Orally active factor Xa inhibitor: Synthesis and biological activity of masked amidines as prodrugs of novel 1,4-diazepane derivatives

Article abstract of DOI:10.1016/j.bmc.2004.07.046

To improve their anticoagulant activity after oral administration, prodrug strategy was applied to fXa inhibitors based on a 1,4-diazepane template by conversion of the amidine group into amidoxime and alkoxycarbonyloxyamidine groups. This study revealed that amidoxime prodrugs bearing an ester moiety are efficient for the expression of oral anticoagulant activity. Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anticoagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effective oral anticoagulant activity in mice.

Full text of DOI:10.1016/j.bmc.2004.07.046

Bioorganic & Medicinal Chemistry 12 (2004) 5415–5426
Orally active factor Xa inhibitor: synthesis and
biological activity of masked amidines as prodrugs
of novel 1,4-diazepane derivatives
Hiroyuki Koshio,* Fukushi Hirayama, Tsukasa Ishihara, Hiroyuki Kaizawa,
Takeshi Shigenaga, Yuta Taniuchi, Kazuo Sato, Yumiko Moritani, Yoshiyuki Iwatsuki,
Toshio Uemura, Seiji Kaku, Tomihisa Kawasaki, Yuzo Matsumoto,
Shuichi Sakamoto and Shin-ichi Tsukamoto
Chemistry Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd, 21 Miyukigaoka,
Tsukuba, Ibaraki 305-8585, Japan
Received 21 June 2004; revised 22 July 2004; accepted 22 July 2004
Available online 19 August 2004
Abstract—Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anti-
coagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among
the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effec-
tive oral anticoagulant activity in mice.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
fXa inhibitor 1, which lacked a strongly basic aceto-
amidine moiety as the P4 part.4 The amidine moiety
possessed by each of these compounds as the P1 part
was considered essential for fXa inhibitory activity. In
addition, each compound contained a polar functional
group, such as a carboxyl moiety. However, such polar
functional groups are considered unfavorable in terms
of their effects on oral absorption and the pharmacoki-
netic properties required by oral antithrombotic agents.
In this paper, we discuss modification of these polar
functional groups by means of a prodrug strategy with
the goal of improving oral anticoagulant activity
(Fig. 1).
Myocardial infarction, stroke, deep vein thrombosis,
and pulmonary embolism are major causes of mortality
in the industrialized world. Therefore, the prevention of
blood coagulation is a major target for new therapeutic
agents. Factor Xa (fXa) is strategically located at the
junction of the intrinsic and extrinsic arms of the coagu-
lation cascade, and it is thought that its inhibition may
be more effective than direct inhibition of thrombin in
interrupting the cascade and that fXa inhibitors may in-
volve a lesser risk of bleeding than thrombin inhibi-
tors.1,2
We have previously reported the potent and selective
bisamidine type fXa inhibitors YM-60828 and YM-
96765, which contained an amidine moiety as their P1
parts and an acetoamidine moiety as their P4 parts.3–5
These compounds displayed effective oral antithrombotic
activity in rats without prolongation of bleeding time.4,5
We also discovered a potent orally active monoamidine
2. Chemistry
The synthesis of the intermediate cyanonapthalene
derivatives 6–12 is illustrated in Scheme 1. Treatment
of 2 with 1-methyl-1,4-diazepane provided 4-substituted
nitrobenzene 3. The intermediate 5 was synthesized by
reduction of the nitro group of 3, followed by reductive
alkylation with 7-formyl-2-naphthonitrile. Acylation of
5 with various sulfonylchlorides gave sulfonamide deriv-
atives 6–8 and 12. The removal of the tert-butoxycarb-
onyl (Boc) protecting group from 8 under acidic
Keywords: Anticoagulants; Enzyme inhibitors; Factor Xa; Amidoxime;
Prodrugs.
*
Corresponding author. Tel.: +81-(0)29-854-1548; fax: +81-(0)29-852-
5387; e-mail: koshio@yamanouchi.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.046

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H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
intermediates 6, 7, 9, 11, and 12 with hydroxylamine
O
O
CO2H
Me
NH
S
N
hydrochloride provided the amidoxime derivatives 14,
16, 18, 21, and 23. Hydrolysis of 21 with 1N NaOH
provided carboxy derivative 19. Hydrogenolysis of 18
in the presence of 10% Pd–C gave the amidine derivative
17.
H2N
H2N
N
NH
NH
O
YM-60828
Schemes 3 and 4 show the synthesis of styrylamidine and
related prodrug derivatives. Intermediate 25 was ob-
tained by the same procedure as used to produce 5 using
cinnamaldehyde 243 instead of 7-formyl-2-naphthonitrile.
In the manner described above, 25 was converted to
sulfonamide derivatives 26 and 27. Compound 26 was
also converted to amidine derivative 29 and amidoxime
derivatives 30 and 31 under the conditions described
above. Hydrolysis of 29 under acidic conditions pro-
vided carboxy derivative 28. The method of preparing
carbamate derivatives 33 and 34 from amidine 29 in-
volved reaction with an appropriate chloroformate in
the presence of aqueous sodium hydroxide. Acylation
of 30 with acetic anhydride gave acetyl amidoxime
derivative 32. Hydrolysis of 30 with 1N NaOH provided
carboxy derivative 35. Esterification with various alcoh-
ols gave 36, 37, and 39. Isopropyl ester derivative 38 was
prepared from benzonitrile derivative 27 under the con-
ditions described above.
O
CO2H
S
O
N
Me
NH
N
N
YM-96765
O
CO2H
S
O
N
H2N
NH
N
N
Me
1
Figure 1. YM-60828 and related compounds.
conditions gave intermediate 9. Sulfonyl carbamate
derivative 8 was alkylated with methanol under Mitsun-
obu conditions to produce 10. Dimethyl sulfamide
derivative 11 was prepared by removal of the Boc pro-
tecting group from 10 followed by alkylation using the
same Mitsunobu conditions. Synthesis of the naphtham-
idine and naphthamidoxime derivatives 13–23 is shown
in Scheme 2. Treatment of intermediates 6, 7, 9, and
12 under Pinner conditions (HCl/EtOH) converted them
to the imidates, which were immediately reacted with
excess ammonium acetate to provide the corresponding
amidine derivatives 13, 15, 20, and 22. Treatment of
3. Results and discussion
Compounds were evaluated for FXa inhibitory activity
according to their IC50 values, and for anticoagulant
activity in vitro by the CT2 values of their prothrombin
times (PT). The CT2 value was defined as the concentra-
tion required to double the clotting time. In addition,
oral anticoagulant activity was evaluated by the ability
of compounds to prolong PT following oral administra-
tion in mice.
a
b
c
O2N
F
O2N
N
H2N
N
N
N
Me
Me
4
3
2
R1
O
O
S
N
H
N
d
NC
NC
N
N
e
N Me
N Me
5
6
7
8
R1=Me
R1=CH2CO2Et
R1=NHBOC
9 R1=NH2
f
10 R1=N(Me)BOC
e, f
11 R1=NMe2
12 R1=NHCO Et
2
Scheme 1. Reagents and conditions: (a) 1-methyl-1,4-diazepane, K2CO3, DMF; (b) H2, 10% Pd–C, EtOH; (c) 7-formyl-2-naphthonitrile,
NaB(OAc)3H, AcOH, 1,2-dichloroethane; (d) R1SO2Cl, pyridine, 1,2-dichloroethane; (e) HCl, CHCl3, EtOAc; (f) MeOH, PPh3, diethyl
azodicarboxylate (DEAD), THF.

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
5417
R1
O
O
S
N
H2N
NH
N
N Me
13 R1=Me
R1
a, b
O
O
15 R1=NH2
S
N
22 R1=NHCO Et
20 R1=CH2CO2Et
2
NC
N
N Me
6,7,9,11,12
R1
O
O
S
N
H2N
R2
c
N
N
N Me
14 R1=Me
16 R1=NH2
R 2=OH
R 2=OH
21 R1=CH2CO2Et R2=OH
19 R1=CH2CO2H R2=OH
d
23 R1=NHCO Et R2=OH
2
18 R1=NMe2
17 R1=NMe2
R2=OH
R2=H
e
Scheme 2. Reagents and conditions: (a) HCl, EtOH; (b) NH4OAc, EtOH for 6, 7, 9, and 12; (c) H2NOHÆHCl, Et3N, EtOH for 6, 7, 9, 11, and 12;
(d) 1N NaOH; (e) H2, 10% Pd–C, Ac2O, AcOH.
b
a
H
N
NC
NC
CHO
NC
24
25
N
N Me
O
S
CO2R1
O
N
N
N Me
26 R1=Et
27 R1=iPr
O
O
O
O
S
N
CO2R1
S
N
CO2Et
H2N
H2N
R2
f
NH
N
N
N
N Me
N Me
33 R2=CO2Et
34 R2=CO2CH2CCl3
29 R1=Et
28 R1=H
e
c, d
26
g
O
S
N
CO2Et
O
H2N
R2
N
N
N Me
30 R2=OH
32 R2=OAc
31 R2=OMe
h
Scheme 3. Reagents and conditions: (a) 4, NaB(OAc)3H, AcOH, 1,2-dichloroethane; (b) ClO2SCH2CO2R1, pyridine; (c) HCl, EtOH; (d) NH4OAc,
EtOH; (e) concd HCl; (f) R2OCOCl, 0.1N NaOH, CHCl3; (g) H2NOHÆHCl for 30, Et3N, EtOH, H2NOMeÆHCl for 31, Et3N, EtOH; (h) Ac2O,
pyridine, DMF.

