Selective endothelin a receptor antagonists. 3. Discovery and structure- activity relationships of a series of 4-phenoxybutanoic acid derivatives

Article abstract of DOI:10.1021/jm9707131

The third in this series of papers describes our further progress into the discovery of a potent and selective endothelin A (ET(A)) receptor antagonist for the potential treatment of diseases in which a pathophysiological role for endothelin has been implicated. These include hypertension, ischemic diseases, and atherosclerosis. In earlier publications we have outlined the discovery and structure-activity relations of two moderately potent series of nonpeptide ET(A) receptor antagonists. In this paper, we describe how a pharmacophore model for ET(A) receptor binding was developed which enabled these two series of compounds to be merged into a single class of 4-phenoxybutanoic acid derivatives. The subsequent optimization of in vitro activity against the ET(A) receptor led to the discovery of (R)-4-[2-cyano-5-(3-pyridylmethoxy)phenoxy]-4-(2- methylphenyl)butanoic acid (12m). This compound exhibits low-nanomolar binding to the ET(A) receptor and a greater than 1000-fold selectivity over the ET(B) receptor. Data are presented to demonstrate that 12m is orally bioavailable in the rat and is a functional antagonist in vitro and in vivo of ET-1-induced vasoconstriction.

Full text of DOI:10.1021/jm9707131

2732
J . Med. Chem. 1998, 41, 2732-2744
Selective En d oth elin A Recep tor An ta gon ists. 3. Discover y a n d
Str u ctu r e-Activity Rela tion sh ip s of a Ser ies of 4-P h en oxybu ta n oic Acid
Der iva tives
Peter C. Astles,* Clive Brealey, Thomas J . Brown, Vincenzo Facchini, Caroline Handscombe, Neil V. Harris,
Clive McCarthy, Iain M. McLay, Barry Porter, Alan G. Roach, Carol Sargent, Christopher Smith, and
Roger J . A. Walsh
Rhoˆne-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex RM10 7XS, U.K.
Received October 20, 1997
The third in this series of papers describes our further progress into the discovery of a potent
and selective endothelin A (ETA) receptor antagonist for the potential treatment of diseases in
which a pathophysiological role for endothelin has been implicated. These include hypertension,
ischemic diseases, and atherosclerosis. In earlier publications we have outlined the discovery
and structure-activity relations of two moderately potent series of nonpeptide ETA receptor
antagonists. In this paper, we describe how a pharmacophore model for ETA receptor binding
was developed which enabled these two series of compounds to be merged into a single class
of 4-phenoxybutanoic acid derivatives. The subsequent optimization of in vitro activity against
the ETA receptor led to the discovery of (R)-4-[2-cyano-5-(3-pyridylmethoxy)phenoxy]-4-(2-
methylphenyl)butanoic acid (12m ). This compound exhibits low-nanomolar binding to the ETA
receptor and a greater than 1000-fold selectivity over the ETB receptor. Data are presented to
demonstrate that 12m is orally bioavailable in the rat and is a functional antagonist in vitro
and in vivo of ET-1-induced vasoconstriction.
In tr od u ction
characterizing ET receptors particularly in isolated
tissues, but their usefulness has been limited where in
vivo studies are concerned. However it is the recent
disclosure of several nonpeptidic orally active antago-
nists6 that has allowed a more extensive exploration of
the role of endothelin in the pathology of animal disease
models and which has led to the commencement of
clinical studies in humans. Some of these nonpeptide
antagonists (Chart 1) include Ro 47-0203 (Bosentan)
(2),11 SB 209670 (3),12 PD 156707 (4),13 and A-127722
(5).14 Other compounds from Bristol Myers Squibb,
Zeneca, Roussel, Merck, Knoll, and Texas Biotechnology
as well as from our own laboratories at Rhone-Poulenc
Rorer15,16 have been disclosed recently.
Endothelin-1 (ET-1) is a 21-amino acid peptide which
possesses potent vasoconstrictor properties.1 Adverse
stimuli (such as ischemia/ hypoxia) can stimulate ET-1
production from both human and animal endothelial
cells as well as other cell types such as smooth muscle
cells, fibroblasts, macrophages, glial cells, mesangial
cells, and various cultured cancer cells.2 A large body
of evidence has accumulated in recent years implicating
a pathophysiological role for ET-1 in numerous clinical
conditions.2 ET-1 belongs to a family of three isopep-
tides (ET-1, ET-2, ET-3) found in humans which exert
their activities via two specific seven-transmembrane,
G-protein-coupled receptors. These receptors have been
cloned and designated ETA and ETB receptors.2 The
ETA receptor is located principally on vascular smooth
muscle and mediates vasoconstriction and smooth muscle
cell proliferation.3 Stimulation of ETB receptors located
on the endothelium evokes the release of NO and PGI2
to cause local vasodilation and prevention of platelet
aggregation.4 However, ETB receptors have also been
identified on vascular smooth muscle and can produce
contraction upon activation.5
Throughout the 1990s the search by the pharmaceu-
tical industry for competitive antagonists of the ET
receptors has been very successful, and consequently
there are presently a large number of reports describing
structurally diverse receptor antagonists with varying
potencies and subtype selectivity.6 Many of the first
compounds that were identified were peptides and
include the selective ETA receptor antagonists BQ 123
(1)7 (Chart 1) and FR 139317,8 the selective ETB
antagonist BQ 788,9 and the mixed ETA/ETB antagonist
PD 142893.10 These peptides have been invaluable in
There is debate about the potential merits of a
selective ETA receptor antagonist compared with those
of a mixed ETA/ETB antagonist.17 Potential advantages
for a selective antagonist of the ETA receptor are (a)
Vasoconstriction in the human cardiovascular system
by ET-1 appears to be predominantly mediated by the
ETA receptor.17,18 The involvement of the ETB receptor
in the contraction of human tissue is the subject of
current research and is only now being elucidated.19 (b)
The mitogenic and proinflammatory effects induced by
ET-1 are mediated by ETA receptor stimulation.3 (c)
Blockade of the ETB receptor would prevent the putative
beneficial effects of ET-1-induced NO and PGI2 release
from the endothelium.4,20 (d) The ETB receptor, pre-
dominantly in the lung, appears to act as a clearance
receptor for ET-1; for example, the ETB-selective an-
tagonist BQ 788 has been shown to reduce the rate of
clearance of exogenous ET-1 from the circulation of the
rat.21 In addition, the mixed receptor antagonist Ro 46-
2005 induces a raised plasma level of ET-1, and this
S0022-2623(97)00713-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/20/1998

Selective Endothelin A Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2733
Ch a r t 1. Endothelin Antagonists
Ch a r t 2. Common Pharmacophoric Features
two series of compounds and combined them into one
series of potent, selective, bioavailable ETA receptor
antagonists.
Ch em istr y
The synthesis of the majority of the compounds
described in this paper is depicted in Scheme 1 and
begins from readily available resorcinol derivatives.
Monoalkylation of the appropriate commercially avail-
able resorcinols 9 with 3-(chloromethyl)thiophene pro-
ceeded under standard conditions and was facilitated
by the presence of a phase-transfer catalyst. Resorcinol
itself (9a , X ) H) yielded the expected mixture of
starting, and mono- and dialkylated material from
which the desired product 10a (X ) H) could be isolated
by chromatography. Where X is electron-withdrawing
(COMe, CO2Me), the alkylation of 9b,c proceeded quite
chemoselectively to yield almost entirely the desired
monoalkylated products 10b,c, respectively. The bu-
tanoic acid side chain was then introduced by a second
alkylation with the 4-chlorobutanoic ester 11 followed
by hydrolysis using methanolic KOH to yield the 4-phe-
noxybutanoic acid derivatives 12a -e (Table 1). The
chloro ester 11 (and analogues) was most efficiently
prepared by the titanium-mediated “homoenolate” reac-
tion25 of silyl ether 13 with an aryl aldehyde (Scheme
1). The synthesis of the non commercially available
4-(2-pyridyl)resorcinol (9e) is outlined in Scheme 2 and
relies on the palladium(II)-mediated coupling of 2-bro-
mopyridine with the organomagnesium compound de-
rived from bromide 14. The resultant biaryl 15 was bis-
demethylated to furnish 9e which was carried through
to target compound 12e as described above.
effect would be expected to compromise effective ETA
blockade.22 It is likely however that the “ideal” subtype
selectivity will depend on the tissue/disease being
targeted and will only become clear following the release
of data from clinical trials in humans. At the outset of
our research however, we sought to identify a novel
series of nonpeptidic ETA-selective ligands. The preced-
ing papers in this series15,16 outlined our lead generation
studies23 based on the known ETA ligands BQ 123 (1)7
and myriceron caffeoyl ester (6)24 and explicated the
discovery and structure-activity relationships of two
moderately potent series of ETA-selective antagonists
exemplified by the pyrazole acid 7 (1000 nM ETA) and
the thienyl derivative 8 (90 nM ETA) (Chart 2). The
work presented in this paper will describe how we
identified common pharmacophoric elements in these
The ester group in 10c (X ) CO2Me) could be further
elaborated to amides and to heterocyclic functionalities
(e.g., oxazole and imidazole) as shown in Scheme 3. Thus
treatment of 10c (X ) CO2Me) with ethanolic ammonia
under high pressure yielded the amide 10d (X )
CONH2) which was taken on to target compound 12d

