Design, synthesis and biological activity of amidinobicyclic compounds (derivatives of DX-9065a) as factor Xa inhibitors: SAR study of S1 and aryl binding sites

Article abstract of DOI:10.1016/j.bmc.2004.02.032

Since factor Xa (fXa) plays a pivotal role in the blood coagulation cascade, inhibition of fXa is thought to be an effective treatment for a variety of thrombotic events. (2S)-2-[4-[[(3S)-1-Acetimidoyl-3-pyrrolidinyl]oxy]phenyl] -3-(7-amidino-2-naphthyl)p

Full text of DOI:10.1016/j.bmc.2004.02.032

Bioorganic & Medicinal Chemistry 12 (2004) 2099–2114
Design, synthesis and biological activity of amidinobicyclic
compounds (derivatives of DX-9065a) as factor Xa inhibitors:
SAR study of S1 and aryl binding sites
Satoshi Komoriya,* Naoaki Kanaya, Takayasu Nagahara, Asako Yokoyama,
Kazue Inamura, Yukio Yokoyama, Shin-ichi Katakura and Tsuyoshi Hara
Tokyo R&D Center, Daiichi Pharmaceutical Co. Ltd, 16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan
Received 3 December 2003; accepted 24 February 2004
Abstract—Since factor Xa (fXa) plays a pivotal role in the blood coagulation cascade, inhibition of fXa is thought to be an effective
treatment for a variety of thrombotic events. (2S)-2-[4-[[(3S)-1-Acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naph-
thyl)propanoic acid hydrochloride pentahydrate (DX-9065a) was previously found in our laboratory as a novel orally active factor
Xa inhibitor. DX-9065a exhibits a strong inhibitory activity toward fXa by occupying the substrate recognition (called S1) sites and
aryl binding sites of fXa. Herein we describe conversions of the amidinonaphthalene and the acetimidoylpyrrolidine moieties of DX-
9065a. Some compounds showed remarkably increased in vitro anti-factor Xa and PRCT activities compared with those of DX-
9065a. The most promising compound 38 showed four times the prolongation of APTT against DX-9065a after oral administration
to rats.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
(fXa). Thrombin indeed plays a pivotal role, that is,
fibrin formation at the final step of the blood coagul-
ation cascade. Thrombin not only converts fibrinogen to
fibrin for clot formation, however, but is also directly
involved in platelet aggregation, protein C activation,
factor XIII activation, and feedback activation of fac-
tors V and VIII. These complicated actions of thrombin
might imply some difficulties of thrombin inhibitors
with its use. Furthermore, fXa, whose major practical
role is the generation of thrombin by the limited pro-
teolysis of prothrombin, holds a central position that
links the intrinsic and extrinsic activation mechanisms in
the final common pathway of coagulation. fXa in
combination with fVa and calcium ion on the
phospholipid surface forms the prothrombinase com-
plex that generates thrombin via the proteolysis of
prothrombin. This activation of thrombin by the com-
plex is a highly amplified process of the blood coagul-
ation cascade. The tenacious role of fXa in the
coagulation cascade encouraged us to study fXa inhib-
itors. We have already reported finding efficacy, and a
tendency for less bleeding of highly selective DX-9065a
type fXa inhibitors2 (Fig. 1).
Thrombotic events are thought to be major causes of
fatal diseases, such as myocardial infarction and cere-
bral infarction. Although several anticoagulants, as well
as antiplatelet agents, are clinically prescribed for the
treatment and prevention of such events nowadays,
warfarin is the only anticoagulant that can be adminis-
tered orally. Sixma described that an ideal anti-throm-
botic should meet six criteria: (a) it should inhibit
thrombosis, (b) it should not affect homeostasis to such
an extent that problems arise, (c) it should have a long
half-life, (d) it should not have serious side effects, (e) it
should be absorbed after oral administration, and (f) it
should have a large therapeutic range.1 From these cri-
teria, warfarin still has several defects regarding its use;
for example, interaction with other drugs or foods and
the necessity of regular monitoring. Therefore, many
groups have been expanding massive efforts in the
search for new orally active anticoagulants, particularly
for inhibitors against thrombin or activated factor Xa
Brandstetter et al. have already reported the structure of
the complex of DX-9065a in des-Gla-fXa.3 Because the
* Corresponding author. Tel.: +81-3-3680-0151; fax: +81-3-5696-8772;
e-mail: komor0ni@daiichipharm.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.032

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S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
18a–b,8;9 with 17 gave the cyano derivatives 20a–b.
O
OH
Pinner reaction of 20a–b, followed by hydrolysis in 2 N
HCl gave the carboxylic acid derivatives 21a–b, respec-
tively. Acetimidation of 21a with ethyl acetimidate gave
22a (Scheme 2).
H2N
NH
N
NH
O
Figure 1. Structure of DX-9065a.
Syntheses of the naphthalene derivatives 27a–c, 28a,
29a, 28d, 29d, and 30d were shown in Scheme 3. Reac-
tion of the phosphonium salt 232 with the a-ketoesters
24a–c using DBU as a base, followed by catalytic
hydrogenation, gave the cyano derivatives 25a–c. Pinner
reaction of the nitriles 25a–c gave the corresponding
amidine derivatives 26. Reaction of 26 with ethyl ace-
timidate hydrochloride in an ethanol solution, followed
by acid hydrolysis gave the carboxylic acid derivatives
27a–c, respectively. The piperidine derivative 26a was
converted into the other imidoyl derivatives with the
corresponding imidate, and then acid hydrolysis gave
the carboxylic acid derivatives 28a and 29a.
amidinonaphthalene moiety shows a good interaction
with the S1 site of fXa on the basis of X-ray analysis, the
planar amidinobicyclic aromatic ring moieties were
thought to be appropriate structures for the S1 site of
fXa. And, the cation hole, which was formed by the
carbonyl oxygen atoms of Lys-97 together with the Glu-
97 side chain, interacted with the acetimidoylpyrroli-
dine. Therefore, we synthesized some amidinobicyclic
planar aromatic compounds and some basic aliphatic
heterocycles to further optimize our fXa inhibitors. As a
consequence of this study, we obtained the 6-amidino-
indole and the piperidinyloxyphenyl structures as highly
interactive moieties for the S1 site and the aryl binding
site of fXa, respectively. The structure of 38 was the
hybridization of the foregoing structures, and com-
pound 38 showed the best anti-fXa activity and
anticoagulant activity with oral administration in the
series.
Reaction of the phosphonium salt 232 with the a-keto-
esters 2 using DBU as a base, followed by catalytic
hydrogenation, gave the nitrile 25d. Pinner reaction of
the nitrile 25d gave the corresponding amidine derivative
26d. The pyrrolidine derivative 26d was converted into
some imidoyl derivatives with the corresponding imi-
date, and then acid hydrolysis gave the carboxylic acid
derivatives 28d, 29d, and 30d.
The a-ketoesters 24a–c were prepared by Mitsunobu
reaction of ethyl 4-hydroxyphenylglyoxalate 312 and the
commercially available alcohols 32a–c (Scheme 3).
2. Chemistry
All compounds reported herein were synthesized as a
mixture of two epimers/enantiomers at a carbon of
carbonyl group. Carboxylic acid derivatives 5a–h, 6a,
and 6h were synthesized in a convergent synthesis as
outlined in Scheme 1. Wittig reaction of the a-keto ester
22 with the phosphonium salts 1a–h using 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) as a base, followed by
catalytic hydrogenation, gave the cyanobicyclic deriva-
tives 3a–h. After Pinner reaction of the compounds 3a–h
(successive reaction of the compounds with saturated
HCl ethanol solution, and then with ammonia in etha-
nol), esters of the obtained amidine derivatives 4a–h
were hydrolyzed to give the carboxylic acid derivatives
5a–h, respectively. Acetimidation of the pyrrolidine
moieties was accomplished by treating 5a or 5h with
ethyl acetimidate hydrochloride in an aqueous solution
or ethanol solution. The phosphonium salts 1a–h (ex-
cept 1c) were synthesized by treating the alcohol deriv-
atives 7a–b,4 12a–b, and 14a–c (12a–b and 14a–c were
prepared by alkylation of esters 11a4 or 13a4 followed by
reduction), with SOCl2/PPh3 or PBr3/PPh3. Synthesis of
the phosphonium salt 1c was accomplished by treating
85 with 96 in ethanol solution, followed by formation of
the phosphonium salt using PPh3 in ClCH2CH2Cl
solution (Scheme 1).
Syntheses of the benzothiophene derivatives 34 and 36,
and the indole derivative 38 were shown in Scheme 4.
These compounds were prepared by six steps from the
phosphonium salt 1a or 1h in the same manner as
described for 27a–c (Scheme 4).
3. Result and discussion
Anti-fXa activities, anti-fIIa activities, and the plasma
recalcification times (PRCT) of the synthesized com-
pounds are shown in Tables 1–3. Further, the antico-
agulant activities (activated partial thromboplastin time;
APTT) of compounds 6h, 27a, 36, and 38 orally
administered to rats are shown in Table 4.
These compounds were a mixture of two epimers at the
carbon where the carboxylic acid was substituted.
Compound 44 is a mixture of DX-9065a and the epimer.
Anti-fXa activity of DX-9065a is two times higher than
that of 44. The epimer of DX-9065a at carbon with
carboxyl moiety showed about 10 times less active
against fXa. Anti-fXa activity was influenced by
the stereo of the carbon where the carboxylic acid
was substituted.2 According to the above observation,
a single stereoisomer would show two times higher
anti-fXa activity than the mixture of two stereo-
isomers.
Syntheses of the benzothiazole derivatives 21a–b and
22a were shown in Scheme 2. Mitsunobu reaction of 157
with 162 gave the malonate derivative 17. Reaction of
19a–b, which were prepared subsequent cyanation of

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2101
CO2Et
2
1)
O
N
Boc
O
Br- or Cl-
CO2Et
Y
X
5
Y
X
DBU, THF, EtOH
1) sat. HCl-EtOH
2) sat. NH3-EtOH
+
NC
NC
PPh3
2) H2, THF, EtOH
Pd-C or PdO(H2O)BaSO4
N
Boc
6
1a:5-CN,X=S,Y=CH
1b:6-CN,X=S,Y=CH
1c:5-CN,X=NH,Y=N
1d:5-CN,X=NH,Y=CH
1e:6-CN,X=NH,Y=CH
1f:6-CN,X=NMe,Y=CH
1g:5-CN,X=NEt,Y=CH
1h:6-CN,X=NEt,Y=CH
O
3
3a:5-CN,X=S,Y=CH
3b:6-CN,X=S,Y=CH
3c:5-CN,X=NH,Y=N
CO2H
Y
X
CO2H
Y
X
2N HCl
reflux
Am
CO2Et
Y
X
Am
Me
NH
NH
Am
N
5a:5-Am,X=S,Y=CH
5b:6-Am,X=S,Y=CH
5c:5-Am,X=NH,Y=N
5d:5-Am,X=NH,Y=CH
5e:6-Am,X=NH,Y=CH
5f:6-Am,X=NMe,Y=CH
5g:5-Am,X=NEt,Y=CH
5h:6-Am,X=NEt,Y=CH
O
NH
O
EtOC(NH)CH3
NaOHaq
O
4
6a:5-Am,X=S,Y=CH
6h:6-Am,X=NEt,Y=CH
1) SOCl2, pyridine
Et2O
+ Cl-
OH
NC
NC
PPh3
S
S
2) PPh3, xylene
reflux
7a:5-CN
1a:5-CN
7b:6-CN
1b:6-CN
NH
9
NC
Cl
N
PPh3
NC
NH2
NH2
NC
EtO
N
+
Cl-
PPh3
HCl
Cl
N
H
ClCH2CH2Cl
N
H
EtOH,reflux
1c
8
10
NC
CaI2
NC
NC
CO2Et
1) PBr3, CH2Cl2
+
OH
Br-
PPh3
N
R
NaBH4
N
R
N
R
2) PPh3, CH2Cl2
reflux
11a:R=H
EtI
11b:R=Et
1d:R=H
NaH
12a:R=H
1g:R=Et
12b:R=Et
CO2Me
1) PBr3, CH2Cl2
CaI2
+
OH
Br-
PPh3
N
R
NC
N
R
NC
N
R
NC
NaBH4
2) PPh3, CH2Cl2
reflux
1e:R=H
13a:R=H
13b:R=Me
13c:R=Et
MeI
NaH
EtI
NaH
1f:R=Me
14a:R=H
14b:R=Me
14c:R=Et
1h:R=Et
Scheme 1.
The result of optimization of the bicyclic aromatic ring
is shown in Table 1. Indole and benzothiophene rings,
five–six membered bicyclic aromatic rings, were con-
sidered to be favorable for the anti-fXa activities and
anticoagulant activities. Especially, 5-amidinobenzothi-
ophene derivative 5a and 6-amidino-1-ethylindole
derivative 5h exhibited good activities comparable to
that of 7-amidinonaphthalene derivative 42. Their ace-
timidoyl derivatives 6a and 6h had high activities, and
compound 6h showed two times higher anti-fXa and
anticoagulant activity in comparison with those of
compound 44 (Table 1).
27a, having an acetimidoylpiperidine moiety showed the
strongest activity among these naphthalene derivatives
27 to 30 and 44.
Since the 5-amidinobenzothiophene derivative and
6-amidino-1-ethylindole derivative showed good activi-
ties in vitro, compounds 34, 36, and 38 were synthesized.
Compound 38 showed anti-fXa and anticoagulant
activities similar to those of compound 27a. In spite of
being a mixture of epimers, the anti-fXa activity and
anticoagulant activity of compounds 27a and 38 were
higher than those of DX-9065a (anti-fXa; 0.07 lM,
PRCT; 0.5 lM) (Table 3). In our previous report, we
separated two epimers of 25d with recrystallization.2
HPLC study revealed that 25d and 44 were 1:1
mixture of epimers.2 All diastereomeric mixtures (in-
clude 25d, 26d, and 44) in this report did not separate
The results of the optimization of aryl binding site fitting
moiety are shown in Table 2. Compounds 28d and 29d,
which have more hydrophobic moieties, showed much
higher activities than those of compound 44. Compound

