Triphenyltin(IV) benzoates with diazenyl/imino scaffold exhibiting remarkable apoptosis mediated by reactive oxygen species

Highlights

• Triphenyltin(IV) benzoates (1–6) with diazo- and imino- scaffolds were synthesized.

• Compounds show cytotoxicity: MDAMB231 and HeLa cells (IC₅₀ ~ 1 μM (1 and 3), ~ 0.8 μM (5).

• The cytotoxic performance was determined by state-of-the art-methods.

• Antitumor activity was achieved by intracellular reactive oxygen species generation.

• Compounds cause delay in cell cycle by inhibiting cells at G₂/M phase.

Abstract

The cytotoxic potency of a series of triphenyltin(IV) compounds of general composition [Ph₃Sn(Lⁿ)] (1–6) has been probed in vitro employing MDA-MB-231 (human breast cancer) and HeLa (human cervical cancer) cell lines, where Lⁿ = L^1–3; isomeric 2/3/4-{(E)-2-[4-(dimethylamino)phenyl]diazenyl}benzoates and L^4–6 are their corresponding isoelectronic imino analogues 2/3/4-[(E)-{[4-(dimethylamino)phenyl]methylidene}amino]benzoates. Compounds 1–6 have been characterized by elemental analysis and their spectroscopic properties were studied using IR and NMR (¹H, ¹³C, ¹¹⁹Sn) techniques. The molecular structures of a pro-ligand 2-[(E)-{[4-(dimethylamino)phenyl]methylidene}amino]benzoic acid (HL⁴) and two representative molecules, Ph₃Sn(L²) 2 and Ph₃Sn(L⁵) 5, have been determined by X-ray crystallography. Structural analyses of 2 and 5 revealed distorted tetrahedral geometries within C₃O donor sets owing to monodentate modes of coordination of the respective carboxylate ligands, close intramolecular Sn…O(carbonyl) interactions notwithstanding. Cytotoxic studies in vitro in MDA-MB-231 and HeLa cell lines revealed high activity, in sub-micromolar range, for all investigated compounds. Among these, 1 and 3 exhibited potent cytotoxicity most effectively towards MDA-MB-231 cells with a IC₅₀ value of 1.19 and 1.44 μM, respectively, whereas 5 showed remarkable activity towards HeLa cells with a IC₅₀ value of 0.88 μM, yet the series of compounds had minimal cytotoxic effect on normal HEK 293 (human embryonic kidney) cell line. The underlying investigation suggested that the compounds exert potent antitumor effect by elevating intracellular reactive oxygen species generation and cause delay in cell cycle by inhibiting cells at G₂/M phase. The results presented herein suggest further development of this class of triphenyltin(IV) compounds-based drugs as potential anti-cancer therapies should be pursued.

Introduction

Cisplatin, cis-diammine-dichloroplatinum(II) (CDDP), a square planar Pt(II) complex remains in the frontline of inorganic chemotherapeutic agents for the treatment for cancer [1], [2], [3], [4]. The clinical effectiveness of CDDP is limited by considerable side effects and the emergence of drug resistance. Consequently, a number of second- and third-generation CDDP analogues including carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin and satraplatin were developed for use in the treatment of various cancers. These advances have spurred a surge of investigations to identify new inorganic agents (non-platinum metals) for use in chemotherapy with improved specificity and decreased toxic side effects. Accordingly, non-platinum metallodrugs such as Ru, Cu, Au, Pd, Fe, Co, Ti, Ga, Ni, Rh, Ir, Sn, Os, Zn, V, Ag, Re, Mo and lanthanide complexes have emerged and been shown to exhibit comparable or better cytotoxic properties accompanied by different specificities towards cancer cells, and by a more favourable pharmacological and toxicological profiles [5], [6]. Review articles describing the application of transition metal complexes as anti-cancer agents are available in the literatures [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23].

