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 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 (10−100×). 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, 100×) and (+)-CBI-TMI (0.02 nM, 50×). 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.