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31 January 2023

Evaluation of Ruthenium(II) N-Heterocyclic Carbene Complexes as Enzymatic Inhibitory Agents with Antioxidant, Antimicrobial, Antiparasitical and Antiproliferative Activity

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1
Department of Biology, College of Science and Arts, Qassim University, Unaizah 51911, Saudi Arabia
2
Department of Science Laboratories, College of Science and Arts, Qassim University, Ar Rass 51921, Saudi Arabia
3
Department of Clinical Nutrition, College of Applied Health Sciences, Qassim University, Ar Rass 51921, Saudi Arabia
4
Department of Chemistry, Faculty of Science and Art, İnönü University, Malatya 44280, Turkey
Molecules2023, 28(3), 1359;https://doi.org/10.3390/molecules28031359
This article belongs to the Special Issue Recent Progress in the Preparation of Novel Heterocyclic Compounds and Their Therapeutic Applications
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Abstract

A series of [RuCl2(p-cymene)(NHC)] complexes were obtained by reacting [RuCl2(p-cymene)]2 with in situ generated Ag-N-heterocyclic carbene (NHC) complexes. The structure of the obtained complexes was determined by the appropriate spectroscopy and elemental analysis. In addition, we evaluated the biological activities of these compounds as antienzymatic, antioxidant, antibacterial, anticancer, and antiparasitic agents. The results revealed that complexes 3b and 3d were the most potent inhibitors against AchE with IC50 values of 2.52 and 5.06 μM mL−1. Additionally, 3d proved very good antimicrobial activity against all examined microorganisms with IZ (inhibition zone) over 25 mm and MIC (minimum inhibitory concentration) < 4 µM. Additionally, the ligand 2a and its corresponding ruthenium (II) complex 3a had good cytotoxic activity against both cancer cells HCT-116 and HepG-2, with IC50 values of (7.76 and 11.76) and (4.12 and 9.21) μM mL−1, respectively. Evaluation of the antiparasitic activity of these complexes against Leishmania major promastigotes and Toxoplasma gondii showed that ruthenium complexes were more potent than the free ligand, with an IC50 values less than 1.5 μM mL−1. However, 3d was found the best one with SI (selectivity index) values greater than 5 so it seems to be the best candidate for antileishmanial drug discovery program, and much future research are recommended for mode of action and in vivo evaluation. In general, Ru-NHC complexes are the most effective against L. major promastigotes.

1. Introduction

Currently, the use of organometallic and inorganic compounds is very common in contemporary medication [1,2]. New organometallic complexes called N-Heterocyclic Carbene (NHC) complexes show promise drug formulation [3,4,5,6,7]. Çetinkaya et al. revealed the results of the first study on the biological functions of NHC complexes [8,9,10,11]. For this reason, several research teams have synthesized functionalized NHC complexes and investigated their biological activities [12,13,14,15,16,17]. In this regard, complexes of the ruthenium (II/III) type have been thoroughly studied as DNA binding, antibacterial, and anticancer agents [18,19,20,21]. In particular, ruthenium-type compounds have been investigated against various cancer cell lines as prospective substitutes for the well-known diamine-dichloroplatinum (II) in the formulation of novel anticancer medicines (cisplatin) [22]. Under physiological circumstances, ruthenium can access the +2 and +3 oxidation states and can bind to cells’ proteins, nucleic acids, sulfur, or oxygen-containing molecules [23,24,25,26,27,28,29]. Additionally, depending on the characteristics of their ligand, ruthenium complexes can optimize the kinetics of their interactions with cell components. As a result, ruthenium complexes ligands exchange rates are near to those of biological processes, making them ideally suited for use in a variety of therapeutic contexts. Therefore, ruthenium compounds may have greater cytostatic activity than platinum-based medications against a variety of cancer cells and may also be helpful against cisplatin-resistant cancer cells. In addition, a brand-new class of highly intriguing physiologically active compounds known as ruthenium complexes has developed. Among the most dangerous human pathogens, Staphylococcus aureus is an eminent human pathogen that can colonize the human host and cause severe life-threatening illnesses [30,31,32,33,34,35,36,37].
Our research group was also investigating metal complexes with anticancer activity against a various types of cancer cell lines [38,39,40]. We are currently investigating new functional NHC ligands that provide a favorable environment for the development and utilization of metal compounds. In this paper, we synthesized and characterized a new series of Ru(I) NHC complexes containing benzimidazoles. The structure of the new compound was characterized by various spectroscopic and analytical methods. Next, enzyme inhibition against AChE and TyrE, antioxidant against 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and β-carotene bleaching test, various biological activities such as antimicrobial activity against Gram-positive, Gram-negative and Candida albicans, antiproliferative activity against colon cancer cell lines (HCT-116) and hepatocellular carcinoma cell lines (HepG-2), as well as antiparasitic activity against Leishmania major and Toxoplasma gondii, and cytotoxicity against Vero cells, were examined.

