Pyrrolidine-3-carboxylic acids as endothelin antagonists. 2. Sulfonamide-based ET(A)/ET(B) mixed antagonists

Article abstract of DOI:10.1021/jm970101g

When the N,N-dialkylacetamide side chain of the highly ET(A)-selective endothelin antagonist ABT-627 (1; [2R,3R,4S]-2-(4-methoxyphenyl)-4-(1,3- benzodioxol-5-yl)-1-[[(N,N-dibutylamino)carbonyl]methyl]pyrrolidine-3- carboxylic acid; A-147627) is replaced by N,S-dialkylsulfonami-doethyl, the resultant analogs retain ET(A) affinity, but exhibit substantial ET(B) affinity as well. Structure-activity studies reveal that modifications in the length of the two alkyl groups, and in the substitution on the anisyl ring, are important in optimizing this 'balanced' antagonist profile. In particular the combination of an N-n-propyl group, an S-alkyl chain between four and six carbons in length, and a fluorine atom ortho to the aromatic OCH₃ provides compounds with sub-nanomolar affinities for both receptor subtypes, and with ET(A)/ET(B) ratios close to 1. A number of these compounds also exhibit oral bioavailabilities (in rats) in the 30-50% range and have substantial plasma half-lives. The balanced receptor-binding profile of these potent and orally bioavailable compounds complements the ET(A) selectivity observed with 1.

Full text of DOI:10.1021/jm970101g

J . Med. Chem. 1997, 40, 3217-3227
3217
P yr r olid in e-3-ca r boxylic Acid s a s En d oth elin An ta gon ists. 2.
Su lfon a m id e-Ba sed ETA/ETB Mixed An ta gon ists
Hwan-Soo J ae,* Martin Winn, Douglas B. Dixon, Kennan C. Marsh,† Bach Nguyen,† Terry J . Opgenorth, and
Thomas W. von Geldern*
Metabolic Diseases Research and Drug Analysis Department, Pharmaceutical Products Research Division, Abbott Laboratories,
Abbott Park, Illinois 60064
Received February 19, 1997X
When the N,N-dialkylacetamide side chain of the highly ETA-selective endothelin antagonist
ABT-627 (1; [2R,3R,4S]-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-[[(N,N-dibutylamino)-
carbonyl]methyl]pyrrolidine-3-carboxylic acid; A-147627) is replaced by N,S-dialkylsulfonami-
doethyl, the resultant analogs retain ETA affinity, but exhibit substantial ETB affinity as well.
Structure-activity studies reveal that modifications in the length of the two alkyl groups, and
in the substitution on the anisyl ring, are important in optimizing this “balanced” antagonist
profile. In particular the combination of an N-n-propyl group, an S-alkyl chain between four
and six carbons in length, and a fluorine atom ortho to the aromatic OCH3 provides compounds
with sub-nanomolar affinities for both receptor subtypes, and with ETA/ETB ratios close to 1.
A number of these compounds also exhibit oral bioavailabilities (in rats) in the 30-50% range
and have substantial plasma half-lives. The balanced receptor-binding profile of these potent
and orally bioavailable compounds complements the ETA selectivity observed with 1.
In tr od u ction
In recent articles in this journal,1-4 our group has
described the identification and optimization of several
novel classes of endothelin receptor antagonists. In
particular4-6 we have reported on a family of pyrroli-
dine-based compounds, exemplified by ABT-627 (1;
[2R,3R,4S]-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-
yl)-1-[[(N,N-dibutylamino)carbonyl]methyl]pyrrolidine-
3-carboxylic acid; A-147627; Figure 1), which are highly
potent and selective for the ETA receptor subtype.
F igu r e 1. ABT-627, an ETA-selective antagonist.
While we have hypothesized7 that ETA selectivity may
confer an advantage for the treatment of ET-induced
required to maintain high ETA selectivity. We were
constrictive and/or proliferative disorders, the relative
curious to explore the effect of “reorienting” these two
merits of selective vs nonselective therapy is still the
alkyl groups by moving the H-bond donor to different
subject of debate; in fact, many of the compounds
positions on the side chain. A notable effect was
currently under development8 exhibit a “balanced”
observed when the carbonyl group of the amide linkage
profile of ETA and ETB activity.
