Potent and selective non-benzodioxole-containing endothelin-A receptor antagonists

Article abstract of DOI:10.1021/jm960077r

The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET) receptor antagonists thus far reported. As part of our own endothelin antagonist program we have developed (2R*,3R*,4S*)-1-(N,N- dibutylacetamido)-4-(1,3-benzodioxol-5-yl)-2-(4-methoxyphenyl)pyrrolidine-3- carboxylic acid (A-127722). This is a potent antagonist, binding to the ET(A) and ET(B) receptor subtypes with affinities (IC₅₀) of 0.4 and 520 nM, respectively, and also contains the aforementioned benzodioxole. While this compound was seemingly optimized at its N-terminus, no effort had been directed toward understanding the contributions to binding affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or 2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p- anisyl, were potent compounds with IC₅₀s in the low-nanomolar range. Simple deletion of either of the two oxygen atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity for the ET(A) receptor. Additionally, the compounds showed enhanced selectivity, binding to the ET(B) receptor subtype in the micromolar range. This paper describes the development of this novel class of compounds.

Full text of DOI:10.1021/jm960077r

322
J . Med. Chem. 1997, 40, 322-330
P oten t a n d Selective Non -Ben zod ioxole-Con ta in in g En d oth elin -A Recep tor
An ta gon ists
Andrew S. Tasker,* Bryan K. Sorensen, Hwan-Soo J ae, Martin Winn, Thomas W. von Geldern,
Douglas B. Dixon, William J . Chiou, Brian D. Dayton, Samuel Calzadila, Lisa Hernandez, Kennan C. Marsh,
J . Ruth WuWong, and Terry J . Opgenorth
Aging and Degenerative Diseases Research, Pharmaceutical Products Division, Abbott Laboratories, D-47V, AP10/ 3,
100 Abbott Park Road, Abbott Park, Illinois 60064
Received September 16, 1996X
The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET)
receptor antagonists thus far reported. As part of our own endothelin antagonist program we
have developed (2R*,3R*,4S*)-1-(N,N-dibutylacetamido)-4-(1,3-benzodioxol-5-yl)-2-(4-methoxy-
phenyl)pyrrolidine-3-carboxylic acid (A-127722). This is a potent antagonist, binding to the
ETA and ETB receptor subtypes with affinities (IC50) of 0.4 and 520 nM, respectively, and also
contains the aforementioned benzodioxole. While this compound was seemingly optimized at
its N-terminus, no effort had been directed toward understanding the contributions to binding
affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or
2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p-anisyl, were potent
compounds with IC50s in the low-nanomolar range. Simple deletion of either of the two oxygen
atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity
for the ETA receptor. Additionally, the compounds showed enhanced selectivity, binding to
the ETB receptor subtype in the micromolar range. This paper describes the development of
this novel class of compounds.
Ch a r t 1
In tr od u ction
Endothelins (ET) are 21-amino acid bicyclic peptides
originally isolated from the supernatant of cultured
porcine endothelial cells.1 ET-1 is one member of a
family of isopeptides encoded by three distinct genes;
ET-2 and ET-3 are also members of this class.2 Some
10-fold more potent than angiotensin II, ET-1 is the
most powerful pressor peptide isolated to date and also
a potent mitogen. These diverse actions are attributed
to the existence of multiple receptor subtypes with
discrete cellular distributions and functions. This sug-
gests an unusual role for these hormones in the patho-
genesis of cardiovascular diseases. Thus far, two sub-
types of the ET receptor have been characterized.3 The
ETA receptor is found principally in vascular smooth
muscle and binds the isopeptides with affinities in the
order ET-1 ∼ ET-2 > ET-3. It regulates vascular tone4
and plays a role in smooth muscle cell proliferation.5
The ETB receptor has equal affinity for the three peptide
isoforms. ETB was originally considered the ‘vasodilator
receptor’ due to its mediation of nitric oxide release.6 It
is now apparent that ETB receptors are involved in
smooth muscle contraction in blood vessels such as the
rabbit saphenous vein7 and the guinea pig bronchus.8
Numerous reports exist in the literature describing both
peptidic and nonpeptidic antagonists. ETA-selective
peptidic agents include BQ 1239 and FR 139317.10 BQ
78811 and IRL 103812 are specific for the ETB receptor.
In addition, a hexapeptide (PD 142893)13 displays
nonselective antagonism at both receptors. Nonselec-
tive, nonpeptidic structures include Ro 47-0203,14 SB
209670,13 and L-749,329.16 The first potent and highly
selective ETA antagonist, PD 156707, was disclosed
recently.17 We18 recently reported a series of potent
endothelin antagonists based on pyrrolidinecarboxylic
acids (Chart 1).
Cytochrome P450 is essential to the phase I metabo-
lism of foreign compounds.19 This enzyme system is a
ubiquitous monooxygenase that effects numerous oxida-
tive processes required by the organism. In certain
instances the benzodioxole group can inhibit and induce
cytochrome P450, its subsequent degradation following
either of two pathways: It can undergo oxidation to
yield formate or carbon monoxide and a catechol, which
is then redox active and may enter into quinone-reactive
oxygen-based toxicity, or alternatively, it may complex
with the heme function by way of a metallocarbene. In
certain instances these complexesscharacterized by their
absorption at 455 nmsexhibit high stability and inhibit
the catalytic cycle of the enzyme. This phenomenon has
been used to advantage with the insecticide synergist
piperonyl butoxide.
With the exception of Ro 47-0203 and BMS-182,874,20
each series of the nonpeptide endothelin antagonists
reported to date contains the benzodioxole group. Seek-
ing to avoid possible complications arising from P450
oxidation, we synthesized a number of compounds with
benzodioxole replacements. These compounds were
potent and displayed high ETA selectivity.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00077-5 CCC: $14.00 © 1997 American Chemical Society

Non-Benzodioxole ETA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 323
Sch em e 1a
a
i
(a) NaH, CO(OEt)2; (b) MeNO2, 50% NaOH, 15 °C, then pour into 6 N aq HCl; (c) nitrostyrene 5, 5 mol % DBU, PrOH, THF; (d) W-2
i
Raney Ni, 4 atm of H2, EtOAc; (e) NaBH3CN, HCl, EtOH, THF, pH 3-4; (f) BrCH2C(O)N(Me)Pr, Pr2NEt, MeCN; (g) 50% aq NaOH,
EtOH.
