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 ^[1][39]. They belong to five different families: imidazolinylidenes, imidazolylidenes, triazolinylidenes, thiazolilylidenes and pyrazolinylidenes (Figure 1) ^[2][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 ^[2][40]. Most importantly, different organometallic-NHC complexes, such as silver, gold, platinum, copper, palladium and selenium demonstrated interesting biological properties ^[3][4][5][6][41,42,43,44], including antimicrobial ^[7][8][9][45,46,47], anticancer ^{[10][11][12][13][14][15][16]}[48,49,50,51,52,53,54], antiparasitic ^[17][55], hemolytic and thrombolytic activities ^[18][19][56,57]. Recent studies for gold and silver-NHC compounds are addressed to breast ^[20][21][58,59], ovarian ^[22][60] and cervical human cancer ^[23][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 ^[24][25][62,63], actin ^[26][64] and tubulin ^[27][65], or triggering the reactive oxygen species-dependent intrinsic apoptotic pathway ^[28][66]. Recently, gold and silver compounds with NHC have been indicated as promising compounds for the treatment of COVID-19 ^[29][67], as they demonstrated a strong inhibition of the S/ACE2 interaction and particularly of the PL^pro enzymatic activity ^[30][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 ^[31][69].

Besides the importance of ruthenium-NHC complexes in organometallic catalysis ^[32][33][70,71] and bioinorganic chemistry, they have been described as effective anticancer and antimicrobial agents ^[2][5][9][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 ^[1][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.

Recent studies regarding the antiproliferative activity of Ru(II)-NHC complexes are summarized and the half-maximal (50%) inhibitory concentration (IC₅₀) values are given, when reported in the literature.

Lam et al., 2018, ^[34][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)(η⁶-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.

Tabrizi et al., 2019, ^[35][73] studied the in vitro antiproliferative potential and cyclooxygenase-2 (COX-2) inhibitory activity of a cyclometalated Ru(II) complex (2) containing ibuprofen (Ibu), 1,3,5-triaza-7-phosphaadamantane (PTA) and a CCC-pincer containing naproxen moiety (CCC-Nap). Antiproliferative studies were carried out on breast cancer (MCF-7 and MDA-MB-231), colon cancer (HT-29) cell lines, healthy breast cell lines (MCF-10A), and human embryonic kidney normal cells (HEK293) by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, after 72 h exposure. The complex 2 was quite potent, about twice as active as cisplatin against breast cancer cells, and around 14 times less active against HT-29 cell lines than cisplatin. Interestingly, the complex 2 inhibition studies against COX-2 revealed that it displayed approximately about 16- and 5-times stronger interactions than the free Ibu and CCC-Nap ligands, respectively. Moreover, it improved the production of reactive oxygen species (ROS) by 10.7-fold compared to H₂O₂, when used as a positive control in MCF-7 cells.

Rana et al., 2021, ^[36][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 (IC₅₀ 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 ^[37][75] investigated a pyrimidine functionalized non-annulated half-sandwich Ru(II)-NHC complex, namely chloro(p-cymene)-1-methyl-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 ^[36][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 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, ^[38][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. IC₅₀ for cisplatin was referred to data of the literature: IC₅₀ = 30.18 ± 2.58 µM (MCF-7), 11.75 ± 1.23 µM (HeLa), 17.29 ± 1.05 µM (HFF-1) ^[39][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.

Paşahan et al., 2022, ^[40][78] recently reported a study on Ru-NHC complexes as potential anticancer agents, examining their antiproliferative activity against rat glioblastoma (C6) and human cervix adenocarcinoma (HeLa) cell lines by ELISA assay. Four complexes, namely 8–11, were more active than cisplatin, used as the standard (IC₅₀ = 136 ± 0.7 mM (C6) and 126 ± 0.6 mM (HeLa)).

Chen et al., 2020, ^[41][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 caspase-dependent 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., ^[42][80] designed four (NHC)Ru(II)(η⁶-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 IC₅₀ of 3.61 µM, and that it can bind the plasmidic DNA without exerting genotoxic effects.

A recent interesting article by Wilke et al., ^[43][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.

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 ^[44][82]. In the past decade, the antimicrobial activity of ruthenium complexes has been reviewed ^[45][83] and studied as antimicrobial agents and alternatives or adjuvants to the more traditional antibiotics ^[46][84]. The recent antimicrobial studies regarding complexes of NHC-ruthenium are summarized in Table 2, and the minimal inhibitory concentrations (MICs) are reported.