The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor.

Article abstract of DOI:10.1016/S0968-0896(01)00418-7

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found tha

Full text of DOI:10.1016/S0968-0896(01)00418-7

Bioorganic & Medicinal Chemistry 10 (2002) 1509–1523
The Discovery of YM-60828: A Potent, Selective and
Orally-Bioavailable Factor Xa Inhibitor
Fukushi Hirayama,a,* Hiroyuki Koshio,a Naoko Katayama,a Hiroyuki Kurihara,a
Yuta Taniuchi,b Kazuo Sato,c Nami Hisamichi,a Yumiko Sakai-Moritani,a
Tomihisa Kawasaki,a Yuzo Matsumotoa and Isao Yanagisawaa
aInstitute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,
Tsukuba, Ibaraki 305-8585, Japan
bClinical Development Department, Yamanouchi Pharmaceutical Co., Ltd., 3-17-1 Hasune, Itabashi, Tokyo 174-8612, Japan
cProject Coordination Department, Yamanouchi Pharmaceutical Co., Ltd., 3-17-1 Hasune, Itabashi, Tokyo 174-8612, Japan
Received 9 October 2001; accepted 22 November 2001
Abstract—Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an
attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we
found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities.
Structure–activity relationship (SAR) of the substituent (R1) on the central aniline moiety suggested that increasing lipophilicity
caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic
substituent in R1 showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this
series was sulfamoylacetic acid derivative 8o (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and
was chosen for clinical development. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
a crucial rolein thrombosis by not only producing fibrin
from fibrinogen for clot formation but also by inducing
Warfarin, the sole orally-effective anticoagulant ther-
apeutic agent currently available, has been widely used
for treatment and/or prevention of thromboembolic
disorders. Warfarin exerts its potent anticoagulant effect
by inhibiting biosynthesis of a vitamin K-dependent
coagulation factor. Dueto its indircet mcehanism,
Warfarin shows slow onset of action, narrow ther-
apeutic range, and interaction with many drugs and
foods. Furthermore, the most serious problem is an
adverse effect on bleeding and must, therefore, be mon-
itored carefully.1ꢀ3 Consequently, development of a
direct acting, orally-available and with no associated
hemorrhaging inhibitor of the coagulation factor is very
important.
platelet aggregation.4ꢀ8 Despite numerous efforts, no
orally effective thrombin inhibitor has been launched as
yet. Recently, inhibition of a thrombin generation
‘‘Factor Xa inhibitor’’ has received much attention as
an alternative to direct inhibition of thrombin. The ser-
ine protease Factor Xa (FXa) occupies the convergence
point of theintrinsic and extrinsic coagulation cascade
and combines with factor Va, Ca2+ and a phospholipid
to form a prothrombinase complex, which generates
thrombin by the proteolysis of prothrombin. Because
the coagulation cascade is a highly amplified process,
FXa is present in blood at a much lower concentration
compared with thrombin, leading to the hypothesis that
a FXa inhibitor is expected to show therapeutic effect
with much smaller doses than a thrombin inhibitor.
Moreover, a specific FXa inhibitor does not affect pla-
telet activation and aggregation caused by thrombin;
therefore, it could have little hemorrhagic risk. Actually
Efforts to discover a specific inhibitor of a protease
along the coagulation cascade have been concentrated
at inhibition of thrombin, a serine protease which plays
theprotein specific FXa inhibitor from thelech (Anti-
stasin)9,10 and thetick (TAP)
11,12
haveben shown to be
effective antithrombotics in animal models without pro-
longation bleeding time when administrated parenterally.
*Corresponding author. Tel +81-298-54-1563; fax +81-298-52-2971;
e-mail: hirayaf@yamanouchi.co.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00418-7

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F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
Scheme 1. (a) 4-Nitrophenol, PPh3, diethyl azodicarboxylate, THF; (b) H2, 10% Pd/C, EtOH; (c) NMe3O, CHCl3; (d)NaB(OAc)3H, CH2Cl2,
AcOH; (e) R12O, basefor 6b and 6j; EtNCO for 6d; R1Cl, base for the other compounds; (f) HCl, EtOH or MeOH; (g) NH3, EtOH for 7n from 6k;
NH4OAc, EtOH or MeOH for the other compounds; (h) ethyl acetimidate hydrochloride, Et3N, EtOH or MeOH; (i) HCl, H2O.
Recently, a variety of non-peptide FXa inhibitors have
been reported in the literature.13ꢀ15 Among them DX-
9065a (Fig. 1) was reported as the first FXa inhibitor
which possessed anticoagulant activity after oral
administration in rats.16,17 We planned to create more
potent and orally effective FXa inhibitors based on the
transformation of three parts of DX-9065a, such as the
(7-amidino-2-naphthyl)methyl moiety, 4-[(1-acetimidoyl-
4-pyrrolidyl)oxy]phenyl moiety, and carboxylic acid
moiety. In order to investigate the variability of SAR, a
nitrogen atom was introduced as a juncture of the three
parts, which resulted in facilitation of the synthesis,
achirality as well as a novel lead compound. In these
strategies, we started to explore a series of N-{(7-ami-
dino-2-naphthyl)methyl}aniline derivatives. In this
paper, we describe the results of our work on the synth-
esis and SAR of potent and selective FXa inhibitors.
Figure 1. Structureof DX-9065a.
After reaction of 5 with various acyl chlorides, sulfonyl
chlorides or isocyanate, the resulting cyano derivatives
were converted to the corresponding amidines 7a–7m
under standard Pinner reaction conditions by bubbling
HCl gas into a solution of thecyano compounds in
ethanol or methanol and subsequent amination of the
formed imidate by treatment with ammonium acetate.
Carboxamide 7n was prepared from 6k using saturated
ammonia solution instead of ammonium acetate. The
piperidine moieties were treated with ethyl acetimidate
hydrochloride and triethylamine in methanol or ethanol
to give the corresponding bis-amidines 8a–8n.
Sulfamidederivatives 8q–8w were also synthesized from
the intermediate 5 as illustrated in Scheme 2. Following
an existing synthetic method19 of methyl chloro-
sulfonylcarbamate, chlorosulfonylisocyanate was allowed
to react with tert-butylalcohol and ethanol to give tert-
butyl and ethyl chlorosulfonylcarbamate 11a and 11b,
which was treated with 5 to afford sulfamides 9a and 9b.
Sulfamides 9a and 9b were alkylated with methanol
under Mitsunobu condition or with an alkylbromide to
Chemistry
Key intermediate 5 was synthesized as depicted in Scheme
1. Mitsunobu condensation of the hydroxypiperidine
derivative 1 and 4-nitrophenol followed by reduction of
thenitro group afforded aniline 2. Reductive alkylation
of 2 with aldehyde 4, which was prepared by oxidation of a
known naphthylbromide 318 with anhydrous trimethyl-
amine N-oxidein CHCl 3, gave the versatile intermediate 5.

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1511
Scheme 2. (a) t-BuOCONHSO2Cl (11a) for 9a, EtOCONHSO2Cl (11b) for 9b, pyridine; (b) MeOH, PPh3, diethyl azodicarboxylate, THF for 10a;
R3Br, K2CO3, DMF for 10b and 10c; (c) HCl, EtOH or MeOH; (d) NH4OAc, EtOH or MeOH; (e) ethyl acetimidate hydrochloride, Et3N, EtOH or
MeOH; (f) HCl, H2O.
afford N,N,N0,N0-tetra-substituted sulfamides 10a–10c.
Thesulfamides 9a, 9b and 10a–10c were converted to
corresponding bis-amidines 8q–8t and 8v by thesame
method described above. Acid derivatives 8o, 8p, 8u and
8w were synthesized from the corresponding esters by
acid hydrolysis.
activity was comparable to that of reference compound,
DX-9065a. This achiral compound 8a is positioned to
be a valuable and novel lead to explore superior FXa
inhibitors.
Some small alkyl groups, methyl or ethyl, were intro-
duced on the juncture –NH– with various linkages such
as amide, urea, sulfonamide or sulfamide, to evaluate
their inhibitory activities (8b–8g, and 8q). Among these
compounds, methanesulfonamide 8f showed 5- and 10-
fold enhancements in potency relative to the lead com-
pounds 8a and DX-9065a, respectively. Sulfonamide
and sulfamidederivatives 8f, 8g and 8q (IC50=3.8–9.7
nM) showed relatively higher potencies than amide, urea
and urethane derivatives 8b–8e (IC50=12.0–31.9 nM).
Schemes 3 and 4 depict the synthesis of benzamidine
derivatives 16, 17 and 22. Intermediate 14 was obtained
according to thesameprocdeureas that for
5 using
cinnamaldehyde 13 instead of naphthyl aldehyde 4.
Aldehyde 13 was prepared from benzaldehyde 12 and
triphenylphosphoranylidene acetaldehyde by Wittig
condensation. Compound 14 was reduced by hydro-
genation in the presence of 10% palladium on carbon to
afford saturated derivative 15. In thesamemannre
described above, 14 and 15 were converted to methane-
sulfonamide derivatives and subsequent transformation
to bis-amidino compounds to give desired benzamidines
16 and 17. Another intermediate 21 was prepared by
alkylation of sulfonamide 18 with bromoacetanilide 20.
Compound 18 and 20 were easily prepared from com-
pound 2 and aniline 19, respectively. Compound 21 was
also converted to bis-amidine derivative 22 under the
same conditions described above.
Removal of the acetimidoyl moiety from the piperidine
ring (7f) resulted in decrease in the activity. In addition,
Table 1. In vitro inhibitory activities (IC50) against factor Xa,
thrombin, and trypsin
Compd
R1
H
R4
IC50 (nM) a
Results and Discussion
Factor Xa Thrombin Trypsin
All compounds prepared were evaluated by IC50 values
for theinhibition of FXa, thrombin, and trypsin enzy-
matic activities. CT2 values for the prolongation of
prothrombin time(PT) arealso tabulated in Table3 as
an indicator of anticoagulant activity in vitro. CT2
values were defined as the concentration required to
doubleclotting time.
8a
8b
8c
8d
8e
8f
8g
C(=NH)CH3 19.5
12.4
C(=NH)CH3 31.9
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
278
748
245
822
265
108
181
171
COMeC(=NH)CH
COEt
CONHEt C(=NH)CH3 16.3
COOEt C(=NH)CH3 12.0
SO2 MeC(=NH)CH
SO2Et C(=NH)CH3 9.7
SO2NHMeC(=NH)CH 6.6
3
3.8
3
8q
7f
DX-9065a
3
SO2 MeH
127
>100,000 1410
39.5ꢁ3.7b >100,000 3170
As shown in Table1, compound 8a, a derivative bearing
no substituent R1 on the juncture–NH– position turned
out to havestrong FXa inhibitory activity and high
degree of selectivity for thrombin. The FXa inhibitory
aHuman purified enzymes were used. IC50 values were determined
duplicated. The variation from the mean value is ꢁ15% or less.
bn=3.