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H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
O
O
O
O
S
N
CO2Et
S
N
CO2H
H2N
HO
H2N
HO
a
N
N
N
N
N Me
N Me
30
35
O
O
S
N
CO2R2
H2N
b
N
HO
N
N Me
36 R2=Me
37 R2=nPr
39 R2=iBu
O
O
O
O
i
i
S
N
CO2 Pr
S
N
CO2 Pr
H2N
HO
c
NC
N
N
N
N Me
N Me
38
27
i
Scheme 4. Reagents and conditions: (a) NaOH, EtOH, H2O; (b) R2OH, HCl, 1,4-dioxane; (c) H2NOHÆHCl, Et3N, PrOH.
It has previously been shown that amidoxime com-
pounds can be used as prodrugs of corresponding ami-
dine compounds and that they can be converted to
parent amidines in vivo by liver microsomal enzymes.6
Weller et al. have demonstrated that amidoxime deriva-
tives are effective as prodrugs of monoamidine fibrino-
gen antagonists, and possess significantly improved
oral bioavailability.7 In the present study, we applied
the amidoxime prodrug strategy to our amidine-contain-
ing fXa inhibitors (Table 1). Firstly, the amidine group
of the least polar methanesulfonamide derivative, 13
(R2 = Me), was modified to an amidoxime moiety (14).
Table 1. Prodrugs of naphthamidine derivatives
R2
O
S
N
O
H2N
N
R1
N
N Me
Compd
R1
R2
IC50 (nM)a
CT2 (lM)b PTc
PT/control PTd
1.0h
0.5h
2.0h
13
14
15
16
17
18
1
H
Me
Me
18
10,132
17
1.1
>284
1.3
1.45
1.08
1.18
1.09
1.01
1.00
2.21
1.15
1.85
2.87
1.48
1.17
NTe
1.16
NTe
NTe
NTe
0.92
2.04
1.31
1.80
2.37
NTe
1.03
NTe
1.27
NTe
NTe
NTe
1.02
1.75
1.14
1.47
1.92
NTe
1.19
OH
H
NH2
NH2
OH
H
1200
19
NT
NMe2
NMe2
1.8
OH
H
2200
6.5
890
NT
CH2CO2H
CH2CO2H
CH2CO2Et
CH2CO2Et
NHCO2Et
NHCO2Et
0.76
>248
0.77
38
19
20
21
22
23
OH
H
12
OH
H
3200
13
1.4
OH
990
NT
a Human purified enzyme were used. IC50 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 compared with that measured using normal mice plasma at 0.5, 1.0, and 2.0h after oral administration (100mg/kg,
n = 3).
e Not tested.

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
Table 2. Prodrugs of styrylamidine derivatives
5419
CO2R2
O
S
N
O
H2N
R1
N
N
N Me
Compd
R1
R2
IC50 (nM)a
CT2 (lM)b PTc
PT/control PTd
1.0h
0.5h
2.0h
28
30
31
32
33
34
H
OH
H
27
7200
15,000
6000
310
1.8
>252
NTe
1.43
2.27
1.35
1.67
1.82
1.43
NTe
2.17
1.40
1.49
1.59
1.45
NTe
2.10
1.27
1.27
1.41
1.18
Et
Et
Et
Et
Et
OMe
OAc
NTe
CO2Et
CO2CH2CCl3
NTe
NTe
600
a–eRefer to Table 1.
This, however, had an adverse effect on oral anticoagu-
lant activity in mice compared with 13. Similar results
were obtained following the conversion of sulfonamide
derivatives 15 to 16 and 17 to 18. We next prepared
the amidoxime derivative 19 of compound 1, this latter
having been reported to show good oral anticoagulant
activity. However, compound 19 showed poor oral anti-
coagulant activity. Ethyl ester derivative 20 showed anti-
coagulant activity in vitro comparable to that of parent
compound 1, but did not provide a substantial increase
in oral anticoagulant activity. Interestingly, masking the
amidine group of compound 20 to give the prodrug 21
resulted in more potent oral anticoagulant activity,
and prolonged PT 2.87-fold at 0.5h, and 1.92-fold at
2.0h, compared to compounds 1 and 20. On the other
hand, prodrug possessing a similar ethoxycarbonyl moi-
ety (23) exhibited poor oral anticoagulant activity and
prolonged PT only 1.17-fold at 0.5h, and 1.19-fold at
2.0h. These results strongly suggested that whether or
not amidoxime derivatives would function effectively
as prodrugs of their parent amidine derivatives and
show potent activity after oral dosing was dependent
on the nature of the substituent incorporated and that
an ester moiety was a suitable substituent.
Further studies for the conversion of promoieties of
amidine and alkyl groups of ethyl esters were conducted
based on styrene derivative 28. Firstly, the ester group
was fixed as ethyl ester and modification of the amidine
group was undertaken. Compound 30, in which the ami-
dine group was replaced by an amidoxime group had in-
creased oral anticoagulant activity compared with
compound 28, and the PT was prolonged by more than
2-fold at all time points investigated after oral adminis-
tration. On the other hand, modification of the amidox-
ime group with an alkyl or acyl group led to a decrease
in oral anticoagulant activity (30 vs 31 and 32). The alk-
oxycarbonyloxyamidine derivatives (33, 34) did not
result in a significant increase in oral anticoagulant
activity compared with 28. These results indicated that
the amidoxime derivative was the most promising pro-
drug for this series of monoamidine fXa inhibitors.
Table 3. Prodrugs of styrylamidine derivatives
CO2R2
O
S
N
O
H2N
N
R1
N
N Me
Compd
R1
R2
IC50 (nM)a
CT2 (lM)b PTc
PT/control PTd
1.0h
0.5h
2.0h
28
36
30
37
38
39
H
H
27
NT
1.8
NT
1.43
1.74
2.27
2.15
1.95
1.82
NTe
2.41
2.17
NTe
1.68
NTe
NTe
2.25
2.10
1.91
1.79
1.66
OH
OH
OH
OH
OH
Me
Et
nPr
iPr
iBu
7200
1200
10,000
3800
>252
NT
NT
NT
a–eRefer to Table 1.