2734 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
Sch em e 1. General Synthesis of 4-Phenoxybutanoic Acid Derivativesa
e
e
e
a
(a) ArCH2Cl, K2CO3, KI, Bu4NBr, MEK; (b) K2CO3, KI, MEK 11; (c) 10% KOH, MeOH; (d) TiCl4, CH2Cl2, [(1-ethoxycyclopropyl)ox-
y]trimethylsilane (13).
Sch em e 2. Synthesis of 4-(2-Pyridyl)resorcinol (9e)a
plified in Scheme 5 for the asymmetric synthesis of the
cyano derivative 12k (Table 1). The keto ester 22 was
prepared by standard transformations, a tert-butyl ester
being employed to suppress lactonization in the subse-
quent reduction step. Asymmetric reduction of 22 to
the chiral hydroxy ester 23 was attempted using a
variety of chiral reagents, both stoichiometric and
catalytic.26 The most successful in terms of yield and
enantiomeric excess of 23 was Brown’s B-chlorodiiso-
pinocampheylborane (DIP-chloride).27 Thus treatment
of ketone 22 with (+)-DIP-chloride in ether at -20 °C
followed by workup with diethanolamine gave the (4R)-
hydroxy ester 23 in >98% ee. The enantiomeric excess
was determined in the following manner using Mosher’s
ester. Esterification of 23 with (+)-Mosher’s acid chlo-
ride/pyridine and 1H NMR analysis of the resultant
crude ester 24 showed the methine CHO proton to
appear as a doublet of doublets at δ 6.06. The presence
of the other possible diastereoisomer derived from the
(S)-enantiomer of 23 was not detected. To check this,
the keto ester 22 was reduced with borane:THF and the
resultant racemic alcohol esterified with (+)-Mosher’s
acid chloride/pyridine. The 1H NMR spectrum of the
diastereomeric mixture clearly showed two distinct
double doublets for the methine CHO protons at δ 6.06
and 6.12.
a
(a) Mg, THF, reflux; (b) (Ph3P)PdCl2, 2-bromopyridine, THF,
reflux; (c) Pyr‚HCl, 160 °C.
as described above. In a similar fashion, the ester 10c
(X ) CO2Me) was refluxed with ethanolamine to give
the amide 10f (X ) CONHCH2CH2OH). This compound
was alkylated on the phenolic oxygen with chloro ester
11 and treated with the Burgess dehydrating agent to
afford the oxazoline 16. Oxidation of the oxazoline ring
was effected using nickel oxide with subsequent ester
hydrolysis producing the oxazole 12f. The amide in-
termediate 17 (X ) CONH2) was transformed to the
corresponding thioamide 18 using Lawesson’s reagent
(Scheme 3). Conversion of this to the S-methyl deriva-
tive was effected with methyl iodide, and subsequent
condensation with aminoacetaldehyde diethyl acetal
followed by acid hydrolysis yielded the imidazole 19. The
imidazole acid 12g was then obtained after saponifica-
tion.
Scheme 4 shows how the acetyl-substituted ester 20
was converted into the pyrazoles 12h ,i. The acetyl
group was deprotonated with sodium hydride and
acylated with either diethyl oxalate or ethyl formate.
Treatment of the resultant 1,3-dicarbonyl compounds
with hydrazine afforded the pyrazoles 21h (R ) CO2-
Et) and 21i (R ) H); ester hydrolysis then yielded the
pyrazole diacid 12h and the pyrazole 12i.
Exposure of chiral alcohol 23 to base unfortunately
resulted in immediate lactonization. This problem was
circumvented by saponification of 23 at this stage and
isolation of the sodium salt 25 (Scheme 5). We were
able to further deprotonate 25 with sodium hydride, and
the resultant dianion was found to react chemoselec-
tively with the appropriately substituted 2-fluorophenol
26 affording the (4R)-phenoxybutanoic acid 12k . The
enantiomeric excess of 12k was found to be >99% ee
by chiral HPLC analysis (see the Experimental Section)
following a single recrystallization. The (S)-enantio-
meric compounds (e.g., 12l) were obtained by utilizing
(-)-DIP-chloride in Scheme 5. The (3-thienyl)methyl
group in fluoride 26 was introduced by alkylating the
commercially available cyanophenol 27 with 3-(chlo-
romethyl)thiophene (Scheme 5). Alkylation of 27 with
Where the target compounds contained nitrile groups
at the X position which were strongly electron-with-
drawing, we were able to take advantage of the nucleo-
philic aromatic substitution reaction of an ortho fluoride
ion with an alkoxide group. This was particularly useful
for the synthesis of enantiomerically pure compounds
utilizing chiral alkoxide anions. This process is exem-

Selective Endothelin A Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2735
Sch em e 3. Synthesis of Oxazole and Imidazole Analogues 12f,ga
a
(a) NH3, EtOH, 100 °C or ethanolamine, reflux; (b) NaH, THF, 11; (c) (methoxycarbonylsulfamoyl)triethylammonium hydroxide (Burgess
reagent), THF, reflux; (d) NiO2, toluene, 100 °C; (e) 10% KOH, MeOH; (f) Lawesson’s reagent, THF; (g) NaH, MeI, THF; (h) (i)
aminoacetaldehyde diethyl acetal, AcOH, THF, reflux, (ii) 5.0 M aq HCl.
Sch em e 4. Synthesis of Pyrazole Analogues 12h ,ia
a
(a) NaH, diethyl oxalate (for 21h ) or ethyl formate (for 21i), toluene, reflux; (b) hydrazine hydrate, EtOH, reflux; (c) 10% KOH,
MeOH.
the appropriate chloromethyl-substituted heteroaromat-
ics allowed this position to be further varied giving rise
to compounds 12m -p (Table 2). These chloromethyl
compounds were either commercially available or pre-
pared using published procedures.28
1,3-substituted phenyl ring with (iii) a pendant aryl-
oxymethoxy, and (iv) a phenyl ring attached through a
two-atom spacer. No direct superimposition was pos-
sible; invariably three of the features fitted reasonably
well but not the fourth. At this stage the concept of
group interaction sites was introduced. It is not neces-
sary to have all features of molecules superimposed for
them to interact with the same binding groups of a
receptor site. A basic receptor center, say a lysine
group, can accept an interaction with an acid group from
many directions. In such a case it would be the
interaction center, and not the groups themselves, which
would be considered to superimpose. To enable testing
of this concept a dummy atom, representing an interac-
tion site, was bonded to an oxygen of each carboxylic
acid, in the direction of the lone pair and at 2.8 Å. The
two molecules were then subjected to conformational
analysis. Prior to the analysis the central phenyl rings
Resu lts a n d Discu ssion
Id en t ifica t ion of t h e P h en oxyb u t a n oic Acid
Ser ies. The previous papers in this series described
the discovery and structure-activity relationships of
two series of selective ETA receptor antagonists exem-
plified by the pyrazole 715 and the ketone 816 (Chart 2).
It was hypothesized that these two series bound to the
same local site in the receptor since they had been
generated from the same 3D database search.23 The
relationship between the two series was not obvious;
however, several features of the molecules seemed to
be common: (i) a carboxylic acid group, (ii) a central

