Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

Pharmacokinetic drivers of toxicity for basic molecules: strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor.

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors.

The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8 mL min(-1) kg(-1), respectively) and high in rats (79.2 mL min(-1) kg(-1)). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5 mg kg(-1) the R/S enantiomer ratio of AUC(inf) was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively. In an oral single-dose dose-ranging study at 200 and 500 mg kg(-1) and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C(min) values and tissue concentrations approximately 24 h after the final dose. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

Identification of 4-aminopyrazolylpyrimidines as potent inhibitors of Trk kinases.

The design, synthesis and biological evaluation of a series of 4-aminopyrazolylpyrimidines as potent Trk kinase inhibitors is reported. High-throughput screening identified a promising hit in the 4-aminopyrazolylpyrimidine chemotype. Initial optimization of the series led to more potent Trk inhibitors. Further optimization using two strategies resulted in significant improvement of physical properties and led to the discovery of 10z (AZ-23), a potent, orally bioavailable Trk A/B inhibitor. The compound offers the potential to test the hypothesis that modulation of Trk activity will be of benefit in the treatment of cancer and other indications in vivo.