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CC-1065, Benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1,6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8,8a-tetrahydro-7-methyl-4- oxocyclopropa[c]pyrrolo[3,2-e]indol-2(1H)-yl)carbonyl]benzo [1,2-b:4,3-b']dipyrrol-3(2H)-yl]carbonyl]-1,6-dihydro-4-hydroxy- 5-methoxy-, (7bR,8aS), binds to the B form of poly(dl-5BrdC).poly(dl-5BrdC) to yield a reversibly bound species whose stability with respect to an irreversibly bound species (presumably the inosine N-3 adduct) is much greater than it is for other DNA polymers. Competitive binding experiments with netropsin, show that this reversibly bound species of CC-1065 contains CC-1065 in the minor groove of the double helix. A review of the CC-1065 binding data obtained on other synthetic DNA polymers suggests that the widely different rates of species conversion shown by these polymers may result from small differences in DNA secondary structure rather than from different alkylating abilities of the adenine or inosine N-3 active site. CC-1065 converts the Z-form of poly(dl-5BrdC).poly(dl-5BrdC) in 3.5 M sodium chloride to the B form and does not bind to the Z form in this solvent system. CC-1065 bound to the B form polymer inhibits the formation of the Z form if the helix is saturated with CC-1065. Regions of the polymer without bound CC-1065 can convert to the Z form with added salt, producing a situation where the polymer contains both the B and Z conformations. In 4.0 M sodium chloride, where the Z conformation is also predominate, the addition of CC-1065 causes chiral aggregates to form, and CC-1065 binds to the aggregates. The addition of dimethylformamide in the absence of CC-1065 or a simple dilution of the 4.0 M sodium chloride polymer solution with water also causes aggregation, indicating that the Z form of this polymer in 4.0 M sodium chloride is unstable with respect to an aggregated form.

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.