Synthesis and biological activity of novel 1,2-disubstituted benzene derivatives as factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.11.005

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fX

Full text of DOI:10.1016/j.bmc.2004.11.005

Bioorganic & Medicinal Chemistry 13 (2005) 1305–1323
Synthesis and biological activity of novel 1,2-disubstituted
benzene derivatives as factor Xa inhibitors
Hiroyuki Koshio,a,* Fukushi Hirayama,a Tsukasa Ishihara,a Ryouta Shiraki,a
Takeshi Shigenaga,a Yuta Taniuchi,b Kazuo Sato,a Yumiko Moritani,a
Yoshiyuki Iwatsuki,a Seiji Kaku,a Naoko Katayama,a Tomihisa Kawasaki,a
a
Yuzo Matsumoto,a Shuichi Sakamotoc andShin-ichi Tsukamoto
aInstitute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
bDrug Development Division, Yamanouchi Pharmaceutical Co. Ltd, 3-17-1 Hasune, Itabashi-Ku, Tokyo 174-8612, Japan
cInstitute for Technology Development, Yamanouchi Pharmaceutical Co. Ltd, 160-2 Akahama, Takahagi, Ibaraki 318-0001, Japan
Received15 October 2004; revised5 November 2004; accepted5 November 2004
Available online 23 November 2004
Abstract—Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the
Yamanouchi compoundlibrary yieldedleadcompound 1 with the ability to inhibit fXa at micromolar concentrations. To improve
its fXa inhibitory activity andits oral anticoagulant activity, the linker between benzamidine andthe central benzene ring was
modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a
(YM-202054), and 41c (YM-203558) exhibitedpotent fXa inhibitory activity andoral anticoagulant activity. In particular,
YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0h. Additionally,
these compounds showed a high degree of selectivity for other serine proteases.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
In the course of the search for fXa inhibitors in our lab-
oratories, we have previously reportedpotent, selective,
andorally active N-[(7-amidino-2-naphthyl)methyl]ani-
line-basedfXa inhibitors, YM-60828 andYM-167065,
respectively, containing an acetimidoylpiperidine moiety
andan N-methyl-1,4-diazepane moiety as the P4 part.2–4
With the hope of discovering a novel class of fXa inhib-
itors having novel frame structures, high-throughput
screening (HTS) of the Yamanouchi compoundlibrary
was conducted, leading to the identification of the 1,2-
dibenzoamidobenzene derivative 1 as an fXa inhibitor
(Fig. 1). Compound 1 showedrather weak inhibitory
activity against fXa (IC50 = 6226nM), but possesseda
novel andunique structure, which resultedin different
from our naphthylamidine-based fXa inhibitors.5–9 We
therefore selected 1 as a leadcompound, andmodified
it in a search for potent andorally active fXa inhibitors.
Thrombotic diseases such as deep vein thrombosis,
stroke, andpulmonary embolism are a major cause of
mortality and morbidity in the developed world. There-
fore, the prevention of bloodcoagulation is a major tar-
get for new therapeutic agents. Factor Xa (fXa) is a
trypsin-like serine protease that forms a prothrombinase
complex with factor Va, Ca2+, andphospholipidto
produce thrombin. This key enzyme functions at the
convergence of the intrinsic andextrinsic coagulation
pathways in a process that involves signal amplification,
one molecule of fXa activating many molecules of pro-
thrombin to thrombin.1 Inhibition of fXa may therefore
be more effective than the inhibition of thrombin itself.
The discovery of orally active, small molecule competi-
tive fXa inhibitors as novel therapies for thrombo-
embolic disorders has been a major focus of the
pharmaceutical industry.
2. Chemistry
Scheme 1 illustrates the synthesis of 1,2-dibenzamido-
benzene derivatives, starting from either 1,2-phenylene-
diamine (3, 11a, 11b) or 2-nitroaniline (8a, 8b).
Keywords: Anticoagulants; Enzyme inhibitors; Factor Xa.
*
Corresponding author. Tel.: +81 029 854 1548; fax: +81 029 852
5387; e-mail: koshio@yamanouchi.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.005

1306
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
Treatment of 1,2-phenylenediamine (2) with 2equiv of
p-anisoyl chloride gave 3. Treatment of 2 with cyano-
benzoic acidfollowedby acylation with 13 gave interme-
diates 10a and 10b. In the 2-nitroaniline approach, the
first side chain was introduced through acylation with
p-anisoyl chloride to give compound 5. The nitro group
was then reduced by hydrogenation to aniline 6. A
secondacylation with cyanobenzoic acidprovidedthe
diacylated intermediates 7a and 7b. Treatment of inter-
mediates 7a, 7b, 10a, and 10b under Pinner conditions
produced the imidates, which were immediately reacted
with excess ammonium acetate to give the correspond-
ing amidine derivatives 8a, 8b, 11a, and 11b. The
benzoic acidderivative 13 was preparedby hydrolysis
of 12.10
Synthesis of intermediates 16a–g is described in Scheme
2. Alkylation with phenol derivatives or reductive alkyl-
ation with benzaldehyde gave the nitrobenzene deriva-
tives 15a–c. Intermediates 16a–c were preparedby
reduction of the nitro group in 15a–c with reduced iron,
followedby acylation with 13. Treatment of 14d with tri-
Figure 1. YM-60828 andYM-167065 andHTS hit compound 1.
O
NH2
MeO
a
HN
H2N
H
N
OMe
O
2
3
O
O
NH2
HN
O
HN
b
O2N
a
O2N
H2N
OMe
OMe
4
5
6
O
4
4
NC
3
c
d, e
H2N
HN
HN
H
N
H
N
OMe
OMe
HN
3
O
O
8a;3-C(NH)NH2
8b;4-C(NH)NH2
7a;3-CN
7b;4-CN
O
NH2
4
NH2
4
NC
3
H
H2N
c
f
NC
HN
N
H
N
3
N
O
N
Me
O
9a;3-CN
9b;4-CN
10a;3-CN
10b;4-CN
2
O
4
H2N
HN
HN
g, h
H
N
N
3
N
Me
N
O
11a;3-C(NH)NH2
11b;4-C(NH)NH2
i
NC
N
HO2C
N
N
Me
Me
12
13
Scheme 1. Reagents andconditions: (a) p-anisoylchloride, pyridine; (b) H2, 10% Pd–C, MeOH; (c) 3-cyanobenzoic acid or 4-cyanobenzoic acid,
HOBt, WSC, DMF; (d) HCl, MeOH; (e) NH4OAc, MeOH; (f) (1) SOCl2, 13, (2) pyridine; (g) HCl, EtOH; (h) NH4OAc, EtOH; (i) 6N NaOH.

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1307
O
NO2
a
d
b, c
b, c
HN
O
NC
NC
O
Br
N
NC
NC
NC
N
Me
14a
14b
15a
16a
16b
O
NO2
NO2
HN
O
N
NC
O
OH
N
Me
15b
O
b, c
e
f
HN
O
NC
NC
N
H
NC
NC
NH2
N
NC
N
H
N
Me
15c
O
14c
14d
16c
HN
HN
g, b, c
-
+
N
+
Br
PPh3Br3
N
Me
N
N
NC
N
N
Me
NC
17
16d
16e
O
g, h, c
HN
NC
j
Me
16f
O
O
NH2
i
HN
HN
H2N
H
N
H2N
N
N
NC
N Me
N Me
18
2
16g
Scheme 2. Reagents andconditions: (a) 2-nitrophenol, K 2CO3, DMF; (b) Fe, NH4Cl, EtOH, H2O; (c) (1) SOCl2, 13, (2) pyridine; (d) 2-
bromomethylnitrobenzene, K2CO3, DMF; (e) 2-nitrobenzaldehyde, NaB(OAc)3H, 1,2-dichloroethane, AcOH; (f) PPh3, benzene; (g) 2-nitrobenz-
aldehyde, DBU, toluene; (h) PdO–BaSO4, H2, EtOH; (i) (1) SOCl2, 13, (2) pyridine; (j) 3-cyanobenzaldehyde, NaB(OAc)3H, 1,2-dichloroethane,
AcOH.
phenylphosphine gave 17. Intermediates 16d–f were pre-
paredby Wittig olefination, reduction of the nitro group
in 16d and 16e or both the nitro group andthe double
bondin 16f, followedby acylation with 13. Treatment
of 2 with 13 followedby reductive alkylation with 3-
cyanobenzaldehyde gave intermediate 16g.
with 4-[4-(tert-butoxycarbonyl)-1,4-diazepan-1-yl]ben-
zoic acidgave 33. Compound 33 was alkylatedwith
ethyl bromoacetate to give 34. Compound 34 was con-
vertedto 35 by reduction with reduced iron followed
by acylation with 3-cyanobenzoylchloride. Following
the removal of the tert-butoxycarbonyl (BOC) protect-
ing group from 35 under acidic conditions, the resulting
1,4-diazepane derivative was converted to the 4-methyl-
1,4-diazepane derivative 30a by reductive alkylation
with formaldehyde.
The synthesis of 21a–c, 24a–c, and 30a–c is outlinedin
Scheme 3. The nitrobenzene derivatives 20a–c and 23b
were synthesizedby bromination of 19a–c followedby
reaction with phenol or aniline. Treatment of 22 with
3-cyanobenzoylchloride gave nitrobenzene derivative
23a. Compounds 20a–c, 23a, and 23b were converted
to corresponding intermediates 21a–c, 24a, and 24b by
the same procedure applied to 16a–c in Scheme 2. Com-
pound 25 was preparedby bromination of 19b, followed
by oxidation with N-methyl morphorine-N-oxide. Ani-
line derivative 26 was preparedby a Wittig reaction
involving 17 and 25 followedby reduction of the nitro
group and the double bond. Compound 26 was acylated
with 13 to give intermediate 24c. Phenol derivative 27
was alkylatedwith ethyl glycolate unedr Mitsunobu
conditions to give 28. Compounds 30b and 30c were syn-
thesizedby the same procedure as 21a. Treatment of 32
The fully assembledbenzonitrile intermediates were then
elaborated to the amidine target molecules under the
conditions described in Scheme 1, as illustratedin
Scheme 4.
3. Results and discussion
Compounds were evaluated for fXa inhibitory activity
according to their IC50 values, andfor anticoagulant
activity in vitro according to the CT2 values of their
prothrombin times (PT). The CT2 value was defined
as the concentration requiredto oduble the clotting

1308
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
Scheme 3. Reagents andconditions: (a) NBS, AIBN, CCl 4; (b) 3-cyanophenol, K2CO3, DMF; (c) Fe, NH4Cl, EtOH, H2O; (d) (1) SOCl2, 13; (2)
pyridine; (e) 3-cyanobenzoylchloride, pyridine; (f) 3-cyanoaniline, K2CO3, DMF; (g) N-methyl morphorine N-oxide, CH3CN; (h) 17, DBU, toluene;
(i) PdO–BaSO4, H2, EtOH; (j) ethyl glycolate, PPh3, DEAD, THF; (k) 4-[4-(tert-butoxycarbonyl)-1,4-diazepan-1-yl]benzoic acid, NBS, PPh3,
pyridine, CH2CH2; (l) ethyl bromoacetate, K2CO3, DMF; (m) (1) 4N HCl–AcOEt, (2) HCHO, NaB(OAc)3H, 1,2-dichloroethane, AcOH.
time. In addition, the oral anticoagulant activity of
compounds was evaluated according to their ability to
prolong PT following oral administration in mice.
para- or meta-position produced a 10- to 30-fold in-
crease in inhibitory activity (8a, 8b).7
Next, the 4-methyl-1,4-diazepane,4 which was discov-
eredin our laboratory as a novel P4 scaffoldfor fXa
inhibitors, was introduced to amidine derivatives 8a
and 8b. The 4-amidine derivative 11b showedsignifi-
cantly decreased inhibitory activity. However, the 3-
amidine derivative 11a showeda 20-foldincrease in
Our observations are in agreement with those of Wiley
andcolleagues ( Table 1). Briefly, the methoxy group
on the central benzene ring was not necessary for activ-
ity (3), andreplacement of the 4-methoxy substituent on
one of the side benzene rings of 3 with an amidine at the

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1309
O
a, b
HN
16a-g
H2N
N
A
N Me
NH
36a A=CH2O
36b A=OCH2
36c A=NHCH2
36d A=trans CH=CH
36e A=cis CH=CH
36f A=CH2CH2
36g A=CH2NH
O
O
HN
4
21a-c
24a-c
30a-c
HN
c
a, b
H2N
2
H2N
2
N
A
N
A
R
N Me
R
N Me
NH
NH
3
3
4
40a A=OCH2
40b A=OCH2
40c A=OCH2
41a A=CONH2 R=3-CO2H
41b A=CH2CH2 R=3-CO2H
41c A=NHCH2 R=3-CO2H
42a A=CONH R=2-OCH2CO2H
42b A=CONH R=3-OCH2CO2H
42c A=CONH R=4-OCH2CO2H
R=2-CO2H
R=3-CO2H
R=4-CO2H
37a A=OCH2 R=2-CO2Et
37b A=OCH2 R=3-CO2Et
37c A=OCH2 R=4-CO2Et
38a A=CONH R=3-CO2Et
38b A=CH2CH2 R=3-CO2Et
38c A=NHCH2 R=3-CO2Et
39aA=CONH R=2-OC2HCO2 Et
39b A=CONH R=3-OC2HCO Et
39c A=CONH R=4-OC2HCO2Et
2
Scheme 4. Reagents: (a) HCl, EtOH; (b) NH4OAc, EtOH; (c) NaOH.
Table 1. Activity of 1,2-dibenzamidobenzenes
1
CompdR
R2
R3
IC50 (nM)a
CT2 (lM)b PTc
PT/control PTd
0.5h
2.0h
1
4-MeO
4-MeO
3-Ame
4-Ame
MeO
H
MeO
MeO
MeO
MeO
6216
4224
127
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
3
8a
8b
H
H
364
11a
3-Ame
4-Ame
H
H
5.8
0.33
1.62
1.40
11b
23,779
6.0
7.2
1.07
2.55
1.02
1.74
YM-60828
0.21
a Human purifiedenzyme were used. IC 50 values represent the averaged of three determinations with the average standard error of the mean <10%.
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 comparedwith that measuredusing normal mice plasma at 0.5, 1.0, and2.0h after oral administration (100mg/kg,
n = 3).
e Am means amidine moiety.
potency comparedwith 8a, anda 1000-foldincrease in
potency comparedwith leadcompound 1. Compound
11a was further evaluatedfor both its in vitro anticoag-
ulant activity andits oral anticoagulant activity. The in
vitro anticoagulant activity of 11a was comparable to
that of YM-60828; however, it showedpoor oral
anticoagulant activity in mice at a dose of 100mg/kg.
Modification of the linker between benzamidine and

