Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

Article abstract of DOI:10.1016/j.bmc.2004.12.033

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.

Full text of DOI:10.1016/j.bmc.2004.12.033

Bioorganic & Medicinal Chemistry 13 (2005) 1487–1496
Optimization of a coagulation factor VIIa inhibitor found
in factor Xa inhibitor libraryq
Kazuyuki Sagi,* Koichi Fujita, Masayuki Sugiki, Mitsuo Takahashi, Shunji Takehana,
Kazumi Tashiro, Takashi Kayahara, Masahiro Yamanashi, Yumiko Fukuda, Seiji Oono,
Akiko Okajima, Seinosuke Iwata, Masataka Shoji and Kuniya Sakurai
Pharmaceutical Research Laboratories, Ajinomoto Company Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
Received 22 September 2004; revised 19 December 2004; accepted 19 December 2004
Available online 18 January 2005
Abstract—An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a,
was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our
factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was
based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF
at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in
the TF-dependent extrinsic pathway.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ingly, there is increasing interest in the generation of
potent and selective small-molecular factor VIIa inhibi-
tors as candidates for novel anticoagulants.7
Tissue factor (TF) is a membrane-bound protein and
does not normally circulate in plasma. Damage to the
normal integrity of the vascular system or activation
of monocytes or endothelial cells results in exposure of
TF to the blood. Then TF forms a complex with factor
VIIa (fVIIa) to activate factor IX to factor IXa and fac-
tor X to factor Xa, which in turn activates prothrombin
to thrombin. Active thrombin then cleaves fibrinogen to
fibrin, which forms the structural basis of all blood clots.
The fVIIa/TF complex is recognized as the primary
initiator of blood coagulation in vivo.2
Because fVIIa is a trypsin-like serine protease, like fXa,
we began our search by screening our library of fXa
inhibitors. From this library, we found that 1a has po-
tent factor VIIa/TF-inhibitory activity. This compound
showed strong inhibition of fVIIa/TF (Ki = 64 nM),
although it also inhibited fXa (Ki = 9.2 nM) and throm-
bin (Ki = 120 lM). Herein, we describe the structure-
based design, synthesis, and in vitro evaluation of 1a
analogues with the aim of increasing the potency and
selectivity for inhibition of fVIIa/TF (Chart 1).
Although tremendous progress has been made in the
discovery of potent and selective inhibitors of thrombin
and factor Xa as anticoagulant drugs,3 less effort has
been focused on the discovery of inhibitors of fVIIa/
TF. In recent years, however, studies using anti-factor
VIIa antibodies,4 active-site-inhibited factor VIIa,5 and
recombinant anticoagulant tissue factor pathway inhib-
itor (TFPI)6 have yielded promising results. Accord-
Catalytic Triad
Region
O
OH
O
Linker
O
O
N
H
N
S4 Pocket H2N
( fXa)
NH2
HN
NH2
Asp170G
(fVIIa)
S1 Pocket
Keywords: Factor VIIa; Inhibitor.
q See Ref. 1.
1a
*
Corresponding author. Tel.: +81 44 210 5826; fax: +81 44 210
5876; e-mail: kazuyuki_sagi@ajinomoto.com
Chart 1.
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.033

1488
K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
2. Inhibitor design based on computational modeling
Trp215-Gly216-Gln217, and the pyruvic acid moiety
interacts with the catalytic triad region composed of
the catalytic triad (His 57, Asp102, and Ser195) and
the oxyanion hole (Gly 193 and Ser195).
A model of the lead compound 1a bound to fVIIa
(1DAN8) was computationally constructed (Fig. 1) by
utilizing the same method as described in our paper on
fXa inhibitors,9 and also the crystal structures of com-
pounds related to trypsin. There is a high degree of
structural similarity of the primary binding site, S1
pocket and catalytic triad region among fVIIa, fXa,
and trypsin. In the calculated docking mode, the ami-
dino group of 1a hydrogen bonds with the carboxyl
group of Asp 18910 located at the bottom of the S1
pocket, the guanidino group hydrogen bonds with
Asp170G, forming a part of the S2/S3 pocket, which is
constructed from Thr99, Pro170I, Asp170G, and
The docking calculation was done assuming formation
of a noncovalent Michaelis–Menten complex before
the formation of a plausible covalent complex.11 The
interaction between the pyruvic acid moiety and the cat-
alytic triad region in this calculation will be actually en-
hanced by the formation of covalent stable tetrahedral
complex between the carbonyl carbon of the pyruvic
acid and the Oc of Ser 195, as observed in the crystal
structure of APPA (4-amidinophenylpyruvic acid)–tryp-
sin complex.12 APPA derivatives such as 1awill be
reversible, covalent inhibitors.
On the basis of a visual inspection of this model together
with the previous work9 on this class of inhibitors, we
expected that greater selectivity versus thrombin and
fXa might be achieved by means of the following struc-
tural modifications. (1) Introduction of an R-carboxy-
ethyl moiety into the linker part of 1a would improve
the selectivity against thrombin. The carboxylic acid
group of the R-carboxyethyl moiety would electrostati-
cally repel the side-chain carboxyl group of Glu 192 of
thrombin, but would interact favorably (electrostatic
attraction) with the side-chain amino group of Lys 192
of fVIIa. (2) Replacement of the guanidinophenyl group
of 1a might improve the selectivity against fXa. The gua-
nidino group of 1a would contribute to the affinity to
fXa, as well as fVIIa, probably owing to hydrogen-
bonding and cation–p stabilization with the S4 region
of fXa. We considered that torsion-angle-restricted ana-
logs of the 4-guanidinophenyl group that match the
optimal torsion angle for fVIIa binding, which is prob-
ably not optimal for fXa, could enhance the affinity
for fVIIa and the selectivity over fXa.
Figure 1. Calculated docking mode of 1a in factor VIIa (1DAN). The
docking calculation was done assuming formation of a noncovalent
Michaelis–Menten complex before the formation of a plausible
covalent complex, as observed in the crystal structure of APPA–
trypsin complex. The active site region of 1DAN was extracted after
removal of the complexed ligand. Both the hydrogen and carbon
atoms of the inhibitor 1a are colored green; Asp189 of the bottom of
the S1 pocket is shown in magenta; Ser 195, His 57, and Asp 102
forming the catalytic triad and Gly193 forming the oxy-anion hole are
in orange; Lys 192 at the top of the S1 pocket is in yellow and Thr 99,
Pro 170I, Asp 170G, and Trp215 forming the S2/S3 pocket are in cyan.
