Synthesis and Biological Evaluation of Highly Active 7-Anilino Triazolopyrimidines as Potent Antimicrotubule Agents

Abstract

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.

Design, Synthesis and Biological Investigation of 2-Anilino Triazolopyrimidines as Tubulin Polymerization Inhibitors with Anticancer Activities

“…The activity of 3d was 4-and 100-fold greater than that of the reference compound CA-4 against A549 and HT-29 cells, respectively, but was one and two orders of magnitude less potent than CA-4 against MDA-MB-231 and HeLa cells, respectively. Compound 3d was 4-30-fold more potent as an antiproliferative agent than the corresponding 5-methyl-2-(p-toluidino)-7-(3 ,4 ,5 -trimethoxyanilino)-[1,2,4]triazolo[1,5-a]pyrimidine counterpart previously published by us (IC 50 0.43-0.030 µM and 0.52-1.55 µM, respectively) [65].…”

“…By the preparation of this latter small series, we evaluated if an increased flexibility of the substituent at the 2-position of the triazolopyrimidine scaffold was correlated with further enhancement of antiproliferative activity. The design of the linker chain in derivatives 3p-t was based on preserving flexibility similar to that of a series of compounds with general structure 6 previously published by us [65], characterized by a common 7-(3 ,4 ,5trimethoxyanilino)-[1,2,4]triazolo[1,5-a]pyrimidine scaffold and modified at its 2-position. Three benzylamino derivatives, 6a (4 -Cl), 6b (4 -Me) and 6c (3 ,4 -methylendioxy), along with the 3 -phenylpropylamino derivative 6d, were the most active compounds identified in this series.…”

“…Hela cells treated with the test compounds were harvested by centrifugation and then lysed with 0.1% (v/v) Triton X-100 containing RNase A at 0 • C. The protein content of the solutions was measured and analyzed as described previously [65]. The antibodies directed against cyclin B, cdc2 (Y15), ATR,Bcl-2, cleaved caspase-9 (D330) and actin were purchased from Cell Signaling.…”

“…Zebrafish (Danio rerio) were obtained, staged and raised as described previously [65] and maintained according to the OPBA of the Istituto di Ricerca Pediatrica guidelines. All procedures were conducted following the recommendations and the guidelines of the Animal Use Ethics Committee concerning the protection of animals used for scientific purposes.…”

“…The [1,2,4]triazolo [1,5-a]pyrimidine scaffold incorporating the 1,2,4-triazole nucleus (Figure 1) was previously identified by us and others as a promising nucleus for the design of new microtubule-destabilizing agents [61][62][63][64][65]. Two different series of compounds that retain the 3,4,5-trimethoxyphenyl ring at the 7-position of the triazolopyrimidine core variably functionalized at its 2-position were evaluated for their antitumor activity [62][63][64].…”