Discovery of TBC11251, a potent, long acting, orally active endothelin receptor-A selective antagonist

Article abstract of DOI:10.1021/jm9700068

Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A antagonists with high potency and selectivity. Replacement of an amide group in this class of compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity while providing a compound with oral bioavailability and longer duration of action. The optimal compound discovered during these studies, 15q (TBC11251), binds competitively to human ET(A) receptors with a K(i) of 0.43 ± 0.03 nM and an IC₅₀ of 1.4 nM (IC₅₀ for ET(B) = 9800 nM). This compound inhibits ET-1- induced stimulation of phosphoinositide turnover with a K(i) of 0.686 nM and a pA₂ of 8.0. The compound has a serum half-life in the rat and the dog of 6-7 h and 60-100% oral bioavailability. This compound is one of the most selective ET(A) antagonists reported and therefore is suitable for additional pharmacological and clinical investigation of the role of ET(A) receptors in diseases.

Full text of DOI:10.1021/jm9700068

1690
J . Med. Chem. 1997, 40, 1690-1697
Discover y of TBC11251, a P oten t, Lon g Actin g, Or a lly Active En d oth elin
Recep tor -A Selective An ta gon ist1
Chengde Wu,*,†,‡ Ming F. Chan,† Fiona Stavros,†,‡ B. Raju,†,‡ Ilya Okun,† Seymour Mong,† Karin M. Keller,§
Tommy Brock,§ Timothy P. Kogan,§ and Richard A. F. Dixon§
ImmunoPharmaceutics Inc. (a subsidiary of Texas Biotechnology Corporation), 11011 Via Frontera,
San Diego, California 92127, and Texas Biotechnology Corporation, 7000 Fannin, Suite 1920, Houston, Texas 77030
Received J anuary 6, 1997X
Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A
antagonists with high potency and selectivity. Replacement of an amide group in this class of
compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity
while providing a compound with oral bioavailability and longer duration of action. The optimal
compound discovered during these studies, 15q (TBC11251), binds competitively to human
ETA receptors with a Ki of 0.43 ( 0.03 nM and an IC50 of 1.4 nM (IC50 for ETB ) 9800 nM).
This compound inhibits ET-1-induced stimulation of phosphoinositide turnover with a Ki of
0.686 nM and a pA2 of 8.0. The compound has a serum half-life in the rat and the dog of 6-7
h and 60-100% oral bioavailability. This compound is one of the most selective ETA antagonists
reported and therefore is suitable for additional pharmacological and clinical investigation of
the role of ETA receptors in diseases.
Sch em e 1. Syntheses of Sulfonamides with Imide,
Urethane, Urea, and Amide Linker Groups
In tr od u ction
In the previous paper, we have reported on a series
of 3-thiophenesulfonamides with an arylamide at the
2-position as endothelin receptor antagonists. Some of
the most interesting amides are summarized in Figure
1. Although these amidosulfonamides have high bind-
ing affinities for ETA in radioligand binding assays (10-
20 nM for 1-3; 2-4 nM for 4-7), the most potent
compounds lack oral bioavailability and have short in
vivo half-lives, perhaps as a result of proteolytic cleav-
age of the amide bond. In order to develop compounds
that maintain the desired in vitro and in vivo activity
of this series but demonstrate improved oral bioavail-
ability and better metabolic properties, we investigated
replacing the proteolytically susceptible amide bond in
compound 1 with more durable linkers. In this paper,
we report the synthesis and structure-activity relation-
ships of various linkers to replace the amide of 1, which
led to the discovery of 15q, a compound which has good
potency, a long duration of action, and good oral
bioavailability.
Syn th esis
Scheme 1 shows the synthesis of compounds with
imide, urethane, urea, and extended amide linkages.
Piperyllic acid was treated with carbonyldiimidazole and
then with the dianion generated by deprotonating 8
with 2 equiv of sodium hydride to yield the imide 9. The
thienyl carboxylic acid 10 was first subjected to Curtius
rearrangement using diphenyl phosphorazidate, and the
resulting isocyanate was treated with either
5-hydroxybenzo[d][1,3]dioxole to give the urethane 11
or treated with 5-aminobenzo[d][1,3]dioxole to give the
urea 12. Alternatively, 10 was coupled with 5-(1-
piperadinylmethyl)benzo[d][1,3]dioxole using carbonyl-
diimidazole as the coupling reagent to yield the amide
13.
The general methodology for synthesizing sulfona-
mides with a ketone linkage is shown in Scheme 2. Acid
10 was first coupled with N,O-dimethylhydroxylamine
using carbonyldiimidazole as the coupling reagent to
give the Weinreb amide 14.2 Compound 14 was then
treated with aryl or benzylic Grignard reagents followed
by acidic workup to give the ketones 15. This general
method also provided for access to the ketone interme-
* 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.
† ImmunoPharmaceutics, Inc.
‡ Present address: Texas Biotechnology Corp., 7000 Fannin, Suite
1920, Houston, TX 77030.
§ Texas Biotechnology Corp.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(97)00006-X CCC: $14.00 © 1997 American Chemical Society

Endothelin Receptor-A Selective Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1691
F igu r e 1. High-affinity amidothiophenesulfonamides ETA receptor antagonists.
