Novel strategy to boost oral anticoagulant activity of blood coagulation enzyme inhibitors based on biotransformation into hydrophilic conjugates

Article abstract of DOI:10.1016/j.bmc.2014.09.059

The blood coagulation cascade represents an attractive target for antithrombotic drug development, and recent studies have attempted to identify oral anticoagulants with inhibitory activity for enzymes in this cascade, with particular attention focused on

Full text of DOI:10.1016/j.bmc.2014.09.059

Bioorganic & Medicinal Chemistry 22 (2014) 6324–6332
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel strategy to boost oral anticoagulant activity of blood
coagulation enzyme inhibitors based on biotransformation
into hydrophilic conjugates
a
a
a
b
Tsukasa Ishihara a, , Yuji Koga , Kenichi Mori , Keizo Sugasawa , Yoshiyuki Iwatsuki ,
⇑
Fukushi Hirayama a
a Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
b Pharmacovigilance, Astellas Pharma Inc., 2-5-1, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8411, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The blood coagulation cascade represents an attractive target for antithrombotic drug development, and
recent studies have attempted to identify oral anticoagulants with inhibitory activity for enzymes in this
cascade, with particular attention focused on thrombin and factor Xa (fXa) as typical targets. We previ-
ously described the discovery of the orally active fXa inhibitor darexaban (1) and reported a unique pro-
file that compound 1 rapidly transformed into glucuronide YM-222714 (2) after oral administration.
Here, we propose a novel strategy towards the discovery of an orally active anticoagulant that is based
on the bioconversion of a non-amidine inhibitor into the corresponding conjugate to boost ex vivo anti-
coagulant activity via an increase in hydrophilicity. Computational molecular modeling was utilized to
select a template scaffold and design a substitution point to install a potential functional group for con-
jugation. This strategy led to the identification of the phenol-derived fXa inhibitor ASP8102 (14), which
demonstrated highly potent anticoagulant activity after biotransformation into the corresponding glucu-
ronide (16) via oral dosing.
Received 1 September 2014
Revised 26 September 2014
Accepted 30 September 2014
Available online 19 October 2014
Keywords:
Factor Xa
Anticoagulant
Conjugation
Docking study
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
anticoagulant and antithrombotic activity due to their overall high
hydrophilicity, associated with the inhibitor’s highly polar amidine
Intravascular clot formation results in thromboembolic diseases
including ischemic stroke, deep venous thrombosis, myocardial
infarction, unstable angina, and pulmonary embolism, all of which
are major causes of morbidity and mortality in the industrialized
world. Enzymes in the blood coagulation cascade, such as thrombin
and fXa, are attractive targets for the prevention of thrombosis, with
numerous efforts devoted to the discovery of inhibitors against
these enzymes that can act as orally active anticoagulant agents.
Approaches to the discovery of oral anticoagulant drugs that
directly inhibit blood coagulation enzymes are classified into two
categories. One involves approaches based on the strategy of ami-
dine prodrugs, represented by the anticoagulants ximelagatran1
and dabigatran etexilate.2 An amidine prodrug is bioconverted into
the corresponding amidine inhibitor after oral administration, and
the amidine group forms a bidentate salt bridge interaction with
the carboxylic acid of Asp189 in the S1 site of the enzyme.
Although amidine-derived inhibitors demonstrate potent in vivo
group, the hydrophilic and basic profile of the amidine moiety also
causes a reduction in membrane permeability and oral bioavail-
ability. Thus, the prodrug strategy seeks to mask the high hydro-
philicity and basicity of the amidine unit to improve the inferior
profile of amidine-derived inhibitors.
The other methodology involves nonamidine-type inhibitors—
eliminating highly hydrophilic and basic amidine groups to
improve the oral bioavailability of inhibitors.3 These efforts have
already led to the discovery of the launched coagulants rivarox-
aban,4 apixaban,5 and edoxaban.6 These nonamidine-type inhibi-
tors possess a small lipophilic substituent, such as a methoxy or
chloro group, which occupies the small hydrophobic pocket
formed by the residues Ala190, Val213, and Tyr228 in the bottom
of the S1 site of the enzyme, or a less polar surrogate for an ami-
dine group, such as an aromatic amine or benzylamine. However,
the removal of the amidine group causes an increase in the overall
lipophilicity of the inhibitor, which often leads to a detrimental
reduction in anticoagulant potency.3 Thus, concerted efforts have
been devoted to managing both anticoagulant activity and phar-
macokinetics profile of orally active anticoagulants.
⇑
Corresponding author. Tel.: +81 29 863 6713; fax: +81 29 854 1519.
E-mail address: tsukasa.ishihara@astellas.com (T. Ishihara).
http://dx.doi.org/10.1016/j.bmc.2014.09.059
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
6325
Here, we propose adoption of a third strategy in the discovery of
blood coagulation enzyme inhibitors with boosted anticoagulant
effects after oral administration, based on non-amidine inhibitors,
via bioconversion to corresponding highly hydrophilic conjugates.
into a corresponding hydrophilic amidine inhibitor with high anti-
coagulant potency is the established methodology for identifica-
tion of orally active anticoagulants.1,2 Using this idea as a model,
we investigated whether the biotransformation of a lipophilic pre-
cursor into the corresponding hydrophilic conjugate could be a
new strategy for discovery of oral anticoagulants.
2. Strategy
A key step for this strategy is the installation of a ‘trigger’ at an
appropriate position of a suitable chemical scaffold. The trigger
refers to a functional group with a potential to act as a substrate
for conjugation. A phenolic hydroxyl group is preferable for the
trigger owing to two reasons: conjugates of hydroxyl groups are
known to be relatively stable compared to those of acyl or amino
groups, and phenolic hydroxyl groups have less oxidizability than
alcoholic hydroxyl groups. Similar to compound 1, the launched
phenol-derived cholesterol absorption inhibitor ezetimibe was
found to rapidly undergo glucuronide conjugation before entering
portal plasma via oral dosing.11 This property indicates a general
feature that phenolic hydroxyl groups can be precursors of highly
hydrophilic glucuronide conjugates after oral administration.