On the other hand, organotin(IV) compounds have displayed diverse medicinal applications such as anti-viral, anti-microbial, anti-parasitic, anti-hypertensive, anti-hyperbilirubinemia and anti-cancer activities, apart from their standard uses as biocides, catalyzers and stabilizers [24], [25], [26], [27], [28], [29]. Compared to standard drugs, the majority of organotin(IV) carboxylates discussed in the literature have shown superior anticancer activity against various cell lines. Additionally, some of them have demonstrated distinct anticancer activity even against CDDP-resistive cancer cells. Organotin(IV) compounds with pronounced medicinal properties also exhibit drawbacks such as reproductive toxicity, neurotoxicity and other toxic effects apart from poor water solubility; however, these can be circumvented by a rational design of the structures of the compounds, but the vital point still remains the activity. In practice, organotin(IV) compounds are dissolved in DMSO and diluted with test medium prior to in vitro testing. Nevertheless, the limited solubility needs to be further enhanced in a way comparable to CDDP, which also shows limited water solubility. It is therefore important to find more effective and safer organotin(IV) compounds for therapeutic use by designing and synthesizing new compounds and to find suitable means of their delivery to the bio-targets. In this pursuit, recently a novel biocompatible strategy of drug delivery employing nanostructured silica-based material loaded with triphenyltin(IV) compound was used. This resulted in an increase in efficacy of the drug and the pattern of action leads to the non-aggressive suppression of melanoma tumor growth with non-recognizable toxicity towards normal tissue [30]. While the potency of a drug is a very important consideration, drug selectivity towards cancer cells is key to ensuring both safety and effectiveness [31]. Additionally, the mechanism of action of organotin(IV) compounds in achieving cell death remains uncertain, and hence additional work is essential to identify the actual apoptotic or necrotic pathways.

In view of the above, careful functionalization of a series of organotin compounds was undertaken. In this endeavour, six pro-ligands (i) isomeric 2-, 3- and 4-{(E)-2-[4-(dimethylamino)phenyl]diazenyl}benzoic acids and their isoelectronic imino counterparts, i.e. (ii) 2-, 3- and 4-[(E)-{[4-(dimethylamino)phenyl]methylidene}amino]benzoic acid, have been designed and their triphenyltin(IV) esters prepared (Fig. 1). In the first three pro-ligands (HL¹−HL³), the aryl moieties are linked by a diazenyl skeleton while in the others (HL^4–6) by an imino group, substitution patterns which offer flexibility in the molecules. The presence of the carboxylate group is vital for aqueous solubility and results in increased cellular accumulation [32], [33]. The incorporation of the triphenyltin(IV) moiety in the molecule offers much higher activities when compared with titanocene derivatives and CDDP [34], [35], [36]. Thus, it is expected that the modification of the carboxylate ligands in the current series by incorporating SnPh₃ can optimize the cytotoxicity [37], [38] while varying the location of the triphenyltin ester can modulate the activity at a specific target. In this context, cytotoxic properties of a series of [Ph₃Sn(Lⁿ)] complexes [Lⁿ = L^1–3 (1–3) and L^4–6 (4–6)], were determined towards the MDA-MB-231 (human breast cancer) and HeLa (human cervical cancer) cell lines. To realize the mechanistic/mode of cell death objective, an additional series of experiments were conducted: (i) the cytotoxic activity was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (ii) the ability of the test compounds to generate ROS (reactive oxygen species) in the cells was examined using DCFH-DA (dichlorodihydro-fluorescein diacetate) dye (iii) apoptosis or mode of cell death was detected by Hoechst 33342/PI (propidium iodide) and annexin V-PI assay and (iv) cell cycle arrest by the compounds were examined by FACS (fluorescence-activated cell sorting) studies. The biological results indicated that the triphenyltin(IV) compounds show potent cytotoxicity which can be modulated either by varying the positions of the triphenyltin(IV) ester across a series or switching from triphenyltin(IV)- diazobenzoato- to -iminobenzoato- ligands. The present studies demonstrated that the triphenyltin(IV) compounds exert their cytotoxic effect by elevating intracellular ROS generation as discussed below.