Review New Antimicrobial Strategies Based on Metal Complexes

Mickaël Claudel, Justine V. Schwarte and Katharina M. Fromm * Department of Chemistry, University of Fribourg, Chemin du Musée 9, CH-1700 Fribourg, Switzerland; [email protected] (M.C.); [email protected] (J.V.S.) * Correspondence: [email protected]; Tel.: +41-26-300-8732; Fax: +41-26-300-9738



Received: 13 August 2020; Accepted: 12 October 2020; Published: 16 October 2020


Abstract: Traditional organic antimicrobials mainly act on specific biochemical processes such as replication, transcription and translation. However, the emergence and wide spread of microbial resistance is a growing threat for human beings. Therefore, it is highly necessary to design strategies for the development of new drugs in order to target multiple cellular processes that should improve their efficiency against several microorganisms, including bacteria, viruses or fungi. The present review is focused on recent advances and findings of new antimicrobial strategies based on metal complexes. Recent studies indicate that some metal ions cause different types of damages to microbial cells as a result of membrane degradation, protein dysfunction and oxidative stress. These unique modes of action, combined with the wide range of three-dimensional geometries that metal complexes can adopt, make them suitable for the development of new antimicrobial drugs.

Keywords: metal complexes; new antimicrobial strategies; metallo-drugs; silver; copper; zinc; iron; ruthenium; gallium; bismuth; vanadium; synergic effects

1. Introduction The search for new active antimicrobial compounds is of growing interest since the current clinical pipeline remains insufficient to tackle the challenge of increasing emergence and spread of antimicrobial resistance. In the United States, each year more than 2.8 million people suffer from an antibiotic-resistant infection, resulting in more than 35,000 deaths [1]. In Europe, antibiotic resistance is responsible for an estimated 33,000 deaths annually [2]. According to the World Health Organization (WHO), the newly approved products have limited clinical benefits over existing treatments and almost 75% of the antimicrobials under clinical development are simply derivatives of already known and used molecules existing on the market, and for which multiple resistance mechanisms are well established [3]. Therefore, there is an urgent need for the development of new antimicrobial agents. Since the discovery of antibiotics by Alexander Fleming in the 1920s, most of the current compounds developed by medicinal chemists around the world are almost exclusively purely organic. Although metals and their complexes have been employed since ancient times, they were generally used for their applications as catalysts or materials, and their properties were often associated with toxicity. However, the use of structurally defined metal complexes in medicine mostly appeared at the beginning of the 20th century with the discovery of the arsenic-containing organometallic complex as the first effective treatment of syphilis (Salvarsan) [4]. Since then, many other metal complexes have been found to be useful in medicinal chemistry, like the development of a famous mercuric-based antiseptic agent (Mercurochrome) or the treatment of rheumatoid arthritis by a gold complex agent (Auranofin) [5]. Nevertheless, the most relevant examples in the field of medicinal chemistry are undoubtedly the platinum-based anticancer drugs cisplatin, oxaliplatin and carboplatin [6]. These complexes are still currently used in nearly 50% of all cancer treatments as chemotherapeutic agents, often in combination

Chemistry 2020, 2, 849–899; doi:10.3390/chemistry2040056 www.mdpi.com/journal/chemistry Chemistry 2020, 2 850 with other drugs. Over the past two decades, several metal-based complexes (based on silver, copper, iron gold, bismuth, gallium, etc.) have been designed and have reached human clinical trials for the treatment of cancer, malaria and neurodegenerative diseases [7–9]. Despite their high potential in such diseases, little attention has been paid to their application as antimicrobial compounds. The vast diversity of metals, types of ligands, and geometries makes metal-based coordination complexes very useful in accessing a highly underexplored chemical space for drug development, and especially for the design of new antimicrobials [10]. Unlike the majority of organic molecules, which possess only one- or two- dimensional topologies, metal complexes can adopt three-dimensional structures available through metal coordination chemistry, the latter offering the possibility to create a wide variety of antimicrobials. Furthermore, metal-based complexes may provide unique modes of action: exchange or release of ligands, redox activation and catalytic generation of toxic species (reactive oxygen species, ROS), as well as depletion of essential substrates [11,12], making them able to abolish enzyme activities, disrupt membrane function or damage DNA.

2. Metal Complex-Based Antimicrobial Compounds Metal ions or metal ion binding components play important roles in biological processes, and their rational design could be used to develop new therapeutic drugs or diagnostic probes. The fact that metal atoms easily lose electrons and form positively charged ions makes them soluble in biological fluids. Due to their electron deficiency, they can readily interact with electron-rich biomolecules such as DNA or proteins, and therefore participate either in a catalytic mechanism or in the stabilization/determination of their tertiary or quaternary structures. Depending on the type of metal ion coordination complexes and organometallics, they offer a wide range of oxidation states, coordination numbers and geometries, leading to a virtually unlimited number of structures and conformations. With the improvement of knowledge and understanding of biological processes, judicious metal–ligand combinations can be designed with appropriate geometry for specific interactions. For example, some of them have already been used to inhibit enzymes, label proteins, image cells, probe biomacromolecules, alter bioavailability or provide contrast as MRI agents [13]. In addition, the broad range of metal–ligand combinations makes it possible to design new entities with various physical properties and chemical reactivities including charge, solubility, rates of ligand exchange, strengths of metal–ligand bonds, Lewis acidity, metal- and ligand-based redox potentials, outer-sphere interactions, and ligand conformations [14]. Therefore, compared to organic drugs, the structural and electronic properties of such complexes offer biological and chemical diversity, making them very attractive in the field of medicinal chemistry, especially as antimicrobial agents with novel modes of action to treat drug-resistant diseases. From this point of view, the transition metals accompanied by some other metals are the most promising for disease treatments, while the heavy lanthanides are more investigated for their radioactive and photoluminescent properties. This is illustrated by the growing amount of this kind of organometallic species deposited on open databases specialized in bioactive compounds. Hence, a study of Blaskovich and co-workers, who investigated the compounds submitted on the Community for Open Antimicrobial Drug Discovery, classified the organometallic compounds according to the nature of their metal element and their activity and toxicity [15]. These graphs (Figure1) therefore reflect the most interesting ions for future work. In this review, we will discuss the major discoveries in the non-traditional field of metal complex-based antibiotic compounds, focusing on the last decade and the most promising elements. In particular, we will focus on silver, copper, zinc, iron, ruthenium, gallium, bismuth and vanadium. Chemistry 2020, 2, x 3

Chemistry 2020, 2 851 Chemistry 2020, 2, x 3

Figure 1. Distribution of organometallic compounds submitted to Co-ADD in function of their metal [15].

In this review, we will discuss the major discoveries in the non-traditional field of metal complexFigureFigure-based 1. 1. DistributionDistribution antibiotic of compounds, organometallic of organometallic focusing compounds compounds on the submitted last submitted decade to Co toand-ADD Co-ADD the in mostfunction in promising function of their of metal elements. their In particular,[15].metal [15]. we will focus on silver, copper, zinc, iron, ruthenium, gallium, bismuth and vanadium. 2.1. Silver 2.1. SilverIn this review, we will discuss the major discoveries in the non-traditional field of metal complexSilver-based and antibiotic its compounds compounds, have longfocusing been on used the aslast antimicrobial decade and the agents. most promising In the 18th elements. and 19th Incenturies, particular,Silver silver and we compoundsitswill compounds focus on have silver, have found copper, long a range been zinc, ofused iron, applications as ruthenium, antimicrobial in medicine, gallium agents., especiallybismuth In the and with18th vanadium respectand 19th to. infectiouscenturies, diseases.silver compounds Colloidal have silver found was used, a range for of example, applications for wound in medicine, antisepsis especially and silver with nitrate respect for 2.1.theto infectious Silver treatment diseases. of burn woundsColloidal [ 16silver]. Even was though used, for the example discovery, for of wound antibiotics antisepsis in the and early silver part nitrate of the for the treatment of burn wounds [16]. Even though the discovery of antibiotics in the early part of 20thSilver century and drastically its compounds reduced have the use long of silver,been numerousused as antimicrobial silver-based compoundsagents. In the are 18th still employedand 19th the 20th century drastically reduced the use of silver, numerous silver-based compounds are still centuriesfor their medicinal, silver compounds and low toxicity have found properties. a range Indeed, of applications around 300 in clinical medicine, trials es involvingpecially with silver-based respect employed for their medicinal and low toxicity properties. Indeed, around 300 clinical trials involving tocompounds infectious diseases. and formulations Colloidal forsilver diverse was used applications, for example are currently, for wound ongoing antisepsis [17]. and One silver example nitrate to silver-based compounds and formulations for diverse applications are currently ongoing [17]. One forillustrate the treatment the efficiency of burn and wounds the potential [16]. Even activity though of silver the compoundsdiscovery of is antibiotics silver sulfadiazine in the early1 (Figure part 2of), example to illustrate® the efficiency and the potential activity of silver compounds is silver theknown 20th ascentury Silvadene drastically, which reduced was approved the use of in silver, 1968 by numerous the Food silver and Drug-based Administration compounds are (FDA) still sulfadiazine 1 (Figure 2), known as Silvadene® , which was approved in 1968 by the Food and Drug employedfor use as afor broad-spectrum their medicinal antibiotic and low toxicity for burn properties. wounds. This Indeed, compound around acts 300 asclinical a reservoir trials involving of silver(I) Administration (FDA) for use as a broad-spectrum antibiotic for burn wounds. This compound acts silverin the-based wound, compounds liberating theseand formulations ions slowly. Thefor diverse interest applications in silver-based are new currently materials ongoi as antimicrobialng [17]. One as a reservoir of silver(I) in the wound, liberating these ions slowly. The interest in silver-based new exampleagents is exponentially to illustrate the increasing efficiency and hasand been the reviewed potential previously activity of [ 18 silver]. compounds is silver materials as antimicrobial agents is exponentially increasing and has been reviewed previously [18]. sulfadiazine 1 (Figure 2), known as Silvadene® , which was approved in 1968 by the Food and Drug Administration (FDA) for use as a broad-spectrum antibiotic for burn wounds. This compound acts as a reservoir of silver(I) in the wound, liberating these ions slowly. The interest in silver-based new materials as antimicrobial agents is exponentially increasing and has been reviewed previously [18].

FigureFigure 2. Silver sulfadiazine.

Even though though silver silver and its andcomplexes its complexeshave shown cytotoxic have shown effects against cytotoxic Gram e-ffpositive/Gramects against- Gram-positivenegative bacteria/Gram-negative and fungi, their bacteria mechanisms and of fungi, action their are not mechanisms well understood. of action However, are notthe most well understood.common one However, described the in mostthe literature commonFigur is onee related2. Silver described tosulfadiazine. a slow in the release literature of the is relatedactive silver(I) to a slow ion, release which of thereacts active with silver(I) the thiol ion, which groups reacts of proteinswith the thiol or with groups key of proteinsfunctional or with groups key of functional enzymes groups [19,20 of], Even though silver and its complexes have shown cytotoxic effects against Gram-positive/Gram- enzymescoordinating [19,20 ligands], coordinating just acting ligands as justa carrier acting for as a the carrier silver(I) for the ion. silver(I) These ion. interactions These interactions lead to lead the negative bacteria and fungi, their mechanisms of action are not well understood. However, the most todenaturation the denaturation of proteins of proteins and and the theimpairing impairing of the of the membrane membrane function function [21,22 [21,22].]. The The magnitude magnitude of common one described in the literature is related to a slow release of the active silver(I) ion, which antimicrobial properties of of silver silver complexes complexes is is then then related related to to the the ease ease with with which which they they participate participate to reacts with the thiol groups of proteins or with key functional groups of enzymes [19,20], toligand ligand exchange exchange reactions. reactions. Additionally, Additionally, in some in some cases, cases, silver silver ions can ions produce can produce ROS, known ROS, known to target to coordinating ligands just acting as a carrier for the silver(I) ion. These interactions lead to the targetmainly mainly lipids, lipids, DNA, DNA, RNA RNA and and proteins, proteins, causing causing severe severe consequences consequences such such as as malfunction malfunction of denaturation of proteins and the impairing of the membrane function [21,22]. The magnitude of membranes, proteins, and the DNA replication machinery [2323,24,24].]. Furthermore, upon treatment with antimicrobial properties of silver complexes is then related to the ease with which they participate to silver, condensed DNA molecules have been observed in the bacterial cytoplasm, thereby leading to a ligand exchange reactions. Additionally, in some cases, silver ions can produce ROS, known to target loss of its ability to replicate, and thus resulting in the death of the bacteria [21]. mainly lipids, DNA, RNA and proteins, causing severe consequences such as malfunction of membranes, proteins, and the DNA replication machinery [23,24]. Furthermore, upon treatment with

Chemistry 2020, 2, x 4

Chemistry 2020, 2 852 silver, condensed DNA molecules have been observed in the bacterial cytoplasm, thereby leading to a loss of its ability to replicate, and thus resulting in the death of the bacteria [21]. EvenEven thoughthough a complete complete understanding understanding of of the the mechanism mechanism of ofsilver silver ions ions fighting fighting bacteria bacteria has hasnot notbeen been reached reached until until present present days, days, it has it has been been suggested suggested that that the the activity activity of ofsilver silver-based-based complexes complexes is isstrictly strictly connected connected to their to their water water solubility solubility and stability, and stability, lipophilicity, lipophilicity, redox redox ability ability and rate and of raterelease of releasesilver ions. silver These ions. properties These properties are governed are governedby the choice by theof suitable choice ligands of suitable and ligandsby slight and modulations by slight modulationsin their electronic in their and electronic steric and effects. steric All eff ofects. these All of factors these factorsare fundamental are fundamental for maintaining for maintaining their theirbioavailability bioavailability over over an extended an extended period period of oftime time and and preventing preventing reinfection reinfection or or resistance. resistance. Therefore Therefore,, takingtaking intointo accountaccount thesethese features,features, aa widewide varietyvariety ofof newnew classesclasses ofof silversilver complexescomplexes havehave garneredgarnered attentionattention forfor theirtheir antimicrobialantimicrobial properties,properties, inin particular,particular, N-heterocyclicN-heterocyclic carbenecarbene (NHC)(NHC) complexes,complexes, phosphinephosphine complexes complexes or N-heterocyclic or N-heterocyclic complexes complexes of silver(I) of si [lver(I)25]. Because [25]. ofBecause their electronic of their properties, electronic NHCproperties, ligands NHC form ligands very stable form bonds very withstable the bonds majority with of the metal majority ions andof metal thus improveions and the thus stability improve of thethe complexes.stability of the In terms complexes. of the functionIn terms ofof substituentsthe function thatof substituents are directed that towards are directed the metallic towards center, the NHCmetallic ligands center are, able NHC to ligands protect metal are able ions to and protect have a metal strong ions impact and on have steric a accessibility. strong impact Thus, on silver steric NHCaccessibility. complexes Thus, are mainlysilver NHC used tocomplexes modulate are the mainly silver release used toand modulate to control the the silver systemic release delivery and of to silver.control For the example, systemic Youngs delivery and of coworkers silver. For synthesized example, in Youngs 2004 several and coworkers pincer Ag(I)-carbene synthesized complexes in 2004 (Figureseveral3 )pincer and tested Ag(I) their-carbene antimicrobial complexes properties (Figure 3 against) and testedEscherichia their coliantimicrobial(E. coli), Staphylococcus properties against aureus (EscherichiaS. aureus), andcoli Pseudomonas(E. coli), Staphylococcus aeruginosa (aureusP. aeruginosa (S. aureus). The), and minimum Pseudomonas inhibitory aeruginosa concentrations (P. aeruginosa (MICs)). ofThe compounds minimum2 inhibitoryand 3 were concentrati evaluatedon ands (MIC showeds) of bettercompounds bacteriostatic 2 and 3 activity were evaluated than AgNO and3, showed even at muchbetter lowerbacteriostatic concentration activity [26 than]. The AgNO success3, even of at this much first lower Ag(I)-NHC concentration antimicrobial [26]. The was success followed of this by thefirst development Ag(I)-NHC antimicrobialof other derivatives was followed in order by to improve the development their biological of other activities derivatives [27]. in Moreover, order to phosphinesimprove their and biological N-heterocyclic activities ligands [27].have Moreover, also been phosphines extensively and screened N-heterocyclic in association ligands with have silver also andbeen some extensively of them screened have shown in association promising with applications silver and against some both of them bacteria have [28 shown] and fungi promising [29]. Theapplications authors proposed against both different bacteria modes [28] of and action fungi behind [29]. The this non-proliferationauthors proposed of different bacteria modes and cells of asaction the Ag(I)behind complexes this non could-proliferation possibly of interfere bacteria with and DNA cells through as the intercalationAg(I) complexes or disrupt could thepossibly cell membrane. interfere with DNA through intercalation or disrupt the cell membrane.

Figure 3. Structures of Ag(I)-NHC complexes synthesized by Youngs and coworkers [26]. Figure 3. Structures of Ag(I)-NHC complexes synthesized by Youngs and coworkers [26]. However, despite the great amount of research undertaken and the number of new silver complexes However, despite the great amount of research undertaken and the number of new silver synthesized, most of them have limited efficacy in vivo because of their rapid clearance, which is complexes synthesized, most of them have limited efficacy in vivo because of their rapid clearance, typical for small molecule-based drugs inside the body. A possible solution to overcome this drawback which is typical for small molecule-based drugs inside the body. A possible solution to overcome this could be to encapsulate those active species into biodegradable nanoparticles for transportation and drawback could be to encapsulate those active species into biodegradable nanoparticles for delivery [30,31]. transportation and delivery [30,31]. Several silver complexes displaying antibacterial properties have been published during these last Several silver complexes displaying antibacterial properties have been published during these decades. Without giving an exhaustive list, which could be very long, the most recent results should be last decades. Without giving an exhaustive list, which could be very long, the most recent results mentioned, which showed the best results for a series of metal–antibiotic complexes against different should be mentioned, which showed the best results for a series of metal–antibiotic complexes against bacterial strains. Thus, while the silver–ampicillin complex shows only a weak inhibition effect against different bacterial strains. Thus, while the silver–ampicillin complex shows only a weak inhibition E. coli and S. aureus compared to ampicillin alone (2- to 4-fold decrease of MIC), the inhibition is strongly effect against E. coli and S. aureus compared to ampicillin alone (2- to 4-fold decrease of MIC), the enhanced against K. pneumonia, A. baumannii and P. aeruginosa with a 20- to 114-fold drop of MIC. inhibition is strongly enhanced against K. pneumonia, A. baumannii and P. aeruginosa with a 20- to 114- It is nevertheless noticed that the MICs for the silver–ampicillin complex are similar to those of silver fold drop of MIC. It is nevertheless noticed that the MICs for the silver–ampicillin complex are similar alone, except for a slight decrease against S. aureus, but the authors noted that one has to consider the

Chemistry 2020, 2 853 silver concentration, which is twice as low for the 1:1 complex compared to the silver alone. Moreover, the non-linear evolution of the MIC with respect to the silver ratio leads the authors to conclude to a synergy between the silver(I) ion and its ligand. This is nicely illustrated by measuring the zones of inhibition of a silver/antibiotic mixture (antibiotic = ampicillin or penicillin G, see Table1), showing an enhancement of the activity with the increase of the silver concentration, whereas silver nitrate alone displays the lowest activity [32]. The authors explain this synergetic effect by a decrease of the kinetic rate, although the Michaelis constant increased due to the presence of silver ions. Overall, the ratio kcat/KM decreases, resulting in a slowdown of the antibiotic hydrolysis by the β-lactamase enzymes expressed by the resistant bacteria. Thus, the more silver ions there are in the mixture, the more the enzymes are downturned, allowing the majority of the antibiotic molecules to cross the bacterial cell wall and to reach their target (the peptidoglycan membrane) before being hydrolyzed.

Table 1. Zone of inhibition measured by disk-diffusion assays for silver–ampicillin and silver–penicillin G complexes with different ratios [32].

E. coli CTX-M-15 (Ec71 Metal Salt Conc [µg/mL] E. coli CR48 (IMP-4) E. coli C53 (NDM-4) E. coli TOP 10 producing CTX-M-15) Compound Zone of inhibition (diameter) [mm] AgNO3 only 1.68 10 7 8 10 Amp/Ag(I) 1:1 0.76 10 8 8.5 10 Amp/Ag(I) 1:5 3.8 8.5 10 Amp/Ag(I) 1:10 7.6 14 9 10 10 Amp only 0 0 0 10 PenG/Ag(I) 1:0.5 0.38 8 7 8 8 PenG/Ag(I) 1:5 1.9 11 7 9 10 PenG/Ag(I) 1:10 3.8 11.5 9 10 10 PenG only 0 0 0 0

Other studies aiming to rehabilitate “old” antibiotics that encounter a lot of resistance by themselves have been carried out in recent years by associating them with silver. This is illustrated for instance in a publication of Morones-Ramirez et al., which showed the growth over 3 h of Escherichia coli in the presence of some antibiotics with and without silver. Whereas the concentrations of ofloxacin and ampicillin were adjusted to be bacteriostatic (no growth, no death) alone, the addition of 15 µM of silver nitrate resulted in 10- to 50-fold bacterial death. Even more, although vancomycin is normally inactive against Gram-negative bacteria, 30 µg/mL associated with 30 µM of silver nitrate divided the bacterial concentration by a factor of 10,000 [33]. Finally, rather than associating antibiotics with silver (I) ions, one can also consider association with silver nanoparticles, numerous examples of which are given in a review by Ziora et al. [34]. Nanoparticles have the advantage of releasing silver ions slowly, making it possible to distribute a continuous dose in the organism. Another way to release silver ions progressively is to embed them with or without antibiotics in particles such as zeolites [35] or into sol–gel coatings. Hence, McHale et al. showed that 0.7% w/w of silver ions in a sol–gel coating was enough to avoid any bacterial growth on surfaces, while 0.3% w/w of silver ions with coumarin led to only a slight increase of Enterobacter cloacea (but the coatings with silver ions alone were more efficient against S. aureus MRSA). Nevertheless, sol–gel coatings doped with silver ions and coumarin displayed silver release on a longer period than sol–gel coatings doped with only silver nitrate [36].

2.2. Copper Many transition metals are associated with numerous biological processes that are indispensable to life processes. They are the most abundantly found trace elements present in biological systems. They can coordinate with C- or N- terminals from proteins in a variety of models, and thereby play a vital role in the conformation and utility of living macromolecules. Thus, these metal ions are nowadays present in several inorganic pharmaceuticals used as drugs against different kinds of diseases, ranging from antibacterial and antifungal to anticancer applications [37,38]. In this context, copper is an essential trace element present as a cofactor in many enzymes as, for example, in superoxide dismutase. In this enzyme and many other metabolic pathways, copper Chemistry 2020, 2 854 acts as a redox agent. Furthermore, free copper ions are reported to have toxic effects against both bacteria and fungi [39]. Based on this behavior, numerous researchers used the coordination of organic molecules to copper in order to improve the antimicrobial activity. They showed different mechanisms of action depending on the geometry of the complexes and the nature of the ligand (Figure4)[ 40,41]. Here, we will discuss some of the most relevant examples described in the literature. For instance, compound 4, based on a tetrahedral mixed-ligand copper(I) bromide complex, was 100-fold more active against both Gram-negative and Gram-positive bacteria (Escherichia coli, Xanthomonas campestris, Bacillus subtilis and Bacillus cereus) compared to the clinical antibiotic ampicillin. The authors showed that the mechanism of action of 4 was based on damage of the bacterial membrane through the generation of reactive oxygen species [42]. Another example showing the effectiveness of copper complexes in order to inhibit the bacterial growth is related to the synthesis of a phthalimide-based copper(II) complex 5 [43]. Due to their planar aromatic rings, phthalimide moieties and derivatives possess different biologically active targets and have been studied for their potent anticancer [44], antimicrobial [45], anti-inflammatory [46] and antimalarial activities [47]. The efficacy of these drugs can be credited to their DNA-interacting capabilities. Then, by coordinating this phthalimide ligand to a copper(II) ion, a complex with square-planar geometry is obtained, which exhibited excellent antibacterial activity against different bacterial strains, especially against Salmonella enterica (IC50 = 0.0019 µg/mL), compared to the ligand itself and the clinical antibiotic ciprofloxacin. This higher antibacterial activity could be attributed to an enhanced interaction of the complex with DNA. Furthermore, a multitude of Schiff base–copper(II) complexes have been synthesized for their antimicrobial properties [48–51]. This class of ligands is very interesting in the sense that they are excellent coordinating molecules with strong donor groups (azomethine or imine group) and can exhibit variety in the structure of their metal complexes. They have also found application in a broad range of biological activities like antibacterial, antifungal, anti-tuberculosis, antimalarial and antiviral properties [52]. Based on these facts, Nazirkar et al. synthesized complexes of Cu(II) coordinated by new Schiff base derivatives with a benzofuran core (compound 6)[53]. They observed that some of their complexes had excellent antibacterial activity against Mycobacteria Tuberculosis (MIC = 1.6 µg/mL) which is almost two-fold higher than that of pyrazinamide and ciprofloxacin (MIC = 3.125 µg/mL), and almost four-fold higher than that of streptomycin (MIC = 6.25 µg/mL). This could be explained for the first time by the presence of active pharmacophores in the molecular structures of the newly synthesized ligands (benzofuran moiety, imine group and well-positioned halogen), which might interfere in the mitosis cell mechanism and lead to a stop of the bacteria growth. However, Cu(II) complexes showed improved antibacterial activities as compared with their parent Schiff base ligands. The authors explained this by an increase of the lipophilicity of the complexes, which occurs after the complexation of the organic residue around the copper ion which favors their transfer across the lipid membrane of the bacterial cell wall. Indeed, according to the Ligand Field Theory (LFT), overlapping of metal orbitals with orbitals from ligands induce a minimization of the positive charge on the metal by gaining the electrons from donor groups of the Schiff base ligands. The delocalization of the electrons from the ligands to the central metal atom enhances the lipophilic nature of the complex. Thus, the degree of lipophilicity of a molecule is a key factor governing the magnitude of antimicrobial activity. Chemistry 2020, 2, x 7

Chemistry 2020, 2 855 metal atom enhances the lipophilic nature of the complex. Thus, the degree of lipophilicity of a molecule is a key factor governing the magnitude of antimicrobial activity.

