Pyrrolidine-3-carboxylic acids as endothelin antagonists. 3. Discovery of a potent, 2-nonaryl, highly selective ET(A) antagonist (A-216546)

Article abstract of DOI:10.1021/jm980217s

Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of A-127722), a 2,4-diaryl substituted pyrrolidine-3- carboxylic acid based endothelin receptor-A antagonist. This compound binds to the ET(A) receptor with an affinity (

Full text of DOI:10.1021/jm980217s

J . Med. Chem. 1998, 41, 3261-3275
3261
P yr r olid in e-3-ca r boxylic Acid s a s En d oth elin An ta gon ists. 3. Discover y of a
P oten t, 2-Non a r yl, High ly Selective ETA An ta gon ist (A-216546)
Gang Liu,*,† Kenneth J . Henry, J r.,*,‡,| Bruce G. Szczepankiewicz, Martin Winn, Natasha S. Kozmina,
Steven A. Boyd, J ames Wasicak,‡ Thomas W. von Geldern, J inshyun R. Wu-Wong, William J . Chiou,
Douglas B. Dixon, Bach Nguyen,§ Kennan C. Marsh,§ and Terry J . Opgenorth
Metabolic Disease Research, Cancer Research, and Drug Analysis Department, Pharmaceutical Products Division,
Abbott Laboratories, Abbott Park, Illinois 60064-3500
Received April 8, 1998
Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of
A-127722), a 2,4-diaryl substituted pyrrolidine-3-carboxylic acid based endothelin receptor-A
antagonist. This compound binds to the ETA receptor with an affinity (Ki) of 0.034 nM and
with a 2000-fold selectivity for the ETA receptor versus the ETB receptor. We have expanded
our structure-activity studies in this series, in an attempt to further increase the ETA
selectivity. When the p-anisyl group of 1 was replaced by an n-pentyl group, the resultant
antagonist 3 exhibited substantially increased ETB/ETA activity ratio, but a decreased ETA
affinity. Structure-activity studies revealed that substitution and geometry of this alkyl group,
and substitution on the benzodioxolyl ring, are important in optimizing this series of highly
ETA selective antagonists. In particular, the combination of a (E)-2,2-dimethyl-3-pentenyl group
and a 7-methoxy-1,3-benzodioxol-5-yl group provided hydrophobic compound 10b with subna-
nomolar affinity for human ETA receptor subtype and with an ETB/ETA activity ratio of over
130000. Meanwhile, synthetic efforts en route to olefinic compounds led to the discovery that
2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u ) replacement of the p-anisyl group of 1
yielded very hydrophilic ETA antagonists with potency and selectivity equal to those of 10b.
On the basis of overall superior affinity, high selectivity for the ETA receptor (Ki, 0.46 nM for
ETA and 13000 nM for ETB), and good oral bioavailability (48% in rats), A-216546 (10a ) was
selected as a potential clinical backup for 1.
In tr od u ction
human studies, ET receptor antagonists have demon-
strated clear benefit in acute myocardial infarction,12
congestive heart failure,13 pulmonary hypertension,14
cerebral vasospasm,15 renal failure,16 and restenosis.17
It is apparent from these studies that ETA receptors
mediate most of the actions of ET-1 associated with
these pathological conditions, while the ETB receptor
may mediate some beneficial effects. These findings
suggest that a selective ETA receptor antagonist would
be useful as a therapeutic agent for chronic treatment
of the aforementioned pathological conditions.
The endothelins (ET-1, ET-2 and ET-3), 21-residue
amino acid peptides discovered in 1988,1,2 are potent,
long-acting constrictors of vascular smooth muscle and
are also potent mitogens.3 ET-1, made primarily by
endothelial cells, is thought to act as an autocrine/
paracrine mediator in the regulation of vascular func-
tions through modulation of tone, cell proliferation,
vascular permeability, and hormone production.4-7
The biological effects of the ETs are mediated by
G-protein-linked receptors. Two subtypes of human
endothelin receptors (ETA and ETB) have been cloned
and are approximately 55% homologous.8,9 The ETA
receptor has greater affinity for ET-1 than ET-3 and is
expressed in vascular smooth muscle cells. Binding of
ET-1 to ETA receptor mediates the vasoconstrictive and
mitogenic effects of ET-1 both in vitro and in vivo.3-7
The ETB receptor has equal affinity for ET-1 and ET-3
and is expressed in endothelial cells. Binding of ET-1
or ET-3 to the ETB receptor may attenuate the vaso-
constrictive effects of local ET-1 by mediating production
of nitric oxide10 and by clearing ET-1 from the circula-
tion.11
Recently, our laboratories have disclosed the design
and synthesis of a novel series of 1,2,4-trisubstituted
pyrrolidine-3-carboxylic acid based ETA receptor selec-
tive antagonists, exemplified by ABT-627 (1, A-147627,
active enantiomer of A-127722).18 This compound com-
petitively inhibits [125I]ET-1 binding to cloned human
ETA and ETB receptors with Ki values of 0.034 and 63.3
nM, respectively. In our efforts to search for a follow-
up compound possessing even greater selectivity for the
ETA receptor, the contributions to binding affinity or
receptor subtype selectivity conferred by the p-anisyl
group of 1 were investigated. When the p-anisyl sub-
stituent of A-127722 was changed to a p-ethylphenyl
group (2), a decrease of ETB affinity was observed with
a slight increase of ETA affinity (Scheme 1). Intrigued
by this observation, we experimented with aliphatic
groups and quickly discovered that the corresponding
n-pentyl group (3) further improved upon the ETB/ETA
activity ratio of 2, though 3 exhibited decreased affinity
Endothelin-1 has been implicated as a contributing
factor in many diseases.3-7 In animal models and
† Address correspondence to D-47V, AP-10, Abbott Laboratories, 100
Abbott Park Road, Abbott Park, IL 60064-3500. E-mail: Gang.Liu@
abbott.com.
‡ Cancer Research.
| Current address: Lilly Research Laboratories, Lilly Corporate
Center, Indianapolis, IN 46285.
§ Drug Analysis Department.
S0022-2623(98)00217-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/28/1998

3262 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Liu et al.
Sch em e 1. Rationale for Developing the Alkyl-Based Highly ETA Selective Antagonists
Sch em e 2a
a
(a) CDI, THF, then magnesium monoethylmalonate; (b) NaH, then n-BuLi, then R′-Br, THF; (c) MeNO2, NH4OAc, AcOH-EtOH,
reflux; (d) cat. t-BuOK, THF; (e) H2, Raney Ni, EtOAc; (f) NaBH3CN, concentrated HCl, THF-EtOH, pH 5; (g) DBU, CH3CN, reflux; (h)
n-Bu2NCOCH2Br, i-Pr2NEt, CH3CN; (i) 6 N aqueous NaOH, EtOH.
for ETA receptor.19 The net result is a 13-fold improve-
ment in ETA selectivity. The distinctive structure and
selectivity feature of 3 served as a valuable lead for
further exploration of this 2-alkylpyrrolidine-based,
highly selective ETA receptor antagonists.
trans,trans isomer 7 predominated. The cis,cis isomer
could be converted to trans,trans isomer by epimeriza-
tion (DBU, CH3CN). Chromatographic separation on
silica gel afforded the essentially pure trans,trans
isomer of the amino ester 7 in ca. 40% yield from the
Michael adduct 6. Alkylation of pyrrolidine 7 with N,N-
dibutylbromoacetamide and subsequent hydrolysis of
the ethyl ester furnished final compounds 8-10 in 80%
yield for in vitro assays.
Ch em istr y
Most of the new compounds in this report were
synthesized by direct analogy to our earlier work on 1,18
as shown in Scheme 2. Various â-ketoesters 4 were
prepared according to the published procedures, through
either reacting the imidazolide of the carboxylic acid
with magnesium monoethylmalonate followed by de-
carboxylation20 (ca. 70% yield) or alkylating an acetoac-
etate dianion with an alkyl bromide21 (20-70% yield).
Condensation of nitromethane with piperonals provided
the corresponding â-nitrostyrenes 5.22 Potassium tert-
butoxide catalyzed Michael addition of â-ketoesters 4
to nitrostyrenes 5 yielded two isomers of the adducts 6
in ca. 80% yield. Raney nickel mediated hydrogenation
of the adduct 6 and reduction of the resultant cyclic
imine with sodium cyanoborohydride provided a mixture
of three diastereomeric pyrrolidines in which the desired
Compounds not suitable for the Raney nickel hydro-
genation conditions (mainly olefin-containing sub-
strates) were synthesized from dioxolanes 11 through
a four-step sequence (Scheme 3). Protection of the
pyrrolidine nitrogen as an allyl carbamate and hydroly-
sis of the acetal provided aldehyde 12. Wittig reaction
of the resulting aldehyde 12 and removal of the ure-
thane yielded the corresponding olefin substituted pyr-
rolidine 13, which was converted to the final antagonist
as described in Scheme 2. Addition of methylmagne-
sium bromide to the aldehyde 12 and dehydration of
the resulting alcohol 14 with Burgess reagent yielded
the trans olefin 15. The alcohol 14 was also oxidized
to ketone 16, which was converted to the terminal olefin
17 with an appropriate Wittig ylide.

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3263
Sch em e 3a
Analogues
a
(a) Allyl chloroformate, py, DMAP, CH2Cl2; (b) 1 N aqueous HCl, THF, reflux; (c) Ph3PdR, THF; (d) Pd(Ph3P)4, Ph3P, pyrrolidine,
CH2Cl2; (e) n-Bu2NCOCH2Br, i-Pr2NEt, CH3CN; (f) 6 N aqueous NaOH, EtOH; (g) MeMgBr, THF; (h) CH3O2CNSO2N(C2H5)3, CH3CN,
heat; (i) PCC, MgSO4, CH2Cl2.
Intermediates 15 and 17 were then converted to the
desired antagonists via steps described previously in
Scheme 2.
ET-3 from the ETB receptor. Confirmatory binding
studies were performed in a similar fashion, using
human ETA and ETB receptor (hETA, hETB) perma-
nently expressed in CHO cells.
For detailed evaluation, a number of compounds have
been prepared in optically pure form. The preparation
of final products 8-10 as single enantiomers was
generally accomplished through resolution of the pyr-
rolidine 7 prior to N-alkylation (Scheme 4). Certain
racemic pyrrolidine esters 7 could be resolved via
formation of chiral salts 18 with (S)-(+)-mandelic acid.
Recrystallization produced material of >90% ee, as
evaluated by chiral HPLC. The diastereomerically pure
salt 18 was then converted to the optically pure final
products as shown in Scheme 2. Alternatively, racemic
pyrrolidine 7 could be converted to N-Boc carboxylic acid
19 in two steps. Acid 19 was then coupled with (S)-(-
)-4-benzyl-2-oxazolidinone through a pentafluorophe-
nylester to give diastereomers of 20a and 20b, which
may be separated by silica flash chromatography. The
desired acyl oxazolidinone 20a was then cleaved with
sodium methoxide. Treatment of the resulting N-Boc
methyl ester with trifluoroacetic acid and neutralizing
the salt with aqueous base provided the optically pure
pyrrolidine 21, which was converted to the final com-
pounds.
The decreased ETB affinity of n-pentyl compound 3
suggested that the alkyl group might be finding a new
hydrophobic binding site only present on the ETA
receptor or the increased hydrophobicity of 3 was less
tolerated at the ETB receptor than at the ETA receptor.
In a separate study, the length of the linear alkyl groups
was investigated.19 The optimal chain length appears
to be five atoms. Further shortening (propyl and butyl)
or lengthening (hexyl and heptyl) of the alkyl chain was
detrimental to both the ETA binding affinity and the
ETB/ETA activity ratio. Only n-pentyl compound 3
provided reasonable affinity for ETA receptor and the
largest separation of ETA and ETB activity. With the
optimal chain length established, the substitution pat-
tern of the aliphatic chain was then explored (Table 1).
Only the smallest alkyl substituent, a methyl group, was
tolerated at position 4 and 2 of the pentyl chain (8a and
8c), but not at position 3 or 1 (8b and 8d ). For the
larger substituents, 3-ethylpentyl group (8e) was 4
times less active and selective than 2-propylpentyl group
(8f), and neither improved upon the parent n-pentyl
compound 3. Fluorination at the terminal position of
the pentyl chain has a slight negative effect on the
affinity and selectivity of the antagonist 8g for the ETA
receptor. Since a methyl group was tolerated at the
2-position of the alkyl chain, a gem-dimethyl group at
the position 2 was introduced to eliminate the extra
stereocenter. This modification (8h ) slightly boosted the
affinity for ETA receptor and decreased the ETB receptor
Str u ctu r e-Activity Rela tion sh ip s
The initial screening for the compounds described in
this study was a measurement of their ability to displace
endothelin from its receptors. We employed a rodent
ETA receptor derived from MMQ cells (rETA); ETB
receptor (pETB) was derived from porcine cerebellar
tissue. IC50 data were recorded by measuring the
displacement of [125I]ET-1 from the ETA receptor or [125I]-