1310
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
Table 2. Effects of linker between the benzamidine and the central benzene ring
CompdA
IC
(nM)a
CT2 (lM)b PTc
PT/control PTd
50
0.5h
2.0h
11a
36a
36b
36c
36d
36e
36f
36g
–CONH–
–CH2O–
–OCH2–
5.8
1236
23.8
0.33
9.2
1.62
1.03
1.51
1.17
1.00
1.01
1.12
1.13
1.40
1.02
1.29
1.07
0.97
1.01
1.04
1.04
0.62
0.24
1.2
NTe
0.93
NTe
–NHCH2–
trans-CH@CH–
cis-CH@CH–
–CH2CH2–
–CH2NH–
9.8
92.0
1668
97.1
903.9
a Human purifiedenzyme were used. IC 50 values represent the averaged of three determinations with the average standard error of the mean <10%.
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 comparedwith that measuredusing normal mice plasma at 0.5, 1.0, and2.0h after oral administration (100mg/kg,
n = 3).
e Not tested.
the central benzene ring was undertaken in order to im-
prove its oral anticoagulant activity. The results are
shown in Table 2.
The fXa inhibitory activity of the trans-olefin derivative
36d, which enforces geometry similar to that imposedby
an amide linker, was 15 times less than that of 11a, and
that of the cis-olefin analogue (36e) was 280 times less
than that of 11a. The activity of the ethylene derivative
36f, considered to have greater flexibility than imparted
by an amide bond, was comparable to that of the trans-
olefin derivative 36d. Among compounds containing
ether or aminomethyl linkers, 36b and 36c, containing
a hetero atom locatednear the benzamidine moiety,
showedfavorable fXa inhibitory activity, and 36c
showedfXa inhibitory activity similar to that of amide
derivative 11a. Compounds 36a and 36g, in which the
hetero atom was locatedin a different position than in
36b and 36c, had50–90 times less activity than 36b
and 36c. However, compounds 36a–g all showedpoor
oral anticoagulant activity in mice.
Figure 2. Binding model of compound 36b to fXa.
enzyme. Previous studies on structure–activity relation-
ships of YM-60828 derivatives have demonstrated that
the presence of a carboxyl group resultedin potent oral
anticoagulant activity in mice.3,4 Therefore, to improve
oral anticoagulant activity, a carboxyl group was intro-
duced to the central benzene ring in ether derivative 36b.
Compounds 40a–c retainedor hada slightly improved
fXa inhibitory activity, andshowedgoodin vitro antico-
agulant activity. Moreover, all compounds showed in-
creasedoral activity as expecte. d In particular,
compound 40b, containing a carboxyl group at the 3-
position in the central benzene ring, showedthe greatest
oral activity.
Figure 2 illustrates the overlayedenergy-minimized
structure of the fXa active site complex of ether deriva-
tive 36b. The analysis indicates that the benzamidine
moiety occupies the S1 pocket, andthat the 1,4-diaze-
pane moiety is in close contact with the S4 pocket de-
finedby the three aromatic amino acisd. Moreover,
the NH group locatedbetween the central benzene ring
andthe phenyl-1,4-diazepane moiety is in a position to
form a hydrogen bond with the backbone carbonyl
group of Gly 216. From the modeling study illustrated
in Figure 2, we considered there was a possibility of
introducing a substituent in the central benzene ring
without markedly affecting fXa inhibitory activity,
thereby changing the physiological profiles of the com-
pounds to improve their oral efficacy by having the cen-
tral benzene ring pointing into solution away from the
Figure 3 illustrates the overlayedenergy-minimized
structure of the fXa active site complex of ether deriva-
tive 40b. Molecular modeling studies indicate that the
carboxyl group at the 3-position of the central benzene
ring is located outside the enzyme pocket and extends

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1311
taken. The amide derivative, the ethylene derivative,
andthe aminomethyl derivative improvedoral anticoag-
ulant activity without decreasing inhibitory activity (41a
vs 11a, 41b vs 36f, and 41c vs 36c). In particular, com-
pound 41c exhibitedthe most potent oral activity, pro-
longing PT 3.25-foldat 0.5h and3.44-foldat 2.0h
(Table 3).
Compounds 40b and 41a–c, which exhibitedgoodfXa
inhibitory activity andoral anticoagulant activity in
mice, were further evaluatedin terms of enzymatic selec-
tivity andin vitro anticoagulant activity using human
plasma. All compounds showed high selectivity for
fXa over trypsin (>300-fold). In particular, they showed
exceptional selectivity against the relatedserine protease
thrombin (>30,000-fold). These compounds also pro-
longedPT to a greater extent in human plasma than
in mouse plasma (Table 4).
Figure 3. Binding model of compound 40b to fXa.
into the solvent. A possible explanation for the fact that
compound 40b has a slightly greater fXa inhibitory
activity than 36b, even though it does not seem to bind
directly to the enzyme, is that the carboxyl group is bet-
ter able to solvate in the aqueous environment sur-
rounding the enzyme. In order to study other acidic
residues and optimum position on the central benzene
ring, an oxymethylcarboxylic acidmoiety was introduce
to amide derivative 11a. Compounds 42a–c showedsim-
ilar properties, andthe 3-subsititutedderivative 42b had
the greatest oral activity. On the other hand, the 4-sub-
sititutedderivative 42c showedno significant increase in
oral anticoagulant activity. Therefore, the carboxyl
group was fixedat the 3-position in the central benzene
ring, andmoidfication with the other linkers that
showedgoodinhibitory activity in Table 2 was under-
4. Conclusion
We have designed and synthesized fXa inhibitors based
on 1,2-dibenzoamidobenzene derivatives from HTS
screening to improve inhibitory activity. The series of
fXa inhibitors containing a 3-benzamidine moiety as
the P1 part anda 4-methyl-1,4-idazepane moiety as
the P4 part exhibitedpotent fXa inhibitory activity.
Additionally, their oral anticoagulant activity was im-
provedby the introduction of a carboxyl group at the
central benzene ring, without affecting their inhibitory
activity. Compounds 40b (YM-203552), 41a (YM-
202054), and 41c (YM-203558) exhibitedpotent fXa
inhibitory activity andshoweda high degree of selectiv-
ity for other serine proteases, andprolongedPT in both
Table 3. Effects of the central benzene ring substituents
CompdA
R
IC
(nM)a
CT2 (lM)b PTc
PT/control PTd
50
0.5h
2.0h
36b
40a
40b
40c
11a
42a
42b
42c
41a
41b
41c
–OCH2–
–OCH2–
H
23.8
15.7
8.6
0.62
0.55
0.29
0.24
0.33
0.43
0.27
0.71
0.35
0.44
0.16
1.51
2.29
2.58
1.72
1.62
1.71
2.41
1.00
2.82
1.90
3.25
1.29
1.84
2.39
1.43
1.40
1.33
1.60
1.01
2.70
1.66
3.44
2-CO2H
3-CO2H
4-CO2H
H
–OCH2–
–OCH2–
13.6
5.8
–CONH–
–CONH–
–CONH–
–CONH–
–CONH–
–CH2CH2–
–NHCH2–
2-OCH2CO2H
3-OCH2CO2H
4-OCH2CO2H
3-CO2H
3-CO2H
3-CO2H
2.8
2.4
6.3
3.2
28.3
3.5
a Human purifiedenzyme were used. IC 50 values represent the averaged of three determinations with the average standard error of the mean <10%.
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 comparedwith that measuredusing normal mice plasma at 0.5, 1.0, and2.0h after oral administration (100mg/kg,
n = 3).

1312
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
Table 4. The in vitro anticoagulant activity andenzyme selectivity
CompdA
IC
(nM)a
CT2 (lM)bPTc
Human
50
fXa
Trypsin
Thrombin
Mice
40b
41a
41b
41c
–OCH2–
8.6
3.2
3300
8600
>100,000
>100,000
>100,000
>100,000
0.29
0.35
0.44
0.16
0.11
0.13
0.14
0.093
–CONH–
–CH2CH2–
–NHCH2–
28.3
3.5
19,000
4000
a Human purifiedenzyme were used. IC 50 values represent the averaged of three determinations with the average standard error of the mean <10%.
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.
mouse and human plasma. Additionally, these com-
pounds exhibited excellent oral anticoagulant activity
in mice. In particular, YM-203558 exhibitedthe most
potent oral activity, prolonging PT more than 3-fold
at 0.5 and2.0h after oral administration to mice at a
dose of 100mg/kg.
(3H, s), 7.02 (2H, d, J = 9.2Hz), 7.18–7.74 (1H, m),
7.67–7.74 (1H, m), 7.97 (2H, dd, J = 1.5, 8.4Hz), 9.00
(1H, dd, J = 1.5, 8.4Hz), 11.3 (1H, s); FAB MS m/e
(M+H)+ 273.
4.1.3. N-(2-Aminophenyl)-4-methoxybenzamide (6). To
the solution of 5 (4.35g, 16mmol) in EtOH (30mL)
was added 10% Pd–C powder (400mg) and stirred in
hydrogen atmosphere at ambient temperature for 15h.
The reaction mixture was filtratedthrough a padof Cel-
ite andconcentratedin vacuo to give 6 (3.82g, quant.
yield) as a brown amorphous powder: 1H NMR
(DMSO-d6) d: 3.88 (3H, s), 6.56–6.63 (1H, m), 6.78
(1H, dd, J = 1.2, 8.1Hz), 6.93–7.00 (1H, m), 7.04 (2H,
d, J = 9.0Hz), 7.13–7.18 (1H, m), 7.97 (2H, d,
J = 9.0Hz); FAB MS m/e (M+H)+ 243.
4.1. Chemistry
1H NMR spectra were measuredwith a JEOL EX90,
EX400, or GX500 spectrometer; chemical shifts are
expressedin d units using tetramethylsilane as the stand-
ard (in NMR description, s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, andbr = broad
peak). Mass spectra were recorded with a Hitachi M-
80 or JEOL JMS-DX300 spectrometer. ODS column
chromatography was performedon YMC gel (ODS-A
120-230/70).
4.1.4. 3-Cyano-N-{2-[(4-methoxybenzoyl)amino]phenyl}-
benzamide (7a). To a stirredsolution of 6 (600mg,
2.46mmol) in DMF (20mL) was added 3-cyanobenzoic
acid(361mg, 2.46mmol), HOBt (450mg, 3.72mmol),
andWSC (720mg, 3.72mmol) at ambient temperature
for 12h. The reaction mixture was diluted with ethyl
acetate andwashedwith H 2O andsaturatedsaline.
The organic layer was dried over Na2SO4 andconcen-
tratedin vacuo. The resulting residues was crystallized
by EtOH (30mL) to give 7a (808mg, 88%) as a white
4.1.1. N,N0-1,2-Phenylenebis(4-methoxybenzamide) (3).
To a stirring solution of o-phenylene diamine (800mg,
7.4mmol) in pyridine (10mL) was added p-anisoyl chlo-
ride (3.1g, 17.8mmol). After 12h, the solvent was
removedin vacuo andthe resiude was partitioned
between chloroform and1M NaOH. The organic layer
was washedwith H 2O andsaturatedsaline. The organic
layer was dried over Na2SO4 andconcentratedin vacuo
to give 2.22g (80%) of white solid: 1H NMR (CDCl3) d:
3.87 (6H, s), 6.77 (2H, d, J = 3.6, 6.0Hz), 6.99 (4H, d,
J = 9.0Hz), 7.34 (2H, dd, J = 3.6, 6.0Hz), 7.98 (4H, d,
J = 9.0Hz), 9.39 (2H, s); EI MS m/e (M)+ 377; Anal.
Calcdfor C 22H20N2O4: C, 70.20; H, 5.36; N, 7.44.
Found: C, 70.18; H, 5.42; N, 7.36.
1
powder: H NMR (DMSO-d6): 3.82 (3H, s), 7.05 (2H,
d, J = 8.7Hz), 7.28–7.34 (2H, m), 7.60–7.70 (2H, m),
7.75 (1H, t, J = 7.8Hz), 7.94 (2H, d, J = 8.7Hz), 8.06
(1H, d, J = 7.8Hz), 8.23 (1H, d, J = 7.8Hz), 8.36 (1H,
s), 9.84 (1H, s), 10.24 (1H, s); FAB MS m/e (M+H)+
372.
4.1.2. 4-Methoxy-N-(2-nitrophenyl)benzamide (5). To a
stirring solution of 2-nitroaniline (2g, 14.5mmol) in
pyridine (20mL) was added p-anisoyl chloride (3.03g,
17.4mmol). After 12h, the solvent was removedin
vacuo andthe residue was partitionedbetween chloro-
form and1M NaOH. The organic layer was washed
with H2O andsaturatedsaline. The organic layer was
dried over Na2SO4 andconcentratedin vacuo to give
4.1.5. 4-Cyano-N-{2-[(4-methoxybenzoyl)amino]phenyl}-
benzamide (7b). Compound 7b was synthesizedfrom 4-
cyanobenzoic acidand 6 according to the same proce-
dure as that for 7a. Compound 7b was obtainedas a
white amorphous powder (87% yield): 1H NMR
(DMSO-d6) d: 3.82 (3H, s), 7.05 (2H, d, J = 8.7Hz),
7.64–7.70 (2H, m), 7.93 (2H, d, J = 8.7Hz), 8.02 (2H,
d, J = 8.7Hz), 8.10 (2H, d, J = 8.7Hz), 9.89 (1H, s),
10.28 (1H, s); FAB MS m/e (M+H)+ 372.
1
3.9g (98%) of yellow solid: H NMR (CDCl3) d: 3.89

