Pyrrolidine-3-carboxylic acids as endothelin antagonists. 4. Side chain conformational restriction leads to ET(B) selectivity

Article abstract of DOI:10.1021/jm990170q

When the dialkylacetamide side chain of the ETA-selective antagonist ABT-627 is replaced with a 2,6-dialkylacetanilide, the resultant analogues show a complete reversal of receptor selectivity, preferring ETB over ETA. By optimizing the aniline substitution pattern, as well as the alkoxy group on the 2-aryl substituent, it is possible to prepare antagonists with subnanomolar affinity for ETB and with selectivities in excess of 4000-fold. A number of these compounds also show promising pharmacokinetic profiles; a useful balance of properties is found in A-192621 (38). Pharmacology studies with A-192621 serve to reveal the role of the ETB receptor in modulating blood pressure; the observed hypertensive response to persistent ETB blockade is consistent with previous postulates and indicates that ETB-selective antagonists may not be suitable as agents for long-term systemic therapy.

Full text of DOI:10.1021/jm990170q

3668
J . Med. Chem. 1999, 42, 3668-3678
P yr r olid in e-3-ca r boxylic Acid s a s En d oth elin An ta gon ists. 4. Sid e Ch a in
Con for m a tion a l Restr iction Lea d s to ETB Selectivity
Thomas W. von Geldern,* Andrew S. Tasker,† Bryan K. Sorensen, Martin Winn, Bruce G. Szczepankiewicz,
Douglas B. Dixon, William J . Chiou, Liming Wang, J erry L. Wessale, Andy Adler, Kennan C. Marsh,‡
Bach Nguyen,‡ and Terry J . Opgenorth
Metabolic Disease Research and Drug Analysis Department, Pharmaceutical Products Research Division,
Abbott Laboratories, Abbott Park, Illinois 60064-6098
Received April 12, 1999
When the dialkylacetamide side chain of the ETA-selective antagonist ABT-627 is replaced
with a 2,6-dialkylacetanilide, the resultant analogues show a complete reversal of receptor
selectivity, preferring ETB over ETA. By optimizing the aniline substitution pattern, as well as
the alkoxy group on the 2-aryl substituent, it is possible to prepare antagonists with
subnanomolar affinity for ETB and with selectivities in excess of 4000-fold. A number of these
compounds also show promising pharmacokinetic profiles; a useful balance of properties is
found in A-192621 (38). Pharmacology studies with A-192621 serve to reveal the role of the
ETB receptor in modulating blood pressure; the observed hypertensive response to persistent
ETB blockade is consistent with previous postulates and indicates that ETB-selective antagonists
may not be suitable as agents for long-term systemic therapy.
In tr od u ction
probing the conformation of the amide side chain of
ABT-627 (1), we made an unexpected observation which
led to a complete reversal of receptor selectivity and led
to the first ETB-selective agent of this class. This
observation has been pursued and has led to the
discovery of A-192621, a potent and orally deliverable
ETB-selective antagonist. As one example of how such
a selective agent can improve our understanding of
endothelin receptor biology, pharmacology studies em-
ploying A-192621 have helped to elucidate the role of
ETB receptors in the maintenance of vascular tone.
Endothelin (ET),1 a 21-amino acid bicyclic peptide, is
the most powerful peptidic constrictor of vascular
smooth muscle reported to date, as well as a potent
mitogen. Produced predominantly by endothelial cells,
it acts in both an autocrine and a paracrine fashion as
a mediator of vascular function. It has been implicated
as a pathogenic factor for a variety of disease states,2a
including asthma,2b coronary vasospasm and myocardial
infarction,2c pulmonary hypertension,2d restenosis,2e and
atherosclerosis,2f in which excessive vasoconstriction or
smooth muscle proliferation plays a role.
Key Obser va tion
Endothelin acts by binding to a family of membrane-
associated G-protein coupled receptors (GPCRs).3 Bind-
ing to the ETA receptor subtype, which predominates
in vascular smooth muscle cells, triggers a cascade of
events which leads, via the hydrolysis of inositol phos-
phates, to the observed vasoconstrictive and prolifera-
tive responses. The results of binding to ETB, which is
the major receptor on endothelial cells, are less clearly
understood. While this receptor mediates constriction
in some tissue beds,4 it has also been linked to vasodi-
lation through the production of nitric oxide5 and to the
clearance of endogenous ET.6
A number of groups, including our own, have reported
the development of potent nonpeptidic endothelin an-
tagonists which bind selectively to the ETA receptor
subtype or are nonselective. These agents have provided
valuable insights into the role of the ETA receptor in
mediating a variety of disease states. There have been
fewer reports,7 however, describing high-quality ETB
antagonists, and correspondingly the role of ETB re-
mains more obscure. In the course of a series of studies
Several previous studies by our group have indicated
the critical role that the N-linked side chain of 1 plays
in determining receptor binding and selectivity (Scheme
1).8,9 Structure-activity studies during the development
of ABT-627 (IC50 ) 0.08 nM against ETA; 1800 times
selective for ETA versus ETB) indicated to us the
importance of the side chain amide carbonyl. We suspect
that this carbonyl oxygen is involved in a hydrogen-
bonding interaction with the ETA receptor which serves
to orient the butyl groups into appropriate hydrophobic
binding pockets. Our work also indicated that the
presence of both alkyl groups are required to maintain
high ETA selectivity. We later learned that, by modifying
the orientation of these two alkyl groups, we can
produce analogues which retain high affinity for ETA
but simultaneously bind to ETB receptors as well. The
result of these studies was A-182086 (2), the most potent
and “balanced” antagonist reported to date (IC50 ) 0.08
nM against ETA; A/B ) 3).
On the basis of the above results, we continued to
explore conformational restriction as a route to improv-
ing the selectivity of compounds of this class. One way
to restrict the motions of these side chain alkyl groups
would be to tie them back as substituents on an
aromatic ring (e.g. compound 3). Fortunately this strat-
* Address correspondence to T. W. von Geldern, D-47H/AP10-LL-
12E, Abbott Laboratories, 100 Abbott Park Rd; Abbott Park, IL 60064-
6098. E-mail: thomas.vongeldern@abbott.com.
‡ Department of Drug Analysis.
† Current address: Amgen Inc., Thousand Oaks, CA 91320.
10.1021/jm990170q CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/13/1999

ETB-Selective Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3669
Sch em e 1. Conceptual Framework for Side Chain Conformational Restriction Strategy
egy is an easy one to test and requires no knowledge of
the preferred substituent orientation, since all of the
required dimethylanilines are commercially available.
We thus prepared a series of compounds (Table 1, 4-9)
which covers all of the possibilities. In general these
compounds are relatively inactive and exhibit modest
selectivity for the ETA receptor subtype. A notable
exception is analogue 7, which carries methyl groups
at the 2- and 6-positions. Surprisingly, this compound
is slightly selective for the ETB over ETA receptor and
has a relatively high affinity for ETB as well (IC50 < 20
nM).
predominates. An additional portion of trans,trans-
material may be derived by epimerization (DBU, tolu-
ene) of the cis,cis-isomer, which is conveniently sepa-
rated by chromatography.
Appropriately substituted anilines are acylated with
bromoacetyl bromide to provide the corresponding bro-
moacetanilides, which are used to alkylate the above
pyrrolidines 14. Under standard saponification condi-
tions (NaOH, MeOH, room temperature, overnight) the
hydrolysis of the trans,trans-ester is dramatically faster
than that of any other isomer, providing the pure trans,-
trans-acid 16. This valuable selective hydrolysis was
first observed during the preparation of our ETA-
selective analogues.5 When necessary, optically active
analogues may be prepared through application of
pyrrolidine core resolution strategies we have described
previously.8,9
Ch em istr y
The core pyrrolidines employed in this study have
been assembled (Scheme 2) by direct analogy to our
earlier work.8 Briefly, the Michael reaction between
â-ketoesters 13 and nitrostyrene 11 provides a mixture
of diastereomeric nitroketones. The ketoesters them-
selves are prepared from the corresponding ketones (by
carbethoxylation) or benzoic acids (through activation
and malonate homologation). Nitrostyrene 11 derives
from Henry reaction of piperonal (10). The diastereo-
meric mixture of nitroketones is reductively cyclized in
two steps (Raney nickel reduction to provide a cyclic
imine, which is further reduced using cyanoborohydride)
to give pyrrolidines 14, generally as a mixture of three
diastereomers in which the desired trans,trans-isomer
Com p ou n d Eva lu a tion
The first line of biochemical analysis for the com-
pounds described in this study is a measurement of their
ability to displace endothelin from its receptor. For the
purpose of screening we employ human ETA and ETB
receptors (hETA, hETB) permanently expressed in CHO
cells. IC50 data are recorded by measuring the displace-
ment of [125I]ET-1 from ETA or of [125I]ET-3 from ETB.
Analogues of particular interest are examined for
their ability to block the ET-1-induced hydrolysis of
Sch em e 2. Synthesis of Arylacetanilide Analoguesa
a
CH3NO2, NH4OAc, HOAc, heat; (ii) NaH, (EtO)2CO, THF; (iii) CDI, THF; MgOOCCHCOOEt, DMF; (iv) DBU, THF-iPrOH; (v) H2,
Ra-Ni, THF-HOAc; NaBH3CN, THF-EtOH, pH 5; (vi) DBU, THF, heat; (vii) BrCH2CONHAr, iPR2NEt, CH3CN; (viii) NaOH, H2O-
EtOH.