3264 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Liu et al.
Sch em e 4a
a
(a) (S)-(+)-mandelic acid, Et2O-hexanes; (b) n-Bu2NCOCH2Br, i-Pr2NEt, CH3CN; (c) 6 N aqueous NaOH, EtOH; (d) Boc2O, Et3N,
CH2Cl2; (e) pentafluorophenol, EDCl, DMF; (f) lithium salt of (S)-(-)-4-benzyl-2-oxazolidinone, THF, then silica gel chromatography; (g)
NaOMe, MeOH; (h) TFA (neat); (i) aqueous NaHCO3, Et2O extraction.
affinity, thus tripling the receptor selectivity observed
with antagonist 3.
antagonist with subnanomolar affinity and over 100000-
fold selectivity for the ETA receptor versus the ETB
receptor.
Double bonds were then introduced to the different
positions of the pentyl chain to increase the rigidity of
the aliphatic chain and to better position the alkyl group
in the binding site (Table 1). Generally, double bonds
between the position 3 and 4 were well tolerated.
Compound 8i, which includes a 4-methyl-3-pentenyl
group, was slightly more potent and selective than
n-pentyl analogue 3. One carbon extension of the chain
(8j) increased the binding affinity, but not the receptor
selectivity. A more rigid derivative of 8i, cyclopropy-
lidenepropyl group (8k ), exhibited a subnanomolar
affinity for ETA, but without much improvement in
selectivity. For the unsymmetric olefins, trans geometry
seemed to have positive effects on increasing the affinity
for ETA receptor, exemplified by (E)-3-pentenyl replace-
ment 8l. Unfortunately, compromised by the slightly
increased ETB affinity, the subnanomolar ETA affinity
of 8l only doubled the receptor selectivity over lead
compound 3. On the other hand, transition from trans
to cis geometry (8m ) resulted in over 6-fold loss of ETA
affinity and 3-fold loss of selectivity. Terminal olefins,
both monosubstituted 8n and disubstituted 8o, have
negative effects on the affinity and selectivity comparing
to the aliphatic chain. Methyl substitution at the
3-position of trans olefin 8p was tolerated. Compound
8q, gem-dimethyl substituted 8i at position 2, seemed
to increase the selectivity for ETA receptor slightly. The
optimal antagonist was realized through combining the
two structural features of 8h and 8l, 2,2-dimethyl
substitution and 3,4-trans olefin, to give 8r , the first
Because of the incompatibility of Raney nickel hydro-
genation conditions with most of the olefinic compounds,
2-dioxolylalkyl substituted pyrrolidines 11 were pre-
pared as the common intermediates for the olefin
analogues (see Scheme 3). Surprisingly, alkylation of
pyrrolidine 11 (n ) 1) with the side chain of 1 and
subsequent hydrolysis of the ester furnished potent (IC50
) 2.3 nM) and highly selective (> 43500×) antagonist
9a (Table 2). The structural difference between diox-
olylethyl and n-pentyl group intrigued us most, sug-
gesting that we might have reached an alternative,
hydrophilic binding site only present on the ETA recep-
tor. The structure-activity relationship of this seren-
dipitously discovered lead was then explored.
Initial derivatization of the 1,3-dioxolane ring offered
disappointing results. Methyl substituents on the di-
oxolane ring (9b) were not tolerated. The 1,3-dioxane
9c was less potent and selective than the 1,3-dioxolane
9a . Compared to propylene linker (9d ), ethylene ap-
peared to be the optimal link between the heterocycles.
Thus this chain length was kept constant for the
ensuing analogues. Interestingly, transition from 1,3-
dioxolane to 1,3-dithioxolane ring structure (9e) resulted
in a 2-fold loss of the potency and over 6-fold decrease
in selectivity for the ETA receptor, which strongly
implicates hydrogen bonding as a factor contributing to
the high selectivity. In contrast to the case of pentenyl
compound 8r , the introduction of a gem-dimethyl group
at the R-carbon of the dioxolane (9f) only marginally

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3265
Ta ble 1. SAR of 2-Acyclic Analogues: Radioligand Binding
IC50 (nM)a
rETA binding
mean (range)
pETB binding
mean (range)
compd
R
B/A ratiob
18800
formula
3
C4H9CH2
2.5
47000
C27H42N2O5‚0.5H2O
(3.3-1.8)
(85000-26000)
8a
8b
8c
8d
8e
8f
(CH3)2CHC2H4CH2
2.5
35800
14300
5000
C28H44N2O5‚0.65TFA
C28H44N2O5‚0.60TFA‚0.25Et2O
C28H44N2O5‚1.0TFA‚0.5H2O
C29H46N2O5‚1.60HCl
(5.5-1.1)
16
(31-8.1)
3.0
(3.3-2.8)
1300
(1800-950)
22
(88-9.5)
6.0
(28-2.3)
4.0
(4.2-3.8)
1.3
(1.3-1.2)
1.3
(1.6-1.2)
0.86
(1.5-0.4)
0.64
(0.83-0.49)
0.78
(0.97-0.70)
4.8
(10.9-2.3)
9.9
(39-4.3)
5.2
(5.7-4.8)
3.3
(6.8-1.7)
2.7
(38500-33000)
80000
(80000)
71000
(73300-68500)
36000
(45800-28900)
72000
(83950-55270)
95000
(95000)
34000
(48300-24400)
62000
(72000-54000)
33200
(38600-28500)
7000
(15800-3900)
10000
(12500-8070)
26200
(52300-19000)
67000
(79900-56700)
60000
(73200-51600)
36000
(36000)
48000
(48000)
C2H5CH(CH3)CH2CH2
C3H7CH(CH3)CH2
23670
28
C4H9C(CH3)2
(C2H5)2CHCH2CH2
3270
C29H46N2O5‚0.3TFA
(C3H7)2CHCH2
15800
8500
C30H48N2O5‚0.35TFA
C27H39N2O5F3‚1.05TFA
C29H46N2O5‚1.05TFA
C28H42N2O5‚0.05H2O‚1.15TFA
C29H44N2O5‚3.80TFA
C28H40N2O5‚1.80TFA
C27H40N2O5‚0.85TFA
C27H40N2O5‚1.30TFA
C27H40N2O5‚1.10TFA‚1.05AcOH
C28H42N2O5‚1.30TFA
C28H42N2O5‚0.70TFA
C30H46N2O5‚1.05TFA
C29H44N2O5‚0.70TFA
8g
8h
8i
F3CC3H7CH2
C3H7C(CH3)2CH2
47690
25500
8100
(CH3)2CdCHCH2CH2
(C2H5)(CH3)CdCHCH2CH2
(CH2)2CdCHCH2CH2
(E)-CH3CHdCHCH2CH2
(Z)-CH3CHdCHCH2CH2
CH2dCC2H4CH2
8j
8k
8l
15600
33600
14000
6060
8m
8n
8o
8p
8q
CH2dC(CH3)C2H4CH2
(E)-CH3CHdC(CH3)CH2CH2
(CH3)2CdCHC(CH3)2CH2
(E)-CH3CHdCHC(CH3)2CH2
6900
14500
23700
>128000
64000
(64000)
>100000
(3.2-2.3)
0.78
(1.1-0.57)
8r (2S,3R,4S)
a
IC50 calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 unless otherwise
b
noted. Expressed as IC50(ETB)/IC50(ETA).
augmented the affinity and the selectivity. This result
is consistent with our hypothesis that the hydrophobic
and hydrophilic substituents bind to different sites of
the ETA receptor.
Dioxolane replacements oxazoles and pyrazole were
also examined. Oxazole 9l showed a decreased ETB
affinity while maintaining the ETA affinity, which led
to an antagonist with over 41700-fold selectivity for the
ETA receptor. Despite an overall increase in potency,
dimethyl substituted oxazole 9m showed a greater
increase of ETB affinity than ETA affinity. Compound
9n , a N-methylated pyrrole, did not offer any improve-
ment over the 1,3-dioxolane. A more dramatic effect
was seen in 2-pyridyl compound 9o, which has subna-
nomolar affinity and almost 40000-fold selectivity for
the ETA receptor over the ETB receptor.
On the basis of the active pyridine compound 9o, a
strategy of heteroatom frame shift was conceived. The
ethylene linker was moved to the nitrogen of the
heterocycle and the original connecting carbon center
was changed to nitrogen or a carbonyl group. Com-
pound 9p , a pyrazole replacement, offered subnanomo-
lar affinity and improved selectivity compared to 1,3-
dioxolane 9a . Succinimide 9q was equally potent and
A pharmacokinetic study of the compound 9a (in rats)
indicated some hydrolysis of the dioxolane to the alde-
hyde in vivo. To eliminate the acid labile dioxolane
moiety, heterocycles mimicking the dioxolane ring struc-
ture were sought. By replacing one of the oxygens of
the dioxolane ring with carbon, furan and pyran deriva-
tives were synthesized. Among this group, the best
compound in terms of affinity and specificity for the ETA
receptor was the 2-tetrahydropyranyl compound 9g,
with slightly improved ETA affinity and decreased
receptor selectivity. Moving the oxygen to the position
4 of the ring resulted in antagonist 9h with much lower
affinity for the ETA receptor. For the five-membered
ring system, 2-furan 9i was equally active and selective
as 3-furan 9j. The fully reduced 2-tetrahydrofuran 9k
was 6 times less active than the tetrahydropyan 9g.

3266 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Ta ble 2. SAR of 2-Heterocyclic Analogues: Radioligand Binding
Liu et al.
IC50 (nM)a
rETA binding
mean (range)
pETB binding
mean (range)
compd
R
B/A ratiob
19000
formula
3
C3H7CH2
2.5
47000
C27H42N2O5‚0.5H2O
(3.3-1.8)
(85000-26000)
9a
9b
9c
9d
9e
9f
(1,3-dioxol-2-yl)CH2
2.3
>100000
>43500
3700
C27H40N2O7‚1.2TFA
C29H44N2O7‚1.35TFA
C28H42N2O7‚1.50TFA
C28H42N2O7‚1.50TFA
(3.1-1.7)
13
(28-5.9)
3.3
(7.6-1.2)
7.0
(25-3.0)
4.5
(6.8-3.0)
1.4
(3.0-0.65)
1.8
(2.0-1.6)
10
(17.6-5.9)
2.7
(2.8-2.7)
2.2
(5.1-0.94)
12
(35-2.6)
2.4
(2.7-2.1)
1.05
(1.11-0.99)
2.5
(3.7-1.9)
0.38
(0.39-0.37)
0.51
(0.90-0.29)
5.0
(4,5-dimethyl-1,3-dioxol-2-yl)CH2
(1,3-dioxan-2-yl)CH2
(1,3-dioxol-2-yl)CH2CH2
(1,3-dithia)CH2
48000
(77500-29800)
62000
(62000)
50000
(66000-37900)
29300
18800
7140
6500
C27H40N2O5S2‚1.15TFA
C29H44N2O7‚1.1TFA‚0.2H2O
C29H44N2O6‚1.4TFA
C29H44N2O6
(33400-25700)
>100000
(1,3-dioxol-2-yl)C(CH3)2
(2-tetrahydropyranyl)CH2
(4-tetrahydropyranyl)CH2
(2-furfuryl)CH2
>71400
26600
>10000
17400
11800
>9090
>41700
28300
17400
37000
56000
>20000
>2440
42500
18600
156000
9g
9h
9i
47800
(60000-38000)
>100000
47000
(86000-18000)
26000
(69400-9980)
>100000
C28H38N2O6‚0.8TFA‚0.15H2O
C28H38N2O6‚0.2TFA
C28H42N2O6‚0.45TFA
C27H37N3O6‚1.70TFA
C29H41N3O6‚1.05TFA
C29H41N3O5‚1.25TFA
C29H39N3O5‚1.75TFA
C27H38N4O5‚0.75TFA
C28H39N3O7‚1.05TFA
C27H41N3O7S1‚0.60TFA
C29H43N3O6‚0.70TFA
C29H39N3O6‚0.70TFA
C28H41N3O6‚1.4TFA
9j
(3-furfuryl)CH2
9k
9l
(2-tetrahydrofuryl)CH2
(1,3-oxazol-2-yl)CH2
>100000
9m
9n
9o
9p
9q
9r
(4,5-dimethyl-1,3-oxazol-2-yl)CH2
(N-methylpyrrol-2-yl)CH2
(2-pyridyl)CH2
29700
(33000-26500)
43600
(77800-24400)
14100
(16200-12200)
28600
(1-pyrazolyl)CH2
(28900-28200)
>100000
(N-succinimido)CH2
(7.1-3.5)
41
(71-25)
2.0
(3.1-1.3)
2.2
(3.0-1.6)
0.42
(N-propylsultamyl)CH2
(2-oxopiperidin-1-yl)CH2
(2-oxopyrid-1-yl)CH2
(2-oxopyrrolidin-1-yl)CH2
>100000
9s
9t
85000
(85000)
41000
(41000)
65400
(65400)
9u
(1.1-0.16)
a
IC50 calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 unless otherwise
b
noted. Expressed as IC50(ETB)/IC50(ETA).
as selective as dioxolane 9a , while sultam 9r yielded a
much weaker and less selective antagonist. Piperidi-
none 9s was equally potent as pyridone 9t, but twice
as selective. The most potent and selective dioxolane
mimic was five-membered 2-pyrrolidinone 9u , which
binds 10 times better than dioxolane 9a to the ETA
receptor without much increase in the ETB affinity. In
general, any five- or six-membered heterocycles with
hydrogen-bonding acceptors at the 2-position seem to
be well tolerated without impairing the ETA receptor
selectivity. These efforts led to the optimal antagonist
9u with subnanomolar affinity for the ETA receptor and
an ETB/ETA activity ratio of over 100000-fold.
Secon d a r y Eva lu a tion s
After synthesizing a series of very potent and highly
selective ETA antagonists with varying hydrophobicity
and hydrophilicity, a small set of compounds were
prepared in enantiomerically pure form to evaluate the
impact of this polarity swing on the pharmacokinetic
profiles of the compounds. These compounds were first
evaluated in our standard screening assay, as well as
in a second set of binding assays employing human ETA
and ETB receptors expressed in CHO cells (Table 3). As
we have observed in previous studies,18,23 the rETA and
pETB receptors used in the screening assays reliably
predict the relative and absolute affinities observed

