Inhibitory effects of ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit and para-acylated phenols on human cancer cells

Abstract

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, Hs578Ts(i)8 as well as of human prostate cancer cells, C4-2B. These cell lines provide a good model system to study migration and invasion, since they represent metastatic cancer. Our studies show that ethacrynic acid analogues with methyl substituents at the aromatic ring demonstrate no inhibitory effect on the migration of both cancer cell lines, whereas a precursor in the synthesis of these ethacrynic acid analogues (II-1, a paraacylated m-cresol) is an excellent inhibitor of the migration of both cancer cell lines.Ethacrynic acid (EA) (Fig. 1) is a loop diuretic and is used to treat high blood pressure and the swelling caused by diseases like liver, kidney, and congestive heart failure. 1,2 EA possesses an α,β-unsaturated carbonyl unit (a Michael acceptor) and can be attacked by nucleophiles (e.g. sulfur-atoms) at the β-carbon. This α,β-unsaturated carbonyl moiety is often employed in the design of SH-enzyme inhibitors (e.g. cysteine proteases). 3-6 EA inhibits the enzyme by binding to the cysteinyl residue in the active site by means of a Michael-like addition and is therefore a good glutathione S-transferase P1-1 (GST P1-1) inhibitor. 7 Several research groups have synthesized EA analogues in order to increase the inhibitory effect on GST P1-1. GST P1-1 is also of importance in controlling multidrug resistance (MDR), proliferation and apoptosis processes and hence can be considered a target for cancer treatment. Along this line, it has been demonstrated that EA exerts antiproliferative effects against tumor cells, but only at higher concentrations (60-100 μM). 2,[8][9][10] Additionally, it has also been shown that EA is cytotoxic towards primary chronic lymphocytic leukemia (CLL) cells. 11 However, the relative lack of potency and the diuretic properties do not support the use of EA as a chemotherapeutic agent. 12 Recently, we Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Our previous studies show that the most potent candidates to inhibit the migration of human breast cancer cells, MCF-7/AZ, are EA analogues with one or two methoxy-groups attached to the aromatic system but lacking the α,β-unsaturated carbonyl unit. We hypothesized that the increase in electron density of the aromatic system, caused by electron donating groups like the methoxy group, is responsible for the anti-migratory properties of the corresponding compounds. 13 To further investigate this hypothes...

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“…4), in contrast to the purified GST preparation (Fig. 6), can be explained based on the proposed mechanism of action of EA, whereby EA inhibits enzyme activity by binding to cysteinyl residues (Bryant et al, 2011). Therefore, in crude cytosol preparations, EA binds to the active site of GST and also reacts with other biomolecules that contain cysteinyl residues.…”

“…Enzyme-catalyzed GTN biotransformation assays for in vitro embryo homogenates were run in triplicate using 6-mL glass septum-sealed vials, flushed with argon to create anaerobic conditions, with constant agitation in the dark at 37 °C for 2 h. Each 1 mL sample contained: GTN (30 µmol), cytosol (5 mg protein) or enzyme preparation (0.50 mg protein), GSH (100 µM), enzyme inhibitor, either ethacrynic acid (200 µM), an &,'-unsaturated ketone that inhibits the enzyme by binding to the cysteinyl residue in the active site (Cameron et al, 1995;Bryant et al, 2011), or diphenyliodonium chloride (100 µM), an inhibitor of flavoenzymes (Bhushan et al, 2003) and Buffer A. Controls were prepared by omitting enzyme or GSH from the reaction mixture.…”

Ethacrynic acid improves the antitumor effects of irreversible epidermal growth factor receptor tyrosine kinase inhibitors in breast cancer

“…EA exhibits selective toxicity to chronic lymphocytic leukemia cells by inhibiting the Wnt/β-catenin signaling pathway [ 8 ]. EA analogues inhibit the migration of human prostate cancer and breast cancer cell lines, which provide a good model system to study migration and invasion since they represent metastatic cancer [ 20 ], [ 21 ]. Zhang et al .…”

Ethacrynic Acid: A Promising Candidate for Drug Repurposing as an Anticancer Agent

“…Clinical studies have reported that ECA has weakly inhibited migration and has been repurposed for the treatment of nonmuscle invasive bladder cancer [ 35 ]. Additionally, new ECA derivatives have been synthesized, which significantly inhibit migration in breast and prostate cancers [ 77 , 78 ].…”