SAR and factor IXa crystal structure of a dual inhibitor of factors IXa and Xa

Article abstract of DOI:10.1016/j.bmcl.2004.08.034

An X-ray crystal structure of compound 3b complexed to fIXa shows a similar binding mode to that previously observed with pyrazole inhibitors in the fXa active site. The best combination of fIXa potency and selectivity in this series was obtained with 5-c

Full text of DOI:10.1016/j.bmcl.2004.08.034

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5263–5267
SAR and factor IXa crystal structure of a dual inhibitor
of factors IXa and Xa
Joanne M. Smallheer,* Richard S. Alexander, Jianmin Wang, Shuaige Wang,
Suanne Nakajima, Karen A. Rossi, Angela Smallwood, Frank Barbera, Debra Burdick,
Joseph M. Luettgen, Robert M. Knabb, Ruth R. Wexler and Prabhakar K. Jadhav
Bristol-Myers Squibb Company, PO Box 5400, Princeton, NJ 08543-5400, USA
Received 16 June 2004; revised 16 August 2004; accepted 16 August 2004
Available online 15 September 2004
Abstract—Modifications to the P4 moiety and pyrazole C3 substituent of factor Xa inhibitor SN-429 provided several new com-
pounds, which are 5–10nM inhibitors of factor IXa. An X-ray crystal structure of one example complexed to factor IXa shows that
these compounds adopt a similar binding mode to that previously observed with pyrazole inhibitors in the factor Xa active site both
with regard to how the inhibitor binds and the position of Tyr99.
Ó 2004 Elsevier Ltd. All rights reserved.
Thromboembolic events such as deep vein thrombosis
(DVT), pulmonary embolism, myocardial infarction,
and thromboembolic stroke are collectively the major
cause of mortality and morbidity in the industrialized
world. Normal hemostasis is maintained as the result
of a complex interplay among the numerous serine pro-
teases that regulate the process of coagulation. The ideal
point of intervention in the coagulation cascade to
achieve an optimum combination of efficacy (anticoagu-
latory effects) and safety (i.e., a low incidence of bleed-
ing) will only be determined after selective inhibitors
of the various coagulation serine proteases have been
identified and compared in vivo. While significant bene-
fit has been demonstrated in the treatment of deep vein
thrombosis using both thrombin and factor Xa inhibi-
tors, in clinical practice one drug/mechanism of action
may not be ideal for all antithrombotic indications.1
Antithrombotic drugs that target the ꢀintrinsicꢁ pathway
of clot propagation, as opposed to the ꢀextrinsicꢁ path-
way of clot initiation may provide a means of preventing
vessel occlusion without blockade of coagulation at an
injury site along the vessel wall. In vivo studies with
active-site blocked factor IXa or antifactor IXa anti-
bodies have demonstrated that good efficacy in animal
models of arterial and venous thrombosis can be
achieved via this mechanism.2,3
High throughput screening of compounds from our
thrombin and factor Xa (fXa) programs yielded a
number of potent, but nonselective, factor IXa (fIXa)
inhibitors. Specifically, compound 1, a subnanomolar
inhibitor of fXa, was identified as an 82nM inhibitor
of fIXa. In comparison, a structurally similar compound
SN-429,4 a picomolar fXa inhibitor, was only a weak
inhibitor of fIXa (Table 1).
This suggested that a re-optimization of the P4 substit-
uent in our existing fXa scaffolds might lead to addi-
tional potent and selective fIXa inhibitors. As a result
of a two amino acid insertion in the fIXa 99-loop, and
the specific interactions of the amino acid side chains
in this loop, which are unique to fIXa, the 99-loop in
fIXa provides an environment in the S4 binding pocket
that should be quite distinct from that of fXa.7 The pub-
lished X-ray crystal structure of porcine fIXa inhibited
with an irreversible inhibitor (D)-Phe-Pro-Asp-chloro-
methylketone (PPACK), which forms a covalent bond
to the catalytic serine, shows an unusual orientation of
Tyr99, which blocks the S3/S4 binding site and allows
binding of proline in the S2 pocket.8a The crystal struc-
ture of human fIXa inhibited with p-aminobenzamidine
also has Tyr99 rotated to partially block access to the S4
pocket.8b In keeping with these observations in the
published crystal structures, it has been shown by
Keywords: Factor IXa; Factor Xa; Pyrazole.
*
Corresponding author. Tel.: +1 609 818 5332; fax: +1 609 818
3450; e-mail: joanne.smallheer@bms.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.034

