 2), inhibit the synthesis of β-1,3-glucan in the fungal cell wall via non-competitive inhibition of enzyme β-1,3-glucan synthase (Fig. 1, in green, right). The absence of cell walls in mammalian cells make the echinocandins minimally toxic, however, their application is limited to a narrow range of fungal pathogenic infections, including Candidiasis and Aspergillosis. Poor oral bioavailability of echinocandins require they be administered by intravenous injection, similar to AmB. 13 4) Allylamines, such as terbinafine (TRB, Fig. 2), act by inhibiting squalene epoxidase (Fig. 1, bottom left)the first enzyme in the biosynthetic pathway of ergosterol (and cholesterol) -resulting in sterol depletion and accumulation of the squalene precursor. This affects membrane structure and function, including nutrient uptake. 14 Although resistance to TRB has not been reported for human pathogens, observation in the corn pathogen U. maydis was first described in 1990. 15 5) Antimetabolites such as 5-flucytosine (5-FC, Fig. 2). 5-FC is a fluorine analogue of cystine that is taken up by fungal cells, converted to 5-fluorouracil, phosphorylated and incorporated into RNA. The inclusion of the antimetabolite results in miscoding and halts protein synthesis. Additionally, phosphorylated fluorouracil is also converted to its deoxynucleoside which inhibits fungal DNA replication by thymidylate synthase (Fig. 1, bottom right). However, the use of 5-FC is limited by its bone marrow toxicity in mammals and the high rate of spontaneous mutation to resistance. Given the increasing incidence of fungal infections and the emerging resistance to existing drugs, it is important to develop structurally novel medicines. 16,17 Fig. 2: Representative drugs of the five main antifungal agent classes approved for human clinical application.

In order to evaluate the therapeutic interest of potential new antifungals, two independent, standardised methods for susceptibility testing of fungi are generally used: 1) broth dilution testing to determine minimum inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC), which are commonly used to assess the fungicidal activities of azoles and echinocandins against Candida or Aspergillus species; and 2) disk diffusion testing/zone of inhibition assays which can quantify the inhibitory effects a compound has on the growth of an organism. These methods are convenient, economical and well suited for testing water-soluble antifungal drugs, such as 5-FC, FCZ and voriconazole (VRC), against yeasts and molds. 18,19

Metal-based drugs

The discovery of the antitumor activity of cisplatin in 1969 stimulated significant interest in the use of metal complexes as diagnostic agents or chemotherapeutic drugs. 20 Metal complexes offer fantastic opportunities in the development of new agents for clinical use due to their specific, unique physicochemical properties.

The wide range of thermodynamic and kinetic properties of metal complexes (e.g. redox activity, coordination number, geometry and ligand versatility as well as capacity for ligand exchange) and the intrinsic medical properties of the metal ions and ligands themselves give rise to many possibilities for the design of therapeutic agents not readily available to purely organic compounds. 21 Many metal compounds including arsenic, bismuth, platinum, ruthenium, gold, copper and silver have been studied in the pharmacological field as anticancer agents, 22 as well as antimicrobial, 23 antinflammatory, 24 antiparasitic, 25 and antimalarial agents. 26 For instance, the organic arsenical Salvarsan ((AsR) n , where R = 3-amino-4-hydroxyphenyl and n = 3 or 5) which was historically used to treat syphilis and trypanosomiasis, however, in recent times it has been removed from clinical use. It is thought that the active form of the drug resulted from an in vivo oxidation process and Salvarsan serves as a slow-release source of RAs(OH) 2 with oxidation from As(I) to As(III), as demonstrated by Nicholson and co-wokers. 27 Although the design of metal complexes with good therapeutic indices is still rather empirical, metal-based therapeutics have the potential for completely new, metalspecific modes of action that may make it difficult for fungal organisms to develop mechanisms of resistance.

In this article, we provide a comprehensive overview of the developments in the design of metal complexes with antifungal activity. The particular case of metal nanoparticles will not be covered in this review.

Azole antifungal drugs

Overview

Azoles are a class of five-membered heterocyclic compounds that contain a nitrogen atom and at least one other non-carbon atom as part of the ring (e.g., nitrogen, sulfur, or oxygen). The first report of antifungal activity of an azole compound, benzimidazole, was described in 1944 by Woolley, 28 however, they were not used in clinical settings until 1958 with the release of the topical ointment, chlormidazole. In the late 1960s, three compounds that firmly established azoles as antifungal agents were introduced: Clotrimazole (CTZ, Fig. 3) was developed by Bayer AG (Germany) and the first imidazole antifungal drug to enter the market; Miconazole (MCZ, Fig. 3) and Econazole (ECO, Fig. 3), both developed by Janseen Pharmaceutica (Belgium). 29 Ketoconazole (KTZ, Fig. 3) was the first orally, bioavailable, broad spectrum imidazole, representing a major advance in treatment of systemic fungal infections. However, adverse gastrointestinal side effects have been frequently reported, causing these to be replaced by triazoles. Imidazoles and triazoles contain two or three nitrogen atoms in the five-membered heterocyclic ring, respectively. In general, triazoles have a broader spectrum of antifungal activity and reduced toxicity when compared with imidazoles. Terconazole (TER, Fig. 4) is a topically active drug for treatment of vaginal candidiasis and dermatomycoses and was the first triazole-based antifungal agent marketed for human use, however, the two leading triazole-based drugs are FCZ (Fig. 4) and Itraconazole (ITC, Fig. 4). FCZ was approved for use as a single-dose oral therapy for vulvovaginal candidiasis in 1994 as well as widespread use as a prophylaxis and treatment of superficial and invasive Candida infections and Cryptococcus infections. The bioavailability of FCZ is higher than other azoles, which may be attributed to its high aqueous solubility and low affinity for plasma proteins. In immuno-compromised and immuno-suppressed patients (such as those living with AIDS or bone marrow transplant recipients) ITC has become the drug of choice for the treatment of paracoccidioidomycosis, blastomycosis, histoplasmosis and sporotrichosis despite it being less effective than FCZ. 30 New triazoles, such as VRC (Fig. 4) and Posaconazole (POS, Fig. 4), 31,32 have also been approved for human use. The emergence of high-level resistance is accompanied by widespread use of azole agents. FCZ resistance in C. spp. has been reported extensively and is attributed to this drug being the most widely used azole. 33 Furthermore, FCZ-resistant clinical isolates of C. neoformans and H. capsulatum and ITCresistant clinical isolates of A. fumigatus have also been reported. 34,35,36 More specialized or comprehensive reviews are available for further details about cellular and molecular mechanisms of azole resistance can be referred. 12,10 Fig. 4: Structures of clinically approved triazole-based antifungal drugs.

Metallocenyl-derivatives of azole antifungal drugs

Iron is an essential micronutrient for most living organisms as it participates in multiple metabolic processes, including synthesis of haem and iron-sulfur cluster-containing proteins, amino acids, DNA, lipids, and sterols. 37,38 Recently, it was also reported that high iron concentrations can alter cell wall architectures in C. albicans, reducing fungal cell survival upon exposure of antifungal agents. 39 Ferrocene is a sandwich structure consisting of two cyclopentadienyl (Cp) rings and iron atom, its discovery initiated the field of organometallic chemistry. The remarkable physicochemical properties of ferrocene have made it a very popular substituent for medicinal chemistry purposes for several reasons: 1) The general lack of toxicity of ferrocene allows it to be injected, inhaled, or taken orally without causing major health problems; 2) the stability of ferrocene in aqueous and aerobic media; 40 3) the robustness of ferrocene allows for a straightforward accessibility of a large variety of derivatives; 4) the lipophilicity of ferrocene moiety makes it easier for ferrocenyl derivatives to cross cell membranes; 5) the redox properties of ferrocene, as it can undergo a one-electron oxidation, yielding the ferrocenium cation. 40,41,42 Ferrocenium salts may generate DNA-damaging hydroxyl radicals in situ from hydrogen peroxide via Fenton-type reactions. Successful studies on ferrocenyl analogues, such as the ferrocenyl hydroxytamoxifen analogue (hydroxyferrocifen), a molecule in which a phenyl ring of tamoxifen is replaced by a ferrocene moiety, was prepared as an oestradiol receptor site-directed cytotoxic agent by Jaouen and co-workers. 43,44 This compound was found to be cytotoxic against breast cancer cells overexpressing the oestradiol receptor as well as those not overexpressing this receptor, contrary to tamoxifen. Using a similar synthetic strategy, Biot et al. obtained ferroquine (FQ) by introducing a ferrocenyl moiety into the carbon chain of chloroquine (CQ). FQ was found to be active in vitro against both CQ-sensitive and CQ-resistant strains of Plasmodium falciparum as well as in vivo against P. berghghei N. and P. yoelii NS.

FQ is currently in phase IIb trials, where the efficacy of an artemisinin-based combination therapy between artesunate and FQ in malaria patients is examined. 45 The first example of implementing this strategy in antifungal research was reported in 2000 by Biot et al., who replaced the 2,4-difluorophenyl ring of FCZ with a ferrocenyl moiety to give the first ferrocenecontaining FCZ derivative (1). Unfortunately, analogue 1 showed no growth inhibition on the studied fungi (C. albicans, C. glabrata, C. parapsilosis, and C. krusei). 46 Fang and co-workers prepared a wide variety of ferrocene derivatives containing 1,2,4-triazole, thiazole or both, such as ferrocenyl-substituted vinyl 1,2,4triazole derivatives (2Z, 2E), 47 ferrocene-triadimefon analogues (3), 48 ferrocene-triadimenol analogues (4), 49 1-ferrocenyl-3-aryl-2-(1H-1,2,4-triazol-1-yl)-prop-2-en-1-one derivatives (5, Fig. 5), 50 and N-substituted benzylidene-4-ferrocenyl-5-(1H-1,2,4-triazol-1-yl)-1,3-thiazol-2-amine derivatives (6, Fig. 6). 51 The fungicidal activity of complexes 2Z, 2E against powdery mildews and brown rusts were evaluated at concentration of 50 parts per million (ppm). For all compounds, the E-isomers exhibited higher inhibitory activities than the Zisomers. Among them, the E-isomers of 2b-e, 2g, 2j, and 2l had a 20-40% increase in the efficacy compared to the Z-isomers against brown rusts, no positive control was given for comparison. The crystal structure of (Z/E)-2d showed the linkage (N-C-C-C) between the triazole ring and substituted aryl group in the E-isomer was linear, however, the backbone of the Z-isomer was distorted. The authors suggest that the E-isomers might be more favourable to bind to the receptor. The antifungal activities of two series of ferrocenetriadimefon (3) and triadimenol (4) derivatives against powdery mildews and brown rusts were studied at a concentration of 50 mg⋅L -1 . Unfortunately, all the tested compounds exhibited lower antifungal activities (∼30% inhibition) than the parent compounds, triadimefon or triadimenol (∼80% inhibition). The crystal structure of 3f showed that the substituted aryl group was spatially repelled and turned toward the nearby triazole group due to the presence of the bulky ferrocene moity. The authors proposed that the bulky ferrocene close to the triazole cycle was responsible for the decrease biological activity. The desired mechanism of action of the triazole derivatives was to target ferrous cytochrome-P450 enzymes. It was hypothesized that binding of triazole to the ferrous ions of cytochrome-P450 enzymes could be replaced by binding to the ferrous ion of ferrocene through intramolecular or intermolecular interactions. In 2006, Fang and co-workers evaluated the antifungal activity of complexes 5 against six fungal strains (G. zeae, A. solani, P. asparagi, P. piricola, C. achidicola and C. cucumerinum). The crystal structure of 5l showed that this series of complexes were in the E-configuration, but via a bent linkage. Most of the complexes displayed fungicidal activities within 30-50% inhibition across all the tested fungi at a concentration of 50 mg⋅L -1 , no positive control was reported for comparison. One year later, a series of N-substituted benzylidene-4-ferrocenyl-5-(1H-1,2,4-triazol-1-yl)-1,3thiazol-2-amine derivatives (6) containing thiazole and 1,2,4-triazole were prepared by Fang and co-workers.

Four fungal stains (P. zeae, A. solani, P. pircola and C. ara) were selected to evaluate the antifungal activities of these complexes at the same concentration. The results showed that the P. pircola strain was more susceptible than others reported with inhibition up to 62%. The author suggested that further structural modifications of ferrocenyl thiazole-containing derivatives would be necessary to increase the activity. strains (Fig. 6). The results were recorded as the percentage inhibition of mycelial growth. Compound 7e showed moderate antifungal activity with >55% inhibition against both strains. By comparison, FCZ recorded inhibition of 75.3% and 74.6% against A. niger and A. flavus, respectively. All other compounds did not display any appreciable antifungal activity. Overall, it could be concluded that the activity of the phenyl analogues were higher than the coumarin analogues against both fungi. 52 In 2016, our group investigated the antifungal activity of ferrocenyl sedaxane derivatives against S. cerevisiae (10a-d, Fig. 8). Sedaxane is a potent broad-spectrum fungicide that targets succinate dehydrogenase, an enzyme involved in fungal oxidative phosphorylation. 54 Compound 10a displayed impressive activity with a half maximal effective concentration (EC 50 ) value of 43 µM, which was higher than underivatized sedaxane and its ruthenocene analogue 10b (EC 50 > 100 µM). Host cytotoxicity was investigated against normal human cells, human fibroblast (MRC-5) and retinal pigment epithelial (RPE1 hTert). Compound 10a was found to be not toxic against both cell lines with half maximal inhibitory concentration (IC 50 ) above 100 µM. A 33% increase in the levels of reactive oxygen species (ROS) in the fungal culture clearly confirmed the importance of a reversible redox couple in 10a, a property not available for the ruthenium analogue 10b and sedaxane alone. However, compounds 10c and 10d, which bore phenyl or phenoxy groups on their pyrazole core, respectively, had much lower antifungal activity. This was attributed to the sterically hindered substituents that blocked the active pyrazole core. Overall, this work demonstrated that the introduction of a ferrocene moiety into known broad-spectrum drugs can produce promising fungicides (10a). In 2019, Singh et al. reported a series of ferrocenyl chalcone derivatives containing a triazole and organosilatrane groups (11-16, Fig. 9) with the aim of merging the pharmacological actions of the constituting moieties into a single molecular scaffold. Two types of ferrocenyl chalcones were designed: Type 1 (11-14) in which the carbonyl group was directly linked to the ferrocene ring, and type 2 (15-16) with a carbonyl group directly attached to the phenyl ring. The antifungal activities of these ferrocene conjugates were evaluated against seven fungal strains. Compound 14 (IC 50 = 31.25 µM) was found to be the most effective against C. albicans, while compound 12 (IC 50 = 31.25 µM) exhibited the highest activity against C. tropicalis.