1512
F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
Scheme 3. (a) Triphenylphosphoranylidene acetaldehyde, benzene; (b) n, NaB(OAc)3H, CH2Cl2, AcOH; (c) H2, Pd/C, MeOH; (d) MeSO2Cl, pyri-
dine; (e) HCl, EtOH; (f)NH4OAc, EtOH; (g) ethyl acetimidate hydrochloride, Et3N, EtOH.
Scheme 4. (a) Methanesulfonyl chloride, pyridine; (b) bromoacetyl bromide, NEt3, 1,2-dichloroethane; (c) K2CO3, CH3CN; (d) HCl, EtOH; (e)
NH4OAc, EtOH; (f) ethyl acetimidate hydrochloride, Et3N, EtOH.
introduction of 3-amidinophenyl structures instead of
the 7-amidino-2-naphthyl moiety afforded styrene deri-
vative 16 with moderate FXa inhibition, but all these
derivatives 16, 17 and 22 reduced the potencies com-
pared with parent compound 8f (Table2).
of DX-9065a in Trypsin20 and FXa,21 which have been
solved by Bode et al. The analysis indicates that the
naphthamidine moiety occupies deeply the S1 pocket,
and theamidinegroup makes a salt bridgeto Asp-189
at the bottom of it. Moreover, the acetimidoylpiperidine
moiety is in close contact to an aryl-binding pocket
defined by the three aromatic amino acids Phe-174, Tyr-
99 and Trp-215, and nitrogen of the acetimidoyl group
forms a water-mediated hydrogen bond to the oxygen
atoms of Thr-98 and Ile-175. These two parts of com-
pound 8g mainly participatein thebinding to FXa and
consequently compound 8g takes a L-shape conforma-
tion. In contrast, the ethanesulfonyl moiety points to
thesolution away from thenezymeand makse little
In an effort to provide understanding of the SARs for
these compounds, the binding conformation of 8g to
FXa was studied. Because of the general difficulty in
obtaining an X-ray crystal structureof FXa bound with
inhibitor, it was modeled based on the crystal structure
of 8g in the complex with the related enzyme, trypsin
(Fig. 2). The generated binding mode was similar to that
contact with FXa. Removing the acetimidoyl group
results in loss of the hydrogen-bonding interaction;
therefore, compound 7f reduces its inhibitory potencies
relative to acetimidoylpiperidine analogue 8f. Similarly,
replacement of the amidinonaphthyl moiety with ben-
zamidine( 16, 17 and 22) reduces the activity, possibly
Table 2. In vitro inhibitory activities (IC50) against factor Xa,
thrombin, and trypsin
due to lowering lipophilic interaction. On the other
1
hand, theroleof thesubstituent R
is not in forming
Compd
R5
IC50 (nM)a
Thrombin
additional interaction with FXa, but the sulfonamide
and sulfamide linkages are most likely to contribute to
stabilization of theL-shapeconformation.
Factor Xa
3.8
Trypsin
108
From the above modeling study, we considered that
there is a possibility to modify the ethyl moiety of sul-
fonamidein 8g without markedly affecting FXa inhibi-
tory potency (IC50), and changethephysiological
profiles of the compounds to improve anticoagulant
activities (PT assay) in vitro as well as oral efficacy ex
vivo. Since we thought the nM level of FXa inhibitory
potency (IC50) was enough to develop an anticoagulant,
our synthetic efforts were focused on the modification of
sulfonamide and sulfamide alkyl moieties.
8f
>100,000
>100,000
>100,000
>100,000
16
17
17.3
258.7
301.0
972
5806
6540
22
With sequential elongation of the alkyl sulfonamide
from oneto four carbons ( 8f–8i), theanticoagulant
aRefer to Table 1.

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1513
Figure 2. Binding model of 8g in factor Xa.
Table 3. In vitro inhibitory activities (IC50) against factor Xa, thrombin, trypsin and anticoagulant potencies (PT, CT2)
Compd
R1
IC50 (nM)a
Thrombin
CT2 (mM)b
PTc
Factor Xa
Trypsin
8f
8g
8 h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
SO2 Me3.8
SO2Et
SO2n-Pr
SO2n-Bu
SO2CF3
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
77,100
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
108
181
173
128
215
147
126
12
0.94ꢁ0.07
1.25ꢁ0.11
1.46ꢁ0.10
2.85ꢁ0.26
10.53ꢁ0.71
0.83ꢁ0.06
3.30ꢁ0.16
1.75ꢁ0.29
0.84ꢁ0.04
0.70ꢁ0.06
1.18ꢁ0.09
0.81ꢁ0.15
0.73ꢁ0.04
1.41ꢁ0.25
0.96ꢁ0.04
0.81ꢁ0.05
1.72ꢁ0.17
0.96e
9.7
7.5
4.4
6.5
8.8
9.7
0.94
4.3
SO2CH2CO2Et
SO2Ph
SO2(4-C6H4)CO2Et
SO2CH2CONH2
SO2CH2CO2H
SO2(4-C6H4)CO2H
SO2NHMe6.6
SO2NH2
154
216ꢁ28d
2.3ꢁ0.5d
0.074
133
171
3.4
2.4
2.8
3.4
134
116
116
148
90
116
3170
SO2NHCO2Et
SO2NHCH2CO2Et
SO2NHCH2CO2H
SO2N(CO2Et)CH2CO2 Me2.0
SO2N(CO2Et)CH2CO2H
8t
8u
8v
8w
5.1
39.5ꢁ3.7f
DX-9065a
4.87ꢁ0.19
aRefer to Table 1.
bThe concentration required to double clotting time (n=3).
cProthrombin timeusing mouseplasma.
dn=7.
en=1.

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F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
activities in the PT assay decrease gradually
(CT2=0.94–2.85 mM; Table 3). Furthermore, the deri-
vatives 8j and 8l bearing trifluoromethanesulfonamide
and benzenesulfonamide showed poor anticoagulant
activities (CT2=10.53 and 3.30 mM, respectively). As
expected from the X-ray crystallographic analysis, these
changes at the sulfonamide moiety didn’t greatly affect
the FXa inhibitory potencies (IC50=3.8–9.7 nM).
Therefore, we speculated that lipophilicities of the alkyl
sulfonamide moieties influenced the anticoagulant
activities in the PT assay by changing their plasma pro-
tein binding. Based on this consideration, polar func-
tional groups were introduced on the alkyl sulfonamide
moiety. Consequently compounds, which had ethoxy-
carbonyl, carbamoyl and calboxyl groups on the
methansulfonamide moiety of 8f, were synthesized (8k,
8n, and 8o). All these compounds possessed potent
anticoagulant activities (CT2=0.70–0.84 mM). In this
way these sulfonamide series of compounds (8f–8l, 8n,
and 8o) showed a relationship between CT2 values in PT
and calculated lipophilicity values (clogP),22 as shown
in Figure3.
bitory activity and the selectivity. These results are dif-
ficult to explain by the expected binding conformation
obtained from 8g in FXa, because this sulfonamide
moiety is suggested to be located out of the binding
pocket and have little contact with the enzyme. There-
fore, these results raise the possibility of a conforma-
tional difference between alkyl sulfonamide derivatives
and benzene sulfonamide derivatives.
In spite of their excellent FXa inhibitory activities, their
anticoagulant activities in the PT assay were not
observed to be so potent (CT2=1.75 and 1.18 mM,
respectively), presumably due to the large lipophilicity
of benzensulfonamide derivatives 8m and 8p
(clogP=4.41 and 4.04, respectively). These results
clearly indicate that high FXa inhibitory activity as well
as moderate hydrophilicity of the compound is quite
important for exhibiting anticoagulant activity in
plasma effectively.
From this consideration, limited sulfamide derivatives
with low clog P values (under 3.0) were synthesized and
evaluated. Almost all compounds prepared in this
strategy showed not only potent FXa inhibitory activ-
ities but also potent anticoagulant activities in the PT
assay (8q–8w).
On the other hand, unexpected results were obtained
with the benzenesulfonamide derivatives. The com-
pounds substituted on the 4-position of benzenesulfon-
amidewith theoxycarbonyl (
8m) or carboxyl (8p)
yielded about 10- and 130-fold enhancement in the FXa
inhibitory potency, compared to the mother analogue
(8l). Particularly quitestrong inhibitory pontecy
(IC50=0.074 nM) and excellent selectivity over trypsin
(ca. 1800-fold) were observed for compound 8p. Since
non-substituted benzenesulfonamide derivative 8l
showed nearly equal FXa inhibitory potency as that of
theothre alkylsulfonamidedreivativse, thecarboxyl
PT-prolonging effects of these advanced inhibitors fol-
lowing oral administration were evaluated in mice. Male
ICR mice were dosed via gastric tube with saline solu-
tion of inhibitors; 0.5 and 2.0 h after oral administra-
tion, blood was collected and platelet poor plasma was
prepared to measure PT. As outlined in Table 4, several
compounds (8f, 8o, 8t, 8u, and 8w) prolonged PT more
than 2-fold at 0.5 h. In contrast, highly lipophilic com-
pounds 8m and 8p showed no activities at the same
conditions. For thesimplealkyl sulfonamidedreiva-
tives, it was observed that the longer the alkyl groups,
group of 8p seemed to enhance strongly the FXa inhi-
thewaekre theoral activitsie ( 8f–8h). Moreover,
compounds having a carboxyl group in its structure
Table 4. Anticoagulant activity after oral administration in mice
Compd
PT/control PTa
0.5 h
2.0 h
8f
8g
8h
8k
8m
8n
8o
8p
8q
8r
8s
8t
8u
2.1
1.5
1.3
1.7
1.0
1.6
2.6
1.0
1.5
1.8
1.9
2.2
2.1
2.0
1.5
1.5
NTb
1.2
1.4
0.7
1.5
1.7
0.8
1.4
1.7
1.8
1.4
1.6
1.6
1.3
8w
DX-9065a
aThe relative prothrombin time compared with that measured using
normal miceplasma at 0.5 and 2.0 h after oral administration (100
mg/kg, n=3).
bNot tested.
Figure 3. The correlation between clogP and CT2 (PT) of sulfonamide
derivatives. clogP was calculated using TSAR ver. 3.0 (Oxford Mole-
cular Group). PT, CT2; see corresponding footnotes b and c in Table 3.

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1515
showed potent activities (8o, 8u, and 8w). Particularly,
compound 8o exhibited strongest oral activity, which
prolonged PT 2.6-fold at 0.5 h, and 1.7-fold at 2.0 h.
Compound 8o was further evaluated by pharmaco-
kinetic studies in guinea pigs and squirrel monkeys,
showing theabsoluteoral bioavailability at 33.4 and
20.4%,23 respectively. Moreover, it exerted antith-
rombotic effect in several thrombosis models in
rats,24ꢀ29 mice,30 and guinea pigs31 with no or little
prolongation of bleeding time. These results suggest
compound 8o (YM-60828) may proveto bea valuable,
orally-activeand potent anti-coagulation drug.
vacuo and crystallized from ethanol to give tert-Butyl 4-
(4-Nitrophenoxy)piperidine-1-carꢂboxylate (166.74 g,
1
77%) as a whitesolid: mp 93–94 C; H NMR (CDCl3)
d 1.48 (9H, s), 1.72–1.86 (2H, m), 1.90–2.03 (2H, m),
3.31–3.44 (2H, m), 3.63–3.77 (2H, m), 4.56–4.66 (1H,
m), 6.96 (2H, d, J=9.0 Hz), 8.20 (2H, d, J=9.0 Hz); EI
Ms m/e (M)+ 322.
To thesolution of thenitro compound (10.0 g, 31
mmol) in EtOH (100 mL) was added 10% Pd/C powder
(1.0 g). The reaction mixture was stirred in a hydrogen
atmosphere at ambient temperature for 1.5 h. The reac-
tion mixture was filtered through a pad of Celite and
concentrated in vacuo to give 2 (8.86 g, 98%) as a pale
brown solid: mp 89–91 ꢂC; H NMR (CDCl3) d 1.46
1
Conclusion
(9H, s), 1.60–1.76 (2H, m), 1.80–1.93 (2H, m), 3.10–3.65
(4H, m), 3.65–3.77 (2H, m), 4.21–4.31 (1H, m), 6.63
(2H, d, J=9.0 Hz), 6.76 (2H, d, J=9.0 Hz); FAB Ms m/
e (M)+ 292.
We have synthesized and evaluated a novel series of N-
[(7-amidino-2-naphthyl)methyl]aniline derivatives as
FXa inhibitors. Among this series, from FXa enzymatic
assay, quite potent and selective inhibitor 8p was dis-
covered. However, despite its excellent FXa inhibitory
activity in vitro, 8p showed mediocre anticoagulant
activity in vitro and poor activity ex vivo after oral
administration in mice. SAR studies indicated that
moderate hydrophilicity was an important factor for
exhibiting anticoagulant activity in plasma effectively.
7-Formylnaphthalene-2-carbonitrile (4). To a stirred
solution of 7-bromomethylnaphthalene-2-carbonitrile 3
(2460 mg, 10 mmol) in acetonitrile (40 mL) at ambient
temperature was added 4-methylmorpholine N-oxide
(2340 mg, 20 mmol). After 5 h, water (60 mL) was
added and the resulting precipitate was filtered, washed
with water and dried in vacuo to give 4 (949 mg, 52%)
as a whitesolid: mp 139–141 ꢂC; H NMR (CDCl3) d
1
From the evaluation of oral administration screening in
mice, compound 8o has been chosen for the best com-
pound as an orally-bioavailableand potent FXa inhi-
bitor. After extensive preclinical evaluation, YM-75466,
the methanesulfonate salt of 8o, is currently undergoing
clinical development.
7.78 (1H, dd, J=1.5, 8.4 Hz), 8.02 (2H, d, J=8.4 Hz),
8.13 (1H, dd, J=1.5, 8.4 Hz), 8.41 (2H, s), 10.21 (1H, s);
FAB Ms m/e (M+H)+ 182.
7-({4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]anilino}-
methyl)naphthalene-2-carbonitrile (5). To a stirred solu-
tion of 2 (1370 mg, 4.7 mmol) and 4 (849 mg, 4.7 mmol)
in methylene chloride (10 mL) and AcOH (2.7 mL) at
ambient temperature was added sodium triacetoxyboro-
hydride (1290 mg, 6.1 mmol). After 45 min the reaction
mixturewas washde with 2 M potassium carbonate
solution, water, and 10% citric acid solution. The
organic layer was dried over Na2SO4 and concentrated
in vacuo. The resulting residue was crystallized from
methanol to give 5 (1698 mg, 79%) as a whitesolid: mp
154–155 ꢂC; 1H NMR (CDCl3) d 1.46 (9H, s), 1.63–1.74
(2H, m), 1.80–1.92 (2H, m), 3.21–3.30 (2H, m), 3.65–
3.77 (2H, m), 4.00 (1H, br), 4.21–4.28 (1H, m), 4.49 (2H,
s), 6.59 (2H, d, J=8.8 Hz), 6.79 (2H, d, J=8.8 Hz), 7.59
(1H, d, J=8.3 Hz), 7.66 (1H, d, J=8.8 Hz), 7.84–7.92
(3H, m), 8.19 (1H, s); FAB Ms m/e (M)+ 457.
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, 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).
tert-Butyl 4-(4-Aminophenoxy)piperidine-1-carboxylate
(2). To a stirred solution of tert-butyl 4-hydroxy-
piperidine-1-carboxylate 1 (162 g, 805 mmol) and 4-
nitrophenol (93.6 g, 673 mmol) in THF (1.3 L) at
ambient temperature was added triphenylphosphine
(211 g, 805 mmol), and diethyl azodicarboxylate (127
mL, 805 mmol). After stirring at ambient temperature
for 21 h, the reaction mixture was concentrated in
vacuo. The residue was dissolved in methylene chloride
and washed with 1 N aqueous NaOH solution, water
and brine. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The resulting residue was sus-
pended with diethylether and insoluble material was
removed by filtration. The filtrate was concentrated in
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7-cyano-2-naphthyl)methyl]acetamide (6b). To a stirred
solution of 5 (150 mg, 0.33 mmol) in pyridine(1 mL) at
ambient temperature was added acetic anhydride (268
mg, 2.6 mmol) and 4-dimethylaminopyridine (10 mg,
0.08 mmol). After 15 h, the reaction mixture was diluted
with ethyl acetate and washed with 10% citric acid
solution and saturated sodium bicarbonate. The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The resulting residue was crystallized from ethanol to
give 6b (139 mg, 84%) as a whitesolid: mp 162–163 ꢂC;
1H NMR (CDCl3) d 1.46 (9H, s), 1.67–1.77 (2H, m),
1.85–1.97 (5H, m), 3.27–3.36 (2H, m), 3.63–3.76 (2H,