2. Results

2.1. Chemistry

2.1.1. Preparation of Benzimidazolium Salts 2a–d

The synthesis of benzimidazolium salts (2a–d) as NHC precursors was carried out as previously described [41] (Scheme 1). By using 1H NMR, 13C NMR, FT-IR, and elemental analysis, the structures of the benzimidazolium salts 2a–d were confirmed.
Scheme 1. General preparation of benzimidazolium salts (2a–d).
The 1H NMR spectra of precursors 2a–d show characteristic downfield shifts in the range δ 9.85–11.83 ppm for the NCHN protons due to the positive charge of the molecules [42].
The assigned structure was further supported by the benzimidazolium salt’s 1H NMR spectra. Sharp singlets representing the C(2)-H resonances were detected at 9.85, 10.34, 11.83, and 11.58 ppm for 2a–c, respectively. Chemical shifts measured by 13C NMR agreed with the suggested structure. At 141.3, 143.0, 143.1, and 152.8 ppm, the imino carbon is a characteristic singlet for the 1H decoupling mode of the benzimidazolium bromides 2a–d. The aliphatic area of the 13C NMR spectra displayed a sequence of peaks in the range of 20.76–35.18 ppm corresponding to resonances of the aliphatic carbon nucleus, whereas aromatic rings were seen in the range of 113.38–152.82 ppm. These numbers are fairly consistent with data that have already been published [43,44].

2.1.2. Preparation of Ruthenium-Carbene Complexes 3a–3d

By transmetallating the corresponding silverNHC derivatives without isolation, the novel [RuCl2(p-cymene)(NHC)] complexes (3a–3d) were prepared using a two-step procedure. By then adding [RuCl2(p-cymene)]2 to the mixture, orange–brown complexes were obtained with high yields (80–90%). In contrast to nonpolar solvents, chloroform, dichloromethane, tetrahydrofuran, and ruthenium carbene complexes (3a–3d) are soluble in these solvents. Scheme 2 provides the synthesis and the structures of Ru(II)-NHCs complexes. Using spectroscopic methods such as 1H NMR, 13C NMR, and IR as well as elemental studies, the structures of complexes 3a–3d were determined.
Scheme 2. General preparation of ruthenium N-heterocyclic carbene complexes 3a–3d.
The aromatic protons of complexes 3a–3d appeared as multiplets between 5.57–5.88 and 7.13–7.69 ppm, the methyl protons appeared between 0.92–1.16 and 2.27–2.61 ppm as singlets. In all complexes (3a–3d), the -CH proton of the p-cymene group was seen as a heptet in the 2.64–2.86 ppm range. (NCH2) exhibited a doublet resonance in the 1 H NMR spectra of (3a–3d) between 4.21 and 5.01 and 5.29 and 5.31 ppm. The carbene carbon in the ruthenium complexes 3a–3d exhibits 13C chemical shifts at 189.2, 187.8, 188.9, and 189.0 ppm, respectively. The values obtained are comparable to those that have been published for other Ru-NHC complexes [45,46]. The ruthenium complexes 3a–3d were also validated by elemental analysis results.

2.2. Biological Evaluation

2.2.1. Enzymatic Inhibitory, AChE and TyrE Inhibitory Activity

The results shown in Table 1 indicate that complexes 3b and 3d were the most potent inhibitors against AchE with IC50 values of 2.52 and 5.06 μM mL−1.
Table 1. Anti-AChRi and anti-TyrEI inhibitory activity of the synthesized compounds IC50 in µM mL−1 of mean ± S.D.

2.2.2. Antioxidant Activity

A significant antioxidant activity, comparable to that of the conventional BHT, was found for compound 3d. For the DPPH, ABTS, and -carotene tests, the IC50 values of this compound were 32.18, 18.17, and 92.25 µM mL−1, respectively (Table 2). For the DPPH, ABTS, and -carotene tests, the standard BHT’s IC50 values were 31.55, 17.41, and 89.55 µM mL−1, respectively (Table 2).
Table 2. Antioxidant activity of the synthesized compounds assessed by DPPH, ABTS antiradical scavenging power and β-carotene bleaching test presented by their IC50 in µM mL−1 of mean ± S.D.

