Structure-activity relationships of N2-aryl-3-(isoxazolylsulfamoyl)-2- thiophenecarboxamides as selective endothelin receptor-A antagonists

Article abstract of DOI:10.1021/jm9608366

We report here that N²-aryl-3-(isoxazolylsulfamoyl)-2- thiophenecarboxamides are potent and selective small molecule ET(A) receptor antagonists. The aryl group was subjected to extensive structural modification. With monosubstitution, the para

Full text of DOI:10.1021/jm9608366

1682
J . Med. Chem. 1997, 40, 1682-1689
Str u ctu r e-Activity Rela tion sh ip s of
2
N -Ar yl-3-(isoxa zolylsu lfa m oyl)-2-th iop h en eca r boxa m id es a s Selective
En d oth elin Recep tor -A An ta gon ists1
Chengde Wu,*,† Ming F. Chan, Fiona Stavros,† B. Raju,*,† Ilya Okun, and Rosario S. Castillo
ImmunoPharmaceutics Inc. (a subsidiary of Texas Biotechnology Corporation), 11011 Via Frontera,
San Diego, California 92127
Received December 9, 1996X
2
We report here that N -aryl-3-(isoxazolylsulfamoyl)-2-thiophenecarboxamides are potent and
selective small molecule ETA receptor antagonists. The aryl group was subjected to extensive
structural modification. With monosubstitution, the para position was most useful in increasing
potency, with methyl being preferred. With disubstitution, 2,4-disubstitution further enhanced
activity with methyl or cyano groups being preferred at the 2-position. In this series, a benzo-
[d][1,3]dioxole group is equivalent to a 4-methyl group in in vitro activity and afforded the
compounds with both in vivo activity and moderate half-lives.
Sch em e 1. Synthesis of the Intermediate Acid 4
In tr od u ction
The potent vasoactive endothelins (ET-1, ET-2, ET-
3), isolated in 1988 by Yanagisawa and co-workers, are
a family of peptides with 21 amino acid residues and
two disulfide bridges.2 The endothelins have been
implicated in many physiological and pathological pro-
cesses such as hypertension, congestive heart failure,
renal failure, cerebral vasospasm, atherosclerosis, res-
tenosis, myocardial infarction, pulmonary disorders, and
subarachnoid hemorrhage.3-9 The endothelins exert
their biological activity through cell surface receptors.
Two receptors subtypes have been cloned: the ET-1
selective ETA receptor and the nonselective ETB
receptor.3,10-14 In the past few years, efforts at various
laboratories have resulted in both peptide and non-
peptide endothelin receptor antagonists being re-
ported: The ETA selective compounds include BQ-
123,15,16 FR-139317,17 BMS-182874,18,19 PD-155080,20
and A-127722.21 The nonselective inhibitors include SB-
209670,22 L-749329,23,24 Ro-46-2005,9 and bosentan,25
while the ETB selective compounds reported are BQ-
788,26 IRL-1038,27 Ro-46-8443,28,29 and IPI-950.30 In
continuation of our work in the sulfonamide ET
antagonists,30-34 we have turned our attention to thiophe-
nesulfonamides.35,36 In this paper, we present the
structure-activity relationships of one particular thiophe-
in a few cases, methyl groups at the 4-position of the
isoxazole ring. We report the discovery that N -aryl-
3-[(4-chloro-3-methyl-5-isoxazolyl)sulfamoyl]-2-thiophen-
ecarboxamides are high-affinity, selective ETA receptor
antagonists.
2
Syn th esis
Most of the sulfonamides were synthesized by cou-
pling the desired aniline with a carboxylic acid 4
(Scheme 1): 5-Amino-3-methylisoxazole 1 was treated
with NCS or NBS in dichloromethane at 0 °C to give
haloisoxazole 2 (87%). The amine was then coupled
with the commercially available 2-(methoxycarbonyl)-
3-thiophenesulfonyl chloride using sodium hydride in
THF at 0 °C. The resulting ester 3 was usually not
isolated but directly hydrolyzed in 1 N NaOH to the
corresponding acid 4 (45%, two steps).
Coupling of the acid 4 with anilines was achieved by
three general methods (Scheme 2): either (A) conversion
of acid 4 using carbonyldiimidazole to the corresponding
acylimidazolide which was then treated with strongly
nucleophilic anilines with or without heating, (B) in the
case of weakly nucleophilic anilines, sodium hydride was
used to deprotonate the aniline at 0 °C prior to the
coupling with the acylimidazolide, or (C) treatment of
a mixture of acid 4 and an aniline in THF with
phosphonitrilic chloride trimer.
2
nesulfonamide series: N -aryl-3-(isoxazolylsulfamoyl)-
2-thiophenecarboxamides. These compounds consist of
three units: an isoxazlole, a thiophene, and an aryl
moiety. The isoxazole and the thiophene moieties are
tethered by a sulfonamide group, while the aryl group
and the thiophene are connected by an amide linkage.
Studies in our laboratory and others have shown that
isoxazoles with dimethyl substituents are preferred for
binding affinity.18,19,34 A halogen substitution on the
4-position has been shown to increase the binding
affinity by 3-5-fold.32,33 Therefore in this study, we
prepared isoxazolyl sulfonamides with chloro, bromo, or,
* Address for correspondence: Texas Biotechnology Corp., 7000
Fannin, Suite 1920, Houston, TX 77030. Phone: (713) 796-8822. Fax:
(713) 796-8232. E-mail: cwu@tbc.com.
† Present address: Texas Biotechnology Corp., 7000 Fannin, Suite
1920, Houston, TX 77030.
Compounds 6h , 6i, and 7 were generated by saponi-
fication of the corresponding esters 5h , 5i, and 5c using
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00836-9 CCC: $14.00 © 1997 American Chemical Society

Endothelin Receptor-A Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1683
Sch em e 3. Additional Synthetic Methodologies
Sch em e 2. General Methods for Synthesizing the
Amidesa
a
Method A: carbonyldiimidazole/ArNH2/THF or DMF. Method
B: (1) carbonyldiimidazole/THF or DMF, (2) ArNH2/NaH. Method
C: phosphonitrilic chloride trimer/ArNH2/Et3N/THF.
1 N NaOH at room temperature in essentially quantita-
tive yields (Scheme 3). The amide 8 was made by
coupling the acid 7 with in situ generated ammonia
(66%). The amide in 5e was reduced with borane‚THF37
to give the corresponding amine 9 (12%). Piperazine
11 was prepared by deprotonating the amide 10 with
sodium hydride followed by heating of the dianion with
3-chloro-6-methoxypiperazine in DMF. The methoxy
group on the piperazine ring was hydrolyzed during
acidic workup to give compound 11(15%).
Scheme 4 shows the syntheses of several anilines
required for generation of the corresponding sulfona-
mides. The aniline 13 used to make 5n n was prepared
by nitrating 5-cyanobenzo[d][1,3]dioxole with nitric acid
(87%) followed by the catalytic hydrogenation of 12 to
give a mixture of the desired aminobenzonitrile 13 and
aminobenzamide 14. The pure aniline 13 was obtained
by recrystallizing the mixture from aqueous methanol
(33%). The (hydroxyalkyl)aniline 17 was prepared by
reduction of the commercial acid 15 with borane‚THF.
The resulting alcohol was nitrated with nitric acid, and
subsequent catalytic hydrogenation gave the corre-
sponding aniline 17 (64%, three steps). The methane-
sulfonamidoaniline 18 was synthesized by mesylating
piperonylamine followed by nitration and catalytic
hydrogenation (18%, three steps). The (cyanomethyl)-
aniline 19 was derived by nitrating 5-(cyanomethyl)-
benzo[d][1,3]dioxole followed by catalytic hydrogenation
(30%).
