Linker Length Modulates DNA Cross-Linking Reactivity and Cytotoxic Potency of C8/C8‘ Ether-Linked C2-exo-Unsaturated Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) Dimers

A C2/C2‘-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer <BO>4b</BO> (DRG-16) with a C8−O(CH<INF>2</INF>)<INF>n</INF><INF></INF>O−C8‘ diether linkage (n = 5) has been synthesized that shows markedly superior in vitro cytotoxic potency (e.g., >3400-fold in IGROV1 ovarian cells) and interstrand DNA cross-linking reactivity (>10-fold) compared to the shorter homologue <BO>4a</BO> (SJG-136; n = 3). In contrast, for the C-ring unsubstituted series, the corresponding n = 5 dimer (<BO>3c</BO>) is generally less cytotoxic and has a lower interstrand cross-linking reactivity compared to its shorter n = 3 homologue (<BO>3a</BO>). Dimer <BO>4b</BO> cross-links DNA with >10-fold efficiency compared to <BO>4a</BO>, and also inhibits the activity of the restriction endonuclease BamH1 more efficiently than either <BO>3a</BO> or <BO>4a</BO>. The C2-exo-unsaturated PBD dimers <BO>4a</BO>,<BO>b</BO> are not only more effective than their C-ring saturated counterparts in terms of induced ΔT<INF>m</INF> shift, but they also exert this effect more rapidly. Thus, while <BO>3a</BO> and <BO>3c</BO> exert 68 and 35% of their maximum effect immediately upon interaction with DNA, this level increases to 76 and 97% for <BO>4a</BO> and <BO>4b</BO>, respectively. Molecular modeling shows a rank order of <BO>4b</BO> (n = 5) > <BO>4a</BO> (n = 3) > <BO>3a</BO> (n = 3) > <BO>3c</BO> (n = 5) in terms of binding energy toward duplexes containing embedded target 5‘-<BO>G</BO>AT<INF>1</INF><INF>-</INF><INF>2</INF>C cross-link sequences, reflecting the superior fit of the C2-exo-unsaturated rather than saturated C-rings of the PBD dimers. A novel synthesis of core synthetic building blocks for PBD dimers via stepwise Mitsunobu reaction and nitration with Cu(NO<INF>3</INF>)<INF>2</INF> is also reported.