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1313
4.1.6. 3-[Amino(imino)methyl]-N-{2-[(4-methoxybenzo-
yl)amino]phenyl}benzamide (8a). HCl gas bubbled
through a solution of 7a (300mg, 0.81mmol) in MeOH
(5mL) andchloroform (10mL) under ꢀ20ꢁC for 20min.
The mixture was allowedto stir for 24h at 5 ꢁC, andthen
concentrated in vacuo. To the crude imidate dissolved in
MeOH (20mL) at ambient temperature was added
ammonium acetate (280mg, 4.1mmol). The reaction
mixture was stirredat ambient temperature for 36h
andconcentratedin vacuo. The resulting resiudes
was chromatographedon silica gel eluting with
chloroform/MeOH (5:1) to give 8a (186mg, 60%) as a
white amorphous powder: 1H NMR (DMSO-d6) d:
2.00 (3H, s), 3.82 (3H, s), 7.04 (2H, d, J = 8.7Hz),
7.25–7.31 (2H, m), 7.64–7.71 (2H, m), 7.77 (1H, t,
J = 8.7Hz), 8.03 (3H, t, J = 8.7Hz), 8.29 (1H, d,
J = 8.7Hz), 8.53 (1H, s), 9.28–9.64 (3H, br); FAB MS
m/e (M+H)+ 389; Anal. Calcdfor C 22H20N4O3ÆC2H4O4:
C, 64.25; H, 5.40; N, 12.20. Found: C, 64.35; H, 5.45; N,
12.02.
the reaction mixture was concentratedin vacuo. The res-
idue was dissolved in pyridine (10mL) and cooled to
0ꢁC. To this solution 9a (900mg, 3.79mmol) was added
andstirredat ambient temperature for 4h. The reaction
mixture was concentratedin vacuo andthe residue was
chromatographedon silica gel eluting with chloroform/
methanol/ammonia (15:1:0.1) to give 10a (822mg, 48%)
1
as a white amorphous powder: H NMR (CDCl3) d:
2.05–2.16 (2H, m), 2.44 (3H, s), 2.63–2.69 (2H, m),
2.78–2.83 (2H, m), 3.58 (2H, t, J = 6.2Hz), 3.66–3.72
(2H, m), 6.73 (2H, d, J = 9.0Hz), 6.87–6.94 (2H, m),
7.31–7.35 (1H, m), 7.42–7.46 (1H, m), 7.62 (1H, t,
J = 7.8Hz), 7.79–7.84 (1H, m), 7.89 (2H, d,
J = 9.0Hz), 8.24–8.32 (2H, m), 8.92 (1H, s), 10.10 (1H,
s); FAB MS m/e (M+H)+ 454.
4.1.11. 4-Cyano-N-(2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide (10b). Compound 10b
was synthesizedfrom 9b according to the same proce-
dure as that for 10a. Compound 10b was obtainedas
a white amorphous powder (65% yield): 1H NMR
(CDCl3) d: 2.05–2.15 (2H, m), 2.40 (3H, s), 2.60–2.68
(2H, m), 2.79–2.84 (2H, m), 3.55 (2H, t, J = 6.0Hz),
3.66–3.70 (2H, m), 6.74 (2H, d, J = 8.8Hz), 6.80–6.96
(1H, m), 7.50–7.56 (1H, m), 7.77 (2H, d, J = 8.8Hz),
7.88 (2H, d, J = 8.8Hz), 8.12 (2H, d, J = 8.8Hz), 8.80
(1H, s), 10.08 (1H, s); FAB MS m/e (M+H)+ 454.
4.1.7. 4-[Amino(imino)methyl]-N-{2-[(4-methoxybenzo-
yl)amino]phenyl}benzamide (8b). Compound 8b was syn-
thesizedfrom 7b according to the same procedure as
that for 8a. Compound 8b was obtainedas a white
amorphous powder (60 % yield): 1H NMR (DMSO-
d6) d: 2.00 (3H, s), 3.82 (3H, s), 7.04 (2H, d,
J = 8.7Hz), 7.26–7.35 (2H, m), 7.60–7.71 (2H, m), 7.94
(2H, d, J = 8.7Hz), 8.01 (2H, d, J = 8.7Hz), 8.17 (2H,
d, J = 8.7Hz), 9.20–9.80 (3H, br); FAB MS m/e
(M+H)+ 389; Anal. Calcdfor C 22H20N4O3ÆC2H4O4: C,
64.25; H, 5.40; N, 12.20. Found: C, 64.28; H, 5.39; N,
12.49.
4.1.12. 3-[Amino(imino)methyl]-N-(2-{[4-(4-methyl-1,4-
diazepan-1-yl)benzoyl]amino}phenyl)benzamide
(11a).
HCl gas bubbledthrough a solution of 10a (350mg,
0.77mmol) in EtOH (15mL) under ꢀ20ꢁC for 20min.
The mixture was allowedto stir for 24h at 5 ꢁC, andthen
concentrated in vacuo. To the crude imidate dissolved in
EtOH (20mL) at ambient temperature was added
ammonium acetate (595mg, 7.70mmol). The reaction
mixture was stirredat ambient temperature for 36h
andconcentratedin vacuo. The resultedresidue was
4.1.8. N-(2-Aminophenyl)-3-cyanobenzamide (9a). To a
stirredsolution of
o-phenylene diamine (2.2g,
20.4mmol) in DMF (20mL) was added 3-cyanobenzoic
acid(1.5g, 10.2mmol), HOBt (2.1g, 15.3mmol), and
WSC (2.9g, 15.3mmol) at ambient temperature for
18h. The reaction mixture was diluted with ethyl acetate
andwashedwith H 2O andsaturatedsaline. The organic
layer was dried over Na2SO4 andconcentratedin vacuo
to give 9a (2.217g, 92%) as a brown amorphous powder:
1H NMR (DMSO-d6) d: 6.60 (1H, t, J = 7.7Hz), 6.79
(1H, dd, J = 1.4, 7.7Hz), 6.99 (1H, t, J = 7.7Hz), 7.17
(1H, d, J = 7.7Hz), 7.73 (1H, t, J = 7.7Hz), 8.05 (1H,
d, J = 7.7Hz), 8.27 (1H, d, J = 7.7Hz), 8.44 (1H, s),
9.83 (1H, s); FAB MS m/e (M+H)+ 238.
chromatographedon ODS-gel eluting with EtOH/H O
2
(10:90). EtOH was removedin vacuo, andthe aqueous
solution was lyophilizedafter being acidifiedwith 1N
HCl. Compound 11a (190mg, 53%) was obtainedas a
white amorphous powder: 1H NMR (DMSO-d6):
2.10–2.22 (1H, m), 2.29–2.43 (1H, m), 2.77 (3H, d,
J = 4.9Hz), 3.00–3.21 (2H, m), 3.36–3.56 (4H, m),
3.70–3.82 (1H, m), 3.86–3.99 (1H, m), 6.83 (2H, d,
J = 8.8Hz), 7.22–7.32 (2H, m), 7.63 (1H, dd, J = 2.0,
7.3Hz), 7.69 (1H, dd, J = 2.0, 7.3Hz), 7.77 (1H, t,
J = 7.8Hz), 7.97 (3H, d, J = 8.8Hz), 8.04 (1H, d,
J = 8.8Hz), 8.31 (1H, d, J = 7.8Hz), 8.53 (1H, s), 9.34
(2H, s), 9.57 (2H, s), 9.97 (1H, s), 10.97 (1H, s), 10.96
(1H, s); FAB MS m/e (M+H)+ 471; Anal. Calcdfor
C27H30N6O2Æ3.1HClÆ2.0H2O: C, 52.34; H, 6.03; N,
13.56; Cl, 17.74. Found: C, 51.99; H, 6.19; N, 13.60;
Cl, 17.84.
4.1.9. N-(2-Aminophenyl)-4-cyanobenzamide (9b). Com-
pound 9b was synthesizedfrom 4-cyanobenzoic acid
and o-phenylene diamine according to the same proce-
dure as that for 9a. Compound 9b was obtainedas a
white amorphous powder (48% yield): 1H NMR
(DMSO-d6) d: 6.60 (1H, t, J = 7.5Hz), 6.77 (1H, d,
J = 7.5Hz), 6.99 (1H, t, J = 7.5Hz), 7.16 (1H, d, J =
7.5Hz), 8.00 (2H, d, J = 8.2Hz), 8.13 (2H, d,
J = 8.2Hz), 9.87 (1H, s); FAB MS m/e (M+H)+ 238.
4.1.13. 4-[Amino(imino)methyl]-N-(2-{[4-(4-methyl-1,4-
diazepan-1-yl)benzoyl]amino}phenyl)benzamide
(11b).
Compound 11b was synthesizedfrom 10b according to
the same procedure as that for 11a. Compound 11b
was obtained as a white amorphous powder (26% yield):
1H NMR (DMSO-d6) d: 2.10–2.22 (1H, m), 2.39–2.45
(1H, m), 2.77 (3H, d, J = 4.9Hz), 3.01–3.20 (2H, m),
4.1.10. 3-Cyano-N-(2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide (10a). The solution of
13 (1.1g, 4.1mmol) in thionylchloride (5mL) stirred at
60ꢁC for 2h. After the reaction mixture was cooled,

1314
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
3.37–3.56 (4H, m), 3.79–3.83 (1H, m), 3.86–3.95 (1H,
m), 6.83 (2H, d, J = 9.2Hz), 7.23–7.32 (2H, m), 7.61–
7.68 (2H, m), 7.92–8.01 (3H, m), 8.21 (2H, d,
J = 8.3Hz), 9.35 (2H, s), 9.57 (2H, s), 10.66 (1H, s),
11.20 (1H, s); FAB MS m/e (M+H)+ 471; Anal. Calcd
for C27H30N6O2Æ3.1HClÆ2.0H2O: C, 52.34; H, 6.03; N,
13.56; Cl, 17.74. Found: C, 52.23; H, 6.29; N, 13.63;
Cl, 17.64.
4.1.18. (3-Cyanobenzyl)(triphenyl)phosphonium bromide
(17). To a stirredsolution of 3-cyanobenzylbromied
(3.01g, 15.4mmol) in benzene (100mL) was added tri-
phenylphosphine (4.04g, 15.4mmol) at 100ꢁC for 14h.
After the reaction mixture was cooled, the reaction mix-
ture was filtratedto give 17 (6.28g, 89%) as a white
solid: 1H NMR (CDCl3) d: 6.72–6.76, 6.99–7.05,
7.16–7.19, 7.24–7.80, 7.95–8.17 (10H, m); EI MS
m/e (M+H)+ 250.
4.1.14. 4-(4-Methyl-1,4-diazepan-1-yl)benzoic acid (13).
The solution of 12 (2.5g, 11.6mmol) in 6M NaOH
(20mL) was refluxed. After 40h, CHCl was added and
4.1.19. N-(2-Aminophenyl)-4-(4-methyl-1,4-diazepan-1-
yl)benzamide (18). To a stirredsolution of o-phenylene-
diamide (4.2g, 30mmol) in DMF (120mL) was added
13 (2.7g, 10mmol), HOBt (1.7g, 15mmol), andWSC
(2.4g, 15mmol) at ambient temperature for 12h. The
reaction mixture was diluted with ethyl acetate and
washedwith H 2O andsaturatedsaline. The organic
layer was dried over Na2SO4 andconcentratedin vacuo.
The resulting residues was chromatographed on silica
gel eluting with chloroform/methanol/NH4OH (7:1:0.1)
to give 18 (2.87g, 89%) as a pale brown amorphous
1
concentratedin vacuo gave 13 (2.71g, 98%): H NMR
(DMSO-d6) d: 2.06–2.24 (1H, m), 2.30–2.45 (1H, m),
2.77 (3H, s), 3.00–3.24 (2H, m), 3.24–3.55 (4H, m),
3.70–4.00 (2H, m), 6.81 (2H, d, J = 9.1Hz), 7.78 (2H,
d, J = 9.1Hz), 11.06 (1H, s), 12.20 (1H, s); FAB MS
m/e (M+H)+ 235.
4.1.15. 3-[(2-Nitrophenoxy)methyl]benzonitrile (15a). To
a stirredsolution of 2-nitrophenol (2.39g, 17.2mmol)
in DMF (30mL) was added 3-cyanobenzylbromide
(3.37g, 17.2mmol), potassium carbonate (2.61g,
18.9mmol) at ambient temperature for 1h. The reaction
mixture was diluted with chloroform and washed with
H2O andsaturatedsaline. The organic layer was dried
over Na2SO4 andconcentratedin vacuo. The resulting
residues was chromatographed on silica gel eluting with
hexane/ethylacetate (1:1) to give 15a (3.96g, 89%) as a
1
powder: H NMR (CDCl3) d: 1.98–2.06 (2H, m), 2.38
(3H, s), 2.54–2.59 (2H, m), 2.69–2.73 (2H, m), 3.49
(2H, t, J = 6.2Hz), 3.55–3.60 (2H, m), 5.45–5.54 (2H,
m), 6.66–6.78 (2H, m), 7.43–7.47 (1H, m), 7.57 (1H, s);
EI MS m/e (M+H)+ 325.
4.1.20. N-{2-[(3-Cyanobenzyl)oxy]phenyl}-4-(4-methyl-
1,4-diazepan-1-yl)benzamide (16a). To the solution of
15a (1.04g, 4.09mmol) in EtOH (40mL) andH 2O
(40mL) was added Fe powder (2.28g, 40.8mmol) and
ammonium chloride (110mg, 2.0mmol) and refluxed
for 0.5h. The reaction mixture was filtratedthrough a
padof Celite andconcentratedin vacuo. The resulting
residue was diluted with chloroform and washed with
H2O andsaturatedsaline. The organic layer was dried
over Na2SO4 andconcentratedin vacuo to give interme-
diate aniline compound. The solution of 13 (1.22g,
4.51mmol) in thionylchloride (40mL) refluxed for 2h
at 60ꢁC. After the reaction mixture was cooled, the reac-
tion mixture was concentratedin vacuo. The residue was
dissolved in pyridine (10mL) and cooled to 0ꢁC. To this
solution aniline intermediate was added and stirred at
ambient temperature for 20h. The reaction mixture
was concentratedin vacuo andthe residue was chroma-
tographedon silica gel eluting with chloroform/metha-
nol (20:1) to give 16a (1.37g, 76%) as a pale yellow
amorphous powder: 1H NMR (CDCl3) d: 2.41–2.55
(2H, m), 2.74 (3H, s), 3.07–3.13 (2H, m), 3.17–3.25
(2H, m), 3.61 (2H, t, J = 6.6Hz), 3.89–3.95 (2H, m),
5.19 (2H, s), 6.77 (2H, d, J = 9.0Hz), 6.95–6.99 (1H,
m), 7.03–7.09 (2H, m), 7.53 (1H, dd, J = 7.7Hz), 7.60–
7.68 (2H, m), 7.78 (2H, d, J = 9.0Hz), 7.83–7.86 (1H,
m), 8.45–8.47 (1H, m), 8.50–8.54 (1H, m); FAB MS
m/e (M+H)+ 441.
1
pale yellow amorphous powder: H NMR (CDCl3) d:
5.38 (2H, s), 7.14–7.19 (1H, m), 7.44–7.46 (1H, m),
7.62–7.71 (2H, m), 7.79–7.85 (2H, m), 7.91–7.94 (2H,
m); EI MS m/e (M+H)+ 254.
4.1.16. 3-[(2-Nitrobenzyl)oxy]benzonitrile (15b). Com-
pound 15b was synthesizedfrom 2-nitrobenzylbromide
and 3-cyanophenol according to the same procedure as
that for 15a. Compound 15b was obtainedas a white
amorphous powder (69% yield): 1H NMR (CDCl3)
d: 5.25 (2H, s), 7.21–7.27 (2H, m), 7.31 (1H, dt,
J = 1.3, 7.5Hz), 7.40–7.45 (1H, m), 7.51–7.57 (1H, m),
7.71 (1H, dt, J = 1.3, 7.9Hz), 7.83 (1H, dd, J = 1.1,
7.9Hz), 8.20 (1H, dd, J = 1.3, 8.2Hz); EI MS m/e
(M+H)+ 254.
4.1.17. 3-[(2-Nitrobenzyl)amino]benzonitrile (15c). To a
stirred solution of 2-nitro benzaldehyde (2.50g,
16.5mmol) and3-cyanoaniline (1.95g, 16.5mmol) in
1,2-dichloromethane (50mL) and AcOH (4.7mL,
82.1mmol) at ambient temperature was added sodium
triacetoxyborohydride (5.26g, 24.8mmol). After 19h,
the reaction mixture was washedwith 10% potassium
carbonate solution andbrine. The organic layer was
dried over Na2SO4 andconcentratedin vacuo. The
resulting residues was chromatographed on silica gel
eluting with hexane/ethylacetate (1:1) to give 15c
4.1.21. N-{2-[(3-Cyanophenoxy)methyl]phenyl}-4-(4-meth-
yl-1,4-diazepan-1-yl)benzamide (16b). Compound 16b
was synthesizedfrom 15b according to the same proce-
dure as that for 16a. Compound 16b was obtainedas
a white amorphous powder (26% yield): 1H NMR
(CDCl3) d: 2.20–2.32 (2H, m), 2.78 (3H, s), 3.16–3.56
(4H, m), 3.75–3.93 (2H, m), 4.06–4.13 (2H, m), 5.21
1
(3.75g, 89%) as a pale yellow amorphous powder: H
NMR (CDCl3) d: 4.75 (2H, d, J = 6.2Hz), 6.75–6.78
(2H, m), 6.98–7.01 (1H, m), 7.19–7.25 (1H, m), 7.45–
7.51 (1H, m), 7.57–7.76 (2H, m), 8.11 (1H, d,
J = 7.7Hz); FAB MS m/e (M+H)+ 254.