Resu lts a n d Discu ssion
Inhibition of endothelin binding to both the ETA and
ETB receptors was determined in an in vitro 125I-labeled
ET-1 competition assay (Table 1). A simple primary
amide had an IC50 of 838 nM for ETA and no detectable
activity against ETB. Phenyl substitution of the amide
caused a 5-fold increase in affinity. However, the
N-methylanilino analog 5c was more than 10-fold less
potent. This was probably due more to steric factors
than to the absence of a proton since the corresponding
ester maintained potent activity.36 Lengthening the
distance between the amide nitrogen and the aromatic
ring by one carbon unit resulted in a moderate decrease
of ETA activity. Introduction of the 3-methoxyl sub-
stituent was preferred, and a comparison between 5e
and 9 showed that the amide carbonyl group was
required for potent ETA activity and selectivity.
substitution in the meta position (5i) was preferred over
the parent, but not ortho substitution (5h ). Realizing
the effectiveness of para substitution, we then synthe-
sized and evaluated a series of compounds with para
alkyl or phenyl substitution. The data indicate that the
para position can tolerate alkyl groups of considerable
size, from a methyl to a tert-butyl group. While all of
them showed better activity than the parent compound,
the methyl substituent remained the most active of the
set. However, when the alkyl group was too long, such
as n-butyl (5p ), the activity was compromised. A phenyl
group (5s) was not well tolerated at the para position.
Phenyl and n-butyl groups at the para position also
lowered ETA selectivity.
Substituents at the ortho, meta, and para positions
of the phenyl ring were investigated. For methoxy and
methyl substitution, all three positions showed improve-
ment in activity over the parent compound 5b with the
para position being the most effective. Carboxylic acid
Since it was known that a halogen substitution at the
4-position of the isoxazole improves the activity 3-5-

1684 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Sch em e 4. Syntheses of Anilines
Wu et al.
Ta ble 1. Effect of Thiophene Substitution on
125I]Endothelin-1 Binding
[
IC50, nM (n)
ETA ETB
entry
R
X
5a CONH2
5b CONHC6H5
5c CON(CH3)C6H5
5d CONHCH2C6H5
Br 838 ( 229 (2) >100000 (2)
Br 166 (1)
Br 1910 (1)
Br 229 (1)
77600 (1)
>100000 (1)
97700 (1)
5e CONH(C6H4-m-OCH3) Br 85 (1)
63100 (1)
5f
CONH(C6H4-o-OCH3)
Br 132 (1)
Br 24 (1)
Br 214 ( 1 (2)
61700 (1)
30200 (1)
19500 ( 3300 (2)
117000 (3)
>100000 (2)
67400 ( 12000 (2)
9120 ( 300 (4)
5g CONH(C6H4-p-OCH3)
5h CONH(C6H4-o-CO2H)
5i
5j
5k CONH(C6H4-m-CH3)
5l CONH(C6H4-p-CH3)
5m CONH(C6H4-m-OH)
5n CONH(C6H4-p-CH2CH3) Br 17 (1)
CONH(C6H4-m-CO2H) Br 62 ( 10 (3)
CONH(C6H4-o-CH3)
Br 27 ( 2 (2)
Br 104 ( 6 (2)
Br 12 ( 9 (4)
Br 146 ( 24 (3) 67500 ( 2500 (3)
7410 (1)
4370 (1)
5o CONH(C6H4-p-
Br 20 (1)
Br 200 (1)
Br 54 (1)
CH(CH3)2)
5p CONH(C6H4-p-
CH2(CH2)2CH3)
5q CONH(C6H4-p-
6170 (1)
5890 (1)
CH(CH3)CH2CH3)
5r
5s
5t
CONH(C6H4-p-C(CH3)3) Br 47 (1)
6920 (1)
8710 (1)
67600 (1)
14100 ( 0 (2)
7590 (1)
CONH(C6H4-p-C6H5)
CONH(C6H4-p-CH3)
Br 977 (1)
CH3 32 (1)
5u CONH(C6H4-p-CH3)
9
Cl 15 ( 5 (2)
CH2NH(C6H4-m-OCH3) Br 933 (1)
Ta ble 2. Effect of Heterocyclic Amides on [125I]Endothelin-1
Binding
IC50, nM (n)
entry
heterocycle
X
ETA
ETB
5v
5w
5-CH3-3-isoxazolyl Cl 57 ( 15 (2)
92100 ( 25400 (2)
5-CH3-1,3,4-
Cl 1290 ( 470 (2) >100000 (2)
thiadiazol-2-yl
11
5x
6-OH-3-pyridazyl Br 65 ( 3 (2)
81400 ( 5300 (2)
44700 ( 1400 (2)
5-CH3-2-pyridyl
Cl 13 ( 1 (2)
in vivo in a DOCA salt model19 of hypertension or ET-1
challenge model.25 While this may be due to a number
of factors, we considered the possibility that the amide
bond was rapidly cleaved in serum. We therefore
decided to investigate the effect of an ortho substituent
in addition to a para substitution to see if this increase
in steric hindrance around the amide would slow down
cleavage of the amide linkage. Initially we investigated
effects on potency of this class of sulfonamides.
fold,32,33 we wanted to investigate this with the more
active compounds we had in hand. As expected, the
methylisoxazole 5t was 3-fold less active than 5l and
5u , while the activity of the chloro- and bromoisoxazolyl
sulfonamides were almost identical. We subsequently
report mainly chloroisoxazolyl sulfonamides since this
series have preferred physical and pharmacological
properties.
Heterocycle-substituted amides are reported in Table
2. Except for the pyridine derivative 5x, the heterocycle-
substituted compounds were less active than the all
carbon aromatic system and were not extensively in-
vestigated due to the additional synthetic challenge
presented.
Although some of the para-substituted phenylami-
nocarbonyl thiophenesulfonamides are quite potent in
in vitro assays, they did not demonstrate good activity
We evaluated several 2,4-disubstituted anilino amides
(Table 3) derived from commercially available anilines.
Although a 2-methoxy group (5y) did not show much
improvement over the corresponding para monosubsti-
tuted analog (5g), 2-methyl (5a a ) and 2-hydroxyl group
(5z) showed marked increase of activity over their
corresponding para monosubstituted analogs (5t, 5g).
