The preparation and evaluation of 6 and 7, analogues of the duocarmycins and CC-1065 in which the subunit linking amide has been replaced with an amidine and thioamide, are described. Consistent with the increased electron- withdrawing properties and conjugation of thioamides relative to amides, 7 showed 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 preferential N2 amidine linkage hydrolysis rather than solvolysis of the alkylation subunit, attributable to preferential N2 vinylogous amide versus amidine conjugation. The natural isomers (+)-6 and (+)-7 exhibited an identical DNA alkylation selectivity as (+)-CBI-TMI and (+)-duocarmycin SA but were less efficient (10-100x). Biological studies of (+)-6 and (+)-7 (0.75 and 1.1 nM, respectively) indicated the analogues retained good cytotoxic activities (L1210), but were less potent than (+)-duocarmycin SA (0.01 nM, 100x) and (+)-CBI-TMI (0.02 nM, 50 x). The enhanced properties of the linking amide versus amidine or thioamide established the N2 amide as the optimal linking unit examined to date and revealed that it provides a beautiful balance between competing amide (reactivity) and vinylogous amide (stability) conjugation.