Andrew D. Westwell

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.

The role of fluorine in medicinal chemistry

The small and highly electronegative fluorine atom can play a remarkable role in medicinal chemistry. Selective installation of fluorine into a therapeutic or diagnostic small molecule candidate can enhance a number of pharmacokinetic and physicochemical properties such as improved metabolic stability and enhanced membrane permeation. Increased binding affinity of fluorinated drug candidates to target protein has also been documented in a number of cases. A further emerging application of the fluorine atom is the use of 18F as a radiolabel tracer atom in the exquisitely sensitive technique of Positron Emission Tomography (PET) imaging. This short review aims to bring together these various aspects of the use of fluorine in medicinal chemistry applications, citing selected examples from across a variety of therapeutic and diagnostic settings. The increasingly routine incorporation of fluorine atom(s) into drug candidates suggests a bright future for fluorine in drug discovery and development. A major challenge moving forward will be how and where to install fluorine in a rational sense to best optimise molecular properties.

2-Arylbenzothiazole as a Privileged Scaffold in Drug Discovery

Substituted 2-arylbenzothiazoles have emerged in recent years as an important pharmacophore in a number of diagnostic and therapeutic settings. Attractive features of drug candidates based on this benzothiazole scaffold include their synthetic accessibility, and synthetic approaches to 2-arylbenzothiazole-based compounds will be reviewed here. Examples of 2-arylbenzothiazoles endowed with diagnostic/therapeutic activity include the 2-(4-aminophenyl)benzothiazole series, which has a remarkable activity profile in both the potential non-invasive diagnosis of Alzheimers disease and as antitumour agents.

Exploring the Structural Requirements for Inhibition of the Ubiquitin E3 Ligase Breast Cancer Associated Protein 2 (BCA2) as a Treatment for Breast Cancer

The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C=S)S-S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and against recombinant BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f) were found to have potent activity (submicromolar IC(50)) in BCA2 positive MCF-7 and T47D cells but were inactive (IC(50) > 10 microM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast epithelial cell line MCF10A. Testing in the isogenic BCA2 +ve MDA-MB-231/ER cell line restored antitumor activity for compounds that were inactive in the BCA2 -ve MDA-MB-231 cell line. In contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive of selective E3 ligase-inhibitory activity and worthy of further development.

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.

2-[(1-Methylpropyl)dithio]-1H-imidazole inhibits tubulin polymerization through cysteine oxidation

2-[(1-Methylpropyl)dithio]-1H-imidazole (IV-2) is a known inhibitor of the thioredoxin system. It causes the oxidation of cysteine residues from both thioredoxin reductase and thioredoxin, with only the latter leading to irreversible inhibition of protein function. Although IV-2 is considered to be the first specific inhibitor of thioredoxin to undergo evaluation in cancer patients (under the name PX-12), it is unclear whether the oxidative ability of IV-2 is limited to proteins of the thioredoxin family. The current study investigated the specificity of IV-2 by examining its interaction with tubulin, a protein in which cysteine oxidation causes loss of polymerization competence. The cellular effects of IV-2 were examined in MCF-7 breast cancer and endothelial cells (human umbilical vein endothelial cells). Immunocytochemistry revealed a loss of microtubule structure with Western blot analysis confirming that treated cells contained a higher proportion of unpolymerized tubulin. Cell-free tubulin polymerization assays showed a dose-dependent inhibition of tubulin polymerization and depolymerization of preformed microtubules, confirming a direct interaction between IV-2 and tubulin. Further investigation of the tubulin interaction, through analysis of sulfhydryl reactivity and disulfide bond formation, suggested that IV-2 acts through the oxidation of cysteines in tubulin. Biochemical assays indicated that the oxidative properties of IV-2 are not limited to thioredoxin and tubulin, as cysteine-dependent proteases were also inhibited. Breast cancer cells with thioredoxin silenced by short interfering RNA remained sensitive to IV-2, albeit at higher antiproliferative GI50 values than in cells with normal thioredoxin function. These findings show that modulation of targets other than thioredoxin contribute to the effects of IV-2 on proliferating cells. [Mol Cancer Ther 2008;7(1):143–51]

Antitumor benzothiazoles. Frontier molecular orbital analysis predicts bioactivation of 2-(4-aminophenyl)benzothiazoles to reactive intermediates by cytochrome P4501A1Part 23. For part 22 see Ref. 1.

