Synthesis and biological activity of novel 1,4-diazepane derivatives as factor Xa inhibitor with potent anticoagulant and antithrombotic activity

Article abstract of DOI:10.1016/j.bmc.2004.02.022

Factor Xa (fXa) is a serine protease involved in the coagulation cascade, which has received great interest as a potential target for the development of new antithrombotic drugs. Herein we report a novel series of fXa inhibitors in which the 1,4-diazepane moiety was designed to interact with the S4 aryl-binding domain of the fXa active site. Compound 13 (YM-96765) showed potent fXa inhibitory activity (IC₅₀=6.8nM) and effective antithrombotic activity without prolonging bleeding time.

Full text of DOI:10.1016/j.bmc.2004.02.022

Bioorganic & Medicinal Chemistry 12 (2004) 2179–2191
Synthesis and biological activity of novel 1,4-diazepane derivatives
as factor Xa inhibitor with potent anticoagulant and antithrombotic
activity
Hiroyuki Koshio,* Fukushi Hirayama, Tsukasa Ishihara, Yuta Taniuchi, Kazuo Sato,
Yumiko Sakai-Moritani, Seiji Kaku, Tomihisa Kawasaki, Yuzo Matsumoto,
Shuichi Sakamoto and Shin-ichi Tsukamoto
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
Received 1 December 2003; revised 19 February 2004; accepted 19 February 2004
Abstract—Factor Xa (fXa) is a serine protease involved in the coagulation cascade, which has received great interest as a potential
target for the development of new antithrombotic drugs. Herein we report a novel series of fXa inhibitors in which the 1,4-diazepane
moiety was designed to interact with the S4 aryl-binding domain of the fXa active site. Compound 13 (YM-96765) showed potent
fXa inhibitory activity (IC50 ¼ 6.8 nM) and effective antithrombotic activity without prolonging bleeding time.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
We have reported in previous publications the discovery
of potent and selective fXa inhibitors YM-608284 and
YM-169964,5 which contain the naphthamidine moiety
for the S1 part and 1-acetoimidoyl-4-piperidyloxy moiety
for the S4 part (Fig. 1). In these studies we have presented
the following structure–activity relationships (SAR):
Warfarin1 has been widely used for many years in
anticoagulant therapy. It exerts its potent anticoagulant
effect by inhibiting the biosynthesis of a vitamin K-
dependent coagulation factor. Its indirect mechanism
results in some disadvantages, such as difficulty in the
control of anticoagulant activity and the adverse effect
of bleeding. Therefore, novel oral anticoagulant agents
are required, which have new mechanisms of action and
are much safer and easier to use than warfarin.
(1) The naphthamidine moiety deeply occupies the S1
pocket and the amidine group makes a salt bridge
to Asp-189. Replacement of naphthylamidine with
benzamidine derivatives results in a minor loss of
fXa potency.
Factor Xa (fXa) is a trypsin-like serine protease that
forms a prothrombinase complex with factor Va, Ca2þ
and phospholipid to produce thrombin. This key
enzyme is at the convergent point of the intrinsic and
extrinsic coagulation pathway. This process involves
signal amplification, with one molecule of fXa activating
many molecules of prothrombin to thrombin.2 Inhibi-
tion of fXa may therefore be more effective than the
inhibition of thrombin itself. Moreover, the risk of
bleeding is expected to decrease because fXa inhibitors
specifically affect coagulation but not platelet function.3
(2) Molecular modeling studies indicate that the acetic
acid moiety is positioned outside the enzyme pocket
O
O
CO2H
Me
S
N
H2N
N
N
NH
NH
NH
O
YM-60828
CO2H
Me
N
H2N
O
NH
O
Keywords: Antithrombotics; Anticoagulants; Enzyme inhibitors; Fac-
tor Xa.
YM-169964
Figure 1. YM-60828 and YM-169964.
* Corresponding author. Tel.: +81-29-854-1548; fax: +81-29-852-5387;
e-mail: koshio@yamanouchi.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.022

2180
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
Table 1. SAR from the P4 moiety modification
ation of 3 provided a complex mixture. Thus biphenyl
derivative 8a was obtained by alkylation of 4, which was
prepared by mesylation of 40-amino-1,10-biphenyl-4-
carbonitrile 3, with 2-bromomethyl-7-cyanonaphthal-
ene.6 The syntheses of the amidine derivatives are shown
in Scheme 2. Treatment of the intermediates 8a–g under
Pinner conditions (HCl/EtOH) afforded the imidates,
which were immediately reacted with excess ammonium
acetate to provide the corresponding amidine derivatives
9a–g. The amidine derivatives 9c, d and f were converted
to bis-amidines 10c, d and f by reaction with ethyl ace-
timidate and triethylamine. Hydrolysis of 9f, g and 10f
under acidic conditions provided the carboxyl deriva-
tives 11, 12 and 13.
O
Me
S
O
N
H2N
NH
R
Compd
R
IC50 (nM)a
Me
NH
1b
3.8
N
O
2b
127
NH
O
4-Alkylated 1,4-diazepane derivatives 16a–c were syn-
thesized as depicted in Scheme 3. Following the removal
of the tert-butoxycarbonyl (Boc) protecting group from
8f under acidic conditions, the resulting 1,4-diazepane
derivative was converted to the 4-substituted 1,4-diaze-
pane derivatives 14a–c by reductive alkylation with
several alkylaldehydes. Compounds 14a–c were also
converted to the amidine derivatives 16a–c in an anal-
ogous procedure to that described above.
9a
84
62
NH2
NH
N
N
N
9b
NH2
NH
10c
10d
121
3. Result and discussion
N
Me
NH
The IC50 value for the inhibition of fXa enzymatic
activity was determined for all the compounds prepared.
The CT2 values for the prothrombin time (PT) were also
determined for selected inhibitors, as an indicator of
in vitro anticoagulant activity. CT2 value was defined
as the concentration required to double clotting time.
Moreover, oral anticoagulant activities were also eval-
uated by prolongation of PT subsequent to oral
administration in mice.
Me
NH
7.9
N
a Human purified enzyme were used. IC50 values represent the average
of three determinations with the average standard error of the mean
<10%.
b Taken from Ref. 4.
Table 1 illustrates the effect of varying the S4 ligand on
in vitro biological activity. Replacing the 1-acetoimi-
doyl-4-piperidyloxy group of compound 1 for a P4 part
with a 4-amidinophenyl group or a 4-amidinopiperidine-
1-yl group conferred a significant decrease in the fXa
inhibitory activity (1 vs 9a and 9b). An additional two-
fold decrease in activity was noted with the 4-acetoimi-
doylpiperadine-1-yl analogue (10c) compared with 9a
and 9b. However, compound 10d, that expanded the
piperazine ring of 10c, substantially improved the
inhibitory activity and afforded inhibitors with similar
potency to 1.
and extends into the solvent. Modification of this
moiety affords no significant loss of fXa inhibition.
However few SAR around the P4 part have been
reported except the observation that removal of the
acetimidoyl moiety from the piperidine ring results in a
decrease in the activity (Table 1, 1 vs 2). Therefore, we
concentrated our investigations on modification of this
section and herein describe a novel series of inhibitors,
which contain a 1,4-diazepane moiety for the P4 part.
2. Chemistry
Potent fXa inhibitor 10d was further evaluated for both
its in vitro anticoagulant activity and oral anticoagulant
activity. In vitro anticoagulant activity of 10d was
comparable to that of YM-60828, however it showed
poor oral anticoagulant activity (Table 2). Previous
studies on YM-60828 have demonstrated that the pres-
ence of a carboxyl group, particularly an acetic acid
moiety gave potent oral anticoagulant activity in mice.4
In view of the effect of the carboxyl group, a sulfonyl
acetic acid derivative 13 was synthesized, which as pre-
The syntheses of the intermediates of naphthoamidine
derivatives 8a–g are illustrated in Scheme 1. Treatment
of 5 with various amines provided the 4-substituted
nitrobenzenes 6b–g. The cyanonaphthalene intermedi-
ates 8b–g were synthesized by reduction of the nitro
group, reductive alkylation of 2-cyanonaphthalene-7-
carboxyaldehyde4 and acylation with several sulfonyl
chlorides. However, the corresponding reductive alkyl-

