2798
D. Vijaykumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2796–2799
yielded the corresponding benzimidazole derivatives 2
and 4. Treatment of ester 8 with p-amino-benzamidine
at 100 ꢁC for 2 h afforded the necessary amide 3. Synthe-
sis of compound 1 was reported earlier.12
By replacing the central six-membered phenol of 1 with
a hydroxy pyrazole, we were successful in producing a
novel triaryl scaffold which selectively and efficaciously
inhibits fIXa. The fVIIa crystal structure with com-
pound 2 was obtained and compared with a published
structure of fIXa. This revealed some key structural fea-
tures which may be responsible for the observed selectiv-
ity seen for fIXa. In addition, the presence of the
heterocycle in the core of the structure possibly helped
improve its physicochemical properties, which is reflect-
ed in an ex vivo clotting assay (Scheme 1).
Figure 2. X-ray crystal structure of 2 in fVIIa (green) superimposed
onto human fIXa with 4-amino-benzamidine (PDB entry code
1rfn:grey). Residues 57, 102, 191–194, 213–216, and 225–228
Acknowledgments
˚
(rms = 0.33 A) were used to overlay the proteins.
The authors thank Ronnell Cabuslay and Ellen Sanford
for providing the in vitro data and Michael J. Green for
scientific input and support.
N
2
HO
a
b
O
O
O
O
O
O
O
O
O
References and notes
5
6
7
c
1. (a) Mckean, M.-L.; Adelman, S. J. Expert Opin. Investig.
Drugs 1998, 7, 687; (b) Weitz, J. I.; Bates, S. M. J. Thromb.
Haemost. 2005, 3, 1843.
2. Bajaj, S. P.; Joist, J. H. Semin. Thromb. Hemost. 1999, 25,
407.
NH
NH
N
N
d
N
HN
H N
NH
OH
OH
2
e
2
8
3. (a) Young, W. B.; Kolesnikov, A.; Rai, R.; Sprengeler, P.
A.; Leahy, E. M.; Shrader, W. D.; Sangalang, J.; Burgess-
Henry, J.; Spencer, J.; Elrod, K.; Cregar, L. Bioorg. Med.
Chem. Lett. 2001, 11, 2253; (b) Rai, R.; Kolesnikov, A.;
Li, Y.; Young, W. B.; Leahy, E.; Sprengeler, P.; Verner,
E.; Shrader, W.; Burgess-Henry, J.; Sangalang, J.; Allen,
D.; Chen, Xi.; Katz, B.; Luong, C.; Elrod, K.; Cregar, L.
Bioorg. Med. Chem. Lett. 2001, 11, 1797; (c) Young, W.
B.; Mordenti, J.; Torkelson, S.; Shrader, W. D.; Kolesni-
kov, A.; Rai, R.; Liu, L.; Hu, H.; Leahy, E. M.; Green, M.
J.; Sprengeler, P. A.; Katz, B.; Janc, J. W.; Marzec, U. M.;
Hanson, S. R.; Bioorg. Med. Chem. Lett., in press; (d)
Young, W. B.; Rai, R.; Shrader, W. D.; Burgess-Henry, J.;
Hu, H.; Elrod, K.; Sprengeler, P. A.; Katz, B.; Sukbunth-
erng, J.; Mordenti, J. Bioorg. Med. Chem. Lett., in press.
4. Inhibition assays for factor Xa and thrombin were
performed as described (Cregar, L.; Elrod, K. C.; Putnam,
D.; Moore, W. R. Arch. Biochem. Biophys. 1999, 366, 125)
with the pH adjusted to 7.4. The fVIIa trypsin and fIXa
assays were performed and analyzed as in the above
reference with the following additional details. Factor
VIIa (Enzyme Research) was incubated at 7 nM and
CH3SO2-D-CHA-But-Arg-pNA (Centerchem) was used as
the substrate. The buffer for the factor VIIa assay was
supplemented with 11 nM relipidated tissue factor and
5 mM CaCl2. Trypsin (Athens Research Institute) was
incubated at 10 nM with variable concentrations of
inhibitor in 50 mM Tris (pH 7.4), 150 mM NaCl,
1.5 mM EDTA, 0.05% Tween 20, and 10% DMSO. The
reaction was initiated with substrate, Tosyl-Gly-Pro-Lys-
pNA (Centerchem) supplied at the Km (25 lM). Factor
IXa (Haematologic Technologies) was incubated at
16.6 nM with variable concentrations of inhibitor in
50 mM Tris (pH 7.4), 100 mM NaCl, 5.0 mM CaCl2,
f
N
NH
OH
H
N
N
NH
N
HN
O
NH
OH
4
N
NH
2
3
Scheme 1. Synthesis of pyrazole based compounds 2–4. Reagents and
conditions: (a) i—(COCl)2, CH2Cl2; ii—Meldrum’s acid, pyridine,
EtOH, 0 ꢁC to rt, overnight; (b) p-TsN3, NEt3, CH3CN, 15 ꢁC, 1 h; (c)
NaH, THF, 0 ꢁC to rt, 24 h; (d) 3,4-diamino-benzamidine, PPA,
140 ꢁC, 4 h; (e) 4-amino-benzamidine, DMF heat; (f) pyridine-3,4-
diamine, PPA, 140 ꢁC 4 h.
and the resulting unique orientation of the distal phenyl
ring described above, as compared to central phenol ring
containing molecules, may allow it to bind further down
in the S1 pocket to match the deeper pocket of fIXa. The
fact that the distal phenyl ring is pulled away from the
S10 pocket may also be beneficial for fIXa binding due
to the shift in the position of the 40s loop. Thus, the side
chain of Phe41 may actually be in a better position for
packing against the distal phenyl ring.
Phenyl acetic acid 5 was converted to b-ketoester 6 via a
reported procedure.9 b-ketoester 6 was treated with 4-
carbamoyl-benzenesulfonyl azide and a base to get the
diazo derivative 7. Compound 7 upon treatment with
sodium hydride10 furnished the requisite pyrazole ester
8. Heating the ester 8 with either diamino-benzamidine11
or pyridine diamine in polyphosphoric acid (PPA)