Azilsartan and its Zn(II) complex. Synthesis, anticancer mechanisms of action and binding to bovine serum albumin

Highlights

• Study of a Zn complex with the designed antihypertensive drug, azilsartan

• Cytotoxicity against human lung cancer A549 cell lines

• No interference on human lung fibroblast MRC5 cell line viability

• Oxidative stress mediated apoptosis via intrinsic pathway evaluated by Western blot.

• Azilsartan and the complex interacted with bovine serum albumin.

Abstract

Azilsartan is the eighth approved member of angiotensin II receptor blockers for hypertension treatment. Considering that some drugs have additional effects when administered, we studied its effects and mechanisms of action on a human lung cancer cell line A549. We have also modified the structure of the drug by complexation with Zn(II) cation and assayed the anticancer effect. The crystal structure of the new binuclear Zn(II) complex, for short [Zn₂(azil)₂(H₂O)₄]·2H₂O (ZnAzil), was determined by X-ray diffraction methods. The zinc ions are bridged by azilsartan ligands through their carboxylate oxygen and oxadiazol nitrogen atoms. The compounds were examined for their cytotoxic effects against human lung fibroblast (MRC5) and human lung cancer (A549) cell lines. Azilsartan displayed low cytotoxic effects at 150 μM concentrations in A549 human lung cancer cells but the higher effect measured for the Zn complex suggested that this compound may act as an anticancer agent. An apoptotic oxidative stress mechanism of action via the mitochondrial-dependent intrinsic pathway has been determined. Besides, the compounds exerted weak cytotoxic effects in the normal lung related cell line MRC5. Binding constants of the complex formed between each compound and bovine serum albumin (BSA) are in the intermediate range, hence suggesting that azilsartan and ZnAzil could be bonded and transported by BSA.

Introduction

Hypertension affects approximately one in three adults in the world and is a major risk factor for cardiovascular disease. One of the eight classes of medications used in the treatment of hypertension are the angiotensin II (Ang II) receptor blockers (ARBs). Azilsartan is the eighth ARB to be approved for the treatment of hypertension and received FDA approval in February 2011 (Zaiken and Cheng, 2011). Azilsartan medoxomil (postassium salt) is the prodrug that undergoes rapid hydrolysis to liberate azilsartan, the active ingredient (Ding et al., 2013). Azilsartan is structurally similar to candesartan, another other antihypertensive pharmaceutical, in which the tetrazole ring has been replaced by a 5-oxo-1,2,4-oxadiazole (Fig. 1). This group is related to the stronger molecular mode of binding to the angiotensin II type 1 receptor (AT1R) and slow dissociation from the receptor, which gave the drug the superior efficacy to lower blood pressure (Tamura et al., 2013). It has been reported that the AT1R has been found in several kind of cancer cells and that Ang II would be involved in tumor angiogenesis (Goldfarb et al., 1994). In fact, AT1R have been found in the human lung cancer cell line A549 (Batra et al., 1994). Ang II can induce neovascularization in experimental systems due to the expression of different growth factors such as angiopoietin 2, vascular endothelial factor, and its receptor (Escobar et al., 2004) and this is an essential process for the growth and metastasis of solid cancerous tumors (Weidner et al., 1991). Besides, ATR1 is overexpressed in ovary, bladder, lung, and breast cancers (Oh et al., 2015) and the inhibition of angiogenesis, growth, and metastasis of tumors is highly dependent on AT1R blockade (Mamoru et al., 2002).

Metal ions play a crucial role in biological systems. Coordination complexes used in medicine with a wide variety of geometries and reactivities are currently an area of intense research in bioinorganic chemistry. It is well known that the biometal-drug complexation modulates the pharmacological actions of therapeutic ligands due to a synergistic effect. (Gaberc-Porekar et al., 2011; Ndagi et al., 2017; Frezza et al., 2010). In particular, Zn(II), an essential trace element constituent of proteins, hormones, peptides and receptors is involved in many important physiological processes, including metabolism, signaling, proliferation, gene expression and apoptosis. Zn(II) ion also has antioxidant properties and it has been shown that its deprivation in diet increases the cellular susceptibility to oxidative stress. For this reason, certain diseases are associated with Zn(II) deficiency, including hypertension (Jurowski et al., 2014). Besides, zinc oxide has been used in the form of topical creams in the treatment of various skin conditions (e.g. diaper rash) (Šikić Pogačar et al., 2017; Bookout et al., 2004). Likewise, its deficiency is associated with cancer. Supplementation with this biometal in the diet has managed to lower tumor risk since it reduces oxidative stress and improves immune function (Ho, 2004). Besides, it has been reported that exogenous zinc stress produces toxic effects in cancer cell lines. Lung cancer A549 cells response to zinc stress and the MTT assay and flow cytometric analysis showed that the ZnSO₄ treatment led to a bi-phasic variation in viability and a slight fluctuation in the apoptosis of A549 cells (Yuan et al., 2012).

In addition, the in vitro cell system for the appropriate cell types is a good laboratory model to investigate the biochemistry and possible mechanisms of action employed by the metal complex to exert its actions. One major challenge in anticancer therapy is to increase the selectivity of current treatments toward cancer cells. The anticancer agents must exert minimal adverse effects on normal tissues with maximal capacity to kill tumor cells and/or inhibit tumor growth. On the other side, it is well known that serum albumin is a transporter for different types of endogenous and exogenous compounds such as drugs, fatty acids and dyes in the bloodstream. The binding of this protein to small molecules such as drugs can significantly affect their absorption, distribution, metabolism and toxicity. It is also well established that numerous small-molecule compounds are capable of binding various target proteins and exhibit their antitumor activity (Zhang et al., 2016) improving their effects in some cases.

Hence, it could be interesting to design a metal coordination complex by the combination of azilsartan with Zn(II) ions, and examine its effectiveness as a potential anticancer agent. Herein, we report the syntheses and characterization of tetraaquobis(μ-2-ethoxy-3-[[4-[2-(5-oxo-1,2,4-oxadiazol-3-yl)phenyl]phenyl]methyl] benzimidazole-4-carboxylate-N-,O-) dizinc(II) dihydrate and its in vitro cytotoxicity and the determination of the possible mechanisms of action against some cancer cells. With comparative purposes, the biological effects of azilsartan were also evaluated. We find that the metal complex showed anticancer action on the A549 cancer cell line generating a synergistic effect in comparison with azilsartan. Our studies showed that the possible mechanisms of action involved reactive oxygen species (ROS) generation and cellular antioxidant status depletion. Additionally, to evaluate the possibility that both compounds could be transported in the bloodstream, the binding affinity of azilsartan and ZnAzil with the serum albumin has been studied.