Synthesis and Cytotoxicity of Dinuclear Silver (I)-N-Heterocyclic Carbene Complexes

Human colon cancer is the third most common and the fourth most frequent cause of cancer deaths worldwide [1]. The marketing chemotherapeutic drugs for the treatment of colon related cancer are Carboplatin, Levamisole, Miltefosine, Semustine and Floxuridine (Chart 1). The chart displays that most of the drugs are organic compounds. Among these, only Carboplatin is a metal based anticancer drug that has been recently approved to treat human colon cancer.


Introduction
Human colon cancer is the third most common and the fourth most frequent cause of cancer deaths worldwide [1]. The marketing chemotherapeutic drugs for the treatment of colon related cancer are Carboplatin, Levamisole, Miltefosine, Semustine and Floxuridine (Chart 1). The chart displays that most of the drugs are organic compounds. Among these, only Carboplatin is a metal based anticancer drug that has been recently approved to treat human colon cancer.
The incorporation of Carboplatin against cancer has recently attracted researchers to further explore the area of metal based anticancer drugs [2,3]. Except transition metals, the organometallic compounds based on coinage metals (Ag, Au, Cu) have now attracted the researchers for medicinal applications [4,5]. Recently, Au(I)-NHC complexes have been tested against HCT 116 cell line (Chart 2), showed significant in vitro anticancer results [6]. However, according to the published studies, Ag(I)-N-Heterocyclic Carbene (NHC) complexes have been rarely studied against this specific cancer. The current study is an effort in this area.
Human colorectal tumor (HCT 116) cell line was purchased from American type culture collection (Rockvill, MD, USA). HCT 116 cell line has been derived from colonic epithelial carcinoma. The cells were maintained in RPMI 1640 containing 10% HIFBS and 1% PS. Cells were cultured in 5% CO 2 -humidifed atmosphere at 37°C. Preparation of cell culture and MTT Assay was performed according to the reported procedure [15,24].

Results and Discussion
Synthesis: The reaction of two equivalents of N-alkylated benzimidazole with 1,4-bis(bromomethylene)benzene in 1,4-dioxane at 100°C for 24 h afforded the para-xylyl linked bis-benzimidazolium salts (III-IV) with good yields. The salts (III-IV) appeared as white precipitates in 1,4-dioxane, filtered and washed with fresh 1,4-dioxane (3×5 ml). The precipitates so obtained were dried at ambient temperature for 24 h. This is in accordance with the previously reported para-xylyl linked bis-benzimidazolium salts having different terminal N-substitutions [14,15]. The salts so obtained were found to be soluble in polar solvents like methanol, ethanol, DMSO, DMF but remained insoluble in non-polar solvents like diethyl ether, dichloromethane and chloroform. Also, the reported salts were found insoluble in water. However, on changing counter ions from halide to hexaflourophosphate (PF 6 ), the solubility of salts affected. For example, with PF6 as counter anions the salts became insoluble in methanol, ethanol but soluble in acetonitrile. However, the salts remained soluble in DMSO and DMF with both types of anions. The change of counter anions changes the polarity of compound. With the halide as counter anion polarity of these salts increased.
Synthesis of dinuclear Ag(I)-NHC (V-VI) complexes using bisbenzimidazolium halides (III-IV) as starting material is of significance importance, because using this method pure form of desired complexes can be obtained with ease. Scheme 1 shows three simple steps for the synthesis, starting from N-alkylated benzimidazoles to PF 6 salts of dinuclear Ag(I)-NHC complexes. As a preliminary confirmation of successful synthesis, it was notice that the melting points of

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Ag(I)-NHC complexes were significantly higher than compared to respective ligands. This is in accordance with the previous the reports [14,15,26,27].

FT-IR spectra of the compounds:
The synthesized bisbenzimidazolium salts do not have many functional groups to be distinguished by FT-IR spectroscopic technique but it is possible to Step-I Step II