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3267
Ta ble 3. Summary Data for Single Enantiomers
binding IC50 (nM)a,b
binding IC50 (nM)a,c
rETA mean
(range)
pETB
mean (range)
B/A
hETA mean
(range)
hETB mean
(range)
B/A
compd
R
X
ratiod
ratiod
formula
8h
C3H7C(CH3)2
H
H
H
H
H
0.67
2900
4300 0.29
(0.37-0.23) (4300-1900)
>128000 1.0 92700
2800
9700 nd
(0.96-0.45) (5900-1600)
0.78
(1.1-0.57)
0.72
8r
(E)-CH3CHdCHC(CH3)2
>100000
92700 C29H44N2O5‚0.70TFA
(1.3-0.77) (92700)
9o
(2-pyridyl)CH2
26000
36000 0.29
32000
110000 C29H39N3O5‚2.25TFA‚
1.10H2O
39600 C27H38N4O5‚0.90TFA
(1.4-0.35) (36000-15000)
0.68 26200
(0.71-0.66) (33000-20600)
0.22 24000
(0.25-0.20) (27700-20600)
OCH3 0.56 16700
(0.86-0.33) (24900-9100)
(E)-CH3CHdCHC(CH3)2 OCH3 0.41 45300
(0.41-0.40) (81000-23000)
(0.68-0.12) (41000-25000)
9p
(1-pyrazolyl)CH2
(2-oxopyrrolidinyl)CH2
C3H7C(CH3)2
38500 1.06
42000
(1.67-0.68) (83800-21100)
9u
110000 0.17
18000
106000 C28H41N3O6‚0.85TFA
31400 C30H48N2O6‚0.7TFA
137000 C30H46N2O6‚0.80TFA
(0.18-0.17) (23000-13800)
10a
10b
29800 0.49
15400
(0.61-0.41) (20000-12400)
110000 0.29
39700
(0.68-0.13) (73300-27000)
a
IC50 calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 unless otherwise
noted. Binding assays recorded as described in Experimental Section, using MMQ cells (rETA), porcine cerebellar tissue (pETB). c Binding
assays recorded as described in Experimental Section, using clonal CHO cell lines (hETA and hETB). Expressed as IC50(ETB)/IC50(ETA).
b
d
Ta ble 4. Summary of Pharmacokinetic Profiles for Single Enantiomers
pharmacokinetic profiles (rats)a
T1/2 iv
T1/2 oral
Cmax
AUC
compd
R
X
(h)
(h)
(µg/mL)
(µg h/mL)
F (%)
8h
8r
C3H7C(CH3)2
(E)-CH3CHdCHC(CH3)2
(2-pyridyl)CH2
(2-oxopyrrolidin-1-yl)CH2
C3H7C(CH3)2
(E)-CH3CHdCHC(CH3)2
H
H
H
H
OCH3
OCH3
2.45
1.6
2.8
1.4
1.6
1.5
3.85
3.58
8.8
nd
2.5
1.48
1.49
0.58
0.016
1.69
0.66
7.06
2.62
0.78
0.009
3.27
2.09
76.8
32.2
21.1
0.3
47.7
27.8
9o (racemic)
9u
10a
10b
3.94
a
T1/2 iv, half-life after intravenous dosing. T1/2 oral, Cmax, AUC, and F are half-life, maximum drug concentration, total drug exposure
(area under the curve) and oral bioavailability after oral dosing in rats. Calculated from raw pharmacokinetic data as described in the
Experimental Section.
against human receptors, with some tendency for this
class of compounds to exhibit a higher B/A ratio for the
human genotypes. Most of the compounds in this group
have subnanomolar potencies against hETA receptors;
B/A ratios vary from 9700 to 137000.
Pharmacokinetic studies (in rats) of these enanti-
omers revealed that the vastly different pharmacoki-
netic profiles of these two series were associated with
their distinctively different physical properties (Table
4). Oral bioavailability of 8h is very good (77%) but with
a very slow absorption rate, which could be explained
by the extremely high hydrophobicity of the compound,
compared to 1. Compound 8r has much lower bioavail-
ability and a much smaller AUC values. On the other
hand, the very hydrophilic series of compounds, exem-
plified by 9o and 9u , exhibit very poor pharmacokinetic
profiles: low oral bioavailabilities and small Cmax and
AUC values. Rapid elimination and a tendency toward
oxidation might contribute to the poor showing of oral
bioavailability of a number of these compounds.
A separate unpublished SAR study by our group has
indicated that the placement of a methoxy group at the
position 7 of the benzodioxolane ring was well tolerated
for the ETA selective antagonists. To improve the
absorption rate of compounds 8h and 8r , slightly more
hydrophilicity was added to the alkyl series by introduc-
ing this modification. Initial biochemical assays indi-
cated improved binding affinity and receptor selectivity
for new analogs 10a (A-216546) and 10b over the parent
compounds (Table 3). In fact, compound 10b is arguably
the most ETA selective antagonist known to date against
human receptors.

3268 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Liu et al.
Ch a r t 1
Ta ble 5. Comparison Data for Representative ETA Selective
Antagonists
The results of pharmacokinetic profile change of these
two compounds were mixed. The hydrophilicity modi-
fication improved the pharmacokinetic profiles of 10a ,
but not of 10b. The absorption rate of 10a increased
with improved hydrophilicity, comparing to compound
8h . Compound 10a , is also well absorbed (oral bioavail-
ability F ) 48%), despite its relatively short iv half-life
(1.6 h). In the phase I single dose human study, ABT-
627 (1) exhibits a much longer elimination half-life (24
h) than that expected from the rat study (3.5 h). Both
in vitro and in vivo metabolism studies have suggested
that one of the major metabolic routes for 1 appears to
be the glucuronidation of the carboxylic acid moiety. The
in vivo studies with [14C]A-127722 indicates that the
drug is recovered through enterohepatic recycling of the
glucuronidate of 1, which explains its prolonged half-
life in human. Apparently, this recycling process is
much more profound in human than in rat. If the same
trend holds, a longer half-life for 10a in human than in
rat is expected. The ability of 10a to inhibit ET-1
stimulated phosphoinositol (PI) hydrolysis was also
determined. Compound 10a effectively inhibits ET-1
evoked PI hydrolysis in rat MMQ cells with an IC50
value of 0.59 nM, showing the compound indeed is a
very potent functional antagonist of endothelin.
Ki (nM)a
hETA hETB
B/A
iv
compd
binding
binding
ratiob
half-lifec (h) Fc (%)
10a
22
23
24
25
26
0.46
0.13
0.17
48
1.1
0.43
13000
175
139
28260
1346
818
1.6
0.8
1.0
N/A
3.3
6.7
48
N/A
41
N/A
66
60
>50000
>1040
111
100
>4300
>10000
a
Human receptor data acquired in
a variety of systems,
collected from a variety of sources. b Expressed as Ki(ETB)/Ki(ETA).
c T1/2 and oral bioavailability (F) data, measured in rats, collected
from a variety of sources.
Con clu sion s
Two novel series of pyrrolidine-3-carboxylic acid based
highly ETA selective antagonists have been identified
through modification of the p-anisyl group of the ETA-
selective endothelin antagonist 1. In the first series,
structure-activity studies revealed that the p-anisyl
group of 1 could be replaced with hydrophobic 2,2-
dimethylpentyl (8h ) and 2,2-dimethylpentenyl (8r )
groups. The resultant analogues retain the high ETA
affinity of 1, but exhibit substantially reduced ETB
activity. In particular, the combination of a 2,2-dim-
ethylpentenyl group, N,N-dibutylacetamide side chain,
and a 7-methoxy-1,3-benzodioxol-5-yl group provides
antagonist 10b with subnanomolar affinity for the ETA
receptor and over 100000-fold selectivity. A number of
these compounds also exhibit oral bioavailabilities (in
rats) in the 28-77% range and have good plasma half-
lives. Of these, compound 10a , exhibits the best com-
bination of biochemical and pharmacological properties,
and has been identified as a potential backup to 1.
For the alkyl heterocycle series, it was found that the
size of the heterocycles and positioning of the hydrogen
bonding acceptors are important in optimizing this
series of highly selective antagonists. In particular,
2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u )
groups have the best combination of potency and
Since 1993, a number of non-peptide ETA selective
antagonists have been reported (Chart 1), including Ro
61-179024 (22), PD 15670725 (23), BMS 18287426 (24),
SB 21724227 (25), and TBC1125128 (26). Except for 24,
all of these compounds exhibit subnanomolar affinities
for the ETA receptor (Table 5). The selectivity of these
antagonists for human ETA receptor is about 1000-fold,
with the exception of 25 and 26. Compound 26 is the
most selective agent (>10000-fold for ETA vs ETB)
reported to date and is well-absorbed orally (60% in
rats). By comparison, compound 10a appears to be as
potent against the ETA receptor as any of the above
antagonists; it also shows the least affinity for ETB
receptor, which translates into the largest ETB/ETA ratio
(over 30000).