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
2181
R1
N
O2N
O2N
H
a
b, c
d
N
NC
NC
F
R
R
R
6b-g
7b-g
8b-g
5
N
N
N
N
N
R1=SO2Me
8b R=
8c R=
7b R=
6b R=
CN
CONH2
CONH2
N
N
R1=SO2Me
R1=SO2Me
N
N
7c R=
7d R=
6c R=
6d R=
NBOC
NBOC
NBOC
NBOC
8d R=
NBOC
NBOC
N
N
N
N
R1=SO2Me
N
N
8e R=
8f R=
7e R=
7g R=
6e R=
6g R=
R1=SO2CH2CO2Et
NBOC
N
N
N
N
N
N
R1=SO2CH2CO2Et
8g R=
N
O
O
Me
Me
S
S
O
HN
O
H2N
N
e
f
NC
CN
CN
CN
3
4
8a
Scheme 1. Reagents and conditions: (a) amine, K2CO3, DMF; (b) H2, 10% Pd–C, EtOH; (c) 7-formyl-2-naphthonitrile, NaB(OAc)3H, 1,2-
dichloroethane, AcOH; (d) MsCl for 8b–8e, pyridine, ClO2SCH2CO2Et for 8f and 8g, pyridine; (e) MsCl, pyridine; (f) 2-bromomethyl-7-naph-
thonitrile, K2CO3, DMF.
dicted exhibited improved oral anticoagulant activity
without affecting its fXa inhibitory activity. As a result,
further modification of the acetoimidoyl moiety of
compound 13 was undertaken. Surprisingly compound
11, in which the acetoimidoyl moiety was absent,
retained fXa inhibitory activity similar to that of com-
pound 13. This result suggested that 1,4-diazepane
derivatives showed different SAR compared with that of
YM-60828 derivatives, which contained a 1-acetoimi-
doyl-4-piperidyloxy moiety. Furthermore, replacing the
acetoimidoyl group with a methyl group provided
compound 16a, which showed comparable activity to
compound 11. Introduction of more bulky substituents
such as the butyl group (16b) or benzyl group (16c),
resulted in a minor loss of fXa inhibitory potency. 4-
Pyridyl substitution at the same position was also
acceptable (12). To evaluate the importance of the bas-
icity of the 1,4-diazepane in fXa inhibitory activity, the
corresponding azepane derivative 9e was prepared.
Compound 9e caused a significant reduction in fXa
inhibitory activity, which suggested that the basicity
of 1,4-diazepane might be important to activity.
same conditions. Compound 13, which exhibited good
oral anticoagulant activity in mice, equivalent to that of
compound 16a, also showed prolongation PT in human
plasma (CT2, PT ¼ 0.12 lM) equally compared with that
of in mice plasma. Compound 13 was further evaluated
enzymatic selectivity and pharmacokinetic profiles.
Compound 13 showed high selectivity for fXa over
trypsin (>100-fold). In particular, it showed exceptional
selectivity against the related serine protease thrombin
(>10,000-fold). Compound 13 was evaluated by a
pharmacokinetic study following oral administration in
dogs and cynomolgus monkeys, showing the absolute
oral bioavailability at 58% and 14.7% (Table 3).
The oral antithrombotic effect in rats thromboplastin-
induced venous thrombosis model and the prolongation
of bleeding time were examined with warfarin and the
hydrogen bromide salt of compound 13 (Fig. 2). Com-
pound 13 showed oral antithrombotic activity in a dose
dependent manner. No difference between the anti-
thrombotic effect (ID50) and the prolongation of bleed-
ing time (ED2, which was the dose that caused a twofold
prolongation of bleeding time in the control group) was
observed with the typical antithrombotic drug warfarin
(ED2/ID50 ¼ 0.71). However, the antithrombotic effect
of compound 13 was notably separate from its effect on
bleeding time (ED2/ID50 ¼ 27). The clinical administra-
tion of agents such as warfarin are strictly controlled to
The oral anticoagulant activity in mice of the inhibitors
(11, 12, 16a), which were nearly equal to 13 in in vitro
anticoagulant activity, were evaluated. Compound 16a
prolonged PT by more than twofold at 0.5 and 1.0 h. In
contrast, compound 12 showed poor activity under the

2182
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
R1
N
R1
a, b
H2N
N
NC
NH
R
R
8a-g
9a-g, 11, 12
c
9a R1=SO2Me
9f R1=SO2CH2CO2Et
11 R1=SO2CH2CO2H
N
N
R=
R=
R=
NH
NH
NH2
NH
c
9b R1=SO2Me
9g R1=SO2CH2CO2Et
12 R1=SO2CH2CO2H
N
N
N
R=
R=
R=
N
N
N
N
NH2
NH
9c R1=SO2Me
N
R=
NH
9d R1=SO2Me
N
R=
NH
9e R1=SO2Me
N
R=
R1
d
N
H2N
9c, d, f
NH
R
10c, d, f, 13
10c R1=SO2Me
N
R=
N
Me
NH
10d R1=SO2Me
R=
N
Me
N
NH
c
10f R1=SO2CH2CO2Et
13 R1=SO2CH2CO2H
Me
NH
Me
NH
N
N
R=
R=
N
N
Scheme 2. Reagents and conditions: (a) HCl, EtOH; (b) NH4OAc, EtOH; (c) cHCl; (d) ethyl acetimidate hydrochloride, Et3N, EtOH.
prevent hemorrhage. These data suggest that compound
13 can exert its antithrombotic effect without signifi-
cantly prolonging bleeding time and is therefore a much
safer agent than warfarin.
based on compound YM-96765 will be reported in
future publications.
4. Conclusion
5. Experimental
5.1. Chemistry
We have designed and synthesized a series of fXa
inhibitors based on a 1,4-diazepane template as the P4
moiety. Among these compounds, compound 13 (YM-
96765) showed nanomolar potency in fXa inhibitory
activity and had excellent oral anticoagulant activity in
mice and good pharmacokinetic profiles in dogs and
cynomolgus monkeys. Furthermore, this compound
showed effective oral antithrombotic activity in rats
without prolonging bleeding time. Further optimization
1H NMR spectra were measured with a JEOL EX90,
EX400 or GX500 spectrometer; chemical shifts are
expressed in d units using tetramethylsilane as the
standard (in NMR description, s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, m ¼ multiplet and br ¼ broad
peak). Mass spectra were recorded with a Hitachi M-80
or JEOL JMS-DX300 spectrometer. ODS column

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
CO2Et CO2Et
2183
O
O
S
N
S
N
O
O
a, b
c, d
NC
NC
N
N
NBOC
NR
14a R=Me
14b R=Bu
14c R=Bn
8f
CO2Et
CO2H
O
O
S
S
O
O
e
N
N
H2N
H2N
NH
NH
N
N
NR
NR
15a R=Me
15b R=Bu
15c R=Bn
16a R=Me
16b R=Bu
16c R=Bn
Scheme 3. Reagents and conditions: (a) TFA, CH2Cl2; (b) HCHO for 14a or EtCHO for 14b or PhCHO for 14c, NaB(OAc)3H, 1,2-dichloroethane,
AcOH; (c) HCl, EtOH; (d) NH4OAc, EtOH; (e) cHCl.
chromatography was performed on YMC gel (ODS-A
120-230/70).
5.5. tert-Butyl 4-(4-nitrophenyl)-1,4-diazepane-1-carboxy-
late (6d)
To a stirred solution of tert-butyl 1,4-diazepane-1-car-
boxylate (1.8 g, 8.99 mmol) in DMF (10 mL) was added
5 (1.62 g, 11.5 mmol), potassium carbonate (1.84 g,
13.3 mmol), and stirring continued at 90 °C for 13 h.
After the reaction mixture was cooled, the reaction
mixture was diluted with ethyl acetate and washed with
H2O and saturated saline. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The resulted
residue was chromatographed on silica gel eluting with
chloroform/MeOH (100/1) to give 6d (2.4 g, 83% yield)
5.2. N-(40-Cyanobiphenyl-4-yl)methanesulfonamide (4)
To a stirred solution of 40-amino-1,10-biphenyl-4-car-
bonitrile (230 mg, 1.18 mmol) in pyridine (12 mL) at
ambient temperature was added methanesulfonyl chlor-
ide (162 mg, 1.42 mmol). After 17 h the reaction mixture
was concentrated in vacuo. The resulting residues was
chromatographed on silica gel eluting with hexane/ethyl
acetate (1/2) to give 4 (320 mg, quant yield) as a col-
1
1
ourless amorphous powder: H NMR (CDCl3) d 3.11
as a yellow amorphous powder: H NMR (CDCl3) d
(3H, s), 6.64 (1H, s), 7.33 (2H, d, J ¼ 8:4 Hz), 7.59 (2H,
d, J ¼ 8:8 Hz), 7.65 (2H, d, J ¼ 8:8 Hz), 7.73 (2H, d,
J ¼ 8:4 Hz); FABMS m=e (M+H)þ 273.
1.40 (9H, s), 1.85–2.10 (2H, m), 3.19–3.35 (2H, m), 3.58–
3.72 (6H, m), 6.71 (2H, d, J ¼ 9:5 Hz), 8.12 (2H, d,
J ¼ 9:5 Hz); FABMS m=e (M+H)þ 321.
5.3. 1-(4-Nitrophenyl)piperidine-4-carboxamide (6b)
5.6. 1-(4-Nitrophenyl)azepane (6e)
Compound 6b was synthesized from 5 and pipridine-4-
carboxamide according to the same procedure as that
for 6d. Compound 6b was obtained as a yellow powder
(87% yield): 1H NMR (CDCl3) d 1.82–1.93 (2H, m),
1.96–2.06 (2H, m), 2.39–2.51 (1H, m), 2.99–3.09 (2H,
m), 3.94–4.03 (2H, m), 6.82 (2H, d, J ¼ 9:4 Hz), 8.11
(2H, d, J ¼ 9:4 Hz); FABMS m=e (M+H)þ 250.
Compound 6e was synthesized from 5 and hexamethyl-
eneimine according to the same procedure as that for 6d.
Compound 6e was obtained as a yellow powder (50%
1
yield): H NMR (CDCl3) d 1.49–1.56 (4H, m), 1.72–
1.81 (4H, m), 3.35–3.45 (4H, m), 6.61 (2H, d, J ¼
9:3 Hz), 8.10 (2H, d, J ¼ 9:3 Hz); FABMS m=e (M+H)þ
221.
5.4. tert-Butyl 4-(4-nitrophenyl)piperazine-1-carboxylate
(6c)
5.7. 1-(4-Nitrophenyl)-4-(pyridin-4-yl)-1,4-diazepine (6g)
Compound 6c was synthesized from 5 and tert-butyl
piperazine-1-carboxylate according to the same proce-
dure as that for 6d. Compound 6c was obtained as a
Compound 6g was synthesized from 5 and 1-(pyridyl-4-
yl)-1,4-diazepine according to the same procedure as
that for 6d. Compound 6g was obtained as a yellow
powder (54% yield): 1H NMR (CDCl3) d 2.02–2.21 (2H,
m), 3.42–3.70 (8H, m), 6.55 (2H, d, J ¼ 3:2 Hz), 6.69
(2H, d, J ¼ 9:5 Hz), 8.14 (2H, d, J ¼ 9:5 Hz), 8.26 (2H,
d, J ¼ 3:2 Hz); FABMS m=e (M+H)þ 299.
1
yellow powder (51% yield): H NMR (CDCl3) d 1.19
(9H, s), 3.34–3.46 (4H, m), 3.55–3.68 (4H, m), 6.80 (2H,
d, J ¼ 9:5 Hz), 8.15 (2H, d, J ¼ 9:5 Hz); FABMS m=e
(M)þ 307.

