Ethenesulfonamide and ethanesulfonamide derivatives, a novel class of orally active endothelin-A receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(01)00187-0

In the previous paper, we described a series of 2-phenylethenesulfonamide derivatives, a novel class of ETA -selective endothelin (ET) receptor antagonists, including the 2-methoxyethoxy derivative 2a and the 2-fluoroethoxy derivative (2b). In this paper, we wish to report further details of structure activity relationships (SARs) of the two regions of the molecule in compound 2b, which were the alkoxy region at the 6-position of the core pyrimidine ring and the 2-phenylethenesulfonamide region. In these modifications, replacement of the 2-fluoroethoxy group with a methoxy group (6e) and replacement of the 2-phenylethenesulfonamide group with a 2-(pyridin-3-yl)ethenesulfonamide group (6l) or 2-phenylethanesulfonamide group (6q) were well tolerated both in the ET_A binding affinity and ET_A selectivity. Among them, compound 6e showed further improvement in oral activity compared to 2b. After oral administration, compound 6e inhibited the big ET-1 induced pressor response in conscious rats at 0.3 mg /kg with a duration of > 6.5 h. Compound 6e also exhibited a potent antagonisti activity in the pithed rats. Copyright

Full text of DOI:10.1016/S0968-0896(01)00187-0

Bioorganic & Medicinal Chemistry 9 (2001) 2955–2968
Ethenesulfonamide and Ethanesulfonamide Derivatives, a Novel
Class of Orally Active Endothelin-A Receptor Antagonists
Hironori Harada,a,* Jun-ichi Kazami,a Susumu Watanuki,a Ryuji Tsuzuki,b
Katsumi Sudoh,a Akira Fujimori,a Masanao Sanagi,a Masaya Orita,a Hideaki Nakahara,a
Jun Shimaya,c Shin-ichi Tsukamoto,a Akihiro Tanakad and Isao Yanagisawaa
aInstitute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
bBulk Manufacturing & Technology Division, Yamanouchi Pharmaceutical Co., Ltd., 160-2 Matsukubo,
Akahama, Takahagi, Ibaraki 318-0001, Japan
cInstitute for Drug Development Research, Yamanouchi Pharmaceutical Co., 3-17-1 Hasune, Itabashi, Tokyo 174-8612, Japan
dCorporate Planning Department, Yamanouchi Pharmaceutical Co., Nihonbashi-Honcho, Chuo-ku, Tokyo 103-0023, Japan
Received 30 March 2001; accepted 19 May 2001
Abstract—In the previous paper, we described a series of 2-phenylethenesulfonamide derivatives, a novel class of ETA-selective
endothelin (ET) receptor antagonists, including the 2-methoxyethoxy derivative 2a and the 2-fluoroethoxy derivative (2b). In this
paper, we wish to report further details of structure–activity relationships (SARs) of the two regions of the molecule in compound
2b, which were the alkoxy region at the 6-position of the core pyrimidine ring and the 2-phenylethenesulfonamide region. In these
modifications, replacement of the 2-fluoroethoxy group with a methoxy group (6e) and replacement of the 2-phenylethenesulfona-
mide group with a 2-(pyridin-3-yl)ethenesulfonamide group (6l) or 2-phenylethanesulfonamide group (6q) were well tolerated both
in the ETA binding affinity and ETA selectivity. Among them, compound 6e showed further improvement in oral activity compared
to 2b. After oral administration, compound 6e inhibited the big ET-1 induced pressor response in conscious rats at 0.3 mg /kg with
a duration of >6.5 h. Compound 6e also exhibited a potent antagonistic activity in the pithed rats. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
the ETB receptor has nearly equipotent affinity for these
three ETs.4
Endothelin (ET), isolated from the conditioned medium
of cultured porcine vascular endothelial cells in 1988, is
a highly potent vasoconstrictive 21-amino acid peptide.1
There are three isoforms (ET-1, ET-2, ET-3). Among
these isoforms, ET-1 is the predominant component of
the three ET-isopeptides and is derived from precursor
big ET-1.2 ET-1 has been believed to be implicated in
the pathogenesis of the various diseases, largely because
of its ability to constrict vascular and nonvascular
smooth muscle.3
A number of ETA- selective and ETA/ETB non-selective
or mixed non-peptide antagonists have been reported
for decades,5 some literature sources indicated that the
ETA-selective antagonists were the promising candidates
for the treatments of cardiovascular diseases such as
hypertension and heart failure.6 So we decided that our
first goal was to discover the ETA-selective antagonists.
In the previous paper,7 we reported on a series of 2-
phenylethenesulfonamide derivatives which have
a
Two subtypes of receptors for ETs, termed ETA recep-
tor and ETB receptor, have been cloned and stably
expressed in mammals, and the ETA receptor appears to
exhibit affinity for ET-1 and ET-2 over ET-3, whereas
hydrogen-bond accepting or donating group in the side
chain at the 6-position of the pyrimidine ring and the
discovery of potent and ETA-selective antagonists (2a–
c) through the modification of the ETA/ETB mixed
antagonist, Ro47-0203 (Bosentan, 1) (Fig. 1).
Although 2b showed high ETA-selectivity in vitro and
potent oral antagonistic activity in vivo, we further
*Corresponding author. Tel.: +81-298-54-1562; fax: +81-298-52-
2971; e-mail: haradah@yamanouchi.co.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00187-0

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
investigated the structure–activity relationships (SARs)
of the alkoxy region at the 6-position of the core pyr-
imidine ring in compound 2b, partly because the toxicity
of the 2-fluoroethanol had been reported.8 We were
concerned that metabolism of 2b might give toxic 2-
fluoroethanol or 2-fluoroacetic acid, and we considered
that we should avoid using the toxic 2-fluoroethanol for
the large scale synthesis. We were also curious to explore
the effect of a hydrogen-bond acceptor, such as a fluorine
atom or a methoxy group, in the alkoxy side chain at the
6-position. So we started examining compounds that
have a simple alkoxy group at the 6-position without
hydrogen-bond acceptor or donor in the alkyl moiety.
butyl methanesulfonamide to give the alcohol (9).
Compound 9 was then mesylated with methanesufo-
nylchloride, followed by elimination of methanesulfonic
acid to give the N-tert-butyl ethenesulfonamide (10).
The tert-butyl group in 10 was removed using TFA to
give the (E)-2-arylethenesulfonamide derivatives 4c–e.
The alkanesulfonamide derivative 4h–j was prepared in
three steps (method C).12 The chloride (11) was con-
verted to the sulfonic acid (12) with Na2SO3. Com-
pound 12 was treated with thionyl chloride, followed by
aqueous annmonia to give the alkanesulfonamide deri-
vative 4h–j.
After we discovered an alternative substituent of a 2-
fluoroethoxy group at the 6-position, we further explored
the details of the SARs of the 2-phenylethensulfonamide
region of the molecule in our class of compounds.
The (E) form of all compounds with an ethenyl group
was confirmed by the coupling constant between vinyl
protons (J>14 Hz) in the H NMR spectrum except 5c,
5e. 5f (see Experimental).
1
Chemistry
Results and Discussion
Schemes 1 and 2 show the syntheses of alkenesulfona-
mide or alkanesulfonamide derivatives.
Compounds have been evaluated in vitro for their affi-
nity toward cloned human ETA and ETB receptors
expressed in COS-1 cells by employing receptor-binding
assays. Functional vascular ET-1 antagonism was
determined in vitro for the ability to inhibit the ET-1
induced contraction of a ring preparation sample of rat
aorta. Some compounds were further examined in vivo
for their ability after intravenous or oral administration
to inhibit an increase in mean arterial blood pressure
(MABP) due to the administration of exogenous big
ET-1 to pithed or conscious rats.
The starting compound (3) was prepared according to
the method reported by Burri et al.9 Nucleophilic sub-
stitution of the pyrimidine derivative 3 with (E)-alkene-
sulfonamide (4a–g and 4k) or alkanesulfonamide (4h–j)
resulted in the chloropyrimidines (5a–k). The chloro-
pyrimidine 5 was treated with alkoxide in DMF or the
corresponding alcohol to give alkoxy analogues (6a–e,
6i–r). Treatment 5 with sodium thioalkoxide in DMF
gave the alkysulfanyl analogue 6f. Compound 5k was
heated with methylamine in aqueous dioxane to give the
methylamino derivative (6g). The hydroxy analogue
(6h) was synthesized by treatment 5k with sodium
hydroxide in aqueous DMF.
The SARs of our novel series of endothelin receptor
antagonists are summarized in Tables 1–3.
Table 1 shows the SARs of the alkoxy region at the 6-
position of the core pyrimidine ring.
In Scheme 2, an outline of the synthesis of the key
intermediates (4a–j) is given.
Removal of a fluorine atom from the 2-fluoroethoxy
derivative 2b afforded the ethoxy derivative 6a and
resulted in a 7-fold loss of affinity for the ETA receptor.