FigureFigure 4.4. Structure of antimicrobial coppercopper complexescomplexes [[4242,,4433,,5533].].

Moreover,Moreover, aa seriesseries ofof coppercopper complexescomplexes withwith sulfonamidesulfonamide ligandsligands havehave alsoalso beenbeen synthesizedsynthesized forfor theirtheir antimicrobialantimicrobial propertiesproperties [[5454].]. FigureFigure5 5 shows shows the the general general structure structure of of sulfonamides sulfonamides and and the the coordinationcoordination versatility of these compounds,compounds, which make them very useful in inorganic chemistry. 4 TheyThey act act as as monodentate monodentate ligands ligands through through the theN atom 4N atomor Nh (nitrogen or Nh (nitrogen from heterocycle from heterocycle R), as bidentate R), as 1 4 throughbidentate the throughN and the N 1hNor and bridging Nh or bridging two metal two ions metal through ions throughN and N4Nh ,and as bidentateNh, as bidentate to one to Cu(II) one 1 4 throughCu(II) through Nh and NhN and and 1N bridging and bridging to an adjacentto an adjacent Cu(II) Cu(II) through through the N.the It 4N. is alsoIt is wellalso knownwell known that sulfonamidesthat sulfonamides are competitive are competitive antagonisms antagonisms of PABA of PABA (p-aminobenzoic (p-aminobenzoic acid) acid) and and then then interfere interfere in the in biosynthesisthe biosynthesis of tetrahydrofolic of tetrahydrofolic acid, which acid, is which quite essential is quite to essential the bacterial to the metabolism. bacterial Nonetheless, metabolism. someNonetheless, studies have some shown studies diff erent have toxicological shown different and pharmacological toxicological and properties pharmacological between sulfonamides properties andbetween their metalsulfonamides complex and counterparts their metal [55 ]. complex For example, counterparts Kremer [ et55 al.]. For evaluated example, the Kremer antimicrobial et al. activityevaluated of theirthe antimicrobial synthesized activity complexes of their and ligandssynthesized against complexes both Gram-positive and ligands andagainst Gram-negative both Gram- bacteriapositive (andS. aureus Gramand-negativeE. coli)[ bacteria54]. They (S. showed aureus and that theirE. coli copper(II)) [54]. They complexes showed withthat their five-membered copper(II) heterocycliccomplexes with ring fivesubstituents-membered (sulfisoxazole heterocyclic7 , ring sulfamethoxazole, substituents (sulfisoxazole sulfamethizole) 7, sulfamethoxazole, were more active thansulfamethizole) the free sulfonamides were more (in active opposition than to the the free copper(II) sulfonamides complexes (in with opposition six-membered to the heterocyclic copper(II) ringcomplexes substituents, with six showing-membered no improvement heterocyclic ofring activity substituents, compared showing to their no title improvement ligand). To understand of activity thecompared microbiological to their title behaviors ligand) of. To these understand complexes, the one microbiological has to consider behaviors knowledge of these about complexes, the activity one of freehas sulfonamides,to consider knowledge where only about the ionicthe activity form is theof free active sulfonamides, antibacterial where species only [56]. the However, ionic form due is to the its lowactive lipophilicity, antibacterial the species penetration [56]. e However,fficiency across due theto its lipoidal low lipophilicity bacterial membrane, the pene istration very low efficiency for this anionicacross the form. lipoidal In addition, bacterial therefore, membrane as mentioned is very low above, for thethis complexationanionic form. of In this addition, kind of ligandstherefore, with as metalmentioned ions could above, be the one complexation possibility to increaseof this kind their of lipophilicity ligands with leading metal toions an could enhanced be one permeation possibility of theto increase drug inside their the lipophilicity cell. In the caseleadi ofng copper(II) to an enhanced complexes, permeation the most of e ffithecient drug ones inside coordinate the cell. through In the thecase heterocyclic of copper(II) N complexes, atom, thus maintainingthe most efficient the anionic ones coordinate form of sulfonamides. through the Furthermore,heterocyclic N intracellular atom, thus Cu(II)maintaining can also the act anionic as a redox form center of sulfonamides. and undergoes Furthermore, reduction to intracellular Cu(I). This Cu(II) latter ion can may also catalyzeact as a theredox reduction center and of O undergoes2 to O2−, while reduction the remaining to Cu(I). copper(II)This latter ions ion couldmay catalyze participate the toreduction the dismutation of O2 to

Chemistry 20202020,, 22, x 8568

O2−, while the remaining copper(II) ions could participate to the dismutation mechanism of O2− to mechanism of O2− to H2O2 [57]. Hence, the reactive oxygen species produced by such redox reactions H2O2 [57]. Hence, the reactive oxygen species produced by such redox reactions cause severe damages cause severe damages to cell and participate synergistically to the high toxicity observed and the to cell and participate synergistically to the high toxicity observed and the increased MIC values increased MIC values obtained with these complexes compared to the free sulfonamides. obtained with these complexes compared to the free sulfonamides.

Figure 5. (a) General structure of sulfonamides showing the labelling of the atoms; (b) structure of Figure 5. (a) General structure of sulfonamides showing the labelling of the atoms; (b) structure of [Cu(sulfisoxazole)2(H2O)4] 2H2O 7- the two oxazole rings and the copper ion are in the same plane [Cu(sulfisoxazole)2(H2O)4]∙2H· 2O 7- the two oxazole rings and the copper ion are in the same plane (c) (c) structure of a six-membered heterocycle substituted sulfonamide with its environment [54]. structure of a six-membered heterocycle substituted sulfonamide with its environment [54]. Besides Schiff bases and sulfonamides, another interesting ligand type could be the cyclams. CyclamBeside derivativess Schiff bases and theirand sulfonamides, zinc- and particularly another interesting copper complexes ligand type were could demonstrated be the cyclams. to be activeCyclam against derivative the Mycobacteriums and their zinc- tuberculosis and particularlybacteria. copper Thus, complexes a series were of cyclam demonstrated ligands bearing to be active one oragainst two the triazole Mycobacterium moieties substitutedtuberculosis bybacteria. a naphthalimide Thus, a series showed of cyclam a good ligands potency bearing against one or some two Mycobacteriumtriazole moietiesstrains substituted with by MICs a naphthalimide in the low micromolarshowed a good range potency (Figure against6). It some was Mycobacterium shown that astrains disubstituted with MIC cyclams in the displayedlow micromolar a higher range activity (Figure than 6). It the was monosubstituted shown that a disubstituted one [58]. Further, cyclam bydisplay investigatinged a higher the activity influence than the of themonosubstituted substituent, it one was [58] observed. Further, that by investigating a simple naphthalene the influence is betterof the substituent, than a naphthalimide it was observed (25 µM that vs a 6.25 simpleµM naphthalene against M. tuberculosis is better than). For a naphthalimide most of these ligands,(25 µM theirvs 6.25 complexation µM against with M. tuberculosis zinc (II) ions). For does most not improve of these theirligands, antimycobacterial their complexation activity, with but zinc their (II) copper ions does not improve their antimycobacterial activity, but their copper analogues display 2- to 4-fold analogues display 2- to 4-fold decrease of their MIC. Finally, the highest difference between ligand alonedecrease and of copper-complexes their MIC. Finally, are the for highest two substituted difference between cyclams whereligand thealone naphthalimide and copper-complexes was changed are for two substituted cyclams where the naphthalimide was changed for a smaller phenyl or benzyl for a smaller phenyl or benzyl part. In both cases, whereas the ligand alone and its zinc-complex have verypart. highIn both MICs case (50s, µwhereasM for the the phenyl-substitution ligand alone and andits zinc more-complex than 100 haveµM very for the high benzyl-substitution), MICs (50 µM for the phenyl-substitution and more than 100 µM for the benzyl-substitution), their copper complexes their copper complexes showed a strong enhancement with a 8- to more than 16-fold decrease of the showed a strong enhancement with a 8- to more than 16-fold decrease of the MICs (6.25 µM in both MICs (6.25 µM in both cases) [59]. cases) [59].

Chemistry 2020, 2 857 Chemistry 2020, 2, x 9

FigureFigure 6. 6.Some Some of of the the triazole-based triazole-based substituted cyclam cyclam ligands ligands [58,59] [58,59. ].

AsAs shown shown in in these these few few examples examples and and accordingaccording to the literature, many many copper copper-based-based complexes complexes havehave been been designed designed over over thethe past few few decades decades,, possessing possessing different different kind kindss of ligan of ligands,ds, substituents substituents and andgeometries geometries that that influence influence their their antimicrobial antimicrobial activities. activities. Regarding Regarding their their structural structural and and electronic electronic properties,properties, those those complexes complexes showed showed a promising a promising action action on microorganisms on microorganisms and are andrelevant are candidates relevant forcandidates further pharmaceutical for further pharmaceutical studies. studies.

2.3.2.3. Zinc Zinc TheThe average average human human body body contains contains 2–3 2– g3 of g zinc,of zinc, which which makes makes it the it the second second most most abundant abundantd-block d- naturalblock metalnatural ion metal in humans ion in humans after iron. after Asiron. it isAs involved it is involved in many in many vital vital cellular cellular reactions reactions at at its its low endogenouslow endogenous concentrations, concentrations, it is an it essentialis an essential element element for most for most living living species species [60,61 [60,61]. Zn]2. +Znions2+ ions have beenhave the been topic the of topic several of several studies, studies, demonstrating demonstrating their keytheir role key in role metalloenzymes in metalloenzymes and /and/oror metal-based metal- pharmaceuticalsbased pharmaceuticals [62–64], especially[62–64], especially as a recognized as a recognized antiseptic [65 antiseptic]. Their antimicrobial[65]. Their antimicrobial activities are activities are explained by two different mechanisms: (i) a direct interaction with microbial explained by two different mechanisms: (i) a direct interaction with microbial membranes leading to membranes leading to membrane destabilization and enhanced permeability [66]; (ii) an interaction membrane destabilization and enhanced permeability [66]; (ii) an interaction with nucleic acids and with nucleic acids and deactivation of enzymes of the respiratory system [67]. Taken together, these deactivation of enzymes of the respiratory system [67]. Taken together, these two modes of action lead two modes of action lead to cell death. to cell death. As Zn2+ ions are very close to Cu2+ ions in terms of size and charge density, their interactions 2+ 2+ withAs Zn O-, Nions- or are S- verydonor close ligands to Cu are ions quite in similar. terms of For size example, and charge novel density, Schiff their base interactions Zn(II) metal with O-,coordination N- or S- donor complexes ligands can are be quite designed similar. for For their example, interesting novel antimicrobial Schiff base properties. Zn(II) metal Yamgar coordination et al. complexessynthesized can be Zn(II) designed complexes for their possessing interesting significant antimicrobial antifungal properties. activities Yamgaret compared al. synthesized with standard Zn(II) complexesFluconazole, possessing that are significant 4 and 10 times antifungal higher against activities Candi comparedda albicans with and standard Aspergillus Fluconazole, niger, respectively that are 4 and[68 10].times In their higher comparative against Candida studies, albicans almost andall metalAspergillus complexes niger ,showed respectively increased [68]. Inactivity their comparativecompared studies,with Schiff almost base all ligand. metal complexesThey attributed showed these increased enhancements activity to the compared greater lipophilic with Schi natureff base of ligand. the Theycomplexes attributed, which these enhancementsfacilitates the penetrationto the greater through lipophilic the nature lipid ofmembrane the complexes, as discussed which facilitates above. theAdditionally penetration, through Sheikhshoaie the lipid et al. membrane showed aspromising discussed antimicrobial above. Additionally, activities, Sheikhshoaieas their square et al. showedpyramidal promising Zn(II) complexes antimicrobial had activities,both bacteriostatic as their and square bactericidal pyramidal effects Zn(II) again complexesst a wide range had bothof bacteriostaticbacteria and and fungi bactericidal [69]. effects against a wide range of bacteria and fungi [69].

Chemistry 2020, 2 858

ChemistryHowever, 2020, 2 according, x to the Irving–Williams series and the Ligand Field Stabilization Energy10 (LFSE), the stability of copper(II) complexes is higher than that of zinc(II) complexes. In general, complexHowever, stability accordingincreases as to the the ionic Irving radius–Williams decreases series across and the the Ligand series. Field Nevertheless, Stabilization unlike Energy Cu2 +, showing(LFSE), a the high stability stability of that copper(II) can be complexes attributed is to higher LFSE obtainedthan that through of zinc(II) the complexes. Jahn–Teller In distortion, general, Zncomplex2+ has low stability stability increases due toas athe lack ionic of radius LFSE fordecreases its d10 acrosselectronic the series. configuration, Nevertheless, and unlike thus Cu has2+, no preferenceshowing fora high any stability specific that ligand can be field attributed geometry. to LFSE These obtained principles through may the be Jahn an– explanationTeller distortion, of the 2+ 10 diffZnerences has obtained low stability between due these to a lack two oflatter LFSE metal for complexes its d electronic in some con studiesfiguration in terms, and of thus antimicrobial has no preference for any specific ligand field geometry. These principles may be an explanation of the activities [51,53,58,59,69], where the stronger affinity of Cu(II) for biomolecules could enhance the differences obtained between these two latter metal complexes in some studies in terms of permeability of the Cu(II) complexes through cell membrane [70]. For instance, considering the large antimicrobial activities [51,53,58,59,69], where the stronger affinity of Cu(II) for biomolecules could panel of complexes synthesized by Nazirkar et al., Cu(II) complexes possessing higher antibacterial enhance the permeability of the Cu(II) complexes through cell membrane [70]. For instance, activity against Mycobacteria Tuberculosis showed stronger efficacy compared to their Zn(II) counterparts considering the large panel of complexes synthesized by Nazirkar et al., Cu(II) complexes possessing 2+ byh aigher factor antibacterial of 62.5 [53]. activity Nonetheless, against theMycobacteria electronic Tuberculosis configuration showed and stronger the ability efficacy of Zn comparedions to haveto notheir geometry Zn(II) preferences counterparts could by a also factor be anof 62.5 advantage [53]. Nonetheless, in some cases the in electronic order to designconfiguration unique and structural the complexesability of thatZn2+ areions not to have suitable no geometry with other preferences metals. For could instance, also be Abuan advantage Ali et al. in synthesized some cases fivein order Zn(II) complexesto design withunique the structural nonsteroidal complexes anti-inflammatory that are not suitable drug with Ibuprofen other metals. in the For presence instance, of Abu N-donor Ali heterocyclicet al. synthesized ligands, five and Zn(II) having complexes different with carboxylate the nonsteroidal coordination anti-inflammatory modes (monodentate, drug Ibuprofen bidentate in andthe bridging presence bidentate, of N-donor see heterocyclic Figure7)[ 71ligands,]. They and determined having different their crystal carboxylate structures coordination by single-crystal modes X-ray(monodentate, diffraction bidentate and have and shown bridging that theirbidentate compounds, see Figure have 7) [ completely71]. They determined different structurestheir crystal and shapes,structures ranging by single from-crystal distorted X-ray tetrahedral diffraction to and hexagonal have shown to squarethat their planar compounds coordination have completely geometries. Theydifferent also investigated structures andin vitroshapes,antibacterial ranging from activities distorted of their tetrahedral complexes to hexagonal against both to square Gram-positive planar (Micrococcuscoordination luteus geometries., Staphylococcus They also aureus investigatedand Bacillus in vitro subtilis antibacterial) and Gram-negative activities of their (Escherichia complexes coli , Klebsiellaagainst pneumoniae both Gramand-positivProteuse (Micrococcus mirabilis) bacteria,luteus, Staphylococcus and the results aureus obtained and Bacillus exhibited subtilis a strong) and influenceGram- of thenegative geometry (Escherichia of the coli complexes, Klebsiella on pneumoniae their antimicrobial and Proteus activities. mirabilis) bacteria, and the results obtained exhibited a strong influence of the geometry of the complexes on their antimicrobial activities.

FigureFigure 7. 7.Structure Structure ofof zinc–Ibuprofenzinc–Ibuprofen complexes complexes [71 [71].].

According to the previously described properties, some new strategies have been developed in order to enhance the antimicrobial properties of such complexes. One of them is focusing on the

Chemistry 2020, 2 859

ChemistryAccording 2020, 2, x to the previously described properties, some new strategies have been developed in11 order to enhance the antimicrobial properties of such complexes. One of them is focusing on the development of new metallo-antibiotics that associate, all in one molecule, metal ions and organic development of new metallo-antibiotics that associate, all in one molecule, metal ions and organic antibiotics. Due to their multicomponent structure, these molecules can interact with several targets antibiotics. Due to their multicomponent structure, these molecules can interact with several targets in the bacteria. The weak interactions between Zn2+ ions and ligands should offer the possibility to in the bacteria. The weak interactions between Zn2+ ions and ligands should offer the possibility to release easily active molecules inside the target. In this context, Boughougal et al. synthesized a new release easily active molecules inside the target. In this context, Boughougal et al. synthesized a new model of a Zn-based complex (compound 8) based on the association of two complementary model of a Zn-based complex (compound 8) based on the association of two complementary antibiotics antibiotics as ligands (sulfadiazine and enrofloxacin), and an antiseptic central Zn(II) cation (Figure as ligands (sulfadiazine and enrofloxacin), and an antiseptic central Zn(II) cation (Figure8)[ 72]. 8) [72]. Structural determination of this complex was carried out using X-ray diffraction on single Structural determination of this complex was carried out using X-ray diffraction on single crystals and crystals and showed a cationic metal heart, where Zn2+ was located in an almost perfect square-base showed a cationic metal heart, where Zn2+ was located in an almost perfect square-base pyramidal − pyramidal geometry. The charge balance is ensured by ClO4 as counter-ions. Antimicrobial geometry. The charge balance is ensured by ClO4− as counter-ions. Antimicrobial experiments wereexperiments also performed, were also and performed the results, and obtained the results showed obtained MIC showed values ofMIC this values latter of complex this latter lower complex than 0.5lowerµg/ mLthan against 0.5 μg/mL different against bacteria different strains bacteria (E. coli, S.strains aureus (E.and coliE., faecalisS. aureus), which and E. was faecalis much), betterwhich than was thosemuch integrating better than only those one integrating type of antibiotic only one (sulfadiazine type of antibiotic or enrofloxacin). (sulfadiazine Combining or enrofloxacin). the cationic chargeCombining of the the compound cationic that charge facilitates of the the compound interaction that with facilitates the lipoidal the bacterial interaction membrane with the leading lipoidal to anbacterial enhanced membrane penetration leading ability to an inside enhanced the cell, penetration the synergetic ability eff ectinside of each the cell, chemical the synergetic entity appears effect toof stronglyeach chemical increase entity the antimicrobial appears to strongly activity ofincrease this model. the antimicrobial Further optimization activity of concerning this model. the Further choice ofoptimization antibiotics bindingconcerning metal the ions choice in order of antibiotics to provide binding an optimal metal association ions in order could to beprovide promising an optimal in the futureassociation for the could development be promising of new in the bioactive future moleculesfor the development to treat multi-drug of new bioactive resistant molecules diseases. to treat multi-drug resistant diseases.

FigureFigure 8. 8.Crystal Crystal structure structure of the of cationic the cationic Zn(II) Zn(II) complex comple8 withx sulfadiazine8 with sulfadiazine and enrofloxacin and enrofloxacin antibiotics usedantibiotics as ligands. used Reprintedas ligands. withReprinted permission with permission from ref. [72 from]. Copyright ref. [72]. Copyright 2020, Royal 2020, Chemical Royal Society.Chemical Society. Moreover, another example showing the great importance of the geometry and the structural configuration of a metallodrug is the complexation of a specific cyclam to Zn(II). Xylylbicyclam is Moreover, another example showing the great importance of the geometry and the structural a potent anti-HIV agent and is in clinical use as a stem-cell mobilizing drug (AMD3100, Figure9, configuration of a metallodrug is the complexation of a specific cyclam to Zn(II). Xylylbicyclam is a Compound 9). Its target is the co-receptor CXCR4, which assists the entry of HIV into cells and anchors potent anti-HIV agent and is in clinical use as a stem-cell mobilizing drug (AMD3100, Figure 9, stem cells in the bone marrow [73]. Additionally, it has been reported that there is a close correlation Compound 9). Its target is the co-receptor CXCR4, which assists the entry of HIV into cells and between the antiviral activity and the binding to the CXCR4 [74]. Therefore, as cyclams are known to be anchors stem cells in the bone marrow [73]. Additionally, it has been reported that there is a close strong metal-chelating agents, some studies have shown that the complexation of AMD3100 to several correlation between the antiviral activity and the binding to the CXCR4 [74]. Therefore, as cyclams metal ions, especially Zn2+, into the cyclam rings enhances the co-receptor binding strength (by a factor are known to be strong metal-chelating agents, some studies have shown that the complexation of of 36 in the case of Zn2+), resulting in good anti-HIV activity [75]. Further studies have also reported AMD3100 to several metal ions, especially Zn2+, into the cyclam rings enhances the co-receptor that there are only specific metallo-macrocycle configurations that can be recognized by the active binding strength (by a factor of 36 in the case of Zn2+), resulting in good anti-HIV activity [75]. Further site of the proteins to specific amino acid sidechains, H-bonding and hydrophobic interactions [76], studies have also reported that there are only specific metallo-macrocycle configurations that can be allowing optimization of drug design. Archibald et al. synthesized, for example, a configurationally recognized by the active site of the proteins to specific amino acid sidechains, H-bonding and restricted analogue of bismacrocyclic cyclam CXCR4 receptor antagonist (Figure9 compound 10) hydrophobic interactions [76], allowing optimization of drug design. Archibald et al. synthesized, for and its Zn(II) complex [77]. They showed that this latter complex adopts only one configuration in example, a configurationally restricted analogue of bismacrocyclic cyclam CXCR4 receptor antagonist (Figure 9 compound 10) and its Zn(II) complex [77]. They showed that this latter complex adopts only one configuration in solution leading to an enhancement of interactions with the protein receptor and an improvement of its anti-HIV activity, which is three times more potent than [Zn2(AMD3100]4+.