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3269
in 100 mL of ether; a solution of 1.6 g (10.5 mmol) of (S)-(+)-
mandelic acid in 60 mL of ether was added, the total volume
was made up to ∼200 mL, and the solution was seeded. The
mixture was stirred slowly overnight. The resultant crystals
were collected by filtration and recrystallized from ether/EtOAc
to give 1.8 g (3.5 mmol, 37%) of a white solid. This material
was partitioned between sodium bicarbonate and ether; the
ether layer was washed with brine, dried over sodium sulfate,
filtered, and concentrated in vacuo to give the enantiomerically
pure product (>98% ee), as indicated by chiral HPLC analysis,
using a Regis Whelk-O column.
selectivity. Unfortunately, poor pharmacokinetic pro-
files are generally exhibited by these very hydrophilic
compounds, which prevent them from being orally
dosed.
The highly selective receptor-binding profile of these
potent and orally bioavailable compounds further im-
proved the ETA selectivity observed with 1. It remains
to be seen whether reduced ETB antagonism will prove
to be advantageous in treating diseases in which en-
dothelin-1 plays a pathogenic role.
Meth od B. tr a n s,tr a n s-Meth yl [2S,3R,4S]-2-(2,2-Di-
m eth ylp en tyl)-4-(7-m eth oxy-1,3-ben zod ioxol-5-yl)p yr r o-
lidin e-3-car boxylate (21, X ) C3H7C(CH3)2CH2, Y ) OCH3).
To a solution of crude amino ester 7 (2.0 g, 5.1 mmol) in 40
mL of dichloromethane with 4 mL (28.7 mmol) of triethylamine
was added 2.0 g (9.2 mmol) of di-tert-butyl dicarbonate, and
the mixture was stirred at ambient temperature for 5 h.
Solvents were then removed in vacuo, and the residue was
taken up in 60 mL of ethanol. Aqueous sodium hydroxide (10
mL of 2.5 N solution, 25 mmol) was added, and the resultant
solution was stirred overnight. Solvents were removed in
vacuo; the residue was taken up in water and extracted with
ether. The aqueous phase was acidified with aqueous 1 N
phosphoric acid and extracted with EtOAc. The organic
extracts were washed with brine, dried over sodium sulfate,
Exp er im en ta l Section
Gen er a l. Unless otherwise specified, all solvents and
reagents were obtained from commercial suppliers and used
without further purification. All reactions were performed
under nitrogen atmosphere unless specifically noted. Flash
chromatography was performed using silica gel (230-400
mesh) from E. M. Science. Proton NMR spectra were recorded
on a General Electric QE300 instrument with Me4Si as an
internal standard and are reported as shift (multiplicity,
coupling constants, proton counts). Mass spectral analyses
were accomplished using different techniques, including direct
chemical ionization (DCI), atmospheric pressure chemical
ionization (APCI), electrospray ionization (ESI), and fast atom
bombardment (FAB), as specified for individual compounds.
Elemental analyses were performed by Robertson Microlit
Laboratories, Madison, NJ , and are consistent with theoretical
values to within 0.4% unless indicated.
filtered, and concentrated to give 1.0 g of a colorless oil.
A
sample of this material (0.734 g, 1.58 mmol) was combined
with 0.35 g (1.9 mmol) of pentafluorophenol and 0.364 g (1.9
mmol) of EDAC in 5 mL of DMF. The resultant solution was
stirred at ambient temperature for 1 h, then was poured onto
50 mL of 0.6 M sodium bicarbonate solution and extracted (3
15 mL) with ether. The combined ether extracts were washed
with brine, dried over magnesium sulfate, filtered, and con-
centrated in vacuo to give a foam, which was dissolved in 5
mL of THF and cooled to 0 °C. Simultaneously, 0.418 g (2.37
mmol) of (S)-(-)-4-benzyl-2-oxazolidinone was combined with
∼0.1 mg of pyreneacetic acid in 5 mL of THF and cooled to 0
C. N-Butyllithium (1.6 M in hexanes) was added to a red
endpoint (persists ∼10 s), and the solution was stirred for 10
min. The solution was transferred into the THF solution of
the pentafluorophenyl ester, and the resultant solution was
stirred at 0 °C for 40 min. Solvents were removed in vacuo;
the residue was taken up in sodium bicarbonate and extracted
with ether (3 × 10 mL). The combined ether extracts were
washed with brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude mixture of diastereomeric
products was separated by flash chromatography on silica gel,
eluting with a gradient from 4:1 f 3:1 f 2:1 hexanes/EtOAc,
giving 423 mg (0.68 mmol) of the faster moving and 389 mg
(0.63 mmol) of the slower moving diastereomer, respectively.
The faster moving diastereomer was dissolved in 2 mL of a
2.0 M solution of sodium methoxide (4.0 mmol) in methanol
(freshly prepared, containing 5% methyl formate by volume)
and stirred at ambient temperature for 16 h. Solvents were
removed in vacuo, and the residue was partitioned between
ether and aqueous 1 N sodium hydroxide. The ether layer was
washed with brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel, eluting with 4:1 hexanes/EtOAc.
The resultant material was dissolved in 5 mL of TFA and
stirred at ambient temperature for 1 h. Solvents were
removed in vacuo; the residue was suspended in sodium
bicarbonate solution and extracted with EtOAc. The organic
phase was washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo to give 98 mg (0.25 mmol,
33% from the N-Boc acid) of the resolved amino ester.
Eth yl 2-(n -Hexa n oyl)-3-(n itr om eth yl)-1,3-ben zod ioxol-
5-p r op ion a te (6, X ) C4H9CH2, Y ) H). To a solution of
ethyl 3-oxo-6-octanoate (5.0 g, 26.9 mmol) in 100 mL of THF
was added 3,4-methylenedioxy-â-nitrostyrene (5.1 g, 26.4
mmol), followed by the addition of potassium tert-butoxide (30
mg, 0.27 mmol). The mixture was stirred at room temperature
for 2 h, during which the solid dissolved and the solution
became reddish. After 2 h, TLC (ethyl acetate-hexanes, 1:5)
indicated complete consumption of the ketoester. The solution
was then concentrated in vacuo, and the residue was flash
chromatographed on silica gel eluting with 20% ethyl acetate
in hexane to produce 8.7 g (23.0 mmol, 85%) of the title
compound 6 as a mixture of diastereomers in a 1:1 ratio.
tr a n s,tr a n s-Eth yl 2-(n -P en tyl)-4-(1,3-ben zod ioxol-5-yl)-
p yr r olid in e-3-ca r boxyla te (7, X ) C4H9CH2, Y ) H). The
nitro ketone 6 (8.7 g, 23.0 mmol) in 150 mL of ethyl acetate
was hydrogenated under 4 atm of hydrogen pressure using a
Raney nickel 2800 catalyst (8.7 g). The Raney nickel was
washed sequentially with methanol, THF, and EtOAc before
use. The catalyst was then removed by filtration, and the
solution was concentrated under reduced pressure. The
resulting crude imine (7.7 g, 23.3 mmol) was dissolved in 25
mL of tetrahydrofuran and 50 mL of ethanol. Sodium cy-
anoborohydride (1.6 g, 25.5 mmol) and 2 mg of bromocresol
green were added. To this blue solution was added dropwise
a solution of 1:2 concentrated HCl in ethanol at such a rate
that the color was kept at light yellow-green. After the yellow
color persisted without additional HCl, the solution was stirred
for an additional 40 min. The solution was then concentrated
in vacuo and partitioned between chloroform and an aqueous
saturated sodium bicarbonate solution. The organic phase was
separated, dried over sodium sulfate, and concentrated under
reduced pressure. The residue was dissolved in 35 mL of
acetonitrile and DBU (4.3 g, 28.2 mmol) was added. The
solution was refluxed overnight. TLC (EtOAc) showed no more
cis,cis isomer. The solvent was removed in vacuo and the
crude product was flash chromatographed on silica gel eluting
with 75:25 ethyl acetate-hexane to give 3.5 g of pure trans,-
trans compound 7 (10.5 mmol, 46% from 6) as a light yellow
oil.
N,N-Dibu tylbr om oa ceta m id e. The bromoacetamide em-
ployed in this study was prepared using the method of Weaver,
as described in our earlier work.
Resolu tion of Com p ou n d s. Meth od A. tr a n s,tr a n s-
Eth yl [2S,3R,4S]-2-(2,2-Dim eth ylpen tyl)-4-(1,3-ben zodiox-
ol-5-yl)pyr r olidin e-3-car boxylate (18, X ) C3H7C(CH3)2CH2,
Y ) H). The amino ester 7 (6.8 g, 18.8 mmol) was dissolved
tr a n s,tr a n s-2-(n -P en tyl)-4-(1,3-ben zodioxol-5-yl)-1-[(N,N-
d i-n -bu tyla m in o)ca r bon ylm eth yl]p yr r olid in e-3-ca r boxy-
lic Acid (3). The pyrrolidine 7 (48 mg, 0.14 mmol) was
combined with 35 mg (0.14 mmol) of the N,N-dibutylbromoac-