In this docking mode the amidino group of 1a makes hydrogen bonds
with both the carboxyl group of Asp 189 and the carbonyl group of
Gly 219 and the pyruvic acid interacts with the catalytic triad and the
oxy-anion hole. The amide NH and the guanidino group of 1a also
make hydrogen bonds with the carbonyl group of Gly 216 and the
carboxyl group of Asp 170G, respectively.
3. Chemistry
The 2-substituted 4-amidinophenylpyruvic acids 1 were
conveniently prepared from optionally substituted benz-
oic acids and the key intermediate 2 (Scheme 1). Amine
2 was acylated with various carboxylic acids to give the
corresponding amide 3. The nitrile group of 3 was
converted into an amidino group, and the target mole-
cules 1 were obtained by simultaneous hydrolysis of
both the enamide and the ester of the 2-acetamideacryl-
ate moiety.
COOMe
NHAc
COOH
O
COOMe
O
R2
R2
NHAc
O
R2
O
O
O
b, c, d
R1
N
H
R1
N
H
a
H2N
CN
CN
H2N
NH
1
2
3
Scheme 1. Synthesis of 2-substituted-4-amidinophenylpyruvic acid. Reagents and conditions: (a) R1–COOH, EDC, HOBt, DMF; (b) HCl, EtOH,
dioxane; (c) (NH4)2CO3, EtOH; (d) HCl aq, 80 °C.

K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
1489
R2
R2
R1
HN
R1
a, b
COOH
N
COOH
4a: R1=H, R2=Me
4b: R1=Me, R2=H
HN
NH2
c
d
COOH
H2N
COOMe
COOEt
N
5
N
H
H2N
H2N
O
e
f
N
COOEt
N
COOEt
N
Cl
6
7
N
COOH
g
h
H2N
8
Scheme 2. Synthesis of benzoic acids. Reagents and conditions: (a) HCl, dioxane; (b) NH2CN, EtOH, 50 °C; (c) BrCN, water; (d) 3 M HCl, 65 °C;
(e) m-CPBA, CH2Cl2; (f) POCl3, CH2Cl2, reflux; (g) (i) phenol, (ii) ammonium acetate; (h) 6 M hydrochloric acid 80 °C.
Some benzoic acids required for novel R1 groups were
prepared as shown in Scheme 2.
was much improved by the incorporation of the R-carb-
oxyethyl moiety on the linker part (1b fVIIa: 41 nM,
thrombin: >150 lM). The earlier results of R1 modified
analogues indicate that the incorporation of R-carboxy-
methyl moiety also improve selectivity versus thrombin,
but not enhance the affinity for fVIIa/TF unlike the R-
carboxyethyl moiety (data not shown). Therefore we
adopted the R-carboxyethyl moiety as the substituent
on the linker part.
4. Biological results and discussion
The data in Table 1 demonstrate that the affinity for
fVIIa/TF of 1a (fVIIa: 64 nM, thrombin: 120 lM) was
slightly enhanced and the selectivity versus thrombin
Table 1. Enzyme-inhibitory activities of benzamidine compounds
COOH
O
O
R2
O
R1
N
H
H2N NH
Compound
R1
R2
Ki (lM)
Selectivity
Over thrombinb
fVIIa/TF
fXa
Thrombin
120
Over fXaa
0.14
N
1a
1b
–H
0.064
0.041
0.0092
0.0058
1900
H2N
H2N
NH2
N
–CH2CH2COOH
–CH2CH2COOH
–CH2CH2COOH
>150
>150
>150
0.1
0.1
0.3
>3700
NH2
CH3
1c
1d
0.41
0.26
0.029
0.073
>370
N
H2N
H2N
NH2
CH3
N
>590
29
NH
1e
1f
–CH2CH2COOH
–CH2CH2COOH
0.26
1.8
4.6
7.5
7
N
0.026
>150
180
>5900
N
H
H2N
(continued on next page)

1490
K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
Table 1 (continued)
Compound
R1
R2
Ki (lM)
Selectivity
Over fXaa Over thrombinb
fVIIa/TF
0.032
fXa
Thrombin
>150
N
1g
–CH2CH2COOH
2.9
90
>4700
N
H
CH3
N
1h
1i
–CH2CH2COOH
–CH2CH2COOH
0.032
0.016
2.9
1.2
30
90
72
930
N
H
N
>150
>9300
H2N
N
1j
–CH2CH2COOH
–H
0.0010
0.041
1.8
>150
47
1800
18
>150,000
1200
S
H2N
H2N
N
1k
0.73
N
1l
–H
0.0032
1.8
60
570
19,000
S
H2N
a (Ki of fXa)/(Ki of fVIIa/TF).
b (Ki of thrombin)/(Ki of fVIIa/TF).
The guanidinophenyl moieties of 1a and 1b appear to
occupy similar sites of fVIIa and fXa. Therefore, to im-
prove selectivity over fXa, the 4-guanidinophenyl group
of 1b was replaced with torsion-angle-restricted analo-
gous groups as described for 1a in the design section.
compounds are equipotent to 1f, and that finding sug-
gests that protonation of these fused-type R1 moieties
would not be required for fVIIa binding because the ba-
sicity of 2-methylbenzimidazole (pKa = 6.2) and benz-
imidazole (5.5) is low.
In the methyl-substituted guanidinophenyl groups (1c
and 1d), the guanidino group and the adjacent phenyl
ring would be preferentially perpendicular to each other
because of the steric constraint between the methyl
group and hydrogens. These compounds show lower
affinity than the parent compound for fVIIa/TF, as well
as fXa. Based on the affinity of 1e (R1 = Ph, 0.26 lM), it
is clear that the guanidino group of 1b (41 nM) greatly
enhances the affinity for fVIIa, whereas the guanidino
group of 1c (0.41 lM) and 1d (0.26 lM) does not im-
prove the affinity.
Next, the 1-nitrogen atom of 1f was replaced with an
ethylene moiety (1i) or a sulfur atom (1j). This ring
expansion approach gave fruitful results. Compound 1i
shows slightly enhanced fVIIa-inhibitory activity
(Ki = 16 nM), and the high selectivity versus thrombin
and fXa is maintained, while 1j showed the strongest
binding affinity to fVIIa/TF (Ki = 1.0 nM) and the best
selectivity versus fXa and thrombin (selectivity over
fXa = 1800, over thrombin >150,000).
Some of the potent novel R1 moieties described above
were introduced into 1a, the compound without R-carb-
oxyethyl substitution on the linker part (1k, 1l). The R1
replacement had a similar effect to that in the case of 1b,
the compound with R-carboxyethyl substitution (see 1i,
1j), indicating that the R-carboxyethyl moiety and R1
moieties have predominantly independent effects from
the viewpoint of structure–activity relationships.
