Discovery of R-142086 as a factor Xa (FXa) inhibitor: Syntheses and structure-activity relationships of cinnamyl derivatives1,2

Article abstract of DOI:10.1248/cpb.57.22

To develop a novel and effective anticoagulant with potent and selective factor Xa (FXa) inhibitory activity, a new series of cinnamyl derivatives with enhanced lipophilicity and prodrug forms were synthesized and their biological activities were evaluate

Full text of DOI:10.1248/cpb.57.22

22
Chem. Pharm. Bull. 57(1) 22—33 (2009)
Vol. 57, No. 1
Discovery of R-142086 as a Factor Xa (FXa) Inhibitor: Syntheses and
Structure–Activity Relationships of Cinnamyl Derivatives1,2)
Tetsuji NOGUCHI,a Naoki TANAKA,*,b Toyoki NISHIMATA,b Riki GOTO,b Miho HAYAKAWA,a
Atsuhiro SUGIDACHI,b Taketoshi OGAWA,a Yoichi NIITSU,b Fumitoshi ASAI,b Tomoko ISHIZUKA,b and
b,3)
Koichi FUJIMOTO
a R&D Division, Daiichi Sankyo Co., Ltd.; 1–16–13 Kitakasai, Edogawa-ku, Tokyo 134–8630, Japan: and b R&D Division,
Daiichi Sankyo Co., Ltd.; 1–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan.
Received July 28, 2008; accepted October 16, 2008; published online October 17, 2008
To develop a novel and effective anticoagulant with potent and selective factor Xa (FXa) inhibitory activity,
a new series of cinnamyl derivatives with enhanced lipophilicity and prodrug forms were synthesized and their
biological activities were evaluated. As a result, we found that cinnamyl derivative (N-{4-[1-(acetimidoyl)-
piperidin-4-yloxy]-3-carbamoylphenyl}-N-[(Z)-3-(3-amidinophenyl)-2-fluoro-2-propenyl]sulfamoyl)acetic acid di-
hydrochloride (26d, R-142086) with a fluorine atom on the double bond exhibited potent anticoagulant activity
and no mutagenic potential. Moreover, orally administered R-142086 exhibited potent anti-FXa activity and anti-
coagulant activity in dogs.
Key words factor Xa inhibitory activity; cinnamyl derivative; anticoagulant; prodrug; Ames test
Warfarin is widely used as a sole oral anticoagulant for the be promising for high oral activity. Herein, we describe our
treatment and prevention of thromboembolic diseases. How- synthetic efforts and further evaluations to discover R-142086
ever, many reports have mentioned the defects of warfarin. It with ex vivo anticoagulant activity in dogs.
requires periodic monitoring because of its narrow therapeu-
Chemistry Cinnamyl derivatives with various imidoyl
tic window and, in addition, warfarin interacts with many dif- group on the piperidine ring were synthesized as shown in
ferent foods and drugs.4—6) Therefore, new anticoagulants Chart 1. Piperidine 313) was reacted with iminoethers14,15)
based on novel mechanisms need to be developed. Recently, under basic conditions, followed by acid hydrolysis to give
blood coagulation factor Xa (FXa) has attracted considerable corresponding bisamidine derivatives 4a—g. Other cinnamyl
attention as a novel anticoagulation target.
FXa acts at the convergence point of the intrinsic pathway 6b) were synthesized by the same methods as compound 4d.
and the extrinsic pathway in a blood coagulation cascade.7)
Monoamidine derivatives were synthesized as shown in
derivatives with a five-membered cyclic imidoyl group (6a,
FXa converts prothrombin into thrombin and leads to fibrin Chart 2. A t-butoxycarbonyl (Boc) group of compound 713)
clot formation. It is thought that the inhibition of FXa effec- was converted to a methyl group (8a) by treatment with
tively diminishes the thrombin generation and, as a result, formaldehyde solution and formic acid. Compound 7 was
leads to the inhibition of clot formation. Indeed, many FXa also deprotected under an acidic condition to give piperidine
inhibitors have been synthesized and their anticoagulant ac- 9. This compound was then subjected to a usual condition to
tivities reported.8,9) In these reports, FXa inhibitors exhibit give acetamide 8b. Piperidine 9 was converted to correspon-
less bleeding risk than warfarin and thrombin inhibitors. We ding N-alkyl or N-aryl compounds (8c—k) by reductive ami-
have also conducted research to discover orally-active novel nation with aldehydes or ketones (method A), or treatment
FXa inhibitors.10—13)
with alkyl- or arylbromide under a basic condition (method
Previously, we found that the series of cinnamyl deriva- B), or Pd-catalyzed amination with arylbromide and phos-
tives represented by 1 and 2 produced potent in vitro FXa in- phine 10 (method C).16) Reduction of the nitro group of com-
hibitory activities and high selectivity. Moreover, these com- pounds 8a—k afforded aniline 11a—k, respectively. An
pounds also exhibited potent ex vivo anticoagulant activities acetyl group of aniline 11b was converted to an ethyl group
in hamsters after oral administration.
(11l) by the treatment with LiAlH4. These anilines 11a,
However, regarding the chemical structure, these com- 11c—l were subjected to methods similar to those of the
pounds are highly hydrophilic with two amidino groups. It other cinnamyl derivatives to give corresponding monoami-
seems that enhancement of these compounds’ lipophilicity dine derivatives 13a, 13c—l. A Boc group of known interme-
may improve the plasma concentration after oral administra- diate 1413) was converted to N-(4-pyridylmethyl)piperidine
tion and the resulting oral anticoagulant activity. Similarly, compound 15 by 2 steps. This compound was also subjected
conversion of these compounds into their prodrug forms may to the similar methods described above to give corresponding
monoamidine derivative 13m.
Prodrugs of monoamidine derivative 16 were synthesized
as shown in Chart 3. Monoamidine derivative 16, synthe-
sized as shown in Chart 2, was converted to its prodrugs
17a—i by the treatment with symmetric carbonates or 4-ni-
trophenyl carbonates.17,18) Benzonitrile 18, also synthesized
as shown in Chart 2, was reacted with hydroxylamine under a
basic condition to give amidoxime 17j.
Fig. 1. Structures of Cinnamyl Derivatives 1 and 2
∗ To whom correspondence should be addressed. e-mail: tanaka.naoki.ri@daiichisankyo.co.jp
© 2009 Pharmaceutical Society of Japan

January 2009
23
Chart 1
Chart 2

24
Vol. 57, No. 1
Chart 3
Chart 4
Cinnamyl derivatives with a substituent on the double compounds were evaluated and expressed as IC50 values. The
bond were synthesized as shown in Chart 4. In the case of in- ex vivo effects on prothrombin time (PT) in hamsters (per os
termediate 21 with a fluorine atom, 3-cyanobenzaldehyde (p.o.)) were also evaluated. Described above, this series of
(19) was subjected to a Horner–Wittig reaction to give (E)-20 cinnamyl derivatives has two amidino groups in their struc-
and (Z)-20 as an undesired mixture ratio (E/Zꢀ93/7). Then, tures. As a result, these compounds have high hydrophilicity.
treated with bromine, the mixture was isomerized to give It seems that enhancement of these compounds’ lipophilicity
(Z)-20 dominantly (E/Zꢀ0.4/99.6).19) This compound was may improve their oral anticoagulant activity. As one of the
treated with NaBH4 to give Z-substituted fluoroallylalcohol approaches for the enhancement of lipophilicity, we at-
21. In the case of intermediate 22 with a methyl group, 3- tempted to remove the acetimidoyl group of these cinnamyl
cyanobenzaldehyde 19 was also reacted with ylide, followed derivatives (Table 1).22) Non-substituted piperidine com-
by Luche reduction20) to give (E)-substituted methylallylalco- pounds with various substituent patterns on the central ben-
hol 22. Substituted allylalcohols 21 and 22 were coupled zene ring (5a, 5b, 27, 3)23) were synthesized and their biolog-
with sulfonamide 23, 24 and 25 by means of a Mitsunobu re- ical activities were evaluated. Most of compounds exhibited
action21), followed by reactions similar to those of the other significantly lower inhibitory activities than those of com-
cinnamyl derivatives to give corresponding cinnamyl deriva- pound 1 and other acetimidoyl derivatives13) in vitro. How-
tives 26a—d.
ever, only chlorobenzene derivative 3 exhibited moderate in
vitro FXa inhibitory activity. It seemed that the substituents
on the benzene ring had a considerable effect on the FXa in-
Results and Discussion
The in vitro FXa and trypsin inhibitory activities of all the hibitory activities of non-substituted piperidine compounds.

January 2009
25
Table 3. Biological Activities of Compounds 16, 17a—j and 13a
Table 1. Biological Activities of Compounds 5a, 5b, 27, 3 and 1
IC50 (nM)
Compd.a)
R
FXa
Trypsin
5a
5b
27
3
H
CONH2
F
230
290
250
31
3300
2100
2100
1800
520
IC50 (nM)
Hamster ex vivo
(p.o., 1 h)
Trypsin CT2 (mg/kg)b)
Compd.a)
R
Cl
FXa
1
7.4
16
–H
–CO2Et
–CO2(4-OMe-Ph)
–CO2t-Bu
–CO2(4-F-Ph)
–CO2CH2OCOEt
–CO2CH2OCOCH2Ph
–CO2CH2OCOc-Hex
–CO2CH2OCOt-Bu
29
14000
1300
20000
900
3200
NAc)
NAc)
NAc)
NAc)
NAc)
97000
NAc)
NAc)
5700
NAc)
2900
56
43
63
a) All the compounds were synthesized and evaluated as their hydrochlorides.
Table 2. Biological Activities of Compounds 13a and 13c—m
17a
17b
17c
17d
17e
17f
17g
17h
17i
ꢁ100
ꢁ100
ꢁ100
ꢁ100
ꢁ100
ꢁ100
ꢁ100
58
960
320
4500
9900
33
12000
16
17j
13a
IC50 (nM)
Hamster ex vivo
(p.o., 1 h)
48
Compd.a)
R
FXa
16
Trypsin
2900
CT2 (mg/kg)b)
a) All the compounds were synthesized and evaluated as their hydrochlorides. b)
The concentration required to double the clotting time. c) Not active (ꢁ100000).
13a
13l
48
77
hibited potent anticoagulant activities (ex vivo). On the other
hand, phenyl derivative 13f exhibited significantly lower FXa
inhibitory activity than those of alkyl derivatives. However,
the introduction of an alkyl chain (13g, h) between the
piperidine ring and a phenyl group of 13f, or conversion of
one carbon atom on the phenyl group into a nitrogen atom
(13i—k) improved the FXa inhibitory activities. Among
them, 4-pyridyl derivative 13k exhibited potent in vitro FXa
inhibitory activity comparable to that of the bisamidine de-
rivative 1. However, in the ex vivo test by oral administration,
compound 13k exhibited almost no oral anticoagulant activ-
ity. From these results, a methyl group (13a) and a cyclo-
pentyl group (13e) were favorable at this position.
10
16
24
8700
8700
8000
13c
13d
ꢁ100
77
13e
12
5400
52
13f
170
15
2300
990
NTc)
NTc)
13g
NTc)
NTc)
NTc)
As another approach to improve the lipophilicity and oral
activity of monoamidine derivatives, we intended to convert
13a to its prodrug form (Table 3). Compound 16 with an
ester group instead of a carboxylic group exhibited oral anti-
coagulant activity similar to that of compound 13a. Next, we
attempted to convert an amidino moiety of compound 16 into
its carbamate forms (17a—i) and amidoxime form (17j).
Among these compounds, carbamate prodrugs 17a, 17b and
amidoxime prodrug 17j exhibited anticoagulant activities (ex
vivo) similar to that of compound 13a. However, as prodrugs,
no advantages were observed. From the results shown in Ta-
bles 2 and 3, the conversion of bisamidine derivatives into
monoamidine derivatives and their prodrugs could not im-
13h
13i
13j
13
20
32
2200
780
590
13k
6.4
650
ꢁ100
13m
41
1600
NTc)
a) All the compounds were synthesized and evaluated as their hydrochlorides. b)
The concentration required to double the clotting time. c) Not tested.
Based on the results shown in Table 1, we synthesized prove the oral anticoagulant activity of bisamidine derivative
monoamidine derivatives with non-amidine substituents i.e. 1.
alkyl, aryl, and arylalkyl groups on the nitrogen atom in the
As an alternative approach to improve the lipophilicity of
piperidine ring of chlorobenzene derivative 3, and evaluated bisamidine derivative 1, additional lipophilic imidoyl moi-
their biological activities (Table 2). Regarding alkyl deriva- eties other than an acetimidoyl group were introduced instead
tives (13a, c, d, e, l), these compounds exhibited potent FXa of an acetimidoyl group of compound 1, and their biological
inhibitory activities (10—24 nM) in vitro. In particular, activities were evaluated (Table 4). On the whole, most of the
methyl derivative 13a and cyclopentyl derivative 13e also ex- imidoyl compounds exhibited potent FXa inhibitory activi-