2102
S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
N
Boc
16
CO2Et
CO2Et
HO
EtO2C
EtO2C
N
Boc
DEAD,PPh3,THF
O
OH
15
17
CO2Et
EtO2C
N
S
N
S
N
S
NBS
AIBN,CCl4
reflux
NC
NC
CH3
NC
N
Boc
Br
NaH,THF
O
19a:5-CN
19b:6-CN
18a:5-CN
18b:6-CN
20a:5-CN
20b:6-CN
Am
CO2H
CO2H
N
S
N
S
1)sat. HCl-EtOH
Am
Me
NH
N
NH
EtOC(NH)CH3
NaOHaq
2)sat. NH3-EtOH
3)2N HCl, reflux
O
O
21a:5-Am
21b:6-Am
22a
Scheme 2.
1)24a-c
CO2Et
CO2Et
Br-
1)HCl-EtOH
2)NH3-EtOH
DBU,THF, EtOH
Am
NC
+
PPh3
2)H2,
PdO(H2O)BaSO4
THF, EtOH
R1
NC
R1
O
26
O
25a:R1=1-Boc-piperidin-4-yl
23
25b:R1=(3R)-1-Boc-pyrrolidin-3-yl
26a:R1=piperidin-4-yl
25c:R1=[(2S)-1-Boc-pyrrolidin-2-yl]methyl
1)EtOC(NH)CH3,
Et3N,EtOH
CO2H
27a:R1=1-ethanimidoyl-piperidin-4-yl
27b:R1=(3R)-1-ethanimidoyl-pyrrolidin-3-yl
Am
27c:R1=[(2S)-1-ethanimidoyl-pyrrolidin-2-yl]methyl
R1
2)2N HCl,reflux
O
1)alkylimino-
methylation
CO2H
R2
28a:R2=C(NH)CH2CH3
29a:R2=C(NH)CH2CH2CH3
26a
N
Am
2)2N HCl,reflux
O
CO2Et
CO2Et
1)HCl-EtOH
Br-
1) 2, DBU
NC
Am
+
N
PPh3
NH
Boc
2)H2,
PdO(H2O)BaSO4
2)NH3-EtOH
NC
25d
26d
O
O
23
1)alkylimino-
methylation
CO2H
28d:R2=C(NH)CH2CH3
29d:R2=C(NH)CH2CH2CH3
30d:R2=C(NH)Ph
Am
N
R2
2)2N HCl,reflux
O
CO2Et
O
CO2Et
Boc
N
32a-c
HO
N
N
Boc
O
HO
HO
DEAD, PPh3,
THF
OH
R1
Boc
32c
32b
O
32a
31
24a:R1=1-Boc-piperidin-4-yl
24b:R1=(3R)-1-Boc-pyrrolidin-3-yl
24c:R1=[(2S)-1-Boc-pyrrolidin-2-yl]methyl
Scheme 3.
on the TLC. It can therefore presumed that all
compounds in Table 1–3 are 1:1 mixture of the
epimers.
tion to rats at the dose of 100 mg/kg. The results
are shown in Table 4. All compounds showed
prolongation of thromboplastin time (APPT) to over
4 h after oral administration. Compound 38
showed four times prolongation of APPT at
30 min after oral administration, and twofold pro-
Some compounds were tested for their ex vivo
anticoagulant activity following their oral administra-

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2103
1)24a
1)HCl-EtOH
2)NH3-EtOH
DBU,
CO2Et
NC
Me
NH
CO2H
CO2H
Am
Cl-
PPh3
THF, EtOH
NC
Boc
N
N
+
S
S
S
S
3)EtOC(NH)CH3,
Et3N,EtOH
4)2N HCl, reflux
2)H2, 10%Pd-C
EtOH
34
O
O
O
33
1a
CO2Et
NC
1)24c
DBU
Am
1)HCl-EtOH
2)NH3-EtOH
S
2)reduction
O
35
N
3)EtOC(NH)CH3,
Et3N,EtOH
N
36
Boc
1)24a, DBU,
THF, EtOH
Me
4)2N HCl, reflux
HN
Br-
2)H2, 10%Pd-
1)EtOC(NH)CH3,
Et3N,EtOH
CO2Et
CO2H
Me
NH
C
+
PPh3
EtOH
N
NH
N
NC
N
N
Am
Am
2) 2N HCl,
reflux
3)HCl-EtOH
4)NH3-EtOH
O
O
37
38
1h
Scheme 4.
Table 1. In vitro anticoagulant and enzyme inhibitory activity (bicyclic aromatic ring optimization)
5
6
CO2H
Y
X
Am
N R1
O
Compound
X
Y
R1
fXa (IC50: lM)
fIIa (IC50: lM)
PRCT (CT2: lM)
39a
40a
41a
42a
5a
5-Am
6-Am
6-Am
7-Am
5-Am
6-Am
5-Am
5-Am
6-Am
6-Am
5-Am
6-Am
5-Am
6-Am
5-Am
7-Am
5-Am
6-Am
5-Am
O
CH
CH
CH
CH
CH
CH
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.5
1.7
230
>1000
100
14
16
O
CH@CH
CH@CH
4.1
0.2
22
>1600
100
3.2
3.1
8.4
9.5
1.45
3.5
2
S
0.35
0.8
5b
5c
S
>1000
380
190
NH
NH
NH
NMe
NEt
NEt
S
0.7
5d
5e
CH
CH
CH
CH
CH
N
0.17
0.27
0.3
>1000
110
5f
5g
1.6
>800
8.4
140
8
5h
0.16
1.1
1.15
8
21a
21b
43a
44a
6a
S
N
8.4
0.5
>1600
15
72
6
O
CH
CH
CH
CH
N
C(NH)CH3
C(NH)CH3
C(NH)CH3
C(NH)CH3
C(NH)CH3
CH@CH
S
0.112
0.37
0.054
0.6
>1200
>1000
6.8
0.96
2.4
0.35
5
6h
22a
NEt
S
>600
a Compounds 39–44 have already been reported.2
longation of APPT even after 4 h against the control
(Table 4).
conversion of these two moieties could increase both
in vitro as well as ex vivo activities. One of the combina-
tions, conversion of the 6-amidino-1-ethylindole moiety
to the S1 site and the piperidine moiety to the aryl
binding site, would be favorable for fXa inhibitors.
4. Conclusion
Compound 38 exhibited potent and highly selective anti-
fXa activity and anticoagulant activity in vitro as well as
ex vivo test in rats.
5. Experimental section
5.1. General
€
Melting points were determined on a Buchi 520 appa-
ratus in glass capillary tubes and are uncorrected.
The optimization of the amidinonaphthalene moiety
and pyrrolidine moiety of DX-9065a showed that a

2104
S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
Table 2. In vitro anticoagulant and enzyme inhibitory activity (aliphatic heterocyclic ring optimization)
CO2H
Am
R2
O
R2
R3
fXa (IC50: lM)
fIIa (IC50: lM)
PRCT (CT2: lM)
44
C(NH)CH3
0.112
0.062
0.056
0.088
>1200
330
200
0.96
0.7
N R3
28d
29d
30d
C(NH)CH2CH3
C(NH)CH2CH2CH3
C(NH)CPh
0.72
5.2
220
R3
N
27a
28a
29a
C(NH)CH3
C(NH)CH2CH3
C(NH)CH2CH2CH3
0.011
0.021
0.054
>1200
300
>1200
0.34
0.54
1.1
27b
27c
C(NH)CH3
C(NH)CH3
0.29
0.2
68
2.5
1.6
N R3
140
N R3
Table 3. In vitro anticoagulant and enzyme inhibitory activity (hybrid optimization)
CO2H
Y
X
Am
R4
O
X
S
Y
R4
fXa (IC50: lM)
fIIa (IC50: lM)
PRCT (CT2: lM)
NH
Me
N
34
36
5-Am
5-Am
CH
0.045
0.044
180
180
0.44
S
CH
CH
0.54
0.3
N
Me
HN
NH
Me
N
38
6-Am
NEt
0.011
2.5
Table 4. Ex vivo anticoagulant activity on oral administration
Test/control APTTa ratio (dose 100 mg/kg n ¼ 4)
Compound
0.5 h
1 h
2 h
4 h
DX-9065a
6h
1.63 0.09
2.69 0.20
2.12 0.08
1.68 0.08
4.07 0.66
1.51 0.11
3.60 0.20
2.18 0.07
1.64 0.08
3.96 0.44
1.48 0.04
2.41 0.06
1.69 0.04
1.59 0.04
3.37 0.17
1.28 0.04
1.66 0.06
1.39 0.09
1.47 0.03
2.19 0.16
27a
36
38
Values are means SE.
a Activated partial thromboplastin time. Methods are described in Section 5.
Column chromatography was performed on Merck sil-
ica gel 60 (particle size 0.060–0.200 or 0.040–0.063 mm)
and on Daiaion HP-20 (highly porous polymer type
synthetic adsorbent; Mitsubishi Chemical Industries).
Preparative HPLC was performed with a reverse-phase
ODS column (Sensyu Pak ODS-H-5301 20 · 300 mm), a
mobile phase of acetonitrile/water (5/95–10/90), and a
flow rate of 10 mL/min. Thin-layer chromatography
(TLC) was performed on Merck pre-coated TLC alu-
minum sheets silica gel 60 F254, and detected by UV
quenching at 254 nm or by spraying with phospho-
molybdic acid or ninhydrin. All analytical samples were
found to be homogeneous on TLC.
1H NMR spectra were recorded on a JEOL FX90Q or a
JEOL JNM-EX400 spectrometer and chemical shifts are