Sch em e 2a
a
t
(a) MeOCHCl2, SnCl4, CH2Cl2; (b) NaH, DMF, 0 °C, then BrCH2CH(OEt)2; (c) 1.5 equiv of PPA, PhH, reflux; (d) 2 equiv of BuLi,
Et2O, -78 °C, then DMF.
Ch em istr y
3-4 h), and the pure trans-trans acid 10 was isolated
by simple extractive workup.
All compounds were prepared in a racemic form as
previously described18 and depicted in Scheme 1. Car-
bethoxylation of 4′-methoxyacetophenone (2) with so-
dium hydride in diethyl carbonate yielded the â-keto
ester 3. Condensation21 of nitromethane with the
requisite aldehyde 4 (vide infra) afforded the nitrosty-
rene 5. Treatment of an equimolar mixture of 3 and 5
with a catalytic amount of DBU yielded nitro ketone 6
as a mixture of diastereomers. The nitro group was
reduced with Raney nickel to form an intermediate
amino ketone that underwent spontaneous cyclization
to pyrroline 7. This compound was rapidly reduced with
sodium cyanoborohydride at pH 3-4 to give pyrrolidine
8 as a mixture of four diastereomers. The cis-cis
isomer was readily separable by flash chromatography.
This compound, however, usually accounted for only
20% of the material and was routinely discarded. The
remaining inseparable mixture of cis-trans, trans-
trans, and trans-cis was alkylated with N-methyl-N-
propylbromoacetamide to yield ester 9 and subsequently
hydrolyzed. We were extremely fortunate in that the
cis-trans epimers were resistant to hydrolysis under
the conditions employed (50% aqueous sodium hydrox-
ide in an equal volume of ethanol, room temperature,
Aldehydes were synthesized by various routes as
delineated in Scheme 2. Indan-5-carboxaldehyde (4d )
was prepared by the method of Mathison,22 employing
stannic chloride in place of titanium tetrachloride.
Indole-6-carboxaldehyde (4e) was prepared by the method
of Rapoport.23 Stannic chloride- catalyzed condensation
of R,R-dichloromethyl methyl ether with 2,3-dihydroben-
zofuran afforded almost exclusively the 5-carboxalde-
hyde 4f. The 7-isomer impurity, estimated at ∼5% by
proton NMR, was readily removed in the subsequent
Henry reaction, at which point the nitrostyrene was
purified by recrystallization. Benzofuran-5-carboxal-
dehyde (4g) was synthesized following a procedure
adapted from Barker.24 Reaction of 2-bromoacetalde-
hyde diethyl acetal with 4-bromophenol afforded ether
11. Subsequent intramolecular Friedel-Crafts reaction
with polyphosphoric acid in refluxing benzene gave
5-bromobenzofuran 12. The crude product was sub-
jected to halogen-metal exchange and subsequent
formylation with DMF in the manner described for 4f
to yield the aldehyde 4g.
Benzofurans bearing the aldehyde function at the 4-
or 6-position were prepared in an analogous fashion

324 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Tasker et al.
starting with 3-bromophenol. The resultant mixture of
4- and 6-bromobenzofurans was carried on as described
(vide supra) to yield the aldehydes 4h ,i, which were
readily separated by flash chromatography. Fluoroben-
zaldehydes and anisaldehydes are commercially avail-
able. The final diarylpyrrolidinecarboxylic acid 10j
bearing the 2,3-dihydro-6-benzofuran pendant was pre-
pared by catalytic hydrogenation25 of the unsaturated
analogue 10i (H2 at 60 psi, Pd(C), AcOH, 16 h, 78%
yield). The 4-substituted benzofuran 10h , proved re-
sistant to reduction. 2-(3-Pyridyl)nitroethylene (5a ) was
prepared by the procedure of Bourguignon.26
5-isomer 14b displays higher selectivity, with an ETB/
ETA ratio of 4300. Interestingly, the 6-substituted
analogue 14d is almost 5-fold less selective. A similar
picture holds for the unsaturated analogues 14a ,c:
Overall activity increases greatly. Reducing the Lewis
basicity of the oxygen atom, however, yields a compound
with enhanced selectivity for the ETA receptor. In
contrast to their saturated analogues, the unsaturated
5- and 6-benzofurans 14a ,c have essentially the same
binding profile. The trend continues with the fluoro-
and methoxy-substituted analogues. All display high
affinity (1-10 nM) and selectivities in the 3000-10000-
fold range. Again the benzodioxan 14l follows the same
pattern, showing increased binding affinity for both
receptor subtypes. The indan 14m possessing no hy-
drogen-bonding capability is only 2-fold weaker with an
IC50 of 1.5 nM and shows great selectivity (5300-fold).
This illustrates that the presence of an electron-rich
aromatic ring is desirable but not essential for activity.
These results suggest the presence of a hydrogen-
bonding site in the ETB receptor situated to interact
with an acceptor at the meta-position of the C(4) phenyl
substituent.