5420
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
Table 3 illustrates the results of investigations of modi-
fication of the ethyl ester moiety in compound 30, which
had good oral anticoagulant activity (Table 2). The
methyl ester derivative (36) had oral anticoagulant activ-
ity greater than the carboxylate 28 and comparable to
that of ethyl ester derivative 30. On the contrary,
replacement of the ethyl group with more bulky substi-
tutes, such as isopropyl (38) or isobutyl (39) groups, pro-
vided less potency than other ester moieties. These
results indicate that the most suitable substituents for
effective prodrug function are methyl or ethyl esters.
(2H, m), 2.66–2.70 (2H, m), 3.38–3.44 (2H, m), 3.47–
3.51 (4H, m), 6.57 (2H, d, J = 9.0Hz), 6.65 (2H, d,
J = 9.0Hz); FAB MS m/e (M+1)+ 206.
4.1.3. 7-({[4-(4-Methyl-1,4-diazepan-1-yl)phenyl]amino}-
methyl)-2-naphthonitrile (5). To a stirred solution of 4
(2.09g, 10.2mmol) and 7-formyl-2-naphthonitrile
(1.85g, 10.2mmol) in 1,2-dichloromethane (100mL)
and AcOH (6.0mL) at ambient temperature was added
sodium triacetoxyborohydride (4.2g, 20mmol). After
2h, the reaction mixture was washed with 10% potas-
sium carbonate solution and brine. The organic layer
was dried over Na2SO4 and concentrated in vacuo to
give 5 (3.77g, quant. yield) as a yellow amorphous pow-
der: 1H NMR(CDCl 3) d 1.93–2.06 (2H, m), 2.37 (3H, s),
2.54–2.59 (2H, m), 2.68–2.71 (2H, m), 3.36–3.43 (2H,
m), 3.45–3.56 (2H, m), 4.47 (2H, s), 6.57–6.66 (4H, m),
7.26 (1H, s), 7.57 (1H, dd, J = 1.5, 8.8Hz), 7.87–7.92
(3H, m), 8.18 (1H, s); FAB MS m/e (M+1)+ 370.
4. Conclusion
By pursuing a prodrug approach, we were able to im-
prove the oral anticoagulant activity of potent fXa
inhibitors such as compounds 1 and 28. In particular,
amidoxime derivatives possessing an ethyl ester moiety
(21 and 30) showed quite potent activity ex vivo after
oral dosing in mice and prolonged PT about 2-fold.
Interestingly, the ester moiety was found to be essential
for the expression of potent oral activity in this series of
prodrugs. Although the mechanistic details remain to be
elucidated, these findings are expected to be useful in
improving the oral anticoagulant activity of amidine
compounds through use of a prodrug strategy.
4.1.4. N-[(7-Cyano-2-naphthyl)methyl]-N-[4-(4-methyl-
1,4-diazepan-1-yl)phenyl]methanesulfonamide (6). To a
stirred solution of 5 (1.35g, 3.64mmol) in 36mL 1,2-di-
chloroethane was added pyridine (963mg, 10.9mmol)
and methanesulfonylchloride (630mg, 5.5mmol) and
stirred at ambient temperature. After 17h, the reaction
mixture was diluted with chloroform and washed with
saturated sodium hydrogencarbonate, water, aqueous
10% citric acid, and water. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The resulting
residues was chromatographed on silica gel eluting with
chloroform/methanol/ammonia (100:3:0.3) to give 6
(1.43g, 87%) as a colorless amorphous powder: 1H
NMR(CDCl 3) d 1.89–1.99 (2H, m), 2.35 (3H, s),
2.49–2.55 (2H, m), 2.61–2.66 (2H, m), 2.98 (3H, s),
3.35–3.42 (2H, m), 3.45–3.51 (2H, m), 4.94 (2H, s),
6.53 (2H, d, J = 9.0Hz), 7.01 (2H, d, J = 9.0Hz), 7.26
(1H, s), 7.57 (1H, dd, J = 1.8, 8.8Hz), 7.70 (1H, dd,
J = 1.8, 8.8Hz), 7.81–7.89 (2H, m), 8.12 (1H, s); FAB
MS m/e (M+1)+ 449.
4.1. Chemistry
1H NMRspectra were measured with a JEOL EX90,
EX400 or GX500 spectrometer; chemical shifts are ex-
pressed in d units using tetramethylsilane as the stand-
ard (in NMRdescription, s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, and br = broad
peak). Mass spectra were recorded with a Hitachi M-
80 or JEOL JMS-DX300 spectrometer. ODS column
chromatography was performed on YMC gel (ODS-A
120-230/70).
4.1.1. 1-Methyl-4-(4-nitrophenyl)-1,4-diazepane (3). To a
stirred solution of 1-methyl-1,4-diazepane (13.67g;
120mmol) in DMF (120mL) was added 4-fluoronitro-
benzene (16.1g, 114mmol), potassium carbonate
(31.5g, 228mmol) at 100°C for 17h. After the reaction
mixture was cooled, the reaction mixture was diluted
with ethyl acetate and washed with H2O and saturated
saline. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The resulted residue was washed
with Et2O to give 3 (19.51g, 69%) as a yellow amor-
phous powder: 1H NMR(CDCl 3) d 1.88–2.08 (2H,
m), 2.39 (3H, s), 2.54–2.59 (2H, m), 2.70–2.74 (2H, m),
3.56–3.68 (4H, m), 6.63 (2H, d, J = 8.8Hz), 8.11 (2H,
d, J = 8.8Hz); FAB MS m/e (M+1)+ 236.
4.1.5. Ethyl ({[(7-cyano-2-naphthyl)methyl][4-(4-methyl-
1,4-diazepan-1-yl)phenyl]amino}sulfonyl)acetate (7). Com-
pound 7 was synthesized from 5 and ethyl (chlorosulfo-
nyl)acetate8 according to the same procedure as that for
6. Compound 7 was obtained as a white amorphous
1
powder (80% yield): H NMR(CDCl ) d 1.39 (3H, t,
3
J = 6.9Hz), 1.86–1.97 (2H, m), 2.32 (3H, s), 2.49 (2H,
t, J = 4.3Hz), 2.61 (2H, t, J = 4.3Hz), 3.37 (2H, t,
J = 4.3Hz), 3.46 (2H, t, J = 4.3Hz), 4.07 (2H, s), 4.34
(2H, q, J = 6.9Hz), 5.02 (2H, s), 6.53 (2H, d,
J = 8.3Hz), 7.19 (2H, d, J = 8.3Hz), 7.48–7.54 (1H,
m), 7.62 (1H, s), 7.65–7.71 (1H, m), 7.77–7.85 (2H, m),
8.03 (1H, s); FAB Ms m/e (M+H)+ 521.
4.1.2. 4-(4-Methyl-1,4-diazepan-1-yl)aniline (4). To the
solution of 3 (2.41g, 10.2mmol) in EtOH (100mL)
was added 10% Pd–C powder (0.24g) and stirred in
hydrogen atmosphere at ambient temperature for 2h.
The reaction mixture was filtrated through a pad of Cel-
ite and concentrated in vacuo to give 4 (2.09g, quant.
yield) as a brown amorphous powder: 1H NMR
(CDCl3) d 1.93–2.02 (2H, m), 2.37 (3H, s), 2.54–2.59
4.1.6. tert-Butyl ({[(7-cyano-2-naphthyl)methyl][4-(4-
methyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)carba-
mate (8). Compound 8 was synthesized from 5 and tert-
butyl (chlorosulfonyl)carbamate according to the same
procedure as that for 6. Compound 8 was obtained as
a white amorphous powder (75% yield): 1H NMR
(CDCl3) d 1.59 (9H, s), 1.86–1.97 (2H, m), 2.37–2.43
(5H, m), 2.59–2.70 (2H, m), 2.74–2.83 (2H, m), 3.02–

