During the course of our ACK
During the course of our ACK1 inhibitor program, a high-throughput screening (HTS) campaign of our internal sample collection was conducted. A series of -aryl pyrimidine-5-carboxamides of the general structure () was identified. Earlier studies of the structure–activity relationship (SAR) of identified Nicotinamide australia as one of the more potent inhibitors within this series in both biochemical and cellular autophosphorylation assays., , (ACK1 =0.02μM, Cell IC=0.1μM). Unfortunately, this compound series generally displayed poor selectivity against several other kinases (data not shown) and a poor pharmacokinetic profile in male Sprague–Dawley rats (: Cl=3.8L/h/kg, AUC=76 μgh/L; 0.4mg/kg iv). The X-ray co-crystal structure of the close analog bound to the ACK1 catalytic kinase domain was subsequently determined at 2.3Å resolution (). Inspection of the three-dimensional structure of the complex revealed a binding mode in which the aminopyrimidine moiety participates in key hinge interactions: the pyrimidine nitrogen forms a hydrogen bond with the NH from Ala208, and the carbonyl of Ala208 H-bonds to the NH of the 2-amino group. The 4-position of the aryloxy moiety is oriented towards a solvent-exposed area, while the dimethyl-phenyl group is perfectly positioned to enter a deep hydrophobic pocket. Hydrophobic interactions within this selectivity pocket, combined with an additional H-bond with the conserved threonine (Thr205) gatekeeper amino-acid residue, are key features found in several potent kinase inhibitors, such as those described for the related tyrosine kinase p56 (LCK). Molecular modeling suggested that the inhibitory activity of series might be improved upon by taking advantage of the additional Thr205 hydrogen bond. Following this rationale, several modifications of were designed which tied the amide carbonyl and the ether oxygen into a cyclic structure, while rotating the NH into a more optimal position to interact with Thr205. The new designs were modeled into the ATP-binding site and the most promising candidates were selected for synthesis. These efforts identified ,-diaryl-1-pyrazolo[3,4-]pyrimidine-3,6-diamines as a novel series of inhibitors of ACK1, of which compound is a representative example (, ). Compounds of the general structure were synthesized as outlined in by a five-step procedure. Chlorination of was achieved with a mixture of PCl and POCl. The resulting acid chloride was coupled with an appropriately substituted aniline in the presence of an acid-scavenger and then treated with 3-methyl-3-methoxybutylhydrazine to produce . Cyclization of with PCl in refluxing toluene gave , and subsequent chloride displacement with an appropriate aniline provided compounds and –. Early exploratory work on the new series established that a functionalized aliphatic chain at N-1 of the ,-diaryl-1-pyrazolo[3,4-]pyrimidine-3,6-diamine core was beneficial for ACK1 inhibition. Also, the replacement of the aromatic groups at either N-3 or N-6 of the 1-pyrazolo[3,4-]pyrimidine-3,6-diamine scaffold with an alkyl group or a hydrogen led to significantly reduced levels of ACK1 inhibition (data not shown). Compound , which possesses a 3-methyl-3-methoxybutyl substituent at N-1 () showed significantly improved ACK1 inhibition over in both biochemical and cellular autophosphorylation assays., Subsequent derivatives – demonstrated that both methyl and chloro were well tolerated at although hydrogen was not. Installation of larger groups at such as methyl (compounds and ) diminishes binding, presumably due to steric crowding at the rear of the small hydrophobic pocket that the N3-substituent occupies. The effects of incorporating a smaller fluorine atom at are less pronounced (compounds and ). The 4-position of the N-phenyl ring points out of the enzyme towards solvent, providing an area to introduce polar solubilizing groups for better cell penetration. As seen in , a variety of N- and O-linked groups were tolerated in this position without dramatically affecting enzyme inhibition. A 3-fluoro atom could also be introduced next to O-linked groups without affecting potency (compounds –). The 3-fluoro was introduced in an attempt to impart metabolic stability to the scaffold by stabilizing one of the two electron-rich aniline moieties present in the molecule. Furthermore, in the case of , the other aniline moiety is simultaneously stabilized by 2,6-dichloro substitution.