We then studied functional groups at the 2-position
with the 4- and 5-positions substituted with methoxy

Endothelin Receptor-A Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1685
Ta ble 5. Synthetic and Physical Data
Ta ble 3. Effect of Di- and Trisubstituted Anilino Amides on
[
125I]Endothelin-1 Binding
synth
%
entry method yield
mp, °C
formulaa
C9H8BrN3O4S2
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
5a a
5bb
5cc
5d d
5ee
5ff
5gg
5h h
5ii
5jj
5k k
5m m
5n n
5p p
7
A
F
C
D
E
95
72
39
30
23
26
17
14
12
10
10
15
18
31
19
18
25
28
26
61
53
8
15
17
16
42
66
13
36
15
45
86
12
21
19
13
49
8
168-170
168-170
51-55
186-190
200-202
74-80
b
C15H12BrN3O4S2
C16H14BrN3O4S2‚0.2TFA
b
C16H14BrN3O4S2
C16H14BrN3O5S2‚0.1TFA
b
E
E
C16H14BrN3O5S2
C16H14BrN3O5S2
b
202-205
185-187
183-185
158 dec
143-145
178-180
42-44
C
C
D
D
D
A
D
D
D
D
D
D
C
C
A
B
B
A
A
A
B
B
A
A
G
C
C
A
A
B
B
B
A
G
H
J
C16H12BrN3O6S2‚0.15HOAc
C16H12BrN3O6S2‚0.15HOAc
b
C16H14BrN3O4S2
C16H14BrN3O4S2
C16H14BrN3O4S2
b
b
IC50, nM (n)
ETB
C15H12BrN3O5S2
C17H16BrN3O4S2
C18H18BrN3O4S2
C19H20BrN3O4S2
C19H20BrN3O4S2
C19H20BrN3O4S2
entry
R1
R2
R3
ETA
b
187-190
b
oil
oil
5y
5z
OCH3
OH
OCH3
CH3
OCH3
H
H
H
15 ( 1 (2)
51900 ( 15800 (2)
8240 ( 1860 (2)
14000 ( 2050 (2)
b
6.6 ( 1 (2)
2.6 ( 0.5 (2)
b
205-208
76-86
205-212 dec C21H16BrN3O4S2
5a a CH3
b
5bb CO2CH3 OCH3 OCH3 549 ( 226 (2) >100000 (2)
7
8
5cc
b
CO2H
CONH2 OCH3 OCH3 10.8 ( 0.4 (2) 61000 ( 31000 (2)
CN OCH3 OCH3 3.9 ( 0.6 (3) 12200 ( 1400 (3)
OCH3 OCH3 1430 ( 390 (2) >100000 (2)
b
185-190
177-179
171-172
192-194
158-160
162-164
68-70
58-62
167-168
53-56
138-140
60-62
158-161
78-82
C17H17N3O4S2
b
C16H14ClN3O4S2
C13H11ClN4O5S2‚0.1TFA
C12H10ClN5O4S3
C15H13ClN4O4S2
C17H16ClN3O6S2‚0.1EtOAc
C16H14ClN3O5S2
C17H16ClN3O5S2
C19H18ClN3O8S2
Ta ble 4. Effect of Substituted Benzo[d][1,3]dioxoles on
125I]Endothelin-1 Binding
[
C18H15ClN4O6S2
C16H12BrN3O6S2
C17H14ClN3O6S2
b
C18H16ClN3O8S2
C20H18ClN3O8S2
C20H18ClN3O9S2
b
b
117-119
190-193
147-150
191-193
228-231
167-168
190-193
192-195
189-192
70-73
C20H19ClN4O7S2
C18H17ClN4O8S3‚0.2TFA
b
C18H14ClN3O7S2
IC50, nM (n)
ETB
10100 ( 1500 (5)
C19H17N3O7S2
C17H11ClN4O6S2
C18H13ClN4O6S2
C18H16ClN3O8S2
entry
R
X
ETA
40
15
87
66
12
15
84
18
5d d
5ee
19a
19b
5ff
5gg
5h h
5ii
5jj
5k k
5m m COCH3
5n n
5p p
H
CH3
(CH2)2OH
(CH2)3OH
O(CH2)2OH
(CH2)2OAc
O(CH2)2OAc
CH2CON(CH3)2 Cl
CH2NHSO2CH3 Cl
Br
19 ( 5 (5)
Cl
Cl
Cl
Cl
Cl
Cl
3.0 ( 0.7 (4) 35500 ( 112300 (4)
5.6 ( 0.8 (2) 16800 ( 850 (2)
6.0 ( 3.8 (3) 9080 ( 1870 (3)
5.1 ( 1.3 (3) 14400 ( 2500 (3)
8
9
11
19a
19b
C18H17ClN4O7S2
C16H16BrN3O4S2‚2NH3‚1.5TFA
K
G
A
56-59
C13H10BrN5O5S2‚0.7TFA‚H2O
b
47-52
C18H16ClN3O7S2
4.5 ( 0 (2)
25700 ( 850 (2)
66-69
C19H18ClN3O7S2
6.1 ( 0.7 (2) 15800 ( 2300 (2)
9.3 ( 2.3 (2) 13800 ( 0 (2)
6.8 ( 0.4 (2) 19800 ( 900 (2)
a
b
Analysis for C, H, N are within 0.4% of theory. C, H, N not
done due to insufficient sample. High-resolution MS within 0.004%
of theory. Sample homogeneity established by two diverse ana-
lytical HPLC systems.
COCH3
Cl
10 ( 2 (5)
32600 ( 1800 (5)
>100000 (2)
CH3 31 ( 6 (2)
Cl
Cl
CN
CH2CN
3.4 ( 0.4 (4) 40400 ( 6400 (5)
3.8 ( 0.3 (2) 25700 ( 850 (2)
) 3.4 ( 0.4 nM, with selectivity of >10000, phosphoi-
nositide hydrolysis Ki ) 1.4 nM, pA2 ) 7.9) had 20%
inhibition of ET-1-induced pressor response in conscious,
autonomically blocked rats (n ) 4, 15 mg/kg iv bolus at
30 min). Compound 5n n (TBC11241) was also active
(5 mg/kg iv) in an acute hypoxia-induced pulmonary
hypertension rat model38,39 and had a half-life of 2.5 h
in the rats (60 mg/kg). The ketone compound 5k k
(TBC11192, ETA IC50 ) 10.1 ( 1.7 nM, selectivity
>3000, phosphoinositide hydrolysis Ki ) 0.59 nM, pA2
) 7.87) had 18% inhibition of ET-1-induced pressor
response in conscious, autonomically blocked rats (n )
4, 15 mg/kg iv bolus, at 30 min) with a serum half-life
of 2.5-3 h (60 mg/kg).
groups. A cyano substituent (5cc) was particularly
active, a primary amido group (8) was about 3-fold less
active, a methyl ester (5bb) was 100-fold less active, and
a carboxylic acid (7) caused the loss of essentially all
activity.
Although these dimethoxy sulfonamides did show
some in vivo activity, their serum half-lives in rats were
very short. This was possibly due at least partially to
P450 enzymatic demethylation reactions. We became
interested in replacing the dimethoxyphenyl group with
a benzo[d][1,3]dioxole group because of the wealth of
published information from various laboratories show-
ing its importance in a variety of endothelin antagonist
scaffolds.20-24 In this benzo[d][1,3]dioxole series, we
continued to investigate the effect of different substi-
tutents at the 2-position (Table 4). The 2-position
tolerated groups such as hydroxylalkyl, acetate, amide,
sulfonamide, ketone, and cyano with a tether of reason-
able length, with methyl and cyano groups being the
best substituents. The cyano compound 5n n (ETA IC50
Con clu sion
We report 3-thiophenesulfonamides as a novel series
of endothelin-A receptor antagonists. The benzene ring
of the anilino amide moiety was convenient for inves-
tigating a wide variety of substitutions. Study of the
SAR of the phenyl ring established that the position
para to the nitrogen was very effective for increasing

1686 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Wu et al.
with 1 N HCl twice before it was dried (MgSO4) and concen-
trated. The residue was recrystallized (acetonitrile/water) to
give 5y (224 mg, 16% yield) as a yellow solid: mp 162-164
°C; 1H NMR (DMSO-d6) δ 10.19 (br s, 1H), 7.82 (d, J ) 8.7
Hz, 1H), 7.74 (d, J ) 5.3 Hz, 1H), 7.35 (d, J ) 5.3 Hz, 1H),
6.62 (d, J ) 2.4 Hz, 1H), 6.52 (dd, J ) 8.7, 2.4 Hz, 1H), 3.76
(s, 3H), 3.74 (s, 3H), 2.00 (s, 3H).
activity when substituted with the appropriate group
such as methyl, methoxy, or methylenedioxy. In the
presence of the required para substitution, additional
ortho substitution was useful to further increase the
potency of the compounds. A combination of ortho
substitution on a benzo[d][1,3]dioxole rendered a series
of compounds with in vitro potency, ETA selectivity, in
vivo activity, and a moderate half-life.
