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  • The suboptimal trypsin selectivity profile of DPC

    2022-05-26

    The suboptimal trypsin selectivity profile of DPC423 prompted us to investigate additional P moieties for potency and selectivity. Glycinamide P () showed low nanomolar binding affinity against fXa which suggested that this P moiety sits deeper into the S pocket of fXa. To further elaborate on this finding, a set of constrained heterocyclic analogs were evaluated (–). Although most of these compounds were less potent, the triazolinone Pasireotide synthesis retained the fXa affinity with a fXa of 0.5nM (). With the exception of , these compounds show significantly weaker affinity for thrombin and trypsin compared with DPC423. We next evaluated the triazolinone P group appended to the bicyclic pyrazole scaffold of apixaban. As shown in , compounds – with a trifluoromethyl substitution on the bicyclic pyrazole core achieved low nanomolar to sub-nanomolar affinity. However, weak in vitro anticoagulant activity was observed with these compounds. This was attributed to higher lipophilicity which contributes to higher protein binding (for a discussion of the effect of plasma protein binding on translation from fXa to in vitro anticoagulant activity see Ref. ). To lower the protein binding of compound , the C-3 pyrazole trifluoromethyl group was replaced with C-3 methyl and C-3 carboxamide moieties, which have previously been shown to impact higher free fraction in related compounds. While the methyl analog lost fXa affinity, the carboxamide compound had a fXa of 0.25nM and improved in vitro anticoagulant potency (PT EC2x=4.4μM). In comparison with razaxaban (fXa =0.19nM and PT EC2x=2.1μM), compound was within twofold in terms of in vitro anticoagulant activity, and despite a threefold difference in fXa affinity, was comparable in the PT assay to apixaban (fXa =0.08nM and PT EC2x=3.8μM). Modeling of the phenyltriazolinone into the S pocket of the fXa active site indicated that the phenyltriazolinone moiety was not expected to fit in the S pocket. The increased size of the triazolinone moiety was predicted to result in steric clashes with Asp189. To better understand the observed results, X-ray structures of compounds , , and – bound to human fXa were determined. All of these compounds bind to fXa in a similar mode as depicted in , which shows the overlay of all five compounds in the fXa active site. The X-ray structures show all compounds maintain the general binding mode previously observed with other pyrazole-based fXa inhibitors except in the S pocket. With all five compounds, the Asp189 side chain in the S binding site has rotated away from its normal location. This movement creates a novel larger pocket to accommodate the phenyltriazolinone. shows the crystal structure of compound with the initial electron density of the ligand. An overlay of the phenyltriazolinone with the aminobenzisoxazole clearly shows that Asp189 has moved and the triazolinone has extended much deeper into the S site (). shows the detailed interactions of the phenyltriazolinone in the S pocket. One of the nitrogen atoms of the triazolinone forms a H-bond with the Gly218 carbonyl. The triazolinone carbonyl forms two H-bonds, one directly with Ala221 nitrogen and another with one of the carboxylic oxygens of Asp189 side chain through a water molecule. Another nitrogen of the triazolinone ring forms a H-bond with the second oxygen of Asp189 side chain which in turn forms a H-bond with Tyr223 OH through an additional water molecule. This network of H-bonds enables the triazolinone moiety to have strong interactions with the enzyme. The selectivity of the triazolinones against trypsin can be rationalized by a change in residue in the S pocket. FXa has Ala190 adjacent to Asp189, whereas trypsin has a bulkier serine residue in this position. These larger S substituents are unable to bind when the serine is present, therefore yielding selectivity over trypsin. Comparison of the S pockets of fXa and thrombin reveals a possible reason for the selectivity of the triazolinones against thrombin. The fXa structure shows the movement of Asp189; however, thrombin would be unable to adopt this conformation due to differences in residues near Asp189 that would create unfavorable steric and electronic interactions with Asp189. The triazolinone P moiety moves Asp189 deeper into the fXa S pocket, and puts it adjacent to Ala221. In thrombin, this residue is Asp221, which partially occludes the pocket and provides an unfavorable electrostatic environment for Asp189 to rotate towards, resulting in improved selectivity against thrombin ().