Sch em e 2. General Synthesis of Ketosulfonamides
diate 15d (Table 1) as well as 15q (Table 3).
5-Methylbenzo[d][1,3]dioxole was treated with aqueous
formaldehyde and concentrated hydrochloric acid in
ethyl ether to give the desired benzyl chloride 16 and
condensation product 17. This mixture of 16 and 17
was used to form the Grignard reagent of 16 without
separation.
In Scheme 3 an outline of the synthesis of several
derivatives of ketone 15d is given. Reduction using
sodium borohydride gave alcohol 18. Treatment of 15d
with hydroxylamine gave oxime 19. Reductive amina-
tion using dimethylamine and sodium cyanoborohydride
produced amine 20. Ketalization under azeotropic
conditions using ethylene glycol gave ketal 21. Addition
of methylmagnesium bromide to ketone 15d gave
alcohol 22.
The resulting ester 23 was brominated to give 24 which
was further treated with cuprous cyanide in hot DMF
to give the benzonitrile 25.4 The methyl ester of 25 was
then exchanged to a tert-butyl ester by saponification,
treatment with thionyl chloride, and reaction of the
resulting acid chloride with tert-butyl alcohol to give 26.
The benzonitrile 26 was then deprotonated using so-
dium hydride and reacted with the acylimidazolide
generated in situ by reacting acid 10 and carbonyldi-
imidazole. The intermediate tert-butyl ester 27 was
concomittantly hydrolyzed and decarboxylated in warm
acid to give the desired cyano ketone 28. The methyl
to tert-butyl ester exchange (25-26) was necessary
because of difficulties in hydrolyzing the methyl ester
in the coupled product: alkaline hydrolysis caused a
retro-aldol reaction to give back acid 10. Compound 29
was synthesized via carbonyldiimidazole-mediated cou-
pling of acid 10 and the anion generated by reacting
5-(cyanomethyl)benzo[d][1,3]dioxole with sodium hy-
dride.
The synthesis of cyano ketones 28 and 29 is shown
in Scheme 4.
5-Benzo[d][1,3]dioxolylacetic acid was esterified in the
presence of 2,2-dimethoxypropane in acidic methanol.3

1692 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Ta ble 1. Effect of Linker Groups on [125I]Endothelin-1 Binding
Wu et al.
a
IC50, nM (n)
entry
L
X
ETA
ETB
1
2
3
4
5
6
7
9
11
12
13
15a
15b
15c
15d
18
19
20
21
22
29
CONH
Br
19 ( 5 (5)
15 ( 5 (2)
10100 ( 1500 (5)
14100 ( 0 (2)
10 ( 2 (5)
32600 ( 1800 (5)
355500 ( 112300 (4)
40400 ( 6400 (5)
14000 ( 2050 (2)
12200 ( 1400 (3)
37200 ( 7800 (2)
5900 ( 700 (3)
3.0 ( 0.7 (4)
3.4 ( 0.4 (4)
2.6 ( 0.5 (2)
3.9 ( 0.6 (3)
887 ( 401 (2)
88 ( 64 (3)
CONHCO
NHCO2
NHCONH
CON(CH2CH2)2NCH2
CO
COCH2
Br
Br
Br
Cl
Br
CH3
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
574 ( 298 (3)
19600 ( 5400 (2)
2000 ( 200 (2)
27 ( 4 (2)
1300 ( 300 (3)
>100000 (2)
15400 ( 3300 (2)
27200 ( 400 (2)
5500 ( 500 (4)
7000 ( 960 (6)
15700 ( 2700 (4)
4200 ( 500 (2)
>100000 (2)
COCH2
COCH2
14 ( 10 (4)
18 ( 6 (6)
CH(OH)CH2
C(NOH)CH2
CH(N(CH3)2)CH2
C(OCH2CH2O)CH2
C(OH)(CH3)CH2
COCH(CN)
56 ( 16 (4)
1100 ( 50 (2)
21000 ( 1300 (2)
123 ( 13 (2)
22200 ( 10400 (2)
2100 ( 100 (2)
2100 ( 70 (2)
>100000 (2)
27300 ( 3000 (2)
a
Values are means ( SEM for separate determinations.
Sch em e 3. Derivatization of Ketone 15d
Str u ctu r e-Activity Rela tion sh ip s
(19), dimethylamino (20), and tertiary alcohol (22) all
drastically decreased the activity. Substitution on the
ketone R-carbon with a cyano group (29) was also
deleterious. This study coupled with the observation
that direct attachment of the aryl group to the thiophene
or when the linker was methylene, ethylene, or vinylene,
the activity was lost to various degrees5 argued that an
acetyl group was the best identified replacement of the
amide group in compound 1.
In this paper we report the investigation of a series
of linker groups as replacements for the amide group
in compound 1 (Figure 1) in order to afford compounds
with improved properties as oral endothelin antagonists.
The results are summarized in Table 1. When the
amide group was replaced by an imide group, the
resultant compound 9 was more than 40-fold less active.
Replacement of the amide group by a urethane (11) or
ureido group (12) gave compounds that were 4- or 30-
fold less active, respectively. Introduction of a long and
bulky linker such as a piperazinylcarbonyl group (13)
was very deleterious to activity, as was the inclusion of
a very short linker such as a ketone group (15a ).