1516
F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
ꢂ
m), 4.37–4.45 (1H, m), 5.02 (2H, s), 6.81 (2H, d, J=8.8
Hz), 6.88 (2H, d, J=8.8 Hz), 7.56–7.65 (3H, m), 7.83
(1H, d, J=8.3 Hz), 7.89 (1H, d, J=8.3 Hz), 8.13 (1H,
s); FAB Ms m/e (M+H)+ 500.
dine(2 mL) at 3 C was added methanesulfonyl chloride
(315 mg, 2.8 mmol). After 15 h, the reaction mixture
was diluted with ethyl acetate and washed with 10%
citric acid solution and saturated aqueous sodium
bicarbonate. The organic layer was dried over Na2SO4
and concentrated in vacuo. The resulting residue was
chromatographed on silica gel eluting with ethyl ace-
tate/n-hexane (20:80–30:70) to give 6f (264 mg, 90%) as
a whiteamorphous powder: 1H NMR (CDCl3) d 1.45
(9H, s), 1.63–1.73 (2H, m), 1.80–1.90 (2H, m), 2.99 (3H,
s), 3.25–3.35 (2H, m), 3.60–3.70 (2H, m), 4.34–4.40 (1H,
m), 4.97 (2H, s), 6.80 (2H, d, J=8.8 Hz), 7.17 (2H, d,
J=8.8 Hz), 7.59 (1H, dd, J=1.5, 8.8 Hz), 7.63–7.72
(2H, m), 7.85 (1H, d, J=8.3 Hz), 7.88 (1H, d, J=8.8
Hz), 8.13 (1H, s); FAB Ms m/e (M+H)+ 536.
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7 - cyano - 2 - naphthyl)methyl]propionamide (6c). To a
stirred solution of 5 (150 mg, 0.33 mmol) in 1,2-
dichloroethane (5 mL) at ambient temperature was
added triethylamine (50 mg, 0.5 mmol) and propionyl
chloride (46 mg, 0.5 mmol). After 8 h, the reaction
mixture was poured into ice water and extracted with
methylene chloride. The organic layer was washed with
brine, dried over Na2SO4 and concentrated in vacuo.
The resulting residue was chromatographed on silica gel
eluting with ethyl acetate/n-hexane (1:12) to give 6c (167
mg, 98%) as a whitesolid: mp 194–195 ꢂC; H NMR
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7 - cyano - 2 - naphthyl)methyl]ethanesulfonamide (6g).
Compound 6g was synthesized from 5 and ethanesulfo-
nyl chlorideaccording to thesameprocedureas that for
6f. Compound 6g was obtained as a white solid (97%
1
(CDCl3) d 1.05 (3H, t J=7.0 Hz), 1.46 (9H, s), 1.54–
2.02 (6H, m), 3.01–3.38 (2H, m), 3.50–3.74 (2H, m),
4.34–4.51 (1H, m), 5.01 (2H, s), 6.91–7.12 (4H, m), 7.45–
7.88 (5H, m), 8.14 (1H, s); FAB Ms m/e (M+H)+ 514.
yield): mp 144–145 ꢂC; H NMR (CDCl3) d 1.43–1.50
1
1-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-1-
[(7-cyano-2-naphthyl)methyl]-3-ethylurea (6d). To a stir-
red solution of 5 (150 mg, 0.33 mmol) in methylene
chloride (2 mL) was added ethyl isocyanate (35 mg, 0.49
mmol). The reaction mixture was stirred at ambient
temperature for 15 h. Then, ethyl isocyanate (117 mg,
1.64 mmol) was added again, stirred for 6 h and the
reaction mixture was concentrated in vacuo. The crude
residue was chromatographed on silica gel eluting with
ethyl acetate/n-hexane (35:65) to give 6d (154 mg, 88%)
as a whiteamorphous powder: 1H NMR (CDCl3) d 1.06
(3H, t, J=7.3 Hz), 1.46 (9H, s), 1.65–1.78 (2H, m),
1.82–1.95 (2H, m), 3.20–3.37 (4H, m), 3.62–3.75 (2H,
m), 4.24 (1H, t, J=5.5 Hz), 4.36–4.44 (1H, m), 4.99 (2H,
s), 6.83 (2H, d, J=7.0 Hz), 6.96 (2H, d, J=7.3 Hz), 7.57
(1H, d, J=8.5 Hz), 7.62–7.68 (2H, m), 7.83 (1H, d
J=8.5 Hz), 7.87 (1H, d, J=8.5 Hz), 8.13 (1H, s); FAB
Ms m/e (M)+ 528.
(12H, m), 1.63–1.73 (2H, m), 1.80–1.91 (2H, m), 3.12
(2H, q, J=7.3 Hz), 3.25–3.36 (2H, m), 3.60–3.70 (2H,
m), 4.33–4.41 (1H, m), 5.00 (2H, s), 6.78 (2H, d, J=6.8
Hz), 7.15 (2H, d, J=6.8 Hz), 7.58 (1H, d, J=8.5 Hz),
7.64 (1H, s), 7.69 (1H, d, J=8.5 Hz), 7.84 (1H, d J=8.3
Hz), 7.88 (1H, d, J=8.3 Hz), 8.13 (1H, s); FAB Ms m/e
(M+H)+ 550.
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7-cyano-2-naphthyl)methyl]propanesulfonamide
(6h).
Compound 6h was synthesized from 5 and propane-
sulfonyl chlorideaccording to thesameprocdeureas
that for 6f. Compound 6hꢂwas obtained as a white solid
1
(71% yield): mp 177–178 C; H NMR (CDCl3) d 1.08
(3H, t, J=7.3 Hz), 1.45 (9H, s) 1.63–1.73 (2H, m), 1.80–
2.00 (4H, m), 3.02–3.09 (2H, m), 3.24–3.34 (2H, m),
3.59–3.69 (2H, m), 4.33–4.41 (1H, m), 4.98 (2H, s), 6.79
(2H, d, J=9.3 Hz), 7.15 (2H, d, J=9.3 Hz), 7.58 (1H,
dd, J=8.3, 1.5 Hz), 7.64 (1H, s), 7.69 (1H, dd, J=8.8,
1.5 Hz), 7.84 (1H, d, J=8.3 Hz), 7.87 (1H, d, J=8.3
Hz), 8.12 (1H, s); FAB Ms m/e (M)+ 563.
Ethyl N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phe-
nyl}-N-[(7-cyano-2-naphthyl)methyl]carbamate (6e). To
a stirred solution of 5 (150 mg, 0.33 mmol) in DMF (2
mL) at ambient temperature was added ethyl chloro-
formate(157 mL, 1.65 mmol) and K 2CO3 (271 mg, 1.98
mmol). After 3.5 h, water was added and the mixture
was extracted with methylene chloride. The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The resulting residue was chromatographed on silica gel
eluting with ethyl acetate/n-hexane (20:80) to give 6e
(169 mg, 97%) as a whiteamorphous powder: 1H NMR
(CDCl3) d 1.15–1.30 (3H, br), 1.46 (9H, s), 1.65–1.80
(2H, m), 1.80–1.95 (2H, m), 3.25–3.37 (2H, m), 3.60–
3.75 (2H, m), 4.20 (2H, q, J=6.8 Hz), 4.35–4.45 (1H,
m), 4.98 (2H, s), 6.79 (2H, d, J=8.8 Hz), 6.90–6.71 (2H,
br), 7.55–7.64 (2H, m), 7.68 (1H, s), 7.84 (1H, d, J=8.3
Hz), 7.89 (1H, d, J=8.9 Hz), 8.15 (1H, s); FAB Ms m/e
(M)+ 529.
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7 - cyano - 2 - naphthyl)methyl]butanesulfonamide (6i).
Compound 6i was synthesized from 5 and butanesulfo-
nyl chlorideaccording to thesameprocedureas that for
6f. Compound 6i was obtained as a white solid (62%
yield): mp 166–167 ꢂC; H NMR (CDCl3) d 0.95 (3H, t,
1
J=7.1 Hz), 1.24–1.36 (2H, m) 1.45 (9H, s), 1.63–1.94
(6H, m), 2.94–3.85 (6H, m), 4.27–4.53 (1H, m), 5.02
(2H, s), 6.71–7.37 (4H, m), 7.42–7.96 (5H, m), 8.07 (1H,
s); FAB Ms m/e (M)+ 577.
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7-cyano-2-naphthyl)methyl]trifluoromethanesulfonamide
(6j). Compound 6j was synthesized from 5 and tri-
fluoromethanesulfonic anhydride according to the same
procedure as that for 6f. Compound 6j was obtained as
a white amorphous powder (54% yield): 1H NMR
(CDCl3) d 1.45 (9H, m), 1.56–1.92 (4H, m), 3.11–3.82
(4H, m), 4.22–4.51 (1H, m), 5.02 (2H, s), 6.65–7.18 (4H,
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7 - cyano - 2 - naphthyl)methyl]methanesulfonamide (6f).
To a stirred solution of 5 (250 mg, 0.55 mmol) in pyri-

