Hawkins, Rachel Marina; (2007) Design, Synthesis and Biological Evaluation of Novel PBD-Heterocycle Conjugates as Potential Transcription Factor Binding Inhibitors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Transcription factor binding sites in disease related genes are potential therapeutic targets for small molecules. This research project describes the solution phase chemical synthesis of a small library of PBD-heterocycle conjugates that bind in the minor groove of DNA and inhibit NF-Y binding. Pyrrolo[2,1-c][l,4]benzodiazepines recognise and covalently bind guanine residues in the minor groove of DNA resulting in antitumour and antibiotic activity. In addition, heterocyclic polyamides have exhibited sequence recognition properties via a non-covalent interaction. By varying the heterocycles used different sequences of DNA can be targeted. Heterocyclic polyamides comprised of two or three heterocycles were assembled in a combinatorial fashion from pyrrole, imidazole and thiazole building blocks. Heterocycles were coupled together using EDCI and DMAP followed by Hoc deprotection. Subsequent coupling cycles were repeated depending on the required polyamide length. The PBD building block synthesis was based on an approach reported by Fukuyama. However, it was discovered that the final step, a saponification, had produced a racemate. The introduction of THP and Alloc protecting groups allowed the successful synthesis of the enantiomerically pure PBD building block. The PBD was coupled to the heterocyclic polyamides and simultaneous Alloc/THP deprotections were performed to yield twenty-one active imine molecules. Finally, a series of assays were performed on the target compounds in order to assess the effect of different heterocycle combinations on biological activity. The assays investigated sequence recognition properties and DNA binding activity using footprinting and thermal denaturation techniques respectively. Functional assays were carried out to investigate the ability of the compounds to disrupt transcription factor binding. Cytotoxicity data using the K562 cell line and the NCI 60 human tumour cell line panel was also obtained. These assays allowed the molecules to be ranked and lead molecules were identified. A promising molecule, RMH041 (Py-Py-Im-PBD), exhibited impressive DNA binding affinity and the potential to inhibit NF-Y transcription factor binding. In conclusion, different combinations of heterocycles attached to the PBD unit significantly affect cytotoxicity, DNA binding affinity and the DNA sequence recognised.

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