Identification of potent water-soluble DNA-dependent protein kinase (DNA-PK) inhibitors using a small-molecule library approach [abstract]

4156 The clinical response to DNA-damaging anticancer therapies may be compromised by cellular DNA repair processes, and agents that impede DNA repair are thus of potential therapeutic interest as chemo- and radio-sensitising agents in the treatment of cancer. The phosphatidylinositol-3-kinase related kinase (PIKK) family member DNA-dependent protein kinase (DNA-PK) is a key player in the non-homologous end-joining pathway of DNA double-strand break (DSB) repair. Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 μM), guided the subsequent development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 13 nM). Although proof-of-principle studies with NU7441 confirmed promising activity in vitro as a chemo- and radio-potentiator in a range of human tumour cell lines, further biological studies were hampered by sub-optimal pharmaceutical properties. Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. This approach predicted the 1-, 8-, and 9-positions on the pendant dibenzothiophen-4-yl substituent of NU7441 as tolerant to substitution without detriment to DNA-PK inhibitory activity. The introduction of suitable functionality (e.g. OH, NH2 CO2H) at these positions provided a platform for the synthesis of focussed libraries of compounds bearing water-solubilising amine substituents. This could be achieved directly at the dibenzothiophene 1-position through nitration and subsequent functional group modification. By contrast, functionalisation of the dibenzothiophene 8- and 9-positions of NU7441 entailed incorporation of the required substituent into a synthetic precursor, prior to construction of the dibenzothiophene ring and coupling to the chromenone scaffold. Interestingly, substitution with a methyl or allyl group at the 3-position of the dibenzothiophen-4-yl ring enabled the separation by chiral hplc of atropisomers, as a consequence of restricted rotation about the dibenzothiophene-chromenone bond, albeit with a marked loss of potency (R = 3-Me, IC50 = 2.5 μM). Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation (dibromoethane or methyl bromoacetate) or N-acylation (chloroacetyl chloride or 2-bromopropionyl chloride) of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of compounds that combined potent DNA-PK inhibition with excellent aqueous solubility (20-40 mg/mL as acid salts), without compromising cellular activity. Prominent amongst these derivatives is KU-0060648 (DNA-PK IC50 = 8.6 nM), which exhibits 20-1000 fold selectivity for DNA-PK over related PIKK enzymes and PI3K family members.