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compare the spectral features of ligands (III-IV) and respective Ag(I)-NHC (IV-V) complexes. Some noticeable changes in specific regions could be observed as primary indicators of a successful synthesis [15].
The modes of the C sp3 -H stretching vibrational bands, both in alkyl benzimidazoles (I-II) and bis-benzimidazolium salts (III-IV), appeared at around 2850 to 3000 cm -1 (C-H aliph ). This range of vibrations is due to the presence of C-H (sp 3 -s) stretching of alkyl chains and methylene (N-CH 2 -Ar) groups. This can be further classified as the vibrational bands in the range 2840-2855 cm -1 appeared for CH 3 stretchings, 2920-2925 cm -1 for CH2 anti-symmetric stretchings, and 2955-2960 cm -1 for CH 2 symmetric stretchings. A vivid and intense band was observed in the range 1350 to 1500 cm -1 assigned to the stretching vibrations of benzimidazole ring due to the presence of -HC=N-module [15,28]. The ring vibrations, other than -HC=N, are intense bands at around 1050 and 1220 cm -1 .
Some interesting FT-IR features to confirm a successful synthesis of Ag(I)-NHC complexes were observed [15]. It was observed that bonding of NHC carbon with silver ion strengthened vibrations in the range 1350 to 1500 cm -1 and a typical "four fingers (f.fs)" pattern appeared for the synthesized Ag(I)-NHC complexes [13,14,24,29]. This region is specific for -C=N (Carom-Nbenzimi) stretching and CH 2 bending vibrations. The contemplated "f.fs" patterns were entirely different than all the respective vibrations in azolium salts and were easily discernible. This information also remained a primary confirmation for the successful syntheses of desired Ag(I)-NHC complexes.
FT-NMR spectra of the compounds: FT-NMR spectra of all the compounds were scrutinized either in DMSO-d 6 or acetonitrile-d 3 over the scan range 0 to 11 δ ppm for 1 H NMR and 0 to 200 δ ppm for 13 C NMR studies. 1 H NMR spectra of the salts (III-IV) evidenced a sharp singlet around 10 ± 0.3 δ ppm assigned to the benzimidazolium ring (NCHN) acidic proton. These signals are in accordance with previous reports [30,31]. Synthesis of Ag(I)-NHC complexes was indicated by the recede from view of this acidic proton peak and notice of observable changes in aromatic (Ar-H) peaks (Supplementary I). The methylene (N-CH 2 -Ar) group, which connects para-xylyl unit with benzimidazolium units, exhibited a sharp singlet at 5.7 δ ppm. Furthermore, the resonance of N-substituted alkyl chain protons appeared in the range 0.8-4.5 δ ppm. Similarly, the structural features of the salts were confirmed by the 13 C NMR data. The spectra of all the salts displayed a distinguished peak in the most down field region at 142 δ ppm assigned to the benzimidazole ring carbon (NCHN). The signals for methylene (N-CH 2 -Ar) carbon appeared at 49.2 δ ppm and for alkyl chain in the range 13.7-46.8 δ ppm [15,24].
After complexation with Ag ions, usually two doublets appear at about 188 δ ppm with Ag-C coupling constants ca. 206 ± 2 Hz and 184 ± 2 Hz [9,14,25,32]. These doubletes have been mostly observed in dimeric complexes of structure [L 2 Ag 2 ] 2+ due to carbene carbon bonding to C-Ag 107 and C-Ag 109 , respectively [15,33]. In some cases, these doublets do not appear. In the current research, this happened for both complexes (see 13 C NMR of VI, Supplementary II). This light be due to bulky N-alkyl substitutions where the NMR signals for Ag-C coupling could not be generated for the analyzed quantity. However, resonances of aromatic carbons, in ligands (III-IV) ranging ≈113-135 δ ppm were observed in the range ≈112-137 δ ppm in respective dinuclear complexes (V-VI) with noticeable splitting. Also, the C-2 carbon (NCN) disappeared in respective complexes indicating its possible downfield movement. Furthermore, the resonances due to N-C-Ar carbon and alkyl chain carbons were observed in the chemical shift regions 51 and 13-49 δ ppm, respectively. These signals are 2-3 δ ppm downfield compared to respective ligands. All these observations indicated the formation of desired complexes (IV-VI).

In vitro anticancer activity: Effect of benzimidazolium salts (ligands)
and respective silver(I)-NHC complexes on proliferation of HCT 116.
In this work, antiproliferative potential of NHC pro-ligands and respective dinuclear Ag(I)-NHC complexes was evaluated against HCT 116 cancerous cells. The results have been compiled as mean percentage inhibition of cell proliferation (±S.D). The compounds (III-VI) were tested against HCT 116, exhibited a strong antiproliferative activity with IC 50 values in the range 0.03-65.9 µg/ml ( Table 1). The results were more pronounced than the standard drug 5-fluorouracil (IC 50 =5.9 µM). Recently, we reported anticancer activity of meta-& para-xylyl linked bis-benzimidazolium salts and respective dinuclear Ag(I)-NHC complexes. We found that in general the complexes are more active against human colon cancer compared to respective ligands. The current results support our previous findings. Furthermore, Benzimidazole, the starting material, was also tested against HCT 116 cell lines and was found almost inactive (IC 50 =>200 μM). Hence the bis-benzimidazolium salts (IC 50 =0.3-65.9 μM) were found to be active against human colon cancer compared to benzimidazole moiety whereas the Ag(I)-NHC complexes of these bis-benzimidazolium salts were further found to be relatively active (IC 50 =0.03-5.6 μM).
The Figure 1 illustrates the anti-proliferative effect of the test compounds. The graphs clearly show that, all the compounds inhibited the proliferation of HCT 116 cells in a dose dependent manner. Figure  2 shows the cell images of cell line with 48 hours control without and with drugs.  the affected cellular morphology of the treated cells. The intercellular junctions were disconnected and cells appeared less intacted. C) Photomicrograph of HCT 116 cells injected with the complex VI showed a strongest cytotoxic effect of the complex (IC 50 =0.03 µM). It can be visualized clearly that the compound affected the proliferation of all most all the cells of the group which rendered the cells to lose their pseudopodial like extensions. D) Treatment with the ligand III showed a moderate cytotoxicity (IC 50 =65.9 µM) than compared to the other compounds tested. E) HCT 116 cells treated with the complex V demonstrated a significant cytotoxicity (IC 50 =5.6 µM) as the population of cells decreased substantially within the 48 hours of treatment. F) Cells treated with the Standard drug 5-fluorouracil (IC 50 =5.9 µM) showed decreased workability.

Conclusions
In conclusion, two new para-xylyl linked bis-benzimidazolium salts and their Ag(I)-NHC complexes were synthesized. The compounds were characterized tested for their possible cytotoxicity on human colorectal cancer (HCT 116). All tested compounds showed dosedependent cytotoxicity against human colon cancer. The IC 50 values for HCT 116 cells were ranged between 0.03 and 65.9 μM. Some of the tested compounds showed IC 50 values significantly better than commercially available drug (5-FU, IC 50 =5.9 μM). Among all, the complex VI was found to be the most active against human cancer cell line (IC 50 =0.03 μM). Based on the results it can be concluded that incorporation of silver ions with organic moieties enhances their anticancer potential.