26
Vol. 57, No. 1
Table 5. Biological Activities of Compounds 2, 6a, 6b, 26a—d and 28
Table 4. Biological Activities of Compounds 1 and 4a—g
IC50 (nM)
Hamster ex vivo
Hamster ex vivo
Compd.a)
R
(p.o., 1 h)
IC50 (nM)
Compd.a) R1
R2
R3
(p.o., 1 h) Ames test
FXa
Trypsin
CT2 (mg/kg)b)
CT2 (mg/kg)b)
FXa Trypsin
1
7.4
6.3
520
14
28
2
H
H
H
6.4
7.1
520
320
34
16
ꢂ
ꢂ
4a
1200
2.2 fold@100 mg
CONH2
6a
H
H
F
H
CONH2
H
14
3100
NTc)
NTc)
38
ꢂ
ꢂ
4b
7.8
860
ꢁ100
4c
4d
4e
4f
13
3500
2200
2000
4000
NTc)
27
6b
9.8 4400
26a
9.3
960
ꢃ
7.0
26b Me
H
10
1300
92
ꢃ
10
47
26c
26d
F
Cl
7.4 3100
ꢁ100
NTc)
ꢃ
16
NTc)
F CONH2
12
3100
33
4g
34
3400
NTc)
a) All the compounds were synthesized and evaluated as their hydrochlorides. b)
The concentration required to double the clotting time. c) Not tested.
a) All the compounds were synthesized and evaluated as their hydrochlorides. b)
The concentration required to double the clotting time. c) Not tested.
ties in vitro. In particular, propioimidoyl (4a), benzimidoyl
(4b), and five-membered cyclic imidoyl (4d) derivatives ex-
hibited potent activities. Regarding cyclic imidoyl derivatives
(4d—g), a tendency was observed indicating that a smaller
ring size was suitable. In the ex vivo anticoagulant activities
of these compounds, compound 4d was as potent as com-
pound 1 (27 mg/kg). From these results, we found that the
acetimidoyl and five-membered cyclic imidoyl groups were
suitable as this part.
Based on the results shown in Table 4, five-membered
cyclic imidoyl derivatives 6a and 6b were synthesized and
their biological activities were compared with those of corre-
sponding acetimidoyl derivatives 28 and 2, which exhibited
favorable results in the previous report13) (Table 5). Com-
pounds 6a and 6b exhibited potent FXa inhibitory activities
in vitro. In addition, these compounds exhibited improved
enzyme selectivity over trypsin. However, we examined the
Fig. 2. Structures of Compounds 29,a) 30a) and YM-75466b)
a) Hydrochloride. b) Methanesulfonate.
mutagenic potential of these cinnamyl derivatives and found exhibited negative results in Ames tests. Compound 26a also
that all the derivatives exhibited positive results in Ames exhibited potent FXa inhibitory activity and oral anticoagu-
tests.24) On the other hand, the reported naphthylamidine de- lant activity similar to those of non-substituted derivative 28
rivative YM-7546625) (Fig. 2) exhibited a negative result in in hamsters. To apply these findings to compounds 1 and 2,
the test. In terms of structural differences between YM- we synthesized 26c and 26d with a fluorine atom and evalu-
75466 and our derivatives, YM-75466 has a naphthyl moiety ated their activities. Both compounds exhibited potent FXa
whereas our derivatives have cinnamyl moieties. Therefore, inhibitory activities in vitro. In the ex vivo tests, however,
we speculated that the cause of the positive result was the ox- compound 26c with a chlorine atom on the central benzene
idation of the double bond. Based on this speculation, we in- ring exhibited almost no oral anticoagulant activity. On the
troduced substituents at the b-position of the cinnamyl moi- other hand, compound 26d with a carbamoyl group at this
ety of compound 28 and their mutagenic potentials as well as position exhibited potent anticoagulant activity in hamsters
biological activities were examined. As a result, both com- (33 mg/kg, p.o.). Moreover, compound 26d exhibited no mu-
pound 26a with a fluorine atom and 26b with a methyl group tagenic potential similar to those of 26a and 26b.

January 2009
27
spectrometer and were reported as d values relative to Me4Si as the internal
standard. The abbreviations of the 1H-NMR peak patterns are as follows:
bsꢀbroad singlet, sꢀsinglet, dꢀdoublet, ddꢀdouble doublet, tꢀtriplet, dtꢀ
double triplet, qꢀquartet and mꢀmultiplet. IR spectra were obtained on a
Jasco FT/IR-6100 spectrometer in KBr pellets. Merck Silica gel 60 (230—
400 mesh) was used in the column chromatography. Tetrahydrofuran, N,N-
dimethylformamide, t-butyl methyl ether, and dimethylsulfoxide are abbre-
viated as THF, DMF, TBME and DMSO, respectively.
Cinnamyl derivatives with potent anticoagulant activities
and no mutagenic potential (1, 26a, 26b, 26d) in addition to
our reported compounds (29, 30) and YM-75466 (Fig. 2)
were subjected to a pharmacokinetic study in dogs in order to
assess the oral absorption of these derivatives (1 mg/kg) (Fig.
3). Compounds 1 and 26a exhibited extremely low plasma
concentrations, whereas compound 26d exhibited an obvi-
ously high plasma concentration superior to those of the
other tested compounds.
(N-[(E)-3-(3-Amidinophenyl)-2-propenyl]-N-{3-chloro-4-[1-(1-imino-
propyl)piperidin-4-yloxy]phenyl}sulfamoyl)acetic Acid Dihydrochloride
(4a) To a solution of ethyl {N-[(E)-3-(3-amidinophenyl)-2-propenyl]-N-[3-
Moreover, compound 26d exhibited a dose-dependent in-
crease in plasma anti-FXa and anticoagulant activities after
oral administration to dogs (Fig. 4).
From these results, compound 26d (R-142086) has potent
biological activities and a high plasma concentration by oral
administration.
In conclusion, we synthesized many cinnamyl derivatives
in order to develop compounds having potent oral anticoagu-
lant activities. As a result, we found that cinnamyl derivatives
with a fluorine atom or a methyl group on the double bond
exhibited oral anticoagulant activities in hamsters and no
mutagenic potential. Among them, 26d (R-142086) exhibited
potent oral anti-FXa and anticoagulant activity in addition to
a high plasma concentration compared to the other indoline
and cinnamyl derivatives in dogs.
chloro-4-(piperidin-4-yloxy)phenyl]sulfamoyl}acetate dihydrochloride
3
(0.770 g, 1.27 mmol) in EtOH (25 ml) were added ethyl propionimidate hy-
drochloride (0.540 g, 3.92 mmol) and Et3N (0.880 ml, 6.35 mmol) and the re-
sulting mixture was allowed to stand at room temperature for 22 h. Ethyl
propionimidate hydrochloride (0.180 g, 1.31 mmol) and Et3N (0.350 ml, 2.52
mmol) were added and the mixture was stirred at room temperature for 4.5
h. A 4 N solution of hydrogen chloride in dioxane (10 ml) was added and the
mixture was concentrated. The resulting residue was purified by a prepara-
tive HPLC (YMC-pack ODS, YMC Corp., H2O/MeCNꢀ4/1) to give an
amorphous solid. This amorphous solid was dissolved in EtOH (10 ml) and
a 4 N solution of hydrogen chloride in dioxane (2 ml) and the mixture was
concentrated. The resulting residue was dissolved in H2O and the solution
was lyophilized to give ethyl (N-[(E)-3-(3-amidinophenyl)-2-propenyl]-N-
{3-chloro-4-[1-(1-iminopropyl)piperidin-4-yloxy]phenyl}sulfamoyl)acetate
dihydrochloride (0.570 g, 0.860 mmol, 67%) as a colorless amorphous solid.
This amorphous solid (0.420 g, 0.633 mmol) was dissolved in a 3 N solution
of hydrogen chloride (15 ml) and stirred at 60 °C for 6.5 h. The reaction mix-
ture was concentrated and the resulting residue was purified by a preparative
HPLC (YMC-pack ODS, YMC Corp., H2O/MeCNꢀ41/9) to give an amor-
phous solid. This amorphous solid was dissolved in a 3 N solution of hydro-
gen chloride (3 ml) and the mixture was concentrated. The resulting residue
was dissolved in H2O and the solution was lyophilized to give 4a (0.370 g,
0.583 mmol, 93%) as a colorless amorphous solid. MS m/z: 562 (MꢂH)ꢂ.
1H-NMR (DMSO-d6) d: 1.15 (3H, t, Jꢀ7.5 Hz), 1.71—1.87 (2H, m), 2.00—
2.12 (2H, m), 2.63 (2H, q, Jꢀ7.5 Hz), 3.59—3.81 (4H, m), 4.30 (2H, s),
4.48 (2H, d, Jꢀ5.5 Hz), 4.81—4.88 (1H, m), 6.46 (1H, dt, Jꢀ16.0, 5.5 Hz),
6.58 (1H, d, Jꢀ16.0 Hz), 7.34 (1H, d, Jꢀ9.0 Hz), 7.43 (1H, dd, Jꢀ2.5, 9.0
Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.60 (1H, d, Jꢀ2.5 Hz), 7.70—7.76 (2H, m),
7.94 (1H, s). IR (KBr) cmꢃ1: 1734, 1671, 1620, 1349, 1156. Anal. Calcd for
C26H32ClN5O5S·2.5HCl·0.4H2O: C, 47.28; H, 5.39; N, 10.60; Cl, 18.79; S,
4.85. Found: C, 47.12; H, 5.33; N, 10.71; Cl, 18.99; S, 4.86.
Experimental
Mass spectra were obtained on a JEOL LCmate spectrometer. 1H-NMR
spectra were obtained on a Varian Mercury 400 or Unity Inova 500 FT-NMR
Similarly, compounds 4b—g were prepared.
4b: MS m/z: 610 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.70—1.78 (1H, m),
1.88—2.02 (2H, m), 2.14—2.22 (1H, m), 3.28—3.50 (2H, m), 3.83—3.90
(1H, m), 3.91—4.01 (1H, m), 4.27 (2H, s), 4.45 (2H, d, Jꢀ5.0 Hz), 4.82—
4.89 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 5.0 Hz), 6.56 (1H, d, Jꢀ16.0 Hz), 7.32
(1H, d, Jꢀ9.0 Hz), 7.40 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.51—7.71 (9H, m), 7.90
(1H, s). IR (KBr) cmꢃ1: 1733, 1673, 1605, 1349, 1155. Anal. Calcd for
C30H32ClN5O5S·2.2HCl·1.0H2O: C, 50.87; H, 5.15; N, 9.89; Cl, 16.02; S,
4.53. Found: C, 50.57; H, 5.02; N, 10.08; Cl, 15.95; S, 4.92.
4c: MS m/z: 562 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.19 (3H, t, Jꢀ7.0
Hz), 1.72—1.88 (2H, m), 1.98—2.09 (2H, m), 3.51—3.79 (6H, m), 4.28
(2H, s), 4.47 (2H, d, Jꢀ6.0 Hz), 4.80—4.87 (1H, m), 6.44 (1H, dt, Jꢀ16.0,
6.0 Hz), 6.57 (1H, d, Jꢀ16.0 Hz), 7.32 (1H, d, Jꢀ9.0 Hz), 7.41 (1H, dd, Jꢀ
Fig. 3. Plasma Concentrations of Compounds 1, 26a, 26b, 26d, 29, 30 and
YM-75466 in Dogs after Oral Administration of Each Compound at a Dose
of 1 mg/kg
Fig. 4. Plasma Anti-FXaa) and Anticoagulant Activities after Oral Administration of R-142086 to Dogs
a) The activity was calibrated using the anti-FXa activity standard enoxaparin.

