Cytotoxic potential of silver, palladium, rhodium, ruthenium, and iridium complexes of a cycloheptyl-substituted lipophilic N-heterocyclic carbene ligand

Abstract Lipophilicity is a crucial parameter for cytotoxicity of metal complexes, in many cases associated with increased activity. In the present study, we synthesized a series of N-heterocyclic carbene (NHC) complexes of different metals with a lipophilic benzimidazolium chloride to investigate and compare their cytotoxicity. Rh– (4), Ru– (5), and Ir–NHC (6) complexes of a cycloheptyl-substituted benzimidazole-based NHC ligand (1) have been prepared by transmetalation reaction between Ag–NHC and the corresponding metal compound. The newly synthesized complexes have been characterized by NMR, IR, LC-MS spectroscopy, and elemental analyses. The anti-growth effects of the newly synthesized Rh–, Ru–, and Ir–NHC complexes and previously reported NHC precursor (1), Ag– (2), and Pd–NHC (3) complexes against human breast (MCF-7), colorectal (Caco-2), ovarian (A2780), and prostate (LNCaP) cancer cell lines have been investigated by MTT assay. The results revealed that the compounds provide stronger anti-growth effects against all cell lines compared to standard drug cisplatin. Among the compounds, benzimidazolium chloride, and Ag–NHC complex showed promising activities.  


