Synthesis and Evaluation of Duocarmycin and CC-1065 Analogues Containing Modifications in the Subunit Linking Amide

The preparation and evaluation of <b>6 </b>and <b>7</b>, 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, <b>7</b> showed increased solvolysis reactivity (<i>t</i><sub>1/2</sub>, 160 h versus 230 h) at pH 3, attributable to a diminished vinylogous amide stabilization of the reacting alkylation subunit. Amidine <b>6</b> proved to be even more unstable (<i>t</i><sub>1/2</sub>, 12 h) despite the diminished electron-withdrawing properties, but underwent preferential N<sup>2</sup> amidine linkage hydrolysis rather than solvolysis of the alkylation subunit, attributable to preferential N<sup>2</sup> vinylogous amide versus amidine conjugation. The natural isomers (+)-<b>6</b> and (+)-<b>7</b> exhibited an identical DNA alkylation selectivity as (+)-CBI-TMI and (+)-duocarmycin SA but were less efficient (10−100×). Biological studies of (+)-<b>6</b> and (+)-<b>7</b> (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 N<sup>2</sup> 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.