of 1 was transposed along the chain (compound 2, Table
In order to maximize our chances for success in the
1; see also Scheme 1). This modification led to a large
field of endothelin-blocking therapeutics, and because
decrease in ETA affinity (from 0.36 to 20 nM) with little
we believe that differing receptor profiles might be
change in ETB affinity (from 520 to 850 nM); the net
optimally effective for the treatment of particular
result is a 35-fold change in ETA selectivity, from 1400×
disease states, we hoped to develop a series of mixed
to 40×. Intrigued by this observation, we experimented
ETA/ETB antagonists to complement our earlier ETA-
with other amide surrogates, and quickly discovered
selective candidate. A fortuitous observation during the
that the corresponding sulfonamide (3) was substan-
studies leading to 1 has provided us access to such a
tially more potent at both ETA and ETB receptors, while
series of compounds; the results of our explorations in
retaining the reduced ETA/ETB activity ratio of 2. The
this area are reported below.
potency/selectivity profile of 3 is comparable to those
of a number of “balanced” compounds which have
Cr itica l Obser va tion
recently been described in the literature;8 more impor-
Structure-activity studies during the development of
1 indicated to us the critical importance of the side chain
amide carbonyl; we suspect that this carbonyl is in-
volved in a hydrogen-bonding interaction with the ETA
receptor which serves to orient the butyl groups into
appropriate hydrophobic binding pockets. Our studies
also indicated that the presence of both alkyl groups are
tantly, it has served as a valuable lead for further
optimization.
Ch em istr y
The core pyrrolidines employed in this study have
been assembled (Scheme 2) by direct analogy to our
earlier work.4 Briefly, the Michael reaction between
â-keto esters 7 and nitrostyrene 5 provides a mixture
of diastereomeric nitro ketones. The keto esters them-
† Drug Analysis Department.
X Abstract published in Advance ACS Abstracts, September 1, 1997.
S0022-2623(97)00101-5 CCC: $14.00 © 1997 American Chemical Society

3218 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
J ae et al.
Sch em e 1. Conceptual Development of Sulfonamide-Based Mixed Antagonists
Sch em e 2. General Synthetic Route to Sulfonamidoethyl-Substituted Pyrrolidinesa
a
(i) CH3NO2, NH4OAc, HOAc, heat; (ii) NaH, (EtO)2CO, THF; (iii) DBU, THF-iPrOH; (iv) H2, Ra-Ni, THF-HOAc; NaBH3CN, THF-
EtOH, pH 5; (v) DBU, THF, heat; (vi) Br(CH2)2Br, cat. NaI, heat; (vii) R1NH2, EtOH, cat. NaI, heat; (viii) R2SO2Cl, iPr2NEt, CH3CN; (ix)
NaOH, H2O-EtOH.
selves are prepared by carbethoxylation of the corre-
sponding ketones 6, which are either purchased (e.g. as
in the case of p-methoxyacetophenone) or assembled
through Friedel-Crafts acetylation. Nitrostyrene 5
derives from Henry reaction of piperonal 4.
The diastereomeric mixture of nitro ketones is reduc-
tively cyclized in two steps (Raney nickel reduction to
provide a cyclic imine, which is further reduced using
cyanoborohydride) to give pyrrolidines 8, generally as
a mixture of three diasteromers in which the desired
trans,trans isomer predominates. An additional portion
of trans,trans material may be derived by epimerization
(DBU, toluene) of the cis,cis isomer, which is conve-
niently separated by chromatography.
To attach the sulfonamidoethyl side chains, amine 8
(as a mixture of stereoisomers) is reacted with 1,2-
dibromoethane under iodide catalysis to give a 1-(bro-
moethyl)pyrrolidine. Displacement of the second bro-
mine atom (also catalyzed by I-) with a primary
amine gives 9. Sulfonylation of the resultant second-
ary amine provides the fully elaborated side chain.
Under standard saponification conditions (NaOH, MeOH,
room temperature, overnight), the hydrolysis of the
trans,trans ester is dramatically faster than that of
any other isomer, providing the pure trans,trans acid
10. This valuable selective hydrolysis was first observed
during the preparation of our ETA-selective analogs.4
In several cases the compounds have been prepared
in optically pure form. The preparation of 10 as a single
enantiomer is accomplished through resolution of the
core pyrrolidine 8 prior to side chain assembly (Scheme
3). In practice, the racemic pyrrolidine ester is con-
verted in two steps to the corresponding Boc acid; as
described above, this hydrolysis step selects for the
trans,trans isomer. Formation of a chiral salt of racemic
acid 11 using (R)-(+)-R-methylbenzylamine provides a
practical and general resolution strategy. In each case
a single recrystallization produces material of >99.5%
ee, as evaluated by chiral HPLC. The diasteromerically
pure salt is washed with acid to remove the chiral amine
(which may be recovered); the resultant optically active
11 is reconverted to amino ester 8 under Fischer
esterification conditions. Optically pure antagonists 10
(see Table 4 for characterization) are prepared by the
steps already described in Scheme 2.
