Synthesis of N-acyl Derivatives of Aminocombretastatin A-4 and Study of their Interaction with Tubulin and downregulation of c-Myc.

Aims In search of new promising anti-cancer agents. Background Six N-acyl derivatives of aminocombretastatin A-4 have been synthesized and evaluated as regards their interaction with tubulin and as c-Myc downregulators. Objective This study is focused on the synthesis and the biological evaluation of N-acyl derivatives of aminocombretastatin A-4 (CA-4). Docking studies were carried out in order to find out whether the synthetic derivatives could bind to tubulin at the colchicine site in a conformation similar to that of CA-4. The effect of the synthetic derivatives on the proliferation of several cancer cells and on non-cancer cells has been measured. In addition, their effect on tubulin polymerization, on cell cycle distribution, on the microtubule network and on c-Myc expression have also been evaluated. Method A set of six N-acyl derivatives were achieved by means of a peptide-type coupling of aminocombretastatin A-4 and the corresponding carboxylic acid. The ability of the synthetic compounds to inhibit cell proliferation was measured by MTT assay against three human carcinoma cell lines (colorectal HT-29, lung A549 and breast adenocarcinoma MCF-7) and one non-tumor cell line (HEK-293). Inhibition of tubulin polymerization was evaluated by turbidimetry time-course measurements. The action of the synthetic derivatives on cell cycle distribution was measured by flow cytometry and their effects on the microtubule network was determined by immunofluorescence microscopy. Finally, the downregulation of the synthetic derivatives on c-Myc protein was quantified by ELISA assay while the effect on cMyc gene was measured by RT-qPCR analysis. Results Derivatives bearing pentanoyl (compound 2), hexanoyl (compound 3), and heptanoyl (compound 4) side chains show antiproliferative activities on the HT-29 line in the low nanomolar range, with values similar to that exhibited by AmCA-4 but far exceeding those of CA-4. Compounds 1 (butanoyl side chain) and 2-3 inhibit tubulin polymerization in vitro in a manner similar to that of CA-4 and AmCA-4 whereas compounds 4, 5 (octanoyl side chain) and 6 (dodecanoyl side chain) may be considered as partial inhibitors of tubulin polymerization. While all derivatives are able to accumulate cells in G2/M phase, compounds with the longest acyl chains (5 and 6) are the least active ones in this particular action. Moreover, compounds 2-3 were the most active ones as c-Myc downregulators. Conclusion Our studies show that the most active compounds in the disruption of the microtubule network are also the most potent ones in the downregulation of c-Myc expression. Other: Compounds 2 and 3 are good candidates for in vivo studies as they combine the best anti-mitotic and c-Myc downregulation activities at low doses.