Design, synthesis and biological activity of YM-60828 derivatives. Part 2: Potent and orally-bioavailable factor Xa inhibitors based on benzothiadiazine-4-one template

Article abstract of DOI:10.1016/S0968-0896(02)00462-5

Compound YM-60828 was previously characterized in our laboratory as a potent, selective and orally-bioavailable Factor Xa (FXa) inhibitor. The L-shape conformation of this compound in the active site of FXa was recognized as an important factor in display

Full text of DOI:10.1016/S0968-0896(02)00462-5

Bioorganic & Medicinal Chemistry 11 (2003) 367–381
Design, Synthesis and Biological Activity of YM-60828
Derivatives. Part 2: Potent and Orally-Bioavailable Factor Xa
Inhibitors Based on Benzothiadiazine-4-one Template
Fukushi Hirayama,* Hiroyuki Koshio, Naoko Katayama, Tsukasa Ishihara,
Hiroyuki Kaizawa, Yuta Taniuchi, Kazuo Sato, Yumiko Sakai-Moritani,
Seiji Kaku, Hiroyuki Kurihara, Tomihisa Kawasaki, Yuzo Matsumoto,
Shuichi Sakamoto and Shin-ichi Tsukamoto
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
Received 16 July 2002; accepted 13 September 2002
Abstract—Compound YM-60828 was previously characterized in our laboratory as a potent, selective and orally-bioavailable
Factor Xa (FXa) inhibitor. The L-shape conformation of this compound in the active site of FXa was recognized as an important
factor in displaying its FXa inhibitory activity. This led to the exploration of conformationally restricted cyclic scaffolds bearing a
similar active conformation. The current study investigated a novel series of benzothiadiazine-4-one based compounds as FXa
inhibitors. Structure–activity relationship (SAR) investigations revealed some potent FXa inhibitors that were selected for further
in vitro and ex vivo anticoagulant studies. Among them, compound 6j (YM-169920) was proved to be most effective anticoagulant
in this series. The synthesis and SAR in addition to docking studies of this class of inhibitors are described.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
We have previously reported a series ofN-[(7-amidino-2-
naphthyl)methyl]aniline derivatives as potent and
orally-bioavailable FXa inhibitors.5 Furthermore, we
revealed compound YM-60828 and its mesylate salt
YM-75466 which displayed optimum properties, pos-
sessing potent efficacies for various thrombosis models
without side effect bleeding.4,6 Moreover, the docking
study of YM-60828 in FXa revealed that the active
conformation of YM-60828 was L-shaped and the
introduction of a rigid scaffolding which could adopt
the active conformation, such as naphthoanilide and
naphthalensulfonanilide templates, has led to discovery
of a series of YM-60828 derivatives with potent FXa
activities (Fig. 1a).7 Our design of potent and selective
FXa inhibitors was focused on developing molecules
with a similar conformationally restricted scaffold
which adopts an active L-shape conformation. The
docking study showed negligible interaction of the car-
boxyl moiety of YM-60828 to FXa.7 We subsequently
synthesized the derivatives of YM-60828 cyclized at the
sulfamoylacetic acid side chain and central benzene ring
(Fig. 1b). In this report we describe the SAR of the
cyclic derivatives of YM-60828 and discuss its con-
formations bound to FXa.
Factor Xa (FXa) is a serine protease which plays a
pivotal role in the sequence of blood coagulation events.
Intrinsic and extrinsic coagulation cascades intersect at
FXa, and activate prothrombin to generate thrombin
through proteolytic cleavage. Thrombin, in turn, pro-
motes blood clot formation by the conversion of fibri-
nogen to insoluble fibrin and activating platelets.
Although small molecule thrombin inhibitors have been
intensely investigated as a treatment for thromboem-
bolic disorders,1 only argatroban2 has been marketed as
a parenteral drug and none of the orally effective
thrombin inhibitors have been successfully developed.
Recently, a number of reports suggested that direct FXa
inhibitors were more suitable antithrombotic agents
compared to direct thrombin inhibitors, based on
abnormal bleeding side effects in the animal thrombotic
models.3,4 Therefore, exploration of FXa inhibitor is
recently quite attractive field for the discovery of
antithrombotic agents.
*Corresponding author. Tel.: +81-298-63-6712; fax: +81-298-52-
5378; e-mail: hirayaf@yamanouchi.co.jp
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00462-5

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F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
Figure 1.
Chemistry
10 via nucleophilic addition of sodium cyanide to the
methanesulfonate intermediate, followed by stepwise
reduction of cyano and nitro groups to afford diamine
12a. Alternatively, alcohol 9 was subjected to a Mitsu-
nobu reaction with phthalimide followed by hydrazine
deprotection and subsequent reduction of the nitro
group to yield diamine 12b. The benzothiadiazepine 13a
and benzothiadiazine 13b were each coupled with
3-((E)-3-hydroxypropenyl)benzonitrile by a Mitsunobu
reaction to afford cyanocinnamyl derivatives 5c and 5d,
respectively. Methyl substituted derivative 5e was pre-
pared by the reaction of 5d with methyl iodide under
basic conditions (K2CO3). Bis-amidines 6c–6e were
obtained from 5c–5e, respectively, in the same manner
as described for 6a and 6b.
The synthesis of benzodiazepine derivatives 6a and 6b
are shown in Scheme 1. Construction of the benzodia-
zepine skeletons 5a and 5b were accomplished by treat-
ment of anthranilic acid derivatives 4a and 4b with
bromoacetylbromide, followed by cyclization with
ammonia. Aniline 3 was prepared from phenol 1 and
alcohol 2 under Mitsunobu condensation conditions
(PPh3, DEAD, THF). The anthranilic acid derivatives
4a and 4b were synthesized by the condensation of
aldehydes 7-formylnaphthalene-2-carbonitrile5 and
(E)-3-cyanocinnamaldehyde5 respectively with 3. Ben-
zodiazepines 5a and 5b were subjected to the standard
Pinner reactions and aminations, followed by treatment
with ethyl acetimidate to give bis-amidines 6a and 6b,
respectively.
The benzothiadiazine-4-one derivatives 6f–6k were pre-
pared as shown in Scheme 3. All compounds were syn-
thesized via key sulfamide intermediates 14a–14f. A
general synthesis of this series of compounds was
exemplified by the preparation of compounds 6f and 6g.
The anthranilic acid derivatives 4a and 4b were reacted
with tert-butyl chlorosulfonylcarbamate5 in pyridine to
give sulfamide derivatives 14a and 14b, respectively. The
conversion of intermediates 14a and 14b to correspond-
ing benzothiadiazine-4-ones 15a and 15b was achieved
by TFA deprotection from the BOC protecting group
The preparative route to benzothiadiazepine (6c) and
benzothiadiazine (6d, 6e) derivatives are shown in
Scheme 2. Rings 13a and 13b were constructed from
diamine derivatives 12a and 12b by treatment with sul-
famide in pyridine under reflux. Compound 9 was pre-
pared by coupling phenol 7 with methanesulfonate 8
which was subsequently used as the common starting
material for the synthesis of the diamines 12a and 12b.
Alcohol 9 was converted to cyano-substituted derivative
Scheme 1. Synthesis of benzodiazepine derivatives: (a) PPh3, diethyl azodicarboxylate (DEAD), THF; (b) aldehydes, NaB(OAc)3H, AcOH, CH2Cl2;
(c) bromoacetylbromide, pyridine, diethylether; (d) NH3, MeOH; (e) HCl, EtOH; (f) NH4OAc, EtOH, MeOH; (g) ethyl acetimidate hydrochloride,
Et3N, EtOH.

F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
369
Scheme 2. Synthesis of benzothiadiazepine and benzothiadiazine derivatives: (a) K2CO3, DMF; (b) methanesulfonylchloride, Et3N, 1,2-dichloro-
.
ethane; (c) NaCN, DMF; (d) phthalimide, PPh3, DEAD, THF; (e) BH3–THF, THF, (f) Fe, NH4Cl, EtOH, H2O; (g) NH2NH2 H2O, EtOH; (h)
sulfamide, pyridine; (i) 3-((E)-3-Hydroxypropenyl)benzonitrile, PPh3, DEAD, THF; (j) MeI, K2CO3, CH3CN; (k) HCl, EtOH; (l) NH4OAc, EtOH;
(m) ethyl acetimidate hydrochloride, Et3N, EtOH.
followed by cyclization under basic conditions (NaOEt,
EtOH). Bis-amidines 6f and 6g were prepared using the
same procedure as described above. Phenylpropyl deri-
vative 6h was prepared through intermediate 14c which
was obtained following reduction of the phenylpropenyl
intermediate 14b. Compound 4c, an intermediate of
phenoxyethyl derivative 6i, was synthesized by the
sized and evaluated for its inhibitory activities against
FXa and selectivities against related serine proteases
thrombin and trypsin. As shown in Table 1, 6a dis-
played a moderate activity and selectivity for FXa
(IC50=24.0 nM, 29-fold against trypsin). These results
indicated that cyclization of YM-60828 at this position
was tolerable, and optimization of this series of com-
pound could lead to some novel series of potent and
selective FXa inhibitors. Since styrene analogue 6b,
which was a readily synthesized and accessible analogue
of 6a, retained the activity and selectivity
(IC50=27.4 nM, 39-fold against trypsin), we com-
menced optimization of the ring structure of 6b. Intro-
duction of the sulfonamide structure, which was also
contained in YM-60828, resulted in a decrease in FXa
inhibitory activity (6c). However, the six-membered ring
analogue 6d enhanced FXa potency. Methylation at the
ring nitrogen (6e) did not affect the activity. The most
potent derivative from this set was compound 6g con-
taining a benzothiadiazine-4-one scaffold, which showed
an 10- and 2-fold enhancement in FXa inhibitory activ-
ity (IC50=2.8 nM) compared to lead compounds 6b and
YM-60828, respectively.
reductive coupling of aniline
3
and phenoxy-
acetaldehyde 17 which was prepared by the oxidative
cleavage of diol 16.8 Different routes were employed to
synthesize phenylcarbamoylmethyl derivative 6j and its
methyl substituted analogue 6k. N-Methyl substituted
phenylcarbamoylmethyl intermediate 4d was readily
synthesized by the coupling of alkylbromide 21 with
aniline 3. However, the corresponding reaction for the
N-demethyl analogue provided a complex mixture.
Thus the trifluoroacetylated derivative of 3 was alky-
lated with benzyl 2-bromoacetate in the presence of
sodium hydride to afford intermediate 18. Reductive
cleavage of the benzyl ester of 18 followed by con-
densation of aminobenzonitrile gave phenylcarba-
moylmethyl intermediate 19. 6j and 6k were obtained
readily from 19 and 4d respectively by an identical pro-
cedure to that described above.
Further modification of the styrene moiety led to iden-
tification of other dispensable P1 groups as shown in
Table 2. Replacement of the styrene with the initial
naphthalene structure retained the activity (6f,
IC50=2.1 nM), however conformationally unrestricted
phenethyl (6h) and phenoxymethyl (6i) derivatives
resulted in a decreased FXa inhibitory activity. The
anilide derivative 6j showed potent activity
(IC50=2.5 nM) equal to that of styrene and naphthalene
derivatives 6g and 6f and exhibited an improved selec-
tivity against trypsin (248-fold for 6j vs 95- and 33-fold
for 6g and 6f). In contrast, the same translation was not
tolerated for the uncyclized derivatives of YM-60828.5
N-Methylation of anilide derivative 6j resulted in a drop
in potency (6k).
Results and Discussion
As a preliminary investigation, cyclic templates which
could potentially exhibit the L-shaped conformation as
observed for modeling study of YM-608287 in the active
site of FXa, were initially screened in the Cambridge
Structural Database based on the distance and the angle
between the two benzene rings (A and B rings). Among
this database search results, a benzodiazepine derivative
BEDZPN10, which possessed a similar L-shape con-
formation to YM-60828, as shown in Fig. 2, was selec-
ted as an appropriate scaffold. Next, benzodiazepine
derivative 6a designed from BEDZPN10 was synthe-