Com p ou n d Eva lu a tion
The first line of biochemical analysis for the com-
pounds described in this study is a measurement of their
ability to displace endothelin from its receptor. For the
purpose of screening, we employ a rodent ETA receptor
derived from MMQ cells (rETA); ETB receptor (pETB) is
derived from porcine cerebellar tissue. IC50 data are

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3219
Sch em e 3. Preparation of Enantiomerically-Enriched Analogsa
a
(i) Boc2O, CH2Cl2; (ii) NaOH, H2O-EtOH; (iii) (S)-(-)-R-methylbenzylamine, EtOAc-Et2O, 1 recrystallization, >99.5% ee; (iv) wash
with 1 N H3PO4; (v) sat. HCl/EtOH; (vi) BrCH2CH2Br, cat. NaI, heat; (vii) R1NH2, cat. NaI, EtOH, heat; (viii) R2SO2Cl, iPr2NEt, CH3CN;
(ix) NaOH, H2O-EtOH.
Ta ble 1. Exploration of Substituents on the Sulfonamidoethyl Side Chain
IC50 (nM)a
compd
R1
R2
C3H7CO
C3H7SO2
C3H7SO2
C3H7SO2
C3H7SO2
C2H5SO2
C4H9SO2
rETA binding
pETB binding
A/B ratio
formula
C29H38N2O6
C28H38N2O7S
C25H32N2O7S
2
3
C4H9
C4H9
CH3
20
2.0
10
850
60
260
36
62
95
9.4
4.1
4.5
57
5.2
6.3
77
34
210
41
43
30
26
22
7.7
79
4.7
7.2
10
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12m
12n
12o
12p
12q
C3H7
i-C4H9
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
1.6
8.1
1.2
2.0
0.57
0.43
5.1
0.78
2.0
5.2
10
C27H36N2O7S
C28H38N2O7S‚0.2TFA
C26H34N2O7S‚0.3TFA
C28H38N2O7S
C5H11SO2
C6H13SO2
C7H15SO2
C29H40N2O7S
C30H42N2O7S‚0.75H2O
C31H44N2O7S‚0.3TFA
C28H38N2O7S
C27H35N2O7ClS
C27H36N2O8S
11
i-C4H9SO2
Cl(CH2)3SO2
CH3O(CH2)2SO2
C6H5CH2SO2
C6H5SO2
4-ClC6H4SO2
4-CH3C6H4SO2
4-CH3OC6H4SO2
6.6
3.1
15
3.4
150
6.5
10
C31H36N2O7S
1.4
6.3
8.3
0.53
C30H34N2O7S‚0.5H2O
C30H33N2O7ClS
C31H36N2O7S
85
66
120
C31H36N2O8S
a
IC50s calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M unless otherwise
noted.
recorded by measuring the displacement of [125I]ET-1
modified side chain roughly isosteric with the original
N,N-dibutylacetamide of 1. The observation of in-
creased ETB affinity in this series suggested that our
assumptions regarding the overlay of these alkyl sub-
stituents were likely to be inaccurate. The suspicion
that one of these alkyl groups might be finding a new
hydrophobic site which we had not previously explored
prompted a reevaluation of the side chain structure-
activity profile (Table 1). A brief exploration of the
N-substituent led to the choice of an n-propyl group (as
in 12b) as one providing an optimal combination of ETA
and ETB activity. Additional decreases in the length of
this substituent (12a , R1 ) CH3) or the presence of a
branch in the side chain (viz., 12c) are detrimental to
ETA binding affinity and offer no improvement in ETA/
ETB ratio over the original n-butyl group. The strong
analogy between this result and that of our earlier
study4 which determined the preferred amide N-alkyl
substituents in the 1 series suggests that the sulfona-
from ETA, or of [125I]ET-3 from ETB. Confirmatory
binding studies are performed in a similar fashion, using
human ETA and ETB receptors (hETA, hETB) perma-
nently expressed in CHO cells.
Analogs of particular interest are examined for their
ability to block the ET-1-induced hydrolysis of inositol
phosphates in MMQ cells (ETA-receptor mediated) or
in ETB-CHO. In both experiments the compounds are
also tested for the ability to function as agonists. This
same subset of compounds of particular interest has also
been examined for their pharmacokinetic properties
using a standard protocol which compares the time
course of plasma drug levels after dosing in rats by
intravenous injection and oral gavage.