2.2.3. Antimicrobial Activity

Figure 1 displays the antibacterial activity of the corresponding ruthenium (II) complexes (3a–3d) and the synthetic NHC ligands (2a–2d). All of the examined indicator organisms were inhibited by complexes 3b and 3d. Inhibition zones caused by complex 3d against S. aureus, L. monocytogenes, E. coli, P. aeruginosa, S. typhimurium, and C. albicans are 26, 28, 29, 27, 27, and 27 mm, respectively (Figure 1).
Figure 1. The antimicrobial activity of the synthesized compounds. The diameters of the inhibition zones were reported in mm.
The MIC ranges from 1.95 to 62.5 µM mL−1 for S. aureus and S. typhimurium, from 3.9 to 62.5 µM mL−1 for L. monocytogenes, and from 1.25 to 31.25 µM mL−1 for C. albicans. Complexes 3b and 3d gave the lowest MIC values. The MIC values for synthetic compound 3b against L. monocytogenes, S. aureus, S. typhimurium, and C. albicans were 15.6, 3.9, 3.9, and 1.25 µM mL−1, respectively (Figure 2). In relation to complex 3d, the MIC values against L. monocytogenes, S. aureus, S. typhimurium, and C. albicans were 3.9, 1.95, 1.95, and 1.25 µM mL−1, respectively (Figure 2). Additionally, Figure 2 shows that complex 3d’s MIC value against L. monocytogenes was the same as that of conventional ampicillin (3.9 g).
Figure 2. Minimum inhibitory concentration (MIC) of synthesized compounds against L. monocytogenes, S. aureus and S. typhimurium.

2.2.4. Antiproliferative Activity

Screening of the selected compounds against human colon carcinoma cancer cell lines and hepatocellular carcinoma cell lines revealed that the compound ruthenium(II) complex 3a had IC50 values (4.12 and 9.21 µM mL−1) in both human cancer cell lines where the mentioned values were approximately equivalent to those of standard vinblastine drugs (3.83 and 6.05 µM mL−1) in cytotoxic activity Table 3.
Table 3. IC50 of the synthesized compounds on colon carcinoma cells (HCT-116) and hepatocellular carcinoma cell lines (HepG-2). IC50 in µM mL−1 of mean ± S.D.

2.2.5. Antiparasitical Activity

Antileishmanial Results
From Table 4, we can observe that all the compounds revealed antileishmanial activity against both the amastigote and promastigote stages. For amastigotes, all the compounds gave IC50 values less than 4.3 µM mL−1, and only Compound 2b showed IC50 values less than 1 µM mL−1 (0.3 µM mL−1). All ruthenium(II) complexes (3a–d) gave IC50 values less than 1 µM mL−1 against L. major promastigotes. However, complexes 3c and 3d are the most active against L. major promastigotes, with SI values over five. There are strong similarities for the cytoxicity results of all compounds, with CC50 values in the range of 1.1 to 2.9 µM mL−1. Therefore, only two compounds, 3a and 3d, can be recommended for future use as antileishmanial agents.
Table 4. Antileishmanial activity of selected compounds against L. major promastigotes and amastigotes in IC50 and CC50 of µM mL−1 of mean ± S.D.
Antitoxoplasmal Results
Table 5 indicates that 4 compounds (ruthenium (II) complexes 3a–d) showed antitoxoplasmic activity with IC50 values ≤ 1.5 µM mL−1, which were 1.3, 1.4, 1.5 and 1.4 µM mL−1, respectively. However, their SI was less than 1.5, which indicates their toxicity for Vero cells that can limit their future uses for drug formulation.
Table 5. Antitoxoplasmal activity of selected compounds against T. gondii in IC50 and CC50 of µM mL−1 of mean ± S.D.

3. Materials and Methods

All procedures were carried out under an inert atmosphere using standard Schlenk line techniques according to our previous work [38,39,40].

