New Arythioindoles Inhibitors of Tubulin Polymerization. 3. Biological Evaluation, SAR and Molecular Modeling Studies

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Giuseppe La Regina,^†,◊ Michael C. Edler,^§ Andrea Brancale,^‡ Sahar Kandil,^‡ Francesco Piscitelli,^† Ernest Hamel,^§ Gabriella De Martino,^† Ruth Matesanz,^¢ José Fernando Díaz,^¢ Anna Ivana Scovassi,^fi Ennio Prosperi,^fi Antonio Lavecchia,° Ettore Novellino,° Marino Artico,^† and Romano Silvestri*^,†

Istituto Pasteur – Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3XF, UK, Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli “Federico II”, Via Domenico Montesano 49, I-80131, Napoli, Italy, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, C/ Ramiro de Maeztu 9, E-28040 Madrid, Spain, Istituto di Genetica Molecolare – Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, I-27100 Pavia, Italy.

Microtubules have essential roles in vital cellular functions, such as motility, division, shape maintenance, and intracellular transport. Drugs that interact with tubulin, the protein subunit of microtubules, cause mitotic arrest, interfering with the dynamic equilibrium of these organelles by either inhibiting tubulin polymerization or blocking microtubule disassembly. Inhibitors of tubulin assembly include colchicine (1), combretastatin A-4 (2a, CAS4) and the Catharanthus alkaloids vincristine and vinblastine (Chart 1). At high concentrations these compounds interact with ab-tubulin dimers at the interface between alpha and beta (Ravelli et al. 2004) and cause microtubule destabilization and apoptosis. Taxoids and epothilones bind as well to ab-tubulin to a lumenal site at the b-subunit (Nogales et al. 1999) (Nettles et al. 2004), and probably to a recently described microtubule stabilizing agents binding site (Buey et al. 2007) in the pore on the microtubule surface formed by two different a and b tubulin subunits. Paclitaxel stimulates microtubule polymerization and stabilization at high concentrations, whereas at lower concentrations the drug inhibits microtubule dynamics with little effect on the proportion of tubulin in polymer.^1-3 Independent of precise mechanism of action, clinical use of antitubulin drugs is associated with problems of drug resistance, toxicity, and bioavailability.⁴

In the last few years, several antitubulin agents that target the colchicine binding site have been intensively investigated as vascular-disrupting antitumor drugs.⁵ For example, combretastatin A-4 phosphate (2b) and ZD6126 (3) stop blood flow through tumor capillaries, probably caused by rapid disruption of endothelial cell morphology, and consequently the tumor is starved of nutrients and rapid tumor cell death occurs.^6,7 These vascular-disrupting agents are currently in ongoing clinical trials for either single- or multi-drug combination antitumor therapy.^7,8

Arylthioindoles (ATIs, general structure 4) are a new class of potent tubulin assembly inhibitors that bind to the colchicine site in the interphase between a- and  b-tubulin.⁹ Structure-activity relationship (SAR) analysis clarified structural requirements for good activity in this class of inhibitors. Essential structural features for an active agent have included (A) a small-size ester function at position 2 of the indole, (B) the 3-arylthio group, (C) the sulfur atom bridge, and (D) a substituent at position 5 of the indole (Chart 1).¹⁰ We carried out molecular modeling studies and dynamics simulations that helped explain  the experimental data. We therefore have used the molecular model in designing new ATI derivatives.¹⁰

Recent studies have focused on the synthesis of aminoderivatives related to CSA4.¹¹ The potent antitubulin activity displayed by these analogues (for example 5,¹² 6,¹³ 7,¹³ and 8¹⁴, Chart 2) attracted our attention. Compounds 5-8 share, as a common structural feature, an amino group located ortho to the bridging group (either carbonyl or cis-ethenyl group). We hypothesized that this ortho-substituted-aniline might resemble the indole nucleus of ATI derivatives (see Chart 2), with the indole ring acting as a bioisostere of the ortho-substituted-aniline. These observations prompted us to design new ATI derivatives 9-28. Predictive docking simulations using our model¹⁰ showed that, despite the absence of the ester moiety at position 2 of the indole ring, most of the compounds should bind in the colchicine binding site of tubulin in the same orientation as the previously studied ATIs. The new ATI derivatives, like those described previously, were potent inhibitors of tubulin polymerization and of the growth of cancer cells, with activities comparable with those of colchicine and combretastatin A-4.  Finally, we should note the recent paper of Hsieh and collaborators,¹⁵ which included a group of 3-aroylthioindoles.  These compounds are significantly different from the ATI's we have prepared, since there are major differences in our SAR findings and those of the Hsieh group.

 

Chemistry

The structures of ATI derivatives 9-31 are shown in Table 1. Compounds 10, 11, 14-26 and 28 were synthesized by a two-step procedure (Scheme 1). O-Ethyl-S-(3,4,5-trimethoxyphenyl)carbonodithioate¹⁶ was transformed into 3,4,5-trimethoxy-thiophenol by heating at 65 °C in aqueous ethanol in the presence of sodium hydroxide. This mixture was made acidic with 6 N HCl, and treated at 25 °C with the appropriate indole while adding dropwise an aqueous iodine - potassium iodide solution. Compounds 12 and 13 were prepared similarly, starting from the corresponding commercially available carbonodithioate. Compound 27 was prepared by treating 26 with (2-bromoethoxy)-tert-butyldimethylsilane in the presence of potassium carbonate in boiling acetone; the intermediate silyloxy derivative was stirred with para-toluensulfonic acid in methanol at room temperature. The 5-ethoxy- (32) and 5-isopropyloxy- (33) indoles were obtained by alkylation of 5-hydroxyindole with iodoethane or 2-iodopropane, respectively, in the presence of potassium carbonate. 5-(2-Benzyloxy)ethoxyindole (34) was prepared by reaction of 5-hydroxyindole with 2-benzyloxyethanol in the presence of diethyl azodicarboxylate and  triphenylphosphine in boiling THF.