Compound 11 was the most potent compound against all fungi strains with IC 50 values ranging from 62.50 to 250 µM. In general, type 1 compounds were more potent than type 2. Nevertheless, these ferrocene conjugates only exhibited moderate activity compared to the positive control, AmB (IC 50 range of 0.195-1.25 µM). 56 In 2019, Zhao and co-workers synthesised six ferrocene-containing 1,3,4-oxadiazoles 18a-f (Fig. 11). Their antifungal activities were tested on Gibberella nicotiancola, Gibberella saubinetii, and F. oxysporium f.sp. niveum. Most of the compounds exhibited excellent antifungal activity, which was higher than the control compound, hymexazol. Specifically, compound 18b had significant activities against G. nicotiancola (EC 50 = 0.022 µg⋅mL -1 i.e. 61 nM) and G. saubinetii (EC 50 = 0.008 µg⋅mL -1 i.e. 22 nM), both of which were lower than hymexazol (EC 50 = 0.032 µg⋅mL -1 and 0.019 µg⋅mL -1 , i.e. 323 nM and 192 nM, respectively). Similarly, compound 18e displayed excellent activities against G. saubinetii and F. oxysporium f.sp. niveum with EC 50 values of 0.029 µg⋅mL -1 (80 nM) and 0.009 µg⋅mL -1 (25 nM ), respectively. Both 18b and 18e can be considered as promising drugs for further investigation. 58 Recently, four FCZ ferrocenyl derivatives with one triazole moiety replaced by different alkyl substituents were developed in our group. The stability of these complexes in deuterated DMSO and H 2 O -common media for preparation of stock solutions for biological screeningwere monitored by NMR spectroscopy and showed that all tested compounds were stable for up to 24 hours in solution. In vitro studies against wild type S. cerevisiae showed that compounds 19a-d (Fig. 11) were more active than FCZ and that compounds 19b-c displayed 9-to 17-fold greater activities than the positive control.

Additionally, the cytotoxicities of these compounds were investigated against human retinal pigment epithelial cells (RPE-1-h Tert) and human fibroblasts (MRC-5). The results showed that 19a-d were more selective towards microorganisms than mammalian cells with selectivity indices (SI) between 5 and 24.

Compound 19b was identified as the most promising antifungal candidate and was further screened against C. albicans, P. paneum, A. glaucus and T. asahi. The antifungal activities of 19b were 400-fold higher than FCZ against P. paneum and also active against azole-resistant fungal strains. In vivo studies were performed on Candida-infected mice with drug doses of 10 mg⋅kg -1 . Compound 19b was found to diminish distal dissemination to the liver and brain and greatly improved inflammatory pathologies in the kidney and colon. 59

Azole-based metal complexes

In 2012, Kumar et ) adopted a square planar geometry. The DNA binding modes of these metal complexes were also studied. Hypochromicity and increases in viscosity and melting temperature indicated that the bonding mode of macrocyclic metal complexes with calf thymus DNA (CT-DNA) was driven by intercalation. DNA intercalation is the insertion of a planar, chromophoric region of a molecule between two stacked base pairs. In this process, DNA primary and secondary structures still remain intact while the DNA tertiary structure (helix) is partially lengthened and thus somewhat unwound compared to the original structure. 60 Furthermore, the DNA photocleavage ability of metal complexes 20-22 were studied in the presence of H 2 O 2 and upon irradiation with 360 nm light. The cleavage efficiency of the Cu(II) complex was found to be higher than those of the Ni(II) and Co(II) complexes. This could be explained by the redox activity of the Cu(I)/Cu(II) couple, which can facilitate the formation of ROS that cause DNA damage via Fenton-type reactions. 61 The antifungal activities of these metal complexes against C. albicans and C. parasilosis ranged 23), [(η 6 -p-cymene)-RuCl(L) 2 ]Cl (24), and [(η 6 -p-cymene)-RuCl(L) 3 ](PF 6 ) 2 (25) (Fig. 13). Of note among these compounds were complex [(η 6 -p-cymene)-RuCl 2 (CTZ)] reported by Sanchez-Delgado and coworkers, 63 and complex [(η 6 -p-cymene)-RuCl 2 (MCZ)] by our group. 64 The stability of these metal-azole complexes in deuterated DMSO was monitored by NMR spectroscopy. Partial DMSO-mediated ligand dissociation was observed in the monoazole complexes, resulting in a mixture of monoazole-ruthenium and DMSO-ruthenium species. However, the bis-and tris-azole complexes were found to be stable in DMSO and aqueous solution with a small amount of decomposition evident after 12 h (<3%). These mono-, bis-, and trisazoles complexes inhibited the growth of Culvularia lunata at low millimolar concentrations, however, the efficiency decreased with increasing number of coordinating ligands. Since these complexes cannot bind to the haem iron within the active site of lanosterol 14α-demethylase -due to blocking of the N3 atom through ruthenium coordination -the authors suggested that the complexes must operate via different modes of action. 65 [Au(PPh 3 )(CTZ)]PF 6 (29b) (Fig. 14) against Sporothrix strains (S. schenckii, S. brasiliensis and S. globose). The coordination geometries of these complexes (26a-b, 27a-b and 28a) were deduced via use of a range of techniques ( 1 H NMR, 2D NMR, UV-vis and IR spectroscopies, conductivity studies, etc.). Data collected for compounds 26a and 26b corresponded to an 18-electron Cu(I) complex with tetrahedral geometry, while the structures of cationic complexes 27a and 27b corresponded to 14-electron Au(I) complexes that most likely adopted linear coordination geometries. Compound 28a corresponded to a 16-electron Pt(II) square planar complex with a trans configuration, which was supported by density functional theory (DFT). Complexes 26a/b and 29a/b, which contained phosphine as an auxiliary ligand, displayed excellent activity with MIC values less than 2 nM against all tested isolates and more active than the parent drugs KTZ and CTZ. In the case of complexes 29a and 29b, both compounds displayed significant fungicidal activity with MFC values ranging from 0.4 to 8 nM against the six tested fungi. The effect of the most active complexes (26a, 26b, 29a and 29b) on the morphology of Sporothrix spp. was analysed by scanning electron microscopy (SEM).

Furthermore, the cytotoxicity tests on mouse fibroblast cells and human red blood cells indicated that these metal complexes were more selective towards fungi than mammalian cells with high SIs (>2536). Metal complexes 26a, 26b and 29b proved to be promising fungistatic drug candidates, while complex 29a was excluded due to its haemolytic effect. 69 albicans clinical isolates. The Zn(II) complex showed 5.4-and 11-fold higher activity than FCZ against C. krusei, and C. parapsilosis. The cytotoxicities of the complexes were tested against human fibroblast cells (MRC-5) and it was found that the metal complexes had increased toxicities when compared to FCZ (11.4 to 12.7 times higher). However, the SI values of the Cu(II) and Zn(II) complexes were 41 and 73, respectively. Considering the yeast-to-hyphae transition is one of the main pathogenic properties of Candida spp, the metal complexes (and FCZ) were exposed to C. albicans ATCC 10231 at subinhibitory concentrations. The results showed that the metal complexes were able to completely inhibit hyphae formation. Additionally, metal complexes showed moderate to good inhibition against biofilm formation of C. albicans ATCC 10231 and C. parapsilosis ATCC 22019. The studies of FCZ and its metal complexes on fluorescent Candida species adhesion to A549 cells (an initial step to invade the host cells) indicated that the Zn(II) complex reduced the adhesive properties of fungal cells, while the Cu(II) complex and FCZ had no effect. Furthermore, the exposure of these compounds at subinhibitory concentration reduced the amount of ergosterol in C. albicans. The Zn(II) complex and uncoordinated FCZ had comparable decreases in ergosterol depletion, however, the Cu(II) complex was found to be more effective. It was postulated that the mode of action could be similar to FCZ itself and that the Cu(II) complexes could be interacting with other active sites to further reduce the amount of ergosterol. Molecular docking studies of the metal complexes with CYP51 showed that Zn(II) complex had higher binding strength to the target enzyme, via electrostatic, steric and internal energy interactions. 70 Navarro and co-workers reported another series of Zn(II) and Cu(II) KTZ-and CTZ-containing complexes, 15). The antifungal activity of these metal complexes was screened against three fungi species, C. albicans, C. neoformans, and S.

namely, [Zn(KTZ) 2 (Ac) 2 ]⋅H 2 O (30a), [Zn(KTZ) 2 Cl 2 ]⋅0.4CH 3 OH (31a), [Zn(KTZ) 2 (H 2 O)(NO 3 )](NO 3 ) (32a), [Cu(KTZ) 2 (Ac) 2 ]⋅H 2 O (33a), [Cu(KTZ) 2 Cl 2 ]⋅3.2H 2 O (34a), [Cu(KTZ) 2 (H 2 O)(NO 3 )](NO 3 )⋅H 2 O (35a), [Zn(CTZ) 2 (Ac) 2 ]⋅4H 2 O (30b), [Zn(CTZ) 2 Cl 2 ] (31b), [Zn(CTZ) 2 (H 2 O)(NO 3 )](NO 3 )⋅4H 2 O (32b), [Cu(CTZ) 2 (Ac) 2 ]⋅H 2 O (33b), [Cu(CTZ) 2 Cl 2 ]⋅2H 2 O (34b) and [Cu(CTZ) 2 (H 2 O)(NO 3 )](NO 3 )⋅2H 2 O (35b) (Fig.
brasiliensis. Against one or more species, all metal complexes were found to be more active than the parent drugs. Amongst all tested compounds, complexes 30a and 32a were the most active against the three fungal species with MIC values ranging from 0.03-0.25 µM. These values were lower than those of KTZ (MIC = 0.125-0.5 µM). Further studies were performed on complexes 30a and 32a. The superficial alterations in S.

brasiliensis morphology after exposure to sublethal concentrations of complexes 30a and 32a were characterised by SEM. Treatment with complexes 30a and 32a resulted in pronounced changes, such as increased cell size, compromised cell walls, and loss of cellular structure when compared to untreated cells.

Cytotoxic studies showed that compounds 30a and 32a were more toxic than KTZ against mammalian cells.

Nevertheless, compounds 30a and 32a displayed similar SI values (>60) towards fungus over LLC-MK2 cells when compared to KTZ. 71 UV-Vis spectra of the metal complexes in solution with DMSO showed that they were stable up to at least 30 minutes. At 20 µM, the neutral Ru-Cl complex (36), ATZ and NaBF 4 displayed no antifungal activities, while, conversely, the [Ru-ACN] + (37) and [Ru-ATZ] + (38) complexes displayed significant inhibition on the growth of all tested strains with MIC values ranging from 2.4 to 9.3 µM. The MIC values of both cationic species were found to be lower than FCZ against C. glabrata and C. krusei, which are known to be FCZ-resistant. It was found that when C. glabrata cells were treated with [Ru-ATZ] + , the compound was taken up in a dosedependent manner and generated significant amounts of intracellular ROS. By comparison, the Ru-Cl complex (36) had ROS levels comparable to the non-treated cells. These findings were consistent with the compounds reported antifungal activities and potentially indicated the importance of the cationic nature of

Polypyridine based metal complexes

Polypyridyl ligands, such as 1,10-phenanthroline (phen) and 2,2'-bipyridine (bipy), have played an important role in the development of coordination chemistry (Fig. 17). 73 As powerful chelating ligands, these ligands coordinate with a variety of transition metal ions to form thermodynamically stable metal complexes. Both the metal complex and the free polypyridyl ligands can possess potent bioactivity and disturb biological systems. When the ligands are active alone, it is usually assumed that the sequestration of trace metals in situ is involved and that the resulting metal complexes are the active species. 74,75 In 2000, McCann et al. prepared a series of metal complexes (Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Ag(I)) of malonic acid (mal) chelated with the phen ligand, namely, [Mn(phen and increased lipid peroxidation (7:1). By contrast, the same measurements conducted for the Zn(II) complex remained largely unchanged relative to the negative control (GSH/GSSG ratio = 22:1 and lipid peroxides = 1:1 vs. 17:1 and 1:1, respectively, for the control). These observations would appear to substantiate the hypothesis that the significant anti-Candida activity of the Cu(II) and Mn(II) complexes may be attributed to their redox activity and potential action as Fenton reagents. 76 On the basis of previous work in their laboratory, Coyle et al. further investigated the modes of action of

) 2 ](mal) 2 ⋅2H 2 O, [Co(phen) 3 ](mal) 2 ⋅2H 2 O, [Cu(phen)(mal) 2 ]⋅2H 2 O, [Ni(phen) 2 ](mal) 2 ⋅2H 2 O, [Zn(phen)(mal) 2 ]⋅2H 2 O,
complexes [Cu(phen)(mal) 2 ]⋅2H 2 O, [Mn(phen) 2 ](mal) 2 ⋅2H 2 O and [Ag 2 (phen) 3 (mal)]⋅2H 2 O
, which are highly toxic against C. albicans. Cells exposed to either phen or metal-phen complexes resulted in significant reductions in cellular respiration, however, despite the drug-treated cells being metabolically hindered, their oxygen-uptake was found to increase, with the exception of the Ag(I) complex. This result could be explained by the fact that cells were using oxygen for processes other than respiration (e.g., formation of ROS).