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
5421
3.10 (2H, m), 5.23 (2H, s), 6.33 (2H, d, J = 9.0Hz), 7.04
(2H, d, J = 9.0Hz), 7.52 (1H, dd, J = 1.8, 9.0Hz), 7.64
(1H, s), 7.78–7.86 (3H, m), 8.07 (1H, s); FAB MS m/e
(M+1)+ 550.
6.50 (2H, d, J = 8.8Hz), 7.05 (2H, d, J = 8.8Hz), 7.54–
7.72 (3H, m), 7.80–7.89 (2H, m), 8.11 (1H, s); FAB
MS m/e (M+1)+ 478.
4.1.10. Ethyl ({[(7-cyano-2-naphthyl)methyl][4-(4-methyl-
1,4-diazepan-1-yl)phenyl]amino}sulfonyl)carbamate (12).
Compound 12 was synthesized from 5 and ethyl (chloro-
sulfonyl)carbamate according to the same procedure as
that for 6. Compound 12 was obtained as a slight green
amorphous powder (91% yield): 1H NMR(DMSO- d6) d
1.23 (3H, t, J = 6.8Hz), 1.88–2.00 (2H, m), 2.53 (3H, s),
2.77–2.89 (2H, m), 2.91–3.00 (2H, m), 3.01–3.09 (2H,
m), 3.19–3.51 (2H, m), 4.03 (2H, q, J = 6.8Hz), 5.10
(2H, s), 6.47 (2H, d, J = 8.8Hz), 7.05 (2H, d,
J = 8.8Hz), 7.70 (1H, dd, J = 1.6, 8.0Hz), 7.74 (1H,
dd, J = 1.6, 8.0Hz), 7.82 (1H, s), 7.96 (1H, d,
J = 8.8Hz), 8.02 (1H, d, J = 8.8Hz), 8.46 (1H, s); FAB
MS m/e (M+1)+ 522.
4.1.7. N-[(7-Cyano-2-naphthyl)methyl]-N-[4-(4-methyl-
1,4-diazepan-1-yl)phenyl]sulfamide (9). To a stirred solu-
tion of 8 (0.24g, 0.44mmol) in 4.8mL chloroform was
added 4N hydrogen chloride in ethyl acetate (4.8mL)
and stirred at ambient temperature for 6h. The reaction
mixture was concentrated in vacuo to give 9 (0.25g,
1
quant. yield) as a white amorphous powder: H NMR
(DMSO-d6) d 2.20–2.45 (2H, m), 2.75 (3H, s), 2.94–
3.23 (2H, m), 3.30–3.39 (4H, m), 3.58–3.69 (2H, m),
4.85 (2H, s), 6.60 (2H, d, J = 8.4Hz), 7.16 (2H, d,
J = 8.4Hz), 7.71–7.77 (2H, m), 7.90 (1H, s), 7.98 (1H,
d, J = 8.8Hz), 8.05 (1H, d, J = 8.8Hz), 8.49 (1H, s);
FAB MS m/e (M+1)+ 450.
4.1.8. tert-Butyl ({[(7-cyano-2-naphthyl)methyl][4-(4-
methyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)methyl-
carbamate (10). To a stirred solution of 8 (1.3g,
2.36mmol) in 50mL tetrahydrofurane was added triphe-
nylphosphine (2.48g, 9.44mmol), methanol (0.38mL;
9.44mmol), and diethylazodicarboxylate (1.49mL,
9.44mmol) and stirred at ambient temperature for 4h.
The reaction mixture was concentrated, added H2O,
and extracted with chloroform. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The
resulting residue was chromatographed on silica gel elut-
ing with chloroform/methanol/ammonia (50:1:0.1) to
give 10 (1.33g, quant. yield) as a yellow amorphous
4.1.11. 7-{[[4-(4-Methyl-1,4-diazepan-1-yl)phenyl](methyl-
sulfonyl)amino]methyl}naphthalene-2-carboximidamide
(13). HCl gas was bubbled through a solution of 6
(1.05g, 2.34mmol) in MeOH (12mL) and chloroform
(21mL) under ꢀ20°C for 20min. The mixture was al-
lowed to stir for 21h at 5°C, and then concentrated in
vacuo. To the crude imidate dissolved in MeOH
(12mL) and 1,4-dioxane (12mL) at ambient tempera-
ture was added ammonium acetate (1.8g, 23mmol).
The reaction mixture was stirred at ambient temperature
for 19h and concentrated in vacuo. The resulted residue
was chromatographed on ODS-gel eluting with MeOH/
H2O (10:90). MeOH was removed in vacuo, and the
aqueous solution was lyophilized after being acidified
with 1N HCl. Compound 13 (629mg, 50%) was ob-
tained as a white amorphous powder: 1H NMR
(DMSO-d6) d 2.02–2.14 (1H, m), 2.29–2.41 (1H, m),
2.70 (3H, d, J = 4.8Hz), 2.98–3.05 (2H, m), 3.09 (3H,
s), 3.25–3.44 (4H, m), 3.62–3.87 (2H, m), 5.00 (2H, s),
6.64 (2H, d, J = 8.8Hz), 7.23 (2H, d, J = 8.8Hz), 7.67
(1H, dd, J = 1.6, 8.4Hz), 7.82–7.87 (1H, m), 7.90 (1H,
s), 8.01 (1H, d, J = 8.4Hz), 8.08 (1H, d, J = 8.4Hz),
8.54 (1H, s), 9.36–9.45 (2H, br), 9.55–9.66 (2H, br);
FAB MS m/e (M+1)+ 466; Anal. Calcd for C25H31N5O2-
SÆ2.5HClÆ1.0H2O: C, 50.06; H, 5.99; N, 12.97; S, 4.95;
Cl, 13.69. Found: C, 50.19; H, 6.12; N, 13.03; S, 4.93;
Cl, 13.84.
1
powder: H NMR(CDCl 3) d 1.63 (9H, s), 1.90–1.99
(2H, m), 2.35 (3H, s), 2.48–2.55 (2H, m), 2.61–2.67
(2H, m), 2.94 (3H, s), 3.37–3.43 (2H, s), 3.45–3.52
(2H, m), 5.15 (2H, s), 6.51 (2H, d, J = 8.8Hz), 7.01
(2H, d, J = 8.8Hz), 7.56 (1H, dd, J = 1.8, 8.8Hz),
7.65–7.72 (2H, m), 7.85 (2H, t, J = 8.8Hz), 8.12 (1H,
s); FAB MS m/e (M+1)+ 564.
4.1.9. N-[(7-Cyano-2-naphthyl)methyl]-N0,N0-dimethyl-
N-[4-(4-methyl-1,4-diazepan-1-yl)phenyl]sulfamide (11).
To a stirred solution of 10 (1.43g, 2.54mmol) in 30mL
chloroform was added trifluoroacetic acid (6.0mL) and
stirred at ambient temperature for 5h. The reaction mix-
ture was diluted with chloroform and washed with satu-
rated sodium hydrogencarbonate and water. The
organic layer was dried over Na2SO4 and concentrated
in vacuo to give intermediate de-protected compound.
To a stirred solution of crude de-protected compound
in 30mL tetrahydrofurane was added triphenylphos-
phine (1.35g, 5.16mmol), methanol (0.21mL;
5.16mmol), and diethylazodicarboxylate (0.81mL,
5.16mmol) and stirred at ambient temperature for
24h. The reaction mixture was concentrated, added
H2O, and extracted with chloroform. The organic layer
was dried over Na2SO4 and concentrated in vacuo. The
resulting residue was chromatographed on silica gel
eluting with chloroform/methanol/ammonia (50:1:0.1)
to give 11 (520mg, 84%) as a yellow amorphous powder:
4.1.12. N-Hydroxy-7-{[[4-(4-methyl-1,4-diazepan-1-yl)-
phenyl](methylsulfonyl)amino]methyl}naphthalene-2-carb-
oximidamide (14). To a stirred solution of 6 (350mg,
0.78mmol) in 15mL MeOH was added triethylamine
(202mg; 2.00mmol), hydroxylamine hydrochloride
(65mg, 0.94mmol) and stirred at 90°C for 24h. The
reaction mixture was concentrated and the resulting res-
idue was chromatographed on silica gel eluting with
chloroform/methanol/ammonia (100:10:1). The organic
solvent was removed in vacuo, and the result residue
was lyophilized after being acidified with 1N HCl. Com-
pound 14 (280mg, 34%) was obtained as a white amor-
1
phous powder: H NMR(DMSO- d6) d 2.03–2.14 (1H,
1H NMR(CDCl ) d 1.89–1.99 (2H, m), 2.35 (3H, s),
m), 2.24–2.39 (1H, m), 2.72 (3H, d, J = 4.9Hz), 2.96–
3.04 (2H, m), 3.09 (3H, s), 3.25–3.42 (4H, m), 3.59–
3.75 (2H, m), 4.99 (2H, s), 6.64 (2H, d, J = 8.8Hz),
3
2.48–2.54 (2H, m), 2.61–2.66 (2H, m), 2.76 (6H, s),
3.36–3.43 (2H, m), 3.45–3.50 (2H, m), 4.89 (2H, s),