On the other hand, the 2-aminobenzimidazolyl group
(1f), which mimics the planar conformation of the 4-
guanidinophenyl group, shows equipotent fVIIa/TF
inhibitory activity (26 nM) and enhanced selectivity
against fXa (selectivity over fXa = 180 compared to
0.1). The rigidity of the fused ring might contribute to
the reduction of the fXa-inhibitory activity, as we had
hoped. However, it should be noted that the reduction
of the basicity of the R1 moiety might also contribute
to the selectivity over fXa. The pKa of 2-aminobenzimi-
dazole is 7.513 and that of guanidinobenzene is 10.8.
The in vitro anticoagulant activities of 1i (Ki = 16 nM),
1l (3.2 nM), and 1j (1.0 nM) are shown in Table 2.
The assays were performed with high drug concentra-
tion to show clearly the differences between the prolon-
gations of the prothrombin time (PT) and the activated
partial thromboplastin time (aPTT). The differences be-
come clearer as the drug concentration is higher in our
assay condition. Inhibition of the TF-dependent extrin-
sic clotting pathway was evaluated in terms of the pro-
longation of the PT. The PT prolongation correlated
with fVIIa-inhibitory activity, and this indicates that
the PT prolongation was mainly owing to fVIIa inhibi-
Compound 1f was considered as a new lead compound
with potent fVIIa/TF-inhibitory activity and selectivity
against fXa and thrombin. In an attempt to further im-
prove the affinity for fVIIa, groups analogous to the 2-
aminobenzimidazolyl group were evaluated as the R1
moiety. First, the 2-amino substituent was replaced with
a methyl group (1g) or a hydrogen atom (1h). These

K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
Table 2. Clotting time prolongation by fVIIa inhibitors
1491
contained 0.1% trifluoroacetic acid. Fractions contain-
ing the desired material were concentrated and lyophi-
lized to obtain the final products as a white solid.
NMR spectra were recorded on a Varian EM-390 at
300 MHz. Keto–enol tautomerism was observed in the
NMR spectra of all amidinophenylpyruvic acids (1).
Mass spectra (ESI) were measured on JEOL JMS-
DX300 instruments. Where analyses are indicated only
by the symbols of the elements, the results obtained were
within 0.4% of the theoretical values. Reagent abbrevia-
tions: HOBT, 1-hydroxybenzotriazole; EDC, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide HCl; TEA, tri-
ethylamine; TFA, trifluoroacetic acid; DMF,
dimethylformamide.
Compound
PTa
aPTTb
1i
1l
1j
9c
2300
3100
4500
1100
360
410
370
990
a Prothrombin time (% of control at the drug concentration of 32 lM).
b Activated partial thromboplastin time (% Of control at the drug
concentration of 32 lM).
c fXa-selective inhibitor. See Ref. 9.
tion. The prolongation of the TF-independent intrinsic
pathway was much smaller, as judged from the prolon-
gation of the aPTT. From a comparison with a selective
fXa inhibitor 3-[2-[2-[1-(4-pyridyl)piperidine-4-carbon-
ylamino]ethoxy]-4-amidinophenyl]propionic acid9 (9)
(Ki: fVIIa/TF = 16 lM, fXa = 43 nM, thrombin >1500
lM), we suppose that these fVIIa inhibitors preferen-
tially inhibit the extrinsic pathway while the selective
fXa inhibitor 9 inhibits both pathways and prolongs
both PT and aPTT, because fXa is located at the conver-
gence of the intrinsic and extrinsic pathways.
6.3. 3-[4-Cyano-2-[2-(4-guanidinobenzoylamino)-
ethoxy]phenyl]-2-acetamidoacrylic acid methyl ester
trifluoroacetate (3a)
A mixture of 3-[4-cyano-2-(2-aminoethoxy)phenyl]-2-
acetamidoacrylic acid methyl ester HCl9 (2a) (3.19 g,
9.39 mmol), 4-guanidinobenzoic acid (2.03 g, 9.39
mmol), triethylamine (1.54 mL, 11.3 mmol), and bromo-
trispyrrolidinophosphonium hexafluorophosphate (5.25
g, 11.3 mmol) in DMF was stirred overnight. The reac-
tion mixture was concentrated in vacuo, and the residue
was purified by reverse-phase HPLC to give 3a (3.32 g,
5. Conclusion
1
The optimization of a lead from our fXa inhibitor
library led to the fVIIa/TF inhibitor 1j, which is active
at nanomolar concentrations. This compound possesses
high selectivity for fVIIa/TF over fXa and thrombin,
and preferentially inhibits the TF-dependent extrinsic
clotting pathway.
61%): H NMR (CD3OD) d 2.03 (3H, s), 3.76 (3H, s),
3.80 (2H, t), 4.31 (2H, t), 7.30–7.48 (5H, m), 7.61 (1H,
d), 7.90 (2H, d); MS (ESI) 465 (MH+).
6.4. 3-[4-Amidino-2-[2-(4-amidinobenzoylamino)ethoxy]-
phenyl]-2-oxopropionic acid bistrifluoroacetate (1a)
Potential advantages of small-molecular fVIIa/TF inhib-
itors as compared with fXa or thrombin inhibitors in the
treatment of thrombotic disorders are not well estab-
lished.14 We intend to examine the separation of anti-
thrombotic efficacy and bleeding side effect by using
our selective fVIIa/TF inhibitor 1j in our intravenous
studies in animal models.
A mixture of 3a (2.0 g, 3.5 mmol), 4.0 M HCl solution in
1,4-dioxane (40 mL) and ethanol (20 mL) was stirred for
4 days and concentrated in vacuo. To the residue,
ethanol (10 mL) and ammonium carbonate (1.66 g,
17.3 mmol) were added. The mixture was stirred over-
night, and concentrated in vacuo. The residue was di-
luted with 6 N HCl (40 mL). The mixture was stirred
at 80 °C, and concentrated in vacuo. Purification by re-
1
verse-phase HPLC gave 1a (960 mg, 45%): H NMR
6. Experimental section
6.1. Molecular modeling
(DMSO-d6) d 3.61–3.73 (2H, m), 4.13–4.30 (2H, m),
6.81 (1H, s), 7.28–7.46 (5H, m), 7.63 (4H, br s), 7.92
(2H, d), 8.33 (1H, d), 8.79 (1H, t), 9.03 (2H, br s),
9.27 (2H, br s) 10.02 (1H, br s); MS (ESI) 427 (MH+).
Anal. Calcd for C20H22N6O5Æ2.0TFAÆ1.5H2O: C,
42.29; H, 3.993; N, 12.33. Found: C, 42.35; H, 4.06;
N, 11.98.
