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Seven dicationic 2,5-diarylfurans have been synthesized, and their interactions with poly(dA-dT) and the duplex oligomer d(CGCCAATTCGCG)2 were evaluated by Tm measurements. The inhibition of topoisomerase II isolated from Giardia lamblia, the inhibition of growth of G. lamblia in cell culture by these furans, and the effectiveness of these compounds against Pneumocystis carinii in the immunosuppressed rat model have been assessed. Strong binding affinities to poly(dA-dT) and to the oligomer were observed for the dicationic furans, and the interaction strength is directly correlated to the biological activity of the compounds. An X-ray structure for the complex of the dicationic amidine derivative, 2,5-bis(4-guanylphenyl)furan (1), with the oligomer demonstrates the snug fit of these compounds with the AATT minor-groove binding site and hydrogen bonds to AT base pairs at the floor of the minor groove. The stronger DNA binding molecules are the most effective inhibitors of topoisomerase II and G. lamblia in cell culture, and there is a correlation for both DNA interaction and topoisomerase II inhibition with the biological activity of these compounds against G. lamblia. Compound 1 is the most effective against P. carinii, it is more active and less toxic than pentamidine on intravenous administration and it is also effective by oral dosage. The results presented here suggest a model for the biological action of these compounds in which the dication first binds in the minor groove of DNA and forms a complex that results in the inhibition of the microbial topoisomerase II enzyme.

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition. O rganic cations that bind in the DNA minor groove have biological activities that range from anti-opportunistic infection to anticancer properties (1-5). Such compounds have also provided a wealth of fundamental information about nucleic acid recognition properties, and they continue to be important models in the study of nucleic acid complexes (1, 2, 6-8). Netropsin ( Fig. 1) was the first minor-groove-binding compound crystallized with a B-form DNA, and the structure of the complex provided clear suggestions about the molecular basis for AT base pair sequence-specific recognition (9). The structure also lead to proposals by the Dickerson and Lown groups for minor-groove binding netropsin analogs, lexitropsins, that could specifically recognize GC base pairs and could, thus, have extended sequence recognition capability (10-12). Initial efforts in the design of analogs of netropsin that could recognize GC base pairs, however, yielded compounds of limited specificity. A breakthrough in this area occurred with the discovery that the monocationic relative of netropsin, distamycin ( Fig. 1), could bind into the minor groove of some AT sequences of DNA as a stacked, antiparallel dimer (7,13,14). Replacement of pyrrole groups in distamycin by imidazole provided lexitropsins with improved GC recognition specificity through dimer complexes, and current design efforts in the pyrrole-imidazole polyamide system have reached a high level of success (15-18). With recent developments by the Dervan group, AT and TA as well as GC and CG base pairs can now be effectively distinguished in DNA sequences by polyamides (15-18).Interestingly, the polyamide system is the only one of the well known minor-groove binding motifs that has yielded conclusive evidence for formation of the stacked-dimer recognition unit, although recent evidence indicates that some monocationic cyanine dyes can form an array of stacked dimers in the DNA minor groove (19). No dications have been found to form the stacked-dimer recognition motif. Netropsin, the first minorgroove-binding agent to be characterized in detail and a dicationic relative of the monocation distamycin (Fig. 1), for example, does n...

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Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-benzimidazoles were screened for their inhibitory and fungicidal activities againstCandida albicans and Cryptococcus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC80s) comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC80 of ≤0.09 μg/ml, and the most potent compound against C. neoformans had an MIC80 of 0.19 μg/ml. Selected compounds were also found to be active againstAspergillus fumigatus, Fusarium solani,Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. It is clear from the data presented here that further studies on the structure-activity relationships, mechanisms of action and toxicities, and in vivo efficacies of these compounds are warranted to determine their clinical potential.

The syntheses of 12 new 2,5-bis[4-(N-alkylamidino)phenyl]furans are reported. The interaction of these dicationic furans with poly(dA-dT) and with the duplex oligomer d(CGCGAATTCGCG)2 was determined by Tm measurements, and the effectiveness of these compounds against the immunosuppressed rat model of Pneumocystis carinii was evaluated. At the screening dose of 10 mumol/kg, 9 of the 14 N-alkylamidino furans described here are more active than the parent compound 1. Substitution of an alkyl group of the amidino nitrogen, except for in 9, 13, and 15, resulted in higher affinity for DNA than the parent compound as judged by the larger delta Tm values and suggests enhanced van der Waals interactions in the bis-amidine-DNA complex. Five of the compounds, 3, 5, 7, 10, and 12, yield cyst counts of less than 0.1% of control when administered at a dosage of 10 mumol/kg. Five compounds, 1, 6, 8, 10, and 12, show significant activity at a dosage of approximately 1 mumol/kg; 12 is the most active derivative, and it is approximately 100 times more effective than pentamidine in this animal model.

ETS transcription factors mediate a wide array of cellular functions and are attractive targets for pharmacological control of gene regulation. We report the inhibition of the ETS-family member PU.1 with a panel of novel heterocyclic diamidines. These diamidines are derivatives of furamidine (DB75) in which the central furan has been replaced with selenophene and/or one or both of the bridging phenyl has been replaced with benzimidazole. Like all ETS proteins, PU.1 binds sequence specifically to 10-bp sites by inserting a recognition helix into the major groove of a 5′-GGAA-3′ consensus, accompanied by contacts with the flanking minor groove. We showed that diamidines target the minor groove of AT-rich sequences on one or both sides of the consensus and disrupt PU.1 binding. Although all of the diamidines bind to one or both of the expected sequences within the binding site, considerable heterogeneity exists in terms of stoichiometry, site–site interactions and induced DNA conformation. We also showed that these compounds accumulate in live cell nuclei and inhibit PU.1-dependent gene transactivation. This study demonstrates that heterocyclic diamidines are capable of inhibiting PU.1 by targeting the flanking sequences and supports future efforts to develop agents for inhibiting specific members of the ETS family.