Furthermore, all compounds appeared to inhibit cytochrome biosynthesis, leading to a reduction in cellular ergosterol content, with the exception of [Mn(phen) 2 ](mal) 2 ⋅2H 2 O which increased ergosterol content. The mode action of phen and its metal complexes have been related to mitochondrial function, reduction of cytochrome b and c synthesis, induction of respiratory uncoupling and increase of cell wall permeability in fungal cells. These are distinct modes of action compared to conventional azole antifungal drugs targeting ergosterol in the fungal cell membrane. Hence these compounds can be considered as a novel group of antifungal agents to be used alone or in combination with existing antifungal drugs. 77 Further studies on the effect of these drugs on the morphology of fungal and mammalian cells, as well as the integrity of cellular DNA were also carried out by Coyle et al. 78 A phen derivative, namely 1,10-phenanthroline-5,6-dione (phendio) and its Cu(II) and Ag(I) complexes - 39) and [Ag(phendio) 2 ]ClO 4 (40) (Fig. 17). The X-ray crystal structure of 40 was obtained, which showed that the Ag(I) adopted a pseudo tetrahedral environment and was chelated by four nitrogens from two phendio ligands. Both of the phendio ligand and its metal complexes showed higher activity than underivatized phen against C. albicans. It was envisioned that the two carbonyl oxygens on the phendio play an important role in its bioactivity. Extensive and non-specific DNA cleavage was found after C.

[Cu(phendio) 3 ](ClO 4 ) 2 ⋅4H 2 O (
albicans was exposed to phendio alone, simple Ag(I) salts and the [Ag(phendio) 2 ]ClO 4 complex (40). Moreover, phendio alone and in complex with Ag(I) (40) caused severe morphological alterations to C. albicans as well as disruption of cell division. 79 In continuation of this work, the antifungal activity of phendio and its Cu(II)

and Ag(I) complexes (39 and 40) against Phialophora verrucose was also assessed. The inhibition of these compounds on conidial growth followed the order (based on IC inhibited the growth of P. verrucose with an MIC value of 5 µM, whereas Cu(ClO 4 ) 2 ⋅6H 2 O was inactive even at 54 µM. It was been speculated that such observations may result from the metal complexes having higher lipophilicities than the simple metal salts. This property was attributed to a reduction in total electron density on the free ligand by coordination with a metal ion and sharing of the positive charge of metal cation with Ndonor atoms, inducing electron delocalization over the chelate ring. The inhibitory modes of action of antifungal agents against the growth of P. verrucose such as ultrastructure alternations, metallopeptidase activity, sterol synthesis and morphological transitions (conidia-into-mycelial transformation) were further evidenced by the inhibitory effect of phendio and its Ag(I) and Cu(II) complexes. 80 Recently, Granato et al.

reported additional mechanisms of action of phendio and its metal complexes (39 and 40) against P. verrucao, focusing on (i) combination therapy with AmB, (ii) biofilm formation and disarticulation, (iii) interaction with human macrophages, and (iv) in vivo studies on infected G. mellonella larvae. Overall, complex 40 was found to be a promising for the treatment of P. verrucose-infected larvae due to its lower toxicity against mice. 81 haemulonii complex were recorded by overall geometric mean of the MIC values (GM-MIC). Among all tested metal complexes, complexes 43 and 45-58 showed significant inhibitory effect on the viability of planktonicgrowing cells of all fungus isolates with GM-MICs less than 10 µM, however, still less effective than the positive control, CFG (0.18 µM). Simple metal salts such as CuCl 2 and MnCl 2 were found to have no appreciable effect on fungal growth. Conversely, inorganic Ag(I) salts (such as silver nitrate) and the free ligands (phen and phendione) significantly inhibited fungal growth. The inhibitory activity of the silver salts was likely attributed to the Ag(I) ion which is known to have significant biocidal activity. However, the Ag(I) complex with phen or phendio induced a more pronounced inhibitory effect compared to the metal ion or parent ligands alone. A similar effect was observed for Cu(II) complex (47) Similar inhibition trends were observed for the planktonic-growing cells, however, the biofilm cells were generally more resistant. 82 Gandra et al. further investigated the in vivo activities of compounds 47-57 against G. mellonella larvae -a commonly used in vivo infection model to understand C. haemulonii infection. The results showed that the metal chelates were capable of: 1) Inhibiting fungal proliferation during in vivo infection; 2) inducing an immune response in the G. mellonella host; and 3) affecting the fungal burden of infected larvae and the virulence of C. haemulonii in a dose-dependent manner. Among the tested compounds, the Mn(II) complex (54) was the most promising antifungal agent for further investigation. 83 In 2010, Raman and co-workers reported mixed-ligand Cu(II) and Zn(II) complexes with an isatin-based Schiff base (L) as a primary ligand, phen/bipy as auxiliary ligands and the general formula [ML(phen) 2 ]Cl 2 (59)/[ML(bipy) 2 ]Cl 2 (60) (Fig. 18). The DNA binding modes of these complexes were verified by absorption hypochromicity and viscosity measurements. The studies of cyclic voltammetry showed that in the absence of DNA the copper complexes had quasi-reversible redox properties. On the addition of DNA, the anodic and cathodic peak currents decreased with increasing DNA concentration indicating slow diffusion of the Cu(II) complex upon complexation with the DNA macromolecules. These results might be attributed to The MIC values of the metal complexes were lower than the free ligand (L), but higher than the reference drug, nystatin. Moreover, the Cu(II) complexes were more active than the Zn(II) complexes against all tested organisms. 84 In 2018, Oliveira and co-workers synthesised two octahedral Cu(II) complexes with bipy and thiouracil as co-ligands (61-62, Fig. 18). to 250 µg⋅mL -1 . Conversely, complex 62 and the free ligands (2-thiouracil and 6-methyl-2-thiouracil) were found to be inactive. In addition, CuCl 2 displayed MIC values of 1000 µg⋅mL -1 on all tested isolates, suggesting that the antifungal activity of complex 61 could be attributed to the synergistic effect of the metal-ligand complex. Further studies showed that complex 61 had no mutagenic potential and slightly inhibited biofilm formation, making it a promising antifungal agent for further study. 85 al. 86 The dinuclear Cu(II) complex (64) was found to exist in a monomeric form in DMSO solution, as determined by ESI-MS. The antifungal activities of two Cu(II) complexes (as well as the Cu(II) salt source, phen, bipy, and free ligand) were screened against three fungal strains (A. brasiliensis, C.albicans, and S. cerevisae).

The Cu(II) complexes were found to be more active than FCZ but less so than free bipy or phen. Both the Cu(II) salt and free ligand displayed low antifungal activities, indicating the importance of the bidentate α-diimine ligands and resultant complexes in promoting the inhibitory effect of the metals. The cytotoxicities of these compounds were quantified by brine shrimp lethality bioassays. The Cu(II) salt and bipy/phen displayed moderate toxicities while the free ligand and compounds 63 and 64 had lower cytotoxicities. Nagashri and co-workers examined the antifungal activities of metal chelates of phen derivatives (M = Cu(II), Co(II), Ni(II), and Zn(II)) (65, Fig. 19)) against C. albicans, A. niger, and A. flavus. Among the tested complexes, the Cu(II) compound was found to be the most active with MIC values ranging from 18 to 25 µg⋅mL -1 ; less than nystatin (10-15 µg⋅mL -1 ). The stabilities of the complexes in DMSO and PBS buffer were monitored by UV-visible spectrophotometery and were found to be stable for up to 24 h with only minor degradation, likely due to ligand hydrolysis under physicological conditions. The inherent binding constants of the complexes with DNA were obtained based on molar extinction coefficients (K b ): 4.11, 3.62, 3.85 and 2.9 × 10 5 for Cu(II), Ni(II), Co(II),

and Zn(II), respectively, indicating that Cu(II) chelate had the highest affinity. 87 In 2021, three Cu(II) complexes containing phen and bio-essential amino acid, L-tryptophan, (66a-c, Fig. 19)) were synthesized by Arumugham and co-workers. The antifungal activities of the metal chelates against A. niger, Rhizopus spp., and Pencillium spp. showed that 66a and 66c were inactive and that only 66b had antifungal activity, which was comparable to positive control, AmB. Recently, the activities of five complexes of Cu(II) with pyridine-4,5-dicarboxylate esters against Candida were evaluated by Glišić and co-workers (67-71, Fig. 20). Complex 67 adopted an elongated octahedral geometry while the geometries of complexes 68-71 were distorted square-pyramids. These metal complexes were also found to be stable in DMSO after 24 and 48 h of incubation and the partition coefficients (LogP) of these compounds were within the ideal range, 0 ≤ LogP ≤ 2. Additionally, ionic interactions, van der Waals interactions, hydrogen bonding and other partial intercalation binding modes may have also been present. 91 In 2014, Abu-Dief and co-workers developed a range of tridentate Schiff base-amino acid ligands that formed octahedral geometries with Fe(II) ions and had the general formula [Fe(HL) 2 ]⋅nH 2 O (76a-e, Fig. 23). The ligands were prepared by condensation of 5-bromosalicylaldehyde with the α-amino acids (L-alanine, Lphenylalanine, L-aspartic acid, L-histidine and L-arginine). Interaction of the Fe(II) complexes with CT-DNA were investigated by UV-Vis absorption, viscosity and agarose gel electrophoresis measurements. The metal complexes were found to bind to DNA via an intercalative mode, exhibiting binding strengths that followed the order 76e > 76d > 76a > 76c > 76b. Antifungal activity assays of the metal complexes on three fungal strains (P. purpurogenium, A. flavus and T. rosium) showed that the metal complexes were more active than the parent ligands. 92 For comparison, nickelocene-amino acid conjugates (77a-c, Fig. 23) were also obtained by reaction of amino acid azides with ethynyl nickelocene using a 1,3-dipolar cycloaddition. These organometallic complexes exhibited moderate antifungal activities against A. niger, A. flavus and A. alternata, but were less active than FCZ. 93 80) (Fig. 24) (E = P or As; B = PPh 3 , AsPh 3 or Py). Octahedral geometries were tentatively proposed for all complexes. 95,96 The antifungal activities of the Ru(II) chalconato complexes against A. niger and Mucor Sp. strains were greater than the parent ligands alone, but still did not reach the efficacy of the standard drug, Bavistin®. The increase in activity of the metal complexes was explained on the basis of increased lipophilicity of the complexes through metal ion coordination. In 2016, a set of ferrocenyl chalcones were prepared from O-alkylated vanillins and acetylferrocene by Muškinja et al.

(81a-f, Fig. 24). The antifungal activities of these complexes were determined against five fungal strains (M.

Sulfonamide-based metal complexes

The development of metal complexes based on sulfonamide was stimulated by the discovery of silver sulfadiazine, which is a topical antibiotic used to prevent infections in burn victims. 98 Chohan et al. prepared a range of bidentate furanyl-derived sulfonamide ligands which were subsequently coordinated with metal ions (Co(II), Cu(II), Ni(II) and Zn(II)) (82a-x, 83a-p, Fig. 25). 99,100 All resultant metal-based compounds adopted octahedral geometries. The non-electrolytic nature of these metal complexes was determined by molar conductance, confirming that the chloride ions were coordinated to the metal ions. IR spectra indicated that the ligands were coordinated to the metal ion through the furanyl oxygen and the azomethine nitrogen. The antifungal activities of these metal complexes were screened against M. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glabrata strains and compared with the reference drugs, MCZ and AmB, all complexes had comparable or higher activity than the parent ligands. Complexes 82a-x only showed ∼30% inhibition at 200 µg⋅mL -1 against the tested fungi, however, most of the complexes 83a-p exhibited significant inhibition (∼80%) at the same concentration. The cytotoxicities of these complexes were tested using a brine shrimp bioassay and, except for some copper complexes, the complexes were found to be non-cytotoxic.

Furthermore, pyrrolyl and thienyl-derived sulfonamide ligands and their metal complexes with Co(II), Cu(II), Ni(II) and Zn(II) ions (84a-t, Fig. 25) were also synthesised by the same authors in a separate article. 101 Shifting of the vibrational modes of the C=N, C-N/S functionalities to lower frequencies, as recorded by IR spectroscopy, suggested that metal coordination occurred through the azomethine nitrogen and pyrrolyl nitrogen/thienyl sulfur. The antifungal activities against the aforementioned six strains showed that most of the complexes possessed significant activity across all tested organisms with inhibition zones greater than 16 mm at 200 µg⋅mL -1 recorded, however, all metal complex activities were still less than the standard drugs MCZ and AmB (∼30 mm). Considering the biological and pharmacological activity of isatin-derived compounds, some isatin bearing sulfonamides and their metal complexes (Co(II), Cu(II), Ni(II) and Zn(II)) were also developed by Chohan and co-workers (88a-x, 89a-p, Fig. 27 and 90a-l, Fig. 28). 105,106,107 Their antifungal activities were assessed against six fungus strains (M. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glabrata) at 200 µg⋅mL -1 .

Most complexes (89a-p and 90i-l) significantly inhibited the growth of the various fungal strains by ∼65 to 70%. Among those tested, the Zn(II) complexes were found to be the most active, while complexes 88a-x only showed moderate activity (approximately 30% inhibition). The furanyl-/thienyl-analogues, 90a-h, exhibited reduced fungicidal activities than the isatin-derivatives 90i-l. Growth inhibition of uncoordinated ligands was enhanced upon metal complexation and was rationalized on the basis of the Overtone's concept and increased liposolubility due to electron delocalization resulting from metal ion coordination. According to the Overtone's concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of lipid-soluble materials, making liposolubility an important factor that influences drug uptake and activity. 108 Chelation considerably reduces the polarity of the metal ion due to partial sharing of its positive charge with the donor groups and possible π-electron delocalization over the whole chelate ring. This process of chelation could enhance the lipophilic nature of central metal atom, subsequently favouring its permeation through the lipid layers of the cell membrane. It has also been observed that the introduction of azomethine linkages can be helpful in increasing biological activity by similar rationale. 109 Complexes 92c-d with more alkyl chains exhibited a greater inhibitory effect (MICs ranging from 8.25 to 16.5 µg⋅mL -1 ) than 92a-b (8.25-66 µg⋅mL -1 ) on the tested fungal strains, of particular note was compound 92d which was as active as the positive control, KTZ.