Resu lts a n d Discu ssion
The lengths of the side chain N- and S-alkyl substit-
uents in 3 (or 2) were originally chosen to make the

3220 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Ta ble 2. Modification of Position 2 Aryl Substituent
J ae et al.
IC50 (nM)a
compd
R3
OCH3
OCH2OCH3
O(CH2)2CH3
OCH3
X
rETA binding
pETB binding
A/B ratio
formula
12f
13a
13b
13c
13d
13e
13f
13g
H
H
H
0.57
0.62
48
4.1
5.2
610
73
7.0
0.85
2.6
7.2
8.4
13
32
10
1.6
2.0
3.8
C30H42N2O8S‚0.25H2O
C31H44N2O7S
OCH3
2.3
C30H42N2O8S‚0.6TFA
C30H40N2O8S‚1.0H2O
C29H39N2O7FS‚0.25H2O
C30H41N2O7S‚0.25H2O
-O(CH2)2O-
0.66
0.52
1.3
OCH3
OCH2CH3
F
F
F
F
18
68
C28H36N2O6F2S
a
IC50s calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M unless otherwise
noted.
mide N-alkyl is sharing one of these previously-
described binding sites.
yls. Replacement of the 4-OCH3 group with 4-(meth-
oxymethoxy) is well tolerated (compound 13a ), but offers
no improvement in either potency or A/B balance;
additionally, the acid lability of this group might prove
to be disadvantageous. The stable isosteric 4-propoxy
replacement (13b) is at least 50 times less potent. The
latter result suggested that the second oxygen atom in
13a might be playing an important role and led us to
find other means for introducing such a moiety. Sub-
stitution at the position adjacent to the methoxy group
provides a useful means for accomplishing this purpose
and for modulating A/B selectivity. The 3,4-dimethox-
yphenyl analog 13c exhibits a slight decrease in ETA
affinity and a larger decrease in ETB; the net result is
a higher A/B ratio. When these two alkoxy groups are
tied together to form a 3,4-benzodioxane moiety (13d ),
both ETA and ETB affinity improve, as does the A/B
ratio. The critical observation in this study results from
introduction of a fluorine atom at the aryl 3-position.
This change produces a substantial improvement in ETB
affinity with little change in ETA binding; the resultant
analog 13e is the first in the series (and the first
reported to date) to exhibit subnanomolar potency
against both receptor subtypes and in addition has an
exceptionally good balance between ETA and ETB. As
follow-up to this result we have subsequently prepared
analogs having a slightly larger alkoxy substituent (13f)
as well as one in which the 4-OCH3 group is completely
replaced by a second fluorine atom (13g). These studies
confirm that a methoxy group is the optimal 4-substitu-
ent. The 3-fluoro-4-ethoxyphenyl analog 13f is slightly
less active (2-4×) at both receptors, retaining an A/B
ratio near unity; with the 3,4-difluorophenyl replace-
ment, the loss in binding affinity for ETA and ETB is
between 20- and 100-fold.
It is possible to substantially improve ETB affinity
(and thereby decrease the A/B ratio) through modifica-
tion of the S-substituent. As the length of this alkyl
chain varies from two (12d ) through six carbons (12g),
the affinity of the resultant analog for the ETB receptor
steadily improves, with little change in ETA binding.
This combination of events translates into better recep-
tor balance; compounds 12e-h have A/B ratios in the
5-10 range. The optimal chain length appears to be
five or six atoms; further extension (as in 12h ) results
in decreases in affinity for both receptors. This struc-
ture-activity profile is markedly different than any
observed in the ABT-627 series; it suggests to us that
this S-substituent has accessed a new hydrophobic
domain that was not available to the amide series.
Importantly, it appears that, unlike the “second” amide
N-alkyl domain of the ETA-selective series, this S-alkyl
binding site is present on both ETA and ETB receptors.
The sulfonamide S-substituent is not limited to linear
alkyl groups. Branching is well tolerated (12i), as is
heteroatom substitution (see in particular 12j; also 12k ).
A benzyl group decreases the activity at both ETA and
ETB receptors by about 1 order of magnitude (12m ),
retaining a low A/B ratio. A phenyl substituent also
reduces affinity for both receptors (12n ); through place-
ment of substituents on the aromatic ring (12o-q), we
are able to substantially improve the ETA activity, but
not ETB. This leads the more active compounds in this
S-aryl series (e.g. 12q) to have unacceptably high
(>100×) A/B ratios.