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1517
m), 7.46–7.97 (5H, m), 8.11 (1H, s); FAB Ms m/e
(M+H)+ 590.
Hz), 7.34 (2H, d, J=8.8 Hz), 7.66, (1H, dd, J=8.8, 1.4
Hz), 7.80, (1H, dd, J=8.3, 1.5 Hz), 7.91 (1H, s), 8.01
(1H, d, J=8.8 Hz), 8.09 (1H, d, J=8.3 Hz), 8.46 (1H s),
9.23 (5H, br); FAB Ms m/e (M+H)+ 453. Anal. calcd
for C24H28N4O3S 2.0HCl 3.2H2O: C, 49.43; H, 6.29; N,
9.61; S, 5.50; Cl, 12.16. Found: C, 49.55; H, 6.32; N,
9.60; S, 5.65; Cl, 12.08.
Ethyl (N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phe-
nyl}-N-[(7-cyano-2-naphthyl)methyl]sulfamoyl)acetate
(6k). Compound 6k was synthesized from 5 and ethyl
(chlorosulfonyl)acetate32 according to thesameproce-
dureas that for 6f. Compound 6k was obtained as a
whitesolid (62% yiled): mp 156–157 ꢂC; 1H NMR
(CDCl3) d 1.39 (3H, t, J=7.2 Hz), 1.45 (9H, s), 1.57–
1.75 (2H, m), 1.78–1.92 (2H, m), 3.24–3.36 (2H, m),
3.59–3.71 (2H, m), 4.05 (2H, s), 4.30–4.42 (3H, m), 5.04
(2H, s), 6.80 (2H, d, J=9.0 Hz), 7.29 (2H, d, J=9.0
Hz), 7.58 (1H, dd, J=1.8, 8.7 Hz), 7.66 (1H, dd, J=1.8,
8.7 Hz), 7.80–7.90 (2H, m), 8.12 (1H, s); FAB Ms m/e
(M+H)+ 608.
.
.
To a stirred solution of 7f (165 mg, 0.28 mmol) in EtOH
(4 mL) at ambient temperature was added ethyl acet-
imidatehydrochloride(90 mg, 0.73 mmol) and triethyl-
amine(157 mL, 1.13 mmol). Themixturewas allowed to
stir for 4 days at ambient temperature, and then con-
centrated in vacuo. The resulting residue was chroma-
tographed on ODS-gel eluting with MeOH/H2O (0:100–
5:95). MeOH was removed in vacuo, and the aqueous
solution was lyophilized after acidification with 1 N
HCl. 8f (135 mg 78%) was obtained as a white amor-
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-N-
[(7-cyano - 2- naphthyl)methyl] -benzenesulfonamide (6l).
Compound 6l was synthesized from 5 and benzene-
sulfonyl chlorideaccording to thesameprocdeureas
that for 6f. Compound 6l was obtained as a white
1
phous powder: H NMR (DMSO-d6) d 1.60–1.74 (2H,
m) 1.94–2.03 (2H, m) 2.27 (3H, s), 3.13 (3H, s), 3.43–
3.55 (2H, m), 3.63–3.73 (1H, m), 3.73–3.80 (1H, m),
4.60–4.65 (1H, m), 5.03 (2H, s), 6.93 (2H, d, J=9.2
Hz), 7.34 (2H, d, J=8.5 Hz), 7.66, (1H, dd, J=8.6,
1.2 Hz), 7.81, (1H, dd, J=8.6, 1.8 Hz), 7.91 (1H, s),
8.01 (1H, d, J=8.6 Hz), 8.09 (1H, d, J=8.5 Hz), 8.47
(1H, s), 8.74 (1H, s), 9.20–9.31 (3H, m), 9.48 (2H, s);
FAB Ms m/e (M+H)+ 494. Anal. calcd for
1
amorphous powder (91% yield): H NMR (CDCl3) d
1.45 (9H, s), 1.60–1.70 (2H, m), 3.23–3.09 (2H, m),
3.23–3.33 (2H, m), 3.60–3.70 (2H, m), 4.30–4.38 (1H,
m), 4.86 (2H, s), 6.69 (2H, d, J=8.8 Hz), 6.86 (2H, d,
J=8.8 Hz), 7.48–7.73 (8H, m), 7.82 (1H, d, J=7.8 Hz),
7.86 (1H, d, J=8.3 Hz), 8.09 (1H, s); FAB Ms m/e
(M)+ 597.
.
.
C26H31N5O3S 2.0HCl 3.0H2O: C, 50.32; H, 6.33; N,
11.28; S, 5.17; Cl, 11.43. Found: C, 50.58; H, 6.22; N,
11.39; S, 5.07; Cl, 11.87.
4-(N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-
N -[(7 -cyano-2- naphthyl)methyl]sulfamoyl)benzoic acid
(6m). Compound 6m was synthesized from 5 and 4-
(chlorosulfonyl)benzoic acid according to the same
procedure as that for 6f. Compound 6m was obtained as
a whitesolid (58% yiled): mp 226–227 ꢂC; 1H NMR
(DMSO-d6) d 1.32–1.47 (11H, m), 1.75–1.86 (2H, m),
3.00–3.15 (2H, m), 3.55–3.66 (2H, m), 4.37–4.47(1H, m),
4.97 (2H, s), 6.80 (2H, d, J=8.8 Hz), 6.96 (2H, d, J=9.3
Hz), 7.67 (1H, d, J=8.8 Hz), 7.74 (1H, dd, J=8.3, 1.5
Hz), 7.81, (2H, d, J=8.3 Hz), 7.89 (1H, s), 8.00 (1H, d,
J=8.3 Hz), 8.07 (1H, d, J=8.8 Hz), 8.15 (2H, d, J=8.3
Hz), 8.52 (1H s), 13.52, (1H, s); FAB Ms m/e (M)+ 597.
7-({4-[(1-Acetimidoyl - 4 - piperidyl)oxy]anilino}methyl)-
naphthalene-2-carboxamidine (8a). Compound 8a was
synthesized from 5 according to thesameprocedureas
that for 8f. Compound 8a was obtained as a pale brown
1
amorphous powder (25% yield): H NMR (DMSO-d6)
d 1.60–1.73 (2H, m), 1.88–1.99 (2H, m), 2.27 (3H, s),
3.42–3.55 (2H, m), 3.63–3.77 (2H, m), 4.35–4.42 (1H,
m), 4.45 (2H, d, J=6.1 Hz), 6.16 (1H, t, J=6.1 Hz),
6.55 (2H, d, J=8.5 Hz), 6.75 (2H, d, J=8.5 Hz), 7.74,
(1H, d, J=8.5 Hz), 7.79, (1H, d, J=8.5 Hz), 7.99 (1H,
s), 8.03 (1H, d, J=8.5 Hz), 8.11 (1H, d, J=8.5 Hz), 8.43
(1H s), 9.0-9.6 (7H, br); FAB Ms m/e (M+H)+ 416.
.
.
Anal. calcd for C25H29N5O 3.2HCl 4.0H2O: C, 49.69;
H, 6.71; N, 11.59; Cl, 18.77. Found: C, 49.69; H, 6.54;
N, 11.84; Cl, 18.98.
General procedure for synthesis of bis-amidine deriva-
tives 8. N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-
[(7-amidino-2-naphthyl)methyl]methanesulfonamide (8f).
HCl gas was bubbled through a solution of 6f (711 mg,
1.33 mmol) in EtOH (15 mL) and CHCl3 (10 mL) under
ꢀ20 ꢂC for 20 min. Themixturewas allowed to stir for
30 h at 5 ꢂC, and then concentrated in vacuo. To the
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino - 2 - naphthyl)methyl]acetamide (8b). Compound 8b
was synthesized from 6b according to thesameproce-
dureas that for 8f. Compound 8b was obtained as a
white amorphous powder (69% yield): 1H NMR
(DMSO-d6) d 1.61–1.76 (2H, m), 1.87 (3H, s), 1.95–2.06
(2H, m), 2.28 (3H, s), 3.43–3.55 (2H, m), 3.63–3.73 (1H,
m), 3.75–3.84 (1H, m), 4.61–4.69 (1H, m), 5.03 (2H, s),
6.96 (2H, d, J=8.5 Hz), 7.15 (2H, d, J=8.5 Hz), 7.59
(1H, d, J=8.5 Hz), 7.81 (1H, d, J=8.5 Hz), 7.85 (1H,
s), 8.01 (1H, d, J=8.5 Hz), 8.11 (1H, d, J=8.5 Hz), 8.48
(1H s), 8.80 (1H, s), 9.30 (2H, s), 9.33 (1H, s), 9.51 (2H,
s); FAB Ms m/e (M+H)+ 458. Anal. calcd for
crudeimidatedissolved in EtOH (15 mL) and CHCl
3
(20 mL) was added ammonium acetate (1.0 g, 13.3
mmol) at ambient temperature. The reaction mixture
was stirred at ambient temperature for 16 h and con-
centrated in vacuo. The resulting residue was chroma-
tographed on ODS-gel eluting with MeOH/H2O (0:100–
5:95). MeOH was removed in vacuo, and the aqueous
solution was lyophilized after acidification with 1 N
HCl. 7f (312 mg 40%) was obtained as an amorphous
powder: 1H NMR (DMSO-d6) d 1.68–1.81 (2H, m),
1.97–2.07 (2H, m), 2.48–2.52 (2H, m), 3.10–3.18 (5H,
m), 4.51–4.61(1H, m), 5.02 (2H, s), 6.92 (2H, d, J=9.2
.
.
C27H31N5O2 2.3HCl 4.1H2O: C, 52.71; H, 6.80; N,
11.38; Cl, 13.23. Found: C, 52.47; H, 6.54; N, 11.60; Cl,
12.88.