The coordinate sets for the protein structures used in
this investigation were 1DAN8 for fVIIa/TF, 1HCG15
for fXa and 1DWC16 for thrombin. Compound 1a was
docked into the fVIIa active site using an in-house dock-
ing program.9,17
6.5. (4R)-4-t-Butoxycarbonylamino-5-hydroxypentanoic
acid benzyl ester (10)
6.2. Chemistry
To the mixture of N-t-butoxycarbonyl-D-glutamic acid
c-benzyl ester (10.9 g, 32.0 mmol), N-methylmorpholine
(3.29 g, 32.5 mmol), and THF (100 mL), ethyl chloro-
formate (3.08 mL, 32 mmol) was added at 0 °C. The
reaction mixture was stirred and then filtered to remove
precipitates. A small amount of crushed ice and NaBH4
(1.23 g, 32.5 mmol) in water was added to the filtrate.
The mixture was stirred for 3 min, diluted with 0.5 N
HCl and stirred for 1 h. EtOAc was added to the
Work-up included drying over magnesium sulfate, filter-
ing, and concentrating in vacuo. Column chromatogra-
phy of intermediates was performed using silica gel
(Merck, particle size 0.063–0.200 mm). Final products
were purified by preparative reverse-phase HPLC, which
was performed on a Waters 600 system with a C-18 re-
verse-phase column (InertsilÒ ODS-3, GL Sciences Inc.)
using a mixture of acetonitrile and water, both of which

1492
K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
1
trated in vacuo to give 2b (4.5 g): H NMR (DMSO-d6)
solution, and then the organic layer was washed with
saturated NaHCO3 and saturated NaCl, and worked
up to give 10 (10.9 g): H NMR (CDCl3) d 1.45 (9H,
s), 1.75–2.00 (2H, m), 2.40–2.50 (2H, m), 3.50–3.70
(3H, m), 4.80 (1H, br), 5.10 (2H, s), 7.35 (5H, s).
d 1.90–2.10 (5H, m), 2.65 (2H, t), 3.60 (1H, br), 3.70
(3H, s), 4.15–4.35 (2H, m), 5.10 (2H, s), 7.30 (1H, s),
7.35 (5H, s), 7.48 (1H, d), 7.60 (1H, s), 7.70 (1H, d),
8.40 (3H, br), 9.75 (1H, s).
1
6.6. (4R)-4-t-Butoxycarbonylamino-5-chloropentanoic
acid benzyl ester (11)
6.10. 2-Acetamido-3-[2-[(2R)-4-benzyloxycarbonyl-2-(4-
guanidinobenzoylamino)butoxy]-4-cyanophenyl]acrylic
acid methyl ester trifluoroacetate (3b)
To a mixture of 10 (10.8 g, 33.4 mmol) and triethyl-
amine (3.4 g, 33.4 mmol) in dichloromethane, metha-
nesulfonyl chloride (3.83 g, 33.4 mmol) was added at
0 °C. The reaction mixture was washed with 1 N HCl
and saturated NaCl, and worked up to give crude
(4R)-4-t-butoxycarbonylamino-5-methanesulfonyloxy-
pentanoic acid benzyl ester. A mixture of the crude
product and lithium chloride (7.2 g, 170 mmol) in
DMF (50 mL) was stirred at 50 °C overnight and con-
centrated in vacuo. The residue was diluted with EtOAc,
washed with water and saturated NaCl, and worked up.
The residue was washed with a mixture of EtOAc and
A mixture of 2b (150 mg, 0.3 mmol), 4-guanidinoben-
zoic acid HCl (65 mg, 0.3 mmol), 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide HCl (EDC HCl)
(58 mg, 0.3 mmol), triethylamine (61 mg, 0.6 mmol), 1-
hydroxybenzotriazole (41 mg, 0.3 mmol), and DMF
(5 mL) was stirred overnight. The reaction mixture
was concentrated in vacuo, and the residue was purified
by reverse-phase HPLC to give 3b (140 mg, 63%): MS
(ESI) 627 (MH+).
6.11. (4R)-4-[(4-Guanidinobenzoyl)amino]-5-[5-amidino-
2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic acid bistri-
fluoroacetate (1b)
1
hexane to give 11 (8.29 g, 73%): H NMR (CDCl3) d
1.40 (9H, s), 1.90–2.00 (2H, m), 2.40–2.50 (2H, m),
3.55–3.70 (2H, m), 3.85–4.00 (1H, m), 4.70 (1H, br),
5.10 (2H, s), 7.35 (5H, s).
A mixture of 3b (140 mg, 0.19 mmol), 4.0 M HCl solu-
tion in 1,4-dioxane (5 mL), and ethanol (1 mL) was stir-
red for 2 days, and concentrated in vacuo. To the
residue, ammonium carbonate (80 mg) and ethanol
(5 mL) were added. The mixture was stirred overnight
and concentrated in vacuo. To the residue, 3 N hydro-
chloric acid was added. The mixture was stirred at
80 °C for 2 h, and concentrated in vacuo. The residue
was purified by reverse-phase HPLC to give 1b (41 mg,
30 %): 1H NMR (DMSO-d6) d 1.80–2.10 (2H, m),
2.25–2.40 (2H, m), 4.00–4.40 (3H + 2H of keto form,
m), 6.80 (1H, s, enol form), 7.25–7.50 (4H + 1H of
keto form, m), 7.65 (4H, br s), 7.95 (2H, d), 8.35 (1H,
d, enol form), 8.45 (1H, d), 9.10 (2H, br), 9.25 (2H,
br), 9.75 (1H, br, enol), 10.10 (1H, s); MS (ESI) 499
(MH+). Anal. Calcd for C23H26N6O7Æ2.0TFAÆ2H2O: C,
42.52; H, 4.229; N, 11.02. Found: C, 42.32; H, 4.59;
N, 11.35.
6.7. (4R)-4-t-Butoxycarbonylamino-5-(5-cyano-2-iodo-
phenoxy)pentanoic acid benzyl ester (12)
A mixture of 11 (8.29 g, 24.3 mmol), 3-hydroxy-4-
iodobenzonitrile (6.9 g, 28.0 mmol), K2CO3 (6.9 g,
50 mmol), and KI (4.15 g, 25 mmol) in DMF (80 mL)
was stirred at 70 °C for 3 days. The reaction mixture
was concentrated in vacuo, and the residue was purified
by chromatography (EtOAc/hexane) to give 12 (5.72 g,
1
43%): H NMR (CDCl3) d 1.45 (9H, s), 2.10 (2H, br),
2.55 (2H, t), 4.05 (3H, br), 4.90 (1H, br), 5.15 (2H, s),
6.95 (1H, s), 7.00 (1H, d), 7.35 (5H, s), 7.90 (1H, d).