5422
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
7.24 (2H, d, J = 8.8Hz), 7.67 (1H, dd, J = 1.5, 8.8Hz),
7.72 (1H, dd, J = 1.5, 8.8Hz), 7.90 (1H, s), 8.00 (1H,
d, J = 8.8Hz), 8.08 (1H, d, J = 8.8Hz), 8.37 (1H, s),
11.02–11.16 (2H, br), 11.28–11.50 (2H, br); FAB MS
m/e (M+1)+ 482; Anal. Calcd for C25H31N5O3SÆ2.4H-
ClÆ1.0H2O: C, 51.14; H, 6.08; N, 11.93; S, 5.46; Cl,
14.49. Found: C, 51.35; H, 6.21; N, 11.65; S, 5.48; Cl,
14.36.
(1H, s), 8.00 (1H, d, J = 8.8Hz), 8.09 (1H, d,
J = 8.8Hz), 9.30–9.40 (2H, br), 9.38–9.57 (2H, br);
FAB MS m/e (M+1)+ 495; Anal. Calcd for C26H34N6O2-
SÆ2.7HClÆ1.0H2O: C, 51.10; H, 6.38; N, 13.75; S, 5.25;
Cl, 15.66. Found: C, 50.83; H, 6.59; N, 13.57; S, 5.23;
Cl, 15.78.
4.1.16.
7-({[(Dimethylamino)sulfonyl][4-(4-methyl-1,4-
diazepan-1-yl)phenyl]amino}methyl)-N-hydroxynaphtha-
lene-2-carboximidamide (18). Compound 18 was synthe-
sized from 11 according to the same procedure as that
for 14. Compound 18 was obtained as a white amor-
phous powder (90% yield): 1H NMR(DMSO- d6) d
2.03–2.12 (1H, m), 2.20–2.32 (1H, m), 2.73 (2H, d,
J = 4.8Hz), 2.78 (6H, s), 2.99–3.10 (4H, m), 3.58–3.70
(2H, m), 4.99 (2H, s), 6.62 (2H, d, J = 8.0Hz), 7.21
(2H, d, J = 8.0Hz), 7.64 (1H, dd, J = 2.0, 8.0Hz), 7.71
(1H, dd, J = 2.0, 8.0Hz), 7.87 (1H, s), 8.00 (1H, d,
J = 8.0Hz), 8.07 (1H, d, J = 8.0Hz), 8.35 (1H, s),
8.80–9.40 (1H, br), 10.81–10.92 (1H, br), 11.18–11.40
(1H, br); FAB MS m/e (M+1)+ 511; Anal. Calcd for
C26H34N6O3SÆ2.0HClÆ2.2H2O: C, 50.11; H, 6.53; N,
13.49; S, 5.15; Cl, 11.38. Found: C, 50.03; H, 6.41; N,
13.12; S, 4.99; Cl, 11.52.
4.1.13. 7-({(Aminosulfonyl)[4-(4-methyl-1,4-diazepan-1-
yl)phenyl]amino}methyl)naphthalene-2-carboximidamide
(15). Compound 15 was synthesized from 9 according to
the same procedure as that for 13. Compound 15 was
obtained as a white amorphous powder (30% yield):
1H NMR(DMSO- d6) d 2.03–2.15 (1H, m), 2.24–2.37
(1H, m), 2.71 (3H, d, J = 4.9Hz), 2.95–3.07 (2H, m),
3.23–3.45 (4H, m), 3.58–3.74 (2H, m), 4.87 (2H, s),
5.53 (2H, s), 6.61 (2H, d, J = 8.8Hz), 7.17 (2H, d,
J = 8.8Hz), 7.73 (1H, dd, J = 1.4, 8.8Hz), 7.81 (1H,
dd, J = 1.4, 8.8Hz), 7.92 (1H, s), 7.99 (1H, d,
J = 8.8Hz), 8.08 (1H, d, J = 8.8Hz), 8.46 (1H, s),
9.31–9.36 (2H, br), 9.52–9.58 (2H, br); FAB MS m/e
(M+1)+ 467; Anal. Calcd for C24H30N6O2SÆ2.8HClÆ1.5-
H2O: C, 48.39; H, 6.60; N, 14.11; S, 5.38; Cl,
16.66. Found: C, 48.40; H, 6.11; N, 13.85; S, 5.07; Cl,
16.54.
4.1.17. ({({7-[(Hydroxyamino)(imino)methyl]-2-naphth-
yl}methyl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}-
sulfonyl)acetic acid (19). To a stirred solution of 21
(550mg, 0.99mmol) in 10mL dioxane was added 1N
NaOHaq (2mL, 2.00mmol) and stirred at ambient tem-
perature for 3h. The reaction mixture was concentrated
in vacuo and the residue was chromatographed on
ODS-gel eluting with 0.001M HCl/CH3CN (100:5).
CH3CN was removed in vacuo, and the aqueous solu-
tion was lyophilized after being acidified with 1N HCl.
Compound 19 (107mg, 19%) was obtained as a white
4.1.14. 7-({(Aminosulfonyl)[4-(4-methyl-1,4-diazepan-1-
yl)phenyl]amino}methyl)-N-hydroxynaphthalene-2-carb-
oximidamide (16). Compound 16 was synthesized from 9
according to the same procedure as that for 14. Com-
pound 16 was obtained as a white amorphous powder
(16% yield): 1H NMR(DMSO- d6) d 2.02–2.13 (2H,
m), 2.20–2.39 (2H, m), 2.71 (3H, d, J = 4.9Hz), 2.95–
3.09 (2H, m), 3.25–3.41 (4H, m), 3.59–3.74 (2H, m),
4.87 (2H, s), 6.63 (2H, d, J = 8.8Hz), 7.17 (2H, d,
J = 8.8Hz), 7.68–7.74 (2H, m), 7.93 (1H, s), 7.98 (1H,
d, J = 8.8Hz), 8.07 (1H, d, J = 8.8Hz), 8.33 (1H, s),
8.98–9.51 (2H, br), 11.10 (1H, s), 11.25–11.50 (1H, br);
FAB MS m/e (M+1)+ 487; Anal. Calcd for C24H30N6O3-
SÆ3.0HClÆ1.0H2O: C, 46.57; H, 5.86; N, 13.58; S, 5.18;
Cl, 17.18. Found: C, 46.48; H, 5.98; N, 13.50; S, 5.16;
Cl, 17.48.
1
amorphous powder: H NMR(DMSO- d6) d 2.03–2.14
(1H, m), 2.29–2.40 (1H, m), 2.71 (3H, d, J = 4.9Hz),
2.98–3.10 (2H, m), 3.26–3.41 (4H, m), 3.62–3.77 (2H,
m), 4.24 (2H, s), 5.02 (2H, s), 6.66 (2H, d, J = 8.8Hz),
7.23 (2H, d, J = 8.8Hz), 7.66 (1H, dd, J = 1.5, 8.8Hz),
7.74 (1H, dd, J = 1.2, 8.8Hz), 7.88 (1H, s), 8.02 (1H,
d, J = 8.8Hz), 8.07 (1H, d, J = 8.8Hz), 8.36 (1H, s),
8.90–9.54 (4H, br); FAB MS m/e (M+1)+ 526; Anal.
Calcd for C26H31N5O5SÆ3.0HClÆ1.8H2O: C, 46.53; H,
5.66; N, 10.44; S, 4.78; Cl, 16.38. Found: C, 46.90; H,
6.04; N, 10.39; S, 4.86; Cl, 16.30.
4.1.15.
7-({[(Dimethylamino)sulfonyl][4-(4-methyl-1,4-
diazepan-1-yl)phenyl]amino}methyl)naphthalene-2-carb-
oximidamide (17). To the solution of 18 (850mg,
0.137mmol) in acetic acid (17mL) and chloroform
(17mL) was added acetic anhydride (0.2mL) and 10%
Pd–C powder (100mg) and stirred in hydrogen atmos-
phere at ambient temperature for 2h. The reaction mix-
ture was filtrated through a pad of Celite and
concentrated in vacuo. The resulted residue was chro-
matographed on ODS-gel eluting with MeOH/H2O
(10:90). MeOH was removed in vacuo, and the aqueous
solution was lyophilized after being acidified with 1N
HCl. Compound 17 (424mg, 51%) was obtained as a
white amorphous powder: 1H NMR(DMSO- d6) d
2.02–2.18 (1H, m), 2.25–2.40 (1H, m), 2.71 (3H, d,
J = 4.8Hz), 2.78 (6H, s), 2.98–3.10 (2H, m), 3.26–3.45
(4H, m), 3.60–3.79 (2H, m), 5.00 (2H, s), 6.62 (2H, d,
J = 8.8Hz), 7.20 (2H, d, J = 8.8Hz), 7.66 (1H, dd,
J = 1.2, 8.8Hz), 7.82 (1H, dd, J = 1.2, 8.8Hz), 7.88
4.1.18. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}sulfon-
yl)acetate (20). Compound 20 was synthesized from 7
according to the same procedure as that for 13. Com-
pound 20 was obtained as a white amorphous powder
(40% yield): 1H NMR(DMSO- d6) d 1.27 (3H, t,
J = 7.2Hz), 2.04–2.16 (1H, m), 2.28–2.40 (1H, m), 2.71
(1.5H, s), 2.72 (1.5H, s), 2.99–3.09 (2H, m), 3.26–3.42
(4H, m), 3.62–3.76 (2H, m), 4.25 (2H, q, J = 7.2Hz),
4.36 (2H, s), 5.02 (2H, s), 6.67 (2H, d, J = 8.4Hz), 7.22
(2H, d, J = 8.4Hz), 7.67 (1H, d, J = 8.4Hz), 7.83 (1H,
d, J = 8.4Hz), 7.90 (1H, s), 8.03 (1H, d, J = 8.4Hz),
8.10 (1H, d, J = 8.4Hz), 8.51 (1H, s), 9.37 (2H, s), 9.56
(2H, s), 11.21 (1H, s); FAB MS m/e (M+1)+ 538; Anal.
Calcd for C28H35N5O4SÆ2.9HClÆ1.5H2O: C, 50.16; H,