1518
F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino - 2 - naphthyl)methyl]propionamide (8c). Compound
8c was synthesized from 6c according to thesamepro-
cedure as that for 8f. Compound 8c was obtained as a
white amorphous powder (27% yield): 1H NMR
(DMSO-d6) d 0.99 (3H, t, J=9.3 Hz), 1.64–1.80 (2H, m)
1.94–2.14 (4H, m), 2.28 (3H, s), 3.02–3.09 (2H, m),
3.62–3.80 (2H, m), 4.60–4.64 (1H, m), 5.03 (2H, s), 6.95
(2H, d, J=9.2 Hz), 7.12 (2H, d, J=8.5 Hz), 7.48 (1H, d,
J=8.5 Hz), 7.78–7.86 (2H, m), 8.00 (1H, d, J=8.4 Hz),
8.11 (1H, d, J=8.6 Hz), 8.48 (1H s), 8.82 (1H, s), 9.30
(3H, s), 9.51 (2H, s); FAB Ms m/e (M+H)+ 472. Anal.
(M+H)+ 508. Anal. calcd for C27H33N5O3S 2.0HCl
.
3.0H2O: C, 51.10; H, 6.51; N, 11.04; S, 5.50; Cl, 11.17.
Found: C, 50.91; H, 6.32; N, 11.17; S, 5.08; Cl, 11.41.
.
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino-2-naphthyl)methyl]-propanesulfonamide (8h). Com-
pound 8h was synthesized from 6h according to the
sameprocdeureas that for
8f. Compound 8h was
obtained as a white amorphous powder (43% yield): 1H
NMR (DMSO-d6) d 1.03 (3H, t, J=7.6 Hz), 1.60–1.73
(2H, m), 1.74–1.85 (2H, m), 1.93–2.03 (2H, m), 2.27
(3H, s), 3.21–3.25 (2H, m), 3.42–3.56 (2H, m), 3.63–3.71
(1H, m), 3.75–3.82 (1H, m), 4.59–4.63 (1H, m), 5.05
(2H, s), 6.92 (2H, d, J=8.5 Hz), 7.33 (2H, d, J=8.5
Hz), 7.66 (1H, dd, J=8.6, 1.2 Hz), 7.82 (1H, dd, J=8.5,
1.8 Hz), 7.89 (1H, s), 8.01 (1H, d, J=8.6 Hz), 8.09 (1H,
d, J=8.5 Hz), 8.48 (1H, s), 8.80 (1H, s), 9.28–9.38 (3H,
m), 9.52 (2H, s); FAB Ms m/e (M+H)+ 522. Anal.
.
.
calcd for C28H33N5O2 2.4HCl 3.5H2O: C, 54.05; H,
6.87; N, 11.26; Cl, 13.68. Found: C, 54.03; H, 6.78; N,
11.06; Cl, 13.50.
1-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-1-[(7-ami-
dino-2-naphthyl)methyl]-3-ethylurea (8d). Compound 8d
was synthesized from 6d according to thesameproce-
dureas that for 8f. Compound 8d was obtained as a
white amorphous powder (62% yield): 1H NMR
(DMSO-d6) d 0.99 (3H, t, J=7.3 Hz), 1.61–1.73 (2H,
m), 1.95–2.05 (2H, m), 2.28 (3H, s), 3.01–3.13 (2H, m),
3.45–3.59 (2H, m), 3.64–3.84 (2H, m), 4.58–4.65 (1H,
m), 4.97 (2H, s), 5.73 (1H, t, J=5.5 Hz), 6.93 (2H, d,
J=8.6 Hz), 7.08 (2H, d, J=9.2 Hz), 7.60 (1H, d, J=8.5
Hz), 7.75–7.86 (2H, m), 8.00 (1H, d, J=8.5 Hz), 8.10
(1H, d, J=9.2 Hz), 8.46 (1H, s), 8.85 (1H, s), 9.23–9.40
(3H, m), 9.51 (2H, s); FAB Ms m/e (M+H)+ 487. Anal.
.
.
calcd for C28H35N5O3S 2.0HCl 3.0H2O: C, 51.85; H,
6.68; N, 10.80; S, 4.94; Cl, 10.93. Found: C, 51.44; H,
6.58; N, 10.79; S, 4.90; Cl, 11.31.
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino - 2 - naphthyl)methyl]butanesulfonamide (8i). Com-
pound 8i was synthesized from 6i according to thesame
procedure as that for 8f. Compound 8i was obtained as
a white amorphous powder (35% yield): 1H NMR
(DMSO-d6) d 0.92 (3H, t, J=7.3 Hz), 1.41–1.46 (2H,
m), 1.62–1.78 (4H, m), 1.91–2.01 (2H, m), 2.27 (3H, s),
3.24 (2H, t, J=7.9 Hz), 3.41–3.55 (2H, m), 3.63–
3.80(2H, m), 4.58–4.62 (1H, m), 5.06 (2H, s), 6.91 (2H,
d, J=9.2 Hz), 7.33 (2H, d, J=9.2 Hz), 7.65 (1H, d,
J=8.5 Hz), 7.80 (1H, d, J=6.7 Hz), 7.90 (1H, s), 8.01
(1H, d, J=8.6 Hz), 8.09 (1H, d, J=8.5 Hz), 8.46 (1H,
s), 8.72 (1H, s), 9.18–9.30 (3H, m), 9.47 (2H, s); FAB
.
.
calcd for C28H34N6O2 2.1HCl 2.5H2O: C, 55.29; H,
6.81; N, 13.82; Cl, 12.24. Found: C, 55.10; H, 6.77; N,
13.81; Cl, 12.36.
Ethyl N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-
[(7 - amidino - 2 - naphthyl)methyl]carbamate (8e). Com-
pound 8e was synthesized from 6e according to the
Ms
m/e
(M+H)+
536.
Anal.
calcd
for
.
.
sameprocdeureas that for
8f. Compound 8e was
C29H37N5O3S 2.1HCl 2.0H2O: C, 53.73; H, 6.70; N,
10.80; S, 4.95; Cl, 11.48. Found: C, 53.96; H, 6.47; N,
10.90; S, 4.65; Cl, 11.56.
obtained as a white amorphous powder (51% yield): 1H
NMR (DMSO-d6) d 1.16 (3H, t, J=7.0 Hz), 1.61–1.77
(2H, m), 1.95–2.06 (2H, m), 2.29 (3H, s), 3.43–3.57 (2H,
m), 3.65–3.75 (1H, m), 3.75–3.83 (1H, m), 4.12 (2H, q,
J=7.0 Hz), 4.58–4.65 (1H, m), 5.03 (2H, s), 6.91 (2H, d,
J=8.5 Hz), 7.15 (2H, d, J=8.5 Hz), 7.60 (1H, d, J=8.5
Hz), 7.82 (1H, d, J=8.5 Hz), 7.86 (1H, s), 8.03 (1H, d,
J=8.5 Hz), 8.11 (1H, d, J=8.5 Hz), 8.50 (1H, s), 8.81
(1H, s), 9.33 (2H, s), 9.35 (1H, s), 9.53 (2H, s); FAB Ms
m/e (M+H)+ 488. Anal. calcd for C28H33N5O3
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino-2-naphthyl)methyl]trifluoromethanesulfonamide (8j).
Compound 8j was synthesized from 6j according to the
sameprocdeureas that for
8f. Compound 8j was
obtained as a white amorphous powder (14% yield): 1H
NMR (DMSO-d6) d 1.60–1.74 (2H, m), 1.92–2.05 (2H,
m), 2.26 (3H, s), 3.44–3.50 (2H, m), 3.67–3.76 (2H, m),
4.63–4.65 (1H, m), 5.25 (2H, s), 6.95 (2H, d, J=9.2 Hz),
7.30 (2H, d, J=9.2 Hz), 7.60 (1H, d, J=6.7 Hz), 7.83
(1H, d, J=7.3 Hz), 7.92 (1H, s), 8.04 (1H, d, J=8.6
Hz), 8.12 (1H, d, J=8.6 Hz), 8.48 (1H, s), 8.68 (1H, s),
9.20 (3H, s), 9.47 (2H, s); FAB Ms m/e (M+H)+ 548.
.
.
2.0HCl 3.0H2O: C, 54.72; H, 6.72; N, 11.40; Cl, 11.54.
Found: C, 54.91; H, 6.60; N, 11.43; Cl, 11.66.
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino - 2 - naphthyl)methyl]ethanesulfonamide (8g):. Com-
pound 8g was synthesized from 6g according to thesame
procedure as that for 8f Compound 8g was obtained as a
white amorphous powder (42% yield): 1H NMR
(DMSO-d6) d 1.33 (3H, t, J=7.3 Hz), 1.59–1.73 (2H, m)
1.93–2.03 (2H, m), 2.27 (3H, s), 3.26 (2H, q, J=7.3 Hz),
3.43–3.54 (2H, m), 3.63–3.71 (1H, m), 3.71–3.80 (1H,
m), 4.58–4.64 (1H, m), 5.07 (2H, s), 6.91 (2H, d, J=9.2
Hz), 7.33 (2H, d, J=9.2 Hz), 7.65 (1H, d, J=8.5 Hz),
7.81 (1H, d, J=8.5 Hz), 7.90 (1H, s), 8.01 (1H, d, J=8.5
Hz), 8.09 (1H, d, J=9.2 Hz), 8.47 (1H, s), 8.74 (1H, s),
9.26 (2H, s), 9.28 (1H, s), 9.50 (2H, s); FAB Ms m/e
.
.
Anal. calcd for C26H28N5O3SF3 2.3HCl 3.6H2O: C,
44.85; H, 5.43; N, 10.06; S, 4.61; Cl, 11.71; F, 8.19.
Found: C, 45.21; H, 5.29; N, 10.43; S, 4.49; Cl, 11.51; F,
7.78.
Ethyl (N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-
[(7 - amidino - 2 - naphthyl)methyl]sulfamoyl)acetate (8k).
Compound 8k was synthesized from 6k according to
thesameprocedureas that for
8f. Compound 8k was
obtained as a white amorphous powder (52% yield): 1H
NMR (DMSO-d6) d 1.25 (3H, t, J=7.3 Hz), 1.61–1.74