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2105
given in ppm (d) from tetramethylsilane as the internal
standard. Mass spectra were performed with a JEOL
JMS-AX505W (EI, CI), a JEOL JMS-HX110 (FD,
FAB), or a JEOL JMS-700 (HRMS) spectrometer. IR
spectra were recorded on a HITACHI 270–30 or
HORIBA FT-720 spectrometer.
several time points. After these blood samples were cen-
trifuged, the platelet-poor plasma samples were used for
measuring their activated thromboplastin times (APTT).
5.6. APTT
Plasma (20 lL) and saline (20 lL) or a saline solution of
the inhibitors were mixed with Platern Plus Activator
(Enzyme Research Laboratories, Inc.) (20 lL) in the
process tube, and the coagulation was started by the
addition of 20 mM CaCl2 (20 lL).
5.2. Anticoagulant activity. Plasma recalcification time (PRCT)
Plasma was mixed with saline (100 lL) or a saline
solution of the inhibitor in a glass tube and incubated
for 2 min at 37 ꢁC. Coagulation was started with the
addition of 20 mM CaCl2 solution (100 lL). Anticoag-
ulant activity was evaluated with the plasma clotting
time doubling concentration (CT2).
5.7. [(5-Cyano-1-benzothien-2-yl)methyl](triphenyl)phos-
phonium chloride 1a
To a stirred and ice-cooled solution of compound 7a
(4.0 g, 21 mmol) in Et2O (100 mL)–pyridine (10 drops)
was added a solution of SOCl2 (5.5 g, 46 mmol) in Et2O
(5.0 mL). After stirring for 2 h at room temperature, the
reaction mixture was poured onto ice-cold water and
extracted with benzene. The separated organic layer was
washed with satd NaHCO3 aq, dried over MgSO4, and
concentrated. The solution of the residue and triphenyl-
phosphine (7.2 g, 28 mmol) in xylene (100 mL) was he-
ated at reflux for 10 h. After cooling the reaction mix-
ture, collecting the precipitate gave a colorless powder
(6.3 g, 64%). Mp > 250 ꢁC. 1H NMR (CDCl3):
d 6.70 (2H, d, J ¼ 15:1 Hz), 7.30–8.10 (19H, m).
MS (FAB): m=z 434 (M)þ. HRMS (FAB) calcd
for C28H21NPS: 434.1132. Found: 434.1136. IR
(ATR): 2813, 2742, 2217, 1434, 1112, 1062, 890,
5.3. Anti-fXa activity
Anti-fXa activities were measured by using chromogenic
substrate S-2222 (KabiVitrum) and human fXa. fXa was
obtained from the activation of factor X (Enzyme Re-
search Laboratories, Inc.) by Russell viper venom. Sal-
ine (20 lL) or a saline solution of the inhibitor and
1 mM S-2222 (100 lL) were mixed with 0.1 M Tris–
0.2 M NaCl buffer pH 8.4 (360 lL). The reaction was
started with the addition of 0.5 unit/mL human fXa
solution (20 lL), and the mixture was incubated for
10 min at 37 ꢁC. The reaction was terminated by the
addition of 60% AcOH (100 lL), and the optical densi-
ties (OD) were measured (405 nm)
831 cmꢀ1
.
Anti-fXa activity (inhibition%) ¼
1 ) (OD of the inhibitor/OD of saline control) · 100.
The IC50 value was obtained by plotting the inhibitor
concentrations against the anti-fXa activity (inhibi-
tion%) on statistical probability paper.
5.8. [(6-Cyano-1-benzothien-2-yl)methyl](triphenyl)phos-
phonium chloride 1b
Starting with 7b and following the procedure for the
preparation of 1a gave 1b (yield, 64%) as colorless nee-
dle crystals. Mp > 250 ꢁC. 1H NMR (CDCl3): d 6.5 (2H,
d, J ¼ 17 Hz), 7.20–8.00 (19H, m). MS (FAB): m=z 434
(M)þ. HRMS (FAB) calcd for C28H21NPS: 434.1132.
Found: 434.1108.
5.4. Anti-fIIa activity
Saline (100 lL; Tris–HCl buffered to pH 7.45) (TBS)
containing fibrinogen (6 mg/mL; Type 1, Daiichi Pure
Chemicals Co., Ltd) was mixed with saline or a saline
solution of the inhibitors (100 lL). After the addition of
a solution of thrombin (100 lL; 4 units/mL: Sankyo Co.,
Ltd) to the above mixture, the clotting time was mea-
sured at 37 ꢁC, and then a calibration curve was pre-
pared. Antithrombin activities (inhibition%) were
obtained by measuring the clotting time using solutions
of inhibitors in saline (100 lL). The IC50 value was
obtained from the percentage of inhibition.
5.9. [(5-Cyano-1H-indol-2-yl)methyl](triphenyl)phospho-
nium bromide 1d
Starting with 11a and following the procedure for the
preparation of 1h gave 1d (yield, 51%) as a colorless
amorphous mass. 1H NMR (CDCl3): d 5.42 (2H, d,
J ¼ 14:2 Hz), 7.50–7.75 (19H, m). MS (FAB): m=z 417
(M)þ. HRMS (FAB) calcd for C28H22N2P: 417.1521.
Found: 417.1549. IR (ATR): 3135, 2215, 1436, 1319,
1112, 804 cmꢀ1
.
5.5. Ex vivo anticoagulant activity on oral administration
Male Wistar rats (200–250 g) were fasted overnight. Syn-
thetic compounds were dissolved in water and adminis-
tered orally to rats with a stomach tube. Fifteen minutes
after administration, rats were anesthetized with sodium
thiopental (100 mg/kg, ip). Blood samples were collected
from the juglar vein (in the presence of trisodium citrate) at
5.10. [(6-Cyano-1H-indol-2-yl)methyl](triphenyl)phos-
phonium bromide 1e
Starting with 13a and following the procedure for the
preparation of 1h gave 1e (yield, 51%) as a colorless
powder. Mp 255–259 ꢁC. 1H NMR (CDCl3): d 5.83 (2H,

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S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
d, J ¼ 14:4 Hz), 7.15–7.80 (19H, m). MS (FAB): m=z 417
(M)þ. HRMS (FAB) calcd for C28H22N2P: 417.1521.
Found: 417.1521. IR (ATR): 3054, 2964, 2215, 1436,
oxy]-1-pyrrolidinecarboxylate (2) (2.6 g, 7.0 mmol) in
dry THF (50 mL)–EtOH (50 mL) was added DBU
(1.1 g, 7.0 mmol) at room temperature. The mixture was
stirred for 1 h at room temperature. After removal of the
solvent, the residue was purified by silica gel column
chromatography with toluene/ethyl acetate (19/1) as an
eluent, providing ethyl 2-(4-{[(3S)-1-(tert-butoxycar-
bonyl)pyrrolidin-3-yl]oxy}phenyl)-3-(5-cyano-1-benzoth-
ien-2-yl)-propenoate as a mixture of E and Z forms. The
mixture of E and Z and 10% palladium on carbon (50%
wet) (5.0 g) in THF (50 mL)–EtOH (50 mL) was shaken
at room temperature under a current of hydrogen
(1 atm). After filtration of the catalyst, followed by
evaporation of the filtrate, the residue was purified by
silica gel column chromatography with toluene/ethyl
acetate (19/1) as an eluent, yielding a viscous oil (2.2 g,
68%). 1H NMR (CDCl3): d 1.17 (3H, t, J ¼ 7:0 Hz), 1.47
(9H, s), 1.90–2.20 (2H, m), 3.10–3.95 (7H, m), 4.10 (2H,
q, J ¼ 7:0 Hz), 4.84 (1H, br), 6.81 (2H, d, J ¼ 9:0 Hz),
7.20 (1H, s), 7.25 (2H, d, J ¼ 9:0 Hz), 7.44 (1H, dd,
J ¼ 9:0, 1.6 Hz), 7.81 (1H, dd, J ¼ 9:0, 1.6 Hz), 7.94
(1H, d, J ¼ 1:6 Hz).
1307, 1110, 817 cmꢀ1
.
5.11. [(6-Cyano-1-methyl-1H-indol-2-yl)methyl](triphen-
yl)phosphonium bromide 1f
Starting with 13b and following the procedure for the
preparation of 1h gave 1f (yield, 77%) as a pale yellow
amorphous mass. 1H NMR (DMSO-d6): d 3.10–3.30
(3H, m), 5.53 (2H, br d, J ¼ 15:4 Hz), 7.20–8.10 (19H,
s). MS (FAB): m=z 431 (M)þ. HRMS (FAB) calcd for
C29H24N2P: 431.1677. Found: 431.1656. IR (ATR):
2892, 2829, 2769, 2223, 1513, 1473, 1438, 1432, 1340,
1182, 1108, 995, 908, 831 cmꢀ1
.
5.12. [(5-Cyano-1-ethyl-1H-indol-2-yl)methyl](triphen-
yl)phosphonium bromide 1g
Starting with 11b and following the procedure for the
preparation of 1h gave 1g (yield, 70%) as a colorless
powder. Mp > 270 ꢁC. 1H NMR (CDCl3+CD3OD): d
1.12 (3H, t, J ¼ 7:2 Hz), 3.75–3.85 (2H, m), 5.29 (2H, br
d, J ¼ 13:9 Hz), 7.30–7.95 (19H, s). MS (FAB): m=z 445
(M)þ. HRMS (FAB) calcd for C30H26N2P: 445.1834.
Found: 445.1841. IR (ATR): 2987, 2842, 2778, 2219,
5.15. Ethyl 2-(4-{[(3S)-1-(tert-butoxycarbonyl)pyrrolidin-
3-yl]oxy}phenyl)-3-(6-cyano-1-benzothien-2-yl)propano-
ate 3b
1481, 1436, 1340, 1110, 921, 800 cmꢀ1
.
Starting with 1b and following the procedure for the
preparation of 3a gave 3b (yield, 81%) as a colorless
viscous oil. 1H NMR (CDCl3):
d 1.17 (3H, t,
5.13. [(6-Cyano-1-ethyl-1H-indol-2-yl)methyl](triphe-
nyl)phosphonium bromide 1h
J ¼ 7:5 Hz), 1.46 (9H, s), 1.90–2.30 (2H, m), 3.20–3.75
(6H, m), 3.80–3.95 (1H, m), 4.10 (2H, q, J ¼ 7:5 Hz),
4.80–5.00 (1H, br), 6.75–7.00 (2H, m), 7.04 (1H, s),
7.15–7.40 (2H, m), 7.50–7.85 (2H, m), 8.04 (1H, s).
To a stirred and ice-cooled solution of compound 13c
(14 g, 61 mmol) in THF (200 mL) was added CaI2 (34 g)
and NaBH4 (10 g). After stirring for 2 h at room tem-
perature, to the mixture were added glacial acetic acid
and AcOEt. The separated organic layer was dried over
Na2SO4. After removal of the solvent, the residue was
purified by silica gel column chromatography with
CHCl3/MeOH (98/2) as an eluent, providing alcohol
14c. To a stirred and ice-cooled solution of the obtained
alcohol 14c in CH2Cl2 (200 mL) was added PBr3
(6.0 mL). After stirring for 1 h at room temperature, to
the mixture was added ice-cold water and the mixture
was neutralized with Na2CO3. The separated organic
layer was dried over Na2SO4. The solution of the residue
and triphenylphosphine (7.2 g, 28 mmol) in CH2Cl2 was
heated at reflux for 1 h. After cooling the reaction mix-
ture, collecting the precipitate gave 1h as a colorless
5.16. Ethyl 2-(4-{[(3S)-1-(tert-butoxycarbonyl)pyrrolidin-
3-yl]oxy}phenyl)-3-(5-cyano-1H-benzimidazol-2-yl)pro-
panoate 3c
To a stirred solution of compound 10 (1.0 g, 5.6 mmol) in
1,2-dichloroethane (30 mL) was added triphenylphos-
phine (2.2 g, 8.4 mmol). The reaction mixture was heated
at 140 ꢁC for 1 h. Removal of the solvent gave crude
phosphonium salt 1c. To a solution of phosphonium salt
1c and tert-butyl (3S)-3-[4-(2-ethoxy-2-oxoacetyl)phen-
oxy]-1-pyrrolidinecarboxylate 2 (2.0 g, 5.6 mmol) in dry
THF (20 mL)–EtOH (20 mL) was added DBU (1.1 g,
7.2 mmol) at room temperature. The mixture was stirred
for 72 h at room temperature. After removal of the sol-
vent, the residue was purified by silica gel column chro-
matography with CHCl3/EtOH (98/2) as an eluent,
providing ethyl 2-(4-{[(3S)-1-(tert-butoxycarbonyl)pyrr-
olidin-3-yl]oxy}phenyl)-3-(5-cyano-1H-benzimidazol-
2-yl)propenoate as a mixture of E and Z forms. The
mixture of E and Z and PdO(H2O)BaSO4 (palladium
oxide hydrate barium sulfate)10 (1.5 g) in THF (50 mL)–
EtOH (50 mL) was shaken at room temperature under a
current of hydrogen (1 atm). After filtration of the cata-
lyst, followed by evaporation of the filtrate, the residue
was purified by silica gel column chromatography with
CHCl3/EtOH (99/1) as an eluent, yielding a colorless
1
powder (17 g, 59%). Mp > 270 ꢁC. H NMR (CDCl3): d
1.00–1.15 (3H, m), 3.75–3.85 (2H, m), 5.75 (2H, br d,
J ¼ 13:7 Hz), 7.25–7.85 (19H, s). MS (FAB): m=z 445
(M)þ. HRMS (FAB) calcd for C30H26N2P: 445.1834.
Found: 445.1812. IR (ATR): 2985, 2840, 2773, 2213,
1714, 1436, 1334, 1187, 1108, 871, 835 cmꢀ1
.
5.14. Ethyl 2-(4-{[(3S)-1-(tert-butoxycarbonyl)pyrrolidin-
3-yl]oxy}phenyl)-3-(5-cyano-1-benzothien-2-yl)propano-
ate 3a
To a solution of phosphonium salt 1a (3.0 g, 6.4 mmol)
and tert-butyl (3S)-3-[4-(2-ethoxy-2-oxoacetyl)phen-

