Microtubules are dynamic polymers and integral components of the cytoskeleton. They play important roles in the regulation of cellular signaling, motility, maintenance of cell shape, secretion, intercellular transport and spindle formation during mitosis.1 As a consequence, small molecules that interfere with the dynamics of microtubules have been recognized as powerful tools for the treatment of cancer.2, 3

A systematic structure-activity relationship (SAR) study starting from morpholino-substituted biheteroaryls with moderate microtubule disrupting activities allowed the optimizations of biological activity, metabolic stability, and drug-like properties. Interestingly, the type of core (aryl, pyridine or pyrimidine) was of importance, as well as the regioisomeric arrangement of the pyrimidine nitrogens. Pyrimidines substituted with four- or five-membered N-heterocycles proved to be superior both in terms of biological activity and metabolic stability. Finally, optimization of the heteroaryl substituent of the pyrimidine derivatives culminated in the identification of a novel series of substituted 4-(pyrimidin-2-yl)morpholines targeting microtubule polymerization in the nM range.

Selected compounds potently inhibit cellular microtubule polymerization with EC50 values of 20-90 nM. This was confirmed by phosphorylation of Histone3, nuclear DNA condensation, and cell cycle arrest in G2/M or induction of cell death across multiple cell lines. Moreover, substituted 4-(pyrimidin-2-yl)morpholines were shown to be poor substrates for P-gp multi-drug resistance pumps, and therefore caused efficiently mitotic arrest and cell death in colchicine-resistant cells.

The co-crystal structure of tubulin with selected compounds showed that 4-(pyrimidin-2-yl)morpholines bind to the colchicine pocket located between the α and β subunits of the αβ-tubulin dimer. Relevant inhibitor contact residues include Lys352, Met259, Ala316, Leu248, Val238, Tyr202 and Cys241 of β-tubulin. Moreover, two water molecules link the morpholine oxygen to the β-tubulin bound GTP. Conformational changes induced by inhibitor binding suggest that free or plus end β-tubulin is targeted by this compound series.

Pre-clinical studies characterized a lead compound selection with excellent stability in human hepatocytes, and human, mouse and rat microsomes. Overall, these compounds qualify as a novel class of microtubule destabilizing agents that target the colchicine-binding site, and which warrant further investigations currently in progress (PK studies, xenograft tumor mouse models).

1) Walczak, C. E, Curr. Opin. Cell Biol. 2000, 12, 52-56.

2) Risinger, A. L.; Giles, F. J.; Mooberry, S. L, Cancer Treat. Rev. 2009, 35, 255-261.

3) Downing, K. H.; Nogales, E., Curr. Opin. Struct. Biol. 1998, 8, 785-791.

Citation Format: Alexander M. Sele, Denise Rageot, Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, Michel O. Steinmetz, Matthias P. Wymann. Novel 4-(pyrimidin-2-yl)morpholines targeting the colchicine-binding site of tubulin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1364.