3.1. Synthesis of Ligands (2a–d)

A mixture of benzimidazolium salt 1 (1 g) and the corresponding benzyl bromide (1eq) in DMF (2 mL) was stirred at 70 °C for 2–3 days. After that time, the white solid formed was washed with diethyl ether (20 mL) and stirred for couple hours. Then, the reaction mixture was filtred through filter paper, and the white solid was dried under vacuum, then crystallized with DCM-ether (1:3) for further purification.
5,6-dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[d]imidazol-3-ium bromide 2a
m.p. 307.7 °C. Yield (96%). ν(CN) = 1440.99 cm−1. 1H NMR (CDCl3, 400MHz) δ (ppm) 2.22(s, 30H, CH3), 2.28(s, 6H, CH3(a,b)), 5.8(s, 4H, CH2(1′,1″)), 7.11(s, 2H, Harom(4,7)),9.85(s, 1H, H2). 13CNMR(CDCl3, 101MHz) δ (ppm) 17.01(CH3), 16.92(C(c,g,c’,g’)), 17.01 (C(d,f,d’,f’)), 17.28(C(e,e’)), 20.77(C(a,b)), 48.10(C1′;1″), 113.41(C4;7), 125.58(C8;9), 130.42(C4′;5′;6′;4″;5″;6″), 133.50(C3′;7′;3″;7″), 133.74(C5;6), 136.88(C2′;2″),141.35(C2). Anal. Calcd for C31H39BrN2:C, 71.66%; H, 7.57%; N, 5.39%%. Found: C, 71.17; H, 7.8; N, 5.4%.
5,6-dimethyl-1,3-bis(2,4,6-trimethylbenzyl)-1H-benzo[d]imidazol-3-ium bromide 2b
m.p. 210.6 °C. Yield (92%). ν(CN) = 1454.84 cm−1. 1H NMR (CDCl3, 400MHz) δ (ppm) 2.15(s, 12H, CH3(a,e,a’,e’)), 2.18(s, 12H, CH3(b,d,b’,d’)), 2.20(s, 6H, CH3(c,c′)), 5.84(s, 4H, CH2), 7.2(d, 2H, Harom(5,6)), 7.34(d, 2H, Harom(4,7)), 10.34(s, 1H, H2). 13C NMR(CDCl3,100MHz) δ (ppm) 16.95(Ca,e,a’,e’), 17.06(Cb,d,b’,d’), 17.30(Cc,c’), 48.58(C1′;1″), 113.72(C4;7), 125.36(C5;6),126.88(C8;9),131.85(C5′;5″),133.49(C4′;6′;4″;6″), 133.82(C3′;7′;3″;7″), 137.07(C2′;2″), 143(C2). Anal. Calcd for C29H33BrN2:C, 71.16%; H, 6.80%; N, 5.72%. Found: C, 71.3; H, 6.9; N, 5.8%.
5,6-dimethyl-1,3-((4-(tert-butyl)-4-methyl-benzyl)-1H-benzo[d]imidazol-3-ium bromide 2c
m.p. 296.5 °C. Yield (89%). ν(CN) = 1427.88 cm−1. 1H NMR (CDCl3, 400MHz) δ (ppm) 1.2 (s, 18H, CH3), 5.75(s, 4H, CH2), 7.33(d, 4H, Harom(4′,6′,4″,6″)),7.39(d, 4H, Harom(3′;7′3″,7″)), 7.46(d, 2H, Harom(5;6)), 7.55(d, 2H, Harom(4;7)), 11.83(s, 1H, H2). 13CNMR(CDCl3,101 MHz) δ (ppm) 31.18(CH3), 34.67(C8′;8”), 51.27(C1′;1”), 113.78(C4;7), 126.34(C4′;6′;4″;6″), 127.09(C5,6), 128.17(C3′;7′;3″;7″), 129.56(C8;9),131.41(C2′;2″), 143(C2),152.46(C5′;5″). Anal. Calcd for C28H33BrN2:C, 70.43%; H, 6.80%; N, 5.72%. Found: C, 70.2; H, 6.9; N, 5.8%.
5,6-dimethyl-1,3-(3,5)-dimethyl-4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazolium bromide (2d)