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
5423
6.15; N, 10.45; S, 4.78; Cl, 15.34. Found: C, 50.45; H,
6.04; N, 10.50; S, 4.78; Cl, 15.15.
3.55 (2H, m), 4.38–4.52 (2H, m), 6.20–6.31 (1H, m),
6.42–6.63 (3H, m), 7.13–7.28 (2H, m), 7.52–7.83 (4H,
m); FAB MS m/e (M)+ 346.
4.1.19. Ethyl ({({7-[(hydroxyamino)(imino)methyl]-2-
naphthyl}methyl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]-
amino}sulfonyl)acetate (21). Compound 21 was synthe-
sized from 7 according to the same procedure as that
for 14. Compound 21 was obtained as a white amor-
phous powder (12% yield): 1H NMR(DMSO- d6) d
1.27 (3H, t, J = 7.3Hz), 2.02–2.14 (1H, m), 2.21–2.38
(1H, m), 2.73 (3H, d, J = 4.4Hz), 2.98–3.10 (2H, m),
3.22–3.70 (6H, m), 4.24 (2H, q, J = 7.3Hz), 4.35 (2H,
s), 5.01 (2H, s), 6.66 (2H, d, J = 8.8Hz), 7.21 (2H, d,
J = 8.8Hz), 7.64 (1H, dd, J = 1.9, 8.8Hz), 7.71 (1H,
dd, J = 1.9, 8.8Hz), 7.89 (1H, s), 8.00 (1H, d,
J = 8.8Hz), 8.07 (1H, d, J = 8.8Hz), 8.35 (1H, s),
8.80–9.42 (2H, br), 10.72–10.89 (1H, br), 11.12–11.42
(1H, br); FAB MS m/e (M+1)+ 554; Anal. Calcd for
C28H35N5O5SÆ2.1HClÆ2.8H2O: C, 49.41; H, 6.32; N,
10.29; S, 4.71; Cl, 10.94. Found: C, 49.77; H, 6.34; N,
10.23; S, 4.61; Cl, 11.16.
4.1.23. Ethyl ({[(2E)-3-(3-cyanophenyl)prop-2-en-1-yl][4-
(4-methyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)ace-
tate (26). Compound 26 was synthesized from 25 and
ethyl (chlorosulfonyl)acetate8 according to the same
procedure as that for 6. Compound 26 was obtained
1
as a white amorphous powder (44% yield): H NMR
(CDCl3) d 1.36 (3H, t, J = 6.9Hz), 1.94–2.05 (2H, m),
2.37 (3H, s), 2.52–2.57 (2H, m), 2.66–2.70 (2H, m),
3.42–3.49 (2H, m), 3.52–3.58 (2H, m), 3.99 (2H, s),
4.30 (2H, q, J = 6.9Hz), 4.45 (2H, d, J = 5.1Hz), 6.20–
6.31 (1H, m), 6.42 (1H, d, J = 16.2Hz), 6.63 (2H, d,
J = 9.0Hz), 7.26–7.29 (2H, m), 7.38–7.59 (4H, m);
FAB MS m/e (M+1)+ 497.
4.1.24. Isopropyl ({[(2E)-3-(3-cyanophenyl)prop-2-en-1-
yl][4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}sulfonyl)-
acetate (27). Compound 27 was synthesized from 25 and
isopropyl (chlorosulfonyl)acetate9 according to the same
procedure as that for 6. Compound 27 was obtained as a
white amorphous powder (49% yield): 1H NMR
(CDCl3) d 1.33 (6H, d, J = 6.3Hz), 1.94–2.06 (2H, m),
2.38 (3H, s), 2.53–2.61 (2H, m), 2.66–2.72 (2H, m),
3.42–3.50 (2H, m), 3.52–3.58 (2H, m), 3.95 (2H, s),
4.45 (2H, d, J = 5.3Hz), 5.10–5.19 (1H, m), 6.21–6.30
(1H, m), 6.42 (1H, d, J = 16.1Hz), 6.63 (2H, d,
J = 7.8Hz), 7.21–7.30 (2H, m), 7.35–7.56 (4H, m);
FAB MS m/e (M+1)+ 511.
4.1.20. Ethyl ({({7-[amino(imino)methyl]-2-naphthyl}-
methyl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}sul-
fonyl)carbamate (22). Compound 22 was synthesized
from 12 according to the same procedure as that for
13. Compound 22 was obtained as a white amorphous
1
powder (48% yield): H NMR(DMSO- d6) d 1.29 (3H,
t, J = 6.0Hz), 2.06–2.16 (1H, m), 2.28–2.40 (1H, m),
2.72 (3H, d, J = 3.2Hz), 2.96–3.11 (2H, m), 3.26–3.42
(4H, m), 3.60–3.78 (2H, m), 4.25 (2H, q, J = 6.0Hz),
5.14 (2H, s), 6.67 (2H, d, J = 8.8Hz), 7.10 (2H, d,
J = 8.8Hz), 7.66 (1H, dd, J = 1.6, 8.8Hz), 7.83 (1H,
dd, J = 1.6, 8.8Hz), 7.91 (1H, s), 8.03 (1H, d,
J = 8.8Hz), 8.10 (1H, d, J = 8.8Hz), 8.49 (1H, s),
9.30–9.39 (2H, br), 9.48–9.59 (2H, br); FAB MS m/e
(M+1)+ 539; Anal. Calcd for C27H34N6O2SÆ2.6HClÆ1.0-
H2O: C, 51.91; H, 6.20; N, 12.11; S, 5.54; Cl, 15.94.
Found: C, 52.04; H, 6.18; N, 12.49; S, 5.30; Cl, 15.90.
4.1.25. ({((2E)-3-{3-[Amino(imino)methyl]phenyl}prop-2-
en-1-yl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]amino}sul-
fonyl)acetic acid (28). A solution of 29 (590mg,
1.15mmol) in concd HCl (9mL) was stirred at ambient
temperature for 24h. The reaction mixture was concen-
trated in vacuo and the residues was chromatographed
on ODS-gel eluting with CH3CN/H2O (4:96). CH3CN
was removed in vacuo, and the aqueous solution was ly-
ophilized after being acidified with 1N HCl. Compound
28 (134mg, 24%) was obtained as a white amorphous
powder: 1H NMR(DMSO- d6) d 1.82–1.98 (2H, m),
2.32 (3H, s), 2.52–2.60 (2H, m), 2.64–2.72 (2H, m),
3.40–3.46 (2H, m), 3.48–3.54 (2H, m), 3.73 (2H, s),
4.43 (2H, d, J = 4.9Hz), 6.38–6.52 (2H, m), 6.65 (2H,
d, J = 9.1Hz), 7.32 (2H, d, J = 9.1Hz), 7.51 (1H, t,
J = 7.8Hz), 7.64 (1H, d, J = 7.8Hz), 7.69 (1H, d,
J = 7.8Hz), 7.83 (1H, s), 8.95–9.25 (2H, br), 10.54–
10.82 (2H, br); FAB MS m/e 486 (M+1)+ 486; Anal.
Calcd for C24H31N5O4SÆ2.4HClÆ2.1H2O: C, 47.18; H,
6.20; N, 11.46; S, 5.25; Cl, 13.93. Found: C, 46.91; H,
6.23; N, 11.40; S, 5.22; Cl, 13.85.
4.1.21. Ethyl ({({7-[(hydroxyamino)(imino)methyl]-2-
naphthyl}methyl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]-
amino}sulfonyl)carbamate (23). Compound 23 was syn-
thesized from 12 according to the same procedure as
that for 14. Compound 23 was obtained as a white
1
amorphous powder (42% yield): H NMR(DMSO- d6)
d 1.23 (3H, t, J = 7.4Hz), 1.93–2.01 (2H, m), 2.55 (3H,
s), 2.89–3.03 (4H, m), 3.10–3.19 (2H, m), 3.29–3.40
(2H, m), 4.07 (2H, q, J = 7.4Hz), 5.07 (2H, s), 6.57
(2H, d, J = 8.8Hz), 7.07 (2H, d, J = 8.8Hz), 7.50 (1H,
dd, J = 1.5, 8.8Hz), 7.69 (1H, s), 7.77–7.83 (3H, m),
8.09 (1H, s), 9.75 (1H, s); FAB MS m/e (M+1)+ 555;
Anal. Calcd for C27H34N6O5SÆ3.0HClÆ3.8H2O: C,
44.27; H, 6.14; N, 11.47; S, 4.38; Cl, 14.52. Found: C,
44.00; H, 6.22; N, 11.42; S, 4.30; Cl, 14.82.
4.1.26. Ethyl ({((2E)-3-{3-[amino(imino)methyl]phenyl}-
prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]-
amino}sulfonyl)acetate (29). Compound 29 was
synthesized from 26 according to the same procedure
as that for 13. Compound 29 was obtained as a white
4.1.22. 3-((1E)-3-{[4-(4-Methyl-1,4-diazepan-1-yl)phen-
yl]amino}prop-1-en-1-yl)benzonitrile (25). Compound 25
was synthesized from 4 and 24 according to the same
procedure as that for 5. Compound 25 was obtained
1
amorphous powder (23% yield): H NMR(DMSO- d6)
1
as a white amorphous powder (35% yield): H NMR
(CDCl3) d 1.95–2.07 (2H, m), 2.39 (3H, s), 2.54–2.62
(2H, m), 2.67–2.74 (2H, m), 3.36–3.45 (2H, m), 3.47–
d 1.24 (3H, t, J = 7.4Hz), 2.06–2.20 (1H, m), 2.28–2.40
(1H, m), 2.76 (3H, d, J = 4.4Hz), 3.03–3.18 (2H, m),
3.32–3.45 (4H, m), 3.65–3.83 (2H, m), 4.21 (2H, q,