Increasing the size of the O-ethyl on 6a to propyl (6b),
isopropyl (6c), and cyclopropylmethyl (6d) led to a large
decrease in the ETA binding affinity. It was suggested to
us that a hydrogen-acceptor such as a fluorine atom
might be important for the binding affinity, but unex-
pectedly the methoxy derivative 6e was found to be
equally potent to the 2-fluoroethoxy derivative 2b, with
A Palladium-catalyzed Heck reaction between ethene-
sulfonamide (7) and arylbromide afforded the (E)-2-ary-
lethenesulfonamide derivatives 4a, b, f, g (method A).10
(E)-2-Arylethenesulfonamide derivatives 4c–e also have
been synthesized via the route shown in method B.11
The aldehyde (8) was reacted with the dianion of N-tert-
an IC
at the ETA receptor of 3.1 nM. In this simple
50
unsubstituted alkoxy series, the groups larger than a
methoxy group were not well tolerated, probably due to
steric factors.
There was a difference in the steric limitations of the
alkoxy group at the 6-position between two series ( 2a–c
vs 6a–e). These results suggest that the alkoxy side
chains with a hydrogen-acceptor in 2a–c may interact
with the hydrogen-bonding pocket on the ETA receptor
that the simple alkoxy side chains with no substituent in
6a–e cannot interact, and this hydrogen bonding led to
an increase in the binding affinity for ETA receptor. In
Figure 1.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
2957
Scheme 1. Ar=aryl, DMF=N,N-dimethylformamide.
Scheme 2. tBu=tert-Butyl, TFA=trifluoroacetic acid, Ms=methanesulfonyl.
contrast to the ETA receptor binding affinity, the affi-
nities for the ETB receptor of two series, except 2c, were
weak and almost of the same order (IC50=2000 nM for
2a, 2500 nM for 2b and 2500 nM for 6a), indicating that
such a hydrogen bonding binding site may not exist on
the ETB receptor. The relatively high potency of 2c for
the ETB receptor suggested that the hydroxyl group in
the alkoxy side chain might be playing an important
role in the ETB binding affinty.
dependent fashion. The pA2 value of 6e was 7.4ꢀ0.3
(n=16) and slightly larger than that of 2b (pA2=6.9)7
(pA2; see Experimental).
We also examined alkylamino and alkylsulfanyl sub-
stitutions at 6-position. The methylsulfanyl derivative 6f
had a slightly decreased affinity for the ETA binding.
The methylamino analogue 6g was also less potent by
30-fold than 6e. The hydroxy analogue 6h was inactive.
These results indicated the importance of the alkoxy
groups for the ETA binding affinity in this series of
compounds. We then focused our attention on investi-
gating the SARs of the 2-phenylethensulfonamide
region of 6e (Tables 2 and 3).
Although the ETA selectivity of 6e was lower than that
of 2b, it was still considerably high (390-fold vs the ETB
receptor). Compound 6e also blocked the contractions
caused by ET-1 in isolated rat aorta in a concentration

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
Replacement of the phenyl group with another aryl or
heteoaryl groups was investigated (Table 2). Replace-
ment of the phenyl group with a naphtyl group (6i and
6j) decreased in the ETA binding affinity by ca. 2- to 4-
fold and led to an increase in ETB binding affinity. The
2-naphtyl derivative 6j showed 8-fold lower ETA selec-
tivity than 6e. A series of pyridine derivatives (6k, 6l and
6m) were synthesized, and the rank of order of potency
in ETA binding was found to be 3-Py (6l)>2-Py
(6k)>4-Py (6m). The 3-pyridyl derivative 6l was the
most potent of the 6-methoxy analogues in the ETA
binding affinity and ETA selectivity, with an IC50 of
1.2 nM for the ETA receptor and an ETB/ETA ratio of
820-fold. Replacement of the phenyl group with a thie-
nyl group (6n and 6o) was also well tolerated both in the
ETA binding affinity and ETA selectivity. This exercise
revealed that aryl and heteroaryl substitutions, except
for the 4-pyridyl group,were well tolerated in the ETA
binding affinity.
matic ring is important and that two carbon units are
optimal.
The X-ray crystal structures of Bosentan (1) and 6e are
shown in Figures 2 and 3. Superimposition of 1 and 6e
in Figure 4(a) revealed that the key functional groups of
1 and 6e occupied nearly similar spatial positions,
except for the 2-methoxyphenyl group. Although the
methoxyphenyl ring of 6e was almost orthogonal to the
plane of the core pyrimidine ring, it was in the opposite
direction to the corresponding methoxyphenyl ring of 1.
In order to find stable conformations of the 2-methoxy-
phenyl group in 6e, molecular mechanics studies of 6e
were performed using the software SYBYL 6.6 (Tripos,
Inc., MO, USA) for modeling on the basis of X-ray
data. Rotating the C4–C3–O3-C13 by 1ꢁ, two energeti-
cally favored conformations, A and B, were observed.
The torsion angle (C4–C3–O3–C13) of conformations A
and B were 56.0 and ꢂ97.0ꢁ, respectively. There was no
substantial difference between the two conformation
energetically, because conformation A was preferred by
only 0.67 kcal/mol. As shown in Figure 4(b), good
superimposition of the 2-methoxy phenyl groups of
conformation B and the crystal structure of 1, is
observed. Therefore, though crystal structures of 1 and
6e did not show good alignment of the methoxyphenyl
Next, replacement of the ethenyl group in the 2-phenyl-
ethensulfonamide region of 6e with alkyl groups was
investigated (Table 3). The benzyl derivative 6p sig-
nificantly lost in activity. Though the phenylpropyl
derivative 6r also showed 14-fold less potent ETA bind-
ing affinity compared to 6e, the phenylethyl derivative
6q was almost equipotent to 6e both in the ETA bind-
ing affinity and ETA selectivity. It is suggested that the
distance between the sulfonamide group and the aro-
Table 2. ETA and ETB receptor binding affinities for ethenesulfona-
mide derivatives
Table 1. ETA and ETB receptor binding affinities for ethenesulfona-
mide derivatives
Compound
R
IC50 (nM)a
Selectivity
c
for ETA
b
b
ETA
ETB
e
6e
3.1ꢀ1.0
1200ꢀ250
390
86
Compound
R
IC50 (nM)a
Selectivity
c
for ETA
b
b
6i
7.4 (6.0)
640 (560)
ETA
ETB
1d
7.3ꢀ 1.4
260ꢀ33
36
420
1200
230
2ae,h
2bf,h
2cg,h
6ae
6b
–OCH2CH2OMe
–OCH2CH2F
–OCH2CH2OH
–OEt
4.8ꢀ2.5 2000 (140)
6j
6.2 (2.7)
8.5 (4.5)
1.2 (0.48)
38 (16)
310 (270)
730 (210)
980 (55)
50
86
2.1
2500ꢀ900
1.6 (0.42) 370 (85)
14ꢀ4.0 2500ꢀ190
120 (108)
180
–OPr
–OisoPr
6k
6l
6c
170 (68)
6d
270 (123)
6ee
6f
6g
6h
–OMe
–SMe
–NHMe
–OH
3.1ꢀ1.0 1200ꢀ250
390
130
81
820
>26
490
650
7.0 (0.6)
96 (80)
900 (220)
7700 (1350)
>1000
6m
6n
>1000
aExperiments were performed twice and the difference between the two
values obtained are shown in parentheses unless otherwise noted.
bCloned human receptor binding.
1.3 (0.5)
1.7 (0.4)
640 (211)
1100 (12)
cExpressed as ETB IC50/ETA IC50
.
dSodium salt, values are meansꢀSEM, n=11.
eValues are meansꢀSEM, n=3.
fValues are meansꢀSEM, n=3 for ETB, n=1 for ETA.
gPotassium salt.
6o
hThe biological data of compounds 2a, 2b and 2c were previously
reported.7
aꢂe
See footnotes in Table 1.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
Table 3. ETA and ETB receptor binding affinities for ethanesulfonamide derivatives
2959
Compound
R
R0
IC50 (nM)a
Selectivity
c
for ETA
b
b
ETA
ETB
6ee
6p
6q
6r
MeO–
HO(CH2)2O–
MeO–
3.1ꢀ1.0
>1000
1200ꢀ250
390
3.1 (1.4)
42 (0.2)
740 (108)
240
MeO–
>1000
>24
aꢂe
See footnotes to Table 1.
groups, it is thought that all key functional groups of 1
and 6e including the methoxyphenyl group align well.
group and the sulfonamide group (6e, 6i–l, 6n, 6o and
6q) showed potent ETA binding affinities, but the com-
pounds with one or three carbon units as the linker (6p
or 6r) showed much lower binding affinities.
The tert-butyl group in 1 was important for ETA bind-
ing affinity because removal of the tert-butyl group led
to ca. a 20-fold loss in the ETA binding affinity.9 It is
noteworthy that the tert-butyl group in 1 corresponds to
the phenyl group of 6e in our superimposition [Fig.
4(b)]. This result indicated that a hydrophobic group
such as alkyl or aryl group at a certain distance from
sulfonamide group might be important for the ETA
binding affinity, corresponding to the result in which the
compounds with two carbon units between the aryl
Though the key functional groups of 1 such as methoxy-
phenoxy, pyrimidinyl and sulfonamide groups corre-
spond to these of 6e, the selectivity for ETA receptor in
our series was generally higher than that of 1. These
results suggested that (a) an aryl group such as a phenyl
group may be more beneficial to ETA binding than a
Figure 2. X-ray crystal structure of Bosentan (1) (a hydrate molecule
was eliminated to simplify the figure).