Therefore, it was concluded that the preferred linker
should contain two carbon atoms, or an equivalent.
Replacing the amide group with an acetyl group gave
compound 15c, which showed comparable activity to 1,
as did compounds 15b and 15d . We next investigated
replacement of the carbonyl group with other function-
ality. Although a hydroxyl group replacement of the
carbonyl (18) only reduced the activity by 2-fold and an
ethylenedioxy ketal replacement of the carbonyl (21)
lowered the potency by 6-fold, replacements with oxime
Replacement of the benzo[d][1,3]dioxole group with
4-methyl (15f), 4-methoxy (15e), and 3-methoxyphenyl
groups (15g) gave slightly reduced activity (Table 2)
compared to that of 15c and 15d . A series of ketones
were synthesized to study the effects of substitution
patterns of dimethyl groups. The rank order of potency
seemed to be 2,4-dimethyl > 2,5-dimethyl > 3,5-di-
methyl (Table 2). This exercise prompted us to synthe-
size 15q and introduce the favorable 2-methyl substitu-
ent in the benzodioxole moiety (Table 3). The results
of 15q, the corresponding dimethylisoxazole 15p , and
the isomeric chloroisoxazolesulfonamide 15r indicate
that the 6-methylbenzo[d][1,3]dioxol-5-yl substitution
rendered compounds of high potency as evidenced by
15q with a pA2 of 8.0 and its equipotent isomer 15r with

Endothelin Receptor-A Selective Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1693
Sch em e 4. Syntheses of Cyano Ketosulfonamides
Ta ble 2. Effect of Phenyl Substitution Patterns on [125I]Endothelin-1 Binding
IC50, nM (n)
entry
R2
R3
R4
R5
X
ETA
ETB
15e
15f
15g
15h
15i
H
H
H
H
CH3
CH3
CH3
H
H
H
OCH3
H
H
H
H
CH3
H
OCH3
CH3
H
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
CH3
CH3
Cl
Br
Cl
CH3
CH3
Cl
Br
Cl
43 ( 3 (2)
10100 ( 500 (2)
9100 ( 1900 (2)
19100 ( 1300 (3)
29200 ( 1400 (2)
17400 ( 10800 (2)
4800 ( 2700 (3)
3500 ( 2200 (2)
17700 ( 2600 (2)
18100 ( 2100 (2)
20 ( 2 (2)
30 ( 18 (3)
27 ( 7 (2)
2.7 ( 0.1 (2)
0.97 ( 0.22 (3)
0.76 ( 0.13 (2)
45 ( 6 (2)
15j
15k
15m
15n
CH3
H
Cl
7.2 ( 0.6 (2)
a pA2 of 8.47. Comparisons showed that the methyl
substitution at the 6-position increased potency by about
15-fold with an increase in pA2 of 1.25. The same is
true with a cyano group substitution with 1.95 higher
pA2 as suggested by the previous paper.
dioxole ketone 15d was active in the same model, had
a moderate half-life, and was 30% orally bioavailable.
Although the 6-position substituted benzo[d][1,3]dioxole
amides 3 and 5 had acceptable durations of action and
8-27% oral bioavailability, the inhibition of blood
pressure potentiation of 3-5 was only 8-18%. Amides
6 and 7 had quite high in vivo potency, but unfortu-
nately they were metabolized rapidly and scarcely
bioavailable. On the other hand, the corresponding
ketones 15p , 15q, and 28 all had desirable in vivo
potency, and 15p and 15q also had a long duration of
action and were highly orally bioavailable. Compound
15r and 28 were not further developed due to toxicity
The general preference of ketones over their corre-
sponding amides in terms of in vivo potency, serum half-
life and oral bioavailability is demonstrated in Table 4.
For the simple benzo[d][1,3]dioxole amide 1 or p-toluene
amide 2, no in vivo activity was observed in an ET-1
challenge rat model; the compounds also had very short
half-lives and were not orally bioavailable at detectable
levels. In contrast, the corresponding benzo[d][1,3]-

1694 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Wu et al.
Ta ble 3. Effect of Isoxazole and Benzo[d][1,3]dioxole 6-Position Substitution on [125I]Endothelin-1 Binding
IC50, nM (n)
phosphoinositol
entry
X
R
ETA
ETB
hydrolysis pA2
15p
15q
15r
15d
28
CH3
Cl
CH3
CH3
3.3 ( 0.2 (2)
1.4 ( 0.5 (5)
0.63 ( 0.1 (2)
21 ( 8 (5)
34300 ( 600 (2)
9800 ( 4400 (5)
3900 ( 60 (2)
7200 ( 1200 (5)
723 ( 209 (3)
NTa
8.0
8.47
6.75
8.
Cl
Cl
H
CN
1.3 ( 0.3 (3)
a
NT: not determined.