Meth od B. N 2-(6-Acetylben zo[d ][1,3]d ioxol-5-yl)-3-[(4-
ch lor o-3-m eth yl-5-isoxa zolyl)su lfa m oyl]-2-th iop h en eca r -
boxa m id e (5k k ). Carbonyldiimidazole (553 mg, 3.41 mmol)
was added to a solution of acid 4a (1.0 g, 3.1 mmol) in
anhydrous DMF (10 mL). The mixture was stirred at room
temperature for 15 min to give mixture I. Sodium hydride
(60% dispersion in mineral oil, 521 mg, 13.02 mmol) was added
to a solution of 6-acetyl-5-aminobenzo[d][1,3]dioxole (1.13 g,
6.2 mmol) in anhydrous DMF (10 mL) at 0 °C. The mixture
was stirred at 0 °C for 15 min to give mixture II. Mixture I
was slowly transferred via cannula into mixture II at 0 °C and
was stirred at 0 °C for 4 h. The reaction mixture was poured
into 2 N HCl (200 mL) and the resulting precipitate was
collected by filtration. The solid was washed with water (2 ×
10 mL) and ethyl ether (2 × 10 mL) to give 5k k (730 mg, 49%
yield) as a dull yellow powder: mp 191-193 °C; 1H NMR
(CDCl3) δ 13.34 (br s, 1H), 8.44 (s, 1H), 7.50 (s, 2H), 7.36 (s,
1H), 6.11 (s, 2H), 2.65 (s, 3H), 2.24 (s, 3H).
Exp er im en ta l Section
Gen er a l. Melting points were determined in capillary
tubes with a Mel-Temp II apparatus and are uncorrected.
Proton NMR (1H NMR) spectra were recorded on a GE QE-
300 Plus spectrometer at 300 MHz. Chemical shifts were
reported in parts per million as δ units relative to tetrameth-
ylsilane or residual solvent as internal standard. IR spectra
were recorded on a Mattson GL-2020 Fourier transform
infrared spectrophotometer. High-resolution mass spectra
were recorded with fast atom bombardment (FAB) ionization
by the University of Minnesota Mass Spectrometry Service
Laboratory (Minneapolis, MN). Elemental analyses were
performed by Desert Analytics (Tucson, AZ) and were within
0.4% of theoretical values unless otherwise indicated. Anhy-
drous solvents were obtained from Aldrich Chemical Co.
(Milwaukee, WI) in Sure-Seal bottles. Unless otherwise
stated, reagents and chemicals were of the highest grade from
commercial sources and were used without further purifica-
tion. ET-1 was obtained from Clinalfa Co. (Laufelfingen,
Switzerland) and ET-3 from American Peptide Co. (Sunnyvale,
Meth od C.
N
2-(4-Meth ylp h en yl)-3-[(3,4-d im eth yl-5-
isoxazolyl)su lfam oyl]-2-th ioph en ecar boxam ide (5t). Phos-
phonitrilic chloride trimer dissolved in THF (5 mL) was added
to a suspension of acid 4c (2.0 g, 6.6 mmol) in THF (5 mL)
and Et3N at 0 °C. The cold bath was removed and the reaction
mixture stirred at room temperature for 2 h. The mixture was
diluted with water (150 mL) and acidified to pH 2 using
concentrated HCl. The mixture was then extracted with
methylene chloride (2 × 100 mL), and the combined organic
layers were washed with 2 N HCl (3 × 100 mL), dried over
MgSO4, and concentrated. The residue was dissolved in ether
and allowed to stand at room temperature to give a precipitate
which was filtered and washed with cold ether to give 5t (1.6
g, 61% yield) as a solid: mp 185-190 °C; 1H NMR (DMSO-d6)
δ 10.62 (br s, 1H), 7.86 (d, J ) 5.2 Hz, 1H), 7.51 (d, J ) 8.1
Hz, 2H), 7.31 (d, J ) 5.1 Hz, 1H), 7.16 (d, J ) 8.1 Hz, 2H),
2.28 (s, 3H), 2.04 (s, 3H), 1.65 (s, 3H).
CA).
[
125I]ET-1 was obtained from Amersham (Arlington
Heights, IL). Flash chromatography was performed on silica
gel 60 (230-400 mesh, E. Merck). Thin layer chromatography
was performed with E. Merck silica gel 60 F-254 plates (0.25
mm) and visualized with UV light, phosphomollybdic acid, or
iodine vapor. Analytical HPLC was performed on Vydac C18
column (4.6 × 250 mm), preparative HPLC on Dynamax-60A
(83-241-c) with acetonitrile:water gradients containing 0.1%
trifluoroacetic acid. The detection wave length was 254 nm.
5-Am in o-4-ch lor o-3-m eth ylisoxa zole (2a ). To a solution
of 5-amino-3-methylisoxazole (1) (5.0 g, 51.0 mmol) in meth-
ylene chloride (40 mL) at 0 °C was slowly added N-chlorosuc-
cinimide (6.8 g, 51.0 mmol). The reaction mixture was stirred
for 1 h at 0 °C and an additional 2 h at room temperature.
The crude mixture was washed with 1 N NaOH, and the
organic layer was dried over MgSO4 and concentrated by
evaporation. The residue was shaken with hexanes (50 mL),
and the resulting white precipitate was filtered to give 2a (5.85
g, 87% yield) as a white solid: mp 65-68 °C; 1H NMR (CDCl3)
δ 4.54 (br s, 2H), 2.17 (s, 3H).
Meth od D. N 2-(4-Eth ylp h en yl)-3-[(4-br om o-3-m eth yl-
5-isoxa zolyl)su lfa m oyl]-2-t h iop h en eca r b oxa m id e (5n ).
4-Ethylaniline (242 mg, 2.0 mmol), (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate (Bop) (442
mg, 1.0 mmol) and diisopropylethylamine (0.15 mL) were
added to acid 4b (368 mg, 1.0 mmol), which had been
suspended in methylene chloride (3 mL). The solution was
stirred for 14 h at room temperature. This was diluted with
methylene chloride (50 mL) and washed with 3 N HCl (3 × 50
mL) followed by 5% sodium carbonate solution (2 × 50 mL).
The combined organic layers were dried (MgSO4) and concen-
trated. The residue was purified by column chromatography
using EtOAc as eluent. Recrystallization from EtOAc/hexanes
3-[(4-Ch lor o-3-m et h yl-5-isoxa zolyl)su lfa m oyl]-2-t h io-
p h en eca r boxylic Acid (4a ). To a solution of 2a (1.82 g, 17.7
mmol) in anhydrous THF (60 mL) at 0 °C was added NaH (60%
dispersion in mineral oil, 1.50 g, 37.4 mmol). The resulting
mixture was stirred for 10 min before the slow addition of
2-(methoxycarbonyl)thiophene-3-sulfonyl chloride (3.0 g, 12.5
mmol). The reaction was stirred at room temperature for 4
h. To work up, the mixture was diluted with hexanes (60 mL)
and the resulting precipitate was filtered and washed with
hexanes. The precipitate was then dissolved in 1 N NaOH
and stirred at room temperature for 1 h. After acidifying to
pH ∼2 with 2 N HCl, the resulting precipitate was filtered,
washed with water, and dried under vacuum. Compound 4a
was obtained (1.82 g, 45% yield) as a yellow powder: mp 166-
1
gave 5n (151 mg, 31% yield) as a solid: mp 187-190 °C; H
NMR (DMSO-d6) δ 11.78 (br s, 1H), 7.73 (d, J ) 5.4 Hz, 1H),
7.63 (d, J ) 8.4 Hz, 2H), 7.38 (d, J ) 5.4 Hz, 1H), 7.20 (d, J )
8.4 Hz, 2H), 2.58 (q, J ) 7.5 Hz, 2H), 1.97 (s, 3H), 1.18 (t, J )
7.5 Hz, 3H).
2
Meth od E. N -(4-Meth oxyph en yl)-3-[(4-br om o-3-m eth -
yl-5-isoxa zolyl)su lfa m oyl]-2-th iop h en eca r boxa m id e (5g).
Compound 5g was synthesized in the same fashion as com-
pound 5n (method D) except that bromotris(pyrrolidino)-
phosphonium hexafluorophosphate (PyBrop) was used instead
of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa-
fluorophosphate (Bop). Compound 5g was obtained in 17%
yield as a solid: mp 202-205 °C; 1H NMR (DMSO-d6) δ 11.74
(br s, 1H), 7.73 (d, J ) 5.1 Hz, 1H), 7.65 (d, J ) 9.0 Hz, 2H),
7.39 (d, J ) 5.1 Hz, 1H), 6.96 (d, J ) 9.0 Hz, 2H), 3.76 (s, 3H),
1.98 (s, 3H).
