Pyrazolo[1,5-a]pyridines: alpha-isoform-selective inhibitors of phosphatidylinositol-3-kinase (PI3K)

Abstract

The Class 1A phosphatidylinositol 3-kinases (PI 3-kinases) operate downstream of receptor tyrosine kinases. By catalyzing the phosphorylation of PIP2 to PIP3 they recruit Akt to the membrane, where it is activated through phosphorylation at Thr308 by PDK1. They are comprised of a common p85 regulatory subunit and one of three catalytic subunits (p110α, -β or -δ). The p110α and p110β isoforms are ubiquitous, whereas the δ isoform is predominantly expressed in leucocytes. The p110α isoform is often over-expressed and/or mutated in tumors, making it a prime target for inhibitory drugs. A wide range of chemotypes have been shown as ATP site inhibitors of the class 1A PI 3-kinases, but the majority of these show little or only modest selectivity towards the p110α isoform. This is also reflected by the initial series of PI 3-kinase inhibitors selected for clinical trial. In a search for more p110α-selective inhibitors, we explored structure-activity relationships around the known class of imidazo[1,2-a]pyridines, exemplified by the compound PIK-75 [(E)-N'-((6-bromoimidazo[1,2-a]pyridin-3-yl)methylene)-N,2-dimethyl-5-nitrobenzenesulfonohydrazide], which showed high potency for p110α (IC50 12 nM) and good selectivity for p110α over the other isoforms (25-fold over p110β and 80-fold over p110δ). An initial study of the heterocyclic unit showed generally tight structure-activity relationships (SAR), but discovered the superiority of the pyrazolo[1,5-a]pyridine chromophore. Studies of analogues of this demonstrated the desirability of small electron-withdrawing substituents at the 5-position for retaining both potency and selectivity. Replacements for the hydrogen bond accepting nitro group on the appended phenyl ring were also sought, with cyano and bromo proving most suitable. More soluble analogues could be prepared by placing weakly basic groups on either the sulfonohydrazide nitrogen or at the 2-position of the appended benzene ring. These compounds broadly retained potency against, and selectivity for, the p110α isoform. Modelling studies showed that the key interactions stabilizing the compounds are H-bonds between the drug N-1 nitrogen atom and the backbone NH of Val-882, and between the nitro group on the arylsulfonyl moiety and the backbone NH of Asp-884 and Ala-885. The high selectivity for the p110α isoform was explained using a p110α homology model derived from the p110γ structure, which identified a hydrogen bond interaction between one of the oxygen atoms of the sulfonyl group and the NH of a histidine (His855) that is unique to the α isoform. Selected compounds were screened in a panel of established (OVCAR3 ovarian, HCT116 colon, H460 lung) and early-passage human melanoma cell lines, typed for PI 3-kinase E545K and H1047R mutations and PTEN, p53 and BRAF expression. Cell lines with similar genetic backgrounds showed broadly similar sensitivities to the compounds. The pyrazolo[1,5-a]pyridines showed good stability in human plasma, but varying microsomal stabilities. Results from current work focused on improving stability, and from efficacy studies in human tumour xenografts, will be reported.