Chemistry 2020, 2 860 solution leading to an enhancement of interactions with the protein receptor and an improvement of 4+ itsChemistry anti-HIV 2020, activity, 2, x which is three times more potent than [Zn2(AMD3100] . 12

Figure 9. Antiviral macrocyclic bicyclams AMD3100 9; constrained analogue of AMD3100 10 [77]. Figure 9. Antiviral macrocyclic bicyclams AMD3100 9; constrained analogue of AMD3100 10 [77]. Rather than using antimicrobial compounds as ligands to obtain metal complexes, some assays Rather than using antimicrobial compounds as ligands to obtain metal complexes, some assays were carried out to associate metal complexes with antimicrobials as cofactors. This is the case, were carried out to associate metal complexes with antimicrobials as cofactors. This is the case, for for instance, for Starzac and her group, who were interested in the antimicrobial properties of instance, for Starzac and her group, who were interested in the antimicrobial properties of thiadiazole thiadiazole complexes of zinc and copper. Over a four members series of metal complexes (two copper complexes of zinc and copper. Over a four members series of metal complexes (two copper complexes complexes coordinating two thiadiazole ligands, and two zinc complexes coordinating one thiadiazole coordinating two thiadiazole ligands, and two zinc complexes coordinating one thiadiazole and one and one acetate), only the two zinc complexes displayed a definite antibacterial effect, with a MIC acetate), only the two zinc complexes displayed a definite antibacterial effect, with a MIC of 1.36 mM of 1.36 mM and 1.06 mM against S. aureus, and of 2.71 mM and 2.12 mM against E. coli, respectively. and 1.06 mM against S. aureus, and of 2.71 mM and 2.12 mM against E. coli, respectively. This is better This is better than the ligand alone (2.39 mM and 4.78 mM), but much higher than current antibacterials than the ligand alone (2.39 mM and 4.78 mM), but much higher than current antibacterials like like kanamycin, which demonstrates MICs of 8.05 and 16.10 µM with respect to the two bacterial kanamycin, which demonstrates MICs of 8.05 and 16.10 µM with respect to the two bacterial strains. strains. Only one of these two zinc complexes was tested together with kanamycin against S. aureus; Only one of these two zinc complexes was tested together with kanamycin against S. aureus; this this induced the fall of their respective MICs, which became 0.34 mM for the thiadiazole–zinc complex, induced the fall of their respective MICs, which became 0.34 mM for the thiadiazole–zinc complex, and 1.03 µM for the kanamycin [78]. Thus, rather than perform antimicrobial assays directly on new and 1.03 µM for the kanamycin [78]. Thus, rather than perform antimicrobial assays directly on new compounds, it is sometimes interesting to use them as cofactors of known antibiotics to determine their compounds, it is sometimes interesting to use them as cofactors of known antibiotics to determine synergistic effects. This will be discussed in more depth in the last chapter of our review (Section3). their synergistic effects. This will be discussed in more depth in the last chapter of our review (Section Therefore, the electronic properties of Zn2+ offer the possibility to design new antimicrobial drugs 3). that may have a wide range of structural geometries and interesting multicomponent arrangements for Therefore, the electronic properties of Zn2+ offer the possibility to design new antimicrobial interacting with several targets in the bacteria. These features could be further investigated in order to drugs that may have a wide range of structural geometries and interesting multicomponent enhance the activity and the selectivity as well as the bioavailability of the Zn(II) complexes. arrangements for interacting with several targets in the bacteria. These features could be further 2.4.investigated Iron in order to enhance the activity and the selectivity as well as the bioavailability of the Zn(II) complexes. As the most abundant transition metal in the human body, iron performs many important functions.2.4. Iron It is primarily involved in the transfer of oxygen from lungs to tissues by forming a complex, known as heme, between its ferrous form (Fe2+) and protoporphyrin IX in hemoglobin and myoglobin. A largeAs number the most of abundant enzymes transitionalso require metal iron in as the a cofactor human for body, their iron functions, performs especially many important for their electronfunctions. transfer It is primarily properties involved (cytochromes, in the iron-sulfur transfer of proteins) oxygen from or for lungs their to ability tissues to transport by forming and a 2+ storecomplex, iron (transferrin,known as heme, ferritin). between its ferrous form (Fe ) and protoporphyrin IX in hemoglobin and myoglobin.Furthermore, A large it number is well knownof enzymes that also iron require is an essential iron as a transition cofactor for metal their ion functions, for the growthespecially of pathogenicfor their electron bacteria, transfer which have properties different (cytochromes, processes for iron iron- acquisitionsulfur proteins) [79]. Taking or for into their account ability that to irontransport can be and coordinated store iron by(transferrin, organic molecules ferritin). presenting antimicrobial activity, the development of new metallo-antibioticsFurthermore, it is basedwell known on the processthat iron of ironis an acquisition essential transition could be ametal possible ion strategy for the ingrowth order toof enhancepathogenic the bacteria, antimicrobial which activity have different of active processes drugs inside for iron the cellacquisition by using [79 iron]. Taking as a carrier. into account Numerous that examplesiron can be were coordinated reported inby theorganic literature molecules about presenting iron complexes antimicrobial with antimicrobial activity, the activity development involving of thisnew strategy. metallo-antibiotics For example, based Tarallo on the et process al. synthesized of iron acquisition new iron–quinoxaline could be a possible derivative strategy compounds in order toto obtain enhance new the and antimicrobial more potent activity therapeutic of active tools drugs against inside tuberculosis the cell [80 by]. using Previous iron studies as a carrier. have alreadyNumerous displayed examples the were antibacterial reported activity in the literature of quinoxaline about derivativesiron complexes [81], with and antimicrobial thus by coordinating activity thoseinvolving ligands this to strategy. iron, the For authors example, expected Tarallo to increase et al. synthesized their bioactivity. new Theiron– resultsquinoxaline showed derivative that the compounds to obtain new and more potent therapeutic tools against tuberculosis [80]. Previous studies have already displayed the antibacterial activity of quinoxaline derivatives [81], and thus by coordinating those ligands to iron, the authors expected to increase their bioactivity. The results showed that the newly developed iron complexes have a significantly higher activity than the free ligands against Mycobacteria Tuberculosis, with MIC values of 0.78 μg/mL and 3.9–6.2 μg/mL

Chemistry 2020, 2 861 newly developed iron complexes have a significantly higher activity than the free ligands against Mycobacteria Tuberculosis, with MIC values of 0.78 µg/mL and 3.9–6.2 µg/mL respectively. These results are also comparable to or better than those obtained for clinical antibiotics such as streptomycin (MIC = 1.00 µg/mL), ciprofloxacin (MIC = 2.00 µg/mL) or gentamicin (MIC = 2.00–4.00 µg/mL). The high potential activity of these iron complexes could be explained by the fact that iron(III) acts as a carrier of bioactive ligands and thereby producing an enhanced concentration of these molecules inside the mycobacterial cells. Additionally, interesting results have attracted attention towards triazole derivatives, which are associated with various biological activities including antimicrobial properties, especially when they are functionalized with amino groups in order to obtain various Schiff bases [82,83]. Kharadi synthesized for instance Fe(III) complexes of 1,2,4-triazole Schiff bases that possess better antimicrobial activity against different Gram-positive and Gram-negative bacteria strains than that of the respective free ligands under identical experimental conditions [84]. The author attributed this bioactivity to the presence of quinolones in the complexes that interfere with enzyme production. Furthermore, as already mentioned above, chelation increases the lipophilic nature of the central metal atom, which in turn favors the permeation of the complex through the membrane of the microorganism and, hence, enhancing its activity. Moreover, another strategy that could be an option to enhance the antimicrobial properties of currently used organic drugs is the design of bio-organometallic derivatives containing either the antimicrobial drug, the active moiety of the drug, or using the metal to mimic a part of the drug [85]. Biot et al. demonstrated, for example, that the addition of a ferrocenyl moiety into the structure of the antimalarial chloroquine, to give the so-called organometallic complex ferroquine (compound 11) allowed additional modes of action compared to the parent organic drug (Figure 10a) [86]. Due to the presence of the ferrocene moiety, ferroquine is active against chloroquine-resistant parasitic strains. This higher efficiency can be explained by the redox properties of iron. Indeed, in addition to having a similar mechanism of action to chloroquine, ferroquine can also produce reactive oxygen species that are able to kill the parasites resistant to chloroquine. Ferroquine is thus one of the most advanced organometallic drug candidates, soon to enter in clinical phase III trials. Other examples have been reported for the preparation of bio-organometallic derivatives having antimicrobial properties and incorporating iron in the molecular structure. Edwards et al. prepared for the first time, in 1975, semi-synthetic derivatives of penicillin and cephalosporin (compounds 12 and 13) in which the conventional phenyl or heteroaromatic group has been replaced by a ferrocene moiety (Figure 10b) [87]. Since then, other ferrocenylamide derivatives of these two β-lactam antibiotics were synthesized and the resulting compounds showed variable degrees of antibacterial activity depending on the proximity of the metal atom and the β-lactam ring. By using the nutrient assimilation machinery of the bacteria, the development of new metallo-antibiotics based on the ability of active organic molecules to easily coordinate iron ions could be a promising approach in order to enhance the antimicrobial activity. Further strategies including the design of new organometallic derivatives of conventional antimicrobial drugs based on structure-relationship methods is still underdeveloped and due to their modes of action that are different from the current antimicrobial agents, they may be applied to tackle multi-drug resistant diseases. Chemistry 2020, 2, x 13 respectively. These results are also comparable to or better than those obtained for clinical antibiotics such as streptomycin (MIC = 1.00 μg/mL), ciprofloxacin (MIC = 2.00 μg/mL) or gentamicin (MIC = 2.00–4.00 μg/mL). The high potential activity of these iron complexes could be explained by the fact that iron(III) acts as a carrier of bioactive ligands and thereby producing an enhanced concentration of these molecules inside the mycobacterial cells. Additionally, interesting results have attracted attention towards triazole derivatives, which are associated with various biological activities including antimicrobial properties, especially when they are functionalized with amino groups in order to obtain various Schiff bases [82,83]. Kharadi synthesized for instance Fe(III) complexes of 1,2,4-triazole Schiff bases that possess better antimicrobial activity against different Gram-positive and Gram-negative bacteria strains than that of the respective free ligands under identical experimental conditions [84]. The author attributed this bioactivity to the presence of quinolones in the complexes that interfere with enzyme production. Furthermore, as already mentioned above, chelation increases the lipophilic nature of the central metal atom, which in turn favors the permeation of the complex through the membrane of the microorganism and, hence, enhancing its activity. Moreover, another strategy that could be an option to enhance the antimicrobial properties of currently used organic drugs is the design of bio-organometallic derivatives containing either the antimicrobial drug, the active moiety of the drug, or using the metal to mimic a part of the drug [85]. Biot et al. demonstrated, for example, that the addition of a ferrocenyl moiety into the structure of the antimalarial chloroquine, to give the so-called organometallic complex ferroquine (compound 11) allowed additional modes of action compared to the parent organic drug (Figure 10a) [86]. Due to the presence of the ferrocene moiety, ferroquine is active against chloroquine-resistant parasitic strains. This higher efficiency can be explained by the redox properties of iron. Indeed, in addition to having a similar mechanism of action to chloroquine, ferroquine can also produce reactive oxygen species that are able to kill the parasites resistant to chloroquine. Ferroquine is thus one of the most advanced organometallic drug candidates, soon to enter in clinical phase III trials. Other examples have been reported for the preparation of bio-organometallic derivatives having antimicrobial properties and incorporating iron in the molecular structure. Edwards et al. prepared for the first time, in 1975, semi- synthetic derivatives of penicillin and cephalosporin (compounds 12 and 13) in which the conventional phenyl or heteroaromatic group has been replaced by a ferrocene moiety (Figure 10b) [87]. Since then, other ferrocenylamide derivatives of these two β-lactam antibiotics were synthesized and the Chemistry resulting2020 , compounds2 showed variable degrees of antibacterial activity depending862 on the proximity of the metal atom and the β-lactam ring.

Figure 10. Bio-organometallic compounds of conventional antibacterial drugs. (a) Schematic structures Figure 10. Bio-organometallic compounds of conventional antibacterial drugs. (a) Schematic of chloroquine and ferroquine, two of the most commonly used antimalarial compounds [86]; structures(b )of semi-synthetic chloroquine derivatives and ferroquine, of penicillin two and of cephalosporin the most commonly [87]. used antimalarial compounds [86]; (b) semi-synthetic derivatives of penicillin and cephalosporin [87]. 2.5. Ruthenium In general, transition metals of the second and third row, with closed electron shells have shown interesting applications in medicinal inorganic chemistry when structurally rigid compounds are required. The replacement of a metal center with its heavier homologs of the same group leads to isostructural complexes with mild changes of the geometrical parameters but that differ significantly in the redox properties or in kinetics. The replacement of the ferrocenyl moiety by a ruthenocene unit to give ruthenoquine, a structural analog of ferroquine presented in the previous section, led for example to a decreased potency due to the absence of redox chemistry [88]. Other studies revealed that the chemical inertness of ruthenium complexes could also be used to define a portion of space with a precise geometry yielding in a structure that has stereo-electronic complementarity with the active site of a target. This concept was employed by the group of Meggers, wherein, mimicking the natural product staurosporin, Ru(II) complexes were designed in order to act as inhibitors of protein kinases [89]. Although ruthenium complexes have been widely studied for their anticancer activity, some interesting antimicrobial properties were also described since the mid-twentieth century by Dwyer [90,91]. Among all the tested transition metals, it was shown that the inert 2+ polypyridylruthenium(II) complex [Ru(Me4phen)3] exhibited remarkable antimicrobial activity in vitro, particularly against Gram-positive strains [92]. The activity of these octahedral complexes is probably due to their ability to interact with nucleic acids through intercalation with aromatic bases, aided by electrostatic attraction between the positive charged-metal complex and the negative charged phosphate groups. Aldrich-Wright and co-workers also reported that their mononuclear Ru(II) 2+ complexes [Ru(2,9-Me2phen)2(dppz)] (compound 14, Figure 11) incorporating DNA intercalating ligands exhibited significant antibacterial activity against Gram-positive bacteria (B. subtilis and S. aureus, MIC = 2–8 µg/mL) [93]. However, these complexes were not active against the Gram-negative bacterium E. coli. The mechanism of action remained unclear but the hypothesis mentioned is that the Ru(II) complexes were not able to cross the outer membrane of E. coli. Chemistry 2020, 2 863 Chemistry 2020, 2, x 15

FigureFigure 11. 11.Structures Structures of of antimicrobial antimicrobial ruthenium ruthenium complexes presenting presenting different different modes modes of of action. action. (a,(ba),b Interaction) Interaction with with DNA DNA through through intercalation; intercalation; ( c(c)) aPDT-active aPDT-active Ru(II)Ru(II) complexcomplex [[9292,,94,95].].

2+ The same was observed by Sun et al., who tested a [Ru(phen )(p-BPIP)]2+ against E. coli, The same was observed by Sun et al., who tested a [Ru(phen2)(p2 -BPIP)] against E. coli, M. M.tetrage tetragenusnus, S., S.aureus aureus, and, and some some moistures. moistures. Antibacterial Antibacterial activity activity was found was only found against only againstboth Gram both- Gram-positivepositive bacteria. bacteria. Further Further scanning scanning electron electronmicroscopy microscopy studies showed studies non showed-smooth non-smooth cell walls, and cell walls,parts and of the parts cytoplasm of the cytoplasm outside of outside the bacteria of the cell. bacteria Finally, cell. it Finally,was demonstrated it was demonstrated that the complex that the complexcauses thecauses fragmentation the fragmentation of the DNA. of the Therefore, DNA. Therefore, it was concluded it was concluded that the that treatment the treatment of Gram of- Gram-positivepositive bacteria bacteria by the byruthenium the ruthenium complex complex resulted resultedin a perforated in a perforated membrane membranedue to its interaction due to its interactionwith the bacterial with the cell bacterial wall, i cellncreasing wall, increasingthe permeability, the permeability, even if the even main if mechanism the main mechanism of action of of 2+ action[Ru(phen of [Ru(phen2)(p-BPIP)]2)(p-BPIP)]2+ (Figure 11(Figure, compound 11, compound 15) is the damage15) is the of damage the DNA of and the DNARNA [ and94]. RNA [94]. Furthermore,Furthermore, ruthenium ruthenium polypyridyl polypyridyl complexes complexes can can also also act actas photosensitizers, as photosensitizers, generating generating ROS uponROS light upon irradiation light irradiation for use for in antibacterialuse in antibacterial photodynamic photodynamic therapy therapy (aPDT). (aPDT). The multi-target The multi- featuretarget offeature ROS not of ROS only not renders only renders aPDT highly aPDT potenthighly potent in killing in killing even multidrug-resistanteven multidrug-resistant bacteria, bacteria, but but also makesalso makes bacteria bacteria difficult difficult to develop to develop any resistance any resistance against against these multiplethese multiple attacks. attacks. However, However, it is a greatit is challengea great challenge to inactivate to inactivate selectively selectively bacterial bacterial cells while cells while leaving leaving mammalian mammalian cells cells una ffunaffected.ected. Inthis In context,this context, ruthenium ruthenium polypyridyl polypyridyl complexes complexes are promising are promising candidates candidates as aPDT as agentsaPDT agents due to due their to overall their positiveoverall charge, positive which charge may, which promote may interactions promote interactions with the negatively with the negativelycharged bacterial charged membrane, bacterial membrane,1 their high 1O2 yields, and excellent chemical stability and photostability. In 2007, their high O2 yields, and excellent chemical stability and photostability. In 2007, Donnelly et al. wereDonnelly the first et to al.describe were these the firstfeatures to by describe analyzing these photophysical features by and analyzing microbiological photophysical behaviors and of theirmicrobiological Ru(II) complexes. behaviors They of showedtheir Ru(II) that complexes upon white. They light showed irradiation, that upon their white compounds light irradiation, presented MICtheir values compounds of 12.5, presented 50 and 12.5MICµ valuesg/mL againstof 12.5, S.50 aureusand ≤12.5, P. aeruginosa μg/mL againstand C. S. albicansaureus, ,P. respectively. aeruginosa ≤ Unfortunately,and C. albicans no, respectively. toxicological Unfortunately, studies against nohuman toxicological cell lines studies were against reported human [96]. cell More lines recently, were Fengreported et al. depicted[96]. More a seriesrecently, of charged Feng et rutheniumal. depicted complexes a series of by charged using quaternary rutheniumammonium-modified complexes by using quaternary ammonium-modified bipyridine as ligand [95]. Their results indicated that the most bipyridine as ligand [95]. Their results indicated that the most highly charged complex (Figure 11, highly charged complex (Figure 11, compound 16), bearing eight positive charges, exhibited the most compound 16), bearing eight positive charges, exhibited the most potent aPDT activity against S. aureus, potent aPDT activity against S. aureus, displaying 6–7 log reduction in bacterial viability (comparable displaying 6–7 log reduction in bacterial viability (comparable to the traditional antibiotic vancomycin to the traditional antibiotic vancomycin at equal concentrations) when irradiated with 470 nm light,

Chemistry 2020, 2 864

Chemistry 2020, 2, x 16 at equal concentrations) when irradiated with 470 nm light, while only minor activity was observed while only minor activity was observed against Gram-negative bacterium E. coli, probably due to its against Gram-negative bacterium E. coli, probably due to its dense and compact outer membrane which dense and compact outer membrane which may hamper the photodegradation. Damages and may hamper the photodegradation. Damages and deformations of cell walls in S. aureus have also been deformations of cell walls in S. aureus have also been observed by scanning electron microscopy observed by scanning electron microscopy (SEM) for the aPDT-treated cells, pointing to the highly (SEM) for the aPDT-treated cells, pointing to the highly negatively charged bacterial surfaces as the negatively charged bacterial surfaces as the target of this class of compounds. Co-culture experiments target of this class of compounds. Co-culture experiments revealed the selective photoinactivation of revealed the selective photoinactivation of compound 16 toward bacterial cells over mammalian cells. compound 16 toward bacterial cells over mammalian cells. The spherical octahedral coordination The spherical octahedral coordination structure and hydrophilic cationic character of the Ru(II) core structure and hydrophilic cationic character of the Ru(II) core may lack the interaction with may lack the interaction with cytoplasmic membranes and therefore could be responsible for their cytoplasmic membranes and therefore could be responsible for their lower affinity toward lower affinity toward mammalian cells. mammalian cells. Another light-mediated strategy was pursued by Smith et al., who designed a photoactive Another light-mediated strategy was pursued by Smith et al., who designed a photoactive ruthenium(II) complex incorporating the anti-tuberculosis drug isoniazid (Figure 12, compound ruthenium(II) complex incorporating the anti-tuberculosis drug isoniazid (Figure 12, compound 17) 17)[97] that could be further released from the Ru(II) core upon 463 nm light irradiation. This results [97] that could be further released from the Ru(II) core upon 463 nm light irradiation. This results in in a selective high potency towards Mycobacterium smegmatis (MIC = 4 µM) over both Gram-positive a selective high potency towards Mycobacterium smegmatis (MIC = 4 μM) over both Gram-positive (B. (B. subtilis) and Gram-negative (E. coli) bacteria within no activity was observed. This was a 5.5-fold subtilis) and Gram-negative (E. coli) bacteria within no activity was observed. This was a 5.5-fold increase in potency compared to the title compound isoniazid. Moreover, this Ru(II) compound was increase in potency compared to the title compound isoniazid. Moreover, this Ru(II) compound was found to be non-toxic to human lung cell line. found to be non-toxic to human lung cell line.

2+ FigureFigure 12. 12.Stepwise Stepwise photoactivationphotoactivation ofof thethe antibacterialantibacterial prodrug prodrug cis-[Ru(bpy) cis-[Ru(bpy)22(INH)(INH)22]]2+ 1177 [[9797]]..

Another approach approach reported reported firstly firstly by Keene by Keene and Collins and inCollins 2011 was in to 2011 investigate was to the investigate antimicrobial the activityantimicrobial of an extensive activity of range an of extensive mono-, di- range and oligonuclear of mono-, inert di- polypyridylruthenium(II)and oligonuclear inert complexespolypyridylruthenium(II) [98]. The results complexes demonstrated [98]. The that results for the demonstrated dinuclear Ru(II) that complexes for the d linkedinuclear by Ru(II) long flexiblecomplexes alkane linked chains by long (compounds flexible alkane18, Figure chains 13 (compound) are highlys active18, Figure against 13) bothare highly Gram-positive active against and Gram-negativeboth Gram-positive bacteria and (with Gram MIC-negative values bacteria that are (with comparable MIC values or better that than are comparable the well-known or better antibiotic than gentamicin),the well-known but areantibiotic considerably gentamicin), less toxic but to are human considerably eukaryotic less cells. toxic In to subsequent human eukaryotic studies, the cells. same In groupsubsequent exhibited studies, that thethe antimicrobialsame group activityexhibited of that these the complexes antimicrobial is correlated activity with of these the level complexes of cellular is uptake.correlated As with the longer the level alkyl of cellular chain length uptake. leads As tothe more longer lipophilic alkyl chain compounds, length leads the authorsto more concluded lipophilic compounds, the authors concluded that the increasing lipophilicity is responsible for the higher uptake [99]. Later, they showed that compound 18 with n = 16 condensed ribosomes when they

Chemistry 2020, 2 865

Chemistry 2020, 2, x 17 that the increasing lipophilicity is responsible for the higher uptake [99]. Later, they showed that existed as polysomes,compound 18 withleadingn = 16 to condensed stop the ribosomes protein whenproduction, they existed and as polysomes,thereby inhibit leading tothe stop bacterial the growth protein production, and thereby inhibit the bacterial growth [100]. [100].