Yield: 92%; M.p. = 235 °C; FT-IR (KBr) ν(CN)(cm−1): = 1565 (C=N); 1359 (C-N) cm−1 ; 1H NMR (CDCl3,300 MHz) δ (ppm):= 11.58 (s, 1H, H2, NCHN); 7.44 (d, 2H, H4″, 6″, arom. CH, 3 JHH = 7.43 Hz); 7.37 (s, 1H, H5′, arom. CH); 7.35 (d, 2H, H3″, 7″, arom. CH, 3 JHH = 7.34 Hz); 7.27 (s, 1H, H3′, arom. CH); 7.01 (s, 2H, H4,7, arom. CH); 6.94 (s, 1H, H7′, arom. CH); 5.76 (s, 2H, H1′,CH2); 5.67 (s, 2H, H1″,CH2); 2.26 (s, 6H, Hd,e, 2 × CH3); 1.25 (s, 9H, Ha,b,c, 3×CH3). 13C NMR (CDCl3, 75MHz) (δ (ppm)):152. 81 (C2, NCN); 142.37 (C4′, 6′, arom.Cq); 139.59 (C2′, arom.Cq); 137.84 (C5″, arom. CH); 133.11 (C2″, arom.Cq); 131.35 (C5, 6, arom.Cq); 130.45 (C8, 9, arom.Cq); 128.53 (C4″, 6″, arom. CH); 126.77 (C3′, 5′, 7′, arom. CH); 126.23 (C3″, 7″, arom. CH); 113.82 (C4, 7, arom. CH); 51.80 (C1′, CH2); 51.43 (C1″, CH2); 35.17 (Cd, e, 2 × CH3); 31.70 (Cc, CH3); 21.75 (Ca, b, 2 × CH3); Anal. Calcd for C27H31BrN2:C, 69.97%; H, 6.74%; N, 6.04%. Found: C, 70.1; H, 6.8; N, 6.1%
Synthesis of ruthenium N-heterocyclic Carbene complexes (3a–3d): The synthesis was performed according to our previous work [38].
5,6-Dimethyl-[1,3-(2,3,4,5,6-pentamethyl-2,4,6-trimethyl)-benzimidazol-2-ylidene](p-cymene) ruthenium(II) chloride, (3a)
Yield: (80%); m.p. = 204 °C. FT-IR(KBr)ν(CN)(cm−1) = 1404 (C-N).1HNMR (300 MHz, CDCl3, δ(ppm)): 7.13 (d, 2H, p-CH3C6H4CH(CH3)2); 7.00 (d, 2H, p-CH3C6H4CH(CH3)2); 6.92 (s, 1H, CH2C6H3(CH3)2-3,5); 6.84 (d, 4H, CH2C6H4(CH3)-4); 6.67 (s, 2H, CH2C6H3(CH3)2-3,5); 6.51 (m, 2H, CH2C6H4(CH3)—4); 5.57 (m, 2H, C6H2(CH3)2-5,6); 5.30 (s, 2H, H1′, CH2); 5.02 (s, 2H, H1″, CH2); 2.64 (p, 1H, H 7‴pCH3C6H4CH(CH3)2); 2.34 (s, 3H, He, CH3); 2.30 (s, 6H, Hc, d, 2 × CH3); 2.19 (s, 6H, Ha, b, 2 × CH3); 1.82 (s, 3H, Hf, CH3); 1.58 (s, 3H, Hi, CH3); 1.15 (s, 6H, Hg, h, 2 × CH3 (p-CH3C6H4CH(CH3)2).13C NMR (CDCl3, 75 MHz) (δ (ppm)): 189.2 (C2, NCN); 138.4; 138.2; 137.0; 135.0; 134.5; 134.3; 132.3; 129.6; 129.0; 125.8; 123.6; 111.9; 107.4; 96.6; 85.2; 52.4 (C1′, CH2); 52.2 (C1″, CH2); 30.6 (C7‴, pCH3C6H4CH(CH3)2); 21.6 (Ch, g, 2 × CH3, p-CH3C6H4CH(CH3)2); 21.2 (Cc, d, 2 × CH3); 22.3 (Ce, f, 2 × CH3); 18.1 (Ca, b, i, 3 × CH3). Anal. Calcd for C41H53RuN2Cl2:C, 66.02%; H, 7.16%; N, 3.76%. Found: C, 66.1; H, 7.3; N, 3.8%.
5,6-Dimethyl-[1,3-(2,3,4,5,6-pentamethyl-2,4,6-trimethyl)-benzimidazol-2-ylidene](p-cymene) ruthenium(II) chloride, (3b)