Ta ble 4. Percent Inhibition in ET-1 Challenge, Serum
Half-Life, and Oral Bioavailability in the Rat of High-Affinity
Amide and Ketone ETA Receptor Antagonists
Ta ble 5. Synthetic and Physical Data
synth
%
entry method yield
mp, °C
formulaa
% inhibition
of ET-1 challenge,
15 mg/kg (n)
oral
bioavailability
(%)
1
9
11
12
B
C
D
D
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
E
15
4
138-140 C16H12BrN3O6S2
90-93
39-43
62-65
221-223
47-49
152-157
oil
35-38
35-38
146-150
oil
C17H12BrN3O7S2
entry
t1/2 (h)
29
12
54
12
65
40
50
2
78
28
65
34
52
28
57
33
14
71
16
67
73
36
67
32
17
19
C16H12BrN3O7S2‚0.3TFA
amides
C
C
16H13BrN4O6S2‚0.1TFA
1
2
not active
not active
18 (4)
14 (3)
8 (3)
<0.5
<0.5
2.5-3.0
NTa
2.5
<0.15
<0.15
0
0
13
21H21ClN4O6S2
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15m
15n
15p
15q
15r
18
C16H11BrN2O6S2‚0.3EtOAc
b
3 (TBC11192)
27
NT
8
NT
2
C18H16N2O6S2
b
4
C17H13BrN2O6S2
C17H13ClN2O6S2
C17H15ClN2O5S2‚0.2EtOAc
C17H15BrN2O4S2
5 (TBC11241)
6
7
33 (3)
50 (3)
b
ketones
C17H15ClN2O5S2
b
15d
15p
41 (6) 60 mg/kg
33 (3)
15q (TBC11251) 41 (6)
1.5
7
6.7
NT
30
100
60
95-100
oil
C18H18N2O4S2
C19H20N2O4S2
b
b
48-54
58-63
45-50
72-76
45-50
42-45
46-50
38-41
142-145
56-59
60-63
39-41
C18H17ClN2O4S2
b
28
52 (3)
NT
C18H17BrN2O4S2
b
C18H17ClN2O4S2
C18H17ClN2O4S2
C19H18N2O6S2
a
NT: not determined.
b
C18H15ClN2O6S2
C18H15ClN2O6S2
C17H15ClN2O6S2‚0.6EtOAc
concerns and instability in alkaline aqueous media,
respectively. It was concluded that 15q was suitable
for further pharmacological and clinical studies.
19
20
21
22
28
29
F
C
C
C
17H14ClN3O6S2‚0.2EtOAc
19H20ClN3O5S2‚1.6TFA
19H17ClN2O7S2
G
H
J
K
C
P h a r m a cok in etics a n d in Vivo Da ta
C18H17ClN2O6S2
The pharmacokinetic data for 15q is expressed as
mean plasma concentration plotted against hours after
dosing for rats (Figure 2) and dogs (Figure 3). Following
bolus intravenous administration of 15q to both rats
and dogs, plasma levels of 15q declined rapidly over the
first 30-60 min and then more slowly over the remain-
ing 24 h observation period. The t1/2 for the terminal
elimination phase was 5.9-7.5 h for the rat and 4-4.8
h for the dog. Orally administered 15q was rapidly
absorbed in both the rat and the dog with a t1/2(abs) of
0.7 and 0.3 h, respectively. Peak plasma concentrations
occurred between 2 and 3 h postdosing in the rat and
between 45 and 90 min in the dog. Following oral
administration the t1/2,elimination ranged from 2 to 5 h in
rats and from 4 to 7 h in dogs. Oral bioavailability,
calculated from comparison of administered dose and
AUC, indicated that the material was 50-60% bioavail-
able in the rat and 90-100% bioavailable in the dog.
Compound 15q has demonstrated efficacy in an in
vivo model of acute hypoxia-induced pulmonary hyper-
tension in rats by both iv and oral routes of administra-
tion with an EC50 of 0.5 and 1 mg/kg, respectively.6
105-108 C18H12ClN3O6S2‚0.2CH3CN
142-145
C
18H12ClN3O6S2‚0.2TFA
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. Homogeneity established by two diverse analyti-
cal HPLC systems.
Furthermore, 15q was shown to both prevent and
reverse chronic hypoxia-induced pulmonary hyperten-
sion following oral administration at a dose of 11 mg/
kg/day in the rat.6
Con clu sion
In extension of the study of amidesulfonamides as
ETA selective endothelin receptor antagonists and to
improve the profile of the compounds particularly
regarding oral bioavailability and half-life, we investi-
gated replacing the amide bond with various linker
groups. This effort resulted in the identification of an
acetyl group as the optimal spacer. Incorporation of
data from the previous paper regarding the preference
for a 2-methyl substituent on the benzodioxole group

Endothelin Receptor-A Selective Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1695
iodine vapor. Analytical HPLC was performed on a Vydac C18
column (4.6 × 250 mm); preparative HPLC, on a Dynamax-
60A (83-241-c) with acetonitrile:water gradients containing
0.1% trifluoroacetic acid. The detection wavelength was 254
nm.
Met h od A. 4-Ch lor o-3-m et h yl-5-[[2-[(b en zo[d ][1,3]-
dioxol-5-yl)acetyl]-3-th ien yl]su lfon am ido]isoxazole (15d).
To a solution of acid 10a (5.0 g, 15.49 mmol) in anhydrous
THF (70 mL) was added 1,1′-carbonyldiimidazole (2.76 g, 17.04
mmol). The mixture was stirred at room temperature for 15
min before the sequential addition of imidazole (2.11 g, 30.98
mmol) and N,O-dimethylhydroxylamine hydrochloride (2.31 g,
23.24 mmol). The mixture was stirred at room temperature
for 4 h. THF was removed under reduced pressure and the
residue partitioned between EtOAc and 1 N hydrochloric acid.
