Chirality and Spectroscopic Changes Induced by the Recognition of Ethyl 5-Amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl Carbamate Analogs by Tubulin

The two chiral isomers of ethyl 5-amino-2-methyl-l,2-di-hydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate, NSC 613863 (R-isomer)-(+) and NSC 613862 (S-isomer)-(-) (CI980) and the three achiral analogs NSC 330770 (2-de-methylated analog A), NSC 337238 and C179 are potent microtubule inhibitors. These ligands interact with tubulin overlapping the colchicine binding site. This study addresses the effects of recognition by tubulin on the conformational properties of the ligands. The near-UV CD (circular dichroism) band of the R-isomer was suppressed, while that of the S-isomer displayed a more intense negative band when these compounds were bound to tubulin. Interestingly, the three other initially achiral compounds became optically active upon binding to tubulin; particularly, analog A exhibited a negative CD band on the order of magnitude of chiral compounds. The CD changes are reversible, highly specific and actually permit measuring the binding of the ligands by tubulin. These CD changes are compatible with the deformation of the bound ligands. Fluorescence emission is strongly enhanced and blue shifted upon binding to tubulin. Water among a solvent series had a specific solvent effect, except on the 1,2-dehydro analogs NSC 337238 and C179, suggesting hydrogen bonding to Nl. The emission of tubulin-bound R-isomer, S-isomer and analog A could be mimicked by solvent viscosity, supporting the notion that the intramolecular rotation between the pyridopyrazine and phenyl rings is frozen upon binding.