Figure 3. X-ray crystal structure of 6e (a ethanol solvent of crystal-
lization was eliminated to simplify the figure).

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
Figure 4. (a) Superimposition of the crystal structures of Bosentan 1 (gray) and 6e; (b) superimposition of the crystal structure of Bosentan 1 (gray)
and conformation B of 6e (green).
tert-butyl group and be similar to a tert-butyl group for
ETB binding(1 versus 2c), and (b) the hydroxyethoxy
group at the 6-position of pyrimidine may be more
beneficial to ETB binding than other alkoxy groups
such as a methoxy group (2c vs 6e). These two factors
may contribute to the ETA selectivity of our series of
compounds.
Figure 5(a) and (b) and Table 4 highlight our in vivo
studies of the selected compounds.
Compounds with a 2-phenylethenesulfonamide moiety
(6e and 6l) and a 2-phenylethanesulfonamide moiety
(6q) inhibited the increase in MABP due to the admin-
istration of exogenous big ET-1 to conscious rats after
oral administration [Fig. 5(a)]. These all showed excel-
lent inhibitory activities at 0.3 mg/kg. Maximum inhibi-
tion of the pressor effect of big ET-1 was 82% for 6e,
76% for 6l and 81% for 6q (0.3 mg/kg). Though the
duration of action of the pyridylethenesulfonamide 6l
was short (<2.5 h), the phenylethenesulfonamide 6e and
the phenylethanesulfonamide 6q showed a longer dura-
tion of action (>6.5 h).
Figure 5(b) and Table 4 show the comparison of the in
vivo activity of 1 (Bosentan), 2b and 6e.
As shown in Figure 5(b), the oral antagonistic activity
of 6e at 1 mg/kg was more potent than that of 1 at
10 mg/kg, and equipotent to that of 2b at 3 mg/kg.
Compounds 1, 2b and 6e also inhibited the increase in
MABP due to the administration of exogenous big ET-1
to anesthetized pithed rats after intravenous adminis-
tration or oral administration (Table 4). In this study,
the ID50 value was defined as the dose of test com-
pounds which caused a 50% inhibition of the pressor
response to big ET-1 in diastolic blood pressure (DBP).
(details are described in the Experimental). Compound
6e showed excellent inhibitory activities after oral
Figure 5. (a) Effect of po administration (0.3 mg/kg) of 6e, 6l and 6q
on pressor response to big ET-1 in conscious normotensive rats: (b)
comparison of the effect of po administration of 1, 2b and 6e on
pressor response to big ET-1 in conscious normotensive rats: Change
in MABP (%): increase in mean arterial blood pressure (MABP) in
conscious rats elicited by iv administration of big ET-1 (0.5 nmol/kg).
1: sodium salt.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
2961
administration with an ID50 value of 1.1 mg /kg. The
oral activity of compound 6e was 32-fold more potent
than that of 1 and 4-fold more potent than that of 2b,
calculated from these ID50 values.
JMS-700T mass spectrometer. Elemental analysis data
were withinꢀ0.4% of the calculated values. All organic
extracts were dried over anhydrous MgSO4. Chromato-
graphic purification.
Figures 6 and 7 show the pharmacokinetic data for 6e
expressed as mean plasma concentration plotted against
hours after dosing of the monopotassium salt of 6e for
rats and dogs. At an oral dose of 0.3 mg/kg for the rat, 6e
had an area under the curve of 2.12 mg/h/mL, a half-life of
2.52h, a peak plasma concentration of 0.50mg/mL, and
the time of peak plasma concentration=0.50h. At an oral
dose of 0.3 mg/kg for the dog, 6e had an area under the
curve of 18.75ꢀ1.22 mg h/mL, a half-life of 7.42ꢀ0.92 h, a
peak plasma concentration of 1.43ꢀ0.08 mg/mL, and the
time of peak plasma concentration=0.69ꢀ0.38 h. The
bioavailability in the oral administration was estimated to
be 89% in the rat and 97ꢀ9% in the dog.
4,6-Dichloro-5-(2-methoxyphenoxy)pyrimidine
(3).
Sodium (12.7 g, 551 mmol) was added to ethanol
(700 mL) and stirred at room temperature until all
sodium was dissolved. Diethyl 2-(2-methoxyphenoxy)-
malonate (51.8 g, 184 mmol) and 2-amidinopyrimidine13
(benznesulfonic acid salt, 51.4 g, 184 mmol) was added
to the solution and heated under reflux for 2.75 h. It was
poured into ice-water. The mixture was acidified with
3 M HCl, and the resulting precipitate was collected by
filtration. The solid was washed with water and ethanol
to give 4,6-dihydroxy-5-(2-methoxyphenoxy)pyrimidine
(32.6 g, 57%): mp. 276–277 ꢁC. H NMR (DMSO-d6) d
1
Conclusion
Investigation of the details of the SARs of our ethene-
sulfonamide derivative 2b led to the discovery of some
potent ETA selective endothelin antagonists, the 2-aryl-
ethenesulfonamide derivatives and the 2-phenylethane-
sulfonamide derivative, including 6e and 6q (YM-
119836). Among this series, the 2-phenylethenesulfona-
mide 6e showed a potent affinity for the ETA receptor
with high ETA selectivity. Compound 6e also had a long
acting oral activity in the inhibition of the pressor
response caused by a big ET-1 infusion in both pithed
and conscious rats. The monopotassium salt of 6e
showed an excellent pharmacokinetic profile with bioa-
vailabilities of 89% in the rat and 97% in the dog
(0.3 mg/kg). The monopotassium salt of 6e (YM598
monopotassium salt) is now in clinical trials.
Figure 6. Pharmacokinetics of 6e in rats.
Experimental
Melting points were determined on a Yanagimoto micro
1
melting point apparatus without correction. H NMR
spectra were recorded on JNM-LA400, LA500, and
A500 spectrometers using tetramethylsilane as an inter-
nal standard. MS spectra were determined with a Hita-
chi M-80 or JEOL JMS-DX300 spectrometer. High
resolution mass spectra were recorded using a JEOL
Table 4. Comparison of the effects of po and iv administration of the
2-phenyethenesulfonamide derivatives on pressor response to big ET-1
in pithed rats
Compound
ID50 (mg/kg)a
iv
Po
6e
2b
1b
0.76 (0.63–0.94)
2.0 (1.5–3.2)
6.1 (4.9–8.0)
1.1 (0.70–2.4)
4.1 (3.0–6.6)
35 (19–220)
aID50 value was defined as the dose of test compounds which caused a
50% inhibition of the pressor response to big ET-1 in diastolic blood
pressure (DBP). n=3–8, ID50 values are expressed as the mean with
95% confidence limits.
bSodium salt.
Figure 7. Pharmacokinetics of 6e in dogs.

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
1
3.86 (3H, s), 6.67 (1H, dd, J=1.2, 8.0 Hz), 6.76–6.81
(1H, m), 6.93 (1H, dt, J=1.6, 8.0 Hz), 7.03 (1H, dd,
J=1.6, 8.0 Hz), 7.73 (1H, t, J=4.8 Hz), 9.07 (2H, d,
J=4.8 Hz), 12.25 (2H, m). FAB-MS m/z: 313 (M++1).
To phosphorous oxychloroide (90 mL) was added 4,6-
an oil: 90 MHz H NMR (CDCl3) d 1.41 (9H, s), 3.29–
3.37 (2H, m), 3.90–4.20(1H, m), 4.62 (1H, s), 5.29 (1H,
t, J=6.3 Hz), 7.26–7.37 (2H, m), 8.50–8.65 (2H, m).
FAB-MS m/z 259 (M++1).
dihydroxy-5-(2-methoxyphenoxy)pyrimidine
(30.0 g,
Step 2: To a ice-cooled solution of the alcohol 9a
(1.58 g, 6.14 mmol) in dichloromethane (CH2Cl2) was
added triethylamine (2.6 mL, 18.7 mmol) and methane-
sulfonylcholoride (0.7 mL, 9.04 mmol). The mixture was
stirred at room temperature for 1 h and then con-
centrated in vacuo. To the residue was added water and
extracted with EtOAc. The organic layer was washed
with brine and concentrated in vacuo. The residue was
chromatographed over silica gel using 3% MeOH in
CHCl3 to give (E)-N-tert-butyl 2-(pyridin-4-yl)-ethene-
sulfonamide (10a) (1.41 g, 96%) as a yellow oil: 90 MHz
1H NMR (CDCl3) d 1.38 (9H, s), 4.39 (1H, s), 6.97 (1H,
d, J=15.7 Hz), 7.26–7.49 (3H, m), 8.64–8.71 (2H, m).
FAB-MS m/z 241 (M++1).