3270 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Liu et al.
etamide in 3 mL of acetonitrile; 0.5 mL (2.9 mmol) of Hu¨nig’s
base was added, and the solution was allowed to stir overnight
at ambient temperature. Solvents were removed in vacuo; the
residue was partitioned between EtOAc and aqueous 1 N
phosphoric acid. The organic layer was washed with sodium
bicarbonate solution and brine, then dried over sodium sulfate,
filtered and concentrated. The residue was purified by flash
chromatography on silica gel, eluting with 2:1 hexanes/EtOAc.
The product was dissolved in 4 mL of ethanol; 1 mL of 2.5 N
aqueous sodium hydroxide (2.5 mmol) was added, and the
resultant solution was stirred overnight at ambient temper-
ature. Solvents were removed in vacuo; the residue was taken
up in water and extracted with ether. The aqueous phase was
acidified to pH 5 with aqueous 1 N phosphoric acid and
extracted with EtOAc. The organic extracts were washed with
brine, dried over sodium sulfate, and filtered. The solvents
were removed in vacuo to give 56 mg (0.12 mmol, 86%) of the
title compound as white foam: 1H NMR (CDCl3, 300 MHz) δ
0.87 (t, J ) 7.5 Hz, 3H), 0.89 (t, J ) 7.5 Hz, 3H), 0.97 (t, J )
7.5 Hz, 3H), 1.21-1.42 (brm, 10H), 1.43-1.78 (brm, 6H), 2.76
(t, J ) 7.0 Hz, 1H), 3.02-3.30 (brm, 6H), 3.40-3.60 (m, 3H),
3.73 (d, J ) 14.0 Hz, 1H), 5.98 (AB, 2H), 6.70 (d, J ) 8.1 Hz,
1H), 6.77 (dd, J ) 1.8, 8.1 Hz, 1H), 6.89 (d, J ) 1.8 Hz, 1H);
MS (DCI/NH3) (M + H)+ at m/ z 475. Anal. Calcd for
tr a n s,tr a n s-2-(3-Eth ylp en tyl)-4-(1,3-ben zod ioxol-5-yl)-
1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]p yr r olid in e-3-
ca r boxylic Acid (8e): white solid; 1H NMR (CDCl3, 300 MHz)
δ 0.81 (t, J ) 7.5 Hz, 6H), 0.90 (t, J ) 7.5 Hz, 3H), 0.96 (t, J
) 7.5 Hz, 3H), 1.10-1.42 (m, 12H), 1.43-1.62 (m, 4H), 1.68-
1.72 (brm, 1H), 2.91 (brt, J ) 7.5 Hz, 1H), 3.14-3.28 (m, 2H),
3.28-3.52 (m, 6H), 3.70 (brd, J ) 6.6 Hz, 1H), 3.92 (brd, J )
16.5 Hz, 1H), 5.92 (dd, J ) 2.0, 4.0 Hz, 2H), 6.71 (d, J ) 8.1
Hz, 1H), 6.79 (dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J ) 1.8 Hz,
1H); MS (DCI/NH3) (M + H)+ at m/ z 503. Anal. Calcd for
C
29H46N2O5‚0.3TFA: C, 66.22; H, 8.69; N, 5.22. Found: C,
66.40; H, 9.08; N, 5.10.
tr a n s,tr a n s-2-(2-P r op ylp en t yl)-4-(1,3-b en zod ioxol-5-
yl)-1-[(N,N-di-n -bu tylam in o)car bon ylm eth yl]pyr r olidin e-
3-ca r boxylic Acid (8f): an amorphous solid; 1H NMR (CDCl3,
300 MHz) δ 0.85 (m, 6H), 0.92 (t, J ) 7.5 Hz, 3H), 0.97 (t, J )
7.5 Hz, 3H), 1.12-1.40 (m, 13H), 1.42-1.68 (m, 6H), 2.90 (m,
1H), 3.14-3.30 (m, 3H), 3.33 (m, 5H), 3.72 (brm, 1H), 3.90
(brm, 1H), 5.93 (dd, J ) 2.0, 4.0 Hz, 2H), 6.73 (d, J ) 8.1 Hz,
1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J ) 1.8 Hz, 1H);
MS (DCI/NH3) (M + H)+ at m/ z 517. Anal. Calcd for
C
30H48N2O5‚0.35TFA: C, 66.24; H, 8.76; N, 5.03. Found: C,
66.26; H, 8.82; N, 4.98.
tr a n s,tr a n s-2-(5,5,5-Tr iflu or open tyl)-4-(1,3-ben zodioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (8g): an amorphous solid; 1H NMR
(CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5
Hz, 3H), 1.31 (septet, J ) 7.5 Hz, 4H), 1.41-1.66 (m, 8H),
1.76-2.15 (m, 4H), 3.10-3.25 (m, 3H), 3.30 (dd, J ) 6.0, 12.6
Hz, 1H), 3.40 (dd, J ) 6.0, 12.6 Hz, 1H), 3.67 (t, J ) 10.8 Hz,
1H), 3.77 (t, J ) 10.8 Hz, 1H), 3.99 (dd, J ) 9.9, 19.2 Hz, 1H),
4.03 (d, J ) 16.5 Hz, 1H), 4.23-4.33 (m, 1H), 4.35 (d, J ) 16.5
Hz, 1H), 5.94 (s, 2H), 6.73 (d, J ) 8.1 Hz, 1H), 6.79 (dd, J )
1.8, 8.1 Hz, 1H), 6.82 (d, J ) 1.8 Hz, 1H); MS (ESI) (M + H)+
at m/ z 529. Anal. Calcd for C27H39N2O5F3‚1.05TFA: C, 53.91;
H, 6.23; N, 4.32. Found: C, 53.99; H, 6.08; N, 4.09.
tr a n s,tr a n s-2-(2,2-Dim eth ylp en tyl)-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (8h ): white solid; 1H NMR (CDCl3,
300 MHz) δ 0.80-0.99 (m, 15H), 1.10-1.37 (m, 8H), 1.43-
1.58 (m, 4H), 1.77-1.97 (m, 2H), 3.48-3.12 (m, 5H), 3.60-
3.69 (m, 1H), 3.75-3.86 (m, 1H), 3.95-4.16 (m, 2H), 4.28-4.4
(m, 2H), 5.94 (s, 2H), 6.74 (d, J ) 8.1 Hz, 1H), 6.8 (dd, J ) 1.8,
8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3) (M + H)+
at m/ z 503. Anal. Calcd for C29H46N2O5‚1.05TFA: C, 60.01;
H, 7.62; N, 4.50. Found: C, 60.21; H, 7.37; N, 4.33.
tr a n s,tr a n s-2-(4-Meth yl-3-pen ten yl)-4-(1,3-ben zodioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (8i): white solid; 1H NMR (CDCl3,
300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5 Hz, 3H),
1.24-1.38 (m, 4H), 1.35-1.57 (m, 4H), 1.57 (s, 3H), 1.64 (s,
3H), 1.83-2.16 (m, 4H), 3.10-3.40 (m, 5H), 3.69-3.88 (m, 2H),
3.95-4.10 (m, 1H), 4.19 (d, J ) 15.9 Hz, 1H), 4.26-4.34 (m,
1H), 4.39 (d, J ) 16.2 Hz, 1H), 5.00-5.07 (m, 1H), 5.94 (s,
2H), 6.73 (d, J ) 8.1 Hz, 1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H),
6.85 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 487.
Anal. Calcd for C28H42N2O5‚0.05H2O‚1.15TFA: C, 58.79; H,
6.90; N, 4.34. Found: C, 58.82; H, 7.05; N, 4.53.
tr a n s,tr a n s-2-(4-Meth yl-4-h exen yl)-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (8j): white solid; 1H NMR (CDCl3,
300 MHz, 1:1 mixture of geometric isomers) δ 0.87-1.01 (m,
6H), 1.23-1.39 (m, 4H), 1.45-1.60 (m, 4H), 1.54 (brs, 3H), 1.66
(brs, 1H), 1.83-2.20 (m, 6H), 1.96 (q, J ) 7.5 Hz, 2H), 3.16
(brt, J ) 7.8 Hz, 2H), 3.22-3.32 (m, 1H), 3.36 (brtm, J ) 7.5
Hz, 2H), 3.69-3.83 (brm, 2H), 4.05 (brdd, J ) 9.6, 18.0 Hz,
1H), 4.23-4.42 (m, 3H), 4.97-5.06 (brm, 1H), 5.96 (s, 2H), 6.77
(brs, 2H), 6.84 (brs, 1H); MS (DCI/NH3) (M + H)+ at m/ z 501.
Anal. Calcd for C29H44N2O5‚3.80TFA: C, 47.07; H, 5.16; N,
3.00. Found: C, 47.14; H, 4.96; N, 2.97.
C
27H42N2O5‚0.5H2O: C, 67.05; H, 8.96; N, 5.79. Found: C,
67.30; H, 8.77; N, 5.68.
The following compounds were prepared using the proce-
dures described above for compound 3.
tr a n s,tr a n s-2-(4-Met h ylp en t yl)-4-(1,3-b en zod ioxol-5-
yl)-1-[(N,N-di-n -bu tylam in o)car bon ylm eth yl]pyr r olidin e-
3-ca r boxylic Acid (8a ): an amorphous solid; 1H NMR (CDCl3,
300 MHz) δ 0.83 (d, J ) 6.9 Hz, 3H), 0.84 (d, J ) 6.9 Hz, 3H),
0.91 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5 Hz, 3H), 1.13-1.90
(m, 15H), 3.00-4.20 (m, 11H), 5.93 (s, 2H), 6.74 (d, J ) 8.1
Hz, 1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz,
1H); MS (APCI) (M + H)+ at m/ z 489. Anal. Calcd for
C
28H44N2O5‚0.65TFA: C, 62.53; H, 8.00; N, 4.98. Found: C,
62.50; H, 8.02; N, 4.96.
tr a n s,tr a n s-2-(3-Met h ylp en t yl)-4-(1,3-b en zod ioxol-5-
yl)-1-[(N,N-di-n -bu tylam in o)car bon ylm eth yl]pyr r olidin e-
3-ca r boxylic Acid (8b): an amorphous solid; 1H NMR (CDCl3,
300 MHz, 1:1 mixture of diastereomers) δ 0.83 (t, J ) 7.5 Hz,
3H), 0.85 (d, J ) 7.5 Hz, 3H), 0.91 (t, J ) 7.5 Hz, 3H), 0.97 (t,
J ) 7.5 Hz, 3H), 1.05-1.22 (m, 2H), 1.22-1.41 (m, 7H), 1.43-
1.68 (m, 5H), 1.89 (m, 1H), 2.94 (t, J ) 6.0 Hz, 1H), 3.15-3.27
(m, 3H), 3.29-3.60 (m, 5H), 3.72 (brd, J ) 6.0 Hz 1H), 3.92
(brd, J ) 13.5 Hz, 1H), 5.93 (dd, J ) 2.0, 4.0 Hz, 2H), 6.73 (d,
J ) 8.1 Hz, 1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J )
1.8 Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 489. Anal. Calcd
for C28H44N2O5‚0.60TFA‚0.25Et2O: C, 66.08; H, 9.02; N, 4.99.
Found: C, 65.93; H, 8.81; N, 4.84.
tr a n s,tr a n s-2-(2-Met h ylp en t yl)-4-(1,3-b en zod ioxol-5-
yl)-1-[(N,N-di-n -bu tyl)am in ocar bon ylm eth yl]pyr r olidin e-
3-ca r boxylic Acid (8c): white solid; 1H NMR (CDCl3, 300
MHz, 1:1 mixture of diastereomers) δ 0.8-1.0 (m, 12H), 1.20-
1.40 (m, 7H), 1.45-1.60 (m, 6H), 1.60-1.74 (m, 1H), 1.80-2.0
(m, 1H), 3.10-3.40 (m, 5H), 3.67-3.78 (m, 1H), 3.80-3.91 (m,
1H), 4.0-4.20 (m, 2H), 4.30-4.50 (m, 2H), 5.93 (d, J ) 1.5
Hz, 2H), 6.73 (dd, J ) 1.8, 8.1 Hz, 1H), 6.79 (ddd, J ) 1.8, 1.8,
8.1 Hz, 1H), 6.86 (dd, J ) 1.8, 3.9 Hz, 1H); MS (DCI/NH3) (M
+ H)+ at m/ z 489. Anal. Calcd for C28H44N2O5‚1.0TFA‚
0.5H2O: C, 58.91; H, 7.58; N, 4.58. Found: C, 58.91; H, 7.58;
N, 4.45.
tr a n s,tr a n s-2-(1,1-Dim eth ylp en tyl)-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (8d ): white solid; 1H NMR (CDCl3,
300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.94 (t, J ) 7.5 Hz, 3H),
0.95 (t, J ) 7.5 Hz, 3H), 1.06 (s, 3H), 1.12 (s, 3H), 1.20-1.42
(m, 10H), 1.44-1.68 (m, 4H), 3.16-3.60 (m, 6H), 3.91 (brdd, J
) 6.0, 11.4 Hz, 1H), 4.08 (d, J ) 16.8 Hz, 1H), 4.27 (m, 1H),
4.33 (d, J ) 9.0 Hz, 1H), 5.87 (brd, J ) 18.0 Hz, 1H), 5.94 (s,
2H), 6.70 (d, J ) 8.1 Hz, 1H), 6.82 (dd, J ) 1.8, 8.1 Hz, 1H),
6.85 (d, J ) 1.8 Hz, 1H); MS (APCI/NH3) (M + H)+ at m/ z
503. Anal. Calcd for C29H46N2O5‚1.60HCl: C, 62.09; H, 8.55;
N, 4.99. Found: C, 62.09; H, 8.21; N, 4.65.
tr a n s,tr a n s-2-(3-Cyclop r op ylid en ep r op yl)-4-(1,3-b en -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
1
p yr r olid in e-3-ca r boxylic Acid (8k ): white solid; H NMR
(CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3271