With optimal lengths established for the N- and
S-substituents on the side chain sulfonamide, compound
12f was chosen as a standard to evaluate modifications
of the pyrrolidine core. In particular we focused (Table
2) on changes in the anisyl ring at position 2 of the
pyrrolidine. Structure-activity studies of compounds
related to 1 had suggested to us that the replacement
of the 4-methoxy substituent with a group other than
alkoxy was detrimental to ETB activity (these studies
are unpublished, but will be reported in due course) and
led us to focus our attention on substituted 4-alkoxyar-
This second SAR study served to establish 13e as a
new standard for the sulfonamide series. The combina-
tion of 3-fluorination on the anisyl ring with a longer
(five-carbon) S-alkyl chain has led to a compound with
IC50 values of 0.52 and 0.88 nM for ETA and ETB
receptors, respectively. Since our earlier study of side
chain substitution had suggested that several other
alkyl groups were well-tolerated as replacements for

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3221
Ta ble 3. Second-Generation Analogs
IC50 (nM)a
compd
R
rETA binding
pETB binding
A/B ratio
formula
13e
14a
14b
14c
14d
14e
14f
14g
C5H11
C4H9
C6H13
i-C4H9
i-C5H11
Cl(CH2)3
CF3(CH2)3
C6H5CH2
0.52
0.54
0.46
0.21
0.46
0.40
0.55
2.7
0.85
1.0
1.5
1.6
1.2
1.0
0.98
2.9
1.6
1.9
3.3
7.6
2.6
2.5
1.8
1.1
C28H37N2O7FS
C30H41N2O7FS
C28H37N2O7FS‚0.25H2O
C29H39N2O7FS
C27H34N2O7ClFS
C28H34N2O7F4S
C31H35N2O7FS‚0.5H2O
a
IC50s calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M unless otherwise
noted.
Ta ble 4. Summary Data for Single Enantiomers
pharmacokinetic profilec (rats)
T1/2 Cmax AUC
iv (h) oral (h) (µg/mL) (µg h/mL) F (%)
functional IC50
,
binding IC50, nM (SEM, n)a,b
nM (n)a,c
T1/2
,
,
compd
R
rETA
pETB
hETA
hETB
rETA
hETB
15a
C4H9
0.10
0.32
0.044 (3) 0.47 (3) 0.38 (2) 0.60 (2)
0.078 (3) 0.28 (3) 0.48 (4) 1.28 (3)
0.077 (2) 0.48 (2) 0.14 (1) 1.12 (1)
6.9
5.0
3.3
7.3
8.1
6.4
9.6
8.9
1.1
2.4
0.7
0.4
1.2
3.7
11.2
2.8
31
54
40
25
25
(0.018, 7) (0.055, 7)
0.13 0.29
(0.015, 6) (0.029, 6)
0.11 0.34
(0.016, 5) (0.046, 5)
0.094 0.20
(0.021, 5) (0.024, 5)
0.22 0.32
(0.055, 5) (0.059, 5)
15b
(A-182086)
15c
C5H11
C6H13
15d
15e
Cl(CH2)3
CF3(CH2)3
0.070 (2) 0.14 (2) 0.55 (1) 0.83 (1) 14.0
0.055 (2) 0.20 (2) 1.66 (1) 1.46 (1) 4.3
2.2
4.3
a
IC50s calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M unless otherwise
b
noted. Binding assays recorded as described in Experimental Section, using MMQ cells (rETA), porcine cerebellar tissue (pETB), or
clonal CHO cell lines (hETA and hETB). c Measurements of phosphoinositide (PI) hydrolysis as described in Experimental Section, using
d
MMQ cells (rETA) or CHO cells containing a human ETA receptor clone (hETB). 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.
S-pentyl, we deemed it worthwhile to reevaluate a
number of these side chain possibilities with the fluorine-
containing core. The results of this fine-tuning (Table
3) largely confirm our initial observations. As the length
of the linear S-alkyl chain varies from four to six carbon
atoms, receptor affinities are largely unchanged, and
the A/B ratio remains low. Branching is tolerated at
the S-substituent (14c,d ) and does not appear to dis-
tinguish between ETA and ETB. Halo substitution is
also acceptable, with both 3-chloropropyl and 4,4,4-
trifluorobutyl chains (compounds 14e and 14f, respec-
tively) resulting in analogs with potencies similar to
13e. In fact, not only 13e but also 14a -f seem likely
(when prepared as single, active enantiomers) to meet
our goal of providing subnanomolar affinities for both
receptors. Only benzylsulfonamide 14g appears to be
significantly less active.