1
170 °C; H NMR (DMSO-d6) δ 7.91 (d, J ) 5.1 Hz, 1H), 7.42
(d, J ) 5.1 Hz, 1H), 2.13 (s, 3H).
Meth od A. N 2-(2,4-Dim eth oxyp h en yl)-3-[(4-ch lor o-3-
m et h yl-5-isoxa zolyl)su lfa m oyl]-2-t h iop h en eca r b oxa m -
id e (5y). To a solution of acid 4a (1.0 g, 3.1 mmol)) in
anhydrous DMF (10 mL) at room temperature was added
carbonyldiimidazole (533 mg, 3.41 mmol). The mixture was
stirred at room temperature for 15 min before the addition of
2,4-dimethoxyaniline (1.22 g, 7.75 mmol). The mixture was
stirred for 5 h. The crude reaction mixture was partitioned
between 1 N HCl and EtOAc. The organic layer was washed
Met h od F .
N
2-P h en yl-3-[(4-b r om o-3-m et h yl-5-isox-
a zolyl)su lfa m oyl]-2-th iop h en eca r boxa m id e (5b). Com-
pound 5b was synthesized in the same fashion as 5n (method
D) except that 1-ethyl-3′-[3-(dimethylamino)propyl]carbodiim-
ide (EDCI) was used instead of Bop. Compound 5b was

Endothelin Receptor-A Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1687
1
obtained in 72% yield as a yellow solid: mp 168-179 °C; H
NMR (DMSO-d6) δ 11.32 (br s, 1H), 8.72 (br s, 1H), 7.76 (d, J
) 4.2 Hz, 1H), 7.65 (d, J ) 10.2 Hz, 2H), 7.31-7.36 (m, 3H),
7.08 (t, J ) 7.3 Hz, 1H), 2.00 (s, 3H).
and the nitro compound was catalytically hydrogenated in the
same manner as for 13 to give 17 (3.2 g, 64% yield): 1H NMR
(CDCl3) δ 6.56 (s, 1H), 6.32 (s, 1H), 5.85 (s, 2H), 3.60 (t, 2H),
5.70 (t, 2H), 1.81 (m, 2H).
Meth od G. N 2-(6-Ca r boxylben zo[d ][1,3]d ioxol-5-yl)-3-
[(4-ch lor o-3-m eth yl-5-isoxa zolyl)su lfa m oyl]-2-th iop h en -
eca r boxa m id e (7). Sodium hydroxide solution (1.5 N, 250
mL) was added to 5bb. The resulting suspension was stirred
at room temperature overnight to give a clear solution. The
solution was acidified using concentrated hydrochloric acid
while cooling. The precipitate was filtered, washed with water
(3 × 50 mL), and dried under vacuum to give 7 (347 mg, 87%
yield) as a yellow powder: mp 192-195 °C; 1H NMR (DMSO-
d6) δ 11.70 (br s, 1H), 7.96 (s, 1H), 7.86 (d, J ) 5.1 Hz, 1H),
7.44 (s, 1H), 7.40 (d, J ) 5.1 Hz, 1H), 3.84 (s, 3H), 3.80 (s,
3H), 2.08 (s, 3H). Compounds 5ff and 19a were synthesized
in the same fashion from 5h h and 5gg, respectively.
5-Am in o-6-(m et h a n esu lfa m id om et h yl)b en zo[d ][1,3]-
d ioxole (18). To a solution of piperonylamine (6.07 g, 38.95
mmol) and triethylamine (5.37 g, 53.12 mmol) in dichlo-
romethane (100 mL) at 0 °C was added methanesulfonyl
chloride (4.14 g, 35.41 mmol). The reaction was stirred at 0
°C for 1 h before the mixture was diluted with dichloromethane
(100 mL) and washed with 1 N HCl (2 × 100 mL). The organic
layer was dried over MgSO4 and concentrated to a gray solid
(8.4 g). This solid was then nitrated, and the resulting nitro
compound was catalytically hydrogenated in the same manner
as for 13 to give 18 (2.3 g, 27% yield): 1H NMR (CDCl3) δ 6.61
(s, 1H), 6.31 (s, 1H), 5.85 (s, 2H), 5.16 (br s, 1H), 4.16 (d, 2H),
2.92 (s, 3H).
5-Am in o-6-(cya n om et h yl)b en zo[d ][1,3]d ioxole (19).
Compound 19 was synthesized in the same manner as for 13
except that the nitration was done in acetic acid instead of in
neat nitric acid to give 19 in 30% overall yield: 1H NMR
(DMSO-d6) δ 7.68 (s, 1H), 6.35 (s, 1H), 5.84 (s, 2H), 4.88 (br s,
2H), 3.66 (s, 2H).
N ,N -D i m e t h y l-6-(5-a m i n o b e n z o [d ][1,3]d i o x o ly l)-
a ceta m id e (20). Compound 20 was synthesized in the same
manner as for 13 from N,N-dimethyl-5-(benzo[d][1,3]dioxolyl)-
acetamide, which in turn was made from 5-(benzo[d][1,3]-
dioxolyl)acetic acid using method A. Compound 20 was
obtained in 88% overall yield: 1H NMR (CDCl3) δ 6.54 (s, 1H),
6.30 (s, 1H), 5.84 (s, 2H), 4.30 (br s, 2H), 3.55 (s, 2H), 3.13 (s,
3H), 2.94 (s, 3H).
Mem br a n e P r ep a r a tion . A membrane preparation con-
taining human ETA receptor was prepared from TE 671 (ATCC
# HTB 139). Cells, grown to confluence, were harvested using
a rubber policeman and centrifuged at 190g for 10 min at 4
°C. The pellet was resuspended in 5 mM HEPES (pH 7.4)
containing 5 mM EDTA and 100 KIU aprotinin and homog-
enized using a Tenbroeck homogenizer. The suspension was
centrifuged at 57800g for 15 min at 4 °C, and the pellet was
resuspended in 5 mL of 5 mM HEPES buffer, pH 7.4,
containing 10 mM MnCl2 to which 5 mL of a 0.001% deoxyri-
bonuclease Type 1 was added. The suspension was mixed,
incubated at 37 °C for 30 min, and then centrifuged at 57800g
for 15 min at 4 °C. The pellet was then washed twice with 5
mM HEPES buffer containing 5 mM EDTA before finally being
resuspended in 30 mM HEPES buffer, pH 7.4, containing
aprotinin (100 KIU/mL) to give a final membrane protein
concentration of 2 mg/mL. Aliquots of membrane were stored
at -70 °C until use. Protein determinations were carried out
using the Pierce BCA assay kit with bovine serum albumin
(BSA) as a standard.
2
Meth od H. N -[3,4-Dim eth oxy-6-(a m in oca r bon yl)p h e-
n yl]-3-[(4-ch lor o-3-m e t h yl-5-isoxa zolyl)su lfa m oyl]-2-
th iop h en eca r boxa m id e (8). Compound 8 was synthesized
in the same fashion as for compound 5y (method A) except
that acid 7 was used instead of acid 4a . Compound 8 was
obtained in 66% yield as a yellow powder: mp 189-192 °C;
1H NMR (DMSO-d6) δ 12.65 (br s, 1H), 8.12 (br s, 1H), 7.96 (s,
1H), 7.85 (d, J ) 5.1 Hz, 1H), 7.57 (br s, 1H), 7.42 (d, J ) 5.1
Hz, 1H), 7.41 (s, 1H), 3.81 (s, 6H), 2.11 (s, 3H).
2
Meth od J . N -(3-Meth oxyp h en yl)-3-[(4-br om o-3-m eth -
yl-5-isoxa zolyl)su lfa m oyl]-2-th iop h en eca r boxa m id e (9).