The organic layer was dried over MgSO4 and concentrated.
The residue was treated with toluene and concentrated again.
The residual red oil was dissolved in dry THF (20 mL) and
treated at 0 °C with a Grignard reagent prepared from
5-(chloromethyl)benzo[d][1,3]dioxole (14.0 g, 82.02 mmol) and
magnesium turnings (2.3 g, 95.69 mmol) in THF (100 mL).
The mixture was stirred at 0 °C for 1 h and at room
temperature for an additional 2 h. The reaction was quenched
with a mixture of concentrated hydrochloric acid (20 mL) and
methanol (50 mL) while cooling. After 10 min of stirring at 0
°C, the mixture was concentrated. The aqueous residue was
partitioned between EtOAc and 1 N hydrochloric acid. The
organic layer was dried over MgSO4 and concentrated, and
the residue was purified via preparative HPLC to give 15d
F igu r e 2. Pharmacokinetics of 15q in rats.
1
(3.0 g, 50% yield) as a yellow solid: mp 35-38 °C; H NMR
(DMSO-d6) δ 7.89 (d, J ) 5.1 Hz, 1H), 7.43 (d, J ) 5.1 Hz,
1H), 6.84 (d, J ) 7.8 Hz, 1H), 6.80 (s, 1H), 6.70 (d, J ) 7.8 Hz,
1H), 5.98 (s, 2H), 4.44 (s, 2H), 2.09 (s, 3H).
Meth od B. N-(4-Ch lor o-3-m eth yl-5-isoxa zolyl)-2-{[4-
(ben zo[d][1,3]dioxol-5-ylm eth yl)piper azin -1-yl]car bon yl}-
th iop h en e-3-su lfon a m id e (13). To a solution of acid 10a
(1.0 g, 3.10 mmol) in anhydrous THF (20 mL) was added 1,1′-
carbonyldiimidazole (603 mg, 3.72 mmol). The mixture was
stirred at room temperature for 10 min before the addition of
1-piperonylpiperazine (2.0 g, 9.30 mmol). The mixture was
stirred at room temperature for 4 h. THF was removed by
evaporation, and the residue was partitioned between 1 N
NaOH and a 1:1 mixture of EtOAc and ethyl ether. The
aqueous layer was treated with saturated NH4Cl to pH 7-8
and was then extracted with EtOAc. The organic layer was
dried over MgSO4 and concentrated, and the residue was
purified via HPLC to give 13 (852 mg, 54% yield) as a white
powder: mp 221-223 °C; 1H NMR (DMSO-d6) δ 9.61 (br s,
1H), 7.61 (d, J ) 5.1 Hz, 1H), 7.18 (d, J ) 5.1 Hz, 1H), 7.06 (s,
1H), 6.99 (m, 2H), 5.73 (s, 2H), 4.25-4.54 (m, 3H), 2.93-3.72
(m, 7H), 1.99 (s, 3H).
F igu r e 3. Pharmacokinetics of 15q in beagle dogs.
led to the eventual discovery of 15q, which retains high
potency and selectivity for the ETA receptor while
having both oral bioavailability and long half-life.
Results of clinical development will be reported in due
course.
Exp er im en ta l Section
Met h od C.
N-(4-Br om o-3-m et h yl-5-isoxa zolyl)-2-
[[(b en zo[d ][1,3]d ioxol-5-ylca r bon yl)a m in o]ca r b on yl]t h -
iop h en e-3-su lfon a m id e (9). 1,1′-Carbonyldiimidazole (213
mg, 1.31 mmol) was added to a solution of piperonylic acid
(182 mg, 1.09 mmol) in dry THF (10 mL). The resulting
mixture was stirred for 15 min. Compound 8 (400 mg, 1.09
mmol) and NaH (175 mg, 60% dispersion in mineral oil, 4.37
mmol) were added sequentially. The mixture was stirred at
room temperature for 8 h. Water was added to destroy the
excess NaH. The volatiles were then evaporated, and the
residue was partitioned between EtOAc and 1 N hydrochloric
acid. The organic layer was dried over MgSO4 and concen-
trated. The residue was recrystallized from EtOAc to give 9
(20 mg, 3.6% yield) as a yellow powder: mp 90-93 °C; 1H NMR
(DMSO-d6) δ 12.50 (br s, 1H), 7.86 (d, J ) 5.3 Hz, 1H), 7.80
(dd, J ) 8.4, 1.5 Hz, 1H), 7.68 (d, J ) 1.5 Hz, 1H), 7.40 (d, J
) 5.3 Hz, 1H), 7.05 (d, J ) 8.4 Hz, 1H), 6.15 (s, 2H), 1.95 (s,
3H).
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,
Compound 29 was synthesized by the same method as 9
except that acid 10a was used instead of piperonylic acid, and
5-(cyanomethyl)benzo[d][1,3]dioxole was used instead of 8:
CA).