Computational molecular modeling simulations could be uti-
lized to identify an appropriate scaffold and where to install the
triggering phenolic hydroxyl group. Figure 2 shows a proposed
binding model of compound 1 and its glucuronide 2 to the active
site of fXa. The sugar residue of compound 2 extends into solvent
space without disturbing the interaction between the ligand and
the enzyme, resulting in fXa inhibitory activity. A novel inhibitor
can therefore be designed using computational molecular model-
ing by selecting a chemical scaffold on which a phenolic hydroxyl
group is directed out of the binding site and into the bulk solvent.
At the time our study was initiated,12 only three representative
scaffolds, compounds 5 (DPC423),13 6,14 and 7,15 had been reported
as non-amidine fXa inhibitors (Fig. 1). Our computational modeling
simulation of these three inhibitors and consideration of reactivity
for glucuronidation led us to presume the followings:
Inhibitors of enzymes in the blood coagulation cascade target
the venous and arterial vascular system, therefore their high distri-
bution in the circulation is preferred. Such a property is consistent
with a pharmacokinetic profile of low distribution volume, and a
general method to decrease volume of distribution is to increase
hydrophilicity of a compound.7 Consequently, an inhibitor with
increased hydrophilicity is expected to improve in vivo antithrom-
botic effect. This concept is consistent with the previous result that
found the fXa inhibitor 3, which contains a combination of highly
hydrophilic amidine and carboxylic acid, displays 30-fold higher
activity in an in vivo thrombosis model during continuous intrave-
nous infusion despite its equipotency to the non-amidine fXa
inhibitor 4 in an in vitro anticoagulant assay (Fig. 1).8 Pinto et al.
also reported the desirability of a pharmacokinetic profile with
low distribution volume towards vascular-targeted agents in the
discovery process of the launched fXa inhibitor apixaban.5
We developed the fXa inhibitor 1 to demonstrate potent antico-
agulant effect after oral administration (Fig. 1).9 Compound 1 was
found to be rapidly metabolized when dosed orally, and the pre-
dominant metabolite detected in the plasma was the correspond-
ing glucuronide 2. A comparison of the lipophilicity of the
compounds 1 and 2 found that the latter was much more hydro-
philic (clogP values of 2.30 for compound 1 and ꢀ1.03 for com-
pound 2),10 which led us to consider whether compound 1’s oral
anticoagulant activity was achieved by it being converted into
the highly hydrophilic glucuronide 2 in vivo. Biotransformation
of a lipophilic amidine prodrug with high membrane permeability
N
N
N
N
H2N
NH
NH
OH
O
HN
O
Cl
H
N
HN
O
R
OH
OH
O
HN
O
H
N
H
N
OGlu =
O
O
COOH
O
O
COOH
; darexaban (1)
; YM-222714 (2)
R = OH
R= OGlu
3
4
N
N
S
O
N
O
F
O
H2N
Cl
HN
O
O
NH
Cl
3
4
N
H
N
N
O
N
N
5
N
S
O
O
Cl
CF3
DPC423 (5)
6
7
Figure 1. Structures of direct fXa inhibitors.

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T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
Figure 2. Docking model of compound 1 and glucuronide 2 complexed with the active site of factor Xa. Carbon atoms of compounds are colored cyan, while the protein
surface is colored white.
Compound 5: The aminomethylphenyl moiety was bound to
ASP189 at the bottom of the S1 pocket and the biphenyl part
lay deep within the S4 binding cleft. Therefore, both aromatic
units were directed away from the bulk solvent (Fig. 3). Although
the 5-position of the central pyrazole ring was oriented towards
the solvent-exposed region, installation of a hydroxyl group onto
this position would be inadequate, because 2,6-disubstituted
aromatic rings are very poor substrates for glucuronidation.16
Compound 6: The naphthalene and pyridine rings deeply occu-
pied the S1 and S4 binding cavities, respectively. All aromatic
cores were shielded by the residues of these pockets and did
not extend into the bulk solvent (Fig. 4).
3-benzyloxy-2-nitrobenzoic acid (8),17 was converted to acid
chloride by treatment with oxalyl chloride and then coupled with
2-amino-5-chloropyridine to afford N1-(2-pyridyl)anthranilamide
9. Compound 9 was transformed to aminophenol 10 via a two-step
sequence involving trifluoroacetic acid (TFA)-mediated deprotec-
tion of benzyl moiety followed by catalytic hydrogenation in the
presence of RaneyÒ nickel. Treatment of aminophenol 10 with
Compound 7: Although the distal pyridine ring was buried in
the S1 pocket and made no contact with the aqueous environ-
ment surrounding the enzyme, the 3- and 4-positions on the
central phenyl motif were oriented into the solvent region
(Fig. 5). Reports revealed that 2-acetylaminophenol possesses
greater reactivity than 3-acetylaminophenols and that
4-hydroxybenzamide is a poor substrate for glucuronidation.16
These investigations led us to select the compound 7 as our
template and introduce a trigger hydroxyl group onto the
3-position of its central phenyl ring.
3. Chemistry
Figure 4. Docking model of compound 6 complexed with the active site of factor
Xa. Carbon atoms of compounds are colored cyan, while the protein surface is
colored white.
Scheme 1 depicts the subsequent steps that were employed to
elaborate the target compounds 13–16. The starting material,
Figure 3. Docking model of compound 5 complexed with the active site of factor
Xa. Carbon atoms of compounds are colored cyan, while the protein surface is
colored white.
Figure 5. Docking model of compound 7 complexed with the active site of factor
Xa. Carbon atoms of compounds are colored cyan, while the protein surface is
colored white.

