Biological Activities of Ruthenium NHC Complexes: An Update

Ruthenium N-heterocyclic carbene (NHC) complexes have unique physico-chemical properties as catalysts and a huge potential in medicinal chemistry and pharmacology, exhibiting a variety of notable biological activities. In this review, the most recent studies on ruthenium NHC complexes are summarized, focusing specifically on antimicrobial and antiproliferative activities. Ruthenium NHC complexes are generally active against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Listeria monocytogenes and are seldom active against Gram-negative bacteria, including Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli and fungal strains of Candida albicans. The antiproliferative activity was tested against cancer cell lines of human colon, breast, cervix, epidermis, liver and rat glioblastoma cell lines. Ruthenium NHC complexes generally demonstrated cytotoxicity higher than standard anticancer drugs. Further studies are needed to explore the mechanism of action of these interesting compounds.


Introduction
Ruthenium (II) complexes represent an important class of organometallic compounds with numerous applications in the fields of homogeneous, heterogeneous and photocatalytic catalysis [1,2], including biological stains, and in the field of therapy [3,4]. Ruthenium complexes endowed with anticancer properties [5] are attracting significant attention, due to their vast and different structural types and their ability to variously bind different ligands with the advantage of lower toxicity than platinum complexes [6][7][8]. These compounds reasonably penetrate more efficiently tumor cells and effectively bind to DNA, from which comes the suggestion of their use in different cancers [9,10], including lung [11], breast [12], ovarian [13][14][15][16] and colorectal ones [8]. Recently, nanomedicine formulations of metal complexes developed for the treatment of cancers are under study [17]. Moreover, numerous other biological activities [18], such as antioxidant, anti-inflammatory [19], and antimicrobial [3,20,21], have been described for ruthenium complexes. Recent studies are also addressed to the use of ruthenium complexes as antivirals for the treatment of COVID-19 [22]. Ruthenium complexes include those with Schiff bases [23][24][25], phosphines [26], carbazole [27], N-heterocyclic carbenes (NHCs), cycloruthenated and halfsandwiched compounds [28]. Considering the huge variety of ruthenium (II) complexes, this review will focus merely on one class of them, namely Ru-NHC complexes. Ru-NHC complexes have promising catalytic potential for a vast range of synthetic applications, including the activity in transfer hydrogenation [29,30] of ketones, metathesis reactions [31], secondary alcohol oxidation [32], N-alkylation of amines, amides, and sulfonamides [33,34] and Oppenauer-type oxidation [35,36]. The high catalytic activity of these compounds is