The antitumor and metabolic activities of 2-(4-aminophenyl)benzothiazoles and their fluorinated analogues cannot be explained or predicted by conventional chemical means. Their mode of anti-cancer action involves metabolism of the benzothiazoles to an as yet unidentified reactive species. This species then forms DNA adducts which provoke cell death. The electronic structures and possible intermediates of these compounds have been computed quantum mechanically. The counter-intuitive patterns of metabolism can only be explained by considering the active intermediate to be a nitrenium ion. The distribution of the highest occupied molecular orbital for the nitrenium species derived from each fluorinated analogue correlates perfectly with the production, or otherwise, of an exportable metabolite. Further related compounds have been analyzed by this method and the predictions of their metabolism have subsequently been verified experimentally.

Design, synthesis and pro-apoptotic antitumour properties of indole-based 3,5-disubstituted oxadiazoles

A series of new indole-based 3,5-disubstituted 1,2,4-oxadiazoles has been designed and synthesised as potential pro-apoptotic antitumour agents, via the base-catalysed condensation reaction between substituted amidoximes and indole esters. Evaluation of antiproliferative activity against the human cancer cell lines COLO 320 (colorectal) and MIA PACA-2 (pancreatic) revealed IC(50) values in the low micromolar range. Selected compounds were able to trigger apoptosis in sensitive cell lines, for example via activation of caspase-3/7, demonstrating that indole-based oxadiazoles possess in vitro antitumour and pro-apoptotic activity.

Oxidative stress in carcinogenesis: new synthetic compounds with dual effects upon free radicals and cancer.

DNA mutation is a very important step in carcinogenesis and elevated levels of oxidative DNA damage have been monitored in a variety of tumors. The discovery of the role of free radicals in cancer has led to a new medical approach. Minimizing oxidative damage may be a significant advance in the prevention or treatment of these diseases, since antioxidants are able to stop the free-radical formation and prevent oxidizing chain reactions. These findings have generated great interest in therapeutic antioxidant-based cancer drug development. The design and development of synthetic compounds, able to scavenge free radicals, could present a significant therapeutic advance, in particular for treating pathological conditions such as cancer. This article will outline the state of the research on the relationship between antioxidant therapy and cancer, describing the new synthetic antioxidant molecules that have anticancer activities. Investigations and association between dietary antioxidants, oxidative stress, and cancer will be also discussed.

The identification and chemical characterization of a new arylcyclohexylamine, methoxetamine, using a novel Emergency Department toxicosurveillance tool

The Emergency Department is frequently the first port of call for patients suffering from recreational drug overdose, alongside pre-hospital and other emergency services. Many drugs are familiar to the seasoned practitioner, but the fluctuant and dynamic nature of both the licit and illicit recreational drugs market is such that there are always new products emerging. Manufacturers, eager to avoid prosecution where possible, have put some effort into developing the ‘legal high market’, using untried and untested compounds bearing labels such as ‘research chemicals’ to bypass the legal ramifications of selling drugs to consumers. One such recently described group of so-called legal highs are the substituted cathinones, of which mephedrone is the best known. We believe that drugs from the arylcyclohexylamine class are becoming the next class of legal highs to significantly spread through the drug-using community, and that both clinicians and analytical chemists should be aware of their existence and effects. A 35-year-old man presented to the Emergency Department at Nevill Hall, Abergavenny (South Wales) in early 2011, having collapsed at his home. He was initially unresponsive on ambulance arrival, and was noted to be bleeding from his left nostril, following insufflation of a white powder (although there was no evidence of further nasal irritation resulting from insufflation). A small empty package was found with the patient, which was labelled with the chemical structure of the compound and the word ‘methoxyphenyl-2-(ethylamine) cyclo-hexanone’. The patient was initially incoherent in his general manner and communication with Emergency Department staff, but returned to a GCS (Glasgow Coma Scale) of 14 over 20min, and a GCS of 15 over 90min. He was hypertensive (blood pressure = 167/ 110mmHg), but not tachycardic (with heart rate and temperature in the normal range), and he had bilateral pupillary mydriasis. Neurological examination was normal, with no focal neurological signs. He described being in contact with both heaven and hell and the spirit of his dead father. His behaviour was ecstatic, but otherwise good-natured. He appeared to be confused about the origins of sounds and colours in his proximity, but denied frank hallucinations. On further enquiry, he was identified as having had previous mental health issues, and was known to have been treated for bipolar disorder using lithium, but had been non-compliant. He