2184
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
Table 2. SAR from the P4 moiety modification
R1
O
S
N
O
H2N
NH
R
Compd
R
R1
IC50 (nM)a
CT2 (lM)b
PT/control PTd
1.0 h
PTc
0.5 h
1.38
2.0 h
1.22
Me
NH
N
N
N
10d
Me
7.9
0.37
1.27
N
Me
NH
13
CH2CO2H
CH2CO2H
CH2CO2H
6.8
6.2
6.5
0.36
0.67
0.76
2.25
1.77
2.21
2.07
2.07
2.04
1.68
1.62
1.75
N
11
NH
N
N
N
N
16a
N
N
Me
16b
16c
CH2CO2H
CH2CO2H
10
1.00
NTe
1.84
NTe
NTe
NTe
NTe
NTe
nBu
Bn
18
N
N
12
9e
CH2CO2H
Me
8.6
0.31
1.21
1.36
1.24
N
N
141
NTe
0.21
NTe
2.55
NTe
2.25
NTe
1.74
YM-60828
6.0
a Refer to Table 1.
b Values represent the concentration required to double clotting time and represent the average of four determination with the average standard error
of the mean <10%.
c Prothrombin time using mice plasma.
d The relative prothrombin time compared with that measured using normal mice plasma at 0.5, 1.0 and 2.0 h after oral administration (100 mg/kg,
n ¼ 3).
e Not tested.
Table 3. Oral pharmacokinetic properties of compound 13
Spaces
Iv t1=2 (min)
CLtot (mL/min/kg)
Oral Cmax (ng/mL)
F (%)
Dogsa
Cynomolgus monkeyb
62
120
11.5
4.0
336
674
58
14.7
a Compound was administered at a dose of 10 mg/kg, po (n ¼ 3) and 0.3 mg/kg, iv (n ¼ 3).
b Compound was administered at a dose of 10 mg/kg, po (n ¼ 3) and 1 mg/kg, iv (n ¼ 3).
5.8. 1-(4-{[(7-Cyano-2-naphthyl)methyl]amino}phenyl)-
piperidine-4-carboxamide (7b)
m), 7.85–8.05 (3H, m), 8.20 (1H, s); FABMS m=e
(M+H)þ 384.
Compound 7b was synthesized from 6b according to the
same procedure as that for 7d. Compound 7b was
obtained as a white amorphous powder (18% yield): H
5.9. tert-Butyl 4-(4-{[(7-cyano-2-naphthyl)methyl]-
amino}phenyl)piperazine-1-carboxylate (7c)
1
NMR (DMSO-d6) d 1.63–1.82 (4H, m), 2.32–2.40 (1H,
m), 3.12–3.19 (2H, m), 3.33–3.56 (2H, m), 4.44 (2H, s),
6.48–6.68 (2H, m), 6.83–6.96 (2H, m), 7.61–7.78 (2H,
Compound 7c was synthesized from 6c according to the
same procedure as that for 7d. Compound 7c was

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
2185
30
100
80
60
40
20
0
Antithrombotic effect
Bleeding time
**
**
**
13 (solid)
warfarin (dot)
20
10
0
*
*
*
*
Mean SEM, n=6
*
*P<0.05, ** P < 0.01
vs the control group
by Dunnett's multiple comparison test
or Steel's test
0.1
1
10
Dose (mg/kg)
100
1000
Figure 2. Antithrombotic activity and bleeding time of 13 (HBr salt) and warfarin after oral administration in rats (n ¼ 6).
1
obtained as a white amorphous powder (25% yield): H
NMR (CDCl3) d 1.54 (9H, s), 2.82–3.15 (4H, m), 3.59
(4H, t, J ¼ 4:9 Hz), 4.89 (2H, s), 6.50–6.71 (2H, m),
6.83–7.01 (2H, m), 7.56–7.80 (2H, m), 7.84–8.03 (3H,
m), 8.25 (1H, s); FABMS m=e (M+H)þ 443.
5.12. 7-[({4-[4-(Pyridin-4-yl)-1,4-diazepan-1-yl]phenyl}-
amino)methyl]-2-naphthonitrile (7g)
Compound 7g was synthesized from 6g according to the
same procedure as that for 7d. Compound 7g was
obtained as a white amorphous powder (62% yield): H
1
NMR (CDCl3) d 2.05–2.16 (2H, m), 3.35–3.48 (4H, m),
3.15–3.58 (2H, m), 3.63–3.68 (2H, m), 4.48 (2H, s), 6.52–
6.58 (2H, m), 6.79 (1H, d, J ¼ 8:9 Hz), 7.35 (1H, d,
J ¼ 8:9 Hz), 7.56–7.60 (1H, m), 7.63–7.69 (2H, m), 7.86–
7.90 (2H, m), 8.20–8.27 (2H, m), 8.70 (1H, s); FABMS
m=e (M+H)þ 434.
5.10. tert-Butyl 4-(4-{[(7-cyano-2-naphthyl)methyl]-ami-
no}phenyl)-1,4-diazepane-1-carboxylate (7d)
To the solution of 6d (14.12 g, 439 mmol) in EtOH
(44 mL) was added 10% Pd–C powder (0.7 g) and stirred
in hydrogen atmosphere at ambient temperature for
16 h. The reaction mixture was filtrated through a pad
of Celite and concentrated in vacuo to give intermediate
aniline compound. To a stirred solution of crude aniline
compound and 7-formyl-2-naphthonitrile (9.55 g,
52 mmol) in 1,2-dichloromethane (440 mL) and AcOH
(26 mL) at ambient temperature was added sodium tri-
acetoxyborohydride (22.3 g, 105 mmol). After 5 h, the
reaction mixture was washed with 10% potassium car-
bonate solution and brine. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The resulted
residue was chromatographed on silica gel eluting with
ethyl acetate/hexane (1/3) to give 7d (15.8 g, 66%) as a
5.13. N-(40-Cyano-1,10-biphenyl-4-yl)-N-[(7-cyano-
2-naphthyl)methyl]methanesulfonamide (8a)
To a stirred solution of 4 (310 mg, 1.14 mmol) in DMF
(11 mL) at ambient temperature was added 7-bromo-
methyl-2-naphthonitrile (281 mg, 1.14 mmol) and
potassium carbonate (318 mg, 2.3 mmol). After 24 h the
reaction mixture was diluted with ethyl acetate and
washed with 10% aqueous potassium carbonate. The
organic layer was dried over MgSO4 and concentrated
in vacuo. The resulting residues was chromatographed
on silica gel eluting with hexane/ethyl acetate (1/1) to
give 8a (570 mg, quant yield) as a colourless amorphous
1
yellow amorphous powder: H NMR (CDCl3) d 1.42
1
(9H, s), 1.80–2.09 (2H, m), 3.07–3.55 (8H, m), 4.47 (2H,
s), 6.61 (4H, s), 7.50–7.71 (2H, m), 7.82–7.94 (3H, m),
8.17 (1H, s); FABMS m=e (M+H)þ 457.
powder: H NMR (CDCl3) d 3.07 (3H, s), 5.01 (2H, s),
7.41 (2H, d, J ¼ 8:8 Hz), 7.52 (2H, d, J ¼ 8:4 Hz), 7.56–
7.61 (3H, m), 7.67–7.73 (4H, m), 7.84–7.89 (4H, m), 8.13
(1H, s); FABMS m=e (M+H)þ 438.
5.11. 7-({[(4-Azepan-1-yl)phenyl]amino}methyl)-2-naph-
thonitrile (7e)
5.14. N-[(7-Cyano-2-naphthyl)methyl]-N-[4-(4-cyano-
piperidin-1-yl)phenyl]methanesulfonamide (8b)
Compound 7e was synthesized from 6e according to the
same procedure as that for 7d. Compound 7e was
obtained as a white amorphous powder (quant yield):
1H NMR (CDCl3) d 1.19–1.58 (4H, m), 1.70–1.79 (4H,
m), 3.35–3.39 (4H, m), 4.66 (2H, s), 6.59 (2H, d,
J ¼ 9:0 Hz), 6.79 (2H, d, J ¼ 9:0 Hz), 7.56 (1H, dd,
J ¼ 1:5, 6.4 Hz), 7.67 (1H, dd, J ¼ 1:5, 6.4 Hz), 7.82–
7.92 (3H, m), 8.17 (1H, s); FABMS m=e (M+H)þ 355.
Compound 8b was synthesized from 7b and meth-
anesulfonyl chloride according to the same procedure as
that for 8d. Compound 8b was obtained as a white
1
amorphous powder (90% yield): H NMR (CDCl3) d
1.89–2.06 (4H, m), 2.75–2.81 (1H, m), 2.98 (2H, s), 3.04–
3.13 (2H, m), 3.33–3.42 (2H, m), 4.97 (2H, s), 6.79 (2H,
d, J ¼ 9:2 Hz), 7.12 (2H, d, J ¼ 9:2 Hz), 7.58 (1H, dd,
J ¼ 1:5, 8.4 Hz), 7.66 (1H, s), 7.67 (1H, dd, J ¼ 1:5,