N-Heterocyclic Carbenes (NHCs)
N-heterocyclic carbenes (NHCs) are a category of electron donor ligands able to form dative metal-ligand bonds, leading to a universal class of compounds in organometallic and coordination chemistry. NHCs typically mimic the chemical properties of phosphines [39]. They belong to five different families: imidazolinylidenes, imidazolylidenes, triazolinylidenes, thiazolilylidenes and pyrazolinylidenes ( Figure 1) [40]. NHCs are suitable for efficient design because they can be synthesized within a few steps, offering the possibility of the N and C functionalization for structure modification. They act as excellent σ-donors, generating a structural variety ranging from linear, square pyramidal, trigonal bipyramidal, tetrahedral and octahedral geometries of metal-NHC complexes. The essential role of NHCs is related to their ability to form complexes with metals. Metal-NHCs are widely used for organic processes, such as the formation of amide linkage, hydrogenation, isomerization, cycloisomerization, cyclopropanation, hydrosilylation, allylation and deallylation, enol-ester synthesis, heterocycle synthesis and C-C alkyne coupling [40]. Most importantly, different organometallic-NHC complexes, such as silver, gold, platinum, copper, palladium and selenium demonstrated interesting biological properties [41][42][43][44], including antimicrobial [45][46][47], anticancer [48][49][50][51][52][53][54], antiparasitic [55], hemolytic and thrombolytic activities [56,57]. Recent studies for gold and silver-NHC compounds are addressed to breast [58,59], ovarian [60] and cervical human cancer [61]. Research has been carried out in order to understand the mechanism of action of gold and silver-NHC complexes as anticancer agents, finding activity against human topoisomerases I and II [62,63], actin [64] and tubulin [65], or triggering the reactive oxygen species-dependent intrinsic apoptotic pathway [66]. Recently, gold and silver compounds with NHC have been indicated as promising compounds for the treatment of COVID-19 [67], as they demonstrated a strong inhibition of the S/ACE2 interaction and particularly of the PL pro enzymatic activity [68]. Moreover, an Ag-N-heterocyclic carbene complex bearing the hydroxyethyl ligand determined the inhibition of human carbonic anhydrase I (hCA I) and hCA II isoenzymes, α-glycosidase and AChE and BChE enzymes, thus suggesting the selection of NHC complexes for further studies in glaucoma, epilepsy and other diseases related to metabolic enzymes [69]. NHCs are suitable for efficient design because they can be synthesized within a few steps, offering the possibility of the N and C functionalization for structure modification. They act as excellent σ-donors, generating a structural variety ranging from linear, square pyramidal, trigonal bipyramidal, tetrahedral and octahedral geometries of metal-NHC complexes. The essential role of NHCs is related to their ability to form complexes with metals. Metal-NHCs are widely used for organic processes, such as the formation of amide linkage, hydrogenation, isomerization, cycloisomerization, cyclopropanation, hydrosilylation, allylation and deallylation, enol-ester synthesis, heterocycle synthesis and C-C alkyne coupling [40]. Most importantly, different organometallic-NHC complexes, such as silver, gold, platinum, copper, palladium and selenium demonstrated interesting biological properties [41][42][43][44], including antimicrobial [45][46][47], anticancer [48][49][50][51][52][53][54], antiparasitic [55], hemolytic and thrombolytic activities [56,57]. Recent studies for gold and silver-NHC compounds are addressed to breast [58,59], ovarian [60] and cervical human cancer [61].
Research has been carried out in order to understand the mechanism of action of gold and silver-NHC complexes as anticancer agents, finding activity against human topoisomerases I and II [62,63], actin [64] and tubulin [65], or triggering the reactive oxygen species-dependent intrinsic apoptotic pathway [66]. Recently, gold and silver compounds with NHC have been indicated as promising compounds for the treatment of COVID-19 [67], as they demonstrated a strong inhibition of the S/ACE2 interaction and particularly of the PL pro enzymatic activity [68]. Moreover, an Ag-N-heterocyclic carbene complex bearing the hydroxyethyl ligand determined the inhibition of human carbonic anhydrase I (hCA I) and hCA II isoenzymes, α-glycosidase and AChE and BChE enzymes, thus suggesting the selection of NHC complexes for further studies in glaucoma, epilepsy and other diseases related to metabolic enzymes [69].

Ruthenium-NHC Complexes
Besides the importance of ruthenium-NHC complexes in organometallic catalysis [70,71] and bioinorganic chemistry, they have been described as effective anticancer and antimicrobial agents [40,43,47]. Patil et al., 2020, published the advances in the design, synthesis, characterization and biomedical applications, particularly the antimicrobial and anticancer activities, of ruthenium NHC-metal complexes and other metals (silver, gold, palladium, rhodium, iridium, and platinum), covering works published from 2015 to 2020 [39]. Several studies regard only the activity of Ru-NHC complexes as antiproliferative agents, whereas the antimicrobial activity is almost always studied along with other activities, including antiproliferative and antioxidant. The two paragraphs below summarized these studies.