2736 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
Sch em e 5. Asymmetric Synthesis of 12k via Fluoride Displacementa
a
(a) Succinic anhydride; (b) isobutylene, CH2Cl2, concentrated H2SO4, (t-Bu)OH; (c) (+)-DIP-chloride, ether, -20 °C; (d) (+)-Mosher’s
acid chloride, pyridine; (e) 5.0 M NaOH; (f) 26, NaH, THF; (g) 3-(chloromethyl)thiophene, K2CO3, KI, Bu4NBr, MEK.
were superimposed and the pendant arylmethoxy group
was locked into a low-energy conformation; the primary
aim of the work was then to compare the positions of
the remaining aryl rings and the acid interaction points.
The analysis was carried out in a 3D box, and distances
of the key groups to the corners of the box were used to
define the conformational space. A comparison of the
molecules was then made looking at the positions of the
aromatic rings and acid interaction points within this
3D box and at the energy cutoff employed; only one
region of conformational space was occupied. Repre-
sentative conformations from this region are shown in
Figure 1; the cationic receptor interaction center is
clearly revealed. From this superimposition the design
of new compounds was a matter of combining features
from both series in a single molecule. The first such
molecule designed was the pyrazole diacid 12h which
incorporated both acid functions enabling a bidentate
interaction with the putative receptor cationic center
(Figure 2). The low-nanomolar activity of this molecule
prompted us to further explore this series of 4-phenoxy-
butanoic acids (see below).
Str u ctu r e-Activity Rela tion sh ip s for th e 4-P h e-
n oxybu ta n oic Acid Ser ies. All test compounds were
assayed in vitro for their ability to antagonize the
binding of [125I]ET-1 to rat aortic A10 cell ETA receptors
and rat cerebellum ETB receptors. Functional activity
F igu r e 1. Superimposition of 7 (blue) and 8 (yellow) in the
unified model.
of selected molecules was measured as the inhibition
of ET-1-induced contraction of isolated rat aortic rings
denuded of the endothelium (an ETA-mediated pro-
cess).29 In all cases, the compounds showed no agonist
activity but antagonized the ET-1-induced contractions
in a concentration-dependent manner; the concentra-
tion-response curves of ET-1 were shifted to the right
in a parallel fashion by increasing concentrations of
compound with no significant reduction in the maximal
response. In vitro data is shown in Tables 1 and 2, the
ETA-selective cyclic peptide BQ 123 (1) and the lead
compounds 7 and 8 are included for purposes of com-
parison.
The pyrazole diacid 12h , designed from the cation
binding model described above, was found to have IC50’s
of 7.0 and 3000 nM in the ETA and ETB receptor binding
assays, respectively. This was an encouraging result
which supported our binding model and represented a
>10-fold improvement in potency at the ETA receptor.
12h was a functional endothelin-1 antagonist having a
pKB of 7.3 on rat aorta, a figure identical to that of the
standard antagonist BQ 123 (1) which was the starting
point for our studies. Unfortunately, 12h possessed
negligible oral bioavailability in the rat following a 30

Selective Endothelin A Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2737
ring was thought to be a possible position to modulate
the pharmacokinetics of this series, and so a number of
additional heterocyclic analogues of 12k were prepared
(Table 2). These compounds (12m -o) were all es-
sentially equiactive and were potent functional antago-
nists on rat aorta (pKB ∼7.0), the exception being the
piperonyl derivative 12p which dropped 10-fold in
binding potency. We introduced this ring as it was
observed to be a key pharmacophore in many other
published endothelin antagonists (Chart 1). The 3-pyr-
idyl derivative 12m (IC50 5.0 nM) was selected for
further in vivo study on account of its superior phar-
macokinetics (see below).
P h a r m a cok in etics of 12m . The pharmacokinetics
of 12m in the rat following iv and oral administration
is illustrated in Figure 3. Single-dose (3 mg/kg) intra-
venous pharmacokinetics of 12m in rats gave area
under the curve (from time 0 to infinity, 19.8% extrapo-
lated) of 5.39 µg h/mL, a terminal phase half-life of 3.6
h, a volume of distribution at steady state of 1.2 L/kg,
and a total plasma clearance of 0.6 L/h/kg. After the
single oral dose (30 mg/kg), a mean peak plasma
concentration of 17.2 ( standard deviation 6.6 µg/mL
was achieved at 1 h, and area under the curve (from
time 0 to infinity, 5.4% extrapolated) was 44.1 µg h/mL.
The terminal phase half-life was 3.3 h. Absolute oral
bioavailability was calculated to be 82%.
F igu r e 2. Pyrazole diacid 12h (green) shown fitting the
unified model.
Effect of 12m on Va socon str iction in th e P ith ed
Ra t. The ability of 12m to block the pressor response
to ET-1 in the pithed rat30 was examined along with
the selective ETA antagonist BQ 123 (1) for purposes of
comparison. Animals were pretreated with the selective
ETB antagonist BQ 788 to ensure pressor effects were
mediated only by the ETA receptor and to abolish the
initial ETB-mediated depressor response to ET-1 ob-
served in this model. The diastolic blood pressure of
the pithed rat increases in a dose-dependent fashion
following bolus injections of ET-1 (see the Experimental
Section). The log dose-response curves to ET-1 are
shifted to the right from vehicle control in a parallel,
dose-related manner following increasing iv doses of
12m and 1 (see Figure 4 and data in Table 3) with no
significant reduction in the maximal reponse.
mg/kg dose (data not shown); the poor absorption being
ascribed to the presence of two anionic acid function-
alities in the molecule.
Synthesis of compound 12i in which the acid is
removed from the pyrazole ring demonstrated that only
one acidic functionality is necessary for low-nanomolar
activity, the IC50 (ETA) being 20 nM. This compound
was still selective, being some 500-fold less active at the
ETB receptor. Removing the pyrazole group altogether
(compound 12a ) reduced potency 40-fold. The effect of
introducing other heterocyclic rings at this position was
explored; the oxazole 12f and the 2-pyridyl derivative
12e were equiactive with 12i. An imidazole ring was
less tolerated, this analogue 12g being 10-fold less
potent than 12i at the ETA receptor. We postulated that
the pyrazole ring was interacting with the receptor as
an H-bond acceptor and proceeded to introduce a series
of groups at this position that would have this property.
Thus the acetyl and amido analogues (12b,d , respec-
tively) were bioisosteric with the pyrazole ring having
IC50’s of ∼30 nM; a carboxylic acid (12c) was however
less tolerated. The most effective H-bond acceptor group
introduced was a nitrile as in 12j which possessed an
IC50 of 7.0 nM (ETA) and was still 500-fold selective.
Preparation of the individual enantiomers of this com-
pound revealed that the (R)-isomer 12k was the euto-
meric component (IC50 5.0 nM). The (S)- isomer 12l was
however only 10-fold less active perhaps reflecting the
flexibility of the propanoic side chain. 12k was exam-
ined in the functional assay and had a pKB of 7.4 on rat
aorta. Most importantly, this compound demonstrated
good oral bioavailability (60%) in the rat (data not
shown).
These experiments in the pithed rat model clearly
demonstrate the ability of 12m to antagonize the
pressor response to ET-1 in vivo in a dose-dependent
manner. 12m appears some 5-fold less potent than BQ
123 (1) in this model, however, despite the two com-
pounds being equipotent in our in vitro binding and
functional assays (see Table 1). This disparity in the
pithed rat model has also been observed for the Shionogi
ETA antagonist 97-13931 and was attributed to plasma
protein binding of the compound. The effect of protein
binding on the in vitro and in vivo activities of this series
of compounds will be the subject of a future publication
from these laboratories.
Con clu sion
The use of molecular modeling to superimpose two
previously discovered series of endothelin antagonists
led to the discovery of a novel class of phenoxybutanoic
acids containing a key H-bond acceptor group. Varia-
tion of this substituent culminated in the synthesis of
The 3-thienyl and 2-methylphenyl substituents present
in this series of antagonists had been identified as
optimum from our earlier studies.16 The heteroaromatic