2186
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
8.2 Hz), 7.80–7.88 (2H, m), 8.13 (1H, s); FABMS m=e
(2H, s), 4.35 (2H, q, J ¼ 7:3 Hz), 5.02 (2H, s), 6.55 (2H,
d, J ¼ 9:0 Hz), 7.20 (2H, d, J ¼ 9:0 Hz), 7.50–7.92 (5H,
m), 8.13 (1H, s); FABMS m=e (M+H)þ 607.
(M+H)þ 444.
5.15. tert-Butyl 4-{4-[[(7-cyano-2-naphthyl)methyl](meth-
ylsulfonyl)amino]phenyl}piperazine-1-carboxylate (8c)
5.19. Ethyl ({[(7-cyano-2-naphthyl)methyl]{4-[4-(pyridin-
4-yl)-1,4-diazepan-1-yl]phenyl}amino}sulfonyl)acetate
(8g)
Compound 8c was synthesized from 7c and meth-
anesulfonyl chloride according to the same procedure as
that for 8d. Compound 8c was obtained as a white
Compound 8g was synthesized from 7g and ethyl
(chlorosulfonyl)acetate according to the same procedure
as that for 8d. Compound 8g was obtained as a white
1
amorphous powder (65% yield): H NMR (CDCl3) d
1.59 (9H, s), 2.80–3.02 (4H, m), 3.16 (3H, s), 3.41–3.59
(2H, s), 6.82–7.05 (2H, m), 7.55–7.78 (2H, m), 8.24 (1H,
s); FABMS m=e (M+H)þ 521.
1
amorphous powder (72% yield): H NMR (CDCl3) d
1.92–2.25 (2H, m), 2.98 (3H, s), 3.30–3.58 (8H, m), 4.95
(2H, s), 6.40–6.68 (4H, m), 7.08 (2H, d, J ¼ 9:0 Hz),
7.56–7.88 (4H, m), 8.07–8.30 (4H, m); FABMS m=e
(M+H)þ 512.
5.16. tert-Butyl 4-{4-[[(7-cyano-2-naphthyl)methyl](meth-
ylsulfonyl)amino]phenyl}-1,4-diazepane-1-carboxylate
(8d)
5.20. 40-[({7-[Amino(imino)methyl]-2-naphthyl}methyl)-
(methylsulfonyl)amino] biphenyl-4-carboximidamide (9a)
To a stirred solution of 7d (400 mg, 0.88 mmol) in
1,2-dichloroethane (5 mL) was added pyridine (208 mg,
2.63 mmol) and methanesulfonyl chloride (201 mg,
1.76 mmol) and stirred at ambient temperature. After
12 h, the reaction mixture was diluted with chloroform
and washed with saturated sodium hydrogencarbonate,
water, aqueous 10% citric acid and water. The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The resulting residue was recrystallized from MeOH to
Compound 9a was synthesized from 8a according to the
same procedure as that for 9d. Compound 9a was
1
obtained as a white amorphous powder (67% yield): H
NMR (DMSO-d6) d 3.22 (3H, s), 5.18 (2H, s), 7.61 (2H,
d, J ¼ 8:8 Hz), 7.72 (1H, dd, J ¼ 1:5, 8.3 Hz), 7.76 (2H,
d, J ¼ 8:8 Hz), 7.82 (1H, dd, J ¼ 1:5, 8.3 Hz), 7.86–7.94
(4H, m), 7.98 (1H, s), 8.03 (1H, d, J ¼ 8:8 Hz), 8.09 (1H,
d, J ¼ 8:8 Hz), 8.50 (1H, s), 9.29 (2H, br s), 9.37 (2H, br
s), 9.47 (2H, br s), 9.54 (2H, br s); FABMS m=e (M+H)þ
472. Anal. Calcd for C26H25N5O2SÆ2.0HClÆ1.5H2O: C,
54.64; H, 5.29; N, 12.25; S, 5.61; Cl, 12.41. Found: C,
54.63; H, 5.40; N, 12.25; S, 5.55; Cl, 12.67.
1
obtain 8d (342 mg, 73%) as a white powder: H NMR
(CDCl3) d 1.30 (4H, s), 1.38 (5H, s), 1.84–1.94 (2H, m),
2.97 (3H, s), 3.15–3.33 (2H, m), 3.44–3.56 (6H, m), 4.94
(2H, s), 6.55 (2H, d, J ¼ 9:2 Hz), 7.02–7.10 (2H, m), 7.58
(1H, dd, J ¼ 1:9, 8.4 Hz), 7.65–7.73 (2H, m), 7.84 (1H,
d, J ¼ 9:9 Hz), 7.88 (1H, d, J ¼ 9:9 Hz), 8.13 (1H, s);
FABMS m=e (M+H)þ 534.
5.21. 1-{4-[({7-[Amino(imino)methyl]-2-naphthyl}methyl)-
(methylsulfonyl)amino]phenyl}piperidine-4-carboximida-
mide (9b)
5.17. N-(4-Azepan-1-ylphenyl)-N-[(7-cyano-2-naphthyl)-
methyl]methanesulfonamide (8e)
Compound 9b was synthesized from 8b according to the
same procedure as that for 9d. Compound 9b was
obtained as a white amorphous powder (12% yield): H
Compound 8e was synthesized from 7e and meth-
anesulfonyl chloride according to the same procedure as
that for 8d. Compound 8e was obtained as a white
1
1
NMR (DMSO-d6) d 1.67–1.89 (4H, m), 2.53–2.64 (3H,
m), 3.33 (3H, s), 3.74–3.82 (2H, m), 5.00 (2H, s), 6.86 (2H,
d, J ¼ 9:2 Hz), 7.24 (2H, d, J ¼ 9:2 Hz), 7.66 (1H, dd,
J ¼ 1:5, 8.4 Hz), 7.80 (1H, dd, J ¼ 1:5, 8.4 Hz), 7.91 (1H,
s), 8.01 (1H, d, J ¼ 8:4 Hz), 8.10 (1H, d, J ¼ 8:4 Hz), 8.47
(1H, s), 8.92 (4H, br s); FABMS m=e (M+H)þ 479. Anal.
Calcd for C25H30N6O2SÆ3.0HClÆ1.3H2O: C, 49.11; H,
5.87; N, 13.75; S, 5.24; Cl, 17.40. Found: C, 49.52; H,
6.34; N, 13.87; S, 5.23; Cl, 17.36.
amorphous powder (62% yield): H NMR (CDCl3) d
1.43–1.54 (4H, m), 1.62–1.77 (4H, m), 2.98 (3H, s), 3.37
(4H, t, J ¼ 5:7 Hz), 4.94 (2H, s), 6.53 (2H, d,
J ¼ 9:3 Hz), 7.02 (2H, d, J ¼ 9:3 Hz), 7.57 (1H, dd,
J ¼ 1:5, 8.4 Hz), 7.67 (1H, s), 7.71 (1H, dd, J ¼ 1:5,
8.4 Hz), 7.82–7.89 (2H, m), 8.13 (1H, s); FABMS m=e
(M+H)þ 432.
5.18. tert-Butyl 4-(4-{[(7-cyano-2-naphthyl)methyl][(2-
ethoxy-2-oxoethyl)sulfonyl]amino}phenyl)-1,4-diazepane-
1-carboxylate (8f)
5.22. 7-({(Methylsulfonyl)[4-(piperazin-1-yl)phenyl]ami-
no}methyl)naphthalene-2-carboximidamide (9c)
Compound 8f was synthesized from 7d and ethyl
(chlorosulfonyl)acetate according to the same procedure
as that for 8d. Compound 8f was obtained as a white
Compound 9c was synthesized from 8c according to the
same procedure as that for 9d. Compound 9c was ob-
tained as a white amorphous powder (32% yield): H
NMR (DMSO-d6) d 3.11–3.19 (7H, m), 3.35–3.40 (4H,
m), 5.00 (2H, s), 6.88 (2H, d, J ¼ 8:5 Hz), 7.25 (2H, d,
1
1
amorphous powder (87% yield): H NMR (CDCl3) d
1.26 (3H, t, J ¼ 7:3 Hz), 1.31 (4H, s), 1.39 (5H, s), 1.75–
2.01 (2H, m), 3.13–3.31 (2H, m), 3.45–3.57 (6H, m), 4.04