28
Vol. 57, No. 1
2.0, 9.0 Hz), 7.52—7.60 (2H, m), 7.68—7.75 (2H, m), 7.89 (1H, s), 8.13
mmol) in dioxane (80 ml) was added a 4 N solution of hydrogen chloride in
dioxane (70 ml), and the resulting mixture was stirred overnight at room
(1H, d, Jꢀ13.5 Hz). IR (KBr) cmꢃ1: 1731, 1698, 1677, 1347, 1155. Anal.
Calcd for C26H32ClN5O5S·2.5HCl·0.2H2O: C, 47.54; H, 5.36; N, 10.66; Cl, temperature. The mixture was concentrated and the resulting residue was
18.89; S, 4.88. Found: C, 47.47; H, 5.38; N, 10.72; Cl, 18.98; S, 4.90.
dissolved in H2O. The mixture was neutralized with NaHCO3 to give crys-
4d: MS m/z: 574 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.73—1.88 (2H, m), tals and the crystals were collected by filtration to give 9 (8.06 g, quant.) as
1
2.00—2.14 (4H, m), 2.97 (2H, t, Jꢀ8.0 Hz), 3.50—3.88 (6H, m), 4.30 (2H, pale yellow needles. H-NMR (CDCl3) d: 1.50—1.60 (2H, m), 1.90—2.00
s), 4.47 (2H, d, Jꢀ5.5 Hz), 4.81—4.88 (1H, m), 6.46 (1H, dt, Jꢀ16.0, 5.5 (2H, m), 2.57—2.68 (2H, m), 2.90—3.00 (2H, m), 3.93—3.99 (1H, m), 7.45
Hz), 6.58 (1H, d, Jꢀ16.0 Hz), 7.34 (1H, d, Jꢀ9.0 Hz), 7.42 (1H, dd, Jꢀ2.5, (1H, d, Jꢀ9.0 Hz), 8.18 (1H, dd, Jꢀ3.0, 9.0 Hz), 8.31 (1H, d, Jꢀ3.0 Hz).
9.0 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.59 (1H, d, Jꢀ2.5 Hz), 7.71—7.76 (2H,
4-(1-Acetylpiperidin-4-yloxy)-3-chloronitrobenzene (8b) To a solu-
m), 7.93 (1H, s). IR (KBr) cmꢃ1: 1734, 1672, 1350, 1155. Anal. Calcd for tion of 3-chloro-4-(piperidin-4-yloxy)nitrobenzene 9 (1.00 g, 3.90 mmol) in
C27H32ClN5O5S·2.4HCl·0.7H2O: C, 48.10; H, 5.35; N, 10.39; Cl, 17.88; S, pyridine (20 ml) was added Ac2O (0.550 ml, 5.82 mmol) and the mixture
4.76. Found: C, 48.06; H, 5.29; N, 10.45; Cl, 18.00; S, 4.69.
was stirred at room temperature for 3 h. H2O was added and the resulting
1
4e: MS m/z: 588 (MꢂH)ꢂ. H-NMR (DMSO-d6) d: 1.64—1.81 (6H, m), mixture was extracted with EtOAc. The organic layer was washed with
1.99—2.08 (2H, m), 2.67—2.72 (2H, m), 3.30—3.37 (2H, m), 3.55—3.78 NaHCO3 solution and brine. The organic layer was dried and concentrated to
(4H, m), 4.28 (2H, s), 4.47 (2H, d, Jꢀ6.0 Hz), 4.80—4.87 (1H, m), 6.44 give 8b (1.05 g, 3.51 mmol, 90%) as a pale yellow solid. H-NMR (CDCl3)
1
(1H, dt, Jꢀ16.0, 6.0 Hz), 6.58 (1H, d, Jꢀ16.0 Hz), 7.32 (1H, d, Jꢀ9.0 Hz),
d: 1.88—2.03 (4H, m), 2.14 (3H, s), 3.50—3.63 (2H, m), 3.68—3.75 (1H,
7.41 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.53—7.59 (2H, m), 7.67—7.74 (2H, m), 7.88 m), 3.91—3.97 (1H, m), 4.78—4.85 (1H, m), 7.01 (1H, d, Jꢀ9.0 Hz), 8.15
(1H, s). IR (KBr) cmꢃ1: 1734, 1675, 1637, 1352, 1156. Anal. Calcd for (1H, dd, Jꢀ2.5, 9.0 Hz), 8.32 (1H, d, Jꢀ2.5 Hz).
C28H34ClN5O5S·2.8HCl·1.2H2O: C, 47.25; H, 5.55; N, 9.84; Cl, 18.93; S,
4.50. Found: C, 46.88; H, 5.89; N, 10.12; Cl, 19.10; S, 5.09.
4-(1-Butylpiperidin-4-yloxy)-3-chloronitrobenzene (8d) (Method A)
To a solution of 3-chloro-4-(piperidin-4-yloxy)nitrobenzene 9 (1.50 g, 5.84
4f: MS m/z: 602 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.52—1.62 (4H, mmol) and butyraldehyde (1.04 ml, 11.5 mmol) in CH2Cl2 (30 ml) were
m), 1.67—1.82 (4H, m), 2.00—2.09 (2H, m), 2.84—2.88 (2H, m), 3.43— added AcOH (0.33 ml, 5.8 mmol) and NaBH3CN (0.18 g, 2.9 mmol) and the
3.49 (2H, m), 3.63—3.91 (4H, m), 4.27 (2H, s), 4.46 (2H, d, Jꢀ5.5 Hz),
4.80—4.86 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 5.5 Hz), 6.57 (1H, d, Jꢀ16.0 Hz),
7.32 (1H, d, Jꢀ9.0 Hz), 7.40 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.51—7.61 (2H, m),
mixture was stirred at room temperature for 3 h. NaBH3CN (0.18 g, 2.9
mmol) was added and the mixture was stirred overnight at room tempera-
ture. The mixture was concentrated and the resulting residue was diluted
7.68—7.75 (2H, m), 7.89 (1H, s). IR (KBr) cmꢃ1: 1734, 1675, 1628, 1351, with EtOAc. The organic solution was washed with H2O, NaHCO3 solution
1156. Anal. Calcd for C29H36ClN5O5S·2.5HCl·1.2H2O: C, 48.72; H, 5.77; and brine. The organic layer was dried and concentrated. The resulting
N, 9.80; Cl, 17.36; S, 4.48. Found: C, 48.93; H, 5.78; N, 9.51; Cl, 17.39; S, residue was chromatographed on a silica gel column (CH2Cl2/MeOHꢀ20/1)
1
4.62.
to give 8d (0.88 g, 2.81 mmol, 48%) as a yellow oil. H-NMR (CDCl3) d:
4g: MS m/z: 630 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.38—1.81 (12H, m), 0.94 (3H, t, Jꢀ7.5 Hz), 1.31—1.39 (2H, m), 1.50—1.57 (2H, m), 1.92—
2.00—2.09 (2H, m), 2.78—2.85 (2H, m), 3.48—3.57 (2H, m), 3.59—3.72 2.04 (4H, m), 2.04—2.15 (2H, m), 2.40—2.48 (2H, m), 2.49—2.57 (2H, m),
(2H, m), 3.73—3.86 (2H, m), 4.27 (2H, s), 4.46 (2H, d, Jꢀ5.5 Hz), 4.80— 2.70—2.80 (2H, m), 4.59—4.66 (1H, m), 6.99 (1H, d, Jꢀ9.0 Hz), 8.13 (1H,
4.88 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 5.5 Hz), 6.57 (1H, d, Jꢀ16.0 Hz), 7.31
(1H, d, Jꢀ9.0 Hz), 7.40 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.51—7.60 (2H, m),
7.64—7.75 (2H, m), 7.87 (1H, s). IR (KBr) cmꢃ1: 1733, 1675, 1627, 1352,
1156. Anal. Calcd for C31H40ClN5O5S·2.1HCl·1.7H2O: C, 50.49; H, 6.22; 2.00—2.15 (2H, m), 2.45—2.60 (2H, m), 2.75—2.90 (3H, m), 4.55—4.63
N, 9.50; Cl, 14.90; S, 4.35. Found: C, 50.40; H, 5.75; N, 9.64; Cl, 14.61; S, (1H, m), 6.98 (1H, d, Jꢀ9.0 Hz), 8.13 (1H, dd, Jꢀ3.0, 9.0 Hz), 8.30 (1H, d,
dd, Jꢀ2.5, 9.0 Hz), 8.30 (1H, d, Jꢀ2.5 Hz).
Similarly, compound 8c was prepared.
8c: 1H-NMR (CDCl3) d: 1.09 (6H, d, Jꢀ6.5 Hz), 1.90—2.00 (2H, m),
4.63.
(N-[(E)-3-(3-Amidinophenyl)-2-propenyl]-N-{4-[1-(4,5-dihydro-3H-
pyrrol-2-yl)piperidin-4-yloxy]phenyl}sulfamoyl)acetic Acid Dihydrochlo-
Jꢀ3.0 Hz).
3-Chloro-4-(1-cyclopentylpiperidin-4-yloxy)nitrobenzene (8e) (Method
B) To a solution of 3-chloro-4-(piperidin-4-yloxy)nitrobenzene 9 (4.00 g,
ride (6a) Ethyl {N-[(E)-3-(3-amidinophenyl)-2-propenyl]-N-[4-(piperidin- 15.6 mmol) in DMF (70 ml) were added cyclopentyl bromide (1.96 ml,
4-yloxy)phenyl]sulfamoyl}acetate dihydrochloride 5a was converted to 6a
by the same procedure as that for 4a. Compound 6a was obtained (45%, 2
18.3 mmol) and K2CO3 (3.23 g, 23.4 mmol) and the mixture was stirred at
100 °C for 7 h. Cyclopentyl bromide (0.700 ml, 6.53 mmol) was added and
steps) as a colorless amorphous solid. MS m/z: 540 (MꢂH)ꢂ. 1H-NMR the mixture was stirred at 100 °C for 2 h and at 120 °C for 5 h. The mixture
(DMSO-d6) d: 1.68—1.80 (2H, m), 2.00—2.13 (4H, m), 2.96 (2H, t, Jꢀ was diluted with EtOAc and washed with brine. The organic layer was dried
8.0 Hz), 3.46—3.72 (5H, m), 3.83—3.92 (1H, m), 4.20 (2H, s), 4.45 (2H, d, and concentrated. The resulting residue was chromatographed on a silica gel
Jꢀ5.5 Hz), 4.67—4.73 (1H, m), 6.45 (1H, dt, Jꢀ16.0, 5.5 Hz), 6.54 (1H, d, column (CH2Cl2/MeOHꢀ30/1—10/1) to give 8e (2.35 g, 7.13 mmol, 46%)
1
Jꢀ16.0 Hz), 7.04 (2H, d, Jꢀ9.0 Hz), 7.39 (2H, d, Jꢀ9.0 Hz), 7.54 (1H, t,
as a yellow oil. H-NMR (CDCl3) d: 1.37—1.48 (2H, m), 1.50—1.61 (2H,
Jꢀ8.0 Hz), 7.71 (2H, d, Jꢀ8.0 Hz), 7.90 (1H, s). IR (KBr) cmꢃ1: 1733, m), 1.65—1.76 (2H, m), 1.85—2.00 (4H, m), 2.00—2.10 (2H, m), 2.45—
1672, 1347, 1155. Anal. Calcd for C27H33N5O5S·2.1HCl·1.6H2O: C, 50.28; 2.55 (2H, m), 2.53—2.60 (1H, m), 2.70—2.81 (2H, m), 4.56—4.64 (1H, m),
H, 5.98; N, 10.86; Cl, 11.54; S, 4.97. Found: C, 50.12; H, 5.65; N, 10.96; Cl, 6.98 (1H, d, Jꢀ9.0 Hz), 8.13 (1H, dd, Jꢀ3.0, 9.0 Hz), 8.30 (1H, d, Jꢀ
11.62; S, 4.99.
3.0 Hz).
Similarly, compounds 8g—i and 8k were prepared.
8g: 1H-NMR (CDCl3) d: 1.88—1.98 (2H, m), 1.98—2.08 (2H, m), 2.37—
Similarly, compound 6b was prepared.
6b: MS m/z: 583 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.80—1.95 (2H, m),
2.00—2.15 (4H, m), 2.96 (2H, m), 3.45—3.55 (1H, m), 3.55—3.75 (4H, m), 2.48 (2H, m), 2.66—2.77 (2H, m), 3.55 (2H, s), 4.54—4.61 (1H, m), 6.97
3.75—3.85 (1H, m), 4.24 (2H, s), 4.47 (2H, d, Jꢀ6.0 Hz), 4.82—4.88 (1H, (1H, d, Jꢀ9.0 Hz), 7.23—7.37 (5H, m), 8.12 (1H, dd, Jꢀ2.5, 9.0 Hz), 8.30
m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.57 (1H, d, Jꢀ16.0 Hz), 7.28 (1H, d, Jꢀ (1H, d, Jꢀ2.5 Hz).
9.0 Hz), 7.52 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.67 (1H, d,
Jꢀ8.0 Hz), 7.72 (1H, d, Jꢀ8.0 Hz), 7.77 (1H, d, Jꢀ2.5 Hz), 7.86 (1H, s). IR
(KBr) cmꢃ1: 1731, 1670. Anal. Calcd for C28H34N6O6S·2.9HCl·1.9H2O: C,
8h: 1H-NMR (CDCl3) d: 1.93—2.03 (2H, m), 2.03—2.13 (2H, m),
2.46—2.59 (2H, m), 2.61—2.71 (2H, m), 2.73—2.88 (4H, m), 4.58—4.63
(1H, m), 6.99 (1H, d, Jꢀ9.0 Hz), 7.17—7.24 (3H, m), 7.24—7.34 (2H, m),
46.54; H, 5.68; N, 11.63; Cl, 14.23; S, 4.44. Found: C, 46.68; H, 5.68; N, 8.13 (1H, dd, Jꢀ3.0, 9.0 Hz), 8.31 (1H, d, Jꢀ3.0 Hz).
11.35; Cl, 14.15; S, 4.71.
8i: 1H-NMR (CDCl3) d: 1.93—2.06 (2H, m), 2.06—2.17 (2H, m), 3.60—
3-Chloro-4-(1-methylpiperidin-4-yloxy)nitrobenzene (8a) To a sus- 3.72 (2H, m), 3.79—3.90 (2H, m), 4.76—4.84 (1H, m), 6.64 (1H, dd, Jꢀ
pension of 4-[1-(t-butoxycarbonyl)piperidin-4-yloxy]-3-chloronitrobenzene 5.0, 7.0 Hz), 6.71 (1H, d, Jꢀ8.5 Hz), 7.04 (1H, d, Jꢀ9.0 Hz), 7.48—7.53
7 (1.50 g, 4.20 mmol) in 90% HCO2H (4.00 g) was added 37% HCHO solu-
tion (2.50 g) and the resulting mixture was stirred at 100 °C for 2 h. The
mixture was neutralized with K2CO3 and extracted with EtOAc. The organic
(1H, m), 8.16 (1H, dd, Jꢀ3.0, 9.0 Hz), 8.20 (1H, dd, Jꢀ2.0, 5.0 Hz), 8.32
(1H, d, Jꢀ3.0 Hz).
8k: 1H-NMR (CDCl3) d: 1.98—2.14 (4H, m), 3.46—3.55 (2H, m),
layer was washed with brine and dried. The organic layer was concentrated 3.58—3.67 (2H, m), 4.80—4.87 (1H, m), 6.72 (2H, d, Jꢀ6.5 Hz), 7.03 (1H,
1
to give 8a (1.12 g, 4.14 mmol, 98%) as a yellow solid. H-NMR (CDCl3) d: d, Jꢀ9.0 Hz), 8.16 (1H, dd, Jꢀ3.0, 9.0 Hz), 8.28 (2H, d, Jꢀ6.5 Hz), 8.32
1.90—2.10 (4H, m), 2.33 (3H, s), 2.35—2.45 (2H, m), 2.60—2.70 (2H, m), (1H, d, Jꢀ3.0 Hz).
4.54—4.61 (1H, m), 6.98 (1H, d, Jꢀ9.0 Hz), 8.13 (1H, dd, Jꢀ3.0, 9.0 Hz),
8.30 (1H, d, Jꢀ3.0 Hz).
3-Chloro-4-(piperidin-4-yloxy)nitrobenzene (9) To a solution of 4-[1-
3-Chloro-4-(1-phenylpiperidin-4-yloxy)nitrobenzene (8f) (Method C)
To a solution of 3-chloro-4-(piperidin-4-yloxy)nitrobenzene 9 (2.68 g, 10.4
mmol), bromobenzene (1.97 g, 12.5 mmol), 2-(di-t-butylphosphino)biphenyl
(t-butoxycarbonyl)piperidin-4-yloxy]-3-chloronitrobenzene 7 (7.91 g, 22.2 10 (0.62 g, 2.1 mmol) and Pd2(dba)3 (0.95 g, 1.0 mmol) in toluene (30 ml)