[
125I]ET-1 was obtained from Amersham (Arlington
1
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, phosphomolybdic acid, or
yellow powder; mp 142-145 °C; H NMR (DMSO-d6) δ 7.89
(d, J ) 5.4 Hz, 1H), 7.43 (d, J ) 5.4 Hz, 1H), 7.30 (d, J ) 1.2
Hz, 1H), 7.12 (dd, J ) 8.4, 1.2 Hz, 1H), 6.98 (d, J ) 8.4 Hz,
1H), 6.05 (s, 2H), 2.11 (s, 3H).

1696 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Met h od D. N-(4-Br om o-3-m et h yl-5-isoxa zolyl)-2-
Wu et al.
was dried over MgSO4 and concentrated to give 21 (74 mg,
67% yield) as a white powder: mp 60-63 °C; 1H NMR (DMSO-
d6) δ 7.91 (br s, 1H), 7.30 (d, J ) 5.4 Hz, 1H), 7.13 (d, J ) 5.4
Hz, 1H), 6.80 (br s, 1H), 6.70 (m, 2H), 5.92 (s, 2H), 3.89-4.05
(m, 4H), 3.55 (s, 2H), 2.24 (s, 3H).
Meth od J . N-(4-Ch lor o-3-m eth yl-5-isoxa zolyl)-2-[2-h y-
d r oxy-2-(b e n zo[d ][1,3]d ioxol-5-ylm e t h yl)-2-p r op yl]t h -
iop h en e-3-su lfon a m id e (22). To a solution of 15d (100 mg,
0.23 mmol) in anhydrous THF at -78 °C was added methyl-
magnesium bromide (378 µL, 3 M in Et2O). The resulting
mixture was stirred at -78 °C for 10 min before it was allowed
to warm to 0 °C and was quenched with saturated NH4Cl(aq).
The mixture was worked up as usual to give 22 (33 mg, 32%
yield) as a pink/orange powder: mp 39-41 °C; 1H NMR
(CDCl3) δ 8.85 (br s, 1H), 7.56 (d, J ) 3.0 Hz, 1H), 7.21 (d, J
) 3.0 Hz, 1H), 6.74 (m, 1H), 6.56 (m, 2H), 5.94 (s, 2H), 3.32
(d, 1H), 3.14 (d, 1H), 2.17 (s, 3H), 1.71 (s, 3H).
Meth od K. N-(4-Ch lor o-3-m eth yl-5-isoxa zolyl)-2-[(6-
cyan oben zo[d][1,3]dioxol-5-yl)acetyl]th ioph en e-3-su lfon a-
m id e (28). Compound 28 was synthesized in the same fashion
as for 9 (method C) except that acid 10a was used instead of
piperonylic acid, and 26 was used instead of 8. The crude
mixture was poured into a 2:2:1 mixture of acetonitrile/water/
concentrated hydrochloric acid, and the resulting mixture was
heated at 40 °C for 12 h. Acetonitrile was then removed by
evaporation, and the aqueous residue was partitioned between
EtOAc and 1 N hydrochloric acid. The organic layer was
concentrated and purified by HPLC to give 28 in 17% yield as
a light dull yellow powder: mp 105-108 °C; 1H NMR (DMSO-
d6) δ 7.95 (d, J ) 5.1 Hz, 1H), 7.47 (d, J ) 5.1 Hz, 1H), 7.42 (s,
1H), 7.04 (s, 1H), 6.17 (s, 2H), 4.83 (s, 2H), 2.06 (s, 3H).
5-(Ch lor om eth yl)-6-m eth ylben zo[d][1,3]dioxole (16). To
a mixture of ether (100 mL) and concentrated hydrochloric acid
(100 mL) at 0 °C were sequentially added 5-methylbenzo[d]-
[1,3]dioxole (10 mL, 81 mmol) and formaldehyde (30% in water,
20 mL, 267 mmol). The mixture was stirred at 0 °C for 1 h
and at room temperature for 4 h. The mixture was diluted
with ether (100 mL), and the organic layer was separated,
dried (MgSO4), and then concentrated. The solid residue was
heated with hexanes (100 mL), and the insolubles were filtered
off. The filtrate was concentrated to give a mixture of 16 and
17 (7:3 by weight with 13 g combined yield) as a white solid.
16: 1H NMR (CDCl3) δ 6.80 (s, 1H), 6.68 (s, 1H), 5.92 (s, 2H),
4.55 (s, 2H), 2.34 (s, 3H). 17: δ 6.68 (s, 2H), 6.40 (s, 2H), 5.90
(s, 4H), 3.70 (s, 2H), 2.18 (s, 6H).
Meth yl (6-Br om oben zo[d ][1,3]d ioxol-5-yl)a ceta te (24).
To a solution of 23 (5 g, 25.8 mmol) in acetic acid (15 mL) was
added dropwise bromine until a red-brown color persisted.
After being stirred at room temperature for 30 min, the
reaction mixture was partitioned between water (200 mL) and
ether (200 mL). The organic layer was washed with water (3
× 200 mL), dried over MgSO4, and concentrated to give 24
(5.9 g, 84% yield) as an oil: 1H NMR (CDCl3) δ 7.02 (s, 1H),
6.76 (s, 1H), 5.98 (s, 2H), 3.71 (s, 3H), 3.69 (s, 2H).
ter t-Bu tyl (6-Cyan oben zo[d][1,3]dioxol-5-yl)acetate (26).