6.8. 2-Acetamido-3-[2-[(2R)-4-benzyloxycarbonyl-2-(t-
butoxycarbonylamino)butoxy]-4-cyanophenyl]acrylic acid
methyl ester (13)
A mixture of 12 (5.72 g, 10.4 mmol), methyl 2-acetami-
doacrylate (3.0 g, 21 mmol), tris(2-methylphenyl)phos-
phine (2.0 g, 6.6 mmol), palladium acetate (II)
(270 mg, 1.1 mmol), and triethylamine (3.54 g, 35 mmol)
in DMF (50 mL) was stirred at 100 °C for 5 h. The reac-
tion mixture was concentrated in vacuo, and the residue
was extracted with EtOAC, washed by the usual meth-
od, and worked up. The crude product was purified by
chromatography (EtOAc/hexane) to give 13 (2.6 g,
6.12. 2-Acetamido-3-[2-[(2R)-4-benzyloxycarbonyl-2-
benzoylamino-butoxy]-4-cyanophenyl]acrylic acid methyl
ester (3e)
A mixture of 2b (90 mg, 0.18 mmol), benzoyl chloride
(28 mg, 0.2 mmol), triethylamine (61 mg, 0.6 mmol),
and DMF (5 mL) was stirred overnight. The reaction
mixture was concentrated in vacuo, and the residue
was diluted with EtOAc. The organic layer was washed
with 1 N HCl, saturated NaHCO3, and saturated NaCl,
and worked up. The residue was purified by chromato-
graphy to give 3e (90 mg, 88%): MS (ESI) 570 (MH+).
1
44%): H NMR (CDCl3) d 1.40 (9H, s), 1.80–2.05 (2H,
m), 2.05 (3H, br), 2.50 (2H, t), 3.85 (3H, s), 4.05 (3H,
br), 4.85 (1H, d), 5.10 (2H, s), 7.12 (1H, s), 7.23 (1H,
d), 7.31–7.48 (8H, m).
6.13. (4R)-4-Benzoylamino-5-[5-amidino-2-(2-carboxy-2-
oxoethyl)phenoxy]pentanoic acid trifluoroacetate (1e)
6.9. 2-Acetamido-3-[2-[(2R)-4-benzyloxycarbonyl-2-
aminobutoxy]-4-cyanophenyl]acrylic acid methyl ester
HCl (2b)
The title compound was prepared from 3e as described
for 1b: 1H NMR (DMSO-d6) d 1.80–2.10 (2H, m),
2.05–2.45 (2H, m), 4.00–4.50 (3H + 2H of keto form,
m), 6.80 (1H, s, enol form), 7.30–7.60 (5H + 1H of keto
A mixture of 13 (5 g, 8.8 mmol) and 4.0 M HCl solution
in 1,4-dioxane (50 mL) was stirred for 2 h, and concen-

K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
1493
form, m), 7.85 (2H, d), 8.30 (1H, d, enol form), 8.40 (1H,
6.18. 2-Aminobenzimidazole–5-carboxylic acid HCl (5)
d), 9.00 (2H, br), 9.25 (2H, br), 9.75 (1H, br, enol); MS
(ESI) 442 (MH+). Anal. Calcd for C22H23N3O7Æ1.0T-
FAÆ0.8H2O: C, 50.58; H, 4.527; N, 7.373. Found: C,
50.25; H, 4.79; N, 7.76.
Part A. To a mixture of 3,4-diaminobenzoic acid methyl-
ester (300 mg, 1.8 mmol) and water (5 mL), a solution of
5 M cyanogen bromide in acetonitrile (0.5 mL) was
added. The reaction mixture was stirred for 20 min,
and filtered. Aqueous ammonia and EtOAc were added
to the filtrate, and the organic layer was washed and
worked up to give 2-aminobenzimidazole–5-carboxylic
acid methyl ester: H NMR (DMSO-d6) d 3.80 (3H, s),
6.50 (2H, br), 7.15 (1H, d), 7.55 (1H, d), 7.70 (1H, s),
10.90 (1H, br).
6.14. 4-Guanidino-3-methylbenzoic acid trifluoroacetate
(4a)
1
A
mixture of 4-amino-3-methylbenzoic acid (2 g,
13.2 mmol) and 4 M HCl solution in dioxane was
stirred for 30 min and concentrated in vacuo. To the
residue, ethanol (30 mL), and cyanamide (1.68 g,
40 mmol) was added, and the mixture was stirred at
50 °C overnight. The reaction mixture was concentrated
in vacuo and purified by reverse-phase HPLC to give 4a
(210 mg, 5%): 1H NMR (DMSO-d6) d 2.25 (3H, s), 7.35
(1H, d), 7.50 (4H, br), 7.85 (1H, d), 7.95 (1H, s), 9.65
(1H, s).
Part B. A mixture of the product of Part A and 3 M
hydrochloric acid was stirred at 65 °C overnight, and
concentrated in vacuo to give 5 (330 mg, 86%): 1H
NMR (DMSO-d6) d 7.45 (1H, d), 7.85 (1H, d), 8.80
(2H, s), 13.00 (1H, br).
6.19. (4R)-4-[(2-Aminobenzimidazole-5-carbonyl)amino]-
5-[5-amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic
acid bistrifluoroacetate (1f)
6.15. (4R)-4-[(4-Guanidino-3-methylbenzoyl)amino]-5-[5-
amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic acid
bistrifluoroacetate (1c)
The title compound was prepared from 5 and 2b as
described for 1b: 1H NMR (DMSO-d6) d 1.80–2.15
(2H, m), 2.25–2.50 (2H, m), 4.05–4.30 (3H + 2H of
keto form, m), 6.80 (1H, s, enol form), 7.35–7.50
(3H + 1H of keto form, m), 7.75 (1H, d), 7.80 (1H, s),
8.30 (1H, d, enol form), 8.45 (1H, d), 8.65 (2H, s),
9.10 (2H, s), 9.25 (2H, s); MS (ESI) 497 (MH+).
Anal. Calcd for C23H24N6O7Æ2.0TFAÆ2.0H2O: C,
42.63; H, 3.975; N, 11.04. Found: C, 42.94; H, 4.22;
N, 10.64.
Part A. Compound 2b (90 mg, 0.18 mmol) and 4a
(50 mg, 0.16 mmol) was treated as described in 3b to
give 2-acetamido-3-[2-[(2R)-4-benzyloxycarbonyl-2-(4-
guanidino-3-methylbenzoylamino)butoxy]-4-cyanophen-
yl]acrylic acid methyl ester trifluoroacetate (3c).
