Differential effects of ethyl 5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate analogs modified at position C-2 on tubulin polymerization, binding, and conformational changes

NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI980) are two chiral isomers of ethyl 5-amino 2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate which have potent antitubulin activity. The S-isomer is a more potent antimitotic compound than the R-isomer, and the two isomers differ markedly in binding to tubulin [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10675-10682]. To understand die origin of such differences, we studied;the interactions of three R- and S-isomer structural analogs which differ in C-2 (the chiral carbon), i.e., C179, NSC 337238, and NSC 330770. C179 is a methylated dehydrogenated achiral compound. It bound to tubulin with an apparent affinity K-a of (2.29 +/- 0.17) x 10(4) M(-1), inhibited tubulin polymerization in vitro at a half-inhibitory concentration (IC50) of 100 mu M, and presented no GTPase activity. The substitution of -CH3 by -H leads to the NSC 337238 compound. It bound to tubulin with a higher affinity [K-a = (2.62 +/- 0.35) x 10(5) M(-1)] and inhibited tubulin polymerization at a lower concentration (IC50 = 14 mu M). It presented no GTPase activity and induced the formation of abnormal polymers at a protein critical concentration (Cr) of 2 mg mL(-1). NSC 330770, a demethylated hydrogenated molecule, interacted strongly with tubulin [K-a = (3.30 +/- 0.56) x 10(6) M(-1)]. It was a potent tubulin polymerization inhibitor (IC50 = 2 mu M) and elicited GTPase activity and the formation of abnormal polymers at a low critical concentration (Cr = 0.5 mg mL(-1)). Competition experiments showed that the three molecules bound on the colchicine site and the ring A and C subsites. The thermodynamic parameters revealed that, for the most active compounds, i.e., the S-isomer and NSC 330770, many structural changes occurred when they interacted with tubulin. Theoretical chemistry calculations by CNDO/2, correlated with the biochemical activities described above, led us to conclude that the position of the methyl group is not important for activity. Thus, NSC 330770 is a powerful inhibitor which has no methyl group in C-2. Finally, for the molecules to have potent antitubulin activity, they require a 1,2-dihydro structure, a large dipole moment value, and an electron density in the C-3, N-4, and C-10 atoms.