Scheme 3. Synthesis of benzothiadiazine-4-one derivatives: (a) tert-butyl chlorosulfonylcarbamate, pyridine; (b) trifluoroacetic acid, CH2Cl2; (c) NaOEt, EtOH; (d) HCl, EtOH; (e) NH4OAc, EtOH; (f)
ethyl acetimidate hydrochloride, Et3N, EtOH; (g) H2, PdO/BaSO4, EtOH; (h) NaIO4, CH2Cl2, H2O; (i) 3, NaB(OAc)3H, AcOH, 1,2-dichloroethane; (j) trifluoroacetic anhydride, pyridine, 1,2-dichlor-
oethane; (k) NaH, benzyl 2-bromoacetate, DMF; (l) H2, Pd/C, MeOH; (m) 3-aminobenzonitrile, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole DMF; (n) K2CO3,
MeOH, H2O; (o) bromoacetylbromide, NaHCO3, EtOAc, H2O; (p) 3, K2CO3, CH3CN.

F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
371
Figure 2. Overlay of FXa-binding conformation of YM-60828 (with white carbons) and crystal structure of BENZEPIN10 (brown).
Table 2. Replacement of styrylamidine
Table 1. In vitro activity of inhibitors based on benzene fused cyclic
templates
Compd
R
IC50 (nM)a
Factor Xa Thrombin Trypsin
Compd.
R
IC50 (nM)a
6a
6b
24.0
27.4
>100,000
693.3
Factor Xa
2.8
Thrombin
Trypsin
6g
6f
>100,000
266.2
>100,000 1075.4
>100,000 1256.3
2.1
74.1
137.6
2.5
>100,000
>100,000
>100,000
>100,000
>100,000
68.4
7169.6
13,699.5
619.3
6c
6d
6e
6g
55.9
16.0
16.9
2.8
6h
6i
>100,000
>100,000
>100,000
>100,000
279.3
212.9
266.2
159.0
6j
6k
456.1
5002.7
YM-60828
6.0
aHuman purified enzymes were used. IC50 values represent the aver-
aged of three or more determinations with the average standard error
of the mean <25%.
aRefer to Table 1.

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F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
The binding models of compounds 6a and 6f in the active
site of FXa was proposed on the basis of X-ray crystal-
lographic analysis of these compounds complexed to the
related enzyme trypsin (Fig. 3). This study revealed that
naphthalene and piperidine moieties of both compounds
docked into S1 and S4 specific pockets, respectively, similar
to that of YM-60828. Interestingly, however, the con-
formations of the benzene fused ring and the piperidine ring
of compound 6f were different from that of YM-60828.
sulfamoylacetic acid of YM-60828, and the carbonyl
oxygen attached to the aniline moiety forms a hydrogen
bond to the side chain amide nitrogen of Gln-192.
Moreover, a further carbonyl oxygen at the diazepine
ring is engaged in hydrogen bonding to the side chain
phenol oxygen of Tyr-99. The piperidine ring of 6a also
has the same conformation as that of YM-60828 and
the nitrogen of the acetimidoyl group forms a hydrogen
bond to the carbonyl oxygen of Thr-98 and Ile-175
through a bridging water molecule. On the other hand,
unlike the sulfamoylacetic acid of YM-60828, the thia-
diazine ring of 6f is distributed along the enzyme surface,
The diazepine ring of 6a directs solvent away from the
enzyme following a similar trend to that shown by the
Figure 3. Stereoviews of the binding models in FXa: (A) overlay of inhibitor 6a (with brown carbons) and YM-60828 (white); (B) overlay of
inhibitor 6f (with brown carbons) and YM-60828 (white).

F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
Table 3. Anticoagulant activities in vitro and ex vivo
373
a
Compd
CT2 (mM)
PT/control PTc
PTb
Mice
Squirrel monkeys
0.5 h
2.0 h
1.0 h
4.0 h
6f
6g
0.081
0.085
0.12
0.21d
2.6
3.7
4.0
2.6
2.0
1.9
3.3
1.7
3.7
7.1
8.3
4.3e
1.8
1.6
3.5
2.0e
6j
YM-60828
aValues represent the concentration required to double clotting time and represent the average of four determinations with the average standard error
of the mean <10%.
bProthrombin time using human plasma.
cThe relative prothrombin time compared with that measured using normal plasma were determined in blood samples taken at the indicated time
points after oral administration at 100 mg/kg in mice, and at 3 mg/kg in squirrel monkeys (n=3).
dn=3.
eThe data for YM-75466 (methansulfonate salt of YM-60828).
and the carbonyl oxygen of the sulfon forms a novel
hydrogen bond to the backbone amide nitrogen of Gly-
218. Further, the conformational change of the piper-
idine ring in 6f affords an additional hydrogen bond
between the piperidine nitrogen and the backbone car-
bonyl oxygen of Glu-97, and the water-mediated
hydrogen bonds of the nitrogen of acetimidoyl group, as
mentioned above, are retained. These different binding
conformations might, in part, explain one order of
magnitude difference of potencies observed for 6a and
6f.
Experimental
Chemistry
1H NMR spectra were measured with a JEOL EX90,
EX400 or GX500 spectrometer; chemical shifts are
expressed in d units using tetramethylsilane as the stan-
dard (in NMR description, s=singlet, d=doublet,
t=triplet, m=multiplet, and br=broad peak). Mass
spectra were recorded with a Hitachi M-80 or JEOL
JMS-DX300 spectrometer. Melting points were mea-
sured with a Yanaco MP-500D melting point apparatus
without correction. ODS column chromatography was
performed on YMC gel (ODS-A 120-230/70).
Potent FXa inhibitors 6f, 6g and 6j were further eval-
uated for both their anticoagulant activity in vitro and
their oral anticoagulant efficacy ex vivo based on the
prolongation of prothrombin time (PT). All of these
compounds showed significantly potent anticoagulant
activities in vitro as shown in Table 3 (CT2, PT=0.081–
0.12 mM), reflecting their potent FXa inhibitory activ-
ities. In the ex vivo oral test, benzamidine derivatives 6g
and 6j demonstrated potent oral efficacies in both mice
and squirrel monkeys. In particular, compound 6j
(YM-169920) effectively prolonged the PT by more than
3-fold, at all time points investigated after oral admin-
istration, in both mice and squirrel monkeys.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]anthra-
nilate (3). To a stirred solution of methyl 2-amino-5-
hydroxybenzoate 1 (0.83 g, 5.0 mmol) and tert-butyl
4-hydroxypiperidine-1-carboxylate 2 (0.86 g, 5.5 mmol)
in tetrahydrofuran (THF) (10 mL) at ambient tempera-
ture was added triphenylphosphine (PPh3) (1.57 mg,
6.0 mmol), and diethyl azodicarboxylate (DEAD)
(0.94 mL, 6.0 mmol). After stirring at ambient tempera-
ture for 4 days, the reaction mixture was concentrated
in vacuo. The residue was dissolved in ethyl acetate
(EtOAc) and the solution was washed with saturated
aqueous NaHCO3 and 10% aqueous citric acid, dried
over Na2SO4 and then concentrated in vacuo. The
resulting residue was chromatographed on silica gel
eluting with EtOAc/n-hexane (Hex) (15:85) to ꢀgive 3
Conclusion
1
We have designed and synthesized a novel series of FXa
inhibitors based on a benzothiadiazine-4-one template.
The docking study of the series of compound 6f in the
active site of FXa revealed that the conformation at the
benzene fused ring and piperidine ring differed from
that of YM-60828. This conformational change affor-
ded novel hydrogen bonding of inhibitor 6f to FXa. In
this series of compound, three derivatives, 6f, 6g and
6j, showed potent FXa inhibitory activities and anti-
coagulant activities in vitro. Among them, compound
6j (YM-169920) exhibited excellent anticoagulant effi-
cacies in both mice and squirrel monkeys after oral
administration ex vivo. Further optimization studies
based on compound YM-60828 will be reported in
future publications.
(716 mg, 41%) as a pale brown solid: mp 96–97 C; H
NMR (CDCl3) d 1.47 (9H, s), 1.63–1.76 (2H, m),
1.80–1.93 (2H, m), 3.22–3.32 (2H, m), 3.62–3.75 (2H,
m), 3.87 (3H, s), 4.23–4.31 (1H, m), 5.47 (2H, bs), 6.63
(1H, d, J=8.5 Hz), 6.96 (1H, dd, J=3.1, 8.5 Hz), 7.41
(1H, d, J=3.1 Hz); FAB MS m/e (M)+ 350.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-[(7-
cyano-2-naphthyl)methyl]anthranilate (4a). To a stirred
solution of
3
(2.70 g, 7.7 mmol) and 7-for-
mylnaphthalene-2-carbonitril5 (1.39 g, 7.7 mmol) in
CH2Cl2 (40 mL) and acetic acid (AcOH) (11 mL) at
ambient temperature was added sodium triacetoxyboro-
hydride (3.27 g 15.4 mmol). After 14 h, the reaction
mixture was washed with saturated aqueous NaHCO3