January 2009
29
was added NaOt-Bu (1.20 g, 12.5 mmol) and the mixture was stirred at
filtered and the filtrate was concentrated. The resulting residue was chro-
80 °C for 2 h. The mixture was filtered and the filtrate was concentrated. The matographed on a silica gel column (CH2Cl2/MeOHꢀ3/1—1/2) to give 11l
resulting residue was diluted with EtOAc and washed with NaHCO3 solution
(448 mg, 1.76 mmol, 58%) as a brown oil. 1H-NMR (CDCl3) d: 1.11 (3H, t,
and brine. The organic layer was dried and concentrated. The resulting Jꢀ7.0 Hz), 1.82—2.04 (4H, m), 2.24—2.34 (2H, m), 2.45 (2H, q, Jꢀ7.0
residue was chromatographed on a silica gel column (hexane/EtOAcꢀ4/1) Hz), 2.73—2.83 (2H, m), 4.11—4.18 (1H, m), 6.51 (1H, dd, Jꢀ3.0, 8.5 Hz),
to give 8f (1.86 g, 5.59 mmol, 54%) as a yellow solid. H-NMR (CDCl3) d: 6.73 (1H, d, Jꢀ3.0 Hz), 6.81 (1H, d, Jꢀ8.5 Hz).
1
2.00—2.10 (2H, m), 2.11—2.21 (2H, m), 3.19—3.29 (2H, m), 3.44—3.53
(2H, m), 4.70—4.77 (1H, m), 6.88 (1H, t, Jꢀ7.5 Hz), 6.95—7.00 (2H, m),
7.03 (1H, d, Jꢀ9.0 Hz), 7.25—7.32 (2H, m), 8.15 (1H, dd, Jꢀ3.0, 9.0 Hz),
8.31 (1H, d, Jꢀ3.0 Hz).
{N-[(E)-3-(3-Amidinophenyl)-2-propenyl]-N-[3-chloro-4-(1-methyl-
piperidin-4-yloxy)phenyl]sulfamoyl}acetic Acid Dihydrochloride (13a)
To a solution of 3-chloro-4-(1-methylpiperidin-4-yloxy)aniline 11a (6.95 g,
28.9 mmol) in CH2Cl2 (150 ml) were added EtO2CCH2SO2Cl (3.88 ml,
28.9 mmol) and pyridine (4.67 ml, 57.7 mmol) and the mixture was stirred at
Similarly, compound 8j was prepared.
8j: 1H-NMR (CDCl3) d: 2.03—2.22 (4H, m), 3.26—3.37 (2H, m), 3.44— room temperature for 5 h. H2O was added and the mixture was extracted
3.54 (2H, m), 4.74—4.81 (1H, m), 7.03 (1H, d, Jꢀ9.0 Hz), 7.18 (1H, dd, Jꢀ with EtOAc. The organic layer was dried and concentrated. The resulting
4.5, 8.5 Hz), 7.22—7.26 (1H, m), 8.12 (1H, dd, Jꢀ1.5, 4.5 Hz), 8.16 (1H,
dd, Jꢀ3.0, 9.0 Hz), 8.32 (1H, d, Jꢀ3.0 Hz), 8.36 (1H, d, Jꢀ3.0 Hz).
3-Chloro-4-(1-methylpiperidin-4-yloxy)aniline (11a) To a solution of
3-chloro-4-(1-methylpiperidin-4-yloxy)nitrobenzene 8a (8.48 g, 31.3 mmol)
residue was chromatographed on a silica gel column (CH2Cl2/MeOHꢀ4/1—
1/1) to give ethyl {N-[3-chloro-4-(1-methylpiperidin-4-yloxy)phenyl]sul-
famoyl}acetate (9.12 g, 23.3 mmol, 81%) as a brown amorphous solid. This
amorphous solid (7.37 g, 18.9 mmol), (E)-3-(3-cyanophenyl)-2-propen-1-ol 12
in AcOH (200 ml) was added tin powder (18.59 g, 156.6 mmol) and the mix- (3.30 g, 20.7 mmol) and PPh3 (5.93 g, 22.6 mmol) were dissolved in CH2Cl2
ture was stirred overnight at room temperature. The mixture was filtered and (200 ml) and the mixture was treated with diethyl azodicarboxylate (DEAD)
the filtrate was concentrated. The resulting residue was neutralized with (3.49 ml, 22.2 mmol). The resulting mixture was stirred overnight at room
K2CO3 solution and the mixture was extracted with EtOAc. The organic temperature and the mixture was concentrated. The resulting residue
layer was dried and concentrated. The resulting residue was chroma- was chromatographed on a silica gel column (EtOAc/MeOHꢀ3/1—1/2)
tographed on a silica gel column (CH2Cl2/MeOHꢀ3/1) to give 11a (6.95 g,
to give ethyl {N-[3-chloro-4-(1-methylpiperidin-4-yloxy)phenyl]-N-[(E)-3-
28.9 mmol, 92%) as a red-brown solid. 1H-NMR (CDCl3) d: 1.82—2.02 (3-cyanophenyl)-2-propenyl]sulfamoyl}acetate (7.29 g, 13.7 mmol, 73%) as
(4H, m), 2.20—2.30 (2H, m), 2.30 (3H, s), 2.68—2.78 (2H, m), 4.09—4.16 an orange amorphous solid. Into a solution of this amorphous solid (938 mg,
(1H, m), 6.51 (1H, dd, Jꢀ3.0, 8.5 Hz), 6.72 (1H, d, Jꢀ3.0 Hz), 6.81 (1H, d, 1.76 mmol) in CH2Cl2 (30 ml) and EtOH (15 ml) was bubbled hydrogen
Jꢀ8.5 Hz).
chloride under ice-cooling, and the resulting mixture was stirred at room
temperature under tightly sealed conditions for 5 h. The reaction mixture
Similarly, compounds 11b—k were prepared.
1
11b: H-NMR (CDCl3) d: 1.78—1.94 (4H, m), 2.11 (3H, s), 3.33—3.43 was concentrated and the resulting residue was dissolved in EtOH (20 ml).
(1H, m), 3.60—3.70 (1H, m), 3.70—3.82 (2H, m), 4.32—4.39 (1H, m), 6.53 The solution was treated with NH4Cl (189 mg, 3.53 mmol) in H2O (10 ml)
(1H, dd, Jꢀ3.0, 8.5 Hz), 6.74 (1H, d, Jꢀ3.0 Hz), 6.81 (1H, d, Jꢀ8.5 Hz).
and NH3 solution (0.350 ml, 5.75 mmol) and the mixture was stirred
overnight at room temperature. The mixture was treated with a 4 N solution
11c: 1H-NMR (CDCl3) d: 1.15 (6H, d, Jꢀ6.5 Hz), 1.80—2.20 (4H, m),
2.61—2.71 (2H, m), 2.91—3.00 (2H, m), 3.00—3.06 (1H, m), 4.24—4.31 of hydrogen chloride in dioxane and concentrated. The resulting residue was
(1H, m), 6.52 (1H, dd, Jꢀ3.0, 8.5 Hz), 6.73 (1H, d, Jꢀ3.0 Hz), 6.80 (1H, d, purified by a preparative HPLC (YMC-pack ODS, YMC Corp., H2O/
Jꢀ8.5 Hz).
MeCNꢀ39/11) to give an amorphous solid. This amorphous solid was dis-
solved in a 1 solution of hydrogen chloride and the mixture was concen-
trated. The resulting residue was lyophilized to give ethyl {N-[(E)-3-(3-
11d: 1H-NMR (CDCl3) d: 0.93 (3H, t, Jꢀ7.5 Hz), 1.30—1.38 (2H, m),
1.55—1.64 (2H, m), 1.92—2.02 (2H, m), 2.08—2.18 (2H, m), 2.58—2.67
N
(2H, m), 2.74—2.84 (2H, m), 2.90—2.98 (2H, m), 4.28—4.34 (1H, m), 6.52 amidinophenyl)-2-propenyl]-N-[3-chloro-4-(1-methylpiperidin-4-
(1H, dd, Jꢀ3.0, 8.5 Hz), 6.73 (1H, d, Jꢀ3.0 Hz), 6.79 (1H, d, Jꢀ8.5 Hz). yloxy)phenyl]sulfamoyl}acetate dihydrochloride (841 mg, 1.35 mmol, 77%)
11e: 1H-NMR (CDCl3) d: 1.48—1.61 (2H, m), 1.61—1.78 (4H, m), as a colorless amorphous solid. This amorphous solid (435 mg, 0.699 mmol)
1.86—2.02 (4H, m), 2.06—2.19 (2H, m), 2.71—2.98 (5H, m), 4.26—4.33 was dissolved in a 3 N solution of hydrogen chloride (20 ml) and stirred at
(1H, m), 6.52 (1H, dd, Jꢀ2.5, 8.5 Hz), 6.73 (1H, d, Jꢀ2.5 Hz), 6.79 (1H, d, 60 °C for 6.5 h. The reaction mixture was concentrated and the resulting
Jꢀ8.5 Hz). residue was purified by a preparative HPLC (YMC-pack ODS, YMC Corp.,
11f: 1H-NMR (CDCl3) d: 1.90—2.01 (2H, m), 2.03—2.12 (2H, m), H2O/MeCNꢀ87/13) to give an amorphous solid. This amorphous solid was
3.02—3.12 (2H, m), 3.50—3.61 (2H, m), 4.23—4.30 (1H, m), 6.53 (1H, dd, dissolved in a 1 N solution of hydrogen chloride (1 ml) and the mixture was
Jꢀ3.0, 8.5 Hz), 6.74 (1H, d, Jꢀ3.0 Hz), 6.81—6.87 (1H, m), 6.84 (1H, d, concentrated. The resulting residue was lyophilized to give 13a (243 mg,
Jꢀ8.5 Hz), 6.96 (2H, d, Jꢀ8.0 Hz), 7.23—7.29 (2H, m).
0.409 mmol, 59%) as a colorless amorphous solid. MS m/z: 521 (MꢂH)ꢂ.
1H-NMR (DMSO-d6) d: 1.90—2.07 (2H, m), 2.13—2.24 (2H, m), 2.68—2.80
11g: 1H-NMR (CDCl3) d: 1.80—1.90 (2H, m), 1.90—2.00 (2H, m),
2.21—2.32 (2H, m), 2.71—2.81 (2H, m), 3.52 (2H, s), 4.08—4.15 (1H, m), (3H, m), 3.00—3.09 (2H, m), 3.30—3.51 (2H, m), 4.28 (2H, s), 4.47 (2H, d,
6.50 (1H, dd, Jꢀ3.0, 8.5 Hz), 6.72 (1H, d, Jꢀ3.0 Hz), 6.80 (1H, d, Jꢀ Jꢀ6.0 Hz), 4.60—4.88 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.58 (1H, d,
8.5 Hz), 7.22—7.28 (1H, m), 7.28—7.36 (4H, m).
Jꢀ16.0 Hz), 7.29—7.33 (1H, m), 7.38—7.43 (1H, m), 7.55 (1H, t, Jꢀ8.0
11h: 1H-NMR (CDCl3) d: 1.83—1.95 (2H, m), 1.95—2.06 (2H, m), Hz), 7.57—7.61 (1H, m), 7.69 (1H, d, Jꢀ8.0 Hz), 7.73 (1H, d, Jꢀ8.0 Hz),
2.32—2.42 (2H, m), 2.58—2.67 (2H, m), 2.77—2.91 (4H, m), 4.12—4.20 7.88 (1H, s). IR (KBr) cmꢃ1: 1732, 1676, 1348, 1155. Anal. Calcd for
(1H, m), 6.52 (1H, dd, Jꢀ3.0, 8.5 Hz), 6.73 (1H, d, Jꢀ3.0 Hz), 6.82 (1H, d, C24H29ClN4O5S·2.1HCl·1.6H2O: C, 46.02; H, 5.52; N, 8.94; Cl, 17.54; S,
Jꢀ8.5 Hz), 7.17—7.24 (3H, m), 7.24—7.32 (2H, m).
5.12. Found: C, 45.95; H, 5.44; N, 8.99; Cl, 17.34; S, 5.31.
11i: 1H-NMR (CDCl3) d: 1.83—1.95 (2H, m), 1.97—2.07 (2H, m),
3.36—3.47 (2H, m), 3.90—4.01 (2H, m), 4.30—4.37 (1H, m), 6.53 (1H, dd,
Jꢀ3.0, 8.5 Hz), 6.59 (1H, dd, Jꢀ5.5, 7.0 Hz), 6.69 (1H, d, Jꢀ8.5 Hz), 6.74
Similarly, compounds 13c—l were prepared.
1
13c: MS m/z: 549 (MꢂH)ꢂ. H-NMR (DMSO-d6) d: 1.22 (6H, d, Jꢀ6.5
Hz), 1.91—2.03 (2H, m), 2.13—2.23 (2H, m), 2.92—3.38 (5H, m), 3.99
(1H, d, Jꢀ3.0 Hz), 6.85 (1H, d, Jꢀ8.5 Hz), 7.45—7.49 (1H, m), 8.17—8.22 (2H, s), 4.48 (2H, d, Jꢀ6.0 Hz), 4.72—4.79 (1H, m), 6.44 (1H, dt, Jꢀ16.0,
(1H, m). 6.0 Hz), 6.55 (1H, d, Jꢀ16.0 Hz), 7.27 (1H, d, Jꢀ9.0 Hz), 7.46 (1H, dd, Jꢀ
11j: 1H-NMR (CDCl3) d: 1.92—2.11 (4H, m), 3.08—3.19 (2H, m), 2.5, 9.0 Hz), 7.54 (1H, t, Jꢀ8.0 Hz), 7.65 (1H, d, Jꢀ2.5 Hz), 7.67 (1H, d, Jꢀ
3.51—3.61 (2H, m), 4.27—4.34 (1H, m), 6.53 (1H, dd, Jꢀ2.0, 9.0 Hz), 6.74
8.0 Hz), 7.71 (1H, d, Jꢀ8.0 Hz), 7.87 (1H, s). IR (KBr) cmꢃ1: 1677, 1344,
(1H, d, Jꢀ2.0 Hz), 6.84 (1H, d, Jꢀ9.0 Hz), 7.16 (1H, dd, Jꢀ4.5, 8.5 Hz), 1151. Anal. Calcd for C26H33ClN4O5S·1.3HCl·2.3H2O: C, 48.95; H, 6.15;
7.18—7.23 (1H, m), 8.08 (1H, d, Jꢀ4.5 Hz), 8.34 (1H, d, Jꢀ2.5 Hz).
N, 8.78; Cl, 12.78; S, 5.03. Found: C, 48.77; H, 6.05; N, 9.09; Cl, 12.76; S,
11k: 1H-NMR (CDCl3) d: 1.85—2.05 (4H, m), 3.30—3.38 (2H, m), 5.31.
3.65—3.73 (2H, m), 4.34—4.41 (1H, m), 6.54 (1H, dd, Jꢀ3.0, 8.5 Hz), 6.69
13d: MS m/z: 563 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 0.90 (3H, t, Jꢀ
(2H, dd, Jꢀ1.5, 5.0 Hz), 6.74 (1H, d, Jꢀ3.0 Hz), 6.83 (1H, d, Jꢀ8.5 Hz),
8.25 (2H, dd, Jꢀ1.5, 5.0 Hz).
3-Chloro-4-(1-ethylpiperidin-4-yloxy)aniline (11l) A solution of 4-(1-
acetylpiperidin-4-yloxy)-3-chloroaniline 11b (820 mg, 3.05 mmol) in THF
(10 ml) was added to a suspension of LiAlH4 (230 mg, 6.06 mmol) in THF
(5 ml) and the mixture was refluxed for 3.5 h. LiAlH4 (115 mg, 3.03 mmol)
7.5 Hz), 1.26—1.35 (2H, m), 1.56—1.65 (2H, m), 1.86—1.98 (2H, m), 2.09—
2.18 (2H, m), 2.83—3.18 (6H, m), 4.03 (2H, s), 4.48 (2H, d, Jꢀ6.0 Hz),
4.67—4.74 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.56 (1H, d, Jꢀ16.0 Hz),
7.27 (1H, d, Jꢀ9.0 Hz), 7.45 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.54 (1H, t, Jꢀ8.0
Hz), 7.64 (1H, d, Jꢀ2.5 Hz), 7.67 (1H, d, Jꢀ8.0 Hz), 7.71 (1H, d, Jꢀ8.0
Hz), 7.86 (1H, s). IR (KBr) cmꢃ1: 1676, 1347, 1153. Anal. Calcd for
was added and the mixture was refluxed for 2 h. Na2SO4·10H2O was added C27H35ClN4O5S·1.4HCl·2.0H2O: C, 49.88; H, 6.26; N, 8.62; Cl, 13.09; S,
and the mixture was stirred overnight at room temperature. The mixture was 4.93. Found: C, 49.58; H, 6.09; N, 8.76; Cl, 13.30; S, 5.04.