5424
H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
J = 7.4Hz), 4.26 (2H, s), 4.41 (2H, d, J = 5.8Hz), 6.38–
6.45 (1H, m), 6.55 (1H, d, J = 5.5Hz), 6.75 (2H, d,
J = 9.3Hz), 7.27 (2H, d, J = 9.3Hz), 7.52 (1H, t,
J = 7.8Hz), 7.59 (1H, d, J = 7.8Hz), 7.70 (1H, d,
J = 7.8Hz), 7.80 (1H, s), 9.37 (2H, s), 9.56 (2H, s),
11.21 (1H, s); FAB MS m/e (M+1)+ 514.
s): FAB MS m/e (M+1)+ 572; Anal. Calcd for
C28H37N5O6SÆ1.5H2O: C, 56.17; H, 6.33; N, 11.70; S,
5.36. Found: C, 56.09; H, 6.69; N, 11.76; S, 5.37.
4.1.30. Ethyl ({((2E)-3-{3-[[(ethoxycarbonyl)amino](imi-
no)methyl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diaze-
pan-1-yl)phenyl]amino}sulfonyl)acetate (33). To a stirred
solution of 29 (530mg, 0.90mmol) in 30mL chloroform
was added chloroethylformate (0.26g, 2.71mmol), 0.1N
NaOH (27.1mL, 2.71mmol) at ambient temperature.
After 24h, the reaction mixture was neutralized with
1N HCl. The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The resulting
residue was chromatographed on silica gel eluting with
chloroform/methanol (10:1). The result residue was ly-
ophilized after being acidified with 1N HCl. Compound
33 (178mg, 34%) was obtained as a white amorphous
4.1.27. Ethyl ({((2E)-3-{3-[(hydroxyamino)(imino)meth-
yl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-1-yl)-
phenyl]amino}sulfonyl)acetate (30). Compound 30 was
synthesized from 26 according to the same procedure
as that for 14. Compound 30 was obtained as a white
1
amorphous powder (31% yield): H NMR(DMSO- d6)
d 1.24 (3H, t, J = 7.4Hz), 2.06–2.20 (1H, m), 2.28–2.40
(1H, m), 2.76 (3H, d, J = 4.4Hz), 3.03–3.18 (2H, m),
3.32–3.45 (4H, m), 3.65–3.83 (2H, m), 4.21 (2H, q,
J = 7.4Hz), 4.26 (2H, s), 4.41 (2H, d, J = 5.8Hz),
6.38–6.45 (1H, m), 6.55 (1H, d, J = 5.5Hz), 6.75 (2H,
d, J = 9.3Hz), 7.27 (2H, d, J = 9.3Hz), 7.52 (1H, t,
J = 7.8Hz), 7.59 (1H, d, J = 7.8Hz), 7.70 (1H, d,
J = 7.8Hz), 7.80 (1H, s), 8.57–9.42 (1H, br), 10.60–
11.58 (1H, br), 11.25–11.42 (1H, br); FAB MS m/e
(M+1)+ 530; Anal. Calcd for C26H35N5O5SÆ2.6HClÆ2.0-
H2O: C, 47.28; H, 6.35; N, 10.60; S, 4.85; Cl, 13.96.
Found: C, 47.12; H, 6.36; N, 10.50; S, 4.91; Cl, 14.24.
powder: 1H NMR(DMSO- d6)
d 1.25 (3H, t,
J = 7.3Hz), 1.33 (3H, t, J = 7.3Hz), 2.10–2.18 (1H, m),
2.29–2.38 (1H, m), 2.77 (3H, d, J = 4.8Hz), 3.04–3.16
(2H, m), 3.34–3.50 (4H, m), 3.73–3.76 (2H, m), 4.21
(2H, q, J = 7.3Hz), 4.26 (2H, s), 4.35 (2H, q,
J = 7.3Hz), 4.42 (2H, d, J = 5.9Hz), 6.42 (1H, dt,
J = 5.9, 15.6Hz), 6.55 (1H, d, J = 15.6Hz), 6.76 (2H,
d, J = 8.7Hz), 7.27 (2H, d, J = 8.7Hz), 7.54 (1H, t,
J = 7.8Hz), 7.65 (1H, d, J = 7.8Hz), 7.74 (1H, d,
J = 7.8Hz), 7.87 (1H, s), 10.52–10.61 (1H, br), 10.43–
11.04 (1H, br), 11.32–11.44 (1H, br): FAB MS m/e
(M+1)+ 586; Anal. Calcd for C29H39N5O6SÆ2.5HClÆ2.5-
H2O: C, 48.25; H, 6.49; N, 9.70; S, 4.44; Cl, 12.28.
Found: C, 48.15; H, 6.42; N, 9.22; S, 4.56; Cl, 12.63.
4.1.28. Ethyl ({((2E)-3-{3-[imino(methoxyamino)methyl]-
phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-1-yl)-
phenyl]amino}sulfonyl)acetate (31). Compound 31 was
synthesized from 26 and methoxyamine instead of hyd-
roxylamine according to the same procedure as that for
14. Compound 31 was obtained as a white amorphous
1
powder (28% yield): H NMR(DMSO- d6) d 1.25 (3H,
4.1.31. Ethyl ({{(2E)-3-[3-(imino{[(trichloromethoxy)car-
bonyl]amino}methyl)phenyl]prop-2-en-1-yl}[4-(4-methyl-
t, J = 7.0Hz), 2.07–2.20 (1H, m), 2.31–2.46 (1H, m),
2.74 (3H, d, J = 4.9Hz), 3.02–3.19 (2H, m), 3.34–3.48
(4H, m), 3.67–3.82 (2H, m), 3.87 (3H, s), 4.20 (2H, q,
J = 7.0Hz), 4.27 (3H, s), 4.41 (2H, d, J = 5.8Hz), 6.44
(1H, dt, J = 5.8, 15.6Hz), 6.64 (1H, d, J = 15.6Hz),
6.76 (2H, d, J = 8.8Hz), 7.28 (2H, d, J = 8.8Hz), 7.49
(1H, t, J = 8.8Hz), 7.64 (2H, t, J = 8.8Hz), 7.86 (1H,
s), 8.50–9.47 (2H, br), 11.29 (1H, s): FAB MS m/e
(M+1)+ 544; Anal. Calcd for C27H37N5O5SÆ2.7HClÆ1.4-
H2O: C, 48.59; H, 6.42; N, 10.48; S, 4.80; Cl, 14.84.
Found: C, 48.63; H, 6.70; N, 10.46; S, 4.78; Cl, 14.52.
1,4-diazepan-1-yl)phenyl]amino}sulfonyl)acetate
(34).
Compound 34 was synthesized from 29 and 2,2,2-tri-
chloroethylchloroformate according to the same proce-
dure as that for 33. Compound 34 was obtained as a
white amorphous powder (28% yield): 1H NMR
(DMSO-d6) d 1.25 (3H, t, J = 7.3Hz), 2.11–2.28 (2H,
m), 2.79 (3H, d, J = 4.9Hz), 3.06–3.18 (4H, m), 3.33–
3.46 (4H, m), 3.60–3.78 (2H, m), 4.20 (2H, q,
J = 7.3Hz), 4.26 (2H, s), 4.41 (2H, d, J = 5.9Hz), 4.97
(2H, s), 6.37 (1H, dt, J = 5.9, 16.1Hz), 6.55 (1H, d,
J = 16.1Hz), 6.76 (2H, d, J = 9.3Hz), 7.13 (2H, d,
J = 9.3Hz), 7.53 (1H, d, J = 7.8Hz), 7.70 (1H, d,
J = 7.8Hz), 7.84 (1H, d, J = 7.8Hz), 7.93 (1H, s),
10.20–10.34 (3H, br): FAB MS m/e (M+1)+ 689; Anal.
Calcd for C29H36N5O6SCl3Æ1.7HClÆ2.3H2O: C, 43.95;
H, 5.38; N, 8.84; S, 4.05; Cl, 21.03. Found: C, 43.70;
H, 5.25; N, 8.39; S, 4.07; Cl, 20.79.
4.1.29. Ethyl ({((2E)-3-{3-[[(acetyloxy)amino](imino)-
methyl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-
1-yl)phenyl]amino}sulfonyl)acetate (32). To a stirred
solution of 30 (517mg, 0.98mmol) in DMF (5mL) and
pyridine (5mL) was added acetic anhydrate (0.99g,
9.7mmol) at ambient temperature. The reaction mixture
was concentrated and the resulting residue was chroma-
tographed on silica gel eluting with chloroform/metha-
nol/ammonia (100:10:1). Compound 32 (162mg, 29%)
4.1.32. ({((2E)-3-{3-[(Hydroxyamino)(imino)methyl]phen-
yl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-1-yl)phenyl]-
amino}sulfonyl)acetic acid (35). To a stirred solution of
30 (181mg, 0.27mmol) in 5mL EtOH and 5mL H2O
was added 1N NaOHaq (2mL, 2.00mmol) and stirred
at ambient temperature for 2h. The reaction mixture
was concentrated in vacuo and the residue was chroma-
tographed on ODS-gel eluting with MeOH/CH3CN
(40:60). CH3CN was removed in vacuo, and the aqueous
solution was lyophilized after being acidified with 1N
HCl. Compound 35 (152mg, 90%) was obtained as a
1
was obtained as a white amorphous powder: H NMR
(DMSO-d6) d 1.24 (3H, t, J = 7.0Hz), 1.92–2.00 (2H,
m), 2.15 (3H, s), 2.38 (3H, s), 2.64–2.71 (2H, m), 2.73–
2.83 (2H, m), 3.21–3.35 (2H, m), 3.52–3.59 (2H, m),
4.20 (2H, q, J = 7.0Hz), 4.26 (3H, s), 4.38 (2H, d,
J = 5.9Hz), 6.30 (1H, dt, J = 5.9, 15.6Hz), 6.49 (1H, d,
J = 15.6Hz), 6.70 (2H, d, J = 8.8Hz), 6.83 (2H, br),
7.22 (2H, d, J = 8.8Hz), 7.37 (1H, t, J = 7.8Hz), 7.48
(1H, d, J = 7.8Hz), 7.58 (1H, d, J = 7.8Hz), 7.71 (1H,