Introduction
Cancer is a major health problem and the fight against cancer continues with different approaches.Chemotherapy is one of the most used methods in cancer treatment, however, tumor resistance and drug-induced toxicity are two important disadvantages of chemotherapy [1].The urgent need for more active and less toxic anticancer agents force scientists to investigate the potential of new compounds and materials as anticancer agents.
Cisplatin is the first metal-based drug used in cancer treatment but it has the limitations mentioned above.The success achieved with cisplatin (and its "second generation" drugs carboplatin and oxaliplatin) paved the way for further investigation of metal-based compounds in this field [2].The ligands used in metal complexes can significantly affect the characteristics of the complexes such as stability, solubility, and lipophilicity which are highly determinant on the anticancer potential.Introducing organic ligands to the structure of the complexes through donor atoms such as N, O, P, S or C yields metal-organic or organometallic complexes which have attracted attention.N-heterocyclic carbenes (NHCs) make highly stable organometallic complexes with metal coordinated through carbene carbon [3].In addition to stability, easy preparation and modification of carbon scaffold, and tunability of electronic and steric properties of NHCs are other reasons of their popularity [4].Although the first NHC complexes were reported in 1968 by Wanzlick [5] and € Ofele [6], independently, and the first isolated free carbene was reported in 1991 by Arduengo [7], pioneering studies on the biological properties of NHC complexes were reported in the early 2000s.In 2004, antimicrobial properties of Ag-NHCs and mitochondrial membrane permeabilization inducing ability of Au-NHCs were reported by Youngs and co-workers [8] and Barnard and co-workers [9], respectively.To date, biological properties of thousands of NHC complexes have been reported [10][11][12][13].Although Pt- [14,15], Au- [16], and Ag-NHCs [17] are biologically most investigated NHC complexes, a remarkable number of studies on biological properties of Pd- [18][19][20], Rh- [21][22][23][24][25], Ru- [26][27][28][29], and Ir-NHCs [30][31][32][33] have also been reported.
Lipophilicity of a drug candidate is crucial for its anticancer potential and mechanism of action [34].Increased lipophilicity may increase passage across the lipid bilayer, and so cellular uptake.There are many studies in which increased lipophilicity is associated with increased anti-proliferative activity [35][36][37][38][39][40][41][42][43][44].Therefore, in the present study, considering both the promising results achieved with NHC complexes and the positive effects of lipophilicity on cytotoxicity, we investigate and compare the cytotoxic potential of a cycloheptyl-substituted benzimidazolium chloride and its Ag-, Pd-, Rh-, Ru-, and Ir-NHC complexes against human breast (MCF-7), colorectal (Caco-2), ovarian (A2780) and prostate (LnCap) cancer cell lines.
were obtained as pure solids after evaporation of dichloromethane and washing with n-hexane.However, Ir-NHC is soluble in n-hexane and it was purified by extraction with n-hexane.All complexes were stable under air both in solid state and in solution, therefore, we did not use argon or nitrogen gases for inert atmosphere during the reaction period or storage.
Characterizations of 4-6 were completed by NMR, IR, and LC-MS spectroscopy, and elemental analyses.Satisfactory elemental analyses (Experimental section) were obtained for 4-6.LC-MS spectra of 4-6 were also supportive for the structures of complexes. 1H, 13 C NMR, IR, and LC-MS spectra of 4-6 and 1 H NMR spectra of re-synthesized 1-3 are given in Supporting Information.Despite our best efforts, we could not obtain suitable crystals for single crystal X-ray analysis, as in our previous studies done with cycloheptyl group; we suggest that this situation may be associated to conformation of cycloheptane ring.
In the 1 H NMR spectrum of 4, the signals of aromatic hydrogens were observed as two doublets and two triplets at 7.16-6.06ppm with coupling constants about 8 Hz.The methylene hydrogens between benzimidazole and phenyl moieties are diastereotopic and observed as doublets at 6.32 and 5.91 ppm with a coupling constant of 14.8 Hz.Among the two methylene groups connecting benzimidazole and cycloheptyl moieties, the hydrogens of the one adjacent to nitrogen were observed as a multiplet at 4.81 ppm, while the remaining ones were observed from 1.99 to 1.42 ppm, overlapped with aliphatic hydrogens of cycloheptane and cod rings.The olefinic hydrogens of cod ring were observed as multiplets at 5.08 (2H), 3.50 (1H), and 3.40 (1H) ppm.The hydrogens of methyl groups attached to phenyl ring were observed as singlets at 2.25 (3H), 2.20 (6H), and 2.18 (6H) ppm.In the 13 C NMR spectrum of 4, the signal of carbene carbon was observed at 196.2 ppm as a doublet with J Rh-C of 50.4 Hz.The signals of ten aromatic carbons were observed from 135.9 to 109.6 ppm, as expected.The olefinic carbons of cod ring coordinated to rhodium were observed as doublets at 109.6, 99.6, 68.9, and 68.0 ppm with J Rh-C of 14.6 and 6.5 Hz.The two methylene carbons adjacent to nitrogen were at 51.4 and 49.1 ppm.The signals of remaining aliphatic carbons were observed at 40.1-16.9ppm.
In the 1 H NMR of 5, the signals of four aromatic hydrogens of benzimidazole were two doublets and two triplets at 7.34-6.12ppm with coupling constants about 8 Hz.The signals of four aromatic hydrogens of p-cymene were observed as multiplets from 5.57 to 4.41 ppm.The hydrogens of methylene connecting the benzimidazole and phenyl moieties are diastereotopic and observed at 6.88 and 5.46 ppm as broad signals.The signals of hydrogens of other methylene carbon adjacent to nitrogen were at 5.31 ppm as a multiplet.While the signals of one hydrogen of isopropyl group in p-cymene were observed as a septet at 3.00 ppm with coupling constant of 6.8 Hz, two methyl hydrogens of isopropyl group in p-cymene were observed as overlapped with aliphatic hydrogen of cycloheptane ring.The signal of hydrogens of other methyl groups in p-cymene were observed as a singlet at 2.13 ppm.The hydrogens of methyl groups attached to phenyl ring were singlets at 2.29 (3H), 2.21 (6H), and 2.16 (6H) ppm.In the 13 C NMR spectrum of 5, the signal of carbene carbon was at 187.9 ppm.The signals of ten aromatic carbons were in the range of 135.8-98.5 ppm, while the signals of four different aromatic carbons of p-cymene were observed from 86.2 to 83.8 ppm.The two methylene carbons adjacent to nitrogen atoms were observed at 52.2 and 50.3 ppm.The signals of remaining aliphatic carbons were observed in the range of 40.0-16.9ppm.
In the 1 H NMR of 6, the signals of aromatic hydrogens of benzimidazole were two doublets and two triplets from 7.17 to 6.08 ppm with coupling constants about 8 Hz.The signals of two diastereotopic methylene hydrogens between benzimidazole and phenyl moieties were doublets at 6.22 and 5.72 ppm with coupling constants of 14.8 Hz.The hydrogens of methylene carbon adjacent to nitrogen were overlapped with two olefinic hydrogens of cod ring at 4.74-4.58ppm.The remaining two olefinic hydrogens of cod ring were observed at 3.14 and 3.00 ppm, as multiplets.The hydrogens of methyl groups attached to phenyl ring were singlets at 2.24 (3H), 2.19 (6H), and 2.17 (6H) ppm.In the 13 C NMR spectrum of 6, the signal of carbene carbon was at 191.1 ppm.The signals of ten aromatic carbons were from 134.8 to 108.7 ppm.The olefinic carbons of cod ring were at 85.0, 84.9, 51.5, and 50.7 ppm.The two methylene carbons adjacent to nitrogen were at 50.1 and 47.6 ppm, while the signals of remaining aliphatic carbons were in the range of 39.0-15.9ppm.