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1519
(2H, m), 1.94–2.04 (2H, m), 2.26 (3H, s), 3.43–3.51 (2H,
m), 3.64–3.78 (2H, m), 4.23 (2H, q, J=7.3 Hz), 4.43
(2H, s), 4.59–4.65 (1H, m), 5.05 (2H, s), 6.94 (2H, d,
J=9.2 Hz), 7.32 (2H, d, J=9.2 Hz), 7.64 (1H, d, J=8.5
Hz), 7.79 (1H, d, J=8.6 Hz), 7.92 (1H, s), 8.01 (1H, d,
J=8.0 Hz), 8.10 (1H, d, J=8.5 Hz), 8.45(1H, s), 8.64
(1H, s), 9.12 (3H, s), 9.43 (2H, s); FAB Ms m/e
(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino - 2 - naphthyl)methyl]sulfamoyl)acetic acid (8o). A
solution of 8k (4.00 g, 5.9 mmol) in 1 N HCl (100 mL)
was stirred under reflux for 1.5 h. The reaction mixture
was concentrated in vacuo and the residue was chroma-
tographed on ODS-gel eluting with CH3CN/H2O (0:100–
1:99). Theproduct containing fractions wreeacidifide
with 1 N HCl (pH=1), combined and concentrated in
vacuo. Theresulting residuewas crystallized from EtOH
to give 8o (1.11 g, 28%) as a whitesolid: mp 203–
(M+H)+ 566. Anal. calcd for C29H35N5O5S 2.2HCl
.
.
1.8H2O: C, 51.35; H, 6.06; N, 10.32; S, 4.73; Cl, 11.50.
Found: C, 51.38; H, 6.14; N, 10.31; S, 4.78; Cl, 11.60.
206 ꢂC, H NMR (DMSO-d6) d 1.59–1.76 (2H, m), 1.92–
1
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino-2-naphthyl)methyl]-benzenesulfonamide (8l). Com-
pound 8l was synthesized from 6l according to thesame
procedure as that for 8f. Compound 8l was obtained as
a white amorphous powder (45% yield): 1H NMR
(DMSO-d6) d 1.58–1.70 (2H, m), 1.91–2.03 (2H, m),
2.27 (3H, s), 3.42–3.49 (2H, m), 3.67–3.77 (2H, m),
4.58–4.61 (1H, m), 4.99 (2H, s), 6.84 (2H, d, J=9.2 Hz),
6.98 (2H, d, J=8.5 Hz), 7.64–7.76 (6H, m), 7.82 (1H, d,
J=10.4 Hz), 7.92 (1H, s), 8.01 (1H, d, J=8.6 Hz), 8.09
(1H, d, J=8.5 Hz), 8.46(1H, s), 8.81 (1H, s), 9.34 (3H,
s), 9.53 (2H, s); FAB Ms m/e (M+H)+ 570. Anal. calcd
2.05 (2H, m), 2.28 (3H, s), 3.40–3.57 (2H, m), 3.64–3.85
(2H, m), 4.30 (2H, s), 4.59–4.68 (1H, m), 5.05 (2H, s), 6.94
(2H, d, J=8.8 Hz), 7.33 (2H, d, J=8.8 Hz), 7.66 (1H, d,
J=8.0 Hz), 7.83(1H, d, J=8.4 Hz), 7.90 (1H, s), 8.02 (1H,
d, J=8.0 Hz), 8.10 (1H, d, J=8.4 Hz), 8.49(1H, s), 8.85
(1H, s), 9.40 (3H, s), 9.55 (2H, s); FAB Ms m/e (M+H)+
538. Anal. calcd for C27H31N5O5S 2.0HCl 3.0H2O: C,
48.80; H, 5.91; N, 10.54; S, 4.82; Cl, 10.67. Found: C,
48.83; H, 5.53; N, 10.55; S, 4.95; Cl, 10.92.
.
.
4-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-
amidino-2-naphthyl)methyl]-sulfamoyl)benzoic acid (8p).
A solution of 8m (1.85 g, 2.55 mmol) in 1 N HCl (200
mL) was refluxed for 7 h. The reaction mixture was
concentrated and dried in vacuo. Compound 8p (1.74 g,
.
.
for C31H33N5O3S 2.1HCl 2.2H2O: C, 55.42; H, 5.93; N,
10.42; S, 4.77; Cl, 11.08. Found: C, 55.16; H, 5.77; N,
10.43; S, 4.84; Cl, 11.12.
1
97%) was obtained as a white amorphous powder: H
Ethyl 4-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N
-[(7-amidino-2-naphthyl)methyl]sulfamoyl)benzoate (8m).
Compound 8m was synthesized from 6m according to
NMR (DMSO-d6) d 1.57–1.72 (2H, m), 1.91–2.02 (2H, m),
2.26 (3H, s), 3.40–3.51 (2H, m), 3.62–3.80 (2H, m), 4.55–
4.62 (1H, m), 5.01 (2H, s), 6.85 (2H, d, J=9.2 Hz), 7.00
(2H, d, J=9.2 Hz), 7.67 (1H, d, J=9.5 Hz), 7.77–7.85 (3H,
m), 7.92 (1H, s), 8.02 (1H, d, J=8.6 Hz), 8.09 (1H, d,
J=8.5 Hz), 8.16 (2H, d, J=8.6 Hz), 8.45 (1H, s), 8.74 (1H,
s), 9.18–9.34 (3H, m), 9.49 (2H, s), 13.53 (1H, s); FAB Ms
thesameprocedureas that for
8f. Compound 8m was
obtained as a white amorphous powder (44% yield): 1H
NMR (DMSO-d6) d 1.37 (3H, t, J=7.0 Hz), 1.57–1.73
(2H, m), 1.92–2.02 (2H, m), 2.26 (3H, s), 3.40–3.50 (2H,
m), 3.62–3.80 (2H, m), 4.39 (2H, q, J=7.0 Hz), 4.55–
4.62 (1H, m), 5.00 (2H, s), 6.85 (2H, d, J=9.2 Hz), 6.99
(2H, d, J=8.6 Hz), 7.66 (1H, d, J=8.6 Hz), 7.81(1H,
dd, J=1.8, 9.2 Hz), 7.85 (2H, d, J=8.5 Hz), 7.92 (1H,
s), 8.02 (1H, d, J=8.6 Hz), 8.09 (1H, d, J=8.5 Hz), 8.18
(2H, d, J=8.6 Hz), 8.44 (1H, s), 8.71 (1H, s), 9.13–9.27
(3H, m), 9.47 (2H, s); FAB Ms m/e (M+H)+ 628. Anal.
m/e (M+H)+ 600. Anal. calcd for C32H33N5O5S 2.1HCl
.
.
1.4H2O: C, 54.79; H, 5.45; N, 9.98; S, 4.57; Cl, 10.61.
Found: C, 54.63; H, 5.34; N, 10.07; S, 4.55; Cl, 10.57.
tert-Butyl N - (N - {4- [(1 - tert - Butoxycarbonyl - 4 -piper-
idyl)oxy]phenyl} - N - [(7 - cyano - 2 - naphthyl)methyl]-
sulfamoyl)carbamate (9a). To a stirred solution of
chlorosulfonylisocyanate (5.0 g, 35.3 mmol) in benzene
(15 mL) at ambient temperature was added tert-butyl
alcohol (2.6 g, 35.3 mmol) dropwise. After 1 h, n-hexane
(30 mL) was added and the resulting precipitate was fil-
tered, washed with n-hexane and dried in vacuo. tert-
Butyl chlorosulfonylcarbamate( 11a) (4.1 g, 54%) was
obtained as a hygroscopic white solid. 1H NMR
(CDCl3) d 1.48 (9H, s). Compound 9a was synthesized
from 5 and 11a according to thesameprocedureas that
for 6f. Compound 9a was obtained as a white amorphous
.
.
calcd for C34H37N5O5S 2.0HCl 1.3H2O: C, 56.40; H,
5.79; N, 9.67; S, 4.43; Cl, 9.79. Found: C, 56.66; H, 6.09;
N, 9.67; S, 4.52; Cl, 10.09.
(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino-2-naphthyl)methyl]sulfamoyl)acetamide (8n). Com-
pound 8n was synthesized from 6k according to the
sameprocdeureas that for
8f, but using saturated
ammonia solution in ethanol instead of ammonium
acetate for conversion of the imidate to the amidine and
ester to amide simultaneously. Compound 8n was
obtained as a white amorphous powder (68% yield): 1H
NMR (DMSO-d6) d 1.60–1.72 (2H, m), 1.94–2.02 (2H,
m), 2.26 (3H, s), 3.42–3.51 (2H, m), 3.64–3.78 (2H, m),
4.07 (2H, s), 4.58–4.64 (1H, m), 5.01 (2H, s), 6.93 (2H,
d, J=8.6 Hz), 7.38 (2H, d, J=9.2 Hz), 7.52 (1H,s), 7.66
(1H, d, J=8.6 Hz), 7.80 (1H, d, J=8.6 Hz), 7.84 (1H,
s), 7.90 (1H, s), 8.00 (1H, d, J=8.60 Hz), 8.08 (1H, d,
J=9.2 Hz), 8.44 (1H, s), 8.84–9.14 (5H, br); FAB Ms m/
1
powder (86% yield): H NMR (CDCl3) d 1.45 (9H, s),
1.57 (9H, s), 1.63–1.73 (2H, m), 1.80–1.91 (2H, m), 3.25–
3.35 (2H, m), 3.60–3.70 (2H, m), 4.33–4.43 (1H, m), 5.21
(2H, s), 6.80 (2H, d, J=9.3 Hz), 7.00 (1H, s), 7.18 (2H,
d, J=8.8 Hz), 7.58 (1H, dd, J=1.7, 8.5 Hz), 7.62–7.70
(2H, m), 7.84 (1H, d, J=6.6 Hz), 7.87 (1H, d, J=8.3
Hz), 8.13 (1H, s); FAB Ms m/e (M)+ 636.
Ethyl N-(N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-
phenyl}-N-[(7-cyano-2-naphthyl)methyl]sulfamoyl)carba-
mate (9b). Ethyl chlorosulfonylcarbamate( 11b) was
synthesized from chlorosulfonylisocyanate and ethanol
e (M+H)+ 537. Anal. calcd for C27H32N6O4S 2.0HCl
.
.
2.0H2O: C, 50.23; H, 5.93; N, 13.02; S, 4.97; Cl, 10.98.
Found: C, 50.10; H, 6.01; N, 12.97; S, 5.05; Cl, 10.58.

1520
F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
according to thesameprocedureas that for 11a. Com-
pound 11b was obtained as a hygroscopic white solid
(88% yield). H NMR (CDCl3) d 1.38 (3H, t, J=9.5
(2H, m), 3.60–3.71 (5H, m), 4.21 (2H, s), 4.34–4.44 (3H,
m), 5.19 (2H, s), 6.78 (2H, d, J=8.8 Hz), 7.13 (2H, d,
J=8.8 Hz), 7.58 (1H, d, J=8.5 Hz), 7.62–7.69 (2H, m),
7.84 (1H, d, J=8.8 Hz), 7.88 (1H, d, J=8.8 Hz), 8.12
(1H, s); FAB Ms m/e (M)+ 680.
1
Hz), 4.39 (2H, q, J=9.5 Hz). Compound 9b was syn-
thesized from 5 and 11b according to thesameproce-
dureas that for 6f. Compound 9b was obtained as a
whiteamorphous powder (72% yield): 1H NMR (CDCl3)
d 1.37 (3H, t, J=7.1 Hz), 1.45 (9H, s), 1.62–1.74 (2H, m),
1.80–1.91 (2H, m), 3.25–3.34 (2H, m), 3.60–3.71 (2H, m),
4.29–4.42 (3H, m), 5.21 (2H, s), 6.80 (2H, d, J=8.8 Hz),
7.08–7.20 (3H, m), 7.58 (1H, dd, J=1.5, 8.3 Hz), 7.62–
7.69 (2H, m), 7.84 (1H, d, J=8.8 Hz), 7.87 (1H, d,
J=8.3 Hz), 8.13 (1H, s); FAB Ms m/e (M)+ 608.
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino-2-naphthyl)methyl]-N’-methylsulfamide (8q). Com-
pound 8q was synthesized from 10a according to the
sameprocdeureas that for
8f. Compound 8q was
obtained as a white amorphous powder (54% yield): 1H
NMR (DMSO-d6) d 1.59–1.73 (2H, m) 1.92–2.03 (2H,
m), 2.27 (3H, s), 2.67 (3H, d, J=4.9 Hz), 3.41–3.55 (2H,
m), 3.63–3.72 (1H, m), 3.72–3.81 (1H, m), 4.56–4.63
(1H, m), 4.96 (2H, s), 6.89 (2H, d, J=9.2 Hz), 7.27 (2H,
d, J=8.5 Hz), 7.39–7.46 (1H, m), 7.66 (1H, d, J=8.6
Hz), 7.81 (1H, dd, J=1.5, 8.8 Hz), 7.89 (1H, s), 8.00
(1H, d, J=8.5 Hz), 8.09 (1H, d, J=8.6 Hz), 8.45 (1H,
s), 8.79 (1H, s), 9.23–9.40 (3H, m), 9.52 (2H, m); FAB
tert-Butyl N - (N - {4-[(1 - tert -Butoxycarbonyl -4 -piper-
idyl)oxy]phenyl}-N-[(7-cyano-2-naphthyl)methyl]sulfa-
moyl)-N-methylcarbamate (10a). To a stirred solution
of 9a (172 mg, 0.27 mmol) in THF (0.7 mL) at 3 ꢂC was
added triphenylphosphine (139 mg, 0.53 mmol), meth-
anol (32 mL, 0.79 mmol) and diethyl azodicarboxylate
(83 mL, 0.53 mmol). After stirring at ambient tempera-
ture for 40 min, the reaction mixture was concentrated
in vacuo. The resulting residue was chromatographed
on silica gel eluting with ethyl acetate/n-hexane (15:85)
to give 10a (153 mg, 87%) as a whiteamorphous pow-
Ms
m/e
(M+H)+
509.
Anal.
calcd
for
.
.
C26H32N6O3S 2.1HCl 2.3H2O: C, 49.83; H, 6.22; N,
13.41; S, 5.12; Cl, 11.88. Found: C, 49.81; H, 6.13; N,
13.54; S, 4.87; Cl, 11.81.
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-ami-
dino - 2 - naphthyl)methyl]sulfamide (8r). Compound 8r
was synthesized from 9a according to thesameproce-
dureas that for 8f. Compound 8r was obtained as a
white amorphous powder (47% yield): 1H NMR
(DMSO-d6) d 1.59–1.74 (2H, m), 1.93–2.03 (2H, m),
2.27 (3H, s), 3.43–3.57 (2H, m), 3.63–3.71 (1H, m),
3.73–3.81 (1H, m), 4.56–4.63 (1H, m), 4.90 (2H, s), 6.89
(2H, d, J=9.2 Hz), 7.20 (2H, s), 7.27 (2H, d, J=9.2
Hz), 7.71 (1H, d, J=7.6 Hz), 7.80 (1H, dd, J=1.8, 8.6
Hz), 7.92 (1H, s), 7.99 (1H, d, J=8.5 Hz), 8.08 (1H, d,
J=8.6 Hz), 8.44 (1H, s), 8.80 (1H, s), 9.24–9.37 (3H, m),
9.51 (2H, m); FAB Ms m/e (M+H)+ 495. Anal. calcd
1
der: H NMR (CDCl3) d 1.45 (9H, s), 1.57–1.73 (11H,
m), 1.80–1.92 (2H, m), 2.93 (3H, s), 3.24–3.35 (2H, m),
3.60–3.72 (2H, m), 4.33–4.42 (1H, m), 5.19 (2H, s), 6.79
(2H, d, J=9.3 Hz), 7.15 (2H, d, J=8.8 Hz), 7.57 (1H,
dd, J=1.5, 8.3 Hz), 7.64–7.70 (2H, m), 7.84 (1H, d,
J=8.3 Hz), 7.87 (1H, d, J=8.3 Hz), 8.13 (1H, s); FAB
Ms m/e (M)+ 650.
Ethyl N-(N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]-
phenyl}-N-[(7-cyano-2-naphthyl)methyl]sulfamoyl)-N-tert
-butoxycarbonyl glycinate (10b). To a stirred solution of
9a (1.5 g, 2.36 mmol) of DMF (15 mL) at ambient
temperature was added ethyl bromoacetate (0.79 mL,
7.08 mmol) and K2CO3 (978 mg, 7.08 mmol). Themix-
ture was allowed to stir for 18 h at ambient temperature,
and then concentrated in vacuo. Water was added, and
the mixture was extracted with ethyl acetate. The com-
bined organic extracts were dried over Na2SO4 and
concentrated in vacuo. The resulting residue was chro-
matographed on silica gel eluting with ethyl acetate/n-
hexane (10:90–17:83) to give 10b (1.26 g, 74%) as a
.
.
for C25H30N6O3S 2.0HCl 2.7H2O: C, 48.73; H, 6.12; N,
13.64; S, 5.20; Cl, 11.51. Found: C, 48.83; H, 6.05; N,
13.67; S, 5.15; Cl, 11.84.
Ethyl N-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-
N - [(7- amidino - 2-naphthyl)methyl]sulfamoyl)carbamate
(8s). Compound 8s was synthesized from 9b according
to thesameprocedureas that for
8f. Compound 8s was
obtained as a white amorphous powder (45% yield): 1H
NMR (DMSO-d6) d 1.27 (3H, t, J=7.3 Hz), 1.60–1.75
(2H, m), 1.95–2.03 (2H, m), 2.27 (3H, s), 3.41–3.57 (2H,
m), 3.63–3.73 (1H, m), 3.73–3.82 (1H, m), 4.23 (2H, q,
J=7.3 Hz), 4.60–4.67 (1H, m), 5.17 (2H, s), 6.96 (2H, d,
J=9.2 Hz), 7.23 (2H, d, J=8.5 Hz), 7.65 (1H, d, J=7.3
Hz), 7.82 (1H, d, J=8.5 Hz), 7.91 (1H, s), 8.03 (1H, d,
J=8.5 Hz), 8.10 (1H, d, J=8.5 Hz), 8.47 (1H, s), 8.81
(1H, s), 9.23–9.40 (3H, m), 9.51(2H, s), 11,52 (1H, s);
FAB Ms m/e (M+H)+ 567. Anal. calcd for
whiteamorphous powdre:
1H NMR (CDCl3) d 1.24
(3H, t, J=7.1 Hz), 1.45 (9H, s), 1.58 (9H, s), 1.60–1.76
(2H, m), 1.80–1.92 (2H, m), 3.24–3.35 (2H, m), 3.60–
3.71 (2H, m), 4.08–4.20 (4H, m), 4.32–4.42 (1H, m), 5.19
(2H, s), 6.77 (2H, d, J=8.8 Hz), 7.15 (2H, d, J=8.8
Hz), 7.57 (1H, d, J=8.3 Hz), 7.62–7.70 (2H, m), 7.83
(1H, d, J=8.3 Hz), 7.87 (1H, d, J=8.3 Hz), 8.12 (1H,
s); FAB Ms m/e (M)+ 722.
.
.
Methyl N-(N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)ox-
y]phenyl}-N-[(7-cyano-2-naphthyl)methyl]sulfamoyl)-N-
ethoxycarbonyl glycinate (10c). Compound 10c was
synthesized from 9b and methyl bromoacetate accord-
C28H34N6O5S 2.0HCl 2.6H2O: C, 48.99; H, 6.05; N,
12.24; S, 4.67; Cl, 10.33. Found: C, 48.71; H, 5.98; N,
12.24; S, 4.68; Cl, 10.73.
ing to thesameprocedureas that for
10b. Compound
Ethyl N-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-
N - [(7 - amidino - 2 - naphthyl)methyl]sulfamoyl)glycinate
(8t). Compound 8t was synthesized from 10b according
10c was obtained as a white amorphous powder (98%
yield): 1H NMR (CDCl3) d 1.38 (3H, t, J=7.1 Hz), 1.45
(9H, s), 1.62–1.74(2H, m), 1.80–1.92 (2H, m), 3.24–3.35
to thesameprocedureas that for
8f. Compound 8t was