Heterocycles-based Metal Complexes

Heterocyclic compounds contain at least one non-carbon atom in a ring and there has been considerable interest in the pharmacological applications of heterocycle-based metal complexes. In 2006, Tripathi et al.

reported a set of diorganotin(IV) dipyrazolinates (93a-l, Fig. 29) by reaction of diorganotin(IV) dichloride with sodium salts of pyrazolines. The bidentate pyrazoline ligands coordinated to Sn(IV) in distorted transoctahedral geometries. Among the tested complexes, complex 93i displayed greater inhibitory effect against A. niger compared to the free pyrazoline ligand and positive control, TRB. 112 A square planar Cu(II) complex with a benzimidazole derivative (94, Fig. 29) was synthesised by Arjmand and co-workers in 2005. Hyperchromicity and decreased viscosity in the presence of DNA suggested that the complex interacted with the biomolecule through only partial intercalation. A cyclic voltammogram of the same complex in the absence of CT-DNA showed a quasi-reversible redox wave for a one-electron transfer process, corresponding to the Cu(I/II) couple. The ratio of the anodic and cathodic peak currents I pa /I pc was 1.33, confirming the quasi-reversible redox process. After addition of DNA to the metal complex solution, the I pa /I pc ratio for the bound complex decreased to 0.67. The decrease in the current was attributed to the diffusion of an equilibrium mixture of free and DNA-bound metal complex to the electrode surface. The ratio of equilibrium constants could be predicted by the shift in the E 1/2 of Cu(I/II) couple following the equation: The ratio of binding constants (K 1+ /K 2+ ) for DNA binding of the Cu(I/II) couple was close to 1 (0.9), indicating that both Cu(II) and Cu(I) forms interacted with CT-DNA to a similar extent. The Cu(II) complex ( 94) was more active than its precursor ligand and the bis(ethylenediamine) Cu(II) complex against A. niger. 114 In 2018, Mansour synthesised three photo-induced CO-releasing pyridylbenzimidazole-based Ru(II) carbonyl complexes, which were functionalized with alkylated sulfonate, phthalimide or morpholine moieties (95a-c, Fig. 31). The protein affinities of these compounds with hen egg-white lysozyme (HEWL) were evaluated using electrospray ionization mass spectrometry (ESI-MS). While the protein adducts were stable in the dark, CO release was detected upon illumination at 365 nm. Compound 95b showed significant antifungal activity against the fungi C. albicans and C. neoformans with similar MIC values of 16 µg⋅mL -1 (24 nM). Using the standard octanol-water partition coefficient method, the lipophilicity of compound 95b (logP 7.4 = 0.8 ± 0.06) was found to be higher than 95a (logD 7.4 = -0.4 ± 0.06) and 95c (logP 7.4 = 0.3 ± 0.03). The hydrophobic nature of 95b enhanced its cellular uptake, therefore increasing its antifungal activity. The cytotoxicity of compound 95b was assessed on noncancerous human embryonic kidney cells (HEK293) and recorded moderate cell viability of 62% at 32 µg⋅mL -1 . Additionally, compound 95b showed good blood compatibility with 10 and 50% haemolysis concentration (HC 10 or 50 ) values greater than 32 µg⋅mL -1 , making compound 95b the most promising fungicide of this series for further investigations. 115 all tested strains. Among all the metal-based compounds, complex 101f, which contained a para-nitro group on the phenyl ring, showed comparable or higher antifungal activity than FCZ against C. glabrata (32 µg⋅mL -1 ),

C. parapsilosis (32 µg⋅mL -1 ) and C. tropicails (16 µg⋅mL -1 ). 119 In continuation of this study, a series of ferrocenyl pyrazoline complexes containing quinolone units (102a-f, Fig. 34) were synthesised by the same authors. The antifungal activities of these compounds were screened against the seven strains of fungi mentioned above.

Interestingly, all compounds exhibited equivalent or higher antifungal activities than FCZ against the fungal organisms, with MIC values ranging from 8 to 64 µg⋅mL In 2018, Damljanović and co-workers synthesised a class of ferrocenyl pyrazolidinone derivatives derived from acryloylferrocene and N,N'-cyclic azomethine imines. The mixture of two diastereoisomers (trans and cis)-5-aryletrahydropyrazolo [1,2-α]pyrazol-1(5H)-ones were separated and isolated as pure substances (103a-n and 104a-n, Fig. 35). All complexes were screened in vitro against bacteria or fungi. Contrary to many antimicrobial agents, which are often more sensitive to bacteria than fungi, the inverse was found to be true of the complexes reported in this article when tested against C. albicans, A. brasiliensis, P. canescens, and F. oxysporum. Compound 103c was the most effective complex of this series (MIC <0.02 mM), compared to the reference drug KTZ (MIC = 0.0753 mM) against C. albicans. The antifungal activities of the ferrocenyl pyrazolidinone derivatives were found to be more significant than the N,N'-cyclic azomethine imine derivatives, but lower than those of acryloylferrocene. Overall, most of the ferrocenyl pyrazolidinone derivatives showed inhibitory effects at low concentrations. 121 Based on earlier observations that the ferrocene-substituted porphyrin RL-91 (106a, Fig. 36) had a considerable impact on C. albicans biofilm formation without photoactivation, 123 Lippert et al. further developed trans-ferrocenyl porphyrin (106b, Fig. 36) by replacing two ferrocenylphenyl moieties with methoxy methylene substituted tert-butylphenyl entities. The modified compound, 106b, possessed higher lipophilicity and lower toxicity against human lung fibroblast cells (MRC5) than 106a. In addition, electrochemical studies showed that the ferrocenyl moieties of porphyrins 106a and 106b were more easily oxidized to ferricenium ions, compared to ferrocene alone. Compound 106b also showed higher inhibition of C. albicans (ATCC10231) than 106a by disk diffusion assay. The interaction of 106b with DNA was investigated using in silico modelling which determined binding to the major DNA groove to be dominant. 124 complexes (110a-f, Fig. 38) against C. albicans. 128 The results showed that all the tested complexes (except 110a) displayed significant inhibition against fungal cells, while the ligand and metal precursors did not.

Among all compounds, the morpholine-substituted complexes, 110b and 110e, showed greater inhibitory zones than FCZ (≥20 mm vs. 18mm for FCZ). In 2021, McGowan and co-workers screened a set of Ru(II) bis(bipy) ferrocenyl β-diketone complexes (111a-o, Fig. 38) against C. albicans and C. neoformans. 129 The results showed that all the tested complexes (except 111a) had >100% inhibition against C. neoformans at a concentration of 32 µg⋅mL -1 . However, compounds 111b-c, 111f-h and 111k-o displayed reduced efficacies against C. albicans with inhibitory zones ranging from 89.90% (111h) to 100.44% (111n). A lack of aromatic substituents on the ferrocenyl β-diketone ligands for complexes 111a, 111d, and 111e were thought to be responsible for their inactivity against C. albicans. Compound 111e was found to be 2-fold more selective towards C. neoformans over human blood cells making it one of the most promising complexes studies. No positive controls were reported.

Guanidine-based metal complexes

Gul et al. investigated the antifungal activity of ferrocenyl guanidines (112a-h, Fig. 39) against three fungal strains, namely F. moniliforme, A. fumigatus and A. flavus. The interactions of these ferrocenyl guanidine compounds with DNA were determined to be predominantly electrostatic modes by cyclic voltammetry and UV-visible studies. Among the tested compounds, complexes 112a-c, which contained electron-withdrawing substituents, possessed inhibitory properties comparable to or greater than the reference drug, TRB (65-96% vs. 92%). However, complexes 112d-h -containing electron-donating substituents -displayed less activity with only 13 to 47% inhibition of fungal growth. Of note, the tested compounds displayed significant stability as they did not decompose for up to seven day in ethanol or DMSO. 130 In 2018, Sadler and co-workers

Cyclotetraphosphazene-based metal complexes

Kilic and co-workers evaluated the antifungal activities of fully substituted monoferrocenylspirocyclotetraphosphazenes with pyrrolidine, morpholine and 1,4-dioxa-8-azaspiro [4,5] µM). 132 Based on these promising results, bis-ferrocenyl pendant-armed dispirocyclotetraphosphazenes (115a-l, Fig. 40) were also synthesised by the same group. Among all tested compounds, 115l was found to In addition, the dose-dependent inhibition of these metal complexes on the activity of the enzyme, alkaline phosphatase, was also observed. 134 Asghar et al. synthesised three ferrocene-containing urea ligands by the deprotection of ferrocene-based thioureas, using alkaline Hg(II) as a sulfur capturing agent (117a-c, Fig. 41). The DNA binding modes of these compounds were studied by UV-vis spectroscopy and cyclic voltammetry which indicated that an electrostatic mode of interaction between the positively charged 117 and the negatively charged DNA phosphate backbone was likely. The antifungal activities of these complexes were determined and compound 117c was found to be the most active compared to the other analogues, with >70% inhibition against all the tested fungal strains (F. moniliforme, A. fumigatus and A. flavus). The enhanced activity might be due to a more pronounced inductive effect of the bromo group at the ortho position than at the meta and para positions. 135 Three ferrocene-containing selenourea ligands (118a-c, Fig. 41) displayed moderate activity (∼50% inhibition) on F. solani and Helmentosporium sativum strains. 136

Schiff bases

Imines and azomethines, commonly known as Schiff bases after German-Italian chemist Hugo Schiff, are the product of aldehyde or ketone condensation with an amine. These species are naturally occurring, for example, in corrin which forms the core of vitamin B 12 and binds Co(II) as a cofactor. Schiff bases have been identified as versatile pharmacophores that possess antifungal, antibacterial, antiviral, anti-inflammatory, antiproliferative, and antiparasitic properties. The electrophilic carbon and nucleophilic nitrogen that form the backbone of Schiff bases facilitate reactions with many nucleophiles and electrophiles. Such reactivity allows interactions with important biological target sites, such as enzymes, proteins, and DNA. Schiff bases have been shown to be excellent ligands for a multitude of metal ions -from main groups to transition metals to the f-block, covering a wide range of complex geometries and oxidation states.

The antifungal properties of some Schiff bases and related compounds are well known, however, many compounds and their metal complexes lacking significant antifungal (or antibacterial, anticancer, etc.)

activities have also been published. Several reports are devoted to the characterisation of complexes, and application seen as an afterthought. Often the discussion of any antifungal activity is justified by the idea that the metal complex of a ligand would increase lipophilicity by delocalisation of the metal ion charge, thereby improving cellular uptake and fungicidal activity. While this may be true in some cases, it does not necessarily indicate a successful or effective antifungal agent has been developed. For example, a series of amino acids (glycine, alanine, phenylalanine, methionine, cysteine) were condensed with salicylaldehyde to form Schiff bases (Fig. 44), and complexed with Co(II), Cu(II), Ni(II), Zn(II) to form 25 compounds (including the free ligands). 142 The compounds were tested against six fungal strains (150 experiments) and ∼77% displayed no antifungal activity while the remaining 23% achieved a maximum inhibitory effect of 35%, relative to positive controls, MCZ and AmB. Similar studies with lacklustre antifungal results are common in the literature, however, there are also positive examples of metal complexes resulting in significant fungicidal activities. Of these investigations, a vast number of tested compounds yielded poor antifungal activities relative to positive controls but some promising insights can be gleaned from the studies as a whole. Comparison of antifungal experiments revealed that nitrogen-containing heterocycles (pyrrole or pyridine) and their derivatives had the most prominent activities. For example, Chohan et al. found that treatment with 200 µg⋅mL -1 solutions of triazole-pyrrole/pyridine oxovanadium complexes (Fig. 46) achieved up to 80% fungal growth inhibition against all tested organisms. Similarly, 75-80% inhibition was achieved against M. canis, F. solani, and C. glabrata for a methyl-pyridinyl compound. Salicylaldehyde-triazole derivatives including p-bromo/chloro/nitro functionalities (Fig. 47C) were found to be effective against selected fungi (C. glabrata, F. solani, A. niger, A. flavus) and achieved ∼70% inhibition. 143,148,149 However, in this instance, the free ligand displayed significant antifungal activity and complexation with the metal ions only increased the activity by 5 to 10%.

Pyrazoloyl-based

Pyrazolyl-Schiff bases formed between 3-methyl-1-phenyl-4-acetylpyrazolin-5-ol and 1,2-diaminobenzene or 2-aminophenol were reported in 2010. 150 Complexation with V(IV), Fe(III), Cu(II), and Co(II) ions yielded eight different complexes. The tridentate ligands coordinated as ONN or ONO donors via the pyrazolyl oxygen, azomethine nitrogen and phenol or amino O/N (Fig. 48). The compounds and free ligands were tested for antifungal efficacy against three species (C. albicans, Rhizopus sp., and A. niger) through disk diffusion experiments at 200 µg⋅mL -1 with AmB as positive control. Interestingly, the free 2-aminophenol Schiff base displayed significant activity against A. niger, resulting in similar inhibition diameters to the positive control.

Complexation of the ligand with Co(II) increased the antifungal properties against Rhizopus spp. where the complex approached the efficacy of AmB, and against both A. niger and C. albicans where inhibitory zone surpassed the positive control. Similarly, the Co(II) complexes with the diaminobenzene derived ligand displayed equivalent inhibitory effects against A. niger, and greater than 80% inhibition against C. albicans. Two Schiff based ligands -phenazone-furfural and aminophenol-nitrobenzaldehyde -were synthesised and used to form mixed ligand complexes with Fe(III) and other divalent first row transition metal ions (Fig. 49). 151 Against the chosen fungal strains, all complexes were found to have lower MIC values than either the free ligands or metal salt sources. The lowest MIC reported was for the Co(II) complex against A. niger (15 µg⋅mL -1 ) and C. albicans (16 µg⋅mL -1 ) which approached the MIC values of the three positive controls (nystatin: 12 and 16 µg⋅mL -1 ; KTZ: 14 and 15 µg⋅mL -1 ; CTZ: 10 and 8 µg⋅mL -1 against A. niger and C. albicans, respectively).