To a solution of 25 (5g, 18.32 mmol) in methanol (100 mL)
was added 1 N NaOH (50 mL). The reaction mixture was
stirred at room temperature for 1.5 h, and methanol was
removed by evaporation. The aqueous residue was acidified
with concentrated hydrochloric acid to pH ∼1 and extracted
with EtOAc. The organic layer was dried over MgSO4 and
concentrated to give a solid. The solid was treated with thionyl
chloride (50 mL), and the mixture was heated under reflux
for 10 min before the volatiles were removed by evaporation.
The residue was dissolved in dichloromethane (15 mL) and
added dropwise to a solution of 2-methyl-2-propanol (6.8 g, 91.6
mmol) and triethylamine (9.3 g, 91.6 mmol) in dichlo-
romethane (100 mL) at 0 °C. The reaction was stirred at room
temperature for 2 h. The mixture was washed with water (3
× 150 mL), and the organic layer was dried over MgSO4 and
concentrated to give 26 (335 mg, 7% yield) as a solid.
[[(ben zo[d][1,3]dioxol-5-yloxy)car bon yl]am in o]th ioph en e-
3-su lfon a m id e (11). Triethylamine (2.28 mL, 16.35 mmol)
and diphenyl phosphorazidate (773 mg, 2.72 mmol) were
sequentially added to a solution of acid 10b (1.0 g, 2.72 mmol)
in anhydrous THF (40 mL). The mixture was stirred for 8 h.
Sesamol (1.54 g, 10.9 mmol) was added, and the mixture was
heated under reflux for 2 h and then allowed to cool to room
temperature. The solvent was removed by evaporation, and
the residue was partitioned between EtOAc and 1 N hydro-
chloric acid. The organic layer was concentrated and purified
by HPLC to give 11 (400 mg, 29% yield) as a beige powder:
1
mp 39-43 °C; H NMR (DMSO-d6) δ 9.94 (br s, 1H), 7.14 (d,
J ) 5.7 Hz, 1H), 6.94-7.03 (m, 3H), 6.74 (dd, J ) 8.3, 2.1 Hz,
1H), 6.08 (s, 2H), 2.10 (s, 3H).
Compound 12 was synthesized by the same method as 11
except that 5-aminobenzo[d][1,3]dioxole was used instead of
sesamol: brown/gray powder; 62-65 °C; 1H NMR (DMSO-d6)
δ 10.10 (br s, 1H), 9.41 (br s, 1H), 7.17 (d, J ) 1.2 Hz, 1H),
7.00 (d, J ) 5.8 Hz, 1H), 6.95 (d, J ) 5.8 Hz, 1H), 6.82-6.88
(m, 2H), 5.99 (s, 2H), 2.11 (s, 3H).
Meth od E. N-(4-Ch lor o-3-m eth yl-5-isoxa zolyl)-2-[1-h y-
d r oxy-2-(ben zo[d ][1,3]d ioxol-5-yl)eth yl]th iop h en e-3-su l-
fon a m id e (18). Lithium borohydride (36.6 mg, 1.68 mmol)
was added slowly to a solution of 15d (74 mg, 0.17 mmol) in
THF (10 mL). The resulting mixture was stirred for 8 h.
Saturated NH4Cl(aq) was added to quench the excess lithium
borohydride, and the resulting mixture was concentrated by
evaporation. The residue was partitioned between EtOAc and
1 N hydrochloric acid, the organic layer was dried over MgSO4
and concentrated to give 18 (67 mg, 67% yield) as a yellow
powder: mp 38-41 °C; 1H NMR (DMSO-d6) δ 7.54 (d, J ) 7.2
Hz, 1H), 7.22 (d, J ) 7.2 Hz, 1H), 6.80 (d, J ) 8.1 Hz, 1H),
6.78 (s, 1H), 6.64 (s, 1H), 5.96 (s, 2H), 5.41 (m, 1H), 2.90 (m,
1H), 2.60 (m, 1H), 2.12 (s, 3H).
Met h od F . N-(4-Ch lor o-3-m et h yl-5-isoxa zolyl)-2-[1-
(h yd r oxyim in o)-2-(b e n zo[d ][1,3]d ioxol-5-yl)e t h yl]t h -
iop h en e-3-su lfon a m id e (19). To a mixture of 15d (100 mg,
0.23 mmol) and hydroxylamine hydrochloride (160 mg, 2.3
mmol) was added water (5 mL). To this stirred heterogeneous
mixture was then added NaOH pellets (320 mg, 8.0 mmol).
The mixture was stirred until it became homogeneous, heated
at 80 °C for 10 min, allowed to cool to room temperature, and
was then poured onto ice mixed with 1.5 mL of concentrated
hydrochloric acid in 100 mL of water. The resulting white
precipitate was filtered and dried in vacuo to give 19 (75 mg,
73% yield) as a yellow powder: mp 142-145 °C; 1H NMR
(DMSO-d6) δ 11.80 (br s, 1H), 7.59 (d, J ) 5.4 Hz, 1H), 7.31
(d, J ) 5.4 Hz, 1H), 6.75 (d, J ) 7.8 Hz, 1H), 6.61 (d, J ) 0.9
Hz, 1H), 6.55 (dd, J ) 7.8, 0.9 Hz, 1H), 5.94 (s, 2H), 4.03 (s,
2H), 2.14 (s, 3H).