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1315
(2H, s), 6.83 (2H, d, J = 9.0Hz), 7.22–7.52 (8H, m), 7.87
(2H, d, J = 9.0Hz), 9.73 (1H, s); FAB MS m/e (M+H)+
441.
17 (1.62g, 3.52mmol) in toluene (30mL) was added
DBU (0.53mL, 3.54mmol) stirredat 80 ꢁC. After 2.0h,
2-nitrobenzaldehyde (510mg, 3.35mmol) in toluene
(20mL) was added and stirred at 80ꢁC for 16h. The pre-
cipitates was removedby filtration andconcentratedin
vacuo. To the solution of the residue in EtOH (5mL)
was added PdO–BaSO4 powder (100mg) and stirred in
hydrogen atmosphere at ambient temperature for 24h.
The reaction mixture was filtratedthrough a padof Cel-
ite andconcentratedin vacuo to give intermediate ani-
line compound. The solution of 13 (851mg, 3.14mmol)
in thionylchloride (18mL) stirred for 2h at 60ꢁC. After
the reaction mixture was cooled, the reaction mixture
was concentrated in vacuo. The residue was dissolved
in pyridine (10mL) and cooled to 0ꢁC. To this solution
aniline intermediate was added and stirred at ambient
temperature for 20h. The reaction mixture was concen-
tratedin vacuo andthe residue was chromatographed
on silica gel eluting with chloroform/MeOH (9:1) to give
16f (764mg, 66%) as a pale brown amorphous powder.
1H NMR (CDCl3) d: 2.12–2.21 (2H, m), 2.67–2.73
(2H, m), 2.88 (3H, s), 3.10–3.32 (6H, m), 3.53 (2H, t,
J = 6.2Hz), 3.75–3.82 (2H, m), 6.84 (2H, d,
J = 9.2Hz), 7.14–7.31 (4H, m), 7.40–7.49 (2H, m),
7.52–7.54 (1H, m), 7.61 (1H, dd, J = 1.7, 6.9Hz), 7.91
(2H, d, J = 9.0Hz), 9.62 (1H, s); FAB MS m/e
(M+H)+ 439.
4.1.22. N-(2-{[(3-Cyanophenyl)amino]methyl}phenyl)-4-
(4-methyl-1,4-diazepan-1-yl)benzamide (16c). Compound
16c was synthesizedfrom 15c according to the same pro-
cedure as that for 16a. Compound 16c was obtainedas a
white amorphous powder (13% yield): 1H NMR
(CDCl3) d: 1.85–1.95 (2H, m), 2.27 (3H, s), 2.42–2.48
(2H, m), 2.60–2.65 (2H, m), 3.51 (2H, t, J = 6.2Hz),
3.55–3.60 (2H, m), 4.30 (2H, d, J = 5.7Hz), 6.73–6.93
(5H, m), 7.14–7.33 (4H, m), 7.45 (1H, d, J = 7.5Hz),
7.81 (2H, d, J = 8.6Hz), 9.66 (1H, s); FAB MS m/e
(M+H)+ 440.
4.1.23. N-{2-[(E)-2-(3-Cyanophenyl)vinyl]phenyl}-4-(4-
methyl-1,4-diazepan-1-yl)benzamide (16d) and N-{2-
[(Z)-2-(3-cyanophenyl)vinyl]phenyl}-4-(4-methyl-1,4-dia-
zepan-1-yl)benzamide (16e). To the solution of 17 (5.5g,
12.0mmol) in toluene (90mL) was added DBU
(1.80mL, 21.0mmol) stirredat 80 ꢁC. After 2.0h, 2-
nitrobenzaldehyde (1.65g, 10.9mmol) in toluene
(50mL) was added and stirred at 80ꢁC for 16h. The pre-
cipitates was removedby filtration andconcentratedin
vacuo. To the solution of the residue in EtOH (40mL)
andH 2O (40mL) was added Fe powder (2.23g,
39.9mmol) andammonium chloride (107mg, 2.0mmol)
refluxedfor 0.5h. The reaction mixture was filtrated
through a padof Celite andconcentratedin vacuo.
The resulting residue was diluted with chloroform and
washedwith H 2O andsaturatedsaline. The organic
layer was dried over Na2SO4 andconcentratedin vacuo
to give intermediate aniline compound. The solution of
13 (1.62g, 5.98mmol) in thionylchloride (20mL) stirred
for 2h at 60ꢁC. After the reaction mixture was cooled,
the reaction mixture was concentratedin vacuo. The res-
idue was dissolved in pyridine (10mL) and cooled to
0ꢁC. To this solution aniline intermediate was added
andstirredat ambient temperature for 20h. The reac-
tion mixture was concentratedin vacuo andthe residue
was chromatographedon silica gel eluting with chloro-
form/MeOH (15:1) to give 16d (951mg, 34%) as a pale
brown amorphous powder and 16e (396mg, 14%) as a
pale brown amorphous powder: 16d; 1H NMR (CDCl3)
d: 1.86–1.94 (2H, m), 2.27 (3H, s), 2.43–2.48 (2H, m),
2.61–2.65 (2H, m), 3.52 (2H, t, J = 6.2Hz), 3.57–3.62
(2H, m), 6.78 (2H, d, J = 9.0Hz), 7.25 (1H, d,
J = 16.5Hz), 7.25–7.37 (2H, m), 7.40–7.44 (1H, m),
7.44 (1H, d, J = 16.5Hz), 7.57 (1H, dd, J = 7.7,
7.9Hz), 7.68–7.73 (1H, m), 7.78–7.90 (4H, m), 7.96–
7.98 (1H, m), 9.73 (1H, s); FAB MS m/e (M+H)+ 437.
4.1.25. N-{2-[(3-Cyanobenzyl)amino]phenyl}-4-(4-methyl-
1,4-diazepan-1-yl)benzamide (16g). To a stirredsolution
of 18 (486mg, 1.5mmol) and3-cyanobenzaledhyed
(197mg, 2.25mmol) in 1,2-dichloromethane (5mL) and
AcOH (0.85mL) at ambient temperature was added so-
dium triacetoxyborohydride (636mg, 3.0mmol). After
5h, the reaction mixture was washedwith 10% potas-
sium carbonate solution andbrine. The organic layer
was dried over Na2SO4 andconcentratedin vacuo.
The resultedresiude was chromatographedon silica
gel eluting with chloroform/MeOH (9:1) to give 16g
(495mg, 70%) as a white amorphous powder: 1H
NMR (CDCl3) d: 1.99–2.08 (2H, m), 2.39 (3H, s),
2.54–2.59 (2H, m), 2.70–2.75 (2H, m), 3.56 (2H, t,
J = 6.2Hz), 3.62–3.66 (2H, m), 4.43 (2H, d,
J = 5.5Hz), 4.84–4.90 (1H, m), 6.60–6.64 (1H, m), 6.74
(2H, d, J = 9.0Hz), 6.77–6.83 (1H, m), 7.05–7.11 (1H,
m), 7.42 (1H, dd, J = 7.7Hz), 7.50–7.55 (1H, m), 7.64
(1H, d, J = 7.9Hz), 7.69 (2H, br s), 7.80 (2H, d,
J = 9.0Hz); FAB MS m/e (M+H)+ 440.
4.1.26. Ethyl 3-[(3-cyanobenzyl)oxy]-2-nitrobenzoate
(20a). To the solution of ethyl 3-methyl-2-nitrobenzoate
(8.4g, 40.2mmol) in CCl4 (200mL) was added NBS
(8.72g, 48.2mmol) andcatalytic AIBN (10mg) and
refluxedfor 24h. The reaction mixture was filtrated
through a padof Celite andconcentratedin vacuo.
To the crude product in CH3CN (50mL) was added
3-cyanophenol (2.34g, 19.7mmol) andpotassium
carbonate (3.32g, 23.6mmol) at ambient temperature
for 48h. The reaction mixture was diluted with chloro-
form andwashedwith H 2O andsaturatedsaline. The
organic layer was dried over Na2SO4 andconcentrated
in vacuo. The resulting residues was chromatographed
on silica gel eluting with hexane/ethylacetate (3:1) to
1
16e; H NMR (CDCl3) d: 2.04–2.15 (2H, m), 2.58 (3H,
s), 2.86–3.09 (4H, m), 3.52 (2H, t, J = 6.2Hz), 3.69–
3.76 (2H, m), 6.63 (1H, d, J = 11.9Hz), 6.74 (1H, d,
J = 11.9Hz), 6.80 (2H, d, J = 9.0Hz), 7.02–7.11 (2H,
m), 7.28–7.34 (1H, m), 7.40 (1H, dd, J = 7.5, 7.9Hz),
7.44–7.53 (2H, m), 7.57–7.59 (1H, m), 7.60–7.64 (1H,
m), 7.79 (2H, d, J = 9.0Hz), 9.58 (1H, s); FAB MS
m/e (M+H)+ 437.
4.1.24. N-{2-[2-(3-Cyanophenyl)ethyl]phenyl}-4-(4-meth-
yl-1,4-diazepan-1-yl)benzamide (16f). To the solution of

1316
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
give 20a (5.946g, 93%) as a yellow amorphous pow-
der: H NMR (CDCl3) d: 1.36 (3H, t, J = 7.0Hz), 4.38
(2H, q, J = 7.0Hz), 5.14 (2H, s), 7.14–7.22 (1H, m), 7.30
(1H, dt, J = 1.0, 7.7Hz), 7.37 (1H, s), 7.40 (1H, s), 7.63
(1H, t, J = 7.7Hz), 7.82 (1H, dd, J = 1.0, 7.7Hz), 7.97
(1H, dd, J = 1.0, 7.7Hz); FAB MS m/e (M+H)+ 327.
7.22–7.33 (3H, m), 7.39–7.42 (1H, m), 7.45–7.48 (1H,
m), 7.74 (2H, d, J = 9.2Hz), 7.86 (1H, dd, J = 1.7,
7.9Hz), 8.55 (1H, d, J = 8.6Hz), 8.70 (1H, d,
J = 1.7Hz); FAB MS m/e (M+H)+ 513.
1
4.1.32. Ethyl 4-[(3-cyanobenzoyl)amino]-3-nitrobenzoate
(23a). To a stirring solution of ethyl 4-amino-3-nitro-
benzoate (6.02g, 28.6mmol) in 1,2-dichloroethane
(30mL) was added pyridine (3.34g, 43mmol) and 3-
cyanobenzoylchloride (3.1g, 17.8mmol). After 12h, the
solvent was removedin vacuo andthe resiude was
washedEtOH to give 23a (10.59g, 47%) as a yellow
4.1.27. Ethyl 4-[(3-cyanobenzyl)oxy]-3-nitrobenzoate
(20b). Compound 20b was synthesizedfrom ethyl 4-
methyl-3-nitrobenzoate according to the same proce-
dure as that for 20a. Compound 20b was obtainedas
a yellow amorphous powder (45% yield): 1H NMR
(CDCl3) d: 1.44 (3H, t, J = 7.1Hz), 4.46 (2H, q,
J = 7.1Hz), 5.56 (2H, s), 7.24–7.35 (4H, m), 7.96 (1H,
d, J = 8.2Hz), 8.34 (1H, dd, J = 1.7, 8.4Hz), 8.83 (1H,
d, J = 1.7Hz); FAB MS m/e (MꢀH)+ 325.
1
powder: H NMR (CDCl3) d: 1.44 (3H, t, J = 7.1Hz),
4.45 (2H, q, J =7.1Hz), 7.72 (1H, dd, J = 7.7, 7.9Hz),
7.92 (1H, dt, J = 1.3, 7.7Hz), 8.17–8.22 (1H, m), 8.31–
8.33 (1H, m), 8.38 (1H, dd, J = 2.0, 8.8Hz), 8.97 (1H,
d, J = 2.0Hz), 9.08 (1H, d, J = 8.8Hz), 11.6 (1H, br s);
FAB MS m/e (M+H)+ 338.
4.1.28. Ethyl 3-[(3-cyanobenzyl)oxy]-4-nitrobenzoate
(20c). Compound 20c was synthesizedfrom ethyl 5-
methyl-4-nitrobenzoate according to the same proce-
dure as that for 20a. Compound 20c was obtainedas a
yellow amorphous powder (37% yield): 1H NMR
(CDCl3) d: 1.42 (3H, t, J = 7.1Hz), 4.32 (2H, q,
J = 7.1Hz), 5.49 (2H, s), 7.26–7.34 (4H, m), 8.15–8.22
(2H, m), 8.50 (1H, s); FAB MS m/e (MꢀH)+ 325.
4.1.33. Ethyl 4-{[(3-cyanophenyl)amino]methyl}-3-nitro-
benzoate (23b). To the solution of ethyl 4-methyl-3-
nitrobenzoate (20.3g, 97mmol) in CCl4 (145mL) was
added NBS (19g, 107mmol) and catalytic AIBN
(50mg) andrefluxedfor 24h. The reaction mixture
was filtratedthrough a padof Celite andconcentrated
in vacuo. To the crude product in CH3CN (90mL)
was added 3-cyanoaniline (7.97g, 67.5mmol) and
potassium carbonate (12.44g, 90mmol), andstirring at
70ꢁC for 3h. The reaction mixture was filtratedand
concentratedin vacuo andthe resultedresiude was
chromatographedon silica gel eluting with hexane/ethyl-
acetate (3:1) to give 23b (12.63g, 39% yield) as a brown
amorphous powder: 1H NMR (CDCl3) d: 1.42 (3H,
t, J = 7.2Hz), 4.43 (2H, q, J = 7.2Hz), 4.63 (1H, t,
J = 5.7Hz), 4.81 (2H, d, J = 6.0Hz), 6.72–6.78
(2H, m), 7.01 (1H, dt, J = 1.3, 7.7Hz), 7.19–7.27 (1H,
m), 7.69 (1H, d, J = 8.0Hz), 8.24 (1H, dd, J = 1.7,
8.0Hz), 8.73 (1H, d, J = 1.7Hz); FAB MS m/e
(M+H)+ 295.
4.1.29. Ethyl 3-[(3-cyanobenzyl)oxy]-2-{[4-(4-methyl-1,4-
diazepan-1-yl)benzoyl]amino}benzoate (21a). Compound
21a was synthesizedfrom 20a according to the same
procedure as that for 16a. Compound 21a was obtained
1
as a white amorphous powder (81% yield): H NMR
(CDCl3) d: 1.38 (3H, t, J = 7.1Hz), 2.21–2.32 (2H, m),
2.56 (3H, s), 2.77–2.84 (2H, m), 2.93–2.98 (2H, m),
3.61 (2H, t, J = 6.4Hz), 3.80 (2H, t, J = 6.4Hz), 4.36
(2H, q, J = 7.1Hz), 5.13 (2H, s), 6.75 (2H, d,
J = 9.1Hz), 7.12–7.21 (3H, m), 7.24–7.34 (2H, m),
7.81–7.85 (1H, m), 7.95 (2H, d, J = 9.1Hz), 8.02 (1H,
dd, J = 1.7, 7.9Hz), 10.73 (1H, s); FAB MS m/e
(M+H)+ 513.
4.1.30. Ethyl 4-[(3-cyanobenzyl)oxy]-3-{[4-(4-methyl-1,4-
diazepan-1-yl)benzoyl]amino}benzoate (21b). Compound
21b was synthesizedfrom 20b according to the same
procedure as that for 16a. Compound 21b was obtained
4.1.34. Ethyl 4-formyl-3-nitrobenzoate (25). To the solu-
tion of ethyl 4-methyl-3-nitrobenzoate (35.6g,
170.3mmol) in CCl4 (500mL) was added NBS (34g,
188mmol) andcatalytic AIBN (10mg) andrefluxedfor
24h. The reaction mixture was filtratedthrough a pad
of Celite and concentrated in vacuo. To the crude prod-
uct in CH3CN (500mL) was added N-methyl morphorine
N-oxide (20.0g, 170.3mmol) at ambient temperature for
1.0h. The reaction mixture was concentratedin vacuo
and the residue was diluted with chloroform and washed
with H2O andsaturatedsaline. The organic layer was
dried over Na2SO4 andconcentratedin vacuo. The
resulting residues was chromatographed on silica gel elut-
ing with hexane/ethylacetate (4:1) to give 25 (10.723g,
1
as a white amorphous powder (86% yield): H NMR
(CDCl3) d: 1.39 (3H, t, J = 7.1Hz), 1.98–2.07 (2H, m),
2.39 (3H, s), 2.54–2.59 (2H, m), 2.70–2.74 (2H, m),
3.55 (2H, t, J = 6.2Hz), 3.61–3.66 (2H, m), 4.39 (2H,
q, J = 7.1Hz), 5.21 (2H, s), 6.69 (2H, d, J = 9.2Hz),
7.22–7.33 (3H, m), 7.39–7.42 (1H, m), 7.45–7.48 (1H,
m), 7.74 (2H, d, J = 9.2Hz), 7.86 (1H, dd, J = 1.7,
7.9Hz), 8.55 (1H, s), 8.70 (1H, d, J = 1.7Hz); FAB
MS m/e (M+H)+ 513.
1
4.1.31. Ethyl 3-[(3-cyanobenzyl)oxy]-4-{[4-(4-methyl-1,4-
diazepan-1-yl)benzoyl]amino}benzoate (21c). Compound
21c was synthesizedfrom 20c according to the same pro-
cedure as that for 16a. Compound 21c was obtainedas a
white amorphous powder (52% yield): 1H NMR
(CDCl3) d: 1.39 (3H, t, J = 7.1Hz), 1.98–2.07 (2H, m),
2.39 (3H, s), 2.54–2.59 (2H, m), 2.70–2.74 (2H, m),
3.55 (2H, t, J = 6.2Hz), 3.61–3.66 (2H, m), 4.39 (2H,
q, J = 7.1Hz), 5.21 (2H, s), 6.69 (2H, d, J = 9.2 H),
28%) as a white amorphous powder: H NMR (CDCl3)
d: 1.46 (3H, t, J = 7.2Hz), 4.48 (2H, q, J = 7.2Hz), 8.00
(1H, d, J = 8.0Hz), 8.42 (1H, d, J = 8.0Hz), 8.75 (1H,
s), 10.46 (1H, s); GC MS m/e (M)+ 224.
4.1.35. Ethyl 3-amino-4-[2-(3-cyanophenyl)ethyl]benzoate
(26). Compound 26 was synthesizedfrom 17 and 25
according to the same procedure as that for 16f. Com-
pound 26 was obtainedas a brown amorphous powder

