Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.03.066

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding elem

Full text of DOI:10.1016/j.bmc.2007.03.066

Bioorganic & Medicinal Chemistry 15 (2007) 4175–4192
Prodrug-based design, synthesis, and biological evaluation
of N-benzenesulfonylpiperidine derivatives as novel,
orally active factor Xa inhibitors
Tsukasa Ishihara,a,* Norio Seki,a Fukushi Hirayama,a Masaya Orita,a Hiroyuki Koshio,a
Yuta Taniuchi,b Yumiko Sakai-Moritani,a Yoshiyuki Iwatsuki,a Seiji Kaku,a
Tomihisa Kawasaki,a Yuzo Matsumotoa and Shin-ichi Tsukamotoc
aDrug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
bDevelopment, Astellas Pharma Inc., 3-17-1, Hasune, Itabashi, Tokyo 174-8612, Japan
cCorporate Strategy, Astellas Pharma Inc., 2-3-11, Nihonbashi-Honcho, Chuo, Tokyo 103-8411, Japan
Received 29 January 2007; revised 20 March 2007; accepted 21 March 2007
Available online 25 March 2007
Abstract—We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase
parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element.
This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity
over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demon-
strated an anticoagulant effect after oral dosing.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
enzymes has emerged as one of the most active areas
of current research in the drug discovery field.
Intravascular clot formation results in thromboembolic
diseases including ischemic stroke, deep vein
thrombosis, myocardial infarction, unstable angina,
and pulmonary embolism, which are major cause of
morbidity and mortality in the industrialized world.
Up to this point, the primary medical strategy for treat-
ing and preventing such diseases has been the use of the
anticoagulants heparin, low molecular weight heparins,
and vitamin K antagonist warfarin. However, all these
drugs have therapeutic limitations. For example, neither
heparin nor low molecular weight heparins can be
administered orally.1 The oral anticoagulant agent
warfarin is being used for a growing number of anti-
thrombotic indications; however, it has a slow onset of
action and also requires individual dose titration and
periodic monitoring due to its indirect mechanism of
action.2 Therefore, the discovery of novel, orally active
anticoagulants that directly inhibit blood coagulation
Recent understanding of the mechanisms of blood coag-
ulation has increased rapidly. Thrombin, which pro-
motes blood clot formation by catalyzing the
conversion of fibrinogen to insoluble fibrin as well as
strongly inducing platelet aggregation,3 plays a central
role in thrombosis. Therefore, thrombin inhibitors have
been extensively studied as potential anticoagulant
agents. The direct blocking of thrombin, however,
shows a tendency to prolong bleeding at levels
approaching the effective dose.4
An attractive alternative to direct thrombin inhibition is
blocking the biosynthesis of thrombin itself. Thrombin
formation from prothrombin in the prothrombinase
complex is catalyzed by factor Xa (fXa). The generation
of thrombin by fXa is a highly amplified process,5 sug-
gesting that inhibition of fXa may be more efficacious
than direct inhibition of thrombin in interrupting the
blood coagulation cascade. Moreover, since fXa inhibi-
tors affect coagulation specifically and do not affect
platelet function, they theoretically have less potential
to increase the risk of bleeding.6 Consequently, fXa
Keywords: Factor Xa; Anticoagulant; Prodrug; Docking study.
*
Corresponding author. Tel.: +81 (0) 29 852 5111; fax +81 (0) 29 852
5387; e-mail: tsukasa.ishihara@jp.astellas.com
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.066

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T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
has emerged as an attractive target for the development
of new therapeutic agents with potential for the treat-
ment of arterial and venous thrombosis.
0.0038 lM (Fig. 1).7 In this research program, we se-
lected compound 1 as a starting point and applied the
following two-step design strategy (Step A and B) to
generate new scaffold fXa inhibitors (Fig. 2).
We have already reported some potent amidine-based
fXa inhibitors, such as compound 1,7 which originated
from Daiichi’s DX-9065a.8 fXa inhibitors with amidine
groups are highly efficacious in animal thrombosis mod-
els,6,9 yet they do not have an appropriate pharmacoki-
netic profile for use as oral antithrombotic agents due to
the presence of highly basic and hydrophilic amidine
units.10 One methodology employed to improve the oral
bioavailability of amidine derivatives is to mask the po-
lar groups, that is, a prodrug strategy. Herein, we report
on our discovery of a novel class of potent and selective
amidine-based fXa inhibitors and also describe the oral
anticoagulant activity of the related prodrugs.
Compound 1 has two amidino groups. One is a benzam-
idine group, and the other is an acetimidoyl group.
N-Hydroxybenzamidine is the well-established biopre-
cursor of benzamidine, for example, the thrombin inhib-
itor ximelagatran,11 the GP IIb-IIIa receptor antagonist
sibrafiban,12 so we planned that the benzamidine group
of compound 1 would be transformed into N-hydroxy-
benzamidine based on prodrug strategy. To the con-
trary, no promoieties for an acetimidoyl group have
been reported to date. These backgrounds led us to elim-
inate the N-acetimidoylpiperidine moiety of compound
1 (Step A). Exploring surrogates for this group, we fo-
cused on compound 2, a member of the first class of
nonamidine fXa inhibitors (Fig. 1).13 Our molecular
modeling study based on the reported X-ray crystal
structure of compound 214 indicated that this inhibitor
was bound to the active site of fXa, with its pyridine
moiety in the S4 pocket bordered by the residues of
Phe174, Tyr99, and Trp215. Similarly, the aceti-
midoylpiperidine unit of the ethanesulfonamide deriva-
tive 10 lay in the S4 binding region.7 We expected that
the replacement of the acetimidoylpiperidine moiety of
analogue 1 with a pyridine ring would afford a potent
fXa inhibitor. Thus we designed compound X, and
undertook further structural modifications using a par-
allel synthesis methodology to obtain suitable S4 bind-
ing elements (compound Y).
2. Inhibitor design
In order to discover potent and orally active fXa inhib-
itors, we adopted a strategy based on the combination of
amidine derivatives and their prodrugs because of the
following three reasons: (1) an amidine derivative is
known to demonstrate potent anti-fXa activity due to
the bidentate salt bridge interaction between its amidine
moiety and the carboxylic acid of Asp189 in the S1 site
of the enzyme, (2) masking the high hydrophilicity of the
polarizable groups on the inhibitor reportedly enhances
its membrane permeability, improves its activity via oral
administration, and reduces drug–food interaction,11
and (3) a prodrug with low inhibitory activity does not
interact with fXa in the gastrointestinal tract, which will
provide a potential advantage for patients with silent
gastrointestinal bleeding.
The second round consisted of the installation of a car-
boxyl group into the amidine-based inhibitor and opti-
mization of the compound involving the prodrug
approach (Step B). Compounds with amidine groups
sometimes cause cardiovascular side effects in terms of
reduced blood pressure and heart rate, probably because
of the high basicity of the amidine group.15 The intro-
duction of an acidic carboxyl group into the amidine
derivative and the consequent reduction of the basicity
of the whole chemical structure are important for the
avoidance of these side effects.16 Moreover, we found
that the installation of a carboxyl group into a prodrug
with a masked amidine moiety was important for the
biotransformation of the masked amidine into ami-
dine.17 Therefore, we decided to construct a molecule
with a carboxyl group. The appropriate position for
the introduction of the carboxyl group was determined
based on computational modeling studies. We also ex-
plored the effects of naphthamidine replacements to en-
hance the fXa selectivity over other serine proteases and
to improve the oral anticoagulant activity (compound Z).
Our previous studies demonstrated that diamidine deriv-
ative 1 was a potent fXa inhibitor with an IC50 value of
COOH
HN
NH
N
NH2
O
DX-9065a
O
R
O
S
N
NH
HN
N
NH2
O
1 (R = Me)
1' (R = Et )
O
3. Chemistry
Cl
N
N
N
The library of piperidine derivatives was prepared using
solid-phase parallel synthesis as outlined in Scheme 1.
Conversion of 7-bromomethylnaphthalene-2-carbonitri-
le (3)18 into the corresponding amine 5 was accom-
plished through a two-step sequence involving a
S
O
O
N
2
Figure 1. Structures of fXa inhibitors.

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4177
Step A. Exploring a P4 surrogate
O
O
Cl
O
S
N
NH
N
HN
N
N
N
S
NH2
O
O
N
O
1
2
R
N
HN
N
NH2
N
X
R
N
HN
N
NH2
X
Y
COOR'
N
L
N
QN
NH2
X
Z
Step B. Installation of a carboxyl group and optimization
Figure 2. Inhibitor design.
substitution reaction with potassium phthalimide fol-
lowed by
reductive amination of the polymer-bound piperidine 9
with various aldehydes, and subsequent acidic cleavage
from the resin.
a
methylamine-promoted deprotection.
Reductive amination of tert-butyl 4-oxopiperidine-
1-carboxylate with the amine 5 and sodium triacetoxy-
borohydride as the reducing agent followed by
mesylation afforded intermediate nitrile 6. Compound 6
was then converted into amidine 7 using the thio-Pinner
procedure19 followed by treatment with ammonium
acetate. Amidine 7 was loaded onto resin 8,20 followed
by acidic removal of its Boc group to yield a polymer-
bound piperidine precursor 9. Treatment of the poly-
mer-bound piperidine 9 with a collection of sulfonyl
chlorides followed by TFA-mediated cleavage from the
polymer afforded a set of 74 N-sulfonylpiperidine deriv-
atives 10. N-Acyl piperidine derivatives were prepared
by the condensation of the polymer-bound piperidine
9 with a library of carboxylic acids using PyBOP
(benztriazol-1-yl-oxytris(pyrrolidino)phosphonium hexa-
fluorophosphate)21 followed by cleavage from the poly-
mer. Two dozen compounds were synthesized in this
fashion. Preparation of a set of 37 N-alkylpiperidine
analogues 10 was accomplished in two steps via
Scheme 2 depicts the stepwise processes that were em-
ployed to elaborate nitriles 17–24, the precursors of target
amidine derivatives. The sulfonylation of 4-piperidone
hydrate (12) with 2,5-dimethoxyphenylsulfonyl chloride
yielded ketone 13. The ketones 1122 or 13 were each
coupled with 7-aminomethylnaphthalene-2-carbonitrile
(5) or (E)-3-(3-aminopropenyl)benzonitrile23 in the pres-
ence of sodium triacetoxyborohydride to provide inter-
mediate amines 14–16. The secondary amines 14–16
were treated with appropriate sulfonyl chlorides to yield
sulfonamides 17, 18, 21, and 23. Amide analogues 19,
22, and 24 were synthesized by acylating the intermediate
amines 15 and 16 with appropriately substituted acid
chlorides. Tertiary amine 20 was obtained via reductive
amination of the secondary amine 15 with formaldehyde.
All samples prepared by the solid-phase techniques were
analyzed by LC–MS to confirm purity and identity of the
compounds.