To a solution of 5e (1.0 g, 2.12 mmol) in anhydrous THF (15
mL) was added BH3‚THF (15 mL, 1 M in THF). The mixture
was heated under reflux for 8 h and then cooled to room
temperature. The volatiles were removed by evaporation, and
methanol was added to the residue. The solution was con-
centrated again and the residue purified by HPLC to give 9
(113 mg, 12% yield) as a light yellow/gray powder: mp 70-73
1
°C; H NMR (DMSO-d6) δ 7.48 (d, J ) 5.6 Hz, 1H), 7.21 (d, J
) 5.6 Hz, 1H), 6.96 (dd, J ) 8.1, 8.2 Hz, 1H), 6.16 (d, J ) 8.1
Hz, 1H), 6.12 (d, J ) 8.2 Hz, 1H), 6.10 (s, 1H), 4.59 (s, 2H),
3.62 (s, 3H), 2.19 (s, 3H).
Met h od K.
N
2-(6-Hyd r oxy-3-p yr id a zyl)-3-[(4-br om o-
3-m eth yl-5-isoxa zolyl)su lfa m oyl]-2-th iop h en eca r boxa m -
id e (11). To a solution of 5a (572 mg, 1.56 mmol) in anhydrous
DMF (10 mL) were sequentially added 3-chloro-6-methoxypy-
ridazine (475 mg, 3.12 mmol) and sodium hydride (60%
dispersion in mineral oil, 194 mg, 4.84 mmol). The resulting
mixture was heated at 100 °C under a nitrogen atmosphere
for 4 h. The reaction mixture was allowed to cooled to room
temperature, poured into 1 N HCl acid, and extracted with
EtOAc. The organic layer was washed with 1 N HCl twice,
dried (MgSO4), and then concentrated. The residue was
purified by HPLC to give 11 (111 mg, 15% yield) as a brown
powder: mp 56-59 °C; 1H NMR (DMSO-d6) δ 12.75 (br s, 1H),
8.45 (d, J ) 9.3 Hz, 1H), 7.94 (d, J ) 9.3 Hz, 1H), 7.87 (d, J )
5.1 Hz, 1H), 7.40 (d, J ) 5.1 Hz, 1H), 1.98 (s, 3H).
5-Am in o-6-cya n oben zo[d ][1,3]d ioxole (13). Piperony-
lonitrile (10 g) was added to nitric acid (70%, 40 mL) at 0 °C
over 30 min. After 30 min at 0 °C and 30 min at 40 °C, ice
was added to the reaction mixture. The resulting precipitate
was filtered to give 12 (11.4 g, 87% yield) as a bright yellow
powder. To a solution of 12 (11.4 g) in EtOAc (400 mL) was
added 10% Pd/C (540 mg). The flask was purged with
hydrogen gas three times from a balloon, and the reaction
mixture was stirred overnight. The solid was filtered off, and
the filtrate was concentrated. The resulting yellow solid
mixture of 13 and 14 (1:4) was recrystallized from methanol/
water to give 13 (3.67 g, 33% yield) as a yellow solid: 1H NMR
(CDCl3) δ 6.74 (s, 1H), 6.26 (s, 1H), 5.94 (s, 2H), 4.26 (br s,
2H).
A membrane preparation containing human ETB receptors
was prepared as described above from COS 7 cells which were
transfected with DNA encoding the human ETB receptor, as
previously described.40,41
Liga n d Bin d in g Stu d ies. Binding studies were performed
in a 30 mM HEPES buffer, pH 7.4, containing 150 mM NaCl,
5 mM MgCl2, and 0.05% bacitracin using 3 mg/tube (ETA) or
0.75 mg/tube (ETB) membrane. Test compounds were dis-
solved in DMSO and diluted with the assay buffer to give a
final concentration of 0.25% DMSO. Competitive inhibition
experiments were performed in triplicate in a final volume of
200 µL containing 4 pM [125I]ET-1 (1.6 nCi). Nonspecific
binding was determined in the presence of 100 nM ET-1.
Samples were incubated for 16-18 h at 24 °C. One milliliter
of PBS was then added and the assay centrifuged at 2000g
for 25 min at 24 °C. The supernatant was decanted and the
membrane bound radioactivity counted on a Genesys gamma
counter.
P h osp h oin ositid e Hyd r olysis in Cells. TE 671 or trans-
fected COS 7 cells were grown to confluence in six-well plates.
Sixteen hours prior to use, the media in each well was replaced
with 2 mL of inositol-free RPMI-164 (IF-RPMI) media contain-
ing 10% inositol-free FCS and 2 mCi [3H]myoinositol, and this
was incubated at 37 °C in the presence of 6% CO2. The media
was aspirated, and the cells were washed twice with PBS. Cells
5-Am in o-6-(3-h yd r oxy-1-p r op yl)b en zo[d ][1,3]d ioxole
(17). To a solution of 3-(benzo[d][1,3]dioxol-5-yl)propanoic acid
(5 g, 25.75 mmol) in anhydrous THF (20 mL) at 0 °C was added
BH3‚THF (51.5 mL, 1.0 M in THF, 51.5 mmol). The mixture
was heated under reflux for 1 h. The solvent was evaporated,
the residue was treated with methanol (20 mL), and the
solution was concentrated again. This process was repeated
six times to give an oil (4.7 g). This oil was then nitrated,

1688 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Wu et al.
(4) Watanabe, T.; Suzuki, N.; Shimamoto, N.; Fujino, M.; Imada,
A. Endothelin in Myocardial Infarction. Nature 1990, 344, 114.
(5) Saito, Y.; Nakao, K.; Mukoyama, M.; Imura, H. Increased Plasma
Endothelin Level in Patients with Essential Hypertension. N.
Engl. J . Med. 1989, 322, 205.
(6) Giaid, A.; Yanagisawa, M.; Langleben, D.; Michel, R.; Levy, R.;
Shennib, H.; Kimra, S.; Masaki, T.; Duguid, W.; Path, F. R. C.;
Stewart, D. J . Expression of Endothelin-1 in the Lungs of
Patients with Pulmonary Hypertension. N. Engl. J . Med. 1993,
328, 1732-1739.
(7) Takahashi, K.; Totsune, K.; Mouri, T. Endothelin in Chronic
Renal Failure. Nephron 1994, 66, 373-379.
(8) Cosentino, F.; Katusic, Z. S. Does Endothelin-1 Play a Role in
the Pathogenesis of Cerebral Vasospasm? Stroke 1994, 25, 904-
908.
(9) Clozel, M.; Breu, V.; Burri, K.; Cassao, J .-M.; Fischli, W.; Gray,
G. A.; Hirth, G.; Loffler, B.-M.; Muller, M.; Neidhart, W.; Ramuz,
H. Pathophysiological Role of Endothelin Revealed by the First
Orally Active Endothelin Receptor Antagonist. Nature 1993, 365,
759-761.
(10) Arai, H.; Hori, S.; Aramori, I.; Ohkubo, H.; Nakanishi, S. Cloning
and Expression of a cDNA Encoding an Endothelin Receptor.
Nature 1990, 348, 730-732.
(11) Sakurai, T.; Yanagisawa, M.; Takuwa, Y.; Miyazaki, H.; Kimura,
S.; Goto, K.; Masaki, T. Cloning of a cDNA Encoding a Non-
isopeptide-selective Subtype of the Endothelin Receptor. Nature
1990, 348, 732-735.
were preincubated for 10 min in 1 mL of lithium buffer (15
µM HEPES, pH 7.4, 145 µM NaCl, 5.4 µM KCl, 1.8 µM CaCl2,
0.8 µM MgSO4, 1.0 µM NaH2PO4, 11.2 µM glucose, 20 µM LiCl)
with or without test compound prior to the addition of 100 µM
of ET-1 at different concentrations. Cells were then incubated
for an additional 45 min. The buffer was discarded, and the
accumulated inositol phosphates were extracted with ice cold
methanol and measured according to the method of Berridge.
