Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines benzo[c]phenanthridine) and 4,benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(π)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 mM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.Benzo[c]phenanthridine derivatives are a class of substances possessing a broad spectrum of pharmacological activities. In the literature many alkaloids with a benzo[c]phenanthridine ring system and interesting biological properties are mentioned. Especially antitumoural, cytotoxic and antileukaemic activities have been described for members of that group of alkaloids like for example fagaronine (Simeon et al. 1989). In our laboratory we developed a very short synthetic route to benzo[c]phenanthridine derivatives with an exocyclic primary amine group in the ortho position to the endocyclic nitrogen Kock et al. 2005). The NCI (National Cancer Institute, Bethesda, USA) results of the in vitro efficacy tests against 60 different tumour cell lines show meangraph midpoints (MG-MID) as averaged activity parameters for the 50% growth inhibitory concentration (GI 50 ) of 0.98 mM for BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and 0.28 mM for BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) ( fig. 1). These results demonstrate a better activity for our compounds than for fagaronine. BP-D7 showed excellent in vivo activity as well

A new and convenient two-step synthesis of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines and 6-aminobenzo[c]phenanthridines (BPs and BP-Ds) was developed recently in the authors' laboratory. These compounds revealed a high antitumoral activity in in vitro and in vivo test systems. In particular, 11-phenyl-substituted derivatives with two or three methoxy groups showed good activity. It was not clear if the dihydro-derivatives (BPs) were transformed enzymatically into the phenanthridines (BP-Ds), thus acting as prodrugs. The in vitro metabolism of several of these cytostatically active 6-aminobenzo[c]phenanthridines was investigated using human and porcine liver microsomes and a range of expressed human cytochrome P450 enzymes. High-performance liquid chromatography and liquid chromatography-mass spectrometry analysis were used for the quantification and structural identification of the observed metabolites. Aromatic hydroxylation was observed to be the major metabolic pathway in addition to a number of other metabolites. The formation of N-hydroxy- and 6-oxo-derivatives was detected only in very small amounts. BP derivatives are not prodrugs of BP-Ds and no significant differences between human and porcine microsomes were observed, confirming the pig as a good model for metabolism studies of these compounds.

The synthesis of various new structures of a library of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines (BP) and 11-substituted 6-aminobenzo[c]phenanthridines (BP-D) is presented. These structures, further synthetic modifications, and the preparation of follow-up products which delivered about 40 new derivatives are described. Their potential as antiproliferative drug candidates was investigated by comparison of NCI 60 developmental therapeutics program (DTP) human tumor cell line screening data based on the results of in vitro tumor cell growth inhibition, including about 40 hitherto unpublished compound test results with up to 60 cancer cell lines. NCI-COMPARE studies helped to suggest the modes of action of the highly active antiproliferative drugs. These findings are supported by in vitro biological investigations showing either inhibition of tubulin polymerization and depolymerization or topoisomerase inhibition. Together with physicochemical parameters of the drug candidates, structure-activity relationships are critically discussed. Tubulin interaction or inhibition of topoisomerase I and IIα/β activity are two rationales that can explain the antiproliferative activity observed in the NCI 60 DTP human tumor cell line screen. However, it can also be reasonably assumed that these compounds address several targets, thus prohibiting the identification of simple structure-activity relationships. The new structures described herein are thought to act as so-called multitarget drugs, thus being of special interest in the area of multidrug resistance.