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
2187
J ¼ 8:5 Hz), 7.68 (1H, d, J ¼ 8:5 Hz), 7.74 (1H, d,
5.26. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-pyridin-4-yl-1,4-diazepan-1-yl)-
phenyl]amino}sulfonyl)acetate (9g)
J ¼ 8:5 Hz), 7.99–8.09 (3H, m), 8.53 (1H, s), 9.50 (4H,
br s); FABMS m=e (M+H)þ 483.
Compound 9g was synthesized from 8g according to the
same procedure as that for 9d. Compound 9g was
obtained as a white amorphous powder (19% yield): H
5.23. 7-{[[4-(1,4-Diazepan-1-yl)phenyl](methylsulfon-
yl)amino]methyl}naphthalene-2-carboximidamide (9d)
1
NMR (DMSO-d6) d 1.27 (3H, t, J ¼ 6:8 Hz), 1.82–1.89
(2H, m), 3.42–3.46 (2H, m), 3.60–3.63 (4H, m), 3.83–
3.86 (2H, m), 4.24 (2H, q, J ¼ 6:8 Hz), 4.33 (2H, s), 4.98
(2H, s), 6.82 (2H, d, J ¼ 9:2 Hz), 7.03–7.10 (2H, m), 7.15
(2H, d, J ¼ 9:2 Hz), 7.60 (1H, dd, J ¼ 1:6, 8.8 Hz), 7.82
(1H, dd, J ¼ 1:6, 8.8 Hz), 7.89 (1H, s), 8.01 (1H, d,
J ¼ 8:8 Hz), 8.11 (1H, d, J ¼ 8:8 Hz), 8.12–8.16 (2H, m),
8.48 (1H, s), 9.31 (2H, s), 9.52 (2H, s), 13.68 (1H, br s);
FABMS m=e (M+H)þ 601.
HCl gas bubbled through a solution of 8d (333 mg,
0.62 mmol) in EtOH (10 mL) under )20 °C for 20 min.
The mixture was allowed to stir for 15 h at 5 °C, and
then concentrated in vacuo. To the crude imidate dis-
solved in EtOH (10 mL) at ambient temperature was
added ammonium acetate (480 mg, 6.22 mmol). The
reaction mixture was stirred at ambient temperature for
24 h and concentrated in vacuo. The resulted residue
was chromatographed on ODS-gel eluting with MeOH/
H2O (5/95). MeOH was removed in vacuo, and the
aqueous solution was lyophilized after being acidified
with 1 N HCl. Compound 9d (249 mg, 82%) was
obtained as a white amorphous powder: 1H NMR
(DMSO-d6) d 1.68–1.75 (2H, m), 2.57–2.65 (2H, m),
2.77–2.84 (2H, m), 3.07 (3H, s), 3.24–3.45 (4H, m), 4.96
(2H, s), 6.57 (2H, d, J ¼ 9:3 Hz), 7.14 (2H, d,
J ¼ 9:3 Hz), 7.90 (1H, s), 8.00 (1H, d, J ¼ 8:3 Hz), 8.09
(1H, d, J ¼ 8:3 Hz), 8.45 (1H, s); FABMS m=e (M+H)þ
452.
5.27. 7-{[[4-(4-Ethanimidoylpiperazin-1-yl)phenyl](meth-
ylsulfonyl)amino]methyl}naphthalene-2-carboximidamide
(10c)
Compound 10c was synthesized from 9c according to
the same procedure as that for 10d. Compound 10c was
1
obtained as a white amorphous powder (14% yield): H
NMR (DMSO-d6) d 2.33 (3H, s), 3.12 (3H, s), 3.24–3.38
(4H, m), 3.62–3.70 (2H, m), 3.73–3.80 (2H, m), 5.03 (2H,
s), 6.93 (2H, d, J ¼ 8:8 Hz), 7.30 (2H, d, J ¼ 8:8 Hz),
7.66 (1H, d, J ¼ 8:8 Hz), 7.86–7.93 (2H, m), 8.01 (1H, d,
J ¼ 8:8 Hz), 8.09 (1H, d, J ¼ 8:8 Hz), 8.55 (1H, s), 9.50
(2H, br s), 9.72 (2H, br s); FABMS m=e (M+H)þ 479.
Anal. Calcd for C25H30N6O2SÆ2.1HClÆ2.5H2O: C, 50.03;
H, 6.23; N, 14.00; S, 5.34; Cl, 12.40. Found: C, 50.33; H,
6.20; N, 14.09; S, 5.37; Cl, 12.48.
5.24. 7-({4-(Azepan-1-ylphenyl)(methylsulfonyl)-
amino}methyl)naphthalene-2-carboximidamide (9e)
Compound 9e was synthesized from 8e according to the
same procedure as that for 9d. Compound 9e was
1
obtained as a white amorphous powder (72% yield): H
NMR (DMSO-d6) d 1.39–1.48 (4H, m), 1.61–1.72 (4H,
m), 3.09 (3H, s), 3.36 (4H, t, J ¼ 5:9 Hz), 4.97 (2H, s),
6.62 (2H, br s), 7.12 (2H, d, J ¼ 7:8 Hz), 7.68 (1H, d,
J ¼ 8:8 Hz), 7.81 (1H, d, J ¼ 8:8 Hz), 7.91 (1H, s), 8.14
(1H, d, J ¼ 8:8 Hz), 8.09 (1H, d, J ¼ 8:8 Hz), 8.47 (1H,
s), 9.27 (2H, br s), 9.49 (2H, br s); FABMS m=e (M+H)þ
451. Anal. Calcd for C25H30N4O2SÆ1.9HClÆ1.0H2O: C,
55.82; H, 6.35; N, 10.42; S, 5.96; Cl, 12.52. Found: C,
55.56; H, 6.43; N, 10.41; S, 6.04; Cl, 12.48.
5.28. 7-{[[4-(4-Ethanimidoyl-1,4-diazepan-1-yl)-phenyl]-
(methylsulfonyl)amino]methyl}naphthalene-2-
carboximidamide (10d)
To a stirred solution of 9d (224 mg, 0.5 mmol) in EtOH
(10 mL) at ambient temperature was added ethyl acet-
imidate hydrochloride (650 mg, 3.3 mmol) and Et3N
(683 mg, 6.7 mmol). The mixture was allowed to stir for
24 h at ambient temperature, and then concentrated
in vacuo. The resulting residues was chromatographed
on ODS-gel eluting with MeOH/H2O (5/95). MeOH
was removed in vacuo, and the aqueous solution was
lyophilized after being acidified with 1 N HCl. Com-
pound 10d (169 mg, 54%) was obtained as a white
5.25. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(1,4-diazepan-1-yl)phenyl]amino}sulfonyl)-
acetate (9f)
Compound 9f was synthesized from 8f according to the
same procedure as that for 9d. Compound 9f was
1
1
amorphous powder: H NMR (DMSO-d6) d 1.77–1.87
obtained as a white amorphous powder (99% yield): H
(2H, m), 2.01 (1.8H, s), 2.24 (1.2H, s), 3.09 (3H, s), 3.44–
3.76 (8H, m), 4.98 (2H, s), 6.65–6.73 (2H, m), 7.16–7.23
(2H, m), 7.63–7.68 (1H, m), 7.78–7.84 (1H, m), 7.89–7.93
(1H, m), 7.99–8.04 (1H, m), 8.09 (1H, d, J ¼ 8:8 Hz),
8.49 (0.4H, s), 8.51 (0.6H, s), 8.61 (0.6H, s), 8.75 (0.4H,
s), 9.24–9.32 (3H, m), 9.51 (2H, s); FABMS m=e (M+H)þ
493. Anal. Calcd for C26H32N6O2SÆ2.2HClÆ2.7H2O: C,
50.25; H, 6.42; N, 13.52; S, 5.16; Cl, 12.55. Found: C,
50.50; H, 6.49; N, 13.59; S, 5.12; Cl, 12.63.
NMR (DMSO-d6) d 1.27 (3H, t, J ¼ 7:0 Hz), 2.01–2.07
(2H, m), 3.03–3.06 (2H, m), 3.11–3.15 (2H, m), 3.41 (2H,
t, J ¼ 6:0 Hz), 3.62–3.65 (2H, m), 4.24 (2H, q, J ¼
7:0 Hz), 4.36 (2H, s), 5.01 (2H, s), 6.68 (2H, d, J ¼
9:2 Hz), 7.20 (2H, d, J ¼ 9:2 Hz), 7.66 (1H, dd, J ¼ 1:7,
8.6 Hz), 7.82 (1H, dd, J ¼ 1:7, 8.6 Hz), 7.90 (1H, s), 8.02
(1H, d, J ¼ 8:6 Hz), 8.10 (1H, d, J ¼ 8:6 Hz), 8.49 (1H,
s), 9.27 (2H, br), 9.52 (2H, s); FABMS m=e (M+H)þ
524.