96.1 mmol) and 2, 4, 6-trimethylpyridine (12.7 mL,
96.1 mmol). The mixture was stirred at 90 ꢁC for 17.5 h,
and it was poured into ice-water. The resulting pre-
cipitate was collected by filtration. The solid was recys-
tallized from ethanol to give 3 (25.1 g, 75%): mp. 153–
154 ꢁC. H NMR (DMSO-d6) d 3.85 (3H, s), 6.85–6.91
1
(1H, m), 6.98 (1H, dd, J=1.6, 8.0 Hz), 7.11–7.16 (1H,
m), 7.17–7.21 (1H, m), 7.72 (1H, t, J=4.8 Hz), 9.08 (2H,
d, J=4.8 Hz). EI–MS m/z 348 (M+).
The syntheses of the intermediate ethenesulfonamide
derivatives 4a–g and alkanesulfonamide derivatives 4h–j
were previously reported10,14,15 except for the com-
pounds described bellow.
Step 3: To 10a (1.36 g, 5.66 mmol) was added tri-
fluoroacetic acid (TFA) (20 mL) and the solution was
stirred at room temperature for 24 h. The solution was
then concentrated in vacuo. To the residue was added
water and neutralized with 1 N aqueous NaOH. It was
extracted with EtOAc. The organic layer was washed
with brine and concentrated in vacuo. The residual solid
was washed ꢁwith EtOAc/Hex to give 4e (0.73 g, 70%):
(E)-2-(Thiophen-3-yl)ethenesulfonamide (4f). To a solu-
tion of triphenylphosphine (130 mg, 0.496 mmol) in
N,N-dimethylformamide (DMF) (10 mL) was added
palladium acetate [Pd(OAc)2, 55 mg, 0.245 mmol] at
room temperature under an argon atmosphere. After
stirring for 5 min, to the mixture was added a solution
of 3-bromothiophen (1.2 mL, 12.7 mmol), ethenesulfo-
namide 7 (1.50 g, 14.0 mmol) and triethylamine (5.0 mL,
35.9 mmol) in DMF (5 mL). The mixture was stirred at
140 ꢁC for 7 h and then concentrated in vacuo. To the
residue was added diluted HCl and extracted with
EtOAc. The organic layer was washed with brine and
concentrated in vacuo. The residue was chromato-
graphed over silica gel using 1:1 ethyl acetate (AcOEt)/
hexane (Hex) to give a solid. The solid was recystallized
from AcOEt/Hex to give 4f (0.75 g, 31%): mp 152–
153 ꢁC. 90 MHz 1H NMR (DMSO-d6) d:7.03(1H,d,
J=15.5 Hz), 7.04 (2H,s), 7.34 (1H, d, J=15.5 Hz), 7.50–
7.97 (3H, m). FAB-MS m/z 190 (M++1).
1
mp 197–198 C (decomp). 90 MHz H NMR (CDCl3) d
7.33 (2H, s), 7.35 (1H, d, J=16.9 Hz), 7.68 (1H, d,
J=16.9 Hz), 7.84–8.00 (2H, m), 8.66–8.90 (2H, m).
FAB-MS m/z 185 (M++1).
(E)-2-(Pyridin-2-yl)ethenesulfonamide (4c). Compound
4c was prepared in the same manner as 4f. Mp 111–
ꢁ
1
112 C (EtOAc/Hex), 400 MHz H NMR (DMSO-d6) d
7.20 (2H, brs), 7.34 (1H, d, J=15.2 Hz), 7.33–7.45 (1H,
m), 7.44 (1H, d, J=15.2 Hz), 7.73 (1H, d, J=18.0 Hz),
7.85–7.90 (1H, m), 8.63–8.65 (1H, m). FAB-MS m/z 185
(M++1).
(E)-2-(Naphthalen-2-yl)ethenesulfonamide (4b). Com-
pound 4b was prepared in the same manner as 4f in
74% yield as a yellow solid: mp 189–190 ꢁC. 90 MHz 1H
NMR (DMSO-d6) d 7.05–7.70 (6H, m, containing 7.27,
d, J=15.5 Hz, 7.55, d, J=15.5 Hz), 7.80–8.30 (5H, m).
EI–MS m/z: 233 (M+).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(naphtalen-1-yl) ethenesulfonamide
(5a). To an ice-cooled solution of (E)-2-(naphtalen-1-
yl)ethenesulfonamide (4a)10 (670 mg, 2.87 mmol) in DMF
(10 mL) was added 60% sodium hydride in mineral oil
(230 mg, 5.75 mmol) and the mixture was stirred for 10 min
at room temperature. To the mixture 4,6-dichloro-5-(2-
methoxyphenoxy)pyrimidine (3) (950mg, 2.72 mmol) was
added, and the mixture was stirred for 4 h at room
temperature. It was poured into ice-water and acidified
with 1 N aqueous HCl, and the resulting precipitate was
collected by filtration. The solid was recrystallized from
ethanol (EtOH) to give 5a (1.19 g, 80%): mp 175–
(E)-2-(Pyridin-4-yl)ethenesulfonamide (4e). Step 1: To a
solution of N-tert-butylmethanesulfonamide (1.50 g,
9.92 mmol) in tetrahydrofuran (THF) (10 mL) was
added a hexane solution of n-butyl lithium (12.0 mL,
19.56 mmol) at ꢂ78 ꢁC under an argon atmosphere.
After stirring for 10 min at 0 ꢁC, 4-pyridinecarboxy-
aldehyde 8a (0.9 mL, 9.58 mmol) in THF (10 mL) was
added to the mixture at ꢂ78 ꢁC. The mixture was stirred
at room temperature for 45 min and poured into water.
The mixture was extracted with EtOAc added to the
mixture. The organic layer was washed with brine and
concentrated in vacuo. The residue was chromato-
graphed over silica gel using 7% methanol (MeOH) in
chloroform (CHCl3) to give N-tert-butyl 2-hydroxy-2-
(pyridin-4-yl)-ethanesulfonamide (9a) (1.61 g, 65%) as
177 ꢁC. H NMR (DMSO-d6) d 3.80 (3H, s), 6.83–6.90
1
(2H, m), 7.06–7.16 (2H, m), 7.54–7.65 (4H, m), 7.91(1H,
d, J=19.3 Hz), 7.98–8.03 (2H, m), 8.05 (1H, d,
J=10.0 Hz), 8.16 (1H, d, J=10.0 Hz), 8.59 (1H, d,
J=19.3 Hz), 8.94 (2H, d, J=6.0 Hz). FAB-MS m/z 544
(M+ꢂ1).
The chloropyrimidine derivatives (5b–j) were prepared
in the same manner as 5a.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
2963
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(naphtalen-2-yl) ethenesulfonamide
(5b). Compound 5b was prepared from (E)-2-(naphtha-
len-2-yl)ethenesulfonamide (4b) in 47% yield: mp 203–
(1H, d, J=4.9 Hz), 8.10 (1H, d, J=14.1 Hz), 9.12 (2H,
d, J=4.9 Hz), 12.01 (1H, brs). FAB-MS m/z
502(M++1).
208 ꢁC (EtOH): H NMR (DMSO-d6) d 3.83 (3H, s),
N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyri-
midin-4-yl]-1-phenylmethanesulfonamide (5h). Compound
5h was prepared from 1-phenylmethanesulfonamide 4h
1
6.68–6.79 (1H, m), 6.80–6.89 (1H, m), 7.00–7.08 (1H,
m), 7.12 (1H, d, J=9.0 Hz), 7.54–7.60 (2H, m), 7.72
(1H, t, J=6.5 Hz), 7.88–8.00 (5H, m), 8.50 (1H, d,
J=20 Hz), 8.13–8.20 (1H, m), 9.13 (2H, d, J=6.0 Hz).
FAB-MS m/z 544 (M+ꢂ1).
(prepared
from
1-phenylmethanesulfonylchloride,
Aldrich Co., Ltd.) in 96% yield: mp 105–106 ꢁC (Et2O).
1H NMR (DMSO-d6) d 3.78 (3H, s), 5.28 (2H, s), 6.50–
6.64 (1H, m), 6.75–6.86 (1H, m), 6.98–7.06 (1H, m), 7.09
(1H, d, J=8.5 Hz), 7.25–7.46 (5H, m), 7.71 (1H, t,
J=5.0 Hz), 9.09 (2H, d, J=5.0 Hz). FAB-MS m/z 484
(M++1).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(pyridin-2-yl) ethenesulfonamide (5c).
Compound 5c was prepared from (E)-2-(pyridine-2-
yl)ethenesulfonamide (4c) in 68% yield: mp 189–190 ꢁC
(EtOH). 1H NMR (DMSO-d6) d 3.80 (3H, s), 6.79–6.90
(2H, m), 7.04–7.17 (2H, m), 7.39–7.46 (1H, m), 7.70
(1H, t, J=6.0 Hz), 7.78 (1H, d, J=10.0 Hz), 7.83–7.98
(3H, m), 8.65 (1H, d, J=10.0 Hz), 9.07 (2H, d,
J=6.5 Hz). FAB-MS m/z: 497 (M++1).