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T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
O
a
b
HCl
NH2
Br
N
NC
NC
NC
O
4
5
3
O
O
O S
N
O S
N
c,d
e
HN
NC
NBoc
O
NBoc
NH2
6
7
O
O S
N
O
O
PP
N
H
f-h
O
PP
N
O
N
H
OH
NH
O
NH2
8
9
O
O S
N
i, j, or k
HN
NR
NH2
TFA
10
Scheme 1. Reagents and conditions: (a) potassium phthalimide, DMF, 90 °C; (b) i—MeNH2, MeOH; ii—HCl, EtOAc; (c) tert-butyl
4-oxopiperidine-1-carboxylate, NaBH(OAc)3, Et3N, AcOH, 1,2-dichloroethane; (d) methanesulfonyl chloride, Et3N, 1,2-dichloroethane, 0 °C;
(e) i—H2S, Et3N, pyridine; ii—MeI, acetone, reflux; iii—AcONH4, MeOH, reflux; (f) 4-nitrophenylchloroformate, Py, CH2Cl2; (g) 7, Et3N, NMP;
(h) HCl/EtOAc, MeOH; (i) i—sulfonyl chloride, i-Pr2EtN, dioxane; ii—TFA, CH2Cl2; (j) i—carboxylic acid, PyBOP, i-Pr2EtN, DMF; ii—TFA,
CH2Cl2; (k) i—aldehyde, NaBH(OAc)3, AcOH, 1,2-dichloroethane; ii—TFA, CH2Cl2.
The preparation of the amidine derivatives 10x and
25–39 is shown in Scheme 3. Conversion of the nitrile
groups to amidines was performed with two general pro-
cedures. The precursor nitriles 21–24 were transformed
into N-hydroxyamidine derivatives 25–28 by treatment
with hydroxylamine. Subsequent in situ activation of
the N-hydroxyamidine groups by formation of the ace-
tate ester and N–O bond cleavage by catalytic hydroge-
nation afforded the corresponding amidine derivatives
32–33.24 Alternatively, the treatment of nitriles17–20,
23, and 24 under the Pinner conditions (HCl/EtOH)
afforded imidates,25 which were immediately reacted
with ammonium acetate to yield amidines 10x, 29–31,
34, and 35. Finally, the ester groups of compounds
32–35 were hydrolyzed under basic conditions to pro-
vide the desired carboxylic acids 36–39.
using a gastric tube. After oral administration of com-
pounds, citrated blood was collected and platelet poor
plasma was prepared to measure PT at a predetermined
time.
5. Results and discussion
Table 1 shows the result for the replacement of the ace-
timidoylpiperidinyloxyphenyl moiety in lead compound
1 with a 4-(piperidino)pyridine group. The resulting
compound 29 exhibited good inhibitory activity against
fXa, which was only twofold less potent than the clinical
candidate DX-9065a. This result led us to extensive
modification of compound 29. The pKa value of highly
basic benzamidine is 11.6, and 4-(piperidino)pyridine is
also highly basic comparable to benzamidine.26 This
might cause the 4-(piperidino)pyridine derivatives to
have poor oral absorption. In addition to that, the 4-
(piperidino)pyridine derivatives were reported to be rap-
idly metabolized by N-oxide formation.27 Based on
these data, we next turned our attention to the investiga-
tion of alternative fragments to the pyridine moiety.
4. Pharmacology
All compounds synthesized were tested for their potency
to inhibit human fXa in a purified enzyme system. Sub-
sequent screening against human thrombin and human
trypsin was carried out for further selected compounds.
The anticoagulant activity for selected compounds was
evaluated by measuring the prolongation of prothrom-
bin time (PT) in mouse and human plasma. Selected
compounds were evaluated for their PT-prolongation ef-
fects after oral administration in mice. Each test drug
was suspended in a 0.5% methylcellulose solution prior
to use and was orally administered to male ICR mice
Over 100 compounds were prepared to scan new P4
ligands by means of parallel solid-phase synthesis, and
the results for a representative set of compounds are
shown in Table 2. N-Alkylsulfonyl derivatives, such as
compound 10b, did not show inhibitory activity, and
N-benzenesulfonyl derivative 10c was found to be a
moderate but novel fXa inhibitor. The introduction of

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4179
O
O
HO
HO
O
a
N
N
S
O O
NH
N
O
HCl
11
13
12
b
b
X
Y
H
N
NC
N
R1
14 X, Y = -CH=CH- R1 = 4-pyridyl
15 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl
16 X, Y = H, H R1 = 2,5-dimethoxyphenylsulfonyl
c, d, or e
X
R2
N
Y
NC
N
R1
17 X, Y = -CH=CH- R1 = 4-pyridyl
R2 = SO2Me
18 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 = SO2Me
19 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 = COMe
20 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 = Me
SO2CH2COOEt
21 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 =
22 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 =
COCH COOEt
2
SO2CH2COOEt
23 X, Y = H, H
24 X, Y = H, H
R1 = 2,5-dimethoxyphenylsulfonyl R2 =
R1 = 2,5-dimethoxyphenylsulfonyl R2 =
COCH COOEt
2
Scheme 2. Reagents and conditions: (a) 2,5-dimethoxybenzenesulfonyl chloride, Et3N, 1,2-dichloroethane; (b) 5 or (E)-3-(3-aminopropenyl)ben-
zonitrile, NaBH(OAc)3, AcOH, 1,2-dichloroethane; (c) sulfonyl chloride, Et3N, 1,2-dichloroethane, 0 °C; (d) acid chloride, Et3N, 1,2-dichloroethane,
0 °C; (e) HCHOaq, NaBH(OAc)3, AcOH, 1,2-dichloroethane.
a methyl or chloro group at the 2-position on the phenyl
ring did not cause a reduction of potency (10d and 10e),
and their incorporation at the 3-position served more
suitably as S4 binding elements (10f and 10g). Substitu-
tion at the 4-position was, however, less well tolerated
(10h and 10i). The 3-position on the phenyl ring toler-
ated various substituents without deleterious effects on
potency (10f, 10g, 10j–10l). In this series, 3-chloro deriv-
ative 10g showed potent inhibitory activity with an IC50
value of 0.049 lM. In contrast, 3-substituted benzyl or
benzoyl analogues were found to be poor inhibitors
(10m–10p). In disubstituted benzenesulfonyl derivatives,
compound with a 2,5-disubstitution pattern maintained
high inhibitory activity (10t). The 2,5-dichloro-
thiophenesulfonyl analogue 10u exhibited comparable
activity to the phenyl derivative 10t, indicating that
the thiophene core would become the isostere of a ben-
zene ring for an S4 binding element. Analogues incorpo-
rating a variety of 2,5-disubstituted benzenesulfonyl
groups were well tolerated (10v–10x), and the 2,5-dim-
ethoxybenzenesulfonyl derivative 10x was found to be
a highly potent fXa inhibitor with an IC50 value of
0.011 lM.
Figure 3 shows the proposed binding model of com-
pound 10x docked into the active site of fXa, and the
important interactions of the inhibitor with the enzyme
are schematically depicted in Figure 4. In this model,
compound 10x is bound in an extended L-shaped con-
formation similar to the reported binding mode of
DX-9065a.28 The naphthamidine moiety of compound
10x occupies the S1 pocket of fXa, and its amidine
group binds via a bidentate charge interaction with
Asp189 at the bottom of the S1 pocket. The 2,5-dim-
ethoxybenzenesulfonyl unit fills the S4 aryl binding
pocket with its m-methoxy substituent stretching along
the cleft in the S4 pocket. Our docking study suggests
that o-methoxy group occupies the cavity of S4 binding
pocket and interacts through hydrophobic contacts.
This binding model also indicates that the central

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T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
X
O
R2
N
Y
a
HON
21—24
N
NH2
S
O O
O
X, Y = -CH=CH- R2 =
X, Y = -CH=CH- R2 =
SO2CH2COOEt
25
26
27
28
COCH COOEt
2
X, Y = H, H
X, Y = H, H
R2 =
R2 =
SO2CH2COOEt
COCH COOEt
2
c for 25 and 26
X
R2
N
Y
b
HN
17—20, 23, 24
N
NH2
HCl
R1
29 X, Y = -CH=CH- R1 = 4-pyridyl
R2 = SO2Me
10x X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 = SO2Me
30 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 = COMe
31 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 = Me
SO2CH2COOEt
32 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 =
33 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 =
COCH COOEt
2
SO2CH2COOEt
34 X, Y = H, H
35 X, Y = H, H
R1 = 2,5-dimethoxyphenylsulfonyl
R1 = 2,5-dimethoxyphenylsulfonyl
=
=
R2
R2
COCH COOEt
2
d for32—35
SO2CH2COOH
36 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 =
37 X, Y = -CH=CH- R1 = 2,5-dimethoxyphenylsulfonyl R2 =
COCH COOH
2
SO2CH2COOH
38 X, Y = H, H
39 X, Y = H, H
R1 = 2,5-dimethoxyphenylsulfonyl
R1 = 2,5-dimethoxyphenylsulfonyl
=
=
R2
R2
COCH COOH
2
Scheme 3. Reagents and conditions: (a) NH2OHÆHCl, Et3N, EtOH, reflux; (b) i–HCl, EtOH, CHCl3, 0 °C; ii—AcONH4, EtOH; (c) H2 (1 kgf/cm2),
10% Pd/C, Ac2O, AcOH; (d) 1 N aq NaOH, dioxane, 0 °C.
Table 1. Effect of modification of P4 element
methanesulfonyl group extends into the solvent and that
no direct interaction is observed between this group and
the enzyme (Fig. 3, right panel). This points toward our
prediction that an additional substitution in the metha-
nesulfonyl group would be tolerated.
O
Me
O
S
N
HN
X
NH2
a
Next we modified the central hinge and the P1 ligand of
compound 10x. The structure–activity relationships
(SAR) for fXa binding are summarized in Table 3.
Replacement of the methanesulfonyl group with an
acetyl group afforded compound 30, which maintained
potent anti-fXa activity. On the other hand, tertiary
amine analogue 31 suffered a 50–fold loss in activity
compared to the methanesulfonyl derivative 10x. Here,
we directed our efforts toward the introduction of a car-
boxyl group into the inhibitor. Our modeling study of
compound 10x within fXa indicated that its analogue,
with an additional substituent on the methanesulfonyl
group, would retain potent inhibitory activity. This
prompted us to apply the installation of carboxyl groups
onto the limbs of the central elements of compounds 10x
Compound
X
IC50 (lM) fXa
NH
1
0.0038b
N
Me
O
N
29
0.090
0.040
N
DX-9065a
a Human purified enzyme was used. The IC50 value represents the
mean of three separate experiments, and the average standard error is
less than 15% of the mean.
b See Ref. 7.

