molecules Article Evaluation of Ruthenium(II) N-Heterocyclic Carbene Complexes as Antibacterial Agents and Inhibitors of Bacterial Thioredoxin Reductase Hilke Burmeister 1, Pascal Dietze 2, Lutz Preu 1 , Julia E. Bandow 2 and Ingo Ott 1,* 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany; [email protected] (H.B.); [email protected] (L.P.) 2 Applied Microbiology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44801 Bochum, Germany; [email protected] (P.D.); [email protected] (J.E.B.) * Correspondence: [email protected] Abstract: A series of ruthenium(II) complexes with N-heterocyclic carbene (NHC) ligands of the general type (arene)(NHC)Ru(II)X2 (where X = halide) was prepared, characterized, and evaluated as antibacterial agents in comparison to the respective metal free benzimidazolium cations. The ruthenium(II) NHC complexes generally triggered stronger bacterial growth inhibition than the metal free benzimidazolium cations. The effects were much stronger against Gram-positive bacte- ria (Bacillus subtilis and Staphylococcus aureus) than against Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa), and all complexes were inactive against the fun- gus Candida albicans. Moderate inhibition of bacterial thioredoxin reductase was confirmed for Citation: Burmeister, H.; Dietze, P.; Preu, L.; Bandow, J.E.; Ott, I. selected complexes, indicating that inhibition of this enzyme might be a contributing factor to the Evaluation of Ruthenium(II) antibacterial effects. N-Heterocyclic Carbene Complexes as Antibacterial Agents and Keywords: antibacterial; N-heterocyclic carbenes; ruthenium; thioredoxin reductase Inhibitors of Bacterial Thioredoxin Reductase. Molecules 2021, 26, 4282. https://doi.org/10.3390/molecules 26144282 1. Introduction Metal complexes with N-heterocyclic carbene (NHC) ligands represent an important Academic Editors: Sotiris Hadjikakou, type of catalysts, however, in addition to their application in chemistry, the biomedical Mario Waser, Christina N. Banti and properties have been evaluated intensively. In particular, inorganic medicinal chemistry Andreas K. Rossos and bioorganometallic chemistry nowadays make intensive use of the relatively high stability of the metal-carbon bonds of these compounds and the convenient synthetic Received: 1 June 2021 access to a broad variety of NHC ligands [1–7]. Accepted: 12 July 2021 Published: 15 July 2021 The design of drug candidates based on metal NHC complexes has been dominated by gold and silver as metals, however, more recently, an increasing number of metals Publisher’s Note: MDPI stays neutral has demonstrated very promising results, including, for example, rhodium [8–12] and with regard to jurisdictional claims in iridium [12,13] species. published maps and institutional affil- We and others have recently reported on ruthenium(II) NHC complexes of the general iations. type (p-cymene)(NHC)Ru(II)X2 (where X = halide) and structurally related complexes as novel anticancer drug candidates [14–20]. For complexes with the general structure (p-cymene)(NHC)Ru(II)Cl2 with different substituents on the NHC nitrogen atoms, the antiproliferative activity was dependent on the lipophilicity of the nitrogen side chains and on cellular uptake. The compounds inhibited thiol or selenol containing enzymes Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. (e.g., thioredoxin reductase, cathepsin B), were generally reactive towards biologically This article is an open access article relevant thiols, and underwent ligand exchange reactions (e.g., the replacement of the NHC distributed under the terms and ligand upon reaction with N-acetylcysteine could be confirmed by NMR). Interestingly, conditions of the Creative Commons the complexes were well tolerated regarding toxicity in zebrafish embryos and mouse Attribution (CC BY) license (https:// models [14,17]. Hartinger et al. demonstrated by means of X-ray crystallography with the creativecommons.org/licenses/by/ model protein hen egg white lysozyme that, upon binding, the p-cymene ligand of the 4.0/). complexes was replaced, while the NHC ligand remained coordinated in this case [21]. Molecules 2021, 26, 4282. https://doi.org/10.3390/molecules26144282 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 11 Molecules 2021, 26, 4282 protein hen egg white lysozyme that, upon binding, the p-cymene ligand of the complexes2 of 11 was replaced, while the NHC ligand remained coordinated in this case [21]. Interestingly, the same group also demonstrated that derivatives with a pyridine in one of the side chain showed enhanced stability towards cysteine [19]. Interestingly, the same group also demonstrated that derivatives