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J. M. Smallheer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5263–5267
Table 1.
Scheme 2. Reagents and conditions: (a) N-heterocycle, CuI, K2CO3,
1,10 phenanthroline, DMF, 110°C, 40–60%; (b) N-heterocycle,
K2CO3, DMSO, 55–85%; (c) H2/Pd/MeOH, 80–95%.
Anilines 4 with o-fluoro-substitution were prepared as
illustrated in Scheme 2 by Ullmann coupling of the
desired N-heterocycle with 2-fluoro-4-iodoaniline.
Des-fluoro anilines were conveniently obtained by direct
nucleophilic aromatic substitution of 4-fluoronitro-
benzene followed by reduction of the nitro group to
the corresponding amine. When the N-heterocycle
employed was an unsymmetrically substituted benzimi-
dazole or azabenzimidazole, mixtures of regioisomers
were obtained and were separated either by silica gel
chromatography at the aniline stage or by reverse phase
HPLC after elaboration of the final targets.11
Compd
IXa Ki5 (nM)
Xa Ki6 (nM)
IXa/Xa
216
> 100,000
1
SN-429
82
1900
0.38
0.013
site-directed mutagenesis that the amino acid sequence
in the 99-loop of fIXa plays a key role in determining
substrate selectivity for small peptide substrates.9
It was hoped that additional structural modifications
based on the differences in amino acid sequences in other
areas of the fXa and fIXa active sites could then be
exploited to introduce additional fIXa potency. In par-
ticular, we were interested in targeting an interaction
with Glu219, since this residue is unique to fIXa whereas
the other coagulation serine proteases all have a con-
served glycine residue at this position. It has been postu-
lated that the relatively poor catalytic efficiency of fIXa
is due in part to a partial collapse of the S1 recognition
pocket, since Glu219 must assume a high energy main
chain conformation to accommodate substrate binding.7
In the human fIXa-p-aminobenzamidine crystal struc-
ture, this conformation is stabilized by a salt bridge be-
tween the side chains of Glu219 and Arg148.8b A direct
interaction between a bound inhibitor and the carboxy-
lic acid of Glu219 might therefore result in increased
affinity through stabilization of the inhibitor–enzyme
complex.
Heterocycles capable of forming ionic interactions or H-
bonds to Glu219 were introduced on the methyl group
at C3 of the pyrazole ring as shown in Scheme 3. Com-
pound 54 was brominated by treatment with N-bromo-
succinimide to give monobromo intermediate 6,
followed by displacement of the bromide with imidaz-
ole, triazole, or tetrazole to provide esters 7, which were
condensed with the appropriate aniline 4 using Weinreb
conditions. A Pinner reaction followed by ammonolysis
provided amidines 8.
Scheme 1 shows the preparation of analogs of 1 from
pyrazole carboxylic acid 2 using previously described
procedures.4,10
Scheme 3. Reagents and conditions: (a) NBS, CCl4, reflux, 35–40%;
(b) imidazole, triazole, or tetrazole, K2CO3, DMF, 60–70%; (c)
(CH3)3Al, CH2Cl2, 4, 20–50%; (d) HCl/MeOH then (NH4)2CO3/
EtOH, 40–60%.
Scheme 1. Reagents and conditions: (a) COCl2, DMF, CH2Cl2; (b )4,
pyridine, DMAP, CH2Cl2, 50–80%; (c) HCl, MeOH then (NH4)2CO3,
EtOH, 50–70%.

