Development and validation of an improved HPLC method for the control of potentially counterfeit isometamidium products.

Isometamidium, a mixture of related substances of which 8-(3-m-amidinophenyl-2-triazeno)-3-amino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (M&B4180A) is the principal active component, is the only chemical agent available for prophylaxis of veterinary trypanosomiasis. A method for the simultaneous quantitation of the major constituents M&B4180A, 3-(3-m-amidinophenyl-2-triazeno)-8-amino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (M&B38897), 7-(m-amidinophenyldiazo)-3,8-diamino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (M&B4250) and 3,8-di(3-m-amidinophenyltriazeno)-5-ethyl-6-phenylphenanthridinium chloride dihydrochloride (M&B4596) is described. The related substances are resolved on a Gemini C18 column (150 mm x 4.6 mm, 5 microm) using a mobile phase composed of a mixture of acetonitrile and 50 mM ammonium formate buffer pH 2.8 (25:75 v/v) at a flow rate of 1 ml/min with UV detection at 320 nm. The method is compatible with electrospray ionisation mass spectrometry and provides a tool for the control of substandard and counterfeit commercial preparations of isometamidium.

Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts.

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.

Potential transition state phosphoramidate inhibitors of beta-tubulin as antifilarial agents.

Transition state phosphoramidate inhibitors of beta-tubulin were designed as potential antifilarial agents. The reaction of 2-aminobenzimidazole with diisopropyl phosphite and carbon tetrachloride at a low temperature gave the unexpected 1-diisopropoxyphosphoryl-2-aminobenzimidazole, which on heating gave the novel benzimidazole derivative, 2-(diisopropoxyphosphoryl)aminobenzimidazole. Both products were fully characterized and the synthetic procedure to both compounds was optimized. The procedure was used to prepare the related 5-benzoyl-2-(diisopropoxyphosphoryl)aminobenzimidazole and 5-benzoyl-2-(diethoxyphosphoryl)aminobenzimidazole (1d). In a preliminary trial against Brugia pahangi compound 1d was found to have no antifilarial activity. This lack of activity may be attributed to its extreme insolubility and thus low bioavailability. The synthesis of analogous, more soluble, phosphorothioate-substituted benzimidazoles using the same methods may yield compounds with greater antifilarial activity.

The effect of 12-alkoxy modification on the in vitro antileukaemic activity of N-methyl 2,3,8,9-tetramethoxybenzo[c]phenanthridinium salts.

Some members of a series of 12-alkyloxy benzo[c]phenanthridines are potent inhibitors of the growth of P388 tumour cells in vitro, with a strong dependence on the nature of the 12-substituent. Analogues with a quaternary nitrogen in the side chain bind strongly to DNA but are less active against the tumour cells. The multi-drug-resistant cell line Pr8/22 shows less sensitivity to the new compounds. K562 Human leukaemia cells undergo differentiation in the presence of the benzo[c]phenanthridine derivatives with a structure-activity relationship which does not correlate well with potency against the P388 cell line.

The role of the iminium bond in the inhibition of reverse transcriptase by quaternary benzophenanthridines.

The quaternary benzo[c]phenanthridines fagaronine, nitidine and O-methylfagaronine have been reviewed as potential antitumour and antiviral agents. Their mode of action has not been established, but their ability to bind with DNA by intercalation is believed to be involved. Of the three synthetic analogues of O-methylfagaronine which we have synthesized, methoxidine and ethoxidine are active against HIV-1 reverse transcriptase (IC50 values 2.8 microM and 2.4 microM respectively) whereas hydroxidine is inactive. One of the prerequisites for the enzyme inhibitory activity of this class of molecule is the presence of an iminium group--it is well known that a positive charge on a polyaromatic nucleus facilitates intercalative binding with DNA. Through UV spectrophotometric and modelling studies, we have shown that the iminium bond plays a more fundamental role in enzyme inhibition through its susceptibility to nucleophilic attack--the inactive analogue hydroxidine has a non-electrophilic iminium bond. Consequently, we have demonstrated that iminium bond electrophilicity is a parameter which needs to be considered in ternary complex formation with reverse transcriptase.

Molecular modelling of the interaction between DCMU and QB-binding site of photosystem II.

The preferred binding orientations for the herbicide DCMU within the QB-binding of the D1 protein model from a photosystem II reaction centre have been determined. Calculation of the intermolecular energy between the herbicide and the binding site has been instrumental in obtaining optimum positions reinforced by experimental results from mutation studies and herbicide binding to analogous bacterial reaction centres. We have shown that two binding sites are possible, one involving a hydrogen bond to the His 215 residue of the QB-binding site and the other to the Ser 264 residue. In both cases, it appears to be the van der Waals forces which are more important for the stabilization of the interactions.