Research paper
A novel class of ethacrynic acid derivatives as promising drug-like potent generation of anticancer agents with established mechanism of action

https://doi.org/10.1016/j.ejmech.2016.05.063Get rights and content

Highlights

  • New ethacrynic acid derivatives were found as efficient antiproliferative agents.

  • Antiproliferative activities increased by substitution of the carboxylic function.

  • Complex mechanism of action was pointing out.

Abstract

The well-known diuretic Ethacrynic acid (EA, Edecrin), showing low anti-proliferative activities, was chemically modified at different positions. The new EA derivatives have been tested in vitro in anti-proliferative assays on both tumor KB (epidermal carcinoma) and leukemia HL60 (promyelocytic) cells suitable targets for anticancer activity. Reduction of the α-β double bond of EA completely abolished anti-cancer activities, whereas introduction of either 2-(4-substituted phenyl)ethanamine (series A) or 4-(4-substituted phenyl)piperazine (series B) moieties generated compounds showing moderate to strong anti-proliferative activities against human cancer cell lines. Several substitutions on the phenyl of these two moieties are tolerated. The mechanism of action of the EA derivatives prepared in this study is more complex than the inhibition of glutathione S-transferase π ascribed as unique effect to EA and might help to overcome tumor resistances.

Introduction

Cancer is the second leading cause of death in North America and Europe [1]. Although an armada of anticancer agents has received FDA approval over the past two decades [2], the inefficiency of their discovery and development is no longer sustainable and the pipeline of new cancer agents is slim. Today, the outcome of patients with advanced metastasis, for instance regarding lung, colorectal, prostate and breast cancers, remains very poor [3]. The development of anticancer agents for several decades was based on the identification of active compounds, with cytostatic or cytotoxic activity on tumor cell lines, but with many side effects. Consequently, new anticancer agents within new chemical families are urgently needed to allow an extension of this cancer-fighting armada. In addition to the discovery and development of new anticancer agents, an improved understanding of each cancer patient’s needs is also essential to enhance the ability of standard treatments to kill cancer cells without significantly affecting normal cells [4].

One important consideration in the development of cancer treatment regimens is resistance against anticancer drugs, which remains a serious obstacle [5]. Indeed, microsomal glutathione transferase 1 (mGST1) and glutathione transferase π (GST π) are often overexpressed in tumors and confer resistance against a number of cytostatic drugs, such as cisplatin and doxorubicin (DOX) [6]. To address this point, the diuretic drug ethacrynic acid (EA, Edecrin) 1, an inhibitor of π class glutathione S-transferase, has been tested against multiple myeloma, and as adjuvant in clinical trials [7]. On the basis of these considerations, Dyson et al. [8] and Osella et al. [9] synthesized and characterized the bifunctional EA-Pt(IV) complex 2 (ethacraplatin) and the EA–Pt(II) complex 2′ (cis-diaminobis(ethacrynato)platinum(II) (Fig. 1), both of them able to release a cytotoxic platinum (II) agent (inducing apoptosis of cancer cell) and two EA moieties (inhibiting glutathione S-transferase (GST) and overcoming drug resistance) via hydrolysis or reduction, respectively. Ethacraplatin 2 resulted to be an excellent inhibitor of GST in several tumor cell lines such as A549 lung adenocarcinoma epithelial cell line, MCF7 and T47D breast tumor cell lines and HT29 colon cancer cell line albeit demonstrating a moderate anti-proliferative activity. Unfortunately, the bifunctional conjugates 2 and 2′ did not offer any advantage over cisplatin for the treatment of malignant pleural mesothelioma (MPM) cells, since the increase of intracellular glutathione (GSH) counteracts the modest inhibition of GST [10]. Recently, Yang et al. constructed biodegradable nanoparticles to codeliver EA and dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt) which is a precursor of oxaliplatin as a promising approach to overcome the drug resistance in cancer chemotherapy [11]. In vitro studies showed that these hybrid nanoparticles could release both EA and DACHPt enhancing of up to ∼5 fold of the anticancer efficacy versus DACHPt alone. Interestingly, in vivo studies showed better anticancer activities than the simple combination of EA and DACHPt.

