4570
H. Hu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4567–4570
assays (pH 7.4) and forms water bridged hydrogen bonds
with Asp 189 of the enzyme to afford desired nanomolar
potency against fVIIa. The 4-chloro-5-azaindole P1, which
has a calculated pKa ꢀ5.7, is not protonated and therefore
has little interaction with Asp 189, which is consistent with
the loss of potency against fVIIa (micro-molar Ki for
fVIIa.TF). The 4-amino-5-azaindole P1, which has a
calculated pKa ꢀ9.9, is mostly protonated and forms a
stronger interaction with Asp 189 through water bridged
hydrogen bonds. In addition, the 4-amino group is also
thought to interact with G219 of the enzyme. Both
interactions are considered to contribute to the improved
potency.
sider such a moiety in their drug development programs,
especially in the challenging arena of developing fVIIa
inhibitors.
References and notes
1. Harker, L. A.; Hanson, S. R.; Wilcox, J. N.; Kelly, A. B.
Haemostasis 1996, 26, 76.
2. Himber, J.; Kirchhofer, D.; Riederer, M.; Tschopp, T. B.;
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1115.
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407.
12. 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
trypsin and fVIIa 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 (Center-
chem) was used as the substrate. The buffer for the
factor VIIa assay was supplemented with 11 nM relip-
idated tissue factor and 5 mM CaCl2. Trypsin (Athens
Research Institute) was incubated at 10 nM with vari-
able 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). Coagulation assays (PT and aPTT) were
carried out at 37 ꢁC in human plasma using a Beckman
Coulter ACL100 in accordance with the manufacturer’s
instructions.
4. Olivero, A. G.; Eigenbrot, C.; Goldsmith, R.; Robarge,
K.; Artis, D. R.; Flygare, J.; Rawson, T.; Sutherlin, D. P.;
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Henry, J.; Spencer, J.; Elrod, K.; Cregar, L. Bioorg. Med.
Chem. Lett. 2001, 11, 2253.
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Marzec, U. M.; Shrader, W. D.; Rai, R.; Kolesnikov, A.;
Liu, L.; Hu, H.; Leahy, E.; Sprengeler, P.; Katz, B.; Janc,
J. W. Bioorg. Med. Chem. Lett. 2006, 16, 2034.
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R.; Hu, H.; Torkelson, S.; Young, W. B.; Sprengeler, P.;
Katz, B.; Yu, C.; Cabuslay, R.; Sanford, E.; Janc, J.
Bioorg. Med. Chem. Lett. 2006, 16, 1596.
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Shrader, W. D.; Stephens, S.; Liu, L.; Pan, L.; Mordenti,
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9. (a) Riggs, J. R.; Hu, H.; Kolesnikov, A.; Leahy, E. M.;
Wesson, K. E.; Shrader, W. D.; Vijaykumar, D.; Wahl, T.
A.; Tong, Z.; Sprengeler, P. A.; Green, M. J.; Yu, C.;
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Rai, R.; Kolesnikov, A.; Sprengeler, P. A.; Torkelson, S.;
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¨
Synthesis 2004, 59.
16. The synthesis of intermediate 14 is shown in Scheme 2.
Commercially available 4-amino-2-chloropyridine 18 was
iodinated to afford a mixture of iodopyridines 19 and 20
(ꢀ6:4). Compound 20 can be easily isolated by chroma-
tography on silica. Treatment of 20 with methanesulfonyl
chloride led to a mixture of monomesylate 14 and bis-N-
mesylated byproduct. Subsequent treatment with NaO-
H(aq)/THF converted the bismesylate to monomesylate
14.
10. The 5-azaindole, which has a calculated pKa ꢀ7.8 (ACD
Labs/pKa DB), is thought to be partially protonated in the
Cl
Cl
Cl
Cl
I
NHMs
I
NH2
N
b, c
N
a
N
N
+
NH2
NH2
I
18
20
14
19
Scheme 2. Reagents and conditions: (a) ICl, KOAc/HOAc, 60–70 ꢁC; (b) MsCl, Et3N, DCM, 0 ꢁC; (c) 10% NaOH, THF.