At the outset of targeting the two Gln side chain

At the outset of targeting the two Gln756 side-chain rotamers, it was unclear which rotational isomer would lead to more potent ASK1 inhibitors. While such a preference might have been anticipated, the cohort of ASK1 inhibitors in Table 3 did not provide any guidance regarding which of the Gln756 amide side chain rotamers is preferred. The results suggest that engaging either the carbonyl or the NH2 group of the Gln756 side chain can lead to ASK1 inhibitors with sub nM biochemical potency as exemplified by sulfonamide 20 and amide 45. It was highlighted that the ASK1 inhibitors described herein were designed to productively engage Gln756. The successful outcome of this effort was confirmed by X-ray crystallography. As a consequence of the rather low occurrence of Gln in this position of the ATP-binding site across the kinome, it was speculated that a productive interaction with this amino PP 2 would lead to selective ASK1 inhibitors. Fig. 8 depicts the selectivity profile of the analogs described in Table 3 in a standard selectivity panel of 40 kinases [40]. Broadly, this set of potent ASK1 inhibitors displays a very high degree of selectivity when assayed at 1 μM in the presence of 1 mM ATP to mimic physiologically relevant conditions. The less potent analogs 18, 36 and 38 display a very clean overall kinome profile in this panel with a low to moderate signal for CHEK2 which increases for the more potent analogs 20–21 and 25. In order to study the pharmacokinetic (PK) profile of this chemotype, compounds 18 and 38 were selected for a rat PK study. Methylsulfone 38 in particular is characterized by the lowest lipophilicity and highest permeability among the ASK1 inhibitors in Table 3 while having low in vitro clearance as measured in rat hepatocytes [41] (Table 4). Compound 18 has comparable lipophilicity and in vitro clearance but poorer permeability. However, following a 1 mg/kg IV dose, both compounds 18 and 38 were found to have moderate in vivo clearance in rat (Table 4, see Supplementary Materials for protocol details). When dosed orally as a solution to minimize kinetic dissolution effects, both compounds 18 and 38 were shown to have low bioavailability (F = 1% and 8% respectively). It may be that though the compounds were dosed as solutions, the low thermodynamic solubility [42] of each compound hindered absorption, but the precise source of the poor bioavailability could not be determined.
Conclusions A careful analysis of the ATP-binding site of ASK1 was carried out which identified the relatively unique Gln756 position and thus provided an intriguing design hypothesis for the identification of selective ASK1 inhibitors. An investigation of how compound 4 binds to ASK1 led to the hypothesis that it is a single point hinge binder via the carbonyl oxygen. This binding hypothesis was later validated by protein crystallography. This hypothesis informed the design of a virtual library of ∼14 K amides which was enumerated in silico and triaged based on the analogs' ability to engage either one of the two rotamers of the Gln756 amide side chain. Several of the 29 amides targeted for synthesis were found to have IC50 values below 1 μM when tested in the full-length ASK1 biochemical kinase assay in the presence of 1 mM ATP as exemplified by amide 18 with an IC50 of 2 nM and LipE of 7.8. Subsequent SAR work led to the discovery of a series of sulfonamides, amides and sulfones with sub-nanomolar potencies when assayed against the ASK1 enzyme. In addition to displaying attractive enzymatic activity, several analogs were found to be highly potent in a cell-based assay measuring inhibition of phosphorylation of p38, a key kinase downstream of ASK1 involved in the regulation of inflammation and oncology pathways. Though potent in the cell-based assay, there was a large right shift between enzyme and cellular potency potentially due to the limited cell permeability as predicted by the low RRCK and over expression of ASK1 in HEK293 cells. In addition to excellent potency, these inhibitors deliver a high degree of kinome selectivity when assessed in a panel of 40 kinases. While lead compounds from this program displayed encouraging in vitro ADME properties oral bioavailability in rat was poor. Further efforts will have to be undertaken to address the series' low bioavailability (as exemplified by compounds 18 and 38). However, the ASK1 inhibitors reported here constitute attractive tool compounds with the potential to support the elucidation of the pharmacological consequences of ASK1 inhibition.