Analysis of ASK crystal structures

Analysis of ASK1 crystal structures as well as an investigation of how Cy5 NHS ester(Et) 4 might bind were undertaken to understand opportunities for engaging Gln756. To this end a docking model derived from PDB 3VW622 was used to predict the binding mode of amide 4 in the ASK1 active site as shown in Fig. 4. Instead of interacting with the hinge through the hydrogen bond donor and acceptor motif of the amino-pyridine moiety, amide 4 was predicted to act as a single-point hinge binder with its amide carbonyl engaging the NH of Val757. In light of a recent analysis on kinome selectivity, a single-point hinge binding motif may be preferable to a two-point hinge binder to increase the probability of achieving kinome selectivity [28]. In addition to the hinge contact, the docking model suggested a productive interaction of the triazole with Lys709 and a non-classical hydrogen-bond interaction between the imidazole C5-H and the carbonyl of Gln756 (3.3 Å). Having identified amide 4 as an inhibitor which features key criteria for its target engagement such as being a single-point hinge binder and displaying an interaction with Gln756, efforts were directed toward expanding the chemical space around this compound. Specifically we sought to maintain the single point hinge binding characteristics while exploring which other moieties might engage Gln756.
Library design A virtual amide library was enumerated by acylating the iPr-triazole-substituted 2-amino-pyridine moiety with available carboxylic acids (Fig. 5). The goal of this library was to identify ASK1 inhibitors which would engage either rotamer of the carboxamide side-chain of Gln756, thus targeting either the NH2 or the C=O group with a range of functional groups. In addition, substituents were sought that provided a vector to access the selectivity channel occupied by the tert-butyl phenyl moiety in amide 1. To this end, a virtual library of more than 14,000 amide analogs was enumerated and docked into the ATP-binding site of ASK1 using a docking model derived from 1 (pdb code 3VW6) wherein each of the two Gln756 rotamers were sampled. This effort led to the prioritization of 29 chemically diverse analogs for synthesis which were expected to deliver highly potent and selective ASK1 inhibitors.
Synthesis of potential inhibitors The compounds in Table 1, Table 2, Table 3 were prepared by amide bond coupling of the appropriately substituted benzoic acid fragments with 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (5). Preparation of the substituted benzoic acid fragments not commercially available is outlined in Scheme 1, Scheme 2. 2-Methoxy-5-(methylcarbamoyl)benzoic acid (9) was prepared by the amide coupling of 3-bromo-4-methoxybenzoic acid (6) with methylamine to afford 7. Palladium catalyzed carbonylation of 7 under 50 psi CO in methanol/DMF (1:1) provided ester 8. The ester hydrolysis was accomplished using lithium hydroxide in aqueous methanol to give compound 9 (Scheme 1). 2-Methoxy-4-methyl-5-sulfamoylbenzoic acid (12) was prepared by treatment of 10 with thionyl chloride and chlorosulfonic acid to afford 11. The sulfonyl chloride was dissolved in ammonium hydroxide and acidified to give 12 (Scheme 2). 16 was prepared by treating 11 with acetic acid at 90 °C followed by reduction with tin metal in hydrochloric acid at 45 °C to give the thiol 13. The thiol was alkylated with iodomethane in acetone with potassium carbonate as base to afford the thioether 14. The thioether was oxidized with potassium permanganate in a biphasic reaction to give 15 followed by ester hydrolysis to provide 16 (Scheme 2). Compounds 18–21 were synthesized by conversion of the appropriate acid to the acid chloride using oxalyl chloride and subsequent addition of 5 in pyridine (Scheme 3). Compounds 25–27 were synthesized in a library format by coupling the commercially available acids with 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine using propylphosphonic anhydride (T3P) with triethylamine at 100 °C (Scheme 4).