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F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
and H2O. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The resulting residue was chro-
matographed on silica gel eluting with EtOAc/Hex
(10:90–80:20) to give 4a (3.93 g, 99%) as a pale yellow
General procedure for synthesis of bis-amidine
derivatives 6
7-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(7-amidino-2-naph-
thyl)methyl]-1H-1,4-benzodiazepine-2,5(3H,4H)-dione
(6a). HCl gas was bubbled through a solution of 5a
(0.47 g, 0.86 mmol) in ethanol (EtOH) (30 mL) under
À20 ꢀC for 20 min. The mixture was allowed to stir for
29 h at 5 ꢀC, and then concentrated in vacuo. To the
crude imidate dissolved in EtOH (20 mL) and MeOH
(30 mL) was added ammonium acetate (1.99 g,
25.8 mmol) at ambient temperature. The reaction mix-
ture was stirred at ambient temperature for 72 h and
concentrated in vacuo. The resulting residue was chro-
matographed on ODS gel eluting with MeOH/H2O
(0:100–2:98). MeOH was removed in vacuo, and the
aqueous solution was lyophilized after acidification with
1 N HCl. 7-[(4-piperidyl)oxy]-1-[(7-amidino-2-naph-
thyl)methyl]-1H-1,4-benzodiazepine-2,5(3H,4H)-dione
(369 mg, 81%) was obtained as a white amorphous
powder: 1H NMR (DMSO-d6) d 1.74–1.86 (2H, m),
2.01–2.09 (2H, m), 2.98–3.07 (2H, m), 3.14–3.22 (2H,
m), 3.55–3.64 (1H, m), 3.87–3.96 (1H, m), 4.62–4.70
(1H, m), 5.17 (1H, d, J=16.2 Hz), 5.41 (1H, d,
J=16.2 Hz), 7.15 (1H, dd, J=3.1, 9.2 Hz), 7.19 (1H, d,
J=3.1 Hz), 7.38–7.48, (2H, m), 7.79 (1H, dd, J=1.8,
8.6 Hz), 7.83 (1H, s), 7.99 (1H, d, J=8.5 Hz), 8.10 (1H,
d, J=9.2 Hz), 8.36 (1H s), 8.81–8.87 (1H, m), 9.22 (1H,
br-s), 9.49 (2H, br-s); FAB MS m/e (M+1)+ 458.
solid: mp 125–126 ꢀC; H NMR (CDCl3) d 1.46 (9H, s),
1
1.61–1.73 (2H, m), 1.79–1.91 (2H, m), 3.21–3.31 (2H,
m), 3.61–3.73 (2H, m), 3.89 (3H, s), 4.21–4.30 (1H, m),
4.62 (2H, s), 6.55 (1H, d, J=9.1 Hz), 6.95 (1H, dd,
J=3.1, 9.1 Hz), 7.52 (1H, d, J=3.0 Hz), 7.58 (1H, d,
J=8.2 Hz), 7.64 (1H, d, J=8.5 Hz), 7.83 (1H, s), 7.87
(1H, d, J=8.5 Hz), 7.89 (1H, d, J=8.5 Hz), 8.05 (1H,
bs), 8.18 (1H, s); FAB MS m/e (M)+ 515.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-
[(E)-3-cyanocinnamyl]anthranilate (4b). Compound 4b
was synthesized from 3 and (E)-3-cyanocinnamalde-
hyde5 according to the same procedure as that for 4a.
Compound 4b was obtained as a brown amorphous
1
powder (99% yield): H NMR (CDCl3) d 1.47 (9H, s),
1.62–1.77 (2H, m), 1.82–1.94 (2H, m), 3.23–3.35 (2H,
m), 3.65–3.77 (2H, m), 3.88 (3H, s), 4.05 (2H, d,
J=5.0 Hz), 4.22–4.32 (1H, m), 6.37 (1H, dt, J=5.0,
16.1 Hz), 6.58 (1H, d, J=16.1 Hz), 6.65 (1H, d,
J=9.1 Hz), 7.04 (1H, dd, J=2.7, 9.1 Hz), 7.39 (1H, t,
J=7.8 Hz), 7.45–7.83 (5H, m); FAB MS m/e (M)+
491.
7-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-1-[(7-cyano-2-
naphthyl)methyl] - 1H - 1,4 - benzodiazepine - 2,5(3H,4H) -
dione (5a). To a stirred solution of 4a (0.90 g, 1.8 mmol)
in diethylether (20 mL) at ambient temperature was
added bromoacetylbromide (0.23 mL, 2.6 mmol) and
pyridine (0.21 mL, 2.6 mmol). After 1 h, the reaction
mixture was filtered. The filtrate was evaporated and
dried in vacuo. The resulting residue was dissolved in
methanol and ammonia gas was bubbled through the
solution at ambient temperature until the solution was
saturated. The reaction mixture was stirred for 5 h at
ambient temperature, and evaporated in vacuo. The
resulting residue was chromatographed on silica gel
eluting with MeOH/CHCl3 (0:100–2:98) to give 5a
(885 mg, 94%) as a white amorphous powder: 1H NMR
(CDCl3) d 1.46 (9H, s), 1.63–1.76 (2H, m), 1.83–1.96
(2H, m), 3.25–3.35 (2H, m), 3.60–3.73 (2H, m), 3.79–
4.10 (2H, m), 4.44–4.51 (1H, m), 5.13 (1H, d,
J=15.8 Hz), 5.34 (1H, d, J=15.8 Hz), 6.69 (1H, t,
J=6.4 Hz), 6.99 (1H, dd, J=3.1, 9.1 Hz), 7.17 (1H, d,
J=9.1 Hz), 7.32 (1H, d, J=3.1 Hz), 7.44 (1H, d,
J=8.5 Hz), 7.58 (1H, d, J=8.5 Hz), 7.66 (1H, s), 7.83
(1H, d, J=8.5 Hz), 7.86 (1H, d, J=8.5 Hz), 8.14 (1H, s);
FAB MS m/e (M)+ 541.
To a stirred solution of the mono-amidine intermediate
(0.30 g, 0.56 mmol) in EtOH (4 mL) at ambient tem-
perature was added ethyl acetimidate hydrochloride
(0.69 g, 5.6 mmol) and triethylamine (Et3N) (0.78 mL,
5.6 mmol). The mixture was allowed to stir for 14 h at
ambient temperature, and then concentrated in vacuo.
The resulting residue was chromatographed on ODS gel
eluting with MeOH/H2O (0:100–2:98). MeOH was
removed in vacuo, and the aqueous solution was lyo-
philized after acidification with 1 N HCl. 6a (0.17 g
1
46%) was obtained as a white amorphous powder: H
NMR (DMSO-d6) d 1.63–1.80 (2H, m), 1.93–2.09 (2H,
m), 2.28 (3H, s), 3.45–4.00 (6H, m), 4.67–4.78 (1H, m),
5.10–5.47 (2H, m), 7.15 (1H, dd, J=3.1, 9.2 Hz), 7.20
(1H, d, J=3.1 Hz), 7.42 (1H, d, J=9.2 Hz), 7.46 (1H, d,
J=8.6 Hz), 7.80 (1H, d, J=8.6 Hz), 7.83 (1H, s), 8.0
(1H, d, J=8.6 Hz), 8.10 (1H, d, J=8.6 Hz), 8.37 (1H, s),
8.77 (1H, s), 8.85 (1H, t, J=6.1 Hz), 9.17–9.36 (3H, m),
9.50 (2H, s); FAB MS m/e (M+1)+ 499. Anal. calcd for
.
.
C28H30N6O3 2.1HCl 3.3H2O: C, 53.00; H, 6.15; N,
13.24; Cl, 11.73. Found: C, 52.79; H, 5.92; N, 13.17; Cl,
11.78.
7-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-1-[(E)-3-cya-
nocinnamyl]-1H-1,4-benzodiazepine-2,5(3H,4H)-dione
(5b). Compound 5b was synthesized from 4b according
to the same procedure as that for 5a. Compound 5b was
obtained as a pale yellow amorphous powder (55%
7-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(E)-3-amidinocinna-
myl] - 1H - 1,4 - benzodiazepine - 2,5(3H,4H) - dione (6b).
Compound 6b was synthesized from 5b according to
the same procedure as that for 6a. Compound 6b was
obtained as a white amorphous powder (33% yield). 1H
NMR (DMSO-d6) d 1.64–1.85 (2H, m), 1.94–2.13 (2H,
m) 2.31 (3H, s), 3.45–3.94 (6H, m), 4.53–4.70 (2H, m),
4.73–4.82 (1H, m), 6.48 (1H, dt, J=4.8, 16.2 Hz), 6.58
(1H, d, J=16.2 Hz), 7.21–7.27 (2H, m), 7.44–7.50 (1H,
m), 7.52–7.60 (1H, m), 7.66–7.78 (2H, m), 7.87–7.93
1
yield): H NMR (DMSO-d6) d 1.40 (9H, s), 1.43–1.59
(2H, m), 1.82–1.95 (2H, m), 3.01–3.25 (2H, m), 3.44–
3.90 (4H, m), 4.56–4.65 (3H, m), 6.46–6.50 (2H, m),
7.16–7.22 (2H, m), 7.39–7.44 (1H, m), 7.52 (1H, t,
J=7.8 Hz), 7.62–7.73 (2H, m), 7.84–7.88 (1H, m), 8.79
(1H, t, J=6.1 Hz); FAB MS m/e (M)À 515.