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1317
(83% yield): 1H NMR (CDCl3) d: 1.38 (3H, t,
J = 7.1Hz), 2.82 (2H, t, J = 8.4Hz), 2.96 (2H, t,
J = 8.4Hz), 4.34 (2H, q, J = 7.1Hz), 6.97 (1H, d, J =
8.4Hz), 7.33–7.41 (4H, m), 7.44–7.52 (2H, m); FAB
MS m/e (M+H)+ 295.
(DMSO-d6) d: 1.20 (3H, t, J = 6.9Hz), 3.31 (2H, s),
4.16 (2H, q, J = 6.9Hz), 7.00 (1H, d, J = 9.2Hz), 7.21
(1H, dd, J = 3.1, 9.2Hz), 7.27 (2H, s), 7.36 (1H, d,
J = 3.1Hz); FAB MS m/e (M+H)+ 240.
4.1.40. Ethyl (4-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}-3-nitrophenoxy)acetate (29). Compound 29 was
synthesizedfrom 28 according to the same procedure
as that for 24c. Compound 29 was obtainedas a white
4.1.36. Ethyl 4-[(3-cyanobenzoyl)amino]-3-{[4-(4-methyl-
1,4-diazepan-1-yl)benzoyl]amino}benzoate (24a). Com-
pound 24a was synthesizedfrom 23a according to the
same procedure as that for 16a. Compound 24a was ob-
1
amorphous powder (95% yield): H NMR (CDCl3) d:
1
tained as a white amorphous powder (55% yield): H
1.32 (3H, t, J = 7.1Hz), 2.33 (3H, s), 2.73–2.80 (4H,
m), 3.02–3.41 (2H, m), 3.57–3.67 (2H, m), 3.89–4.03
(2H, m), 4.29 (2H, q, J = 7.1Hz), 4.68 (2H, s), 6.76
(2H, d, J = 8.9Hz), 7.24–7.36 (1H, m), 7.72 (1H, d,
J = 2.9Hz), 7.89 (2H, d, J = 8.9Hz), 8.95 (1H, d,
J = 9.1Hz), 11.06 (1H, s); FAB MS m/e (M+H)+ 457.
NMR (CDCl3) d: 1.40 (3H, t, J = 7.1Hz), 2.00–2.10
(2H, m), 2.41 (3H, br s), 2.57–2.62 (2H, m), 2.73–2.77
(2H, m), 3.62 (2H, t, J = 6.3Hz), 3.67–3.72 (2H, m),
4.40 (2H, q, J = 7.1Hz), 6.76 (2H, d, J = 9.2Hz), 7.57
(1H, t, dd, J = 7.5, 7.9Hz), 7.72 (1H, dt, J =1.5, 7.9Hz),
7.85 (1H, d, J = 9.0Hz), 8.0–8.08 (4H, m), 8.30–8.35
(1H, m), 8.40–8.42 (1H, m); FAB MS m/e (M+H)+ 525.
4.1.41. Ethyl {4-[(3-cyanobenzoyl)amino]-3-nitrophen-
oxy}acetate (31). Compound 31 was synthesizedfrom
28 according to the same procedure as that for 23a.
Compound 31 was obtainedas a white amorphous pow-
der (43% yield): 1H NMR (CDCl3) d: 1.33 (3H, t,
J = 7.1Hz), 4.31 (2H, q, J = 7.1Hz), 4.71 (2H, s), 7.37
(1H, dd, J = 2.9, 9.3Hz), 7.68 (1H, t, J = 7.8Hz), 7.77
(1H, d J = 2.9Hz), 7.86–7.91 (1H, m), 8.14–8.19 (1H,
m), 8.27–8.30 (1H, m), 8.88 (1H, d, J = 9.3Hz); FAB
MS m/e (M+H)+ 370.
4.1.37. Ethyl 4-{[(3-cyanophenyl)amino]methyl}-3-{[4-(4-
methyl-1,4-diazepan-1-yl)benzoyl]amino}benzoate (24b).
Compound 24b was synthesizedfrom 23b according to
the same procedure as that for 16a. Compound 24b
was obtained as a white amorphous powder (67% yield):
1H NMR (CDCl3) d: 1.39 (3H, t, J = 7.4Hz), 1.97–2.06
(2H, m), 2.38 (3H, s), 2.53–2.59 (2H, m), 2.68–2.73 (2H,
m), 3.51 (2H, t, J = 6.4Hz), 3.57–3.63 (2H, m), 4.34–4.42
(5H, m), 6.58 (2H, d, J = 8.8Hz), 6.96–7.01 (2H, m),
7.12 (1H, d, J = 7.8Hz), 7.31 (1H, t, J = 7.8Hz), 7.40
(1H, d, J = 8.3Hz), 7.65 (2H, d, J = 8.7Hz), 7.81 (1H,
dd, J = 1.5, 7.8Hz), 8.67 (1H, d, J = 2.0Hz), 8.85 (1H,
s); FAB MS m/e (M+H)+ 512.
4.1.42. Ethyl (3-[(3-cyanobenzoyl)amino]-4-{[4-(4-methyl-
1,4-diazepan-1-yl)benzoyl]amino}phenoxy)acetate (30c).
Compound 30c was synthesizedfrom 29 according to
the same procedure as that for 16a using 3-cyanobenzoic
acidinsteadof 13. Compound 30c was obtainedas a
white amorphous powder (50% yield): 1H NMR
(CDCl3) d: 1.28 (3H, t, J = 7.1Hz), 1.98–2.08 (2H, m),
2.34 (3H, s), 2.54–2.60 (2H, m), 2.68–2.76 (2H, m),
3.56 (2H, t, J = 6.2Hz), 3.61–3.67 (2H, m), 4.23 (2H,
q, J = 7.1Hz), 4.34 (2H, s), 6.59 (1H, dd, J = 2.9,
9.0Hz), 6.73 (2H, d, J = 9.0Hz), 7.15–7.21 (2H, m),
7.26 (1H, s), 7.60 (1H, t, J = 7.7Hz), 7.80 (1H, dt,
J = 1.3, 7.7Hz), 7.86 (2H, d, J = 9.0Hz), 8.21–8.26
(1H, m), 8.33 (1H, t, J = 1.3Hz), 8.57 (1H, s), 10.16
(1H, s); FAB MS m/e (M+H)+ 556.
4.1.38. Ethyl 4-[2-(3-cyanophenyl)ethyl]-3-{[4-(4-methyl-
1,4-diazepan-1-yl)benzoyl]amino}benzoate (24c). The
solution of 13 (2.94g, 9.59mmol) in thionylchloride
(10mL) stirredfor 2h at 60 ꢁC. After the reaction mix-
ture was cooled, the reaction mixture was concentrated
in vacuo. The residue was dissolved in pyridine
(10mL) andcooledto 0 ꢁC. To this solution 25 (2.35g,
7.79mmol) was added and stirred at ambient tempera-
ture for 20h. The reaction mixture was concentratedin
vacuo andthe residue was chromatographedon silica
gel eluting with chloroform/methanol (20:1) to give 24c
(3.681g, 90%) as a white amorphous powder: 1H
NMR (CDCl3) d: 1.37 (3H, t, J = 7.2Hz), 2.42–2.53
(2H, m), 2.76 (3H, s), 2.94 (4H, s), 3.14–3.21 (2H, m),
3.24–3.29 (2H, m), 3.60 (2H, t, J = 6.4Hz), 3.88–3.95
(2H, m), 4.34 (2H, q, J = 7.2Hz), 6.73 (2H, d,
J = 9.0Hz), 7.18–7.22 (1H, m), 7.28–7.35 (3H, m),
7.39–7.44 (1H, m), 7.69–7.72 (1H, m), 7.75 (2H, d,
J = 9.0Hz), 7.86 (1H, dd, J = 1.7, 8.0Hz), 8.24 (1H, d,
J = 1.5Hz); FAB MS m/e (M+H)+ 511.
4.1.43. Ethyl (4-[(3-cyanobenzoyl)amino]-3-{[4-(4-methyl-
1,4-diazepan-1-yl)benzoyl]amino}phenoxy)acetate (30b).
Compound 30b was synthesizedfrom 31 according to
the same procedure as that for 16a. Compound 30b
was obtained as a white amorphous powder (57% yield):
1H NMR (CDCl3) d: 1.27 (3H, t, J = 6.9Hz), 1.97–2.05
(2H, m), 2.33 (3H, s), 2.50–2.57 (2H, m), 2.62–2.76 (2H,
m), 3.55 (2H, t, J = 6.1Hz), 3.62–3.68 (2H, m), 4.22 (2H,
q, J = 6.9Hz), 4.44 (2H, s), 6.57–6.61 (1H, m), 6.72
(2H, d, J = 9.0Hz), 7.12–7.20 (2H, m), 7.22 (1H, s),
7.60 (1H, t, J = 7.6Hz), 7.73–7.82 (1H, m), 7.86 (2H, d,
J = 9.0Hz), 8.22–8.25 (1H, m), 8.90 (1H, t, J = 1.3Hz),
8.56 (1H, s), 10.20 (1H, s); FAB MS m/e (M+H)+ 556.
4.1.39. Ethyl (4-amino-3-nitrophenoxy)acetate (28). To a
stirring solution of 4-amino-3-nitrophenol (1.54g,
10.0mmol) in tetrahydofuran (20mL) was added gly-
colic acidethyl ester (0.95mL, 10mmol), triphenylphos-
phine (2.89g, 11mmol), andDEAD (1.7mL, 11mmol).
After 24h, the reaction mixture was concentrated
in vacuo andthe residue was chromatographedon silica
gel eluting with hexane/ethylacetate (2:1) to give 28
4.1.44.
tert-Butyl
4-(4-{[(2-hydroxy-6-nitrophenyl)-
amino]carbonyl}phenyl)-1,4-diazepane-1-carboxylate (33).
To the solution of tert-butyl 4-(4-carboxyphenyl)-1,4-
diazepane-1-carboxylate (5.11g, 16mmol) in methylene-
chloride (50mL) was added triphenylphosphine (4.31g,
1
(1.37g, 57%) as a white amorphous powder: H NMR