Part B. The product of Part A was treated as described
1
for 1b to give 1c (13 mg, 11%): H NMR (DMSO-d6) d
1.80–2.10 (2H, m), 2.25 (3H, s), 2.25–2.40 (2H, m),
4.00–4.30 (2H + 2H of keto form, m), 4.40 (1H, m),
6.80 (1H, s, enol form), 7.30–7.60 (8H + 1H of keto
form, m), 7.75 (1H, d), 7.85 (1H, s), 8.30 (1H, d, enol
form), 8.55 (1H, d), 9.15–9.52 (4H, m), 9.70–9.85
(1H + 1H of enol form, m); MS (ESI) 513 (MH+). Anal.
Calcd for C24H28N6O7Æ2.0TFAÆ2.0H2O: C, 43.30; H,
4.412; N, 10.82. Found: C, 43.65; H, 4.37; N, 10.91.
6.20. 2-Aminoquinoline-6-carboxylic acid HCl (8)
Part A. A mixture of quinoline-6-carboxylic acid (4.9 g,
28.3 mmol), a solution of 4.0 M HCl in dioxane
(40 mL), and ethanol (80 mL) was stirred at 70 °C over-
night. The reaction mixture was concentrated in vacuo,
and the residue was diluted with EtOAc, washed and
worked up to give quinoline-6-carboxylic acid ethyl
ester: 1H NMR (CDCl3) d 1.45 (3H, t), 4.45 (2H,
q), 7.45 (1H, dd), 8.15 (1H, d), 8.25–8.35 (2H, m), 8.60
(1H, s), 9.00 (1H, d).
6.16. 4-(1-Methylguanidino)benzoic acid trifluoroacetate
(4b)
The title compound was prepared from 4-(1-methyl-
amino)benzoic acid as described for 4a: 1H NMR
(DMSO-d6) d 3.30 (3H, s), 7.50 (2H, d), 7.55 (4H, br),
8.05 (2H, d).
Part B. To a mixture of the product of Part A and
dichloromethane (80 mL), m-chloroperbenzoic acid
(6.2 g, 36 mmol) was added at 0 °C. The reaction mix-
ture was stirred at ambient temperature overnight,
washed with 10% sodium sulfite, saturated NaHCO3
and saturated NaCl, and worked up. To the residue,
dichloromethane (70 mL) and phosphoryl chloride
(35 mL) were added and the mixture was stirred at
50 °C overnight. The reaction mixture was concentrated
in vacuo, and the residue was extracted with dichloro-
methane, washed and worked up. The crude product
was purified with chromatography (EtOAc/hexane) to
give 2-chloroquinoline-6-carboxylic acid ethyl ester (7)
6.17. (4R)-4-[(4-(1-Methylguanidino)benzoyl)amino]-5-[5-
amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic acid
bistrifluoroacetate (1d)
The title compound was prepared from 4b and 2b as de-
scribed for 3b and 1b: 1H NMR (DMSO-d6) d 1.82–2.10
(2H, m), 2.25–2.43 (2H, m), 3.30 (3H, s), 4.07–4.30
(2H + 2H of keto form, m), 4.32–4.48 (1H, m), 6.78
(1H, s, enol form), 7.30–7.60 (8H + 1H of keto form,
m), 8.00 (2H, d), 8.31 (1H, d, enol form), 8.55 (1H, d),
9.15–9.52 (4H, m), 9.70–9.82 (1H, br, enol); MS (ESI)
513 (MH+). Anal. Calcd for C24H28N6O7Æ2.0TFAÆ2.0-
H2O: C, 43.30; H, 4.412; N, 10.82. Found: C, 43.55;
H, 4.52; N, 10.85.
1
(1.87 g, 28%): H NMR (CDCl3)) d 1.40 (3H, t), 4.45
(2H, d), 7.46 (1H, d), 8.06 (1H, d), 8.21 (1H, d), 8.34
(1H, d), 8.58 (1H, s).

1494
K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
Part C. A mixture of 7 (1.87 g, 8 mmol) and phenol
(5.5 g) was stirred at 70 °C for 10 min. To the mixture,
ammonium acetate (5.5 g) was added and the reaction
mixture was stirred for 1 h. EtOAc and 1 N NaOH were
added to the mixture, and the organic layer was washed
with saturated NaCl and worked up. The crude product
was purified with chromatography (EtOAc/hexane/etha-
nol) to give 2-aminoquinoline-6-carboxylic acid ethyl
6.24. (4R)-4-[(2-Aminobenzothiazole-6-carbonyl)amino]-
5-[5-amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic
acid bistrifluoroacetate (1j)
Part A. A mixture of 2-aminobenzothiazole-6-carbox-
ylic acid ethyl ester (500 mg, 2.25 mmol) and 4 M hydro-
chloric acid (20 mL) was stirred at 80 °C for 5 h. The
reaction mixture was concentrated in vacuo, and the res-
idue was washed with a mixture of EtOAc and hexane to
give 2-aminobenzothiazole-6-carboxylic acid HCl
(545 mg): 1H NMR (DMSO-d6) d 7.60 (1H, d), 8.00
(1H, d), 8.50 (1H, s), 9.70 (2H, br).
1
ester (330 mg, 19%): H NMR (CDCl3) d 1.40 (3H, t),
4.40 (2H, q), 5.00 (2H, br), 6.75 (1H, d), 7.64 (1H, d),
7.95 (1H, d), 8.16 (1H, d), 8.37 (1H, s).
Part D. A mixture of the product of Part C and 6 M
hydrochloric acid (30 mL) was stirred at 80 °C, and con-
centrated in vacuo. The residue was washed with EtOAc
to give 8 (330 mg, 96%): 1H NMR (DMSO-d6) d 7.16 (1H,
d), 7.78 (1H, d), 8.23 (1H, d), 8.50 (1H, d), 8.55 (1H, s).
Part B. The title compound 1j was prepared from the 2-
aminobenzothiazole-6-carboxylic acid HCl and 2b as de-
scribed for 3b and 1b: 1H NMR (DMSO-d6) d 1.80–2.15
(2H, m), 2.25–2.50 (2H, m), 4.00–4.50 (3H + 2H of keto
form, m), 6.80 (1H, s, enol form), 7.30–7.55 (3H + 1H of
keto form, m), 7.75 (1H, d), 7.95 (2H, s), 8.15 (1H, s),
8.25–8.40 (1H + 1H of enol form, m), 9.00 (2H, s),
9.25 (2H, s), 9.75 (1H, s, enol); MS (ESI) 514 (MH+).
