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A further investigation aiming to generate new potential antitumor agents led us to synthesize a new series of twenty-two compounds characterized by the presence of the 7-(3′,4′,5′-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine pharmacophore modified at its 2-position. Among the synthesized compounds, three were significantly more active than the others. These bore the substituents p-toluidino (3d), p-ethylanilino (3h) and 3′,4′-dimethylanilino (3f), and these compounds had IC50 values of 30–43, 160–240 and 67–160 nM, respectively, on HeLa, A549 and HT-29 cancer cells. The p-toluidino derivative 3d was the most potent inhibitor of tubulin polymerization (IC50: 0.45 µM) and strongly inhibited the binding of colchicine to tubulin (72% inhibition), with antiproliferative activity superior to CA-4 against A549 and HeLa cancer cell lines. In vitro investigation showed that compound 3d was able to block treated cells in the G2/M phase of the cell cycle and to induce apoptosis following the intrinsic pathway, further confirmed by mitochondrial depolarization and caspase-9 activation. In vivo experiments conducted on the zebrafish model showed good activity of 3d in reducing the mass of a HeLa cell xenograft. These effects occurred at nontoxic concentrations to the animal, indicating that 3d merits further developmental studies.

Two different series of fifty-two compounds, based on 3′,4′,5′-trimethoxyaniline (7a–ad) and variably substituted anilines (8a–v) at the 7-position of the 2-substituted-[1,2,4]triazolo [1,5-a]pyrimidine nucleus, had moderate to potent antiproliferative activity against A549, MDA-MB-231, HeLa, HT-29 and Jurkat cancer cell lines. All derivatives with a common 3-phenylpropylamino moiety at the 2-position of the triazolopyrimidine scaffold and different halogen-substituted anilines at its 7-position, corresponding to 4′-fluoroaniline (8q), 4′-fluoro-3′-chloroaniline (8r), 4′-chloroaniline (8s) and 4′-bromoaniline (8u), displayed the greatest antiproliferative activity with mean IC50′s of 83, 101, 91 and 83 nM, respectively. These four compounds inhibited tubulin polymerization about 2-fold more potently than combretastatin A-4 (CA-4), and their activities as inhibitors of [3H]colchicine binding to tubulin were similar to that of CA-4. These data underlined that the 3′,4′,5′-trimethoxyanilino moiety at the 7-position of the [1,2,4]triazolo [1,5-a]pyrimidine system, which characterized compounds 7a–ad, was not essential for maintaining potent antiproliferative and antitubulin activities. Compounds 8q and 8r had high selectivity against cancer cells, and their interaction with tubulin led to the accumulation of HeLa cells in the G2/M phase of the cell cycle and to apoptotic cell death through the mitochondrial pathway. Finally, compound 8q significantly inhibited HeLa cell growth in zebrafish embryos.

A series of eleven 4-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were designed and synthesized and their biological activities were evaluated. Synthesis involved the Gewald reaction to synthesize ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate ring, and SNAr reactions. Compound 4 was 1.6- and ~7-fold more potent than the lead compound 1 in cell proliferation and microtubule depolymerization assays, respectively. Compounds 4, 5 and 7 showed the most potent antiproliferative effects (IC50 values < 40 nM), while compounds 6, 8, 10, 12 and 13 had lower antiproliferative potencies (IC50 values of 53–125 nM). Additionally, compounds 4–8, 10 and 12–13 circumvented Pgp and βIII-tubulin mediated drug resistance, mechanisms that diminish the clinical efficacy of paclitaxel (PTX). In the NCI-60 cell line panel, compound 4 exhibited an average GI50 of ~10 nM in the 40 most sensitive cell lines. Compound 4 demonstrated statistically significant antitumor effects in a murine MDA-MB-435 xenograft model.

Induction of apoptosis is a common chemotherapeutic mechanism to kill cancer cells The thiazole system has been reported over the past decades as a building block for the preparation of anticancer agents. A novel series of 2-arylalkylamino-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)-thiazole derivatives designed as dual inhibitors of tubulin and cyclin-dependent kinases (CDKs) were synthesized and evaluated for their antiproliferative activity in vitro against two cancer cell lines and, for selected highly active compounds, for interactions with tubulin and cyclin-dependent kinases and for cell cycle and apoptosis effects. Structure-activity relationships were elucidated for various substituents at the 2-position of the thiazole skeleton. Among the synthesized compounds, the most active analogues were found to be the p-chlorobenzylamino derivative 8e as well as the p-chloro and p-methoxyphenethylamino analogues 8f and 8k, respectively, which inhibited the growth of U-937 and SK-MEL-1 cancer cell lines with IC50 values ranging from 5.7 to 12.2 μM. On U-937 cells, the tested compounds 8f and 8k induced apoptosis in a time and concentration dependent manner. These two latter molecules did not affect tubulin polymerization (IC50 > 20 μM) nor CDK activity at a single concentration of 10 μM, suggesting alternative targets than tubulin and CDK for the compounds.

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3′,4′,5′-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3′,4′,5′-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 μM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.

Monoterpenoid indole alkaloids are structurally diverse natural products found in plants of the family Apocynaceae. Among them, vincristine and its derivatives are well known for their anticancer activity. Bousigonia mekongensis, a species in this family, contains various monoterpenoid indole alkaloids. In the current study, fourteen known aspidosperma-type monoterpenoid indole alkaloids (1–14) were isolated and identified from a methanol extract of the twigs and leaves of B. mekongensis for the first time. Among them, compounds 3, 6, 9, and 13 exhibited similar antiproliferative activity spectra against A549, KB, and multidrug-resistant (MDR) KB subline KB-VIN cells with IC50 values ranging from 0.5–0.9 μM. The above alkaloids efficiently induced cell cycle arrest at the G2/M phase by inhibiting tubulin polymerization as well as mitotic bipolar spindle formation. Computer modeling studies indicated that compound 7 likely forms a hydrogen bond (H-bond) with α- or β-tubulin at the colchicine site. Evaluation of the antiproliferative effects and SAR analysis suggested that a 14,15-double bond or 3α-acetonyl group is critical for enhanced antiproliferative activity. Mechanism of action studies demonstrated for the first time that compounds 3, 4, 6, 7, and 13 efficiently induce cell cycle arrest at G2/M by inhibiting tubulin polymerization by binding to the colchicine site.