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2107
amorphous form (0.32 g, 11%). 1H NMR (CDCl3): d 1.14
(3H, t, J ¼ 7:0 Hz), 1.48 (9H, s), 1.90–2.30 (2H, m), 3.05–
3.90 (7H, m), 4.12 (2H, q, J ¼ 7:0 Hz), 4.00–4.30 (1H, br),
4.70–4.95 (1H, br), 6.79 (2H, d, J ¼ 8:8 Hz), 7.19 (2H, d,
J ¼ 8:8 Hz), 7.35–8.10 (3H, m). MS (FD): m=z 505
(M+H)þ.
(2H, br), 3.00–4.80 (7H, m), 5.00–5.20 (1H, br), 6.93
(2H, d, J ¼ 9:0 Hz), 7.34 (2H, d, J ¼ 9:0 Hz), 7.91 (2H,
s), 8.28 (1H, s), 9.36 (2H, br), 9.61 (2H, br), 9.40–10.10
(2H, br). MS (FAB): m=z 394 (M+H)þ. HRMS (FAB)
calcd for C21H24N5O3: 394.1879. Found: 394.1859. IR
(KBr): 1725, 1680, 1509, 1435 cmꢀ1. Anal. Calcd for
C21H23N5O3Æ3HClÆ1.5H2O: C, 47.60; H, 5.52; N, 13.22.
Found: C, 47.34; H, 5.80; N, 13.31.
5.17. 3-(5-Amidino-1-benzothien-2-yl)-2-{4-[(3S)-pyrroli-
din-3-yloxy]phenyl}propanoic acid 5a
5.20. 3-(5-Amidino-1H-indol-2-yl)-2-{4-[(3S)-pyrrolidin-
3-yloxy]phenyl}propanoic acid 5d
A solution of 3a (2.2 g, 4.2 mmol) in dry EtOH (100 mL)
was saturated with HCl gas with ice cooling and was left
to stand for 24 h at room temperature. After distilling off
the solvent and HCl, the resulting residue was dissolved
in ethanolic ammonia solution (14% w/v), and the whole
mixture was left to stand for 24 h at room temperature.
After removal of the solvent, the resulting residue was
purified by HP-20 column chromatography (acetoni-
trile/H2O, 5/95). After the addition of a small amount of
concentrated HCl to selected fractions, the solvents were
removed to give ethyl 3-(5-amidino-1-benzothien-2-yl)-
2-{4-[(3S)-pyrrolidin-3-yloxy]phenyl}propanoate as a
colorless amorphous solid (1.9 g, 83%). A solution of
ethyl 3-(5-amidino-1-benzothien-2-yl)-2-{4-[(3S)-pyrro-
lidin-3-yloxy]phenyl}propanoate (1.2 g, 2.2 mmol) in
2 N HCl was heated at reflux for 30 min. After evapo-
ration of the solvent, the residue was purified by pre-
parative HPLC. After the addition of a small amount of
concentrated HCl to selected fractions, the solvents were
removed to give a colorless amorphous solid (0.70 g,
62%). 1H NMR (DMSO-d6): d 1.90–2.40 (2H, br), 3.00–
4.10 (7H, m), 5.14 (1H, br), 6.93 (2H, d, J ¼ 8:2 Hz),
7.28 (1H, s), 7.33 (2H, d, J ¼ 8:2 Hz), 7.70 (1H, d,
J ¼ 8:8 Hz), 8.09 (1H, d, J ¼ 8:8 Hz), 8.26 (1H, s), 9.24
(2H, br), 9.47 (2H, br), 9.00–10.20 (2H, br). IR (KBr):
To a solution of phosphonium salt 1d (5.0 g, 10 mmol)
and tert-butyl (3S)-3-[4-(2-ethoxy-2-oxoacetyl)phen-
oxy]-1-pyrrolidinecarboxylate 2 (3.7 g, 10 mmol) in dry
THF (50 mL)–MeOH (50 mL) was added DBU (1.8 g,
12 mmol) at room temperature. The mixture was stirred
for 2 h at room temperature. After removal of the sol-
vent, the residue was purified by silica gel column
chromatography with CH2Cl2/acetone (99/1) as an elu-
ent, providing the ester as a mixture of E and Z forms.
The mixture of E and Z and PdO(H2O)BaSO4 (palla-
dium oxide hydrate barium sulfate) (5.0 g) in THF
(50 mL)–MeOH (100 mL) was shaken at room temper-
ature under a current of hydrogen (1 atm). After filtra-
tion of the catalyst, followed by evaporation of the
filtrate, the residue was purified by silica gel column
chromatography with CH2Cl2/acetone (98/2) as an elu-
ent, providing an ester compound. A solution of the
ester compound in dry EtOH (150 mL)–CH2Cl2
(100 mL) was saturated with HCl gas with ice cooling
and left to stand for 24 h at room temperature. After
distilling off the solvents and HCl, the resulting residue
was dissolved in ethanolic ammonia solution (14% w/v)
(100 mL), and the whole mixture was left to stand for
24 h at room temperature. After removal of the solvent,
a solution of the resulting residue in 2 N HCl (200 mL)
was heated at reflux for 30 min. After evaporation of the
solvent, the resulting residue was purified by HP-20
column chromatography (acetonitrile/H2O, 7/93). After
the addition of a small amount of concentrated HCl to
selected fractions, the solvents were removed to give a
colorless amorphous solid (2.3 g, 47%). 1H NMR
(DMSO-d6): d 2.00–2.35 (2H, m), 2.90–3.80 (6H, m),
4.00–4.30 (1H, m), 5.00–5.30 (1H, br), 6.37 (1H, s), 7.00
(2H, d, J ¼ 8:0 Hz), 7.40 (2H, d, J ¼ 8:0 Hz), 7.60 (2H, s),
8.10 (1H, s), 8.90–10.10 (6H, m), 11.6 (1H, s). MS (FAB):
m=z 393 (M+H)þ. HRMS (FAB) calcd for C22H25N4O3:
1674,
1509,
1239 cmꢀ1
.
Anal.
Calcd
for
C22H23N3O3SÆ2HClÆ1.5H2O: C, 51.87; H, 5.54; N, 8.25.
Found: C, 52.10; H, 5.92; N, 8.32.
5.18. 3-(6-Amidino-1-benzothien-2-yl)-2-{4-[(3S)-pyrrol-
idin-3-yloxy]phenyl}propanoic acid 5b
Starting with 3b and following the procedure for the
preparation of 5a gave 5b (yield, 34%) as a colorless
amorphous solid. 1H NMR (DMSO-d6): d 2.00–2.30
(2H, m), 3.00–4.05 (7H, m), 5.09 (1H, br s), 6.96 (2H, d,
J ¼ 8:3 Hz), 7.20–7.40 (3H, m), 7.60–7.80 (1H, m), 8.42
(1H, s), 9.00–9.80 (6H, m). MS (FAB): m=z 410
(M+H)þ. HRMS (FAB) calcd for C22H24N3O3S:
410.1538. Found: 410.1541. IR (ATR): 1725, 1680,
393.1927. Found: 393.1934. IR (KBr): 1720, 1670 cmꢀ1
.
1509,
1435 cmꢀ1
.
Anal.
Calcd
for
5.21. 3-(6-Amidino-1H-indol-2-yl)-2-{4-[(3S)-pyrrolidin-
3-yloxy]phenyl}propanoic acid 5e
C22H23N3O3SÆ2HClÆH2O: C, 52.80; H, 5.44; N, 8.40.
Found: C, 53.00; H, 5.64; N, 8.57.
Starting with 1e and following the procedure for the
preparation of 5d gave 5e (yield, 10%) as a brown
amorphous solid. 1H NMR (DMSO-d6): d 1.90–2.30
(2H, m), 2.70–4.50 (4H, m), 5.16 (1H, br s), 6.36 (1H, s),
7.00 (2H, d, J ¼ 8:0 Hz), 7.25–7.80 (4H, m), 7.96 (1H, s),
9.20–9.50 (6H, m), 11.80 (1H, s). MS (FAB): m=z 393
(M+H)þ. HRMS (FAB) calcd for C22H25N4O3: 393.1927.
Found: 393.1934. IR (KBr): 3064, 1668, 1542, 1509cmꢀ1.
5.19. 3-(5-Amidino-1H-benzimidazol-2-yl)-2-{4-[(3S)-
pyrrolidin-3-yloxy]phenyl}propanoic acid 5c
Starting with 3c and following the procedure for the
preparation of 5a gave 5c (yield, 51%) as a colorless
amorphous solid. 1H NMR (DMSO-d6): d 1.98–2.28