Ru(II)-NHC Complexes with Antiproliferative Activity
Recent studies regarding the antiproliferative activity of Ru(II)-NHC complexes are summarized and the half-maximal (50%) inhibitory concentration (IC 50 ) values are given, when reported in the literature. Lam et al., 2018, [72] investigated several halide-substituted benzimidazolium-derived NHC of Ru II /Os II complexes, using NHCs that were symmetrically and non-symmetrically methyl-and benzyl-substituted, and reported their inhibition of the selenoenzyme thioredoxin reductase (TrxR) and antiproliferative activities. The anticancer activity was studied against human colon (HCT-116), human cervical (SiHa) and human breast cancer (NCI-H460) cells. The diiodido(1,3-dibenzylbenzimidazol-2-ylidene)(η 6 -p-cymene)ruthenium(II) complex 1 is a potent TrxR inhibitor and an antiproliferative agent. The authors found out that there was no clear correlation between the two activities, thus, it was suggested that TrxR inhibition was unlikely to be the main mode of action.
Rana et al., 2021, [74] described the synthesis and study of two pyridine and pyrimidine Ru(II)-NHC complexes, functionalized annulated NHC 3 and 4 with a half-sandwich geometry, and their in vitro cytotoxicity studies against lung (A549), colon (HCT-116) and breast (MCF-7) cancer cell lines and non-cancerous 3T3 cells (embryonic fibroblast isolated from a mouse), using cisplatin as the reference (IC 50 were 64; 23.2; 14 and 64 µM, respectively) via the MTT assay. Both compounds were more active than the reference, with the exception of 3 against HCT-116 cancer cells, and, overall, compound 4 was more active than compound 3. Moreover, in silico studies, predicting the binding-sites and atomic interactions of lead molecules with B cell CLL/lymphoma (BCL-2) (PDB entry: 4lvt) and DNA dodecamer (PDB entry: 1bna), suggested that the order of reactivity of the molecules is 4 > 3. Subsequently, the same group [75] investigated a pyrimidine functionalized non-annulated half-sandwich Ru(II)-NHC complex, namely chloro(p-cymene)-1methyl-3-pyrimidylimidazolideneruthenium(II)-hexafluorophosphate 5, derived from the non-annulated NHC precursor 1-methyl-3-pyrimidylimidazolium-hexafluorophosphate. The half-sandwich geometry of the molecule was established with single crystal X-ray diffraction. As well, this compound was more active than cisplatin against the cell lines used [74]. Particularly, 5 and 4 were 3-to 4-fold more active than 3 and cisplatin. Predicting binding affinities for 5 and 4 were −7.1 and −7.0 kcal/mol -1 , respectively, for BCL-2 and −7.3 and −8.2 kcal/mol -1 , respectively, for DNA. DNA cleavage activity of complex 5 confirmed the ability of ruthenium to perform a direct double-strand breaking. Moreover, molecular docking analysis suggested that complex 5 binds, with the highest binding affinity, a hydrophobic pocket in BLC-2, different from that of complexes 3 and 4. The opposite Antibiotics 2023, 12, 365 4 of 18 was found in the contact simulation of the complexes with a DNA strand: complexes 4 and 5 are superimposed, while complex 3 binds the other side of the DNA strand. However, the binding affinity predicted for 4 was higher than 5.
Rodriguez-Prieto et al., 2021, [76] reported the synthesis of three spherical carbosilane metallodendrimers of different generations holding Ru-NHC complexes. Compounds 6 and 7, which belong to the first-and second-generation dendrimers, respectively, are shown in Table 1, while the third-generation dendrimer is not shown. These compounds showed cytotoxic activity similar, or even better, than cisplatin against four cancer cell lines, namely advanced prostate (PC3), breast (HCC1806), cervix (HeLa), human liver (HEPG2) and the non-tumoral fibroblast (HFF-1) cell line, as determined by MTT assay. IC 50 for cisplatin was referred to data of the literature: IC 50 = 30.18 ± 2.58 µM (MCF-7), 11.75 ± 1.23 µM (HeLa), 17.29 ± 1.05 µM (HFF-1) [77]. The complexes have been proposed as possible candidates for cancer treatment, due to their combined double action, i.e., antitumoral and carrier for anticancer siRNA. These compounds were capable of forming dendriplexes by promoting the entrance of Mcl-1-FITC (myeloid cell leukemia-1 fluorescein labelled) small interfering RNA (siRNA) to HEPG2 cancer cells, protecting the siRNA from RNAse. Moreover, the cellular uptake of the three complexes was studied by confocal microscopy with Mcl-1-FITC siRNA. Particularly, the second-generation dendrimer 7 was more active than first-generation dendrimer 6. Compound 7 displayed promising antitumoral properties, being selective, even more than cisplatin, against cancer cell lines, with respect to the normal ones. The different activity was related to the inability of first-generation dendrimer 6 to interact with the siRNA. The compounds were internalized into the cells by endocytosis and internalization increased by generation of the dendritic system. A recent interesting article by Wilke et al., [81] described the study of the ruthenium complex HB324 (15), which showed promising potential as a novel anticancer agent in vitro. This complex showed good effects, even in low micromolar concentrations, especially regarding proliferation inhibition and apoptosis induction via the mitochondrial pathway on human B-cell precursor leukemia Nalm-6 cells. Moreover, of particular interest is the upregulation of the Harakiri resistance protein, which inhibits the anti-apoptotic and death repressor proteins BCL-2 and BCL-xL. Finally, compound 15 showed synergistic activity with various established anticancer drugs, including vincristine, and overcame the resistance in several cell lines, such as neuroblastoma cells. Table 1. Antiproliferative activity of Ru(II)-NHC complexes.