Resu lts a n d Discu ssion
At the outset of this work we were concerned over the
potential of the benzodioxole moiety in certain com-
pounds to undergo metabolism by cytochrome P450. The
presence of a methylene bonded by two oxygens is
apparently critical for this process to occur. Our initial
studies focused on replacing this group with simple
carbocyclic analogues. Naphthalene, bonded at either
the 1- or 2-position (Table 1, entries 10a ,b), resulted in
a dramatic reduction in binding affinity; this was
equally true for the phenyl analogue 10c. A better
substitution was the isosteric 5-indan 10d , which was
50-fold less potent than benzodioxole 1. The hydrogen
bond-donating 6-indole 10e was exceptionally deleteri-
ous. We felt, therefore, that a hydrogen-bond acceptor
was necessary for good potency. Simple deletions of
each oxygen atom in turn yielded a series of dihydro-
benzofurans: The 5-substituted dihydrobenzofuran 10f
and the 6-isomer 10j were the most potent compounds
with IC50s of 130 and 137 nM, respectively. Benzofuran
10i displayed somewhat lower affinity. Driven by these
results, we sought to design simpler compounds. The
p-methoxyphenyl analogue 10k possessed activity mid-
way between the benzofurans and their saturated
analogues. A surprising result is observed with the
m-methoxyl 10l: It would appear the binding pocket has
rather strict steric requirements, and the freely rotating
methyl group suffers an unfavorable interaction with
the receptor. Adding an additional hydrogen bond
acceptor at the para-position (entries 10m ,n ) restores
the binding affinity. Especially interesting is the 3,4-
difluorobenzene 10o. This substituent is barely larger
than the phenyl 10c yet has a binding affinity almost
10-fold higher. The benzodioxan 10p yields surprising
results: It is somewhat isosteric with the 2-naphthyl
yet displays greatly increased binding affinity. Fur-
thermore, the compound shows increased ETB activity.
All the analogues described are, however, significantly
weaker antagonists than the benzodioxole 1.
Pharmacokinetic studies in conscious rats were per-
formed on a number of the most potent compounds. The
results are summarized in Table 3. All compounds were
well absorbed and had oral bioavailabilities in the 30-
65% range.
Con clu sion s
At the outset of this work we wanted to examine the
role played by the benzodioxole moiety in endothelin
receptor binding. Additionally we were concerned with
the propensity of benzodioxole-containing compounds to
undergo cytochrome P450-mediated metabolism, wherein
the substrate becomes irreversibly bound to the enzyme.
This type of metabolism may lead to drug-drug inter-
actions or nonlinear pharmacokinetics. With this in
mind we sought to develop compounds devoid of this
functionality. We have since attempted to induce
A-127722 to form such P450-carbene complexes (de-
tected by a shift in the ultraviolet absorbance from 450
to 455 nm) and have been unable to find any direct
evidence for such an event. Nonetheless, these benzo-
dioxole replacement studies have proved to be interest-
ing for other reasons. We have developed a series of
agents that function as orally active, highly potent
antagonists for the ETA receptor; more importantly
several of these compounds display increased selectivity
over the ETB receptor. Compounds lacking the meta-
oxygen on the C(4) phenyl group provide this increased
selectivity without sacrificing potency.
During this work it was discovered18 that increasing
the side chain length from N-methyl-N-propyl to N,N-
dibutyl enhanced the potency of 1 15-fold, and yields
compound 13 (A-127722, Chart 2) that displays 1000-
fold selectivity with receptor binding affinities of 0.4 and
520 nM for the ETA and ETB subtypes, respectively.
Accordingly, the most potent compounds from the N-
methyl-N-propylamide series were resynthesized incor-
porating the extended chain. Table 2 summarizes the
biological data. The increased binding affinity is more
striking for the non-benzodioxoles: Dihydrobenzofurans
14b,d show a 300-fold increase in binding affinity, and
both are extremely potent ETA receptor antagonists with
IC50s of 0.6 and 1 nM, respectively. Moreover, the
Exp er im en ta l Section
Gen er a l. Compounds were prepared in a fashion analogous
to ref 18 substituting the appropriate aldehyde. All solvents
and reagents were reagent grade unless otherwise specified
and were purchased from Aldrich Chemical Co. Flash chro-
matography was performed on silica gel (230-400 mesh) from
E. M. Science. Proton NMR spectra were recorded on a
General Electric QE300 instrument. Chemical shifts are
reported in ppm downfield from tetramethylsilane as an
internal standard. Elemental analyses were obtained from
Robertson Microlit Laboratories, Madison, NJ . Abbrevia-
tions: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, N,N-
dimethylformamide; THF, tetrahydrofuran.

Non-Benzodioxole ETA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 325
Ta ble 1. Biological Data of N-Methyl-N-propyl-Substituted Pyrrolidine Amides
a
b
Compounds gave satisfactory analyses within (0.4% of theoretical calculations unless otherwise stated. Number of determinations
in parentheses.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
Hz), 7.86 (2H, d, J ) 8 Hz), 7.75 (1H, d, J ) 8 Hz), 7.49 (3H,
m), 7.34 (2H, dd, J ) 3, 9 Hz), 6.83 (2H, dd, J ) 9, 2 Hz), 4.50
(1H, m), 3.94 (1H, dd, J ) 9, 2 Hz), 3.78 (3H, s), 3.65 (1H, m),
3.49 (1H, d, J ) 14 Hz), 3.40-2.93 (5H, m), 2.91 (2.83) (3H,
m et h oxyp h en yl)-4-(1-n a p h t h yl)p yr r olid in e-3-ca r b oxy-
lic a cid (10a ): substituting naphthalene-1-carboxaldehyde;
1H NMR (300 MHz, CDCl3) (rotamer) δ 8.29 (1H, bd, J ) 8

326 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Tasker et al.
hyde; 1H NMR (300 MHz, CDCl3) (minor rotamer) δ 7.65 (1H,
bd), 7.60 (1H, d, J ) 2 Hz), 7.55 (1H, d, J ) 8 Hz), 7.35 (3H,
m), 6.85 (2H, dd, J ) 8, 3 Hz), 6.75 (1H, dd, J ) 3, 2 Hz), 3.83
(2H, m), 3.79 (3H, s), 3.60-3.0 (7H, m), 2.91 (2.83) (s, 3H),
1.51 (2H, sept, J ) 7 Hz), 0.83 (0.78) (3H, t, J ) 7 Hz); MS
Ch a r t 2
(DCI/NH3) m/ z 451 (MH+), 350, 263. Anal. (C26H30
N2O5‚0.5H2O) C, H, N.