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S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
5.22. 3-(6-Amidino-1-methyl-1H-indol-2-yl)-2-{4-[(3S)-
pyrrolidin-3-yloxy]phenyl}propanoic acid 5f
2.00–2.50 (5H, m), 3.10–4.20 (7H, m), 4.96 (1H, br), 6.93
(2H, d, J ¼ 7:9 Hz), 7.29 (1H, s), 7.34 (2H, d,
J ¼ 7:9 Hz), 7.73 (1H, d, J ¼ 8:3 Hz), 8.10 (1H, d,
J ¼ 8:3 Hz), 8.30 (1H, s), 8.50–9.30 (1H, br), 9.37 (2H,
br), 9.54 (3H, br). MS (FAB): m=z 451 (M+H)þ. HRMS
(FAB) calcd for C24H27N4O3S: 451.1804. Found:
Starting with 1f and following the procedure for the
preparation of 5d gave 5f (yield, 50%) as a colorless
amorphous solid. 1H NMR (DMSO-d6): d 2.00–2.40
(2H, m), 3.00–3.90 (6H, m), 3.79 (3H, s), 4.00–4.30 (1H,
m), 5.15–5.35 (1H, m), 6.50 (1H, s), 7.10 (2H, d,
J ¼ 7:9 Hz), 7.25–7.75 (4H, m), 8.35 (1H, s), 8.90–9.90
(6H, m). MS (FAB): m=z 407 (M+H)þ. HRMS (FAB)
calcd for C23H27N4O3: 407.2083. Found: 407.2091. IR
451.1784. IR (KBr): 1674, 1509, 1449 cmꢀ1
.
5.26. 3-(6-Amidino-1-ethyl-1H-indol-2-yl)-2-{4-[(3S)-1-
ethanimidoylpyrrolidin-3-yloxy]phenyl}propanoic acid 6h
(KBr): 1720, 1665, 1615 cmꢀ1
. Anal. Calcd for
A solution of compound 5h (1.2 g, 2.3 mmol) in EtOH–
SOCl2 (2.0 mL) was heated at reflux for 1 h. After
evaporation of the solvent, the residue was purified by
HP-20 column chromatography (acetonitrile/H2O, 10/
90–20/80), providing an ester derivative. The obtained
ester derivative was dissolved in EtOH (30 mL). To the
stirred and ice-cooled solution were added triethylamine
(1.0 mL, 7.2 mmol) and ethyl acetimidate hydrochloride
(0.20 g, 1.6 mmol). After stirring for 3 h at room tem-
perature, the solvent was distilled off. A solution of the
obtained residue in 2 N HCl (50 mL) was heated at
reflux for 30 min. After evaporation of the solvent, the
residue was purified by HP-20 column chromatography
(acetonitrile/H2O, 5/95–10/90). After the addition of a
small amount of concentrated HCl to selected fractions,
the solvents were removed to give a colorless amorphous
solid (0.95 g, 75%). 1H NMR (DMSO-d6): d 1.31 (3H, t,
J ¼ 7:1 Hz), 2.15–2.35 (2H, m), 2.29 (3H, d,
J ¼ 12:7 Hz), 3.10–3.20 (1H, m), 3.40–4.00 (5H, m),
4.10–4.20 (1H, m), 4.20–4.40 (2H, m), 5.19 (1H, d,
J ¼ 25:9 Hz), 6.38 (1H, s), 6.90–7.05 (2H, m), 7.35–7.45
(2H, m), 7.48 (1H, d, J ¼ 8:9 Hz), 7.62 (1H, d,
J ¼ 8:5 Hz), 8.16 (1H, s), 8.57 (1H, d, J ¼ 35:6 Hz), 9.05
(2H, s), 9.30 (2H, s), 9.37 (2H, d, J ¼ 15:1 Hz). MS
(FAB): m=z 461 (M+H)þ. IR (ATR): 3052, 1668, 1610,
1508, 1463, 1340, 1234, 1172, 1087, 821 cmꢀ1. Anal.
Calcd for C26H31N5O3Æ2HClÆH2O: C, 56.52; H, 6.39; N,
12.68. Found: C, 56.31; H, 6.43; N, 12.68.
C23H26N4O3Æ2HClÆ0.5H2O: C, 56.56; H, 5.98; N, 11.47.
Found: C, 56.89; H, 5.99; N, 11.50.
5.23. 3-(5-Amidino-1-ethyl-1H-indol-2-yl)-2-{4-[(3S)-
pyrrolidin-3-yloxy]phenyl}propanoic acid 5g
Starting with 1g and following the procedure for the
preparation of 5d gave 5g (yield, 6.8%) as a colorless
amorphous solid. 1H NMR (DMSO-d6): d 1.10–1.40
(3H, m), 1.95–2.30 (2H, m), 2.90–4.60 (9H, m), 5.10–
5.30 (1H, br), 6.37 (1H, s), 6.92 (2H, d, J ¼ 8:0 Hz),
7.30–7.80 (4H, m), 8.17 (1H, s), 8.90–10.10 (6H, m). MS
(FAB): m=z 421 (M+H)þ. HRMS (FAB) calcd for
C24H29N4O3: 421.2240. Found: 421.2249. IR (ATR):
3392, 1722, 1668, 1610, 1552, 1510, 1468, 1404, 1348,
1238, 1178 cmꢀ1
.
Anal. Calcd for C24H28N4O3Æ
2HClÆ1.5H2O: C, 55.38; H, 6.39; N, 10.77. Found: C,
55.35; H, 6.17; N, 10.65.
5.24. 3-(6-Amidino-1-ethyl-1H-indol-2-yl)-2-{4-[(3S)-
pyrrolidin-3-yloxy]phenyl}propanoic acid 5h
Starting with 1h and following the procedure for the
preparation of 5d gave 5h (yield, 46%) as a colorless
amorphous solid. 1H NMR (DMSO-d6): d 1.10–1.40
(3H, m), 1.95–2.30 (2H, m), 2.90–3.80 (6H, m), 3.90–
4.45 (3H, m), 5.00–5.20 (1H, br), 6.37 (1H, s), 6.92 (2H,
d, J ¼ 8:0 Hz), 7.30–7.70 (4H, m), 8.11 (1H, s), 8.90–
10.10 (6H, m). MS (FAB): m=z 421 (M+H)þ. HRMS
(FAB) calcd for C24H29N4O3: 421.2240. Found:
421.2245. IR (KBr): 1713, 1668, 1614 cmꢀ1. Anal. Calcd
for C24H28N4O3Æ2HClÆ2H2O: C, 54.44; H, 6.47; N,
10.58. Found: C, 54.17; H, 6.50; N, 10.53.
5.27. (5-Cyano-1H-benzimidazol-2-yl)methyl chloride 10
To a stirred solution of compound 8 (3.42 g, 25.7 mmol)
in dry ethanol (100 mL) was added chloroacetimino
ethyl ether hydrochloride (3.42 g, 25.7 mmol). The
reaction mixture was heated at reflux for 3 h. After re-
moval of the solvent, the residue was partitioned be-
tween AcOEt and water. The separated organic layer
was dried over MgSO4. After concentration of the sol-
vent, the precipitate was collected and dried to give a
colorless powder (2.7 g, 55%). Mp 144–146 ꢁC. 1H NMR
(CDCl3): d 4.83 (2H, s), 7.48 (2H, d, J ¼ 7:1 Hz), 7.57
(2H, d, J ¼ 7:1 Hz), 7.95 (1H, s). IR (KBr): 2224, 1626,
1455, 1422, 1311 cmꢀ1. No further purification was at-
tempted on this compound, which was used directly in
the next step.
5.25. 3-(5-Amidino-1-benzothien-2-yl)-2-{4-[(3S)-1-ethan-
imidoylpyrrolidin-3-yloxy]phenyl}propanoic acid 6a
To a stirred and ice-cooled solution of compound 5a
(2.0 g, 3.8 mmol) in H2O (40 mL) was added ethyl ace-
timidate hydrochloride (2.4 g, 19 mmol) in small por-
tions while the reaction mixture was maintained at
pH 7.5–8.5 with 1 N NaOH aq. After stirring for further
15 min with cooling in an ice bath, the reaction mixture
was adjusted to pH 1.0 and concentrated to dryness. The
obtained residue was purified by HP-20 column chro-
matography (acetonitrile/H2O, 5/95). After the addition
of a small amount of concentrated HCl to selected
fractions, the solvents were removed to give a colorless
amorphous solid (0.70 g, 33%). 1H NMR (DMSO-d6): d
5.28. Ethyl 5-cyano-1-ethyl-1H-indole-2-carboxylate 11b
Starting with 11a and following the procedure for the
preparation of 13c gave 11b (yield, 95%) as a colorless

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2109
amorphous mass. 1H NMR (CDCl3): d 1.41 (3H, t,
J ¼ 7:2 Hz), 1.43 (3H, t, J ¼ 7:2 Hz), 4.40 (2H, q,
J ¼ 7:2 Hz), 4.64 (2H, q, J ¼ 7:2 Hz), 7.36 (1H, s), 7.46
(1H, d, J ¼ 8:8 Hz), 7.50–7.60 (1H, m), 8.00–8.10 (1H,
m). MS (FAB): m=z 243 (M+H)þ. HRMS (FAB) calcd
for C14H15N2O2: 243.1134. Found: 243.1142. IR (KBr):
J ¼ 7:1 Hz), 4.52 (1H, s), 4.75–4.95 (1H, m), 6.82 (2H, d,
J ¼ 10 Hz), 7.28 (2H, d, J ¼ 10 Hz). MS (FAB): m=z 444
(M+Na)þ. IR (KBr): 2977, 1731, 1691, 1509, 1402, 1365,
1241, 1216, 1162, 1114, 1 9129 cmꢀ1. Anal. Calcd for
C22H31NO7: C, 62.69; H, 7.41; N, 3.32. Found: C, 62.84;
H, 7.53; N, 3.28.
2220, 1714 cmꢀ1
.
5.32. 2-(Bromomethyl)-1,3-benzothiazole-5-carbonitrile
19a
5.29. Methyl 6-cyano-1-methyl-1H-indole-2-carboxylate
13b
To a solution of 2-methyl-1,3-benzothiazole-6-carbo-
nitrile 18a (7.46 g, 43 mmol) in CCl4 (250 mL) were ad-
Starting with 13a and following the procedure for the
preparation of 13c gave 13b (yield, 87%) as a colorless
amorphous mass. H NMR (DMSO-d6): d 3.92 (3H, s),
4.10 (3H, s), 7.42 (1H, s), 7.52 (1H, dd, J ¼ 8:0, 1.5 Hz),
7.98 (1H, d, J ¼ 8:0 Hz), 8.30–8.40 (1H, br). MS (FAB):
m=z 215 (M+H)þ. HRMS (FAB) calcd for C12H11N2O2:
215.0821. Found: 215.0808. IR (KBr): 2220, 1730,
1720 cmꢀ1. Anal. Calcd for C12H10N2O2: C, 67.28; H,
4.71; N, 13.08. Found: C, 67.23; H, 4.97; N, 12.99.
ded
NBS
(7.62 g,
43 mmol)
and
2,20-
1
azobisisobutylonitrile (0.15 g, 0.91 mmol). The reaction
mixture was refluxed and irradiated with light for
1 night. The resultant suspension was filtered, and the
filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography with tol-
uene as an eluent, yielding colorless prismatic crystals
(2.18 g, 20%). Mp 185–186 ꢁC. 1H NMR (CDCl3): d 4.83
(2H, s), 7.67 (1H, dd, J ¼ 9:1, 1.7 Hz), 7.97 (1H, d,
J ¼ 9:1 Hz), 8.15 (1H, d, J ¼ 1:7 Hz). IR (KBr):
2232 cmꢀ1. Anal. Calcd for C9H5BrN2S: C, 42.71; H,
1.99; N, 11.07. Found: C, 42.55; H, 1.98; N, 11.00.
5.30. Methyl 6-cyano-1-ethyl-1H-indole-2-carboxylate
13c
To a stirred and ice-cooled solution of compound 13a
(15 g, 75 mmol) in DMF (100 mL) was added NaH (60%
in oil) (3.1 g). After stirring for 20 min at room temper-
ature, to the reaction mixture was added EtI (6.8 mL,
85 mmol) followed by stirring for 2 h at room tempera-
ture. To the mixture were added water and AcOEt/tolu-
ene. The separated organic layer was dried over Na2SO4.
After removal of the solvent, the residue was purified by
silica gel column chromatography with CHCl3 as an
eluent, yielding a colorless amorphous mass (14 g, 83%).
1H NMR (CDCl3): d 1.42 (3H, t, J ¼ 7:5 Hz), 3.97 (3H,
s), 4.65 (2H, q, J ¼ 7:5 Hz), 7.30–7.45 (2H, m), 7.70–7.90
(2H, m). MS (FAB): m=z 229 (M+H)þ. HRMS (FAB)
calcd for C13H13N2O2: 229.0977. Found: 229.0975. IR
5.33. 2-(Bromomethyl)-1,3-benzothiazole-6-carbonitrile
19b
Starting with 18b and following the procedure for the
preparation of 19a gave 19b (yield, 49%) as pale yellow
prismatic crystals. Mp 117–119 ꢁC. 1H NMR (CDCl3): d
4.84 (2H, s), 7.77 (1H, dd, J ¼ 10, 1.7 Hz), 8.02 (1H, d,
J ¼ 10 Hz), 8.20 (1H, d, J ¼ 1:7 Hz). IR (KBr):
2224 cmꢀ1. Anal. Calcd for C9H5BrN2S: C, 42.71; H,
1.99; N, 11.07. Found: C, 42.52; H, 2.00; N, 11.20.
5.34. 3-(5-Amidino-1,3-benzothiazol-2-yl)-2-{4-[(3S)-
pyrrolidin-3-yloxy]phenyl}propanoic acid 21a
(KBr): 2220, 1720, 1512 cmꢀ1
.
To a stirred and ice-cooled solution of compound 17
(6.3 g, 15 mmol) in THF (80 mL) was added NaH (60%
in oil) (0.60 g). After stirring for a few minutes at room
temperature, to the reaction mixture was added THF
(100 mL) solution of 2-(bromomethyl)-1,3-benzothiaz-
ole-5-carbonitrile 19a (3.2 g, 13 mmol) and stirred for
3 days at room temperature. To the mixture were added
ice-cooled concentrated HCl and AcOEt. The separated
organic layer was washed with water and dried over
Na2SO4. After removal of the solvent, the residue was
purified by silica gel column chromatography with
CHCl3 as an eluent, yielding a yellow oil 20a (7.7 g,
quant.). A solution of 20a (7.7 g, 14 mmol) in dry EtOH
(250 mL) was saturated with HCl gas with ice cooling
and left to stand for 24 h at room temperature. After
distilling off the solvents and HCl, the resulting residue
was dissolved in ethanolic ammonia solution (14% w/v),
and the whole was left to stand for 24 h at room tem-
perature. After removal of the solvent, a solution of the
resulting residue in 2 N HCl (100 mL) was heated at
reflux for 1.5 h. After evaporation of the solvent, the
5.31. Diethyl 2-(4-{[(3S)-1-(tert-butoxycarbonyl)pyrroli-
din-3-yl]oxy}phenyl)malonate 17
To a stirred solution of diethyl 2-(4-hydroxyphen-
yl)malonate 15 (4.7 g, 19 mmol), tert-butyl (3R)-3-
hydroxy-1-pyrrolidinecarboxylate 16 (4.7 g, 25 mmol) and
triphenylphosphine (6.58 g, 25 mmol) in anhydrous THF
(150 mL) was added diethyl azodicarboxylate (4.37 g,
25 mmol) at room temperature. The resultant solution
was stirred for 1 day. Additional tert-butyl-(3R)-3-
hydroxy-1-pyrrolidinecarboxylate 16 (1.0 g, 5.3 mmol)
and triphenylphosphine (1.6 g, 6.1 mmol) and diethyl
azodicarboxylate (1.0 g, 5.7 mmol) were added to the
reaction mixture. The resultant solution was stirred for a
further 1 day at room temperature. After removal of the
solvent, the residue was purified by silica gel column
chromatography with the solvent system toluene/AcOEt
1
(19/1), providing a colorless oil (5.5 g, 69%). H NMR
(CDCl3): d 1.25 (6H, t, J ¼ 7:1 Hz), 1.49 (9H, s), 2.00–
2.10 (2H, m), 3.35–3.70 (4H, m), 4.20 (2H, q,

