A series of novel [3a,4]dihydropyrazolo[1,5a]-pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R 3 ) and back-pocket (R 4 ) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. KEYWORDS: PI3K-β inhibitor, PTEN-null, phosphatidylinositol-3-kinase, pyrazolopyrimidine, structure−activity relationship T he phosphatidylinositol 3-kinases (PI3Ks) are members of a larger family of lipid kinases that phosphorylate the inositol moiety of membrane-associated phosphatidyl inositols. 1 PI3Ks catalyze the conversion of phosphatidylinositol-4,5-diphosphate (PIP 2 ) to phosphatidylinositol-3,4,5-triphosphate (PIP 3 ), which allows recruitment of proteins containing the PIP 3 -binding pleckstrin homology (PH) domain to the plasma membrane. The PI3K class I family comprises four isoforms (α, β, δ, and γ), with the α and β isoforms most widely expressed. Each is a heterodimer consisting of an isoform-specific p110 catalytic subunit and a regulatory subunit.PI3Ks are an important mediator of receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) signal transduction and are involved in the regulation of cellular metabolism, survival, and proliferation. 2 Aberrant activation of the PI3K pathway is strongly associated with malignant transformation, through either overexpression of PI3Kα, mutations that lead to constitutive activity of PI3Kα, or loss of function of the tumor suppressor PTEN (phosphatase and tensin homologue), which opposes PI3K function by converting PIP 3 to PIP 2 . 3−9 While the PI3K isoforms share the same substrate specificity, they have distinct biochemical roles and cellular functions. 10 In particular, there is considerable evidence that in PTEN-null tumors, malignant transformation is primarily driven by the PI3Kβ isoform. 11−15 The development of PI3Kβ-selective inhibitors is therefore an attractive target for the treatment of PTEN-null tumors.We have previously disclosed imidazopyrimidones (1) and triazolopyrimidones (2), as shown in Figure 1, which are potent and selective inhibitors of PI3Kβ. 16,17 However, the compounds were not suitable for in vivo studies due to high clearance in rat and mouse. Both 1 and 2 contain an embedded benzylamine moiety, a potential site for metabolism, which might contribute to the high clearance. We therefore designed a pyrazolo-pyrimidine scaffold, in which the benzyl group is attached to a carbon atom rather than a nitrogen, in an attempt to improve clearance.Pyrazolopyrimidines were generally prepared according to previously reported methods 18−20 as shown in Scheme 1. An α-cyanocarbonyl compound I is alkylated 21 with a benzyl bromide II and then condensed with hydrazine to afford the key aminopyrazole intermediate IV. The aminopyrazole is then condensed with a β-carbonyl-containing ester V to afford the 7-hydroxypyrazolopyrimi...