Salicylaldehyde-based ligands

Salicylaldehydes are a commonly used carbonyl in the synthesis of Schiff bases, likely due to their own bioactivity and the stabilising effect that conjugation between the azomethine nitrogen and the aromatic core provides. A study of binuclear Ru(III) complexes coordinated by bis(salicylaldehyde) ligands bridged by a range of flexible and rigid spacers was reported but the greatest inhibitory effects against A. flavus, F.

oxysporum, and R. solani were 19% (Fig. 51). 153 Schiff bases formed between salicylaldehyde, aniline and o-/m-/p-toluidine were cmplexed with Ru(II) in mixed ligand systems (Fig. 52A). The resultant compounds were tested against A. flavus at concentrations of 100 and 200 ppm with Bavistin as a positive control. The antifungal results of only four complexes and the free ligands were reported, with the highest achieved inhibition was only 65%. 154 Similar salicylaldehyde-based compounds that differed by the inclusion of methylamine, cycloamine or 2aminopyridine were complexed with Ru(III) ions to form mixed ligand systems with triphenyl arsane/phosphane and halogen ions (Fig. 52B). The compounds were tested against A. flavus and Flusarium spp. at concentrations of 100 and 200 ppm. Similarly, only a subset of the antifungal measurements were reported, but the compounds achieved up to 76% inhibition of F. spp. and 68% of A. flavus. 155 A mixed-ligand Cu(II) complex was synthesised with Schiff base salicylalanine derivatives, phen, and dodecylamine (Fig. 53A). Characterisation of the complex included determination of its micelle forming properties, interactions with DNA, and antimicrobial activity. 156 The two fungal strains used in the agar well diffusion studies were A. and C. albicans. Interestingly, after incubation of C. albicans with 3 mg⋅mL -1 solutions of the complex for 24 and 48 h, the inhibitory diameter exceeded that of the positive control (KTZ). For the treatment of A. the inhibition zone diameter approached the positive control (25 ± 0.54 and 29 ± 0.58 mm for 24 and 48 h, respectively, compared to 30 ± 0.58 mm for KTZ after 24 h -48 h measurement not reported). Potentially, the results could be due to the combined effect of the different complex components used, which were explained to have been chosen for the bioactivity of Schiff bases, copper ions and phen as well as the surfactant properties of dodecylamine. similar Schiff bases and phen (Fig. 53B). 157 The two synthesised Schiff bases differed by the paraaminobenzene species used (either chloro or nitro) that were condensed with N-acetylaceto-o-toluidine. The free ligand and metal complexes (Cu(II), Ni(II) and Zn(II)) were characterised by a range of spectroscopic and spectrometric techniques and the ligand was determined to have NNNN coordination via two azomethine nitrogen atoms and two phen ligands. The chloro and nitro Cu(II) complexes were the most potent against all organisms against five strains of fungi. The MIC values of these complexes approached or reached the MICs of FCZ. Such results showed promise for the use of the metal complexes to treat fungal infections, while at the same time raising questions about their modes of action. The authors did not address the biochemical possibilities behind the observed differences between the metal ions used, however, the redox properties of the Cu(I/II) couple could provide additional fungicidal effects through the generation of ROS. 55A). 159 The complexes differed by their ancillary ligands, which depended on the organotin precursor used, e.g., dimethyl, dibutyl, diphenyl, dioctyl Sn(IV) halides and oxides. The compounds were characterised by standard analytical methods: IR spectroscopy, mass spectrometry, 1 H and 13 C, as well as 119 Sn NMR spectroscopic measurements.

Single crystal structures of the free ligand, dimethyl, and diphenyltin(IV) complexes were also reported. The structures confirmed that the Schiff base acted as a tridentate ONO ligand, coordinating via the phenol oxygen, azomethine nitrogen and enolized aldehyde. The metal complexes and the parent ligand were tested against six fungal strains via agar tube dilution. The Schiff base ligand alone showed significant levels of activity against F. solani and M. canis with approximately 65 and 90% inhibition, respectively (relative to the positive control). Against the same strains of fungi, complexation with the organotin species decreased activities significantly. For most other tin complexes studied, however, complexation of the Schiff base with the metal resulted in an observable increase of the fungal inhibition. The most significant activity enhancement, relative to the free ligand, was observed for the dibutyl, diphenyl, and dioctyltin(IV)-Schiff base complexes against A. flavis, which resulted in approximately 90, 80 and 85% increases in efficacy, respectively.

While organotin complexes have shown promising antifungal activity, the inclusion of a heavy metal in the drugs raises concerns regarding mammalian toxicity of the compounds as well as tin-based metabolites leaching into the environment, particularly in agricultural or veterinary fungal treatments. Sharma et al. developed a series of linked di-indole ligands that differed by the bridging moiety (Fig. 56B). 163 The ligands chelated M(III) ions (M = Ru, Rh, Ir) through the carbonyl oxygen and azomethine groups. At a concentration of 1000 ppm, all metal complexes achieved at least 60% inhibition of A. niger and F. oxysporium against the three tested fungal strains. 164 Additionally, the Cu(II) complex was also found to have superior antioxidant activity compared to the other five complexes and more than double that of the free ligand.

Ferrocenyl-containing ligands

As has been mentioned above, improved drug bioactivity can sometimes be achieved by the inclusion of ferrocene moieties into drug molecules 165 as the ferrocene motif possesses its own fungicidal activity. In this vein, many groups have sought to complex Schiff-functionalised ferrocenyl moieties with metal ions.

Mahmoud et al. condensed L-alanine with 2-acetyl ferrocene and complexed a range of first row transition metal ions (Fig. 58A). It was found that more than 70% of the compounds had no antifungal activity when tested against A. fumigatus and C. albicans by zone of inhibition experiments. 166 However, of the 30% of compounds that elicited a fungicidal response, the Co(II) complex exceeded the positive control (KTZ) -20 mm⋅mg -1 vs. 17 mm⋅mg -1 , respectively -while the Cd(II) complex achieved an inhibitory zone equal to that of KTZ. In another report, the same group synthesised, characterised and tested mixed ligand ferrocenylsalicylaldehyde (L, Fig. 58B), phen first row transition metals complexes. 167 One example refuting the theory that metal chelation of an organic species improves bioactivity was found in the work of Abou-Hussein et al. 168 Mono and diacetyl ferrocene species were condensed with oaminothiolbenzene (Fig. 58C) in either 1:1 or 1:2 molar ratios, depending on the ferrocenyl species used.

When tested against F. oxysporium, both free ligands possessed appreciable antifungal activities, however, all ligand-metal complexes with Ru(III), V(IV) or U(VI) showed no improvement in fungicidal response or a decrease in activity.

A very similar o-aminophenol-monoacetyl ferrocene Schiff base ligand (Fig. 58D) complexed with first row transition metal ions and Cd(II) elicited more significant fungicidal responses. 169 The compounds were tested against four fungal and four bacterial strains in disk diffusion experiments, with AmB as positive control against the fungi. The Cu(II) complex displayed significant antifungal activities against A. fumigatus, S.

racemosum and G. candidum by reaching or exceeding the inhibition zones of AmB. Other promising results

were recorded for the Fe(III), Co(II) and Zn(II) complexes which all approached the positive control result against the tested fungi. None of the tested complexes had considerable effect against C. albicans, relative to AmB.

A diacetyl-ferrocene-ethanolamine ligand (Fig. 58E) was coordinated to Co(II), Cu(II), Ni(II) and Zn(II) and tested against the six strains of fungi. 101 The Zn(II) metal complex performed best with zone of inhibition areas approaching those of both MCZ and AmB (except against C. albicans). Against C. albicans none of the compounds displayed significant inhibitory effects, relative to the positive controls.

Condensation of diacetylferrocene with 2-furoic/thiophene/salicylic hydrazide (Fig. 59) yielded a family of Schiff base ligands which were used to form metal complexes with Co(II), Ni(II), Cu(II) and Zn(II). 170 A total of 15 compounds (including the free ferrocene-containing ligands) were tested against T. longifusus, C. albicans, A. flavus, M. canis, F. solani, C. glabrata for antifungal activities. Most metal complexes performed slightly better than their parent ligands with some displaying inhibition values greater than 70%. However, the Co(II)salicylaldehyde complex achieved 96% inhibition of C. albicans relative to AmB.

Miscellaneous Schiff bases

Coumarin-based ligands appended with heterocycles (pyrimidine, triazine, thiazole, thiadiazole) and hydrazine(thio)carbamides (Fig. 60) via Schiff base condensation were complexed with first row transition metals and tested against six pathogenic fungi. 171 When tested against C. albicans, the thiazole-Co(II) complex had an inhibition zone greater than MCZ and 96% inhibition relative to AmB. Against A. flavus, the Zn(II) complexes of the hydrazinecarboxamide-coumarin and thiazole-coumarin derivatives had the greatest inhibitory effects. Against M. canis, all complexes with the hydrazidethiocarboxamide derivative as a ligand had >80% inhibition and a similar result was observed for the pyrimidine-Zn(II), Ni(II) and Cu(II) complexes. Coordination of Ru(III) with bis(aminophenol), bis(aminothiophenol)glyoxal or bis(aminophenol)benzyl (Fig.

61A

) was investigated and the antifungal efficacies of the resultant complexes tested against Fusarium spp.

at 200 and 150 µg⋅mL -1 . 172 Unfortunately, no complex/ligand tested recorded greater than 22% inhibition of the organism. No positive control drug was stated in the article and percent inhibition was only determined relative to a 'control plate'. to 4-7 µg⋅mL -1 ). However, the metal complex MIC values were still four to five times higher than the positive control (nystatin).

Schiff bases formed between substituted aromatic aldehydes and ethanolamine (Fig. 62C) and subsequently complexed with Co(II), Cu(II), Ni(II) and Zn(II) were tested for antifungal properties against T. longifusus, C. albicans, A. flavus, M. canis, F. solani, and C. glabrata. 176 In general, all metal complexes with the bromo-and nitro-derivatised ligands had greater antifungal responses than the naphthyl and salicylaldehyde compounds.

This was reflected in the average zone of inhibition distances (averaged over all experiments with a particular compound), which were ∼19-22 mm for the bromo/nitro complexes and 15-17 mm for the napthyl/salicylaldehyde complexes. The standard drugs had inhibitory zones greater then 25 mm.

Indandione condensed with methionine (Fig. 62D) was reported to have MIC values ranging from 9.6 µg⋅mL -1 against C. albicans to 12.5 µg⋅mL -1 against A. flavus. 177 However, when complexes were formed with Co(II), Cu(II), Ni(II) or Zn(II) the MIC values against the four tested fungal strains increased, indicating they were less toxic against the organisms. FCZ was measured to have inhibitory concentrations between 6.1 and 9.4 µg⋅mL -1 .

Condensation of 2'-methylacetoacetoanilide with 2-amino-3-hydroxypyridine yielded an ONO chelating ligand (Fig. 62E). 178 Subsequent complexation with Co(II), Ni(II), Cu(II) or Zn(II) and determination of MIC values against A. niger, R. bataicola, R. stolonifer, and C. albicans showed that, while the metal coordination improved the antifungal effect, the resulting MIC values were at least 10-40 times higher than nystatin.

A macrocyclic compound formed through Schiff condensation of 2,4-diaminotoluene and benzil (Fig. 63A)

was coordinated with Mn(II), Fe(II), Co(II) or Ni(II). 179 Testing against C. albicans by zone of inhibition experiments found that the Mn(II) and Fe(II) complexes approached the positive control, gentamycin, achieving ∼80% inhibition of organism growth. The Co(II) and Ni(II) compounds reached between 60 and 70% inhibition. A series of macrocyclic NNNN and NNOO donor ligands with different bridging spacers (Fig. 63B) were coordinated to Ru(II) ions and tested against A. flavus and Fusarium spp. at 0.5 and 1.0 mg⋅mL -1 by zone of inhibition experiments. 180 The free ligands all had moderate inhibitory effects and increased as a result of Ru(II) complexation. Despite this, no compound achieved significant inhibition relative to the positive controls, AmB or Bavistin. Similarly, a range of phenyl-pyrazoyl TSC derivatives, differing by the TSC-terminal N-substituent (Fig. 65B)

were complexed with Cu(II) ions to give compounds with general formula of [Cu(L)Br 2 /Cl 2 ], depending on the metal salt source used. 185 The antifungal activities of these complexes against A. niger and P. variotii were determined by zone of inhibition experiments. Against both organisms, most of the bromo/chloro Nderivatives performed equally as well as their parent ligand or otherwise had decreased inhibitory effects.

However, the Cu(II)-piperidinyl chloro derivative had a slight increase in inhibitory effect against A. niger (∼1-2 mm), and P. variotii (∼5-10 mm). Conversely, inclusion the bromo ligand in the complexes resulted in decreased inhibition against both organisms. solution of the complex, the zones of inhibition ranged from 6 to 18 mm for three of the organisms; no measurements were reported for A. niger. When tested against P. notatum the Ru(II)-nitro derivative approached the positive control, griseofulvin, with ∼85% inhibition. Additionally, the hydroxyderivative achieved ∼75% inhibition against Colleotrichum while all other fungal strains were only inhibited by ∼60%. 86%, respectively and also displayed significant antibacterial properties, inhibiting all bacterial strains by 85-96%.

Heteroaromatic TSC ligands

Mono and di-coordinated titanocene/zirconocene complexes of 2-acetylpyridine TSC (Fig. 68A 2-acetylpyridine N-diethyl TSC (Fig. 68B) chloro/bromo Cu(II) complexes tested against seven fungal organisms by disk diffusion yielded inhibition zones ranging from 9 to 17 mm. 192 No inhibition zones for the positive control (nystatin) were reported, however, against various organisms, identical MIC values were recorded for both the bromo complex and nystatin (31.5-62.5 µg.mL -1 ).

2-Acetylpyridine and 2-benzoylpyridinyl TSC (Fig. 68B) used as ligands to form organo Sn(IV) complexes from n-butylSnCl 3 managed to approach, and exceed, some MIC values of the positive control against Candida spp.