2110
S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
residue was purified by HP-20 column chromatography
(acetonitrile/H2O, 5/95) and purified by preparative
HPLC. After the addition of a small amount of con-
centrated HCl to selected fractions, the solvents were
removed to give a colorless amorphous solid (1.1 g,
17%). 1H NMR (DMSO-d6): d 2.00–2.30 (2H, br), 2.80–
4.40 (7H, m), 5.00–5.20 (1H, br), 6.95 (2H, d,
J ¼ 8:3 Hz), 7.34 (2H, d, J ¼ 8:3 Hz), 7.70–7.90 (1H, m),
8.29 (1H, d,J ¼ 8:3 Hz), 8.35–8.45 (1H, m), 9.15–9.70
(6H, m). MS (FAB): m=z 411 (M+H)þ. IR (KBr): 3030,
piperidinecarboxylate 32a (5.2 g, 26 mmol) and triphen-
ylphosphine (10 g, 39 mmol) in anhydrous THF (40 mL)
was added diethyl azodicarboxylate (6.7 g, 39 mmol) at
room temperature. The resultant solution was stirred for
24 h. After removal of the solvent, the residue was
purified by silica gel column chromatography with the
solvent system hexane/AcOEt (7/3–1/1), providing a
yellow oil (5.6 g, 58%). 1H NMR (CDCl3): d 1.35 (3H, t,
J ¼ 6 Hz), 1.49 (9H, s), 1.80–2.00 (4H, m), 3.20–4.00
(4H, m), 4.46 (2H, q, J ¼ 6 Hz), 4.60–4.80 (1H, m), 7.01
(2H, d, J ¼ 8 Hz), 8.04 (2H, d, J ¼ 8 Hz). MS (FAB):
m=z 378 (M+H)þ. HRMS (FAB) calcd for C20H28NO6:
378.1917. Found: 378.1872. IR (ATR): 2975, 2935,
2871, 1673, 1594, 1421, 1365, 1257, 1232, 1207, 1159,
1678,
1600,
1510 cmꢀ1
.
Anal.
Calcd
for
C21H22N4O3SÆ2HClÆH2O: C, 50.31; H, 5.23; N, 11.17.
Found: C, 50.17; H, 5.58; N, 11.12.
1022, 970, 848 cmꢀ1
.
5.35. 3-(6-Amidino-1,3-benzothiazol-2-yl)-2-{4-[(3S)-
pyrrolidin-3-yloxy]phenyl}propanoic acid 21b
5.38. tert-Butyl (3R)-3-[4-(2-ethoxy-2-oxoacetyl)phen-
oxy]-1-pyrrolidinecarboxylate 24b
Starting with 19b (3.3 g, 13 mmol) and following the
procedure for the preparation of 21a gave 21b (510 mg,
7.8%) as a pale brown amorphous solid. 1H NMR
(DMSO-d6): d 2.00–2.30 (2H, m), 3.00–4.35 (7H, m),
5.00–5.20 (1H, br), 6.95 (2H, d, J ¼ 8:3 Hz), 7.35 (2H, d,
J ¼ 8:3 Hz), 7.90 (2H, dd,J ¼ 8:3, 1.3 Hz), 8.13 (1H, d,
J ¼ 8:3 Hz), 8.60 (1H, d, J ¼ 1:3 Hz), 9.20–9.80 (6H, m).
MS (FAB): m=z 411 (M+H)þ. HRMS (FAB) calcd for
C21H23N4O3: 411.1491. Found: 411.1513. IR (KBr):
Starting with 32b and following the procedure for the
preparation of 24a gave 24b (yield, 80%) as a yellow oil.
1H NMR (CDCl3): d 1.40 (3H, t, J ¼ 7:2 Hz), 1.46 (9H,
s), 2.00–2.30 (2H, m), 3.40–3.70 (4H, m), 4.43 (2H, q,
J ¼ 7:2 Hz), 4.90–5.10 (1H, m), 6.95 (2H, d, J ¼ 9:0 Hz),
7.99 (2H, d, J ¼ 9:0 Hz). MS (EI): m=z 363 (M+H)þ.
HRMS (EI) calcd for C19H25NO6: 363.1682. Found:
363.1691.
3010,
1677,
1608 cmꢀ1
.
Anal.
Calcd
for
C21H22N4O3SÆ2HClÆ1.5H2O: C, 49.41; H, 5.33; N, 10.98.
Found: C, 49.23; H, 5.29; N, 10.95.
5.39. tert-Butyl (2S)-2-[4-(2-ethoxy-2-oxoacetyl)phen-
oxy]methyl-1-pyrrolidinecarboxylate 24c
5.36. 3-(5-Amidino-1,3-benzothiazol-2-yl)-2-{4-[(3S)-1-
ethanimidoylpyrrolidin-3-yloxy]phenyl}propanoic acid
22a
Starting with 32c and following the procedure for the
preparation of 24a gave 24c (yield, 46%) as a pale yellow
1
oil. H NMR (CDCl3): d 1.41 (3H, t, J ¼ 10:7 Hz), 1.47
To a stirred and ice-cooled solution of compound 21a
(0.70 g, 1.4 mmol) in H2O (15 mL) was added ethyl
acetimidate hydrochloride (4.0 g, 3.2 mmol) in small
portions while the reaction mixture was maintained at
pH 7.5–8.5 with 1 N NaOH aq. After stirring for a fur-
ther 3 h with cooling on an ice bath, the reaction mixture
was adjusted to pH 1.0 and concentrated to dryness. The
obtained residue was purified by HP-20 column chro-
matography (acetonitrile/H2O, 5/95) and then pre-
parative HPLC (acetonitrile/H2O). After the addition of
a small amount of concentrated HCl to selected frac-
tions, the solvents were removed to give a colorless
amorphous solid (0.49 g, 63%). 1H NMR (DMSO-d6): d
2.26, 2.30 (total 3H, each s), 2.80–4.35 (9H, m), 5.10–
5.30 (1H, br), 6.99 (2H, d, J ¼ 7:9 Hz), 7.31 (2H, d,
J ¼ 7:9 Hz), 7.88 (2H, d, J ¼ 7:9 Hz), 8.25 (1H, d,
J ¼ 7:9 Hz), 8.40–8.70 (2H, m), 9.33, 9.55 (total 4H, br
s). IR (KBr): 3100, 1674, 1510 cmꢀ1. Anal. Calcd for
C23H25N5O3SÆ2HClÆ2.2H2O: C, 48.97; H, 5.61; N, 12.42.
Found: C, 49.19; H, 5.28; N, 12.07.
(9H, s), 1.65–2.15 (4H, br), 3.20–3.50 (2H, br), 3.80–4.30
(3H, m), 4.43 (2H, q, J ¼ 10:7 Hz), 7.0 (2H, d,
J ¼ 9:5 Hz), 7.95 (2H, d, J ¼ 9:5 Hz). MS (FAB): m=z
378 (M+H)þ. HRMS (FAB) calcd for C20H28NO6:
378.1917. Found: 378.1888. IR (ATR): 2975, 2879,
1733, 1675, 1596, 1571, 1509, 1388, 1365, 1307, 1263,
1203, 1157, 1106, 1016, 977, 842 cmꢀ1
.
5.40. Ethyl 2-(4-{[(3S)-1-(tert-butoxycarbonyl)pyrrolidin-
3-yl]oxy}phenyl)-3-(7-cyano-2-naphthyl)propanoate 25d
To
16.5 mmol) and tert-butyl (3S)-3-[4-(2-ethoxy-2-oxoace-
tyl)phenoxy]-1-pyrrolidinecarboxylate (5.00 g,
a solution of phosphonium salt 23 (8.40 g,
2
13.8 mmol) in dry THF (100 mL)–EtOH (100 mL) was
added DBU (2.51 g, 16.5 mmol) at room temperature.
The mixture was stirred for 3 h at room temperature.
After removal of the solvent, the residue was purified by
silica gel column chromatography with hexane/ethyl
acetate (7/3) as an eluent, providing ethyl 2-(4-{[(3S)-1-
(tert-butoxycarbonyl)pyrrolidin-3-yl]oxy}phenyl)-3-(7-
cyano-2-naphthyl)propenoate as a mixture of E and Z
forms. The mixture of E and Z and PdO(H2O)BaSO4
(palladium oxide hydrate barium sulfate) (2.0 g) in THF
(80 mL)–EtOH (80 mL) was shaken at room tempera-
ture under a current of hydrogen (1 atm). After filtration
5.37. tert-Butyl 4-[4-(2-ethoxy-2-oxoacetyl)phenoxy]-1-
piperidinecarboxylate 24a
To a stirred solution of ethyl 2-(4-hydroxyphenyl)-2-
oxoacetate 31 (5.0 g, 26 mmol), tert-butyl 4-hydroxy-1-

