Pharmacophore based drug design and synthesis of oxindole bearing hybrid as anticancer agents

Highlights

• Benzimidazole substituted oxindolinone hybrids as VEGFR-2 and EGFR inhibitors.

• The IC₅₀ value of 5b was found 6.81 and 13 nM against VEGFR-2 and EGFR, respectively.

• The cytotoxicity was found as GI₅₀ at 0.9 µM against epidermoid carcinoma cells.

• Mechanistically 5b governs through VEGF/EGF, p53, bcl/bax and MMP9 signalling proteins.

• Comparatively 5b was found more potent and safer than other dual inhibitors.

• The dual inhibition was facilitated due to conformational switch in designed molecules.

Abstract

Dual TK inhibitors have shown significant clinical effects against many tumors, but with unmanageable side effects. Design approach and selectivity of these inhibitors plays substantial role in their potency and side-effects. Understanding the homology of binding sites in targeted receptors, and involvement of signaling proteins after the inhibition might help in producing less toxic but effective inhibitors. Herein, we designed benzylideneindolon-2-one derivatives based on homology modeling in binding sites of VEGFR-2 and EGFR receptors as dual- inhibitor potent anticancer compounds with high selectivity. The benzylideneindolon-2-one derivatives were found to possess conformational switch in form of oxindole, substituted at 2-benzimidazole. Within synthesized compounds, 5b was found most active in in-vitro enzyme inhibition assay against VEGFR-2 and EGFR with highest IC₅₀ value of 6.81 ± 2.55 and 13.04 ± 4.07 nM, respectively. Interestingly, cytotoxicity studies revealed selective toxicity of compound 5b against proliferation of A-431 cell lines (over expressed VEGFR-2 and EGFR) with GI₅₀ value of 0.9 ± 0.66 µM. However, the compounds showed mild to moderate activity in all other cancer cell line in the range of 0.2–100 μM. Further mode of action studies by flow cytometry and western blot on A-431 indicated that they work via apoptosis at S- phase following Bcl/Bax pathway, and cell migration via MMP9. 5b not only suppressed tumor growth but also improved vandetanib associated with weight loss toxicity. Moreover, 5b was found safer than sunitinib and erlotinib with LD₅₀ of 500 mg/kg body weight. These results propose 5b as potential anti-tumor drug with safer profile of conventional inhibitors of VEGFR-2 and EGFR for solid tumors.

Introduction

Cancer is amongst prominent causes of mortality and morbidity across the world. A large number of cancers are marked by significant up-regulation of RTKs (Receptor tyrosine kinases) and are commonly used as prognostic markers. These cell surface receptors are utilized by a broad array of polypeptide growth factors, cytokines and hormones [1]. It has been observed that VEGFR-2 (Vascular endothelial growth factor receptor-2) and EGFR (Endothelial growth factor receptor) are two main RTKs which are over-expressed in various cancers like prostate, pancreatic, stomach, glial and ovarian cancers etc [2], [3]. For several decades, these two receptors have been studied as a valid target to design small molecule inhibitors as anti-cancer therapy [4], [5]. Mechanistically, VEGFR-2 (expressed predominantly on endothelial cells of vessels) up-regulates and dimerizes with the over-expressed vascular endothelial growth factor (VEGFA₁₆₅), secreted from neoplastic and inflammatory cells [6], [7], [8]. VEGFA₁₆₅ is reported to migrate and enhance neovascularization at distant tumors by tumor-host communication [9]. Similarly, an over-expressive EGFR when gets activated by endothelial growth factor (EGF), facilitates cell proliferation and differentiation in tumors [5]. However, individual inhibition of any of them does not furnish desired anti-cancer effect; rather they induce recurrence of malignant tumors upon discontinuation, as suggested in some clinical trials [10], [11], [12]. One more correlation between these receptors has been reported which indicates stimulation of VEGFA₁₆₅ leads to increase in the EGFR expression, however after attaining peak concentration, VEGFA₁₆₅ had a sharp fall due to negative feedback mechanism in almost 5–6 min [11], [13]. This signifies that EGFR and VEGFR-mediated pathways have common downstream signaling proteins that might contribute to their reactivation even after inhibition [13], [14], [15]. Numerous dual tyrosine kinase inhibitors (TKIs), for VEGFR-2 and EGFR, like Vandetanib, axitinib, AEE788, sorafenib, ZD6474 etc, are successfully approved by FDA [16], [17], but suffers repeated dose toxicity upon withdrawal and tumor recurrence [18].

Therefore, in order to design better therapeutics, we have reported novel molecules as dual inhibitors of EGFR and VEGFR-2 and investigated the downstream signaling pathways. Here, we have designed 2-benzimidazo substituted oxindolinone hybrid molecules (‘DFG-out’, Type-II inhibitors) using 3D-QSAR and selectivity profiling was done by authenticated simulation technique, MD-based MM/GBSA (for phosphorylation and flexibility detection). In the work undertaken, the most active compound was found to be potent in enzyme inhibition assay it was also found selective against five cancer cell lines (A431, PC-3, MCF-7, A459, HepG2) without affecting normal cell lines (PNT2, HEK293T), through apoptosis mechanism (V-FITC/Propidium iodide staining). The relationship study was also done between MAPK/P38 pathway and dual inhibition of VEGFR-2 and EGFR in overexpressed A431 cancer cells by western blot technique. Finally, we determined the LD₅₀ (500 mg/Kg) as a safety profile and in vivo therapeutic anticancer activity of most active compound 5b in rat model. The comparative study between 5b and clinically used RTK inhibitors proved that the newly synthesized compound (5b) is more effective with negligible toxicity.