The development of new and potent anticancer agents based on EA will undoubtedly offer new opportunities to tackle cancer and some reports in this direction appeared recently reviewed by us and others [12], [13]. In order to improve the poor anti-proliferative activity of EA [14], we designed original EA derivatives based on modifications of the EA core structure and tested the resulting derivatives for their capacity to inhibit cell growth in vitro. For this initial screening, we tested the chemicals on three cell lines: two actively dividing cell lines derived from human cancer, human KB carcinoma and human leukemia HL60, and the non-dividing quiescent endothelial progenitor cells (EPC) from Cyprinus carpio. Cell number and cellular NADH content evaluated after 72 h chemical exposure provided relevant information concerning viability. The two different tumor cell lines have been selected as representative ‘models’ of the two common types of cancer: solid tumor cancers (e.g. breast, lung, colon, etc.) and blood-based cancers (e.g. leukemia, lymphoma, myeloma etc.). According to a recent analysis, the percentage of new patients annually diagnosed for solid tumor cancers and blood-based cancers is 34% and 9%, respectively, whereas the percentage of cancer related deaths is 43% and 9%, respectively [15].

Herein, we report the design, preparation and anti-proliferative activity of original EA derivatives active upon cancer cell lines and exemplifying a promising class of potent anticancer agents. The preparation of EA derivatives and their synthetic pathways are described in Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5, Scheme 6 and their mode of action was proposed. The mechanism of action of the EA derivatives prepared in this study is more complex that the potent inhibition of π class glutathione S-transferase attributed to EA and could potentially overcome tumor resistances.

Section snippets

Chemistry

The initial strategic design of these EA derivatives can be divided into two parts as shown in Fig. 2: part 1: modification of the 3-methylenepentan-2-one unit; and part 2: modification of the carboxylic acid unit. In this study, we decided not to modify the core of the EA.

As a first strategic attempt to identify a strongly toxic EA derivative and to investigate SAR, we targeted the α,β unsaturated carbonyl moiety of EA (part 1) as shown in Scheme 1.

The synthesis of compounds 3 and 4 was

Biological evaluation and discussion

First, based on a MTS (multiple target screening) assay, we examined the in vitro anti-proliferative effects of the 26 EA derivatives prepared on both KB (epidermal carcinoma) and HL60 (promyelocytic) cells. In addition, we tested these compounds against the non-dividing quiescent endothelial progenitor cells (EPC) to figure a selectivity index (SI) and might suggest that these chemicals are specifically acting on cells with a rapid proliferation.

Selective reduction of the ethylene double bond

Conclusions

The starting point of this original study is the diuretic drug ethacrynic acid which displays by itself a modest anti-proliferative activity at high doses (>60 μM) [10a] and is believed to have no direct effect on cell death but acts as inhibitor of π class glutathione S-transferase, in line with its role of adjuvant in chemotherapy. This low intrinsic anti-proliferative activity could be markedly enhanced through the introduction of an additional lateral chain affording original EA

Experimental section

Syntheses were carried out using standard high vacuum and dry-argon techniques. All chemicals were purchased from Across, Aldrich, Fluka, and used without further purification. The solvents were freshly dried and distilled according to standard procedures prior to use. 1H, 13C, and 31P NMR spectra were recorded with Bruker AV300, DPX300, AV400, spectrometers. All 13C NMR and 31P NMR spectra were generally recorded decoupled {1H}. The signal of the non-deuterated solvent served as internal

Acknowledgements

Thanks are due to the CNRS (Toulouse, France) for financial support and to the Euro-Mediterranean University of Fes (UEMF, Fes, Morocco) for granting N.E.B and J.C.

References (31)

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    In the meantime, several analogs of EA were synthesized in order to improve the anti-proliferative activity and / or the capacity to inhibit the GST p. So far, the structure-activity relationship studies clearly showed that all the modifications or reduction of the unsaturated double bond and / or the carbonyl unit abolished the anti-proliferative activity [21]. However, carboxylic group modifications by formation of simple amides or their analogs [21–24], oxadiazole [25,26], thiazole analogs [27], esters [11,28,29], as well as sulfonamides [30], showed a significant improvement of the anti-proliferative activities with IC50 ranging between 25 nM and 20 μM vs. ~50 μM for EA.

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