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2111
of the catalyst, followed by evaporation of the filtrate,
the residue was purified by silica gel column chroma-
tography with hexane/ethyl acetate (7/3) as an eluent,
perature. After removal of the solvent, the resulting
residue was purified by HP-20 column chromatography
(acetonitrile/H2O, 10/90). The solvents were removed to
give a colorless amorphous solid (3.00 g, 78%). 1H NMR
(DMSO-d6+HCl): d 1.01 (3H, t, J ¼ 7:1 Hz), 1.75–1.90
(2H, m), 2.05–2.15 (2H, m), 3.00–3.10 (2H, br), 3.15–
3.25 (3H, m), 3.54–3.50 (1H, m), 3.90–4.00 (2H, m), 4.08
(1H, t, J ¼ 7:8 Hz), 4.55–4.65 (1H, br), 6.95 (2H, d,
J ¼ 8:3 Hz), 7.29 (2H, d, J ¼ 8:3 Hz), 7.61 (1H, d,
J ¼ 8:3 Hz), 7.79 (1H, d, J ¼ 8:8 Hz), 7.84 (1H, s), 7.95
(1H, d, J ¼ 8:3 Hz), 8.08 (1H, d, J ¼ 8:8 Hz), 8.40 (1H,
s), 9.00–9.20 (2H, br), 9.26 (2H, s), 9.52 (2H, s). MS
(FAB): m=z 446 (M+H)þ. HRMS (FAB) calcd for
C27H32N3O3: 446.2444. Found: 446.2462. IR (KBr):
3348, 3112, 2948, 2800, 2736, 1716, 1670, 1608, 1508,
1454, 1374, 1248, 1214, 1170, 1134, 1108, 1062, 1026,
1
yielding a colorless viscous oil (6.24 g, 90%). H NMR
(CDCl3): d 1.10 (3H, t, J ¼ 7:0 Hz), 1.47 (9H, s), 2.00–
2.20 (2H, m), 3.00–3.30 (1H, m), 3.40–3.70 (4H, m),
3.80–4.20 (4H, m), 4.84 (1H, m), 6.80–8.20 (10H, m).
MS (EI): m=z 514 Mþ. HRMS (EI) calcd for
C31H34N2O5: 514.2468. Found: 514.2483.
5.41. Ethyl 2-(4-{[1-(tert-butoxycarbonyl)piperidin-4-
yl]oxy}phenyl)-3-(7-cyano-2-naphthyl)propanoate 25a
Starting with 24a and following the procedure for the
preparation of 25d gave 25a (yield, 80%) as a yellow
amorphous solid. 1H NMR (CDCl3): d 1.11 (3H, t,
J ¼ 6:0 Hz), 1.49 (9H, s), 1.70–2.00 (4H, m), 3.00–4.10
(9H, m), 4.45 (1H, m), 6.80–8.10 (10H, m). MS (FAB):
m=z 529 (M+H)þ. HRMS (FAB) calcd for C32H37N2O5:
529.2702. Found: 529.2655. IR (ATR): 2975, 2931,
2867, 2225, 1725, 1687, 1506, 1427, 1365, 1249, 1236,
1014, 856, 844, 820 cmꢀ1
.
5.45. Ethyl 3-(7-amidino-2-naphthyl)-2-{4-[(3R)-pyrrol-
idin-3-yloxy]phenyl}propanoate 26d
1170, 1149, 1132, 1081, 1049, 835 cmꢀ1
.
Starting with 25d and following the procedure for the
preparation of 26a gave 26d (yield, 56%) as a colorless
1
amorphous solid. H NMR (DMSO-d6): d 1.01 (3H, t,
J ¼ 7:0 Hz), 2.00–2.20 (2H, m), 3.10–3.80 (7H, m), 3.98
(2H, q, J ¼ 7:0 Hz), 5.10 (1H, m), 6.93 (2H, d,
J ¼ 9:0 Hz), 7.32 (2H, d, J ¼ 9:0 Hz), 7.50–8.10 (5H, m),
8.44 (1H, s), 9.41 (2H, br), 9.59 (2H, br), 9.30–10.00
(2H, br). MS (FAB): m=z 432 (M+H)þ. IR (KBr): 3382,
5.42. Ethyl 2-(4-{[(3R)-1-(tert-butoxycarbonyl)pyrrolidin-
3-yl]oxy}phenyl)-3-(7-cyano-2-naphthyl)propanoate 25b
Starting with 24b and following the procedure for the
preparation of 25d gave 25b (yield, 81%) as a yellow oil.
1H NMR (CDCl3): d 1.11 (3H, t, J ¼ 7:0 Hz), 1.47 (9H,
s), 2.00–2.20 (2H, m), 3.00–4.00 (7H, m), 4.06 (2H, q,
J ¼ 7:0 Hz), 4.85 (1H, m), 6.80–8.20 (10H, m). MS
(FAB): m=z 515 (M+H)þ. HRMS (FAB) calcd for
C31H35N2O5: 515.2546. Found: 515.2533. IR (ATR):
2975, 2875, 2225, 1720, 1681, 1508, 1411, 1365, 1245,
1725, 1680, 1611, 1506, 1239, 1059, 966, 915, 843 cmꢀ1
.
Anal. Calcd for C26H29N3O3Æ2HClÆ1.7H2O: C, 58.36; H,
6.49; N, 7.85. Found: C, 58.13; H, 6.49; N, 7.92.
5.46. 3-(7-Amidino-2-naphthyl)-2-{4-[(3R)-1-ethanimi-
doylpyrrolidin-3-yloxy]phenyl}propanoic acid 27b
1228, 1170, 1106, 1068, 898, 836, 813 cmꢀ1
.
A solution of 25b (4.6 g, 9.0 mmol) in dry EtOH
(150 mL) was saturated with HCl gas with ice cooling
and left to stand for 20 h at room temperature. After
distilling off the solvents and HCl, the resulting residue
was dissolved in ethanolic ammonia solution (14% w/v),
and the whole was left to stand for 23 h at room tem-
perature. After removal of the solvent, one portion
(2.6 g) of the resulting residue (5.0 g) was purified by HP-
20 column chromatography (acetonitrile/H2O, 10/90),
providing ethyl 3-(7-amidino-2-naphthyl)-2-{4-[(3R)-
pyrrolidin-3-yloxy]phenyl}propanoate (1.0 g, 2.0 mmol).
With stirring and ice cooling, to EtOH (15 mL) solution
of ethyl 3-(7-amidino-2-naphthyl)-2-{4-[(3R)-pyrrolidin-
3-yloxy]phenyl}propanoate (1.0 g, 2.0 mmol) were
added triethylamine (0.60 g, 6.0 mmol) and ethyl ace-
timidate hydrochloride (0.49 g, 4.0 mmol). After stirring
for 14 h at room temperature, the solvent was distilled
off. A solution of the obtained residue in 2 N HCl
(50 mL) was heated at reflux for 30 min. After evapo-
ration of the solvent, the residue was purified by HP-20
column chromatography (acetonitrile/H2O, 5/95–10/90)
and then preparative HPLC (acetonitrile/H2O). After
the addition of a small amount of concentrated HCl to
selected fractions, the solvents were removed to give a
yellow amorphous solid (0.50 g, 19%). 1H NMR
5.43. Ethyl 2-(4-{[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-
2-yl]methoxy}phenyl)-3-(7-cyano-2-naphthyl)propanoate
25c
Starting with 24c and following the procedure for the
preparation of 25d gave 25c (yield, 68%) as a yellow oil.
1H NMR (CDCl3): d 1.18 (3H, t, J ¼ 7:0 Hz), 1.47 (9H,
s), 1.90–2.10 (4H, m), 3.20–3.60 (2H, m), 3.90–4.50 (8H,
m), 6.80–8.20 (10H, m). MS (FAB): m=z 529 (M+H)þ.
HRMS (FAB) calcd for C32H37N2O5: 529.2702. Found:
529.2715. IR (ATR): 2975, 2933, 2875, 2225, 1727, 1685,
1509, 1388, 1243, 1164, 1106, 1033, 840 cmꢀ1
.
5.44. Ethyl 3-(7-amidino-2-naphthyl)-2-[4-(piperidin-4-yl-
oxy)phenyl]propanoate 26a
A solution of 26a (3.93 g, 7.43 mmol) in dry EtOH
(120 mL) was saturated with HCl gas with ice cooling
and left to stand for 24 h at room temperature. After
distilling off the solvents and HCl, the resulting residue
was dissolved in ethanolic ammonia solution (13% w/v),
and the whole was left to stand for 24 h at room tem-

2112
S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
(DMSO-d6): d 2.00–2.40 (5H, m), 2.90–4.10 (7H, m),
5.20 (1H, m), 6.93 (2H, d, J ¼ 8 Hz), 7.33 (2H, d,
J ¼ 8 Hz), 7.56 (2H, d, J ¼ 8 Hz), 7.70–8.20 (4H, m),
8.45 (1H, s), 8.50–8.80 (2H, m), 9.45 (2H, br), 9.63 (2H,
br). MS (FAB): m=z 445 (M+H)þ. IR (KBr): 3424, 1674,
1632, 1506, 1378, 1240, 1178, 1090, 850 cmꢀ1. Anal.
Calcd for C26H28N4O3Æ2HClÆ 0.5H2O: C, 59.32; H, 5.93;
N, 10.64. Found: C, 59.15; H, 6.20; N, 10.24.
58.06; H, 6.32; N, 10.03. Found: C, 57.94; H, 6.32; N,
9.77.
5.50. 3-(7-Amidino-2-naphthyl)-2-{4-[(1-propanimidoyl-
piperidin-4-yl)oxy]phenyl}propanoic acid 28a
Starting with 26a and ethyl propanimidate and follow-
ing the procedure for the preparation of 28d gave 28a
1
(yield, 8.0%) as a colorless amorphous solid. H NMR
(400 MHz, DMSO-d6): d 1.15 (3H, t, J ¼ 7:3 Hz), 1.73
(2H, br s), 2.03 (2H, br s), 2.61 (2H, q, J ¼ 7:3 Hz),
3.10–3.60 (4H, m), 3.75–3.85 (2H, m), 3.98 (1H, t,
J ¼ 7:8 Hz), 4.68 (1H, br s), 6.95 (2H, d, J ¼ 8:8 Hz),
7.30 (2H, d, J ¼ 8:8 Hz), 7.61 (1H, d, J ¼ 8:3 Hz), 7.78
(1H, d, J ¼ 8:3 Hz), 7.86 (1H, s), 7.96 (1H, d,
J ¼ 8:8 Hz), 8.08 (1H, d, J ¼ 8:3 Hz), 8.79 (1H, br s),
9.23 (1H, br s), 9.26 (2H, br s), 9.53 (2H, br s). MS
(FAB): m=z 473 (M+H)þ. HRMS (FAB) calcd for
C28H33N4O3: 473.2553. Found: 473.2564. IR (KBr):
5.47. 3-(7-Amidino-2-naphthyl)-2-[4-(1-ethanimidoyl-
piperidin-4-yloxy)phenyl]propanoic acid 27a
Starting with 25a and following the procedure for the
preparation of 27b gave 27a (yield, 16%) as a yellow
amorphous solid. 1H NMR (DMSO-d6): d 1.50–2.10 (4H,
m), 2.31 (3H, s), 3.00–4.20 (7H, m), 4.60–4.80 (1H, br),
6.95 (2H, d, J ¼ 9:0 Hz), 7.31 (2H, d, J ¼ 9:0 Hz), 7.50–
8.50 (6H, m), 8.93 (2H, br s), 9.45 (2H, br s), 9.62 (2H, br
s). MS (FAB): m=z 459 (M+H)þ. IR (KBr): 3064, 1678,
3040,
1670,
1608 cmꢀ1
.
Anal.
Calcd
for
1608, 1504, 1376, 1240, 1178, 1032, 912, 850, 714 cmꢀ1
.
C28H32N4O3Æ2HClÆ1.5H2O: C, 58.74; H, 6.51; N, 9.79.
Found: C, 58.57; H, 6.44; N, 9.43.
Anal. Calcd for C27H30N4O3Æ2HClÆ1.5H2O: C, 58.07; H,
6.32; N, 10.03. Found: C, 57.79; H, 6.39; N, 9.99.
5.51. 3-(7-Amidino-2-naphthyl)-2-{4-[(1-butanimidoyl-
piperidin-4-yl)oxy]phenyl}propanoic acid 29a
5.48. 3-(7-Amidino-2-naphthyl)-2-{4-[(2S)-1-ethanimi-
doylpyrrolidin-2-ylmethoxy]phenyl}propanoic acid 27c
Starting with 26a and ethyl butanimidate and following
the procedure for the preparation of 28d gave 29a (yield,
53%) as a colorless amorphous solid. 1H NMR (400
MHz, DMSO-d6): d 0.96 (3H, t, J ¼ 7:3 Hz), 1.50–1.60
(2H, m), 1.71 (2H, br s), 2.03 (2H, br s), 2.50–2.60 (2H,
m), 3.10–3.20 (1H, m), 3.70–3.85 (1H, m), 3.95–4.05 (1H,
m), 4.68 (1H, br s), 6.96 (2H, d, J ¼ 8:8 Hz), 7.30 (2H, d,
J ¼ 8:8 Hz), 7.61 (1H, d, J ¼ 8:3 Hz), 7.80 (1H, d,
J ¼ 8:3 Hz), 7.87 (1H, s), 7.96 (1H, d, J ¼ 8:3 Hz), 8.08
(1H, d, J ¼ 8:3 Hz), 8.42 (1H, s), 8.86 (1H, s), 9.32 (3H,
Starting with 25c and following the procedure for the
preparation of 27b gave 27c (yield, 24%) as a yellow
amorphous solid. 1H NMR (DMSO-d6): d 1.90–2.60
(7H, m), 3.00–4.70 (7H, m), 5.20 (1H, m), 6.92 (2H, d,
J ¼ 9 Hz), 7.31 (2H, d, J ¼ 9 Hz), 7.80–8.10 (5H, m),
8.45 (1H, s), 8.50–8.70 (2H, m), 9.44 (2H, br), 9.64 (2H,
br). MS (FAB): m=z 459 (M+H)þ. IR (KBr): 3388, 1674,
1512, 1376, 1242, 1178, 848 cmꢀ1. Anal. Calcd for
C27H30N4O3Æ2HClÆH2O: C, 59.02; H, 6.24; N, 10.20.
Found: C, 59.14; H, 6.42; N, 9.74.
s), 9.58 (2H, s). IR (KBr): 3100, 1674, 1620, 1506 cmꢀ1
.
Anal. Calcd for C29H34N4O3Æ2HClÆ1.5H2O: C, 59.38; H,
6.70; N, 9.55. Found: C, 59.40; H, 6.92; N, 9.32.
5.49. 3-(7-Amidino-2-naphthyl)-2-{4-[(3S)-1-propanimi-
doylpyrrolidin-3-yloxy]phenyl}propanoic acid 28d
5.52. 3-(7-Amidino-2-naphthyl)-2-{4-[(3S)-1-butanimi-
doylpyrrolidin-3-yloxy]phenyl}propanoic acid 29d
To a stirred and ice-cooled solution of compound 26d
(500 mg, 1.07 mmol) and ethyl propanimidate hydro-
chloride (294 mg, 2.14 mmol) in dry EtOH (10 mL) was
added triethylamine (0.45 mL, 3.21 mmol). After stirring
for 16 h at room temperature, the solvent was distilled
off. A solution of the obtained residue in 2 N HCl
(15 mL) was heated at reflux for 30 min. After evapo-
ration of the solvent, the residue was purified by HP-20
column chromatography (acetonitrile/H2O, 3/97–20/80)
and then preparative HPLC (acetonitrile/H2O). After
the addition of a small amount of concentrated HCl to
selected fractions, the solvents were removed to give a
colorless amorphous solid (350 mg, 59%). 1H NMR
(DMSO-d6): d 0.95–1.10 (3H, m), 2.00–4.10 (12H, m),
5.00–5.20 (1H, m), 6.80 (2H, d, J ¼ 8 Hz), 7.26 (2H, d,
J ¼ 8 Hz), 7.30–8.10 (5H, m), 8.33 (1H, s), 8.40–8.70
(1H, m), 9.00–9.30 (1H, m), 9.32 (1H, br), 9.50 (1H, br).
IR (KBr): 3076, 1674, 1630, 1608, 1506, 1242, 1178,
850 cmꢀ1. Anal. Calcd for C27H30N4O3Æ2HClÆ1.5H2O: C,
Starting with 26d and ethyl butanimidate and following
the procedure for the preparation of 28d gave 29d (yield,
57%) as a colorless amorphous solid. 1H NMR (DMSO-
d6): d 0.60–4.00 (17H, m), 4.90–5.10 (1H, m), 6.79 (2H,
d, J ¼ 8:0 Hz), 7.21 (2H, d, J ¼ 8:0 Hz), 7.30–8.10 (5H,
m), 8.30 (1H, br), 8.34 (1H, s), 8.40–8.70 (1H, m), 9.35
(1H, br), 9.55 (1H, br). IR (KBr): 3052, 1672, 1625,
1608, 1504, 1238, 1176, 848 cmꢀ1. Anal. Calcd for
C28H32N4O3Æ2HClÆ2H2O: C, 57.83; H, 6.59; N, 9.63.
Found: C, 57.83; H, 6.47; N, 9.49.
5.53. 3-{7-Amidino-2-naphthyl}-2-[4-({(3S)-1-[imino-
(phenyl)methyl]pyrrolidin-3-yl}oxy)phenyl]propanoic acid
30d
Starting with 26d and ethyl benzimidate and following
the procedure for the preparation of 28d gave 30d (yield,