-
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyp h en yl)-4-(6-ben zo-2,3-d ih yd r ofu r a n yl)p yr r oli-
d in e-3-ca r boxylic a cid (10j): prepared by catalytic hydro-
genation of 9i (4 atm of H2, AcOH, followed by preparative
HPLC); 1H NMR (300 MHz, CDCl3) (minor rotamer) δ 7.49
(7.47) (2H, d, J ) 8 Hz), 7.19 (1H, d, J ) 8 Hz), 7.00 (1H, m),
7.82 (3H, m), 5.40 (1H, dd, J ) 11, 7 Hz), 4.58 (2H, t, J ) 8
Hz), 4.18 (1H, m) 4.10 (1H, m), 3.88 (1H, m), 3.79 (3H, s), 3.60
(1H, m), 3.35 (1H, m), 3.19 (2H, t, J ) 8 Hz), 3.00 (4H, m),
2.91 (2.78) (s, 3H), 1.53 (1.40) (2H, sept, J ) 7 Hz), 0.88 (0.78)
(3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 453 (MH+), 352. Anal.
(C26H32N2O5‚1.25TFA) C, H, N.
(2R*,3R*,4S*)-1-(N-Meth yl-N-p r op yla ceta m id o)-2,4-bis-
(4-m eth oxyp h en yl)p yr r olid in e-3-ca r boxylic a cid (10k ):
substituting 4-methoxybenzaldehyde; 1H NMR (300 MHz,
CDCl3) (minor rotamer) δ 7.37 (2H, d, J ) 7.5 Hz), 7.32 (2H,
d, J ) 7.5 Hz), 6.86 (4H, m), 3.83 (1H, m), 3.81 (3H, s), 3.79
(3H, s), 3.64 (1H, m), 3.48-2.97 (6H, m), 2.87 (2.83) (3H, s),
2.85 (1H, m), 1.45 (2H, m), 0.84 (0.74) (3H, t, J ) 7.5 Hz); MS
(DCI/NH3) m/ z 441 (MH+). Anal. (C25H32N2O5‚0.5H2O) C, H,
N.
s), 1.48 (2H, sept, J ) 7 Hz), 0.83 (0.77) (3H, t, J ) 7 Hz); MS
(DCI/NH3) m/ z 461 (MH+), 415, 348, 302, 279. Anal.
(C28H32N2O4‚0.5AcOH) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m et h oxyp h en yl)-4-(2-n a p h t h yl)p yr r olid in e-3-ca r b oxy-
lic a cid (10b): substituting naphthalene-2-carboxaldehyde;
1H NMR (300 MHz, CDCl3) (rotamer) δ 7.82 (4H, m), 7.69 (1H,
m), 7.47 (2H, m), 7.37 (2H, dd, J ) 7.5, 2 Hz), 6.85 (2H, dd, J
) 7.5, 2 Hz), 3.90 (1H, d, J ) 8 Hz), 3.78 (3H, s), 3.57 (1H, m),
3.52-2.97 (6H, m), 2.93 (2.85) (3H, s), 2.90 (1H, m), 1.52 (2H,
m), 0.86 (0.76) (3H, t, J ) 7.5 Hz); MS (DCI/NH3) m/ z 461
(MH+). Anal. (C28H32N2O4‚0.5H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyph en yl)-4-(2-ph en yl)pyr r olidin e-3-car boxylic acid
(10c): substituting benzaldehyde; 1H NMR (300 MHz, CDCl3)
(minor rotamer) δ 7.53 (4H, d, J ) 6 Hz), 7.40-7.20 (3H, m),
6.88 (2H, d, J ) 8 Hz), 3.90 (1H, m), 3.79 (3H, s), 3.70-2.95
(8H, m), 2.90 (2.79) (3H, s), 1.50 (2H, sept, J ) 7 Hz), 0.87
(0.72) (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 411 (MH+). Anal.
(C24H30N2O4‚2.00H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yl)a cet a m id o-2-(4-
m e t h oxyp h e n yl)-4-(5-in d a n yl)-p yr r olid in e -3-ca r b ox-
ylic a cid (10d ): substituting indan-5-carboxaldehyde; 1H
NMR (300 MHz, CDCl3) (minor rotamer) δ 7.25-7.1 (5H, m),
6.78 (2H, d, J ) 8 Hz), 3.89 (1H, d, J ) 8 Hz), 3.75 (3H, s),
3.50-2.90 (6H, m), 2.88 (6H, t, J ) 6 Hz), 2.82 (2.80) (3H, s),
2.04 (2H, t, J ) 8 Hz), 1.48 (2H, sept, J ) 7 Hz), 0.83 (0.73)
(3H, t, J ) 7 Hz); MS (FAB) m/ z 473 (MNa+), 451 (MH+). Anal.