3670 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
von Geldern et al.
Ta ble 1.
a
IC50 values calculated using a mean of at least 2 measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M unless
otherwise noted.

ETB-Selective Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3671
inositol phosphates in ETB-CHO. In these experiments
the compounds are simultaneously tested for the ability
to function as agonists. This same subset of compounds
of particular interest has also been examined for their
pharmacokinetic properties using a standard protocol
which compares the time course of plasma drug levels
after dosing in rats by intravenous injection and oral
gavage.
molecules, once the essential nature of the profile (e.g.
ETA-selective, balanced) had been established through
modifications of the side chain group. Having now
identified a new class of antagonist, ETB-selective by
virtue of a 2,6-dialkylanilide moiety, we began a short
series of studies to optimize this profile through a
similar strategy (Table 2). As in past studies, this
exercise proved fruitful. Sequential homologation of the
p-methoxy substituent to ethoxy (31, IC50 ) 3.6 nM) and
propoxy (32, IC50 ) 12 nM) led to small but significant
losses of ETB affinity (∼10-fold total decrease) over the
series, with a substantially larger decrease (>200-fold
total) at ETA. As a result, the extended versions were
increasingly less active, but more selective for ETB.
Replacement of the linear alkyl group with isopropyl
(33) caused a loss of activity; however, the p-methoxy-
ethoxy group of 34 was well-tolerated and led for the
first time to an antagonist with nanomolar ETB affinity
and >1000-fold selectivity. This latter result led us to
examine the roles of the two oxygen atoms on this para-
substituent; indeed, when the proximal ether is removed
(to give 35), potency and selectivity remain high.
Resu lts a n d Discu ssion
We initially chose to probe the question of substitution
pattern on the aniline ring using two methyl groups,
largely due to the ready availability of all possible
dimethylanilines. Having determined that 2,6-substitu-
tion led to an intriguing result, we immediately followed
up with a study of alkyl chain length. Our previous
studies leading to ABT-627 had indicated that this
parameter could be of critical importance in determin-
ing not only receptor affinity but also selectivity as
well. In the event, one-carbon extension of the 2-sub-
stituent to give 17 led to slight improvements in both
potency and ETB selectivity (IC50 ) 4.0 nM; A/B ) 4).
While branching (to give 18) did not provide further
benefit, a similar one-carbon homologation of the 6-
methyl group was accompanied by additional improve-
ments in potency and selectivity; the resultant 2,6-
diethyl analogue 19 exhibits 1.4 nM affinity for ETB and
is >30-fold selective. Further homologation of one (20)
or both (21) alkyl groups leads to a reversion to ETA
selectivity. Thus, it appears that a four-atom spacing
between the amide nitrogen and the alkyl terminus is
preferred for both alkyl groups. Interestingly, a similar
four-atom spacing (albeit with a dramatically different
geometry) provides the di-n-butylamide which defines
the ETA selectivity of ABT-627. Placement of a branched
alkyl chain at both 2- and 6-positions (22) leads to a
20-fold decrease in activity, though selectivity remains
high. Methylation of the amide nitrogen has a dramatic
impact; the resultant analogue 23 is modestly ETA-
selective and has lost affinity for both ETA and ETB.
Other substituents are tolerated at the 2- and 6-posi-
tions as well, though none is the equal of ethyl.
Sequential isosteric replacement of each ethyl group
with methoxy (24, 25) causes a loss of 4-10-fold in ETB
binding affinity in each case; while the first of these
replacements has little effect on selectivity, this is
eroded as well in the 2,6-dimethoxy analogue. In similar
fashion, 2,6-dibromo analogue 26 is a poorer ligand for
ETB as well (IC50 ) 10 nM). Substitution at the
4-position is well-tolerated in some circumstances. Ad-
dition of a 4-fluorine atom provides a compound (27)
which retains the activity/selectivity profile of analogue
19; a small 4-alkyl group (28, 29) decreases ETB
selectivity 2-fold with little effect on ETB binding, while
4-carboxy substitution (30) leads to a substantial de-
crease in activity. In the end, none of these modifications
provides a significant improvement over the 2,6-diethyl-
acetanilide. It is clear from the above results that there
is a very limited hydrophobic space on the ETB receptor
which is efficiently occupied by this conformationally
restricted side chain.
Preliminary pharmacokinetic analysis of a number of
the compounds described above, as racemates (data not
shown), suggested a smaller subset (19, 31, 32, 34)
which warranted further study as single enantiomers.
In general, the relevant pyrrolidine cores may be
resolved through one of several general strategies we
have previously described. The active enantiomers were
identified and characterized in some detail (Table 3).
Because our primary goal for this effort is to identify
an orally deliverable agent which can help to address
the role of ETB receptor blockade in disease therapy,
we felt that it was important to emphasize selectivity
over potency in choosing a candidate for further evalu-
ation in disease models. This analysis led us to pick
compound 38 (A-192621) for scaleup and additional
studies.
To confirm that these compounds are antagonists,
several of the compounds in Table 3 have been evalu-
ated in a functional assay measuring ET receptor-
mediated hydrolysis of inositol phosphates. The com-
pounds show a high level of antagonist activity in this
assay and exhibit no agonist effect even at high doses
(data not shown), confirming that as a class they are
pure, functional antagonists of the ETB receptor.
P h a r m a cology of ETB Block a d e. To determine
whether the in vitro profile described above for A-192621
was manifest in vivo, we began by examining the effects
of acute dosing of the compound in an ETA/ETB pseu-
doefficacy model (Figure 1). Briefly, a number of groups
have demonstrated that a bolus injection of ET-1
produces a biphasic response on blood pressure. A rapid,
sharp, and transient period of hypotension, which has
been demonstrated to be mediated through ETB-induced
release of nitric oxide, is followed by a long period of
elevated pressure which has a slower onset and which
is mediated through the ETA receptor. A single oral dose
of A-192621 (30 mg/kg, by gavage), given 1 h prior to
repeating this study, completely blocks the ET-1-
induced depressor effect, a result which is expected with
a potent ETB antagonist. As would be predicted for a
selective agent, we observe no decrease in the pressor
response. In fact, this pressor effect appears to have a
Our previous studies had highlighted the importance
of the 2-aryl ring in “tuning” the activity and selectivity
profile for this family of pyrrolidine-based ET antagonist

3672 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Ta ble 2. Effects of Modifying the Phenoxyalkyl Substituent
von Geldern et al.
a
IC50 values calculated using a mean of at least 2 measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M unless
otherwise noted.
more rapid onset, as well as a substantially longer
duration of action. The apparently more rapid onset may
be an unmasking effect which results from removal of
the early depressor component. The increased half-life
of this response probably reflects the postulated role of
ETB as a clearance receptor. With a primary clearance
pathway removed through ETB blockade, exogenously
administered ET-1 is expected (and has been demon-
strated10) to have an increased circulating half-life.
The above results suggest the possibility that long-
term treatment with an effective ETB-selective antago-
nist like A-192621 might produce hypertension in a
normal animal as well. Such a hypertensive liability
would substantially limit the utility of these agents in
chronic therapy. To test this possibility, we performed
a study in which normal rats received A-192621 (30 mg/
kg/day in food) for 3 days. The results (Table 4) confirm
that, while no significant effects on food or water intake
were observed, a substantial increase in mean arterial
pressure (23 mmHg) results, along with a significant
decrease in heart rate (12%). Importantly, the addition
of an ETA-selective antagonist rapidly returns the
elevated blood pressure to normal values.11 ET-1 levels
increase 5-fold in the drug-treated group, again con-
firming the role of ETB as a clearance receptor and
strongly suggesting that the hypertensive effect is
related to an increase in the amount of circulating
endothelin.
imparts ETB selectivity to this family of pyrrolidine-
based endothelin antagonists. Optimization of substit-
uents on the aniline ring and of the alkoxy moiety on
the 2-aryl group led to compounds with subnanomolar
affinities for ETB and selectivities of up to 4000-fold
for ETB versus ETA. Some of the antagonists in this
series are orally deliverable. A series of analogues were
prepared in optically active form for detailed evaluation,
and compound 38, A-192621, was chosen for further
study based on a combination of biochemical and
pharmacokinetic properties. Pharmacology studies of
A-192621 in rats confirm that this compound does
indeed function as an ETB-selective agent in vivo. Three-
day oral dosing in food leads to a substantial increase
in blood pressure, indicating that the ETB receptor plays
a role in the maintenance of basal tone, presumably by
acting as a scavenger for circulating endothelins. This
latter result seems to suggest that long-term ETB
antagonism may only be acceptable as a therapeutic
modality in the context of simultaneous ETA blockade.
Exp er im en ta l Section
Unless otherwise specified, all solvents and reagents were
obtained from commercial suppliers and used without further
purification. THF was dried over sodium and purified by
distillation. All reactions were performed under nitrogen
atmosphere unless specifically noted. Flash chromatography
was done using silica gel (230-400 mesh) from E.M. Science.
1H NMR spectra were recorded at 300 MHz; all values are
referenced to tetramethylsilane as internal standard and are
reported as shift (multiplicity, coupling constants, proton
count). Mass spectral analysis is accomplished using fast atom
bombardment (FAB-MS) or direct chemical ionization (DCI-
MS) techniques. All elemental analyses are consistent with
Con clu sion s
An unexpected observation during the course of a
series of conformational studies has led us to the
observation that a 2,6-dialkylacetanilide side chain

ETB-Selective Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3673

3674 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
von Geldern et al.