Hz, 3H), 1.01 (s, 4H), 1.31 (septet, J ) 7.5 Hz, 4H), 1.51 (sextet,
J ) 7.5 Hz, 4H), 1.95-2.41 (m, 4H), 3.16 (t, J ) 7.5 Hz, 2H),
3.24-3.43 (m, 3H), 3.73-3.89 (brm, 2H), 4.03 (dd, J ) 10.5,
19.5 Hz, 1H), 4.21 (d, J ) 16.5 Hz, 1H), 4.34 (m, 1H), 4.36 (d,
J ) 18.0 Hz, 1H), 5.70 (brm, 1H), 5.94 (s, 2H), 6.74 (d, J ) 8.1
Hz, 1H), 6.79 (dd, J ) 1.8, 8.1 Hz, 1H), 6.86 (d, J ) 1.8 Hz,
1H); MS (DCI/NH3) (M + H)+ at m/ z 485. Anal. Calcd for
C28H40N2O5‚1.80TFA: C, 55.02; H, 6.11; N, 4.06. Found:
C, 55.12; H, 6.18; N, 4.13.
(DCI/NH3) (M + H)+ at m/ z 487. Anal. Calcd for C28H42N2O5‚
0.70TFA: C, 62.34; H, 7.60; N, 4.95. Found: C, 62.49; H, 7.43;
N, 4.73.
tr a n s,tr a n s-2-(2,2,4-Tr im eth yl-3-p en ten yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
p yr r olid in e-3-ca r boxylic Acid (8q): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.94 (t, J ) 7.5
Hz, 3H), 1.11 (s, 3H), 1.13 (s, 3H), 1.24-1.37 (m, 4H), 1.46-
1.59 (m, 4H), 1.61 (d, J ) 1.2 Hz, 3H), 1.69 (d, J ) 1.2 Hz,
3H), 2.04-2.11 (m, 2H), 3.10-3.20 (m, 2H), 3.30-3.39 (m, 3H),
3.67-3.82 (m, 2H), 3.95-4.08 (m, 1H), 4.32 (m, 2H), 4.37-
4.47 (m, 1H), 4.99 (s, 1H), 5.95 (s, 2H), 6.73 (d, J ) 8.1 Hz,
1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.84 (d, J ) 1.8 Hz, 1H);
MS (DCI/NH3) (M + H)+ at m/ z 515. Anal. Calcd for
tr a n s,tr a n s-2-[3(E)-P en ten yl]-4-(1,3-ben zod ioxol-5-yl)-
1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]p yr r olid in e-3-
ca r boxylic Acid (8l): white solid; 1H NMR CDCl3, 300 MHz)
δ 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5 Hz, 3H), 1.32 (septet,
J ) 7.5 Hz, 4H), 1.51 (sextet, J ) 7.5 Hz, 4H), 1.60 (dd, J )
6.0, 14.7 Hz, 3H), 1.88-2.21 (m, 4H), 3.10-4.43 (m, 5H), 3.72-
(dd, J ) 10.2, 22.8 Hz, 1H), 3.77 (dd, J ) 12.0, 21.0 Hz, 1H),
3.95-4.10 (m, 2H), 4.26 (m, 1H), 4.29 (d, J ) 16.5 Hz, 1H),
5.23-5.38 (m, 1H), 5.48 (qd, J ) 6.0, 21.0 Hz, 1H), 5.94 (s,
2H), 6.83 (d, J ) 8.1 Hz, 1H), 6.89 (dd, J ) 1.8, 8.1 Hz, 1H),
6.98 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 473.
Anal. Calcd for C27H40N2O5‚0.85TFA: C, 60.52; H, 7.23; N,
4.92. Found: C, 60.63; H, 7.27; N, 4.88.
C
30H46N2O5‚1.05TFA: C, 60.77; H, 7.48; N, 4.42. Found: C,
60.83; H, 7.20; N, 4.43.
tr a n s,tr a n s-[2S,3R,4S]-2-[2,2-Dim eth yl-3(E)-p en ten yl]-
4-(1,3-ben zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bo-
n ylm eth yl]p yr r olid in e-3-ca r boxylic Acid (8r ): white solid;
1H NMR (CDCl3, 300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t,
J ) 7.5 Hz, 3H), 0.98 (s, 3H), 1.00 (s, 3H), 1.32 (septet, J )
7.5 Hz, 4H), 1.54 (sextet, J ) 7.5 Hz, 4H), 1.60 (d, J ) 5.7 Hz,
3H), 1.80-1.94 (m, 1H), 1.96-2.06 (m, 1H), 3.12-3.32 (m, 3H),
3.35 (td, J ) 3.0, 9.6 Hz, 2H), 3.60-3.69 (m, 2H), 3.87-4.20
(m, 3H), 4.14 (brd, J ) 15.0 Hz, 1H), 5.30 (d, J ) 15.6 Hz,
1H), 5.39 (qd, J ) 6.0, 15.6 Hz, 1H), 5.94 (s, 2H), 6.74 (d, J )
8.1 Hz, 1H), 6.82 (dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J ) 1.8 Hz,
1H); MS (DCI/NH3) (M + H)+ at m/ z 501. Anal. Calcd for
tr a n s,tr a n s-2-[3(Z)-P en ten yl]-4-(1,3-ben zod ioxol-5-yl)-
1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]p yr r olid in e-3-
ca r boxylic Acid (8m ): white solid; 1H NMR (CDCl3, 300
MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5 Hz, 3H), 1.20-
1.40 (m, 4H), 1.45-1.63 (m, 4H), 1.57 (dd, J ) 1.5, 6.9 Hz,
3H), 1.88-2.24 (m, 4H), 3.15 (td, J ) 1.8, 7.8 Hz, 2H), 3.25-
3.42 (m, 3H), 3.68-3.88 (m, 2H), 4.03 (dd, J ) 9.0, 11.1 Hz,
1H), 4.12 (d, J ) 16.8 Hz, 1H), 4.32 (brm, 1H), 4.35 (d, J )
16.8 Hz, 1H), 5.29 (brm, 1H), 5.49 (dd, J ) 6.0, 11.1 Hz, 1H),
5.94 (s, 2H), 6.74 (d, J ) 8.1 Hz, 1H), 6.81 (dd, J ) 1.8, 8.1
Hz, 1H), 6.88 (d, J ) 1.8 Hz, 1H); MS (APCI) (M + H)+ at m/ z
473. Anal. Calcd for C27H40N2O5‚1.10TFA‚1.05AcOH: C,
57.10; H, 6.96; N, 4.25. Found: C, 57.17; H, 6.70; N, 3.97.
C
29H44N2O5‚0.70TFA: C, 62.90; H, 7.76; N, 4.83. Found: C,
62.72; H, 7.83; N, 4.82.
tr a n s,tr a n s-2-[2-(1,3-Dioxol-2-yl)et h yl]-4-(1,3-b en zo-
d ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]-
p yr r olid in e-3-ca r boxylic Acid (9a ): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5
Hz, 3H), 1.23-1.38 (m, 4H), 1.52 (sextet, J ) 7.5 Hz, 4H),
1.85-1.95 (m, 2H), 2.02-2.17 (m, 2H), 3.18 (dd, J ) 6.0, 9.0
Hz, 2H), 3.30 (dd, J ) 9.0, 18.0 Hz, 2H), 3.35 (m, 1H), 3.79
(dd, J ) 3.6, 6.9 Hz, 1H), 3.83-3.88 (m, 3H), 3.97 (dd, J ) 4.8,
6.0 Hz, 1H), 4.05 (q, J ) 9.6 Hz, 2H), 4.30-4.40 (m, 1H), 4.37
(s, 2H), 4.87 (t, J ) 4.1 Hz, 1H), 5.94 (s, 2H), 6.73 (d, J ) 8.1
Hz, 1H), 6.79 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz,
1H); MS (APCI) (M + H)+ at m/ z 505. Anal. Calcd for
tr a n s,tr a n s-2-(4-P en t en yl)-4-(1,3-b en zod ioxol-5-yl)-1-
[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]p yr r olid in e-3-
ca r boxylic Acid (8n ): white solid; 1H NMR (CDCl3, 300 MHz)
δ 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5 Hz, 3H), 1.24-1.38
(m, 4H), 1.42-1.65 (m, 6H), 1.80-2.03 (m, 2H), 2.09 (q, J )
7.0 Hz, 2H), 3.12-3.44 (m, 5H), 3.72 (t, J ) 11.3 Hz, 1H), 3.84
(t, J ) 10.5 Hz, 1H), 3.98 (d, J ) 12.0 Hz, 1H), 4.03 (d, J )
16.8 Hz, 1H), 4.30-4.40 (m, 1H), 4.37 (d, J ) 16.8 Hz, 1H),
4.96 (m, 1H), 5.01 (dd, J ) 2.4, 11.7 Hz, 1H), 5.71 (tdd, J )
3.3, 6.9, 18.0 Hz, 1H), 5.95 (s, 2H), 6.74 (d, J ) 8.1 Hz, 1H),
6.81 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz, 1H); MS
(DCI/NH3) (M + H)+ at m/ z 473. Anal. Calcd for C27H40N2O5‚
1.05TFA: C, 59.01; H, 6.99; N, 4.73. Found: C, 58.91; H, 6.72;
N, 4.50.
C
27H40N2O7‚1.20TFA: C, 55.05; H, 6.47; N, 4.37. Found: C,
55.12; H, 6.44; N, 4.27.
tr a n s,tr a n s-2-[2-(4,5-Dim eth yl-1,3-d ioxol-2-yl)eth yl]-4-
(1,3-ben zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon yl-
m eth yl]p yr r olid in e-3-ca r boxylic Acid (9b): white powder;
1H NMR (CDCl3, 300 MHz, mixture of diastereomers) δ 0.89-
0.99 (m, 6H), 1.15 (t, J ) 7.5 Hz, 3H), 1.20-1.38 (m, 7H), 1.44-
1.60 (m, 4H), 1.75-1.95 (m, 2H), 1.97-2.26 (m, 2H), 3.13-
3.40 (m, 4H), 3.62-3.70 (m, 1H), 3.70-3.88 (m, 2H), 4.05 (m,
1H), 4.16 (m, 1H), 4.25-4.45 (m, 4H), 4.88 (t, J ) 4.4 Hz) and
5.06 (td, J ) 1.2, 4.4 Hz, 1H in total), 5.96 (s, 2H), 6.77 (brm,
2H), 6.83 (brs, 1H); MS (DCI/NH3) (M + H)+ at m/ z 533. Anal.
Calcd for C29H44N2O7‚1.35TFA: C, 55.45; H, 6.66; N, 4.08.
Found: C, 55.47; H, 6.47; N, 4.03.
tr a n s,tr a n s-2-[2-(1,3-Dioxa n -2-yl)et h yl]-4-(1,3-b en zo-
d ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]-
p yr r olid in e-3-ca r boxylic Acid (9c): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.90 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5
Hz, 3H), 1.23-1.41 (m, 6H), 1.42-1.62 (m, 6H), 1.62-1.75 (m,
2H), 2.66 (dd, J ) 6.9, 8.4 Hz, 1H), 2.83-2.94 (m, 3H), 3.07
(td, J ) 7.8, 15.0 Hz, 1H), 3.16 (dd, J ) 3.9, 9.6 Hz, 2H), 3.38-
3.69 (m, 3H), 3.72(d, J ) 8.4 Hz, 1H), 3.76 (dd, J ) 3.0, 10.2
Hz, 2H), 4.07 (dd, J ) 4.2, 11.1 Hz, 2H), 4.52 (t, J ) 4.5 Hz,
1H), 5.91 (dd, J ) 2.0, 4.0 Hz, 2H), 6.68 (d, J ) 8.1 Hz, 1H),
6.73 (dd, J ) 1.8, 8.1 Hz, 1H), 6.86 (d, J ) 1.8 Hz, 1H); MS
(DCI/NH3) (M + H)+ at m/ z 519. Anal. Calcd for C28H42N2O7‚
1.50TFA: C, 53.99; H, 6.36; N, 4.06. Found: C, 54.06; H, 6.50;
N, 3.99.
tr a n s,tr a n s-2-(4-Meth yl-4-pen ten yl)-4-(1,3-ben zodioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (8o): white solid; 1H NMR (CDCl3,
300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5 Hz, 3H),
1.31 (septet, J ) 7.5 Hz, 4H), 1.53 (sextet, J ) 7.5 Hz, 4H),
1.67 (s, 3H), 1.87-2.0 (m, 2H), 2.04 (t, J ) 7.2 Hz, 2H), 3.17
(brtd, J ) 3.0, 9.0 Hz, 2H), 3.37 (dd, J ) 8.4, 18.0 Hz, 2H),
3.38 (dd, J ) 6.0, 13.8 Hz, 2H), 3.75 (t, J ) 11.4 Hz, 1H), 3.86
(t, J ) 9.0 Hz, 1H), 4.04 (dd, J ) 11.1, 21.0 Hz, 1H), 4.11 (d,
J ) 16.8 Hz, 1H), 4.37 (d, J ) 16.8 Hz, 1H), 4.40 (brm, 1H),
4.64 (s, 1H), 4.72 (s, 1H), 5.95 (s, 2H), 6.75 (d, J ) 8.1 Hz,
1H), 6.82 (d, J ) 1.8, 8.1 Hz, 1H), 6.89 (d, J ) 1.8 Hz, 1H);
MS (DCI/NH3) (M + H)+ at m/ z 487. Anal. Calcd for
C28H42N2O5‚1.30TFA: C, 57.89; H, 6.87; N, 4.41. Found: C,
57.97; H, 6.84; N, 4.48.
tr a n s,tr a n s-2-[3-Met h yl-3(E)-p en t en yl]-4-(1,3-b en zo-
d ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]-
p yr r olid in e-3-ca r boxylic Acid (8p ): white amorphous solid;
1H NMR (CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.97 (t,
J ) 7.5 Hz, 3H), 1.22-1.40 (m, 5H), 1.44-1.61 (m, 8H), 1.82
(brm, 1H), 2.02 (m, 3H), 3.05-3.30 (m, 5H), 3.38 (m, 1H), 3.55
(brm, 2H), 3.85 (m, 3H), 4.12 (brd, J ) 15.0 Hz, 1H), 5.21 (dd,
J ) 6.0, 12.0 Hz, 1H), 5.93 (s, 2H), 6.73 (d, J ) 8.1 Hz, 1H),
6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J ) 1.8 Hz, 1H); MS
tr a n s,tr a n s-2-[3-(1,3-Dioxol-2-yl)p r op yl]-4-(1,3-ben zo-
d ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]-
p yr r olid in e-3-ca r boxylic Acid (9d ): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5