T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
6327
Cl
Cl
NO2
NO2
O
NH2
X
O
a,b
c,d
HOOC
OBn
OBn
OH
N
N
N
H
N
H
8
9
10 X = H
e or f
11
12
Cl
Br
N
N
Cl
Cl
OH
HN
X
O
OH
O
HN
X
O
O
O
g or h
i
OH
OH
N
N
N
N
H
H
O
COOH
15 X = Cl
16 Br
13 X = Cl
14 Br
Scheme 1. Synthesis of Phenol–Derived Inhibitors and their Glucuronide Conjugates. Reagents and conditions: (a) SOCl2, cat. N,N-dimethylformamide (DMF), ethyl acetate,
50 °C; (b) 2-amino-5-chloropyridine, pyridine, CH3CN; (c) pentamethylbenzene, TFA, 40 °C; (d) H2 (1 kgf/cm2), RaneyÒ-Ni, ethanol; (e) N-chlorosuccinimide, DMF, 50 °C; (f) N-
bromosuccinimide, DMF, ꢀ15 °C; (g) 1-isopropylpiperidine-4-carboxylic acid (17), 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,
DMF; (h) 1-isopropylpiperidine-4-carboxylic acid, SOCl2, cat. DMF, pyridine; (i) i. acetobromo-
methanol; ii. Na2CO3, H2O.
a-D-glucuronic acid methyl ester, 1,8-diazabicyclo-[5.4.0]-7-undecene, CHCl3,
N-chlorosuccinimide and N-bromosuccinimide gave 5-halo ana-
logues 11 and 12, respectively, which were then transformed into
the N2-acylanthranilamides 13 and 14, respectively. Glycosylation
of physicochemical property indicated that the phenol 14 showed
comparable membrane permeability to compound 7.
The phenol 14 and its glucuronide 16 were subjected to further
biological evaluation. Examination of anticoagulant effects via
intravenous bolus injection revealed that the glucuronide 16
exhibited superior potency compared to the deshydroxy analogue
7 at about one-tenth the dose (Fig. 6). Compound 16 was 800-fold
more hydrophilic compared to compound 7 (measured logD7.4 val-
ues of 1.8 for compound 7 and ꢀ1.1 for compound 16), which is
consistent with our hypothesis that a highly hydrophilic profile
is preferable for a blood coagulation enzyme inhibitor when tar-
geting the vascular system and that a highly hydrophilic conju-
gated inhibitor will demonstrate potent ex vivo anticoagulant
effect.
of phenol derivatives 13 and 14 with acetobromo-a-D-glucuronic
acid methyl ester in the presence of 1,8-diazabicyclo-[5.4.0]-
7-undecene followed by basic hydrolysis gave the desired
b-glucuronides 15 and 16, respectively. A 1H nuclear magnetic res-
onance (NMR) spectrum of compound 15 showed a signal at
d = 5.12 ppm with an axial–axial coupling of 6.8 Hz in agreement
with its b-configuration of the anomeric position.18 The structure
of the b-configuration of compound 16 was also supported by an
anomeric doublet at d = 5.12 ppm with a coupling of 6.9 Hz charac-
teristics of the expected axial–axial arrangement.
To examine pharmacokinetic profile, the phenol 14 was admin-
istered orally, and the plasma levels of this compound and the cor-
responding glucuronide 16 were monitored over time (Fig. 7).
Phenol 14 was smoothly bioconverted into the glucuronide conju-
gate 16, as markedly high concentrations of compound 16 were
detected in plasma while both compound 14 and the correspond-
ing sulfate conjugate were below the limit of quantification
(10 ng/mL). Figure 8 displays the anticoagulant activity in cyno-
molgus monkeys after oral administration of the phenol 14 and
the deshydroxy analogue 7. Compound 14 showed an order of
magnitude more effective than compound 7 in this assay. These
results indicated that the phenolic hydroxyl group on compound
14 was subjected to rapid glucuronide-conjugation after oral dos-
ing and that the resultant conjugate efficiently demonstrated
potent ex vivo anticoagulant activity.
4. Results and discussion
Table 1 shows fXa inhibitory activity of phenol derivatives and
the corresponding glucuronide conjugates 13–16. As our molecular
modeling study suggested that substitution onto the 3-position of
the central phenyl ring would not interrupt binding with the
enzyme due to the location towards the solvent–exposed region,
none of investigated substituents resulted in any detrimental
effects on inhibitor affinity. Although chlorinated phenol 13 and
its glucuronide 15 demonstrated slightly less inhibitory activity
against fXa than the deshydroxy parent compound 7, the bromi-
nated phenol 14 and the corresponding glucuronide 16 displayed
potent activity comparable to compound 7. We therefore focused
our attention to the brominated pair 14 and 16. The phenol 14
and the corresponding glucuronide 16 displayed a potent inhibitory
activity against fXa that was comparable to the deshydroxy ana-
logue 7. Confirmatory tests of compounds 14 and 16 revealed that
both inhibitors demonstrated an in vitro PT-prolongation effect
5. Conclusion
with CT2 values of 0.31 and 0.28
l
M, respectively. Subsequent
In this report, we propose a novel methodology for discovery of
orally active inhibitors of the blood coagulation enzymes, which is
based on biotransformation of non-amidine inhibitors into more
hydrophilic conjugates. For the discovery of fXa inhibitors, we
designed the phenol derivative 14 by selecting a template scaffold
screening against human thrombin for selectivity within the coag-
ulation cascade and human trypsin for general specificity against
serine proteases was carried out, and both compounds were found
to be highly selective fXa inhibitors for these enzymes. Examination

6328
T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
Table 1
In vitro enzyme inhibitory activity, anticoagulant activity, and physicochemical property
N
Cl
O
R
OH
O
HN
X
O
OH
OH
N
H
N
OGlu =
O
COOH
PAMPAd (10ꢀ6 cm/s)
a
b
Compd
X
R
IC50
(
l
M)
CT2
PTc
(lM)
fXa
Thrombin
Trypsin
7
Cl
Cl
Cl
Br
Br
H
OH
OGlu
OH
OGlu
0.0058
0.0082
0.015
0.0040
0.0049
NTe
NTe
0.22
NTe
>30
NTe
NTe
>30
NTe
13
15
14
16
NTe
NTe
NTe
>100
>100
NTe
>100
>100
NTe
0.31
0.28
a
Inhibitory activity against human purified enzymes. IC50 value are represented by the average of three separate determinations with an average standard error of the
mean of <20%.
b
CT2 values are defined as the concentration required to double clotting time and represent the average of three separate determinations, with the average standard errors
of the mean being <10%.
c
Prothrombin time using cynomolgus monkey plasma.
pION membrane lipid was used at donor buffer, pH 6.5 (n = 2).