Ta ble 4. Effects of Long-Term ETB Blockadea
MAP (mmHg)
ET-1 level (pg/mL)
control
A-192621-treated
127
150
4
19
a
Normal (Sprague-Dawley) rats received ETB-selective an-
tagonist A-192621 in food (30 mpk/day) for 21 days. The resultant
substantial rise in MAP occurs concommitantly with a 5 times
increase in circulating levels of ET-1, suggesting that the ETB
receptor serves a clearance role.
romethane was added dropwise, followed by 1.0 equiv of
diisopropylethylamine. The solution turned dark and a pre-
cipate formed, as the reaction mixture was warmed to ambient
temperature over 1.5 h. The mixture was taken up in heptane
and extracted three times with water; the organic phase was
dried and purified by silica gel chromatography. All anilines
are commercially available or were prepared according to
literature precedent, except as described below.
F igu r e 1. Pseudoefficacy data: effect on mean arterial
pressure (MAP) of a bolus dose of ET-1 (0.3 nmol/kg, given at
T ) 60 min) to anesthetized rats in the absence (open
diamonds) or presence (solid diamonds) of a prior dose of
A-192621 (administered orally at T ) 0). The ETB antagonist
completely blocks the transient hypotensive spike, unveiling
an earlier phase of the hypertensive response. The hyperten-
sive phase also persists longer, presumably due to reduced
ETB-mediated clearance of ET-1.
2,6-Dieth yl-4-flu or oa n ilin e. To an ice-cooled solution of
1.00 g (9.00 mmol) of 4-fluoroaniline in 20 mL of CH2Cl2 was
added a solution of 5:1 CH2Cl2:Br2 until an orange color
persisted. During the reaction, a precipitate formed. The
reaction was extracted with water (1 × 10 mL), saturated
aqueous NaHCO3 solution (1 × 10 mL), and then brine (1 ×
10 mL), dried over MgSO4, filtered, and concentrated to 2.04
g of a solid. TLC (5% EtOAc/hexanes) showed only one spot,
representing 2,6-dibromo-4-fluoroaniline. To a mixture of 122
mg (0.15 mmol) of (dppf)PdCl2 and 3.48 g (10.7 mmol) of CsCO3
was added a solution of 480 mg (1.78 mmol) of 2,6-dibromo-
4-fluoroaniline in 10 mL of DMF, all under N2. Next, 4 mL of
1.0 M triethylborane in hexanes was added. The reaction was
heated at 50 °C for 22 h, then poured into 50 mL of water,
and extracted with diethyl ether (3 × 10 mL). The combined
ether layers were back-extracted with saturated aqueous
NaHCO3 solution (1 × 10 mL) and brine (1 × 10 mL), dried
over MgSO4, filtered, and concentrated to an oil. The product
was purified via silica gel chromatography, eluting with 20%
EtOAc/hexanes to give 192 mg (64%) of a colorless oil.
2-Eth yl-6-m eth oxya n ilin e. Potassium ethylmalonate (3.68
g) was combined with 2.29 g of magnesium chloride in 12 mL
of DMF; the reaction mixture was heated at 60 °C for 4 h.
The resultant mixture was cooled to ambient temperature.
Simultaneously, 3-methoxy-2-nitrobenzoic acid (3.4 g) was
dissolved in 12 mL of DMF; 3.06 g of 1,1-carbonyldiimidazole
was added (gas evolves), and the resultant solution (after
stirring at ambient temperature for 4 h) was added to the
malonate mixture. The resultant slurry was stirred at ambient
temperature for 14 h. Solvents were removed in vacuo; the
residue was taken up in EtOAc, washed sequentially with 1
N H3PO4, bicarbonate, and brine, and concentrated in vacuo.
The crude product (3.2 g) was dissolved in 50 mL of concen-
trated sulfuric acid and stirred at ambient temperature for
48 h. The reaction mixture was poured onto 300 mL of ice and
extracted twice with EtOAc. The organic extracts were washed
sequentially with water, bicarbonate, and brine and concen-
trated in vacuo. The crude product was heated neat at 160 °C
for 3 h. The resultant dark brown residue was extracted with
EtOAc. The organic extracts were concentrated. The crude
product was dissolved in 15 mL of ethanol, sodium borohydride
(450 mg) was added, and the resultant solution was stirred at
ambient temperature for for 2 h. The solvents were removed
in vacuo; the residue was taken up in 10% aqueous HCl and
stirred for 15 min. The mixture was extracted with EtOAc;
the organic extracts were washed sequentially with bicarbon-
ate and brine and concentrated in vacuo. The crude product
was purified by flash chromatography on silica gel, eluting
with 1:1 EtOAc/hexanes, to give 1.08 g (32% overall) of the
title compound as a colorless oil. A sample of this material
(310 mg) was dissolved in 10 mL of THF; 1.5 mL of H3PO4
was added, followed by 50 mg of 10% palladium-on-charcoal.
The resultant mixture was purged with nitrogen, then placed
under a balloon of hydrogen, and stirred overnight. Bicarbon-
ate was added carefully, and the mixture was filtered through
a pad of Celite. The filtrate was extracted with EtOAc; the
theoretical values to within (0.4% unless indicated. Melting
points were measured on a Thomas-Hoover apparatus and are
uncorrected.
Abbr evia tion s: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
EtOAc, ethyl acetate; TFA, trifluoroacetic acid.
Gen er a l Syn th esis of Cor e Com p ou n d s 14. In general,
ketoesters 13 and core pyrrolidines 14 were prepared, and
pyrrolidines 14 were resolved, according to the procedures we
have described previously5 and employing commercially avail-
able starting materials. An exception is described below.
4-(2-Meth oxyeth yl)ben zoic Acid (12, R3 ) CH2CH2OCH3,
Y ) OH). A three-necked 50-mL flask was charged with 800
mg (20 mmol) of 60% NaH in mineral oil. The suspension was
washed and decanted with hexanes three times, while keeping
a positive pressure of N2 over the NaH. Next, 5 mL of THF
was added, and the suspension was cooled with an ice bath. A
solution of 2.01 g (10.0 mmol) of 4-bromophenethanol in 5 mL
of THF was added via cannula, then the ice bath was removed,
and the reaction was stirred for 10 min. After this time 700
mL (11.2 mmol) of methyl iodide was added, and the reaction
was stirred at ambient temperature. After 1 h, the reaction
was cooled with an ice bath, and the excess NaH was quenched
by addition of 1 mL of H2O. The mixture was poured into 60
mL of water and extracted with diethyl ether. The combined
ether layers were back-extracted with brine (1 × 20 mL), dried
over MgSO4, filtered, and concentrated in vacuo to 2.08 g (97%)
of 1-bromo-4-(2-methoxyethyl)benzene as a colorless oil. To a
solution of 1.03 g (4.78 mmol) of this compound in 10 mL of
THF were added 400 mg of Mg and 1 crystal of I2. The mixture
was warmed to reflux for 5 min and then cooled to ambient
temperature. The resultant Grignard reagent was transferred
via syringe to a 50-mL three-necked flask under N2 equipped
with a CO2 inlet. The CO2 inlet was opened, and the red color
faded to pale yellow. After 1 h, the reaction was concentrated
in vacuo. The residue was taken up in 20 mL of H2O, acidified
with 12 M HCl, and extracted with diethyl ether (3 × 10 mL).
The combined ether layers were extracted with 2 M NaOH (3
× 5 mL); then the combined basic layers were extracted with
ether (2 × 5 mL). The basic layer was acidified with 12 M HCl
and then extracted with ethyl acetate (2 × 5 mL). The
combined ethyl acetate layers were back-extracted with brine
(1 × 5 mL), dried over MgSO4, filtered, and concentrated to
427 mg of a solid (50%).
Gen er a l Syn th esis of Br om oa ceta n ilid es. A solution of
aniline (3 mmol) in 1.5 mL of dichloromethane was cooled on
ice; 1.05 equiv of bromoacetyl bromide in 1 mL of dichlo-

ETB-Selective Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3675
organic extracts were washed with bicarbonate and brine and
concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel, eluting with 1:1 ether/hexanes,
to give 102 mg (43% yield) of the title compound as a colorless
oil.
) 10 Hz, 1H), 3.18 (dd, J ) 9, 10 Hz, 1H), 3.45 (d, J ) 17 Hz,
1H), 3.50 (dd, J ) 3, 10 Hz, 1H), 3.70 (m, 1H), 3.79 (s, 3H),
3.93 (d, J ) 9 Hz, 1H), 5.95 (dd, J ) 1, 2 Hz, 2H), 6.75 (d, J )
8 Hz, 1H), 6.83 (dd, J ) 1, 8 Hz, 1H), 6.9 (m, 3H), 7.0 (m, 2H),
7.36 (brd d, J ) 9 Hz, 2H), 7.78 (d, J ) 7 Hz, 1H), 8.60 (brd s,
1H); MS (DCI/NH3) (M + H)+ at m/z 503. Anal. Calcd for
C29H30N2O6‚0.8H2O: C, 67.38; H, 6.16; N, 5.42. Found: C,
67.42; H, 5.95; N, 5.13.