Met h od G. N-(4-Ch lor o-3-m et h yl-5-isoxa zolyl)-2-[1-
(d im e t h yla m in o)-(b e n zo [d ][1,3]d io x o l-5-y l)e t h y l]t h -
iop h en e-3-su lfon a m id e (20). To a suspension of dimethyl-
amine hydrochloride (420 mg, 5.15 mmol) in methanol (1.7 mL)
was added KOH (170 mg, 3.04 mmol). The suspension was
stirred until a homogeneous milky mixture formed. Following
the addition of 15d (100 mg, 0.23 mmol), the mixture was
stirred at room temperature for 30 min before the addition of
sodium cyanoborohydride (240 mg, 3.82 mmol). The resulting
mixture was stirred at room temperature for 1 h before it was
quenched with acetic acid. The crude mixture was then
purified by HPLC to give 20 (38 mg, 36% yield) as a white
powder: mp 56-59 °C; 1H NMR (DMSO-d6) δ 9.53 (br s, 1H),
7.67 (d, J ) 5.1 Hz, 1H), 7.28 (d, J ) 5.1 Hz, 1H), 6.81 (br s,
1H), 6.70 (m, 2H), 5.93 (s, 2H), 5.64 (m, 1H), 3.50 (m, 1H),
3.20 (m, 1H), 2.85 (s, 6H), 1.99 (s, 3H).
Met h od H . N-(4-Ch lor o-3-m et h yl-5-isoxa zolyl)-2-[2-
(ben zo[d][1,3]dioxol-5-ylm eth yl)-1,3-dioxol-2-yl]th ioph en e-
3-su lfon a m id e (21). To a 100 mL round bottom flask were
sequentially added 15d (100 mg, 0.23 mmol), p-toluenesulfonic
acid monohydrate (140 mg, 0.74 mmol), ethylene glycol (10
mL), and benzene (15 mL). The resulting mixture was heated
under reflux while water was removed azeotropically for 7 h.
The mixture was allowed to cool to room temperature and
partitioned between benzene and water. The organic layer
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)

Endothelin Receptor-A Selective Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1697
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 then the pellet
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.
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 (Challenge).
The degree in inhibition was calculated as
100 × (controlmax. pressor response
-
antagonistmax. pressor response)/controlmax. pressor response
P h a r m a cok in etic Assa ys. All surgical procedures were
performed under aseptic conditions.
Ra ts. Adult Sprague-Dawley rats were anesthetized with
a ketamine-based anaesthetic (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, 20-50 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.
Dogs. Under aseptic conditions three male beagle dogs (8-
10 kg) underwent chronic cannulation. Catheters were intro-
duced into the thoracic aorta via the carotid artery and into
the superior vena cava via the right jugular veins. Catheters
were exteriorized between the scapulae, capped, and secured
under a mesh jacket. Animals were allowed to recover for 7
days prior to receiving 15q either intravenously, 1 mL/kg via
the right saphenous vein, or by oral gavage (2 mL/kg followed
by an aqueous flush). Animals receiving drug by gavage were
fasted overnight prior to drug administration. Seven days
later animals receiving 15q intravenously were dosed with 15q
by oral gavage and vice versa. In all cases serial blood samples
were collected via the jugular cannula catheter over the next
24 h following dosing. Samples were stored frozen until
assayed using an HPLC based assay.
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.7,8
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 2 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 4 °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
incubated at 37 °C in the presence of 6% CO2. The media was
aspirated, and the cells were washed twice with PBS. Cells
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
1H NMR, IR, and
Su p p or tin g In for m a tion Ava ila ble:
high-resolution mass spectral data for compounds 1, 9, 11-
13, 15a -r , 18-22, 28 and 29 (6 pages). Ordering information
is given on any current masthead page.
Refer en ces
(1) Presented in part: Wu, C.; Raju, B.; Okun, I.; Stavros, F.; Chan,
M. F. 2-Benzylcarbonylthiophene-3-sulfonamides Are Orally
Active ETA Selective Endothelin Antagonists. The 211th ACS
National Meeting, New Orleans, LA, March 24-28, 1996,
Abstract MEDI 148.
(2) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815-
1818.
(3) Rachele, J . R. J . Org. Chem. 1963, 28, 2898.
(4) Friedman, L.; Shechter, H. J . Org. Chem. 1961, 26, 2522-2524.
(5) Raju, B.; Okun, I.; Stavros, F.; Chan, M. F. Amide Bond
Surrogates:
a Study in Thiophenesulfonamides Endothelin
Antagonists. The 211th ACS National Meeting, New Orleans,
LA, March 24-28, 1996, Abstract MEDI 147.
(6) Chen, S. J .; Brock, T.; Stavros, F.; Okun, I.; Wu, C.; Chan, F.;
Mong, S.; Dixon, R. A. F.; Oparil, S.; Chen, Y. F., Experimental
Biology ‘96, Washington, DC, April 14-17, 1996.
(7) 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).
(8) Berridge, J . M.; Downes, C. P.; Hanley, M. R. Biochem. J . 1982,
206, 587-595.
J M9700068