30
Vol. 57, No. 1
13e: MS m/z: 575 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.48—1.63 (2H, m),
Ethyl (N-{3-Chloro-4-[1-(4-pyridylmethyl)piperidin-4-yloxy]phenyl}-
N-[(E)-3-(3-cyanophenyl)-2-propenyl]sulfamoyl)acetate (15) To a solution
of ethyl (N-{4-[1-(t-butoxycarbonyl)piperidin-4-yloxy]-3-chlorophenyl}-N-
[(E)-3-(3-cyanophenyl)-2-propenyl]sulfamoyl)acetate 14 (1.25 g, 2.02 mmol)
in EtOH (15 ml) was added a 4 N solution of hydrogen chloride in dioxane
(15 ml) and the resulting mixture was stirred at room temperature for 4 h.
The mixture was concentrated and the resulting residue was diluted with
EtOAc. The organic solution was washed with NaHCO3 and brine, dried
and concentrated to give ethyl {N-[3-chloro-4-(piperidin-4-yloxy)phenyl]-N-
[(E)-3-(3-cyanophenyl)-2-propenyl]sulfamoyl}acetate (1.10 g, quant.) as a
pale yellow oil. This oil (1.10 g) was dissolved in DMF (30 ml) and treated
with 4-(bromomethyl)pyridine hydrobromide (590 mg, 2.33 mmol) and
K2CO3 (590 mg, 4.27 mmol). The mixture was stirred overnight at room
temperature and the mixture was diluted with EtOAc. The mixture was
washed with brine, dried and concentrated. The resulting residue was chro-
matographed on a silica gel column (EtOAc/MeOHꢀ10/1) to give 15
(970 mg, 1.60 mmol, 75%) as a pale yellow amorphous solid. 1H-NMR
(CDCl3) d: 1.36 (3H, t, Jꢀ7.0 Hz), 1.86—1.95 (2H, m), 1.95—2.04 (2H,
m), 2.33—2.43 (2H, m), 2.65—2.74 (2H, m), 3.53 (2H, s), 3.98 (2H, s), 4.31
(2H, q, Jꢀ7.0 Hz), 4.40—4.47 (1H, m), 4.46 (2H, d, Jꢀ6.5 Hz), 6.22 (1H,
dt, Jꢀ16.0, 6.5 Hz), 6.41 (1H, d, Jꢀ16.0 Hz), 6.92 (1H, d, Jꢀ9.0 Hz), 7.28
(2H, d, Jꢀ6.0 Hz), 7.31 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.40 (1H, t, Jꢀ8.0 Hz),
7.49—7.54 (2H, m), 7.53 (1H, d, Jꢀ2.5 Hz), 7.55 (1H, s), 8.54 (2H, d, Jꢀ
6.0 Hz).
(N-[(E)-3-(3-Amidinophenyl)-2-propenyl]-N-{3-chloro-4-[1-(4-pyridyl-
methyl)piperidin-4-yloxy]phenyl}sulfamoyl)acetic Acid Trihydrochlo-
ride (13m) Ethyl (N-{3-chloro-4-[1-(4-pyridylmethyl)piperidin-4-yloxy]-
phenyl}-N-[(E)-3-(3-cyanophenyl)-2-propenyl]sulfamoyl)acetate 15 was
converted to 13m by the same procedure as that for 13a. Compound 13m
was obtained (18%, 3 steps) as a colorless amorphous solid. MS m/z: 598
(MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.97—2.16 (2H, m), 2.16—2.40 (2H, m),
3.05—3.16 (2H, m), 3.30—3.46 (2H, m), 4.28 (2H, s), 4.47 (2H, d,
Jꢀ6.0 Hz), 4.52—4.92 (3H, m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.57 (1H, d,
Jꢀ16.0 Hz), 7.28—7.33 (1H, m), 7.38—7.44 (1H, m), 7.54 (1H, t,
Jꢀ8.0 Hz), 7.59 (1H, s), 7.68—7.74 (2H, m), 7.90 (1H, s), 8.14—8.22 (2H,
m), 8.88—8.93 (2H, m). IR (KBr) cmꢃ1: 1731, 1675, 1347, 1154. Anal.
Calcd for C29H32ClN5O5S·3.2HCl·1.7H2O: C, 46.73; H, 5.22; N, 9.40; Cl,
19.98; S, 4.30. Found: C, 46.65; H, 5.22; N, 9.45; Cl, 20.00; S, 4.44.
Ethyl (N-[3-Chloro-4-(1-methylpiperidin-4-yloxy)phenyl]-N-{(E)-3-[3-
(etoxycarbonylamino)(imino)methylphenyl]-2-propenyl}sulfamoyl)-
acetate Dihydrochloride (17a) To a solution of ethyl {N-[(E)-3-(3-
amidinophenyl)-2-propenyl]-N-[3-chloro-4-(1-methylpiperidin-4-
yloxy)phenyl]sulfamoyl}acetate dihydrochloride 16 (0.420 g, 0.675 mmol)
in H2O (5 ml) were added ethyl 4-nitrophenyl carbonate (0.140 g, 0.663
mmol) in CH2Cl2 (5 ml) and NaHCO3 (0.110 g, 1.31 mmol) and the mixture
was stirred at room temperature for 3 h. NaHCO3 solution was added and the
mixture was extracted with EtOAc. The organic layer was washed with
NaHCO3 solution, dried and concentrated. The resulting residue was chro-
matographed on a silica gel column (CH2Cl2/EtOHꢀ1/1) to give an amor-
phous solid. This amorphous solid was dissolved in EtOH (5 ml) and a 1 N
solution of hydrogen chloride (1.4 ml) and the mixture was concentrated.
The resulting residue was dissolved in H2O and the solution was lyophilized
to give 17a (0.360 g, 0.519 mmol, 78%) as a colorless amorphous solid. MS
m/z: 621 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.23 (3H, t, Jꢀ7.0 Hz), 1.33
(3H, t, Jꢀ7.0 Hz), 1.90—2.07 (2H, m), 2.15—2.25 (2H, m), 2.69—2.78
(3H, m), 3.00—3.10 (2H, m), 3.29—3.37 (1H, m), 3.40—3.50 (1H, m), 4.19
(2H, q, Jꢀ7.0 Hz), 4.35 (2H, q, Jꢀ7.0 Hz), 4.42 (2H, s), 4.47 (2H, d, Jꢀ
6.0 Hz), 4.60—4.89 (1H, m), 6.42 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.59 (1H, d, Jꢀ
16.0 Hz), 7.29—7.33 (1H, m), 7.38—7.43 (1H, m), 7.54 (1H, t, Jꢀ8.0 Hz),
7.57—7.62 (1H, m), 7.66 (1H, d, Jꢀ8.0 Hz), 7.75 (1H, d, Jꢀ8.0 Hz), 7.86
(1H, s). IR (KBr) cmꢃ1: 1742, 1674, 1354, 1157. Anal. Calcd for
C29H37ClN4O7S·2.3HCl·1.5H2O: C, 47.58; H, 5.82; N, 7.65; Cl, 15.98; S,
4.38. Found: C, 47.36; H, 5.93; N, 7.71; Cl, 15.94; S, 4.54.
1.63—1.76 (2H, m), 1.76—1.88 (2H, m), 1.93—2.10 (4H, m), 2.15—2.35
(2H, m), 2.91—3.13 (2H, m), 3.20—3.59 (3H, m), 4.26 (2H, s), 4.47 (2H, d,
Jꢀ6.0 Hz), 4.64—4.93 (1H, m), 6.45 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.58 (1H, d,
Jꢀ16.0 Hz), 7.31 (1H, d, Jꢀ9.0 Hz), 7.42 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.55 (1H,
t, Jꢀ8.0 Hz), 7.61 (1H, d, Jꢀ2.5 Hz), 7.69 (1H, d, Jꢀ8.0 Hz), 7.73 (1H, d,
Jꢀ8.0 Hz), 7.90 (1H, s). IR (KBr) cmꢃ1: 1732, 1676, 1348, 1155. Anal.
Calcd for C28H35ClN4O5S·2.0HCl·1.7H2O: C, 49.55; H, 6.00; N, 8.26; Cl,
15.67; S, 4.72. Found: C, 49.57; H, 5.81; N, 8.48; Cl, 15.56; S, 4.95.
13f: MS m/z: 583 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.88—2.08 (2H, m),
2.10—2.32 (2H, m), 3.04—3.91 (4H, m), 4.28 (2H, s), 4.47 (2H, d, Jꢀ6.0
Hz), 4.79—4.85 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.58 (1H, d, Jꢀ
16.0 Hz), 7.09—7.14 (1H, m), 7.26—7.49 (4H, m), 7.32 (1H, d, Jꢀ9.0 Hz),
7.42 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.60 (1H, d, Jꢀ2.5
Hz), 7.68 (1H, d, Jꢀ8.0 Hz), 7.74 (1H, d, Jꢀ8.0 Hz), 7.88 (1H, s). IR (KBr)
cmꢃ1: 1733, 1676, 1349, 1155. Anal. Calcd for C29H31ClN4O5S·2.0HCl·
1.9H2O: C, 50.46; H, 5.37; N, 8.12; Cl, 15.41; S, 4.65. Found: C, 50.79; H,
5.07; N, 7.93; Cl, 15.22; S, 4.78.
13g: MS m/z: 597 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.90—2.08 (2H, m),
2.12—2.26 (2H, m), 2.92—3.02 (2H, m), 3.20—3.50 (2H, m), 4.20—4.38
(2H, m), 4.25 (2H, s), 4.46 (2H, d, Jꢀ6.0 Hz), 4.59—4.85 (1H, m), 6.42 (1H,
dt, Jꢀ16.0, 6.0 Hz), 6.56 (1H, d, Jꢀ16.0 Hz), 7.27 (1H, d, Jꢀ9.0
Hz), 7.39 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.40—7.50 (3H, m), 7.54 (1H, t, Jꢀ8.0 Hz),
7.55—7.65 (3H, m), 7.66 (1H, d, Jꢀ8.0 Hz), 7.72 (1H, d, Jꢀ8.0 Hz), 7.85 (1H,
s). IR (KBr) cmꢃ1: 1732, 1675, 1349, 1154. Anal. Calcd for C30H33ClN4O5S·
1.9HCl·1.8H2O: C, 51.56; H, 5.55; N, 8.02; Cl, 14.71; S, 4.59. Found: C,
51.43; H, 5.54; N, 8.03; Cl, 14.52; S, 4.92.
13h: MS m/z: 611 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.96—2.08 (2H,
m), 2.18—2.28 (2H, m), 3.00—3.14 (4H, m), 3.20—3.50 (4H, m), 4.26 (2H,
s), 4.47 (2H, d, Jꢀ6.0 Hz), 4.80—4.87 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 6.0
Hz), 6.58 (1H, d, Jꢀ16.0 Hz), 7.22—7.39 (6H, m), 7.42 (1H, dd, Jꢀ2.5, 9.0
Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.60 (1H, d, Jꢀ2.5 Hz), 7.67 (1H, d, Jꢀ8.0 Hz),
7.73 (1H, d, Jꢀ8.0 Hz), 7.87 (1H, s). IR (KBr) cmꢃ1: 1732, 1675, 1349,
1154. Anal. Calcd for C31H35ClN4O5S·1.9HCl·1.6H2O: C, 52.50; H, 5.70;
N, 7.90; Cl, 14.50; S, 4.52. Found: C, 52.52; H, 5.49; N, 7.96; Cl, 14.44; S,
4.62.
13i: MS m/z: 584 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.71—1.82 (2H, m),
2.01—2.12 (2H, m), 3.63—3.75 (2H, m), 3.85—3.97 (2H, m), 4.28 (2H, s),
4.47 (2H, d, Jꢀ6.0 Hz), 4.80—4.87 (1H, m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz),
6.58 (1H, d, Jꢀ16.0 Hz), 6.87—6.92 (1H, m), 7.30—7.40 (1H, m), 7.33
(1H, d, Jꢀ9.0 Hz), 7.41 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.55 (1H, t, Jꢀ8.0 Hz),
7.59 (1H, d, Jꢀ2.5 Hz), 7.68 (1H, d, Jꢀ8.0 Hz), 7.73 (1H, d, Jꢀ8.0 Hz),
7.88 (1H, s), 7.90—7.96 (1H, m), 8.02 (1H, d, Jꢀ6.0 Hz). IR (KBr) cmꢃ1
:
1733, 1676, 1349, 1155. Anal. Calcd for C28H30ClN5O5S·1.8HCl·2.0H2O:
C, 49.04; H, 5.26; N, 10.21; Cl, 14.48; S, 4.68. Found: C, 48.75; H, 4.87; N,
10.24; Cl, 14.34; S, 4.80.
13j: MS m/z: 584 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.69—1.81 (2H,
m), 1.97—2.08 (2H, m), 3.37—3.48 (2H, m), 3.62—3.72 (2H, m), 4.29 (2H,
s), 4.47 (2H, d, Jꢀ6.0 Hz), 4.77—4.84 (1H, m), 6.45 (1H, dt, Jꢀ16.0,
6.0 Hz), 6.58 (1H, d, Jꢀ16.0 Hz), 7.33 (1H, d, Jꢀ9.0 Hz), 7.41 (1H, dd,
Jꢀ2.5, 9.0 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.59 (1H, d, Jꢀ2.5 Hz), 7.69 (1H, d,
Jꢀ8.0 Hz), 7.74 (1H, d, Jꢀ8.0 Hz), 7.77 (1H, dd, Jꢀ5.5, 9.0 Hz), 7.89 (1H,
s), 8.04 (1H, dd, Jꢀ2.0, 9.0 Hz), 8.15 (1H, d, Jꢀ5.5 Hz), 8.48 (1H, d,
Jꢀ2.0 Hz). IR (KBr) cmꢃ1: 1731, 1675, 1348, 1154. Anal. Calcd for
C28H30ClN5O5S·2.2HCl·1.8H2O: C, 48.27; H, 5.18; N, 10.05; Cl, 16.28; S,
4.60. Found: C, 48.19; H, 4.97; N, 10.13; Cl, 16.13; S, 4.76.
13k: MS m/z: 584 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.70—1.80 (2H,
m), 1.99—2.09 (2H, m), 3.64—3.75 (2H, m), 3.80—3.90 (2H, m), 4.26 (2H,
s), 4.47 (2H, d, Jꢀ6.0 Hz), 4.83—4.90 (1H, m), 6.45 (1H, dt, Jꢀ16.0, 6.0
Hz), 6.58 (1H, d, Jꢀ16.0 Hz), 7.22 (2H, d, Jꢀ7.5 Hz), 7.33 (1H, d, Jꢀ9.0
Hz), 7.42 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.59 (1H, d, Jꢀ
2.5 Hz), 7.69 (1H, d, Jꢀ8.0 Hz), 7.73 (1H, d, Jꢀ8.0 Hz), 7.89 (1H, s), 8.24
(2H, d, Jꢀ7.5 Hz). IR (KBr) cmꢃ1: 1731, 1675, 1347, 1154. Anal. Calcd for
C28H30ClN5O5S·1.7HCl·2.3H2O: C, 48.92; H, 5.32; N, 10.19; Cl, 13.92; S,
4.66. Found: C, 49.11; H, 5.08; N, 10.22; Cl, 13.87; S, 4.72.
Similarly, compounds 17b—i were prepared.
17b: MS m/z: 699 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.23 (3H, t,
Jꢀ7.0 Hz), 1.83—1.93 (1H, m), 2.00—2.18 (2H, m), 2.20—2.27 (1H, m),
2.71—2.82 (3H, m), 3.00—3.10 (2H, m), 3.30—3.50 (2H, m), 3.77 (3H, s),
4.19 (2H, q, Jꢀ7.0 Hz), 4.42 (2H, s), 4.48 (2H, d, Jꢀ6.0 Hz), 4.58—4.87
(1H, m), 6.39 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.59 (1H, d, Jꢀ16.0 Hz), 6.99 (2H,
d, Jꢀ9.0 Hz), 7.17 (2H, d, Jꢀ9.0 Hz), 7.29—7.33 (1H, m), 7.39—7.43 (1H,
m), 7.51 (1H, t, Jꢀ7.5 Hz), 7.58—7.63 (1H, m), 7.69 (1H, d, Jꢀ7.5 Hz),
7.80 (1H, d, Jꢀ7.5 Hz), 7.97 (1H, s). IR (KBr) cmꢃ1: 1740, 1671, 1354,
1161. Anal. Calcd for C34H39ClN4O8S·2.0HCl·2.0H2O: C, 50.53; H, 5.61;
N, 6.93; Cl, 13.16; S, 3.97. Found: C, 50.24; H, 5.75; N, 6.98; Cl, 12.90; S,
13l: MS m/z: 535 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.24 (3H, t, Jꢀ
7.5 Hz), 1.92—2.05 (2H, m), 2.14—2.24 (2H, m), 2.99—3.10 (2H, m),
3.02—3.49 (4H, m), 4.15 (2H, s), 4.48 (2H, d, Jꢀ6.0 Hz), 4.72—4.79 (1H,
m), 6.44 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.57 (1H, d, Jꢀ16.0 Hz), 7.29 (1H, d,
Jꢀ9.0 Hz), 7.43 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.54 (1H, t, Jꢀ8.0 Hz), 7.62
(1H, d, Jꢀ2.5 Hz), 7.68 (1H, d, Jꢀ8.0 Hz), 7.72 (1H, d, Jꢀ8.0 Hz), 7.88
(1H, s). IR (KBr) cmꢃ1: 1731, 1676, 1348, 1154. Anal. Calcd for
C25H31ClN4O5S·2.2HCl·1.4H2O: C, 46.88; H, 5.67; N, 8.75; Cl, 17.71; S,
5.01. Found: C, 46.75; H, 5.70; N, 8.48; Cl, 17.57; S, 5.37.