(C27H34N2O4‚2.5H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m et h oxyp h en yl)-4-(6-in d olyl)p yr r olid in e-3-ca r b oxylic
a cid (10e): substituting indole-6-carboxaldehyde; 1H NMR
(300 MHz, CDCl3) (minor rotamer) δ 8.43 (1H, bs), 7.57 (1H,
d, J ) 8 Hz), 7.43 (1H, s), 7.31 (2H, dd, J ) 6, 3 Hz), 7.22 (1H,
d, J ) 8 Hz), 7.1 (1H, t, J ) 3 Hz), 6.78 (2H, dd, J ) 6, 3 Hz),
6.45 (1H, m), 3.93 (1H, dd, J ) 6, 3 Hz), 3.80 (1H, m), 3.73
(3H, s), 3.60-2.90 (6H, m), 2.86 (2.82) (3H, s), 1.47 (2H, sept,
J ) 7 Hz), 0.83 (0.73) (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z
450 (MH+). Anal. (C26H31N3O4‚0.75H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyp h en yl)-4-(5-ben zo-2,3-d ih yd r ofu r a n yl)p yr r oli-
d in e-3-ca r boxylic a cid (10f): substituting 2,3-dihydroben-
zofuran-5-carboxaldehyde; 1H NMR (300 MHz, CDCl3) (minor
rotamer) δ 7.33 (1H, d, J ) 8 Hz), 7.28 (1H, m), 7.19 (1H, m),
6.87 (1H, d, J ) 8 Hz), 6.73 (1H, d, J ) 8 Hz), 4.56 (1H, t, J )
8 Hz), 3.83 (1H, d, J ) 10 Hz), 3.80 (3H, s), 3.63 (1H, m), 3.4-
3.0 (9H, m), 2.87 (2.84) (3H, s), 1.51 (2H, sept, J ) 7 Hz), 0.88
(0.78) (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 453 (MH+), 426,
352, 294. Anal. (C26H32N2O5‚0.25H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m et h oxyp h en yl)-4-(4-b en zofu r a n yl)p yr r olid in e-3-ca r -
boxylic a cid (10h ): substituting benzofuran-4-carboxalde-
hyde; 1H NMR (300 MHz, CDCl3) (minor rotamer) δ 7.59 (1H,
t, J ) 3 Hz), 7.4-7.2 (6H, m), 6.8 (2H, d, J ) 8 Hz), 4.03 (1H,
m), 3.94 (1H, dd, J ) 8, 3 Hz), 3.77 (3H, s), 3.61 (1H, dd, J )
8 7 3 Hz), 3.42 (1H, dd, J ) 11, 5 Hz), 3.40-2.90 (5H, m), 2.82
(2.81) (3H, s), 1.50 (2H, sept, J ) 7 Hz), 0.82 (0.75) (3H, t, J )
7 Hz); MS (DCI/NH3) m/ z 451 (MH+), 336, 160. Anal.
(C26H30N2O5‚AcOH) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyp h en yl)-4-(3-m eth oxyp h en yl)p yr r olid in e-3-ca r -
boxylic a cid (10l): substituting 3-methoxybenzaldehyde; 1H
NMR (300 MHz, CDCl3) (minor rotamer) δ 7.33 (2H, d, J )
7.5 Hz), 7.24 (1H, t, J ) 7.5 Hz), 7.05 (2H, m), 6.85 (2H, dd, J
) 7.5, 2 Hz), 6.76 (1H, m), 3.83 (1H, m), 3.81 (3H, s), 3.79 (3H,
s), 3.64 (1H, m), 3.48-2.97 (6H, m), 2.87 (2.83) (3H, s), 2.85
(1H, m), 1.45 (2H, m), 0.84 (0.74) (3H, t, J ) 7.5 Hz); MS (DCI/
NH3) m/ z 441 (MH+). Anal. (C25H32N2O5‚0.5H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyp h en yl)-4-(3,4-d im eth oxyp h en yl)p yr r olid in e-3-
ca r boxylic a cid (10m ): substituting 3,4-dimethoxybenzal-
dehyde; 1H NMR (300 MHz, CDCl3) (minor rotamer) δ 7.33
(2H, d, J ) 7.5 Hz), 7.07 (1H, d, J ) 2.0 Hz), 6.98 (1H, m),
6.85 (1H, d, 7.5 ) Hz), 6.82 (2H, d, 7.5 ) Hz), 3.91 (3H, s),
3.86 (3H, s), 3.83 (1H, m), 3.79 (3H, s), 3.64 (1H, m), 3.50-
2.95 (6H, m), 2.87 (1H, m), 2.85 (2.83) (3H, s), 1.45 (2H, m),
0.84 (0.74) (3H, t, J ) 7.5 Hz); MS (DCI/NH3) m/ z 471 (MH+).
Anal. (C26H34N2O6‚0.5H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyp h en yl)-4-(2,4-d im eth oxyp h en yl)p yr r olid in e-3-
ca r boxylic a cid (10n ): substituting 2,4-dimethoxybenzalde-
hyde; 1H NMR (300 MHz, CDCl3-CD3OD) (minor rotamer) δ
7.61 (1H, d, J ) 8Hz), 7.30 (2H, d, J ) 8Hz), 6.82 (2H, d, J )
8Hz), 6.55 (1H, d, J ) 8Hz), 6.45 (1H, d, J ) 3Hz), 3.90 (1H,
m), 3.81 (3H, s), 3.79 (3H, s), 3.77 (3H, s), 3.70-2.90 (8H, m),
2.85 (3H, s), 1.50 (2H, sept, J ) 7 Hz), 0.87 (0.77) (3H, t, J )
7 Hz). MS (DCI/NH3) m/ z 471 (MH+). Anal. (C26H34N2O6‚0.75
H2O) C, H, N.
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m eth oxyph en yl)-4-(3,4-diflu or oph en yl)pyr r olidin e-3-car -
boxylic a cid (10o): substituting 3,4-difluorobenzaldehyde; 1H
NMR (300 MHz, CDCl3) (minor rotamer) δ 7.60-7.3 (4H, m),
7.13 (1H, q, J ) 9 Hz), 6.90 (2H, d, J ) 8 Hz), 3.90 (1H, m),
3.79 (3H, s), 3.60-2.95 (6H, m), 2.92 (2.78) (3H, s), 1.55 (2H,
sept, J ) 7 Hz), 0.88 (0.73) (3H, t, J ) 7 Hz). MS (DCI/NH3)
m/ z 447 (MH+). Anal. (C24H28F2N2O4‚1.80H2O) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(5-b en zofu r a n yl)p yr r olid in e-3-ca r b oxy-
lic a cid (14a ): substituting benzofuran-5-carboxaldehyde; 1H
NMR (300 MHz, CDCl3) δ 7.64 (1H, bd), 7.59 (1H, d, J ) 2
Hz), 7.43 (2H, m), 7.33 (2H, d, J ) 8 Hz), 6.85 (2H, d, J ) 8
Hz), 6.73 (1H, dd, J ) 3, 1 Hz), 3.82 (1H, d, J ) 11 Hz), 3.89
(1H, d, J ) 9Hz) 3.79 (3H, s), 3.53 (1H, dd, J ) 10, 3 Hz), 3.44
(2H, m), 3.30 (1H, m), 3.20-2.95 (5H, m), 2.82 (1H, d, J ) 14
Hz), 1.43 (3H, m), 1.23 (3H, m), 1.08 (2H, m), 0.87 (3H, t, J )
(2R*,3R*,4S*)-1-(N-Met h yl-N-p r op yla cet a m id o)-2-(4-
m et h oxyp h en yl)-4-(6-b en zofu r a n yl)p yr r olid in e-3-ca r -
b oxylic a cid (10i): substituting benzofuran-6-carboxalde-

Non-Benzodioxole ETA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 327
Ta ble 2. Biological Data of N,N-Dibutyl-Substituted Pyrrolidine Amides
a
b
Compounds gave satisfactory analyses within (0.4% of theoretical calculations unless otherwise stated. Number of determinations
in parenthesis.
