Synthesis of novel CYP1 activated anticancer prodrugs

CYP1A1 and CYP1B1 are members of the cytochrome P450 enzyme superfamily which catalyses the oxidative metabolism of endogenous and exogenous compounds. CYP1A1 and CYP1B1 are known to be over-expressed in a wide variety of human tumours and provide a target for tumour-selective activation of anticancer prodrugs. CYP1B1 in particular has also been detected in pre-malignant tissue as well as metastatic cancer. The aim of this project was to develop anticancer prodrugs that show low toxicity to normal cells, but are selectively activated, at the site of a tumour, by CYP1 enzymes yielding cytotoxic metabolites that result in the demise of cancer cells whilst leaving normal cells unharmed. Initial compounds were designed by mapping onto the framework of 17β-estradiol. The latter is the only known endogenous substrate for CYP1B1 and undergoes regioselective hydroxylation to yield the known carcinogen 4-hydroxyestradiol. The compounds synthesised were evaluated for their anticancer activity and structure-activity relationship studied by in vitro MTT cytotoxicity assay, using a human breast cell line panel which has been characterised for CYP1 expression. This panel consists of drug resistant MCF7 breast tumour cell line with very low levels of CYP1 expression, MCF7 treated with 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) which induces CYP1A1 expression, MDA-MB-468 (MDA468) breast tumour cell line which constitutively expresses both CYP1A1 and CYP1B1 and MCF10A, a non-tumour cell line which has no basal CYP expression. Lead compounds that showed selectivity and high differential toxicity between tumour cells which express the target CYP1 enzymes versus non-expressing cell lines were identified. Inhibitor studies using known CYP1 inhibitors, acacetin and α-naphthoflavone were carried out to provide supporting evidence that the test compounds had been activated by CYP1 enzymes. Metabolism studies using isolated CYP enzymes provided evidence of CYP enzyme substrate selectivity. Metabolite structures were initially proposed based on MS data (molecular ions and fragmentation patterns). These were confirmed by authentic standards synthesis and co-elution studies. Four novel series of compounds evolved from this project and their structure-activity relationship was studied. The series consists of stilbenes, chalcones, indanones as conformationally restricted trans configured chalcones and heterocyclic analogues, 2-pyridones, pyridines, 2-substituted pyridines and pyrimidine. Based on CYP1B1 estradiol 4-hydroxylase activity implicated in cancer and cancer preventative agent resveratrol (trans-4,3‟,5‟-trihydroxystilbene) structure, a series of cis- and trans-stilbene prodrugs and chalcone prodrugs were designed to undergo selective aromatic hydroxylation to form active tyrosine kinase inhibitors or antimitotic agents. A lead compound DMU 212 (trans 3’,4‟,5‟4-tetramethoxy stilbene) prepared by the Wittig reaction was identified from the stilbene series and found to be highly toxic to the MDA468 cancer cells (IC50 0.001µM) and significantly less toxic to normal breast MCF10A cells (IC50 4 µM) revealing a De Montfort University iii 4300-fold differential cytotoxicity. DMU 212 was preferentially metabolized by CYP1A1 rather than by CYP1B1 to the active catechol metabolite DMU 214 a predicted tyrosine kinase inhibitor and two para-O-demethylated metabolites DMU 291 and DMU 281. Lead chalcones, prepared by the Claisen-Schmidt condensation, included DMU 135 (3,4-methylenedioxy 3‟,4‟5‟-trimethoxychalcone) and DMU 407 (2‟,4‟-dimethoxy-3,4-methylenedioxychalcone) which showed low toxicity to MCF10A cells (IC50 2.3 µM and 14 µM respectively) but were highly toxic to MDA468 cells (IC50 0.006 µM and 0.44 µM respectively). Preferential metabolism of DMU 407 by CYP1A1 generated the active O-demethylated catechol metabolite DMU 409. However chalcones once in solution were found to undergo rapid photoisomerisation under ambient light conditions resulting in a mix of cis and trans isomers. The problem of photoisomerisation was overcome by synthesis of indanones as conformationally constrained chalcone analogues. A lead indanone DMU 5407 (5,7- dimethoxyphenyl-3-(3,4-methylenedioxyphenyl) indanone) was non-toxic to MCF10A cells (IC50 > 100µM) but highly toxic to the MDA468 cells (IC50 0.05 µM). 4,6-Diaryl-2-pyridone and ionisable 2,4-diarylpyridine series was synthesised to increase prodrug activity and/or selectivity for a particular CYP1 isozyme as a result of the greater rigidity of the heterocyclic ring system. A lead pyridone DMU 943 (6-(2,4-dimethoxyphenyl)-4-(3,4- methylenedioxyphenyl)-1H-pyidin-2-one) was prepared from the corresponding chalcone DMU 407 by reaction with 2-acetamidoacetamide. This pyridone showed low toxicity to MCF10A cells (IC50 40 µM) and high toxicity to MDA468 cells (IC50 0.04 µM). Preferential CYP1A1 metabolism generated active O-demethylated catechol metabolite DMU 1137 (4-(3,4- dihydroxyphenyl)-6-(2,4-dimethoxyphenyl)-1H-pyridin-2-one). A lead pyridine DMU 981 (2- (2,4-dimethoxyphenyl)-4-(3,4-methylenedioxyphenyl) pyridine), which was prepared by deoxygenation of pyridone DMU 943, was completely non-toxic to the MCF10A cells but was highly toxic to all the cancer cell lines examined (MDA468 IC50 0.08 µM and TCDD treated and untreated MCF7 IC50 0.3 µM respectively) with a remarkable 1250-fold tumour selectivity. CYP1A1 metabolism of DMU 981 generated active O-demethylated catechol metabolite DMU 5341 (4-(3,4-dihydroxyphenyl)-2-(2,4-dimethoxyphenyl)pyridine). A pyrimidine analogue DMU 1140 (6-(3,4-dimethoxyphenyl)-4-(3,4-methylenedioxypheny) pyrimidine) also showed exciting activities comparable to DMU 981 (MDA468 IC50 0.08 µM and TCDD treated and untreated MCF7 IC50 0.2 µM respectively) revealing greater than 1250-fold tumour selectivity. The mechanism of activation seems to involve the dealkylation of the methylenedioxy group by selective CYP1 isozyme to generate a toxic catechol metabolite. In conclusion, promising new candidates for tumour selective cancer therapy have been identified and three of these compounds (DMU 212, DMU 943 and DMU 981) are currently being taken forward for further development