F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
375
(1H, m), 8.80–8.96 (2H, m), 9.32 (2H, s), 9.40–9.50 (3H,
m); FAB MS m/e (M+1)+ 475. Anal. calcd for
C26H30N6O3 2.4HCl 3.1H2O: C, 50.54; H, 6.30; N,
13.60; Cl, 13.77. Found: C, 50.87; H, 6.85; N, 13.73; Cl,
13.80.
12.4 mmol). After stirring at ambient temperature for
4 h, the reaction mixture was concentrated in vacuo.
H2O was added to the residue and the solution was
extracted with diethylether. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The resulting
residue was chromatographed on silica gel eluting with
EtOAc/Hex (20:80–25:75) to give 11 (4.50 g, 83%) as a
.
.
tert-Butyl 4-[(3-hydroxymethyl-4-nitro)phenoxy]piperidine-
1-carboxylate (9). To a stirred solution of 3-hydroxy-
methyl-4-nitrophenol 7 (4.50 g, 26.6 mmol) in N,N-di-
methylformamide (DMF) (40 mL) was added potassium
carbonate (5.70 g, 41.2 mmol), and tert-butyl 4-
methanesulfonyloxypiperidine-1-carboxylate 8 (11.13 g,
39.8 mmol). After stirring at 100 ꢀC for 2 h, the reaction
mixture was cooled to ambient temperature and then
concentrated in vacuo. The residue was dissolved in
EtOAc and washed with 0.2 N aqueous NaOH, 10%
aqueous citric acid. The organic layer was dried over
Na2SO4 and concentrated in vacuo. The resulting resi-
due was chromatographed on silica gel eluting with
EtOAc/Hex (17:83) to give a solid. The solid was crys-
tallized from toluene to give 9 (4.20 g, 45%) as a white
yellow solid: mp 122–123 ꢀC; H NMR (CDCl3) d 1.44
1
(9H, s), 1.62–1.73 (2H, m), 1.78–1.91 (2H, m), 3.20–3.29
(2H, m), 3.58–3.70 (2H, m), 4.42–4.50 (1H, m), 5.33
(2H, s), 6.61 (1H, d, J=2.5 Hz), 6.85 (1H, dd, J=2.5,
9.3 Hz), 7.76–7.84 (2H, m), 7.89–7.96 (2H, m), 8.20 (1H,
d, J=9.3 Hz); FAB MS m/e (M+1)+ 482.
tert-Butyl 4-{[4-amino-3-(2-aminoethyl)]phenoxy}piperi-
dine-1-carboxylate (12a). To a stirred solution of 10
(7.71 g, 21.4 mmol) in THF (120 mL) was added 1.0 M
solution of boran-THF complex in THF (85.4 mL,
85.4 mmol) and the mixture was refluxed for 2 h. After
cooling, MeOH (24 mL) was added to the reaction mix-
ture and it was stirred for 10 min. Then the reaction
mixture was concentrated in vacuo. The resulting resi-
due was dissolved in 15% aqueous citric acid (200 mL)
and EtOAc (50 mL). After stirring for 10 min at ambient
temperature, the solution was made basic with 1 N
aqueous NaOH and extracted with AcOEt. It was dried
over MgSO4 and concentrated in vacuo to give tert-
butyl 4-{[3-(2-aminoethyl)-4-nitro]phenoxy}piperidine-
solid: mp 141–142 ꢀC; H NMR (CDCl3) d 1.47 (9H, s),
1
1.71–1.85 (2H, m), 1.89–2.05 (2H, m), 2.56 (1H, t,
J=6.4 Hz), 3.33–3.47 (2H, m), 3.62–3.75 (2H, m), 4.59–
4.70 (1H, m), 5.00 (2H, d, J=6.4 Hz), 6.89 (1H, dd,
J=2.4, 8.3 Hz), 7.23 (1H, d, J=2.4 Hz), 8.18 (1H, d,
J=8.3 Hz); FAB MS m/e (M+1)+ 353.
1
tert-Butyl 4-[(3-cyanomethyl-4-nitro)phenoxy]piperidine-
1-carboxylate (10). To a stirred solution of 9 (2.10 g,
5.97 mmol) in 1,2-dichloroethane (20 mL) at 3 ꢀC was
added Et3N (0.92 mL, 6.57 mmol) and methanesulfo-
nylchloride (0.49 mL, 6.27 mmol). After stirring at
ambient temperature for 1 h, the reaction mixture was
washed with 10% aqueous citric acid, saturated aqu-
eous NaHCO3 and dried over Na2SO4. The solution
was concentrated in vacuo to give tert-butyl 4-[(3-
methanesulfonyloxymethyl-4-nitro)phenoxy]piperidine-
1-carboxylate (2.50 g, 97%) as a brown solid which was
used without further purification: FAB MS m/e
(M+1)+ 431.
1-carboxylate (6.69 g, 86%). H NMR (CDCl3) d 1.47
(9H, s), 1.65–1.84 (2H, m), 1.88–2.06 (4H, m), 3.07–3.22
(2H, m), 3.29–3.43 (2H, m), 3.62–3.76 (2H, m), 4.54–
4.66 (1H, m), 6.79–6.90 (2H, m), 8.06 (1H, d,
J=8.7 Hz); FAB MS m/e (M+1)+ 366.
To a stirred solution of the resulting product above
(2.19 g, 6.0 mmol) in EtOH (30 mL) and H2O (10 mL) at
ambient temperature was added ammonium chloride
(0.16 g, 3 mmol) and iron powder (3.35 g, 60 mmol). The
mixture was refluxed for 40 min. After cooling, the
reaction mixture was filtered through a pad of Celite
and concentrated in vacuo. The resulting residue was
dissolved in EtOAc, and the solution was washed with
1 N aqueous NaOH. It was dried over MgSO4 and
concentrated. The crude product mixture was chroma-
tographed on ODS gel eluting with MeOH/H2O (50:50–
60:40) to give 12a (0.80 g, 40%) as a brown viscous oil.
1H NMR (DMSO-d6) d 1.46 (9H, s), 1.65–1.76 (2H, m),
1.83–1.93 (2H, m), 2.33 (4H, br-s), 2.67 (2H, t,
J=6.8 Hz), 3.00 (2H, t, J=6.8 Hz), 3.22–3.31 (2H, m),
3.63–3.75 (2H, m), 4.23–4.30 (1H, m), 6.59–6.68 (3H,
m); FAB MS m/e (M+1)+ 336.
To a stirred solution of sodium cyanide (6.32 g,
129 mmol) in DMF (500 mL) at 3 ꢀC was added the
solution of resulting product above (5.56 g, 12.9 mmol)
in DMF (200 mL) dropwise. After stirring at 3 ꢀC for
1.5 h, the reaction mixture was diluted with H2O and
extructed with diethylether. The organic solution was
dried over Na2SO4, and concentrated in vacuo. The
resulting residue was chromatographed on silica gel
eluting with EtOAc/toluene (9:91) to give 10 (3.92 g,
ꢀ
1
84%) as a pale yellow solid: mp 121–122 C; H NMR
(CDCl3) d 1.48 (9H, s), 1.72–1.86 (2H, m), 1.90–2.03
(2H, m), 3.34–3.46 (2H, m), 3.63–3.76 (2H, m), 4.25
(2H,s), 4.60–4.69 (1H, m), 6.97 (1H, dd, J=2.4, 8.8 Hz),
7.20 (1H, d, J=2.4 Hz), 8.25 (1H, d, J=8.8 Hz); FAB
MS m/e (M+1)+ 362.
tert-Butyl 4-[(4-amino-3-aminomethyl)phenoxy]piperidine-
1-carboxylate (12b). To a stirred solution of 11 (3.79 g,
7.88 mmol) in EtOH (60 mL) was added hydrazine
monohydrate (1.91 mL, 39.4 mmol) and the mixture was
heated at 50 ꢀC for 17 h. After cooling, the reaction
mixture was filtered and concentrated in vacuo. Result-
ing residue was dissolved in CHCl3 and the solution was
washed with saturated aqueous NaHCO3, dried over
MgSO4 and concentrated in vacuo to give tert-Butyl 4-
[(3 - aminomethyl - 4 - nitro)phenoxy]piperidine - 1 - carb-
tert-Butyl
4-[(3-phthalimidomethyl-4-nitro)phenoxy]pi-
peridine-1-carboxylate (11). To a stirred solution of 9
(3.97 g, 11.3 mmol) and phthalimide (1.82 g, 12.4 mmol)
in THF (50 mL) at ambient temperature was added
PPh3 (3.25 g, 12.4 mmol) and DEAD (1.95 mL,
1
oxylate (2.74 g, 99%): H NMR (CDCl3) d 1.48 (9H, s),