2738 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
Ta ble 1. Variation of H-Bond Acceptor Group X: Physical Properties and in Vitro Activity
a
b
Satisfactory microanalyses were obtained ((0.4%) for C, H, N, S unless otherwise stated. Inhibition of [125I]ET-1 binding in vitro to
rat A10 cell ETA receptors (mean of n experiments ( SEM; both values if performed in duplicate). c Inhibition of [125I]ET-1 binding in
d
vitro to rat cerebellum ETB receptors (single experiment). Antagonism of ET-1-mediated contraction of isolated de-endothelialized rat
aortic rings (mean of n experiments ( SEM). e C: calcd, 67.9; found, 68.4. f C: calcd, 66.9; found, 65.3.
the cyano derivative 12m (RPR 111844) which exhibited
an IC50 of 5.0 nM at the rat ETA receptor and 1000-fold
selectivity over the ETB receptor. This level of potency
and selectivity was maintained when the compound was
screened against human ETA and ETB receptors ex-
pressed on CHO cells (data not shown). 12m also
demonstrated in vivo functional activity in a rat model
of ET-1-induced vasoconstriction. The promising phar-
macokinetics of 12m (>80% in rat, terminal t1/2 ) 3.3
h) has prompted us to examine the effects of this
compound in preclinical models of cardiovascular dis-
ease.
Exp er im en ta l Section
Ch em ica l Meth od s. Reagents, starting materials, and
solvents were purchased from common commercial suppliers
and used as received or purified by standard methods. All
organic solutions were dried over magnesium sulfate and
concentrated at reduced pressure under aspirator vacuum
using a Buchi rotary evaporator. Reaction products were
purified, when necessary, by flash chromatography on silica
gel (40-63 µm) eluting with the solvent system indicated.
Yields are not optimized. Melting points were determined on
a Gallenkamp 595 apparatus and are uncorrected. 1H NMR
spectra were acquired on a Varian VXR-400 spectrometer;
peak positions are reported in parts per million relative to

Selective Endothelin A Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2739
Ta ble 2. Variation of Heterocycle: Physical Properties and in Vitro Activity
a-d
See footnotes in Table 1.
F igu r e 3. Pharmacokinetics of 12m in the rat.
internal tetramethylsilane on the δ scale. Elemental analyses
were performed by the Physical Chemistry Department at
Rhone-Poulenc Rorer. The structure and purity of all com-
pounds were confirmed by microanalytical and/or spectroscopic
methods. Satisfactory microanalyses (( 0.4%) were obtained
for C, H, N unless otherwise stated.
(R/S)-4-[3-(3-Th ien ylm et h oxy)p h en oxy]-4-(2-m et h yl-
p h en yl)bu ta n oic Acid (12a ). A suspension of 10a (X ) H)
(3 g, 15 mmol), 4-chloro-4-(2-methylphenyl)butyric acid ethyl
ester (11) (4.53 g, 20 mmol), potassium carbonate (4.14 g, 30
mmol), and potassiun iodide (0.5 g) in methyl ethyl ketone (100
mL) was stirred and heated to reflux for 48 h. After cooling
the reaction mixture was evaporated to dryness and parti-
tioned between ethyl acetate (200 mL) and water (200 mL).
The organic phase was washed with water (100 mL), dried,
and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel, eluting with ethyl acetate and
cyclohexane (5:95 v/v) to give (R/ S)-4-[(3-benzyloxy)phenoxy]-
4-phenylbutanoic acid ethyl ester (1.0 g, 17%) as a yellow oil.
This material (1.0 g, 2.94 mmol) was dissolved in methanol
(50 mL) and treated with 10% aqueous potassium hydroxide
solution (5 mL), and the reaction mixture was stirred and
heated to reflux for 30 min. After cooling, the reaction mixture
was partitioned between ethyl acetate (100 mL) and 1.0 M
hydrochloric acid (50 mL). The organic layer was washed with
water (50 mL), dried, and concentrated. The residue was
recrystallized from cyclohexane to give 12a (0.55 g, 57%) as a
beige solid, mp 100-4 °C. 1H NMR (CDCl3): 2.35 (m, 2H), 2.41
(s, 3H), 2.65-2.75 (m, 2H), 4.85 (br s, 2H), 5.46 (m, 1H), 6.80
(m, 2H), 7.1-7.25 (m, 5H), 7.34 (m, 1H), 7.38-7.49 (m, 2H),
7.52 (m, 1H). Anal. (C22H22O4S) C, H, N.
3-(3-Th ien ylm eth oxy)p h en ol (10a : X ) H). A suspen-

2740 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
was then added to the suspension, keeping the temperature
below 10 °C. After complete addition, the reaction mixture
was allowed to warm to room temperature and stirred for a
further 2 h. The reaction mixture was quenched with water
(100 mL) and extracted with ethyl acetate (5 × 100 mL). The
combined organics were then washed with brine (100 mL),
dried, and evaporated in vacuo to leave a brown oil, which was
purified by fractional distillation to give 11 (23.1 g, 51%) as a
pale-yellow oil, bp 115-20 °C/1 mbar. 1H NMR (CDCl3): 1.28
(t, J ) 8 Hz, 3H), 2.38 (m, 2H), 2.40 (s, 3H), 2.55 (m, 2H), 4.15
(q, J ) 8 Hz, 2H), 5.25 (dd, J ) 12, 4 Hz, 1H), 7.23-7.28 (m,
3H), 7.50 (m, 1H).
(R/S)-4-[2-Acet yl-5-(3-t h ien ylm et h oxy)p h en oxy]-4-(2-
m eth ylp h en yl)bu ta n oic Acid (12b). A mixture of 2,4-
dihydroxyacetophenone (9b: X ) COCH3) (5 g, 33 mmol),
3-thienyl chloride (5.3 g, 40 mmol), potassium carbonate (5.3
g, 40 mmol), potassium iodide (0.1 g, 0.6 mmol), and tetra-n-
butylammonium bromide (0.1 g) in methyl ethyl ketone (100
mL) was stirred and heated to reflux for 6 h. After cooling,