J. M. Smallheer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5263–5267
5265
of fIXa to fXa potency, was improved by replacement
of the C3 pyrazole methyl group in 1 with a trifluoro-
methyl group in compound 3a. However, unlike many
similar fXa compounds, where an additional modest po-
tency enhancement was realized by the introduction of
an o-fluoro substitution on the inner phenyl ring,4 com-
pound 3b had comparable fIXa potency and selectivity
to 3a. In the S4 pocket, an additional nitrogen at differ-
ent positions around the phenyl ring of the benzimidaz-
ole in examples 3c–f had little effect on the fIXa potency,
while slightly improving the fXa potency, for an overall
detrimental effect of the selectivity for fIXa over fXa.
The addition of a 5- or 6-chloro substituent in com-
pounds 3g and 3h, however, provided a 2–3-fold
improvement in fIXa affinity and significantly improved
the ratio of fIXa/fXa potency. Trifluoromethyl com-
pound 3i had similar potency and selectivity to the
chloro compounds whereas methyl, amino, and nitro
compounds 3j–l had potency similar to the unsubsti-
tuted benzimidazoles and displayed decreasing selectiv-
ity with respect to fXa. The 5,6-dichloro compound
3m was both less potent and less selective for fIXa com-
pared with either of the monochloro analogs. Replacing
the benzimidazole with indole 3n resulted in loss of
potency with respect to both enzymes, while the
benzimidazolone analog 3o was 15-fold less active
toward fIXa while maintaining good fXa potency. Trun-
cating the benzimidazole ring to imidazole in 3p also
Scheme 4. Reagents and conditions: (a) MeONH2ÆHCl, EtOH, sieves,
87%; (b) 3-cyanophenylhydrazine, HOAc, reflux, 68%.
Compounds with a 3-pyridyl ring attached directly to
the pyrazole core were prepared from ethyl nicotinoyl-
pyruvate12 as shown in Scheme 4 by treatment with
methoxyamine hydrochloride followed by condensation
of the resulting ethyl 2-(methoxyimino)-4-oxo-4-(pyr-
idin-3-yl)butanoate with 3-cyanophenylhydrazine in
acetic acid at reflux. This gave a 9:1 mixture in favor
of the desired pyrazole regioisomer 9.13 Further elabora-
tion of 9 was carried out as described above for the con-
version of 7 to 8.
Compounds were assayed for inhibitory activity against
fIXa and fXa using purified human enzymes by pub-
lished methods, which measure the inhibition of enzy-
matic hydrolysis of small peptidic chromogenic
substrates.5,6 Potency against fIXa, as well as the ratio
Table 2. Analogs of compound 1
Compda
Z
X
R
IXa Ki (nM)b
Xa Ki (nM)b
IXa/Xa
1
CH3
CF3
H
H
F
1-Benzimidazolyl
1-Benzimidazolyl
1-Benzimidazolyl
82
11
0.38
0.094
0.10
0.024
0.036
0.044
0.028
0.42
0.25
0.29
0.27
0.12
0.08
0.27
5.4
216
117
130
329
220
180
464
10
3a
3b
3c
3d
3e
3f
CF3
CF3
13
F
1-(4-Azabenzimidazolyl)
1-(5-Azabenzimidazolyl)
1-(6-Azabenzimidazolyl)
1-(7-Azabenzimidazolyl)
1-(5-Chlorobenzimidazolyl)
1-(6-Chlorobenzimidazolyl)
1-(5-Trifluoromethyl-benzimidazolyl)
1-(5-Methylbenzimidazolyl)
1-(5-Aminobenzimidazolyl)
1-(5-Nitrobenzimidazolyl)
1-(5,6-Dichlorobenzimidazolyl)
1-Indolyl
7.9
8
CF3
CF3
F
F
7.9
13
CF3
CF3
F
3g
3h
3i
F
4.2
5.1
6.4
10
CF3
CF3
F
20
22
F
3j
CF3
CF3
F
37
92
3k
3l
F
11
11
CF3
CF3
F
138
96
3m
3n
3o
3p
8a
8b
8c
8d
8e
8f
F
26
CF3
CF3
F
1700
190
72
283
>3000
791
207
232
77
H
H
H
H
H
H
H
H
1-(2-Oxobenzimidazolyl)
1-Imidazolyl
1-Benzimidazolyl
0.058
0.091
0.58
0.19
0.26
0.31
0.20
0.65
CF3
1-Imidazolylmethyl
1-(1,2,4-Triazolylmethyl)
1-(1,2,3-Triazolyl)methyl
1-Tetrazolylmethyl
3-Pyridyl
120
44
1-Benzimidazolyl
1-Benzimidazolyl
20
11
1-Benzimidazolyl
1-Benzimidazolyl
1-(5-Chlorobenzimidazolyl)
35
28
5.5
15
3-Pyridyl
23
a All of the compounds in Table 2 were isolated as TFA salts by reverse phase HPLC and gave satisfactory spectral and analytical data.
b Biological assays were carried out as described in Refs. 5 and 6.

