Synthesis, Biological Evaluation, X-ray Structure, and Pharmacokinetics of Aminopyrimidine c-jun-N-terminal Kinase (JNK) Inhibitors

Abstract

Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors, with good cell potency, and good brain penetration properties. The structure activity relationships (SAR) around a series of aminopyrimidines was evaluated utilizing biochemical and cell- based assays to measure JNK inhibition, and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC50 values < 100 nM. Moreover, compound 9l showed an IC50= 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rat, along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.

Mitochondrial c-Jun N-terminal Kinase (JNK) Signaling Initiates Physiological Changes Resulting in Amplification of Reactive Oxygen Species Generation

“…Previous work in our laboratory demonstrated that JNK signaling contributes to the generation of ROS during stress. We found that INS-1 insulinoma rat cells treated with streptozotocin produced ROS, and use of a highly selective small molecule inhibitor of JNK (SR-3562 or Compound 9l) prevented ROS production (19). Congruent with our results, JNK-dependent ROS generation was observed in TNF-␣ stimulated murine embryonic fibroblasts (MEFs) lacking functional NF-B signaling (20).…”

“…It has been proposed that JNK can induce ROS generation (19,20,37), and that this amplification of superoxide production is part of the cellular response to mitochondrial factors released early in cellular stress (37). This observation is born out in our experiment involving the incubation of active JNK with mitochondria from unstressed and stressed JNK null MEFs.…”

“…1B, i). Pre-treatment of cells with 500 nM SR-3562 (19) prior to DMSO treatment resulted in a slight increase in DHE fluorescence (Fig. 1B, ii), whereas pretreatment with 500 nM SR-3562 prevented ROS accumulation in anisomycin-stressed HeLa cells (Fig.…”

“…1A). Because JNK activation occurred between 5 and 45 min, we chose to monitor ROS generation during the first 45 min of anisomycin treatment, as previous work from our laboratory had demonstrated that JNK-selective small molecule inhibitors, such as SR-3562 (or compound 9l) could inhibit ROS generation in a dose-dependent manner with an IC 50 of ϳ1 nM in INS-1 cells treated with streptozotocin (19). Following 35 min of DMSO treatment or anisomycin stress, HeLa cells were stained with DHE to detect whole cell superoxide generation.…”

Enzyme Kinetics and Interaction Studies for Human JNK1β1 and Substrates Activating Transcription Factor 2 (ATF2) and c-Jun N-terminal kinase (c-Jun)

“…Based on the structural information and binding analysis done by other authors for several JNK variants (35)(36)(37)(38)(39)(40)(41)(42), we expected Langmuirian kinetics with 1:1 stoichiometry for the studied interactions. Some of the factors that can cause deviations from pseudo-first order approximation of binding data include: mass transfer effects, immobilized ligand density, inhomogeneity of immobilized ligand or soluble analyte, immobilization chemistry, and rebinding of dissociated analyte (43).…”