Structure
Compd.  A recent interesting article by Wilke et al., [81] described the study of the ruthenium complex HB324 (15), which showed promising potential as a novel anticancer agent in vitro. This complex showed good effects, even in low micromolar concentrations, especially regarding proliferation inhibition and apoptosis induction via the mitochondrial pathway on human B-cell precursor leukemia Nalm-6 cells. Moreover, of particular interest is the upregulation of the Harakiri resistance protein, which inhibits the anti-apoptotic and death repressor proteins BCL-2 and BCL-xL. Finally, compound 15 showed synergistic activity with various established anticancer drugs, including vincristine, and overcame the resistance in several cell lines, such as neuroblastoma cells. Table 1. Antiproliferative activity of Ru(II)-NHC complexes.

Structure
Compd.             * G1 arrest, which means a proliferation inhibition equal to 100% or above, indicating occurring cell death; ** AC50 is the concentration necessary to induce apoptosis in half of the cell population.

Antimicrobial Ru(II)-NHC Complexes Possessing Other Additional Abilities (Antiproliferative, Antioxidant and Anticholinesterase)
Antimicrobic resistance is a cause of great concern worldwide and significantly affects humanity's capacity to prevent and treat a growing number of bacterial and fungal infections [82]. In the past decade, the antimicrobial activity of ruthenium complexes has been reviewed [83] and studied as antimicrobial agents and alternatives or adjuvants to * G1 arrest, which means a proliferation inhibition equal to 100% or above, indicating occurring cell death; ** AC 50 is the concentration necessary to induce apoptosis in half of the cell population.
Chen et al., 2020, [79] reported the synthesis and in vitro antiproliferative activity evaluation of a small panel of NHC-coordinated ruthenium(II) arene complexes. The compounds showed cytotoxic activities against the human ovarian A2780 cancer cells. The highest cytotoxic activities were found for 12 and 13, which were about 2-fold more potent than cisplatin. Furthermore, these compounds induced apoptosis in a caspasedependent manner, primarily through intracellular ROS overproduction and cell cycle arrest at the G1 phase. Moreover, in a preclinical metastatic model of A2780 tumor xenograft, administration of 12 and 13 resulted in a marked inhibition of tumor progression and metastasis. A significant alleviated systemic toxicity was observed in animals for both complexes in comparison with cisplatin.
Sari et al., [80] designed four (NHC)Ru(II)(η 6 -p-cymene) complexes, bearing 2-morpholinoethyl and 4-vinylbenzyl substituents to the benzimidazole core and different alkyl/aryl groups to the second nitrogen atom, and studied their cytotoxic activity against MCF-7 breast cancer cells and their DNA-binding properties. The authors individuated the compound 14 as lead, showing an IC 50 of 3.61 µM, and that it can bind the plasmidic DNA without exerting genotoxic effects.
A recent interesting article by Wilke et al., [81] described the study of the ruthenium complex HB324 (15), which showed promising potential as a novel anticancer agent in vitro. This complex showed good effects, even in low micromolar concentrations, especially regarding proliferation inhibition and apoptosis induction via the mitochondrial pathway on human B-cell precursor leukemia Nalm-6 cells. Moreover, of particular interest is the upregulation of the Harakiri resistance protein, which inhibits the anti-apoptotic and death repressor proteins BCL-2 and BCL-xL. Finally, compound 15 showed synergistic activity with various established anticancer drugs, including vincristine, and overcame the resistance in several cell lines, such as neuroblastoma cells.