Not tested.
d
e
200
150
100
800
700
600
500
400
300
200
100
compound 7 10 mg/kg
compound 14 10 mg/kg
compound 14 1 mg/kg
0
2
4
6
8
Compound 7
Compound 16
Time (hr)
1 mg/kg
0.1 mg/kg
Figure 8. Anticoagulant activity in cynomolgus monkeys after oral administration
of compounds 7 and 14. Relative prothrombin time (PT) compared to that measured
using normal cynomolgus monkey plasma (mean standard deviation, n = 3).
Figure 6. Anticoagulant activity of compounds 7 and 16 after intravenous bolus
injection in cynomolgus monkeys. Relative prothrombin time (PT) compared to that
measured using normal cynomolgus monkey plasma at 5 min after single intrave-
nous bolus dosing (mean + standard deviation, n = 3).
and introducing a potential functional group at a selected position
for its bioconversion into a highly hydrophilic conjugate, using
molecular modeling simulations. The phenol 14 and the corre-
sponding glucuronide conjugate 16 showed fXa inhibitory activity
comparable to that of the deshydroxy analogue 7, which was con-
sistent with our hypothesis that a substituent at the 3-potition of
the central ring extended into the solvent sphere without interfer-
ing against inhibitor binding. Although the fXa inhibitors 7 and 16
demonstrated equivalent activity according to in vitro anticoagu-
lant assays, pharmacodynamics of these two compounds following
intravenous bolus injection resulted in remarkable differences,
with the latter demonstrating 10-fold more effective in ex vivo
anticoagulant assay. Pharmacokinetic data clarified the rapid
transformation of the phenol 14 into the corresponding glucuro-
nide 16 after oral dosing. These results indicated that biotransfor-
mation of compound 14 into the highly hydrophilic conjugate 16
boosted ex vivo anticoagulant activity. Compound 14 (ASP8102)
was selected for advancement to further biological evaluation as
an orally active antithrombotic agent.
4000
Compound 16
3000
2000
1000
0
0
2
4
6
8
Time (hr.)
Figure 7. Plasma concentration–time profiles of compound 16 in cynomolgus
monkeys after oral administration of compound 14 at dose of 10 mg/kg
(mean standard deviation, n = 3). Plasma concentrations of compound 14 and its
a
sulfate conjugate were below the limit of quantification (10 ng/mL).

T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
6329
We believe that our novel methodology is not limited to study
for the discovery of fXa inhibitors. Worldwide efforts are ongoing
for in the quest to identify orally active inhibitors against the coag-
ulation cascade enzymes, such as thrombin, fXa, factor VIIa, factor
VIIIa, and so on. We believe that our conjugation strategy, which
selects a template compound and appropriate position for install-
ing a trigger functional group using computer molecular modeling,
will generate new orally active anticoagulants. Results of our
continuing efforts to identify orally active fXa inhibitors based on
this novel strategy will be published in due course.
N,N-dimethylformamide (1.2 L). The mixture was filtered through
a pad of CeliteÒ and the filtrate was concentrated in vacuo. A
mixture of this material and ethyl acetate (215 mL) was stirred
at 70 °C for 1 h. The resulting precipitate was filtered off and dried
in vacuo to yield the title compound as a beige solid (49.5 g, 74%):
1H NMR (400 MHz, DMSO-d6) d 5.93 (2H, s), 6.44 (1H, t, J = 8.0 Hz),
6.82 (1H, d, J = 8.0 Hz), 7.27 (1H, d, J = 8.0 Hz), 7.93 (1H, dd, J = 3.2
and 8.8 Hz), 8.14 (1H, d, J = 8.8 Hz), 8.41 (1H, d, J = 3.2 Hz), 9.60
(1H, s), 10.46 (1H, s); FAB-MS (monoisotopic) m/z 264 [M+H]+.
6.1.3. 2-Amino-5-chloro-N-(5-chloro-2-pyridyl)-3-hydroxyben-
zamide (11)
6. Experimental section
6.1. Chemistry
A mixture of compound 10 (5.50 g, 20.9 mmol) and N-chloro-
succinimide (2.92 g, 21.9 mmol) in N,N-dimethylformamide
(100 mL) was stirred at 50 °C for 1.5 h. Additional N-chlorosuccin-
imide (280 mg, 20.9 mmol) was added and the whole was stirred
at 50 °C for 1 h. The mixture was filtered through a pad of CeliteÒ
and the filtrate was concentrated in vacuo. The residue was diluted
with ethyl acetate and washed with H2O. The organic layer was
dried over magnesium sulfate and concentrated in vacuo. The
residue was triturated with CHCl3, filtered off, and dried in vacuo
to yield the title compound as a brown solid (4.40 g, 69%): 1H
NMR (400 MHz, DMSO-d6) d 6.04(2H, br s), 6.80 (1H, s), 7.36 (1H,
s), 7.93 (1H, d, J = 8.8 Hz), 8.10 (1H, d, J = 8.8 Hz), 8.42 (1H, s),
10.16 (1H, s), 10.67 (1H, s); FAB-MS (monoisotopic) m/z 298
[M+H]+.