2188
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
1
5.29. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-ethanimidoyl-1,4-diazepan-1-yl)phenyl]-
amino}sulfonyl)acetate (10f)
tained as a white amorphous powder (62% yield): H
NMR (DMSO-d6) d 1.49–1.86 (2H, m), 2.03 (2H, s),
2.26 (1H, s), 3.49–3.79 (8H, m), 4.26 (2H, s), 5.00 (1.4H,
s), 5.01 (0.6H, s), 6.70–6.74 (2H, m), 7.18–7.22 (2H, m),
7.63–7.67 (2H, m), 7.84–7.90 (1H, m), 8.02 (0.3H, d,
J ¼ 8:4 Hz), 8.03 (0.7H, d, J ¼ 8:4 Hz), 8.10 (1H, d,
J ¼ 8:4 Hz), 8.54 (0.3H, s), 8.57 (0.7H, s), 8.75 (0.7H, s),
8.92 (0.3H, s), 9.45 (2H, s), 9.48 (1H, s), 9.62 (0.6H, s),
9.63 (1.4H, s); FABMS m=e (M+H)þ 537. Anal. Calcd
for C27H32N6O4SÆ3.1HClÆ1.5H2O: C, 47.92; H, 5.67; N,
12.42; S, 4.73; Cl, 16.24. Found: C, 47.91; H, 5.93; N,
12.31; S, 4.73; Cl, 16.46.
Compound 10f was synthesized from 9f according to the
same procedure as that for 10d. Compound 10f was
obtained as a white amorphous powder (17% yield): H
1
NMR (DMSO-d6) d 1.25–1.29 (3H, m), 1.79–1.85 (2H,
m), 2.03 (2H, s), 2.25 (1H, s), 3.47–3.75 (8H, m), 4.21–
4.27 (2H, m), 4.37 (2H, s), 5.00 (2H, s), 6.68–6.73 (2H,
m), 7.17–7.21 (2H, m), 7.62–7.66 (1H, m), 7.82 (1H, d,
J ¼ 8:8 Hz), 7.90 (1H, s), 8.03 (1H, dd, J ¼ 4:0, 8.8 Hz),
8.10 (1H, d, J ¼ 8:8 Hz), 8.49 (0.4H, s), 8.51 (0.6H, s),
8.64 (0.6H, s), 8.76 (0.4H, s), 9.29 (2H, s), 9.33 (1H, s),
9.53 (2H, s); FABMS m=e (M+H)þ 565.
5.33. Ethyl ({[(7-cyano-2-naphthyl)methyl][4-(4-methyl-
1,4-diazepan-1-yl)phenyl]amino}sulfonyl)acetate (14a)
5.30. ({({7-[Amino(imino)methyl]-2-naphthyl}methyl)[4-
(1,4-diazepan-1-yl)phenyl]amino}sulfonyl)acetic acid (11)
To a solution of 8f (610 mg, 1.01 mmol) in 1,2-dichlo-
romethane (11 mL) at ambient temperature was added
trifluoroacetic acid (1.1 mL). After stirring at ambient
temperature for 3 h, the reaction mixture was concen-
trated in vacuo. The resulting residues was dissolved in
chloroform and washed with 10% aqueous potassium
carbonate. The organic layer was dried over MgSO4 and
concentrated in vacuo. To a stirred solution of the de-
protected intermediate in 1,2-dichloromethane (10 mL)
at ambient temperature was added acetic acid (0.6 mL,
10 mmol), 35% aqueous formaldehyde (850 mg,
9.9 mmol) and sodium triacetoxyborohydride (420 mg,
1.97 mmol). After 2 h, the reaction mixture was washed
with 10% aqueous potassium carbonate. The organic
layer was dried over MgSO4 and concentrated in vacuo.
The resulted residue was chromatographed on silica gel
eluting with chloroform/MeOH/aqueous NH3 (100/5/
0.5) to give 14a (480 mg, 92%) as a white amorphous
A solution of 9f (250 mg, 0.42 mmol) in concd HCl (9 mL)
was stirred at ambient temperature for 15 h. The reaction
mixture was concentrated in vacuo and the residues was
chromatographed on ODS-gel eluting with CH3CN/H2O
(10/90). CH3CN was removed in vacuo, and the aqueous
solution was lyophilized after being acidified with 1 N HCl.
Compound 11 (187 mg, 78%) was obtained as a white
amorphous powder: 1H NMR (DMSO-d6) d 1.99–2.06
(2H, m), 3.03–3.08 (2H, m), 3.12–3.16 (2H, m), 3.41 (2H, t,
J ¼ 6:0 Hz), 3.61–3.65 (2H, m), 4.23 (2H, s), 5.01 (2H, s),
6.68 (2H, d, J ¼ 9:1 Hz), 7.21 (2H, d, J ¼ 9:1 Hz), 7.66
(1H, dd, J ¼ 1:6, 8.6 Hz), 7.79–7.82 (1H, m), 7.91 (1H, s),
8.02 (1H, d, J ¼ 8:6 Hz), 8.10 (1H, d, J ¼ 8:6 Hz), 8.47
(1H, s), 9.12 (2H, br s), 9.23 (2H, br s), 9.48 (2H, s); FABMS
m=e (M+H)þ 496. Anal. Calcd for C25H29N5-
O4SÆ3.3HClÆ3.0H2O: C, 48.23; H, 5.57; N, 11.25; S, 4.29; Cl,
15.66. Found: C, 48.26; H, 5.65; N, 11.50; S, 4.45; Cl, 15.60.
1
powder: H NMR (CDCl3) d 1.39 (3H, t, J ¼ 6:9 Hz),
1.86–1.97 (2H, m), 2.32 (3H, s), 2.49 (2H, t, J ¼ 4:3 Hz),
2.61 (2H, t, J ¼ 4:3 Hz), 3.37 (2H, t, J ¼ 4:3 Hz), 3.46
(2H, t, J ¼ 4:3 Hz), 4.07 (2H, s), 4.34 (2H, q,
J ¼ 6:9 Hz), 5.02 (2H, s), 6.53 (2H, d, J ¼ 8:3 Hz), 7.19
(2H, d, J ¼ 8:3 Hz), 7.48–7.54 (1H, m), 7.62 (1H, s),
7.65–7.71 (1H, m), 7.77–7.85 (2H, m), 8.03 (1H, s);
FABMS m=e (M+H)þ 521.
5.31. {({7-[Amino(imino)methyl]-2-naphthyl}methyl){4-
[4-(pyridin-4-yl)-1,4-diazepan-1-yl]phenyl}amino)sulfon-
yl}acetic acid (12)
Compound 12 was synthesized from 9g according to the
same procedure as that for 11. Compound 12 was ob-
1
tained as a white amorphous powder (23% yield): H
NMR (DMSO-d6) d 1.87–1.89 (2H, m), 3.42–3.45 (2H,
m), 3.59–3.63 (4H, m), 3.83–3.86 (2H, m), 4.22 (2H, s),
4.98 (2H, s), 6.69 (2H, d, J ¼ 8:8 Hz), 7.03–7.09 (2H, m),
7.15 (2H, d, J ¼ 8:8 Hz), 7.61 (1H, dd, J ¼ 1:6, 8.4 Hz),
7.83 (1H, dd, J ¼ 1:6, 8.3 Hz), 7.89 (1H, s), 8.01 (1H, d,
J ¼ 8:4 Hz), 8.11 (1H, d, J ¼ 8:4 Hz), 8.12–8.15 (2H, m),
8.50 (1H, s), 9.37 (2H, s), 9.55 (2H, s), 13.76 (1H, br s);
FABMS m=e (M+H)þ 573. Anal. Calcd for
C30H32N6O4SÆ2.4HClÆ2.5H2O: C, 47.80; H, 5.84; N,
11.15; S, 5.10; Cl, 13.55. Found: C, 47.79; H, 5.59; N,
11.15; S, 5.08; Cl, 13.53.
5.34. Ethyl ({[4-(4-butyl-1,4-diazepan-1-yl)phenyl][(7-
cyano-2-naphthyl)methyl]amino}sulfonyl)acetate (14b)
Compound 14b was synthesized from 8f and butyral-
dehyde according to the same procedure as that for 14a.
Compound 14b was obtained as a white amorphous
1
powder (87% yield): H NMR (CDCl3) d 0.87 (3H, t,
J ¼ 7:5 Hz), 1.17–1.31 (2H, m), 1.34–1.45 (5H, m), 1.82–
1.93 (2H, m), 2.40 (2H, t, J ¼ 7:5 Hz), 2.51 (2H, t,
J ¼ 5:1 Hz), 2.66 (2H, t, J ¼ 5:1 Hz), 3.37 (2H, t,
J ¼ 5:1 Hz), 3.43 (2H, t, J ¼ 5:1 Hz), 4.08 (2H, s), 4.34
(2H, q, J ¼ 6:9 Hz), 5.03 (2H, s), 6.53 (2H, d,
J ¼ 9:3 Hz), 7.19 (2H, d, J ¼ 9:3 Hz), 7.42–7.53 (1H, m),
7.60 (1H, s), 7.65–7.69 (1H, m), 7.70–7.82 (2H, m), 7.99
(1H, s); FABMS m=e (M+H)þ 563.
5.32. ({({7-[Amino(imino)methyl]-2-naphthyl}methyl)[4-
(4-ethanimidoyl-1,4-diazepan-1-yl)phenyl]amino}sulfon-
yl)acetic acid (13)
Compound 13was synthesized from 10f according to the
same procedure as that for 11. Compound 13 was ob-