the reaction mixture was partitioned between ethyl acetate
(200 mL) and 1.0 M aqueous hydrochloric acid (200 mL). The
organics were washed with water (100 mL), dried, and
concentrated to dryness. The residue was recrystallized (with
charcoal) from cyclohexane to afford 10b (X ) COCH3) as a
colorless solid (2 g, 24%), mp 99-102 °C. 1H NMR (CDCl3):
2.58 (s, 3H), 5.10 (s, 2H), 6.51 (m, 2H), 7.14 (dd, J ) 4, 2 Hz,
1H), 7.35 (m, 2H), 7.65 (m, 1H).
A portion of this material (1.0 g, 4 mmol), 4-chloro-4-
phenylbutyric acid ethyl ester (11) (1.13 g, 5 mmol), potassium
carbonate (1.03 g, 7.5 mmol), and potassium iodide (0.1 g) in
methyl ethyl ketone (100 mL) was stirred and heated to reflux
for 48 h. After cooling, the reaction mixture was partitioned
between ethyl acetate (100 mL) and water (100 mL); the
organics were washed with water (50 mL), dried, and concen-
trated to dryness. The residue was purified by flash chroma-
tography on silica gel, eluting with a mixture of ethyl acetate
and cyclohexane (1:9 v/v) to give (R/ S)-4-[2-acetyl-5-(3-thien-
ylmethoxy)phenoxy]-4-(2-methylphenyl)butanoic acid ethyl es-
ter (20) (1.0 g, 55%) as a colorless oil. 1H NMR (CDCl3): 1.27
(t, J ) 8 Hz, 2H), 2.25 (m, 2H), 2.47 (s, 3H), 2.50-2.60 (m,
2H), 2.73 (s, 3H), 4.10 (q, J ) 8 Hz, 2H), 4.90 (m, 2H), 5.49
(m, 1H), 6.13 (m, 1H), 6.50 (m, 1H), 7.01 (m, 1H), 7.15 (m,
1H), 7.19 (m, 3H), 7.30 (m, 2H), 7.79 (m, 1H).
This material (1.0 g, 2.21 mmol) was dissolved in methanol
(50 mL), treated with 10% aqueous potassium hydroxide
solution (2 mL), and heated to reflux for 1 h. After cooling,
the reaction mixture was evaporated to dryness, and the
residue was partitioned between ethyl acetate (50 mL) and
1.0 M hydrochloric acid (50 mL). The organic layer was
washed with water (50 mL), dried, and concentrated in vacuo
to leave a residue, which was recrystallized from ethyl acetate
and cyclohexane to give 12b (0.4 g, 43%) in a form of a white
solid, mp 116-21 °C. 1H NMR (CDCl3): 2.36 (m, 2H), 2.41 (s,
3H), 2.65-2.75 (m, 2H), 2.72 (s, 3H), 4.90 (m, 2H), 5.51 (m,
1H), 6.14 (m, 1H), 6.50 (m, 1H), 7.03 (m, 1H), 7.15-7.20 (m,
4H), 7.30 (m, 1H), 7.38 (m, 1H), 7.81 (m, 1H). Anal.
(C24H24O5S) C, H, N.
2-(Am in oca r bon yl)-5-(3-th ien ylm eth oxy)p h en ol (10d :
X ) CONH2). A solution of 2-(methoxycarbonyl)-5-(3-thien-
ylmethoxy)phenol (10c: X ) CO2Me) (15 g, 57 mmol) in
ethanolic ammonia solution (150 mL) was placed in a bomb
and heated on a steam bath for 24 h. After cooling, the solvent
was evaporated, and the residue was taken up in dichlo-
romethane (100 mL), filtered, and concentrated to dryness.
Purification of the residue by flash chromatography on silica
gel, eluting with a mixture of ethyl acetate and dichlo-
romethane (1:4 v/v), gave 10d (X ) CONH2) (3.75 g, 26%) as
a white solid, mp 174-7 °C. 1H NMR (DMSO-d6): 5.13 (s,
2H), 6.50 (m, 2H), 7.19 (m, 1H), 7.58 (m, 2H), 7.77 (br m, 1H),
7.79 (m, 1H), 8.21 (br s, 1H).
F igu r e 4. Effects of 12m on ET-1-induced changes in diastolic
blood pressure in the pithed rat (pretreated with BQ 788 (3
mg/kg)).
Ta ble 3. Effects of 12m and BQ 123 (1) on ET-1-Induced
Changes in Diastolic Blood Pressure in the Pithed Rat
Pretreated with BQ 788 (3 mg/Kg)
compd
iv dose (µm/kg)
no. of rats (n)
shifta
12m
12m
12m
12m
1
2.5
7.5
25
75
2.5
25
6
5
6
6
6
4
nsb
2.27c
10.48c
36.58c
15.06c
57.79c
1
a
Degree of rightfold shift of log dose of ET-1-response curve
(mean of n experiments). b Not significant. c Significantly different
from vehicle-treated animals.
sion of resorcinol (9a : X ) H) (18.3 g, 166 mmol), benzyl
chloride (21 g, 166 mmol), potassium carbonate (23.3 g, 169
mmol), potassium iodide (1.0 g), and tetra-n-butylammonium
bromide (1.0 g) in methyl ethyl ketone (500 mL) was stirred
and heated to reflux for 5 h. After completion, the reaction
mixture was filtered and evaporated to dryness. Water (500
mL) was added to the residue, and the reaction mixture was
extracted with diethyl ether (3 × 300 mL). The organic phase
was washed with water (250 mL), and the product was
extracted with 10% aqueous sodium hydroxide (3 × 50 mL).
Solid carbon dioxide was added until the pH of the solution
was 5.0, and a brown oil was liberated. This was extracted
with diethyl ether (3 × 100 mL), dried, and concentrated in
vacuo to leave an oil. Purification by flash chromatography
on silica gel, eluting with a mixture of ethyl acetate and
cyclohexane (1:9 v/v), gave 10a (X ) H) as a colorless solid (9
g, 27%). 1H NMR (CDCl3): 5.00 (s, 2H), 6.80 (m, 2H), 7.34 (m,
1H), 7.38-7.49 (m, 3H), 7.52 (m, 1H).
4-Ch lor o-4-(2-m eth ylp h en yl)bu tyr ic Acid Eth yl Ester
(11). A solution of o-tolualdehyde (22.8 g, 190 mmol) in dry
dichloromethane (50 mL) was cooled to 0 °C and titanium(IV)
tetrachloride (190 mL of a 1.0 M solution in dichloromethane,
190 mmol) was added dropwise giving a yellow suspension
which was stirred at this temperature for 15 min. [(1-
Ethoxycyclopropyl)oxy]trimethylsilane (13) (30 g, 170 mmol)
2-(2,4-Dim eth oxyp h en yl)p yr id in e (15). To a mechani-
cally stirred suspension of magnesium turnings (8.11 g, 333
mmol) in dry tetrahydrofuran (300 mL), containing a few
crystals of iodine under gentle reflux, was added dropwise a

