Synthesis and evaluation of duocarmycin and CC-1065 analogues containing modifications in the subunit linking amide

The preparation and evaluation of 6 and 7, analogues of the duocarmycins an d CC-1065 in which the subunit linking amide has been replaced with an amid ine and thioamide, are described. Consistent with the increased electron-wi thdrawing properties and conjugation of thioamides relative to amides, 7 sh owed increased solvolysis reactivity (t(1/2), 160 h versus 230 h) at pH 3, attributable to a diminished vinylogous amide stabilization of the reacting alkylation subunit. Amidine 6 proved to be even more unstable (t(1/2), 12 h) despite the diminished electron-withdrawing properties, but underwent pr eferential N-2 amidine linkage hydrolysis rather than solvolysis of the alk ylation subunit, attributable to preferential N-2 vinylogous amide versus a midine conjugation. The natural isomers (+)-6 and (+)-7 exhibited an identi cal DNA alkylation selectivity as (+)-CBI-TMI and (+)-duocarmycin SA but we re less efficient (10-100x). Biological studies of (+)-6 and (+)-7 (0.75 an d 1.1 nM, respectively) indicated the analogues retained good cytotoxic act ivities (L1210), but were less potent than (+)-duocarmycin SA (0.01 nM, 100 x) and (+)-CBI-TMI (0.02 nM, 50x). The enhanced properties of the linking a mide versus amidine or thioamide established the N-2 amide as the optimal l inking unit examined to date and revealed that it provides a beautiful bala nce between competing amide (reactivity) and vinylogous amide (stability) c onjugation.