Anal. Calcd for C23H23N5O7SÆ2.0TFAÆ1.0H2O: C,
42.69; H, 3.582; N, 9.219. Found: C, 42.82; H, 3.91;
N, 9.40.
6.21. (4R)-4-[(2-Aminoquinoline-6-carbonyl)amino]-5-[5-
amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic acid
bistrifluoroacetate (1i)
The title compound was prepared from 8 and 2b as de-
scribed for 3b and 1b: 1H NMR (DMSO-d6) d 1.80–2.10
(2H, m), 2.30–2.50 (2H, m), 4.10–4.30 (2H + 2H of keto
form, m), 4.45 (1H, br), 6.75 (1H, s, enol form), 7.10
(1H, d), 7.35–7.50 (2H + 1H of keto form, m), 7.65
(1H, d), 8.15 (1H, d), 8.25–8.45 (2H + 1H of enol form,
d), 8.65 (1H, d), 8.90 (2H, br), 9.10 (2H, br), 9.25 (2H,
br); MS (ESI) 508 (MH+). Anal. Calcd for
C25H25N5O7Æ2.0TFAÆ1.33H2O: C, 45.85; H, 3.936; N,
9.220. Found: C, 45.49; H, 4.27; N, 9.57.
6.25. 3-[4-Amidino-2-[2-(2-aminoquinoline-6-carbonyl)-
aminoethoxy]phenyl]-2-oxopropionic acid bistrifluoro-
acetate (1k)
Part
bonyl)amino-ethoxy]phenyl]-2-acetamidoacrylic
A.
3-[4-Cyano-2-[2-(2-aminoquinoline-6-car-
acid
methyl ester trifluoroacetate (3k) was prepared from 8
and 2a as described for 3b.
6.22. (4R)-4-[(2-Methylbenzimidazole-5-carbonyl)amino]-
5-[5-amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic
acid bistrifluoroacetate (1g)
Part B. The title compound 1k was prepared from 3k as
described for 1a: 1H NMR (DMSO-d6) d 3.60–3.80 (2H,
m), 4.10–4.40 (2H + 2H of keto form, m), 6.80 (1H of
enol, s), 7.10 (1H, d), 7.35–7.54 (2H + 1H of keto form,
m), 7.70 (1H, d), 8.15 (1H, d), 8.30–8.44 (2H + 1H of
enol form, m), 8.97 (1H, br), 9.05 (2H, br), 9.30 (2H,
br); MS (ESI) 436 (MH+). Anal. Calcd for
C22H21N5O5Æ2.0TFAÆ2.0H2O: C, 44.64; H, 3.890; N,
10.01. Found. C, 44.75; H, 3.94; N, 9.94.
The title compound was prepared from 2-methylbenzim-
idazole-5-carboxylic acid and 2b as described for 3b and
1b: 1H NMR (DMSO-d6) d 1.85–2.15 (2H, m), 2.25–2.50
(2H, m), 2.75 (3H, s), 4.10–4.30 (2H + 2H of keto form,
m), 4.45 (1H, br), 6.80 (1H, s, enol form), 7.35–7.55
(2H + 1H of keto form, m), 7.73 (1H, d), 7.87–7.96
(1H, m), 8.16 (1H, s), 8.32 (1H, d, enol form), 8.49–
8.64 (1H, m), 9.10 (2H, br), 9.30 (2H, br); MS (ESI)
496 (MH+). Anal. Calcd for C24H25N5O7Æ2.0TFAÆ2.0-
H2O: C, 44.27; H, 4.113; N, 9.220. Found: C, 44.26;
H, 4.22; N, 9.26.
6.26. 3-[4-Amidino-2-[2-(2-aminobenzthiazole-6-carbon-
yl)aminoethoxy]phenyl]-2-oxopropionic acid bistrifluoro-
acetate (1l)
The title compound was prepared from 2-aminobenz-
thiazole-6-carboxylic acid and 2a as described for 3b
and 1b: 1H NMR (DMSO-d6) d 3.60–3.80 (2H, m),
4.10–4.40 (2H + 2H of keto form, m), 6.80 (1H, s, enol
form), 7.25–8.00 (5H + 1H of keto form, m), 8.15 (1H,
d), 8.30 (1H of enol form, d), 9.00 (2H, s), 9.30 (2H,
s), 9.65 (1H, s, enol); MS (ESI) 442 (MH+). Anal. Calcd
for C20H19N5O5SÆ2.0TFAÆ3.5H2O: C, 39.34; H, 3.852;
N, 9.560. Found: C, 39.13; H, 4.00; N, 9.94.
6.23. (4R)-4-[(Benzimidazole-5-carbonyl)amino]-5-[5-
amidino-2-(2-carboxy-2-oxoethyl)phenoxy]pentanoic acid
bistrifluoroacetate (1h)
The title compound was prepared from benzimidazole-
5-carboxylic acid and 2b as described for 3b and 1b:
1H NMR (DMSO-d6) d 1.80–2.15 (2H, m), 2.15–2.80
(2H, m), 4.00–4.30 (2H + 2H of keto form, m), 4.45
(1H, br), 6.80 (1H, s, enol form), 7.35–7.55 (2H + 1H
of keto form, m), 7.70 (1H, d), 7.86 (1H, d), 8.20 (1H,
s), 8.32 (1H, d, enol form), 8.52 (1H, d), 8.77 (1H, s),
9.00 (2H, br), 9.28 (2H, br); MS (ESI) 482 (MH+). Anal.
Calcd for C23H23N5O7Æ2.0TFAÆ5.0H2O: C, 40.55; H,
4.412; N, 8.758. Found: C, 40.40; H, 4.29; N, 8.81.
6.27. Enzyme assays
Human fVIIa and fXa were obtained from Enzyme
Research Laboratories Inc. Human TF was obtained
from American Diagnostica Inc. Human thrombin was

K. Sagi et al. / Bioorg. Med. Chem. 13 (2005) 1487–1496
1495
3. Much effort has been focused on thrombin or fXa
inhibitors: (a) Coburn, C. A. Exp. Opin. Ther. Pat. 2001,
11(5), 721–738; (b) Betz, A. Exp. Opin. Ther. Pat. 2001,
11(6), 1007.