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J. M. Smallheer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5263–5267
Figure 1. (a) X-ray crystal structure of compound 3b (in orange) complexed with porcine fIXa (in green). Also shown are the alternate orientations of
Tyr99 from the published PPACK-fIXa (in white) and the p-aminobenzamidine-fIXa (in magenta) crystal structures. (b) Overlay of the fIXa-3b
crystal structure (in green) with the DMP-423-fXa structure (in pink).
decreased fIXa potency without adversely affecting the
fXa potency. The best combination of fIXa potency
and selectivity in this series was thus obtained with 5-
chlorobenzimidazole as the P4 substituent (3g).
Figure 1bshows the overlay of the fIXa- 3b crystal struc-
ture with the crystal structure of DMP423, a potent and
selective fXa inhibitor, complexed with fXa.10 With the
exception of the area of the 99-loop, the backbones of
the two enzymes are very similar, and the two inhibitors
bind in the active sites in essentially the same way. In
both crystal structures, Tyr99 blocks the S2 pocket
and forms the bottom of the hydrophobic S4 binding
site. The phenyl ring of the benzimidazole of 3b is in
close proximity to residues Tyr177, Asn97, and Lys98
at the base of the fIXa S4 pocket. In view of the differ-
ences in amino acid sequence in this region (i.e., the cor-
responding residues in fXa are Thr177, Glu97, and
Thr98) and the more restricted conformation of the fIXa
99-loop,8b more extensive SAR in this region may lead
to improved selectivity.
Substitution of heterocycles at the 3-position of the pyr-
azole ring to interact with Glu219 did not provide the
significant increase in potency or selectivity expected
from a strong ionic or H-bonding interaction with the
Glu side chain carboxylic acid. The tetrazole 8d and
pyridine 8e did show improved potency and selectivity
as compared to compound 1, in a similar range to sub-
stituted benzimidazoles 3h–j. Combining the 3-pyri-
dylpyrazole substitution with a 5-chlorobenzimidazole
P4 in compound 8f, however, did not result in additional
potency over that observed for either substitution alone.
The compounds in Table 2 also showed modest selectiv-
ity (5–40-fold) for fIXa over thrombin and trypsin
(unpublished results). No in vivo evaluations were car-
ried out on this series of compounds.
In conclusion, we have identified several pyrazole com-
pounds, which are potent dual inhibitors of fIXa and
fXa, and have shown that the fIXa/fXa potency ratio
can be modulated by changes to the P4 moiety as well
as in the region where these molecules might interact
with Glu219. The X-ray crystal structure of reversible
inhibitor 3b complexed with porcine fIXa provides addi-
tional structural insights, which can be used in the future
design of more potent and selective fIXa inhibitors.
The crystal structure of porcine fIXa complexed with
compound 3b is shown in Figure 1a.14 The inhibitor is
anchored into the active site by the bidentate interaction
between the benzamidine group and Asp189 of fIXa. A
hydrogen bond is observed between N2 of the pyrazole
ring and the backbone nitrogen of Gln192. The amide
carbonyl is also engaged in a hydrogen bond with the
backbone nitrogen of Gly216. The overall binding mode
of this inhibitor is quite similar to that observed with
reversible fXa inhibitors complexed in fXa10,15 in that
the inhibitor optimizes interactions in the S1 pocket
and S4 region of the protein using a linker that engages
in hydrogen bonds with backbone residues of the
enzyme. In contrast to the previously published fIXa
structures, the fIXa-3b structure shows an orientation
of the Tyr99 residue that is more consistent with the ori-
entation observed in fXa crystal structures and that
accommodates binding of the inhibitor in the S4 pocket,
such that the benzimidazole-phenyl group is situated in
the hydrophobic box surrounded by residues Trp215,
Tyr99, and Phe174.
Acknowledgements
We would like to thank Dr. Chong-Hwan Chang for
assistance with refinements of the final crystal structure
for publication. We also thank Mr. Eugene Amparo and
Mr. Michael Orwat for their synthetic assistance.
References and notes
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by flash chromatography using a gradient elution from 0%
to 3% MeOH in CH2Cl2.
F
F
N
N
H2N
N
H2N
N
N
N
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4c
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˚
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