The design, synthesis and evaluation of some duocarmycin prodrugs

The duocarmycins are anti-tumour antibiotics that derive their biological activity through sequence-selective alkylation of the minor groove of duplex DNA. A series of duocarmycin analogues including deshydroxy CI (l,2,7,7a-tetrahydrocyclopropa[c] indol-4-one) and CBI (l,2,9,9a-tetrahydrocyclopropa[c]benz[c]indol-4-one) derivatives were synthesised as potential bio-oxidatively activated prodrugs via Cytochrome P450 mixed function oxidase mediated hydroxylation. A number of isoforms of cytochrome P450 are expressed in tumours and could be used as the basis for tumour selective deshydroxy duocarmycin prodrug activation. Synthesis of the deactivated alkylation subunits of the prodrugs utilised an intramolecular 5-exo-trig free-radical cyclisation onto a tethered vinyl chloride. Upon oxidative activation, the resulting chloroalkane reacts to form the spirocyclopropane critical for DNA alkylation. The extended structure was synthesised through deprotection and coupling with 5-methoxyindole-2-carboxylic acid. Synthesis of enantiomerically pure deshydroxy derivatives was initiated and involved a key Pd(II)catalysed intramolecular cross-coupling reaction followed by hydroboration-oxidation and chlorination to form the 3-chloro-l,2,3,4-tetrahydroquinoline ring expanded and related analogues. The required analogues were synthesised efficiently and in high yields with confirmation by NMR, mass spectrometry and elemental analysis. The oxidative metabolism of deshydroxy Boc-CI, CI-MI, Boc-CBI and CI-MI in rat and human NADPH supplemented liver microsomes as a rich source of cytochrome P450 enzymes and using HPLC analysis showed the formation of numerous hydroxylation metabolites including metabolites that co-eluted with authentic hydroxylated duocarmycins. In A2780 human ovarian cancer and U20S osteosarcoma cell lines, the IC50 values were greater than 500nM for all deshydroxy duocarmycin derivatives investigated. In comparison duocarmycin analogues had IC50 values at least 250-fold lower (A2780 IC50 for CI-MI was 2.6nM and 0.5nM for CBI-MI). Incubation of selected deshydroxy duocarmcyins with NADPH supplemented rat liver microsomes restored their DNA alkylation properties. Specifically, DNA cleavage studies with aliquots of the metabolite and double stranded supercoiled DNA (plasmid ΦF174 RF 1) showed the formation of open circular (nicked) DNA. The results show the potential of deshydroxy duocarmycin as prodrugs that are activated via cytochrome P450 mediated hydroxylation to restore their DNA covalent interactions and potent cytotoxicity