Compound was synthesized by reacting

Compound 31 was synthesized by reacting 5-amino-2-methoxybenzoate (28) with ethanesulfonyl chloride in the presence of pyridine in DCM to give sulfonamide 29. Hydrolysis of 29 by aqueous sodium hydroxide afforded the Imeglimin 30 which was converted to the acid chloride by heating in thionyl chloride. Residual thionyl chloride was removed under reduced pressure and the crude acid chloride reacted with 5 in acetonitrile at room temperature. The resulting precipitate was recrystallized from methanol to provide compound 31 (Scheme 5). Compound 36 was synthesized by the palladium catalyzed carbonylation of 3-bromo-4-methoxybenzonitrile (32) in methanol under 60 psi CO to give ester 33. Hydrolysis with lithium hydroxide and amide coupling with 5 using Mukaiyama's reagent [29] afforded the nitrile 35 which was treated with hydrogen peroxide and potassium carbonate to provide 36 (Scheme 6). Compound 38 was synthesized by converting the commercially available acid 2-methoxy-5-(methylsulfonyl)benzoic acid (37) to the acid chloride with thionyl chloride and reacting with 5 in pyridine at room temperature (Scheme 7). Compound 45 was synthesized by bromination of methyl-2-hydroxy-4-methyl benzoate (39) followed by phenol alkylation with methyl iodide to give 41. Palladium catalyzed cyanation with zinc cyanide was used to obtain 42. Ester hydrolysis using sodium hydroxide followed by amide coupling and treatment of the nitrile with hydrogen peroxide and potassium carbonate afforded the primary amide 45 (Scheme 8).
In-vitro assays Inhibitor efficacy was evaluated using a homogeneous time-resolved fluorescence (HTRF) assay that measures the phosphorylation of a peptide substrate by recombinant human full-length ASK1. In order to best reflect the physiological intracellular environment, a 1 mM concentration of ATP in the assay was employed. As some compounds were potent and approached the limits of the assay as defined by the concentration of enzyme (3 nM), the Kiapp was calculated by fitting the enzyme inhibition versus compound concentration data using the Morrison equation (equation (9.6) in Copeland [30]) or the competitive inhibition equation (equation (8.1) in Copeland [30]) for less potent inhibitors (see Experimental Section). This approach allows a more accurate determination of affinity for the most highly potent inhibitors approaching the functional limitations of the assay [31]. For SAR interpretation purposes, the Kiapp values were converted to IC50 values using the Cheng-Prusoff equation (equation (3) in Cheng and Prusoff [32]) employing an experimentally determined ATP Km of 48 μM [33]. To evaluate intracellular inhibitor potency, a new cell assay was developed. Human full-length ASK1 was over-expressed in HEK293 cells containing a stress-activated luciferase reporter downstream of the transcription factor Activator Protein-1 (AP-1). AP-1 acts downstream of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), hence the expectation was that activation of overexpressed ASK1 would lead to a robust reporter cell line for ASK1 kinase activity via MMK4 or MKK7 [7]. It was discovered that upon expression, full-length ASK1 was constitutively active in the HEK293/AP-1luc cells, and did not require exogenous stimulation for activation, as evidenced by the autophosphorylation of ASK1 at Thr838 (Fig. 6, lanes 1 and 2). This basal autophosphorylation was minimally detected in cells overexpressing kinase-inactive ASK1 K709R (Fig. 6, lane 3). ASK1 autophosphorylation was blocked by ASK1 selective inhibitors such as 38 dosed at 10 μM (Fig. 6, lane 4). Despite the constitutive activation of ASK1 in the overexpressing cells, AP-1-induced luciferase expression was not detected in cells. This was supported by the failure to detect phosphorylated JNK (data not shown). Instead, in this cell system, constitutively activated ASK1 resulted in elevated phosphorylation of p38, the other ASK1-dependent substrate via MKK3 and MKK6 [7].