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1521
obtained as a white amorphous powder (33% yield): 1H
NMR (DMSO-d6) d 1.21 (3H, t, J=7.3 Hz), 1.60–1.73
(2H, m), 1.92–2.02 (2H, m), 2.27 (3H, s), 3.42–3.55 (2H,
m), 3.63–3.70 (1H, m), 3.70–3.80 (1H, m), 3.83 (2H, d,
J=6.1 Hz), 4.14 (2H, q, J=7.3 Hz), 4.56–4.62 (1H, m),
4.94 (2H, s), 6.89 (2H, d, J=9.2 Hz), 7.28 (2H, d, J=9.2
Hz), 7.65 (1H, d, J=8.5 Hz), 7.80 (1H, d, J=8.6 Hz),
7.89 (1H, s), 7.99 (1H, d, J=8.5 Hz), 8.04–8.10 (2H, m),
8.43 (1H, s), 8.74 (1H, s), 9.20–9.31 (3H, m), 9.48 (2H,
s); FAB Ms m/e (M+H)+ 581. Anal. calcd for
m), 4.01 (2H, s), 4.34 (2H, q, J=7.0 Hz), 4.59–4.68 (1H,
m), 5.19 (2H, s), 6.96 (2H, d, J=9.2 Hz), 7.23 (2H, d,
J=9.2 Hz), 7.64 (1H, d, J=8.5 Hz), 7.82 (1H, d, J=8.5
Hz), 7.92 (1H, s), 8.03 (1H, d, J=8.5 Hz), 8.11 (1H, d,
J=8.6 Hz), 8.46 (1H, s), 8.74 (1H, s), 9.20–9.40 (3H, m),
9.48 (2H, s), 13.01 (1H, br-s); FAB Ms m/e (M+H)+
625. Anal. calcd for C30H36N6O7S 2.0HCl 2.6H2O: C,
48.40; H, 5.85; N, 11.29; S, 4.31; Cl, 9.52. Found: C,
48.62; H, 5.75; N, 11.37; S, 4.41; Cl, 9.66.
.
.
.
.
C29H36N6O5S 2.2HCl 2.5H2O: C, 49.34; H, 6.17; N,
11.90; S, 4.54; Cl, 11.05. Found: C, 49.24; H, 6.04; N,
12.06; S, 4.56; Cl, 10.97.
(E)-3-Cyanocinnamaldehyde (13). A solution of 3-cy-
anobenzaldehyde (12, 14.53 g, 110.8 mmol) and triphe-
nylphosphoranylidene acetaldehyde (33.72 g, 110.8
mmol) in benzene was refluxed for 16 h. To the reaction
mixture was added triphenylphosphoranylidene acet-
aldehyde (6.74 g, 37.0 mmol) again and the reaction
mixture was further refluxed for 19 h. After the reaction
mixture was concentrated in vacuo, the crude residue
was chromatographed on silica gel eluting with ethyl
acetate/n-hexane (10:90–30:70) to give 13 (13.49 g, 78%)
N-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-
amidino - 2 - naphthyl)methyl]sulfamoyl)glycine (8u). A
solution of 8t (109 mg, 0.15 mmol) in concd HCl (50
mL) was stirred at ambient temperature for 2 h. The
reaction mixture was concentrated in vacuo and the
residue was chromatographed on ODS-gel eluting with
MeOH/H2O (0:100–12:88). MeOH was removed in
vacuo, and the aqueous solution was lyophilized after
acidification with 1 N HCl. 8u (88 mg 85%) was
obtained as a white amorphous powder: 1H NMR
(DMSO-d6) d 1.60–1.71 (2H, m), 1.90–2.02 (2H, m),
2.26 (3H, s), 3.40–3.55 (2H, m), 3.61–3.81 (4H, m),
4.56–4.62 (1H, m), 4.94 (2H, s), 6.88 (2H, d, J=9.2 Hz),
7.28 (2H, d, J=8.6 Hz), 7.64 (1H, d, J=8.5 Hz), 7.78
(1H, d, J=8.5 Hz), 7.81–7.93 (2H, m), 7.97 (1H, d,
J=8.6 Hz), 8.07 (1H, d, J=8.6 Hz), 8.42 (1H, s), 8.71
(1H, s), 9.26 (1H, s), 9.30–9.50 (4H, m), 12.8 (1H, s);
FAB Ms m/e (M+H)+ 553. Anal. calcd for
as a paleylelow solid: mp 101–102
ꢂC; 1H NMR
(CDCl3) d 6.75 (1H, dd, J=7.3, 16.1 Hz), 7.45 (1H, d,
J=16.1 Hz), 7.57 (1H, t, J=7.9 Hz), 7.73 (1H, dt,
J=1.1, 7.9 Hz), 7.78–7.84 (2H, m), 9.75 (1H, d, J=7.3
Hz); EI Ms m/e (M)+ 157.
3-((E)-3-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]anili-
no}propenyl)benzonitrile (14). Compound 14 was syn-
thesized from 2 and 13 according to thesameprocedure
as that for 5. Compound 14 was obtained as a pale yel-
low viscous oil (59% yield): 1H NMR (DMSO-d6) d
1.46 (9H, s), 1.60–2.00 (4H, m), 3.22–3.31 (2H, m),
3.66–3.76 (2H, m), 3.93 (2H, d, J=5.4 Hz), 4.22–4.31
(1H, m), 6.40 (1H, dt, 16.1, 5.4 Hz), 6.57–6.64 (3H, m),
6.81 (2H, d, J=9.3 Hz), 7.41 (1H, t, J=7.8 Hz), 7.50
(1H, d, J=7.8 Hz), 7.57 (1H, d, J=7.8 Hz), 7.76 (1H,
s); FAB Ms m/e (M)+ 433.
.
.
C27H32N6O5S 1.9HCl 2.7H2O: C, 48.36; H, 5.91; N,
12.53; S, 4.78; Cl, 10.05. Found: C, 48.21; H, 5.68; N,
12.52; S, 4.89; Cl, 9.66.
Methyl N-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}
-N-[(7-amidino-2-naphthyl)methyl]sulfamoyl)-N-ethoxy-
carbonylglycinate (8v). Compound 8v was synthesized
from 10c according to thesameprocedureas that for 8f,
but using methanol as a solvent instead of ethanol.
Compound 8v was obtained as a white amorphous
3-(3-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]anilino}-
propyl)benzonitrile (15). To a solution of 14 (540 mg,
1.24 mmol) in EtOH (12 mL) was added 10% Pd/C
powder (50 mg), the reaction mixture was stirred in a
hydrogen atmosphere at ambient temperature for 1 h. The
reaction mixture was filtered through a pad of Celite and
concentrated under reduced pressure. The residue was
chromatographed on silica gel eluting with ethyl acetate/
n-hexane (75:25) to give 15 (334 mg, 62%) as a colorless
1
powder (46% yield): H NMR (DMSO-d6) d 1.30 (3H,
t, J=7.0 Hz), 1.60–1.75 (2H, m), 1.95–2.05 (2H, m),
2.27 (3H, s), 3.41–3.53 (2H, m), 3.62 (3H, s), 3.65–3.72
(1H, m), 3.75–3.82 (1H, m), 4.17 (2H, s), 4.35 (2H, q,
J=7.0 Hz), 4.60–4.67 (1H, m), 5.19 (2H, s), 6.96 (2H, d,
J=9.2 Hz), 7.23 (2H, d, J=9.2 Hz), 7.63 (1H, d, J=8.6
Hz), 7.83 (1H, d, J=8.6 Hz), 7.91 (1H, s), 8.04 (1H, d,
J=8.6 Hz), 8.11(1H, d, J=8.6 Hz), 8.47 (1H, s), 8.79
(1H, s), 9.26–9.35 (3H, m), 9.52 (2H, s); FAB Ms m/e
1
viscous oil: H NMR (CDCl3) d 1.46 (9H, s), 1.61–1.78
(2H, m), 1.80–1.99 (4H, m), 2.77 (2H, t, J=7.8 Hz), 3.11
(2H, t, J=6.8 Hz), 3.22–3.31 (2H, m), 3.65–3.76 (2H, m),
4.21–4.29 (1H, m), 6.53 (2H, d, J=8.8 Hz), 6.79 (2H, d,
J=8.7 Hz), 7.39 (1H, t, J=7.8 Hz), 7.44 (1H, d, J=7.8
Hz), 7.47–7.52 (2H, m); FAB Ms m/e (M)+ 435.
(M+H)+ 639. Anal. calcd for C31H38N6O7S 2.1HCl
.
.
2.3H2O: C, 49.20; H, 5.95; N, 11.11; S, 4.24; Cl, 9.84.
Found: C, 49.05; H, 5.80; N, 11.23; S, 4.29; Cl, 10.16.
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(E)-3-
(3 - amidinophenyl)allyl]methanesulfonamide (16). Com-
pound 16 was synthesized from 14 according to the
N-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-[(7-
amidino -2- naphthyl)methyl]sulfamoyl)-N -ethoxycarbo-
nylglycine (8w). Compound 8w was synthesized from 8v
sameprocdeureas that for
8f. Compound 16 was
1
according to thesameprocedureas that for
8u. Com-
obtained as a white amorphous powder (9% yield): H
NMR (DMSO-d6) d 1.65–1.81 (2H, m), 1.98–2.10 (2H,
m), 2.28 (3H, s), 3.04 (3H, s), 3.40–3.57 (2H, m), 3.66–
3.82 (2H, m), 4.42 (2H, d, J=5.9 Hz), 4.65–4.73 (1H,
m), 6.45 (1H, dt, J=5.9, 16.1 Hz), 6.58 (1H, d, J=16.1
pound 8w was obtained as a white amorphous powder
(80% yield): 1H NMR (DMSO-d6) d 1.30 (3H, t, J=7.0
Hz), 1.61–1.73 (2H, m), 1.95–2.05 (2H, m), 2.27 (3H, s),
3.41–3.53 (2H, m), 3.62–3.72 (1H, m), 3.72–3.82 (1H,