1318
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
16.4mmol) andNBS (3.2g, 18mmol) at 0 ꢁC andstirred
at ambient temperature. After 5min, 2-amino-3-nitro-
ohenol (2.47g, 16mmol) andpyrinide (1.6mL,
19.8mmol) in methylenechloride (20mL) was added
andstirred12h. The reaction mixture was concentrated
in vacuo andthe resultedresidue was chromatographed
on silica gel eluting with chloroform/MeOH (90:1) to
give 33 (1.867g, 25% yield) as a yellow amorphous pow-
organic layer was dried over MgSO4 andconcentrated
in vacuo. The resultedresidue was chromatographed
on silica gel eluting with chloroform/MeOH (20:1)
to give 30a (2.508g, 82%) as a white amorphous powder:
1H NMR (CDCl3) d: 1.31 (3H, t, J = 7.1Hz), 1.98–
2.08 (2H, m), 2.38 (3H, s), 2.53–2.57 (2H, m), 2.69–
2.75 (2H, m), 3.57 (2H, t, J = 6.2Hz), 3.61–3.67 (2H,
m), 4.29 (2H, q, J = 7.1Hz), 4.76 (2H, s), 6.75 (2H, d,
J = 8.9Hz), 6.83 (1H, dd, J = 1.0, 8.2Hz), 7.24–7.31
(1H, m), 7.57 (1H, t, J = 7.7Hz), 7.74–7.81 (2H, m),
8.02 (2H, d, J = 8.9Hz), 8.22–8.28 (1H, m), 8.32–8.36
(1H, m), 9.48 (1H, s), 10.80 (1H, s); FAB MS m/e
(M+H)+ 556.
1
der: H NMR (CDCl3) d: 1.39 (4H, s), 1.43 (5H, s),
1.94–2.06 (2H, m), 3.24–3.40 (4H, m), 3.56–3.74 (4H,
m), 6.70 (2H, d, J = 8.9Hz), 7.10–7.26 (2H, m), 7.56–
7.62 (1H, m), 7.91 (2H, d, J = 8.9Hz), 8.97 (1H, s);
FAB MS m/e (M+H)+ 457.
4.1.45. tert-Butyl 4-[4-({[2-(2-ethoxy-2-oxoethoxy)-6-
nitrophenyl]amino}carbonyl)phenyl]-1,4-diazepane-1-
carboxylate (34). To a stirredsolution of 33 (1.86g,
4.08mmol) in CH3CN (50mL) was added ethyl bromo-
acetate (0.57mL, 5.12mmol), potassium carbonate
(733mg, 5.12mmol), andstirring at ambient tempera-
ture for 48h. The reaction mixture was filtratedandcon-
centratedin vacuo andthe resultedresiude was
chromatographedon silica gel eluting with chloro-
form/MeOH (100:1) to give 34 (1.995g, 91% yield) as
a yellow amorphous powder: 1H NMR (CDCl3) d:
1.29 (3H, t, J = 7.1Hz), 1.38 (4H, s), 1.44 (5H, s),
3.17–3.25 (2H, m), 3.28–3.36 (2H, m), 3.54–3.67 (4H,
m), 4.27 (2H, q, J = 7.1Hz), 4.74 (2H, s), 6.74 (2H, d,
J = 9.0Hz), 7.11–7.25 (2H, m), 7.62 (1H, dd, J = 1.4,
8.2Hz), 7.90 (2H, d, J = 9.0Hz), 8.97 (1H, s); FAB
MS m/e (M+H)+ 541.
4.1.48. N-[2-({3-[Amino(imino)methyl]benzyl}oxy)phen-
yl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (36a). HCl
gas bubbledthrough a solution of
16a (307mg,
0.70mmol) in EtOH (10mL) andchloroform (10mL)
under ꢀ20ꢁC for 20min. The mixture was allowedto
stir for 24h at 5ꢁC, andthen concentratedin vacuo.
To the crude imidate dissolved in EtOH (10mL) at
ambient temperature was added ammonium acetate
(270mg, 3.50mmol). The reaction mixture was stirred
at ambient temperature for 36h andconcentratedin
vacuo. The resultedresidue was chromatographedon
ODS-gel eluting with EtOH/H2O (2:98). EtOH was
removedin vacuo, andthe aqueous solution was lyoph-
ilizedafter being acidifiedwith 1N HCl. Compound 36a
(101mg, 25%) was obtainedas a pale yellow amorphous
powder: 1H NMR (DMSO-d6) d: 2.12–2.22 (1H, m),
2.31–2.45 (1H, m), 2.78 (3H, d, J = 4.3Hz), 3.02–
3.20 (2H, m), 3.39–3.55 (4H, m), 3.74–3.82 (1H, m),
3.93 (1H, dd, J = 2.9, 16.6Hz), 5.26 (2H, s), 6.83
(2H, d, J = 8.8Hz), 7.24–7.32 (2H, m), 7.34–7.42 (2H,
m), 7.45–7.53 (4H, m), 7.90 (2H, d, J = 8.8Hz), 9.23,
9.40 (each 2H, 2s), 9.82 (1H, s), 11.1 (1H, br s);
FAB MS m/e (M+H)+ 458; Anal. Calcdfor
C27H31N5O2Æ2.2HClÆ2.2H2O: C, 56.16; H, 6.56; N,
12.13; Cl, 13.51. Found: C, 56.04; H, 6.59; N, 12.14;
Cl, 13.41.
4.1.46. tert-Butyl 4-[4-({[2-[(3-cyanobenzoyl)amino]-6-(2-
ethoxy-2-oxoethoxy)phenyl]amino}carbonyl)phenyl]-1,4-
diazepane-1-carboxylate (35). Compound 35 was synthe-
sizedfrom 34 according to the same procedure as that
for 16a using 3-cyanobenzoic acidinsteadof 13. Com-
pound 35 was obtainedas a white amorphous powder
(quant yield): 1H NMR (CDCl3) d: 1.31 (3H, t,
J = 7.0Hz), 1.35 (4H, s), 1.43 (5H, s), 1.92–2.02 (2H,
m), 3.16–3.35 (4H, m), 3.54–3.70 (4H, m), 4.29 (2H, q,
J = 7.0Hz), 4.77 (2H, s), 6.76 (2H, d, J = 9.1Hz), 6.83
(1H, dd, 1.2, 8.0Hz), 7.26–7.35 (1H, m), 7.54–7.60
(1H, m), 7.75–7.80 (2H, m), 8.20 (2H, d, J = 9.1Hz),
8.25–8.26 (1H, m), 8.30–8.45 (1H, m), 9.52 (1H, s),
10.77 (1H, s); FAB MS m/e (M+H)+ 642.
4.1.49. N-[2-({3-[Amino(imino)methyl]phenoxy}methyl)-
phenyl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (36b).
Compound 36b was synthesizedfrom 16b according to
the same procedure as that for 36a. Compound 36b
was obtainedas a pale yellow amorphous powedr
(46% yield): 1H NMR (DMSO-d6) d: 2.14–2.23 (1H,
m), 2.32–2.45 (1H, m), 2.80 (3H, d, J = 4.4Hz), 3.06–
3.23 (2H, m), 3.42–3.58 (4H, m), 3.79 (1H, dd, J = 9.6,
16.4Hz), 3.97 (1H, dd, J = 2.9, 16.1Hz), 5.28 (2H, s),
6.84 (2H, d, J = 9.1Hz), 6.97–7.02 (1H, m), 7.12–7.19
(2H, m), 7.63 (1H, t, J = 7.8Hz), 7.78–7.87 (3H, m),
7.86 (2H, d, J = 9.1Hz), 8.13 (1H, s), 9.24 (1H, s),
9.41, 9.48 (each 2H, 2s) 11.08 (1H, s); FAB MS m/e
(M+H)+ 458; Anal. Calcdfor C 27H31N5O2Æ2.2HClÆ3.0-
H2O: C, 54.80; H, 6.68; N, 11.83; Cl, 13.18. Found: C,
54.61; H, 6.42; N, 11.86; Cl, 13.16.
4.1.47. Ethyl (3-[(3-cyanobenzoyl)amino]-2-{[4-(4-methyl-
1,4-diazepan-1-yl)benzoyl]amino}phenoxy)acetate (30a).
To a solution of 35 (3.48mg, 5.43mmol) in ethyl acetate
(20mL) at ambient temperature was added 4M HCl
ethyl acetate solution (5mL). After stirring at ambient
temperature for 2h, the reaction mixture was concen-
trated in vacuo. The resulting residue was dissolved in
chloroform andwashedwith 10% aquarius potassium
carbonate. The organic layer was dried over MgSO4
andconcentratedin vacuo. To a stirredsolution of the
deprotected intermediate in 1,2-dichloromethane
(10mL) at ambient temperature was added acetic acid
(1.53mL, 54.3mmol), 35% aquarius formaldehyde
(1.53mL, 54.3mmol) and sodium triacetoxyborohydride
(1.8g, 8.15mmol). After 2h, the reaction mixture was
washedwith 10% aqueous potassium carbonate. The
4.1.50.
N-{2-[({3-[Amino(imino)methyl]phenyl}amino)-
methyl]phenyl}-4-(4-methyl-1,4-diazepan-1-yl)benzamide
(36c). Compound 36c was synthesizedfrom 16c accord-
ing to the same procedure as that for 36a. Compound
36c was obtainedas a pale yellow amorphous powder

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1319
(43% yield): 1H NMR (DMSO-d6) d: 2.12–2.22 (1H, m),
2.35–2.46 (1H, m), 2.77 (3H, d, J = 4.4Hz), 3.03–3.20
(2H, m), 3.38–3.56 (4H, m), 3.80 (1H, dd, J = 10.0,
16.0Hz), 3.95 (1H, dd, J = 3.0, 6.6Hz), 4.40 (2H, s),
6.84 (2H, d, J = 9.0Hz), 6.98 (1H, dd, J = 1.0, 8.3Hz),
7.05 (1H, d, J = 7.8Hz), 7.14 (1H, s), 7.19 (1H, dt,
J = 1.0, 7.3Hz), 7.25–7.33 (2H, m), 7.37 (1H, d,
J = 7.8Hz), 7.41 (1H, d, J = 6.8Hz), 7.97 (2H, d,
J = 9.0Hz), 9.21, 9.34 (each 2H, 2s), 9.96 (1H, s), 11.4
(1H, br s); FAB MS m/e (M+H)+ 457; Anal. Calcdfor
C27H32N6OÆ4.6HClÆ3.0H2O: C, 47.81; H, 6.33; N,
12.39; Cl, 24.04. Found: C, 47.74; H, 6.57; N, 12.43;
Cl, 24.30.
(1H, s), 11.3 (1H, br s); FAB MS m/e (M+H)+ 456;
Anal. Calcdfor C 28H33N5OÆ2.6HClÆ2.0H2O: C, 57.35;
H, 6.81; N, 11.94; Cl, 15.72. Found: C, 57.29; H, 7.09;
N, 11.90; Cl, 15.68.
4.1.54.
N-[2-({3-[Amino(imino)methyl]benzyl}amino)-
phenyl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (36g).
Compound 36g was synthesizedfrom 16g according to
the same procedure as that for 36a. Compound
36g was obtainedas a pale yellow amorphous pow-
der (28% yield): 1H NMR (DMSO-d6) d: 2.13–
2.24 (1H, m), 2.29–2.38 (1H, m), 2.79 (3H, d,
J = 4.9Hz), 3.03–3.21 (2H, m), 3.39 –3.57 (4H, m),
3.77 (1H, dd, J = 9.8, 16.2Hz), 3.90–3.98 (1H, m), 4.44
(2H, s), 5.76–5.86 (1H, br), 6.53 (1H, d, J = 8.3Hz),
6.61 (1H, t, J = 7.3Hz), 6.85 (2H, d, J = 8.8Hz), 6.96–
6.99 (1H, m), 7.10–7.13 (1H, m), 7.55 (1H, dd, J = 7.3,
7.8Hz), 7.68 (1H, d, J = 7.8Hz), 7.76 (1H, d, J
=7.8Hz), 7.92 (1H, br s), 7.96 (2H, d, J = 8.8Hz),
9.15, 9.37 (each 2H, 2s), 9.61 (1H, s), 10.90–11.00 (1H,
br); FAB MS m/e (M+H)+ 457; Anal. Calcdfor
C27H32N6OÆ2.0HClÆ2.2H2O: C, 56.98; H, 6.80; N,
14.77; Cl, 12.46. Found: C, 57.07; H, 6.68; N, 14.49;
Cl, 12.19.
4.1.51. N-[2-((E)-2-{3-[Amino(imino)methyl]phenyl}vinyl)-
phenyl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (36d).
Compound 36d was synthesizedfrom 16d according to
the same procedure as that for 36a. Compound 36d
was obtainedas a pale yellow amorphous powedr
(51% yield): 1H NMR (DMSO-d6) d: 2.14–2.23 (1H,
m), 2.36–2.47 (1H, m), 2.79 (3H, d, J = 4.9Hz), 3.05–
3.21 (2H, m), 3.40–3.57 (4H, m), 3.77–3.85 (1H, m),
3.92–3.99 (1H, m), 6.86 (2H, d, J = 9.1Hz), 7.29 (1H,
d, J = 16.4Hz), 7.28–7.37 (1H, m), 7.41 (1H, 1.0,
7.8Hz), 7.50 (1H, d, J = 16.4Hz), 7.60 (1H, t,
J = 7.8Hz), 7.71 (1H, d, J = 7.8Hz), 7.84 (1H, t,
J = 7.3Hz), 7.98 (2H, d, J = 9.1Hz), 7.99 (1H, s), 9.35,
9.50 (each 2H, 2s), 9.90 (1H, s), 11.3 (1H, br s); FAB
MS m/e (M+H)+ 454; Anal. Calcdfor C 28H31N5OÆ2.5H-
ClÆ2.5H2O: C, 57.02; H, 6.58; N, 11.87; Cl, 15.03.
Found: C, 57.35; H, 6.91; N, 11.82; Cl, 15.43.
4.1.55. Ethyl 3-({3-[amino(imino)methyl]phenoxy}meth-
yl)-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzo-
ate (37a). Compound 37a was synthesizedfrom
21a according to the same procedure as that for 36a.
Compound 37a was obtainedas a white amorphous
1
powder (19% yield): H NMR (DMSO-d6) d: 1.12 (3H,
t, J = 7.4Hz), 2.09–2.25 (2H, m), 2.64 (3H, s), 2.96–
3.43 (4H, m), 3.52 (2H, t, J = 5.8Hz), 3.73–3.86
(2H, m), 4.13 (2H, q, J = 7.4Hz), 5.27 (2H, s), 6.83
(2H, d, J = 9.3Hz), 7.28 (2H, dd, J = 2.4, 7.8Hz),
7.37–7.53 (4H, m), 7.74 (1H, d, J = 7.8Hz), 7.81 (1H,
dd, J = 1.0, 7.8Hz), 7.90 (2H, d, J = 8.8Hz), 9.24 (2H,
s), 9.40 (2H, s), 9.95 (1H, s); FAB MS m/e (M+H)+
530.
4.1.52. N-[2-((Z)-2-{3-[Amino(imino)methyl]phenyl}vinyl)-
phenyl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (36e).
Compound 36e was synthesizedfrom 16e according to
the same procedure as that for 36a. Compound 36e
was obtainedas a pale yellow amorphous powedr
(46% yield): 1H NMR (DMSO-d6) d: 2.12–2.22 (1H,
m), 2.34–2.47 (1H, m), 2.77 (3H, d, J = 4.9Hz), 3.03–
3.20 (2H, m), 3.38–3.55 (4H, m), 3.73–3.83 (1H, m),
3.89–3.97 (1H, m), 6.67 (1H, d, J = 12.2Hz), 6.77 (1H,
d, J = 12.2Hz), 6.82 (2H, d, J = 9.1Hz), 7.06–7.11
(2H, m), 7.29–7.34 (2H, m), 7.37 (1H, dd, J = 7.3,
7.8Hz), 7.45 –7.50 (2H, m), 7.62 (1H, d, J = 7.8Hz),
7.66 (1H, s), 7.83 (2H, d, J = 9.1Hz), 9.27, 9.36 (each
2H, 2s), 9.71 (1H, s), 11.3 (1H, br s); FAB MS m/e
(M+H)+ 454; Anal. Calcdfor C 28H31N5OÆ2.3HClÆ3.0-
H2O: C, 56.86; H, 6.70; N, 11.84; Cl, 13.79. Found: C,
57.02; H, 6.58; N, 11.55; Cl, 13.56.
4.1.56. Ethyl 4-({3-[amino(imino)methyl]phenoxy}meth-
yl)-3-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzo-
ate (37b). Compound 37b was synthesizedfrom 21b
according to the same procedure as that for 36a. Com-
pound 37b was obtainedas a white amorphous powder
(19% yield): 1H NMR (DMSO-d6) d: 1.32 (3H, t,
J = 7.4Hz), 2.04–2.22 (2H, m), 2.62 (3H, s), 2.89–3.14
(4H, m), 3.54 (2H, t, J = 6.3Hz), 3.72–3.83 (2H, m),
4.34 (2H, q, J = 7.4Hz), 5.34 (2H, s), 6.83 (2H, d,
J = 8.9Hz), 7.31 (1H, dd, J = 2.0, 7.8Hz), 7.41 (1H,
d, J = 7.8Hz), 7.48–7.54 (2H, m), 7.66 (1H, d,
J = 8.9Hz), 7.84 (1H, dd, J = 2.0, 7.8Hz), 7.92 (2H, d,
J = 8.9Hz), 8.06 (1H, d, J = 2.0Hz), 9.20 (2H, s), 9.40
(2H, s); FAB MS m/e (M+H)+ 530.
4.1.53.
N-[2-(2-{3-[Amino(imino)methyl]phenyl}ethyl)-
phenyl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (36f).
Compound 36f was synthesizedfrom 16f according to
the same procedure as that for 36a. Compound 36f
was obtainedas a pale yellow amorphous powedr
(28% yield): 1H NMR (DMSO-d6) d: 2.14–2.22 (1H,
m), 2.35–2.49 (1H, m), 2.78 (3H, d, J = 4.8Hz), 2.92
(4H, s), 3.05–3.21 (2H, m), 3.39–3.57 (4H, m), 3.80
(1H, dd, J = 10.0, 15.9Hz), 3.95 (1H, dd, J = 2.7,
16.4Hz), 6.85 (2H, d, J = 9.1Hz), 7.18 (1H, dt, J = 1.5,
7.3Hz), 7.24 (1H, dt, J =1.5, 7.3Hz), 7.26–7.31 (2H,
m), 7.45–7.48 (2H, m), 7.61–7.65 (1H, m), 7.72 (1H, s),
7.96 (2H, d, J = 9.1Hz), 9.32, 9.42 (each 2H, 2s), 9.74
4.1.57. Ethyl 3-({3-[amino(imino)methyl]phenoxy}meth-
yl)-4-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzo-
ate (37c). Compound 37c was synthesizedfrom 21c
according to the same procedure as that for 36a. Com-
pound 37c was obtainedas a white amorphous powder
(21% yield): 1H NMR (DMSO-d6) d: 1.32 (3H, t,
J = 7.4Hz), 2.21–2.21 (1H, m), 2.30–2.45 (1H, m), 2,78