The total cell protein in each well was measured using the
Pierce BCA assay after solubilizing the cells in 0.1 M NaOH.
Con sciou s, Au ton om ica lly Block ed Ra t P r essor As-
sa ys (ET-1 Ch a llen ge Assa y). Male Sprague-Dawley rats
(250-350 g) were anesthetized with a short-acting barbiturate
(methohexital: 50 mg/kg ip). Cannula were inserted into the
femoral artery and vein exteriorized. The catheter in the
femoral artery was connected to a P23XL Spectromed pressure
transducer attached to a PO-NE-MAH Digital Acquisition and
Analysis system. Animals were placed in a Brainridge re-
strainer and allowed to recover for 60 min prior to the start of
the experiment. Autonomic blockade was established by
intravenous administration of atropine methyl nitrate (3 mg/
kg) and propranalol (2 mg/kg). Blood pressure was allowed
to stabilize for 30 min prior to administration of vehicle (1 M
Tris base or sodium bicarbonate). Thirty minutes later
animals were challenged by intravenous bolus administration
of ET-1 (1 mg/kg, control). Ninety minutes later, when the
mean arterial pressure (MAP) had returned to baseline,
antagonist was administered by intravenous bolus injection
followed 30 min later by a second ET-1 challenge. The degree
in inhibition was calculated as
(12) Sakamoto, A.; Yanagisawa, M.; Sakurai, T.; Takuwa, Y.; Yanag-
isawa, H.; Masaki, T. Cloning and Functional Expression of
Human cDNA for the ETB Endothelin Receptor. Biochem.
Biophys. Res. Commun. 1991, 178, 656-663.
(13) Hosoda, K.; Nakao, K.; Arai, H.; Suga, S.; Ogawa, Y.; Mukoyama,
M.; Shirakami, G.; Saito, Y.; Nakanishi, S.; Imura, H. Cloning
and Expression of Human Endothelin-1 Receptor cDNA. FEBS
Lett. 1991, 187, 23-26.
(14) Ogwa, Y.; Nakao, K.; Arai, H.; Nagakawa, O.; Hosoda, K.; Suga,
S.; Nakanishi, S.; Imura, H. Molecular Cloning of a Non-
isopeptide Selective Human Endothelin Receptor. Biochem.
Biophys. Res. Commun. 1992, 178, 248-255.
100 × (controlmax. pressor response
-
antagonistmax. pressor response)/controlmax. pressor response
(15) Ishikawa, K.; Fukami, T.; Nagase, T.; Fujita, K.; Hayama, T.;
Niyama, K.; Mase, T.; Ihara, M.; Yano, M. Cyclic Pentapeptide
Endothelin Antagonists with ETA Selectivity. Potency and
Solubility Enhancing Molifications. J . Med. Chem. 1992, 35,
2139-2142.
(16) Ihara, M.; Noguchi, K.; Saeki, T.; Fukoroda, T.; Tsuchida, S.;
Kimura, S.; Fukami, F.; Ishikawa, K.; Nishikibe, M.; Yano, M.
Biological Profiles of Highly Potent Novel Endothelin Antago-
nists Selective for ETA Receptor. Life Sci. 1991, 50, 247-255.
(17) Sogabe, K.; Nirei, H.; Shoubo, M.; Nomoto, A.; Ao, S.; Notsu, Y.;
Ono, T. Pharmacological Profile of FR 139317, a Novel, Potent
Endothelin ETA Receptor Antagonist. J . Pharmacol. Exp. Ther.
1993, 264, 1040-1046.
(18) Stein, P. D.; Hunt, J . T.; Floyd, D. M.; Moreland, S.; Dickenson,
K. E. J .; Mitchell, C.; Liu, E. C.; Webb, M. L.; Murugesan, N.;
Dickey, J .; McMullen, C.; Zhang, R.; Lee, V. G.; Serafino, R.;
Delaney, C.; Schaeffer, T. R.; Kozlowski, M. The Discovery of
Sulfonamide Endothelin Antagonists and the Development of
the Orally Active ETA Antagonist 5-(Dimethylamino)-N-(3,4-
dimethyl-5-isoxazolyl)-1-naphthalenesulfonamide. J . Med. Chem.
1994, 37, 329-331.
(19) Webb, M.; Bird, J .; Liu, E.; Rose, P. N.; Serafino, R.; Stein, P.;
Moreland, S. BMS-182874 is a Selective, Nonpeptide Endothelin
ETA Receptor Antagonist. J . Pharmacol. Exp. Ther. 1995, 272,
1124-1134.
P h a r m a cok in etics in Ra ts. All surgical procedures were
performed under aseptic conditions. Adult Sprague-Dawley
rats were anesthetized with a ketamine-based anesthetic
(containing ketamine, xylazine, and promace), and the jugular
vein was cannulated. The cannula were channeled under the
skin, exteriorized between the scapulae, and protected by a
spring tether (BioResearch, Montreal, Quebec). Rats were
allowed to recover for 48 h prior to use. The compound, 60
mg/kg, was administered at a dose of 1 mL/kg iv or 10 mL/kg
po, and serial blood samples (100 µL) were withdrawn from
the caudal vein over the next 24 h. Blood samples were
immediately centrifuged and the plasma frozen and stored at
-20 °C until assay. Samples were assayed by HPLC following
extraction into acetonitrile.
Ack n ow led gm en t. We acknowledge the support
and helpful scientific discussions with Drs. Richard A.
F. Dixon, Timothy P. Kogan, Tommy Brock, and Sey-
mour Mong. We thank Karin M. Keller for some of the
HPLC and mass spectrometry work. We thank Drs. Erik
J . Verner, Adam Kois, and Yalamoori Venkatachalap-
athi for helpful discussions.
(20) Doherty, A. M.; Patt, W. C.; Edmunds, J . J .; Berryman, K. A.;
Reisdorph, B. R.; Plummer, M. S.; Shahripour, A.; Lee, C.;
Cheng, X. M.; Walker, D. M.; Haleen, S. J .; Keiser, J . A.; Flynn,
M. A.; Welch, K. M.; Hallak, H.; Taylor, D. G.; Reynolds, E. E.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR, IR, and
high-resolution mass spectral data for compounds 5a -z, 5a a -
p p , 7-11, and 19a ,b (10 pages). Ordering information is given
on any current masthead page.
Discovery of
a Novel Series of Orally Active Non-peptide
Endothelin-A Receptor Selective Antagonists. J . Med. Chem.
1995, 38, 1259-1263.
(21) Winn, M.; von Geldern, T. W.; Opgenorth, T. J .; J ae, H.-S.;
Tasker, A. S.; Boyd, S. A.; Kester, J . A.; Mantei, R. A.; Bal, R.;
Sorensen, B. K.; Wu-Wong, J . R.; Chiou, W. J .; Dixon, D. B.;
Novosad, E. I.; Hernandez, L.; Marsh, K. C. 2,4-Diarylpyrroli-
dine-3-carboxylic Acids-Potent ETA Selective Endothelin Recep-
tor Antagonists. 1. Discovery of A-127722. J . Med. Chem. 1996,
39, 1039-1048.
Refer en ces
(1) Presented in part: Wu, C.; Raju, B.; Castillo, R.; Okun, I.;
Stavros, F.; Chan, M. F. 2-Arylaminocarbonylthiophene-3-sul-
fonamides as ETA Selective Endothelin Antagonists. The 211th
ACS National Meeting, New Orleans, LA, March 24-28, 1996,
Abstract MEDI 146.
(2) Yanagisawa, M.; Kurihara, H.; Kimura, S.; Tomobe, Y.; Koba-
yashi, M.; Mitsui, Y.; Goto, K.; Masaki, T. A. Novel Potent
Vasoconstrictor Peptide Produced by Vascular Endothelin Cells.