Figure 13. Structure of dinuclear ruthenium(II) complexes [98]. Figure 13. Structure of dinuclear ruthenium(II) complexes [98]. Although the mode of action of polypyridylruthenium(II) complexes is not completely understood, DNA binding is normally considered as the major interaction leading to the antimicrobial activity. Although the mode of action of polypyridylruthenium(II) complexes is not completely However, some recent interesting studies have shown that these complexes could behave as understood,effi cient DNA photoactivable binding prodrug is normally delivery systems,considered and both as photodynamic the major antimicrobial interaction therapy leading to the antimicrobialand activity. photorelease However, antimicrobial some therapy recent are promising interesting strategies studies for overcoming have shown bacterial that infections. these complexes could behaveTaking as into efficient account these photoactivable recent investigations, prodrug the design delivery of the next generation systems, of and ruthenium-based both photodynamic antimicrobial agents are on the way. Nevertheless, there are only limited data available at this stage antimicrobialconcerning therapyin vivo andefficiency photorelease of these compounds, antimicrobial and future research therapy will have are to promising focus on this area. strategies for overcoming bacterial infections. Taking into account these recent investigations, the design of the 2.6. Gallium next generation of ruthenium-based antimicrobial agents are on the way. Nevertheless, there are only limited data availableDepriving bacteria at this of stage essential concerning nutrients, such in as vivo iron, isefficiency a viable strategy of these for the development compounds, of and future new antimicrobials. As mentioned earlier, iron plays a vital role for all forms of life, including bacteria, research willas ahave cofactor to of focus many vitalon this enzymes. area. With the emergence of multi drug resistant pathogens, gallium has gained interest due to its ability to interact with proteins involved in iron metabolism of a wide range of 2.6. Galliumbacteria based on an intriguing “Trojan horse” strategy [101,102]. The ionic radius and charge density of Ga3+ (r = 62 pm, ρ = 3.01) is effectively almost identical to Fe3+ (r = 55 pm, ρ = 4.30), and many Deprivingbiological bacteria systems of involving essential in microorganismsnutrients, such are as not iron, able to is distinguish a viable easilystrategy these for two the metal development 3+ of new antiions.microbials. Therefore, itAs has mentioned been shown in earlier, different iron studies plays that Ga a vitalions canrole inhibit for theall growth forms of of many life, including bacterial and fungal species by interfering with iron-dependent metabolic pathways [103,104]. As the bacteria, asinsertion a cofactor of Ga of3+ manyinto the vital active enzymes. site of Fe-dependent With the proteins emergence and enzymes of multi renders drug them resistant inactive, pathogens, gallium hasit gained is worth mentioninginterest due that to unlike its ability Fe3+, Ga 3to+ cannotinteract be reducedwith proteins under physiological involved conditions in iron metabolism in of a wide rangebiological of bacteria systems [based105]. It ison also an important intriguing to note “Trojan that gallium horse” is resistant strategy to known [101 bacterial,102]. eThefflux ionic radius pumps, preventing the development of bacterial resistance. 3+ 3+ and charge densityThe antimicrobial of Ga (r properties = 62 pm, of galliumρ = 3.01) were is discovered effectively in 1931 almost by Levaditi identical et al. who to reportedFe (r = 55 pm, ρ = 4.30), and manythat gallium biological tartrate eradicatedsystems syphilisinvolving in rabbits in microorganisms and Trypanosoma evansi arein not mice able [106]. to However, distinguish easily these two metalwith the ions. introduction Therefore, and use it ofhas antibiotics, been shown gallium in has different been overlooked studies until that recently. Ga3+ Since ions then, can inhibit the growth of manygallium bacterial compounds, and ranging fungal from species simple gallium by interfering salts to more with complex iron structures-dependent have advanced metabolic in pathways preclinical and clinical investigations [107]. They can be grouped as first-, second- and third-generation 3+ [103,104]. Asgallium the compounds,insertion of which Ga become into increasinglythe active diverse site of with Fe the-dependent binding ability proteins of gallium and to di ffenzymeserent renders them inactive, it is worth mentioning that unlike Fe3+, Ga3+ cannot be reduced under physiological conditions in biological systems [105]. It is also important to note that gallium is resistant to known bacterial efflux pumps, preventing the development of bacterial resistance. The antimicrobial properties of gallium were discovered in 1931 by Levaditi et al. who reported that gallium tartrate eradicated syphilis in rabbits and Trypanosoma evansi in mice [106]. However, with the introduction and use of antibiotics, gallium has been overlooked until recently. Since then, gallium compounds, ranging from simple gallium salts to more complex structures have advanced in preclinical and clinical investigations [107]. They can be grouped as first-, second- and third- generation gallium compounds, which become increasingly diverse with the binding ability of gallium to different ligands. In this context, gallium nitrate represents the first generation of gallium compounds, as it was the first drug to enter in clinical trials, approved by the Food and Drug Administration (FDA) for the treatment of calcium-associated hypercalcemia and commercialized since 2012 with the brand name Ganite® . This latter gallium formulation has been shown to possess promising activity against different bacterial strains such as P. aeruginosa, A. baumanii and M. tuberculosis [107]. Other gallium(III) salts such as GaCl3 or Ga(III)-citrate, Ga(NO3)3 also exhibited

Chemistry 2020, 2 866 ligands. In this context, gallium nitrate represents the first generation of gallium compounds, as it was the first drug to enter in clinical trials, approved by the Food and Drug Administration (FDA) for the treatment of calcium-associated hypercalcemia and commercialized since 2012 with the brand name Ganite®. This latter gallium formulation has been shown to possess promising activity against different bacterial strains such as P. aeruginosa, A. baumanii and M. tuberculosis [107]. Other gallium(III) salts such as GaCl3 or Ga(III)-citrate, Ga(NO3)3 also exhibited potent suppressive activity on P. aeruginosa biofilm formation in vitro and in murine lung infection models [108]. The second generation of gallium compounds led from simple gallium salts to gallium complexes with organic anions, like gallium citrate or gallium maltolate (compound 19, Figure 14). This latter compound consists of Ga3+ bound to three maltol ligands in a stable coordination geometry with increased solubility and reduced the probability of forming toxic precipitates, allowing better bacterial cell growth inhibition and apoptosis than the first generation of gallium-based compounds, which could be explained by a greater cellular uptake [109]. DeLeon et al. demonstrated that gallium maltolate promoted the survival of all P. aeruginosa-infected, thermally injured mice at significantly lower Ga concentrations than Ga(NO3)3, while it appeared to be well tolerated at all of the doses delivered, as no gross clinical signs of toxicity were observed [110]. The authors also showed a considerable inhibition of the growth of both S. aureus and A. baumannii pathogens, exhibiting the high potential of gallium maltolate to treat both Gram-positive and Gram-negative bacteria in wounds. They hypothesized that the increased lipophilicity of the gallium maltolate formulation and its reduced tendency to form insoluble gallate precipitates could be the reason for the observed greater efficacy over Ga(NO3)3. In addition, by relying on the antimicrobial activity of gallium compounds against a variety of microorganisms, Aridis Pharmaceuticals developed a novel formulation based on gallium citrate (Panaecin™) that recently entered clinical trials for evaluation of its efficacy in certain infections. The third generation of gallium complexes in preclinical evaluation includes gallium bound to ligands such as hydroxamic acid, protoporphyrins, pyridine, hydrazones and others [111–115]. Different studies revealed for example that the heme-mimetic gallium protoporphyrin IX (GaPPIX, compound 20, Figure 14) is likely to exploit heme-uptake routes to enter bacterial cells, where it could inhibit the iron metabolism, resulting in the perturbation of vital cellular functions [116]. According to this, it has been shown that GaPPIX possesses a good antibacterial activity against several bacterial species, including S. aureus, A. baumannii, M. smegmatis or P. aeruginosa [114,116–118]. Because Ga(NO3)3 and Ga porphyrin disrupt different pathways of bacterial ion acquisition and use, a combination of these two types of gallium compounds would result in enhanced antimicrobial activity. This concept was used by Choi et al. who described the in vitro synergistic effect against both Gram-positive (methicillin-resistant S. aureus, MRSA) and Gram-negative bacteria (K. pneumoniae and P. aeruginosa) by combining these two latter components, while a significant reduction of the bacterial populations in K. pneumoniae and P. aeruginosa biofilms have been observed [119]. The same synergistic strategy was employed by Banin et al., who used a combination of a gallium(III) complex (GaDFO, compound 21, Figure 14), where the ligand desferrioxamine is a strong siderophore involved in P. aeruginosa iron metabolism with the anti-Pseudomonas antibiotic gentamicin [120]. Their in vivo studies showed that such combination was able to reduce the bacterial infiltration and final scar size by about 50% in a rabbit keratitis model compared to topical application of gentamicin alone. Moreover, it is also important to note that most studies exhibited the antimicrobial activity of gallium compounds employed iron-poor media, usually through the addition of an iron chelator. Indeed, high concentrations of iron have shown to reduce gallium activity. This was shown by Hijazi et al. who provided a comparative study of Ga(NO3)3, gallium maltolate and gallium protoporphyrin IX, belonging to the first, second and third generation of Ga(III) formulations, respectively [104]. They investigated the antimicrobial activity of these three compounds against ESKAPE species (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) under different growth conditions (standard culture medium Mueller Hinton broth, MHB; iron-depleted MHB, DMHB and RPMI-1640 supplemented with 10% human serum, RPMI-HS) containing different iron proportions. Chemistry 2020, 2, x 18

potent suppressive activity on P. aeruginosa biofilm formation in vitro and in murine lung infection models [108]. The second generation of gallium compounds led from simple gallium salts to gallium complexes with organic anions, like gallium citrate or gallium maltolate (compound 19, Figure 14). This latter compound consists of Ga3+ bound to three maltol ligands in a stable coordination geometry with increased solubility and reduced the probability of forming toxic precipitates, allowing better bacterial cell growth inhibition and apoptosis than the first generation of gallium-based compounds, which could be explained by a greater cellular uptake [109]. DeLeon et al. demonstrated that gallium maltolate promoted the survival of all P. aeruginosa-infected, thermally injured mice at significantly lower Ga concentrations than Ga(NO3)3, while it appeared to be well tolerated at all of the doses delivered, as no gross clinical signs of toxicity were observed [110]. The authors also showed a considerable inhibition of the growth of both S. aureus and A. baumannii pathogens, exhibiting the high potential of gallium maltolate to treat both Gram-positive and Gram-negative bacteria in wounds. They hypothesized that the increased lipophilicity of the gallium maltolate formulation and its reduced tendency to form insoluble gallate precipitates could be the reason for the observed greater efficacy over Ga(NO3)3. In addition, by relying on the antimicrobial activity of gallium compounds against a variety of microorganisms, Aridis Pharmaceuticals developed a novel formulation based on gallium citrate (Panaecin™) that recently entered clinical trials for evaluation of its efficacy in certain infections. The third generation of gallium complexes in preclinical evaluation includes gallium bound to ligands such as hydroxamic acid, protoporphyrins, pyridine, hydrazones and others [111–115]. Different studies revealed for example that the heme-mimetic gallium protoporphyrin IX (GaPPIX, compound 20, Figure 14) is likely to exploit heme-uptake routes to enter bacterial cells, where it could inhibit the iron metabolism, resulting in the perturbation of vital cellular functions [116]. According to this, it has been shown that GaPPIX possesses a good antibacterial activity against several bacterial species, including S. aureus, A. baumannii, M. smegmatis or P. aeruginosa [114,116–118]. Because Ga(NO3)3 and Ga porphyrin disrupt different pathways of bacterial Chemistryion acquisition2020, 2 and use, a combination of these two types of gallium compounds would result in867 enhanced antimicrobial activity. This concept was used by Choi et al. who described the in vitro Thesynergistic authors showed effect against that the both antibacterial Gram-positive properties (methicillin of the- galliumresistantcompounds S. aureus, MRSA) depend and strongly Gram on- negative bacteria (K. pneumoniae and P. aeruginosa) by combining these two latter components, while the media. All ESKAPE species were resistant to the more labile compounds Ga(NO3)3 and gallium a significant reduction of the bacterial populations in K. pneumoniae and P. aeruginosa biofilms have maltolate in MHB and DMHB (MIC > 32 µM), while GaPPIX showed some bactericidal activity been observed [119]. The same synergistic strategy was employed by Banin et al., who used a under these conditions against S. aureus (MIC = 0.06–0.12 µM) and A. baumannii (MIC = 16–32 µM) combination of a gallium(III) complex (GaDFO, compound 21, Figure 14), where the ligand strains. Conversely, in RPMI-HS, the presence of serum albumin, which interferes with GaPPIX but desferrioxamine is a strong siderophore involved in P. aeruginosa iron metabolism with the anti- not with Ga(NO ) or gallium maltolate indicates that among the three Ga(III) compounds tested, Pseudomonas antibiotic3 3 gentamicin [120]. Their in vivo studies showed that such combination was the FDA-approved gallium nitrate and the orally active gallium maltolate were the most effective able to reduce the bacterial infiltration and final scar size by about 50% in a rabbit keratitis model under conditions that better simulate the low iron content in in vivo environment. compared to topical application of gentamicin alone.

Figure 14. Structures of antimicrobial gallium(III) complexes [109,116,120]. Figure 14. Structures of antimicrobial gallium(III) complexes [109,116,120]. Recently, Pandey et al. introduced and assessed the gallium(III) complex of ciprofloxacin- functionalized siderophore desferrichrome as a potential theranostic conjugate [121]. They demonstrated the ability of their compound to act both as a potential therapeutic system for bacterial infection using an in vitro assay and a tracer-based approach employing the radioactive 67Ga. Their results highlighted first the good antibacterial activity of their theranostic gallium conjugate against both Gram-negative (E. coli, MIC = 0.23 µM) and Gram-positive (S. aureus, P. aeruginosa and K. pneumoniae with MIC = 1.9, 3.8 and 12.5 µM respectively) strains in iron-deficient media. Secondly, the authors showed that their radiolabeled-67Ga complexes were able to quantify time-dependent in vitro uptake in bacteria, and in vivo pharmacokinetics in mice. In conclusion, several studies have demonstrated the antimicrobial activity of Ga(III) both in vitro and in vivo. These promising results raise the hope that gallium will confirm its efficacy in clinical trials and will become a valuable therapeutic option in order to cure untreatable bacterial infections. The recent development of gallium conjugates as new theranostic platforms also opens the door for future investigations in bacterial infections by combining both diagnostic and therapeutic tools.

2.7. Bismuth Since the 18th century, bismuth compounds have been used in medicine for the treatment of syphilis, colitis, wound infection and quartan malaria, but mostly for gastrointestinal disorders [122]. Indeed, Bi(III) is well known to exhibit remarkably low toxicity against humans as it is well tolerated at high doses, while being potently toxic against bacteria. Three bismuth-based drugs, namely bismuth subsalicylate (BSS, Pepto-Bismol®), colloidal bismuth subcitrate (CBS, De-Nol®) and ranitidine bismuth citrate (RBC, Pylorid®), have been used clinically in combination with antibiotics to treat infection associated with Helicobacter pylori (Figure 15), a bacterium that leads to the generation of gastritis, ulcers in the gastrointestinal tract, and gastric cancers [123]. Clarithromycin or metronidazole used as the antibiotics of choice to kill this bacterium induce actually a decrease in bactericidal efficiency over time with an increase bacterial resistance. This acquired resistance can then be partially overcome through coadministration of some antibiotics (metronidazole, tetracycline hydrochloride, amoxicillin, Chemistry 2020, 2 868 clarithromycin) with CBS or RBC, so-called bismuth-based triple or quadruple therapy [124,125]. Generally, the antiulcer activity of bismuth-containing medicines is explained by the precipitation of bismuth, probably as BiOCl and bismuth citrate complexes, within the ulcer crater resulting in the Chemistryformation 2020 of, 2, a x protective coating, which contributes to the healing of the lesion [122]. 20

FigureFigure 15. 15. StructuresStructures of ofclinically clinically used used bismuth(III) bismuth(III) compounds compounds to treat to treat infection infection associated associated with with H. pyloriH. pylori. (a).( Bismutha) Bismuth subsalicylate; subsalicylate; (b) (colloidalb) colloidal bismuth bismuth subcitrate; subcitrate; (c) ( cranitidine) ranitidine bismuth bismuth citrate citrate [123 [123]. ].

EveEvenn if if the the exact exact molecular molecular mechanisms mechanisms describing describing the the anti anti--H.H. pylori pylori activityactivity of of Bi Bi-based-based drugs drugs havehave not not been been fully fully understood, understood, the the biological biological targets targets of of such such compounds compounds are are clearly clearly related related to to their their bindingbinding with with specific specific proteins proteins and and enzymes enzymes [ [126126]].. Recent Recent stru structuralctural studies studies of of bismuth bismuth complexes complexes havehave indicated indicated that that Bi(III) Bi(III) has has a avariable variable coordination coordination number number (from (from 3 3 to to 10), 10), an an irregular irregular coordination coordination chemistrychemistry and and an an acidic acidic behavior, behavior, properties properties which which render render this this metal metal ion ion highly highly effective effective to to interact interact withwith a a wide rangerange ofof proteins.proteins. Among Among all all of of them, them, transferrin transferrin and and lactoferrin lactoferrin proteins proteins used used by H. by pylori H. pylorifor iron for iron acquisition acquisition have have been been shown shown to to be be the the specific specific targets targets ofof different different Bi Bi-based-based compounds. compounds. TheThe binding binding of of bismuth bismuth to to these these proteins proteins can can prevent prevent the the acquisitio acquisitionn of of iron iron into into the the pathogens, pathogens, resultingresulting in in the the perturbation perturbation of of their their biological biological pathways pathways and and their their death death [ [126126]].. It It is is also also well well known known thatthat Bi(III) Bi(III) has has a a high high affinity affinity with with thiolate thiolate ligands ligands,, and and thiolation thiolation of of Bi(III) Bi(III) is is thought thought to to be be one one of of the the majormajor bioche biochemicalmical fate fatess of of bismuth bismuth in in biological biological fluids fluids and and cells. cells. Bi(III) Bi(III) readily readily binds binds to to the the cysteine cysteine residuesresidues in amino amino acids acids and and peptides, peptides, making making Bi-based Bi-based drugs drugs particularly particularly effective effective for inhibiting for inhibiting several severalenzymes enzymes from H. from pylori H., for pylori example,, for example, ureases orureases cytosolic or cytosolic alcohol dehydrogenasealcohol dehydrogenase (ADH) [ 127 (ADH),128]. [Urease,127,128] a. Urease, dinuclear a Ni(II)dinuclear enzyme Ni(II) that enzyme converts that urea converts into ammonia urea into and ammonia carbonic and acid, carbonic is crucial acid, for theis crucialbacteria for to the grow bacteria in the to highly grow in acidic the highly stomach acidic environment. stomach environment. Zhang et al. Zhang demonstrated, et al. demonstrated for instance,, that their bismuth complexes, Bi(EDTA), Bi(Cys) and RBC, inhibited urease activity effectively through for instance, that their bismuth complexes, Bi(EDTA),3 Bi(Cys)3 and RBC, inhibited urease activity effectivelyboth competitive through and both non-competitive competitive and inhibition non-competitive modes. inhibition The authors modes. suggest The thatauthors Bi(III) suggest binds that to a Bi(III)cysteine binds residue to a cysteine of the enzyme residue located of the atenzyme the entrance located of at the the urease entrance active of sitethe ure [127ase]. Additionally, active site [127 CBS]. Additionally,has also been CBS shown has toalso inhibit been alcoholshown dehydrogenaseto inhibit alcohol (ADH) dehydrogenase in H. pylori (ADH). ADH in is H. a zinc-containing pylori. ADH is aenzyme zinc-containing responsible enzyme for the responsible oxidation offor alcohols the oxidation to acetaldehydes, of alcohols to which acetaldehydes, are toxic to which gastric are cells toxic and to gastric cells and causing mucosal damage. Jin et al. proposed that the inhibition of ADH by Bi(III) is probably due to its direct interference with the zinc(II) binding sites through a non-competitive process involving interactions with thiol groups [128]. There are an increasing number of novel Bi(III) complexes that have been developed recently as potential agents for the treatment of H. pylori. The group of Andrews designed, for example, numerous bismuth-based complexes with excellent anti- H. pylori activity based on aminoarenesulfonate ligands possessing MIC values (MIC = 0.049 μg/mL)

Chemistry 2020, 2 869 causing mucosal damage. Jin et al. proposed that the inhibition of ADH by Bi(III) is probably due to its direct interference with the zinc(II) binding sites through a non-competitive process involving interactions with thiol groups [128]. There are an increasing number of novel Bi(III) complexes that have been developed recently as potential agents for the treatment of H. pylori. The group of Andrews designed, for example, numerous bismuth-based complexes with excellent anti-H. pylori activity based on aminoarenesulfonate ligands possessing MIC values (MIC = 0.049 µg/mL) much lower than those obtained for BSS (MIC = 12.5 µg/mL), CBS (MIC = 12.5 µg/mL) and RBC (MIC = 8 µg/mL) for the strains 251 and B128 [129]. Furthermore, several bismuth complexes developed over the years have been accessed for other antibacterial and antifungal activities. For instance, Lessa et al. designed different Bi(III) thiosemicarbazone complexes, as this class of thiol/thione compound is reported to have wide pharmacological applications like antiparasitic, antibacterial, anticancer and antiviral properties [130]. The authors demonstrated that upon coordination to Bi(III), the antibacterial activities of both thiosemicarbazones and bis(thiosemicarbazones) increased against the Gram-positive bacteria, especially against S. aureus, where the activity of some of their complexes (MIC = 5.5–6.1 µM) was 15 to 64 times more potent than their corresponding free ligands. The same group also reported the antimicrobial activity of hydrazone derivatives Bi(III) complexes against different bacterial (S. aureus, E. faecalis, S. epidermidis, P. aeruginosa) and fungal (C. albicans) strains [131]. In this study, the ligands investigated were found to be inactive against the panel of microorganisms previously mentioned. However, upon coordination to Bi(III), the antimicrobial activity increased significantly against all Gram-positive bacteria and to a lesser extent against Gram-negative P. aeruginosa. Among all of the bismuth-based complexes developed, two of them were shown to be more active against S. aureus (MIC = 0.2 and 0.3 µM) than tetracycline (MIC = 7.2 µM), a clinical antibiotic used as positive control. Additionally, one of their Bi(III) hydrazone complexes exhibited better activity (MIC = 44 µM) against C. albicans than fluconazole (MIC = 59 µM), a well-known antifungal agent. Unfortunately, despite these very promising results, no further experiments were performed by the authors in order to explain the mechanism of action of such bismuth compounds on their antimicrobial activities. Recently, Sun and coworkers demonstrated that CBS and related bismuth compounds could be novel and potent inhibitors of metallo-β-lactamases (MBLs), especially NDM-1 (New Delhi MBLs), VIM-2 (Verona integron-encoded MBLs) and IMP-4 (imipenemases) [132]. MBLs are zinc(II)-containing enzymes that activate a nucleophilic water molecule to cleave the β-lactam ring, conferring bacterial resistance to currently used organic antibiotics. In contrast, Bi(III) compounds inhibit at micromolar levels the enzyme through an irreversible replacement of two Zn(II) ions by one Bi(III) ion in the active site, leading to the abolition of MBL activity both in vitro and in vivo. The authors explained this unique mechanism by the intrinsic properties of Bi(III), which are the relatively large size of the metal ion and its high coordination numbers, and its thiol-philic feature, which makes interactions with the cysteine residues in the active site easy. Thus, the inhibition of MBLs by Bi(III) compounds restores the antimicrobial activity of β-lactam antibiotics (meropenem, MER), whereas CBS itself showed no or minor growth inhibition toward either NDM-1-positive or -negative bacteria. Then, the combined use of CBS with MER led to a synergistic effect that may represent a new approach for the discovery of MBLs therapies [132]. Other studies have also revealed that bismuth drugs could have unexpected medicinal applications due to the ability of Bi(III) to exert its action through binding to the key enzymes, and thereby disrupt key pathological pathways in the pathogen. As mentioned above, Bi(III) has a high affinity with thiolate ligands, that makes enzymes possessing cysteine residues in their active sites potential targets for this metal ion. This feature was used by Yang et al., who tested different bismuth compounds in order to evaluate their antiviral activities against severe acute respiratory syndrome coronavirus (SARS-CoV) [133]. They showed that RBC (Figure 15c) was particularly efficient as a strong inhibitor of the SARS-CoV helicase, an enzyme containing a cysteine-rich Zn(II)-binding domain, which blocks virus replication. There have also been recent reports on the development of new Bi compounds that can Chemistry 2020, 2, x 22 infantum chagasi and L. amazonensis, which are associated with visceral and cutaneous leishmaniasis, respectively [135]. The authors showed that this Bi(III) complex was slightly more active than dppz alone, and at least 77 and 2400 timesChemistry 2020more, 2 active than potassium antimonyl870 tartrate used as reference

successfully be used to treat leishmaniasis, a disease caused by some parasites [134]. Those compounds in WT and Sb(III)-resistant strains,were e ffectively respectively. found to be potential alternatives toBecause current antimony(V)-based the antileishmanial starting drugs reagent BiCl3 had very little given by their close proximity in the periodic table, their similar biological chemistry in terms of their effects and their modes of action, while providing lower mammalian cell toxicities and opportunities activity against Leishmania, it was ofsuggested oral delivery. Andrews and Demichelithat are thethe two main metal working groups ion investigating alone the use was not sufficient to have anti- of bismuth complexes on Leishmania. For example, Lizarazo-Jaimes et al. reported the synthesis of the [Bi(dppz)Cl3] complex (compound 22, Figure 16) and tested its antileishmanial activity against leishmaniasis activity, but rather improvedwild-type (WT) and Sb-resistant the (SbR) strainsability of L. infantum of chagasi dppzand L. amazonensis to, which inhibit are the growth of promastigotes associated with visceral and cutaneous leishmaniasis, respectively [135]. The authors showed that this Bi(III) complex was slightly more active than dppz alone, and at least 77 and 2400 times more active through complexation. The authorsthan potassium further antimonyl tartrate proposed used as reference in WT and Sb(III)-resistantthat the strains, respectively. leishmanicidal activity of the dppz Because the starting reagent BiCl3 had very little activity against Leishmania, it was suggested that the metal ion alone was not sufficient to have anti-leishmaniasis activity, but rather improved the ability of complex may occur via its interactiondppz to inhibit with the growth ofparasite promastigotes through complexation.DNA The through authors further proposed intercalation and/or a modulation that the leishmanicidal activity of the dppz complex may occur via its interaction with parasite DNA of the hydrophilicity profile of thethrough compound. intercalation and/or a modulation of the hydrophilicity profile of the compound.