Yield: (88%); m.p. = 184 °C. FT-IR(KBr)ν(CN)(cm−1) = 1418 (C-N).1HNMR(300 MHz, CDCl3, δ(ppm)): 7.69 (d, 2H, p-CH3C6H4CH(CH3)2); 7.54 (d, 2H, p-CH3C6H4CH(CH3)2); 7.26 (s, 1H, CH2C6H3(CH3)2-3,5); 6.89 (s, 2H, CH2C6H3(CH3)2); 6.63 (s, 2H, C6H2(CH3)2); 5.88 (s, 1H, CH2C6H(CH3)4); 4.21 (s, 4H, H1′,1″, 2 × CH2); 2.86 (p, 1H, H 7‴, p-CH3C6H4CH(CH3)2); 2.27 (s, 18H, Ha, b, c, d, f, g, 6 × CH3); 2.20 (s, 3H, Hi, CH3); 1.95 (s, 3H, Hl, CH3); 1.32 (s, 6H, He,h, 2 × CH3); 0.92 (s, 6H, Hj, k, 2 × CH3 (p-CH3C6H4CH(CH3)2). 13C NMR (CDCl3, 75 MHz) (δ (ppm)): 187.8 (C2, NCN); 138.4; 135.4; 135.2; 134.5; 133.5; 133.2; 132.3; 131.6; 131.3; 130.0; 128.9; 124.6; 123.4; 107.1; 96.2; 54.0 (C1′, CH2); 51.8 (C1”, CH2); 31.0 (C7‴, p-CH3C6H4CH(CH3)2); 21.5 (Cj, k, 2 × CH3, p CH3C6H4CH(CH3)2); 20.8 (Cc, d, 2 × CH3); 20.2 (Ci, CH3); 18.4 (Cf, g, 2 × CH3); 16.3 (Ca, b, 2 × CH3); 15.4 (Ce, h, l, 3 × CH3). Anal. Calcd for C41H53RuN2Cl2:C, 66.02%; H, 7.16%; N, 3.76%. Found: C, 66.1; H, 7.3; N, 3.8%.
5,6-Dimethyl-[1,3-(4-(tert-butyl)-4-methyl)-benzimidazol-2-ylidene] (p-cymene) ruthenium (II) chloride, (3c)
Yield: (87%); m.p. = 224 °C. FT-IR(KBr)ν(CN)(cm−1) = 1609 (C-N). 1HNMR(300 MHz, CDCl3, δ(ppm)): 7.36 (d, 4H, CH2C6H4C(CH3)3-4); 7.03 (d, 4H, CH2C6H4C(CH3)3-4); 6.82 (s, 2H, C6H2(CH3)2-5,6); 6.55 (d, 2H, p CH3C6H4CH(CH3)2); 5.62 (d, 2H, p- CH3C6H4CH(CH3)2); 5.31 (s, 2H, H 1′, CH2); 5.02 (s, 2H, H 1″, CH2); 2.61(s, 6H, Hd, g, 2 × CH3); 2.19(s, 6H, Ha, b, 2 × CH3); 1.81 (s, 3H, Hi, CH3); 1.66 (s, 1H, H 7‴, p-CH3C6H4CH(CH3)2); 1.31 (s, 15H, Hc, e, f, h, l, 5 × CH3); 1.12 (d, 6H, Hj, k, 2 × CH3 (pCH3C6H4CH(CH3)2). 13CNMR (CDCl3, 75 MHz) (δ (ppm)): 188.9 (C2, NCN); 150.4; 134.9; 134.4; 132.3; 125.8; 125.5; 111.9; 106.8; 97.3; 85.5; 52.3 (C1′,1″, 2 × CH2); 41.1 (C7‴, p-CH3C6H4CH(CH3)2); 34.6 (Cd, g, 2 × CH3, CH2C6H4C(CH3)3); 31.4 (Cc, e, f,h, 4 × CH3); 30.5 (Ci, CH3); 22.6 (Ca, b, 2 × CH3); 20.3 (Cj, k, 2 × CH3). DART-TOF-MS: m/z = 453, 426, 291. Anal. Calcd for C38H46RuN2Cl2:C, 64.95%; H, 6.60%; N, 3.99%. Found: C, 65.1; H, 6.7; N, 4.1%.
5,6-Dimethyl-[1,3-(3,5)dimethyl-4-methyl)-benzimidazol-2-ylidene](p-cymene) ruthenium(II) chloride, (3d)
Yield: (90%); m.p. = 210 °C. FT-IR(KBr)ν(CN)(cm−1)= 1409 (C-N). 1HNMR(300 MHz, CDCl3, δ(ppm)): 14 (d, 4H, CH2C6H4(CH3)-4); 7.00 (d, 4H, CH2C6H4(CH3)); 6.79 (s, 2H, C6H2(CH3)2); 6.46 (d, 2H, p-CH3C6H4CH(CH3)2); 5.68 (d, 2H, p-CH3C6H4CH(CH3)2); 5.29 (s, 2H, H 1′, CH2); 5.03 (s, 2H, H1″, CH2); 2.68 (p, 1H, H 7‴, p-CH3C6H4CH(CH3)2); 2.34 (s, 6H, Hc, d, 2 × CH3); 2.18 (s, 6H, Ha, b, 2 × CH3); 1.84 (s, 3H, He, CH3); 1.16 (d, 6H, Hf, g, 2 × CH3 (p-CH3C6H4CH(CH3)2). 13CNMR (CDCl3, 75 MHz) (δ (ppm)): 189.0 (C2, NCN); 137.0; 134.9; 134.4; 132.3; 129.6; 125.9; 111.9; 107.6; 97.0; 85.30; 52.5 (C1′,1″, 2 × CH2); 30.6 (C7‴, p-CH3C6H4CH(CH3)2); 22.6 (Cf, g, 2 × CH3); 21.2 (Cc, d, 2 × CH3); 20.3 (Ce, CH3); 18.3 (Ca, b, 2 × CH3); 14.2 (CH, CH3).