2-Eth yl-6-p r op yla n ilin e. To a stirred solution of 2-ethy-
laniline (5.00 g., 41.3 mmol) in tetrahydrofuran (50 mL) at
ambient temperature were added benzyl bromide (4.91 mL,
41.3 mmol) and triethylamine (6.90 mL, 49.5 mmol). The
reaction was heated at reflux overnight. Reaction was diluted
with ethyl ether and washed with water and brine. Organic
phase was dried with sodium sulfate, and solvents were
removed in vacuo. Residue was purified on silica gel eluting
with 98:2 hexanes-ethyl acetate to yield N-benzyl-2-ethyla-
niline as a yellow oil (2.20 g, 25% yield). This material was
combined with allyl bromide (1.80 mL, 20.9 mmol) and sodium
carbonate (663 mg, 6.26 mmol) in 4:1 ethanol/water (20 mL)
at ambient temperature. Reaction was stirred overnight at
ambient temperature. Reaction was diluted with ethyl ether
and washed with water and brine. Organic phase was dried
with sodium sulfate, and solvents were removed in vacuo.
Residue was purified on silica gel eluting with 99:1 hexanes-
ethyl ether to yield N-allyl-N-benzyl-2-ethylaniline as a color-
less oil (1.90 g, 73% yield). To a stirred solution of this
compound (1.00 g, 4.0 mmol) in anhydrous o-xylene (8 mL) at
-78 °C under a nitrogen atmosphere was added zinc chloride
(654 mg, 4.80 mmol). Reaction was allowed to warm to ambient
temperature and then heated at reflux overnight. Reaction was
cooled to 0 °C and quenched with 1 N sodium hydroxide. This
was diluted with ethyl ether, and layers were separated.
Organic phase was then washed with brine and dried with
sodium sulfate. Solvents were removed in vacuo. Residue was
purified on silica gel eluting with hexane to 99:1 hexanes-
ethyl acetate to 97:3 hexanes-ethyl acetate to yield the desired
product in 70% purity. This partially purified material, dis-
solved in 5 mL of acetic acid, was added to a flask under a
nitrogen atmosphere containing 10% palladium-on-carbon
catalyst (100 mg). Mixture was stirred vigorously for 3 h at
ambient temperature under an atmosphere of hydrogen.
Catalyst was filtered off through Celite and washed with ethyl
acetate. Solvents were removed in vacuo. Residue was purified
on silica gel eluting with 97:3 hexanes-ethyl acetate to 9:1
hexanes-ethyl acetate to yield the desired product (126 mg,
19% yield).
Com p ou n d 6, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,5-d im eth ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300 MHz)
δ 2.24 (s, 3H), 2.29 (s, 3H), 2.97 (d, J ) 17 Hz, 1H), 3.07 (t, J
) 10 Hz, 1H), 3.19 (dd, J ) 9, 10 Hz, 1H), 3.45 (d, J ) 17 Hz,
1H), 3.50 (dd, J ) 3, 10 Hz, 1H), 3.70 (m, 1H), 3.79 (s, 3H),
3.93 (d, J ) 9 Hz, 1H), 5.95 (dd, J ) 1, 2 Hz, 2H), 6.74 (d, J )
8 Hz, 1H), 6.83 (dd, J ) 1, 8 Hz, 1H), 6.9 (m, 4H), 7.05 (d, J )
8 Hz, 1H), 7.36 (brd d, J ) 9 Hz, 2H), 7.78 (s, 1H), 8.63 (brd
s, 1H); MS (APCI) (M + H)+ at m/z 503. Anal. Calcd for
C
29H30N2O6‚0.8H2O: C, 67.38; H, 6.16; N, 5.42. Found: C,
67.72; H, 5.89; N, 5.25.
Com p ou n d 7, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d im eth ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300 MHz)
δ 2.10 (s, 6H), 3.00 (d, J ) 18 Hz, 1H), 3.13 (m, 2H), 3.46 (d,
J ) 18 Hz, 1H), 3.55 (dd, J ) 5,10 Hz, 1H), 3.72 (m, 1H), 3.81
(s, 3H), 3.95 (d, J ) 10 Hz, 1H), 5.93 (d, J ) 2 Hz, 1H), 5.95
(d, J ) 2 Hz, 1H), 6.75 (d, J ) 9 Hz, 1H), 6.82 (dd, J ) 2, 8 Hz,
1H), 6.88 (d, J ) 2 Hz, 1H), 6.91 (d, J ) 9 Hz, 2H), 7.08 (m,
3H), 7.37 (d, J ) 9 Hz, 2H), 8.32 (brd s, 1H); MS (DCI, NH3)
(M + H)+ at m/z 503. Anal. Calcd for C29H30N2O6‚0.5H2O: C,
68.09; H, 6.11; N, 5.48. Found: C, 67.98; H, 6.02; N, 5.33.
Com p ou n d 8, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[3,4-d im eth ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300 MHz)
δ 2.15 (s, 3H), 2.22 (s, 3H), 2.95 (d, J ) 17 Hz, 1H), 3.05 (t, J
) 10 Hz, 1H), 3.18 (dd, J ) 9, 10 Hz, 1H), 3.45 (d, J ) 17 Hz,
1H), 3.50 (dd, J ) 3, 10 Hz, 1H), 3.70 (m, 1H), 3.79 (s, 3H),
3.93 (d, J ) 9 Hz, 1H), 5.95 (dd, J ) 1, 2 Hz, 2H), 6.75 (d, J )
8 Hz, 1H), 6.83 (dd, J ) 1, 8 Hz, 1H), 6.9 (m, 3H), 7.0 (m, 2H),
7.36 (brd d, J ) 9 Hz, 2H), 7.65 (d, J ) 7 Hz, 1H), 8.68 (brd s,
1H); MS (DCI/NH3) (M + H)+ at m/z 503. Anal. Calcd for
C
29H30N2O6‚0.8H2O: C, 67.38; H, 6.16; N, 5.42. Found: C,
67.24; H, 5.94; N, 5.20.
Com p ou n d 9, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[3,5-d im eth ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300 MHz)
δ 2.29 (s, 6H), 2.89 (d, J ) 17 Hz, 1H), 2.98 (t, J ) 10 Hz, 1H),
3.18 (dd, J ) 9, 10 Hz, 1H), 3.4 (m, 2H), 3.66 (m, 1H), 3.79 (s,
3H), 3.91 (d, J ) 9 Hz, 1H), 5.99 (dd, J ) 1, 2 Hz, 2H), 6.75
(brd s, 1H), 6.80 (d, J ) 8 Hz, 1H), 6.87 (dd, J ) 1, 8 Hz, 1H),
6.9 (m, 3H), 7.03 (d, J ) 1 Hz, 1H), 7.11 (brd s, 2H), 7.34 (d,
J ) 7 Hz, 1H), 8.74 (brd s, 1H); MS (DCI/NH3) (M + H)+ at
m/z 503. Anal. Calcd for C29H30N2O6‚0.5H2O: C, 68.09; H, 6.11;
N, 5.48. Found: C, 67.93; H, 6.01; N, 5.19.
4-Br om o-2,6-d ieth yla n ilin e. To a stirred solution of 2,6-
diethylaniline (10.0 g, 67.0 mmol) in acetic acid (50 mL) at
ambient temperature was added bromine (10.4 mL, 201 mmol).
The reaction was stirred overnight at ambient temperature.
The reaction mixture was diluted with diethyl ether (200 mL)
and washed with 5% sodium bisulfite (4 × 50 mL) and brine.
The organic phase was dried with sodium sulfate, and the
solvents were removed in vacuo. The residue was chromato-
graphed on silica gel, eluting with 9:1 hexanes-ethyl acetate
to give the title compound (3.28 g, 21% yield).
Gen er a l Syn th esis of An ta gon ists. Preparation of final
products was accomplished via alkylation of cores 14 with
bromoacetanilides, followed by selective hydrolysis, as we have
described previously.8
Com p ou n d 17, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2-eth yl-6-m eth ylp h en yl]a ceta m i-
d o)p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300
MHz) δ 1.10 (t, J ) 9 Hz, 3H), 2.10 (s, 3H), 2.44 (q, J ) 9 Hz,
2H), 3.02 (d, J ) 18 Hz, 1H), 3.14 (m, 2H), 3.47 (d, J ) 18 Hz,
1H), 3.55 (dd, J ) 6,12 Hz, 1H), 3.73 (m, 1H), 3.82 (s, 3H),
3.95 (d, J ) 9 Hz, 1H), 5.92 (d, J ) 2 Hz, 1H), 5.94 (d, J ) 2
Hz, 1H), 6.76 (d, J ) 9 Hz, 1H), 6.82 (dd, J ) 2, 8 Hz, 1H),
6.87 (d, J ) 2 Hz, 1H), 6.92 (d, J ) 9 Hz, 2H), 7.20-7.10 (m,
3H), 7.38 (d, J ) 9 Hz, 2H), 8.32 (brd s, 1H); MS (DCI, NH3)
(M + H)+ at m/z 517. Anal. Calcd for C30H32N2O6‚0.5H2O: C,
68.56; H, 6.33; N, 5.33. Found: C, 68.58; H, 6.29; N, 5.13.
