Synthesis and investigation of antiproliferative activity of Ru-NHC complexes against C6 and HeLa cancer cells

The 2-methylpyridine, 2-diethylaminoethyl, and isopentyl linked a series of symmetric and unsymmetric benzimidazolium salts 2a–e were prepared and used in the synthesis of silver-N-heterocyclic carbene (NHC) complexes (3a–e). The Ru(II)-NHC complexes (4a–h) were synthesized via transmetalation reaction from 3a–e. 4a–h complexes were converted to Ru(II)-NHC.HCl complexes (5ah) by HCl solution of diethyl ether and characterized by different spectroscopic techniques such as 1H and 13C NMR, LC/MS-Q-TOF, FT-IR, elemental analysis, and melting point detection. We examined the effect of the structural difference of complexes on anticancer activity via different arenes and metal centers. Antiproliferative activity of 5a–h and 3a was tested against human cervix adenocarcinoma (HeLa) and rat glioblastoma (C6) cell lines by ELISA assay. The IC50 value of 5b, 5c and 5e complexes exhibited good cytotoxic activity than cisplatin on C6 (14.2 ± 0.5 mM; 16.2 ± 0.4 mM; 24.2 ± 0.7 mM, respectively) and HeLa (11.1 ± 0.5 mM; 13.7 ± 0.3 mM; 22.8 ± 0.8 mM, respectively) cell lines.

. Recently, our group reported cytotoxic properties of the Ag-NHC complexes on HeLa, HT-29, and L929 cell lines [87]. Among the NHCs, benzimidazole-based silver, gold and ruthenium-NHC complexes have been studied intensely due to the benzimidazole structure being a component of many biological structures [88][89][90][91][92][93][94]. In our previous study, Ru-NHC complexes showed good antiproliferative activity on Caco-1 and MCF-7 cell lines [16]. Encouraged by these results, we thought it would be helpful to examine the anticancer activities of the similar ruthenium complexes against different types of cancer cell lines to determine the affinity between them.
Herein, we synthesized and investigated the anticancer activity of eight Ru-NHC complexes and one of the Ag-NHC complexes with good lipophilic and hydrophilic properties on C6 and HeLa cell lines by a proliferation BrdU enzymelinked immunosorbent assay (ELISA) (Scheme 2). These water-soluble Ru-NHC complexes displayed pronounced anticancer activity on C6 and HeLa cancer cells.