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4181
Table 2. Effect of modification of P4 element
Table 2 (continued)
a
Compound
X
IC50 (lM) fXa
O
O
Me
S
N
Cl
Cl
HN
10s
10t
0.19
O2S
Cl
N
NH2
X
a
0.065
0.10
Compound
X
IC50 (lM) fXa
Cl
Cl
O2S
Cl
10a
10b
H
SO2Me
>100
>100
S
10u
10v
10w
10x
O2S
10c
10d
0.85
0.95
MeO
O2S
O2S
0.039
0.058
0.011b
Cl
O2S
MeO
Me
Me
O2S
MeO
10e
0.35
O2S
Cl
OMe
O2S
a Human purified enzyme was used. IC50 values are determined by a
single experimental run.
b Activity of resynthesized sample. The IC50 value represents the mean
of three separate experiments, and the average standard deviation is
less than 10% of the mean.
10f
10g
10h
10i
10j
0.25
0.049
>100
1.6
Me
O2S
O2S
Cl
Me
and 30. As anticipated, these modifications maintained
anti-fXa activity, and carboxylic acid derivatives 36
and 37 were revealed as potent inhibitors with IC50 val-
ues of 0.013 and 0.0081 lM, respectively. We also eval-
uated the related styrylamidine analogues 38 and 39
which would have stereochemical geometry similar to
naphthamidines. Compounds 38 and 39 were potent
inhibitors, comparable to naphthamidine derivatives
36 and 37, respectively, which led us to conclude that
a styrene unit functioned as the isostere of the naphtha-
lene ring system.
O2S
Cl
F
O2S
O2S
0.80
10k
10l
0.17
0.20
1.7
CN
Ph
O2S
O2S
Subsequent screening against human thrombin for
selectivity within the coagulation cascade and human
trypsin for general specificity against serine proteases
was carried out for the potent fXa inhibitors. The
results are also listed in Table 3. All compounds tested
demonstrated excellent selectivity against thrombin
(>7000-fold) and trypsin (>100-fold). The styrylami-
dine-based inhibitors 38 and 39 were more selective
versus trypsin than the naphthamidine analogues 36
and 37.
10m
10n
Cl
2.3
CN
Cl
10o
10p
>100
>100
O
The installation of a carboxyl group into a fXa
inhibitor reportedly leads to an increase of selectivity
over thrombin.29 The novel fXa inhibitors reported
here, however, demonstrated high selectivity versus
thrombin, even if they had no carboxyl groups. This
selectivity profile can be explained by our molecular
modeling studies. Figure 5 shows the proposed bind-
ing model of compound 10x docked into the active
site of fXa and its subsequent superimposition onto
that of thrombin. These docking simulations revealed
that the central methanesulfonyl group would hit the
CN
O
Cl
10q
10r
>100
0.56
O2S
O2S
Cl
Cl
Cl

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T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
Figure 3. Proposed binding model of compound 10x within the active site of fXa. Left and right panels correspond to top and side views of the
complex, respectively. The inhibitor is colored by atom type and the protein surface is colored cyan. Coordinates of fXa are available on the
Brookhaven database (Accession code: 1FAX).
CT2 > 100 lM). Although compounds 25–27 did not ex-
hibit anticoagulant activity after oral administration in
mice, compound 28 did display evident prolongation
of PT (Table 5). Figure 6 depicts the time course for
the PT-prolongation effect of compound 28, which indi-
cated the anticoagulant effects peaked at 30 min after
oral dosing. Its parent amidine 39 showed no anticoag-
ulant effect when dosed orally. These results demon-
strated that prodrug 28 was absorbed after oral dosing
and rapidly converted into the corresponding active
compound in vivo.
6. Conclusion
Figure 4. Diagram representing the key interactions between com-
pound 10x and fXa.
In this paper, we have designed and synthesized a new
class of potent, selective, and orally active fXa inhibitors
based on prodrug exploration. Replacement of the S4
binding element in lead compound 1 with a 4-(piperidi-
thrombin-specific insertion loop (Tyr60A-Pro60B-
Pro60C-Trp60D), thus conferring high selectivity
against thrombin.30 Extensive modeling studies indi-
cated that analogous inhibitors bound to the active
site of fXa in a similar fashion as compound 10x,
so we propose that this novel class of fXa inhibitors
demonstrates high selectivity over thrombin due to
steric repulsion of their central moieties and the resi-
dues of the 60-insertion loop of thrombin.
no)pyridine and extensive structural modifications using
solid-phase parallel synthesis methodology led to the
identification of novel piperidine derivatives with substi-
tuted benzenesulfonyl groups as P4 ligands (e.g., com-
pound 10x, fXa IC50 = 0.011 lM). Subsequent SAR
studies demonstrated that the methanesulfonyl to acetyl
transformation at the center of the molecule or the
naphthamidine to styrylamidine conversion as the P1
ligand yielded equipotent inhibitors. We installed a car-
boxyl group on either the central methanesulfonyl or the
acetyl group based on molecular modeling studies which
indicated that the central units were directed out of the
fXa binding site into the solvent environment. These
modifications did not cause any reduction of anti-fXa
activity. The modeling studies also demonstrated that
the central groups would hit the thrombin-specific
60-insertion loop, which is consistent with high selectiv-
ity over thrombin (>7000-fold). The related N-hydroxy-
amidine prodrug 28, which had no in vitro activity,
demonstrated an evident PT-prolongation effect in mice
when administered orally, while its parent amidine 39
showed no oral anticoagulant potency. These results
indicated that the prodrug 28 was absorbed via
oral dosing and bioconverted into the corresponding
active compound. We anticipate that further structural
The four potent and selective carboxylic acid derivatives
36–39 were selected for evaluation in our advanced pro-
filing assays. To evaluate the potential efficacy of these
compounds, in vitro anticoagulation tests were con-
ducted using human and mouse plasma. Table 4 shows
the concentrations of inhibitors required to double the
prothrombin time (PT) in plasma clotting. All com-
pounds indicated potent anticoagulant activities, which
were comparable to those of DX-9065a. We finally
examined the anticoagulant potency after oral dosing
of their related N-hydroxyamidine prodrugs 25–28 to
assess their ability as orally active anticoagulants. Con-
firmatory in vitro tests showed that none of the
N-hydroxyamidine prodrugs had any fXa inhibitory
activity or anticoagulant potency (IC50 > 10 lM,

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
Table 3. Effects of modifications of the central moiety and P1-element
4183
L
OMe
N
Y
N
S
O O
OMe
a
Compound
Y
L
IC50 (lM)
fXa
Thrombin
Trypsin
HN
10x
Ms
0.011
>100
1.1
NH2
HN
HN
30
31
Ac
0.012
0.59
>100
NT
2.5
NH2
NH2
Me
NT
HN
HN
HN
HN
O2S
O
COOH
36
37
38
39
0.013
0.0081
0.010
>100
>100
>100
2.7
3.7
5.3
NH2
NH2
NH2
NH2
COOH
COOH
O2S
O
COOH
0.013
0.040
>100
>100
6.0
3.2
DX-9065a
a See the corresponding footnotes in Table 1.
Figure 5. Stereographical representation of the basis for the selectivity profile of compound 10x. Compound 10x is docked in the active site of fXa
(PDB Accession code: 1FAX) and transferred onto that of thrombin (PDB Accession code: 1KTT). All molecules are colored by atom type, except
the carbon atoms of fXa, which are cyan, and those of thrombin, which are orange.

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T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
Table 4. In vitro anticoagulant activities
250
a
Compound
CT2 (lM) PT
Human
Mouse
200
36
37
0.55
0.56
0.47
0.55
0.55
1.7
1.6
1.2
38
39
150
1.4
4.9
DX-9065a
a CT2 is defined as the concentration of test compound required to
double PT. The value represents the mean of three separate experi-
ments, and the average standard errors are less than 10% of the
mean.
100
0
1
2
3
4
Time (h)
Figure 6. Anticoagulant activity of compound 28 after oral dosing in
mice. Animals were dosed via oral gavage at 100 mg/kg. Values are
expressed as means SEM (n = 3).
modifications of amidine 39 and its prodrug 28 will lead
to more potent and orally active fXa inhibitors. Our
continuing efforts to improve oral anticoagulant activity
will be the subject of future publications.
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 ana-
lyzer (S and Cl), and were within 0.4% of theoretical
values unless otherwise indicated. Melting points were
measured with a Yanaco MP-500D melting point appa-
ratus without correction. ODS column chromatography
7. Experimental
7.1. Chemistry
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 tet-
ramethylsilane as an internal standard (in the NMR
Table 5. Ex vivo anticoagulant activity after oral dosing in mice
L
OMe
N
Y
N
S
O O
OMe
Compound
Y
L
Prolongation effect PTa (%)
HON
O2S
O
COOEt
25
101
109
116
0
5
4
NH2
NH2
NH2
NH2
HON
HON
HON
HN
COOEt
COOEt
26
27
28
39
O2S
O
COOEt
COOH
187 37
O
104
4
NH2
DX-9065a
151 16
a Mean percent change of PT at 0.5 h after oral administration compared to that measured using normal mice plasma. Animals were dosed via oral
gavage at 100 mg/kg. Each value is expressed as the mean SEM (n = 3). All N-hydroxyamidine derivatives prepared had little inhibitory activity
against factor Xa (IC50 > 10 lM) and a low in vitro anticoagulant effect (PT CT2 > 100 lM).