Com p ou n d 18, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2-isop r op yl-6-m eth ylp h en yl]a c-
etam ido)pyr r olidin e-3-car boxylic acid: 1H NMR (300 MHz,
CDCl3) δ 1.07 (d, J ) 8 Hz, 3H), 1.16 (d, J ) 8 Hz, 3H), 2.09
(s, 3H), 2.86 (m, 1H), 3.02 (d, J ) 18 Hz, 1H), 3.15 (m, 2H),
3.50 (m, 2H), 3.72 (m, 1H), 3.83 (s, 3H), 3.96 (d, J ) 10 Hz,
1H), 5.92 (d, J ) 2 Hz, 1H), 5.94 (d, J ) 2 Hz, 1H), 6.75 (d, J
) 9 Hz, 1H), 6.82 (dd, J ) 2, 8 Hz, 1H), 6.86 (d, J ) 2 Hz, 1H),
Com p ou n d 4, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,3-d im eth ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300 MHz)
δ 2.16 (s, 3H), 2.30 (s, 3H), 2.97 (d, J ) 17 Hz, 1H), 3.07 (t, J
) 10 Hz, 1H), 3.18 (dd, J ) 9, 10 Hz, 1H), 3.45 (d, J ) 17 Hz,
1H), 3.50 (dd, J ) 3, 10 Hz, 1H), 3.70 (m, 1H), 3.79 (s, 3H),
3.93 (d, J ) 9 Hz, 1H), 5.95 (dd, J ) 1, 2 Hz, 2H), 6.75 (d, J )
8 Hz, 1H), 6.83 (dd, J ) 1, 8 Hz, 1H), 6.9 (m, 3H), 6.97 (d, J )
8 Hz, 1H), 7.08 (t, J ) 7 Hz, 1H), 7.36 (brd d, J ) 9 Hz, 2H),
7.64 (d, J ) 7 Hz, 1H), 8.67 (brd s, 1H); MS (DCI/NH3) (M +
H)+ at m/z 503. Anal. Calcd for C29H30N2O6‚0.5H2O: C, 68.09;
H, 6.11; N, 5.48. Found: C, 68.13; H, 5.91; N, 5.29.
Com p ou n d 5, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,4-d im eth ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3, 300 MHz)
δ 2.23 (s, 3H), 2.28 (s, 3H), 2.95 (d, J ) 17 Hz, 1H), 3.05 (t, J

3676 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
von Geldern et al.
6.92 (d, J ) 9 Hz, 2H), 7.07 (dd, J ) 2, 9 Hz, 1H), 7.18 (m,
2H), 7.39 (d, J ) 9 Hz, 2H), 8.35 (brd s, 1H); MS (DCI, NH3)
(M + H)+ at m/z 531. Anal. Calcd for C31H34N2O6‚0.5H2O: C,
69.00; H, 6.54; N, 5.19. Found: C, 69.27; H, 6.67; N, 5.21.
Com p ou n d 19, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth ylp h en yl]a ceta m id o)p y-
δ 2.85 (brd d, J ) 18 Hz, 1H), 3.03 (m, 2H), 3.49 (brd d, J )
15 Hz, 1H), 3.70 (m, 2H), 3.71 (s, 6H), 3.81 (s, 3H), 3.88 (brd,
J ) 10 Hz, 1H), 5.93 (s, 2H), 6.56 (d, J ) 9 Hz, 2H), 6.75 (d,
J ) 9 Hz, 1H), 6.86 (d, J ) 2 Hz, 1H), 6.90 (d, J ) 9 Hz, 2H),
6.99 (d, J ) 2 Hz, 1H), 7.17 (t, J ) 9 Hz, 1H), 7.39 (brd d, J )
9 Hz, 2H), 8.18 (brd s, 1H); MS (DCI, NH3) (M + H)+ at m/z
535. Anal. Calcd for C29H30N2O8‚0.75AcOH: C, 63.20; H, 5.74;
N, 4.83. Found: C, 63.18; H, 5.34; N, 4.79.
Com p ou n d 26, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ibr om op h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz, CDCl3)
δ 3.01 (brd d, J ) 18 Hz, 1H), 3.13 (m, 2H), 3.55 (brd d, J )
15 Hz, 1H), 3.73 (m, 2H), 3.81 (s, 3H), 3.98 (brd d, J ) 10 Hz,
1H), 5.93 (s, 2H), 6.76 (d, J ) 9 Hz, 1H), 6.86 (m, 1H), 6.91 (d,
J ) 9 Hz, 2H), 7.02 (t, J ) 9 Hz, 1H), 7.40 (brd d, J ) 10 Hz,
2H), 7.58 (d, J ) 9 Hz, 2H), 8.58 (brd s, 1H); MS (DCI, NH3)
(M + H)+ at m/z 633. Anal. Calcd for C27H24 Br2N2O6‚0.3H2O:
C, 50.85; H, 3.89; N, 4.39. Found: C, 50.45; H, 3.48; N, 4.22.
Com p ou n d 27, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth yl-4-flu or op h en yl]a ceta -
m id o)p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz,
DMSO) δ 1.01 (t, J ) 7.6 Hz, 3H), 2.39 (q, J ) 7.5 Hz, 4H),
2.83 (t, J ) 9.7 Hz, 1H), 2.91 (d, J ) 15.9 Hz, 1H), 3.09 (t, J
) 9.7 Hz, 1H), 3.19 (d, J ) 15.9 Hz, 1H), 3.47 (m, 1H), 3.59
(m, 1H), 3.76 (s, 3H), 3.90 (d, J ) 9.8 Hz, 1H), 5.98 (s, 2H),
6.80 (d, J ) 7.8 Hz, 1H), 6.84 (dd, J ) 1.4, 8.1 Hz, 1H), 6.91
(m, 2H), 6.92 (d, J ) 9.5 Hz, 2H), 7.27 (d, J ) 1.0 Hz, 1H),
7.52 (m, 2H), 9.24 (s, 1H); MS (DCI) (M + H)+ at m/z 549.
Anal. Calcd for C31H33FN2O6‚0.3EtOAc: C, 67.26; H, 6.20; N,
4.87. Found: C, 67.23; H, 6.21; N, 4.80.
1
r r olid in e-3-ca r boxylic a cid : H NMR (300 MHz, CDCl3) δ
1.08 (t, J ) 7 Hz, 6H), 2.42 (q, J ) 7 Hz, 4H), 3.01 (d, J ) 16
Hz, 1H), 3.13 (t, J ) 10 Hz, 1H), 3.15 (dd, J ) 6, 8 Hz, 1H),
3.45 (d, J ) 16 Hz, 1H), 3.56 (dd, J ) 5, 11 Hz, 1H), 3.70 (ddd,
J ) 3, 5, 6 Hz, 1H), 3.82 (s, 3H), 3.96 (d, J ) 10 Hz, 1H), 5.93
(d, J ) 3 Hz, 1H), 5.94 (d, J ) 3 Hz, 1H), 6.75 (d, J ) 8 Hz,
1H), 6.92 (d, J ) 8 Hz, 2H), 7.11 (d, J ) 8 Hz, 2H), 7.21 (dd,
J ) 6, 8 Hz, 1H), 7.39 (d, J ) 8 Hz, 2H), 7.82 (dd, J ) 2, 8 Hz,
1H), 7.89 (d, J ) 3 Hz, 1H), 8.24 (brd s, 1H); MS (DCI, NH3)
(M + H)+ at m/z 531. Anal. Calcd for C31H34N2O6: C, 70.17;
H, 6.46; N, 5.28. Found: C, 69.88; H, 6.42; N, 5.09.
Com p ou n d 20, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2-eth yl-6-p r op ylp h en yl]a ceta m i-
d o)p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz,
CDCl3) δ 0.93 (t, J ) 8 Hz, 3H), 1.15 (t, J ) 8 Hz, 3H), 1.60
(m, 2H), 2.51 (m, 4H), 3.75-3.40 (m, 4H), 3.79 (s, 3H), 3.89
(m, 2H), 4.67 (m, 1H), 5.96 (s, 2H), 7.00-6.70 (m, 7H), 7.41
(m, 3H), 8.84 (brd s, 1H); MS (DCI, NH3) (M + H)+ at m/z 545.
Anal. Calcd for C32H36N2O6‚1.0TFA: C, 62.00; H, 5.66; N, 4.25.
Found C, 61.81; H, 5.90; N, 4.20.
Com p ou n d 21, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ip r op ylp h en yl]a ceta m id o)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz, CDCl3)
δ 0.92 (t, J ) 8 Hz, 3H), 0.96 (t, J ) 8 Hz, 3H), 1.63 (m, 4H),
2.52 (m, 4H), 3.25-2.90 (m, 3H), 3.44 (m, 2H), 3.72 (m, 1H),
3.80 (s, 3H), 3.92 (m, 1H), 5.93 (m, 2H), 6.75 (d, J ) 8 Hz,
1H), 6.83 (m, 2H), 6.88 (d, J ) 9 Hz, 2H), 7.00 (m, 2H), 7.37
(brd d, J ) 9 Hz, 2H), 7.73 (m, 1H), 8.62 (brd s, 1H); MS (DCI,
NH3) (M + H)+ at m/z 559. Anal. Calcd for C33H38N2O6‚
0.5H2O: C, 69.82; H, 6.92; N, 4.93. Found: C, 69.96; H, 6.93;
N, 4.63.