Cytotoxicity and stability studies
The anti-growth effects of 1-6 against MCF-7, Caco-2, A2780 and LNCaP cancer cell lines were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.Cisplatin was used as standard for comparison.The % cell viability results after 24 h of treatment at four different concentrations (0. Except from Pd-NHC (3), all compounds showed anti-growth effect against all cell lines at all concentrations.Pd-NHC had almost no anti-growth effect against MCF-7 and LNCaP, but surprisingly performed the strongest anti-growth effect against A2780.When the compounds are compared to cisplatin, benzimidazolium chloride (1), Ag-NHC (2), and Ru-NHC (5) had stronger activity than cisplatin against all cell lines.Additionally, Rh-(4) and Ir-NHC (6) complexes (both containing cod ring) had stronger or equal anti-growth effect against MCF-7 and Caco-2 compared to cisplatin.If we compare the tested compounds, it can be said that benzimidazolium chloride and Ag-NHC are the most active compounds.
The stability of a metal complex in the test medium is important for the activity and mechanism of action of the complex.Therefore, we tested the stabilities of the Ag-( 2 Lipophilicity plays a crucial role in anti-growth effect but compounds with increased lipophilic character may have low solubility in aqueous media.Therefore, the balance between lipophilicity and solubility is a key parameter for a drug candidate.If the results obtained in this study are evaluated by this view of point, the stronger antigrowth effects of benzimidazolium chloride and Ag-NHC, which have relatively lower lipophilic character compared to other complexes, may be associated to their better solubility and stability in aqueous medium and possible solubility problems of Pd-, Rh-, Ru-, and Ir-NHC complexes.

Conclusion
Rh, Ru, and Ir complexes of a cycloheptyl-substituted benzimidazolium chloride were synthesized and characterized.Anti-growth effects of newly synthesized complexes, benzimidazolium chloride and its previously reported Ag and Pd complexes were tested against human breast, colorectal, ovarian and prostate cancer cell lines.
Especially benzimidazolium chloride and Ag-NHC complex showed promising antigrowth activity against four cell lines, and they deserve further studies.Finally, we must note that, we believe the results obtained in this study are informative for comparison of the most known and mostly investigated types of Ag-, Pd-, Rh-, Ru-, and Ir-NHC complexes.

General remarks for synthesis and characterization of the complexes
The reagents and solvents were purchased from commercial sources and used without purification.[RhCl(cod)] 2 , [RuCl 2 (p-cymene)] 2 , and [IrCl(cod)] 2 dimers were synthesized according to known procedures.The cycloheptyl-substituted benzimidazolium chloride (1) [45], Ag-NHC (2) [45], and Pd-NHC (3) [46] were reported in our previous studies and re-synthesized in this study for biological assay.Melting points were determined in an open capillary tube and were uncorrected using a Gallenkamp MPD350.BM3.5 apparatus.C, H, and N analyses were carried out using a LECO CHNS-932 elemental analyser.FT-IR spectra were recorded with an ATR (attenuated total reflectance) sampling accessory using a Perkin Elmer Spectrum 400 spectrophotometer.NMR spectra were recorded using a Bruker Ascend TM 400 Avance III HD operating at 400 MHz ( 1 H) and 100 MHz ( 13 C).Chemical shifts were expressed in part per million (ppm) and referenced to residual solvent peaks.LC-MS/MS spectra were recorded using a Shimadzu 8040 LC-MS spectrophotometer.
), Rh-(4), and Ru-NHC (5) complexes in DMSO-d 6 and DMSO-d 6 /D 2 O mixture by 1 H NMR spectroscopy (see Figures S31-33 for spectra).The spectra recorded after 24 h in both DMSO-d 6 and DMSO-d 6 /D 2 O show that Ag-and Rh-NHC complexes preserve the structure in both solvent systems in which cytotoxicity studies were carried out; some signals in the spectra of Ru-NHC in the presence of D 2 O broadened, perhaps by coordination of water or DMSO.