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4185
was performed on YMC gel (ODS-A 120-230/70). All
reagents purchased were used without further
purification.
1.39 (9H, s), 1.56–1.62 (2H, m), 1.67–1.77 (2H, m),
2.63–2.74 (2H, m), 2.91 (3H, s), 3.86–3.97 (1H, m),
4.02–4.20 (2H, m), 4.56 (2H, s), 7.62 (1H, d, J =
8.4 Hz), 7.72 (1H, d, J = 8.4 Hz), 7.85–7.94 (3H, m),
8.22 (1H, s); MS (FAB) m/z 444 [M+H]+.
7.1.1. N-(7-Cyanonaphthalen-2-ylmethyl)phthalimide (4).
A mixture of 7-bromomethylnaphthalene-2-carbonitrile
(3)18 (40.1 g, 163 mmol), potassium phthalimide
(30.2 g, 163 mmol), and DMF (200 mL) was stirred at
90 °C for 15 h. The reaction mixture was cooled to
ambient temperature. To the mixture was then added
H2O (40 mL) and the whole was stirred for 0.5 h. The
resulting precipitate was filtered off. A mixture of this
material and EtOH (750 mL) was stirred under reflux
for 10 min. The mixture was cooled to ambient temper-
ature, and the precipitate was filtered off and dried in va-
cuo to yield the title compound as a colorless
7.1.4. tert-Butyl 4-[({7-[Amino(imino)methyl]-2-naph-
thyl}methyl)(methylsulfonyl)amino]piperidine-1-carboxyl-
ate (7). Hydrogen sulfide was bubbled through a
solution of compound 6 (4.53 g, 10.2 mmol) in triethyl-
amine (14 mL) and pyridine (80 mL) for 0.5 h, and the
whole was then stirred at ambient temperature for
15 h. The solvents were removed in vacuo and the resi-
due was redissolved in acetone (150 mL). To the solu-
tion was added iodomethane (12.7 mL, 204 mmol) at
ambient temperature and the whole was heated to reflux
for 3 h. The reaction mixture was allowed to cool to
ambient temperature and the solvent removed in vacuo.
The residue was then redissolved in MeOH (150 mL),
and to the solution was added ammonium acetate
(3.93 g, 51.0 mmol). This mixture was heated to reflux
for 40 min. The reaction mixture was allowed to cool
to ambient temperature and the solvent removed in va-
cuo. The residue was dissolved in H2O (100 mL), made
alkaline with 1 N aqueous NaOH solution at 0 °C, and
then extracted with CHCl3 (100 mL). The organic layer
was dried over magnesium sulfate, and concentrated in
vacuo. The residue was purified using column chroma-
tography on silica gel (eluent: CHCl3/MeOH = 70:30
by volume) to yield the title compound as a beige amor-
1
amorphous powder (9.71 g, 19%): H NMR (DMSO-
d6) d 5.03 (2H, s), 7.58 (1H, d, J = 8.4 Hz), 7.69–7.76
(3H, m), 7.83–7.89 (4H, m), 7.93 (1H, s), 8.21 (1H, s);
MS (FAB) m/z 313 [M+H]+.
7.1.2. 7-(Aminomethyl)-2-naphthonitrile hydrochloride (5).
A mixture of compound 4 (15.6 g, 50.0 mmol), MeOH
(200 mL), and methylamine in MeOH (40%, 60 mL)
was stirred at ambient temperature for 21 h. The mix-
ture was diluted with H2O (200 mL) and extracted with
ethyl acetate (200 mL). The organic layer was dried over
anhydrous sodium sulfate. To this solution was added
4 N hydrogen chloride in ethyl acetate (15 mL) and the
whole was stirred for 0.5 h. The resulting precipitate
was filtered off and dried in vacuo to yield the title com-
pound as a colorless solid (8.05 g, 83%): mp 266–268 °C;
1H NMR (DMSO-d6) d 4.23 (2H, s), 7.82–7.88 (2H, m),
8.11–8.17 (3H, m), 8.55 (3H, br s), 8.60 (1H, s); MS
(FAB) m/z 183 [M+H]+.
1
phous powder (3.24 g, 69%): H NMR (CDCl3) d 1.39
(9H, s), 1.52–1.69 (2H, m), 1.71–1.79 (2H, m), 2.64–
2.73 (2H, m), 2.88 (3H, s), 3.86–3.96 (1H, m), 3.99–
4.23 (2H, m), 4.56 (2H, s), 7.60 (1H, d, J = 8.3 Hz),
7.71 (1H, d, J = 8.8 Hz), 7.85–7.89 (3H, m), 8.09 (1H,
s); MS (FAB) m/z 461 [M+H]+.
7.1.3. tert-Butyl 4-[[(7-Cyano-2-naphthyl)methyl](meth-
ylsulfonyl)amino]piperidine-1-carboxylate (6). A mixture
of compound 5 (4.64 g, 21.2 mmol), tert-butyl 4-oxopi-
peridine-1-carboxylate (4.23 g, 21.2 mmol), triethyl-
amine (4.35 g, 43.0 mmol), AcOH (12.7 g, 212 mmol),
and 1,2-dichloroethane (100 mL) was stirred at ambient
temperature for 0.5 h. To this mixture was added NaB-
H(OAc)3 (6.75 g, 31.8 mmol) at ambient temperature,
and the whole was stirred for 24 h. To the reaction mix-
ture were added 10% aqueous sodium bicarbonate
(200 mL) and 1 N aqueous NaOH solution (100 mL),
and the mixture was extracted with CHCl3 (100 mL).
The organic layer was washed with brine, dried over
magnesium sulfate, and concentrated in vacuo. The
resulting material was dissolved in 1,2-dichloroethane
(80 mL) and combined with triethylamine (6.44 g,
63.6 mmol). To this solution was added methanesulfo-
nyl chloride (4.86 g, 42.4 mmol) at 0 °C and the whole
was stirred for 4 h. To the reaction mixture was added
H2O (150 mL) and extracted with ethyl acetate
(150 mL). The organic layer was washed with 10% aque-
ous sodium bicarbonate followed by 10% citric acid and
then brine, dried over magnesium sulfate, and concen-
trated in vacuo. The residue was triturated with MeOH
and the resulting precipitate was filtered off to yield the
title compound as a colorless solid (4.13 g, 44% from
compound 5): mp 204–206 °C; 1H NMR (CDCl3) d
7.1.5. Polymer-bound 7-{[(Methylsulfonyl)(piperidin-4-
yl)amino]methyl}naphthalene-2-carboximidamide (9). To
a mixture of polymer-bound benzyl alcohol 820 (1.63 g,
1.95 mmol;
substitution
1.20 mmol/g),
pyridine
(10 mL), and CH2Cl2 (30 mL) was added 4-nitrophenyl-
chloroformate (1.97 g, 9.75 mmol) at 0 °C, and the
whole was shaken at ambient temperature for 12 h.
The mixture was filtered, washed sequentially with
CH2Cl2 and pyridine, and then dried to yield polymer-
bound nitrophenylcarbonate. A mixture of this resin,
compound 7 (1.80 g, 3.91 mmol), triethylamine (1.98 g,
19.6 mmol), and NMP (20 mL) was agitated at ambient
temperature for 12 h. The mixture was filtered off,
washed sequentially with NMP, DMF, CH2Cl2, and
MeOH, and then dried to yield polymer-bound N-Boc
piperidine precursor. To a mixture of this resin and
MeOH (15 mL) was added 4 N hydrogen chloride in
ethyl acetate (15 mL) at ambient temperature, and the
whole was shaken for 2 h. The mixture was filtered off,
washed sequentially with MeOH and CH2Cl2, and dried
to yield polymer-bound piperidine intermediate 9.
7.1.6. Solid-phase synthesis of library compounds 10: N-
sulfonylpiperidine derivatives. Polymer 9 was partitioned
into reaction vials that each contained polymer (30 mg,
0.030 mmol), and treated with sulfonyl chloride

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T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
(0.10 mmol) and diisopropylethylamine (0.026 mL,
0.15 mmol) in dioxane (1.0 mL) at ambient temperature.
The reaction mixture was agitated for 12 h and washed
sequentially with MeOH, pyridine, and CH2Cl2. The
reaction mixture in each vessel was then treated with
2.0 mL of 90% aqueous trifluoroacetic acid at ambient
temperature for 4 h. The cleavage mixture was collected
in vials. The resin was washed with MeOH, and each
eluent in the respective vials was combined. The
solutions were evaporated in vacuo to afford N-sul-
fonylpiperidine derivatives 10, which were analyzed for
purity using reversed-phase HPLC and characterized
by MS.
chromatography on ODS gel (eluent: CH3CN/0.001 N
hydrochloric acid = 90:10 by volume). The organic sol-
vent was removed by evaporation, and freeze-drying of
the aqueous product solution yielded the title compound
as a colorless amorphous powder (149 mg, 45%): 1H
NMR (DMSO-d6) d 1.48–1.59 (2H, m), 1.69–1.76 (2H,
m), 2.57–2.65 (2H, m), 3.08 (3H, s), 3.63–3.67 (2H, m),
3.72 (3H, s), 3.74 (3H, s), 3.76–3.82 (1H, m), 4.57 (2H,
s), 7.13–7.20 (3H, m), 7.72 (1H, d, J = 8.3 Hz), 7.83
(1H, d, J = 8.8 Hz), 8.04–8.07 (2H, m), 8.14 (1H, d,
J = 8.3 Hz), 8.50 (1H, s), 9.25 (2H, s), 9.50 (2H, s);
MS (FAB) m/z 561 [M+H]+; Anal. Calcd for
C26H32N4O6S2ÆHClÆ0.6H2O: C, 51.37; H, 5.67; N, 9.22;
Cl, 5.83; S, 10.55. Found: C, 51.04; H, 5.88; N, 9.19;
Cl, 5.79; S, 10.46.
7.1.7. Solid-phase synthesis of library compounds 10: N-
acylpiperidine derivatives. Polymer 9 was partitioned
into reaction vials that each contained polymer
(20 mg, 0.020 mmol), and treated with carboxylic acid
(0.10 mmol), PyBOP (52 mg, 0.10 mmol), and diisopro-
pylethylamine (0.020 mL, 0.10 mmol) in DMF (1.0 mL)
at ambient temperature. The reaction mixture was agi-
tated for 12 h and washed sequentially with DMF,
MeOH, and CH2Cl2. The reaction mixture in each vessel
was then treated with 2.0 mL of 90% aqueous trifluoro-
acetic acid at ambient temperature for 4 h. The cleavage
mixture was collected in vials. The resin was washed
with MeOH, and each eluent in the respective vials
was combined. The solutions were evaporated in vacuo
to afford N-acylpiperidine derivatives 10, which were
analyzed for purity using reversed-phase HPLC and
characterized by MS.
7.1.10. 1-[(2,5-Dimethoxyphenyl)sulfonyl]piperidin-4-one
(13). To a stirred mixture of 4-piperidone hydrochloride
hydrate (12) (520 mg, 3.84 mmol), 2,5-(dimethoxyphe-
nyl)sulfonyl chloride (820 mg, 3.46 mmol), and 1,2-
dichloroethane (38 mL) was added triethylamine
(1.16 g, 11.5 mmol) at ambient temperature, and the
whole was stirred for 20 h. The reaction mixture was
washed with 10% citric acid followed by brine, dried
over magnesium sulfate, and then concentrated in vacuo
to yield the title compound as a colorless amorphous
1
powder (890 mg, 76%): H NMR (CDCl3) d 2.53 (4H,
t, J = 6.0 Hz), 3.59 (4H, t, J = 6.0 Hz), 3.81 (3H, s),
3.89 (3H, s), 6.97 (1H, d, J = 9.0 Hz), 7.08 (1H, dd,
J = 3.3 and 9.0 Hz), 7.48 (1H, d, J = 3.3 Hz); MS
(FAB) m/z 300 [M+H]+.
7.1.8. Solid-phase synthesis of library compounds 10: N-
alkylpiperidine derivatives. The polymer 9 was parti-
tioned into reaction vials that each contained polymer
(30 mg, 0.030 mmol), and treated with aldehyde
(0.60 mmol), NaBH(OAc)3 (64 mg, 0.60 mmol), and
acetic acid (0.20 mL) in CH2Cl2 (2.0 mL) at ambient
temperature. The reaction mixture was agitated for
12 h and washed sequentially with CH2Cl2 and MeOH.
The reaction mixture in each vessel was then treated
with 3.0 mL of 90% aqueous trifluoroacetic acid at
ambient temperature for 4 h. The cleavage mixture
was collected in vials. The resin was washed with
MeOH, and each eluent in the respective vials was com-
bined. The solutions were evaporated in vacuo to afford
N-alkylpiperidine derivatives 10, which were analyzed
for purity using reversed-phase HPLC and characterized
by MS.
7.1.11. 7-{[(1-Pyridin-4-ylpiperidin-4-yl)amino]methyl}-2-
naphthonitrile (14). Compound 5 (7.13 g, 32.6 mmol)
was suspended in CHCl3 (100 mL) and washed with
10% aqueous potassium carbonate. The organic layer
was dried over magnesium sulfate and concentrated in
vacuo to yield 7-(aminomethyl)-2-naphthonitrile
(5.94 g, quant). To a stirred mixture of 4-(4-oxopiperi-
din-1-yl)pyridine (11)22 (270 mg, 1.53 mmol), 7-(amino-
methyl)-2-naphthonitrile (279 mg, 1.53 mmol), AcOH
(0.90 mL), and 1,2-dichloroethane (15 mL) was added
NaBH(OAc)3 (634 mg, 3.00 mmol) at ambient tempera-
ture, and the whole was stirred for 1 h. To the reaction
mixture was added 10% aqueous potassium carbonate
(50 mL), and the mixture was extracted with CHCl3
(50 mL). The organic layer was dried over magnesium
sulfate and concentrated in vacuo to yield the title com-
pound as a colorless amorphous powder (350 mg, 67%):
1H NMR (CDCl3) d 1.30–1.75 (2H, m), 1.94–2.18 (2H,
m), 2.72–3.15 (3H, m), 3.76–4.06 (2H, m), 4.10 (2H, s),
6.71 (2H, d, J = 6.6 Hz), 7.56–8.00 (5H, m), 8.26–8.33
(3H, m); MS (FAB) m/z 343 [M+H]+.
7.1.9. 7-{[{1-[(2,5-Dimethoxyphenyl)sulfonyl]piperidin-4-
yl}(methylsulfonyl)amino]methyl}naphthalene-2-carbox-
imidamide hydrochloride (10x). Gaseous anhydrous
hydrogen chloride was bubbled through a solution of
compound 18 (300 mg, 0.552 mmol) in EtOH/CHCl3
(11 mL, 1:1 by volume) at ꢀ20 °C for 20 min. The reac-
tion mixture was allowed to warm to 5 °C and stirred for
22 h. The solvent was removed under reduced pressure,
and the residue was redissolved in EtOH (11 mL). To
this solution was added ammonium acetate (424 mg,
5.5 mmol) at ambient temperature, and the whole was
stirred for 24 h. The reaction mixture was concentrated
in vacuo, and the residue was purified using column
7.1.12. 7-[({1-[(2,5-Dimethoxyphenyl)sulfonyl]piperidin-4-
yl}amino)methyl]-2-naphthonitrile (15). To a stirred mix-
ture of compound 13 (1.02 g, 3.41 mmol), 7-(amino-
methyl)-2-naphthonitrile (621 mg, 3.41 mmol), AcOH
(2.0 mL), and 1,2-dichloroethane (34 mL) was added
NaBH(OAc)3 (1.44 g, 6.80 mmol) at ambient tempera-
ture, and the whole was stirred for 1 h. To the reaction
mixture was added 10% aqueous potassium carbonate
(50 mL), and the resulting mixture was extracted with