N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyri-
midin-4-yl]-2-phenylethanesulfonamide (5i). Compound 5i
was prepared from 2-phꢁenylethanesulfonamide (4i)14 in
1
82% yield: mp 198–199 C (EtOH). H NMR (DMSO-
d6) d 3.00–3.10 (2H, m), 3.82 (3H, s), 4.16–4.38 (2H, m),
6.82 (1H, d, J=9.5 Hz), 6.85–6.91 (1H, m), 7.05–7.30
(7H, m), 7.69 (1H, t, J=6.0 Hz), 9.04 (2H, d,
J=6.0 Hz), 11.88(1H, s). FAB-MS m/z 496 (M+ꢂ1).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(pyridin-3-yl)ethenesulfonamide (5d).
Compound 5d was prepared from (E)-2-(pyridine-3-
yl)ethenesulfonamide (4d)10 in 62% yield: mp 189–
N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyri-
midin-4-yl]-3-phenylpropanesulfonamide (5j). Compound
5j was prepared from 2-phenylpropanesulfonamide
ꢁ
1
190 C (EtOH), H NMR (DMSO-d6) d 3.81 (3H, s),
6.81 (1H, d, J=10.0 Hz), 6.83–6.89 (1H, m), 7.05–7.10
(1H, m), 7.13 (1H, d, J=10.0 Hz), 7.53 (1H, dd, J=6.0,
10.0 Hz), 7.71 (1H, t, J=10.0 Hz), 7.92 (1H, d,
J=19.5 Hz), 7.97 (1H, d, J=19.5 Hz), 8.21–8.25 (1H,
m), 8.62 (1H, dd, J=2.0, 6.0 Hz), 8.91 (1H, d,
J=2.0 Hz), 9.07 (2H, d, J=6.0 Hz). FAB-MS m/z 497
(M++1).
(4j)15 in 82% yield: mp 194–195 ꢁC (EtOH). H NMR
1
(DMSO-d6)
d 1.90–2.10 (2H, m), 2.70 (2H, t,
J=7.0 Hz), 3.79 (3H, s), 3.82–4.00 (2H, m), 6.80 (1H, d,
J=8.0 Hz), 6.83–6.90 (1H, m), 7.05–7.17 (7H, m), 7.68
(1H, t, J=4.4 Hz), 9.02 (2H, d, J=4.4 Hz), 11.76 (1H,
s). FAB-MS m/z 512 (M++1).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(pyridin-4-yl)ethenesulfonamide (5e).
Compound 5e was prepared from (E)-2-(pyridine-4-
yl)ethenesulfonamide (4e)10 in 21% yield: mp 173–
174 C (decomp) (EtOH). H NMR (DMSO-d6) d 3.85
(3H, s), 6.66 (1H, dd, J=2.0, 7.0 Hz), 6.83–6.87 (1H,
m), 7.05–7.09 (1H, m), 7.12–7.16 (1H, m), 7.54–7.77
(3H, m), 8.62 (1H, d, J=7.5 Hz), 8.68–8.77 (1H, m),
8.95 (2H, d, J=6.5 Hz), 9.02–9.08 (2H, m). FAB-MS m/
z 497 (M++1).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (5k). See
ref 7.
ꢁ
1
(E)-N-[6-Ethoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
(6a).
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide
Sodium (181 mg, 8.07 mmol) was added to ethanol
(10 mL) and stirred at room temperature until all
sodium was dissolved. (E)-N-[6-chloro-5-(2-methoxy-
phenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylet-
henesulfonamide (5k) (400 mg, 0.807 mmol) was added
to the solution and stirred at room temperature for 2 h.
It was poured into ice-water and acidified with 1 M
aqueous HCl. The mixture was extracted with EtOAc
and the organic layer was concentrated in vacuo. The
residue was chromatographed over silica gel using 40:1
CHCl3/MeOH to give an oil. It was crystallized from
ether (Et2O) to give 6a (310 mg, 76%): mp 173–174 ꢁC.
1H NMR (DMSO-d6) d 1.05–1.11 (3H, m), 3.82 (3H, s),
4.36–4.38 (2H, m), 6.74 (1H, d, J=7.9 Hz), 6.83 (1H, t,
J=7.3 Hz), 7.03–7.06 (1H, m), 7.09 (1H, d, J=7.3 Hz),
7.40–7.55 (3H, m), 7.55–7.80 (3H, m), 7.82 (1H, d,
J=15.0 Hz), 7.99 (1H, d, J=15.0 Hz), 9.08 (2H, d,
J=5.0 Hz), 11.43 (1H, s). FAB-MS m/z 506 (M++1).
Anal. calcd for C25H23N5O5S: C, 59.40; H, 4.59; N,
13.85; S, 6.34. Found: C, 59.29; H, 4.51; N, 13.68; S, 6.31.
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)-
pyrimidin-4-yl]-2-(thiophen-3-yl) ethenesulfonamide (5f).
Compound 5f was prepared from (E)-2-(thiophen-3-
yl)ethenesulfonamide (4f) in 69% yield: mp 181–182 ꢁC
1
(EtOH). H NMR (DMSO-d6) d 3.80 (3H, s), 6.81 (1H,
d, J=9.5 Hz), 6.84–6.91 (1H, m), 7.06–7.16 (2H, m),
7.44–7.56 (1H, m), 7.57–7.68 (2H, m), 7.71(1H, t,
J=6.0 Hz), 7.84–8.02 (1H, m), 8.04 (1H, d, J=3.1 Hz),
9.10 (2H, d, J=6.0 Hz), 12.08 (1H, brs). FAB-MS m/z
502 (M++1).
(E)-N-[6-Chloro-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)-
pyrimidin-4-yl]-2-(thiophen-2-yl) ethenesulfonamide (5g).
Compound 5g was prepared from (E)-2-(thiophen-2-
yl)ethenesulfonamide (4g)10 in 38% yield: mp 166–
167 ꢁC (EtOH). H NMR (DMSO-d6) d 3.80 (3H, s),
1
6.82 (1H, d, J=7.8 Hz), 6.84–6.90 (1H, m), 7.05–7.19
(3H, m), 7.52–7.60 (2H, m), 7.73 (1H, t, J=4.9 Hz), 7.77
The compounds 6(b–e) were prepared in the same man-
ner as 6a.

2964
H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
(E)-N-[5-(2-Methoxyphenoxy)-6-propoxy-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (6b).
(1H, d, J=16.0 Hz), 7.29–7.34 (1H, m), 7.38–7.44 (2H,
m), 7.56–7.61 (2H, m), 7.62 (1H, t, J=4.8 Hz), 8.18 (1H,
d, J=16.0 Hz), 9.03 (2H, d, J=4.8 Hz), FAB-MS m/z
530 (M++1). Anal. calcd for C24H20N5O5SK: C, 54.43;
H, 3.81; N, 13.22; S,6.05; K, 7.38. Found: C, 54.45; H,
3.81; N, 13.31; S, 6.08.
330 mg (79%), crystallized from Et2O: mp 161–162 ꢁC.
1H NMR (DMSO-d6) d 0.62 (3H, t, J=7.5 Hz), 1.40–
1.54 (2H, m), 3.81 (3H, s), 4.18–4.34 (2H, m), 6.76 (1H,
d, J=7.5 Hz), 6.82–6.85 (1H, m), 7.02–7.06 (1H, m),
7.09 (1H, d, J=7.5 Hz), 7.36–7.54 (3H, m), 7.60–7.78
(3H, m), 7.82 (1H, d, J=15.0 Hz), 8.98 (1H, d,
J=15.0 Hz), 9.07 (2H, d, J=4.0 Hz), 11.45 (1H, s).
FAB-MS m/z 520 (M++1). Anal. calcd for
C26H25N5O5S: C, 60.10; H, 4.85; N, 13.48; S, 6.17.
Found: C, 59.89; H, 4.87; N, 13.25; S, 6.09.
(E)-N-[5-(2-methoxyphenoxy)-6-methylsulfanyl-2-(pyrimi-
din-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (6f).
A 15% solution of sodium thiomethoxide in water
(10 mL, 21.4 mmol) was added to 5k (992 mg,
2.00 mmol) in DMF (10 mL), and the mixture was stir-
red at room temperature for 2 h. It was poured into ice-
water and acidified with 1 N aqueous HCl. The mixture
was extracted with EtOAc and the organic layer was
concentrated in vacuo. The residue was chromato-
graphed over silica gel using 40:1 CHCl3/MeOH to give
an oil. It was crystallized from MeOH to give 6f
(E)-N-[6-Isopropyloxy-5-(2-methoxyphenoxy)-2-(pyrimi-
din-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (6c).
57 mg (14%), crystallized from Et2O: mp 145–147 ꢁC.
1H NMR (DMSO-d6) d: 1.05 (6H, d, J=7.0 Hz), 3.81
(3H, s), 5.24–5.42 (1H, m), 6.70–6.94 (2H, m), 6.98–7.16
(2H, m), 7.36–7.55 (3H, m), 7.60–7.88 (4H, m), 7.98
(1H, d, J=2.0 Hz), 9.07 (2H, d, J=5.5 Hz), 11.38
(1H, s). FAB-MS m/z 520 (M++1). Anal. calcd for
ꢁ
1
(790 mg. 78%): mp 173–174 C. H NMR (DMSO-d6)
d 2.56 (3H, s), 3.87 (3H, s), 6.71 (1H, d, J=7.4 Hz),
6.82–6.85 (1H, m), 7.04–7.08 (1H, m), 7.20 1H, d,
J=7.4 Hz), 7.40–7.50 (3H, m), 7.67–7.78 (4H, m),
7.82 (1H, d, J=15.2 Hz), 9.13 (2H, d, J=4.9 Hz).