3272 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Liu et al.
Hz, 3H), 1.32 (septet, J ) 7.5 Hz, 4H), 1.52 (sextet, J ) 7.5
Hz, 6H), 1.65-1.75 (m, 2H), 1.85-2.12 (m, 2H), 3.17 (dd, J )
6.3, 9.3 Hz, 2H), 3.21-3.45 (m, 3H), 3.78 (dd, J ) 3.6, 6.9 Hz,
1H), 3.80-3.97 (m, 5H), 4.03 (q, J ) 9.7 Hz, 1H), 4.16 (d, J )
16.5 Hz, 1H), 4.33 (m, 1H), 4.37 (d, J ) 16.5 Hz, 1H), 4.82 (t,
J ) 4.5 Hz, 1H), 5.95 (s, 2H), 6.75 (d, J ) 8.1 Hz, 1H), 6.81
(dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J ) 1.8 Hz, 1H); MS (APCI)
(M + H)+ at m/ z 519. Anal. Calcd for C28H42N2O7‚1.50TFA:
C, 53.99; H, 6.36; N, 4.06. Found: C, 54.06; H, 6.26; N, 4.05.
4H), 1.42-1.60 (m, 4H), 1.90 (m, 1H), 2.03 (m, 1H), 2.39-2.60
(m, 2H), 2.96 (m, 1H), 3.15-3.50 (m, 8H), 3.65-3.85 (m, 2H),
5.91 (s, 2H), 6.24 (s, 1H), 6.70 (d, J ) 8.1 Hz, 1H), 6.79 (d, J
) 8.1 Hz, 1H), 6.90 (s, 1H), 7.20 (s, 1H), 7.32 (s, 1H); MS (DCI/
NH3) (M + H)+ at m/ z 499 Anal. Calcd for C28H38N2O6‚
0.20TFA: C, 65.42; H, 7.38; N, 5.37. Found: C, 65.25; H, 7.41;
N, 5.27.
tr a n s,tr a n s-2-[(2-Tetr a h yd r ofu r a n yl)eth yl]-4-(1,3-ben -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
p yr r olid in e-3-ca r boxylic Acid (9k ): white powder; 1H NMR
(CDCl3, 300 MHz, a 1:1 mixture of two diastereomers) δ 0.86-
1.00 (m, 6H), 1.22-1.40 (m, 4H), 1.40-1.62 (m, 6H), 1.63-
1.77 (m, 1H), 1.80-2.07 (m, 5H), 2.95-3.62 (m, 8H), 3.68-
4.20 (m, 6H), 5.92 (s, 2H), 6.72 (dd, J ) 1.8, 8.1 Hz, 1H), 6.80
(dm, J ) 8.1 Hz, 1H), 6.86 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3)
(M + H)+ at m/ z 503. Anal. Calcd for C28H42N2O6‚0.45TFA:
C, 62.66; H, 7.72; N, 5.06. Found: C, 62.62; H, 7.53; N, 4.93.
tr a n s,tr a n s-2-[2-(1,3-Oxa zol-2-yl)et h yl]-4-(1,3-b en zo-
d ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]-
p yr r olid in e-3-ca r boxylic Acid (9l): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.91 (t, J ) 7.5 Hz, 3H), 0.93 (t, J ) 7.5
Hz, 3H), 1.22-1.36 (m, 4H), 1.43-1.60 (m, 4H), 2.35-2.60
(brm, 2H), 3.08-3.35 (m, 7H), 3.65 (dd, J ) 9.0, 12.3 Hz, 1H),
3.88 (t, J ) 11.3 Hz, 1H), 4.07 (dd, J ) 9.9, 20.0 Hz, 1H), 4.40
(brm, 2H), 4.57 (d, J ) 16.5 Hz, 1H), 5.92 (s, 2H), 6.76 (m,
3H), 7.08 (s, 1H), 7.64 (s, 1H); MS (ESI) (M + H)+ at m/ z 500.
Anal. Calcd for C27H37N3O6‚1.70TFA: C, 52.66; H, 5.63; N,
6.06. Found: C, 52.75; H, 5.80; N, 6.09.
tr a n s,tr a n s-2-[2-(4,5-Dim eth yl-1,3-oxa zol-2-yl)eth yl]-4-
(1,3-ben zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon yl-
m eth yl]p yr r olid in e-3-ca r boxylic Acid (9m ): white powder;
1H NMR (CDCl3, 300 MHz) δ 0.89 (t, J ) 7.5 Hz, 3H), 0.92 (t,
J ) 7.5 Hz, 3H), 1.21-1.37 (m, 4H), 1.43-1.57 (m, 4H), 2.11
(s, 3H), 2.21 (s, 3H), 2.31-2.47 (m, 2H), 2.95-3.41 (m, 7H),
3.62 (dd, J ) 9.0, 12.3 Hz, 1H), 3.83 (t, J ) 12.3 Hz, 1H), 4.10
(dd, J ) 10.2, 19.8 Hz, 1H), 4.30-4.40 (brm, 1H), 4.37 (d, J )
16.5 Hz, 1H), 4.57 (d, J ) 16.5 Hz, 1H), 5.91 (s, 2H), 6.71 (dd,
J ) 1.8, 8.1 Hz, 2H), 6.76 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3)
(M + H)+ at m/ z 528. Anal. Calcd for C29H41N3O6‚1.05TFA:
C, 57.70; H, 6.55; N, 6.49. Found: C, 57.89; H, 6.33; N, 6.41.
tr a n s,tr a n s-2-[2-(1,3-Dith ia )eth yl]-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (9e): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5
Hz, 3H), 1.24-1.40 (m, 4H), 1.45-1.61 (m, 4H), 1.89 (q, J )
7.4 Hz, 2H), 1.97-2.10 (m, 1H), 2.15-2.25 (m, 1H), 3.13-3.45
(m, 9H), 3.75-3.93 (m, 2H), 4.03 (dd, J ) 9.6, 18.9 Hz, 1H),
4.25 (d, J ) 15.9 Hz, 1H), 4.28-4.39 (m, 1H), 4.44 (d, J ) 15.9
Hz, 1H), 4.50 (t, J ) 6.5 Hz, 1H), 5.96 (s, 2H), 6.76 (d, J ) 8.1
Hz, 1H), 6.79 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz,
1H); MS (APCI) (M + H)+ at m/ z 537. Anal. Calcd for
C
27H40N2O5S2‚1.15TFA: C, 52.69; H, 6.21; N, 4.19. Found: C,
52.68; H, 5.97; N, 4.00.
tr a n s,tr a n s-2-[2,2-Dim eth yl-2-(1,3-d ioxol-1-yl)eth yl]-4-
(1,3-ben zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon yl-
m eth yl]p yr r olid in e-3-ca r boxylic Acid (9f): white powder;
1H NMR (CDCl3, 300 MHz) δ 0.82-1.00 (m, 12H), 1.24-1.40
(m, 4H), 1.43-1.64 (m, 5H), 1.76-1.84 (m, 1H), 2.93-3.00 (m,
1H), 3.15-3.47 (m, 6H), 3.60-3.70 (m, 3H), 3.74-3.95 (m, 5H),
4.48 (s, 1H), 5.94 (m, 2H), 6.72 (d, J ) 8.1 Hz, 1H), 6.83 (dd,
J ) 1.8, 8.0 Hz, 1H), 6.94 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3)
(M + H)+ at m/ z 533 Anal. Calcd for C29H44N2O7‚1.10TFA‚
0.2H2O: C, 56.63; H, 6.93; N, 4.23. Found: C, 56.60; H, 6.96;
N, 4.25.
tr a n s,tr a n s-2-[2-(2-Tet r a h yd r op yr a n yl)et h yl]-4-(1,3-
ben zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm e-
th yl]p yr r olid in e-3-ca r boxylic Acid (9g): an amorphous
white solid; 1H NMR (CDCl3, 300 MHz, 1:1 mixture of two
diastereomers) δ 0.89 (t, J ) 7.5 Hz, 3H), 0.91 (t, J ) 7.5 Hz,
3H), 1.30 (septet, 5H), 1.42-1.66 (m, 8H), 1.71 (brm, 1H), 1.85
(brm, 1H), 1.96-2.23 (brm, 2H), 3.10-3.29 (m, 4H), 3.29-3.52
(m, 3H), 3.54-3.81 (m, 3H), 4.01 (q, J ) 9.0 Hz, 1H), 4.12-
4.25 (m, 2H), 4.43 (d, J ) 9.0 Hz, 1H), 4.50 (d, J ) 2.7 Hz,
1H), 5.94 (s) and 5.95 (s, 2H in total), 6.76 (s, 2H), 6.81 (s,
1H); MS (APCI) (M + H)+ at m/ z 517. Anal. Calcd for
tr a n s,tr a n s-2-[2-(N-Meth ylp yr r ol-2-yl)eth yl]-4-(1,3-ben -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
p yr r olid in e-3-ca r boxylic Acid (9n ): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5
Hz, 3H), 1.22-1.37 (m, 4H), 1.42-1.56 (m, 4H), 2.11-2.36 (m,
2H), 2.55-2.80 (m, 2H), 3.04-3.15 (m, 2H), 3.26-3.40 (m, 3H),
3.50 (s, 3H), 3.74 (d, J ) 9.6 Hz, 2H), 3.88 (d, J ) 16.5 Hz,
1H), 4.02 (dd, J ) 9.0, 18.6 Hz, 1H), 4.21 (d, J ) 16.5 Hz, 1H),
4.32-4.43 (m, 1H), 5.86-5.89 (m, 1H), 5.91 (s, 2H), 6.00 (t, J
) 4.5 Hz, 1H), 6.52 (t, J ) 3.0 Hz, 1H), 6.72 (d, J ) 8.1 Hz,
1H), 6.80 (dd, J ) 1.8, 8.0 Hz, 1H), 6.87 (d, J ) 1.8 Hz, 1H);
MS (DCI/NH3) (M + H)+ at m/ z 512. Anal. Calcd for
C
29H44N2O6‚1.40TFA: C, 56.48; H, 6.77; N, 4.14. Found: C,
56.46; H, 6.99; N, 3.83.
tr a n s,tr a n s-2-[2-(4-Tet r a h yd r o-2H -p yr a n yl)et h yl]-4-
(1,3-ben zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon yl-
m eth yl]p yr r olid in e-3-ca r boxylic Acid (9h ): an amorphous
white solid; 1H NMR (CDCl3, 300 MHz) δ 0.90 (t, J ) 7.5 Hz,
3H), 0.97 (t, J ) 7.5 Hz, 3H), 1.17-1.42 (m, 8H), 1.42-1.66
(m, 8H), 1.66-1.81 (m, 1H), 2.73 (t, J ) 7.2 Hz, 1H), 3.06 (dd,
J ) 9.6, 18.6 Hz, 2H), 3.11-3.60 (m, 9H), 3.68 (d, J ) 13.5
Hz, 1H), 3.94 (dd, J ) 2.7, 12.0 Hz, 2H), 5.91 (s, 2H), 6.71 (d,
J ) 8.1 Hz, 1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J )
1.8 Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 517. Anal. Calcd
for C29H44N2O6: C, 67.42; H, 8.58; N, 5.42. Found: C, 67.39;
H, 8.72; N, 5.30.
tr a n s,tr a n s-2-[2-(2-F u r fu r yleth yl)]-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (9i): an amorphous white solid; 1H
NMR (CDCl3, 300 MHz) δ 0.89-0.96 (m, 6H), 1.21-1.38 (m,
4H), 1.42-1.57 (m, 4H), 2.19 (m, 1H), 2.31 (m, 1H), 2.63-2.85
(m, 2H), 3.08-3.39 (m, 5H), 3.69 (d, J ) 10 Hz, 2H), 3.90-
4.01 (m, 2H), 4.12-4.21 (m, 2H), 5.92 (s, 2H), 6.03 (d, J ) 3.0
Hz, 1H), 6.24 (dd, J ) 2.0, 3.0 Hz, 1H), 6.75 (d, J ) 8.1 Hz,
1H), 6.79 (dd, J ) 1.8, 8.1 Hz, 1H), 6.88 (d, J ) 1.8 Hz, 1H),
7.25 (d, J ) 1.8 Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 499.
Anal. Calcd for C28H38N2O6‚0.80TFA‚0.15H2O: C, 60.00; H,
6.65; N, 4.73. Found: C, 60.00; H, 6.69; N, 4.55.
C
29H41N3O5‚1.25TFA: C, 57.83; H, 6.51; N, 6.42. Found: C,
57.68; H, 6.69; N, 6.49.
tr a n s,tr a n s-2-[2-(2-P yr id yl)eth yl]-4-(1,3-ben zod ioxol-5-
yl)-1-[(N,N-di-n -bu tylam in o)car bon ylm eth yl]pyr r olidin e-
3-ca r boxylic Acid (9o): an amorphous white solid; 1H NMR
(CDCl3, 300 MHz) δ 0.91 (t, J ) 7.5 Hz, 6H) 1.22-1.37 (m,
4H), 1.43-1.60 (m, 4H), 2.51 (brs, 2H), 3.13-3.46 (m, 7H), 3.62
(dd, J ) 9.6, 13.8 Hz, 1H), 3.84 (t, J ) 12.6 Hz, 1 H), 4.04 (dd,
J ) 10.5, 20.1 Hz, 1 H), 4.18-4.29 (m, 1H), 4.45 (s, 2H), 5.91
(s, 2H), 6.66 (d, J ) 8.1 Hz, 1H), 6.75 (dd, J ) 1.8, 8.1 Hz,
1H), 6.80 (d, J ) 1.8 Hz, 1H), 7.51 (t, J ) 6.9 Hz, 1H), 7.70 (d,
J ) 9.0 Hz, 1H), 8.06 (t, J ) 6.9 Hz, 1H), 8.65 (d, J ) 6.0 Hz,
1H); MS (DCI/NH3) (M + H)+ at m/ z 510. Anal. Calcd for
C
29H39N3O5‚1.75TFA: C, 55.04; H, 5.79; N, 5.92. Found: C,
55.08; H, 5.64; N, 5.81.
tr a n s,tr a n s-2-[2-(1-P yr a zolyl)eth yl]-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (9p ): an amorphous solid; 1H NMR
(CDCl3, 300 MHz) δ 0.91 (t, J ) 7.5 Hz, 6H), 1.21-1.38 (m,
4H), 1.42-1.59 (m, 4H), 2.43-2.69 (m, 2H), 3.12-3.36 (m, 5H),
3.40-3.61 (t, J ) 10.5 Hz, 1 H), 3.72-3.83 (t, J ) 10.5 Hz,
1H), 3.98-4.55 (m, 6H), 5.91 (s, 2H), 6.28 (t, J ) 3.0 Hz, 1H),
tr a n s,tr a n s-2-[2-(3-F u r fu r yl)eth yl]-4-(1,3-ben zod ioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (9j): an amorphous white solid; 1H
NMR (CDCl3, 300 MHz) δ 0.87-0.96 (m, 6H), 1.21-1.39 (m,