7 Hz), 0.82 (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 507 (MH+),
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(6-b en zofu r a n yl)p yr r olid in e-3-ca r b oxy-
lic a cid (14c): substituting benzofuran-6-carboxaldehyde; 1H
NMR (300 MHz, CDCl3) δ 7.63 (1H, bd), 7.59 (1H, d, J ) 2
Hz), 7.53 (1H, d, J ) 8 Hz), 7.36 (3H, m), 6.85 (2H, d, J ) 8
Hz), 6.73 (1H, dd, J ) 3, 1 Hz), 3.82 (1H, d, J ) 11 Hz), 3.89
(1H, d, J ) 9 Hz) 3.79 (3H, s), 3.53 (1H, dd, J ) 10, 3 Hz),
3.44 (2H, m), 3.30 (1H, m), 3.20-2.95 (5H, m), 2.80 (1H, d, J
) 14 Hz), 1.43 (3H, m), 1.23 (3H, m), 1.08 (2H, m), 0.87 (3H,
t, J ) 7 Hz), 0.82 (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 507
(MH+), 336, 290, 172. Anal. (C30H38N2O5‚0.75H2O) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(6-ben zo-2,3-d ih yd r ofu r a n yl)p yr r olid in e-
350, 336, 172. Anal. (C30H38N2O5) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(5-ben zo-2,3-d ih yd r ofu r a n yl)p yr r olid in e-
3-ca r boxylic a cid (14b): substituting 2,3-dihydrobenzofuran-
1
5-carboxaldehyde; H NMR (300 MHz, CDCl3) δ 7.31 (2H, d,
J ) 8 Hz), 7.27 (1H, d, J ) 2 Hz), 7.18 (1H, dd, J ) 7, 3 Hz),
6.86 (2H, d, J ) 8 Hz), 6.72 (1H, d, J ) 8 Hz), 4.56 (2H, t, J )
7 Hz), 3.78 (3H, s), 3.62 (1H, m), 3.50-3.25 (4H, m), 3.17 (2H,
t, J ) 7 Hz), 3.15-2.90 (5H, m), 2.79 (1H, d, J ) 14 Hz), 1.43
(3H, m), 1.26 (3H, m), 1.08 (2H, m), 0.87 (3H, t, J ) 7 Hz),
0.81 (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 509 (MH+). Anal.
(C30H40N2O5‚0.25H2O) C, H, N.

328 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Tasker et al.
Ta ble 3. Pharmacokinetic Data of N,N-Dibutyl-Substituted Pyrrolidine Amides
3-ca r boxylic a cid (14d ): prepared by catalytic hydrogenation
of 12c (4 atm of H2, AcOH, followed by preparative HPLC);
1H NMR (300 MHz, CDCl3) δ 7.40 (2H, d, J ) 8 Hz), 7.16 (1H,
d, J ) 8 Hz), 6.97 (1H, dd, J ) 8, 2 Hz), 6.89 (3H, m), 5.90
(1H, bs), 4.57 (2H, t, J ) 9 Hz), 4.93 (2H, m), 3.80 (3H, s),
3.70-3.58 (2H, m), 3.40 (1H, m), 3.30-2.90 (8H, m), 1.40 (2H,
m), 1.29 (3H, m), 1.08 (2H, m), 0.92 (3H, t, J ) 7 Hz), 0.82
(3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z 509 (MH+), 338. Anal.
(C30H40N2O5‚0.85TFA) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(4-b en zofu r a n yl)p yr r olid in e-3-ca r b oxy-
lic a cid (14e): substituting N,N-dibutyl bromoacetamide; 1H
NMR (300 MHz, CDCl3) δ 7.62 (1H, d, J ) 3 Hz), 7.39 (1H, dt,
J ) 8, 2 Hz), 7.34 (3H, m), 7.26 (1H, d, J ) 2 Hz), 7.23 (1H, d,
J ) 8 Hz), 6.84 (2H, d, J ) 8 Hz), 4.02 (1H, ddd, J ) 8, 6, 4
Hz), 3.89 (1H, d, J ) 9 Hz), 3.79 (3H, s), 3.67 (1H, dd, J ) 10,
3 Hz), 3.44 (2H, m), 3.35-3.15 (3H, m), 3.00 (2H, m), 2.84 (1H,
d, J ) 14 Hz), 1.43 (3H, m), 1.23 (3H, m), 1.08 (2H, m), 0.87
(3H, t, J ) 7 Hz), 0.82 (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z
507 (MH+), 350. Anal. (C30H38N2O5) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(4-flu or op h en yl)p yr r olid in e-3-ca r b oxylic
a cid (14i): substituting 4-fluorocarboxaldehyde; 1H NMR (300
MHz, CDCl3) δ 7.50 (1H, m), 7.42 (1H, dd, J ) 7, 3 Hz), 7.36
(2H, d, J ) 8 Hz), 7.01 (3H, t, J ) 8 Hz), 6.87 (1H, d, J ) 8
Hz), 3.83 (1H, m), 3.8 (3H, s), 3.67 (1H, m), 3.47 (3H, m), 3.30-
2.90 (5H, m), 2.82 (1H, d, J ) 14 Hz), 1.43 (2H, m), 1.28 (4H,
m), 1.08 (2H, m), 0.90 (3H, t, J ) 7 Hz), 0.82 (3H, t, J ) 7 Hz);
MS (DCI/NH3) m/ z 485 (MH+). Anal. (C28H37FN2O4) C, H,
N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(3,4-d iflu or op h en yl)p yr r olid in e-3-ca r boxy-
lic a cid (14j): substituting 3,4-difluorobenzaldehyde; 1H NMR
(300 MHz, CDCl3) δ 7.35 (1H, m), 7.30 (2H, d, J ) 8 Hz), 7.20-
7.00 (2H, m), 6.87 (2H, d, J ) 8 Hz), 3.78 (3H, s), 3.79 (1H,
m), 3.62 (1H, m), 3.50-3.30 (3H, m), 3.23 (1H, m), 3.15-2.90
(4H, m), 2.78 (1H, d, J ) 14 Hz), 1.43 (2H, m), 1.27 (4H, m),
1.08 (2H, m), 0.85 (3H, t, J ) 7 Hz), 0.80 (3H, t, J ) 7 Hz); MS
(DCI/NH3) m/ z 503 (MH+). Anal. (C28H36F2N2O4‚1H2O) C, H,
N.