FAB-MS m/z 508 (M++1). Anal. calcd for
.
C25H23N5O5S 0.25H2O: C, 59.59; H, 4.90; N, 13.36;
S, 6.12. Found: C, 59.30; H, 4.97; N, 13.33; S,
6.14.
ꢃ
C24H21N5O4S2 1.2H2O: C, 54.47; H, 4.46; N, 13.23;
S, 12.12. Found: C, 54.69; H, 4.55; N, 13.10; S,
12.34.
(E)-N-[6-Cyclopropylmethoxy-5-(2-methoxyphenoxy)-2-
(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonami-
date (6d). 336 mg (78%), crystallized from Et2O : mp
ꢁ
1
134–135 C H NMR (DMSO-d6) d 0.10–0.20 (2H, m),
0.30–0.42 (2H, m), 0.96–1.08 (1H, m), 3.82 (3H, s), 4.19
(2H, d, J=6.0 Hz), 6.76 (1H, d, J=8.0 Hz), 6.82–6.90
(1H, m), 6.98–7.08 (1H, m), 7.10 (1H, d, J=7.0 Hz),
7.38–7.52 (3H, m), 7.60–7.76 (3H, m), 7.81 (1H, d,
J=15.0 Hz), 7.98 (1H, d, J=15.0 Hz), 9.07 (2H, d,
J=4.0 Hz), 11.39 (1H, s). FAB-MS m/z 532 (M++1).
(E)-N-[5-(2-methoxyphenoxy)-6-methylamino-2-(pyrimi-
din-2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (6g).
A mixture of 5k (1.00 g, 2.02 mmol) and methylamine
(40% in water, 30 mL) and dioxane (20 mL) was stirred
at 60 ꢁC for 8 days. It was concentrated in vacuo, and
the residue was chromatographed over silica gel using
40:1 CHCl3/MeOH to give an oil. It was crystallized
from MeOH to give 6g (730 mg. 74%): mp 203–206 ꢁC.
1H NMR (DMSO-d6) d 2.88 (0.78H, d, J=5.5 Hz),
3.01 (2.22H, d, J=5.5 Hz), 3.84 (2.22H, s), 3.89
(0.78H, s), 6.60–7.20 (5H, m), 7.30–7.85 (7.74H, m),
8.20 (0.26H, d, J=20.0 Hz), 9.04 (0.52H, d,
J=6.0 Hz), 9.16 (1.48H, d, J=6.0 Hz), 10.64 (0.26H,
s), 13.33 (0.74H, s). FAB-MS m/z 491 (M++1).
Anal. calcd. for C24H22N6O4S: C, 58.76; H, 4.52; N,
17.13; S, 6.54. Found: C, 58.49; H, 4.47; N, 17.04; S,
6.34.
.
Anal. calcd for C27H25N5O5S 0.25H2O: C, 60.49; H,
4.79; N, 13.06; S, 5.98. Found: C, 60.45; H, 4.52; N,
12.97; S, 5.89.
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2
- yl)pyrimidin - 4 - yl] - 2 - phenylethenesulfonamide (6e).
273 mg (69%). recrystallized from MeOH: mp 101–
102 ꢁC. H NMR (DMSO-d6) d 3.83 (3H, s), 3.89 (3H,
1
s), 6.67 (1H, d, J=7.3 Hz), 6.77–6.86 (1H, m), 6.99–7.06
(1H, m), 7.09 (1H, d, J=7.3 Hz), 7.40–7.50 (3H, m),
7.65–7.77 (3H, m), 7.81 (1H, d, J=19.0 Hz), 7.98 (1H,
d, J=19.0 Hz), 9.08 (2H, d, J=6.0 Hz), 11.48 (1H, s).
FAB-MS m/z 492 (M++1). Anal. calcd for
C24H21N5O5S: C, 58.65; H, 4.31; N, 14.25; S,6.52.
Found: C, 58.53; H, 4.28; N, 14.21; S, 6.40.
(E)-N-[6-Hydroxy-5-(2-methoxyphenoxy)-2-(pyrimidin-
2-yl)pyrimidin-4-yl]-2-phenylethenesulfonamide (6h). A
mixture of 5k (500 mg, 1.01 mmol) and 1 N sodium
hydroxide in water (5.0 mL) and DMF (10 mL) was
stirred at 110 ꢁC for 2 days. It was poured into ice-water
and acidified with 1 N aqueous HCl. The resulting pre-
cipitate was collected by filtration and chromato-
graphed over silica gel using 40:1 CHCl3/MeOH to give
a solid. It was washed with EtOH to give 6h (214 mg,
44%): mp 221–223 ꢁC. 1H NMR (DMSO-d6) d 3.84
(3H, s), 6.75–6.97 (2H, m), 6.98–7.01 (1H, m), 7.06 (1H,
d, J=7.0 Hz), 7.36–7.54 (3H, m), 7.68–7.84 (4H, m),
7.91 (1H, d, J=16.0 Hz), 9.11 (2H, d, J=5.0 Hz), 11.30
(1H, s), 12.78 (1H, s). FAB-MS m/z: 478 (M++1).
Anal. calcd for C23H19N5O5Sꢃ 0.2H2O: C, 57.46; H,
4.06; N, 14.56; S, 6.67. Found: C, 57.35; H, 3.98; N,
14.62; S, 6.49.
Preparation of monopotassium salt of 6e. A mixture of
6e (50.05 g, 100 mmol) and 85%KOH (6.6 g, 100 mmol)
in EtOH (1 L) was heated under reflux for 2 h. Water
(100 mL) was added to the mixture. After the mixture
was heated for 1 h, it was filtered. The filtrate was stirred
at room temperature for 12 h, then the resulting crystal
was collected by filtration to give 49.35 g (93%) of
monopotassium salt of 6e: mp 198–201 ꢁC. H NMR
1
(DMSO-d6) d 3.80 (3H, s), 3.85 (3H, s), 6.40 (1H, dd,
J=1.6, 8.0 Hz), 6.73 (1H, dt, J=1.6, 8.0 Hz), 6.75 (1H,
dt, J=1.6, 8.0 Hz), 7.01 (1H, dd, J=1.6, 8.0 Hz), 7.12

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
2965
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2
-yl)pyrimidin-4-yl]-2-(naphthalen-2-yl)ethenesulfonamide
(6i). Sodium (110 mg, 4.78 mmol) was added to metha-
nol (10 mL) and stirred at room temperature until all
sodium was dissolved. (E)-N-[6-chloro-5-(2-methoxy-
phenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-(naphtha-
len-2-yl)ethenesulfonamide (5b) (260 mg, 0.476 mmol)
was added to the solution. The mixture was stirred at
room temperature for 1 day and at 80 ꢁC for 3 days. It
was poured into ice-water and acidified with 1 N aqu-
eous HCl. The mixture was extracted with EtOAc. The
organic layer was washed with brine and concentrated
in vacuo. The residual solid was recrystallized from
EtOH to give 6i (147 mg, 57%): mp 164–168 ꢁC. 1H
NMR (DMSO-d6) d 3.83 (3H, s), 3.89 (3H, s), 6.68
(1H, d, J=9.5 Hz), 6.78–6.86 (1H, m), 6.99–7.06
(1H, m), 7.09 (1H, d, J=10.0 Hz), 7.56–7.62 (2H,
m), 7.69–7.76 (1H, m), 7.88–8.03 (5H, m), 8.08 (1H,
d, J=20.0 Hz), 8.21 (1H, brs), 9.13 (2H, d,
J=6.0 Hz), 11.52 (1H, s). FAB-MS m/z 540 (M+ꢂ1).
Anal. calcd for C28H23N5O5Sꢃ 0.1H2O: C, 61.89; H,
4.30; N, 12.89; S, 5.90. Found: C, 61.76; H, 4.16; N,
12.81; S, 5.92.
brs), 9.06 (2H, d, J=6.0 Hz), 11.66 (1H, s). FAB-MS m/
z 493 (M++1). Anal. calcd for C23H20N6O5Sꢃ 0.5H2O:
C, 55.08; H, 4.22; N, 16.76; S, 6.39. Found: C, 55.27; H,
4.14; N, 16.39; S, 6.39.
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(pyridin-4-yl)ethenesulfonamide (6m).
76 mg (51%), crystallized from Et2O: mp 150–160 ꢁC.
1H NMR (DMSO-d6) d 3.82 (3H, s), 3.90 (3H, s), 6.68
(1H, d, J=10.0 Hz), 6.77–6.84 (1H, m), 6.98–7.05 (1H,
m), 7.06–7.11 (1H, m), 7.67–7.80 (4H, m), 8.16 (1H, d,
J=20.0 Hz), 8.67 (2H, d, J=7.0 Hz), 9.08 (2H, d,
J=6.0 Hz). FAB-MS m/z: 491 (M+ꢂ1). HRMS calcd
for C23H21N6O5S m/z 493.1294 (M++1), found
493.1306.