(C. albicans, krusei, glabrata, and parapsilosis). 193 Of note were the pyridinyl-4-orthonitro phenyl complex which had an MIC against C. glabrata that was half that of FCZ (5.16 µM vs. 21.32 µM) and the pyridinyl-4orthofluoro phenyl complex which had similar potency (3.74 µg mL -1 , i.e. 6.28 µM). Given the relatively high MIC of FCZ against C. krusei (104.5 µg mL -1 0.341 mM), all complexes and free ligands tested were found to be more potent against this organism, despite the values ranging from 6.32 µg mL -1 to 53.68 µg mL -1 (20.64 µM to 175.27 µM).

A 3-acetylpyridine 4N-(2-pyridyl) TSC ligand (Fig. 69) was complexed with Cu(II), Hg(II), Ni(II), UO 2 (IV), VO(IV)

and Zn(II) and tested against 'yeast', as reported by the authors. 194 The free ligand, Cu(II), Hg(II), and Ni(II) complexes had zones of inhibition ranging from 18 to 28 mm, with the Hg(II) and Ni(II) being the most potent.

experiments (bacterial and fungal). As such, compared with the positive control, all complexes had equal or greater fungal zones of inhibition. Quinoline-TSC ligands with a terminal N-methyl or pyridinyl group were complexed with a range of divalent first row transition metal ions (Fig. 71A). 197 The ligand coordinated all metal ions as a tridentate ONS ligand, however, the pyridinyl derivatives was identified to form binuclear species where the tautomerized TSC-thiol formed a µ 2 -bridge between two metal centres. Against both evaluated fungal organisms (A. niger and Cladosporidium spp.), the Cu(II)-quinoline TSC-methyl derivative outperformed FCZ in disk diffusion experiments. Similarly, the Cu(II)-pyridinyl complex performed best for that class of ligands, although it did not exceed the positive control.

For a thiophene-2,3-bis(TSC) ligand (Fig. 71B the free ligand. Conversely, against S. cerevisiae, the same complex achieved ∼91% inhibition over the same time period, a 15% increase on the efficacy of the free ligand (76%). All percentages were calculated relative to an unspecified control.

A tetraazamacrocyclic ligand comprising an ortho carbonyl hydrazide and indole-dione was complexed with Co(II), Ni(II) and Cu(II) ions (Fig. 72C). 201 When tested against C. albicans, the Cu(II) macrocyclic chloro complex exceeded AmB (14.8 vs. 12.5 mm inhibition zone) and the equivalent Ni(II) complex exceeded the positive control against S. cerevisiae (14.2 vs. 13.6 mm).

A benzoyl-pyridinyl hydrazide in complex with Ni(II) (Fig. 73A) recorded an inhibitory zone of 29 mm after 48 h incubation with C. albicans, far in excess of the AmB control (21 mm). 202 The measurement was recorded at a concentration of 50 µg complex per disk, however, doubling of the concentration to 100 µg per disk resulted in an approximate halving of the inhibitory zone; all experiments were conducted over 24 and 48 h.

Strangely, for some of the complexes (including Co(II) and Cu(II) complexes) at the given concentrations, the initial 24 h measurement appeared significant, only to decrease at the 48 h time point. Whether this was a result of complex stability or perhaps an initial fungistatic inhibition which the organism could overcome was not addressed. A substituted formazan Cr(II) complex reported by Khan et al. (Fig. 73B) was found to have high activity against five fungal strains. 203 In disk diffusion studies, the complex reached zones of inhibition ranging from 29 ± 0.09 mm against C. albicans and up to 39 ± 0.01 mm against A. niger.

Benzoyl and salicylylhydrazones (Fig. 73C) in complex with Mn(II), Co(II), Ni(II) and Cu(II) were synthesised and characterised by Deepa et al. 204 Not only were DMSO solution of the complexes more active fungicidal agents than the parent ligands at 500 ppm, many of the complexes recorded zones of inhibition >25 mm against four strains of pathogenic fungi. Whether the 10 mm diameter of the holes bored into the agar plates was subtracted from the reported values, however, was not specified. Additionally, the DMSO negative control recorded between 10-14 mm inhibition zones for all organisms.

Miscellaneous NO donor ligands

A quinolinol-benzotriazole ligand in complex with Cu(II), Ni(II), Co(II), Mn(II) and Zn(II) (Fig. 74A) was found to inhibit the growth of seven fungal strains at 1000 ppm. 205 Most compounds were found to inhibit growth from 63 to 89% relative to an unspecified positive control. The free ligand was also found to have significant antifungal properties with 70% inhibition of C. glabrata. The Zn(II) complexes of a methoxy and diol phenyl-triazole Schiff base (Fig. 74B) were found to be effective against all fungal strains tested with percent inhibitions ranging from 73-82% for the methoxy derivative and 59-76% for the diol derivative. 206 Complexes with other first row transition metals (Cu(II), Ni(II), Co(II), Mn(II), Zn(II)) were also tested and recorded inhibition of fungal growth approaching 70%, relative to MCZ.

A study by Madzivire et al. investigated a range of Ru(III) mono-substituted ketoiminates complexes (Fig. 75A) and found that, for the 2-ethoxyethanol derivative, the metal centre was reduced to Ru(II). The ketoiminato carbonyl complex that formed was suggested to be the active compound and could possibly act as a carbon monoxide releasing molecule (CORM). 207 While selectivity towards C. albicans over other fungal strains was identified, only an inhibitory effect of ∼44% was determined.

Carboximide-based complexes

Relatively few papers concerning the antifungal properties of carboxamide metal complexes (Fig. 76) have been reported and, unfortunately, several of the articles reported don't include positive controls for relevant comparison of results. Similarly, the 4-bromofuran derivatives approached 70% inhibition against C. glabrata for all metal complexes. 210 Pyridine derivatised carboxamide ligands (Fig. 76C) complexed with Cu(II), Co(II) and Zn(II) at a concentration of 10 µg⋅mL -1 were found to reduce the growth of F. calmorium by only 28% relative to the DMSO negative control. 211 No positive control was reported.

A coumarin-based carboxamide (Fig. 76D) complexed with a range of M(II) ions showed no activity against C.

albicans except for the Co(II) and Ni(II) complexes, which approached and exceeded the positive control, KTZ, Sn(cyclohexane) 3 Cl. 216,217 The Sn(IV)-derivatives were tested for antifungal activities against C. albicans by agar well diffusion with nystatin as positive control (concentration used was ∼22 mM). Most complexes were required to be dissolved in a mixed solvent system (dichloromethane/pentane) and were tested at a range of concentrations (0.25 mM to 3.2 mM, with two-fold dilutions). The n-butyl Sn(IV) complex exceeded the positive control zone of inhibition at a concentration of 0.4 mM (approximately 50 times more dilute) and the Sn(IV) chloride complex exceeded the positive control by 5.5 mm at a concentration of 1.6 mM. While effective in the inhibition of C. albicans, the use of Sn(IV)-derived drugs raises concerns for environmental pollution and mammalian toxicity. A series of ferrocene-derived thiocarbamate species (differing by naphthyl, pyridyl or furanyl pendant groups) were complexed with Co(II), Cu(II), Ni(II) and Zn(II) (Fig. 78C)) with coordination exclusively occurring through the sulfur donors. 218 Tetrahedral species were reported for the latter three metal ions, and an octahedral complex for the Co(II) ion. Antifungal efficacies were evaluated through determination of the complex and ligand MIC values against C. albicans and A. niger with FCZ as a positive control. All metal complexes were reported to have MIC values 5-20 times higher than the positive control, however, the Ni(II)

3-pyridyl complex achieved an MIC equal to that of FCZ for C. albicans and only 10 µg⋅mL -1 higher than for A.

niger.

Another dithiocarbamate appended with aliphatic groups (Fig. 78D) was synthesised and used to form tetrakis Ru(III) complexes. MIC values against seven different A. species were determined (AmB as positive control). 219 The lowest MIC values were reported for the dimethyl and diethyl Ru(III) complexes (4 µg⋅mL -1 , i.e. 4.98 µM) which was ∼15 times less potent than AmB (0.32 µM).

Comparable ruthenium pentakis dithiocarbamate complexes with similar aliphatic decoration (Fig. 78E) were tested against six species of Candida, P. bradiliensis, C. neoformans and S. schenkii by MICs determination. 220 While all Ru(III) complexes were more effective than the free ligand against the organisms, none approached the efficacies of FCZ or AmB.

Liu et al. synthesised a range of ferrocenyl-chalcone-dithiocarbamate Schiff bases metal complexes (Cu(II), Hg(II), Zn(II), Fe(II), Cd(II), Ni(II), Pb(II)) (Fig. 79). [221][222][223] All metal complexes (except Hg(II)) were determined to have NNOOSS coordination through the two azomethine nitrogen atoms, enolized thiols of the Schiff base ligand and two water molecules; the Hg(II) was found to have NNSS coordination, lacking the axial waters.

The antifungal effects of the synthesised complexes were tested against C. albicans, A. fumigatus, A. niger, A. flavus, and S. cerevisiae. All metal complexes had efficacies against the fungal strains of 50% or greater activity relative to nystatin. While complexation of the metal ions appeared to generally increase the activities of the ferrocenyl Schiff bases, the free ligands also had appreciable fungicidal properties, likely due to the organometallic nature of the ferrocene ligands. Of the myriad complexes tested, the three Zn(II) derivatives containing the phenyl, para-phenyl-dimethylamine, and para-phenyl-bromo groups showed the greatest efficacies against all fungal strains tested (∼80% relative to positive control). articles reporting murine-based experiments are disappointingly scarce, and as without those the true efficacies of these compounds cannot be fully ascertained. Such highly needed experiments will allow for the accurate assessment of the potential of these metal-based compounds to help fighting the global societal and economic burdens of pathogenic fungal infections.

Kevin

  Cariou graduated from Chimie ParisTech in 2002, he received his PhD in 2006 from the University Pierre and Marie Curie (now Sorbonne Université) in Paris (France) under the supervision of Prof. M. Malacria and L. Fensterbank, where he studied platinum-and gold-catalysed transformations with a joint CNRS-Sanofi BDI fellowship. From 2007 to 2009, including one-year as a Lavoisier fellowship holder, he worked as a postdoctoral researcher in the group of Prof. A. J. Frontier at the University of Rochester (NY, USA) in the field of total synthesis. He was appointed as Chargé de Recherche in 2009 at the CNRS at the ICSN in the team Synthesis and Methodology Applied to Research in Therapeutics (SMART) led by Dr R. H. Dodd. He obtained his Habilitation à Diriger les Recherches (HDR) in 2015. From 2017 to 2019, he was the leader of the SMART team before moving to Chimie ParisTech in 2020. His research interest lie in the development of new synthetic methods, with a focus on I(III) reagents, nitrogen-rich building blocks and organometallic derivatives, and their application toward the synthesis of biologically active molecules, in particular antiinfectious.

  albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, C. kefyr and C. gulliermondii), Aspergillus (A. flavus, A. niger and A. flumigatus), Cryptococcus (C. neoformans), Fusarium (F. oxysporum, F.

Fig. 3 :

 3 Fig. 3: Structures of clinically approved imidazole-based antifungal drugs.

Fig. 5 :

 5 Fig. 5: Ferrocenyl 1,2,4-triazole-containing derivatives assessed for antifungal activity.

Fig. 6 :

 6 Fig. 6: Ferrocenyl thiazole-containing derivatives assessed for antifungal activity.

Fig. 7 :

 7 Fig. 7: Ferrocenyl pyrazole-containing derivatives assessed for antifungal activity.

55

 55

Fig. 8 :

 8 Fig. 8: Ferrocenyl azole-containing derivatives assessed for antifungal activity.

Fig. 9 :

 9 Fig. 9: Ferrocenyl and silatrane-containing azole derivatives assessed for antifungal activity.

Fig. 10 :

 10 Fig. 10: Ferrocenyl azole-containing derivatives assessed for antifungal activity.

Fig. 11 :

 11 Fig. 11: Ferrocenyl azole-containing derivatives assessed for antifungal activity.

from 2 .

 2 5 to 5 mg⋅mL -1 , as determined by MIC measurements. The Cu(II) and Co(II) complexes exhibited better activity than the Ni(II) complex against C. albicans with MIC values of 2.5 mg⋅mL -1 , no positive controls were reported.62

Fig. 12 :

 12 Fig. 12: Metal-based thiadiazole-containing complexes assessed for antifungal activity.

Fig. 13 :

 13 Fig. 13: Organoruthenium complexes containing CTZ, TCZ and MCZ assessed for antifungal activity.

Fig. 14 :

 14 Fig. 14: KTZ-and CTZ-based metal complexes assessed for antifungal activity.

Fig. 15 :

 15 Fig. 15: KTZ-and CTZ-based metal complexes assessed for antifungal activity.

72 Fig. 16 :

 7216 Fig. 16: Ruthenium-cyclopentadienyl complexes assessed for antifungal activity.

  and [Ag 2 (phen) 3 (mal)]⋅2H 2 O. The Mn(II), Cu(II) and Ag(I) complexes displayed remarkable inhibition of C. albicans with MIC values ranging from 1.25 to 2.5 µg⋅cm -3 . By comparison, the MIC values increased progressively from the Zn(II) complexes (>5 µg⋅cm -3 ) and the Co(II), Ni(II) complexes (20 µg⋅cm -3 ). The MIC values of the different Ag(I) complexes, Ag(CH 3 COO), [Ag 2 (mal)] and [Ag 2 (phen) 3 (mal)]⋅2H 2 O were similar, indicating that the active species was likely the Ag(I) cation itself. To understand the difference between the effective complexes (Mn(II), Cu(II) and Ag(I)) and ineffective complexes (Zn(II), Co(II) and Ni(II)), [Cu(phen)(mal) 2 ]⋅2H 2 O and [Zn(phen)(mal) 2 ]⋅2H 2 O were chosen to represent each group in oxidative stress studies. Reduced glutathione (GSH) is known to be an important antioxidant molecule in yeast cells, and the ratio to oxidized glutathione (GSSG) can give valuable insights into the ability of a compound to induce oxidative stress. The Cu(II) complex induced significant cellular oxidative stress, as shown by decreased ratios of reduced and oxidized glutathione (GSH/GSSG) = 5:1

Fig. 17 :

 17 Fig. 17: Polypyridine metal complexes assessed for antifungal activity.

[

  CuL(phen) 2 ]Cl 2, which stabilizes the duplex (GC pairs) through intercalation. No reversible redox process was found in the Zn(II) complex. The DNA cleavage abilities of these metal complexes in the presence of H 2 O 2 were determined by the conversion of supercoiled DNA to nicked DNA and linear DNA. These observations were likely the result of ROS production. The antifungal activities of the isatin-based ligand and its metal complexes were screened against five fungal strains (A. niger, F. solani, C. lunata, R. bataticola, C. albicans).

