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the S4 pocket formed by the aromatic residues Trp 215, Tyr 99, and Phe 174 (Figure 7). It was proposed that
this collection of aryl residues forms the basis for a cation-π interaction that has recently been well
documented in other cases [66]. Interestingly, this cation-π site is absent in thrombin's equivalent “aryl
binding site” where the corresponding residues are Trp 215, Leu 99, and Ile 174—residues that cannot
provide the π-electrons necessary for stabilization of the cation in the inhibitor. The apparent preference
for FXa inhibitors to have cations in the P3,P4 position may be directly related to the ability of the
Factor Xa S4 site to stabilize these cations via a cation-π interaction. Factor Xa appears to be unique
among the coagulation factors in providing this electron-rich S4 pocket (Table 4).
The initial discovery of bisamidine structures as potential Factor Xa inhibitors was actually made much
earlier with the finding that compounds such as 4 showed an almost 300-fold preference for Factor Xa
over thrombin with a K i of 13 nM (FXa) [67],
As with the DX-9065 analogs and compounds 2 and 3, the Factor Xa potency was relatively insensitive
to the positioning of the amidino groups (4,4' versus 3,3') while replacing the 7-membered cycloalkyl
ring with the 5 or 6 membered ring analogs reduced potency by about 10 fold. Model building
compound 4 and docking into Factor Xa [69], again using the x-ray benzamidine:thrombin complex [65]
as a template, shows that the second aryl amidino group can be positioned into the S4 aromatic pocket of
Factor Xa in a conformation closely related to the mode of binding proposed for DX9065a (Figure 8)
[64].
In an effort to compare the relative efficacy of thrombin versus Factor Xa inhibitors, Markwardt et al.
[67] synthesized a set of amidinoaryl compounds with moderate potency as Factor Xa inhibitors (5).
Table 4 S4 Residues in Selected Serine Proteases
Residue Position a Factor Xa Thrombin Factor VIIa Trypsin
99 Tyr Leu Thr Leu
174 Phe Ile Pro Gly
215 Trp Trp Trp Trp
aChymotrypsin numbering system. Sequence alignments by comparison of x-ray structures (sequence
for Factor VIIa).
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