3.2. Biological Activities

3.2.1. Enzymatic Inhibitory Assay

Acetylcholinesterase Inhibitory (AChEI)
According to the Ellman et al. published spectrophotometric method of electric eel AChE [47], AChEI activity was measured. Acetylthiocholine iodide (ATCI) was used as the reaction’s substrate, and the antiacetylcholinesterase activity was measured using 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB).
Antityrosinase Activity
According to Rangkadilok et al. [48], the TyrE inhibitory activity was measured spectrophotometrically using L-tyrosine as the substrate in a 96-well microplate.

3.2.2. Antioxidant Activity

Three methods were used for the assessing antioxidant properties of the selected compounds, which are 2.2-diphenyl-1-picrylhydrazyl (DPPH), 2.2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals scavenging, and β-carotene linoleic acid bleaching assay. Antioxidant activity was expressed as IC50 (the concentration that causes 50% of inhbition effect). The control compound was butylated hydroxytoluene (BHT), which is a potent antioxidant.
DPPH Radical Scavenging Activity
Briefly, various amounts of produced compounds were diluted with ultrapure water after being dissolved in dimethylsulfoxide (DMSO)/water (1/9; v/v) (1, 0.5, 0.250, 0.125, 0.0625, 0.03125 mg mL−1). The samples were then combined with 500 mL of a 4% (w/v) solution of the DPPH radical in ethanol. The combination was incubated for 30 min. at room temperature and in the dark [49]. Spectrophotometric analysis was used to calculate the scavenging capacity by comparing the decrease in absorbance at 517 nm to a blank.
ABTS Assay
ABTS radical scavenging activity was conducted by referring to the method of Re et al. [50].
β-Carotene Bleaching Assay
β-Carotene bleaching test was conducted following the method described by Pratt’s [51].
All the assays used for antioxidant determination (DPPH, ABTS, and β-carotene bleaching assay) were performed simultaneously three times in the same conditions. The results obtained in µM mL−1 average of the three experiments.

3.2.3. Antimicrobial Activity

Microorganisms, media and growth conditions, agar well diffusion method for inhibition zone determination (IZ) and minimum inhibitory concentration (MIC) were performed according to the literature work [52,53].
The synthesized compounds were examined in vitro for their antimicrobial activity against Six standard microorganisms of ATCC, two Gram positive bacteria S. aureus and Listeria monocytogenes, three Gram negative bacteria Esherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium and the fungus C. albicans. Bacteria were cultured in Luria-Bertani (LB) medium, while Sabouraud agar was used for culturing C. albicans and the assay conducted according to our previous techniques [53]. The results were the average of 3 readings.

3.2.4. In Vitro Anticancer Proliferation Studies

The selected compound was investigated for its cytotoxic properties against HCT-116 and HepG-2 (cancer cell lines of ATCC, Rockville, MD, USA). Vinblastine was applied as reference drug. The assay was conducted according to the methods described by Mossman [54] and our recently published data [55].
The results presented IC50 (The concentration that causes 50% inhibitory of cell viability) of µM mL−1 from the average of 3 reading.