Com p ou n d 31, tr a n s,tr a n s-2-(4-Eth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth ylp h en yl]a ceta m id o)p y-
1
r r olid in e-3-ca r boxylic a cid : H NMR (300 MHz, CDCl3) δ
1.08 (t, J ) 9 Hz, 6H), 1.42 (t, J ) 9 Hz, 3H), 2.43 (q, J ) 9
Hz, 4H), 3.02 (d J ) 18 Hz, 1H), 3.15 (m, 2H), 3.60-3.40 (m,
2H), 3.71 (m, 1H), 4.10-3.90 (m, 3H), 5.92 (d, J ) 2 Hz, 1H),
5.94 (d, J ) 2 Hz, 1H), 6.73 (d, J ) 9 Hz, 1H), 6.83 (dd, J ) 2,
8 Hz, 1H), 6.86 (d, J ) 2 Hz, 1H), 6.90 (d, J ) 9 Hz, 2H), 7.11
(d, J ) 10 Hz, 2H), 7.21 (m, 1H), 7.36 (d, J ) 9 Hz, 2H), 8.26
(brd s, 1H); MS (DCI, NH3) (M + H)+ at m/z 545. Anal. Calcd
for C32H36N2O6‚0.5 H2O: C, 69.42; H, 6.74; N, 5.06. Found:
C, 69.52; H, 6.52; N, 4.89.
Com p ou n d 22, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d iisop r op ylp h en yl]a ceta m i-
d o)p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz,
CDCl3) δ 1.05 (d, J ) 8 Hz, 6H), 1.16 (d, J ) 8 Hz, 6H), 2.84
(m, 2H), 3.01 (d, J ) 18 Hz, 1H), 3.14 (m, 2H), 3.50 (d, J ) 18
Hz, 1H), 3.55 (dd, J ) 6, 12 Hz, 1H), 3.73 (m, 1H), 3.83 (s,
3H), 3.96 (d, J ) 10 Hz, 1H), 5.91 (d, J ) 2 Hz, 1H), 5.93 (d,
J ) 2 Hz, 1H), 6.74 (d, J ) 9 Hz, 1H), 6.83 (dd, J ) 2, 8 Hz,
1H), 6.85 (d, J ) 2 Hz, 1H), 6.93 (d, J ) 9 Hz, 2H), 7.15 (d, J
) 9 Hz, 2H), 7.29 (m, 1H), 7.39 (d, J ) 9 Hz, 2H), 8.29 (brs,
1H); MS (DCI, NH3) (M + H)+ at m/z 559. Anal. Calcd for
Com p ou n d 32, tr a n s,tr a n s-2-(4-P r op oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth ylp h en yl]a ceta m id o)p y-
1
r r olid in e-3-ca r boxylic a cid : H NMR (300 MHz, CDCl3) δ
1.04 (t, J ) 7 Hz, 3H), 1.08 (t, J ) 7 Hz, 6H), 1.82 (sext, J )
7 Hz, 2H), 2.43 (q, J ) 7 Hz, 4H), 3.01 (d, J ) 16 Hz, 1H), 3.13
(t, J ) 10 Hz, 1H), 3.15 (dd, J ) 6, 8 Hz, 1H), 3.48 (d, J ) 16
Hz, 1H), 3.55 (dd, J ) 5, 11 Hz, 1H), 3.70 (ddd, J ) 3, 5, 6 Hz,
1H), 3.85 (q, J ) 7 Hz, 2H), 3.96 (d, J ) 10 Hz, 1H), 5.92 (d,
J ) 3 Hz, 1H), 5.93 (d, J ) 3 Hz, 1H), 6.75 (d, J ) 8 Hz, 1H),
6.90 (d, J ) 8 Hz, 2H), 7.11 (d, J ) 8 Hz, 2H), 7.21 (dd, 6, 8
Hz, 1H), 7.37 (d, J ) 8 Hz, 2H), 7.83 (dd, J ) 2, 8 Hz, 1H),
7.86 (d, J ) 3 Hz, 1H), 8.27 (brd s, 1H); MS (DCI, NH3) (M +
H)+ at m/z 559. Anal. Calcd for C33H38N2O6‚0.25H2O: C, 70.38;
H, 6.89; N, 4.97. Found: C, 70.49; H, 6.85; N, 4.68.
C
33H38N2O6‚0.5H2O: C, 69.82; H, 6.92; N, 4.93. Found: C,
69.69; H, 6.63; N, 4.89.
Com p ou n d 23, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
b en zod ioxol-5-yl)-1-(N-m et h yl-N-[2,6-d iet h ylp h en yl]a c-
eta m id o)-p yr r olid in e-3-ca r boxylic a cid : 1H NMR (CDCl3,
300 MHz) δ 1.03 (t, J ) 7 Hz, 3H), 1.13 (t, J ) 7 Hz, 3H),
2.1-2.4 (m, 4H), 2.46 (d, J ) 16 Hz, 1H), 2.84 (t, J ) 9 Hz,
1H), 2.97 (d, J ) 16 Hz, 1H), 3.06 (t, J ) 10 Hz, 1H), 3.11 (s,
3H), 3.5-3.6 (m, 2H), 3.77 (s, 3H), 3.93 (d, J ) 10 Hz, 1H),
5.93 (dd, J ) 1, 2 Hz, 2H), 6.7-6.8 (m, 3H), 7.0-7.2 (m, 6H);
MS (DCI, NH3) (M + H)+ at m/z 545. Anal. Calcd for
Com p ou n d 33, tr a n s,tr a n s-2-(4-Isop r op oxyp h en yl)-4-
(1,3-ben zodioxol-5-yl)-1-(N-[2,6-dieth ylph en yl]acetam ido)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz, CDCl3)
δ 1.09 (t, J ) 7 Hz, 6H), 1.33 (dd, J ) 2, 7 Hz, 6H), 2.47 (q, J
) 7 Hz, 4H), 3.55 (m, 2H), 3.80 (m, 3H), 4.64 (septet, J ) 7
Hz, 1H), 5.92 (d, J ) 3 Hz, 1H), 5.93 (d, J ) 3 Hz, 1H), 6.78
(d, J ) 8 Hz, 1H), 6.91 (dd, J ) 2, 8 Hz, 1H), 6.99 (brd d, J )
9 Hz, 2H), 7.05 (d, J ) 2 Hz, 1H), 7.11 (dd, J ) 1, 8 Hz, 1H),
7.13 (s, 1H), 7.22 (dd, J ) 8, 9 Hz, 1H), 7.51 (brd d, J ) 9 Hz,
2H); MS (ESI+) (M + H)+ at m/z 559. Anal. Calcd for
C
32H36N2O6: C, 70.57; H, 6.66; N, 5.14. Found: C, 70.26; H,
6.52; N, 4.88.
Com p ou n d 24, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2-eth yl-6-m eth oxyp h en yl]a ceta -
m id o)p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz,
CD3OD) δ 1.10 (t, J ) 8 Hz, 3H), 2.48 (d, J ) 8 Hz, 2H), 3.4-
3.9 (m, 7H), 3.73 (s, 3H), 3.84 (s, 3H), 5.93 (s, 2H), 6.80 (d, J
) 8 Hz, 1H), 6.86 (d, J ) 8 Hz, 2H), 6.93 (dd, J ) 2, 8 Hz, 1H),
7.03 (brd d, J ) 9 Hz, 2H), 7.07 (d, J ) 2 Hz, 1H), 7.23 (t, J )
8 Hz, 1H), 7.53 (brd d, J ) 9 Hz, 2H); MS (APCI) (M + H)+ at
m/z 533. Anal. Calcd for C30H32N2O7‚0.7TFA: C, 61.59; H, 5.38;
N, 4.57. Found: C, 61.27; H, 5.44; N, 4.61.
C
33H38N2O6‚0.7TFA: C, 64.71; H, 6.11; N, 4.39. Found: C,
64.54; H, 5.78; N, 4.21.
Com p ou n d 34, tr a n s,tr a n s-2-(4-[2-Meth oxyeth oxy]p h e-
n yl)-4-(1,3-ben zod ioxol-5-yl)-1-(N-[2,6-d ieth ylp h en yl]a c-
etam ido)pyr r olidin e-3-car boxylic acid: 1H NMR (300 MHz,
CDCl3) δ 1.08 (t, J ) 7 Hz, 6H), 2.43 (q, J ) 7 Hz, 4H), 3.00
(d, J ) 11 Hz, 1H), 3.05-3.15 (m, 2H), 3.44 (s, 3H), 3.46 (d, J
) 11 Hz, 1H), 3.45-3.55 (m, 1H), 3.65-3.75 (m, 1H), 3.75-
Com p ou n d 25, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zodioxol-5-yl)-1-(N-[2,6-dim eth oxyph en yl]acetam ido)-
p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz, CDCl3)

ETB-Selective Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3677
3.80 (m, 2H), 3.93 (d, J ) 7 Hz, 1H), 4.12-4.17 (m, 2H), 5.94
(dd, J ) 2, 4 Hz, 2H), 6.75 (d, J ) 8 Hz, 1H), 6.82 (dd, J ) 2,
9 Hz, 1H), 6.87 (d, J ) 2 Hz, 1H), 6.95 (d, J ) 8 Hz, 1H), 7.10
(d, J ) 6 Hz, 2H), 7.19-7.24 (m, 1H), 7.37 (d, J ) 8 Hz, 2H),
8.29 (s, 1H); MS (APCI+) (M + H)+ at m/z 575. Anal. Calcd
for C33H38N2O7: C, 68.97; H, 6.67; N, 4.87. Found: C, 68.92;
H, 6.83; N, 4.77.