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4187
CHCl3 (50 mL). The organic layer was dried over
magnesium sulfate and concentrated in vacuo to yield
the title compound as a colorless amorphous powder
(1.59 g, quant): 1H NMR (CDCl3) d 1.41–1.56 (4H,
m), 1.92–2.01 (2H, m), 2.57–2.66 (1H, m), 2.71–2.82
(2H, m), 3.79 (3H, s), 3.86 (3H, s), 3.99 (2H, s), 6.94
(1H, d, J = 9.0 Hz), 7.04 (1H, dd, J = 3.0 and 9.0 Hz),
7.43 (1H, d, J = 3.0 Hz), 7.58 (1H, d, J = 8.7 Hz), 7.62
(1H, d, J = 8.4 Hz), 7.79 (1H, s), 7.85 (1H, d,
J = 8.4 Hz), 7.89 (1H, d, J = 8.7 Hz), 8.18 (1H, s); MS
(FAB) m/z 466 [M+H]+.
J = 8.4 Hz), 7.69 (1H, d, J = 8.4 Hz), 7.77–7.92 (3H, m),
8.19 (1H, s); MS (FAB) m/z 544 [M+H]+.
7.1.16. N-[(7-Cyano-2-naphthyl)methyl]-N-{1-[(2,5-dimeth-
oxyphenyl)sulfonyl]piperidin-4-yl}acetamide (19). The title
compound was obtained as a colorless amorphous pow-
der from 500 mg (1.07 mmol) of compound 15, 126 mg
(1.61 mmol) of acetyl chloride, and 326 mg (3.21 mmol)
of triethylamine in a manner identical to that described
above for compound 18 (490 mg, 90%): 1H NMR
(CDCl3) d 1.52–1.86 (4H, m), 2.10 (3H, s), 2.59–2.74
(2H, m), 3.69–3.94 (9H, m), 4.68 (2H, s), 6.83–7.03 (2H,
m), 7.36 (1H, d, J = 3.0 Hz), 7.48 (1H, d, J = 6.9 Hz),
7.65 (1H, d, J = 9.3 Hz), 7.80–7.95 (3H, m), 8.19 (1H, s);
MS (FAB) m/z 508 [M+H]+.
7.1.13. 3-[(E)-3-({1-[(2,5-Dimethoxyphenyl)sulfonyl]pip-
eridin-4-yl}amino)prop-1-en-1-yl]benzonitrile (16). The ti-
tle compound was obtained as a colorless amorphous
powder from 0.680 g (2.27 mmol) of compound 13,
0.360 g (2.28 mmol) of (E)-3-(3-aminopropenyl)benzo-
nitrile,23 954 mg (4.50 mmol) of NaBH(OAc)3, and
1.31 mL (22.8 mmol) of AcOH in a manner identical
to that described above for compound 15 (762 mg,
76%): 1H NMR (CDCl3) d 1.38–1.53 (2H, m), 1.56–
1.72 (2H, m), 1.87–1.99 (2H, m), 2.58–2.67 (1H, m),
2.72–2.82 (2H, m), 3.46 (2H, d, J = 6.0 Hz), 3.80 (3H,
s), 3.87 (3H, s), 6.33 (1H, dt, J = 15.6 and 6.0 Hz),
6.51 (1H, d, J = 15.6 Hz), 6.94 (1H, d, J = 9.0 Hz),
7.25 (1H, dd, J = 3.3 and 9.0 Hz), 7.38–7.63 (5H, m);
MS (FAB) m/z 442 [M+H]+.
7.1.17. 7-{[{1-[(2,5-Dimethoxyphenyl)sulfonyl]piperidin-
4-yl}(methyl)amino]methyl}-2-naphthonitrile (20). The
title compound was obtained as a colorless amorphous
powder from 500 mg (1.07 mmol) of compound 15,
0.550 mL (5.35 mmol) of 35% aqueous HCHO, 454 mg
(2.14 mmol)
of
NaBH(OAc)3,
and
0.610 mL
(10.7 mmol) of AcOH in a manner identical to that de-
scribed above for compound 15 (443 mg, 86%): 1H
NMR (CDCl3) d 1.64–1.78 (2H, m), 1.84–1.92 (2H,
m), 2.22 (3H, s), 2.49–2.68 (3H, m), 3.73 (2H, s), 3.80
(3H, s), 3.87 (3H, s), 3.91–4.00 (2H, m), 6.94 (1H, d,
J = 8.7 Hz), 7.05 (1H, dd, J = 3.0 and 8.7 Hz), 7.44
(1H, d, J = 3.0 Hz), 7.56 (1H, d, J = 8.4 Hz), 7.63 (1H,
d, J = 8.4 Hz), 7.76 (1H, s), 7.83 (1H, d, J = 8.4 Hz),
7.88 (1H, d, J = 8.4 Hz), 8.17 (1H, s); MS (FAB) m/z
480 [M+H]+.
7.1.14. N-[(7-Cyano-2-naphthyl)methyl]-N-(1-pyridin-4-
ylpiperidin-4-yl)methanesulfonamide (17). To a mixture
of compound 14 (170 mg, 0.496 mmol), triethylamine
(0.87 g, 8.61 mmol), and 1,2-dichloroethane (12 mL)
was added methanesulfonyl chloride (986 mg,
8.61 mmol) at 0 °C and the whole was stirred for 5 h.
To the reaction mixture was added 10% aqueous potas-
sium carbonate (20 mL) followed by extraction with
CHCl3 (20 mL). The organic layer was dried over mag-
nesium sulfate and concentrated in vacuo. The residue
was purified using column chromatography on silica
gel (eluent: CHCl3/MeOH/c.NH3 = 100:5:0.5 by vol-
ume) to yield the title compound as a colorless amor-
phous powder (110 mg, 53%): 1H NMR (CDCl3) d
1.78–1.95 (4H, m), 2.73–2.98 (2H, m), 3.00 (3H, s),
3.78–3.93 (1H, m), 3.95–4.12 (2H, m), 4.62 (2H, s),
6.61 (2H, d, J = 6.6 Hz), 7.57–8.00 (5H, m), 8.20–8.30
(3H, m); MS (FAB) m/z 421 [M+H]+.
7.1.18. Ethyl [([(7-cyano-2-naphthyl)methyl]{1-[(2,5-dimeth-
oxyphenyl)sulfonyl]piperidin-4-yl}amino)sulfonyl]acetate
(21). The title compound was obtained as a colorless
amorphous powder from 1.61 g (3.46 mmol) of com-
pound 15, 711 mg (3.81 mmol) of ethyl (chlorosulfo-
nyl)acetate,31 and 385 mg (3.81 mmol) of triethylamine
in a manner identical to that described above for com-
1
pound 18 (1.74 g, 82%): H NMR (CDCl3) d 1.31 (3H,
t, J = 6.9 Hz), 1.60–1.75 (2H, m), 1.85–1.93 (2H, m),
2.58–2.68 (2H, m), 2.77 (6H, s), 3.83–3.91 (5H, m), 4.24
(2H, q, J = 6.9 Hz), 4.61 (2H, s), 6.88 (1H, d,
J = 9.0 Hz), 7.03 (1H, dd, J = 3.3 and 9.0 Hz), 7.35 (1H,
d, J = 3.3 Hz), 7.63 (1H, d, J = 8.4 Hz), 7.70 (1H, d,
J = 8.4 Hz), 7.85 (1H, s), 7.89 (1H, d, J = 8.4 Hz), 7.93
(1H, d, J = 8.4 Hz), 8.23 (1H, s); MS (FAB) m/z 616
[M+H]+.
7.1.15. N-[(7-Cyano-2-naphthyl)methyl]-N-{1-[(2,5-dimeth-
oxyphenyl)sulfonyl]piperidin-4-yl}methanesulfonamide (18).
To a stirred mixture of compound 15 (500 mg,
1.07 mmol), triethylamine (325 mg, 3.21 mmol), and
1,2-dichloroethane (11 mL) was added methanesulfonyl
chloride (135 mg, 1.18 mmol) at 0 °C, and the whole
was stirred for 3 h. The reaction mixture was then washed
with 10% citric acid, followed by brine, dried over magne-
sium sulfate, and concentrated in vacuo. The residue was
purified using column chromatography on silica gel
(eluent: CHCl3/MeOH = 100:5 by volume) to yield the
title compound as a colorless amorphous powder
7.1.19. Ethyl 3-([(7-cyano-2-naphthyl)methyl]{1-[(2,5-dimeth-
oxyphenyl)sulfonyl]piperidin-4-yl}amino)-3-oxopropano-
ate (22). The title compound was obtained as a colorless
amorphous powder from 1.28 g (2.75 mmol) of com-
pound 15, 456 mg (3.03 mmol) of ethyl malonyl chloride,
and 306 mg (3.03 mmol) of triethylamine in a manner
identical to that described above for compound 18
(1.42 g, 89%): 1H NMR (CDCl3) d 1.25 (2H, t,
J = 7.2 Hz), 1.32 (1H, t, J = 7.2 Hz), 1.61–1.77 (4H, m),
2.57–2.75 (2H, m), 3.37 (1.3H, s), 3.62 (0.7H, s), 3.77–
3.97 (9H, m), 4.16 (1.3H, q, J = 7.2 Hz), 4.26 (0.7H,
q, J = 7.2 Hz), 4.69 (1.3H, s), 4.74 (0.7H, s), 6.91–6.96
(1H, m), 7.02–7.08 (1H, m), 7.38 (1H, d, J = 3.3 Hz),
1
(420 mg, 72%): H NMR (CDCl3) d 1.68–1.78 (4H, m),
2.55–2.68 (2H, m), 2.90 (3H, s), 3.74–3.91 (9H, m), 4.55
(2H, s), 6.90 (1H, d, J = 8.7 Hz), 7.03 (1H, dd, J = 3.0
and 8.7 Hz), 7.34 (1H, d, J = 3.0 Hz), 7.59 (1H, d,