1H NMR spectra were recorded on a JEOL JNM-LA300 or a JEOL
JNM-EX400 spectrometer and the chemical shifts are expressed in
d (ppm) values with tetramethylsilane as an internal standard (in
NMR description, s = singlet, d = doublet, t = triplet, m = multiplet,
and br = broad peak). Mass spectra were recorded on a JEOL JMS-
LX2000 spectrometer. For salts, assignments of ion peaks are based
on the basic component. The elemental analyses were performed
with a Yanaco MT-5 microanalyzer (C, H, and N) and a Yokogawa
IC-7000S ion chromatographic analyzer (halogens), and were
within 0.4% of theoretical values. Melting points were measured
using a Yanaco MP-500D melting point apparatus without correc-
tion. C-18 reversed-phase silica gel (ODS) column chromatography
was performed on YMC gel (ODS-A 120–230/70). Analytical high-
performance liquid chromatography (HPLC) experiments were
performed with an ODS column (Tosoh Co. TSK-GEL 80TM,
i.d. = 0.46 cm, l = 15 cm). The HPLC conditions were as follows:
flow rate, 1.0 mL/min; detection wavelength, 254 nm. All reagents
purchased were used without further purification.
6.1.4. 2-Amino-5-bromo-N-(5-chloro-2-pyridyl)-3-hydroxyben-
zamide (12)
To a mixture of compound 10 (5.27 g, 20.0 mmol) and N,N-
dimethylformamide (60 mL) was added N-bromosuccinimide
(3.56 g, 20.0 mmol) portionwise at ꢀ15 °C and the whole was stir-
red for 1.5 h. Additional N-bromosuccinimide (356 mg, 2.00 mmol)
was added at ꢀ15 °C and the whole was stirred for 2 h. To the mix-
ture were added H2O (120 mL) and ethyl acetate (120 mL) at
ꢀ15 °C, and the whole was warm to ambient temperature and stir-
red for 10 min. The mixture was filtered through a pad of CeliteÒ.
The organic layer was separated, decolorized with activated char-
coal, filtered through a pad of CeliteÒ, and then concentrated in
vacuo to yield the title compound as a beige solid (5.70 g, 83%):
1H NMR (400 MHz, DMSO-d6) d 6.06 (2H, br s), 6.90 (1H, d,
J = 2.0 Hz), 7.47 (1H, d, J = 2.0 Hz), 7.93 (1H, dd, J = 2.4 Hz, 8.8 Hz),
8.10 (1H, d, J = 8.8 Hz), 8.42 (1H, d, J = 2.4 Hz), 10.14 (1H, br s),
10.68 (1H, br s); FAB-MS (monoisotopic) m/z 342 [M+H]+.
6.1.1. 3-Benzyloxy-N-(5-chloro-2-pyridyl)-2-nitrobenzamide (9)
To a mixture of 3-benzyloxy-2-nitrobenzoic acid (8)17 (77.2 g,
283 mmol), 10 drops of N,N-dimethylformamide, and ethyl acetate
(300 mL) was added thionyl chloride (101 g, 849 mmol) at ambient
temperature and the whole was stirred at 50 °C for 3 h. The reac-
tion mixture was cooled to ambient temperature and then concen-
trated in vacuo. The residue was dissolved in acetonitrile (200 mL).
To this solution was added a solution of 2-amino-5-chloropyridine
(36.4 g, 283 mmol), pyridine (22.4 g, 849 mmol) in acetonitrile
(350 mL) at 0 °C and the whole was stirred at ambient temperature
for 20 h. The reaction mixture was concentrated in vacuo. The res-
idue was diluted with CHCl3 (1.0 L), and washed with 1N hydro-
chloric acid, 1N aqueous sodium hydroxide solution, and then
brine. The organic layer was dried over magnesium sulfate and
concentrated in vacuo to yield the title compound as a colorless
solid (98.0 g, 90%): 1H NMR (300 MHz, DMSO-d6) d 5.35 (2H, s),
7.32–7.48 (6H, m), 7.60–7.69 (2H, m), 7.96 (1H, dd, J = 2.6 and
9.0 Hz), 8.08 (1H, d, J = 9.0 Hz), 8.45 (1H, d, J = 2.6 Hz), 11.47 (1H,
s); FAB-MS (monoisotopic) m/z 384 [M+H]+.
6.1.5. 40-Chloro-20-[(5-chloro-2-pyridyl)carbamoyl]-60-hydroxyl-
1-isopropylpiperidine-4-carboxanilide hydrochloride (13)
A mixture of 1-isopropylpiperidine-4-carboxylic acid (17)15b
(515 mg, 3.00 mmol), 3 drops of N,N-dimethylformamide, and
thionyl chloride (3.0 mL) was stirred at 80 °C for 0.5 h. The mixture
was cooled to ambient temperature and concentrated in vacuo.
