Geoffrey Wells

Synthesis and biological properties of benzothiazole, benzoxazole, and chromen-4-one analogues of the potent antitumor agent 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610, NSC 721648).

New fluorinated 2-aryl-benzothiazoles, -benzoxazoles, and -chromen-4-ones have been synthesized and their activity against MCF-7 and MDA 468 breast cancer cell lines compared with the potent antitumor benzothiazole 5. Analogues such as 9a, b and 12a, d yielded submicromolar GI50 values in both cell lines; however, none of the new compounds approached 5 in terms of antitumor potency. For 5, binding to the aryl hydrocarbon receptor appeared to be necessary but not sufficient for growth inhibition.

Elucidation of thioredoxin as a molecular target for antitumor quinols

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (beta-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 micromol/L) or 2 (500 nmol/L and 1 micromol/L) determined that of > or =10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 micromol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.

Antitumour benzothiazoles. Part 10: the synthesis and antitumour activity of benzothiazole substituted quinol derivatives.

The synthesis of a series of new antitumour agents, the benzothiazole substituted quinol ethers and esters, is reported via the hypervalent iodine mediated oxidation of hydroxylated 2-phenylbenzothiazoles. The products were found to be active in vitro against human colon and breast cancer cell lines with IC50 values in the nanomolar range.

Antitumor Quinol PMX464 Is a Cytocidal Anti-trypanosomal Inhibitor Targeting Trypanothione Metabolism

Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class (“quinols”) and identified several with potent trypanocidal activity (EC50 < 100 nm). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)2), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)2, TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and l-cysteine bound in a 2:1 ratio with PMX464 with Kd values of 6 and 27 μm, respectively, whereas T(SH)2 bound more tightly in a 1:1 ratio with a Kd value of 430 nm. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)2 is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.

Synthesis of antitumour (1H-1,2,3-triazol-4-yl)-4-hydroxycyclohexa-2,5-dien-1-ones by copper-catalysed Huisgen cycloadditions

4-Ethynyl-4-hydroxycyclohexa-2,5-dien-1-one 5 undergoes cycloaddition reactions with a range of substituted azides in the presence of copper salts to form 1,4-disubstituted triazoles 8-11 bearing the 4-hydroxycyclohexa-2,5-dien-1-one (quinol) pharmacophore; one example of an isomeric 1,5-disubstituted triazole 12 was formed from 5 and benzyl azide in the presence of a ruthenium catalyst. Compounds were screened for growth-inhibitory activity against five cancer cell lines of colon, breast and lung origin, but were overall less potent than the benzothiazolyl- and indolyl-substituted quinols 2 and 3.

Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,β-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3)

Anti-cancer drug discovery efforts to directly inhibit the signal transducer and activator of transcription 3 (STAT3) have been active for over a decade following the discovery that 70% of cancers exhibit elevated STAT3 activity. The majority of research has focused on attenuating STAT3 activity through preventing homo-dimerization by targeting the SH2 or transcriptional activation domains. Such dimerization inhibitors have not yet reached the market. However, an alternative strategy focussed on preventing STAT3 DNA-binding through targeting the DNA-binding domain (DBD) offers new drug design opportunities. Currently, only EMSA and ELISA-based methods have been implemented with suitable reliability to characterize STAT3 DBD inhibitors. Herein, we present a new orthogonal, fluorescence polarization (FP) assay suitable for high-throughput screening of molecules. This assay, using a STAT3127-688 construct, was developed and optimized to screen molecules that attenuate the STAT3:DNA association with good reliability (Z’ value > 0.6) and a significant contrast (signal-to-noise ratio > 15.0) at equilibrium. The assay system was stable over a 48 hour period. Significantly, the assay is homogeneous and simple to implement for high-throughput screening compared to EMSA and ELISA. Overall, this FP assay offers a new way to identify and characterize novel molecules that inhibit STAT3:DNA association.

Influence of steric crowding on hydrogen bonding in anti-cancer quinols

The quinol 4-(1-benzenesulfonyl-1H-6-fluoroindol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) is strongly active against cancer cells [1]. Two “Michael acceptor” electrophilic β-carbons on the quinol ring are believed necessary for optimal antitumor activity, and disruption of thioredoxin signaling is a possible mechanism of action. As a model for the possible product, the adduct (2) with two molecules of ethanethiol was prepared. These molecules have just one classical hydrogen bond (HB) donor group, the quinol OH, but a surfeit of acceptors, namely one C=O and two SO2 oxygen atoms (designated O15, O17 and O18) as well as O14, the OH itself. In (1) paired molecules form a R2 (14) ring by O14-H...O17 HB with H...O distance 2.15 Ǻ and O-H...O angle 156°. In (2) the two EtS groups on the same side of the quinol ring as O14 interfere with this motif. Instead, a C(7) chain is formed by less optimal O14-H...O17 HB (2.34 Ǻ and 134°). In apparent compensation, an intramolecular C-H...O HB to O17 is shorter and straighter in (2). In both structures all O atoms accept C-H...O HB. The unit cell dimensions are dissimilar, but a motif persists: one phenyl CH and one indole CH group bite onto the same O atom, O15 in (1) but O18 in (2).