W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 561–566
565
chain carboxylate. The P4-amidine is held in the S4
binding pocket by pi-cation interaction with Tyr99,
Phe174, and Trp215. However, contrary to the docking
results with factor Xa, the best binding conformation of
compound 45 predicted by Gold displays an inverted
binding mode, with the benzamidino group in the S4 site
of trypsin (Fig. 2b). A less favorable conformer does
indeed show normal binding (not shown here); however,
this conformation of 45 is significantly different from
that of compound 41 in both the S1 and S4 binding
pockets. This observation may explain why compound
45 is a poor trypsin inhibitor.
compound 41 is 0.78 nM. It doubles the thrombin gen-
eration time of rabbit plasma at a concentration of 0.5
mM. The half lives of compound 41 are 0.5 h in rats and
0.9 h in rabbits following intravenous administration at
2 mg/kg. The bioavailability in rats is 4.6% at an oral
dose of 2 mg/kg in 0.5% methylcellulose. This low oral
bioavalibility is characteristic of diamidine compounds.
The antithrombotic effect of compound 41 in our rabbit
deep vein thrombosis model is dose responsive.14 It
shows a 7, 44, and 99% inhibition of thrombosis at the
dose of 25, 50, and 100 mg/kg, respectively. The rabbit
bleeding time is prolonged by 1.7-fold and the plasma
concentration of compound 41 is 200 nM at the 100 mg/
kg dose.
Selected compounds were subjected to more detailed in
vitro and in vivo tests. For example, the Ki value of
In summary, starting with the aniline-based lead com-
pound 2, we have demonstrated that the naphthyl moiety
is tolerant of replacement with a variety of benzene-fused
heterobicycles or biaryls. Compounds 10, 14, and espe-
cially 15 which contain an indole, biphenyl or phenyl-
isoxazole moiety as P1, respectively, display improved
trypsin selectivity. Improvements in potency and trypsin
selectivity were observed when a carboxyl group was
attached to the aniline nitrogen as in the biphenyl and
benzothiophenyl compounds 22 and 23, similar to that
observed for YM-60828. Considerable potency drop
was observed in compounds with a biphenylsulfon-
amide moiety or a pyrrolidinylcarbonyl phenyl group as
the P4 motifs. The benzoxazinone-based compounds
41–45 are active factor Xa inhibitors. The improved
trypsin selectivity profile of the aniline series of com-
pounds is retained by the corresponding benzox-
azinones. Compound 41 shows strong antithrombotic
effect in a dose responsive manner. The compounds
with an amidinobiaryl group as the P1 moiety paved the
way to the convenient introduction of benzamidine
surrogates such as aminoisoquinolines and amino-
benzoisoxazoles, which offer possibility to achieve good
oral bioavailability.15 The details will be reported in due
course.
References and Notes
1. Davie, E. W.; Fujikawa, K.; Kisiel, W. Biochemistry 1991,
30, 10363.
2. (a) Scarborough, R. M. J. Enz. Inhib. 1998, 14, 15. (b) Zhu,
B.-Y.; Scarborough, R. M. Curr. Opin. Cardiov. Pulmon. Ren.
Investig. Drugs 1999, 1, 63. (c) Zhu, B.-Y.; Scarborough, R. M.
Annu. Rep. Med. Chem. 2000, 35, 83.
3. Nagahara, T.; Yokoyama, Y.; Inamura, K.; Katakura, S.;
Komoriya, S.; Yamaguchi, H.; Hara, T.; Iwamoto, M. J. Med.
Chem. 1994, 37, 1200.
4. Hirayama, F.; Koshio, H.; Taniuchi, Y.; Sato, K.; Hisa-
michi, N.; Sakai, Y.; Katayama, N.; Kurihara, H.; Kawasaki,
T. 214th National Meeting of the American Chemical Society,
Las Vegas, NV, Sept. 7–11, 1997; American Chemical Society:
Washington, DC, 1997; MEDI-049.
5. Brandstetter, H.; Kuhne, A.; Bode, W.; Huber, R.; von der
Saal, W.; Wirthensohn, K.; Engh, R. A. J. Biol. Chem. 1996,
271, 2998.
6. For preliminary accounts of our studies on factor Xa
inhibitors with a bicyclic template: Zhu, B. Y.; Li, W.; Huang,
W.; Kane-Maguire, K.; Marlowe, C. K.; Song, Y.; Zhang, P.;
Wang, L.; Fan, J.; Wong, P.; Tran, K.; Sinha, U.; Xing, L.;
Figure 2. Compounds 41 (green) and 45 (yellow) docked in the factor
Xa active site (a, top) and trypsin active site (b, bottom).