Fig. 18 :

 18 Fig. 18: Polypyridyl metal complexes assessed for antifungal activity.

Fig. 19 :

 19 Fig. 19: Polypyridyl metal complexes assessed for antifungal activity.

3 .

 3 Complexes 68 and 71 both showed significant inhibition of C. albicans (ATCC 10231) with MIC values of 31.25 µg⋅mL -1 . Additionally, complex 71 was also found to be relatively non-toxic against human fibroblasts MRC-5 (IC 50 = 70 µg⋅mL -1 ). C. albicans filamentation and biofilm formation in the presence of complexes 67-71 at subinhibitory concentrations were also investigated. Complexes 67 and 70 almost completely inhibited the formation of fungal hyphae and efficiently inhibted biofilm formation. The affinities of these complexes with bovine serum albumin and CT-DNA were confirmed by fluorescence spectrophoresis or gel electrophoresis.88

Fig. 20 :

 20 Fig. 20: Polypyridyl metal complexes assessed for antifungal activity.

Fig. 21 :

 21 Fig. 21: Polypyridyl metal complexes assessed for antifungal activity.

4 .

 4 Amino acid-based metal complexes In 2006, Chohan et al. reported a series of amino acid-derived ligands (L 1 -L 5 ) and their Co(II), Cu(II), Ni(II), and Zn(II) metal complexes with the general formula[M(L) 2 (H 2 O) 2 ] (74) and [M(L)(H 2 O) 4 ]Cl (75, Fig.22). Ligands (L 1 -L 5 ) were synthesised by condensation of β-diketones with the respective amino acid (e.g., glycine, phenylalanine, valine and histidine). The bidentate ligands complexed the metal ion via the azomethine nitrogen and deprotonated oxygen, adopting octahedral geometries. Remarkable increases in antifungal activities upon metal ion coordination against one or more tested fungal strains (M. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glaberata) were reported. The increase in the activity of the metal complexes was explained on the basis of increased lipophilicity of the metal complexes relative to the metal ions or ligands alone, possibly allowing for increased cellular uptake. The cytotoxicities of these complexes were determined using a brine shrimp bioassay with all metal complexes being less cytotoxic than the metalfree ligands.90

Fig. 22 :

 22 Fig. 22: Amino acid-based metal complexes assessed for antifungal activity.

Fig. 23 :

 23 Fig. 23: Amino acid-based metal complexes assessed for antifungal activity.

  mucedo, T. viride, A. niger, C. albicans and P. italicum). The ferrocenyl chalcones were found to have moderate antifungal activities with the MIC values ranging from 0.312 to 10 mg⋅mL -1 . The C. albicans strain was the most sensitive to these compounds, while A. niger was the most resistant.97

Fig. 24 :

 24 Fig. 24: Chalcone-based metal complexes assessed for antifungal activity.

Fig. 25 :

 25 Fig. 25: Sulfonamide-based metal complexes assessed for antifungal activity.

Fig. 26 :

 26 Fig. 26: Sulfonamide-based metal complexes assessed for antifungal activity.

Fig. 27 :

 27 Fig. 27: Sulfonamide-based metal complexes assessed for antifungal activity.

Fig. 28 :

 28 Fig. 28: Sulfonamide-based metal complexes assessed for antifungal activity.

Fig. 29 :

 29 Fig. 29: Metal complexes based on heterocycles assessed for antifungal activity.

Fig. 30 :

 30 Fig. 30: Binding equilibrium of the Cu(I/II) couple with CT-DNA.

Fig. 31 : 116 Fig. 32 :

 3111632 Fig. 31: Heterocyclic metal complexes assessed for antifungal activity.

Fig. 33 :

 33 Fig. 33: Heterocyclic metal complexes assessed for antifungal activity.

- 1 .

 1 Compound 102d, which contained a para-chloro group on the phenyl ring, showed the highest antifungal activity with MIC values of 8 µg⋅mL -1 against C. dubliniensis and C. tropicalis. 120

Fig. 34 :

 34 Fig. 34: Heterocyclic metal complexes assessed for antifungal activity.

Fig. 35 :

 35 Fig. 35: Heterocyclic metal complexes assessed for antifungal activity.

Fig. 36 :

 36 Fig. 36: Heterocyclic metal complexes assessed for antifungal activity.

7 .

 7 β-diketone/dithione-based metal complexesIn 2002, hexacoordinated binuclear Ru(III) complexes with the general formula {[RuX 2 (EPh 3 ) 2 ] 2 (bis-β-dk)} (X = Cl or Br; bis-β-dk = thiobis(β-diketone)) (107, Fig.37) were prepared by Natarajan and co-workers. The antifungal activities of these complexes were screened in vitro against A. flavus, F. oxysporium and Rhizoctonia solani. Under identical experimental conditions, the ruthenium complexes displayed two-fold greater activities than the parent ligands, but were still less effective than the standard drug, Bavistin.125 In 2009, Jayabalakrishman and co-workers prepared a series of binuclear Ru(II) complexes containing bis-βdiketones (108, Fig.37). The binuclear Ru(II) complexes showed two to three-fold higher activities against C. albicans and A. niger than the free ligands or metal salts alone. The improved antifungal activities on the metal complexes were attributed to increased lipophilicity and improved penetration of the cellular membrane by the compounds.126 In 2006, Chohan and co-workers investigated the antifungal activities of ferrocene derivatives incorporating dithiothione or dithioketone moieties (109, Fig.37) against M. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glabrata. The ferrocene derivatives exhibited excellent inhibitory effects with inhibition zone ranging from 24 mm to 32 mm against all tested fungal species. The values were similar or greater than standard drugs, MCZ and AmB, which had inhibition zones of 20-30 mm.127

Fig. 37 :

 37 Fig. 37: Metal complexes based on β-diketone/dithione were assessed for antifungal activity.

Fig. 38 :

 38 Fig. 38: Metal complexes based on β-diketone/dithione were assessed for antifungal activity.

Fig. 39 )

 39 Fig.39). Evaluation of the antifungal activities of the series of complexes against C. albicans and C.

Fig. 39 :

 39 Fig. 39: Metal complexes based on guanidine assessed for antifungal activity.

  decane (DASD) (114a-i, Fig. 40) against three yeast strains (C. albicans, C. krusei and C. tropicalis). Six out of nine compounds were found to be more active against C. albicans than the standard drug, KTZ. In addition, compounds 114c and 114f (MFC ≤19.5 µM) were 7-fold more active than KTZ against C. krusei and C. tropicalis (MFC ≥153

Fig. 40 :

 40 Fig. 40: Metal complexes based on cyclotetraphosphazene assessed for antifungal activity.

Fig. 41 :

 41 Fig. 41: Metal complexes based on urea assessed for antifungal activity.

  Chohan and co-workers used bis-(1,1'-disubstituted ferrocenyl)thiocarbohydrazone and bis-(1,1'disubstituted ferrocenyl)carbohydrazone as ligands to chelate Co(II), Ni(II), Zn(II), and Cu(II). The Co(II), Ni(II),and Zn(II) complexes had the general formula [M(L)Cl 2 ] and adopted distorted octahedral geometries, while the Cu(II) which formed a [Cu(L)]Cl 2 complex with square-planar geometry (119a-h, Fig.42). All compounds were tested against six fungal strains (M. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glaberata).

Fig. 42 :

 42 Fig. 42: Various metal complexes assessed for antifungal activity.

Fig. 43 :

 43 Fig. 43: Transition metal complexes derived from AEPD assessed for antifungal activity.

Fig. 44 :Fig. 45 :

 4445 Fig. 44: Salicylaldehyde amino acid Schiff base ligands and coordination complexes formed by various first-row transition metals.

Fig. 46 :

 46 Fig. 46: Oxovanadyl complexes with triazole-pyridinyl and -pyrrole derivatised Schiff base ligands.

Fig. 47 :

 47 Fig. 47: Triazole-containing Schiff based ligands complexed with a range of metals (not shown) and assessed for antifungal activity.

Fig. 48 :

 48 Fig. 48: Pyrazoyl-diamino/aminophenol ligands complexed with V(IV), Fe(III), Co(II), and Cu(II).

Fig. 49 :

 49 Fig. 49: Mixed ligand complexes formed between a range of transition metal ions and Schiff base-type ligands.

Fig. 50 :

 50 Fig. 50: Mixed 2,2'-bipy/pyrazoyl Mn(IV) complexes, derivatised with glycine, ethylenediamine and ethanolamine.

Fig. 51 :

 51 Fig. 51: Schiff bases, based on salicylaldehyde, which were coordinated to a range of metals (not shown) and assessed for antifungal activity.

Fig. 52 :

 52 Fig. 52: Ru(II/III) complexes formed between salicylaldehyde-Schiff base derivatives.

Fig. 53 :

 53 Fig. 53: Mixed ligand complexes formed between synthesised Schiff base ligands and 1,10-phen.

Fig. 54 :

 54 Fig. 54: Schiff bases complexed with a range of metals (not shown) and assessed for antifungal activity.

Fig. 55 :

 55 Fig. 55: A Complexes formed between a bromosalicylic hydrazine Schiff base ligand and various organo Sn(IV) sources. B Complex formed between Ln(III) ions and a threonine-bromosalicylic Schiff base ligand; structure not displayed in original article by Logu et al., but was drawn by the authors of this review from the originally reported spectroscopic data.

Fig. 56 :

 56 Fig. 56: Indole-containing Schiff base ligands which were coordinated to a range of metals (not shown) and assessed for antifungal properties.

after 96 h

 96 incubation, as determined by poison agar experiments. F. oxysporium exhibited a higher susceptibility to the tested complexes. All compounds displayed a dose-dependent response as the concentration was increased from 100 to 1000 ppm. Indole/quinoline-based Schiff base Cu(II) complexes were reported by Karekal et al. (Fig. 57) and found to have MIC values similar to FCZ against C. oxysporum and A. niger (12.5 µg⋅mL -1 ), but not C. albicans (25 µg⋅mL -1 ). Other compounds (including Co(II), Cd(II), Ni(II), and Hg(II) complexes) had similar MIC values

Fig. 57 :

 57 Fig. 57: Postulated complex geometry formed between the indole-quinoline derived Schiff base ligand and various divalent first-row transition metal ions.

Fig. 58 :

 58 Fig. 58: Ferrocene-containing Schiff base ligands complexed with a range of metals (not shown) and assessed for antifungal properties.

Fig. 59 :

 59 Fig. 59: Dicarbohydrazone ferrocene Schiff bases derivatised with various aromatic groups and complexed with first row transition metal ions.

Fig. 60 :

 60 Fig. 60: Coumarin-based Schiff base ligands which were coordinated to a range of metal ions -Ni(II), Zn(II), Cu(II) -and assessed for antifungal activity.

Fig. 62 :

 62 Fig. 62: Schiff base ligands complexed with metal ions (not shown) and assessed for antifungal activity.

Fig. 63 :

 63 Fig. 63: Macrocyclic tetradentate N 4 and N 2 O 2 Schiff base ligands complexed with metal ions (not shown) and assessed for antifungal activity.

Fig. 64 :

 64 Fig. 64: Aliphatic TSCs complexed with a range of metals (not shown) and assessed for antifungal activity.

Fig. 65 :

 65 Fig. 65: Non-heterocyclic TSCs complexed with a range of metals (not shown) and assessed for antifungal activity.

Fig. 66 :

 66 Fig. 66: Derivatised aromatic TSC ligands complexed with divalent transition metals and tested for antifungal activities.

Fig. 67 :

 67 Fig. 67: Mixed ligand metal complexes with TSC and phen ligands.

Fig. 68 :

 68 Fig. 68: Heterocyclic TSC ligands complexed with a range of metals and assessed for antifungal properties.

Fig. 69 :Fig. 70 :

 6970 Fig. 69: Heterocyclic TSC ligands complexed with a range of metals and assessed for antifungal properties. Antifungal properties of tridentate (ONS) dehydroacetic acid-TSC Ru(II) mixed ligand complexes (Fig. 70A) reported by Kannan et al. were assessed by disk diffusion. 195 Against C. albicans and A. niger, no complex

)

  in complex with Ni(II), Cu(II) and Cd(II) ions, Alomar et al.reported MIC values ranging from 8-62 µg⋅mL -1 against C. albicans, C. glabrata, and A. fumigatus.198 While the positive control (AmB) was far more potent against the latter two organisms (0.125 and 0.5 µg⋅mL -1 , respectively), the Cd(II) complexes (either bromo or chloro, depending on the metal salt source) had the same MIC values as AmB against A. fumigatus (8.0 µg⋅mL -1 ).

Fig. 71 :

 71 Fig.71: Heterocyclic TSC metal complexes assessed for antifungal activity.

Fig. 73 :

 73 Fig. 73: Diazene/hydrazine-containing ligands complexed with a range of metals and assessed for antifungal properties.

Fig. 74 :

 74 Fig. 74: NO coordinating ligands complexed with a range of metals and assessed for antifungal properties.

Fig. 75 :

 75 Fig. 75: NO coordinating ligands complexed with a range of metals and assessed for antifungal properties.

Fig. 76 :

 76 Fig. 76: Carboxamide-based ligands complexed with a range of metals and assessed for antifungal properties.

Fig. 77 :

 77 Fig. 77: Thiocarbamide-containing ligands complexed with organo Sn(IV) compounds and assessed for antifungal properties.