376
F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
1.70–1.85 (4H, m), 1.89–2.20 (2H, m), 3.32–3.45 (2H,
m), 3.62–3.76 (2H, m), 4.15 (2H, s), 4.62–4.67 (1H, m),
6.85 (1H, dd, J=2.4, 9.0 Hz), 7.12 (1H, d, J=2.4 Hz),
8.11 (1H, d, J=9.0 Hz), FAB MS m/e (M+1)+ 352.
m), 7.69 (1H, d, J=7.6 Hz), 7.76 (1H, d, J=7.6 Hz),
7.94 (1H, s); FAB MS m/e (MÀ1)À 537.
6-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-1-[(E)-3-cya-
nocinnamyl]-3,4-dihydro-1H-2,1,3-benzothiadiazine 2,2-
dioxide (5d). Compound 5d was synthesized from 13b
according to the same procedure as that for 5c. Com-
pound 5d was obtained as a pale yellow amorphous
Compound 12b was synthesized from the above inter-
mediate according to the same procedure as that for
12a. Compound 12b was obtained as a brown viscous
1
1
oil (57% yield): H NMR (CDCl3) d 1.46 (9H, s), 1.62–
powder (50% yield): H NMR (DMSO-d6) d 1.40 (9H,
1.76 (2H, m), 1.80–1.93 (2H, m), 3.21–3.32 (2H, m),
3.65–3.76 (2H, m), 3.84 (2H, s), 4.20–4.31 (1H, m),
6.57–6.63 (1H, m), 6.66–6.72 (2H, m); FAB MS m/e
(M+1)+ 322.
s), 1.40–2.05 (2H, m), 1.77–1.92 (2H, m), 3.08–3.22 (2H,
m), 3.59–3.69 (2H, m), 4.40–4.56 (5H, m), 6.55 (1H, dt,
J=5.2, 15.6 Hz), 6.67 (1H, d, J=15.6 Hz), 6.84 (1H, d,
J=2.8 Hz), 6.88 (1H, dd, J=2.8, 8.8 Hz), 6.97 (1H, d,
J=8.8 Hz), 7.52 (1H, t, J=8.0 Hz), 7.61–7.75 (3H, m),
7.90 (1H, s) ; FAB MS m/e (M)+ 524.
7-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-1,3,4,5-tetra-
hydro-2,1,3-benzothiadiazepine 2,2-dioxide (13a). To a
stirred solution of 12a (0.77 g, 2.3 mmol) in pyridine
(15 mL) was added sulfamide (1.54 g, 16.0 mmol) and
the mixture was refluxed for 2 h. After cooling, the
reaction mixture was concentrated in vacuo. The resi-
dual pyridine was removed by adding toluene and then
evaporating to dryness. The residue was dissolved in
CHCl3 and the solution was washed with H2O, dried
over Na2SO4 and concentrated in vacuo. The resulting
residue was crystallized from benzene to give 13a
(0.54 g, 60%) as a white solid: mp 165–166 ꢀC; 1H NMR
(CDCl3) d 1.47 (9H, s), 1.67–1.80 (2H, m), 1.83–1.96
(2H, m), 2.95–3.03 (2H, m), 3.28–3.39 (2H, m), 3.44–
3.53 (2H, m), 3.62–3.73 (2H, m), 4.37–4.46 (1H, m),
4.48–4.57 (1H, m), 6.32 (1H, s), 6.69 (1H, d, J=2.7 Hz),
6.75 (1H, dd, J=2.7, 8.4 Hz), 7.03 (1H, d, J=8.4 Hz);
FAB MS m/e (M)+ 397.
6-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-1-[(E)-3-cya-
nocinnamyl]-3-methyl-3,4-dihydro-1H-2,1,3-benzothiadia-
zine 2,2-dioxide (5e). To a stirred solution of 5d (0.43 g,
0.82 mmol) in CH3CN (10 mL) at ambient temperature
was added K2CO3 (0.34 g, 2.46 mmol) and methyliodide
(0.23 g, 1.63 mmol). After stirring at ambient tempera-
ture for 22 h, the reaction mixture was filtered and dried
in vacuo. The resulting residue was dissolved in EtOAc
and the solution was washed with H2O, dried over
Na2SO4 and concentrated in vacuo to give 5e (446 mg)
as a pale yellow amorphous powder which was used
1
without further purification: H NMR (CDCl3) d 1.46
(9H, s), 1.63–1.76 (2H, m), 1.81–1.93 (2H, m), 2.79 (3H,
s), 3.22–3.37 (2H,m), 3.62–3.73 (2H, m), 4.32–4.41 (1H,
m), 4.57–4.62 (4H, m), 6.36 (1H, dt, J=5.4, 15.6 Hz),
6.64–6.71 (2H, m), 6.79–6.87 (2H, m), 7.41 (1H, t,
J=7.3 Hz), 7.51 (1H, d, J=7.3 Hz), 7.57 (1H, d,
J=7.3 Hz), 7.63 (1H, s); FAB MS m/e (M)+ 538.
6-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-3,4-dihydro-
1H-2,1,3-benzothiadiazine 2,2-dioxide (13b). Compound
13b was synthesized from 12b according to the same
procedure as that for 13a. Compound 13b was
obtained as a pale brown solid (83% yield): mp 230–
7-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(E)-3-amidinocinna-
myl]-1,3,4,5-tetrahydro-2,1,3-benzothiadiazepine 2,2-di-
oxide (6c). Compound 6c was synthesized from 5c
according to the same procedure as that for 6a. Com-
pound 6c was obtained as a white amorphous powder
(57% yield): 1H NMR (DMSO-d6) d 1.62–1.80 (2H, m),
1.97–2.10 (2H, m), 2.29 (3H, s), 2.85–2.96 (2H, m),
3.14–3.23 (2H, m), 3.43–3.58 (2H, m), 3.61–3.87 (2H, m),
4.29 (2H, d, J=6.4 Hz), 4.61–4.69 (1H, m), 6.52 (1H, d,
J=15.7 Hz), 6.66 (1H, dt, J=6.4, 15.7 Hz), 6.79–6.80
(2H, m), 7.17 (1H, d, J=8.3 Hz), 7.56 (1H, t, J=7.8 Hz),
7.64 (1H, t, J=6.8 Hz), 7.71 (1H, d, J=7.8 Hz), 7.75 (1H,
d, J=7.8Hz), 7.97 (1H, s), 8.82 (1H, s), 9.25 (2H, s), 9.36
(1H, s), 9.45 (2H, s); FAB MS m/e (M+1)+ 497. Anal.
ꢀ
1
231 C; H NMR (DMSO-d6) d 1.40 (9H, s), 1.38–1.55
(2H, m), 1.80–1.92 (2H, m), 3.10–3.23 (2H, m), 3.56–
3.68 (2H, m), 4.36 (2H, d, J=7.8 Hz), 4.33–4.46 (1H,
m), 6.64 (1H, d, J=9.0 Hz), 6.88–6.86 (2H, m), 7.15
(1H, t, J=7.8 Hz), 9.81 (1H, s); FAB MS m/e (M)+
383.
7-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-1-[(E)-3-cya-
nocinnamyl]-1,3,4,5-tetrahydro-2,1,3-benzothiadiazepine
2,2-dioxide (5c). To a stirred solution of 13a (374 mg,
0.94 mmol) and 3-((E)-3-hydroxypropenyl)benzonitrile
(179 mg, 1.13 mmol) in THF (6 mL) at ambient tem-
perature was added PPh3 (345 mg, 1.32 mmol) and
DEAD (0.21 mL, 1.32 mmol). After stirring at ambient
temperature for 4.5 h, the reaction mixture was diluted
with EtOAc and washed with H2O. The organic layer
was dried over Na2SO4 and concentrated in vacuo. The
resulting residue was chromatographed on silica gel
eluting with EtOAc/Hex (40:60) to give 5c (375 mg,
74%) as a white amorphous powder: 1H NMR (DMSO-
d6) d 1.40 (9H, s), 1.40–1.55 (2H, m), 1.80–1.92 (2H, m),
2.80–2.92 (2H, m), 3.05–3.28 (4H, m), 3.60–3.70 (2H,
m), 4.26 (2H, d, J=6.4 Hz), 4.42–4.55 (1H, m), 6.48
(1H, d, J=16.4 Hz), 6.59 (1H, dd, J=6.4, 16.4 Hz),
6.75–6.82 (2H, m), 7.12–7.18 (1H, m), 7.47–7.57 (2H,
.
.
calcd for C25H32N6-O3S 2.1HCl 3.0H2O: C, 47.87; H,
6.44; N, 13.40; S, 5.11; Cl, 11.87. Found: C, 48.00; H, 6.48;
N, 13.54; S, 5.23; Cl, 12.02.
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(E)-3-amidinocinna-
myl]-3,4-dihydro-1H-2,1,3-benzothiadiazine 2,2-dioxide
(6d). Compound 6d was synthesized from 5d according
to the same procedure as that for 6a. Compound 6d was
obtained as a white amorphous powder (47% yield): 1H
NMR (DMSO-d6) d 1.63–1.79 (2H, m), 1.95–2.08 (2H,
m), 2.29 (3H, s), 3.46–3.60 (2H, m), 3.65–3.85 (2H, m),
4.46 (2H, d, J=7.6 Hz), 4.54 (2H, d, J=5.3 Hz), 4.56–
4.65 (1H, m), 6.60 (1H, dt, J=5.3, 16.1 Hz), 6.73 (1H, d,
J=16.1 Hz), 6.86–6.94 (2H, m), 7.98 (1H, d, J=9.1 Hz),

