Dalton Transactions View Article Online PAPER View Journal | View Issue Ruthenium(II) p-cymene complexes of a benzimidazole-based ligand capable of VEGFR2 Cite this: Dalton Trans., 2017, 46, 8539 inhibition: hydrolysis, reactivity and cytotoxicity studies† Sudipta Bhattacharyya, Kallol Purkait and Arindam Mukherjee * The design of Ru or other metal-based anticancer agents may achieve better and faster optimization if the ligands used are also designed to have standalone functions. In this scenario, even after dissociation from the metal complex under adverse conditions, the ligand would have anti-cancer properties. In our work, we have generated a bispyrazole-containing benzimidazole ligand with potency against vascular endothelial growth factor receptor 2 (VEGFR2), which is known to have roles in vasculogenesis/angio- 6 genesis. This ligand was used to obtain ternary Ru(II) p-cymene complexes with the formulations [(η -p- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. cymene)Ru(HL)(Cl)](Cl) (1), [(η6-p-cymene)Ru(HL)(Br)](Br) (2) and [(η6-p-cymene)Ru(HL)(I)](I) (3). 1H NMR data supports that hydrolysis of the complex is governed by halide substitution, and the extent of hydrolysis 3 −1 followed the trend 3 > 1 > 2. All the complexes have low affinity towards DNA bases (average Kb ∼ 10 M for CT DNA); however, all the complexes are cytotoxic in nature, with IC50 values less than 15 μM. The pres- ence of excess glutathione (GSH) liberates HL from the complexes in solution. The ability of the Ru complex to impair mitochondrial function and reduce the cellular GSH pool is thought to be the reason that it retains activity in the presence of GSH despite the ability of GSH to degrade the complexes. The chloride analogue 1 shows the best in vitro cytotoxicity against a prostate cancer cell line (LNCaP), with an Received 15th March 2017, IC50 of 6.4 μM. The complexes show anti-proliferative activity by the mitochondria-mediated intrinsic apop- This article is licensed under a Accepted 3rd June 2017 totic pathway. Docking studies showed that HL has high affinity towards vascular endothelial growth factor DOI: 10.1039/c7dt00938k receptor 2 (VEGFR2). The complexes show anti-metastatic activity (in vitro) at almost non-toxic dosages, rsc.li/dalton and the effect is sustained even 48 h after removal of the complexes from the culture media. Open Access Article. Published on 05 June 2017. Downloaded 6/12/2019 7:07:05 AM. – Introduction ference of Ru with Fe metabolism,2 4 (ii) availability of different oxidation states, (iii) slow kinetics of various RuII/III RuII complexes are well-investigated anticancer agents whose complexes with different biological targets (viz. DNA, protein, 1 mechanism of action is being probed. Research on Ru(II/III) etc.), and (iv) tuning of reactivity and mechanistic pathways by complexes has disseminated much important information varying the bidentate ligands, arenes and halides. A few Ru(III) regarding the cytotoxicity of these complexes, viz. (i) inter- compounds, [ImH]trans-[RuCl4(Im)(dmso-S)] (NAMI-A, Im = imidazole), [IndH]trans-[RuCl4(Ind)2] (KP1019, Ind = 1H-inda- zole), and [Na]trans-[RuCl4(Ind)2] (NKP1339), have progressed 5–10 Department of Chemical Sciences, Indian Institute of Science Education and to clinical trials. The ruthenium arene PTA (RAPTA) Research Kolkata, Mohanpur Campus, Mohanpur, Nadia-741246, West Bengal, complexes (where PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1] India. E-mail: [email protected] decane) are a potent class of complexes that have been studied † Electronic supplementary information (ESI) available: Experimental details of in detail and exhibit excellent activity against metastasis and interaction with CT DNA, plasmid DNA interaction, cell lines and culture con- 11–13 angiogenesis. Among other Ru(II) organometallic half- ditions, cell viability assay (MTT), determination of intracellular reactive oxygen species (ROS), DNA ladder assay for apoptosis detection, detection of Caspase sandwich ternary complexes, Ru(II)-p-cymene complexes are activation, ruthenium accumulation inside cancer cells by ICP-MS analysis, in targeted by many research groups because of their high cyto- ovo assay, western blotting, selected bond angle and bond distances of HL, 1 toxicity, photoactivity, and resistance to glutathione 1 – and 2 obtained from X-ray crystallography, NMR characterization spectra ( H, (GSH);8,11,14 34 also, these compounds enable the introduction 13C, HMQC), UV-vis and 1H NMR spectra showing solution stability of com- of new bidentate ligands while retaining a labile halide plexes, detailed procedure of biological experiments. CCDC 1507224–1507226. For ESI and crystallographic data in CIF or other electronic format see DOI: attached to the metal centre. In addition, in few cases, chan- 35,36 10.1039/c7dt00938k ging the halide also alters the pathway of action. This journal is © The Royal Society of Chemistry 2017 Dalton Trans.,2017,46, 8539–8554 | 8539 View Article Online Paper Dalton Transactions Thus, the design of a bidentate ligand that would promote of