3.2.5. Antiparasitical Assessment

Leishmania Major Cell Isolation, Culture Conditions, and Assays
This assay was carried out according to the methods mentioned in our previously published article [56]. L. major promastigotes were isolated locally from an indoor patient in 2016, liquid nitrogen was used for the preservation of the parasites, and BALB/c mice were used for the maintenance of the parasites and production of L. major amastigotes. Phenol red-free RPMI 1640 medium (Invitrogen, USA) with 10% FBS was used for the culture and in vitro evaluation, while amphotericin B (AmB) was used as reference drug. The result was expressed in IC50 values (the concentration that causes 50% inhibition of the viable parasites) of three independent readings, followed by the selectivity index (SI) calculation by dividing CC50 (toxic concentration that causes 50% inhibition of cell growth) over IC50 of the same compound [56].
Toxoplasma Gondii Cell Line, Culture Conditions, and Assay
This assay was carried out according to the methods mentioned in our previously published article [50]. Vero cells line (ATCC® CCL81™, USA) were used for the serial passage and cultivation of T. gondii tachyzoites RH strain, complete RPMI 1640 medium with heat-inactivated 10% FBS was used for the culture and in vitro evaluation, while atovaquone (ATO) was used as reference drug. The results were expressed in IC50 of three independent readings, followed by the selectivity index (SI) calculation by dividing CC50 over IC50 of the same compound [56].

3.2.6. In Vitro Cytotoxicity Assay

MTT colorimetric technique was carried out for cytotoxicity evaluation according to the methods mentioned in our previously published article [57]. An amount of 96 well plates with complete were used for the culture of the cells. FLUOstar OPTIMA spectrophotometer was applied for colorimetric analysis and in vitro evaluation. Cytotoxic effects were expressed by CC50 values (concentration that caused a 50% reduction in viable cells), from three independent experiments [56].

4. Conclusions

In summary, ruthenium(II)-NHC complexes 3a–3d have been easily prepared by the reaction of silver(I)- NHC complexes as a carbene transfer reagent with [RuCl2(p-cymene)]2 in dichloromethane at room temperature in good yields. The molecular structures of the benzimidazolium salts (2a–2d) and the Ru(II)–N-heterocyclic carbene (NHC) complexes 3a–3d were characterized by elemental analysis and 1H- and 13C-NMR spectra.
The results of the enzymatic inhibitory study against AChE and TyrE revealed that complexes 3b and 3d are the most effective inhibitors against AchE, with respective IC50 values of 2.52 and 5.06 µM mL−1 and 19.88 and 24.95 µM mL−1. These results confirm that NHC metallic complexes have potent antibacterial properties [58]. Important antioxidant activity was observed for Complex 3. The synthesized NHC ligands (2a–2d) and their corresponding ruthenium(II) complexes (3a–3d) were screened against HCT-116 and HepG-2, and the results revealed that ruthenium(II) complex 3a exhibited cytotoxic activity approximately equivalent to that of standard vinblastine, so we can suggest ruthenium(II) complex 3a can be used in the formulation of drugs that stimulate cancer treatment against human colon carcinoma cancer and liver hepatocellular carcinoma cancer after further pharmacological and clinical trials investigations. Regarding the last experiment of studying the ruthenium (II) complex as an antiparasitical agent against L. major and T. gondii, compounds 3c and 3d were found to have extremely potent antileishmania effects, with a SI over five, while all tested compounds had less antitoxoplasmic activity. These findings were similar to our previous investigation with NHC palladium complexes as well as the similar ruthenium complexes [53,57]. We propose that 3d can be used as a drug candidate for many antimicrobial, anticancer, and antiparasite bioactivities, and further investigation for mode of action detection and in vivo evaluation is highly recommended.

Author Contributions

N.H. conceptualized the project’s primary principles, drafted the analysis methods, conducted the scientific investigation, formal analysis, data curation, and acquired funding. I.S.A.N., W.S.K., and T.A.K. worked on the project’s concept, design, and monitoring and evaluation throughout the project. N.G. and I.Ö. reviewed and edited the first draft of the paper, which was written by N.H. and I.S.A.N. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript except the suppot for the project number (QU-IF-02-02-27782) from Deputyship for Research& Innovation, Ministry of Education, Saudi Arabia.

Institutional Review Board Statement

In the present work the instructions and rules of the committee of research ethics, Deanship of Scientific Research, Qassim University, permission number 20-03-20 was applied.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education, Saudi Arabia for funding this research work through the project number (QU-IF-02-02-27782). The authors also thank to Qassim University for technical support.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

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