with a pyridine in one of Notably, the probably first report on biological activity of (arene)(NHC)Ru(II)X2 the side chain showed enhanced stability towards cysteine [19]. complexes describes their application as antibacterial agents [22]. However, the literature Notably, the probably first report on biological activity of (arene)(NHC)Ru(II)X2 complexeson such application describes theirof the application complexes asappears antibacterial limited agents to very [22 few]. However, reports [22–25]. the literature on suchIn applicationorder to shed of the more complexes light on appears the po limitedtential toof very ruthenium few reports NHC [22 complexes–25]. as antibacterialIn order agents, to shed we more synthesized light on theand potential evaluated of their ruthenium bacterial NHC cell growth complexes inhibition. as an- tibacterialFurthermore, agents, the inhibition we synthesized of bacterial and evaluated thioredoxin their reductase bacterial was cell studied growth as inhibition. a possible Furthermore,mechanism of the action. inhibition The target of bacterial compounds thioredoxin are based reductase on the ( wasp-cymene)(NHC)Ru(II)Cl studied as a possible 2 scaffold of our previous work [14] and contain substituents with different inductive and mechanism of action. The target compounds are based on the (p-cymene)(NHC)Ru(II)Cl2 scaffoldmesomeric of our effects previous on the work NHC [ 14moiety.] and containThe substi substituentstuents are thus with expected different to inductive influence and the mesomericdonor capacity effects of on the the NHC NHC ligand, moiety. and The with substituents this, the reactivity are thusexpected and biological to influence activity the of donorthe organometallics. capacity of the NHC ligand, and with this, the reactivity and biological activity of the organometallics. 2. Synthesis and Characterization 2. SynthesisThe (p-cymene)(NHC)Ru(II)Cl and Characterization 2 target compounds were obtained based on an establishedThe (p-cymene)(NHC)Ru(II)Cl two-step procedure (Scheme2 target 1) compounds. In the first were step, obtained the benzimidazoles based on an estab- 1a–1f lishedwere alkylated two-step with procedure benzyl (Scheme bromide1 ).resulting In the firstin the step, benzimidazolium the benzimidazoles bromides1a– 1f2a–were2f. In alkylatedthe second with step, benzyl the bromidetarget compounds resulting in3a the–3fbenzimidazolium were obtained in bromides a one-pot2a reaction–2f. In the by secondactivating step, 2a the–2f target with silver compounds oxide followed3a–3f were by obtained transmetalation in a one-pot with reaction [(p-Cym)RuCl by activating2]2. The 2arespective–2f with silvercomplexes oxide followedrequired by adjustments transmetalation regarding with [(p -Cym)RuClthe reaction2]2 . Theperiod respective of the complexestransmetalation required reaction adjustments in step 2 regarding and the is theolation reaction procedure. period ofThus, the to transmetalation obtain complexes re- action3d–3f inwith step halide 2 and substituents, the isolation ex procedure.tended reaction Thus, toperiods obtain of complexes up to 7 days3d– 3fwerewith required. halide substituents,Complex 3f was extended isolated reaction by column periods chromatography of up to 7 days over were silica required. in contrast Complex to the3f otherwas isolatedcomplexes, by column which chromatographycould be obtained over in high silica purity in contrast by filtration to the other over complexes,celite followed which by couldcrystallization. be obtained in high purity by filtration over celite followed by crystallization. SchemeScheme 1. 1.Synthesis Synthesis of of the the (NHC)( (NHC)(pp-Cym)Ru(II)Cl-Cym)Ru(II)Cl2 complexes;2 complexes; (a) (a) K K2CO2CO3,3, benzyl benzyl bromide, bromide, (b) (b) Ag Ag2O,2O, (c)(c) [( [(pp-Cym)RuCl-Cym)RuCl2]22.]2... 1 13 TheThe obtained obtained target target compounds compounds were were characterized characterized by by H-1H- and and 13C-NMRC-NMR and and mass mass spectroscopy.spectroscopy. The The high high purities purities were were confirmed confirmed by by elemental elemental analyses. analyses. SeveralSeveral diagnosticdiagnostic featuresfeatures confirmingconfirming complexcomplex formationformation cancan bebe observedobserved inin the the 1 NMRNMR spectra.spectra. TheThe hydrogenhydrogen signalsignal ofof thethe benzimidazolebenzimidazole C2C2 carboncarbon inin the the H-NMR1H-NMR spectra, which appears in the range of 11.6–12.1 ppm in the spectra of the benzimidazolium spectra, which appears in the range of 11.6–12.1 ppm in the spectra13 of the bromidesbenzimidazolium2a–2f, is missingbromides in 2a the–2f spectra, is missing