1320
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
(3H, d, J = 3.4Hz), 3.03–3.20 (2H, m), 3.35–3.57 (4H,
m), 3.73–3.97 (2H, m), 4.32 (2H, q, J = 7.4Hz), 5.36
(2H, s), 6.84 (2H, d, J = 9.1Hz), 7.34 (1H, dd, J = 2.0,
7.8Hz), 7.44 (1H, d, J = 7.8Hz), 7.49–7.54 (2H, m),
7.77 (1H, d, J = 9.1Hz), 7.97 (1H, dd, J = 2.0, 9.1Hz),
8.13 (1H, d, J = 2.0Hz), 9.22 (2H, s), 9.40 (2H, s);
FAB MS m/e (M+H)+ 530.
8.3Hz), 6.95 (1H, d, J = 8.3Hz), 7.30 (1H, t,
J = 8.3Hz), 7.48 (1H, d, J = 7.8Hz), 7.76 (1H, t, J =
7.8Hz), 7.94 (2H, d, J = 8.8Hz), 7.99 (2H, d,
J = 8.8Hz), 8.19 (1H, d, J = 8.3Hz), 8.37 (1H, s), 9.50
(2H, s), 9.61 (1H, s), 10.31 (1H, s), 10.62–10.78 (1H,
br s); FAB MS m/e (M+H)+ 573.
4.1.62. Ethyl (4-({3-[amino(imino)methyl]benzoyl}amino)-
3-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phen-
oxy)acetate (39b). Compound 39b was synthesizedfrom
30b according to the same procedure as that for 36a.
Compound 39b was obtainedas a white amorphous
4.1.58. Ethyl 4-({3-[amino(imino)methyl]benzoyl}amino)-
3-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzo-
ate (38a). Compound 38a was synthesizedfrom 24a
according to the same procedure as that for 36a. Com-
pound 38a was obtainedas a white amorphous powder
(54% yield): 1H NMR (DMSO-d6) d: 1.34 (3H, t,
J = 7.3Hz), 2.10–2.21 (1H, m), 2.26–2.40 (1H, m), 2.78
(3H, s), 3.04–3.22 (2H, m), 3.40–3.56 (2H, m), 3.68–
3.80 (1H, m), 3.88–3.97 (1H, m), 4.35 (2H, q,
J = 7.3Hz), 6.85 (2H, d, J = 9.2Hz), 7.78 (1H, t,
J = 7.8Hz), 7.86 (2H, s), 7.98–8.05 (3H, m), 8.27 (1H,
s), 8.31 (1H, d, J = 8.3Hz), 8.52 (1H, s), 9.27 (2H, s),
9.51 (2H, s), 10.21 (1H, s), 10.86 (1H, s); FAB MS m/e
(M+H)+ 543.
1
powder (52% yield): H NMR (DMSO-d6) d: 1.22 (3H,
t, J = 7.4Hz), 2.10–2.22 (1H, m), 2.26–2.40 (1H, m),
2.79 (3H, s), 3.01–3.20 (2H, m), 3.35–3.49 (4H, m),
2.59–3.78 (1H, m), 3.82–3.98 (1H, m), 4.20 (2H, q,
J = 7.4Hz), 4.80 (2H, s), 6.80–6.89 (3H, m), 7.36 (1H,
d, J = 2.9Hz), 7.47 (1H, d, J = 8.8Hz), 7.74–7.81 (1H,
m), 7.91 (2H, d, J = 8.8Hz), 8.02 (1H, d, J 7.8Hz),
8.29 (1H, d, J = 7.8Hz), 8.49 (1H, s), 9.23 (2H, s), 9.51
(2H, s), 9.82 (1H, s), 10.52 (1H, s), 10.54–10.60 (1H,
br s); FAB MS m/e (M+H)+ 573.
4.1.59. Ethyl 4-(2-{3-[amino(imino)methyl]phenyl}ethyl)-
3-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzo-
ate (38b). Compound 38b was synthesizedfrom 24b
according to the same procedure as that for 36a. Com-
pound 38b was obtainedas a white amorphous powder
(84% yield): 1H NMR (DMSO-d6) d: 1.33 (3H, t,
J = 6.9Hz), 2.09–2.26 (2H, m), 2.65 (3H, s), 2.92–3.42
(8H, m), 3.54 (2H, t, J = 6.4Hz), 3.73–3.78 (2H, m),
4.32 (2H, q, J = 6.9Hz), 6.84 (2H, d, J = 8.8Hz), 7.41–
7.49 (3H, m), 7.58–7.64 (1H, m), 7.72 (1H, s), 7.76
(1H, dd, J = 1.0, 7.8Hz), 7.90 (1H, d, J = 1.9Hz), 7.95
(1H, d, J = 8.8Hz), 9.22 (2H, s), 9.37 (2H, s), 9.86
(1H, s); FAB MS m/e (M+H)+ 528.
4.1.63. Ethyl (3-({3-[amino(imino)methyl]benzoyl}amino)-
4-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phen-
oxy)acetate (39c). Compound 39c was synthesizedfrom
30c according to the same procedure as that for 36a.
Compound 39c was obtainedas a white amorphous
1
powder (49% yield): H NMR (DMSO-d6) d: 1.23 (3H,
t, J = 6.9Hz), 2.11–2.20 (1H, m), 2.30–2.39 (1H, m),
2.77 (3H, d, J = 4.9Hz), 3.02–3.20 (4H, m), 3.88–4.05
(2H, m), 4.20 (2H, q, J = 6.9Hz), 4.80 (2H, s), 6.80–
6.89 (3H, m), 7.29 (1H, d, J = 2.9Hz), 7.51 (1H, d,
J = 8.0Hz), 7.77 (1H, t, J = 7.8Hz), 7.94–8.05 (3H, m),
8.28 (1H, d, J = 7.8Hz), 8.53 (1H, s), 9.37 (2H, s), 9.56
(2H, s), 9.89 (1H, s), 10.70 (1H, s), 10.97 (1H, s); FAB
MS m/e (M+H)+ 573.
4.1.60. Ethyl 4-[({3-[amino(imino)methyl]phenyl}amino)-
methyl]-3-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}-
benzoate (38c). Compound 38c was synthesizedfrom 24c
according to the same procedure as that for 36a. Com-
pound 38c was obtainedas a white amorphous powder
(73% yield): 1H NMR (DMSO-d6) d: 1.31 (3H, t,
J = 7.3Hz), 2.14–2.30 (2H, m), 2.65–2.78 (2H, m), 3.54
(2H, t, J = 5.8Hz), 3.75–3.80 (4H, m), 4.31 (2H, q,
J = 7.3Hz), 4.42 (2H, d, J = 5.8Hz), 6.77–6.87 (4H,
m), 6.90–6.97 (2H, m), 7.26 (1H, t, J = 7.9Hz), 7.45
(1H, d, J = 8.3Hz), 7.77 (1H, dd, J = 2.0, 8.3Hz), 7.92
(2H, d, J = 8.3Hz), 8.00 (1H, d, J = 2.0Hz), 8.89 (2H,
s), 9.17 (2H, s), 9.98 (1H, s), 10.60 (1H, s); FAB MS
m/e (M+H)+ 529.
4.1.64. 3-({3-[Amino(imino)methyl]phenoxy}methyl)-2-
{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzoic
acid (40a). To a stirredsolution of
37a (896mg,
1.69mmol) in 20mL EtOH was added 1N NaOHaq
(6.8mL, 6.80mmol) andstirredat ambient temperature
for 3h. The reaction mixture was concentratedin vacuo
andthe residue was chromatographedon ODS-gel elut-
ing with 0.002M HCl/CH3CN (100:5). CH3CN was
removedin vacuo, andthe aqueous solution was
lyophilizedafter being acidifiedwith 1N HCl. Com-
pound 40a (872mg, 98%) was obtainedas a white amor-
1
phous powder: H NMR (DMSO-d6) d: 2.12–2.22 (1H,
m), 2.36–2.48 (1H, m), 2.77 (3H, d, J = 4.4Hz), 3.03–
3.21 (2H, m), 3.39–3.58 (4H, m), 3.80 (1H, dd,
J = 10.2, 16.1Hz), 3.90–4.00 (1H, m), 5.23 (2H, s),
6.85 (2H, d, J = 8.8Hz), 7.26 (1H, dd, J = 2.2, 7.8Hz),
7.39 (1H, d, J = 7.8Hz), 7.41 (1H, d, J = 7.8Hz), 7.48
(1H, d, J = 2.0Hz), 7.51 (1H, d, J = 7.8Hz), 7.72 (1H,
d, J = 7.1Hz), 7.85 (1H, d, J = 7.1Hz), 7.91 (2H, d,
J = 8.8Hz), 9.33, 9.45 (each 2H, 2s), 10.09 (1H s),
11.37 (1H, br s); FAB MS m/e (M+H)+ 502; Anal. Calcd
for C28H31N5O4Æ2.7HClÆ2.3H2O: C, 52.43; H, 6.02; N,
10.92; Cl, 14.92. Found: C, 52.89; H, 6.44; N, 11.09;
Cl, 15.28.
4.1.61. Ethyl (3-({3-[amino(imino)methyl]benzoyl}amino)-
2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phen-
oxy)acetate (39a). Compound 39a was synthesizedfrom
30a according to the same procedure as that for 36a.
Compound 39a was obtainedas a white amorphous
1
powder (79% yield): H NMR (DMSO-d6) d: 1.20 (3H,
t, J = 7.3Hz), 2.10–2.21 (1H, m), 2.26–2.37 (1H, m),
2.79 (3H, d, J = 4.8Hz), 3.20–3.23 (2H, m), 3.38–3.57
(4H, m), 3.70–3.81 (1H, m), 3.88–4.00 (1H, m), 4.16
(2H, q, J = 7.3Hz), 4.82 (2H, s), 6.84 (2H, d, J =

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1321
4.1.65. 4-({3-[Amino(imino)methyl]phenoxy}methyl)-3-
{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzoic
acid (40b). Compound 40b was synthesizedfrom 37b
according to the same procedure as that for 40a.
Compound 40b was obtainedas a white amorphous
8.3Hz), 7.45–7.48 (2H, m), 7.62–7.67 (1H, m), 7.73–
7.79 (2H, m), 7.88 (1H, d, J = 1.4Hz), 7.99 (2H, d,
J = 9.3Hz), 9.35, 9.45 (each 2H, 2s), 9.91 (1H, s),
11.38 (1H, br s); FAB MS m/e (M+H)+ 500; Anal. Calcd
for C29H33N5O3Æ3.0HClÆ2.1H2O: C, 53.85; H, 6.26; N,
10.83; Cl, 16.44. Found: C, 54.15; H, 6.67; N, 10.89;
Cl, 16.51.
1
powder (60% yield): H NMR (DMSO-d6) d: 2.02–2.10
(2H, m), 2.75–2.84 (2H, m), 2.88–2.98 (2H, m), 3.20–
3.48 (3H, br), 3.53 (2H, t, J = 6.1Hz), 3.67–3.74 (2H,
m), 5.31 (2H, s), 6.81 (2H, d, J = 8.8Hz), 7.31 (1H,
dd, J = 2.4, 8.3Hz), 7.40 (1H, d, J = 7.8Hz), 7.46–7.48
(1H, m), 7.52 (1H, dd, J = 7.8, 8.3Hz), 7.62 (1H, d,
J = 8.3Hz), 7.82 (1H, dd, J = 2.0, 8.3Hz), 7.89 (2H, d,
J = 8.8Hz), 8.03 (1H, d, J = 1.4Hz), 9.26–9.39 (4H,
br), 9.90(1H, s); FAB MS m/e (M+H)+ 502; Anal. Calcd
for C28H31N5O4Æ1.4HClÆ2.5H2O: C, 56.27; H, 6.30; N,
11.72; Cl, 8.30. Found: C, 56.14; H, 6.51; N, 11.74; Cl,
8.17.
4.1.69. 4-[({3-[Amino(imino)methyl]phenyl}amino)meth-
yl]-3-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benz-
oic acid (41c). Compound 41c was synthesizedfrom 38c
according to the same procedure as that for 40a. Com-
pound 41c was obtainedas a white amorphous powder
(93% yield): 1H NMR (DMSO-d6) d: 2.13–2.23 (2H, m),
2.67 (3H, s), 3.00–3.22 (4H, m), 3.53 (2H, t, J = 6.1Hz),
3.77–3.83 (2H, m), 4.44 (2H, d, J = 5.3Hz), 6.82–6.86
(4H, m), 6.94 (1H, d, J = 7.3Hz), 6.98 (1H, br s), 7.26
(1H, t, J = 8.1Hz), 7.44 (1H, d, J = 7.8Hz), 7.75 (1H,
dd, J = 1.9, 8.1Hz), 7.94 (2H, d, J = 9.2Hz), 7.98 (1H,
d, J = 1.9Hz), 9.07, 9.22 (each 2H, 2s), 9.98 (1H, s);
FAB MS m/e (M+H)+ 501; Anal. Calcdfor
C28H32N6O3Æ1.8HClÆ1.4H2O: C, 56.86; H, 6.24; N,
14.21; Cl, 10.79. Found: C, 56.93; H, 6.52; N, 14.26;
Cl, 10.43.
4.1.66. 3-({3-[Amino(imino)methyl]phenoxy}methyl)-4-{[4-
(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzoic acid
(40c). Compound 40c was synthesizedfrom 37c accord-
ing to the same procedure as that for 40a. Compound
40c was obtainedas a white amorphous powder (63%
yield): 1H NMR (DMSO-d6) d: 2.12–2.24 (1H, m),
2.30–2.45 (1H, m), 2.79 (3H, s), 3.05–3.22 (2H, m),
3.52–3.57 (4H, m), 3.73–3.98 (2H, m), 5.34 (2H, s),
6.85 (2H, d, J = 8.8Hz), 7.33 (1H, dd, J = 2.2, 7.8Hz),
7.42 (1H, d, J = 7.8Hz), 7.49–7.52 (1H, m), 7.54 (1H,
t, J = 7.8Hz), 7.72 (1H, d, J = 7.8Hz), 7.91 (2H, d,
J = 8.8Hz), 7.92–7.96 (1H, m), 8.12 (1H, d,
J = 2.0Hz), 9.17, 9.38 (each 2H, 2s), 9.94 (1H, s),
10.98 (1H, br s), 12.94 (1H, br s); FAB MS m/e
(M+H)+ 502; Anal. Calcdfor C 28H31N5O4Æ2.1HClÆ2.6-
H2O: C, 53.81; H, 6.18; N, 11.21; Cl, 11.91. Found: C,
53.78; H, 6.19; N, 11.18; Cl, 11.63.
4.1.70. (3-({3-[Amino(imino)methyl]benzoyl}amino)-2-{[4-
(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phenoxy)ace-
tic acid (42a). Compound 42a was synthesizedfrom 39a
according to the same procedure as that for 40a. Com-
pound 42a was obtainedas a pale yellow amorphous
1
powder (67% yield): H NMR (DMSO-d6) d: 1.90–2.04
(2H, m), 2.42 (3H, s), 2.62–2.73 (2H, m), 2.82–2.90
(2H, m), 3.38–3.46 (2H, m), 3.57–3.68 (2H, m), 4.50
(2H, s), 6.76 (2H, d, J = 8.8Hz), 7.10 (1H, d,
J = 7.8Hz), 7.27 (1H, t, J = 8.3Hz), 7.60 (1H, d, J =
7.8Hz), 7.75 (1H, t, J = 7.8Hz), 7.96 (1H, d,
J = 7.8Hz), 8.03 (2H, d, J = 8.8Hz), 8.14 (1H, d, J =
7.8Hz), 8.27 (1H, s), 9.18–9.56 (2H, br), 10.04–10.46
(1H, br), 10.55 (1H, s), 12.10 (1H, s); FAB MS m/e
(M+H)+ 545; Anal. Calcdfor C 29H32N6O3Æ0.6HClÆ4.2-
H2O: C, 54.24; H, 6.44; N, 13.09; Cl, 3.31. Found: C,
54.08; H, 6.22; N, 12.98; Cl, 3.51.
4.1.67. 4-({3-[Amino(imino)methyl]benzoyl}amino)-3-{[4-
(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzoic acid
(41a). Compound 41a was synthesizedfrom 38a accord-
ing to the same procedure as that for 40a. Compound
41a was obtainedas a white amorphous powder (63%
yield): 1H NMR (DMSO-d6) d: 1.94–2.06 (2H, m),
2.44 (3H, br s), 2.65–2.93 (4H, m), 3.51 (2H, t,
J = 6.1Hz), 3.62–3.69 (2H, m), 6.80 (2H, d, J =
9.0Hz), 7.79 (1H, t, J = 7.8Hz), 7.83 (2H, s), 7.91 (2H,
d, J = 9.0Hz), 8.01 (1H, d, J = 7.8Hz), 8.23 (1H, s),
8.28 (1H, d, J = 7.8Hz), 8.45 (1H, s), 9.16–9.56 (3H,
br), 10.00 (1H, s), 10.69 (1H, s); FAB MS m/e
(M+H)+ 515; Anal. Calcdfor C 28H30N6O4Æ1.4HClÆ
3.2H2O: C, 53.96; H, 6.11; N, 13.48; Cl, 7.96. Found:
C, 53.98; H, 6.22; N, 13.78; Cl, 7.93.
4.1.71. (4-({3-[Amino(imino)methyl]benzoyl}amino)-3-{[4-
(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phenoxy)ace-
tic acid (42b). Compound 42b was synthesized
from 39b according to the same procedure as that for
40a. Compound 42b was obtainedas a pale yellow
1
amorphous powder (75% yield): H NMR (DMSO-d6)
d: 2.10–2.22 (1H, m), 2.31–2.44 (1H, m), 2.51 (3H, d,
J = 4.9Hz), 3.02–3.19 (2H, m), 3.36–3.55 (4H, m),
3.69–3.73 (2H, m), 4.70 (2H, s), 6.79–6.86 (3H,
m), 7.35 (1H, d, J = 3.0Hz), 7.47 (1H, d, J =
8.3Hz), 7.76 (1H, d, J = 7.8Hz), 7.97 (2H, d,
J = 8.8Hz), 8.04 (1H, d, J = 7.8Hz), 8.31 (1H, d,
J = 7.8Hz), 8.60 (1H, s), 9.42 (2H, s), 9.59 (2H, s),
9.91 (1H, s), 9.71 (1H, s), 10.71 (1H, s), 10.04–11.05
(1H, br); FAB MS m/e (M+H)+ 545; Anal. Calcdfor
C29H32N6O3Æ2.3HClÆ3.1H2O: C, 50.90; H, 5.97; N,
12.28; Cl, 11.92. Found: C, 50.61; H, 5.93; N, 12.08;
Cl, 11.98.
4.1.68. 4-(2-{3-[Amino(imino)methyl]phenyl}ethyl)-3-{[4-
(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzoic acid
(41b). Compound 41b was synthesizedfrom 38b accord-
ing to the same procedure as that for 40a. Compound
41b was obtainedas a white amorphous powder (23%
yield): 1H NMR (DMSO-d6) d: 2.14–2.24 (1H, m),
2.37–2.50 (1H, m), 2.78 (3H, d, J = 4.9Hz), 2.92–2.99
(2H, m), 3.01–3.23 (4H, m), 3.40–3.58 (4H, m), 3.81
(1H, dd, J = 9.8, 16.1Hz), 3.97 (1H, dd, J = 2.7,
16.1Hz), 6.86 (2H, d, J = 9.3Hz), 7.41 (1H, d, J =

