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    • Crystal structures were obtained of

    2023-01-04

    Crystal structures were obtained of sulfonamide 18 and amide 19 as a derivative of amide 36 (Fig. 7A and B). The precise rotameric orientation of amide 36 was of significant interest to understand the compound's interaction with the protein. As it would be difficult to assign the rotomer of 36, the N-methyl amide 19 was prepared and utilized for the X-ray crystal structure experiment along with sulfonamide 18. Fig. 7 shows the X-ray co-crystal structures for sulfonamide 18 and N-Me amide 19 bound to the ATP-binding site of ASK1. The crystal structures confirmed that the analogs in this series are in fact single-point hinge binders as predicted by our docking model. The amide carbonyl of the inhibitors engages the backbone NH of Val757. As intended, the chemotypes interact with either rotamer of the carboxamide of Gln756 depending on whether a STF-118804 synthesis donor (Fig. 7A) or acceptor (Fig. 7B) is proximal to the carboxamide side-chain residue. In addition to the Gln756 interaction, the inhibitors also engage Gly759, either through its backbone NH in case of the sulfonamide 18 or through its backbone carbonyl in case of the substituted amide 19. Having confirmed that these ASK1 inhibitors act as single point hinge binders demonstrating the predictive capabilities of the docking model, X-ray protein crystal structure information was used to guide efforts to design more potent inhibitors. As previously described, sulfonamides such as 18 as well as amides such as 19 interact with Gly759. It was hypothesized that the strength of this hydrogen bond could be enhanced by shielding it with a lipophilic substituent on the 4-position of the methoxy-substituted benzamide ring [39]. To test this hypothesis the corresponding 4-methyl analogs of 18, 36 and 38 were prepared (Table 3). Improvements in enzyme and cell potencies were observed when comparing sulfonamides 18 with 20 as well as amides 36 with 45. The potency increased as a result of the addition of the methyl group in the case of the amides 45 and 36 was essentially lipophilic neutral as evidenced by the LipE values of 8.0 vs 7.8. In contrast, sulfonamide 20 benefitted significantly more from the additional methyl substituent beyond the increase in logD: its LipE value improved by a full unit when compared to sulfonamide 18. This level of potency improvement clearly indicates that the 4-Me group in the context of the sulfonamide substituent has a beneficial impact beyond increasing the lipophilicity. However one must be careful in attributing the cause of the improved activity. Torsion scans of the sulfonamides 18 and 20 and amides 36 and 45 were undertaken using quantum mechanics (QM) as well as molecular mechanics (MM) to assess the effect of the methyl group on the energy of the bound conformation (see Supplementary Materials, Figures SI-2 and SI-3 for corresponding dihedral plots). The torsion scans suggest that the addition of the methyl group ortho to the sulfonamide to afford 20 may have little effect (QM) to a 0.8 kcal stabilization (MM) of the conformational energy. In the case of the amide, ortho methylation is predicted to destabilize the bound conformation by 0.8 kcal (QM) to 0.21 kcal (MM). Thus in the case of the sulfonamides the beneficial effect of the methyl group may not have been compromised by torsion strain, whereas in the case of the amide 45 the conformational effect may have negated any benefit of shielding the H-bond. Sulfone 38 did not benefit from the addition of the methyl group, as sulfone 21 is essentially equipotent to 38 in the enzyme inhibition assay and only shows a twofold improvement in cell potency. While the additional methyl group in analogs 20 and 45 had a beneficial impact on the enzyme potency of up to 15 fold and cell potency of up to 9 fold it did not have a significant impact on the ratio of enzymatic to cellular IC50. The sulfonamides, which are characterized by the lowest permeability as measured by their RRCK values, display the largest shift between the two assays. Conversely, the sulfones, which have the highest permeability, also have the lowest enzyme to cell ratio. In an absolute sense, amide 45 shows the best cell potency among this cohort of ASK1 inhibitors, presumably a composite effect of its enzyme potency and its cell permeability.