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Unfused tricyclic aromatic ring systems 1-6 with one or two cationic side chains have been synthesized and their interactions with DNA and synthetic polymers probed with a variety of techniques. Molecular mechanics calculations indicate that the torsional angle between ring planes in the minimum energy conformation of the tricyclic molecules can range from 0 degree to as high as 50 degrees depending on the type of rings and substituents. Viscometric titrations with linear and supercoiled DNA, linear dichroism, and NMR studies indicated that all compounds with torsional angles of approximately 20 degrees or less bind to DNA by intercalation. The more highly twisted intercalators caused significant perturbation of DNA structure. Unfused intercalators with twist angles of approximately 20 degrees have reduced binding constants, suggesting that they could not form an optimum interaction with the DNA base pairs. Unfused intercalators with twist less than 20 degrees formed strong complexes with DNA. The structures of these unfused intercalators are more analogous to typical groove-binding molecules, and an analysis of their interaction with DNA provides a better understanding of the subtle differences between intercalation and groove-binding modes for aromatic cations. The results indicate that intercalation and groove-binding modes should be viewed as two potential wells on a continuous energy surface. The results also suggest design strategies for intercalators that can optimally complement DNA base pair propeller twist or that can induce bends in DNA at the intercalation site.

Sixteen unfused heterobiaromatic and biphenyl compounds substituted with an amino side chain (protonated in water) have been tested for (i) binding with DNA and (ii) their effect on the digestion of the DNA double helix by a bleomycin-iron complex. Only the DNA intercalating molecules amplify the digestion of DNA. One 2,2'-bipyridine derivative tested is an inhibitor of the bleomycin reaction because it removes ferrous ion from the bleomycin complex. Polarity of the intercalating unfused biaromatic system is of primary importance for effective binding of the molecule with native DNA and, at the same time, for its amplification activity. The molecules that have the biaromatic system polarized extensively in the direction of the side cationic chain, so that the intercalating sites constitutes a positive part of the dipole, show strong binding with DNA and good amplification activity. For strong intercalative forces that determine the amplification activity, it is important that both the heteroaromatic subsystems of the molecule have positive ends of their dipoles positioned away from the side chain. This work provides general guidelines for synthesis of new highly effective bleomycin amplifiers.

Three simple and independent tests have been introduced for studying the effect of DNA intercalating compounds on the bleomycin-mediated digestion of DNA in vitro. These methods are based on hyperchromic changes of DNA solution, changes in viscosity of DNA solution, and HPLC quantitative analysis of the four bases released from digested DNA. All three tests give comparable results. However, the viscometric method is technically the simplest and at the same time the most sensitive. The amplification of the bleomycin-mediated degradation of DNA by three unfused heteropolyaromatic intercalator molecules, namely N-[2''-(dimethylamino)ethyl]-4-thien-2'-ylpyrimidin-2-amine (1N), N,N-dimethyl-2-[(4'-thien-2''-ylpyrimidin-2'-yl)thio] ethylamine (1S), and newly synthesized 2,5-bis[2'-[[2''-(dimethylamino)ethyl]thio]pyrimidin-4'yl]thiophene (2) correlates well with the respective DNA binding constants for these compounds and is concentration dependent. The amplification activity of these compounds increases with increasing concentrations. The strongly binding compound 2 is the best amplifier of bleomycin in vitro found so far. Fused heteropolyaromatic systems, like ethidium bromide, are modest amplifiers of bleomycin at low concentrations but strongly inhibit the bleomycin chemistry at high concentrations.

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