(2R*,3R*,4S*)-1-(N,N-Dibu tylacetam ido)-2,4-bis(4-m eth -
oxyp h en yl)p yr r olid in e-3-ca r boxylic a cid (14f): substitut-
ing 4-methoxybenzaldehyde; 1H NMR (300 MHz, CDCl3) δ 7.38
(2H, d, J ) 8 Hz), 7.30 (2H, d, J ) 8 Hz), 6.87 (4H, dd, J ) 7,
3 Hz), 3.78 (3H, s), 3.76 (3H, s), 3.63 (1H, m), 3.50-3.20 (4H,
m), 3.15-2.90 (5H, m), 2.78 (1H, d, J ) 14 Hz), 1.43 (3H, m),
1.27 (3H, m), 1.09 (2H, m), 0.87 (3H, t, J ) 7 Hz), 0.81 (3H, t,
J ) 7 Hz); MS (DCI/NH3) m/ z 497 (MH+). Anal. (C29H40N2O5)
C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(3-p yr id yl)-p yr r olid in e-3-ca r b oxylic a cid
(14k ): substituting pyridine-3-carboxaldehyde; 1H NMR (300
MHz, CDCl3) δ 8.82 (1H, bs), 8.73 (1H, bd, J ) 9 Hz), 8.62
(1H, bd, J ) 7 Hz), 7.78 (1H, bdd, J ) 9, 3 Hz), 7.38 (2H, d, J
) 10 Hz), 6.90 (2H, d, J ) 10 Hz), 4.39 (1H, d, J ) 12 Hz),
3.95 (1H, m), 3.80 (3H, s), 3.79 (1H, m), 3.68 (1H, d, J ) 18
Hz), 3.50-3.30 (3H, m), 3.25-2.90 (6H, m), 1.47 (2H, m), 1.31
(4H, m), 1.20 (2H, m), 0.92 (3H, t, J ) 7 Hz), 0.83 (3H, t, J )
7 Hz); MS (DCI/NH3) m/ z 468 (MH+). Anal. (C27H37N3O4‚1.65
TFA) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(1,4-b en zod ioxa n -6-yl)p yr r olid in e-3-ca r -
boxylic a cid (14l): substituting benzodioxan-6-carboxalde-
hyde; 1H NMR (300 MHz, CDCl3) δ 7.32 (1H, d, J ) 9 Hz),
6.98 (1H, d, J ) 3 Hz), 6.91 (1H, dd, J ) 9, 3 Hz), 6.85 (2H, d,
J ) 9 Hz), 6.78 (1H, d, J ) 9 Hz), 4.28 (4H, m), 3.79 (3H, s),
3.75 (1H, d, J ) 9 Hz), 3.59-3.26 (5H, m), 3.09-2.95 (4H, m),
2.75 (1H, d, J ) 12 Hz), 1.48-1.21 (6H, m), 1.08 (2H, m), 0.88
(3H, t, J ) 7 Hz), 0.80 (3H, t, J ) 7 Hz); MS (DCI/NH3) m/ z
468 (MH+). Anal. (C30H40N2O6‚0.25H2O) C, H, N.
(2R*,3R*,4S*)-1-(N,N-Dibu tyla ceta m id o)-2-(4-m eth ox-
yp h en yl)-4-(2,4-d im eth oxyp h en yl)p yr r olid in e-3-ca r box-
ylic a cid (14g): substituting 2,4-dimethoxybenzaldehyde; 1H
NMR (300 MHz, CDCl3) δ 7.37 (2H, d, J ) 8 Hz), 7.20 (1H, d,
J ) 8 Hz), 6.92 (2H, d, J ) 8 Hz), 6.60 (1H, d, J ) 3 Hz), 6.49
(1H, dd, J ) 6, 2 Hz), 5.35 (1H, d, J ) 8 Hz), 4.20 (3H, m),
4.10 (3H, s), 3.83 (3H, s), 3.81 (3H, s), 3.75 (3H, m), 3.17 (2H,
hep, J ) 7 Hz), 3.05 (2H, t, J ) 7 Hz), 1.30 (4H, m), 1.07 (4H,
m), 0.87 (3H, t, J ) 7 Hz), 0.80 (3H, t, J ) 7 Hz); MS (DCI/
.
NH3) m/ z 527 (MH+). Anal. (C30H42N2O6 1.30TFA) C, H,N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(3-flu or op h en yl)p yr r olid in e-3-ca r b oxylic
a cid (14h ): substituting 3-fluorocarboxaldehyde; 1H NMR (300
MHz, CDCl3) δ 7.30 (2H, d, J ) 8 Hz), 7.22 (2H, m), 6.91 (1H,
m), 6.86 (2H, d, J ) 8 Hz), 3.79 (1H, m), 3.78 (3H, s), 3.68
(1H, m), 3.55-3.37 (3H, m), 3.29 (1H, m), 3.15-2.90 (5H, m),
2.78 (1H, d, J ) 14 Hz), 1.43 (2H, m), 1.25 (4H, m), 1.07 (2H,
m), 0.87 (3H, t, J ) 7 Hz), 0.80 (3H, t, J ) 7 Hz); MS (DCI/
NH3) m/ z 485 (MH+). Anal. (C28H37FN2O4) C, H, N.