Selective Endothelin A Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2741
solution of 1-bromo-2,4-dimethoxybenzene (48.02 mL, 333
mmol) in dry tetrahydrofuran (100 mL). The reaction mixture
was stirred and heated to reflux for a further 30 min. After
cooling, the above reagent was added dropwise to a refluxing
solution of 2-bromopyridine (50.3 g, 333 mmol) and bis-
(triphenylphosphine)palladium(II) chloride (3.51 g, 5 mmol)
in dry tetrahydrofuran (100 mL). The reaction mixture was
refluxed for a further 6 h, then filtered, and concentrated to
dryness in vacuo; 150 mL of water was added, and the reaction
mixture was extracted with ethyl acetate (3 × 50 mL), dried,
and evaporated to dryness to give a black oil. This was
purified by flash chromatography on silica gel, eluting with a
mixture of diethyl ether and pentane (1:2 v/v) to give 15 (28
g, 39%) as a light-yellow solid, after trituration with pentane,
mp 58-60 °C. 1H NMR (CDCl3): 3.63 (s, 3H), 3.41 (s, 3H),
6.48 (dd, J ) 8, 2 Hz, 1H), 6.54 (d, J ) 2 Hz, 1H), 7.14 (m,
1H), 7.68 (d, J ) 8 Hz, 1H), 7.75 (m, 2H), 8.41 (m, 1H). Anal.
(C13H13NO2) C, H, N.
(s, 3H), 2.58-2.61 (m, 2H), 4.10 (q, J ) 8 Hz, 2H), 4.85 (m,
2H), 5.40 (m, 1H), 6.08 (m, 1H), 6.24 (m, 1H), 7.00 (m, 1H),
7.18 (m, 4H), 7.32 (m, 2H), 7.41 (m, 1H), 9.60 (br s, 1H).
To a portion of this material (0.2 g, 0.41 mmol) in dry
tetrahydrofuran (2 mL) and glacial acetic acid (0.05 mL, 0.82
mmol) was added aminoacetaldehyde diethyl acetal (0.055 mL,
0.41 mmol), and the mixture was heated on a steam bath for
2 h. After the mixture cooled, 5.0 M hydrochloric acid (0.166
mL, 0.82 mmol) was added, and the reaction mixture was
allowed to stir at room temperature for 2 h. The reaction
mixture was basified to pH 8.0 with solid sodium bicarbonate,
diluted with 10 mL water, extracted with ethyl acetate (3 ×
10 mL), dried, and concentrated to dryness. The residue was
purified by flash chromatography on silica gel, eluting with
ethyl acetate and pentane (1:1 v/v) to give 19 (0.07 g, 35%) in
a form of a white solid. 1H NMR (CDCl3): 1.18 (t, J ) 8 Hz,
3H), 2.20 (m, 1H), 2.42 (s, 3H), 2.50 (m, 3H), 4.10 (m, 2H),
4.93 (m, 2H), 5.58 (m, 1H), 6.30 (m, 1H), 6.60 (m, 1H), 7.03
(m, 1H), 7.16 (m, 1H), 7.20 (m, 4H), 7.22 (m, 2H), 7.30 (m,
1H), 8.21 (m, 1H). Anal. (C27H28N2O4S) C, H, N.
(R/S)-4-[2-(Th ioim idato-2)-5-(3-th ien ylm eth oxy)ph en yl]-
4-(2-m eth ylp h en yl)bu ta n oic Acid Eth yl Ester (18: X )
CSNH2). A solution of (R/ S)-4-[2-(aminocarbonyl)-5-(3-thien-
ylmethoxy)phenyl]-4-(2-methylphenyl)butanoic acid ethyl ester
(17) (X ) CONH2) (4.99 g, 11 mmol) in dry tetrahydrofuran
(100 mL) was cooled in an ice bath, and Lawesson’s reagent
(2.22 g, 5.5 mmol) was added dropwise. The yellow suspension
was allowed to warm to room temperature and was stirred
for 5 h to result in a clear solution. The reaction mixture was
concentrated to dryness and purified by flash chromatography,
eluting with a mixture of ethyl acetate and pentane (1:2 v/v).
Trituration of the oily residue with diethyl ether afforded 18
(X ) CSNH2) (2.42 g, 47%) as a light-green solid, mp 134-5
°C. 1H NMR (CDCl3): 1.20 (t, J ) 8 Hz, 3H), 2.18-2.30 (m,
2H), 2.42 (s, 3H), 2.53-2.58 (m, 2H), 4.12 (q, J ) 8 Hz, 2H),
4.95 (m, 2H), 5.58 (m, 1H), 6.21 (m, 1H), 6.58 (m, 1H), 7.01
(m, 1H), 7.20 (m, 4H), 7.25 (m, 1H), 7.30 (m, 1H), 7.94 (br s,
1H), 8.62 (m, 1H), 9.20 (br s, 1H).
(()-5-{2-[3-Ca r boxy-1-(2-m eth ylp h en yl)p r op oxy]-4-(3-
th ien ylm eth oxy)ph en yl}pyr azole-3-car boxylic Acid Hem i-
h yd r a te (12h ). A mixture of 20 (18.89 g, 44 mmol) and
diethyl oxalate (19.27 g, 132 mmol) in dry toluene (100 mL)
was added dropwise to a stirred solution of sodium hydride
(1.76 g of a 60% dispersion w/w in mineral oil, 44 mmol) in
dry toluene (400 mL) under a nitrogen atmosphere. The
reaction mixture was heated to reflux for 16 h to result in a
pale-red solution. After the mixture cooled, the toluene was
evaporated, and ethanol (500 mL) and glacial acetic acid (10.57
g, 176 mmol) followed by hydrazine hydrate (2.2 g, 44 mmol)
were added. The reaction mixture was heated to reflux for a
further 90 min and cooled, and the ethanol removed in vacuo.
The residue was partitioned between ethyl acetate (250 mL);
and water (250 mL), the organic layer was washed with brine
(200 mL), dried, and concentrated to dryness to give an oil.
This was purified by flash chromatography on silica gel,
eluting with pentane and methyl tert-butyl ether (2:1 v/v) to
yield diester 21h (7.2 g, 36%) as a pale-yellow glass. 1H NMR
(CDCl3): 1.21 (t, J ) 8 Hz, 3H), 1.42 (t, J ) 8 Hz, 3H), 2.24
(m, 1H), 2.28 (m, 1H), 2.42 (s, 3H), 2.58 (m, 2H), 4.10 (q, J )
8 Hz, 2H), 4.42 (q, J ) 8 Hz, 2H), 4.95 (m, 2H), 5.60 (m, 1H),
6.39 (m, 1H), 6.60 (m, 1H), 7.03 (m, 1H), 7.10 (s, 1H), 7.18 (m,
4H), 7.20 (m, 1H), 7.34 (m, 1H), 7.58 (m, 1H).
2-(2,4-Dih yd r oxyp h en yl)p yr id in e (9e). A mixture of 15
(14 g, 65 mmol) and pyridine hydrochloride (121 g, 1000 mmol)
was heated to 160 °C for 7 h. After cooling, the residue was
dissolved in dichloromethane (300 mL) and washed with 2.0
M hydrochloric acid (3 × 300 mL). The combined organics
were washed with water (150 mL), dried, and concentrated in
vacuo to furnish an oil. This was purified by flash chroma-
tography on silica gel, eluting with ethyl acetate and pentane
(1:1 v/v) to give 9e (8 g, 66%) as a white solid, mp 170-172
°C. 1H NMR (CDCl3): 6.48 (dd, J ) 8, 2 Hz, 1H), 6.54 (d, J )
2 Hz, 1H), 7.14 (m, 1H), 7.68 (d, J ) 8 Hz, 1H), 7.75 (m, 2H),
8.41 (m, 1H), 12.00 (br, 1H). Anal. (C11H9NO2) C, H, N.
(R/S)-4-[2-(Oxa zol-2-yl)-5-(3-th ien ylm eth oxy)p h en oxy]-
4-(2-m eth ylp h en yl)bu ta n oic Acid (12f). A solution of 10f
(X ) CONHCH2CH2OH) (2.5 g, 5 mmol) in dry tetrahydrofuran
(50 mL) was treated with Burgess reagent (1.3 g, 5.5 mmol),
and the mixture was heated to reflux for 30 min. After cooling,
the reaction mixture was partitioned between ethyl acetate
(100 mL) and water (100 mL); the organic layer was dried and
evaporated to dryness. The residue was purified by flash
chromatography on silica gel, eluting with ethyl acetate and
cyclohexane (1:1 v/v) to give (R/ S)-4-[2-(oxazolin-2-yl)-5-(3-
thienylmethoxy)phenoxy]-4-(2-methylphenyl)butanoic acid eth-
yl ester (16) (1.5 g, 63%) as a colorless oil. A stirred solution
of 16 (0.9 g, 1.88 mmol) in toluene (50 mL) was treated with
nickel peroxide (5.04 g, 56 mmol) and heated at 100 °C for 24
h. The reaction mixture was filtered through
a pad of
diatomaceous earth and concentrated to dryness. The residue
was subjected to purification by flash chromatography on silica
gel, eluting with a mixture of ethyl acetate and cyclohexane
(15:85 v/v) to give (R/ S)-4-[2-(oxazol-2-yl)-5-(3-thienylmethoxy)-
phenoxy]-4-(2-methylphenyl)butanoic acid ethyl ester as a
colorless oil (140 mg, 16%). This material was hydrolyzed as
for example 12a above to afford 12f (70 mg, 54%) as a colorless
solid, mp 112-114 °C, after recrystallization from ethyl
acetate/hexane. 1H NMR (DMSO-d6): 2.05 (m, 2H), 2.40 (s,
3H), 2.41 (m, 1H), 2.58 (m, 1H), 5.00 (m, 2H), 5.62 (m, 1H),
6.38 (s, 1H), 6.64 (m, 1H), 7.10-7.20 (m, 4H), 7.37 (m, 2H),
7.43 (m, 1H), 7.56 (m, 1H), 7.78 (m, 1H), 8.20 (1H). Anal.
(C25H23NO5S) C, H, N.
(R/S)-4-[2-(Im idazol-2-yl)-5-(3-th ien ylm eth oxy)ph en oxy]-
4-(2-m eth ylp h en yl)bu ta n oic Acid Eth yl Ester (19). To a
stirred solution of 18 (X ) CSNH2) (1.63 g, 3.5 mmol) in dry
tetrahydrofuran (25 mL) was added sodium hydride (0.14 g of
a 60% dispersion w/w in mineral oil, 3.5 mmol), followed by
methyl iodide (0.33 mL, 5.2 mmol). The suspension was
stirred at room temperature for 3.5 h to form a clear orange
solution, and the reaction mixture was concentrated to dry-
ness. The residue was dissolved in ethyl acetate (20 mL),
washed with water (2 × 10 mL), dried, and concentrated to
dryness. The residue was purified by flash chromatography
on silica gel, eluting with a mixture of ethyl acetate and
pentane (1:3 v/v) to yield (R/ S)-4-[2-(methylthioimidato-2)-5-
(3-thienylmethoxy)phenyl]-4-(2-methylphenyl)butanoic acid eth-
yl ester (0.91 g, 54%) as a yellow solid, mp 97-9 °C. 1H NMR
(CDCl3): 1.20 (t, J ) 8 Hz, 3H), 2.18 (m, 2H), 2.42 (s, 3H), 2.50
This material was hydrolyzed as for example 12a above to
give 12h (6.22 g, 90%) as a white solid, after recrystallization
from ethanol, mp 209-210 °C. 1H NMR (DMSO-d6): 2.10 (m,
2H), 2.40 (s, 3H), 2.50 (m, 2H), 4.98 (m, 2H), 5.60 (m, 1H),
6.30 (m, 1H), 6.62 (m, 1H), 7.05 (m, 1H), 7.10-7.25 (m, 5H),
7.41 (m, 1H), 7.52 (m, 1H), 7.70 (m, 1H). Anal. (C26H24N2O6S‚
0.5H2O) C, H, N.
(R )-4-[2-Cya n o-5-(3-t h ie n ylm e t h oxy)p h e n oxy]-4-(2-
m eth ylp h en yl)bu ta n oic Acid (12k ). A solution of sodium
(R)-4-hydroxy-4-(2-methylphenyl)butanoate (25) (3.0 g, 15
mmol) in dry tetrahydrofuran (50 mL) was treated portionwise
with sodium hydride (1.5 g of a 60% dispersion w/w in mineral