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3273
6.67 (d, J ) 8.1 Hz, 1H), 6.75 (dd, J ) 1.8, 8.1 Hz, 1H), 6.81
(d, J ) 1.8 Hz, 1H), 7.50 (d, J ) 3.0 Hz, 1H), 7.56 (d, J ) 3.0
Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 499. Anal. Calcd
for C27H38N4O5‚0.75TFA: C, 58.60; H, 6.69; N, 9.59. Found:
C, 58.53; H, 6.45; N, 9.67.
0.86 (t, J ) 7.5 Hz, 3H), 0.93 (t, J ) 7.5 Hz, 3H), 0.96 (t, J )
7.5 Hz, 3H), 1.09-1.38 (m, 8H), 1.45-1.59 (m, 4H), 1.84-2.00
(m, 2H), 3.15 (dd, J ) 6.9, 10.0 Hz, 2H), 3.30-3.42 (m, 3H),
3.72 (t, J ) 10.5 Hz, 1H), 3.86 (t, J ) 10.5 Hz, 1H), 3.88 (s,
3H), 4.02 (q, J ) 10.0 Hz, 1H), 4.12 (d, J ) 16.8 Hz, 1H), 4.29
(d, J ) 16.8 Hz, 1H), 4.41 (brm, 1H), 5.94 (s, 2H), 6.52 (d, J )
1.8 Hz, 1H), 6.67 (d, J ) 1.8 Hz, 1H); MS (ESI) (M + H)+ at
m/ z 533. Anal. Calcd for C30H48N2O6‚0.7TFA: C, 61.57; H,
8.01; N, 4.57. Found: C, 61.59; H, 8.20; N, 4.63.
tr a n s,tr a n s-2-[2-(Su ccin im ido)eth yl]-4-(1,3-ben zodioxol-
5-yl)-1-[(N ,N -d i-n -b u t yla m in o)ca r b on ylm e t h yl]p yr r o-
lid in e-3-ca r boxylic Acid (9q): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5
Hz, 3H), 1.23-1.41 (m, 4H), 1.44-1.63 (m, 4H), 2.10-2.25 (m,
1H), 2.37-2.48 (m, 1H), 2.69 (s, 4H), 3.20 (t, J ) 7.8 Hz, 2H),
3.26-3.40 (m, 3H), 3.50-3.73 (m, 3H), 3.81 (t, J ) 10.5 Hz,
1H), 3.94 (dd, J ) 9.0, 19.2 Hz, 1H), 4.09-4.21 (m, 2H), 4.33
(d, J ) 16.8 Hz, 1H), 5.92 (s, 2H), 6.72 (d, J ) 8.1 Hz, 1H),
6.81 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz, 1H); MS
(DCI/NH3) (M + H)+ at m/ z 530. Anal. Calcd for C28H39N3O7‚
1.05TFA: C, 55.68; H, 6.62; N, 6.47. Found: C, 55.77; H, 6.02;
N, 6.19.
tr a n s,tr a n s-2-[2-(P r op ylsu lta m -1-yl)eth yl]-4-(1,3-ben -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
p yr r olid in e-3-ca r boxylic Acid (9r ): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5
Hz, 3H), 1.23-1.38 (m, 4H), 1.44-1.61 (m, 4H), 2.10-2.42 (m,
4H), 3.02-3.26 (m, 7H), 3.26-3.44 (m, 5H), 3.73 (brt, J ) 10.0
Hz, 1H), 3.90 (q, J ) 8.0 Hz, 1H), 3.93-4.10 (m, 2H), 4.26 (d,
J ) 15.3 Hz, 1H), 5.94 (s, 2H), 6.73 (d, J ) 8.1 Hz, 1H), 6.81
(dd, J ) 1.8, 8.1 Hz, 1H), 6.92 (d, J ) 1.8 Hz, 1H); MS (DCI/
NH3) (M + H)+ at m/ z 552. Anal. Calcd for C27H41N3O7S1‚
0.60TFA: C, 54.62; H, 6.76; N, 6.78. Found: C, 54.74; H, 6.74;
N, 6.69.
tr a n s,tr a n s-2-[2-(2-Oxop ip er id in -1-yl)eth yl]-4-(1,3-ben -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
p yr r olid in e-3-ca r boxylic Acid (9s): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.91 (t, J ) 7.5 Hz, 3H), 0.93 (t, J ) 7.5
Hz, 3H), 1.22-1.38 (m, 4H), 1.42-1.60 (m, 4H), 1.70-1.87 (m,
4H), 2.10-2.23 (brm, 1H), 2.23-2.50 (m, 3H), 3.05 (t, J ) 9.0
Hz, 1H), 3.19-3.40 (m, 7H), 3.40-3.60 (m, 2H), 3.61-3.74 (m,
1H), 3.75-3.99 (m, 3H), 4.22 (brs, 1H), 5.92 (m, 2H), 6.72 (d,
J ) 8.1 Hz, 1H), 6.78 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J )
1.8 Hz, 1H); MS (DCI/NH3) (M + H)+ at m/ z 530. Anal. Calcd
for C29H43N3O6‚0.70TFA: C, 59.91; H, 7.23; N, 6.89. Found:
C, 59.92; H, 7.23; N, 6.80.
tr a n s,tr a n s-[2S,3R,4S]-2-[2,2-Dim eth yl-3(E)-p en ten yl]-
4-(7-m eth oxy-1,3-ben zodioxol-5-yl)-1-[(N,N-di-n -bu tylam i-
n o)ca r bon ylm eth yl]p yr r olid in e-3-ca r boxylic Acid (10b):
white solid; 1H NMR (CDCl3, 300 MHz) δ 0.92 (t, J ) 7.5 Hz,
3H), 0.95 (t, J ) 7.5 Hz, 3H), 0.97 (s, 3H), 0.99 (s, 3H), 1.24-
1.40 (m, 4H), 1.46-1.60 (m, 4H), 1.60 (d, J ) 5.4 Hz, 3H), 1.92
(dd, J ) 6.6, 15.0 Hz, 1H), 2.04 (d, J ) 15.0 Hz, 1H), 3.17 (td,
J ) 3.0, 11.4 Hz, 2H), 3.25-3.40 (m, 3H), 3.55-3.75 (m, 2H),
3.87 (s, 3H), 3.99 (q, J ) 9.0 Hz, 1H), 4.11-4.30 (m, 3H), 5.29
(d, J ) 15.6 Hz, 1H), 5.38 (qd, J ) 6.0, 15.6 Hz, 1H), 5.94 (s,
2H), 6.50 (d, J ) 1.8 Hz, 1H), 6.63 (d, J ) 1.8 Hz, 1H); MS
(DCI/NH3) (M + H)+ at m/ z 531. Anal. Calcd for C30H46N2O6‚
0.80TFA: C, 61.03; H, 7.58; N, 4.50. Found: C, 61.04; H, 7.65;
N, 4.46.
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 are 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, and 5 µg/mL Pepstatin A, and 0.025% Bacitracin]
using a micro ultrasonic 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 of protein. In competition binding studies,
membranes (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 are calculated using an average of at least
two separate determinations.
tr a n s,tr a n s-2-[2-(2-Oxop yr id -1-yl)et h yl]-4-(1,3-b en zo-
d ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r b on ylm et h yl]-
p yr r olid in e-3-ca r boxylic Acid (9t): white powder; 1H NMR
(CDCl3, 300 MHz) δ 0.89 (t, J ) 7.5 Hz, 3H), 0.94 (t, J ) 7.5
Hz, 3H), 1.21-1.39 (m, 4H), 1.41-1.61 (m, 4H), 2.38 (brm, 2H),
3.09-3.24 (m, 3H), 3.32 (t, J ) 8.4 Hz, 2H), 3.54 (brm, 1H),
3.73 (t, J ) 11.4 Hz, 1H), 3.88 (dd, J ) 8.4, 18.3 Hz, 1H), 3.94-
4.10 (m, 2H), 4.15-4.30 (m, 3H), 5.96 (s, 2H), 6.31 (td, J )
1.5, 6.9 Hz, 1H), 6.58 (d, J ) 9.0 Hz, 1H), 6.72 (d, J ) 8.1 Hz,
1H), 6.77 (dd, J ) 1.8, 8.1 Hz, 1H), 6.84 (d, J ) 1.8 Hz, 1H),
7.40 (td, J ) 1.8, 6.3 Hz, 1H), 7.60 (dd, J ) 1.5, 6.0 Hz, 1H);
MS (DCI/NH3) (M + H)+ at m/ z 526. Anal. Calcd for
C
29H39N3O6‚0.70TFA: C, 60.31; H, 6.61; N, 6.94. Found: C,
60.17; H, 6.59; N, 6.91.
tr a n s,tr a n s-2-[2-(2-Oxopyr r olidin -1-yl)eth yl]-4-(1,3-ben -
zod ioxol-5-yl)-1-[(N,N-d i-n -bu tyla m in o)ca r bon ylm eth yl]-
p yr r olid in e-3-ca r boxylic Acid (9u ): an amorphous white
1
solid; H NMR (CDCl3, 300 MHz) δ 0.91 (t, J ) 7.5 Hz, 3H),
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 of [3H]-
myoinositol in RPMI for 16 h. The cells were washed with
PBS and 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 terminated by the addition of 1.5 mL of 1:2 (v/v) chloro-
form-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 of as described by
Berridge.29 The upper aqueous phase (1 mL) was retained,
and a small portion (100 µL) was counted. The rest of the
0.94 (t, J ) 7.5 Hz, 3H), 1.23-1.38 (m, 4H), 1.44-1.60 (m, 4H),
2.05 (t, J ) 6.9 Hz, 2H), 2.12-2.25 (m, 1H), 2.38 (td, J ) 4.2,
8.4 Hz, 2H), 2.47-2.61 (m, 1H), 3.17 (dd, J ) 6.0, 8.7 Hz, 2H),
3.24 (t, J ) 9.0 Hz, 1H), 3.32 (t, J ) 7.8 Hz, 2H), 3.38-3.48
(m, 3H), 3.52 (t, J ) 9.0 Hz, 1H), 3.66 (t, J ) 6.9 Hz, 1H), 3.96
(m, 2H), 4.14 (m, 1H), 4.38 (brs, 2H), 5.93 (s, 2H), 6.74 (d, J )
8.1 Hz, 1H), 6.89 (dd, J ) 1.8, 8.1 Hz, 1H), 6.87 (d, J ) 1.8 Hz,
1H); MS (DCI/NH3) (M + H)+ at m/ z 516. Anal. Calcd for
C
28H41N3O6‚1.40TFA: C, 54.78; H, 6.33; N, 6.22. Found: C,
54.69; H, 6.33; N, 6.14.
tr a n s,tr a n s-[2S,3R,4S]-2-(2,2-Dim eth ylpen tyl)-4-(7-m eth -
oxy-1,3-b en zod ioxol-5-yl)-1-[(N,N-d i-n -b u t yla m in o)ca r -
bon ylm eth yl]p yr r olid in e-3-ca r boxylic Acid (10a ): white
1
solid; H NMR (CDCl3, 300 MHz) δ 0.81 (s, 3H), 0.84 (s, 3H),

3274 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Liu et al.
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1
Analytical Department for assistance in acquiring H
NMR and mass spectra.
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