January 2009
4.45.
31
solved in a 1 N solution of hydrogen chloride and the mixture was concen-
17c: MS m/z: 649 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.35 (3H, t, Jꢀ trated. The resulting residue was dissolved in H2O and the solution was
7.0 Hz), 1.54 (9H, s), 1.87—1.96 (2H, m), 1.97—2.06 (2H, m), 2.32 (3H, s), lyophilized to give 17j (148 mg, 0.232 mmol, 15%) as a colorless amorphous
2.34—2.44 (2H, m), 2.64—2.73 (2H, m), 3.99 (2H, s), 4.30 (2H, q, Jꢀ solid. MS m/z: 565 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.23 (3H, t, Jꢀ7.0
7.0 Hz), 4.37—4.44 (1H, m), 4.44 (2H, d, Jꢀ6.5 Hz), 6.22 (1H, dt, Jꢀ16.0, Hz), 1.86—2.27 (4H, m), 2.71—2.80 (3H, m), 2.98—3.11 (2H, m), 3.40—
6.5 Hz), 6.42 (1H, d, Jꢀ16.0 Hz), 6.91 (1H, d, Jꢀ9.0 Hz), 7.27—7.32 (1H, 3.52 (2H, m), 4.19 (2H, q, Jꢀ7.0 Hz), 4.37—4.49 (4H, m), 4.57—4.89 (1H,
m), 7.34 (1H, t, Jꢀ8.0 Hz), 7.45 (1H, d, Jꢀ8.0 Hz), 7.52 (1H, d, Jꢀ2.5 Hz), m), 6.40 (1H, dt, Jꢀ15.5, 6.0 Hz), 6.57 (1H, d, Jꢀ15.5 Hz), 7.30 (1H, t, Jꢀ
7.66 (1H, d, Jꢀ8.0 Hz), 7.78 (1H, s). IR (KBr) cmꢃ1: 1740, 1655, 1365, 9.0 Hz), 7.38—7.69 (5H, m), 7.77 (1H, s). IR (KBr) cmꢃ1: 1737, 1666,
1163. Anal. Calcd for C31H41ClN4O7S·0.1HCl·0.7H2O: C, 55.95; H, 6.44; 1352, 1156. Anal. Calcd for C26H33N4O6S·2.2HCl·2.0H2O: C, 45.83; H,
N, 8.42; Cl, 5.86; S, 4.82. Found: C, 56.24; H, 6.27; N, 8.27; Cl, 5.85; S, 5.80; N, 8.22; Cl, 16.65; S, 4.71. Found: C, 45.50; H, 5.78; N, 8.59; Cl,
4.79.
16.80; S, 4.66.
17d: MS m/z: 687 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.36 (3H, t, Jꢀ
(Z)-3-(3-Cyanophenyl)-2-fluoro-2-propen-1-ol (21) To a solution of
7.0 Hz), 1.86—1.95 (2H, m), 1.95—2.04 (2H, m), 2.31 (3H, s), 2.32—2.40 ethyl 2-(diethylphosphono)-2-fluoroacetate (9.82 g, 40.5 mmol) in THF (160
(2H, m), 2.60—2.72 (2H, m), 3.99 (2H, s), 4.31 (2H, q, Jꢀ7.0 Hz), 4.36—
4.44 (1H, m), 4.46 (2H, d, Jꢀ6.5 Hz), 6.26 (1H, dt, Jꢀ16.0, 6.5 Hz), 6.47
(1H, d, Jꢀ16.0 Hz), 6.91 (1H, d, Jꢀ9.0 Hz), 7.04—7.12 (2H, m), 7.13—
7.20 (2H, m), 7.31 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.42 (1H, t, Jꢀ8.0 Hz), 7.53
(1H, d, Jꢀ2.5 Hz), 7.54 (1H, d, Jꢀ8.0 Hz), 7.75 (1H, d, Jꢀ8.0 Hz), 7.86
ml) was added NaH (2.12 g, 48.6 mmol, as a 55% (w/w) dispersion in mineral
oil) at ꢃ15 °C and the mixture was stirred at ꢃ15 °C for 10 min. 3-Cyanoben-
zaldehyde 19 (5.58 g, 42.6 mmol) in THF (40 ml) was added and the mixture
was stirred at ꢃ15 °C for 3 h. The reaction mixture was quenched with
NH4Cl solution and the mixture was extracted with EtOAc. The organic
(1H, s). IR (KBr) cmꢃ1: 1739, 1668, 1355, 1162. Anal. Calcd for layer was washed with brine, dried and concentrated. The resulting residue
C33H36ClFN4O7S·0.9HCl·0.5H2O: C, 54.37; H, 5.24; N, 7.69; Cl, 9.24; F, was chromatographed on a silica gel column (hexane/EtOAc/CH2Cl2ꢀ5/2/3)
2.61; S, 4.40. Found: C, 54.40; H, 5.38; N, 7.34; Cl, 9.25; F, 2.69; S, 4.54.
to give a mixture of (E)- and (Z)-20 (6.07 g, 27.7 mmol, 65%) as a colorless
17e: MS m/z: 679 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.15 (3H, t, Jꢀ solid. This solid (6.07 g, 27.7 mmol) was dissolved in MeCN (100 ml), and
7.5 Hz), 1.36 (3H, t, Jꢀ7.0 Hz), 1.87—2.05 (4H, m), 2.32 (3H, s), 2.33— the solution was treated with bromine (3 drops, catalytic amount). The re-
2.43 (4H, m), 2.63—2.72 (2H, m), 3.99 (2H, s), 4.31 (2H, q, Jꢀ7.0 Hz),
sulting mixture was stirred at room temperature for 1 h and the mixture was
4.36—4.47 (3H, m), 5.88 (2H, s), 6.25 (1H, dt, Jꢀ15.5, 7.0 Hz), 6.45 (1H, d, concentrated to give (Z)-20 (6.03 g, 27.5 mmol, 99%) as a pale yellow solid.
Jꢀ15.5 Hz), 6.91 (1H, d, Jꢀ9.0 Hz), 7.28—7.42 (2H, m), 7.50—7.54 (2H, This solid (6.01 g, 27.4 mmol) was suspended in EtOH (100 ml) and the sus-
m), 7.72 (1H, d, Jꢀ8.0 Hz), 7.82 (1H, s). IR (KBr) cmꢃ1: 1741, 1667, 1354, pension was treated with NaBH4 (2.07 g, 54.7 mmol). The mixture was
1158. Anal. Calcd for C31H39ClN4O9S·0.1HCl·0.3H2O: C, 54.10; H, 5.81; stirred at 40 °C for 1 h. NH4Cl solution was added and the mixture was ex-
N, 8.14; Cl, 5.67; S, 4.66. Found: C, 53.81; H, 5.71; N, 8.42; Cl, 5.61; S, tracted with EtOAc. The organic layer was washed with brine, dried and
4.79.
concentrated. The resulting residue was chromatographed on a silica gel col-
17f: MS m/z: 741 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.36 (3H, t, Jꢀ umn (hexane/EtOAc/CH2Cl2ꢀ4/3/3) to give 21 (4.12 g, 23.3 mmol, 85%) as
1
7.5 Hz), 1.86—2.05 (4H, m), 2.29—2.42 (5H, m), 2.61—2.72 (2H, m), 3.69 a colorless solid. H-NMR (CDCl3) d: 4.32 (2H, dd, Jꢀ5.5, 12.5 Hz), 5.82
(2H, s), 3.99 (2H, s), 4.31 (2H, q, Jꢀ7.5 Hz), 4.36—4.49 (3H, m), 5.89 (2H, (1H, d, Jꢀ37.5 Hz), 7.45 (1H, t, Jꢀ8.0 Hz), 7.53 (1H, d, Jꢀ8.0 Hz), 7.70
s), 6.25 (1H, dt, Jꢀ15.5, 7.0 Hz), 6.45 (1H, d, Jꢀ15.5 Hz), 6.91 (1H, d, Jꢀ (1H, d, Jꢀ8.0 Hz), 7.81 (1H, s).
9.5 Hz), 7.23—7.42 (7H, m), 7.50—7.54 (2H, m), 7.71 (1H, d, Jꢀ8.0 Hz),
(E)-3-(3-Cyanophenyl)-2-methyl-2-propen-1-ol (22) A solution of 3-
7.81 (1H, s). IR (KBr) cmꢃ1: 1741, 1667, 1354, 1156. Anal. Calcd for cyanobenzaldehyde 19 (5.00 g, 38.1 mmol) and 2-(triphenylphosphoranyli-
C36H41ClN4O9S·0.1HCl·0.3H2O: C, 57.63; H, 5.60; N, 7.47; Cl, 5.20; S, dene)propionaldehyde (15.8 g, 49.6 mmol) in toluene (170 ml) was stirred at
4.27. Found: C, 57.39; H, 5.38; N, 7.48; Cl, 5.29; S, 4.57.
70 °C for 7 h and the reaction mixture was concentrated. The resulting
17g: MS m/z: 733 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.18—1.51 (8H, m), residue was chromatographed on a silica gel column (CH2Cl2) to give a yel-
1.60—1.80 (3H, m), 1.86—2.05 (6H, m), 2.28—2.43 (6H, m), 2.61—2.72 low solid. This yellow solid was recrystallized from Et2O and hexane (1/9)
(2H, m), 3.99 (2H, s), 4.30 (2H, q, Jꢀ7.0 Hz), 4.36—4.48 (3H, m), 5.87 to give (E)-3-(3-cyanophenyl)-2-methyl-2-propenal (5.19 g, 30.3 mmol, 80
(2H, s), 6.25 (1H, dt, Jꢀ16.5, 6.0 Hz), 6.45 (1H, d, Jꢀ16.5 Hz), 6.91 (1H, d, %) as colorless crystals. These crystals (4.69 g, 27.4 mmol) were dissolved
Jꢀ9.0 Hz), 7.30 (1H, dd, Jꢀ3.0, 9.0 Hz), 7.39 (1H, t, Jꢀ8.0 Hz), 7.50—7.54 in CH2Cl2 (30 ml) and EtOH (60 ml) and the mixture was treated with
(2H, m), 7.72 (1H, d, Jꢀ8.0 Hz), 7.82 (1H, s). IR (KBr) cmꢃ1: 1740, 1667, NaBH4 (518 mg, 13.7 mmol) and CeCl3·7H2O (3.57 g, 9.58 mmol). The
1356, 1157. Anal. Calcd for C35H45ClN4O9S·0.1HCl·0.2H2O: C, 56.77; H, mixture was stirred at 0 °C for 2 h and then NaBH4 (518 mg, 13.7 mmol) was
6.19; N, 7.57; Cl, 5.27; S, 4.33. Found: C, 56.66; H, 6.24; N, 7.39; Cl, 5.39; added and the resulting mixture was stirred at 0 °C for 1 h and quenched
S, 4.47.
with NH4Cl solution. The mixture was then extracted with EtOAc and the
17h: MS m/z: 707 (MꢂH)ꢂ. H-NMR (DMSO-d6) d: 1.22 (9H, s), 1.35 organic layer was washed with brine. The organic layer was dried and con-
(3H, t, Jꢀ8.0 Hz), 1.87—2.05 (4H, m), 2.29—2.44 (5H, m), 2.61—2.73 centrated. The resulting residue was chromatographed on a silica gel column
(2H, m), 3.99 (2H, s), 4.30 (2H, q, Jꢀ8.0 Hz), 4.36—4.49 (3H, m), 5.87 (hexane/EtOAcꢀ1/1) to give 22 (4.50 g, 26.0 mmol, 95%) as a colorless oil.
(2H, s), 6.25 (1H, dt, Jꢀ16.5, 6.0 Hz), 6.45 (1H, d, Jꢀ16.5 Hz), 6.91 (1H, d, 1H-NMR (CDCl3) d: 1.87 (3H, s), 4.21 (2H, s), 6.52 (1H, s), 7.40—7.57
Jꢀ9.0 Hz), 7.30 (1H, dd, Jꢀ3.0, 9.0 Hz), 7.40 (1H, t, Jꢀ8.0 Hz), 7.50—7.54 (4H, m).
1
(2H, m), 7.72 (1H, d, Jꢀ8.0 Hz), 7.82 (1H, s). IR (KBr) cmꢃ1: 1741, 1668,
1356, 1156. Anal. Calcd for C33H43ClN4O9S·0.3HCl·0.3H2O: C, 54.78; H,
(N-{4-[1-(Acetimidoyl)piperidin-4-yloxy]-3-carbamoylphenyl}-N-
[(Z)-3-(3-amidinophenyl)-2-fluoro-2-propenyl]sulfamoyl)acetic Acid Dihy-
6.12; N, 7.74; Cl, 6.37; S, 4.43. Found: C, 54.77; H, 6.13; N, 7.70; Cl, 6.31; drochloride (26d) DEAD (0.860 ml, 5.48 mmol) was added to a solution
S, 4.73. of ethyl (N-{4-[1-(t-butoxycarbonyl)piperidin-4-yloxy]-3-cabamoylphenyl}-
17i: MS m/z: 705 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.35 (3H, t, Jꢀ sulfamoyl)acetate 25 (2.20 g, 4.53 mmol), (Z)-3-(3-cyanophenyl)-2-fluoro-2-
7.5 Hz), 1.83—2.05 (4H, m), 2.19 (3H, s), 2.27—2.43 (5H, m), 2.61—3.73 propen-1-ol 21 (800 mg, 4.51 mmol) and PPh3 (1.50 g, 5.72 mmol) in
(2H, m), 3.99 (2H, s), 4.31 (2H, q, Jꢀ7.5 Hz), 4.35—4.49 (3H, m), 4.93 CH2Cl2 (50 ml) and the resulting mixture was stirred at room temperature
(2H, s), 6.24 (1H, dt, Jꢀ15.5, 6.5 Hz), 6.45 (1H, d, Jꢀ15.5 Hz), 6.91 (1H, d, for 2.5 h. The mixture was concentrated and the resulting residue was chro-
Jꢀ9.0 Hz), 7.28—7.41 (2H, m), 7.49—7.54 (2H, m), 7.71 (1H, d, Jꢀ8.0 matographed on a silica gel column (hexane/EtOAcꢀ1/2—1/4) to give ethyl
Hz), 7.80 (1H, s). IR (KBr) cmꢃ1: 1739, 1659, 1354, 1155. Anal. Calcd for (N-{4-[1-(t-butoxycarbonyl)piperidin-4-yloxy]-3-carbamoylphenyl}-N-[(Z)-
C32H37ClN4O10S·0.2HCl: C, 53.95; H, 5.26; N, 7.86; Cl, 5.97; S, 4.50. 3-(3-cyanophenyl)-2-fluoro-2-propenyl]sulfamoyl)acetate (3.40 g, quant.) as
Found: C, 54.03; H, 5.26; N, 7.71; Cl, 5.94; S, 4.44.
Ethyl (N-{(E)-3-[3-(Amino)(hydroxyimino)methylphenyl]-2-propenyl}-
N-[3-chloro-4-(1-methylpiperidin-4-yloxy)phenyl]sulfamoyl)acetate
a pale yellow amorphous solid. Into a solution of this amorphous solid
(2.90 g, 4.50 mmol) in CH2Cl2 (30 ml) and EtOH (30 ml) was bubbled hy-
drogen chloride under ice-cooling and the resulting mixture was stirred at
Dihydrochloride (17j) To a solution of ethyl {N-[3-chloro-4-(1-methyl- room temperature under tightly sealed conditions for 75 min. The reaction
piperidin-4-yloxy)phenyl]-N-[(E)-3-(3-cyanophenyl)-2-propenyl]sul- mixture was concentrated and the resulting residue was dissolved in EtOH
famoyl}acetate 18 (800 mg, 1.50 mmol) in EtOH (20 ml) were added hy- (30 ml) and H2O (5 ml). The solution was treated with NH4Cl (540 mg,
droxylamine hydrochloride (350 mg, 5.04 mmol) and Na2CO3 (240 mg,
2.26 mmol) and the mixture was stirred overnight at room temperature and
10.1 mmol) and NH3 solution (1.20 ml, 19.8 mmol) and the mixture was
allowed to stand at room temperature overnight. The mixture was concen-
refluxed for 9 h. The mixture was concentrated and the resulting residue was trated and the resulting residue was purified by a preparative HPLC (YMC-
purified by a preparative HPLC (YMC-pack ODS, YMC Corp., H2O/ pack ODS, YMC Corp., H2O/MeCNꢀ17/3) to give ethyl {N-[(Z)-3-(3-
MeCNꢂ13/7) to give an amorphous solid. This amorphous solid was dis-
amidinophenyl)-2-fluoro-2-propenyl]-N-[3-carbamoyl-4-(piperidin-4-