4. Biemond, B. J.; Levi, M.; Ten Cate, H.; Soule, H. R.;
Morris, L. D.; Foster, D. L. Thromb. Haemost. 1995, 73,
223.
obtained from Sigma Chemical Co. The chromogenic
substrates used were S-2288 (Daiichi Pure Chemical)
for fVIIa/TF, S-2222 (Daiichi Pure Chemical, Japan)
for fXa, and S-2238 (Daiichi Pure Chemical) for
thrombin. fVIIa/TF was assayed in a buffer contain-
ing 0.05 M Tris, 0.15 M NaCl, 5 mM CaCl2 0.1% BSA
at pH 7.4, and fVIIa/TF was preformed by incubat-
ing at room temperature in the buffer. fXa and throm-
bin were assayed in a buffer containing 0.1 M Tris,
0.2 M NaCl, 0.1% PEG-6000, and 0.02% Tween 20 at
pH 8.4.
5. Arnljots, B.; Ezban, M.; Hedner, U. J. Vasc. Surg. 1997,
25, 341.
6. Bajaj, M. S.; Bajaj, S. P. Thromb. Haemost. 1997, 78, 471.
7. (a) Robinson, L. A.; Saiah, M. K. Annu. Rep. Med. Chem.
2002, 37, 85–94; (b) Parlow, J. J.; Case, B. L.; Dice, T. A.;
Fenton, R. L.; Hayes, M. J.; Jones, D. E.; Neumann, W.
L.; Wood, R. S.; Lachance, R. M.; Girard, T. J.;
Nicholson, N. S.; Clare, M.; Stegeman, R. A.; Stevens,
A. M.; Stallings, W. C.; Kurumbail, R. G.; South, M. S. J.
Med. Chem. 2003, 46(19), 4050–4062; (c) Klingler, O.;
Matter, H.; Schudoku, M.; Bajaj, S. P.; Czech, J.; Lorenz,
M.; Nestler, H. P.; Schreuder, H.; Wildgoose, P. Bioorg.
Med. Chem. Lett. 2003, 13, 1463–1467; (d) 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, 2253–2256; (e) Kohrt, J. T.; Filipski, K. J.; Rapundalo,
S. T.; Cody, W. L.; Edmunds, J. J. Tetrahedron Lett. 2000,
41, 6041–6044.
8. Banner, D. W.; DꢀArcy, A.; Chene, C.; Winkler, F. K.;
Guha, A.; Konigsberg, W. H.; Nemerson, Y.; Kirchhofer,
D. Nature 1996, 380, 41.
9. Sagi, K.; Nakagawa, T.; Yamanashi, M.; Makino, S.;
Takahashi, M.; Takayanagi, M.; Takenaka, K.; Suzuki,
N.; Oono, S.; Kataoka, N.; Ishikawa, K.; Shima, S.;
Fukuda, Y.; Kayahara, T.; Takehana, S.; Shima, Y.;
Tashiro, K.; Yamamoto, H.; Yoshimoto, R.; Iwata, S.;
Tsuji, T.; Sakurai, K.; Shoji, M. J. Med. Chem. 2003,
46(10), 1845–1857.
The final substrate concentrations in the reaction were
800, 200, and 100 lM for S-2288, S-2222, and S-2238,
respectively. All enzyme assays were carried out at room
temperature in 96-well microtiter plates with final con-
centrations of 5.0 nM for fVIIa/TF, 4.5 nM for fXa,
and 1.1 nM for thrombin. Compounds at appropriate
dilutions were added to wells containing buffer and
enzyme and preincubated for 10 min. The enzyme
reactions were initiated by the addition of substrate,
and color development, due to the release of p-nitroan-
iline from each chromogenic substrate, was monitored
continuously at 405 nm on a microplate reader (Model
3550-UV, Bio-Rad). The measured initial velocities were
used to determine the amount of inhibitor required to
reduce the control velocity by 50%; this concentration
was defined as the IC50 of the inhibitor. The apparent
Ki values of compounds were then calculated according
to the relationship: Ki = IC50/(1 + Km/S) or (IC50)1/
(IC50)2 = (Ki)1/(Ki)2.18
6.28. Coagulation assay
10. Residues are numbered with chymotrypsin-based num-
bering: Bode, W.; Mayr, I.; Baumann, U.; Huber, R.;
Stone, S. R.; Hofsteenge, J. EMBO J. 1989, 8(11), 3467–
3475.
Prothrombin time (PT) and activated partial thrombo-
plastin time (aPTT) were measured with coagulometer
CA3000 (Sysmex, Japan). Citrated human plasma,
which was collected from healthy volunteers, was used
in the assays. Plasma (45 lL) was mixed with 5 lL of
a compound dilution followed by the automatic addi-
tion of 100 lL of prothrombin reagent (Thromboplastin
C plus, Dade Behring) for the PT measurement, or
50 lL of activated cephaloplastin reagent (Dade Beh-
ring) and 50 lL of 20 mM CaCl2 for the aPTT measure-
ment. The prolongation of the clotting time in the
presence of a test compound versus its absence was used
to quantify the anticoagulant effect.
11. In our study on fXa inhibitors, the crystal structure of an 2-
substituted APPA–trypsin complex shows that although
the bonding linkage is formed between the pyruvic acid
moiety and the Ser 195, the overall docking mode including
the pyruvic acid is still similar to that of the calculated
noncovalent 2-substituted APPA–fXa complex. Therefore
we consider the overall docking mode of 1a will be similar
to the noncovalently calculated binding mode: Takahashi,
M.; Sagi, K.; Yamanashi, M.; Makino, S.; Suzuki, N.;
Takayanagi, M.; Nakagawa, T.; Ishikawa, K.; Shoji, M.
13th European Symposium on Quantitative Structure–
Activity Relationships, Dusseldorf, August 2000.
¨
12. Marquart, M.; Walter, J.; Deisenhofer, J.; Bode, W.;
Huber, R. Acta Crystallogr. 1983, B39, 480–490.
13. pKa values described in this paper were taken from
Dictionary of Organic Compounds on CD-ROM version
10:2 Chapman & Hall, or Yamanaka, H.; Hino, T.;
Nakagawa, M.; Sakamoto, T. Chemistry of Heterocyclic
Compounds; Kodansha Scientific: Japan, ISBN 4-06-
190754-9, 1988.
Acknowledgements
The authors thank members of the Analytical Chemistry
Department for mass spectral measurements.
14. There are very few publications that address therapeutic
index (efficacy/bleeding) for a small-molecule inhibitor of
TF/fVIIa: Suleymanov, O. D.; Szalony, J. A.; Salyers, A.
K.; Lachance, R. M.; Parlow, J. J.; South, M. S.; Wood,
R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther. 2003,
306(3), 1115–1121.
15. Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.;
Park, C. H.; Bode, W.; Huber, R.; Blankenship, D. T.;
Cardin, A. D.; Kisiel, W. J. Mol. Biol. 1993, 232, 947–966.
References and notes
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