Figure 16. Structure of antileishmanial-Bi(III) complex [Bi(dppz)Cl3][135].

Overall, bismuth-based compounds are routinely used for the treatment of gastrointestinal Figure 16. Structuredisorders of and antileishmanial were recently found to possess potential-Bi(III) antimicrobial, antiviral complex and antileishmanial [Bi(dppz)Cl3] [135]. properties. It is likely that proteins are the key biomolecular targets of Bi(III), particularly proteins possessing thiol-rich domains or metal binding sites with attractive coordination environments for Bi. Due to the low toxicity of Bi(III) salts and the development of novel bismuth-based compounds with considerable and varied biological activity, there is no doubt that Bi has the potential to play new and Overall, bismuth-based compoundsimportant roles in medicinal are chemistry in routinely the upcoming years. used for the treatment of gastrointestinal disorders and were recently found to possess potential antimicrobial, antiviral and antileishmanial properties. It is likely that proteins are the key biomolecular targets of Bi(III), particularly proteins possessing thiol-rich domains or metal binding sites with attractive coordination environments for Bi. Due to the low toxicity of Bi(III) salts and the development of novel bismuth-based compounds with considerable and varied biological activity, there is no doubt that Bi has the potential to play new and important roles in medicinal chemistry in the upcoming years.

2.8. Vanadium Vanadium is present as cofactors in certain enzymes, such as haloperoxidases and in some kinds of nitrogenases expressed by nitrogen metabolizing bacteria. It is even found at high concentrations (0.15 M) in the blood of some marine animals like sea squirts [136]. Moreover, the structure of the 3− 3− vanadate VO4 is very close to that of phosphate PO4 . In addition, there are other similarities, such as in esterification-type reactions; vanadate can therefore be considered a competitor of phosphate. Knowing the great importance of phosphate in several very different biochemical pathways (kinases, ATP, etc.), vanadate is becoming an interesting target to study, and that is even more true when − 2− looking at their pKa (7.2 for phosphate means a similar ratio for H2PO4 and HPO4 , while it is 8.2 for − vanadate, meaning that H2VO4 is the main species at the physiological pH) and at the coordination (phosphorous can typically not be coordinated by more than five ligands, while vanadium can accept six). Altogether, it was demonstrated that if vanadate easily replaces the phosphate in an enzyme, it can form stable complexes with the enzyme’s target, inhibiting the enzyme [135]. As a small aside about our antimicrobial topic, two consequences of this similarity with phosphate are in the treatment of diabetes and cancers: Vanadium is able to interfere in the metabolism of glucose, making it possible to reduce the acute as well as the secondary consequences of diabetes by mimicking insulin. Indeed, diabetes appears when the insulin-receptors of cells no longer recognize insulin. Yet, the linking of insulin to its receptors normally results in the phosphorylation of a tyrosine residue, which starts a chain reaction. In the absence of insulin, or in case of non-response, the tyrosine is dephosphorylated by another enzyme. Thus, when diabetics are treated with vanadate, it can bind to the active site of this last enzyme and inhibit the dephosphorylation, leading to an activation of the insulin signaling pathway even when the insulin receptor does not react to insulin [137]. Moreover, intervening in the phosphorylation reactions, vanadate therefore causes some issues for cells that have a high need for phosphate. This is the case for tumor cells, which present an uncontrolled development. Hence, uptake of vanadium complexes has been shown to induce cellular death in different kinds of tumors.

Chemistry 2020, 2 871

2.8. Vanadium Vanadium is present as cofactors in certain enzymes, such as haloperoxidases and in some kinds of nitrogenases expressed by nitrogen metabolizing bacteria. It is even found at high concentrations (0.15 M) in the blood of some marine animals like sea squirts [136]. Moreover, the structure of the 3 3 vanadate VO4 − is very close to that of phosphate PO4 −. In addition, there are other similarities, such as in esterification-type reactions; vanadate can therefore be considered a competitor of phosphate. Knowing the great importance of phosphate in several very different biochemical pathways (kinases, ATP, etc.), vanadate is becoming an interesting target to study, and that is even more true when looking 2 at their pKa (7.2 for phosphate means a similar ratio for H2PO4− and HPO4 −, while it is 8.2 for vanadate, meaning that H2VO4− is the main species at the physiological pH) and at the coordination (phosphorous can typically not be coordinated by more than five ligands, while vanadium can accept six). Altogether, it was demonstrated that if vanadate easily replaces the phosphate in an enzyme, it can form stable complexes with the enzyme’s target, inhibiting the enzyme [135]. As a small aside about our antimicrobial topic, two consequences of this similarity with phosphate are in the treatment of diabetes and cancers: Vanadium is able to interfere in the metabolism of glucose, making it possible to reduce the acute as well as the secondary consequences of diabetes by mimicking insulin. Indeed, diabetes appears when the insulin-receptors of cells no longer recognize insulin. Yet, the linking of insulin to its receptors normally results in the phosphorylation of a tyrosine residue, which starts a chain reaction. In the absence of insulin, or in case of non-response, the tyrosine is dephosphorylated by another enzyme. Thus, when diabetics are treated with vanadate, it can bind to the active site of this last enzyme and inhibit the dephosphorylation, leading to an activation of the insulin signaling pathway even when the insulin receptor does not react to insulin [137]. Moreover, intervening in the phosphorylation reactions, vanadate therefore causes some issues for cells that have a high need for phosphate. This is the case for tumor cells, which present an uncontrolled development. Hence, uptake ofChemistry vanadium 2020, complexes2, x has been shown to induce cellular death in different kinds of tumors. Another23 proposed mechanism of action is the generation of ROS in tumoral cells, and it was also shown thatAno vanadocene-derivedther proposed mechanism compounds of action (by is analogy the generation with ferrocene) of ROS are in abletumoral to interact cells, and with it the was DNA, also formingshown that a stable vanadocene complex-derived leading tocompounds apoptosis (see(by Figureanalogy 17 with). Other ferrocene) metallocenes are able show to interact similar with activity, the particularlyDNA, forming titanocene, a stable complex molybdenocene, leading to and apoptos nioboceneis (see [136 Figure,137 ].17). Other metallocenes show similar activity, particularly titanocene, molybdenocene, and niobocene [136,137].

FigureFigure 17.17. InteractionInteraction ofof metallocenesmetallocenes withinwithin thethe DNA,DNA, XX representsrepresentshalogen halogenatoms atoms[ [137137].].

ToTo summarize, summarize, vanadium vanadium participates participates inin thethe regulation regulation of of the the phosphate phosphate pathways, pathways, and and isis consideredconsidered toto bebe anan essentialessential oligooligo element,element, butbut maymay becomebecome toxictoxic inin tootoo largelarge quantities,quantities, explainingexplaining whywhy these these compounds compounds staystay aside aside for for the the momentmoment [ [136136,138,138]] Nevertheless,Nevertheless, to to overcome overcome the the low low biodisponibilitybiodisponibility ofof orallyorally takentaken mineralmineral salts,salts, asas onlyonly aa veryvery lowlow amountamount ofof saltsalt successessuccesses usuallyusually toto passpassthe the gastrointestinalgastrointestinal tracttractinto intothe the bloodblood systemsystem duedue toto lowlow lipophilicity,lipophilicity, thethe hopehope isis onon vanadiumvanadium complexes, which are currently a main part of the research about this element. The goal is therefore to maximize the biodisponibility with a non-toxic ligand, forming stable complexes in the gastrointestinal tract that are able to dissociate once in the bloodstream [136,139]. To come back to our antimicrobial focus, there is ongoing research surrounding the use of vanadium complexes against some parasites. One of the first studies reporting results in this domain was published in the early 2000s, where Barti et al. tested some protozoicidal organic molecules as ligands for different transition metals, like molybdenum, vanadium, and tungsten. All the complexes showed a lower IC50 against an amoeba (Entamoeba histolytica) than their respective ligands alone, and the best results were found for a vanadium complex of 2-(salicylideneimine)benzimidazole (Figure 18, compound 23), exhibiting an IC50 value of 2.35 µM (compared to 9.20 µM for the title ligand and 2.99 µM for the molybdenum complex; tungsten complex n.d.) [140]. The research continued (and is continuing) around some other parasites, mainly against tropical parasites, and particularly against Trypanosoma cruzi. This protozoan parasite causes Chagas disease, resulting in 10,000 deaths per year and 8 million infected people. Several publications dealing with vanadium complexes for potential antitrypanosomal treatment are available, and some of this work was done by the group of Dinorah Gambino. They proposed several vanadium complexes based on salicylaldehyde- and polypyridyl-derivatives ligands [141–143]. Another example is a vanadium complex of aminophen and bromosalycilaldehyde (Figure 18, compound 24), whose IC50 was shown to be in the low micromolar range (0.27–3.8 µM) [144,145] against T. cruzi, similar to the standard drug nifurtimox, but a live/dead assay only resulted in a trypanostatic effect, with the parasites recovering to normal growth after the cutting off treatment. Moreover, the compound did not present toxicity against murine macrophages serving as a mammalian model. Indeed, its IC50 is around 50 µM, almost 200 times higher than against T. cruzi, reflecting a high selectivity. Investigation around the mechanism of action showed that whereas only 2.4% of the dissolved complex was taken up by the parasites (which is a similar amount to what was observed for other metallodrugs such as cisplatin), a high concentration on vanadium was observed within the DNA and the RNA (0.089 ng of vanadium/µg of DNA, and 0.006 ng of vanadium/µg of RNA), suggesting a strong interaction with DNA. A deficiency of the mitochondria is hypothesized as well, due to the high level in the cytoplasm

Chemistry 2020, 2 872

complexes, which are currently a main part of the research about this element. The goal is therefore to maximize the biodisponibility with a non-toxic ligand, forming stable complexes in the gastrointestinal tract that are able to dissociate once in the bloodstream [136,139]. To come back to our antimicrobial focus, there is ongoing research surrounding the use of vanadium complexes against some parasites. One of the first studies reporting results in this domain was published in the early 2000s, where Barti et al. tested some protozoicidal organic molecules as ligands for different transition metals, like molybdenum, vanadium, and tungsten. All the complexes showed a lower IC50 against an amoeba (Entamoeba histolytica) than their respective ligands alone, and the best results were found for a vanadium complex of 2-(salicylideneimine)benzimidazole (Figure 18, compound 23), exhibiting an IC50 value of 2.35 µM (compared to 9.20 µM for the title ligand and 2.99 µM for the molybdenum complex; tungsten complex n.d.) [140]. The research continued (and is continuing) around some other parasites, mainly against tropical parasites, and particularly against Trypanosoma cruzi. This protozoan parasite causes Chagas disease, resulting in 10,000 deaths per year and 8 million infected people. Several publications dealing with vanadium complexes for potential antitrypanosomal treatment are available, and some of this work was done by the group of Dinorah Gambino. They proposed several vanadium complexes based on salicylaldehyde- and polypyridyl-derivatives ligands [141–143]. Another example is a vanadium complex of aminophen and bromosalycilaldehyde (Figure 18, compound 24), whose IC50 was shown to be in the low micromolar range (0.27–3.8 µM) [144,145] against T. cruzi, similar to the standard drug nifurtimox, but a live/dead assay only resulted in a trypanostatic effect, with the parasites recovering to normal growth after the cutting off treatment. Moreover, the compound did not present toxicity against murine macrophages serving as a mammalian model. Indeed, its IC50 is around 50 µM, almost 200 times higher than against T. cruzi, reflecting a high selectivity. Investigation around the mechanism of action showed that whereas only 2.4% of the dissolved complex was taken up by the parasites (which is a similar amount to what was observed for other metallodrugs such as cisplatin), a high concentration on Chemistry 20vanadium20, 2, x was observed within the DNA and the RNA (0.089 ng of vanadium/µg of DNA, and 0.006 ng 24 of vanadium/µg of RNA), suggesting a strong interaction with DNA. A deficiency of the mitochondria is of some hypothesized organites asused well, in due the to the mitochondria. high level in the cytoplasm Moreover, of some the organites analysis used of in the the mitochondria. protein expression concludedMoreover, to overexpression the analysis of of the transporters protein expression and drug concluded efflux, to overexpressionand of some ofproteins transporters involved and in the drug efflux, and of some proteins involved in the transcription. This is coherent with the presence of transcriptionvanadium. This in DNA,is coherent the deficiency with of the mitochondria, presence of and vanadium overexpression in of DNA some, proteinsthe deficiency involved of the mitochondria,in reduction and /oxidationoverexpression pathways of and some hydrolysis, proteins suggesting involved the in vanadium reduction/oxidation complex to cause pathways some and hydrolysis,redox suggesting disorders [ 144the,145 vanadium]. complex to cause some redox disorders [144,145].

Figure 18. Proposed structures for compound 23, and V(IV)-O(Brsal)(aminophen) 24 [140,144,145]. Figure 18. Proposed structures for compound 23, and V(IV)-O(Brsal)(aminophen) 24 [140,144,145].

Vanadium complexes were also developed against Leishmania species, another parasite responsible for leishmaniosis, and close to T. cruzi. The next example deals with a vanadium–stilbene complex (Figure 19, compound 25), which is again based on a salicylic acid moiety like the previous examples. These “salen-derivative” ligands have some advantages, e.g., the ability to form very stable complexes. X-ray analysis of the complex showed a slightly distorted square pyramidal structure around the vanadium cation with oxygen atoms being slightly closer to vanadium than the nitrogen atoms, which can be explained by the strong Lewis acid properties of V(IV), and the anionic character of phenol compared to the imine [146]. The complex displays an IC50 of 3.51 µM against L. amazonensis, which is slightly higher than for other vanadyl polypyridyl complexes. The mechanism of action was proposed to involve the mitochondria, and authors underline the great interest of this target, as the mitochondria of parasite work differently from those of mammalian, resulting in a higher selectivity, and moreover, the parasite being unicellular species, they have only one mitochondrion [147]. As V(IV) was demonstrated to be subject to oxidation, our last, but not least, example is V(V) complexes, active against an amoeba, Entamoeba histolytica (see Figure 19 compounds 26–28). Its activity is similar to those seen before, with an IC50 value of around 0.09–0.85 µM, while the standard drug for amoebas, Metronizadol, has 1.68 µM [148].

Figure 19. Structures of 25 V(IV)O(sal-HBPD) and compounds 26–28 [146–148].

Vanadium complexes can be efficient against viruses and bacteria too. In particular, oxovanadium complexes of thiourea, [149] polyoxovanadates, [150], and oxovanadium porphyrins showed efficiency against HIV, with more than 97% of inhibition at 5 µM for the compound 29 (Figure 20). They act by linking to the HIV-1 reverse transcriptase, and a complementary computational study

Chemistry 2020, 2, x 24 of some organites used in the mitochondria. Moreover, the analysis of the protein expression concluded to overexpression of transporters and drug efflux, and of some proteins involved in the transcription. This is coherent with the presence of vanadium in DNA, the deficiency of the mitochondria, and overexpression of some proteins involved in reduction/oxidation pathways and hydrolysis, suggesting the vanadium complex to cause some redox disorders [144,145].

Figure 18. Proposed structures for compound 23, and V(IV)-O(Brsal)(aminophen) 24 [140,144,145].

Vanadium complexes were also developed against Leishmania species, another parasite responsible for leishmaniosis, and close to T. cruzi. The next example deals with a vanadium–stilbene complexChemistry 2020 (Figure, 2 19, compound 25), which is again based on a salicylic acid moiety like the previous873 examples. These “salen-derivative” ligands have some advantages, e.g., the ability to form very stable complexes. X-ray analysis of the complex showed a slightly distorted square pyramidal structure Vanadium complexes were also developed against Leishmania species, another parasite responsible around the vanadium cation with oxygen atoms being slightly closer to vanadium than the nitrogen for leishmaniosis, and close to T. cruzi. The next example deals with a vanadium–stilbene complex atoms, which can be explained by the strong Lewis acid properties of V(IV), and the anionic character (Figure 19, compound 25), which is again based on a salicylic acid moiety like the previous examples. of phenol compared to the imine [146]. The complex displays an IC50 of 3.51 µM against L. These “salen-derivative” ligands have some advantages, e.g., the ability to form very stable complexes. amazonensis, which is slightly higher than for other vanadyl polypyridyl complexes. The mechanism X-ray analysis of the complex showed a slightly distorted square pyramidal structure around the of action was proposed to involve the mitochondria, and authors underline the great interest of this vanadium cation with oxygen atoms being slightly closer to vanadium than the nitrogen atoms, which target, as the mitochondria of parasite work differently from those of mammalian, resulting in a can be explained by the strong Lewis acid properties of V(IV), and the anionic character of phenol higher selectivity, and moreover, the parasite being unicellular species, they have only one compared to the imine [146]. The complex displays an IC50 of 3.51 µM against L. amazonensis, which is mitochondrion [147]. slightly higher than for other vanadyl polypyridyl complexes. The mechanism of action was proposed As V(IV) was demonstrated to be subject to oxidation, our last, but not least, example is V(V) to involve the mitochondria, and authors underline the great interest of this target, as the mitochondria complexes, active against an amoeba, Entamoeba histolytica (see Figure 19 compounds 26–28). Its of parasite work differently from those of mammalian, resulting in a higher selectivity, and moreover, activity is similar to those seen before, with an IC50 value of around 0.09–0.85 µM, while the standard the parasite being unicellular species, they have only one mitochondrion [147]. drug for amoebas, Metronizadol, has 1.68 µM [148].

FFigureigure 19. StructuresStructures of of 2255 V(IV)O(salV(IV)O(sal-HBPD)-HBPD) and compounds 2266–2288 [1[14646–141488]]..

As V(IV) was demonstrated to be subject to oxidation, our last, but not least, example is V(V) Vanadium complexes can be efficient against viruses and bacteria too. In particular, complexes, active against an amoeba, Entamoeba histolytica (see Figure 19 compounds 26–28). Its activity oxovanadium complexes of thiourea, [149] polyoxovanadates, [150], and oxovanadium porphyrins µ showedis similar efficiency to those seenagain before,st HIV, with with an more IC50 thanvalue 97 of% aroundof inhibition 0.09–0.85 at 5 µMM, for while the compound the standard 29 drug (Figure for µ 20amoebas,). They act Metronizadol, by linking to has the 1.68HIV-1M[ reverse148]. transcriptase, and a complementary computational study Vanadium complexes can be efficient against viruses and bacteria too. In particular, oxovanadium complexes of thiourea, [149] polyoxovanadates, [150], and oxovanadium porphyrins showed efficiency against HIV, with more than 97% of inhibition at 5 µM for the compound 29 (Figure 20). They act by linking to the HIV-1 reverse transcriptase, and a complementary computational study proposed a binding to the CD4 protein, blocking the entry of the virus to the cells [151]. Some polyoxovanadates incorporating silicon, tungsten, and/or boron show activity even at less than 1 µM (K5[SiVW11O40] and K7[BVW11O40]) [150]. Chemistry 2020, 2, x 25 proposed a binding to the CD4 protein, blocking the entry of the virus to the cells [151]. Some Chemistry 2020, 2 874 polyoxovaChemistrynadates 2020, 2, xincorporating silicon, tungsten, and/or boron show activity even at less than25 1 µM (K5[SiVW11O40] and K7[BVW11O40]) [150]. proposed a binding to the CD4 protein, blocking the entry of the virus to the cells [151]. Some polyoxovanadates incorporating silicon, tungsten, and/or boron show activity even at less than 1 µM (K5[SiVW11O40] and K7[BVW11O40]) [150].

FigureFigure 20. 20. V(IV)OV(IV)O-porphyrin-porphyrin complexcomplex29 29[151 [151]. ]. Figure 20. V(IV)O-porphyrin complex 29 [151]. Hydrazone complexes of vanadium(V) 30–32 (see Figure 21) with an octahedral geometry were HydrazoneHydrazone complexes complexes of ofvanadium(V) vanadium(V) 3 300––3232 (see FigureFigure 2 12)1 with) with an anoctahedral octahedral geometry geometry were were obtained by Wu and co-workers. These three V(V) complexes show antibacterial activity against obtainedobtained by Wuby Wu and and co -workersco-workers. These. These three three V(V) V(V) complexes complexes show show antibacterial antibacterial activity activity against against B.subtilis, S. aureus, and E. coli, with MICs between 1.2 and 37.5 µg/mL, which is much better than their B.subtilisB.subtilis, S. aureus, S. aureus, and, and E. coliE. coli, with, with MIC MICss between between 1.21.2 andand 37.5 37.5 µg/mL, µg/mL, which which is much is much better better than than respective title ligands (9.4 to >150 µg/mL, Table2). It may be noted that complex 30 shows the lowest their respective title ligands (9.4 to >150 µg/mL, Table 2). It may be noted that complex 30 shows the theirpotency, respective illustrating title ligands the key (9.4 importance to >150 µg/mL of the substitution, Table 2). It of may the aromaticbe noted ring that [152 complex]. 30 shows the lowestlowest potency, potency, illustrating illustrating the the key key im impportanceortance ofof the substitutionsubstitution of ofthe the aro aromaticmatic ring ring [152 ][.1 52].

FigureFigure 21. 21. StructuresStructures ofof complexescomplexes 3030–32.. R represents represents an an ethanol ethanol or or a a methanol methanol ligand ligand [1 [15252].]. Figure 21. StructuresTable 2. MIC of complexes (µg/mL) of the30– complexes32. R represents30–32, and an ofethanol their title or a ligands methanol [152 ].ligand [152].

Tested Material B. subtilis S. aureus E. coli P. fluorescence

ligand of 30 75 37.5 >150 >150 ligand of 31 18.8 37.5 75 >150 ligand of 32 9.4 18.8 18.8 >150 30 18.8 18.8 37.5 >150 31 2.3 1.2 18.8 >150 32 1.2 2.3 9.4 >150 Penicillin G 2.3 4.7 >150 >150

Chemistry 2020, 2, x 26

Table 2. MIC (µg/mL) of the complexes 30–32, and of their title ligands [152].

Tested Material B. subtilis S. aureus E. coli P. fluorescence ligand of 30 75 37.5 >150 >150 ligand of 31 18.8 37.5 75 >150 ligand of 32 9.4 18.8 18.8 >150 30 18.8 18.8 37.5 >150 31 2.3 1.2 18.8 >150 32 1.2 2.3 9.4 >150 Penicillin G 2.3 4.7 >150 >150

Schiff base complexesChemistry 2020, 2 of vanadium also display antibacterial activity, as it was875 experimented by Khaleghi et al. Results in measuring the inhibition zone are, however, dependent on the tested bacteria, as the two vanadiumSchiff base complexes complexes of vanadium (see also Figure display antibacterial 22) were activity, not active as it was at experimented all against by Gram-negative Khaleghi et al. Results in measuring the inhibition zone are, however, dependent on the tested bacteria, bacteria P. aeruginosaas the two, E. vanadium coli and complexes K. pneumonia (see Figure 22. )They were not nevertheless active at all against showed Gram-negative higher bacteria activity than the vanadium sulfateP. aeruginosa and , theE. coli ciprofloxacinand K. pneumonia. They stand neverthelessard against showed higher the activity Gram than- thepositive vanadium bacteria Listeria sulfate and the ciprofloxacin standard against the Gram-positive bacteria Listeria monocytigenes, E. faecalis monocytigenes, E. andfaecalis against and the fungus againstC. albicans the fungus(respective C. MIC: albicans 0.62, 1.25 and(respective 2.5 mg/mL). Finally,MIC: the0.62, vanadium 1.25 and 2.5 mg/mL). Finally, the vanadiumcomplexes complexes impaired also theimpaired formation ofalso biofilms the of formatio some bacterian [153of ].biofilms of some bacteria [153].