4188
T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
7.46–7.66 (2H, m), 7.69 (0.7H, s), 7.74 (0.3H, s), 7.82–7.95
(2H, m), 8.18 (0.3H, s), 8.19 (0.7H, s); MS (FAB) m/z 580
[M+H]+.
compound was obtained as a colorless amorphous
powder from 1.01 g (1.74 mmol) of compound 22,
242 mg (3.48 mmol) of hydroxylamine hydrochloride,
and 439 mg (4.35 mmol) of triethylamine in a manner
identical to that described above for compound 25
(840 mg, 79%): 1H NMR (DMSO-d6) d 1.17 (3H, t,
J = 7.3 Hz), 1.63–1.70 (4H, m), 2.65–2.74 (2H, m), 3.55
(2H, br s), 3.70–3.74 (5H, m), 3.75 (3H, s), 4.01–4.13
(3H, m), 4.65 (2H, s), 5.87 (2H, br s), 7.10 (1H, d,
J = 8.8 Hz), 7.14 (1H, dd, J = 2.9 and 8.8 Hz), 7.22
(1H, d, J = 2.9 Hz), 7.38 (1H, d, J = 8.8 Hz), 7.70 (1H,
s), 7.76–7.84 (3H, m), 8.15 (1H, s), 9.46 (1H, s); MS
(FAB) m/z 613 [M+H]+; Anal. Calcd for C30H36N4O8-
SÆ0.6H2O: C, 57.79; H, 6.01; N, 8.99; S, 5.14. Found:
C, 57.64; H, 5.99; N, 8.82; S, 5.07.
7.1.20. Ethyl [([(E)-3-(3-cyanophenyl)prop-2-en-1-yl]{1-
[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}amino)sul-
fonyl]acetate (23). The title compound was obtained as a
colorless amorphous powder from 1.02 g (2.31 mmol) of
compound 16, 473 mg (2.54 mmol) of ethyl (chloro-
sulfonyl)acetate, and 257 mg (2.54 mmol) of triethyl-
amine in a manner identical to that described above
1
for compound 18 (1.01 g, 74%): H NMR (CDCl3) d
1.29 (3H, t, J = 7.2 Hz), 1.84–1.97 (4H, m), 2.63–2.73
(2H, m), 3.75–3.82 (4H, m), 3.86 (3H, s), 3.93 (2H, s),
3.96–4.02 (2H, m), 4.04 (2H, d, J = 6.3 Hz), 4.21 (2H,
q, J = 7.2 Hz), 6.22 (1H, dt, J = 15.9 and 6.3 Hz), 6.57
(1H, d, J = 15.9 Hz), 6.94 (1H, d, J = 9.0 Hz), 7.06
(1H, dd, J = 3.0 and 9.0 Hz), 7.40–7.47 (2H, m), 7.53–
7.65 (2H, m), 7.64 (1H, s); MS (FAB) m/z 592 [M+H]+.
7.1.24.
Ethyl
{[((E)-3-{3-[(amino)(hydroxyimino)-
methyl]phenyl}prop-2-en-1-yl){1-[(2,5-dimethoxyphenyl)sul-
fonyl]piperidin-4-yl}amino]sulfonyl}acetate (27). The title
compound was obtained as a colorless amorphous pow-
der from 320 mg (0.541 mmol) of compound 23, 75 mg
(1.35 mmol) of hydroxylamine hydrochloride, and
137 mg (1.35 mmol) of triethylamine in a manner identi-
cal to that described above for compound 25 (235 mg,
7.1.21. Ethyl 3-([(E)-3-(3-cyanophenyl)prop-2-en-1-yl]{1-
[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}amino)-3-
oxopropanoate (24). The title compound was obtained as
a colorless amorphous powder from 740 mg (1.68 mmol)
of compound 16, 858 mg (5.70 mmol) of ethyl malonyl
chloride, and 1.15 g (11.4 mmol) of triethylamine in a
manner identical to that described above for compound
18 (490 mg, 53%): 1H NMR (CDCl3) d 1.25 (3H, t,
J = 7.2 Hz), 1.54–1.58 (2H, m), 1.74–1.81 (2H, m),
2.66–2.74 (2H, m), 3.44 (1.4H, s), 3.51 (0.6H, s), 3.80
(3H, s), 3.86 (2.1H, s), 3.88 (0.9H, s), 3.93–4.09 (5H,
m), 4.17 (2H, q, J = 7.2 Hz), 6.16–6.25 (1H, m), 6.49
(1H, d, J = 15.9 Hz), 6.93–6.98 (1H, m), 7.04–7.08
(1H, m), 7.41–7.62 (5H, m); MS (FAB) m/z 556 [M+H]+.
1
76%): H NMR (DMSO-d6) d 1.20 (3H, t, J = 7.3 Hz),
1.66–1.83 (4H, m), 2.61–2.70 (2H, m), 3.67–3.78 (6H,
m), 3.80 (3H, s), 3.98 (2H, d, J = 6.3 Hz), 4.14 (2H, q,
J = 7.3 Hz), 4.33 (2H, s), 5.88 (2H, br s), 6.24 (1H, dt,
J = 16.1 and 6.3 Hz), 6.57 (1H, d, J = 16.1 Hz), 7.14–
7.22 (3H, m), 7.34 (1H, t, J = 7.8 Hz), 7.42 (1H, d,
J = 7.8 Hz), 7.68 (1H, d, J = 7.8 Hz), 7.73 (1H, s), 9.21
(1H, br s); MS (FAB) m/z 625 [M+H]+; Anal. Calcd
for C27H36N4O9S2Æ0.5H2O: C, 51.17; H, 5.88; N, 8.84;
S, 10.12. Found: C, 51.26; H, 5.91; N, 8.60; S, 9.96.
7.1.22. Ethyl {[({7-[(amino)(hydroxyimino)methyl]-2-
naphthyl}methyl){1-[(2,5-dimethoxyphenyl)sulfonyl]pip-
eridin-4-yl}amino]sulfonyl}acetate (25). A mixture of
compound 21 (1.73 g, 2.81 mmol), hydroxylamine hydro-
chloride (389 mg, 5.60 mmol), triethylamine (627 mg,
6.20 mmol), and EtOH (28 mL) was stirred at 80 °C for
14 h. The reaction mixture was allowed to cool and the
solvent was removed in vacuo. The residue was dissolved
in CHCl3 (30 mL) and washed with brine (20 mL). The or-
ganic layer was dried over magnesium sulfate and concen-
trated in vacuo. The residue was purified using column
chromatography on silica gel (eluent: CHCl3/MeOH/
c.NH3 = 100:3:0.3 by volume) to yield the title compound
as a colorless amorphous powder (1.10 g, 60%): 1H NMR
(DMSO-d6) d 1.22 (3H, t, J = 7.3 Hz), 1.37–1.50 (2H, m),
1.69–1.76 (2H, m), 2.58–2.66 (2H, m), 3.62–3.66 (2H, m),
3.68 (3H, s), 3.71 (3H, s), 3.74–3.86 (1H, m), 4.19 (2H, q,
J = 7.3 Hz), 4.44 (2H, s), 4.55 (2H, s), 5.98 (2H, br s), 7.06
(1H, d, J = 8.3 Hz), 7.10–7.17 (2H, m), 7.51 (1H, d,
J = 8.3 Hz), 7.85 (1H, d, J = 8.3 Hz), 7.86 (1H, s), 7.89
(1H, d, J = 8.3 Hz), 7.93 (1H, d, J = 8.3 Hz), 8.45 (1H,
s), 9.81 (1H, s); MS (FAB) m/z 649 [M+H]+; Anal. Calcd
for C29H36N4O9S2Æ0.5H2O: C, 52.96; H, 5.67; N, 8.52; S,
9.75. Found: C, 52.96; H, 5.64; N, 8.36; S, 9.73.
7.1.25. Ethyl 3-[((E)-3-{3-[(amino)(hydroxyimino)methyl]-
phenyl}prop-2-en-1-yl){1-[(2,5-dimethoxyphenyl)sulfo-
nyl]piperidin-4-yl}amino]-3-oxopropanoate (28). The title
compound was obtained as a colorless amorphous
powder from 330 mg (0.594 mmol) of compound 24,
83 mg (1.19 mmol) of hydroxylamine hydrochloride,
and 150 mg (1.49 mmol) of triethylamine in a manner
identical to that described above for compound 25
(236 mg, 67%): 1H NMR (DMSO-d6) d 1.15 (3H, t,
J = 6.9 Hz), 1.67–1.81 (4H, m), 2.68–2.76 (2H, m),
3.49 (2H, s), 3.75 (3H, s), 3.76–3.83 (5H, m), 3.97–
4.03 (3H, m), 4.08 (2H, q, J = 6.9 Hz), 5.50 (2H, br
s), 6.36 (1H, dt, J = 16.1 and 5.4 Hz), 6.51 (1H, d,
J = 16.1 Hz), 7.15 (2H, s), 7.27 (1H, s), 7.30 (1H, t,
J = 7.8 Hz), 7.36 (1H, d, J = 7.8 Hz), 7.53 (1H, d,
J = 7.8 Hz), 7.66 (1H, s), 9.21 (1H, br s); MS (FAB)
m/z 589 [M+H]+; Anal. Calcd for C28H36N4O8SÆ0.6-
H2O: C, 56.10; H, 6.25; N, 9.35; S, 5.35. Found: C,
56.09; H, 6.25; N, 9.17; S, 5.30.
7.1.26. 7-{[(Methylsulfonyl)(1-pyridin-4-ylpiperidin-4-yl)-
amino]methyl}naphthalene-2-carboximidamide
hydro-
chloride (29). The title compound was obtained as a
colorless amorphous powder from 110 mg (0.262 mmol)
of compound 17 and 200 mg (2.60 mmol) of ammonium
acetate in a manner identical to that described above for
7.1.23. Ethyl 3-[({7-[(amino)(hydroxyimino)methyl]-2-
naphthyl}methyl){1-[(2,5-dimethoxyphenyl)sulfonyl]pip-
eridin-4-yl}amino]-3-oxopropanoate (26). The title
1
compound 10x (63 mg, 47%): H NMR (DMSO-d6) d
1.61–1.72 (2H, m), 1.84–1.90 (2H, m), 3.13 (3H, s),