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(thiophen-3-yl)ethenesulfonamide (6n).
243 mg (88%), recrystallized from EtOAc: mp 128–
133 ꢁC. H NMR (DMSO-d6) d 3.82 (3H, s), 3.88 (3H,
1
s), 6.65 (1H, d, J=9.5 Hz), 6.82 (1H, t, J=9.5 Hz), 7.02
(1H, t, J=9.5 Hz), 7.09 (1H, d, J=9.5 Hz), 7.53 (1H, d,
J=6.0 Hz), 7.62–7.78 (3H, m), 7.88 (1H, d, J=20.0 Hz),
8.00 (1H, brs), 9.09 (2H, d, J=6.0 Hz), 9.09 (2H, d,
J=6.0 Hz), 11.41 (1H, s). FAB-MS m/z: 510 (M++1).
HRMS calcd for C25H24N5O6S m/z 498.0906 (M++1),
found 498.0889.
The (E)-2-arylethenesulfonamide derivatives (6j–n) were
prepared in the same manner as 6i using the corre-
sponding chloropyrimidines (5a, 5c–g).
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2
-yl)pyrimidin-4-yl]-2-(naphthalen-1-yl)ethenesulfonamide
(6j). 300 mg (quant). recrystallized from EtOH: mp
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(thiophen-2-yl)ethenesulfonamide (6o).
To the solution of N-[6-chloro-5-(2-methoxyphenoxy)-
2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-(thiophen-2-yl)etha-
nesulfonamide (5g) (502 mg, 1.00 mmol) in DMF
(10 mL) was added sodium methoxide (540 mg,
10.0 mmol), and the mixture was stirred at room tem-
perature for 1 h. It was poured into ice-water and acid-
ified with 1 N HCl. The resulting precipitate was
collected by filtration and recrystallized from MeOH to
give 6o (219 mg, 44%): mp 145–146 ꢁC. 1H NMR
(DMSO-d6) d 3.83 (3H, s), 3.90 (3H, s), 6.66 (1H, d,
J=9.5 Hz), 6.82 (1H, t, J=9.5 Hz), 7.03 (1H, t,
J=9.5 Hz), 7.09 (1H, d, J=9.5 Hz), 7.16 (1H, t,
J=5.0 Hz), 7.56–7.61 (1H, m), 7.65–7.81 (3H, m),
8.03 (1H, d, J=19.0 Hz), 9.10 (2H, d, J=6.5 Hz),
11.48 (1H, s). FAB-MS m/z: 498 (M++1). HRMS
calcd for C25H24N5O6S m/z 498.0906 (M++1), found
498.0923.
110–113 ꢁC. H NMR (DMSO-d6) d 3.83 (3H, s), 3.89
1
(3H, s), 6.69 (1H, d, J=9.5 Hz), 6.78–6.86 (1H, m),
6.99–7.06 (1H, m), 7.10 (1H, d, J=10.0 Hz), 7.53–7.64
(4H, m), 7.96–8.07 (4H, m), 8.13 (1H, d, J=10.0 Hz),
8.52 (1H, d, J=19.0 Hz), 8.93 (2H, d, J=6.0 Hz),
11.51 (1H, s). FAB-MS m/z 540 (M+ꢂ1). Anal. calcd
for C28H23N5O5Sꢃ 1.2H2O: C, 59.71; H, 4.55; N,
12.43; S, 5.69. Found: C, 61.04; H, 4.77; N, 12.07; S,
5.54.
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(pyridin-2-yl)ethenesulfonamide (6k).
235 mg (79%), crystallized from EtOH: mp 176–177 ꢁC.
1H NMR (DMSO-d6) d 3.83 (3H, s), 3.89 (3H, s), 6.67
(1H, d, J=9.5 Hz), 6.77–6.86 (1H, m), 6.99–7.05 (1H,
m), 7.09 (1H, d, J=9.0 Hz), 7.42 (1H, dd, J=1.5,
9.0 Hz), 7.62–7.71 (1H, m), 7.75 (1H, d, J=8.5 Hz),
7.82–7.93 (2H, m), 7.97 (1H, d, J=17.5 Hz), 8.65 (1H,
d, J=4.5 Hz), 9.06 (2H, d, J=4.0 Hz), 11.52 (1H, s).
FAB-MS m/z 493 (M++1). Anal. calcd for
C23H20N6O5S: C, 56.09; H, 4.09; N, 17.06; S, 6.51.
Found: C, 56.04; H, 4.05; N, 17.19; S, 6.64.
N-[6-(2-Hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimi-
din-2-yl)pyrimidin-4-yl]-1-phenylmethanesulfonamide (6p).
Sodium (380 mg, 16.5 mmol) was added to ethylene gly-
col (9.2 mL, 165 mmol) and stirred at 60 ꢁC until all
sodium was dissolved. N-[6-chloro-5-(2-methoxy-
phenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenyl-
methanesulfonamide (5h) (800 mg, 1.065 mmol) was
added to the solution and stirred at 80 ꢁC for 2.5 h. It
was poured into ice-water. The mixture was acidified
with 1 N HCl, and the resulting precipitate was collected
by filtration. The solid was chromatographed over silica
gel using 40:1 CHCl3/MeOH to give an oil. It was crys-
tallized from Et2Oto give 6p (430 mg, 51%): mp 194–
(E)-N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-
yl)pyrimidin-4-yl]-2-(pyridin-3-yl)ethenesulfonamide (6l).
264 mg (83%), recrystallized from EtOH,: mp 135–
137 ꢁC. H NMR (DMSO-d6) d 3.83 (3H, s), 3.90 (3H,
1
s), 6.68 (1H, d, J=9.5 Hz), 6.78–6.85 (1H, m), 6.89–7.06
(1H, m), 7.09 (1H, dd, J=2.0, 10.0 Hz), 7.50 (1H, dd,
J=6.0, 10.0 Hz), 7.70 (1H, t, J=6.0 Hz), 7.86 (1H, d,
J=20.0 Hz), 8.02 (1H, d, J=20.0 Hz), 8.18 (1H, d,
J=10.0 Hz), 8.61 (1H, dd, J=2.0, 6.0 Hz), 8.89 (1H,
ꢁ
1
196 C. H NMR (DMSO-d6) d 3.45–3.60 (2H, m), 3.76
(3H, s), 4.41 (2H, t, J=5.0 Hz), 4.62–4.82 (1H, m), 5.32

2966
H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
(2H, s), 6.64 (1H, d, J=8.0 Hz), 6.80 (1H, t, J=8.0 Hz),
6.91–7.08 (2H, m), 7,24–7.48 (5H, m), 7.67 (1H, t,
J=5.0 Hz), 9.07 (2H, d, J=5.0 Hz), 11.04 (1H, s). FAB-
Vessel contraction (inhibition of big ET-1 induced
contraction of ring preparation sample of rat aorta)
MS
m/z
510
(M++1).
Anal.
calcd
for
Male Wistar rats were anesthetized with sodium pento-
barbital (60 mg/kg ip) and the thoracic aorta was
quickly removed and placed in a Krebs–Henseleit solu-
tion (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2,
1.2 mM KH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2,
11.1 mM glucose). The endothelium was removed by
gentle rubbing of the intimal surface using a small cot-
ton ball, and each ring was suspended in a 10 mL iso-
lated organ chamber (siliconized) containing gassed
(95% O2–5% CO2) and warmed (37 ꢁC) Krebs–Hense-
leit solution. Vessel segments were attached to an iso-
metric force transducer linked to a physiographic
recorder for monitoring tension change. Baseline ten-
sion was set at 1.0 g and the tissues were allowed to
equilibrate for 60 min. The tissues were contracted with
phenylephrine (1 mM) followed by challenge with acet-
ylcholine (1 mM). A negative relaxant response to acet-
ylcholine confirmed the absence of the endothelium.
The rings were stimulated to contract with 60 mM KCl
repeatedly until the contractile response to KCl became
stable before starting the experiments. Cumulative con-
centration–response curves to ET-1 were performed in
the presence or absence of test compounds after a 30-
min pretreatment period. Contractile responses were
expressed as a percentage of the response elicited by
60 mM KCl. The effective concentration of ET-1 caus-
ing a 50% maximum response (EC50) in the presence or
absence of test compounds was determined by regres-
sion analysis. Dose ratios were determined, and the
results analyzed for competitiveness. The pA2 value was
estimated by plotting the log of (dose ratioꢂ1) as a
function of the negative log of concentration of the test
compounds.
C25H23N5O6Sꢃ 0.2H2O: C, 56.57; H, 4.55; N, 13.74; S,
6.29. Found: C, 56.21; H, 4.52; N, 13.76; S, 6.33.
N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)-
pyrimidin-4-yl]-2-phenylethanesulfonamide (6q). To the
solution of N-[6-chloro-5-(2-methoxyphenoxy)-2-(pyr-
imidin-2-yl)pyrimidin-4-yl]-2-phenylethanesulfonamide
(5i) (450 mg, 0.904 mmol) in DMF (10 mL) was added
sodium methoxide (488 mg, 9.04 mmol), and the mixture
was stirred at room temperature for 16 h. It was poured
into ice-water. The mixture was acidified with 1 N HCl,
and the resulting precipitate was collected by filtration.