Figure 22. Schiff-base complex of vanadium(V) [153]. Figure 22. Schiff-base complex of vanadium(V) [153]. 2.9. Other Metal-Based Complexes as Antimicrobial Agents In recent decades, many other metal-based complexes have been reported in the literature due to 2.9. Other Metal-Basedtheir very Complexes promising antimicrobial as Antimicrobial properties. Here,Agents we will discuss the most relevant examples. As mentioned in the early stage of this review, gold has garnered attention for its medicinal In recent decades,properties many since the other 19th century metal in- treatingbased syphilis, complexes tuberculosis have and been inflammatory reported rheumatoid in the literature due to their very promisingdiseases. Auranofinantimicrobial (compound properties.33, Figure 23), forHere, example, we is awill gold-based discuss FDA-approved the most arthritis relevant examples. drug and is currently used in clinical trials for its anticancer applications [18]. Recently, it has been As mentionedrepurposed in the as aearly potential stage antimicrobial of this agent review, and was found gold to be ehasffective garnered against many attention Gram-positive for its medicinal properties since bacteria, the 19th including century multidrug in resistant treating strains, syphilis, as well as M. tuberculosis tuberculosis [154 ]. and However, inflammatory almost no rheumatoid activity was observed toward Gram-negative species. The exact molecular mechanism of auranofin is diseases. Auranofinstill unclear (compound but it is postulated 33, Figure that its activity 23), isfor provided example, by the inhibition is a gold of the-based bacterial thioredoxinFDA-approved arthritis drug and is currentlyreductase used (Trx), anin essentialclinical enzyme trials for maintainingfor its anticancer the thiol–redox applications balance and protecting [18]. againstRecently, it has been reactive oxidative species, through the interaction with the redox-active selenocysteine or cysteine repurposed as a residuespotential that are antimicrobial present in the active siteagent [155]. and The ine wasffectiveness found of auranofin to be toward effectiv Gram-negativee against many Gram- positive bacteria,bacteria including was suggested multidrug to be the result resistant of their glutathione strains, system as that well is able as to compensateM. tuberculosis for the loss [154]. However, of the reducing ability of Trx [154]. An alternative hypothesis was also proposed, suggesting that the almost no activityouter was membranes observed of Gram-negative toward bacteria Gram were-negative able to prevent species. auranofin The accumulation exact asmolecular an effective mechanism of auranofin is stillbarrier unclear [156]. In but this context, it is possometulated groups have that decided its to design activity auranofin is analogsprovided in order by to improve the inhibition of the the antimicrobial properties of this gold-based drug. For example, Wu et al. prepared 40 auranofin bacterial thioredoxinanalogs reductase by modulating (Trx), the structures an essential of the thiol andenzyme phosphine for ligands, maintaining and tested their the activities thiol–redox balance and protecting againstagainst ESKAPE reactive pathogens, oxidative establishing species, a structure–activity through relationship the interaction [157]. They demonstrated with the redox-active that the Gram-negative activity of auranofin could be effectively modulated by altering the thiol selenocysteine orand cysteine phosphine residues ligands, while that some are compounds present exhibited in the bacterial active inhibition site (MIC) [155]. and The killing ineffectiveness of auranofin toward(MBC) Gram activities-negative up to 65-fold bacteria higher than was that suggested of auranofin (compounds to be the34 –result36 Figure of 23 ).their Moreover, glutathione system preliminary in vitro cytotoxicity experiments performed on A549 cells (human lung epithelial cells) that is able to compensate for the loss of the reducing ability of Trx [154]. An alternative hypothesis was also proposed, suggesting that the outer membranes of Gram-negative bacteria were able to prevent auranofin accumulation as an effective barrier [156]. In this context, some groups have decided to design auranofin analogs in order to improve the antimicrobial properties of this gold- based drug. For example, Wu et al. prepared 40 auranofin analogs by modulating the structures of the thiol and phosphine ligands, and tested their activities against ESKAPE pathogens, establishing

Chemistry 2020, 2, x 27 a structure–activity relationship [157]. They demonstrated that the Gram-negative activity of auranofin could be effectively modulated by altering the thiol and phosphine ligands, while some compounds exhibited bacterial inhibition (MIC) and killing (MBC) activities up to 65-fold higher than that of auranofin (compounds 34–36 Figure 23). Moreover, preliminary in vitro cytotoxicity experiments performed on A549 cells (human lung epithelial cells) revealed that the active analogues had mammalian cellChemistry toxicities2020, 2 either similar to or lower than that of auranofin. These876 findings suggest that the properties of auranofin can be optimized for additional antimicrobial applications. revealed that the active analogues had mammalian cell toxicities either similar to or lower than Furthermore, otherthat of studies auranofin. have These shown findings suggest that auranofinthat the properties is effic of auranofinacious can in be a optimized mouse for model of MRSA systemic infectionadditional [156], antimicrobialdemonstrating applications. its ability Furthermore, in in other vivo studies experiments. have shown that It auranofin was also is demonstrated efficacious in a mouse model of MRSA systemic infection [156], demonstrating its ability in in vivo that auranofin canexperiments. be used It was in also combination demonstrated that with auranofin traditional can be used in antibiotics combination with (ciprofloxacin, traditional linezolid, gentamicin) as anantibiotics alterna (ciprofloxacin,tive strategy linezolid, in gentamicin) order to as provide an alternative a significantstrategy in order protection to provide a from mortality significant protection from mortality associated with MRSA infections and to reduce the potential associated with MRSAemergence infections of drug resistance and [156 ]. to Taken reduce together, the those potential studies strongly emergence suggest that auranofin of drug has resistance [156]. Taken together, significant those promise studies to be strongly repurposed as suggest an effective antimicrobial that auranofin agent for the has treatment significant of systemic promise to be bacterial infections. repurposed as an effective antimicrobial agent for the treatment of systemic bacterial infections.

Figure 23. Structure of auranofin (33) and its antimicrobial analogues [157]. Figure 23. Structure of auranofin (33) and its antimicrobial analogues [157]. Only limited reports on the antimicrobial properties of organometallic iridium(III)-based complexes have been published in the literature. In 2013, Keene and Collins compared the bacteriostatic Only limitedand reports bactericidal onactivities the of antimicrobialchloride-containing ruthenium(II) properties and of iridium(III) organometallic complexes against iridium(III)-based complexes have four been strains published of bacteria [158 ]. in They the have literature. shown that unlike In Ru(II)-polypyridyl 2013, Keene complexes, and Collins the Ir(III) compared the counterparts were only bacteriostatic, rather than bactericidal. The authors suggested that the origin bacteriostatic andof the bactericidal difference in activities activities probably comes of chloride from the higher-containing overall charge ruthenium(II)of iridium compounds and iridium(III) complexes against(+4 fourbefore aquation strains of of the bacteria chloride ligands [158 compared]. They to havethe +2 for shown the ruthenium that counterparts),unlike Ru(II)-polypyridyl which probably prevents their crossing through the membrane. Additionally, the chloride anions complexes, the Ir(III)of the iridium counterparts complexes were were found only to be bacteriostatic more labile than for, rather the corresponding than bactericidal. ruthenium The authors suggested that thecomplexes, origin which of the might difference also affect their in accumulation activities within probably the bacteria, comes and thereby from their the bactericidal higher overall charge activity. Nevertheless, further studies based on cyclometallated polypyridyl iridium complexes of iridium compoundsrevealed promising (+4 before antibacterial aquation activities against of the both Gram-positive chloride and ligands Gram-negative compared bacteria and to the +2 for the ruthenium counterparts),demonstrated that which their mechanisms probably of action are prevents mainly attributed their to their crossing ability to interact through with DNA the membrane. through intercalation or to act as photosensitizers, generating ROS upon light irradiation [158–162]. Additionally, theFurthermore, chloride theanions third-row of transition the iridium metal ion complexes Ir(III) is also supposed were to found be relatively to kineticallybe more inert labile than for the corresponding rutheniumand therefore iscomplexes, likely to reach its which drug target might sites with also its initial affect ligands their still bound. accumulation This could offer within the the bacteria, and thereby their bactericidal activity. Nevertheless, further studies based on cyclometallated polypyridyl iridium complexes revealed promising antibacterial activities against both Gram- positive and Gram-negative bacteria and demonstrated that their mechanisms of action are mainly attributed to their ability to interact with DNA through intercalation or to act as photosensitizers, generating ROS upon light irradiation [158–162]. Furthermore, the third-row transition metal ion Ir(III) is also supposed to be relatively kinetically inert and therefore is likely to reach its drug target sites with its initial ligands still bound. This could offer the opportunity to design novel metallo- antibiotics associating, all in one molecule, metal ions and active organic molecules. For instance, Chen et al. introduced biguanide ligands, including the antidiabetic drug metformin, into organoiridium cyclopentadienyl complexes (compound 37, Figure 24) [162]. Several of these complexes exhibited potent activity against both Gram-negative and Gram-positive bacteria, including MRSA with MICs as low as 0.125 μg/mL, which is four times more potent than the clinically

Chemistry 2020, 2, x 28 used antibiotic vancomycin. However, all biguanide complexes have little activity towards the P. aeruginosa strain (MIC > 32 μg/mL). The authors hypothesized that this inefficiency was probably due to the membrane permeability and the presence of a very effective efflux system, making this bacterial strain particularly resistantChemistry to2020 , many2 antibiotics. Additionally, some compounds877 also displayed high activity against the fungal strains C. albicans and C. neoformans, with MICs values as low as 0.25 opportunity to design novel metallo-antibiotics associating, all in one molecule, metal ions and active μg/mL, which is 32 timesorganic more molecules. active For instance, than Chen the et al. reference introduced biguanide compound ligands, including fluconazole. the antidiabetic Moreover, when co- drug metformin, into organoiridium cyclopentadienyl complexes (compound 37, Figure 24)[162]. administered with vancomycin,Several of these complexes three exhibited complexes potent activity showed against both Gram-negative synergistic and Gram-positive activity and can restore the bacteria, including MRSA with MICs as low as 0.125 µg/mL, which is four times more potent than activity of the antibioticthe against clinically used vancomycin antibiotic vancomycin.-resistant However, all biguanide Enterococci complexes. have It littlewas activity also shown that some of these compounds couldtowards disrupt the P. aeruginosa S. aureusstrain (MIC biofilm> 32 µg/mL). Theformation. authors hypothesized Another that this ineffi ciencyimportant was point is that these probably due to the membrane permeability and the presence of a very effective efflux system, making novel Ir(III)-biguanide this complexes bacterial strain particularly exhibit resistant low to many toxicity antibiotics. toward Additionally, some mammalian compounds also cells, indicating high displayed high activity against the fungal strains C. albicans and C. neoformans, with MICs values as selectivity. Furthermore,low the as 0.25 authorsµg/mL, which isinv 32 timesestigated more active than the the referencepotential compound targets fluconazole. and Moreover, the mechanism of action when co-administered with vancomycin, three complexes showed synergistic activity and can restore of their complexes. Theythe activity suggested of the antibiotic that against their vancomycin-resistant organometallicEnterococci. It was complexes also shown that some could enter the bacteria of these compounds could disrupt S. aureus biofilm formation. Another important point is that (without disrupting the thesecell novel walls) Ir(III)-biguanide and complexesthen undergo exhibit low toxicity ligand toward mammalian exchange cells, indicating reaction high with thiol-containing selectivity. Furthermore, the authors investigated the potential targets and the mechanism of action biomolecules, displacingof theirthe complexes. biguanide They suggested ligand, that their which organometallic might complexes interfere could enter thewith bacteria important cell enzymes. (without disrupting the cell walls) and then undergo ligand exchange reaction with thiol-containing As the biguanide ligandbiomolecules, on its displacing own the had biguanide no ligand, antimicrobial which might interfere with effect, important it cell is enzymes. supposed that the overall As the biguanide ligand on its own had no antimicrobial effect, it is supposed that the overall structure structure of the complexof is the complexrequired is required in in order order to induce to ainduce biological action. a biological action.

Figure 24. Structure of antimicrobial biguanide Ir(III) complex [162].

Figure 24.Cobalt Structure is an essential metalof antimicrobial that is found in very low biguanide abundance in theIr(III) body and complex is well known [162]. to possess biochemical roles, especially as an integral part of vitamin B12, a cofactor involved in different human key processes such as in fatty acids metabolism, in the synthesis and regulation of DNA, and in energy production. Cobalt in biological systems exists almost exclusively as Co(II) Cobalt is an essentialor Co(III), metal though that Co(I) and is Co(IV) found are known in as very well [163 ].low In coordination abundance complexes, these in twothe latter body and is well known predominant oxidation states make them particularly suitable for therapeutic applications. Co(III) ions to possess biochemical roles, especially as an integral part of vitamin B12, a cofactor involved in different human key processes such as in fatty acids metabolism, in the synthesis and regulation of DNA, and in energy production. Cobalt in biological systems exists almost exclusively as Co(II) or Co(III), though Co(I) and Co(IV) are known as well [163]. In coordination complexes, these two latter predominant oxidation states make them particularly suitable for therapeutic applications. Co(III) ions form predominantly octahedral inert complexes, while Co(II) ions can induce the complexation of four, five or six ligands and their d7 electronic configuration makes Co(II)-based complexes particularly labile. The biological activity of cobalt complexes was first reported by Dwyer et al. in 1952, who exhibited in vitro micromolar bactericidal activity of their compounds, while a low systemic toxicity was observed in mice [89]. Since then, different studies have shown that cobalt(III) complexes could be efficiently used for antibacterial, antifungal and antiviral applications [164]. Most of the time, their antimicrobial properties were attributed to the greater lipophilic nature of the complexes as compared to the parent ligands, which improves their penetration across the cell membrane and thereby their activity. Furthermore, it has been shown that a platform based on both Co(II) and Co(III) metal ions can be designed with labile ligands that can undergo exchange with biomolecule moieties such as amino acid side chains. To date, the only cobalt-based complex that has reached phase II clinical trials is Doxovir (CTC-96, compound 38, Figure 25), a Co(III) Schiff base complex used for topical treatment of herpes simplex virus type 1, and it was demonstrated that this compound could also be used to treat viral eye infections [165]. Although the entire mechanism of action of this drug is not fully understood, its promising antiviral activity involved the inhibition of membrane fusion events required for viral entry into cells, through ligand exchange with proteins

Chemistry 2020, 2 878

form predominantly octahedral inert complexes, while Co(II) ions can induce the complexation of four, five or six ligands and their d7 electronic configuration makes Co(II)-based complexes particularly labile. The biological activity of cobalt complexes was first reported by Dwyer et al. in 1952, who exhibited in vitro micromolar bactericidal activity of their compounds, while a low systemic toxicity was observed in mice [89]. Since then, different studies have shown that cobalt(III) complexes could be efficiently used for antibacterial, antifungal and antiviral applications [164]. Most of the time, their antimicrobial properties were attributed to the greater lipophilic nature of the complexes as Chemistry 2020, 2, x compared to the parent ligands, which improves their penetration across the cell membrane and 29 thereby their activity. Furthermore, it has been shown that a platform based on both Co(II) and Co(III) metal ions can be designed with labile ligands that can undergo exchange with biomolecule [166]. It was thenmoieties suggested such as aminothat acidthe sideCo(III) chains. Schiff To date, base the only complex cobalt-based undergoes complex that has a reacheddissociative exchange of its labile 2-methylimidazolephase II clinical trials is axialDoxovir ligand (CTC-96, compound and interacts38, Figure 25 with), a Co(III) proteins Schiff base by complex coordinating used histidine for topical treatment of herpes simplex virus type 1, and it was demonstrated that this compound residues [167]. Thecould largealso be used difference to treat viral eyein infections reactivity [165]. betweenAlthough the entire the mechanism Co(III) and of action Co(II) of this allows specific activation mechanismsdrug is not and fully understood, makes cobalt its promising an idealantiviral candidate activity involved for the use inhibition in redox of membrane-activated prodrugs fusion events required for viral entry into cells, through ligand exchange with proteins [166]. It was [168,169]. It is wellthen known suggested that that the the Co(III) ligand Schiff baseexchange complex undergoesrates of a dissociativeCo(III) complexes exchange of its are labile several orders of magnitude lower2-methylimidazole than those axial of ligand the and Co(II) interacts complexe with proteinss by[1 coordinating70]. An histidine appropriately residues [167 ]. designed Co(III) The large difference in reactivity between the Co(III) and Co(II) allows specific activation mechanisms complex can thenand undergo makes cobalt bio an- idealreductive candidate activation for use in redox-activated in order prodrugs to produce [168,169 ].a Itcobalt is well known species that can acts as an effective redoxthat the-responsive ligand exchange drug rates of carrier. Co(III) complexes This areprinciple several orders can of magnitudebe applied lower thanto the those selective of release of the Co(II) complexes [170]. An appropriately designed Co(III) complex can then undergo bio-reductive bioactive ligands,activation which in order means to produce that a a cobalt complex species that can can be acts administered as an effective redox-responsive as an inert drug and biologically inactive Co(III) complex,carrier. This and principle then can be applied converted to the selective to a labile release ofand bioactive active ligands, Co(II) which complex. means that Such a strategy a complex can be administered as an inert and biologically inactive Co(III) complex, and then be has been widely convertedstudied to in a labile the and past active decades Co(II) complex. for Suchanticancer a strategy hasapplic been widelyations studied [171 in,17 the2 past]. However, to the best of our knowledge,decades for no anticancer reports applications have been [171,172 published]. However, to thein bestthe of literature our knowledge, concerning no reports have the use of cobalt been published in the literature concerning the use of cobalt complexes for the delivery of antimicrobial complexes for thedrugs delivery through aof bio-reductive antimicrobial activation. drugs This way through could be furthera bio explored-reductive by researchers activation. for the This way could be further exploreddevelopment by researchers of new Co-based for complexes the development that are able to induce of new selective Co release-based of active complexes ligands in that are able to induce selective releasespecific chemical of active environments, ligands such in as specific in bacteria. chemical environments, such as in bacteria.

Figure 25. Structure of cobalt-based complex Doxovir used for topical treatment of HSV-1 [166]. Figure 25. Structure of cobalt-based complex Doxovir used for topical treatment of HSV-1 [166].

Despite nickel being considered in very low quantities as an oligo-element, essential for animals and vegetable growth, it is classified in high quantities as carcinogenic for humans, explaining maybe why nickel complexes have not been investigated as much for their antibacterial properties as other metal ion complexes. Indeed, there is currently no nickel-based drug available, and only a few publications have investigated the antibacterial effects of nickel complexes further than fast in vitro assays giving the inhibition zone or the MIC. Nevertheless, some examples of the use of Schiff bases as a ligand for potential antibacterial nickel complexes can be found in the literature. Chalabian F. et al. studied for instance the activity of two nickel–Schiff base complexes 39–40 (Figure 26). Compared to the antibiotic standard gentamycin, the metal complexes showed higher inhibition area against Streptococcus pyogenes, but less good results against Bacillus anthracis and Staphylococcus aureus. It was also noticed that if the title ligand and the metal complexes had identical MICs against the two first bacteria strains, the ligand alone displayed the highest MIC against S. aureus (25 mg/mL), while the complexes showed lowest (6.25 mg/mL) [173].

Figure 26. Structures of Schiff base complexes of nickel(II) [173].

Another kind of nickel ligand is the thiocarbamide. However, although respectable, the activity of the hexakis thiocarbamide nickel(II) nitrate [Ni[CS(NH2)2]6](NO3)2 is lower than other known

Chemistry 2020, 2, x 29

[166]. It was then suggested that the Co(III) Schiff base complex undergoes a dissociative exchange of its labile 2-methylimidazole axial ligand and interacts with proteins by coordinating histidine residues [167]. The large difference in reactivity between the Co(III) and Co(II) allows specific activation mechanisms and makes cobalt an ideal candidate for use in redox-activated prodrugs [168,169]. It is well known that the ligand exchange rates of Co(III) complexes are several orders of magnitude lower than those of the Co(II) complexes [170]. An appropriately designed Co(III) complex can then undergo bio-reductive activation in order to produce a cobalt species that can acts as an effective redox-responsive drug carrier. This principle can be applied to the selective release of bioactive ligands, which means that a complex can be administered as an inert and biologically inactive Co(III) complex, and then be converted to a labile and active Co(II) complex. Such a strategy has been widely studied in the past decades for anticancer applications [171,172]. However, to the best of our knowledge, no reports have been published in the literature concerning the use of cobalt complexes for the delivery of antimicrobial drugs through a bio-reductive activation. This way could be further explored by researchers for the development of new Co-based complexes that are able to induce selective release of active ligands in specific chemical environments, such as in bacteria.

Chemistry 2020, 2 879

Figure 25. Structure of cobalt-based complex Doxovir used for topical treatment of HSV-1 [166]. Despite nickel being considered in very low quantities as an oligo-element, essential for animals and vegetableDespite nickel growth, being it is considered classified in in high very quantities low quantities as carcinogenic as an oligo- forelement, humans, essential explaining for animals maybe whyand nickel vegetable complexes growth, have it is classified not been in investigated high quantities as much as carcinogenic for their antibacterial for humans, propertiesexplaining asmaybe other metalwhy nickel ion complexes. complexes Indeed,have not therebeen investigated is currently as no much nickel-based for their antibacterial drug available, properties and only as other a few publicationsmetal ion complexes. have investigated Indeed, the there antibacterial is currently eff noects nickel of nickel-based complexes drug available, further and than only fast ina few vitro assayspublications giving thehave inhibition investigated zone the or theantibacterial MIC. Nevertheless, effects of nicke somel examplescomplexes of further the use than of Schi fastff inbases vitro as aassays ligand giving for potential the inhibition antibacterial zone or nickel the MIC. complexes Nevertheless, can be foundsome examples in the literature. of the useChalabian of Schiff bases F. et al . studiedas a ligand forinstance for potential the activityantibacterial of two nickel nickel–Schi complexffesbase can be complexes found in 39the– 40literature.(Figure Chalabian26). Compared F. et toal. the studied antibiotic for instance standard the gentamycin, activity of two the nickel metal–Schiff complexes base complexes showed higher 39–40 ( inhibitionFigure 26). areaCompared against Streptococcusto the antibiotic pyogenes standard, but less gent goodamycin, results the againstmetal complexBacilluses anthracis showedand higherStaphylococcus inhibition aureusarea against. It was alsoStreptococcus noticed that pyogenes if the, titlebut less ligand good and results the metal against complexes Bacillus anthr hadacis identical and Staphylococcus MICs against aureus the two. It was first bacteriaalso noticed strains, that the if ligandthe title alone ligand displayed and the metal the highest complex MICes had against identicalS. aureus MICs(25 against mg/mL), the two while first the complexesbacteria strains, showed the lowest ligand (6.25 alone mg displayed/mL) [173 the]. highest MIC against S. aureus (25 mg/mL), while the complexes showed lowest (6.25 mg/mL) [173].

FigureFigure 26. 26. StructuresStructures ofof SchiSchiffff base complexes of of nickel(II) nickel(II) [1 [17373]]..

AnotherAnother kind kind of of nickel nickel ligandligand isis thethe thiocarbamide.thiocarbamide. However, However, although although respectable respectable,, the the activity activity ofof the the hexakis hexakis thiocarbamide thiocarbamide nickel(II) nickel(II) nitrate nitrate [Ni[CS(NH [Ni[CS(NH22))22]6](NO](NO3)32) 2isis lower lower than than other other known known antibiotics, with inhibition zones, for instance, only 0.4 to 0.7 times those of chloramphenicol [174]. It would nevertheless be interesting to test these compounds against resistant bacteria, as they probably do not target the same essential metabolic pathways. Rather than developing completely new therapeutic agents, other scientists have tried to improve the efficiency of current antibiotics by using them as ligands. Hence, by comparing the activity of Ceclor with its Co- and Ni- complexes against both Gram-negative and Gram-positive bacteria strains with the disk-diffusion method, it was observed that both metal derivatives (Figure 27) showed higher inhibition areas that the title Ceclor, and the Ni complex had even better results than the Co complex against Streptococcus pyogenes and Escherichia coli [175]. If the role of cobalt and nickel is not described in the paper, it is known that Ceclor, as a β-lactam antibiotic, acts by impairing the cross-linking of the peptidoglycans forming the bacterial cell wall. A second example is the nickel complexes of the kefzol, another β-lactam antibiotic. They displayed a higher inhibition zone than the antibiotic alone against Staphyloccocus aureus, Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. It was also noticed that the NiL2 complex had better efficiency than the NiL complex, which was in accordance with the antibiotic nature of the ligand [176]. Chemistry 2020, 2, x 30 antibiotics, with inhibition zones, for instance, only 0.4 to 0.7 times those of chloramphenicol [174]. It would nevertheless be interesting to test these compounds against resistant bacteria, as they probably do not target the same essential metabolic pathways. Rather than developing completely new therapeutic agents, other scientists have tried to improve the efficiency of current antibiotics by using them as ligands. Hence, by comparing the activity of Ceclor with its Co- and Ni- complexes against both Gram-negative and Gram-positive bacteria strains with the disk-diffusion method, it was observed that both metal derivatives (Figure 27) showed higher inhibition areas that the title Ceclor, and the Ni complex had even better results than the Co complex against Streptococcus pyogenes and Escherichia coli [175]. If the role of cobalt and nickel is not described in the paper, it is known that Ceclor, as a β-lactam antibiotic, acts by impairing the cross-linking of the peptidoglycans forming the bacterial cell wall. A second example is the nickel complexes of the kefzol, another β-lactam antibiotic. They displayed a higher inhibition zone than the antibiotic alone against Staphyloccocus aureus, Escherichia coli, Klebsiella pneumonia, and Proteus mirabilisChemistry. It was2020 also, 2 noticed that the NiL2 complex had better efficiency than the NiL complex880 , which was in accordance with the antibiotic nature of the ligand [176].