Com p ou n d 35, tr a n s,tr a n s-2-(4-[2-Meth oxyeth yl]p h e-
n yl)-4-(1,3-ben zod ioxol-5-yl)-1-(N-[2,6-d ieth ylp h en yl]a c-
etam ido)pyr r olidin e-3-car boxylic acid: 1H NMR (300 MHz,
DMSO) δ 1.00 (t, J ) 7.5 Hz, 6H), 2.40 (q, J ) 7.5 Hz, 4H),
2.66 (t, J ) 8.3 Hz, 1H), 2.79 (t, J ) 6.8 Hz, 2H), 2.87 (d, J )
15.6 Hz, 1H), 3.00 (t, J ) 9.2 Hz, 1H), 3.20 (d, J ) 15.9 Hz,
1H), 3.24 (s, 3H), 3.42-3.47 (m, 1H), 3.53 (t, J ) 6.9 Hz, 1H),
3.60 (m, 1H), 3.91 (d, J ) 9.5 Hz, 1H), 5.94 (s, 2H), 6.76 (d, J
) 7.8 Hz, 1H), 6.84 (dd, J ) 1.4, 8.1 Hz, 1H), 7.08 (d, J ) 7.5
Hz, 2H), 7.17 (d, J ) 6.4 Hz, 3H), 7.22 (d, J ) 1.4 Hz, 1H),
7.51 (d, J ) 8.1 Hz, 2H), 9.21 (s, 1H); MS (DCI) (M + H)+ at
m/z 559. Anal. Calcd for C33H38N2O6‚0.2H3PO4: C, 68.54; H,
6.73; N, 4.84. Found C, 68.28; H, 6.46; N, 4.82.
Com p ou n d 28, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth yl-4-m eth ylp h en yl]a ce-
ta m id o)p yr r olid in e-3-ca r boxylic a cid . Ethyl trans,trans-
2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(4-bromo-2,6-
d ie t h ylp h e n yl)a m in oca r b on ylm e t h yl)p yr r olid in e -3 -
carboxylate was prepared according to the procedures described
above. This compound (200 mg, 0.31 mmol), in solution in 8
mL of THF, was added to a mixture (purged with nitrogen) of
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1
complex with dichloromethane) (13 mg) and cesium carbonate
(307 mg, 0.942 mmol) in anhydrous N,N-dimethylformamide
(2 mL) at ambient temperature. After stirring the mixture for
10 min at ambient temperature, 1.0 M trimethylborane (0.471
mL, 0.471 mmol) in tetrahydrofuran was added. The reaction
was stirred overnight at 65 °C under nitrogen. The reaction
mixture was diluted with ethyl acetate (100 mL) and washed
with water (2 × 30 mL) and brine. The organic phase was dried
with sodium sulfate, and the solvents were removed in vacuo
. The residue was chromatographed on silica gel eluting with
3:1 hexanes-ethyl acetate to give ethyl trans,trans-2-(4-
methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(N-[2,6-diethyl-4-
methylphenyl]acetamido)pyrrolidine-3-carboxylate (110 mg,
60% yield). This material was hydrolyzed as described above
to give the title compound: 1H NMR (300 MHz, CDCl3) δ 1.07
(t, J ) 9 Hz, 6H), 2.28 (s, 3H), 2.39 (q, J ) 9 Hz, 4H), 3.00 (d,
J ) 18 Hz, 1H), 3.13 (m, 2H), 3.45 (d, J ) 18 Hz, 1H), 3.55
(dd, J ) 2, 9 Hz, 1H), 3.72 (m, 1H), 3.81 (s, 3H), 3.95 (d, J )
10 Hz, 1H), 5.91 (d, J ) 2 Hz, 1H), 5.93 (d, J ) 2 Hz, 1H), 6.75
(d, J ) 9 Hz, 1H), 6.82 (dd, J ) 2, 8 Hz, 1H), 6.86 (d, J ) 2 Hz,
1H), 6.92 (m, 4H), 7.38 (d, J ) 9 Hz, 2H), 8.20 (brd s, 1H); MS
(DCI, NH3) (M + H)+ at m/z 545. Anal. Calcd for C32H36N2O6‚
0.5 H2O: C, 69.42; H, 6.74; N, 5.06. Found: C, 69.43; H, 6.57;
N, 4.94.
9 Hz, 2H), 7.27 (m, 2H), 7.54 (d, J ) 9 Hz, 2H), 7.68 (brd s,
2H); MS (DCI, NH3) (M + H)+ at m/z 575. Anal. Calcd for
C32H34N2O8‚0.5H2O: C, 65.85; H, 6.04; N, 4.80. Found: C,
66.03; H, 5.84; N, 4.67.
P r ep a r a tion of En a n tiom er ica lly P u r e An ta gon ists.
Core pyrrolidines 14 were resolved into individual enantiomers
as follows: for R3 ) OCH3 or OEt, through preparation of a
salt with mandelic acid, followed by multiple recrystallizations;
for R3 ) OPr or OCH2CH2OCH3, through preparative HPLC
on a chiral Regis Whelk-O2 column. Alkylation and hydrolysis
of the resulting chiral cores led to compounds 36-39, which
were analyzed by chiral HPLC using a Regis analytical
Whelk-O column. Compounds 36-39 were all determined to
be >99% enantiopure.
Recep tor Bin d in g Assa ys. All samples were kept at 4 °C
throughout the process of membrane isolation. MMQ cells
(prolactin-secreting rat pituitary cells known to contain ETA
receptors), porcine cerebellar tissues (known to contain ETB
receptors), or Chinese hamster ovary cells (CHO) permanently
transfected with the human ETA or ETB receptor were homog-
enized in 25 mL of 10 mM Hepes (pH 7.4) containing 0.25 M
sucrose and a protease inhibitor cocktail (50 mM EDTA, 0.1
mM PMSF, 5 µg/mL pepstatin A, and 0.025% bacitracin) using
a microultrasonic cell disruptor (Kontes). The mixture was
centrifuged at 1000g for 10 min. The supernatant was collected
and centrifuged at 60000g for 60 min. The precipitate was
resuspended in 20 mM Tris, pH 7.4, containing protease
inhibitor cocktail and centrifuged again. The final membrane
pellet was resuspended in 20 mM Tris, pH 7.4, containing
protease inhibitors and stored at -80 °C until used. Protein
content was determined by the Bio-Rad dye-binding protein
assay.
Binding assays were performed in 96-well microtiter plates
pretreated with 0.1% BSA. Membranes were diluted ∼100-
fold in buffer B (20 mM Tris, 100 mM NaCl, 10 mM MgCl2,
pH 7.4, with 0.2% BSA, 0.1 mM PMSF, 5 µg/mL pepstatin A,
0.025% bacitracin, and 50 mM EDTA) to a final concentration
of 0.2 mg/mL protein. In competition binding studies, mem-
branes (0.02 mg) were incubated with 0.1 nM [125I]ET-1 (for
ETA assay in MMQ or CHO cells) or [125I]ET-3 (for ETB assay
in porcine cerebellum or CHO cells) in buffer B (final volume
0.2 mL) in the presence of increasing concentrations of the test
compound for 3 h at 25 °C. After incubation, unbound ligand
was separated from bound ligand by a vacuum filtration
method using glass-fiber filter strips in PHD cell harvesters
(Cambridge Technology, Inc., MA), washing the filter strips
three times with saline (1 mL). Nonspecific binding was
determined in the presence of 1 µM unlabeled ET-1. IC50 values
were calculated using an average of at least two separate
determinations.
P h osp h oin ositol Hyd r olysis Assa ys. ETA: MMQ cells
(0.4 × 106 cells/mL) were labeled with 10 µCi/mL [3H]-
myoinositol in RPMI for 16 h. The cells were washed with PBS,
then incubated with buffer A (140 mM NaCl, 5 mM KCl, 2
mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 25 mM Hepes, pH
7.4) containing protease inhibitors and 10 mM LiCl for 60 min.
The cells were incubated with test compounds for 5 min and
then challenged with 1 nM ET-1. ET-1 challenge was termi-
nated by the addition of 1.5 mL of 1:2 (v/v) chloroform-
methanol. Total inositol phosphates were extracted after
adding chloroform and water to give final proportions of 1:1:
0.9 (v/v/v) chloroform-methanol-water as described by Ber-
ridge.9 The upper aqueous phase (1 mL) was retained, and a
small portion (100 µL) was counted. The rest of the aqueous
sample was analyzed by batch chromatography using anion-
exchange resin AG1-X8 (Bio-Rad).
The following compounds were prepared using the above
procedure.