Antimicrobial Ru(II)-NHC Complexes Possessing Other Additional Abilities (Antiproliferative, Antioxidant and Anticholinesterase)
Antimicrobic resistance is a cause of great concern worldwide and significantly affects humanity's capacity to prevent and treat a growing number of bacterial and fungal infections [82]. In the past decade, the antimicrobial activity of ruthenium complexes has been reviewed [83] and studied as antimicrobial agents and alternatives or adjuvants to the more traditional antibiotics [84]. The recent antimicrobial studies regarding complexes of NHC-ruthenium are summarized in Table 2 [87] with complex 19 on HCT-116 colorectal cancer cells demonstrated that it triggers apoptosis via a rapid and consistent activation of the ROS-p38 MAPK pathway. Following this, the same group [88] reported a research, similar to the previous one, regarding the antimicrobial and antiproliferative activity of a bis-naphthalimide NHC-ruthenium (II) complex (20). It showed a slight antimicrobial effect against Gram-positive bacteria, whereas it was not cytotoxic against the cell lines studied.  [93] studied a series of benzimidazolium cations and the corresponding organometallics of the type (p-cymene)(NHC)Ru(II)Cl2 as antibacterials against Gram-positive (B. subtilis, S. aureus DSM 20231, S. aureus ATCC 43300) and Gramnegative (E. coli, A. baumannii, P. aeruginosa) strains in a microtiter plate assay, according to CLSI guidelines, using ciprofloxacin as a reference drug. The complex (1,3-dibenzyl-5bromo-1H-benzimidazol-2-ylidene)-dichlorido-(η 6 -p-cymene)ruthenium(II) 28 was the most active compound against Gram-positive bacteria, while it showed low activity against Gram-negative bacteria. This compound showed moderate inhibition of bacterial TrxR (E. coli). Thus, the inhibition of TrxR is unlikely a major mechanism of the antibacterial activity of the ruthenium NHC complexes. Table 2. Antimicrobial and other activities (antiproliferative, antioxidant and anticholinesterase) of Ru(II)-NHC complexes.

Conclusions
NHCs have been recognized as a class of strong donating ligands, which may stabilize diverse metal complexes of catalytic importance. Among these, the NHC ruthenium complexes have been, and still are, a fruitful research, filed for various applications: catalytic, photochemical and biological. This review provides an overview of the most recent studies on ruthenium NHCs complexes with biological activities, focusing on the antimicrobial and antiproliferative properties. Most of these compounds showed interesting antimicrobial activity against Gram-positive bacteria and some are also active against Gramnegative bacteria and fungi. The antiproliferative activity was also demonstrated for several compounds belonging to this class against several types of cancer cell lines.

Conclusions
NHCs have been recognized as a class of strong donating ligands, which may stabilize diverse metal complexes of catalytic importance. Among these, the NHC ruthenium complexes have been, and still are, a fruitful research, filed for various applications: catalytic, photochemical and biological. This review provides an overview of the most recent studies on ruthenium NHCs complexes with biological activities, focusing on the antimicrobial and antiproliferative properties. Most of these compounds showed interesting antimicrobial activity against Gram-positive bacteria and some are also active against Gramnegative bacteria and fungi. The antiproliferative activity was also demonstrated for several compounds belonging to this class against several types of cancer cell lines.