The solid was chromatographed over silica gel using
40:1 CHCl3/MeOH to give an oil. It was crystallized
from Et2O/EtOH to give 6q (294 mg, 66%): mp 147–
149 ꢁC. H NMR (DMSO-d6) d 3.00–3.10 (2H, m), 3.83
1
(3H, s), 3.91 (3H, s), 4.20–4.28 (2H, m), 6.67 (1H, d,
J=8.0 Hz), 6.80–6.87 (1H, m), 7.01–7.06 (1H, m), 7.11
(1H, d, J=7.5 Hz), 7.16–7.22 (1H, m), 7.26 (2H, t,
J=7.0 Hz), 7.26–7.34 (2H, m), 7.65 (1H, t, J=5.0 Hz),
9.02 (2H, d, J=5.0 Hz), 11.20 (1H, s). FAB-MS m/z 492
(M+ꢂ1). Anal. calcd for C24H23N5O5S: C, 58.41; H,
4.70; N, 14.19; S, 6.50. Found: C, 58.28; H, 4.54; N,
14.26; S, 6.59.
The propanesulfonamide derivative (6r) was synthesized
according to the same procedure as for 6q.
N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)-
pyrimidin-4-yl]-3-phenylpropanesulfonamide (6r). 290 mg
(73%) crystallized from Et2O: mp 186–187 ꢁC. 1H NMR
(DMSO-d6)
d 1.96–2.07 (2H, m), 2.71 (2H, t,
J=7.4 Hz), 3.82 (3H, s), 3.84–3.92 (5H, m), 6.65 (1H, d,
J=7.8 Hz), 6.83 (1H, t, J=7.8 Hz), 7.00–7.06 (1H, m),
7.07–7.20 (6H, m), 7.64 (1H, t, J=4.9 Hz), 9.01 (2H, d,
J=4.9 Hz), 11.10 (1H, s). FAB-MS m/z 508 (M++1).
Anal. calcd for C25H25N5O6S: C, 59.16; H, 4.96; N,
13.80; S, 6.32. Found: C, 58.91; H, 4.84; N, 13.83; S,
6.36.
Functional assay in vivo (inhibition of pressor response to
big ET-1): pithed rats
In vivo antagonistic activity in pithed rats was evaluated
according to the method of Clozel et al. described pre-
viously.5g Briefly, male Wistar rats were pithed by
inserting a steel rod under sodium pentobarbital anes-
thesia and artificially ventilated with room air. The right
common carotid artery and the left femoral vein were
cannulated for blood pressure measurements and iv
injection of drugs, respectively. After stabilization of the
blood pressure, various doses of (1 mL/kg) the test
compounds or vehicle (distilled water) were injected.
Five min later, big ET-1 (1.6 nmol/kg) was injected
intravenously. In another series of experiments, the oral
activities of the test compounds were assessed. Varying
doses of (5 mL/kg) test compounds or vehicle (0.5%
methyl cellulose) were administered by gastric gavage
with a cannula. About 20 min later, the rats were anes-
thetized with sodium pentobarbital, and 30 min later,
pithed and ventilated. After stabilization of the blood
pressure, a dose of 1.6 nmol/kg of big ET-1 was injected
intravenously. In this study, the ID50 value was defined
as the dose of the test compounds which caused a 50%
inhibition of the pressor response to big ET-1 in dia-
stolic blood pressure (DBP).
Binding assay
For competition studies, [125I]ET-1 (200 pM) was added
to each membrane preparation, which was incubated
with various concentrations of compounds in 250 mL of
assay buffer containing 50 mM Tris–HCl, pH 7.4,
10 mM MgCl2 and 0.01% BSA. Binding reactions were
initiated by the addition of the membrane preparations.
After the incubation period (180 min, room tempera-
ture), the reaction was terminated by the addition of
3 mL of ice-cold Tris buffer (50 mM Tris–HCl, pH 7.4,
10 mM MgCl2 and 0.01% BSA) followed by rapid fil-
tration through Whatman GF/C filters. The filters were
rinsed twice and the radioactivity retained on the filters
was counted using a gamma counter at 60% efficiency.
Each assay was performed in duplicate and nonspecific
binding was assessed in the presence of 100 nM unla-
beled ET-1. The IC50 values were calculated with a non-
linear regression analysis.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2955–2968
2967
Functional assay in vivo (inhibition of pressor response to
big ET-1): conscious normotensive rats
I>3s (I). Crystal data for 1: Suitable crystals
(C27H29N5O6Sꢃ H2O) for X-ray diffraction studies were
formed from EtOH/H2O. crystal system, monoclinic;
space group, P21/a, a=15.030(1) A, b=15.047(2) A,
c=12.420(1) A, b=95.153(6)ꢁ, V=2797.4(4) A3;
Dc=1.352 g/cm3; Z=4, R=0.050, RW=0.081 for 3886
reflections with I>3s (I). We will deposit the crystal-
lographic data for these structure with the Cambridge
Crystallographic Data Centre after this manuscript is
accepted.
Male Wistar rats were anesthetized with sodium pento-
barbital (60 mg/kg ip). The right common carotid artery
and the left jugular vein were cannulated with a poly-
ethylene tube for determination of blood pressure and
heart rate and for iv administration of big ET-1
(0.5 nmol/kg). The animals were allowed to recover for
2–3 days after the operation, during which time they
were housed in individual cages with free access to rat
chow and water. After an appropriate equilibration
period, bolus iv doses of big ET-1 were administered to
determine control responses and patency of the cathe-
ters. Each rat was treated with a single po dose of
antagonist or vehicle (0.5% methyl cellulose), and any
changes in blood pressure were noted. The percentage
of the pressor response to big ET-1 challenges during
the subsequent 6.5 h and at 24 h were used as a measure
of big ET-1 inhibition.
Molecular modeling study. Molecular modeling studies
were performed using the software SYBYL 6.6 (Tripos,
Inc., MO, USA) and displayed using the software
insightII (Biosym/MSI, CA, USA) on a Silicon Gra-
phics O2 R12000 workstation.
Acknowledgements
The authors are grateful to Dr. Toshio Okazaki and Dr.
Shuichi Sakamoto for their advice. We thank Miss
Akiko Koakutsu for the pharacological study. We also
thank members of the Division of Analytical Research
for performing instrumental analyses.
Pharmacokinetics
The pharmacokinetic behavior of 6e was evaluated in
male rats and dogs. Compound 6e was administered as
its potassium salt. Groups of rats (n=3 per group) and
dogs (n=4 per group) received either a 0.3 mg/kg iv
dose administered as a bolus in the vein or a 0.3 mg/kg
oral dose administered by gavage. Heparinized blood
samples were obtain from a vein of each rat 0.1 (iv only)
0.25, 0.5, 1, 2, 3, 4, 6, 8 and 10 h after dosing and dog
0.1 (iv only) 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10 and 24 h after
dosing. The samples were analyzed by reverse-phase
HPLC following liquid–liquid extraction and solid-
phase extraction from the plasma. For the calculation of
pharmacokinetic parameters, the WinNonlin Standard
Network Edition Version 1.5 was used as software.
(Plasma sample) blood was collected from the vein
using a heparinized syringe. The blood sample was cen-
trifuged to obtain plasma. (Analytical condition for
HPLC) column: TSKgel, ODS-80Ts (4.6ꢄ250 mm,
Tosoh), Column temperature: 35 ꢁC, Mobile phase:
50 mM phosphate buffered solution (pH 2.0)/acetoni-
trile (45:55), Flow rate: 1.0 mL/min, Detection: UV
(270 nm).
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Med. Chem. 1996, 39, 1039. (f) Riechers, H.; Albrecht, H.-P.;
Amberg, W.; Baumann, E.; Bernard, H.; Bohm, H.-J.; Klinge,
D.; Kling, A.; Muller, S.; Raschak, M.; Unger, L.; Walker, N.;
Wernet, W. J. Med. Chem. 1996, 39, 2123. (g) Clozel, M.;
Breu, V.; Gray, G. A.; Kalina, B.; Loffler, B. M.; Burri, K.;
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X-ray crystallographic analysis. The reflection data for 1
were collected on a Rigaku AFC-7R diffractometer with
graphite-monochromated MoKa radiation (l=0.7107 A)
and the reflection data for 6e were collected on a Rigaku
AFC-5R diffractometer with graphite-monochromated
CuKa radiation. (l=1.5418 A). The structures were
solved by direct methods using the SIR92 program. The
structures were then refined by a full-matrix least-squares
procedure with anisotropic temperature factors for the
non-hydrogen atoms and isotropic temperature factors for
the hydrogen atoms. Crystal data for 6e: Suitable crystals
(C24H21N5O5Sꢃ C2H6O) for X-ray diffraction studies
were formed from EtOH/H2O. crystal system, triclinic;
space group, P1, a=11.976 (1) A, b=12.493 (1) A,
c=10.3458 (6) ꢁA, a=98.753 (6)ꢁ, b=105.243
(6)ꢁ, g=113.538(6) , V=1310.0(2) A3; Dc=1.363 g/cm3;
Z=2, R=0.048, RW=0.096 for 4408 reflections with

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