2742 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
oil, 37 mmol) at room temperature. After complete addition,
the reaction mixture was stirred at ambient temperature for
1 h and warmed to 55 °C, and 26 (3.0 g, 13 mmol) was added
in one portion. The reaction mixture was stirred, heated to
55 °C for 15 h, cooled, diluted with 1 N hydrochloric acid (200
mL), and extracted with ethyl acetate (4 × 100 mL). The
combined organic extracts were dried and concentrated to
leave an oily residue which was boiled in isopropyl ether (50
mL), filtered while hot, and allowed to cool. The yellow solid
which separated was collected and recrystallized from a
mixture of ethyl acetate and hexane to give 12k (1.5 g, 24%)
as a white fluffy solid, mp 143 °C, [R]D ) -53° (c ) 0.01,
CHCl3). 1H NMR (CDCl3): 2.34 (m, 2H), 2.41 (s, 3H), 2.62 (m,
1H), 2.78 (m, 1H), 4.89 (m, 2H), 5.48 (dd, J ) 12, 4 Hz, 1H),
6.15 (d, J ) 2 Hz, 1H), 6.50 (dd, J ) 12, 2 Hz, 1H), 7.00 (dd,
J ) 4, 1 Hz, 1H), 7.14-7.20 (m, 4H), 7.31 (dd, J ) 6, 4 Hz,
1H), 7.38 (m, 1H), 7.42 (d, J ) 8 Hz, 1H). Anal. (C23H21NO4S)
C, H, N. Chiral HPLC on a Chiralcel OD column (heptane/
ethanol/glacial acetic acid, 980/20/2, flow rate 1 mL/min)
showed 12k to have a >98% ee. A typical retention time for
the (R)-isomer 12k was 81 min and for the (S)-isomer 12l, 91
min.
2-F lu or o-4-(3-th iop h en eylm eth oxy)ben zon itr ile (26). A
mixture of 4-cyano-3-fluorophenol (27) (4.8 g, 35 mmol),
3-thienyl chloride (5.3 g, 40 mmol), potassium carbonate (5.3
g, 4 mmol), potassium iodide (0.1 g, 0.6 mmol) and tetra-n-
butylammonium bromide (0.1 g) in methyl ethyl ketone (100
mL) was stirred and heated to reflux for 18 h. After cooling,
the reaction mixture was filtered, concentrated, taken up in
ethyl acetate (500 mL), and washed with 1.0 M aqueous
hydrochloric acid (200 mL) and 1.0 M aqueous sodium hy-
droxide (200 mL), followed by water (200 mL). The organic
layer was dried and concentrated in vacuo to leave an oil which
was passed through a short pad of silica gel with the aid of
dichloromethane. The filtrate was evaporated to leave an oil,
which crystallized on standing. Recrystallization from iso-
propyl ether gave 26 (6 g, 50%) as a white solid, mp 63-5 °C.
1H NMR (CDCl3): 5.11 (s, 2H), 6.80 (m, 2H), 7.12 (m, 1H), 7.34
(m, 1H), 7.38 (m, 1H), 7.52 (m, 1H).
ter t-Bu tyl 4-(2-Meth ylp h en yl)-4-oxobu ta n oa te (22). To
a cooled (-78 °C) solution of succinic anhydride (75 g, 750
mmol) in dry tetrahydrofuran (800 mL) was added a solution
of o-tolylmagnesium chloride (800 mL of a 1.0 M solution in
toluene, 800 mmol), and the reaction mixture stirred at this
temperature for 2 h. The reaction mixture was warmed to
room temperature, poured into 1.0 M hydrochloric acid (1 L),
and extracted with diethyl ether (4 × 500 mL). The combined
organics were washed with water (2 × 500 mL), dried, and
concentrated to leave 4-(2-methylphenyl)-4-oxobutanoic acid
as a colorless oil (110 g, 75%). 1H NMR (CDCl3): 2.41 (s, 3H),
2.80 (t, J ) 8 Hz, 2H), 3.30 (t, J ) 8 Hz, 2H), 7.4-7.60 (m,
4H), 10.0 (br, 1H).
A mixture of 4-(2-methylphenyl)-4-oxobutanoic acid (40 g,
208 mmol), concentrated sulfuric acid (4 mL), and tert-butyl
alcohol (4 mL, 42 mmol) in dichloromethane (200 mL) was
cooled to 0 °C, and isobutylene (∼400 mL) was condensed into
the reaction. The reaction mixture was stirred at 0 °C until
the TLC showed complete reaction (∼8 h) and the excess
isobutylene was allowed to evaporate. Saturated sodium
bicarbonate (200 mL) was carefully added with vigorous
stirring. The organic layer was separated, filtered through a
pad of silica gel with the aid of dichloromethane, and concen-
trated to give 22 (50 g, 97%) as a pale-yellow oil. 1H NMR
(CDCl3): 1.46 (s, 9H), 2.40 (s, 3H), 2.76 (t, J ) 8 Hz, 2H), 3.28
(t, J ) 8 Hz, 2H), 7.4-7.60 (m, 4H),
ether (50 mL), before addition of diethanolamine (10 g, 95
mmol). The reaction mixture was stirred vigorously at ambi-
ent temperature for 5 h, poured into pentane (500 mL) and
filtered through a pad of diatomaceous earth. The filtrate was
concentrated and pumped under high vacuum (0.5 mmHg)
with warming to 50 °C, and the residue was purified by flash
chromatography on silica gel, eluting with
a mixture of
methanol and dichloromethane (5:95 v/v) to give 23 (10 g, 84%)
as a colorless oil, bp 175 °C/0.2 mmHg, [R]D ) +49° (c ) 0.01,
CHCl3). 1H NMR (CDCl3): 1.46 (s, 9H), 2.00 (m, 2H), 2.34 (s,
3H), 2.41 (m, 2H), 4.99 (dd, J ) 8, 4 Hz, 1H), 7.1-7.25 (m,
3H), 7.49 (m, 1H). The ee was determined to be >99% by a
1H NMR method in CDCl3 using the derivative 24 formed from
(+)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride (see
the Chemistry section).
Sod iu m (R)-4-Hyd r oxy-4-(2-m eth ylp h en yl)bu ta n oa te
(25). A solution of 23 (0.5 g, 2 mmol) in tetrahydrofuran (3
mL) and methanol (3 mL) was treated with 5.0 M aqueous
sodium hydroxide (0.42 mL) and stirred at ambient temper-
ature for 8 h. The reaction was concentrated to dryness, the
residue was stirred for 30 min in 2-propanol (30 mL), and the
resultant precipitate was collected and washed with isopropyl
ether to give 25 (300 mg, 70%) in a form of a white solid, mp
>250 °C, [R]D ) +34° (c ) 0.01, MeOH). 1H NMR (DMSO-
d6): 1.65 (m, 2H), 2.11 (m, 2H), 2.22 (s, 3H), 4.75 (dd, J ) 8,
4 Hz, 1H), 7.05 (m, 2H), 7.23 (m, 1H), 7.48 (m, 1H).
Biologica l Meth od s. (i) Ra d ioliga n d Bin d in g Assa ys
(IC50 Deter m in a tion ): P r ep a r a tion of ETA Recep tor s.
A10 cells, a rat aortic smooth muscle cell line (ATCC number
CRL-1476), were grown to confluence in Dulbecco’s modified
Eagle’s medium containing 10% v/v fetal bovine serum. Two
days after the final medium change cells were scraped from
the base of the flask and centrifuged at 1000g for 10 min at 4
°C. The resulting pellets were washed twice in 50 mM Hepes
buffer (pH 7.3) containing calcium chloride (1 mM) and
magnesium chloride (5 mM) and resuspended at a density of
140 000 cells/mL in 50 mM Hepes buffer (pH 7.3). Aliquots
of 5 mL were snap-frozen using a mixture of methanol and
solid carbon dioxide and stored at -20 °C until required. For
use in the binding assay, cells were diluted to the required
density with 50 mM Hepes buffer (pH 7.3).
P r ep a r a tion of ETB Recep tor s. Rats were killed by
cervical dislocation, and the cerebellum tissue was removed
into ice-cold 50 mM Tris buffer (pH 7.4) containing sucrose
(0.25 M), ethylenediaminetetraacetic acid (3 mM), aprotinin
(0.5 µg/mL), pepstatin A (10 µg/mL), leupeptin (10 µg/mL), and
phenylmethanesulfonyl fluoride (0.1 mM). After homogeniza-
tion using a glass/Teflon manual homogenizer, the samples
were centrifuged at 4 °C for 17 min at 1000g and the resulting
supernatants retained. This material was centrifuged at
40000g for 35 min at 4 °C, the pellets were resuspended in 50
mM Tris buffer (pH 7.4), and the protein content was deter-
mined. Aliquots of 100 µL were snap-frozen using a mixture
of methanol and solid carbon dioxide and stored at -20 °C
until required. Samples were diluted as necessary with 50
mM Tris buffer (pH 7.4) containing 0.1% w/v bovine serum
albumin for use in the assay.
Bin d in g Assa y. Binding assays were performed in Milli-
pore 0.22-µm 96-well multiscreen plates and consisted of 20
pM [125I]ET-1, test compound or vehicle, and A10 cells or
cerebellum protein in a final volume of 250 µL. Nonspecific
binding was measured using 500 nM unlabeled ET-1. [125I]-
ET-1 was prepared in 50 mM Tris buffer (pH 7.4) containing
0.1% w/v BSA, and test compounds were prepared in the same,
supplemented with dimethyl sulfoxide at 5% v/v final assay
concentration. Reactions were started by the addition of cells
or cerebellum protein and allowed to proceed for 2 h at 37 °C
before being terminated by vacuum filtration. The filters were
then washed and collected for ψ-counting.
ter t-Bu tyl (R)-4-Hyd r oxy-4-(2-m eth ylph en yl)bu ta n oa te
(23). A solution of 22 (12 g, 48 mmol) in dry diethyl ether (50
mL) was dried over 10 g of 4 Å molecular sieves for 3 h and
was then transferred to a dry flask and placed under an argon
atmosphere. The solution was cooled to -20 °C, treated
rapidly with (+)-B-chlorodiisopinocampheylborane ((+)-DIP-
chloride; 25 g, 78 mmol), and sealed under an atmosphere of
argon. The reaction mixture was stored at -20 °C for 48 h,
allowed to warm to room temperature, and diluted with diethyl
Da ta An a lysis. Data from binding assays was corrected
for nonspecific binding and expressed as a percentage of total
[
125I]ET-1 binding in the presence of vehicle. IC50 values were
obtained by curve fitting. In
a minority of cases when
duplicate IC50 values were not obtained, the results of IC50