This material was added to a mixture of compound 11 (520 mg,
1.74 mmol) and pyridine (6.0 mL) at 0 °C and the whole was stirred
at ambient temperature for 12 h. The reaction mixture was concen-
trated in vacuo and the residue was subjected to chromatography
over silica gel eluting with CHCl3/MeOH/c.NH3 (100:10:1 by
volume) to yield a colourless solid (417 mg). This material was sus-
pended in EtOH (4.0 mL). To this suspension was added 1N
hydrochloric acid (0.4 mL) at ambient temperature and the whole
was stirred for 0.5 h. The resulting precipitate was filtered off
and dried in vacuo to yield the title compound as a colorless solid
(308 mg, 21%): mp 249–252 °C (dec.); 1H NMR (400 MHz,
DMSO-d6) d 1.04 (1.8H, d, J = 6.3 Hz), 1.24 (4.2H, d, J = 6.3 Hz),
1.81–2.18 (4H, m), 2.63–3.26 (4H, m), 3.34–3.44 (2H, m), 7.05
(1H, s), 7.13 (1H, s), 7.91–7.96 (1H, m), 8.09–8.13 (1H, m),
6.1.2. 2-Amino-N-(5-chloro-2-pyridyl)-3-hydroxybenzamide (10)
A mixture of compound 9 (98.0 g, 255 mmol), pentamethylben-
zene (49.1 g, 332 mmol), and trifluoroacetic acid (250 mL) was stir-
red at 40 °C for 23 h. The reaction mixture was cooled to ambient
temperature and then filtered. The filtrate was concentrated in
vacuo. The residue was diluted with ethyl acetate (1.0 L) and
washed with saturated sodium bicarbonate. The organic layer
was dried over magnesium sulfate and concentrated in vacuo to
yielded a colourless solid. To a mixture of this material, ethyl ace-
tate (400 mL), and ethanol (400 mL) was added RaneyÒ nickel in
ethanol (40 mL) and the whole was stirred under hydrogen at
atmospheric pressure for 29 h. To the reaction mixture was added

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T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
8.37–8.41 (1H, m), 9.45 (0.7H, s), 9.53 (0.3H, s), 9.59 (0.7H, br s),
10.19 (0.3H, br s), 10.49–10.51 (1H, m), 10.60 (0.7H, s), 10.80
(0.3H, s); FAB-MS (monoisotopic) m/z 451 [M+H]+; Anal. calcd for
6.1.8. 5-Chloro-3-[(5-chloro-2-pyridyl)carbamoyl]-2-[(1-isopro-
pylpiperidine-4-carbonyl)amino]phenyl b-D-glucopyranosidur-
onic acid trifluoroacetate (15)
C
21H24N4O3Cl2ꢁHClꢁ0.5H2O: C, 50.77; H, 5.27; N, 11.28; Cl, 21.41.
In a manner identical to that described above for compound 16,
from 150 mg (0.332 mmol) of 40-bromo-20-[(5-chloro-2-pyridyl)
carbamoyl]-60-hydroxyl-1-isopropylpiperidine-4-carboxanilide,
152 mg (1.00 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene,
Found: C, 50.63; H, 5.29; N, 11.32; Cl, 21.57.
6.1.6. 40-Bromo-20-[(5-chloro-2-pyridyl)carbamoyl]-60-hydroxyl-
1-isopropylpiperidine-4-carboxanilide hydrochloride (14)
A mixture of compound 12 (2.39 g, 7.00 mmol), compound 17
(1.32 g, 7.70 mmol), 1-hydroxybenzotriazole hydrate (1.42 g, 10.5
mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (2.02 g, 10.5 mmol). triethylamine (1.06 g, 10.5 mmol),
and N,N-dimethylformamide (35 mL) was stirred at ambient tem-
perature for 22 h. To the mixture were added H2O (105 mL) and
ethyl acetate (105 mL) at ambient temperature and the whole
was stirred for 2 h. The resulting precipitate was filtered off,
washed with H2O and then ethyl acetate, and dried in vacuo to yield
1.67 g of 40-bromo-20-[(5-chloro-2-pyridyl)carbamoyl]-60-hydro-
xyl-1-isopropylpiperidine-4-carboxanilide. This material was sus-
pended in EtOH (60 mL). To this suspension was added 1N
hydrochloric acid (5.0 mL) at ambient temperature and the whole
was stirred for 30 h. The resulting precipitate was filtered off and
dried in vacuo to yield the title compound as a colorless solid
(1.35 g, 36%): mp 262–265 °C (dec.); 1H NMR (400 MHz, DMSO-
d6) d 1.04 (1.8H, d, J = 6.8 Hz), 1.24 (4.2H, d, J = 6.3 Hz), 1.74–2.12
(4H, m), 2.60–3.45 (6H, m), 7.15–7.19 (1H, m), 7.23–7.27 (1H, m),
7.89–7.97 (1H, m), 8.07–8.14 (1H, m), 8.35–8.41 (1H, m), 9.39–
9.55 (1.7H, m), 9.98–10.10 (0.3H, br s), 10.44–10.50 (1H, m),
10.62 (0.7H, s), 10.81 (0.3H, s); FAB-MS (monoisotopic) m/z 495
[M+H]+; Anal. calcd for C21H24N4O3BrClꢁHClꢁ0.8H2O: C, 46.14; H,
4.90; N, 10.25; Br, 14.62; Cl, 12.97. Found: C, 46.24; H, 4.51; N,
10.33; Br, 14.26; Cl, 13.16.
397 mg (1.00 mmol) of acetobromo-a-D-glucuronic acid methyl
ester, and 114 mg (1.08 mmol) of sodium carbonate was obtained
86 mg (30%) of the title compound as a colorless amorphous pow-
der: 1H NMR (400 MHz, DMSO-d6) d 1.02–1.04 (1.2H, m), 1.22
(4.8H, d, J = 6.4 Hz), 1.63–2.13 (4H, m), 2.63–2.70 (1H, m), 2.86–
3.14 (2H, m), 3.36–3.46 (6H, m), 4.01–4.05 (1H, m), 5.12 (1H, d,
J = 6.9 Hz), 5.18–5.54 (3H, br s), 7.43–7.45 (1H, m), 7.47–7.51 (1H,
m), 7.92–7.95 (1H, m), 8.08–8.14 (1H, m), 8.38–8.42 (1H, m),
8.80–9.00 (1H, br s), 9.44 (0.2H, s), 9.48 (0.8H, s), 10.79 (0.8H, s),
10.93 (0.2H, s), 12.85 (1H, br s); FAB-MS (monoisotopic) m/z 627
[M+H]+; Anal. calcd for C27H32N4O9Cl2ꢁ1.7TFAꢁ2.5H2O: C, 42.15; H,
4.50; N, 6.47; Cl, 8.18; F, 11.18. Found: C, 42.12; H, 4.16; N, 6.55;
Cl, 8.43; F, 11.27. Analytical HPLC tR = 5.1 min, 98.7% pure (eluent;
acetonitrile/0.01 M aqueous HClO4 = 2:8).
