Despite these efforts to prevent oxidative metabolic degrada

Despite these efforts to prevent oxidative metabolic degradation, and showed only marginal improvements in metabolic stability and still exhibited high plasma clearances as shown in . High molecular weight as well as relative high log values could be contributing factors to the poor PK profiles of and related derivatives. We were pleased to discover several compounds with reduced molecular weight where is hydrogen which retain high levels of biochemical and cellular potency (, compounds and ). At this stage, a thorough investigation of the N-1 substituent was desired prior to readdressing the issue of metabolic stability for these truncated analogs. A high-resolution X-ray co-crystal structure of complexed to ACK1 was obtained (). As predicted in our earlier modeling, two hydrogen bonds to the hinge region of the ACK1 ATP-binding site along with an additional H-bond to the carbonyl oxygen of Thr205 serve to anchor into the binding pocket. In addition, the dimethyl-phenyl group makes a deeper contact in the protease inhibitor selectivity pocket as compared to the amide series . Moreover, two regions of are exposed to solvent. The polar piperazine moiety directly faces the solvent, creating a stabilizing interaction that aids in inhibitor binding or recognition. Also, the terminus of the N-1 side chain is exposed to solvent by protruding through a lipophilic channel present in the phosphate-binding region of the active site. The latter observation served to guide subsequent SAR work: It was hoped that binding could be improved by strengthening the solubilizing interaction between the terminus of the N-1 side chain and exposed solvent while maintaining a low overall molecular weight by keeping the N-6 phenyl ring unsubstituted. A variety of -(aryl)--(phenyl)-1-pyrazolo[3,4-]pyrimidine-3,6-diamines possessing polar solubilizing groups linked to N-1 by a one- to three-carbon spacer were synthesized and tested (). While most of the cyclic amine derivatives – (two-carbon spacer) were potent inhibitors of ACK1, inhibition was weakened when an amide linker was employed (compounds and ). Non-cyclic amino groups attached to two- and three-carbon linkers were also investigated. While the ,-dimethylaminoethyl derivative exhibited slightly reduced cellular potency, the ,-dimethylaminopropyl derivatives and strongly inhibited ACK1 ( of 5nM and cellular IC of 20nM for ). Replacing the dimethylamine with the more lipophilic diethylamine and pyrrolidine groups (compounds and ) led to reduced cellular activities compared to . Oxygenated carbon chains at were also investigated (, compounds –). The diols and exhibited excellent biochemical and cellular potencies that were comparable to those of and . However, ACK1 inhibition was weakened in converting the primary alcohol of into a dimethylamine (compound ). Compounds , and were each synthesized by a five-step process from and an appropriately mono-substituted hydrazine as described above (). Compound was further transformed into either or by ester hydrolysis followed by BOPCl-mediated amide formation (). The synthesis of derivatives - is detailed in . Dihydroxylation of either or with a mixture of OsO and NMO generated and , respectively. Reductive amination of the aldehyde derived from diol or produced –, respectively. Compound was transformed into by selective tosylation of the primary alcohol followed by epoxide formation with NaOMe. Finally, was obtained from in low yield by exposure to dimethylamine in ethanol. The biological data in show that structural manipulation did not significantly affect ACK1 inhibitory activity (). However, a moderate improvement in PK properties was observed for a key analog. The male Sprague–Dawley rat pharmacokinetic profile of , one of the most potent analogs in , showed a significant improvement in oral bioavailability relative to earlier analogs (=30%; 4mg/kg po dose). Modulation of selectivity for ACK1 over other related kinases was possible for this series by way of small structural changes. As an example, compound displayed good selectivity against the related kinases KDR, Tie-2, and Jak3, but very potently inhibits BTK and LCK, which possess high structural similarity to ACK1 (). However, modification of the structure of to include a single fluorine atom on the N-6 phenyl ring (compound ) eliminated binding to KDR, Tie-2, and Jak3, while greatly diminishing binding to both LCK and BTK relative to ACK1.