FigureFigure 27. Proposed 27. Proposed structures structures of of nickel complexes complexes with with kefzol kefzol [176]. [176].

Moreover, nickel is present as cofactors in a half-dozen of bacterial enzymes, most of them Moreoverinvolved, in nickel the production is present or use as of cofactors gases. A non-exhaustive in a half-dozen list of enzymes of bacterial using enzymes, nickel ions includes most of them involvedthe in urease the which production catalyzes or the use hydrolysis of gases. of urea, A non a kind-exhaustive of superoxide list dismutase, of enzymes which using uses nickel nickel ions includesions the against urease reactive which oxidative catalyzes species, the hydrolysis the glyoxylase of Iurea of Leishmania, a kind majorof superoxide, which needs dismutase nickel ion , which uses nickelto act ions as Lewis against acid reactive in the conversion oxidative of species methylglyoxal, the glyoxylase to lactate (in I of other Leishmania eukaryotic major organisms,, which needs nickel ionthe nickel to act ion as is Lewis replaced acid by zinc), in the and conversion the NiFe hydrogenase, of methylglyoxal which catalyzes to lactate the reversible (in other redox eukaryotic reaction between protons and dihydrogen. Originally, one subunit of the Methyl-CoM reductase used organisms, the nickel ion is replaced by zinc), and the NiFe hydrogenase, which catalyzes the a nickel ion in its +1 oxidation state, and one of its discussed mechanisms includes an intermediate reversiblestage redox with areaction Ni(III) ion between [177,178 ].protons Thus, bacteria and dihydrogen. need nickel ions Originally, for normal growth,one subunit and it hasof the also Methyl- CoM reductasebeen shown used that thea nickel calprotectin, ion ina chelator its +1 secreted oxidation by the state, human and immune one system,of its discussed can confine nickel mechanisms includesions. an interm Due to theseediate higher stage nickel with requirements a Ni(III) ion for [1 bacteria77,178] compared. Thus, bacteria to humans, need some nickel studies ions have for normal growth,therefore and it has focused also on been the chelationshown that of the the nickel calprotectin, ions naturally a chelator present insecreted the human by bodythe human to impair immune system,bacterial can confine growth, nickel rather ions. than investigating Due to thes thee antibacterialhigher nickel properties requirements of this element. for bacteria From this compared point to of view, and knowing the great importance of metal ions in general for the human proteins, the need humans,to some have a studies very selective have chelatortherefore lead focused to the testing on the of chelation dimethylglyoxime of the nickel as a potential ions naturally antibacterial present in the humancompound. body to Dimethylglyoxime impair bacterial is growth a bidentate, rather ligand, tha andn investigating two molecules the coordinate antibacterial Ni(II) withproperties a of this element.square From planar this geometry point (Figure of view, 28, compoundand knowing41). Inthe vitro greatassays importance on one Klebsiella of metal pneumonia ions instrain general for the humanand twoproteins,Salmonella the entericaneed to serovar have Typhimuriuma very selectivestrains chelator resulted lead in MICs to th ine the testing low millimolar of dimethylglyoxime range, as a potentialand the conclusionantibacterial that compound. the dimethylglyoxime Dimethylglyox has a bacteriostaticime is a ebidentateffect rather beingligand, bactericidal and two [179 molecules]. As expected, the presence of dimethylglyoxime inhibits the hydrogenase activity of the two Salmonella coordinate Ni(II) with a square planar geometry (Figure 28, compound 41). In vitro assays on one and impairs the urease enzyme of a Klebsiella species. Furthermore, while some other toxicology tests Klebsiellaare pneumonia required before strain considering and two dimethylglyoximeSalmonella enterica as serovar a drug candidate, Typhimurium preliminary strains assays resulted showed in MICs in the lowthat millimolar high quantities range, of theand chelators the conclusion are not toxic that for the mice dimethylglyoxime and larvae, but better has reduce a bacteriostatic mortality for effect rather mice being and bacteric insects inidal case [ of17Salmonella9]. As expected,infection, even the if some presence traces are of measurable dimethylglyoxime in the liver of inhibits the the hydrogenasemice [179 activity]. of the two Salmonella and impairs the urease enzyme of a Klebsiella species.

Chemistry 2020, 2, x 31

Furthermore, while some other toxicology tests are required before considering dimethylglyoxime as a drug candidate, preliminary assays showed that high quantities of the chelators are not toxic for mice and larvae, but betterChemistry 2020 reduce, 2 mortality for mice and insects in case of Salmonella881 infection, even if some traces are measurable in the liver of the mice [179].

Figure 28. Structure of dimethylglyoxime and nickel-dimethylglyoxime complex [179].

Figure 28. StructureNickel chelatorsof dimethylglyoxime are also used in the case of and allergies. nickel Indeed,-dimethylglyoxime this element is the most common complex [179]. cause of metal allergy by skin contact and accounts for more cases than all other metals together. Thus, in the case of eczema caused by nickel, the use of chelators can help to treat dermatitis by Nickel chelators aresequestering also theused responsible in the metal ionscase [180 of]. allergies. Indeed, this element is the most common In opposition to nickel, manganese ions are present in several enzymes as cofactors, both in cause of metal allergyhumans by skin and pathogens. contact Then, and chelating accounts the cellular manganese for more could be cases tricky without than disturbing all other metals together. the metal cation homeostasis of the host organism. Therefore, the research rather tried to look at Thus, in the case of eczemathe antibacterial caused effect of manganeseby nickel, complexes. the Although use noof drug chelators based on a manganese can help complex to treat dermatitis by is currently under commercialization, preliminary evaluations of antibacterial properties of some sequestering the responsiblecomplexes havemetal been published.ions [180 Thus,]. Chaudhary et al. synthesized a series of manganese(II) complexes 42. The ligands are constituted of two diaminopyridine or diethylenetriamine molecules In opposition to linkednickel, on each manganese side by an amide bond ions to a malonic, are succinic,present glutaric, in or adipicseveral acid linker. enzymes They result as cofactors, both in humans and pathogens.in macrocycles Then, containingchelat sixing nitrogen the atomscellular able to coordinate mangan the manganeseese could ion, even be if furthertricky without disturbing analysis shows that only those belonging to the amide function coordinate the cation in a plane, the metal cation homeostasisaccompanied of by twothe chlorides host onorganism. both sides to reach Therefore, an octahedral geometry the research [181]. Antibacterial rather tried to look at the assays showed only a slightly improved activity of the macrocycles against Pseudomonas cepacicola, antibacterial effect of manganeseStaphylococcus aureus, andcomplexes.Xanthomonas compestris Althoughcompared to n theo activity drug of the based ligand moieties on alone.a manganese complex is For instance, by considering the ligand constituted of two diaminopyridine and two adipic acid currently under commercialization,molecules (compound series 42 preliminary, model structure on left, evaluationn = 4, Figure 29),s 3.5 andof 4.1 antibacterial mM of respectively properties of some adipic acid and DMAP are needed to get a 3 mm inhibition zone against P. cepacicola, while the complexes have beenmanganese published. complex displays Thus, a 3-fold Chaudhar larger inhibition zoney et for only al. 0.9 synthesized mM. By comparison, at 0.9a mM series of manganese(II) complexes 42. The ligandsthe standard are streptomycin constituted results in aof 3 mm two inhibition diaminopyridine zone. Moreover, there is a greater or diethylenetriamine enhancement molecules of some of the complexes (mainly those containing a DMAP moiety) over their building blocks at higher linked on each side byconcentration. an amide By multiplying bond theto concentration a malonic, by a factor succinic, of 2, the respective glutaric inhibition, or zones adipic of acid linker. They DMAP, adipic acid and the Mn complex are 4 mm, 4 mm and 16 mm. However, the improvements are result in macrocycles slightercontaining against S. aureus six(4 mm,nitrogen 6 mm, 15 mm, atoms whereas itable is 17 mm to for streptomycin)coordinateand X. compestristhe manganese ion, even if (4 mm, 6 mm, 12 mm; 5 mm for the streptomycin). Another issue is the results of the toxicity tests were further analysis showsnevertheless that only performed those in vivo onbelonging rats, showing a lackto ofthe fertility amide [181]. function coordinate the cation in a plane, accompanied by two chlorides on both sides to reach an octahedral geometry [181]. Antibacterial assays showed only a slightly improved activity of the macrocycles against Pseudomonas cepacicola, Staphylococcus aureus, and Xanthomonas compestris compared to the activity of the ligand moieties alone. For instance, by considering the ligand constituted of two diaminopyridine and two adipic acid molecules (compound series 42, model structure on left, n = 4, Figure 29), 3.5 and 4.1 mM of respectively adipic acid and DMAP are needed to get a 3 mm inhibition zone against P. cepacicola, while the manganese complex displays a 3-fold larger inhibition zone for only 0.9 mM. By comparison, at 0.9 mM the standard streptomycin results in a 3 mm inhibition zone. Moreover, there is a greater enhancement of some of the complexes (mainly those containing a DMAP moiety) over their building blocks at higher concentration. By multiplying the concentration by a factor of 2, the respective inhibition zones of DMAP, adipic acid and the Mn complex are 4 mm, 4 mm and 16 mm. However, the improvements are slighter against S. aureus (4 mm, 6 mm, 15 mm, whereas it is 17 mm for streptomycin) and X. compestris (4 mm, 6 mm, 12 mm; 5 mm for the streptomycin). Another issue is the results of the toxicity tests were nevertheless performed in vivo on rats, showing a lack of fertility [181].

Chemistry 2020, 2, x 31

Furthermore, while some other toxicology tests are required before considering dimethylglyoxime as a drug candidate, preliminary assays showed that high quantities of the chelators are not toxic for mice and larvae, but better reduce mortality for mice and insects in case of Salmonella infection, even if some traces are measurable in the liver of the mice [179].

Figure 28. Structure of dimethylglyoxime and nickel-dimethylglyoxime complex [179].

Nickel chelators are also used in the case of allergies. Indeed, this element is the most common cause of metal allergy by skin contact and accounts for more cases than all other metals together. Thus, in the case of eczema caused by nickel, the use of chelators can help to treat dermatitis by sequestering the responsible metal ions [180]. In opposition to nickel, manganese ions are present in several enzymes as cofactors, both in humans and pathogens. Then, chelating the cellular manganese could be tricky without disturbing the metal cation homeostasis of the host organism. Therefore, the research rather tried to look at the antibacterial effect of manganese complexes. Although no drug based on a manganese complex is currently under commercialization, preliminary evaluations of antibacterial properties of some complexes have been published. Thus, Chaudhary et al. synthesized a series of manganese(II) complexes 42. The ligands are constituted of two diaminopyridine or diethylenetriamine molecules linked on each side by an amide bond to a malonic, succinic, glutaric, or adipic acid linker. They result in macrocycles containing six nitrogen atoms able to coordinate the manganese ion, even if further analysis shows that only those belonging to the amide function coordinate the cation in a plane, accompanied by two chlorides on both sides to reach an octahedral geometry [181]. Antibacterial assays showed only a slightly improved activity of the macrocycles against Pseudomonas cepacicola, Staphylococcus aureus, and Xanthomonas compestris compared to the activity of the ligand moieties alone. For instance, by considering the ligand constituted of two diaminopyridine and two adipic acid molecules (compound series 42, model structure on left, n = 4, Figure 29), 3.5 and 4.1 mM of respectively adipic acid and DMAP are needed to get a 3 mm inhibition zone against P. cepacicola, while the manganese complex displays a 3-fold larger inhibition zone for only 0.9 mM. By comparison, at 0.9 mM the standard streptomycin results in a 3 mm inhibition zone. Moreover, there is a greater enhancement of some of the complexes (mainly those containing a DMAP moiety) over their building blocks at higher concentration. By multiplying the concentration by a factor of 2, the respective inhibition zones of DMAP, adipic acid and the Mn complex are 4 mm, 4 mm and 16 mm. However, the improvements are slighter against S. aureus (4 mm, 6 mm, 15 mm, whereas it is 17 mm for streptomycin) and X. compestris (4 mm, 6 mm, 12 mm; 5 mm for the streptomycin). Another issue is the results ofChemistry the 2020 to,xicity2 tests were nevertheless performed in vivo on rats, showing882 a lack of fertility [181].

Chemistry 2020, 2, x 32

Figure 29. Structures of the manganese complexes series 42 [181].

Manganese is known for its antifertility properties, and other kinds of manganese complexes encountered the same issue. Bansal et al. developed ligands based on an imine obtained by the condensation of a salicylanilide with a sulphathiazole. One can notice the sulfonamide moiety, which has already been discussed in theFigure copper 29. Structures part of the(Section manganese complexes2.2). In series addition42 [181]. to the slight antifungal activity of 43 (see Figure 30) by inhibiting the growth of Aspergillus niger, the manganese complex synthesized Manganese is known for its antifertility properties, and other kinds of manganese complexes showed good inhibitionencountered results the same against issue. Bansal Gram et al.- developednegative ligands bacteria, based on with an imine a obtained2- to 9 by-fold the bigger inhibition condensation of a salicylanilide with a sulphathiazole. One can notice the sulfonamide moiety, which zone compared to thehas already streptomycin been discussed instandard, the copper part and (Section compared 2.2). In addition to to the the slight ligand antifungal alone. activity This is explained by the authors by improvedof 43 (see Figure lipophilicity 30) by inhibiting due the growth to the of Aspergillus chelation niger, the of manganese the cation. complex Indeed, synthesized chelation causes a showed good inhibition results against Gram-negative bacteria, with a 2- to 9-fold bigger inhibition decrease in the polarityzone compared around to the streptomycinthe cation. standard, Then, and comparedthe complex to the ligand can alone. interact This is explained more by efficiently with the bacteria cell wall andthe authors pass by through improved lipophilicity it more due easily. to the chelation The diameter of the cation. Indeed,of the chelation inhibition causes a area was however decrease in the polarity around the cation. Then, the complex can interact more efficiently with the similar to the standardbacteria for cell wallStaphylococcus and pass through itaureus more easily., a Gram The diameter-positive of the inhibition bacteria. area was Finally, however despite these data, similar to the standard for Staphylococcus aureus, a Gram-positive bacteria. Finally, despite these data, it was observed thatit was the observed manganese that the manganese complex complex has has the lowest lowest results results compared tocompared complexes between to complexes between the same ligands andthe same tin ligands or silicon and tin or [1 silicon82]. [ 182].

43 Figure 30. Structure of the ligand and proposed structure for the manganese complex [182]. Figure 30. Structure of the ligand and proposed structure for the manganese complex [182]. Anacona and Bastardo also developed a manganese(II) complex with a hexaaza macrocycle Schiff base ligand (Figure 31). Based on a phenanthroline, two diaminonaphthoquinones, and dibromoethane, Anacona andthey Bastardo obtained no crystalsalso deve suitableloped for diffraction; a manganese(II) therefore, they assumed, complex based on the similarity with a with hexaaza macrocycle the literature, that the ligand formed a planar and tetradentate base around the cation, completed Schiff base ligandby (Figure two water molecules 31). Based or two bromine on anions a phenanthroline, to obtain an octahedral configuration. two diaminonapht Antimicrobial hoquinones, and dibromoethane, theyassays obtained show no effect no on Pseudomonascrystals aeruginosasuitableand forE. coli diffraction, a small effect on; thereforeS. aureus, but with, they a less assumed, based on good inhibition than sodium penicillinate except at 2 µg/mL, and higher inhibition of Bacillus cereus the similarity withthan the sodium literature penicillinate, that [183]. the ligand formed a planar and tetradentate base around the cation, completed by two water molecules or two bromine anions to obtain an octahedral configuration. Antimicrobial assays show no effect on Pseudomonas aeruginosa and E. coli, a small effect on S. aureus, but with a less good inhibition than sodium penicillinate except at 2 µg/mL, and higher inhibition of Bacillus cereus than sodium penicillinate [183].

Figure 31. Schiff base ligand for manganese [183].

2.10. Mixing Metal into Complexes To further improve the antimicrobial activity of metal complexes, one solution could be to mix different metals. In addition, this way has the frequent advantage of decreasing the toxicity of the metal ions by reducing their respective MICs; thus, the synthesis and antimicrobial analysis of a series of metal and dimetal complexes based on a salen ligand Cu, Ag, Zn and Bi were performed (Figure 32). Indeed, thanks to their N2O2 site and their numerous possible derivatives, salen-derivatives are versatile and can eventually form several compartments able to accommodate different metal ions.

Chemistry 2020, 2, x 32

Figure 29. Structures of the manganese complexes series 42 [181].

Manganese is known for its antifertility properties, and other kinds of manganese complexes encountered the same issue. Bansal et al. developed ligands based on an imine obtained by the condensation of a salicylanilide with a sulphathiazole. One can notice the sulfonamide moiety, which has already been discussed in the copper part (Section 2.2). In addition to the slight antifungal activity of 43 (see Figure 30) by inhibiting the growth of Aspergillus niger, the manganese complex synthesized showed good inhibition results against Gram-negative bacteria, with a 2- to 9-fold bigger inhibition zone compared to the streptomycin standard, and compared to the ligand alone. This is explained by the authors by improved lipophilicity due to the chelation of the cation. Indeed, chelation causes a decrease in the polarity around the cation. Then, the complex can interact more efficiently with the bacteria cell wall and pass through it more easily. The diameter of the inhibition area was however similar to the standard for Staphylococcus aureus, a Gram-positive bacteria. Finally, despite these data, it was observed that the manganese complex has the lowest results compared to complexes between the same ligands and tin or silicon [182].

43 Figure 30. Structure of the ligand and proposed structure for the manganese complex [182].

Anacona and Bastardo also developed a manganese(II) complex with a hexaaza macrocycle Schiff base ligand (Figure 31). Based on a phenanthroline, two diaminonaphthoquinones, and dibromoethane, they obtained no crystals suitable for diffraction; therefore, they assumed, based on the similarity with the literature, that the ligand formed a planar and tetradentate base around the cation, completed by two water molecules or two bromine anions to obtain an octahedral configuration. Antimicrobial assays show no effect on Pseudomonas aeruginosa and E. coli, a small Chemistry 2020, 2 883 effect on S. aureus, but with a less good inhibition than sodium penicillinate except at 2 µg/mL, and higher inhibition of Bacillus cereus than sodium penicillinate [183].

FigureFigure 31. SchiffSchiff basebase ligand ligand for manganese [18 [1833]].. 2.10. Mixing Metal into Complexes 2.10. Mixing Metal into Complexes To further improve the antimicrobial activity of metal complexes, one solution could be to mix To further improve the antimicrobial activity of metal complexes, one solution could be to mix different metals. In addition, this way has the frequent advantage of decreasing the toxicity of the different metals. In addition, this way has the frequent advantage of decreasing the toxicity of the metal ions by reducing their respective MICs; thus, the synthesis and antimicrobial analysis of a series metal ions by reducing their respective MICs; thus, the synthesis and antimicrobial analysis of a series of metal and dimetal complexes based on a salen ligand Cu, Ag, Zn and Bi were performed (Figure 32). of metal and dimetal complexes based on a salen ligand Cu, Ag, Zn and Bi were performed (Figure Indeed, thanks to their N2O2 site and their numerous possible derivatives, salen-derivatives are 32). Indeed, thanks to their N2O2 site and their numerous possible derivatives, salen-derivatives are versatileChemistry 20 and20, 2 can, x eventually form several compartments able to accommodate different metal ions.33 versatile and can eventually form several compartments able to accommodate different metal ions. Here, the complexes demonstrated large inhibition zones for the bimetallic complexes, comparable to, Here, the complexes demonstrated large inhibition zones for the bimetallic complexes, comparable even if slightly smaller than, those caused by the pure salts, whereas the monometallic complexes did notto, displayeven if slightly any inhibition. smallerAccording than, those to caused the authors, by the this pure lack salts, of activity whereas was the due monometallic to the solubility complexes of the compounds.did not display Indeed, any inhibition. the bimetallic According complexes to the are authors, all charged, this displayinglack of activity higher was solubility due to the across solubility agar andof the therefore compounds. better activity Indeed, using the bimetallic the agar di complexesffusion technique. are all charged, In contrast, displaying monometallic higher complexes solubility areacross all neutral, agar and making therefore their better dissolution activity inusing the agarthe agar diffi dicult.ffusion Another technique. reason In could contrast be the, monometallic strength of thecomplexes salen ligand, are all drastically neutral, making limiting their the dissolution progressive in release the agar of itsdifficult metal. ion,Another while reason the second could onebe the is lessstrength strongly of the coordinated. salen ligand, This drastically is confirmed limiting by the the biological progressive analysis: release the of antimicrobial its metal ion, activities while the of thesecond bimetallic one is compounds less strongly are coordinated. similar to those This ofis confirmed the second by metal the ion,biological suggesting analysis: that the antimicrobial coordination hidesactivities the firstof the one bimetallic to its environment, compounds reducing are similar its biodisponibility to those of the [second184]. metal ion, suggesting that the coordination hides the first one to its environment, reducing its biodisponibility [184].

Figure 32. Monometallic and bimetallic complexes based on salen ligand [184]. Figure 32. Monometallic and bimetallic complexes based on salen ligand [184]. As a last but not least example in this review about metal complexes, it is possible to gather several As a last but not least example in this review about metal complexes, it is possible to gather different metal ions. By combining manganese with rhenium and iron or ruthenium, Bandow et al. several different metal ions. By combining manganese with rhenium and iron or ruthenium, Bandow designed hetero triorganometallic compounds FcPNA and RcPNA (Figure 33, compounds 44 and 45) et al. designed hetero triorganometallic compounds FcPNA and RcPNA (Figure 33, compounds 44 containing a cymantrene residue where the manganese possess a tetrahedral symmetry, a ferrocene or and 45) containing a cymantrene residue where the manganese possess a tetrahedral symmetry, a ruthenocene, and a (dipicolyl)Re(CO)3 group. With a peptide-mimic moiety, a positively charged moiety, ferrocene or ruthenocene, and a (dipicolyl)Re(CO)3 group. With a peptide-mimic moiety, a positively charged moiety, and three organometallic residues constituted of rather lipophilic ligands, the compounds shared similarities with the lipidic membrane of bacteria, allowing good permeability. Hence, by determining the MICs of the two complexes and the small organometallic residues by themselves, it was shown that FcPNA had better efficiency than RcPNA, and that the building blocks did not show any activity at high concentrations (512 µg/mL). Thus, FcPNA was demonstrated to have 4–6 times lower MICs than amoxicillin, similar MICs to norfloxacin, and 5–6 times higher MICs than vancomycin against Bacillus subtilis and Staphylococcus aureus. The best results were against resistant S. aureus MRSA, where the MIC for FcPNA was divided by 100 compared to amoxicillin. The authors report that it also has a good activity against vancomycin-intermediate S. aureus. Moreover, it seems that FcPNA is bactericidal rather than bacteriostatic. Finally, no lysis of the bacteria membrane (leading to the dissolution of the bacteria) was observed, suggesting that the compound’s target is into the bacteria, and that the complex is able to pass through the membrane. Nevertheless, the drawback is that it is also able to interact with proteins, as was demonstrated by measuring the MIC of FcPNA in the presence of bovine serum albumin. The 5-fold increase indicates a strong binding of the FcPNA with the serum. Thus, cytotoxicity studies give dispersed results, with more than 90% survival for CAco-2, L6.C11, or HEpG2 cells, when MCF7 cells have only a 10% viability in the presence of FcPNA. Finally, neither complex demonstrated any activity against E. coli, A. baumannii, or P. aeruginosa, even at the limit of solubility (25 µg/mL) [185]. This is in keeping with the interactions of the complexes with the bacteria membrane, as the three last bacteria are Gram- negative strains, and thus harbor two membranes surrounding a peptidoglycan cell wall in

Chemistry 2020, 2 884

and three organometallic residues constituted of rather lipophilic ligands, the compounds shared similarities with the lipidic membrane of bacteria, allowing good permeability. Hence, by determining the MICs of the two complexes and the small organometallic residues by themselves, it was shown that FcPNA had better efficiency than RcPNA, and that the building blocks did not show any activity at high concentrations (512 µg/mL). Thus, FcPNA was demonstrated to have 4–6 times lower MICs than amoxicillin, similar MICs to norfloxacin, and 5–6 times higher MICs than vancomycin against Bacillus subtilis and Staphylococcus aureus. The best results were against resistant S. aureus MRSA, where the MIC for FcPNA was divided by 100 compared to amoxicillin. The authors report that it also has a good activity against vancomycin-intermediate S. aureus. Moreover, it seems that FcPNA is bactericidal rather than bacteriostatic. Finally, no lysis of the bacteria membrane (leading to the dissolution of the bacteria) was observed, suggesting that the compound’s target is into the bacteria, and that the complex is able to pass through the membrane. Nevertheless, the drawback is that it is also able to interact with proteins, as was demonstrated by measuring the MIC of FcPNA in the presence of bovine serum albumin. The 5-fold increase indicates a strong binding of the FcPNA with the serum. Thus, cytotoxicity studies give dispersed results, with more than 90% survival for CAco-2, L6.C11, or HEpG2 cells, when MCF7 cells have only a 10% viability in the presence of FcPNA. Finally, neither complex demonstrated any activity against E. coli, A. baumannii, or P. aeruginosa, even at the limit of solubility (25 µg/mL) [185]. This is in keeping with the interactions of the complexes Chemistrywith 20 the20, bacteria2, x membrane, as the three last bacteria are Gram-negative strains, and thus harbor 34 two membranes surrounding a peptidoglycan cell wall in opposition with B. subtilis and S. aureus, which oppositionare Gram-positive with B. subtilis bacteria and and S have. aureus only, awhich single cytoplasmicare Gram-positive membrane bacteria constituted and of have phospholipids. only a single cytoplasmic membrane constituted of phospholipids.