F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
377
7.56 (1H, t, J=7.6 Hz), 7.67–7.82 (3H, m), 7.90 (1H, s),
8.80 (1H, s), 9.20 (2H, s), 9.32 (1H, s), 9.43 (2H, s); FAB
MS m/e (M+1)+ 483. Anal. calcd for C24H30-
N6O3S 2.0HCl 2.3H2O: C, 48.29; H, 6.18; N, 14.08; S,
5.37; Cl, 11.88. Found: C, 48.10; H, 5.94; N, 14.21; S,
5.29; Cl, 12.20.
(12 mL) at 3 ꢀC was added trifluoroacetic acid (12 mL).
After 1 h, the reaction mixture was concentrated in
vacuo. To the resulting residue in EtOH (30 mL) was
added sodium ethoxide (NaOEt) (0.46 g, 2.98 mmol)
and the mixture was stirred for 30 min at 3 ꢀC. Then
NaOEt (0.10 g, 1.49 mmol) was added to the mixture
again and the mixture was stirred at 3 ꢀC for 50 min. To
the resulting mixture was further added NaOEt (0.10 g,
1.49 mmol) and the mixture was stirred for 2 h. The
mixture was concentrated in vacuo and the residue was
chromatographed on ODS gel eluting with MeOH/H2O
(0:100–100:0) to give 15a (0.60 g, 87%) as a white
.
.
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(E)-3-amidinocinna-
myl]-3-methyl-3,4-dihydro-1H-2,1,3-benzothiadiazine
2,2-dioxide (6e). Compound 6e was synthesized from 5e
according to the same procedure as that for 6a. Com-
pound 6e was obtained as a white amorphous powder
(54% yield): 1H NMR (DMSO-d6) d 1.63–1.81 (2H, m),
1.96–2.09 (2H, m), 2.30 (3H, s), 2.70 (3H, s), 3.44–3.60
(2H, m), 3.66–3.88 (2H, m), 4.58–4.65 (5H, m), 6.58
(1H, dt, J=5.4, 16.1 Hz), 6.77 (1H, d, J=16.1 Hz), 6.89
(1H, d, J=2.5 Hz), 6.96 (1H, dd, J=2.5, 9.2 Hz), 7.02
(1H, d, J=9.2 Hz), 7.57 (1H, t, J=7.8 Hz), 7.72 (1H, d,
J=7.8 Hz), 7.77 (1H, d, J=7.8 Hz), 7.89 (1H, s), 8.87
(1H,s), 9.27 (2H,s), 9.39 (1H, s), 9.45 (2H, s); FAB MS
m/e (M+1)+ 497. Anal. calcd for C25H32-
1
amorphous powder: H NMR (DMSO-d6) d 1.63–1.76
(2H, m), 1.92–2.03 (2H, m), 2.88–3.06 (2H, m), 3.10–
3.21 (2H, m), 4.43–4.51 (1H, m), 5.10 (2H, s), 6.72 (1H,
d, J=9.3 Hz), 6.91 (1H, dd, J=2.9, 8.8 Hz), 7.49 (1H, d,
J=3.0 Hz), 7.74 (1H, dd, J=1.9, 8.8 Hz), 7.79 (1H, dd,
J=1.5, 8.8 Hz), 7.99–8.09 (3H, m), 8.50 (1H, s); FAB
MS m/e (M+1)+ 463.
1-[(E)-3-Cyanocinnamyl]-6-[(4-piperidyl)oxy]-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (15b). Compound
15b was synthesized from 14b according to the same
procedure as that for 15a. Compound 15b was obtained
.
.
N6O3S 2.0HCl 2.4H2O: C, 49.00; H, 6.38; N, 13.71; S,
5.23; Cl, 11.57. Found: C, 48.96; H, 6.66; N, 13.69; S,
5.18; Cl, 11.86.
as a pale yellow solid (86% yield): mp 162–163 ꢀC; H
1
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-(tert -
butoxycarbonylsulfamoyl) - N - [(7 - cyano - 2 - naphthyl)-
methyl]anthranilate (14a). To a stirred solution of 4a
(0.98 g, 1.89 mmol) in pyridine (15 mL) at ambient tem-
perature was added tert-butyl chlorosulfonylcarbamate5
(0.81 g, 3.76 mmol). After 1 h, the reaction mixture was
concentrated in vacuo. The resulting residue was dis-
solved in EtOAc and the solution was washed with 10%
aqueous citric acid, saturated aqueous NaHCO3 and
H2O, then concentrated in vacuo. The residue was
chromatographed on silica gel eluting with EtOAc/Hex
(30:70) to give 14a (1.07 g, 82%) as a brown amorphous
NMR (DMSO-d6) d 1.45–1.60 (2H, m), 1.84–1.99 (2H,
m), 2.62–2.77 (2H, m), 2.93–3.08 (2H, m), 4.29–4.40
(1H, m), 4.50 (2H, d, J=5.0 Hz), 6.53 (1H, dt, J=5.0,
16.2 Hz), 6.68 (1H, d, J=16.2 Hz), 6.91 (1H, d,
J=8.7 Hz), 7.01 (1H, dd, J=3.1, 8.7 Hz), 7.45 (1H, d,
J=3.1 Hz), 7.50 (1H, t, J=7.8 Hz), 7.67 (1H, d,
J=7.8 Hz), 7.72 (1H, d, J=7.8 Hz), 7.87 (1H, s); FAB
MS m/e (M+1)+ 439.
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(7-amidino-2-naph-
thyl)methyl]-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-di-
oxide (6f). Compound 6f was synthesized from 15a
according to the same procedure as that for 6a. Com-
pound 6f was obtained as a white amorphous powder
(31% yield): 1H NMR (DMSO-d6) d 1.63–1.79 (2H, m),
1.93–2.05 (2H, m), 2.27 (3H, s), 3.40–4.80 (4H, m),
4.61–4.65 (1H, m), 5.23 (2H, s), 7.02 (1H, d, J=9.2 Hz),
7.13 (1H, d, J=9.2 Hz), 7.51 (1H, s), 7.74 (1H, d,
J=8.5 Hz). 7.82 (1H, d, J=8.5 Hz), 8.01 (1H, s), 8.06
(1H, d, J=8.5 H), 8.12 (1H, d, J=8.5 Hz), 8.42 (1H, s),
8.66 (1H, s), 9.15 (2H, s), 9.21 (1H, s), 9.45 (2H, s); FAB
1
powder: H NMR (CDCl3) d 1.46 (9H, s), 1.53 (9H, s),
1.62–1.75 (2H, m), 1.80–1.92 (2H, m), 3.26–3.39 (2H,
m), 3.57–3.69 (2H, m), 3.84 (3H, s), 4.38–4.47 (1H, m),
4.92 (1H, br-s), 5.29 (1H, br-s), 6.85 (1H, dd, J=3.0,
8.5 Hz), 7.01 (1H, d, J=8.5 Hz), 7.35 (1H, d, J=3.0 Hz),
7.59 (1H, dd, J=1.8, 8.5 Hz), 7.63 (1H,s), 7.69 (1H, dd,
J=1.8, 8.5 Hz), 7.82 (1H, d, J=8.5 Hz), 7.88 (1H, d,
J=8.5 Hz), 7.92 (1H, s), 8.10 (1H, s); FAB MS m/e
(MÀ1)À 693.
MS
m/e
(M+1)+
521.
Anal.
calcd
for
.
.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-
(tert-butoxycarbonylsulfamoyl)-N-[(E)-3-cyanocinnamyl]-
anthranilate (14b). Compound 14b was synthesized from
4b according to the same procedure as that for 14a.
Compound 14b was obtained as a brown amorphous
C26H28N6O4S 1.8HCl 3.0H2O: C, 48.77; H, 5.64; N,
13.13; S, 5.01; Cl, 9.97. Found: C, 49.07; H, 5.55; N,
13.27; S, 5.07; Cl, 9.81.
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[(E)-3-(3-amidino-
phenyl)allyl]-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-di-
oxide (6g). Compound 6g was synthesized from 15b
according to the same procedure as that for 6a. Com-
pound 6g was obtained as a white amorphous powder
(25% yield): 1H NMR (DMSO-d6) d 1.67–1.83 (2H, m),
1.97–2.11 (2H, m), 2.31 (3H, s), 3.48–3.65 (2H, m),
3.67–3.88 (2H, m), 4.66 (2H, d, J=5.3 Hz), 4.74–4.82
(1H, m), 6.56 (1H, dt, J=5.3, 15.6 Hz), 6.72 (1H, d,
J=15.6 Hz), 7.38–7.42 (2H, m), 7.51–7.54 (1H, m), 7.57
(1H, t, J=7.9 Hz), 7.70–7.76 (2H, m), 7.89 (1H, s), 8.86
(1H, s), 9.29 (2H, s), 9.41 (1H, s), 9.45 (2H, s); FAB MS
1
powder (99% yield): H NMR (CDCl3) d 1.47 (9H, s),
1.53 (9H, s), 1.70–1.80 (2H, m), 1.86–1.95 (2H, m),
3.30–3.39 (2H, m), 3.60–3.71 (2H, m), 3.89–3.92 (5H,
m), 4.45–4.53 (1H, m), 6.33 (1H, d, J=15.9 Hz), 6.46
(1H, dt, J=6.9, 15.9 Hz), 7.03 (1H, dd, J=3.0, 8.7 Hz),
7.24–7.27 (1H, m), 7.36–7.42 (2H, m), 7.48–7.55 (3H,
m), 8.60 (1H, br); FAB MS m/e (MÀ1)À 669.
1-[(7-Cyano-2-naphthyl)methyl]-6-[(4-piperidyl)oxy]-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (15a). To a
stirred solution of 14a (1.03 g, 1.49 mmol) in CH2Cl2