H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
5425
white amorphous powder: 1H NMR(DMSO- d6) d 1.98–
2.19 (1H, m), 2.26–2.40 (1H, m), 2.76 (3H, d,
J = 3.5Hz), 3.04–3.16 (2H, m), 3.32–3.50 (4H, m),
3.63–3.85 (2H, m), 4.14 (2H, s), 4.42 (2H, d,
J = 5.9Hz), 6.41 (1H, dt, J = 5.9, 16.1Hz), 6.53 (1H, d,
J = 16.1Hz), 6.75 (2H, d, J = 9.3Hz), 7.27 (2H, d, J =
9.3Hz), 7.50 (1H, t, J = 7.8Hz), 7.58 (1H, d, J =
7.8Hz), 7.66 (1H, d, J = 7.8Hz), 7.78 (1H, s), 10.82–
10.95 (1H, br), 10.72–11.40 (2H, br), 13.21–13.63 (1H,
br); FAB MS m/e (M+1)+ 502.
J = 7.8Hz), 7.70 (1H, d, J = 7.8Hz), 7.80 (1H, s),
8.90–9.21 (1H, br), 11.27–11.35 (1H, br), 11.71–11.83
(1H, br): FAB MS m/e (M+1)+ 544; Anal. Calcd for
C27H37N5O5SÆ2.5HClÆ2.3H2O: C, 48.22; H, 6.64; N,
10.41; S, 4.77; Cl, 12.12. Found: C, 48.60; H, 6.43; N,
10.09; S, 4.77; Cl, 12.44.
4.1.36. Isobutyl ({((2E)-3-{3-[(hydroxyamino)(imino)-
methyl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-
1-yl)phenyl]amino}sulfonyl)acetate (39). Compound 39
was synthesized from 35 and 2-methylpropanol accord-
ing to the same procedure as that for 36. Compound 39
was obtained as a white amorphous powder (70% yield):
1H NMR(DMSO- d6) d 0.92 (6H, d, J = 6.8Hz), 1.84–
1.96 (1H, m), 2.08–2.20 (1H, m), 2.25–2.37 (1H, m),
2.77 (3H, d, J = 4.4Hz), 3.05–3.17 (2H, m), 3.34–3.50
(4H, m), 3.65–3.74 (2H, m), 3.95 (2H, d, J = 6.8Hz),
4.29 (2H, s), 4.41 (2H, d, J = 5.3Hz), 6.43 (1H, dt,
J = 5.3, 16.2Hz), 6.55 (1H, d, J = 16.2Hz), 6.76 (2H,
d, J = 8.8Hz), 7.27 (2H, d, J = 8.8Hz), 7.53 (1H, t,
J = 7.3Hz), 7.58 (1H, d, J = 7.3Hz), 7.70 (1H, d,
J = 7.3Hz), 7.79 (1H, s), 8.92–9.21 (1H, br), 10.68–
10.80 (1H, br), 11.20–11.33 (1H, br); FAB MS m/e
(M+1)+ 558; Anal. Calcd for C28H39N5O5SÆ3.3HClÆ2.0-
H2O: C, 47.10; H, 6.54; N, 9.81; S, 4.49; Cl, 16.38.
Found: C, 47.21; H, 6.72; N, 9.61; S, 4.95; Cl, 16.21.
4.1.33. Methyl ({((2E)-3-{3-[(hydroxyamino)(imino)-
methyl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-
1-yl)phenyl]amino}sulfonyl)acetate (36). To a stirred
solution of 35 (100mg, 0.19mmol) in 10mL MeOH
was added 4N HCl 1,4-dioxane solution (10mL) and
stirred at ambient temperature for 12h. The reaction
mixture was concentrated in vacuo and 36 (73mg,
1
64%) was obtained as a white amorphous powder: H
NMR(DMSO- d6) d 2.08–2.50 (2H, m), 2.78 (2H, d,
J = 4.4Hz), 3.08–3.17 (2H, m), 3.36–3.50 (4H, m),
3.60–3.68 (2H, m), 4.29 (3H, s), 4.40 (2H, d,
J = 5.8Hz), 6.42 (1H, dt, J = 5.8, 16.2Hz), 6.55 (1H, d,
J = 16.2Hz), 6.75 (1H, d, J = 7.8Hz), 7.27 (2H, d,
J = 7.8Hz), 7.56 (1H, t, J = 7.8Hz), 7.59 (1H, d,
J = 7.8Hz), 7.68 (1H, d, J = 7.8Hz), 7.78 (1H, s),
8.80–9.50 (1H, br), 10.32–10.48 (1H, br), 11.02–11.30
(1H, br); FAB MS m/e (M+1)+ 516; Anal. Calcd for
C25H33N5O5SÆ2.9HClÆ2.0H2O: C, 45.63; H, 6.12; N,
10.65; S, 4.88; Cl, 15.64. Found: C, 45.49; H, 6.06; N,
10.37; S, 5.11; Cl, 15.32.
4.2. Biology
4.2.1. Chromogenic assay. The hydrolysis rates of syn-
thetic substrates were assayed by continuously measur-
ing absorbance at 405nm at 37°C with a microplate
reader (Model 3550, Bio-Rad, USA). Reaction mixtures
(125lL) were prepared in 96-well plates containing
chromogenic substrates and an inhibitor in either
0.05M Tris–HCl, pH8.4, 0.15M NaCl. Reactions were
initiated with a 25lL portion of the enzyme solution.
Enzymes and substrates were used as follows: factor
Xa and S-2222. The concentration of an inhibitor re-
quired to inhibit enzyme activity by 50% (IC50) was cal-
culated from dose–response curves in which the logit
transformation of residual activity was plotted against
the logarithm of inhibitor concentration.
4.1.34. Propyl ({((2E)-3-{3-[(hydroxyamino)(imino)-
methyl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-
1-yl)phenyl]amino}sulfonyl)acetate (37). Compound 37
was synthesized from 35 and 1-propanol according to
the same procedure as that for 36. Compound 37 was
obtained as a white amorphous powder (63% yield):
1H NMR(DMSO- d6) d 0.92 (3H, t, J = 7.3Hz), 1.58–
1.67 (2H, m), 2.08–2.34 (2H, m), 2.78 (3H, s), 3.02–
3.20 (2H, m), 3.26–3.40 (4H, m), 3.64–3.82 (2H, m),
4.12 (2H, t, J = 7.3Hz), 4.26 (2H, s), 4.40 (2H, d,
J = 5.9Hz), 6.26 (1H, dt, J = 5.9, 15.6Hz), 6.50 (1H, d,
J = 15.6Hz), 6.75 (2H, d, J = 8.8Hz), 7.27 (2H, d,
J = 8.8Hz), 7.44 (1H, t, J = 7.3Hz), 7.54–7.60 (2H, m),
7.75 (1H, s), 10.40–10.83 (3H, br); FAB MS m/e
(M+1)+ 544; Anal. Calcd for C27H37N5O5SÆ1.5HClÆ2.5-
H2O: C, 50.40; H, 6.81; N, 10.88; S, 4.98; Cl, 8.27.
Found: C, 50.51; H, 6.65; N, 10.57; S, 5.59; Cl, 8.37.
4.2.2. Plasma clotting time assays. Citrated blood sam-
ples from mice were collected. Platelet-poor plasma
was prepared by centrifugation at 3000rpm for 10min
and stored at ꢀ40°C until use. Plasma clotting times
were performed using a KC10A coagulometer (Amelung
Co., Lehbrinsweg, Germany) at 37°C. Prothrombin
time (PT) and activated partial thromboplastin time
(APTT) were measured using Orthobrain thromboplas-
tin and thrombofax (Ortho Diagnostic Systems Co.,
Tokyo, Japan), respectively. Coagulation times for each
test sample were compared with coagulation times meas-
ured using a distilled water control. The concentration
required to double the clotting time (CT2) was estimated
from each individual concentration–response curve.
Each measurement was performed three times, and rep-
resented as the mean value.
4.1.35. Isopropyl ({((2E)-3-{3-[(hydroxyamino)(imino)-
methyl]phenyl}prop-2-en-1-yl)[4-(4-methyl-1,4-diazepan-
1-yl)phenyl]amino}sulfonyl)acetate (38). Compound 38
was synthesized from 27 according to the same proce-
dure as that for 14. Compound 38 was obtained as a
white amorphous powder (58% yield): 1H NMR
(DMSO-d6) d 1.25 (6H, d, J = 6.3Hz), 2.08–2.16 (1H,
m), 2.25–2.34 (1H, m), 2.76 (3H, d, J = 4.9Hz), 3.04–
3.16 (2H, m), 3.35–3.44 (4H, m), 3.62–3.76 (2H, m),
4.22 (2H, s), 4.41 (2H, d, J = 5.4Hz), 4.98–5.04 (1H,
m), 6.42 (1H, dt, J = 5.4, 16.2Hz), 6.55 (1H, d,
J = 16.2Hz), 6.76 (2H, d, J = 8.8Hz), 7.27 (2H, d,
J = 8.8Hz), 7.53 (1H, t, J = 7.8Hz), 7.59 (1H, d,
4.2.3. Ex vivo studies. Male mice weighing 30–37g was
used in these studies. Fasted animals for overnight for

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H. Koshio et al. / Bioorg. Med. Chem. 12 (2004) 5415–5426
Y.; Hirayama, F.; Koshio, H.; Matsumoto, Y. Br. J.
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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,
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the oral studies were used. In mice, the test drug was dis-
solved in saline and administered to animals orally at
100mg/kg using a gastric tube. Citrated blood was col-
lected from the vena cava 1min after intravenous injec-
tion or 30min after oral administration. Platelet-poor
plasma was prepared by centrifugation for measurement
of PT or APTT. All data were expressed as relative fold
values, compared with the vehicle group.
5. (a) 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;
(b) Kawasaki, T.; Sato, K.; Sakai, Y.; Hirayama, F.;
Koshio, H.; Taniuchi, Y.; Matsumoto, Y. Thromb. Hae-
most. 1998, 79, 410; (c) Sato, K.; Taniuchi, Y.; Kawasaki,
T.; Hirayama, F.; Koshio, H.; Matsumoto, Y. Eur. J.
Pharmacol. 1998, 347, 231; (d) Sato, K.; Kawasaki, T.;
Hisamichi, N.; Taniuchi, Y.; Hirayama, F.; Koshio, H.;
Ichihara, M.; Matsumoto, Y. Eur. J. Pharmacol. 1998, 350,
87; (e) Sato, K.; Kaku, S.; Hirayama, F.; Koshio, H.;
Matsumoto, Y.; Kawasaki, T.; Iizumi, Y. Eur. J. Pharma-
col. 1998, 352, 59; (f) Sato, K.; Hisamichi, N.; Kawasaki,
T.; Hirayama, F.; Koshio, H.; Matsumoto, Y.; Iizumi, Y.
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Acknowledgements
The authors deeply acknowledge Dr. Minoru Okada for
useful discussion and helpful support for preparing this
manuscript, and are also grateful to the staff of Division
of Analytical Science Laboratories for elemental analy-
sis and spectral measurements.
References and notes
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