Fig. 78 :

 78 Fig. 78: Dithiocarbamate-based ligands complexed with a range of metals (not shown) and assessed for antifungal properties.

Fig. 79 :

 79 Fig. 79: Dithiocarbamate chalcones derivatised with ferrocene and a range of aromatic functional groups and complexed with various metal ions.

21, Fig 12) adopted octahedral geometries

  al. introduced thiadiazoles into tetra-azamacrocycles to coordinate metal centres such as Ni(II), Co(II) and Cu(II). The Ni(II) and Co(II) complexes(20 and

	while the Cu(II) complex (22, Fig 12

  Navarro et al. reported a series of metal-based KTZ and CTZ derivatives (M = Au, Cu and Ru),66,67,68 these metal complexes displayed excellent activity against the epimastigote form of T. cruzi. Hence, in 2018 the same authors explored the inhibitory effects of five new metal-azole complexes [Cu(PPh 3 ) 2 (KTZ) 2 ]NO 3 (26a),

	[Cu(PPh 3 ) 2 (CTZ) 2 ]NO 3 (26b), [Au(KTZ) 2 ]Cl (27a), [Au(CTZ) 2 ]Cl (27b), and [Pt(KTZ) 2 Cl 2 ] (28a) and three
	previously reported metal-azole complexes, namely, [Pt(CTZ) 2 Cl 2 ] (28b), [Au(PPh 3 )(KTZ)]PF 6 (29a) and

  and Mn(II) complexes (49-51 and 53-54), indicating that the metal complexes were superior antifungal agents than the ligands themselves.

	Cytotoxicity studies demonstrated that Mn(II) complexes were more selective towards lung epithelial cells
	(A549) than fungal cells with the highest SI values (>236.40 to 48.19). Additionally, water-soluble
	[Ag 2 (phen) 3 (udda)]⋅3H 2 O (58) (uddaH 2 = undecanedioic acid) displayed a relatively high SI value of 56.11.
	Since biofilm formation is an important virulence factor for pathogenic fungi, as it can protect the organism
	from the host's immune response, further examination on potent metal complexes (GM-MIC values <10 µM)

on biofilm-growing cells were carried out. The Cu(II) complex

(47)

, Mn(II) complexes 51, 53 and 54 and Ag(I) complex 56 also exhibited significant inhibition on the growth of biofilm-growing cells with GM-MIC values less than 10 µM, the remaining metal complexes exhibited GM-MIC values ranging from 12.04 to 28.42 µM.

  The stability of the metal complexes in H 2 O/DMSO (95/5% v/v) solution were assessed via UV-Vis spectroscopy and showed that these compounds were stable for up to 36 h of incubation. Complex 61 displayed significant fungistatic and fungicidal activities against 21 clinical isolates of Candida, with MIC values ranging from 31.25 to 125 µg⋅mL -1 and MFC values ranging from 31.25

  Four fungal strains (A. fumigatus, S. racemosum, G. candidum and C. albicans) were treated with solutions of the metal complexes in zone of inhibition experiments. It was shown that all metal complexes were more potent antifungal agents than the free ligand. One standout result was that of the [Fe(III)(L)(phen)(H 2 O) 2 ]Cl complex which, across all four fungal strains, achieved the same inhibition diameter as AmB. Several of the other complexes approached the positive control, but none to the same extent.

Acknowledgements

The authors would like to acknowledge the artistic contribution of Marie Huynh who created the Graphical Abstract for this article.

awarded a China Scholarship Council to carry out her PhD studies under the supervision of Prof. Gilles Gasser at Chimie ParisTech, PSL University.

Funding Information

This work was financially supported by the Swiss National Science Foundation (Grant Sinergia CRSII5_173718, to G.G.), an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679) and has received support under the program Investissements d'Avenir launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). Y. L. thanks the China Scholarship Council for financial support. The work was further supported by a Feodor Lynen Research Fellowship from the Alexander von Humboldt Foundation (S.K.) as well as an Early Postdoc. Mobility Fellowship from the Swiss National Science Foundation (S.K., Grant P2 BSP2_181760).

be the most active agent MFC of 15.63 µM against C. albicans, much higher than KTZ (MFC = 312.5 µM). 133 However, when compared to the antifungal activities of bis-ferrocenyl-and mono-ferrocenylcyclotetraphoshazenes with the same substituents, bis-ferrocenyl-cyclotetraphoshazenes were less effective than the mono-substituted compounds. The DNA interactions of these compounds were studied using agarose gel electrophoresis. It was observed that bis-ferrocenyl phosphazene derivatives 115a, 115c and 115d decreased DNA mobility, while the corresponding mono compounds, cleaved DNA to linear form III through different interaction with DNA. The cytotoxicities of these complexes were studied against L929 fibroblast and DLD-1 colon cancer cell lines. Most of the compounds were found to be less toxic than cisplatin and doxorubicin.

The metal complexes showed moderate antibacterial activities and significant antifungal activities compared to the ligands alone, but did not perform as well as the positive controls, imipenum, MCZ and AmB. 137 In 2006, Ramesh and co-workers reported a series of orthometallated Ru(II) complexes (120a-e, Fig. 42) derived from reaction of 2-(arylazo)phenol ligands with the Ru(II) precursor, [RuH(Cl)(CO)(PPh 3 ) 3 . The crystal structures of 120a and 120b revealed a distorted octahedral environment around the ruthenium centre. In vitro studies against C. albicans and A. niger demonstrated that the metal complexes had higher activities than the respective free ligands, while RuCl 3 ⋅3H 2 O and the Ru(II) carbonyl precursor complex displayed no antifungal effects under identical experimental conditions. A. niger displayed higher sensitivity to these metal complexes than C. albicans. 138 In 2014, Al-Noaimi et al. synthesised two heterotrimetallic Ru(II)-Pd(II)-Ru(II) complexes, with the general formula [Ru II Cl 2 (PPh 3 ) 2 (triamine)] 2 [Pd II X 2 ] (X = Cl, OAc) (121a-b, Fig. 42).

Complexes 121a-b showed significant antifungal activities against A. niger, C. albicans and P. digitatum with inhibition zones greater than 19 mm, and moderate activities against F. oxysporum (<16.2 mm). As positive control, nystatin displayed inhibition zones in the range of 20.8 to 33.7 mm against the tested fungi. 139 A range of rare earth element complexes (Pr(III), Sm(III), Gd(III), Tb(III), Er(III), Yb(III)) with threoninebromosalicylaldehyde Schiff base ligands (Fig. 55B) were tested for their efficacies against Candida and Aspergillus spp. 160 Interestingly, at 60 µg⋅mL -1 treatments, all compounds except for Pr(III) complex and the free ligand recorded >90% inhibition against Candida relative to KTZ. Similarly, >80% inhibition of Aspergillus was achieved for all complexes except the Pr(III) and Gd(III) compounds. While the sustainability of including rare earth elements in fungal treatments is questionable, the luminescence properties of such complexes could open doors to a greater understanding of metal complex uptake and distribution in in vitro fungal cultures by means of confocal microscopy and X-ray fluorescence spectroscopy.

Indole-based ligands

Several indole-containing Schiff base ligands have been synthesised to incorporate the bioactivities of the indole moiety, imine species and metal ions into one drug. A series of mixed ligand Ru(III) indole-inspired Schiff base complexes with phosphane/arsane, and bromide/chloride ligands (Fig. 56A) were synthesised, characterised and tested for bioactivity against B. cineria and A. niger by disk diffusion at concentrations of 0.25, 0.5 and 1.0% w/v. 161 While the antifungal results initially appeared promising against both organisms tested -almost all diameters of inhibition exceeded that of the positive control -examination of the positive control drug raised questions regarding experimental design. The synergistic antibiotic 'co-trimoxazole' is used to treat severe bacterial infections (being sulfonamide-based) but does not appear to be commonly used against fungal infections. In the cases where it is prescribed for antifungal treatment, the drug acts as a facilitator for entry of the active antifungal drug into the cell by increasing cell permeability. 162 It is unclear whether the authors of the paper assumed co-trimoxazole was synonymous with CTZ (an azole based antifungal commonly used to treat yeast infections) or if this was perhaps a misspelling (despite the same name appearing twice in the paper), but without a suitable positive control for comparison, the fungal inhibition studies do not provide much insight into the antifungal activities of this series of complexes. A salen-based ligand (Fig. 61B) coordinated with Cu(II), Cr(III) or La(III) was found to have zero inhibitory effect against A. flavus, however, the free ligand had moderate antifungal activity. 173 Conversely, against C. albicans, both the free ligand and the La(III) complex had zero inhibitory effect, but the Cu(II) and Cr(III) complexes reached inhibition of approximately 10 and 15 mm per mg of complex, respectively. No compound tested approached AmB.

Various length diamines (ethyl, propyl or butyl) were condensed with 2-acetylthiophene to form a series of ligands (Fig. 62A) and complexes with first row transition metals were formed. 174 By disk diffusion it was determined that >70% inhibition was reached by the Co(II)-butyldiamine complex (against F. solani), Cu(II)propyldiamine complex (against C. albicans), Co(II)-ethyldiamine complex (against T. longifusus), Zn(II)ethyldiamine complex (against A. flavus), and the Ni(II)-butyldiamine complex (against M. canis). Against C. glabrata, the highest inhibition measured was 62% for the Zn(II)-propyldiamine complex. All inhibitions were reported as percentages relative to MCZ and AmB.

NO donor ligand-based complexes 14.1 Diazene/hydrazine-based ligands

Against C. albicans and A. niger, a Pd(II) complex with (E)-4-((2-hydroxyphenyl)diazenyl)-3-phenyl-1Hpyrazol-5(4H)-one (Fig. 72A) was found to have moderate activity (8.2 ± 0.2 and 10.1 ± 0.1 mm, respectively). 199 The free ligand and two other metal complexes (Ni(II) and Pt(II)) were tested, but no results recorded. Additionally, FCZ was listed as a positive control against the fungal organisms, however no measurements were provided in the article. A range of isonicotinic acid hydrazide derivatives were synthesised and complexed with VO 2 (V) ions (Fig. 72B) by Maurya and Rajput. 200 Of the reported complexes, only one was tested for antifungal activity: N-(4'butyrylidene-3'-methyl-1'-phenyl-2'-pyrazolin-5'-one)-isonicotinic acid hydrazide. Against A. niger, the compound inhibited ∼19% of fungal growth after 7 days, which was a decrease of ∼40% when compared to respectively. 212 The antibacterial properties of these complexes were far more prominent than the antifungal properties.

Thiophene-containing hydrazine carboxamide Ni(II) and Cu(II) complexes (Fig. 76E) were tested for antifungal activities at 250 µg⋅mL -1 and found to have a maximum inhibitory diameter against A. flavus of 16 mm, and 10 mm against A. niger. 213 No positive control was reported.

Thiocarbamate-based complexes

A range of derivatised dithiocarbamate ligands in complex with dimethyl Sn(IV) (Fig. 77A) were found to approach 90% inhibition of R. solani at 1.0 mM and close to 100% inhibition of B. cinerea at 240 µM. 214 The positive controls used in the studies were mancozeb and ziram which are a mixture of Zn(II)/Mn(II) dithiocarbamate complexes and a Zn(II) dithiocarbamate, respectively. Dimethyl Sn(IV) was found to only inhibit the fungal strains by ∼15%. Inhibitory properties of the free ligand were not reported due to poor aqueous stability.

Another study was published reported a similar derivatised dithiocarbamate ligand in complex with di-nbutyl Sn(IV) (Fig. 77B). Some of the fluorinated complexes approached 75% inhibition of C. gloeosporioides at concentrations of 98 µM and had IC 50 of approximately 6 µM. 215

Conclusion

With the global rise of antifungal resistance to the commonly used drugs, there is an urgent demand for the discovery of new, effective, safe, and cheap drugs to be used as alternative treatments against multi-drug resistant pathogenic fungi. In this line of thinking, metal complexes based on bioactive ligands and known drugs hold great potential as board-spectrum pathogenic fungicides. Varied physicochemical properties, such as redox activity, coordination geometry, biocompatibility and microbicidal activity are available across the Periodic Table. These metal ion properties, along with intrinsic bioactivities of the free ligand may yield improved antifungal activities via multiple modes of action, potentially bypassing rapid resistance of organisms. Despite a lot of research concerning metal-based antifungal agents being published recently, the rational design of molecular structures to yield antifungal activity is often still empirical. Mechanistic studies are limited to DNA interaction studies and in many cases not related to the specific targeting of fungi. More in-depth studies to understand the modes action of metal-based antifungal agents are needed, in particular with respect to fungi-specific targets such as ergosterol, CYP51, yeast-to-hyphae transitions, antibiofilm formation, adhesion of fungal cells, metallopeptidases, morphological transitions and mutagenic potential.

Standardisation of antifungal results relative to positive controls (appropriate for the fungal strains studied) would also further help to advance the field.

After complexation of organic molecules with metal ions, improvements in antifungal activities were often observed compared to the free ligand, but more than often not rationalized. Of the reported metal complexes, organometallic compounds such as ferrocene derivatives exhibited significant antifungal activities. Additionally, cationic species -e.g. cationic Ru-cyclopentadienyl complexes -were found to be more effective than their neutral counterparts on several instances. Complexes containing metal ions such as Cu(II), Zn(II), Mn(II) and Ni(II) and ligands that included triazole, phen, salicylaldehyde-/thiosemicarbazide-/thiocarbamate-based Schiff base moieties also exhibited significant antifungal activities.

Overall, we hope this review will help to inspire and direct researchers aiming to improve the clinical profiles of metal-based antifungal agents. Particularly regarding cytotoxicity and in vivo studies as the number of

AUTHOR INFORMATION Corresponding Author

* e-mail: kevin.cariou@chimieparistech.psl.eu; gilles.gasser@chimieparistech.psl.eu

ORCID:

Yan Lin: 0000-0001-7808-5444 Harley Betts: 0000-0002-1149-2675 Sarah Keller: 0000-0003-2667-9157 Kevin Cariou: 0000-0002-5854-9632 Gilles Gasser: 0000-0002-4244-5097