Com p ou n d 29, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth yl-4-eth ylp h en yl]a ceta -
m id o)p yr r olid in e-3-ca r boxylic a cid : 1H NMR (300 MHz,
CDCl3) δ 1.08 (t, J ) 9 Hz, 6H), 1.22 (t, J ) 9 Hz, 3H), 2.40 (q,
J ) 9 Hz, 4H), 2.60 (q, J ) 9 Hz, 2H), 3.00 (d, J ) 18 Hz, 1H),
3.13 (m, 2H), 3.46 (d, J ) 18 Hz, 1H), 3.52 (dd, J ) 2, 9 Hz,
1H), 3.71 (m, 1H), 3.82 (s, 3H), 3.95 (d, J ) 10 Hz, 1H), 5.91
(d, J ) 2 Hz, 1H), 5.93 (d, J ) 2 Hz, 1H), 6.75 (d, J ) 9 Hz,
1H), 6.82 (dd, J ) 2, 8 Hz, 1H), 6.84 (d, J ) 2 Hz, 1H), 6.91 (d,
J ) 9 Hz, 2H), 6.95 (s, 2H), 7.38 (d, J ) 9 Hz, 2H), 8.22 (brd
s, 1H); MS (DCI, NH3) (M + H)+ at m/z 559. Anal. Calcd for
C
33H38N2O6‚0.25 H2O: C, 70.38; H, 6.89; N, 4.97. Found: C,
ETB: Chinese hamster ovary cells (CHO) permanently
transfected with the human ETB receptor were grown to
confluence in 24-well tissue culture plates and labeled with 5
µCi/well [3H]myoinositol in F-12 media + 10% FBS + 1xP/S/
F. The adherent cells were washed gently with PBS and then
incubated in 200 µL of buffer A containing protease inhibitors
and 10 mM LiCl for 60 min at 37 °C in a CO2 incubator. Test
compounds were then added followed by the addition of 1 nM
70.18; H, 7.14; N, 4.63.
Com p ou n d 30, tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-
ben zod ioxol-5-yl)-1-(N-[2,6-d ieth yl-4-ca r boxyp h en yl]a c-
etam ido)pyr r olidin e-3-car boxylic acid: 1H NMR (300 MHz,
DMSO) δ 1.04 (t, J ) 9 Hz, 6H), 2.44 (q, J ) 9 Hz, 4H), 3.00-
2.80 (m, 3H), 3.45-3.00 (m, 2H), 3.62 (m, 1H), 3.76 (s, 3H),
3.92 (d, J ) 9 Hz, 1H), 5.98 (s, 2H), 6.83 (m, 2H), 6.93 (d, J )

3678 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
von Geldern et al.
(2) (a) Opgenorth, T. J . Endothelin Receptor Antagonism. Adv.
Pharmacol. 1995, 33, 1-65. (b) Mattoli, S.; Soloperto, M.; Marini,
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ET-1 and incubated for 30 min at 37 °C. The cells were then
solubilized by the addition of 50 µL of 1 N NaOH and
neutralized by the addition of 50 µL of 1 N HCl. The solubilized
cell suspension was transferred to glass tubes and extracted
by the addition of 1.5 mL of 1:2 (v/v) chloroform-methanol.
Total inositol phosphates were extracted and analyzed by
batch chromatography on anion-exchange resin as above. All
IC50 values were calculated using an average of at least two
separate determinations.
P h a r m a cok in etic An a lysis. The pharmacokinetic behav-
ior of compound 16h was evaluated in male Sprague-Dawley
rats. Briefly, the test compound was prepared as a 10 mg/mL
solution in an ethanol:propylene glycol:D5W (20:30:50, by
volume) vehicle containing 1 mol equiv of sodium hydroxide.
Groups of rats (n ) 4/group) received either a 10 mg/kg (1 mL/
kg) intravenous dose administered as a slow bolus in the
jugular vein or a 10 mg/kg (1 mL/kg) oral dose administered
by gavage. Heparinized blood samples (∼0.4 mL/sample) were
obtained from a tail vein of each rat 0.1 (iv only), 0.25, 0.5, 1,
1.5, 2, 3, 4, 6, 9, and 12 h after dosing. The samples were
analyzed by reverse-phase HPLC following liquid-liquid
extraction from the plasma. Initial estimates of the pharma-
cokinetic parameters (e.g. the maximum concentration Cmax
)
for NONLIN8410 were obtained with the program CSTRIP.11
Area under the curve (AUC) values were calculated by the
trapezoidal rule over the time course of the study. Assuming
dose proportionality and correcting for the differences in
dosing, a comparison of the AUC following oral dosing with
that obtained following an intravenous dose provided an
estimate of the bioavailability (F).
(5) Clozel, M.; Gray, G. A.; Breu, V.; Loffler, B.-M.; Osterwalder, R.
Endothelin ET-B Receptor Mediates Both Vasodilation and
Vasoconstrction in vivo. Biochem. Biophys. Res. Commun. 1992,
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Receptors in Rats. Biochem. Biophys. Res. Commun. 1994, 199,
1461-1465.
P seu d oeffica cy Stu d ies. Briefly, rats were anesthetized
with methoxyflurane (Penthrane, USP), and a peripheral vein
and artery were catheterized with polyurethane tubing (Mi-
crorenathane, Braintree Scientific, Braintree, MA) for drug or
ET peptide administration and for measurement of mean
systemic arterial pressure (MAP), respectively. Following
recovery to consciousness (>45 min), rats were dosed orally
by gavage with either vehicle or A-192621 (dose volumes ) 2
mL/kg) and placed unrestrained in individual cages. Their
arterial catheters were coupled to fluid-filled strain-gauge
transducers connected to a digital data acquisition system
(Mi2, Malvern, PA) for determination of blood pressure and
heart rate (HR). ET-1 (0.3 nmol/kg, i.v. in 0.1% BSA-saline)
was administered as a bolus 1 h after the oral dose of A-192621
(30 mg/kg) or vehicle. Baseline blood pressures and HR
(sampled at 2-5-min intervals) were averaged for 30 min prior
to infusion of the ET peptide. The peak value of the transient
vasodepressor response to an ET peptide was determined from
the calibrated strip chart of recording using the algorithm:
MAP ) 1/3(systolic - diastolic) + diastolic. The pressor
response to the ET peptide was quantified as the peak change
in MAP relative to baseline: peak ∆MAP ) maximum change
of MAP following ET-1 challenge. The time-dependent vaso-
pressor effect was calculated from the AUV of MAP versus
time and expressed as a percent change from baseline in each
animal: AUCbp ) % ∆MAP × time.
(7) (a) Ishikawa, K.; Ihara, M.; Noguchi, K.; Mase, T.; Mino, N.;
Saeki, T.; Fukuroda, T.; Fukami, T.; Ozaki, S.; Nagese, T.;
Nishikibe, J .; Yano, M. Biochemical and Pharmacological Profile
of a Potent and Selective Endothelin B-Receptor Antagonist BQ-
788. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4892-4896. (b) Chan,
M. F.; Kois, A.; Raju, B.; Castillo, R. S.; Verner, E. J .; Wu, C.;
Venkatachalapathi, Y. V.; Okun, I.; Stavros, F. D.; Balaji, V. N.
The Discovery of Non-Peptide, Low Molecular Weight, ETB
Selective Endothelin Antagonists. ACS National Meeting, Ana-
heim, CA, April 1995; MEDI 168. (c) Breu, V.; Clozel, M.; Burri,
K.; Hirth, G.; Neidhart, W.; Ramuz, H. In vitro characterization
of Ro 46-8443, the first non-peptide antagonist selective for the
endothelin ETB receptor. FEBS Lett. 1996, 383, 37-41.
(8) Winn, M.; von Geldern, T. W.; Opgenorth, T. J .; J ae, H.-S.;
Tasker, A. S.; Boyd, S. A.; Kester, J . A.; Mantei, R. A.; Bal, R.;
Sorensen, B. K.; Wu-Wong, J . R.; Chiou, W.; Dixon, D. B.;
Novosad, E. I.; Hernandez, L.; Marsh, K. C. 2,4-Diarylpyrroli-
dine-3-carboxylic AcidssPotent ETA Selective Endothelin Recep-
tor Antagonists. 1. Discovery of A-127722. J . Med. Chem. 1996,
39, 1039-1048.
(9) J ae, H. S.; Winn, M.; Dixon, D. B.; Marsh, K. C.; Nguyen, B. N.;
Opgenorth, T. J .; von Geldern, T. W. Pyrrolidine-3-carboxylic
acids as Endothelin Antagonists. II. Sulfonamide-based ETA/ETB
Mixed Antagonists. J . Med. Chem. 1997, 40, 3217-3227.
(10) Fukuroda, T.; Fukikawa, T.; Ozaki, S.; Ishikawa, K.; Yano, M.;
Nishikibe, M. Clearance of circulating endothelin-1 by ETB
receptors in rats. Biochem. Biophys. Res. Commun. 1994, 199,
1461-1465.
Lon g-Ter m Tr ea tm en t Stu d y. In a subchronic study,
A-192621 (30 mg/kg/day) or vehicle (tap water) was adminis-
tered to rats for 3 days in the diet (mixed in meal, #5002, PMI
Nutrition International, St. Louis, MO). On the third day of
dosing, rats were instrumented and caged as described above.
MAP and HR were determined over 90 min (averaged), after
which blood samples were drawn for determination of plasma
ET-1 and drug concentrations.
(11) Adler, A. L.; Wessale, J . L.; Calzadilla, S. C.; Dixon, D. B.;
Dayton, B. D.; Opgenorth, T. J . Characterization of hypertension
induced by chronic ET-B receptor blockade in conscious rats.
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Ack n ow led gm en t. The authors thank the Abbott
1
Analytical Department for assistance in acquiring H
NMR and mass spectra.
(14) Sedman, A. J .; Wagner, J . G. CSTRIP - A FORTRAN Computer
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