DNA minor groove binders, Hoechst 33258 and Hoechst 33342, have been reported to protect against radiation-induced DNA-strand breakage, but their mutagenicity and cytotoxicity limit their use as protectors of normal tissue during radiotherapy and as biological radioprotectors during accidental radiation exposure. On the basis of these observations, two new nontoxic disubstituted benzimidazoles were synthesized, one having two methoxy groups (5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl]benzimidazole, 5) and another having a methoxy and a hydroxyl group (5-(4-methylpiperazin-1-yl)-2-[2'[2''-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl]benzimidazole, 6) ortho to each other on the phenyl ring. The radiomodifying effects of these nontoxic ligands were investigated with a human glioma cell line exposed to low linear energy transfer radiation by determining cell survival and cell proliferation compared with effects of the parent compound, Hoechst 33342. Cytotoxicity assayed by analyzing clonogenicity, cell growth, and metabolic viability showed that both 5 and 6 were nontoxic at 100 microM after 72 h of exposure, whereas Hoechst 33342 resulted in lysis of 77% of these cells in 24 h. Macrocolony assay (clonogenicity) showed that 73%, 92%, and 10% of the cells survived when treated with 100 microM 5, 6, and Hoechst 33342, respectively. Both 5 and 6 did not affect the growth of BMG-1 cells. At 10 microM, 5 and 6 showed 82% and 37% protection against radiation-induced cell death (macrocolony assay) while 100% protection was observed against growth inhibition. Disubstitution of the phenyl ring has not only reduced cytotoxicity but also enhanced DNA-ligand stability, conferring high degree of radioprotection.

DNA minor groove ligands provide a paradigm for double-stranded DNA recognition, where common structural motifs provide a crescent shape that matches the helix turn. Since minor groove ligands are useful in medicine, new ligands with improved binding properties based on the structural information about DNA-ligand complexes could be useful in developing new drugs. Here, two new synthetic analogues of AT specific Hoechst 33258 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole (DMA) and 5-(4-methylpiperazin-1-yl)-2-[2'[2''-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl] benzimidazole (TBZ) were evaluated for their DNA binding properties. Both analogues are bisubstituted on the phenyl ring. DMA contains two ortho positioned methoxy groups, and TBZ contains a phenolic group at C-4 and a methoxy group at C-3. Fluorescence yield upon DNA binding increased 100-fold for TBZ and 16-fold for DMA. Like the parent compound, the new ligands showed low affinity to GC-rich (K approximately 4 x 10(7) M(-1)) relative to AT-rich sequences (K approximately 5 x 10(8) M(-1)), and fluorescence lifetime and anisotropy studies suggest two distinct DNA-ligand complexes. Binding studies indicate expanded sequence recognition for TBZ (8-10 AT base pairs) and tighter binding (DeltaT(m) of 23 degrees C for d (GA(5)T(5)C). Finally, EMSA and equilibrium binding titration studies indicate that TBZ preferentially binds highly hydrated duplex domains with altered A-tract conformations d (GA(4)T(4)C)(2) (K= 3.55 x 10(9) M(-1)) and alters its structure over d (GT(4)A(4)C)(2) (K = 3.3 x 10(8) M(-1)) sequences. Altered DNA structure and higher fluorescence output for the bound fluorophore are consistent with adaptive binding and a constrained final complex. Therefore, the new ligands provide increased sequence and structure selective recognition and enhanced fluorescence upon minor groove binding, features that can be useful for further development as probes for chromatin structure stability.