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4189
3.16–3.22 (2H, m), 4.10–4.18 (1H, m), 4.24–4.28 (2H,
m), 4.62 (2H, s), 7.14 (2H, d, J = 7.2 Hz), 7.70 (1H, d,
J = 9.6 Hz), 7.84 (1H, dd, J = 1.6 and 8.4 Hz), 8.02–
8.04 (2H, m), 8.11 (1H, d, J = 8.4 Hz), 8.14–8.18 (2H,
m), 8.52 (1H, s), 9.39 (2H, s), 9.58 (2H, s), 13.79 (1H,
br s); MS (FAB) m/z 438 [M+H]+; Anal. Calcd for
C23H27N5O2SÆ2.3HClÆ1.4H2O: C, 50.54; H, 5.92; N,
12.81; Cl, 14.92; S, 5.87. Found: C, 50.79; H, 6.24; N,
13.09; Cl, 14.96; S, 5.69.
3.78–3.86 (1H, m), 4.19 (2H, q, J = 7.3 Hz), 4.49
(2H, s), 4.62 (2H, s), 7.12–7.19 (3H, m), 7.71 (1H, d,
J = 8.8 Hz), 7.86 (1H, d, J = 8.8 Hz), 8.05 (1H, s), 8.07
(1H, d, J = 8.8 Hz), 8.15 (1H, d, J = 8.8 Hz), 8.50 (1H,
s), 9.33 (2H, s), 9.54 (2H, s); MS (FAB) m/z 633
[M+H]+; Anal. Calcd for C29H36N4O8S2ÆHClÆH2O: C,
50.68; H, 5.72; N, 8.15; Cl, 5.16; S, 9.33. Found: C,
50.86; H, 5.71; N, 8.02; Cl, 4.95; S, 9.11.
7.1.30. Ethyl 3-(({7-[amino(imino)methyl]-2-naphthyl}-
methyl){1-[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}-
amino)-3-oxopropanoate hydrochloride (33). The title
compound was obtained as a colorless amorphous pow-
der from 0.430 g (0.702 mmol) of compound 26 and
143 mg (1.40 mmol) of acetic anhydride in a manner
identical to that described above for compound 32
(321 mg, 72%): 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.3 Hz), 1.63–1.71 (4H, m), 2.66–2.74 (2H, m), 3.58
(2H, br), 3.69–3.75 (5H, m), 3.77 (3H, s), 4.03–4.14
(3H, m), 4.70 (2H, s), 7.12–7.17 (2H, m), 7.22 (1H, d,
J = 2.4 Hz), 7.58 (1H, d, J = 8.8 Hz), 7.82 (1H, d,
J = 8.3 Hz), 7.85 (1H, s), 7.98 (1H, d, J = 8.3 Hz), 8.07
(1H, d, J = 8.8 Hz), 8.44 (1H, s), 9.26 (4H, br s); MS
(FAB) m/z 597 [M+H]+; Anal. Calcd for C30H36N4O7-
SÆHClÆH2O: C, 55.34; H, 6.04; N, 8.60; Cl, 5.44; S,
4.92. Found: C, 55.13; H, 6.07; N, 8.48; Cl, 5.44; S, 4.86.
7.1.27. N-({7-[Amino(imino)methyl]-2-naphthyl}methyl)-
N-{1-[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}acet-
amide hydrochloride (30). The title compound was
obtained as a colorless amorphous powder from
330 mg (0.650 mmol) of compound 19 and 500 mg
(6.50 mmol) of ammonium acetate in a manner identical
to that described above for compound 10x (121 mg,
1
33%): H NMR (DMSO-d6) d 1.59–1.70 (4H, m), 2.10
(3H, s), 2.67–2.74 (2H, m), 3.65–3.75 (5H, m), 3.77 (3H,
s), 4.18–4.20 (1H, m), 4.67 (2H, s), 7.11–7.15 (2H, m),
7.22 (1H, d, J = 2.9 Hz), 7.56 (1H, d, J = 8.3 Hz), 7.80–
7.84 (2H, m), 7.98 (1H, d, J = 8.7 Hz), 8.07 (1H, d,
J = 8.3 Hz), 8.47 (1H, s), 9.25 (4H, s); MS (FAB) m/z
525 [M+H]+; Anal. Calcd for C27H32N4O5SÆHClÆH2O:
C, 56.00; H, 6.09; N, 9.67; Cl, 6.12; S, 5.54. Found: C,
55.87; H, 6.29; N, 9.64; Cl, 6.47; S, 5.49.
7.1.28. 7-{[{1-[(2,5-Dimethoxyphenyl)sulfonyl]piperidin-
4-yl}(methyl)amino]methyl}naphthalene-2-carboximida-
mide hydrochloride (31). The title compound was
obtained as a colorless amorphous powder from
400 mg (0.845 mmol) of compound 20 and 655 mg
(8.50 mmol) of ammonium acetate in a manner identical
to that described above for compound 10x (242 mg,
50%): 1H NMR (DMSO-d6) d 1.54–1.72 (2H, m),
1.94–2.18 (2H, m), 2.28–2.42 (2H, m), 2.66–2.74 (2H,
m), 2.93 (3H, br s), 3.76–3.86 (9H, m), 7.16–7.19 (2H,
m), 7.28 (1H, s), 7.73–7.85 (2H, m), 8.00–8.18 (3H, m),
8.46 (1H, s), 9.16 (4H, br s); MS (FAB) m/z 497
[M+H]+; Anal. Calcd for C26H32N4O4SÆ1.3HClÆ2.5H2O:
C, 53.01; H, 6.55; N, 9.51; Cl, 7.82; S, 5.44. Found: C,
52.88; H, 6.30; N, 9.30; Cl, 7.96; S, 5.92.
7.1.31. Ethyl [(((E)-3-{3-[amino(imino)methyl]phenyl}-
prop-2-en-1-yl){1-[(2,5-dimethoxyphenyl)sulfonyl]piperi-
din-4-yl}amino)sulfonyl]acetate hydrochloride (34). The
title compound was obtained as a colorless amorphous
powder from 400 mg (0.676 mmol) of compound 23
and 524 mg (6.80 mmol) of ammonium acetate in a
manner identical to that described above for compound
1
10x (380 mg, 91%): H NMR (DMSO-d6) d 1.20 (3H, t,
J = 7.3 Hz), 1.68–1.84 (4H, m), 2.61–2.68 (2H, m), 3.70–
3.76 (6H, m), 3.81 (3H, s), 4.02 (2H, d, J = 5.9 Hz), 4.14
(2H, q, J = 7.3 Hz), 4.36 (2H, s), 6.41 (1H, dt, J = 16.2
and 5.9 Hz), 6.66 (1H, d, J = 16.2 Hz), 7.19–7.22 (3H,
m), 7.59 (1H, t, J = 7.8 Hz), 7.72 (1H, d, J = 7.8 Hz),
7.78 (1H, d, J = 7.8 Hz), 7.91 (1H, s), 9.25 (2H, s),
9.44 (2H, s); MS (FAB) m/z 609 [M+H]+; Anal. Calcd
for C27H36N4O8S2ÆHClÆH2O: C, 48.90; H, 5.93; N,
8.45; Cl, 5.35; S, 9.67. Found: C, 48.74; H, 5.86; N,
8.23; Cl, 5.54; S, 9.49.
7.1.29. Ethyl [(({7-[amino(imino)methyl]-2-naphthyl}-
methyl){1-[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}-
amino)sulfonyl]acetate hydrochloride (32). To a mixture
of compound 25 (710 mg, 1.09 mmol), acetic anhydride
(224 mg, 2.19 mmol), and AcOH (11 mL) was added
Pd/C (10 w/w%, 70 mg), and the mixture was stirred un-
der hydrogen pressure (1 kg/cm2) at ambient tempera-
ture for 8 h. The reaction mixture was filtered through
a pad of CeliteÒ and the filtrate was concentrated in va-
cuo. The residue was purified using column chromatog-
raphy on ODS gel (eluent: EtOH/H2O = 50:50 by
volume). The solvent was removed by evaporation and
the residue was dissolved in EtOH (10 mL). To this solu-
tion was added 4 N HCl/AcOEt (1.0 mL) at ambient
temperature, and the whole was stirred for 10 min.
The mixture was concentrated in vacuo to yield the title
compound as a colorless amorphous powder (518 mg,
7.1.32. Ethyl 3-(((E)-3-{3-[amino(imino)methyl]phenyl}-
prop-2-en-1-yl){1-[(2,5-dimethoxyphenyl)sulfonyl]piperi-
din-4-yl}amino)-3-oxopropanoate hydrochloride (35). The
title compound was obtained as a colorless amorphous
powder from 490 mg (0.882 mmol) of compound 24
and 678 mg (8.80 mmol) of ammonium acetate in a
manner identical to that described above for compound
1
10x (400 mg, 74%): H NMR (DMSO-d6) d 1.19 (3H, t,
J = 6.8 Hz), 1.71–1.79 (4H, m), 2.67–2.76 (2H, m), 3.51
(2H, s), 3.76 (3H, s), 3.77–3.82 (2H, m), 3.83 (3H, s),
3.98–4.05 (3H, m), 4.14 (2H, q, J = 6.8 Hz), 6.36 (1H,
dt, J = 16.1 and 5.4 Hz), 6.58 (1H, d, J = 16.1 Hz),
7.16–7.17 (2H, m), 7.26 (1H, d, J = 1.4 Hz), 7.55 (1H,
t, J = 7.8 Hz), 7.68–7.72 (2H, m), 7.87 (1H, s), 9.23
(4H, s); MS (FAB) m/z 573 [M+H]+; Anal. Calcd for
C28H36N4O7SÆHClÆH2O: C, 53.62; H, 6.27; N, 8.93; Cl,
1
71%): H NMR (DMSO-d6) d 1.24 (3H, t, J = 7.3 Hz),
1.39–1.51 (2H, m), 1.72–1.79 (2H, m), 2.56–2.65
(2H, m), 3.59–3.66 (2H, m), 3.71 (3H, s), 3.72 (3H, s),