Nature 1988, 332, 311-415.
(3) Inoue, A.; Yanagisawa, M.; Kimura, S.; Kasuya, Y.; Miyauchi,
T.; Goto, K.; Masaki, T. The Human Endothelin Family: Three
Structurally and Pharmacologically Distinct Isopeptides Pre-
dicted by Three Separate Genes. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 2863-2867.
(22) Elliott, J . D.; Lago, M. A.; Cousins, R. D.; Gao, A.; Leber, J . D.;
Erhard, K. F.; Nambi, P.; Elshourbagy, N. A.; Kumar, C.; Lee,
J . A.; Bean, J . W.; DeBrosse, C. W.; Eggleston, K. S.; Brooks, D.
P.; Feuerstein, G.; Ruffolo, R. R.; Weinstock, J .; Gleason, J . G.;
Peishoff, C. E.; Ohlstein, E. H. 1,3-Diarylindan-2-carboxylic
acids, Potent and Selective Non-peptide Endothelin Receptor
Antagonists. J . Med. Chem. 1994, 37, 1553-1557.
(23) Walsh, T. F.; Fitch, K. J .; Chakravarty, K.; Williangs, D. L.;
Murphy, K. A.; Nolan, N. A.; O’Brien, J . A.; Lis, E. V.; Pettibone,
D. J .; Kivlighn, S. D.; Gabel, R. A.; Zingaro, G. J .; Krause, S.

Endothelin Receptor-A Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1689
M.; Siegl, P. K. S.; Clineschmidt, B. V.; Greenlee, W. J . Discovery
of L-749,329, a Highly Potent, Orally Active Antagonist of
Endothelin Receptors. The 208th ACS National Meeting, Wash-
ington, DC, August 21-25, 1994, Abstract MEDI 145.
(24) Walsh, T. F. Progress in the Development of Endothelin Receptor
Antagonists. In Annual Reports in Medicinal Chemistry; Bristol,
J . A., Ed.; Academic Press: New York, 1995; Vol. 30, pp 91-
100.
(25) Clozel, M.; Breu, V.; Gray, G. A.; Kalina, B.; Loffler, B.; Burri,
K.; Cassal, J .; Hirth, G.; Muller, M.; Neidhart, W.; Ramuz, H.
Pharmacological Characterization of Bosentan, a New Potent
Orally Active Non-peptide Endothelin Receptor Antagonist. J .
Pharmacol. Exp. Ther. 1994, 270, 228-235.
(26) Ishikawa, K.; Ihara, M.; Noguchi, K.; Mase, T.; Mino, N.; Saeki,
T.; Fukuroda, T.; Fukami, T.; Ozaki, S.; Nagase, T.; Nishikibe,
J .; Yano, M. Biochemical and Pharmacological Profile of a Potent
and Selective Endothelin B-Receptor Antanonist BQ-788. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 4892-4896.
(27) Urade, Y.; Fujitani, Y.; Watakabe, T.; Umemura, I.; Takai, M.;
Okada, T.; Sakata, K.; Karaki, H. An Endothelin B Receptor
Selective Antagonist: IRL 1038, (Cys11-Cys15)-endothelin-1
(11-21). FEBS Lett. 1992, 311, 12-16.
(28) Clozel, M.; Breu, V. The Role of ET-B Receptors in Normotensive
and Hypertensive Rats as Revealed by the Non-peptide Selective
ET-B Receptor Antagonist Ro 46-8443. FEBS Lett. 1996, 383
(1-2), 42-45.
(29) Breu, V.; Clozel, M.; Burri, K.; Hirth, G.; Neidhart, W.; Ramuz,
H. In Vitro Characterisation of Ro 46-8443, the First Non-peptide
Antagonist Selective for the Endothelin ET-B Receptor. FEBS
Lett. 1996, 383 (1-2), 37-41.
(30) Chan, M. F.; Kois, A.; Raju, B.; Castillo, R. S.; Verner, E. J .;
Wu, C.; Venkatachalapathi, Y. V.; Okun, I.; Stavros, F. D.; Balaji,
V. N. The Discovery of Non-peptide, Low Molecular Weight, ETB
Selective Endothelin Antagonists. The 209th ACS National
Meeting, Anaheim, California, April 1995, Abstract MEDI 168.
(31) Chan, M. F.; Kois, A.; Raju, B.; Castillo, R. S.; Verner, E. J .;
Wu, C.; Venkatachalapathi, Y. V.; Okun, I.; Stavros, F. D.; Balaji,
V. N. N-(5-Isoxazolyl)biphenylsulfonamides, N-(3-Isoxazolyl)-
biphenylsulfonamides and Derivatives thereof that Modulate the
Activity of Endothelin. US Patent 5,464,853, Nov 7, 1995.
(32) Chan, M. F.; Raju, B.; Kois, A.; Verner, E. J .; Wu, C.; Castillo,
R.; Balaji, V. N. N-(4-Halo-Isoxazolyl)sulfonamides and Deriva-
tives thereof that Modulate the Activity of Endothelin. US Patent
5514691, May 7, 1996.
(33) Chan, M. F.; Raju, B.; Kois, A.; Castillo, R. S.; Verner, E. J .;
Wu, C.; Hwang, E.; Okun, I.; Stavros, F.; Balaji, V. N. Halogen
Substitution at the Isoxazole Ring Enhances the Activity of
N-(Isoxazolyl)sulfonamide Endothelin Antagonists. Bioorg. Med.
Chem. Lett. 1996, 6 (20), 2393-2398.
(34) Verner, E. J .; Kois, A.; Raju, B.; Wu, C.; Yalamori, V.; Castillo,
R.; Hwang, E.; Okun, I.; Stavros, F. D.; Balaji, V. N.; Wolff, M.
F.; Chan, M. F. Design and Synthesis of Benzenesulfonamides
as Endothelin Antagonists. The 208th ACS National Meeting,
Washington, D.C., August 21-25, 1994, Abstract MEDI 215.
(35) Raju, B.; Wu, C.; Kois, A.; Verner, E.; Okun, I.; Stavros, F.; Chan,
M. F. Thiophenesulfonamides as Endothelin Receptor Antago-
nists. Bioorg. Med. Chem. Lett. 1996, 6 (22), 2651-2656.
(36) Raju, B.; Wu. C.; Castillo, R.; Okun, I.; Stavros, F.; Chan, M. F.
2-Aryloxycarbonylthiophene-3-sulfonamides are ETA Selective
Endothelin Antagonists. The 211th ACS National Meeting, New
Orleans, LA, March 24-28, 1996, Abstract MEDI 145.
(37) Brown, H. C.; Heim, P. J . Am. Chem. Soc. 1964, 86, 3566-3567.
(38) DiCarlo, V. S.; Chen, S. J .; Meng, Q. C.; Durand, J .; Yano, M.;
Chen, Y. F.; Oparil, S. ETA-receptor Antagonist Prevents and
Reverses Chronic Hypoxia-induced Pulmonary Hypertension in
Rat. Am. J . Physiol. 1995, 269 (5 Pt 1), L690-697.
(39) Oparil, S.; Chen, S. J .; Meng, Q. C.; Elton, T. S.; Yano, M.; Chen,
Y. F. Endothelin-A Receptor Antagonist Prevents Acute Hypoxia-
induced Pulmonary Hypertension in the Rat. Am. J . Physiol.
1995, 268 (1 Pt 1), L95-100.
(40) Stavros, F. D.; Hasel, C. W. O.; Okun, I.; Baldwin, J .; Freriks,
K. COS-7 Cells Stable Transfected to Express the Human ET-B
Receptor Provide
a Useful Screen for Endothelin Receptor
Antagonists. J . Cardiovasc. Pharmacol. 1993, 22 (suppl. 8).
(41) Berridge, J . M.; Downes, C. P.; Hanley, M. R. Biochem. J . 1982,
206, 587-595.
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