Results and discussion
The synthesis pathway for the Ag-NHC and Ru-NHC complexes is presented in Scheme 2. The Ag-NHC complexes 3b and 3d were synthesized in good yields of 75% and 88%, respectively, by the reported procedure [95][96][97]. The Ru-NHC complexes were synthesized by transmetalation reaction in DCM from 3a-e complexes, respectively. Transmetalation is one of the most general methods for preparing a wide range of transition metal complexes due to its mild reaction conditions and generating air-stable intermediates. The transmetalation reaction of Ag(I)-NHC with corresponding Ru(II)-arene dimer under the exclusion of light at room temperature led to corresponding Ru-NHC complexes. The 5b, 5d, 5f, and 5h (Ru-NHC.nHCl) complexes were synthesized in moderate to good yields of 84%, 75%, 88%, and 85%, respectively, by adding HCl-diethyl ether solution to the DCM solution of the 4b, 4d, 4f, and 4h complexes. Synthesized complexes are well soluble in polar solvents such as H 2 O, DCM, DMF, DMSO, CH 3 OH. The stability of 5c, 5e, and 5g complexes was tested by 1 H NMR spectroscopy and it was seen that Ru(II)-NHC complexes showed high stability without structural decomposition against oxygen and moisture during two weeks (Figures 1, S1, and S2). Structural descriptions of the complexes were performed by 1 H NMR, 13 C NMR, HRMS ( Figure S3-S10), elemental analysis, and melting point determination.
The resonance of the C2 proton and C2 carbon of 2b and 2d in the 1 H and 13 C NMR were observed at 11.64, 11.08 152.7, and 144.1 ppm in CDCl 3 , respectively. The loss of the C2 proton in 1 H NMR and downfield shift of the C2 carbon to a new area in 13 C NMR spectra of Ag-NHC indicate the formation of Ag-NHC complexes. However, the C2 carbon of 3b and 3d was not observed in 13 C NMR spectra. We think the fast interconversion in the NMR time scale between the mono-carbene and bis-carbene structures causes the C2 carbon to be invisible in 13 C NMR spectra. According to Lin and coworkers [98], since the carbene-silver bond is labile in solution, the resonance of the carbene carbon, which is expected to be observed in the 13 C NMR, may not be observed. In the 13 C NMR spectrum of 5d, 5d, 5f, and 5h complexes, the carbene carbons dramatically shift downfield to 187.7, 183. 5, 193.5, and 193.5 ppm in the 13 C NMR spectra indicating the formation of 5d, 5d, 5f, and 5h complexes, respectively. The LCMS-QTOF spectra were verified in the 5b, 5d, and 5h complexes. The calculated and experimental LCMS-QTOF values are compatible with each other and confirm the proposed complex structures. NMR spectra of newly synthesized compounds and HRMS spectra are given in the supporting information part.
Cytotoxic activities of synthesized Ru-NHC complexes were investigated on C6 and HeLa cell lines. NHC complexes except showed moderate (5d), good (3a, 5a, 5f, 5h) and excellent (5b, 5c, 5e, 5g) activity when compared to cisplatin, which exhibited an IC 50 value of 136 ± 0.74 mM and 126 ± 0.57 mM against C6 and HeLa, respectively. However, when the structures of the complexes are examined, it is seen that structural differences cause antiproliferative activity differences in different cancer cell types. For example, N-substituents on the NHC and type of arene group led    37.3 ± 0.9; 5h, 90.6 ± 0.7 mM) antiproliferative activity. However, the displacement of the p-cymene arene group by hexamethyl benzene increases the antiproliferative activity of 5f. Complexes 5c and 5g, which are structurally identical except the arene group, showed a difference in the antiproliferative activity on C6 and HeLa cells. In both cell lines, the 5c complex showed much better antiproliferative activity than the 5g complex. The situation in the antiproliferative activities of the 5a and 5h complexes also changes in line with this trend, and complex 5h showed slightly better activity than complex 5a. The type of arene ligand also affected the antiproliferative activities of Ru(II)-NHC complexes because of the s-donor-p-acceptor ability of arene's and NHC's [60,99]. This work gives us some useful info about the effect of the metal center's genus on antiproliferative activity. Complex 3a is an analog of complex 5a except for the metal genus. When the antiproliferative activities of these two complexes are compared in the same cancer cells, it is seen that complex 5a has shown better activity. This result may be an indicator of how important the metal genus is in anticancer activity. The exact mode of action (MOA) of Ru-based complexes is unknown; as a result, a lot of Ru-containing drugs are still under development. Ru-complexes can imitate the iron-binding to serum transferrin which solubilizes and transports iron in the plasma thereby inhibiting their toxic delivery of iron. Additionally, numerous oxidation states, kinetics and different MOA provide many advantages over Pt-based complexes. For example, at physiological conditions, the Ru is known to be stable II, III, and IV oxidation states. The slow ligand exchange rates of the Ru-compounds make them suitable for biological applications. The good cytotoxicity of the Ru-complexes is due to their strong binding with DNA. Studies showed that some Ru-compounds could produce mutagenic effects, inhibit the replication of DNA, induce SOS repair, and decrease the synthesis of RNA thereby suggesting a DNA interaction [100]. In addition, according to our previous work [16], molecular docking calculations of similar Ru-NHC complexes showed anticancer activity by binding to DNA.
These observations point out that (a) the modification or fine-tune of the steric and electronic properties of NHCs through the N-substituents is crucial, (b) the arene type and metal center genus have a significant influence on the antiproliferative activity of complexes, and (c) complexes have properties that facilitate their cellular uptake into cells.

Conclusions
A series of Ru-NHC complexes have been prepared, spectroscopically characterized, and antiproliferative activity of complexes was examined on C6 and HeLa cells by a proliferation BrdU ELISA assay. The cytotoxic activities of complex 5b and 5c on C6 and HeLa cell lines are 7-9 times better than those of cisplatin and 2-10 times better than their analogous ruthenium complexes. Complexes 5b, 5c, and 5e have shown excellent low micromolar activity against C6 and HeLa cell lines. Additionally, other ruthenium and silver complexes have shown better activity on every concentration than cisplatin except complex 5d. The lower IC 50 values of the Ru-NHC complexes 5b, 5c, 5e are most likely to be attributed to the better solubility in H 2 O due to asymmetric NHCs. In addition, better solubility of complexes in H 2 O enhanced cellular uptake of complexes into the cell. This finding indicates that type of N-substituents on NHC and arene groups may improve the activity and selectivity. In this manner, the availability of effective drugs will lead to powerful medical treatment, and consequently, the number of surgical treatments will decrease, and life processes will increase.

Acknowledgments
This work was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project No: 114Z036).

Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.