Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNA-interactive agent with highly efficient cross-linking ability and potent cytotoxicity

A novel sequence-selective pyrrolobenzodiazepine (PBD) dimer 5 (SJG-136) ha s been developed that comprises two C2-exo-methylene-substituted DC-81 (3) subunits tethered through their C8 positions via an inert propanedioxy link er. This symmetric molecule is a highly efficient minor groove interstrand DNA cross-linking agent (XL50 = 0.045 muM) that is 440-fold more potent tha n melphalan, Thermal denaturation studies show that, after 18 h incubation with calf thymus DNA at a 5:1 DNA/ligand ratio, it increases the T-m value by 33.6 degreesC, the highest value so far recorded in this assay. The anal ogous dimer 4 (DSB-120) that lacks substitution/ unsaturation at the C2 pos ition elevates melting by only 15.1 degreesC under the same conditions, ill ustrating the effect of introducing C2-exo-unsaturation which serves to fla tten the C-rings and achieve a superior isohelical fit within the DNA minor groove. This behavior is supported by molecular modeling studies which ind icate that (i) the PBD units are covalently bonded to guanines on opposite strands to form a cross-link, (ii) 5 has a greater binding energy compared to 4, and (iii) 4 and 5 have equivalent binding sites that span six base pa irs. Dimer 5 is significantly more cytotoxic than 4 in a number of human ov arian cancer cell lines (e.g., IC50 values of 0.0225 nM vs 7.2 nM, respecti vely, in A2780 cells). Furthermore, it retains full potency in the cisplati n-resistant cell line A2780cisR (0.024 nM), whereas 4 loses activity (0.21 muM) with a resistance factor of 29.2, This may be due to a lower level of inactivation of 5 by intracellular thiol-containing molecules. A dilactam a nalogue (21) of 5 that lacks the electrophilic N10-C11/N10'-C11' imine moie ties has also been synthesized and evaluated. Although unable to interact c ovalently with DNA, 21 still stabilizes the helix (DeltaT(m) = 0.78 degrees C) and has significant cytotoxicity in some cell lines (i.e., IC50 = 0.57 m uM in CH1 cells), presumably exerting its effect through noncovalent intera ction with DNA.