benzimidazole-containing ligands are active anti-cancer – therapeutic effects may be of great benefit for better and agents.28,29,43 48 However, the detailed mechanism of action quicker optimization of such compounds. In addition, even if has been probed for only a few Ru(II) p-cymene complexes – the complex dissociates in the cell under adverse conditions, bearing benzimidazole ligands.28,48 51 The well-characterized the ligand would exhibit the potential to open up multiple complexes (1–3) were studied for their hydrolytic behaviour, – pathways of action against cancer.37 39 From this standpoint, the effects of their halides, and their reactivities in the pres- we investigated bispyrazole-benzimidazole-based ligands to ence and absence of glutathione. The cytotoxicities and the design a ligand that would inhibit vascular endothelial growth pathways of action of the complexes were probed. factor receptor 2 (VEGFR2). Cancer becomes a greater threat when it spreads from its origin to various areas of the body by the lymph system or the Results and discussion bloodstream (metastasis). The spread is followed by the growth and development of blood and lymphatic vessels, Syntheses known as vasculogenesis/angiogenesis. Vascular endothelial The benzimidazole-based ligand (HL) was synthesized using a growth factors (VEGFs) stimulate vasculogenesis/angiogenesis. literature procedure.52 Because o-phenylenediamine is one of VEGFs bind to three types of VEGF receptors (VEGFR1-3) in the synthetic precursors, special care was taken to protect it their tyrosine kinase domains. Among these, inhibition of from photo-degradation. It must be noted that during the last VEGFR2 is a prime target to inhibit angiogenesis; a successful step of ligand synthesis, while extracting the ligand from clinical drug in this regard is axitinib. There are many mole- excess hexane containing toluene, the ligand adhered to the cules in clinical trials, viz. Rumacinib (NCT00627042), surface of the vessel; however, allowing the mixture to stand Foretinib (NCT00725764) and Lucitanib (NCT02109016), with for an hour with stirring allowed the microcrystalline com- the same mechanism. pound to be readily isolated. After that, further washing with ff Creative Commons Attribution-NonCommercial 3.0 Unported Licence. The above idea provided us with the impetus to design Ru(II) hexane followed by vacuum drying a orded ligand that was complexes (Chart 1) with the formulations [(η6-p-cymene) sufficiently pure for the syntheses of the complexes and other Ru(HL)(Cl)](Cl) (1), [(η6-p-cymene)Ru(HL)(Br)](Br) (2) and [(η6- analytical purposes. p-cymene)Ru(HL)(I)](I) (3) using a bispyrazole-benzimidazole The Ru(II) complexes were prepared from HL using different 6 ligand (HL) with potential to inhibit VEGFR2. The design of halide-based Ru(II)(p-cymene) salts prepared from the [(η -p- 18 the benzimidazole-containing bispyrazole ligand (HL)was cymene)2RuCl2]2 precursor (Scheme 2). The reactions were inspired by the known VEGFR2 inhibitor SU5416,40 as demon- performed in dry dichloromethane under reflux conditions strated in Scheme 1. Bis-pyrazole Ru(II) complexes are seldom with ligand and metal precursor in a 1 : 1 mole ratio. The studied for their anticancer activity. Recent studies have shown respective obtained complexes were dried and washed succes- This article is licensed under a their potential against topoisomerase-II41 and demonstrated sively with hexane and diethyl ether to eliminate any excess 42 their cytotoxicity against cancer. Ru(II) p-cymene complexes ligand. The complexes were then re-dissolved in DCM and again precipitated by addition of hexane. The microcrystalline Open Access Article. Published on 05 June 2017. Downloaded 6/12/2019 7:07:05 AM. complexes thus obtained were pure enough for all studies, as per the characterization data. Hence, in the respective com- plexes, the coordinated halide and the counter anion are the same. Characterizations The ligand (HL) and the metal complexes (1–3) were analysed extensively using 1H and 13C NMR experiments (Fig. S1–S11†). The change in chemical environment upon complexation was Chart 1 A general representation of the design of Ru(II) metal complexes. Scheme 1 Representative structure of SU5416 and our ligand (HL). Scheme 2 Synthetic scheme of the ligand and its metal complexes. 8540 | Dalton Trans.,2017,46, 8539–8554 This journal is © The Royal Society of Chemistry 2017 View Article Online Dalton Transactions Paper evident from their chemical shift values (tabulated in Tables in proximity to the bound nitrogen of the benzimidazole ring. S1 and S2† for 1–3). A significant change in the chemical shift The H9 of the aromatic ring is involved in H-bonding with the of the proton signal of the non-coordinated benzimidazole counter halide; thus, it shows halide dependency in its chemi- –NH was found upon complexation because the –NH proton is cal shift. The decrease in the electronegativity of the halide on deshielded due to the coordination of the neighbouring nitro- moving from 1 to 3 resulted in upfield shifts of the p-cymene gen with Ru(II).