378
F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
m/e (M+1)+ 497. Anal. calcd for C24H28N6-
O4S 2.1HCl 2.0H2O: C, 47.32; H, 5.64; N, 13.80; S,
5.26; Cl, 12.22. Found: C, 47.12; H, 5.62; N, 13.82; S,
5.31; Cl, 12.15.
14a. Compound 14d was obtained as a pale yellow
1
.
.
amorphous powder (88% yield): H NMR (CDCl3) d
1.47 (9H, s), 1.49 (9H, s), 1.65–1.83 (2H, m), 1.86–2.00
(2H, m), 3.31–3.44 (2H, m), 3.61–3.74 (2H, m), 3.85
(3H, s), 4.10–4.35 (4H, m), 4.48–4.58 (1H, m), 6.98–7.09
(2H, m), 7.22 (1H, dt, J=7.2, 1.5 Hz), 7.33 (2H, d,
J=9.0 Hz), 7.42 (1H, d, J=3.0 Hz), 7.59 (1H, br-s);
FAB MS m/e (MÀ1)À 673.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-(tert-
butoxycarbonylsulfamoyl)-N-[3-(3-cyanophenyl)propyl]-
anthranilate (14c). To a stirred suspension of PdO/
9
BaSO4 (0.40 g) in EtOH (30 mL) at ambient tempera-
ture was added 14b (1.00 g, 1.49 mmol) and the mixture
was treated with hydrogen at 1 atm for 24 h. The reac-
tion mixture was filtered through Celite, and the filtrate
was concentrated in vacuo. The resulting residue was
chromatographed on silica gel eluting with EtOAc/Hex
(30:70) to give 14c (0.90 g, 90%) as a brown amorphous
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[2-(3-amidinophe-
noxy)ethyl]-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-di-
oxide (6i). Compound 6i was synthesized from 14d
according to the same procedure as that for 6f. Com-
pound 6i was obtained as a white amorphous powder
(23% yield): 1H NMR (DMSO-d6) d 1.68–1.86 (2H, m),
1.98–2.13 (2H, m), 2.32 (3H, s), 3.48–3.67 (2H, m),
3.67–3.89 (2H, m), 4.20–4.31 (4H, m), 4.76–4.85 (1H,
m), 7.17 (1H, dd, J=2.2, 8.5 Hz), 7.33 (1H, s), 7.39–7.46
(2H, m), 7.46–7.52 (2H, m), 7.60 (1H, d, J=8.8 Hz),
8.86 (1H, s), 9.27 (2H, s), 9.39 (1H, s), 9.42 (2H, s); FAB
MS m/e (M+1)+ 501. Anal. calcd for C23H28N6-
1
powder: H NMR (CDCl3) d 1.47 (9H, s), 1.50 (9H, s),
1.66–2.00 (6H, m), 2.63–2.75 (2H, m), 3.29–3.45 (2H,
m), 3.60–4.00 (7H, m), 4.40–4.59 (1H, m), 7.00–7.67
(7H, m); FAB MS m/e (MÀ1)À 671.
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-[3-(3-amidinophenyl)-
propyl]-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide
(6h). Compound 6h was synthesized from 14c accord-
ing to the same procedure as that for 6f. Compound 6h
was obtained as a pale brown amorphous powder (16%
.
.
O5S 2.1HCl 3.0H2O: C, 43.77; H, 5.76; N, 13.31; S,
5.08; Cl, 11.80. Found: C, 43.52; H, 5.45; N, 13.18; S,
4.88; Cl, 12.08.
1
yield): H NMR (DMSO-d6) d 1.61–1.83 (2H, m), 1.83–
Methyl
N-benzyloxycarbonylmethyl-5-[(1-tert-butoxy-
2.18 (4H, m), 2.33 (3H, s), 2.73 (2H, t, J=7.5 Hz), 3.48–
3.94 (6H, m), 4.74–4.86 (1H, m), 7.44 (2H, s), 7.48–7.60
(3H, m), 7.68–7.74 (2H, m), 8.98 (1H, s), 9.37 (2H, s),
9.48 (2H, s), 9.54 (1H, s); FAB MS m/e (M+1)+ 499.
Anal. calcd for C24H30N6O4S 2.4HCl 2.0H2O: C, 46.33;
H, 5.90; N, 13.51; S, 5.15; Cl, 13.68. Found: C, 46.52; H,
5.65; N, 13.11; S, 4.81; Cl, 13.68.
carbonyl-4-piperidyl)oxy]-N-(2,2,2-trifluoroacetyl)anthra-
nilate (18). To a stirred solution of 3 (7.13 g, 30.4 mmol)
in 1,2-dichloroethane (150 mL) at ambient temperature
was added pyridine (4.73 mL 60.5 mmol) and tri-
fluoroacetic anhydride (4.25 mL, 30.5 mmol). After stir-
ring at ambient temperature for 3 h, the mixture was
poured into water and extracted with CHCl3. The
organic extracts were washed with water and dried over
Na2SO4, and concentrated to give methyl 5-[(1-tert-
butoxycarbonyl-4-piperidyl)oxy]-N-(2,2,2-trifluoroacetyl)-
anthranilate (9.43 g) which was used without further
purification: FAB MS m/e (M+1)+ 447.
.
.
3-[(2-Oxo)ethoxy]benzonitrile (17). To a stirred suspen-
sion of silica gel (Kieselgel-60, 230–400 mesh, 50 g) in
CH2Cl2 (400 mL) at ambient temperature was added
sodium periodate (0.65 M aqueous solution, 50 mL,
32.5 mmol) and 168 (0.31 M solution in CH2Cl2, 77 mL,
23.6 mmol). After stirring at ambient temperature for
20 min, the reaction mixture was chromatographed on
silica gel directly eluting with CHCl3/MeOH (100:0–
95:5) to give a crude purified 17 (4.36 g) as a colorless
viscous oil, which was used without further purification:
GC MS m/e (M)+ 161.
To a suspension of NaH (60% in paraffin liquid, 0.24 g,
6.0 mmol) in DMF (30 mL) at 3 ꢀC was added the above
anthranilic acid derivative (2.34 g) and the mixture was
stirred at ambient temperature for 50 min. Benzyl
2-bromoacetate (1.22 mL, 7.86 mmol) was then added to
the mixture at 3 ꢀC and the mixture was stirred at
ambient temperature for 14 h. The reaction mixture was
diluted with water and the solution was extracted with
EtOAc. The organic layer was washed with brine, dried
over Na2SO4 and concentrated in vacuo. The resulting
residue was chromatographed on silica gel eluting with
EtOAc/Hex (15:85) to give 18 (2.42 g, 81%) as a pale yel-
low viscous oil: 1H NMR (CDCl3) d 1.47 (9H, s),
1.67–1.82 (2H, m), 1.87–2.00 (2H, m), 3.32–3.44 (2H, m),
3.62–3.75 (2H, m), 3.79 (1H, d, J=17.4Hz), 3.87 (3H, s),
4.49–4.58 (1H, m), 5.06 (1H, d, J=17.4 Hz), 5.20 (2H, d,
J=2.1 Hz), 7.03 (1H, dd, J=2.7, 8.7 Hz), 7.31–7.38 (5H,
m), 7.51–7.57 (2H, m); FAB MS m/e (M+1)+ 595
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-[2-
(3-cyanophenoxy)ethyl]anthranilate (4c). Compound 4c
was synthesized from 3 and 17 according to the same
procedure as that for 4a. Compound 4c was obtained as
a pale yellow solid (49% yield): mp 117–118 ꢀC; 1H
NMR (CDCl3) d 1.47 (9H, s), 1.62–1.78 (2H, m), 1.82–
1.94 (2H, m), 3.23–3.35 (2H, m), 3.59–3.78 (4H, m), 3.85
(3H, s), 4.20 (2H, t, J=5.4 Hz), 4.23–4.33 (1H, m), 6.72
(1H, d, J=9.0 Hz), 7.07 (1H, dd, J=2.7, 9.0 Hz), 7.13–
7.19 (2H, m), 7.23–7.28 (1H, m), 7.33–7.41 (1H, m), 7.50
(1H, d, J=2.7 Hz), 7.60–7.80 (1H, br-s); FAB MS m/e
(M)+ 495.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-{[(3-
cyanophenyl)carbamoyl]methyl}-N-(2,2,2-trifluoroacetyl)-
anthranilate (19). To a stirred suspension of 10% Pd/C
powder (0.20 g) in MeOH (50 mL) at ambient tempera-
ture was added 18 (2.60 g, 4.37 mmol) and the mixture
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-(tert -
butoxycarbonylsulfamoyl)-N-[2-(3-cyanophenoxy)ethy-
l]anthranilate (14d). Compound 14d was synthesized
from 4c according to the same procedure as that for