1322
H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
4.1.72. (3-({3-[Amino(imino)methyl]benzoyl}amino)-4-{[4-
(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phenoxy)ace-
tic acid (42c). Compound 42c was synthesizedfrom 39c
according to the same procedure as that for 40a. Com-
pound 42c was obtainedas a pale yellow amorphous
of residual activity was plotted against the logarithm
of inhibitor concentration.
4.3.2. Plasma clotting time assays. Citratedbloodsam-
ples from mice was collected. Platelet-poor plasma was
preparedby centrifugation at 3000rpm for 10min and
storedat ꢀ40ꢁC until use. Plasma clotting times were
performedusing a KC10A coagulometer (Amelung
Co., Lehbrinsweg, Germany) at 37ꢁC. Prothrombin
time (PT) andactivatedpartial thromboplastin time
(APTT) were measuredusing Orthobrain thromboplas-
tin andthrombofax (Ortho Diagnostic Systems Co.,
Tokyo, Japan), respectively. Coagulation times for each
test sample were comparedwith coagulation times meas-
uredusing a distilledwater control. The concentration
requiredto double the clotting time (CT 2) was estimated
from each individual concentration–response curve.
Each measurement was performedthree times, andrep-
resentedas the mean value.
1
powder (87% yield): H NMR (DMSO-d6) d: 1.13–1.16
(2H, m), 2.69 (3H, s), 3.07–3.40 (4H, m), 3.51 (2H, t,
J = 6.3Hz), 3.75–3.84 (2H, m), 4.66 (2H, s), 6.78–6.87
(3H, m), 7.27 (1H, d, J = 3.0Hz), 7.48 (1H, d,
J = 8.8Hz), 7.77 (1H, t, J = 8.8Hz), 7.92–8.04 (3H, m),
8.27 (1H, d, J = 8.8Hz), 8.52 (1H, s), 9.53 (3H, s), 9.87
(1H, s), 10.66 (1H, s); FAB MS m/e (M+H)+ 573; Anal.
Calcdfor C 29H32N6O3Æ2.5HClÆ3.0H2O: C, 52.95; H,
6.21; N, 12.78; Cl, 13.47. Found: C, 53.03; H, 6.18; N,
12.65; Cl, 13.62.
4.2. Docking simulation
We choose the Crystal structure of Human factor
Xa/ZK-807834 complex (PDB code 1FJS)11 for docking
simulation among many structure of factor Xa/ligand
complex available today because of its high resolu-
tive experiment andchemically similar feature of
ZK-807834 to our compounds. Initially, all but
HOH605 water molecules andZK-807834 were removed
from 1FJS ligand-binding site and hydrogen atoms were
buildandrelaxedby a minimization protocol using
4.3.3. Ex vivo studies. Male mice weighing 30–37g
was used in these studies. In mice, the test drug was
dissolved in saline and administered to animals intra-
venously at 1mg/kg via tail vein or orally at 100mg/kg
using a gastric tube. Citratedbloodwas collectedfrom
the vena cava 1min after intravenous injection or
30min after oral administration. Platelet-poor plasma
was preparedby centrifugation for measurement of PT
or APTT. All data were expressed as relative fold values,
comparedwith the baseline value the vehicle group in
mice.
TRIPOS force fieldin
As HOH605 is considered to be important for ligand
SYBYL program package.12
˚
binding, this water molecule and atoms within 12A
of the ligand-binding site were used to calculate the
13
docking interaction energy. The program GOLD was
then usedto perform a odcking simulation of
compounds 36b and 40b. In the genetic algorithm
dockings, an initial population size of 100 individuals
with nich size of 2 on each 5 islands, a maximum
number of 1.0 · 105 genetic operations (crossover,
migration, mutation), a cross-over weight of 95, a
migration weight of 10, a mutate weight of 95, anda
selection pressure of 1.1 were employed. Though all
protein atoms except for terminating NH andOH group
were fixed during docking simulations, resulted confor-
mations of the ligands were well converged to ZK-
807834 binding region and much the same interaction
as ZK-807834 were formedbetween our compounds
andfactor Xa.
Acknowledgements
The authors deeply acknowledge Dr. Minoru Okada for
useful discussion and helpful support for preparing this
manuscript, andare also grateful to the staff of Division
of Analytical Science Laboratories for elemental analy-
sis andspectral measurements.
References and notes
1. Rosenberg, J. S.; Beeler, D. L.; Rosenberg, R. D. J. Biol.
Chem. 1975, 250, 1607.
2. (a) Sato, K.; Kawasaki, T.; Taniuchi, Y.; Hirayama,
F.; Koshio, H.; Matsumoto, Y. Eur. J. Pharmacol. 1997,
339, 141; (b) Kawasaki, T.; Sato, K.; Hirayama, F.;
Koshio, H.; Taniuchi, Y.; Matsumoto, Y. Thromb.
Haemost. 1998, 79, 859; (c) Sato, K.; Kawasaki, T.;
Hisamichi, N.; Taniuchi, Y.; Hirayama, F.; Koshio, H.;
Matsumoto, Y. Br. J. Pharmacol. 1998, 132, 92; (d)
Taniuchi, Y.; Sakai, Y.; Hisamichi, N.; Kayama, M.;
Mano, Y.; Sato, K.; Hirayama, F.; Koshio, H.; Matsu-
moto, Y.; Kawasaki, T. Thromb. Haemost. 1998, 79, 543;
(e) Kawasaki, T.; Sato, K.; Sakai, Y.; Hirayama, F.;
Koshio, H.; Taniuchi, Y.; Matsumoto, Y. Thromb.
Haemost. 1998, 79, 410; (f) Sato, K.; Taniuchi, Y.;
Kawasaki, T.; Hirayama, F.; Koshio, H.; Matsumoto,
Y. Eur. J. Pharmacol. 1998, 347, 231; (g) Sato, K.;
Kawasaki, T.; Hisamichi, N.; Taniuchi, Y.; Hirayama, F.;
Koshio, H.; Ichihara, M.; Matsumoto, Y. Eur. J.
Pharmacol. 1998, 350, 87; (h) Sato, K.; Kaku, S.;
Hirayama, F.; Koshio, H.; Matsumoto, Y.; Kawasaki,
4.3. Biology
4.3.1. Chromogenic assay. The hydrolysis rates of syn-
thetic substrates were assayedby continuously measur-
ing absorbance at 405nm at 37ꢁC with a microplate
reader (Model 3550, Bio-Rad, USA). Reaction mixtures
(125lL) were preparedin 96-well plates containing
chromogenic substrates andan inhibitor in either
0.05M Tris–HCl, pH8.4, 0.15M NaCl. Reactions were
initiatedwith a 25 lL portion of the enzyme solution.
Enzymes andsubstrates were usedas follows: factor
Xa andS-2222; thrombin andS-2238; trypsin andS-
2222. The concentration of an inhibitor requiredto inhi-
bit enzyme activity by 50% (IC50) was calculatedfrom
dose-response curves in which the logit transformation

H. Koshio et al. / Bioorg. Med. Chem. 13 (2005) 1305–1323
1323
T.; Iizumi, Y. Eur. J. Pharmacol. 1998, 352, 59; (i) Sato,
K.; Hisamichi, N.; Kawasaki, T.; Hirayama, F.; Koshio,
H.; Matsumoto, Y.; Iizumi, Y. Jpn. J. Pharmacol. 1998,
78, 191.
Rats, A. M.; Milot, G.; Shuman, R. T.; Smith, T.; Smith,
G. F.; Tebbe, A. L.; Tinsley, J. M.; Towner, R. D.;
Wilson, A.; Wilson, A.; Yee, Y. K. J. Med. Chem. 2000,
43, 883.
3. 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.
4. Koshio, H.; Hirayama, F.; Ishihara, T.; Taniuchi, Y.;
Sato, K.; Sakai-Moritani, Y.; Kaku, S.; Kawasaki, T.;
Matsumoto, Y.; Sakamoto, S.; Tsukamoto, S. Bioorg.
Med. Chem. 2004, 12, 2179.
5. Wiley andco-workers have also discovered 1 from HTS
screening aroundthe same time andreportedseveral
potent fXa inhibitors by modification of 1.6–9
6. Masters, J. J.; Franciskovich, J. B.; Tinsley, J. M.;
Campbell, C.; Campbell, J. B.; Craft, T. J.; Froelich, L.
L.; Gifford-Moore, D. S.; Hay, L. A.; Herron, D. K.;
Klimkowski, V. J.; Kurz, K. D.; Mets, J. T.; Rats, A. M.;
Shuman, R. T.; Smith, G. F.; Smith, T.; Towner, R. D.;
Wiley, M. R.; Wilson, A.; Yee, Y. K. J. Med. Chem. 2000,
43, 2087.
8. Herron, D. K.; Goodson, T., Jr.; Wiley, M. R.; Weir, L.
C.; Kyle, J. A.; Yee, Y. K.; Tebbe, A. L.; Tinsley, J. M.;
Mendel, D.; Masters, J. J.; Franciskovich, J. B.; Sawyer, J.
S.; Beight, D. W.; Rats, A. M.; Milot, G.; Hall, S. E.;
Klimkowski, V. J.; Wikel, J. H.; Eastwood, B. J.; Towner,
R. D.; Gifford-Moore, D. S.; Craft, T. J.; Smith, G. F.
J. Med. Chem. 2000, 43, 859.
9. Yee, Y. K.; Tebbe, A. L.; Linebarger, J. H.; Beight, D. W.;
Craft, T. J.; Gifford-Moore, D.; Goodson, T., Jr.; Herron,
D. K.; Klimkowski, V. J.; Kyle, J. A.; Sawyer, J. S.; Smith,
G. F.; Tinsley, J. M.; Towner, R. D.; Weir, L.; Wiley,
M. R. J. Med. Chem. 2000, 43, 873.
10. Kagawa, H.; Sagawa, M.; Tsunoda, A.; Kaji, M. Jpn.
Kokai Tokkyo Koho JP 06347846 A2, Application:
JP 93-140736 19930611.
11. Adler, M.; Davey, D. D.; Phillips, G. B.; Kim, S.-H.;
Jancarik, J.; Rumennik, G.; Light, D. R.; Whitlow, M.
Biochemistry 2000, 39, 12534.
7. Wiley, M. R.; Weir, L. C.; Briggs, S.; Bryan, N. A.; Buben,
J.; Campbell, C.; Chirgadze, N. Y.; Conrad, R. C.; Craft,
T. J.; Ficorilli, J. V.; Franciskovich, J. B.; Froelich, L. L.;
Gifford-Moore, D. S.; Goodson, T., Jr.; Herron, D. K.;
Klimkowski, V. J.; Kurz, K. D.; Kyle, J. A.; Masters, J. J.;
12. SYBYL 6.0, Tripos Associates Inc., 1699 South Hanley
Road, Suite 303, St. Louis, Missouri 63144, USA (314 647
1099).
13. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor,
R. J. Mol. Biol. 1997, 267, 727.