(2R *,3R *,4S *)-1-(N ,N -Dib u t yla ce t a m id o)-2-(4-m e t h -
oxyp h en yl)-4-(5-in d a n yl)p yr r olid in e-3-ca r b oxylic a cid
(14m ): substituting indan-5-carboxaldehyde; 1H NMR (300M
Hz, CDCl3) δ 7.30 (3H, m), 7.28 (2H, m) 6.85 (2H, d, J ) 8
Hz), 3.81 (1H, m), 3.79 (3H, s), 3.68 (1H, m), 3.40 (3H, m),
3.32 (1H, m), 3.14 (4H, m), 3.91-3.80 (6H, m), 2.08 (1H, t, J
) 7 Hz), 2.07 (1H, t, J ) 7 Hz), 1.43 (2H, m), 1.25 (4H, m),

Non-Benzodioxole ETA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 329
1.11 (2H, m), 0.88 (3H, t, J ) 7 Hz), 0.81 (3H, t, J ) 7 Hz); MS
(DCI/NH3) m/ z 525 (MH+). Anal. (C31H42N2O4) C, H, N.
Recep tor Bin d in g Assa ys. Membranes prepared from
MMQ cells (prolactin secreting rat pituitary cells known to
contain predominantly ETA receptors) or porcine cerebellar
tissues (known to contain ETB receptors) grown in 20 culture
dishes (150 mm in diameter) were collected by washing and
scraping the cells in phosphate-buffered saline (PBS) contain-
ing 50 mM EDTA. The cell pellet was then homogenized in
50 mL of 10 mM HEPES (pH 7.4) containing 0.25 M sucrose
and protease inhibitors (50 mM EDTA, 0.1 mM PMSF, and 5
µg/mL pepstatin A) by a microultrasonic cell disrupter (Kon-
tes). 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 buffer B (20 mM
Tris, 100 mM NaCl, 10 mM MgCl2, pH 7.4, containing the
aforementioned inhibitors) and centrifuged again at 60000g
for 60 min. During the preparation of cell membranes, the
sample was kept at 4 °C to avoid degradation of membrane
proteins. The final pellet was resuspended in buffer B and
stored at -80 °C until used. Protein content was determined
by the Bio-Rad dye-binding protein assay.
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 pharmacokinetic param-
eters for NONLIN8427 were obtained with the program
CSTRIP.28 AUC values were calculated by the trapezoidal rule
over the time course of the study. The terminal-phase rate
constant (b) was utilized in the extrapolation of the AUC from
12 h to infinity to provide an AUC0-∞ value. The plasma
clearance (CLp) was calculated by dividing the dose by the
AUC. 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).
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[
125I]ET-1 binding to the membrane receptor was performed
in 96-well microtiter plates precoated with 0.1% BSA. Mem-
branes prepared from cells (5-10 µg) were incubated with 0.1
nM [125I]ET-1 in binding buffer (buffer B containing 0.2% BSA)
at 25 °C for the indicated periods of time. The total incubation
volume for each well was 0.2 mL. At the end of the incubation,
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vacuum filtration using glass-fiber filter strips in a PHD cell
harvester (Cambridge Technology, Inc., MA) followed by
washing the filter strips with saline (1 mL) three times.
Nonspecific binding was determined in the presence of 1 µM
ET-1. In a saturation binding study, various concentrations
of [125I]ET-1 (from 0.002 nM to 1 nM) were incubated with 10
µg membrane protein at 25 °C for 4 h. In the competition
binding study, 10 µg/well of membrane proteins were incu-
bated with 0.1 nM [125I]ET-1 in the presence of increasing
concentrations of unlabeled test ligands for 3 h at 25 °C.
Ra t id Absor p tion Mod el. Male Sprague-Dawley rats
weighing approximately 225 g were fasted overnight. After
anesthetization with inactin (100 mg/kg, ip), the pyloric
sphincter was ligated, and PE50 catheters were surgically
implanted in the carotid artery and portal vein. The test
compounds were dosed (10 mg/kg) by injection into the
duodenum (t ) 0), and portal and carotid blood samples were
drawn at 10, 30, and 60 min. On completion of the protocol,
the animals were sacrificed by inactin overdose.
Plasma samples (200 µL) and an equal volume of an internal
standard were extracted with dichloromethane:ethanol (8:2,
5 mL). The organic phase was transferred to a 10 mL conical
centrifuge tube, the solvent was evaporated using a stream of
dry air, and the residue was redissolved in mobile phase.
Recovery of samples following extraction varied 40-80%.
Samples were analyzed by HPLC using a Regis Little Champ
column (50 × 4.6 mm i.d., Spherisorb S3ODSII, 3 µm).
Chromatography was performed using a 10 min linear gradi-
ent usually consisting of 38-48% acetonitrile, 5% methanol,
and 57-47% 10 mM tetramethylammonium perchlorate (aque-
ous) containing 0.1% trifluoroacetic acid. UV detection of
sample analyte was measured at 226 nm. Drug concentrations
(µg/mL) were calculated from standard curves formulated in
plasma from 0.01 to 50 µg/mL in triplicate. Portal blood drug
levels were used to calculate the area under the curve (id AUC)
using the trapezoidal rule. The limit of detection by these
methods was 15-20 ng/mL.
P h a r m a cok in etics in Ra ts. The pharmacokinetic behav-
ior of the test compound was evaluated in male Sprague-
Dawley rats. The compound was prepared as a 10 mg/ml
solution in an ethanol:propylene glycol:D5W (20:30:50, by vol)
vehicle containing 1 mol equiv of sodium hydroxide. Groups
of rats (n ) 4/group) received either a 5 mg/kg (0.5 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,

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