32
Vol. 57, No. 1
yloxy)phenyl]sulfamoyl}acetate (1.04 g, 1.85 mmol, 41%) as a colorless ment was performed three times. The concentration required to double the
amorphous solid. This amorphous solid (1.00 g, 1.78 mmol) was dissolved clotting time (CT2) was estimated by linear regression analysis using two
in EtOH (30 ml) and the mixture was treated with ethyl acetimidate hy- data points, the two mean values of the concentrations closest to the pre-
drochloride (450 mg, 3.64 mmol) and Et3N (1.00 ml, 7.21 mmol). The result-
ing mixture was stirred overnight at room temperature and the reaction mix-
dicted 2-fold PT.
Pharmacokinetic Study Each compound was orally administered to 3
ture was concentrated. The resulting residue was purified by a preparative dogs at a dose of 1 mg/kg. The compounds were dissolved in saline and used
HPLC (YMC-pack ODS, YMC Corp., H2O/MeCNꢀ41/9) to give an amor- as the dosing solutions. Blood samples were collected at each time. The
phous solid. This amorphous solid was dissolved in a 1 N solution of hydro-
gen chloride (5.6 ml) and the mixture was concentrated to give ethyl (N- centrations of the compounds were determined by LC-MS/MS using multi-
{4-[1-(acetimidoyl)piperidin-4-yloxy]-3-carbamoylphenyl}-N-[(Z)-3- ple reaction monitoring in positive electrospray ionization mode on a Micro-
(3-amidinophenyl)-2-fluoro-2-propenyl]sulfamoyl)acetate dihydrochloride mass Quattro LC mass spectrometer coupled with a Waters Alliance 2790
blood was transferred into tubes containing sodium citrate. The plasma con-
(1.06 g, 1.57 mmol, 88%) as a colorless amorphous solid. This amorphous
solid (910 mg, 1.35 mmol) was dissolved in a 3 N solution of hydrogen chlo-
ride (20 ml) and the mixture was stirred at 70 °C for 3 h. The reaction mix-
HPLC. Each compound was separated on an Atlantis HILIC Silica column.
Plasma Anti-FXa and Anticoagulant Assay in Dogs The plasma anti-
FXa activity was assessed by an amidolytic assay using a fluorogenic FXa
ture was concentrated and the resulting residue was purified by a preparative substrate, Z-Pyr-Gly-Arg-MCA3, and the activity (IU/ml) was calibrated
HPLC (YMC-pack ODS, YMC Corp., H2O/MeCNꢀ9/1) to give an amor- using an anti-FXa activity standard, enoxaparin, a low molecular weight he-
phous solid. This amorphous solid was dissolved in a 1 N solution of hydro-
parin. Frozen plasma samples were thawed rapidly in a 37 °C water bath and
gen chloride (4 ml) and the mixture was concentrated. The resulting residue placed on ice. An aliquot each of 5 ml plasma sample, 40 ml of buffer A
was lyophilized to give 26d (240 mg, 0.370 mmol, 27%) as a colorless amor-
(0.1 M Tris–HCl–0.2 M NaCl, pH 8.4) and 5 ml of 2 U/ml AT-III (final con-
phous solid. MS m/z: 575 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.79—1.92 centration (f.c.) of 0.1 U/ml) were added to each well of a 96-well plate and
(2H, m), 2.01—2.12 (2H, m), 2.30 (3H, s), 3.48—3.86 (4H, m), 4.27 (2H, the plate was briefly shaken. After the addition of 30 ml of 0.33 IU/ml FXa
s), 4.62 (2H, d, Jꢀ16.5 Hz), 4.83—4.90 (1H, m), 5.98 (1H, d, Jꢀ39.0 Hz),
(f.c. 0.1 IU/ml) and 20 ml of 1 mM Z-Pyr-Gly-Arg-MCA (f.c. 0.2 mM) to each
7.30 (1H, d, Jꢀ9.5 Hz), 7.51—7.61 (2H, m), 7.69 (1H, d, Jꢀ8.0 Hz), 7.74— reaction mixture, the plate was briefly shaken and incubated for 10 min at
7.83 (3H, m). IR (KBr) cmꢃ1: 1672, 1352, 1158. Anal. Calcd for room temperature under light-shielded conditions. The reaction was stopped
C26H31FN6O6S·2.0HCl·0.5H2O: C, 47.56; H, 5.22; N, 12.80; Cl, 10.80; F, by adding 50 ml of 60% (v/v) acetic acid solution to each well and the fluo-
2.89; S, 4.88. Found: C, 47.26; H, 5.08; N, 12.68; Cl, 10.87; F, 2.90; S, 4.82.
Similarly, compounds 26a—c were prepared.
rescent intensity (excitation 380 nm, emission 440 nm) was measured by a
multilabel counter (Wallac 1420 ARVOsx, PerkinElmer, Inc.). Anti-FXa ac-
26a: MS m/z: 532 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.68—1.81 (2H, m), tivity (IU/ml) in each plasma sample was calibrated with two-fold serial di-
1.99—2.11 (2H, m), 2.29 (3H, s), 3.47—3.57 (2H, m), 3.67—3.75 (1H, m), lutions (0.000—2.000 anti-FXa IU/ml plasma) of enoxaparin in pooled rat
3.78—3.85 (1H, m), 4.20 (2H, s), 4.59 (2H, d, Jꢀ15.5 Hz), 4.68—4.74 (1H, plasma using 4-parameter analysis (SOFTmax PRO 3.1.1, Molecular De-
m), 5.95 (1H, d, Jꢀ38.0 Hz), 7.06 (2H, d, Jꢀ9.0 Hz), 7.42 (2H, d, Jꢀ9.0
Hz), 7.59 (1H, t, Jꢀ8.0 Hz), 7.68 (1H, d, Jꢀ8.0 Hz), 7.76 (1H, d, Jꢀ8.0 Hz),
vices Corp.).
PT was used to determine the ex vivo anticoagulant activities of the test
7.81 (1H, s). IR (KBr) cmꢃ1: 1673, 1627, 1350, 1157. Anal. Calcd for compound. Plasma samples (50 ml) were each added to test tubes and placed
C25H30FN5O5S·2.3HCl·2.6H2O: C, 45.34; H, 5.71; N, 10.57; Cl, 12.31; F, in a coagulometer (KC-10A micro, Heinrich Amelung GmbH), and 100 ml
2.87; S, 4.84. Found: C, 45.26; H, 5.59; N, 10.64; Cl, 12.36; F, 2.84; S, 5.05.
of PT reagent (Thromboplastin C plus, Dade Behring Marburg GmbH) was
26b: MS m/z: 528 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.63—1.82 (2H, added to the plasma to start the clotting after 1 min incubation at 37 °C.
m), 1.88 (3H, s), 1.99—2.11 (2H, m), 2.30 (3H, s), 3.44—3.90 (4H, m), 4.19
(2H, s), 4.37 (2H, s), 4.67—4.75 (1H, m), 6.33 (1H, s), 7.05 (2H, d, Jꢀ9.0
Hz), 7.41 (2H, d, Jꢀ9.0 Hz), 7.48 (1H, d, Jꢀ8.0 Hz), 7.53—7.58 (2H, m),
7.67 (1H, d, Jꢀ8.0 Hz). IR (KBr) cmꢃ1: 1672, 1627, 1345, 1156. Anal.
Calcd for C26H33N5O5S·2.5HCl·1.2H2O: C, 48.76; H, 5.97; N, 10.94; Cl,
13.84; S, 5.01. Found: C, 48.75; H, 6.16; N, 10.87; Cl, 13.94; S, 4.84.
26c: MS m/z: 566 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.69—1.87 (2H, m),
1.97—2.11 (2H, m), 2.29 (3H, s), 3.46—3.81 (4H, m), 4.30 (2H, s), 4.61
(2H, d, Jꢀ16.5 Hz), 4.79—4.88 (1H, m), 5.98 (1H, d, Jꢀ38.5 Hz), 7.34 (1H,
d, Jꢀ9.0 Hz), 7.42 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.55—7.61 (2H, m), 7.69 (1H, d,
Jꢀ7.5 Hz), 7.76 (1H, d, Jꢀ7.5 Hz), 7.81 (1H, s). IR (KBr) cmꢃ1: 1736,
1675, 1625, 1351, 1157. Anal. Calcd for C25H29ClFN5O5S·2.2HCl·1.3H2O:
C, 44.84; H, 5.09; N, 10.46; Cl, 16.94; F, 2.84; S, 4.79. Found: C, 44.58; H,
5.07; N, 10.25; Cl, 17.00; F, 3.02; S, 4.77.
Acknowledgments We wish to thank Ms. Ikuko Shimada, Ms. Naoko
Suzuki and Ms. Yumiko Fujisawa for their expert synthetic and technical as-
sistance, as well as Dr. Ken-ichi Otsuguro for his many helpful discussions.
References and Notes
1) Fujimoto K., Tanaka N., Shimada I., Asai F., WO 02/081448 (2002).
2) Fujimoto K., Tanaka N., Shimada I., Asai F., Inoue K., Okada J., WO
02/089803 (2002).
3) Present address: General Administration Department, Daiichi Sankyo
Business Associe Co., Ltd.
4) Harder S., Thurmann P., Clin. Pharmacokinet., 30, 416—444 (1996).
5) Hirsh J., N. Engl. J. Med., 324, 1865—1875 (1991).
6) Wells P. S., Holbrook A. M., Crowther N. R., Hirsh J., Ann. Intern.
Med., 121, 676—683 (1994).
7) Davie E. W., Fujisawa K., Kisiel W., Biochemistry, 30, 10363—10370
(1991).
Biology. Anti-FXa and Trypsin Assay The hydrolysis of chro-
mogenic substrates was assayed by continuously measuring the absorbance
at 405 nm at 37 °C with a microplate reader (SPECTRA max PLUS 384,
Molecular Devices, CA, U.S.A.). Reaction mixtures (90 ml) were prepared in
96-well plates containing enzymes and compounds in reaction buffer (50 mM
Tris–HCl–150 mM NaCl, pH 8.4). Reactions were initiated by the addition of
10 ml of substrate and monitored for 5 min. The concentration required to in-
8) Hara T., Yokoyama A., Tanabe K., Ishihara H., Iwamoto M., Thromb.
Haemost., 74, 635—639 (1995).
9) Sato K., Kawasaki T., Hisamichi N., Taniuchi Y., Hirayama F., Koshio
H., Matsumoto Y., Br. J. Pharmacol., 123, 92—96 (1998).
hibit enzyme activity by 50% (IC50) was estimated from the dose–response 10) Noguchi T., Tanaka N., Nishimata T., Goto R., Hayakawa M., Sugi-
curves. The enzymes and substrates used were as follows: human FXa
(0.5 IU, Enzyme Research Laboratories, Inc., IN, U.S.A.) and S-2222 (4 mM,
dachi A., Ogawa T., Asai F., Matsui Y., Fujimoto K., Chem. Pharm.
Bull., 54, 163—174 (2006).
Daiichi Pure Chemical, Japan); human trypsin (750 mU, Athens Research & 11) Noguchi T., Tanaka N., Nishimata T., Goto R., Hayakawa M., Sugi-
Tech., Inc., GA, U.S.A.) and S-2222 (4 mM, Daiichi Pure Chemical, Japan).
Coagulation Assay Citrated blood samples were collected from healthy
dachi A., Ogawa T., Asai F., Ozeki T., Fujimoto K., Chem. Pharm.
Bull., 55, 393—402 (2007).
male volunteers and male hamsters (Japan SLC). Platelet-poor plasma was 12) Noguchi T., Tanaka N., Nishimata T., Goto R., Hayakawa M., Sugi-
prepared by centrifugation at 2000ꢄg for 10 min and stored at ꢃ20 °C until
dachi A., Ogawa T., Asai F., Fujimoto K., Chem. Pharm. Bull., 55,
use. The plasma clotting times were determined using a COAGMASTER II
1494—1504 (2007).
(Sankyo, Japan) and an ACL9000 (Instrumentation Laboratories, MA, 13) Noguchi T., Tanaka N., Nishimata T., Goto R., Hayakawa M., Sugi-
U.S.A.). The prothrombin time (PT) was measured using SIMPLASTIN
EXCEL (Organon Teknika, NC, U.S.A.) and HemosILTM RecombiPlastin
dachi A., Ogawa T., Asai F., Fujimoto K., Chem. Pharm. Bull., 56,
758—770 (2008).
(Instrumentation Laboratories, MA, U.S.A.). The activated partial thrombo- 14) Meyers A. I., Knaus G., Kamata K., Ford M. E., J. Am. Chem. Soc.,
plastin time (APTT) was measured using Platelin LS (Organon Teknika, NC,
U.S.A.) and HemosILTM SynthASil (Instrumentation Laboratories, MA,
U.S.A.). The coagulation times for each compound were compared with the
98, 567—576 (1976).
15) Bredereck H., Effenberger F., Henseleit E., Angew. Chem., 75, 790—
791 (1963).
coagulation times measured using a distilled water control. Each measure- 16) Guram A. S., Rennels R. A., Buchwald S. L., Angew. Chem. Int. Ed.

