Thus phosphorylation of p was used to measure the

Thus, phosphorylation of p38 was used to measure the intracellular potency of ASK1 inhibitors. In this assay, HEK293/AP-1luc Mevastatin receptor expressing human full-length ASK1 were incubated with compound for 18 h and then lysed and the level of phospho-p38 was quantified using the HTRF assay [33].
Results and discussion Gratifyingly, several of the 29 prepared library analogs incorporating a variety of chemically diverse substituents were found to display IC50 values in the recombinant ASK1 enzyme assay below 1 μM. Table 1highlights four key analogs from this initial library with the enzymatic potency, as well as lipophilicity (SFlogD) and lipophilic efficiency (LipE) values shown [34,35]. Analogs 18 and 25–26 were attractive in that they provided functional groups with synthetically straightforward design opportunities to explore further potency enhancements and adjustment of physicochemical properties. Among the four analogs shown in Table 1, sulfonamide 18 stood out with respect to its enzymatic potency and LipE. This analog is about 30 times more potent than the reversed ethyl sulfonamide 25 which is reflected also in the LipE value of 18 being 2 units higher than that of 25. Primary sulfonamide 18 was the only analog in the library of 29 compounds which carries a methoxy group in the ortho-position to the amide. It was hypothesized that this substituent led to a significant enhancement in potency as a result of rigidifying the biaryl amide through an intra-molecular hydrogen-bond that lowered the rotational flexibility of the molecule thus bringing the ground state conformation close to the bioactive conformation. To ascertain whether the o-OMe substituent would consistently lead to a reproducible increase in potency, the methoxy analogs corresponding to reverse sulfonamide 25 and primary amide 26 were prepared and tested for their ability to inhibit ASK1 (Table 2). Indeed, the putative intramolecular hydrogen bond established in analogs 31 and 36 resulted in a >200 fold and >500 fold enzyme potency improvement compared to 25 and 26, respectively. It was recognized that the additional hydrogen bond donor substituents on analogs such as 18, 31 and 36 may have a detrimental effect on permeability. Methyl sulfone 38 was designed to minimize the H-bond donor count while still displaying a functional group that could potentially engage Gln756 as predicted in our docking model. Despite a 3–15 fold reduction in potency in the ASK1 enzyme assay compared to the sulfonamides 18 and 31 as well as amide 36, the sulfone moiety of inhibitor 38 (IC50 = 5 nM), remained an attractive functional group to be considered for the design of ASK1 inhibitors as the permeability as measured by RRCK cells (low efflux MDCK cells) [36] was increased. Overall, the methoxy containing inhibitors are in a desirable lipophilicity range with SFlogD below three [34,37] (Table 2) as measured in the shake flask logD (SFlogD) assay [38] and have excellent LipE values. These compounds are also characterized by low to moderate molecular weight and acceptable RRCK permeability [36]. As a result of their desirable physicochemical and ADME profile, these analogs were advanced to the cell-based assay described above. When evaluated in the cellular assay, analogs 18, 31, 36 and 38 are characterized by a significant shift in potency compared to the biochemical enzymatic IC50 values (Table 2). As indicated above, the kinase assay was carried out in the presence of 1 mM ATP to approximate intracellular ATP concentrations. As a result, a modest enzyme-to-cell shift was expected, especially since the analogs appear to have sufficient cell-permeability as predicted by the measured RRCK values. We postulate that this unexpected shift is due the artificial nature of the engineered cells over expressing ASK1. To identify compounds with improved enzymatic and cellular potency using structure-based design, several X-ray co-crystal structures of these inhibitors bound in the active site of ASK1 were obtained. To this end, efforts were focused on sulfonamides, amides and sulfones represented by examples 18, 36 and 38. The reverse sulfonamide chemotype 31 was deprioritized in order to avoid the potential of forming a reactive p-quinone intermediate under conditions of oxidative metabolism.