4190
T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
5.65; S, 5.11. Found: C, 53.89; H, 6.25; N, 8.77; Cl, 5.66;
S, 5.09.
7.1.36. 3-(((E)-3-{3-[Amino(imino)methyl]phenyl}prop-2-
en-1-yl){1-[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}-
amino)-3-oxopropanoic acid hydrochloride (39). The title
compound was obtained as a colorless amorphous pow-
der from 460 mg (0.755 mmol) of compound 35 and
1.8 mL of 1 N aqueous NaOH solution in a manner
identical to that described above for compound 36
7.1.33. [(({7-[Amino(imino)methyl]-2-naphthyl}methyl){1-
[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}amino)sulfo-
nyl]acetic acid hydrochloride (36). To a stirred solution
of compound 32 (270 mg, 0.403 mmol) in dioxane
(4.0 mL) and H2O (2.7 mL) was added 1 N aqueous
NaOH solution (1.3 mL) at 0 °C, and the whole was stir-
red for 4 h. To this mixture was added 1 N hydrochloric
acid (1.3 mL) at 0 °C and the whole was allowed to
warm to ambient temperature. The solvent was removed
in vacuo and the residue was purified using column
chromatography on ODS gel (eluent: CH3CN/0.001 N
hydrochloric acid = 50:50 by volume). The organic sol-
vent was removed by evaporation, and freeze-drying of
the aqueous product solution yielded the title compound
as a colorless amorphous powder (148 mg, 57%): 1H
NMR (DMSO-d6) d 1.38–1.50 (2H, m), 1.72–1.80 (2H,
m), 2.55–2.63 (2H, m), 3.56–3.65 (2H, m), 3.71 (3H, s),
3.72 (3H, s), 3.75–3.85 (1H, m), 4.33 (2H, s), 4.62 (2H,
s), 7.11–7.18 (3H, m), 7.72 (1H, d, J = 8.8 Hz), 7.84
(1H, d, J = 8.8 Hz), 8.05 (1H, s), 8.07 (1H, d,
J = 8.8 Hz), 8.15 (1H, d, J = 8.8 Hz), 8.49 (1H, s), 9.35
(2H, s), 9.50 (2H, s), 13.37 (1H, br s); MS (FAB) m/z
605 [M+H]+; Anal. Calcd for C27H32N4O8S2Æ0.9HClÆ-
H2O: C, 49.47; H, 5.37; N, 8.55; Cl, 4.87; S, 9.78. Found:
C, 49.30; H, 5.36; N, 8.55; Cl, 4.93; S, 9.76.
1
(249 mg, 57%): H NMR (DMSO-d6) d 1.68–1.79 (4H,
m), 2.68–2.76 (2H, m), 3.75 (3H, s), 3.76–3.81 (4H, m),
3.82 (3H, s), 3.99–4.04 (3H, m), 6.34 (1H, dt, J = 16.1
and 5.3 Hz), 6.54 (1H, d, J = 16.1 Hz), 7.15–7.17 (2H,
m), 7.26 (1H, s), 7.49 (1H, t, J = 7.8 Hz), 7.64–7.69
(2H, m), 7.85 (1H, s), 8.72 (4H, s); MS (FAB) m/z 545
[M+H]+; Anal. Calcd for C26H32N4O7SÆ0.8HClÆH2O:
C, 52.77; H, 5.93; N, 9.47; Cl, 4.79; S, 5.42. Found: C,
52.76; H, 5.94; N, 9.69; Cl, 4.46; S, 5.31.
7.2. Pharmacology
7.2.1. In vitro chromogenic assay. The hydrolysis rates of
the synthetic substrates were assayed by continuously
measuring absorbance at 405 nm at 37 °C with a micro-
plate spectrophotometer (Spectramax 340PC, Molecular
Device Co., California, USA). The reaction mixtures
(40 lL) were prepared in 96-well plates containing chro-
mogenic substrates and an inhibitor in 20 mM Hepes,
0.01% BSA, 5 mM CaCl2, 0.15 M NaCl, pH 7.4. Reac-
tions were initiated with 10 lL of enzyme solution.
Enzymes and substrates were used as follows: fXa
and S-2222; thrombin and S-2238; trypsin and S-2222.
The concentration of inhibitor required to inhibit en-
zyme activity by 50% (IC50) was calculated by regression
analysis.
7.1.34. 3-(({7-[Amino(imino)methyl]-2-naphthyl}methyl)-
{1-[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}amino)-
3-oxopropanoic acid hydrochloride (37). The title
compound was obtained as a colorless amorphous pow-
der from 0.240 g (0.379 mmol) of compound 33 and
1.2 mL of 1 N aqueous NaOH solution in a manner iden-
tical to that described above for compound 36 (147 mg,
64%): 1H NMR (DMSO-d6) d 1.58–1.67 (4H, m),
2.66–2.75 (2H, m), 2.87 (2H, s), 3.69–3.76 (5H, m), 3.77
(3H, s), 4.04–4.16 (1H, m), 4.67 (2H, s), 7.11–7.17 (2H,
m), 7.22 (1H, d, J = 2.5 Hz), 7.56 (1H, d, J = 8.3 Hz),
7.80–7.82 (2H, m), 7.98 (1H, d, J = 8.3 Hz), 8.07 (1H, d,
J = 8.8 Hz), 8.47 (1H, s), 9.25 (4H, br s); MS (FAB) m/z
569 [M+H]+; Anal. Calcd for C28H32N4O7SÆHClÆH2O:
C, 53.97; H, 5.66; N, 8.99; Cl, 5.69; S, 5.15. Found: C,
54.17; H, 5.73; N, 8.72; Cl, 5.61; S, 5.02.
7.2.2. In vitro plasma clotting time assay. PT was mea-
sured using a KC10A coagulometer (Amelung Co., Leh-
bringsweg, Germany). Fifty microliters of pooled citrated
mouse plasma was incubated for 1 min at 37 °C with
50 lL of diluted compound, followed by the addition of
50 lL of PT reagent (Hemoliance Brain Thromboplastin,
Instrumentation Laboratory Company, Lexington, MA,
USA) to initiate clot formation. The concentration
required to double clotting time (CT2) was estimated from
each individual dose–response curve.
7.2.3. Ex vivo anticoagulant assays in mice. Male ICR
mice weighing 20–35 g were fasted overnight. Inhibitors
were suspended in a 0.5% methylcellulose solution and
administered orally to the mice at 100 mg/kg using a
gastric tube. At a predetermined time after oral admin-
istration of the inhibitor, blood (0.9 mL) was collected
from the abdominal vena cava into syringes containing
0.1 mL of 3.8% citrate. Platelet poor plasma was then
prepared by centrifugation to measure PT. All data were
expressed as relative values compared with the vehicle
group.
7.1.35. [(((E)-3-{3-[Amino(imino)methyl]phenyl}prop-2-
en-1-yl){1-[(2,5-dimethoxyphenyl)sulfonyl]piperidin-4-yl}-
amino)sulfonyl]acetic acid hydrochloride (38). The title
compound was obtained as a colorless amorphous pow-
der from 360 mg (0.558 mmol) of compound 34 and
1.2 mL of 1 N aqueous NaOH solution in a manner iden-
tical to that described above for compound 36 (233 mg,
1
68%): H NMR (DMSO-d6) d 1.67–1.85 (4H, m), 2.59–
2.67 (2H, m), 3.69–3.76 (6H, m), 3.81 (3H, s), 4.03 (2H,
d, J = 5.8 Hz), 4.23 (2H, s), 6.42 (1H, dt, J = 16.2 and
5.8 Hz), 6.66 (1H, d, J = 16.2 Hz), 7.19–7.22 (3H, m),
7.59 (1H, t, J = 7.8 Hz), 7.70 (1H, d, J = 7.8 Hz), 7.78
(1H, d, J = 7.8 Hz), 7.90 (1H, s), 9.23 (2H, s), 9.42 (2H,
s), 13.33 (1H, br s); MS (FAB) m/z 581 [M+H]+; Anal.
Calcd for C25H32N4O8S2Æ1.1HClÆ0.7 H2O: C, 47.41; H,
5.49; N, 8.85; Cl, 6.16; S, 10.13. Found: C, 47.70; H,
5.55; N, 8.76; Cl, 5.95; S, 9.72.
7.3. Molecular modeling studies
Docking of the inhibitor into the active site of fXa
was performed using the GOLD program32 (version
2.1) as follows. The X-ray crystal structure of fXa was
retrieved from the Brookhaven Protein Data Bank

T. Ishihara et al. / Bioorg. Med. Chem. 15 (2007) 4175–4192
4191
(http://www.rcsb.org/pdb, PDB Accession code: 1FAX)28
and used as the target for a docking. The structure of the
ligand was constructed using Sybyl 6.8 and energetically
minimized with molecular mechanics using a Tripos
force field.33 The docking run was carried out using
standard default settings with a population size of 100,
a maximum number of 100,000 operations, and a muta-
tion crossover rate of 95. The fitness function that was
implemented in GOLD basically consisted of H-bond-
ing, complex energy, and ligand internal energy terms.
Based on the GOLD fitness score, the bound conforma-
tion with the highest fitness score was considered to be
the best bound conformation. The X-ray crystal struc-
ture of thrombin was also taken from the Brookhaven
Protein Data Bank (PDB Accession code: 1KTT),16b
and the coordinates of the thrombin were transferred
to those of fXa (PDB Accession code: 1FAX) through
superposition of the conserved residues of thrombin
and fXa.
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Acknowledgments
The authors express our gratitude to Dr. Junya Ohmori
for helpful support in the preparation of the manuscript,
and we are also grateful to the staff of the Division of
Analytical Science Laboratories for the elemental analy-
sis and spectral measurements.
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