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
2189
5.35. Ethyl ({[4-(4-benzyl-1,4-diazepan-1-yl)phenyl][(7-
cyano-2-naphthyl)methyl]amino}sulfonyl)acetate (14c)
d, J ¼ 8:8 Hz), 8.50 (1H, s), 9.34 (2H, s), 9.54 (2H, s),
11.41 (1H, s); FABMS m=e (M+H)þ 634.
Compound 14c was synthesized from 8f and benzalde-
hyde according to the same procedure as that for 14a.
Compound 14c was obtained as a white amorphous
5.39. ({({7-[Amino(imino)methyl]-2-naphthyl}methyl)-
[4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)-
acetate (16a)
1
powder (88% yield): H NMR (CDCl3) d 1.38 (3H, t,
J ¼ 7:2 Hz), 1.84–1.92 (2H, m), 2.51–2.57 (2H, m), 2.61
(2H, t, J ¼ 5:1 Hz), 3.36–3.45 (4H, m), 3.56 (2H, s), 4.06
(2H, s), 4.34 (2H, q, J ¼ 7:2 Hz), 5.02 (2H, s), 6.52 (2H,
d, J ¼ 9:0 Hz), 7.16–7.35 (10H, m), 7.75–7.84 (2H, m),
8.02 (1H, s); FABMS m=e (M+H)þ 597.
Compound 16a was synthesized from 15a according to
the same procedure as that for 11. Compound 16a was
obtained as a white amorphous powder (40% yield): H
1
NMR (DMSO-d6) d 2.73 (3H, s), 3.01–3.11 (2H, m),
3.31–3.33 (6H, m), 3.52–3.55 (2H, m), 4.23 (2H, s), 5.02
(2H, s), 6.66 (2H, d, J ¼ 8:8 Hz), 7.22 (2H, d,
J ¼ 8:8 Hz), 7.67 (1H, d, J ¼ 8:8 Hz), 7.80 (1H, dd,
J ¼ 2:0, 8.8 Hz), 7.92 (1H, s), 8.02 (1H, d, J ¼ 8:8 Hz),
8.10 (1H, d, J ¼ 8:8 Hz), 8.47 (1H, s), 9.20 (2H, s), 9.47
(2H, s), 10.73 (1H, s); FABMS m=e (M+H)þ 510. Anal.
Calcd for C26H31N5O4SÆ2.9HClÆ1.5H2O: C, 50.16; H,
6.15; N, 10.45; S, 4.78; Cl, 15.34. Found: C, 50.45; H,
6.04; N, 10.50; S, 4.78; Cl, 15.15.
5.36. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}-
sulfonyl)acetate (15a)
Compound 15a was synthesized from 14a according to
the same procedure as that for 9d. Compound 15a was
1
obtained as a white amorphous powder (40% yield): H
NMR (DMSO-d6) d 1.27 (3H, t, J ¼ 7:2 Hz), 2.04–2.16
(1H, m), 2.28–2.40 (1H, m), 2.71 (1.5H, s), 2.72 (1.5H,
s), 2.99–3.09 (2H, m), 3.26–3.42 (4H, m), 3.62–3.76 (2H,
m), 4.25 (2H, q, J ¼ 7:2 Hz), 4.36 (2H, s), 5.02 (2H, s),
6.67 (2H, d, J ¼ 8:4 Hz), 7.22 (2H, d, J ¼ 8:4 Hz), 7.67
(1H, d, J ¼ 8:4 Hz), 7.83 (1H, d, J ¼ 8:4 Hz), 7.90 (1H,
s), 8.03 (1H, d, J ¼ 8:4 Hz), 8.10 (1H, d, J ¼ 8:4 Hz),
8.51 (1H, s), 9.37 (2H, s), 9.56 (2H, s), 11.21 (1H, s);
FABMS m=e (M+H)þ 538.
5.40. ({({7-[Amino(imino)methyl]-2-naphthyl}methyl)-
[4-(4-butyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)acetic
acid (16b)
Compound 16b was synthesized from 15b according to
the same procedure as that for 11. Compound 16b was
1
obtained as a white amorphous powder (20% yield): H
NMR (DMSO-d6) d 0.87 (3H, t, J ¼ 7:4 Hz), 1.22–1.31
(2H, m), 1.59–1.71 (2H, m), 2.06–2.14 (1H, m), 2.32–
2.43 (1H, m), 2.95–3.05 (4H, m), 3.25–3.47 (4H, m),
3.69–3.74 (2H, m), 4.24 (2H, s), 5.02 (2H, s), 6.66 (2H, d,
J ¼ 9:2 Hz), 7.22 (2H, d, J ¼ 9:2 Hz), 7.67 (1H, dd,
J ¼ 1:2, 8.8 Hz), 7.82 (1H, dd, J ¼ 1:2, 8.8 Hz), 7.91
(1H, s), 8.02 (1H, d, J ¼ 8:8 Hz), 8.10 (1H, d,
J ¼ 8:8 Hz), 8.49 (1H, s), 9.32 (2H, s), 9.53 (2H, s), 10.96
(1H, s); FABMS m=e (M+H)þ 552. Anal. Calcd for
C28H35N5O4SÆ2.3HClÆ2.5H2O: C, 52.54; H, 6.87; N,
9.88; S, 4.52; Cl, 11.51. Found: C, 53.04; H, 6.90; N,
9.43; S, 4.28; Cl, 11.26.
5.37. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-butyl-1,4-diazepan-1-yl)phenyl]amino}-
sulfonyl)acetate (15b)
Compound 15b was synthesized from 14b according to
the same procedure as that for 9d. Compound 15b was
obtained as a white amorphous powder (26% yield): H
1
NMR (DMSO-d6) d 0.85–0.89 (3H, m), 1.23–1.30 (5H,
m), 1.59–1.72 (2H, m), 2.06–2.13 (1H, m), 2.31–2.41
(1H, m), 2.95–3.05 (4H, m), 3.25–3.45 (4H, m), 3.68–
3.73 (2H, m), 4.25 (2H, t, J ¼ 7:2 Hz), 4.69 (2H, s), 5.02
(2H, s), 6.66 (2H, d, J ¼ 8:8 Hz), 7.22 (2H, d,
J ¼ 8:8 Hz), 7.66 (1H, dd, J ¼ 1:6, 8.8 Hz), 7.82 (1H, dd,
J ¼ 1:6, 8.8 Hz), 7.91 (1H, s), 8.02 (1H, d, J ¼ 8:8 Hz),
8.10 (1H, d, J ¼ 8:8 Hz), 8.48 (1H, s), 9.28 (2H, s), 9.51
(2H, s), 10.88 (1H, s); FABMS m=e (M+H)þ 580.
5.41. ({({7-[Amino(imino)methyl]-2-naphthyl}methyl)[4-
(4-benzyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)acetic
acid (16c)
Compound 16c was synthesized from 15c according to
the same procedure as that for 11. Compound 16c was
obtained as a white amorphous powder (18% yield): H
5.38. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-benzyl-1,4-diazepan-1-yl)phenyl]amino}-
sulfonyl)acetate (15c)
1
NMR (DMSO-d6) d 2.08–2.16 (1H, m), 2.38–2.49 (1H,
m), 2.92–3.02 (2H, m), 3.24–3.46 (4H, m), 3.72–3.77 (2H,
m), 4.23 (2H, s), 4.25–4.35 (2H, m), 5.02 (2H, s), 6.65
(2H, d, J ¼ 8:8 Hz), 7.21 (2H, d, J ¼ 8:8 Hz), 7.41–7.44
(3H, m), 7.60–7.64 (2H, m), 7.67 (1H, dd, J ¼ 1:5,
8.8 Hz), 7.82 (1H, dd, J ¼ 1:2, 8.8 Hz), 7.90 (1H, s), 8.02
(1H, d, J ¼ 8:8 Hz), 8.10 (1H, d, J ¼ 8:8 Hz), 8.49 (1H,
s), 9.32 (2H, s), 9.53 (2H, s), 11.35 (1H, s); FABMS m=e
(M+H)þ 536. Anal. Calcd for C32H35N5O4SÆ2.5-
HClÆ3.2H2O: C, 53.55; H, 6.33; N, 9.18; S, 4.20; Cl, 11.62.
Found: C, 53.46; H, 6.08; N, 9.19; S, 4.22; Cl, 11.24.
Compound 15c was synthesized from 14c according to
the same procedure as that for 9d. Compound 15c was
obtained as a white amorphous powder (26% yield): H
NMR (DMSO-d6) d 1.27 (3H, t, J ¼ 7:6 Hz), 2.07–2.16
(1H, m), 2.39–2.49 (1H, m), 2.91–3.02 (2H, m), 3.24–
3.42 (4H, m), 3.72–3.76 (2H, m), 4.22–4.34 (4H, m), 4.35
(2H, s), 5.02 (2H, s), 6.66 (2H, d, J ¼ 8:8 Hz), 7.20 (2H,
d, J ¼ 8:8 Hz), 7.41–7.44 (3H, m), 7.83 (1H, dd, J ¼ 1:2,
8.8 Hz), 7.90 (1H, s), 8.02 (1H, d, J ¼ 8:8 Hz), 8.10 (1H,
1