Figure 33. Structure of the heterotriorganometallic complexes, M = Fe (FcPNA) 44 or M = Ru (RcPNA) Figure 33. Structure of the heterotriorganometallic complexes, M = Fe (FcPNA) 44 or M = Ru (RcPNA) 45 [185]. 45 [185]. By comparing the protein expression of B. subtilis in the presence of the complexes to its proteomic libraryBy comparing under normal the conditions, protein expression Bandow et al. of observedB. subtilis which in the proteins presence were increasingly of the complexes expressed. to its proteomicThese werelibrary linked under to thenormal cell envelopeconditions, stress Bandow (among et othersal. observed PspA, stabilizingwhich proteins the cell were membrane), increasingly expressed.the energy These metabolism were linked (proteins to involvedthe cell envelope in the fatty stress acid biosynthesis, (among others often PspA, observed stabilizing as a response the cell membrane),to the use the of antibioticsenergy metabolism targeting the (proteins membrane), involved and the in general the fatty stress acid response. biosynthesis All these, often elements observed as a ledresponse the authors to the to use suggest of antibiotics that the complexes targeting target the membrane the cell membrane), and the by gen embeddingeral stress in it.response. Finally, All theseno elements major di fflederence the wasauthors observed to suggest for bacteria that the cultivating complexes with target FcPNA the compared cell membrane to those by cultivated embedding with RcPNA, except for an oxidative stress due to FcPNA. That only FcPNA can be responsible for in it. Finally, no major difference was observed for bacteria cultivating with FcPNA compared to oxidation can be linked to the different redox properties of the ferrocene and the ruthenocene [185]. those cultivated with RcPNA, except for an oxidative stress due to FcPNA. That only FcPNA can be responsible3. Synergy for of oxidation Metal Complexes can be linkedwith Antimicrobials to the different redox properties of the ferrocene and the ruthenoceneIn this [18 part,5]. we will not present many literature examples, some of which have been previously discussed in their related metal chapter. The aim of this chapter is rather to develop the idea of 3. Synergyassociating of Metal antimicrobials Complexes and metalwith complexesAntimicrobials against pathogen bacteria. In this part, we will not present many literature examples, some of which have been previously discussed in their related metal chapter. The aim of this chapter is rather to develop the idea of associating antimicrobials and metal complexes against pathogen bacteria. The ability of metal complexes to open new routes in therapy may be exploited against resistant bacteria. A second manner to associate metal complexes with antimicrobials is to exploit existing antimicrobial compounds as ligands for metal cations. Indeed, as shown in a lot of the previous examples [32,72,73,77,78,80,84,86,87,97,175,176], the metal complexes displayed higher activity than their ligands or their building-blocks themselves. It is therefore suggested that metal–antibiotic combinations could lead to synergistic effects in terms of their respective antimicrobial properties. A reason for this is found by focusing on the chemical properties of both partners. Among other things, a great issue for the design of new antibiotics is balancing their lipophilic and hydrophilic properties. Indeed, for a drug to be effective in vivo, one important property is to be sufficiently soluble to be brought through the blood to its targeted bacteria, but at the same time to be sufficiently lipophilic to pass through the membrane; those of bacteria as well as the gastrointestinal wall for oral drugs. Thus, some potential antibiotics having a promising activity in vitro do not pass the in vivo step due to having a biodisponibility that is too low. Metal salts are often too polar to interact with the lipid membranes, as well as some organic molecules, designed in this way, they are able to link to their target, generally through H-bonds or electrostatic interactions, and therefore with a lot of polar functions. On the face of it, it would seem there is no reason to combine two “too polar” moieties to decrease the overall hydrophilicity. Nevertheless, as explained previously with the ligand field theory, the complexation of the metal ion with a ligand tends to attenuate the positive charge of the cation by sharing it on a larger area through the orbitals and to decrease the overall electron density of the ligand [55,56,68,84,99,110,164,182]. This is why orienting the research to the synthesis of metal ion-antibiotic complexes could lead to an enhancement of their usual efficiency through an improvement of the biodisponibility and antimicrobial activity [176].

Chemistry 2020, 2 885

The ability of metal complexes to open new routes in therapy may be exploited against resistant bacteria. A second manner to associate metal complexes with antimicrobials is to exploit existing antimicrobial compounds as ligands for metal cations. Indeed, as shown in a lot of the previous examples [32,72,73,77,78,80,84,86,87,97,175,176], the metal complexes displayed higher activity than their ligands or their building-blocks themselves. It is therefore suggested that metal–antibiotic combinations could lead to synergistic effects in terms of their respective antimicrobial properties. A reason for this is found by focusing on the chemical properties of both partners. Among other things, a great issue for the design of new antibiotics is balancing their lipophilic and hydrophilic properties. Indeed, for a drug to be effective in vivo, one important property is to be sufficiently soluble to be brought through the blood to its targeted bacteria, but at the same time to be sufficiently lipophilic to pass through the membrane; those of bacteria as well as the gastrointestinal wall for oral drugs. Thus, some potential antibiotics having a promising activity in vitro do not pass the in vivo step due to having a biodisponibility that is too low. Metal salts are often too polar to interact with the lipid membranes, as well as some organic molecules, designed in this way, they are able to link to their target, generally through H-bonds or electrostatic interactions, and therefore with a lot of polar functions. On the face of it, it would seem there is no reason to combine two “too polar” moieties to decrease the overall hydrophilicity. Nevertheless, as explained previously with the ligand field theory, the complexation of the metal ion with a ligand tends to attenuate the positive charge of the cation by sharing it on a larger area through the orbitals and to decrease the overall electron density of the ligand [55,56,68,84,99,110,164,182]. This is why orienting the research to the synthesis of metal ion-antibiotic complexes could lead to an enhancement of their usual efficiency through an improvement of the biodisponibility and antimicrobial activity [176]. Finally, it is possible to resort to metal complexes as cofactors of antibiotics. As discussed in some examples of this review, metal complexes sometimes display higher antimicrobial activity than the standard antimicrobials, particularly against resistant bacteria such as S. aureus MRSA [32,119,156,161,185]. Therefore, it could be interesting to administer the metal complex together with an antimicrobial to improve both of their effects [32,78]. When they do not have the same targets, this multi-therapy approach leads to reaching the bacteria independently on both ends. The metal complex can act for instance by increasing the porosity of the bacterial cell wall, which 2+ facilitates the entry of the antibiotic into the bacteria (see, for instance, the [Ru(phen2)(p-BPIP)] [94]), as well as by protecting the antibiotic against degradation by inactivating the involved enzymes (see for instance some bismuth complexes, where the Bi(III) ion may replace the zinc ions of the metallo-β-lactamase enzymes [133]). Therefore, co-administration of different compounds could be useful either against resistant organisms, or to avoid the emergence of resistance by non-resistant organisms, as it is more complicated to develop two different resistance pathways simultaneously [156,186]. Inducing synergy between the effects of the metal ion and its ligands themselves, resulting in not only the addition of their antimicrobial properties but even their multiplication, may be a good therapeutic option. The presence (or the absence) of synergy is caused by the fractional inhibitory concentration index (FICI): below 0.5, the FICI indicates synergy; between 0.5 and 4.0, it indicates an absence of synergy; and over 4.0, it indicates an antagonism between both parts of the complex. The FICI for combination therapy of n compounds is calculated by the addition over n of the ratio MIC of compound x in combination/MIC of compound x alone [32] Thus, the combination of metal ions with known antibiotics, more than just adding their antimicrobial properties, may improve both of them, as illustrated by the example of silver–ampicillin complexes described previously [32,33]. To improve the synergy and reduce the possibility of resistance emergence, it could be interesting to look on the resistance pathway developed by the bacteria against metal ions and antibiotics. Hence, to resist rifampicin or zinc, bacteria protect themselves by a slight modification of their targets, develop efflux pumps against chloramphenicol, cadmium, or nickel, or decrease the permeability against silver, manganese, or tetracyclines [146]. Therefore, it would be worthwhile studying the main resistance Chemistry 2020, 2 886 pathways of an antibiotic or a metal cation, and associating it with a cation or an antibiotic with a different resistance system to limit their inhibition. However, this could lead to the development of common resistance mechanisms [146].

4. Conclusions and Future Perspectives The field of metallodrugs in medicinal inorganic chemistry has grown constantly for around 50 years. However, to date, the proportion of metallodrugs that have reached clinical trials is still very low as compared to the traditional small organic or biological drug molecules. It seems that there is also a lack of public acceptance of the use of metals in the clinic and the “toxicity associated with metals” may hamper the development of metallodrugs for further pharmaceutical applications. Nevertheless, the wide range of metals and their combination with many types of ligands provide a great structural variety (ranging from linear to octahedral and even beyond) and a far more diverse stereochemistry than organic compounds. The rational design of ligands offers medicinal chemists proper control of the organometallic kinetic properties, such as the rate of ligand exchange, which is particularly useful for their interaction with biomolecule moieties such as DNA, enzymes or proteins that are present in the cell. By tuning the ligands, they can also adjust the lipophilicity of the overall metal complex, providing it with the ability to cross the cell membrane, and thus having a considerable impact on its biological activity and its pharmacokinetics. By using the nutrient assimilation machinery of the cell, they were able to design new metallodrugs possessing very promising activities through interaction with growing cell processes, while other strategies include the development of new organometallic derivatives of conventional organic or biological drug molecules based on structure-relationship methods. Additionally, more and more toxicological studies have been performed alongside, and the results point out that metals or metal-based drug molecules are not necessarily less biocompatible than conventional small organic molecules. In this review article, we focused our attention on the most prominent metal-based complexes developed in the last couple of years for their antimicrobial applications (see an overview in Table3). We exposed different examples encountered in the literature in order to attract the attention of the readers on the fact that every metal complex displayed different biological properties and mechanisms of action against bacteria, fungi and viruses. The treatment of bacterial infections is still challenging because of a decline in the current arsenal of useful antibiotics and the slow rate of new drug development. Therefore, research and development in the area of antimicrobial metallodrugs is a potential strategy to overcome the global rise of antibiotic resistance. Nevertheless, in many instances, the mechanisms of antimicrobial metal toxicity remain uncertain, and the identification of bacterial targets and uptake pathways are key issues for microbiologists and toxicologists. There are only limited in vivo data currently available for metal complexes, hindering further development of such promising compounds. Hopefully, future studies will be focused on the design of novel strategies for targeting toxic metals in order to have a better understanding of the metal complexes behavior in living organisms. An alternative approach could be the development of nanostructured antimicrobials, which possess new chemical, physical and biological properties due to their small size and large surface area. Even though this new class of compounds has already shown very promising results in terms of their higher bioactive entities released, easier interaction with biomolecules and enhanced selectivity, their development is still at the early stage and further toxicological and mechanistic studies are also required. To conclude, the future is bright for this field of research, and we believe that in the upcoming years, more metal-based antimicrobial compounds will be able to reach clinical trials and the market. Chemistry 2020, 2 887

Table 3. Overview of metal complexes discussed in this review article.

Metal Compound Antimicrobial Activity Media a Molecular Target/Mode of Action Cytotoxicity b In Vivo c MRSA, Acinetobacter spp. Proteus mirabilis, E coli, Interference with multiple 1 [187] K. pneumoniae, E. cloacae, Serratia marcescens, P aeruginosa Mueller Hinton agar [187] No toxic Yes (human) Ag cellular processes (MIC90 = 50–100 µg/mL)) [188] DNA intercalation, bacterial 2,3 [26] E. coli, S. aureus, P. aeruginosa (n.d.) LB n.d. n.d. membrane [28,29] Damage of the bacterial membrane 4 [42] E. coli, X. campestris, B. subtilis, B. cereus (n.d.) LB n.d. n.d. through the generation of ROS Cu P. aeruginosa (IC50 = 41.1 µM), S. enterica (IC50 = 3.5 nM), 5 [43] S. pneumoniae (IC50 = 15.2 µM), E. faecalis (IC50 = 20.6 µM), Nutrient broth DNA intercalation No toxic n.d. K. pneumonia (IC50 = 35.2 µM), E. coli (IC50 = 8.0 µM) M. Tuberculosis (MIC = 1.9 µM), S. aureus, E. coli, Interference in the mitosis 6 [53] MH n.d. n.d. B. subtilis (n.d.) cell mechanism S. aureus, E. coli Sulfamides are competitive inhibitor 7 [54] MH + Trypticase soy broth n.d. n.d. (MIC = 16 µg/mL) which inhibit folic acid synthesis Association of an antiseptic central 8 [72] E. coli, S. aureus, E. faecalis (MIC < 0.4 µM) BHI Zn(II) cation with two types of antibiotic n.d. n.d. Zn as ligands leads to a synergetic effect 9 [77] HIV-1 IIIB (EC50 = 0.008 µM) MT-4 cells CXCR4 co-receptor No toxic n.d. 10 [77] HIV-1 IIIB (EC50 = 0.0025 µM) MT-4 cells CXCR4 co-receptor Some toxicity n.d. SGE2, FG2, FG4, FG3, FCM6, FCM17, Active against chloroquine-resistant 11 [86] Culture-adapted parasites n.d. Yes (mouse) Fe FG1 (IC50 = 0.12–0.36 µM) parasitic strains by producing ROS 12,13 [87] n.d. - β-lactamase n.d. n.d. B. subtilis, S. aureus (MIC = 2.2–8.6 µM) 14 [93] LB, BHI DNA intercalation n.d. Yes (fungi) Not active against E. coli S. aureus, M. tetragenus (MIC > 15–15 µM) Damage of DNA and RNA Ru 15 [94] LB n. d. n. d. Not active against E. coli Alteration of cell walls aPDT 16 [96] S. aureus (n.d.) LB No toxic n.d. Damages and deformations of cell walls M. smegmatis at 10 µM Dark, yellow or red LED = 94–100% survival; green led 4%; Release of INH, Non-toxic even 17 [97] blue LED < 1% an anti-tuberculosis drug with blue LED At 22 µM, red LED = 2%survival, others = 0% survival S. aureus, MRSA (MIC = 0.6 µM), E. coli (1.2–2.5 µM), 18 [98] CAMBH RNA, ribosomes Some toxicity n.d. P. aeruginosa (5.0–10.0 µM) 19 [108,109] P. aeruginosa, S. aureus, A. baumannii (n.d.) LB Fe metabolism No toxicity Yes (mouse) Ga A. baumannii (MIC = 31.7 µM) [114], CAMBH, nutrient broth, Fe metabolism Yes (mouse, 20 [114,116–118] S. aureus (MIC = 2.5 µM), M. smegmatis (MIC = 0.6 µM), No toxicity LB + Tween 80, DCAA Cytochromes G. mellonella larvae) Y. enterocolitica (MIC = 0.6 µM) [115], P. aeruginosa (n.d.) 21 [120] P. aeruginosa (MIC = 0.032 mM) TSB Fe metabolism No toxicity Yes (rabbit) ETEC, S. Typhimurium, S. sonnei, BSS [123] TSA, BHI + 10% L-cysteine [189] Multiple targets No toxicity Yes C. difficile (MIC = 5.5–22.1 mM) [187] Bi Multiple targets including ADH NDM-1 (IC = 2.81 µM), VIM-2 (IC = 3.55 µM), CBS [123] 50 50 HEPES/Na [125] inhibition in H. pylori [128] No toxicity Yes IMP-4 (IC = 0.70 µM) [132] 50 and MBLs [132] Multiple targets including urease RBC [123] SCV helicase protein (IC50 = 0.3 µM) [133] Tris-HCl [133] activity inhibition [127], SARS-CoV No toxicity Yes helicase inhibition [133] L. infantum chagasi (WT and SbR) (IC = 0.59–0.61 µM), α-MEM supplemented with 10% (v/v) DNA intercalation and/or modulation of 22 [135] 50 Some toxicity n.d. L. amazonensis (WT and SbR) (IC50 = 1.07–1.12 µM) heat inactivated fetal calf serum the hydrophilicity profile Chemistry 2020, 2 888

Table 3. Cont.

Metal Compound Antimicrobial Activity Media a Molecular Target/Mode of Action Cytotoxicity b In Vivo c E. hystolitica 23 [140] PEHPS medium n.d. n.d. n.d. (IC50 = 2.359.60 µM) T.cruzi (trypanostatic, 5 µM reduce proliferation to 25%; 24 [145] BHI Affect cell shape and motility n.d. n.d. V IC50 = 3.76 µM) Murine L. amazonensis RPMI +10% inactivated FBS or Warren’s 25 [147] Oxidative stress macrophage: n.d. (IC50 = 3.51–6.65 µM) medium + 10% inactivated FBS IC50 = 24.32 µM E. hystolytica Non toxic 26–28 [148] TYIS-33 n.d. n.d. (IC50 = 0.09–8.55 µM) (IC50 = 100 µM) inhibition of HIV-1 RT and binding to HIV-1(BaL) 29 [150] lysis buffer, TBS CD4 => Block virus entrance into n.d. n.d. 5 µM Inhibit 97% of the virus hosts cells 30–32 [120] B. subtilis, S.aureus, E.coli (MIC = 1.2–37.5 µg/mL) MH n.d. n.d. n.d. A. baumannii (MIC = 47 µM), P. aeruginosa (MIC = 377 µM), E. cloacae (MIC = 189 µM), K. pneumoniae (MIC = 377 µM), 33 [155] CAMBH Trx inhibition Some toxicity Yes (mouse) [154] S. aureus (MIC = 0.04 µM), E. faecium (MIC = 0.2/0.09 µM), Au E. coli (MIC = 24 µM) A. baumannii (MIC = 10 µM), P. aeruginosa (MIC = 41 µM), E. cloacae (MIC = 5/10 µM), K. pneumoniae (MIC = 20 µM), 34 [157] CAMBH n.d. Some toxicity n.d. S. aureus (MIC = 0.03 µM), E. faecium (MIC = 0.2/0.3 µM), E. coli (MIC = 10 µM) A. baumannii (MIC = 6/13 µM), P. aeruginosa (MIC = 101 µM), E. cloacae (MIC = 3 µM), K. pneumoniae (MIC = 13 µM), 35 [157] CAMBH n.d. Some toxicity n.d. S. aureus (MIC = 0.09 µM), E. faecium (MIC = 0.2/0.4 µM), E. coli (MIC = 6 µM) A. baumannii (MIC = 6/3 µM), P. aeruginosa (MIC = 23/91 µM), E. cloacae (MIC = 3 µM), K. pneumoniae (MIC = 11 µM), 36 [157] CAMBH n.d. Some toxicity n.d. S. aureus (MIC = 0.3 µM), E. faecium (MIC = 0.3 µM), E. coli (MIC = 1/6 µM) S. aureus (MIC = 1.4 µM), B. subtilis (MIC = 0.3 µM), Ir 37 [162] S. pyogenes (MIC = 0.17 µM), S. epidermidis (MIC = 0.7 µM), CAMBH Biguanine ligand release Some toxicity n.d. E. faecalis (MIC = 1.4 µM) DMEM supplemented with 5% bovine Perturbation of endocytosis pathways Co 38 [166] HSV-1 (MIC 94 µM) [165] Some toxicity Yes (rabbit) ≥ calf serum required for viral entry into cells S. pyogenes, B. anthracis (MIC = 25 mg/mL); 39 [174] MH n.d. n.d. n.d. S. aureus (MIC = 12.5 mg/mL) Ni S. pyogenes, B. anthracis (MIC = 25 mg/mL); 40 [174] MH n.d. n.d. n.d. S. aureus (MIC = 6.25 mg/mL) Sequestration of nickel(II)—Inhibition of Not toxic 41 [180] Bacteriostatic against S. typhimurium and K. pneumoniae LB n.d. hydrogenase and urease enzymes (mice, larvae) 42 [182] At 1 mg/mL, better than streptomycin against X. compestris peptone, beef extract, NaCl (1:1:1) n.d. n. d. fertility issue (rats) Mn 43 [183] Better than streptomycin against K. aerogenous, P. cepacicola peptone, beef extract, NaCl (1:1:1) n.d. n. d. fertility issue (rats) Chemistry 2020, 2 889

Table 3. Cont.

Metal Compound Antimicrobial Activity Media a Molecular Target/Mode of Action Cytotoxicity b In Vivo c MCF7, NRK-52E, CCRF-CEM yes S. aureus (1.4 µM); S. aureus MRSA (1.4 µM); embed into the membrane, (<57% viability); 44 [185] B. subtilis (1.4 µM) but binds to serum MH n.d. Mix oxidative stress Caco-2, L6.C11, no effect on Gram-negative bacteria HepG2 no (>90% viability) S. aureus (2.7 µM); S. aureus MRSA (4 µM); 45 [187] MH embed into the membrane n.d. n.d. B. subtilis (21 µM) a Growth media used for antimicrobial experiments; b if the cytotoxicity was evaluated against human cells; c if the compounds were evaluated in vivo (animal model). n.d.: not determined. LB: Lysogeny Broth; MH: Mueller-Hinton broth; BHI: Brain Heart Infusion medium; HIV: Human Immunodeficiency Virus; MT-4 cells: Human leukaemia T cell lymphoblasts; SCE2, FG2, FG4, FG3, FCM6, FCM17, FG1: parasitic strains; aPDT: antimicrobial Photodynamic Therapy; CAMBH: cation-adjusted Mueller-Hinton broth; DCAA: iron-free Casamino Acids medium; TSB: Tryptic Soy Broth; ETEC: Enterotoxigenic Escherichia Coli; TSA: Trypticase Soy Agar; ADH: Alcohol Dehydrogenase; MBLs: Metallo-β-lactamases; SCV: SARS coronavirus; Tris: tris((hydroxymethyl)aminomethane); SARS: Severe Acute Respiratory Syndrome; WT: Wild-Type; SbR: Antimony-Resistant; α-MEM: Minimum Essential culture Medium; Trx: thioredoxin reductase; HSV-1: Herpes Simplex Virus type 1; DMEM: Dulbecco’s Minimal Essential Medium; p-BPIP: 2-(4-bromophenyl)imidazo [4,5-f][1,10]phenanthroline. Chemistry 2020, 2 890

Author Contributions: K.M.F. conceptualized the manuscript and contributed to the correction and final shaping of it, while M.C. searched literature and wrote the first version part of the manuscript and J.V.S. searched literature and contributed to the writing and the corrections of the second version (in particular, chapters on vanadium and synergic effects). All authors have read and agreed to the published version of the manuscript. Funding: This research acknowledges funding from an Innosuisse project “GACAPS”. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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