6.2. Pharmacology
6.2.1. In vitro assay for inhibition of factor Xa
The hydrolysis rates of synthetic substrates were assayed by
continuously measuring absorbance at 405 nm at 37 °C with a
microplate reader (model 3550, Bio-Rad, U.S.). Reaction mixtures
(125
substrates (S-2222) and an inhibitor in either 0.05 M Tris-HCl, pH
8.4, or 0.15 M NaCl. Reactions were initiated with 25 L of enzyme
lL) were prepared in 96-wellplates containing chromogenic
l
solution. The concentration of inhibitor required to inhibit enzyme
activity by 50% (IC50) was calculated from dose–response curves in
which the logit transformation of residual activity was plotted
against the logarithm of inhibitor concentration.
6.1.7. 5-Bromo-3-[(5-chloro-2-pyridyl)carbamoyl]-2-[(1-isopro-
pylpiperidine-4-carbonyl)amino]phenyl b-D-glucopyranosidur-
onic acid trifluoroacetate (16)
6.2.2. Enzyme selectivity
To a stirred mixture of 40-bromo-20-[(5-chloro-2-pyridyl)carba-
moyl]-60-hydroxyl-1-isopropylpiperidine-4-carboxanilide (1.00 g,
2.02 mmol), methanol (20 mL), and CHCl3 (20 mL) was added 1,8-
diazabicyclo[5.4.0]undec-7-ene (921 mg, 6.05 mmol) at ambient
temperature. After 30 min, to the reaction mixture was added acet-
Reaction mixtures were prepared in 96-well plates containing
the chromogenic substrate and test compound. The reaction was
initiated by the addition of enzyme, and the color was continu-
ously monitored at 405 nm using a microplate reader SpectraMax
340PC (Molecular Devices, CA, U.S.) at 37 °C. Each enzyme was
used at final concentration as follows: 0.20 UmLꢀ1 thrombin and
1.0 UmLꢀ1 trypsin. The enzymatic activities were assessed by the
amidolysis of the following chromogenic substrates for the corre-
sponding protease: S-2222 for trypsin and S-2238 for thrombin.
The rate of substrate hydrolysis (mOD minꢀ1) was measured at
37 °C. The mode of inhibition was estimated from a Lineweaver–
Burk plot. The Ki was determined from a Dixon plot by plotting
the reciprocal of the initial reaction velocities at different substrate
concentrations against different inhibitor concentrations.
obromo-a-D-glucuronic acid methyl ester (2.41 g, 6.06 mmol) at
ambient temperature, and the whole was stirred for 16 h. To the
reaction mixture were added sodium carbonate (1.07 g, 10.1 mmol)
and H2O (20 mL) at ambient temperature, and the whole was stirred
for 23 h. The reaction mixture was concentrated in vacuo. The resi-
due was diluted with H2O, washed with CHCl3, and then extracted
with n-butanol. The organic layer was concentrated in vacuo. The
residue was diluted with H2O (5 mL), neutralized with acetic acid
and concentrated in vacuo. The residue was subjected to chroma-
tography over ODS gel eluting with CH3CN/0.1% aqueous trifluoro-
acetic acid (4:10 by volume) to give the title compound as a
colorless amorphous powder (502 mg, 28%): 1H NMR (400 MHz,
DMSO-d6) d 1.02–1.05 (1.2H, m), 1.23 (4.8H, d, J = 6.9 Hz), 1.66–
2.13 (4H, m), 2.62–3.46 (9H, m), 4.02–4.05 (1H, m), 5.12 (1H, d,
J = 6.8 Hz), 5.40 (3H, br s), 7.31–7.33 (1H, m), 7.38–7.40 (1H, m),
7.91–7.95 (1H, m), 8.08–8.13 (1H, m), 8.40 (0.75H, d, J = 2.5 Hz),
8.41 (0.25H, d, J = 2.5 Hz), 8.76–8.92 (1H, m), 9.46 (0.2H, s), 9.49
(0.8H, s), 10.79 (0.8H, s), 10.93 (0.2H, s), 12.88 (1H, br s); FAB-MS
(monoisotopic) m/z 671 [M+H]+; Anal. calcd for C27H32N4O9-
BrClꢁ1.7TFAꢁ2H2O: C, 40.49; H, 4.21; N, 6.21; Br, 8.86; Cl, 3.93; F,
10.94. Found: C, 40.48; H, 3.98; N, 6.30; Br, 8.72; Cl, 4.05; F, 10.68.
Analytical HPLC tR = 5.5 min, 98.6% pure (eluent; acetonitrile/
0.01 M aqueous HClO4 = 2:8).
6.2.3. Prothrombin time assays in vitro
After collection of citrated blood samples from cynomolgus
monkey, platelet-poor plasma was prepared by centrifugation at
3000 rpm for 10 min and stored at ꢀ40 °C until use. Plasma clot-
ting times were measured using a KC10A coagulometer (Amelung
Co., Lehbrinksweg, Germany) at 37 °C. Prothrombin time (PT) was
measured using Orthobrain thromboplastin (OrthoDiagnostic Sys-
tems Co., Tokyo, Japan), and values for each test sample were com-
pared with coagulation times of a distilled water control. The
concentration required to double the clotting time (CT2) was esti-
mated from each individual concentration–response curve. Each
measurement was performed three times and represented as the
mean value.

T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
6331
6.2.4. Ex vivo studies
model of factor Xa. The solvent molecules were removed from
1HCG so as not to prevent inhibitors from appropriate binding.
Because the solvent molecules including ions in 1QL9 played an
important role for reproducing the binding mode of compound 6
by docking simulations, the solvent molecules of 1QL9 were
inserted into 1HCG by superposing 1QL9 over 1HCG based on the
structure alignment of the two proteins. To summarize, the protein
coordinates of 1HCG and the solvent coordinates of 1QL9 were
used for the docking simulations. The binding pocket was defined
as the cavity formed by the residues within the twice of the radius
of gyration of the compound 6 in 1QL9 (10.0 angstrom) from the
center of mass of the compound 6 in 1QL9. The docking program,
Gold, was used for the docking simulations. The molecular visual-
ization was produced by MOE.