S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
2113
17%) as a colorless amorphous solid. 1H NMR (DMSO-
d6): d 2.00–4.10 (10H, m), 4.85–5.25 (1H, m), 6.70–8.10
(14H, m), 8.32 (1H, s), 9.10–9.50 (4H, m). IR (KBr):
3044, 1672, 1606, 1504, 1238, 1178, 848 cmꢀ1. Anal.
Calcd for C31H30N4O3Æ2HClÆ2H2O: C, 60.48; H, 5.91; N,
9.10. Found: C, 60.70; H, 6.14; N, 9.12.
s), 1.70–2.10 (4H, br), 3.20–3.50 (3H, m), 3.60–4.50 (7H,
m), 6.80–7.10 (4H, m), 7.10–7.50 (3H, m), 7.60–8.05
(2H, m). MS (FAB): m=z 535 (M+H)þ. HRMS (FAB)
calcd for C30H35N2O5S: 535.2267. Found: 535.2239. IR
(ATR): 2975, 2225, 1720, 1697, 1513, 1402, 1365, 1272,
1245, 1216, 1164, 1103, 1031, 892, 829 cmꢀ1
.
5.54. Ethyl 2-(4-{[1-(tert-butoxycarbonyl)piperidin-4-
yl]oxy}phenyl)-3-(5-cyano-1-benzothien-2-yl)propanoate
33
5.57. 3-(5-Amidino-1-benzothien-2-yl)-2-{4-[(2S)-1-ethan-
imidoylpyrrolidin-2-ylmethoxy]phenyl}propanoic acid 36
Starting with 36 and following the procedure for the
preparation of 27b gave 36 (yield, 63%) as a pale yellow
amorphous solid. 1H NMR (DMSO-d6): d 2.05 (4H, br),
2.25 and 2.43 (total 3H, each s), 3.00–4.50 (8H, m),
6.80–7.00 (2H, m), 7.15–7.30 (3H, m), 7.65 (1H, d,
J ¼ 8:9 Hz), 8.10 (1H, d, J ¼ 8:9 Hz), 8.25 (1H, s), 8.55
(2H, br s), 9.25 (2H, br s), 9.45 (2H, br s). MS (FAB):
m=z 465 (M+H)þ. HRMS (FAB) calcd for
C25H29N4O3S: 465.1960. Found: 465.1967. IR (ATR):
To
10.2 mmol) and tert-butyl 4-[4-(2-ethoxy-2-oxoacet-
yl)phenoxy]-1-piperidinecarboxylate 24a (3.51 g,
a solution of phosphonium salt 1a (4.81 g,
10.3 mmol) in dry THF (40 mL)–EtOH (40 mL) was
added DBU (1.69 g, 11.1 mmol) at room temperature.
The mixture was stirred for 3 h at room temperature.
After removal of the solvent, the residue was purified
by silica gel column chromatography with CH2Cl2
as an eluent, providing ethyl 2-(4-{[1-(tert-butoxycar-
bonyl)piperidin-4-yl]oxy}phenyl)-3-(5-cyano-1-benzoth-
ien-2-yl)propenoate as a mixture of E and Z forms. The
mixture of E and Z and 10%Pd on carbon (50% wet)
(9.0 g) in EtOH (100 mL) was shaken at room temper-
ature under a current of hydrogen (1 atm). After filtra-
tion of the catalyst, followed by evaporation of the
filtrate, the residue was purified by silica gel column
chromatography with hexane/ethyl acetate (7/3) as an
eluent, to yield a yellow amorphous solid (1.92 g,
3132,
1672,
1612 cmꢀ1
.
Anal.
Calcd
for
C25H28N4O3SÆ2HClÆ1.5H2O: C, 53.19; H, 5.89; N, 9.92.
Found: C, 53.19; H, 5.79; N, 9.92.
5.58. 3-(6-Amidino-1-ethyl-1H-indol-2-yl)-2-[4-(1-ethan-
imidoylpiperidin-4-yloxy)phenyl]propanoic acid 38
To
21.7 mmol) and tert-butyl 4-[4-(2-ethoxy-2-oxoace-
tyl)phenoxy]-1-piperidinecarboxylate 24a (7.50 g,
a solution of phosphonium salt 1h (11.4 g,
1
39%). H NMR (CDCl3): d 1.10 (3H, t, J ¼ 6:0 Hz),
1.50 (9H, s), 1.70–2.00 (4H, m), 3.20–4.00 (7H, m),
4.15 (2H, q, J ¼ 6:0 Hz), 4.30–4.60 (1H, m), 6.80–8.10
(8H, m). MS (FAB): m=z 535 (M+H)þ. HRMS
(FAB) calcd for C30H35N2O5S: 535.2267. Found:
535.2244. IR (ATR): 2956, 2863, 2227, 1729, 1689,
1506, 1425, 1365, 1245, 1226, 1201, 1164, 1124, 1027,
20.6 mmol) in dry THF (150 mL)–MeOH (150 mL) was
added DBU (3.44 g, 22.6 mmol) at room temperature.
The mixture was stirred for 17.5 h at room temperature.
After removal of the solvent, the residue was purified by
silica gel column chromatography with hexane/ethyl
acetate (2/1) as an eluent, providing ethyl 2-{4-[1-(tert-
butoxycarbonyl)piperidin-4-yloxy]phenyl}-3-(6-cyano-1-
ethyl-1H-indol-2-yl)propenoate as a mixture of E and Z
forms. The mixture of E and Z and PdO(H2O)BaSO4
(palladium oxide hydrate barium sulfate) (1.5 g) in
MeOH (250 mL) was shaken at room temperature under
a current of hydrogen (1 atm). After filtration of the
catalyst, followed by concentration of the filtrate, the
residue was purified by silica gel column chromatogra-
phy with hexane/ethyl acetate (4/1) as an eluent, pro-
viding an ester as a colorless amorphous solid. A
solution of the ester in dry EtOH (130 mL) was satu-
rated with HCl gas with ice cooling and left to stand for
20 h at room temperature. After distilling off the sol-
vents and HCl, the resulting residue was dissolved in
ethanolic ammonia solution (14% w/v), and the whole
was left to stand for 23 h at room temperature. After
removal of the solvent, the resulting residue was purified
by HP-20 column chromatography (acetonitrile/H2O,
10/90), providing ethyl 3-(6-amidino-1-ethyl-1H-indol-2-
yl)-2-[4-(piperidin-4-yloxy)phenyl]propanoate 37. With
stirring and ice cooling, to EtOH (37 mL) solution of
compound 37 were added triethylamine (2.26 g,
22.4 mmol) and ethyl acetimidate hydrochloride (1.79 g,
14.5 mmol). After stirring for 17 h at room temperature,
the reaction mixture was distilled off the solvent. A
912, 813 cmꢀ1
.
5.55. 3-(5-Amidino-1-benzothien-2-yl)-2-[4-(1-ethan-
imidoylpiperidin-4-yloxy)phenyl]propanoic acid 34
Starting with 33 and following the procedure for the
preparation of 27b gave 34 (yield, 6.8%) as a pale brown
amorphous solid. 1H NMR (DMSO-d6): d 1.65–2.10
(4H, m), 2.32 (3H, s), 3.20–4.00 (7H, m), 4.60–4.70 (1H,
m), 6.96 (2H, d, J ¼ 8:3 Hz), 7.30 (3H, m), 7.69 (1H, d,
J ¼ 8:3 Hz), 8.10 (1H, d, J ¼ 8:3 Hz), 8.26 (1H, s), 8.95
(2H, br s), 9.32 (2H, br s), 9.52 (2H, br s). MS (FAB):
m=z 465 (M+H)þ. IR (KBr): 3424, 1674, 1510, 1448,
1242,
1178,
1062 cmꢀ1
.
Anal.
Calcd
for
C25H28N4O3SÆ2HClÆ1.5H2O: C, 53.19; H, 5.89; N, 9.92.
Found: C, 53.60; H, 5.45; N, 9.45.
5.56. Ethyl 2-(4-{[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-
2-yl]methoxy}phenyl)-3-(5-cyano-1-benzothien-2-yl)pro-
panoate 35
Starting with 24c and following the procedure for the
preparation of 33 gave 35 (yield, 57%) as a viscous oil.
1H NMR (CDCl3): d 1.17 (3H, t, J ¼ 7:6 Hz), 1.47 (9H,

2114
S. Komoriya et al. / Bioorg. Med. Chem. 12 (2004) 2099–2114
solution of the obtained residue in 2 N HCl (120 mL)
was heated at reflux for 1.5 h. The reaction mixture was
concentrated to dryness to give a residue. After evapo-
ration of the solvent, the residue was purified by HP-20
column chromatography (acetonitrile/H2O, 15/85) and
then preparative HPLC (acetonitrile/H2O). After the
addition of a small amount of concentrated HCl to se-
lected fractions, the solvents were removed to give a pale
yellow amorphous solid (0.56 g, 8.1%). 1H NMR
(DMSO-d6): d 1.30 (3H, t, J ¼ 7:0 Hz), 1.73–2.10
(4H, m), 2.31 (3H, s), 3.05–3.15 (1H, m), 3.30–3.80 (5H,
m), 4.05 (1H, t), 4.20–4.35 (2H, m), 4.70 (1H, br s),
6.38 (1H, s), 6.97 (2H, d, J ¼ 8:5 Hz), 7.37 (2H,
d, J ¼ 8:3 Hz), 7.48 (1H, d, J ¼ 8:3 Hz), 7.61 (1H,
d, J ¼ 8:3 Hz), 8.14 (1H, s), 8.86 (1H, br), 9.15–
9.50 (5H, m). IR (KBr): 1672, 1614, 1510, 1466,
1342 cmꢀ1. Anal. Calcd for C27H33N5O3Æ2HClÆH2O: C,
57.24; H, 6.58; N, 12.36. Found: C, 57.42; H, 6.81; N,
12.33.
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