2190
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
6. Biology
baseline value (dogs and monkeys) or the vehicle group
(mice).
6.1. Chromogenic assay
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,
USA). Reaction mixtures (125 lL) were prepared in 96-
well plates containing chromogenic substrates and an
inhibitor in either 0.05 M Tris–HCl, pH 8.4, 0.15 M
NaCl. Reactions were initiated with a 25 lL portion of
the enzyme solution. Enzymes and substrates were used
as follows: factor Xa and S-2222; thrombin and S-2238;
trypsin and S-2222. The concentration of an 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.
6.4. Thromboplastin-induced venous thrombosis model in
rats
Male SD rats weighing 280–320 g were used. In the
intravenous studies, the test drug (HBr salt of 13, hep-
arin or vehicle) was administered to non-fasted animals
under anesthesia with urethane (0.96 g/kg ip) by con-
tinuous iv infusion using an infusion pump (Harvard
apparatus) 2 h before the thrombus formation. In the
oral studies, the test drug (HBr salt of 13, warfarin or
vehicle) was orally administered to fasted animals using
a gastric tube 1 h (HBr salt of 13 and vehicle) or 18 h
(warfarin or vehicle) before the thrombus formation.
Thrombus formation was induced by the method of
Reyers et al. Under anesthesia with urethane, the
abdomen was surgically opened and the inferior vena
cava was carefully isolated. Venous thrombosis was
induced by injection of 25 lg/kg thromboplastin (Ortho
Diagnostic Systems Co., Tokyo, Japan) into the right
femoral vein. One minute after the injection of throm-
boplastin, two tight ligations 1 cm apart were made with
cotton thread on the inferior vena cava just below the
left renal venous branch. Ten minutes after the ligation,
the thrombosed segment was longitudinally opened. The
thrombus was gently removed, and dissolved in 2 mL of
0.5 N NaOH. The thrombus protein content was mea-
sured by photometry using a due binding assay kit (Bio-
Rad, Hercules, CA) and bovine serum albumin (BSA) as
a protein standard. The experiments were performed on
groups of six animals each. Data represent percentages
of inhibition compared with the thrombus wet weight
of the saline group. Data represent the means SEM.
6.2. Plasma clotting time assays
Citrated blood samples from mice and dogs were col-
lected. Platelet-poor plasma was prepared by centrifu-
gation at 3000 rpm for 10 min and stored at )40 °C until
use. Plasma clotting times were performed using a
KC10A coagulometer (Amelung Co., Lehbrinsweg,
Germany) at 37 °C. Prothrombin time (PT) and acti-
vated partial thromboplastin time (APTT) were mea-
sured using Orthobrain thromboplastin and thrombofax
(Ortho Diagnostic Systems Co., Tokyo, Japan),
respectively. Coagulation times for each test sample
were compared with coagulation times measured using a
distilled water control. The concentration required to
double the clotting time (CT2) was estimated from each
individual concentration–response curve. Each mea-
surement was performed three times, and represented
as the mean value.
6.5. Template bleeding time in rats
Male SD rats weighing 300–330 g were used. In the oral
studies, the test drug (HBr salt of 13, warfarin or vehi-
cle) was orally administered to fasted animals using a
gastric tube 1 h (HBr salt of 13 and vehicle) or 18 h
(warfarin or vehicle) before the measurement of bleeding
time. A template bleeding device (Simplateâ, Organon
Teknika, Tokyo) was placed on the right planta and
triggered. Blood flowing from the incision was gently
wiped away with filter paper every 30 s. Bleeding time
was measured as time elapsed until bleeding stopped.
When template bleeding time was prolonged beyond
30 min, measurement was stopped and the bleeding time
was recorded as 30 min. The experiments were per-
formed on groups of six animals each. Data represent
the means SEM.
6.3. Ex vivo studies
Male mice weighing 30–37 g and male beagle dogs
weighing 8.6–12.6 kg were used in these studies. In all
animal species used, non-fasted animals for intravenous
studies and fasted animals for overnight for the oral
studies were used. In mice, the test drug was dissolved in
saline and administered to animals intravenously at
1 mg/kg via tail vein or orally at 100 mg/kg using a
gastric tube. Citrated blood was collected from the vena
cava 1 min after intravenous injection or 30 min after
oral administration. In dogs, the test drug was dissolved
in saline or 0.5% methylcellulose and administered to
animals intravenously at 0.3 mg/kg via cephalic vein or
orally at 10 mg/kg using a gastric tube. At a predeter-
mined time before and after the drug administration,
citrated blood was collected from the cephalic vein. At a
predetermined time before and after the drug adminis-
tration, citrated blood was collected from the femoral
vein. Platelet-poor plasma was prepared by centrifuga-
tion for measurement of PT or APTT. All data were
expressed as relative fold values, compared with the
6.6. Statistical analyses
Statistical analysis was performed by Dunnett multiple
comparison test for thrombus formation or Steelꢀs test
for bleeding time, compared with the vehicle group. A
p value of less than 0.05 was considered significant.

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 2179–2191
2191
S.; Chu, V.; Brown, K.; Colussi, D.; Czekaj, M.; Perrone,
M.; Leadley, R., Jr. Eur. J. Pharmacol. 2000, 389, 201; (d)
Sato, K.; Kawasaki, T.; Taniuchi, Y.; Hirayama, F.;
Koshio, H.; Matsumoto, Y. Eur. J. Pharmacol. 1997, 339,
141; (e) Kawasaki, T.; Sato, K.; Hirayama, F.; Koshio, H.;
Taniuchi, Y.; Matsumoto, Y. Thromb. Haemostasis 1998,
79, 859; (f) Sato, K.; Kawasaki, T.; Hisamichi, N.;
Taniuchi, Y.; Hirayama, F.; Koshio, H.; Matsumoto, Y.
Br. J. Pharmacol. 1998, 132, 92.
Acknowledgements
The authors deeply acknowledge Dr. Yuuji Mano for
pharmacokinetic studies. The authors also grateful to
the staff of Division of Analytical Science Laboratories
for elemental analysis and spectral measurements.
References and notes
4. Hirayama, F.; Koshio, H.; Katayama, N.; Kurihara, H.;
Taniuchi, Y.; Sato, K.; Hisamichi, N.; Sakai-Moritani, Y.;
Kawasaki, T.; Matsumoto, Y.; Yanagisawa, I. Bioorg.
Med. Chem. 2002, 10, 1509.
5. Hirayama, F.; Koshio, H.; Ishihara, T.; Watanuki, S.;
Hachiya, S.; Kaizawa, H.; Kuramochi, T.; Katayama, N.;
Kurihara, H.; Taniuchi, Y.; Sato, K.; Sakai-moritani, Y.;
Kaku, S.; Kawasaki, T.; Matsumoto, Y.; Sakamoto, S.;
Tsukamoto, S. Bioorg. Med. Chem. 2002, 10, 2597.
6. Buhlmayer, P.; Criscione, L.; Fuher, W.; Furet, P.; de
Gasparo, M.; Whitebread, S. J. Med. Chem. 1991, 34, 3105.
1. (a) Harder, S.; Thurmann, P. Clin. Pharmacokinet. 1996,
30, 416; (b) Hirsh, J. New Engl. J. Med. 1991, 324, 1865.
2. Rosenberg, J. S.; Beeler, D. L.; Rosenberg, R. D. J. Bio.
Chem. 1975, 250, 1607.
3. (a) Sikto, G. R.; Ramjit, D. R.; Stabilito, I.; Lehman, D.;
Lynch, J. J.; Vlasuk, G. P. Circulation 1992, 85, 805; (b)
Morishima, Y.; Tanabe, K.; Terada, Y.; Hara, T.; Kunit-
ada, S. Thromb. Haemostasis 1997, 78, 1366; (c) Heran, C.;
Morgan, S.; Kasiewski, C.; Bostwick, J.; Bentley, R.; Klein,