January 2009
Engl., 34, 1348—1350 (1995).
17) Folkmann M., Lund F. J., Synthesis, 1990, 1159—1166 (1990).
18) Alexander J., Bindra D. S., Glass J. D., Holahan M. A., Renyer M. L.,
Rork G. S., Sitko G. R., Stranieri M. T., Stupienski R. F., Veerapanane
H., Cook J. J., J. Med. Chem., 39, 480—486 (1996).
19) Eddarir S., Francesch C., Mestdagh H., Rolando C., Tetrahedron Lett.,
31, 4449—4452 (1990).
20) Gemal A. L., Luche J. L., Tetrahedron Lett., 22, 4077—4080 (1981).
21) Mitsunobu O., Synthesis, 1981, 1—28 (1981).
33
(MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.23 (3H, t, Jꢀ7.0 Hz), 1.87—1.97
(2H, m), 2.08—2.17 (2H, m), 3.01—3.11 (2H, m), 3.15—3.24 (2H,
m), 4.20 (2H, q, Jꢀ7.0 Hz), 4.38 (2H, s), 4.47 (2H, d, Jꢀ6.0 Hz),
4.77—4.84 (1H, m), 6.45 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.58 (1H, d, Jꢀ
16.0 Hz), 7.24 (1H, d, Jꢀ9.0 Hz), 7.47—7.74 (5H, m), 7.90 (1H, s).
IR (KBr) cmꢃ1: 1736, 1671, 1658. Anal. Calcd for C26H33N5O6S·
2.2HCl·1.2H2O: C, 48.38; H, 5.87; N, 10.85; Cl, 12.08; S, 4.97.
Found: C, 48.74; H, 6.30; N, 11.11; Cl, 12.09; S, 5.04. 27: MS m/z:
519 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.23 (3H, t, Jꢀ7.0 Hz), 1.79—
1.91 (2H, m), 2.04—2.15 (2H, m), 3.00—3.11 (2H, m), 3.13—3.24
(2H, m), 4.19 (2H, q, Jꢀ7.0 Hz), 4.40 (2H, s), 4.47 (2H, d, Jꢀ7.0 Hz),
4.64—4.71 (1H, m), 6.37—6.48 (1H, m), 6.58 (1H, d, Jꢀ16.0 Hz),
7.25 (1H, dd, Jꢀ2.5, 9.0 Hz), 7.31 (1H, t, Jꢀ9.0 Hz), 7.43 (1H, dd,
Jꢀ2.5, 12.5 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.66—7.71 (1H, m), 7.73
(1H, d, Jꢀ8.0 Hz), 7.88 (1H, s). IR (KBr) cmꢃ1: 1737, 1675. Anal.
Calcd for C25H31FN4O5S·2.0HCl·2.0H2O: C, 47.85; H, 5.94; N, 8.93;
Cl, 11.30; F, 3.03; S, 5.11. Found: C, 48.02; H, 5.70; N, 8.86; Cl,
22) In fact, the cLogP values of cinnamyl derivatives were increased by the
removal of the acetimidoyl group or the conversion of carboxyl group
into its ethyl ester form. For example, the cLogP values of compounds
1, 31 and 3 were 0.13ꢅ0.70, 1.12ꢅ0.71, and 2.29ꢅ0.50, respectively.
11.60; F, 3.03; S, 4.92. 3: MS m/z: 535 (MꢂH)ꢂ. H-NMR (DMSO-
1
d6) d: 1.23 (3H, t, Jꢀ7.0 Hz), 1.82—1.94 (2H, m), 2.05—2.16 (2H,
m), 3.03—3.12 (2H, m), 3.13—3.24 (2H, m), 4.19 (2H, q, Jꢀ7.0 Hz),
4.41 (2H, s), 4.47 (2H, d, Jꢀ6.5 Hz), 4.74—4.81 (1H, m), 6.44 (1H,
dt, Jꢀ16.0, 6.5 Hz), 6.57 (1H, d, Jꢀ16.0 Hz), 7.30 (1H, d, Jꢀ9.5 Hz),
7.41 (1H, dd, Jꢀ2.5, 9.5 Hz), 7.55 (1H, t, Jꢀ8.0 Hz), 7.59 (1H, d,
Jꢀ2.5 Hz), 7.69 (1H, d, Jꢀ8.0 Hz), 7.73 (1H, d, Jꢀ8.0 Hz), 7.88 (1H,
s). IR (KBr) cmꢃ1: 1737, 1675. Anal. Calcd for C25H31ClN4O5S·
1.8HCl·2.0H2O: C, 47.16; H, 5.83; N, 8.80; Cl, 15.59; S, 5.04. Found:
C, 47.31; H, 5.67; N, 8.53; Cl, 15.84; S, 4.86.
23) These compounds were synthesized in the previous report (see ref.
11). The physical data of these compounds are as follows. 5a: MS m/z:
501 (MꢂH)ꢂ. 1H-NMR (DMSO-d6) d: 1.23 (3H, t, Jꢀ7.0 Hz), 1.78—
1.90 (2H, m), 2.05—2.15 (2H, m), 2.98—3.09 (2H, m), 3.13—3.23
(2H, m), 4.20 (2H, q, Jꢀ7.0 Hz), 4.34 (2H, s), 4.45 (2H, d, Jꢀ6.0 Hz),
4.62—4.69 (1H, m), 6.45 (1H, dt, Jꢀ16.0, 6.0 Hz), 6.55 (1H, d, Jꢀ
16.0 Hz), 7.04 (2H, d, Jꢀ8.5 Hz), 7.39 (2H, d, Jꢀ8.5 Hz), 7.55 (1H,
t, Jꢀ8.0 Hz), 7.69 (1H, d, Jꢀ8.0 Hz), 7.72 (1H, d, Jꢀ8.0 Hz), 7.89
(1H, s). IR (KBr) cmꢃ1: 1737, 1675. Anal. Calcd for C25H32N4O5S·
1.9HCl·2.4H2O: C, 48.97; H, 6.36; N, 9.14; Cl, 10.99; S, 5.23. Found:
C, 49.29; H, 6.20; N, 8.84; Cl, 11.01; S, 4.98. 5b: MS m/z: 544
24) Ames B. N., Gurney E. G., Miller J. A., Bartsch H., Proc. Nat. Acad.
Sci. U.S.A., 69, 3128—3132 (1972).
25) Hirayama F., Koshio H., Katayama N., Kurihara H., Taniuchi Y., Sato
K., Hisamichi N., Sakai-Moritani Y., Kawasaki T., Matsumoto Y.,
Yanagisawa I., Bioorg. Med. Chem., 10, 1509—1523 (2002).