Design and DNA binding of model anti-cancer drugs

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Science, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

This thesis describes the design and measurement of the DNA binding properties in the presence of formaldehyde of several acridine derivatives with primary amino-containing side chains. An aim of this work was to emulate the activation of Adriamycin by formaldehyde and consequent binding to DNA, using simpler, easier to synthesise compounds. Two derivatives of the anti-tumour agent N-[2-(dimethylamino)ethyl] acridine-4-carboxamide (designated C2-DACA and C2-DACA dimer in this thesis) were studied. The length of the side chain was varied, and the tertiary amine was modified to a primary amine. This was to facilitate interaction with formaldehyde and subsequent covalent binding of the formaldehyde-activated drug to DNA. The DNA-binding abilities of a derivative N-(2-aminoethyl)acridine-4- carboxamide.oxalate, C2-DACA, and its dimer were investigated using radiolabelled DNA electrophoretic crosslinking assays, and binding studies based on absorbance, but did not show DNA binding. Molecular modelling showed that the side chain of C2-DACA was too short and under considerable steric hindrance once intercalated and covalently bound to DNA. The length of the side chain proved to be insufficient to reach the preferred binding site. Molecular modelling also showed that extending the chain by 2 or 3 carbons would allow favourable interactions between the intercalating acridine derivative and DNA, and allow the side chain to covalently bind to the DNA. The compound arising from the molecular modelling N-(4-aminobutyl) acridine-4-carboxamide, C4-DACA, was synthesised in a similar manner to C2-DACA. DNA binding experiments including an in vitro transcription assay showed that formaldehyde-activated C4- DACA bound to DNA. The energetics of Schiff base and aminal formation were calculated using quantum mechanic molecular orbital methods. Formaldehyde was found to be the most energetically favourable aldehyde in both enthalpy and entropy. The overall binding reaction of a primary amine reacting with formaldehyde, and subsequently with guanine to produce an aminal was exothermic. Most of this energy came from the Schiff base formation, with an additional smaller amount from the subsequent reaction of the imine with the exocyclic amine of guanine.