1522
F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
Hz), 7.01 (2H, d, J=9.2 Hz), 7.38, (2H, d, J=8.8 Hz),
7.54 (1H, t, J=7.8 Hz), 7.66 (1H, d, J=7.8 Hz), 7.72
(1H, d, J=7.8 Hz), 7.86 (1H, s), 8.64 (1H s), 9.02 (2H,
s), 9.18 (1H, s), 9.33 (2H, s); FAB Ms m/e (M+H)+
470. Anal. calcd for C24H31N5O3S 2.1HCl 1.6H2O: C,
50.13; H, 6.36; N, 12.18; S, 5.58; Cl, 12.95. Found: C,
50.05; H, 6.35; N, 12.29; S, 5.68; Cl, 13.09.
2-(N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl}-N-(me-
thylsulfonyl)amino)-N-(3-amidinophenyl)acetamide (22).
Compound 22 was synthesized from 21 according to the
sameprocdeureas that for
8f. Compound 22 was
1
.
.
obtained as a white amorphous powder (8% yield): H
NMR (DMSO-d6) d 1.63–1.80 (2H, m), 1.99–2.10 (2H,
m), 2.30 (3H, s), 3.13 (3H, s), 3.45–3.60 (2H, m), 3.70–
3.82 (2H, m), 4.52 (2H, s), 4.68–4.76 (1H, m), 7.04 (2H,
d, J=9.2 Hz), 7.42–7.50, (3H, m), 7.54 (1H, t, J=7.9
Hz), 7.84 (1H, d, J=8.6 Hz), 8.11 (1H s), 9.29 (6H, br-
s); FAB Ms m/e (M+H)+ 487. Anal. calcd for
N-{4-[(1-Acetimidoyl-4-piperidyl)oxy]phenyl} - N - [3 - (3 -
amidinophenyl)propyl]methanesulfonamide (17). Com-
pound 17 was synthesized from 15 according to the
.
.
sameprocdeureas that for
8f. Compound 17 was
C23H30N6O4S 1.9HCl 2.0H2O: C, 46.67; H, 6.11; N,
14.20; S, 5.42; Cl, 11.38. Found: C, 47.07; H, 6.18; N,
14.22; S, 4.44; Cl, 11.10.
1
obtained as a white amorphous powder (3% yield): H
NMR (DMSO-d6) d 1.62–1.83 (4H, m), 2.00–2.13 (2H,
m), 2.31 (3H, s), 2.63–2.74 (2H, m), 2.95 (3H, s), 3.48–
3.88 (6H, m), 4.68–4.77 (1H, m), 7.05 (2H, d, J=8.8
Hz), 7.34, (2H, d, J=8.8 Hz), 7.47–7.54 (2H, m), 7.60–
7.65 (2H, m), 8.79 (1H, s), 9.16 (2H, s), 9.27–9.37 (3H,
br); FAB Ms m/e (M+H)+ 472. Anal. calcd for
X-ray crystallographic experiment
Crystals of 8g/bovine pancreatic trypsin complex were
prepared using the same method as reported previously.33
TheX-ray diffraction data werecollected with a Rigaku
R-AXIS IIc image-plate system. The data set covers 80%
of theoretically calculated number of reflections up to
1.85 A. Thestructural analysis of theinhibitor complex
was achieved by the Patterson search method based on
a molecular model of the bovine pancreatic trypsin/
NAPAP complex (1ppc.pdb). Model building, electron
density calculation, and model refinement were carried
out using program O34 and CNX2000 (Accelrys Inc.).
The model has been refined to a crystallographic R-
valueof 20.8% (R free=23.5%) with good stereo-
chemistry (r.m.s.d. of bonds=0.006 A and angles=1.3ꢂ
from ideality). We will deposit the crystallographic data
for this structurewith theCambridgeCrystallographic
Data Center after this manuscript is accepted.
.
.
C24H33N5O3S 2.7HCl 2.0H2O: C, 47.56; H, 6.60; N,
11.55; S, 5.29; Cl, 15.79. Found: C, 47.79; H, 6.96; N,
11.74; S, 5.53; Cl, 15.70.
N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}me-
thanesulfonamide (18). Compound 18 was synthesized
from 2 according to thesameprocedureas that for
6f.
Compound 18 was obtained as a white solid (79%
yield): mp 145–146 ꢂC; 1H NMR (CDCl3) d 1.47 (9H, s),
1.68–1.82 (2H, m), 1.83–1.98 (2H, m), 2.96 (3H, s),
3.28–3.39 (2H, m), 3.63–3.74 (2H, m), 4.39–4.57 (1H,
m), 6.44 (1H, s), 6.89 (2H, d, J=8.8 Hz), 7.19 (2H, d,
J=8.8 Hz); FAB Ms m/e (M)+ 370.
2-Bromo-N-(3-cyanophenyl)acetamide (20). To a stirred
solution of 3-aminobenzonitrile (19, 2.00 g, 16.9 mmol)
and triethylamine (7.12 mL, 51.1 mmol) in 1,2-dichloro-
ethane (20 mL) at 3 ꢂC was added bromoacetyl bromide
(2.54 mL, 25.4 mmol). After stirring at ambient tem-
perature for 3 h, the reaction mixture was poured into
water and extracted with CHCl3. Theorganic layer was
washed with brine, dried over Na2SO4 and concentrated
in vacuo. The residue was chromatographed on silica
gel eluting with ethyl acetate/n-hexane (1:3–1:2) to give
20 (1.15 g, 28%) as a palebrown solid: mp 124–125 ꢂC;
1H NMR (CDCl3) d 4.04 (2H, s), 7.44–7.50 (2H, m),
7.70–7.77 (1H, m), 7.97 (1H, s), 8.23 (1H, br-s); FAB
Ms m/e (M+1)+ 239, 241.
Modeling study
Compound 8g was initially placed in the active site of
factor Xa by superimposing the crystal structure of the
8g/trypsin complex on to that of factor Xa35 (1 hcg.pdb)
using corresponding Ca atoms. The EGF-like domain
of factor Xa and some water molecules around the
active site were then removed from the model because
they seem to have unsuitable contacts with the inhibitor.
After manual adjustment of the position of the side
chains, energy minimization of the complex model was
performed with program DISCOVER (Accelrys Inc.).
During theminimization, only 8g, water molecule and
sidechain atoms within 10 A from theinhibitor were
allowed to move.
2-(N-{4-[(1-tert-Butoxycarbonyl-4-piperidyl)oxy]phenyl}-
N - (methylsulfonyl)amino)-N-(3-cyanophenyl)acetamide
(21). To a stirred solution of 18 (1.80 g, 4.8 mmol) and
20 (1.15 g, 4.8 mmol) in acetonitrile (35 mL) at ambient
temperature was added K2CO3 (750 mg, 5.4 mmol).
After the reaction mixture was refluxed for 6 h, it was
filtered and concentrated in vacuo. The residue was
chromatographed on silica gel eluting with ethyl ace-
tate/n-hexane (1:2–1:1) to give 21 (1.24 g, 49%) as a pale
brown amorphous powder: 1H NMR (CDCl3) 1.47 (9H,
s), 1.67–1.82 (2H, m), 1.83–1.98 (2H, m), 3.05 (3H, s),
3.28–3.40 (2H, m), 3.62–3.73 (2H, m), 4.01–4.50 (3H,
m), 6.93 (2H, d, J=9.3 Hz), 7.37 (2H, d, J=8.8 Hz),
7.42–7.47 (2H, m), 7.65–7.68 (1H, m), 7.98 (1H, s), 8.21
(1H, s); FAB Ms m/e (M- H)ꢀ 527.
Biology
Chromogenic assay. The hydrolysis rates of synthetic
substrates were assayed bꢂy continuously measuring
absorbanceat 405 nm at 37 C with a microplate reader
(Model 3550, Bio-Rad, Richmond, USA). Reaction
mixtures (125 mL) were prepared in 96-well plates con-
taining chromogenic substrates and an inhibitor in
either 0.05 M Tris–HCl, pH 8.4, 0.15 M NaCl. Reac-
tions were initiated with a 25 mL of enzyme solution.
Enzymes and substrates were used as follows: factor Xa
and chromogenic Xa; thrombin and S-2238; trypsin and

F. Hirayama et al. / Bioorg. Med. Chem. 10 (2002) 1509–1523
1523
S-2222. The concentration of an inhibitor required to
inhibit enzyme activity by 50% (IC50) was calculated
from dose–response curves in which the logit transfor-
mation of residual activity was plotted against the
logarithm of inhibitor concentration.
11. Schaffer, L. W.; Davidson, J. T.; Vlasuk, G. P.; Siegl,
P. K. S. Circulation 1991, 84, 1741.
12. Wong, P. C.; Crain, E. J., Jr.; Nguan, O.; Watson, C. A.;
Racanelli, A. Thromb. Res. 1996, 83, 117.
13. Rai, R.; Sprengeler, P. A.; Elrod, K. C.; Young, W. B.
Curr. Med. Chem. 2001, 8, 101.
14. Zhu, B. Z.; Scarborough, R. M. Ann. Rep. Med. Chem.
2000, 35, 83.
15. Ewing, W. R.; Pauls, H. W.; Spada, A. P. Drugs Future
1999, 24, 771.
16. Nagahara, T.; Yokoyama, Y.; Inamura, K.; Katakura, S.;
Komoriya, S.; Yamaguchi, H.; Hara, T.; Iwamoto, M. J. Med.
Chem. 1994, 37, 1200.
17. Hara, T.; Yokoyama, A.; Ishihara, H.; Yokoyama, Y.;
Nagahara, T.; Iwamonto, M. Thromb. Haemost. 1994, 71, 314.
18. Buhlmayer, P.; Criscione, L.; Fuhrer, W.; Furet, P.; de
Gasparo, M.; Stutz, S.; Whitebread, S. J. Med. Chem. 1991,
34, 3105.
Plasma clotting time assay. Prothrombin time(PT) was
performed using a KC10A coagulometer (Amelung Co.,
Lehbringsweg, Germany). Fifty mL of citrated mice
pool plasma was incubated for 1 min at 37 ꢂC with 50
mL of diluted compound, followed by the addition of 50
mL of PT reagent (Ortho Diagnostic Systems Co.,
Tokyo, Japan) to initiateclot formation. Thecon-
centration required to double clotting time (CT2) was
estimated from each individual dose–response curve.
Anticoagulant assays in mice. MaleICR miceweighing
20–30 g were fasted overnight. Inhibitors were dissolved
in saline and administered orally to the mice at 100 mg/
kg using a gastric tube. Several times after oral admin-
istration of the inhibitor, blood (0.9 mL) was collected
from the abdominal vena cava into syringes containing
0.1 mL of 3.8% citrate, and platelet poor plasma was
prepared by centrifugation to measure PT.
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Acknowledgements
The authors deeply acknowledge Dr. Shuichi Sakamoto
for useful discussion and helpful support for preparing
this manuscript, and arealso grateful to thestaff of the
Division of Analytical Science Laboratories for the ele-
mental analysis and spectral measurements.
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