The test drug was dissolved or suspended in 10% HP-b-CyD/5%
mannitol or 0.5% methyl cellulose and administered intravenously
via saphenous vein or orally using a gastric tube to cynomolgus
monkeys (mass range: 3.9–4.9 kg). Animals were last fed more
than 12 h before drug administration. Citrated blood was collected
from the femoral vein, and platelet-poor plasma was prepared by
centrifugation for measurement of PT. All data were expressed as
relative-fold values, compared with the baseline value of vehicle-
treated cynomolgus monkeys.
6.2.5. Pharmacokinetic study
Compound 14 suspended in 0.5% methylcellulose was orally
administered to male cynomolgus monkeys (mass range: 4.0–
4.5 kg, HAMRI CO., Ltd or ShinNihon Sangyo, Ltd, n = 3) at a dose
of 10 mg/kg under fasted conditions. Blood samples were collected
at 1, 2, 4, 6, 8, 12, and 24 h after administration and centrifuged to
obtain the plasma fraction. The plasma samples were purified by
solid phase extraction using Oasis HLB 96 well plate (Waters,
Japan). After extraction, the elute was evaporated to dryness, and
the residue was reconstituted in mobile phase and injected into
an LC–MS/MS apparatus to determine the plasma concentrations
of compounds 14 and 16. Dose and plasma concentrations were
expressed as free form. Compound 13 was used as the analytical
internal standard.
Acknowledgment
The authors deeply acknowledge Drs. Shuichi Sakamoto, Tom-
ihisa Kawasaki, and Takeshi Shigenaga for useful discussion. The
authors are grateful to Ms. Yumiko Moritani for performing phar-
macological experiments. The authors also thank to Dr. Takeshi
Kadokura for pharmacokinetic analysis and Dr. Katsuhiro Yamano
for assistance in preparing this manuscript, and the staff of the
Division of Analytical & Pharmacokinetics Research Laboratories
for the elemental analysis and spectral measurements.
6.2.6. PAMPA
References and notes
The PAMPA Evolution instrument from pION Inc. was used in
this study. In PAMPA, a ‘sandwich’ is formed from a 96-well micro-
titer plate (pION Inc., part no. 110243) and a 96-well filter plate
(Millipore, IPVH) such that each composite well is divided into
two chambers: donor at the bottom and acceptor at the top, sepa-
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Klimkowski, V. J.; Kurz, K. D.; Metz, J. T.; Ratz, A. M.; Shuman, R. T.; Smith, G. F.;
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rated by a 125 lm thick microfilter disk (0.45 lm pores) and
coated with a 20% (w/v) dodecane solution of a lecithin mixture
(pION Inc., part no. 110669). Drug samples were introduced as
10 mM DMSO stock solutions in a 96-well polypropylene microti-
ter plate. The robotic liquid handling system draws a 5 lL aliquot
of the DMSO stock solution and mixes it into an aqueous buffer
solution including 10% (v/v) of DMSO so that the final typical sam-
ple concentration is 50 lM. The drug solutions were filtered using
a 96-well filter plate (Corning, PVDF) and added to the donor
compartments. The donor solutions were adjusted in pH 6.5
(NaOH-treated universal buffer, pION Inc., part no. 110151), while
the acceptor solution had the same pH 7.4 (pION Inc., part no.
110139). The plate sandwich was formed and allowed to incubate
at 25 °C for 2 h in a humidity-saturated atmosphere. On comple-
tion of the prescribed incubation time, the sandwich plates were
separated and both the donor and acceptor compartments were
assayed for the amount of material present by comparison with
the UV spectrum (270–400 nm) obtained from reference stan-
dards. Mass balance was used to determine the amount of material
remaining in the membrane barrier, and permeability (Pe) was cal-
culated using PAMPA Evolution software (pION Inc.).
10. The clogP values are calculated using the ACD/logP version 9.0 (Advanced
Chemistry Development, Inc.). The sulfate conjugate of compound 1 was more
lipophilic than the glucuronide conjugate 2 (clogP = 0.63).
11. (a) van Heek, M.; France, C. F.; Compton, D. S.; McLeod, R. L.; Yumibe, N. P.;
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Gifford-Moore, D. S.; Goodson, T.; Halligan, N. G.; Hay, L.; Herron, D. K.;
Jackson, C. V.; Joseph, S. P.; Klimlowski, V. J.; Kurz, K. D.; Linebarger, J. H.;
Masters, J. J.; McCowan, J. R.; Mendel, D.; Muegge, L. P.; Murphy, A. T.; Pineiro-
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Docking simulations of factor Xa inhibitors were performed to
get insight into the appropriate position for the trigger phenolic
hydroxyl group, because no complex structure of factor Xa with
the nonamidine-type inhibitors was available when we started this
study, although some X-ray structures of factor Xa with or without
amidine-type inhibitors were reported. As the X-ray structure of
rat trypsin with compound 6 (PDB ID: 1QL9) was available, we
started the computational molecular modeling of factor Xa and
its inhibitors based on the trypsin X-ray structure. The X-ray struc-
ture of apo-form factor Xa (PDB ID: 1HCG) was selected as the

6332
T. Ishihara et al. / Bioorg. Med. Chem. 22 (2014) 6324–6332
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Denny, C. P.; Franciskovich, J. B.; Goodson, T.; Hall, S. E.; Herron, D. K.; Joseph, S.
P.; Klimlowski, V. J.; Masters, J. J.; Mendel, D.; Milot, G.; Pineiro-Nunez, M. M.;
Sawyer, J. S.; Shuman, R. T.; Smith, G. F.; Tebbe, A. L.; Tinsley, J. M.; Weir, L. C.;
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