Selective Endothelin A Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2743
measurements were confirmed by retesting a single concentra-
tion selected to fall on the concentration-response curve.
(ii) In Vitr o F u n ction a l Assa y (p KB Deter m in a tion ):
This was carried out as previously described15 using de-
endothelialized rat aortic rings.
was carried for all unfrozen bonds within -X using 3-point
rotation about sp3-sp3 bonds, 6-point rotation about sp3-sp2
bonds, and 2-point rotation about conjugated bonds and the
bond connecting the acid dummy oxygen to the acid group
carbon. Energy minimization of all conformations generated
was carried out using the torsional minimizer setting. Con-
formations generated were then plotted using the following
distance variables: acid group dummysD1, D2, D3, and D4;
aryl group dummysD1, D2, D3, and D4; and the ortho
hydrogen of the Aryl groupsD1, D2, D3, and D4. The ortho
hydrogen was introduced to allow the fixing of the ring plane.
Four corners of the box were used to ensure complete speci-
fication of the 3D position of each group. The plots for each
molecule studied were combined in the order 8 then 7 using
the AND logical operator. A tolerance of 10% was used along
with an energy cutoff of 8 kcal above the found minimum for
each conformation. The conformational space containing the
remaining conformations for the three compounds was then
visualized; the remaining conformations were listed and
retrieved from the conformational database. Display of the
compounds is shown in Figure 1. This superimposition was
used for all subsequent design work.
(iii) In Vivo F u n ction a l Assa y (P ith ed Ra t Mod el): The
experimental methods have been described in detail by RPR
researchers.30 A brief protocol is as follows: Male Sprague-
Dawley rats (250-350 g) were anesthetized with isoflurane,
pithed with a stainless steel rod, and artificially respired. A
jugular vein and carotid artery were cannulated for adminis-
tration of vehicle or test compound and measurement of blood
pressure (via a pressure transducer). BQ 788 (3 mg/kg) and
test compound (2.5, 7.5, 25, 75 µg/kg) were given iv 10 min
prior to measurement of the dose- pressor response curve
following bolus injection of ET-1 (0.01-10 nmol/kg). Test
compounds caused a rightward, parallel dose-dependent shift
of the dose-reponse curve from vehicle control. The shift from
vehicle for each antagonist was calculated using the dose of
ET-1 which caused a 40 mmHg change in diastolic blood
pressure.
P h a r m a cok in etics in th e Ra t. The pharmacokinetics of
12m were investigated following the administration of single
doses by the oral and intravenous routes in male Sprague-
Dawley rats. 12m was formulated as a 7.5 mg/mL solution/
suspension in 1% w/v carboxymethyl cellulose/0.2% v/v Tween
80 in deionized water for the oral route and as a 3.75 mg/mL
solution in dimethyl sulfoxide for the intravenous route. Three
individual rats per time point (selected randomly from 57
animals, mean body weight 331 g, standard deviation 22 g)
were dosed by gavage at 30 mg/kg (4 mL of vehicle/kg) or at 3
mg/kg (0.8 mL of vehicle/kg) by slow bolus injection into the
jugular vein while under light isoflurane anesthesia. Hep-
arinized blood samples were taken by cardiac puncture from
individual rats postsacrifice by carbon dioxide asphyxiation
at 0.5, 1, 2, 3, 5, 8, 12, 16, and 24 h postdose for the oral groups
and at 0.08, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, and 8 h postdose for
the intravenous groups. Plasma samples obtained from blood
by centrifugation (4 °C) were analyzed by reverse-phase HPLC
using an internal standard (1 mL/min 45% methanol/35%
water, pH 3/20% tetrahydrofuran, 25-cm × 4-mm i.d. Lichro-
cartRP column, UV detection 250 nm). Retention times of 12m
and internal standard were 6.5 and 11 min, respectively. The
limit of detection was 15 ng of 12m /mL of plasma. Pharma-
cokinetic parameters were derived from the mean plasma
concentrations at each time point using the SIPHAR PK
computer program (SIPHAR, SIMED, France).
Molecu la r Mod elin g. All structures were initially created
using Concord 3D builder (distributed by Tripos Inc., 1699
Hanley Rd, Suite 303, St. Louis, MO 63144). The remaining
modeling was carried out within Chem-X (developed and
distributed by Chemical Design Ltd., Roundway House, Crom-
well Park, Chipping Norton, Oxfordshire OX7 5SR, U.K.).
Charges were first set using the Gasteiger method. The acid
group interaction points were introduced by conversion of the
existing carboxylic acid hydrogen atom to a dynamic dummy
atom and extension of the dummy-oxygen bond length to 2.8
Å. The theoretical VDW radius for this dummy was set to
zero as was its charge. The charges on the remaining oxygens
were modified using the equation ((e1 + e2) - e3)/2, where e1
and e2 were the original charges on the oxygens and e3 was
the charge on the original attached hydrogen. This procedure
allowed removal of the hydrogen and equivalencing of the
charges on the oxygens but prevented the creation of a full
negative charge which would have a distorting effect on energy
calculations carried out in vacuo. The molecules were super-
imposed using the central phenyl template and the 1,3-
attachment points. The pendant arylmethoxy groups were
frozen in an identical low-energy conformation consistent with
SAR. A further dummy was placed at the center of the
remaining aryl ring. The 3D box was created around the
molecules using a command procedure which placed dummies
at defined coordinate points; four of these dummies were
designated D1, D2, D3, and D4. The conformational analysis
Ack n ow led gm en t. The authors wish to thank the
following people for their technical contributions to the
work described in this paper: David Neighbour, Adnan
Al-Shaar, Rajesh Patel, Shelly Darnborough, Imtiaz
Ahmed, J anet Archer, Lisa Sandow, Robert Petheram,
Devandan Chatterjee, J effrey Phillips, Melanie Wong,
Peter Lockey, Andrea Stoppard, Roman Brazdil, Declan
Flynn, Mark Birrell, Michael Podmore, Mark Vine,
Anne Stevens, and Sanj Deverajan.
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