F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
379
was treated with hydrogen at 1 atm for 3 h. The reaction
mixture was filtered through Celite, and the filtrate was
concentrated in vacuo to give N-{2-methoxycarbonyl-4-[(1-
tert-butoxycarbonyl-4-piperidyl)oxy]}phenyl-N-(2,2,2-tri-
fluoroacetyl)glycine (2.16 g) which was used without
further purification: FAB MS m/e (MÀ1)À 503.
(1H, s), 8.80 (1H, s), 9.18 (2H, s), 9.32–9.42 (3H, m),
11.02 (1H, s); FAB MS m/e (M+1)+ 514. Anal. calcd
for C23H27N7O5S 2.2HCl 1.1H2O: C, 45.02; H, 5.16; N,
15.98; S, 5.23; Cl, 12.71. Found: C, 45.34; H, 5.30; N,
15.77; S, 4.75; Cl, 12.83.
.
.
2-Bromo-N-(3-cyanophenyl)-N-methylacetamide (21). To
a stirred solution of 3-methylaminobenzonitrile (20,
5.00 g, 29.6 mmol) and NaHCO3 (9.20 g, 110 mmol) in
EtOAc (35 mL) and H2O (35 mL) at ambient tempera-
ture was added bromoacetyl bromide (5.90 mL,
68.2 mmol). After stirring vigorously at ambient tem-
perature for 15 min, the reaction mixture was diluted
with diethyl ether. The organic layer was separated and
the aqueous layer was extracted with EtOAc. The com-
bined organic layer was dried over Na2SO4 and con-
centrated in vacuo. The resulting residue was
recrystallized from EtOAc/Hex to give 21 (6.91 g, 92%)
To a stirred solution of above glicine derivative (2.10 g)
and 3-aminobenzonitrile (0.49 g, 4.16 mmol) in DMF
(10 mL) at 3 ꢀC was added 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (0.88 g, 4.58 mmol)
and 1-hydroxybenzotriazole (0.56 g, 4.58 mmol). After
stirring at ambient temperature for 17 h, The reaction
mixture was diluted with water and the solution was
extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4 and concentrated in
vacuo. The resulting residue was chromatographed on
silica gel eluting with EtOAc/Hex (20:80–35:75) to give
19 (1.67 g, 65%) as a white amorphous powder: 1H
NMR (CDCl3) d 1.47 (9H, s), 1.69–1.85 (2H, m), 1.87–
2.02 (2H, m), 3.32–3.46 (2H, m), 3.60–3.75 (2H, m), 3.88
(3H, s), 4.25 (1H, d, J=15.6 Hz), 4.49–4.68 (2H, m),
7.13 (1H, dd, J=3.0, 9.0 Hz), 7.36–7.49 (3H, m), 7.57
(1H, d, J=3.0 Hz), 7.74 (1H, dt, J=6.6, 2.4 Hz), 7.99–
8.03 (1H, m), 8.98 (1H, s); FAB MS m/e (M+1)+
605.
ꢀ
1
as a white solid: mp 115–116 C; H NMR (CDCl3) d
3.33 (3H, s), 3.63 (2H, s), 7.55–7.75 (4H, m),; FAB MS
m/e (M+1)+ 253, 255.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-
{[(3-cyanophenyl)(methyl)carbamoyl]methyl}anthranilate
(4d). To a stirred solution of 3 (0.80 g, 3.14 mmol) and
21 (1.00 g, 2.85 mmol) in acetonitrile (50 mL) at ambient
temperature was added K2CO3 (0.43 g, 3.14 mmol).
After the reaction mixture was refluxed for 12 h, it was
filtered and concentrated in vacuo. The residue was
chromatographed on silica gel eluting with EtOAc/Hex
(33:77) to give 4d (0.52 g, 35%) as a pale brown amor-
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-(tert -
butoxycarbonylsulfamoyl) - N - {[(3 - cyanophenyl)carba-
moyl]methyl}anthranilate (14e). A mixture of 19 (1.50 g,
2.48 mmol) and K2CO3 (0.68 g, 4.96 mmol) in MeOH
(10 mL) and H2O (10 mL) was stirred for 24 h. The
mixture was concentrated to remove MeOH and to the
resulting aqueous solution was added brine followed by
extraction with EtOAc. The organic extracts were dried
over Na2SO4 and concentrated in vacuo. The residue
was chromatographed on silica gel eluting with MeOH/
CHCl3 (3:97) to give a crude purified methyl 5-[(1-tert-
butoxycarbonyl - 4 - piperidyl)oxy] - N - {[(3 - cyanophe-
nyl)carbamoyl]methyl}anthranilate (0.97 g, 77%) which
was used without further purification: FAB MS m/e
(M)+ 508.
1
phous powder: H NMR (CDCl3) 1.46 (9H, s), 1.60–
1.76 (2H, m), 1.80–1.93 (2H, m), 3.22–3.36 (6H, m),
3.63–3.80 (3H, m), 3.87 (3H, s), 4.21–4.31 (1H, m), 6.98
(1H, dd, J=3.3, 8.7 Hz), 7.47–7.73 (6H, m); FAB MS
m/e (M)+ 522.
Methyl 5-[(1-tert-butoxycarbonyl-4-piperidyl)oxy]-N-
(tert-butoxycarbonylsulfamoyl)-N-{[(3-cyanophenyl)(me-
thyl)carbamoyl]methyl}anthranilate (14f). Compound
14f was synthesized from 4d according to the same pro-
cedure as that for 14a. Compound 14f was obtained as a
brown amorphous powder (95% yield): 1H NMR
(CDCl3) d 1.47 (18H, s), 1.63–1.81 (2H, m), 1.82–2.00
(2H, m), 3.26–3.44 (7H, m), 3.60–3.71 (2H, m), 3.81
(3H, br-s), 4.46–4.59 (1H, m), 7.04 (1H, dd, J=9.0,
3.0 Hz), 7.35 (1H, d, J=2.7 Hz), 7.38–7.71 (4H, m), 7.81
(1H, d, J=9.3 Hz); FAB MS m/e (M+1)+ 702.
Compound 14e was synthesized from above inter-
mediate according to the same procedure as that for
14a. Compound 14e was obtained as a pale brown
1
amorphous powder (56% yield): H NMR (CDCl3) d
1.47 (9H, s), 1.51 (9H, s), 1.66–1.83 (2H, m), 1.83–2.00
(2H, m), 3.30–3.44 (2H, m), 3.58–3.70 (2H, m), 4.02
(3H, s), 4.37–4.56 (2H, m), 5.16 (1H, d, J=17.4 Hz),
7.05 (1H, dd, J=3.0, 8.7 Hz), 7.28–7.43 (4H, m), 8.08
(1H, dt, J=7.5, 2.1 Hz), 8.11–8.15 (1H, m), 10.91 (1H,
s); FAB MS m/e (MÀ1)À 686.
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-{[(3-amidinophe-
nyl)(methyl)carbamoyl]methyl} - 1H - 2,1,3 - benzothiadia-
zin-4(3H)-one 2,2-dioxide (6k). Compound 6k was
synthesized from 14f according to the same procedure
as that for 6f. Compound 6k was obtained as a pale
brown amorphous powder (9% yield): 1H NMR
(DMSO-d6) d 1.60–1.85 (2H, m), 1.92–2.12 (2H, m),
2.30 (3H, s), 3.00–4.00 (7H, m), 4.40–4.64 (2H, m),
4.73–4.82 (1H, m), 7.34 (2H, s), 7.48 (1H, s), 7.60–8.00
(4H, m), 8.73 (1H, s), 9.27 (3H, s), 9.53 (2H, s); FAB
MS m/e (M+1)+ 528. Anal. calcd for C24H29N7-
6-[(1-Acetimidoyl-4-piperidyl)oxy]-1-{[(3-amidinophenyl)-
carbamoyl]methyl}-1H-2,1,3-benzothiadiazin-4(3H)-one
2,2-dioxide (6j). Compound 6j was synthesized from
14e according to the same procedure as that for 6f.
Compound 6j was obtained as a white amorphous
1
powder (30% yield): H NMR (DMSO-d6) d 1.68–1.85
.
.
(2H, m), 1.97–2.12 (2H, m), 2.30 (3H, s), 3.49–3.65 (2H,
m), 3.68–3.86 (2H, m), 4.75–4.89 (3H, m), 7.38–7.47
(3H, m), 7.50–7.58 (2H, m), 7.78–7.84 (1H, m), 8.10
O5S 2.2HCl 2.5H2O: C, 44.15; H, 5.59; N, 15.02; S,
4.91; Cl, 11.95. Found: C, 44.29; H, 5.98; N, 14.83; S,
4.26; Cl, 11.56.

380
F. Hirayama et al. / Bioorg. Med. Chem. 11 (2003) 367–381
X-ray crystallographic experiment
Laboratory, Lexington, MA, USA) to initiate clot for-
mation. The concentration required to double clotting
time (CT2) was estimated from each individual dose–
response curve.
Crystals of the bovine pancreatic trypsin in complex
with inhibitors were prepared using the same method as
reported previously.10 The crystals belonged to the
space group P212121 and were isomorphous to those
reported for YM-60828.7 The X-ray diffraction data
were collected with the Rigaku R-AXIS IIc image-plate
system and the structural analysis of the inhibitor com-
plex was achieved by the Patterson search method based
on a molecular model of the bovine pancreatic trypsin/
NAPAP complex (1PPC). Model building, electron
density calculation, and model refinement were carried
out using program O11 and CNX2000 (Accelrys Inc.).
Diffraction data and refinement statistics for inhibitor/
trypsin complexes are as follows. 6a: resolution range,
Ex vivo studies. Male ICR mice weighing 20–30 g and
squirrel monkeys of both sexes weighing 660–775 g were
fasted overnight. Inhibitors were dissolved in saline and
administered orally to the mice at 100 mg/kg, and to
squirrel monkeys at 3 mg/kg using a gastric tube. Sev-
eral times after oral administration of the inhibitor,
citrated blood was collected from the abdominal vena
cava (mice) or the femoral vein (squirrel monkeys), and
platelet poor plasma was prepared by centrifugation to
measure PT. All data were expressed as relative fold
values, compared with the baseline value (squirrel mon-
keys) or the vehicle group (mice).
46.86–2.2 A; data completeness, 74.9%; R-factor/Rfree
18.1%/21.0%; r.m.s.d. of bonds, 0.009 A; r.m.s.d. of
,
angles 1.5ꢀ. 6f: resolution range, 46.91–2.5 A; data
completeness, 86.8%; R-factor/Rfree
,
27.8%/29.4%;
Acknowledgements
r.m.s.d. of bonds, 0.006 A; r.m.s.d. of angles 1.3ꢀ. We
deposit the crystallographic data for these structures in
the Protein Data Bank.
The authors deeply acknowledge the staff of the Divi-
sion of Analytical Science Laboratories for the ele-
mental analysis and spectral measurements.
Modeling study
The ZK-807834 inhibited factor Xa X-ray coordinate
(1FJS)12 was employed in this study. Each inhibitor/
trypsin complex was superimposed on to ZK-807834/
factor Xa complex using coordinate of Ca atoms
around active site. After manual adjustment based on
the same method as reported for YM-60828,7 energy
minimization of each inhibitor/water/factor Xa complex
model was performed using CFF91 force field imple-
mented in the program DISCOVER (Accelrys Inc.). All
atoms within 10 A from inhibitor were allowed to move
during the minimization.
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Biology
Chromogenic assay. The hydrolysis rates of synthetic
substrates were assayed by continuously measuring
absorbance at 405 nm at 37 ꢀC with a microplate reader
(Model 3550, Bio-Rad, Richmond, USA). Reaction
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liance
Brain
Thromboplastin,
Instrumentation

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