A more detailed evaluation was undertaken on a
smaller set of compounds prepared in optically enriched
form (Table 4). This subset (compounds 15a -e) was
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. As we
have observed in previous studies,3,4 the rETA and pETB
receptors used in the screening assays accurately predict
the relative and absolute affinities observed against
human receptors, with some tendency for this class of
compounds to exhibit higher affinities for the human
genotypes. In fact, 15a -e all have subnanomolar
potencies against both hETA and hETB receptors; A/B

3222 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Ch a r t 1. Representative “Balanced” ETA/B Antagonists
J ae et al.
Ta ble 5. Comparison Data for Representative Mixed
Antagonists
ratios vary from 2 (15d ) to 10 (15a ). To confirm that
the compounds are antagonists, 15a -e have been
evaluated in a set of functional assays measuring ETR-
mediated hydrolysis of inositol phosphates. The potency
in these assays correlates well with receptor affinity,
and the compounds show no agonist effect at high doses
(data not shown), confirming that as a class they are
pure, functional antagonists at both ETA and ETB.
Because it was difficult to identify an optimal com-
pound in this series based purely on receptor binding
properties, pharmacokinetic profiles (in rats) were
acquired for all analogs in Table 4. These studies
suggest that, in addition to their superior biochemical
profiles, this class of balanced ET antagonists may be
readily deliverable through an oral route. Oral bio-
availabilities for 15a -e are generally high, and the
compounds have plasma half-lives in the range 5-10
h. While it is difficult to find a clear distinction between
compounds 15, it appears that 15b (A-182086) may offer
the best combination of potency, ETA/ETB balance, and
pharmacokinetic properties.
It is instructive to evaluate 15b against other bal-
anced ET antagonists which have appeared in the recent
literature (Chart 1; comparison data in Table 5). Roche’s
Bosentan8a (16) has been extensively studied and is
currently under evaluation in human subjects. Take-
da’s TAK-0448b (17) is another nonselective peptide;
balanced nonpeptide antagonist series have been re-
ported by SmithKline Beecham (SB209670, 18; and
SB217242, 19),8c and by Merck (L-754,142; 20),8d among
others. The most potent of these are 17, 18, and 20,
which exhibit subnanomolar affinities for human ETA
receptor.
IC50 (nM)a
hETA
binding
hETB
binding
A/B
iv half-lifec
(h)
compd
ratiob
Fc (%)
30-80
168a
6.5 (Ki)
0.24
0.20 (Ki)
1.1 (Ki)
0.26
340 (Ki)
130
18 (Ki)
110 (Ki)
4.4
53
540
90
100
17
4-8
178b
0.5-1.0
188c(1)
198c(2)
208d
2.0
2.0
5.0
60
60
54
15b
0.078
0.28
3.6
a
Human receptor data acquired in a variety of systems; IC50
b
values are given unless Ki is explicitly stated. Expressed as
IC50(ETB)/IC50(ETA). T1/2 and oral bioavailability (F) data, mea-
c
sured in rats, collected from a variety of sources.
antagonists; it also shows a balance between receptors
closer than has previously been described. The subna-
nomolar ETB potency of this compound is particularly
noteworthy; to our knowledge, 15b is the most potent
ETB antagonist reported to date. The compound is also
well absorbed (oral bioavailability ) 54%) and has a
substantially longer iv half-life (5 vs 2 h) than 20. It is
difficult to make direct comparisons between data
recorded by different groups under differing assay
conditions (for example, it is known9 that the presence
or absence of serum albumin in binding assay buffer
can substantially impact the binding of various ET
antagonist molecules to ETR); however, it is clear that
15b has superior properties as a balanced antagonist
of endothelin receptors.
Con clu sion s
When the N,N-dialkylacetamide side chain of the
highly ETA-selective endothelin antagonist ABT-627 (1)
is replaced by (N,S-dialkylsulfonamido)ethyl, the result-
ant analogs retain ETA affinity, but exhibit substantially
improved ETB activity as well. Structure-activity
studies reveal that modifications in the length of the
two alkyl groups, and in the substitution on the anisyl
While the first two of these are useful only as
parenteral agents, 20 is well-absorbed orally (60% in
rats) and is also the best “balanced” agent (17-fold
difference between ETA and ETB activities) reported to
date. By comparison 15b appears to be substantially
more potent at the ETA receptor than any of the above

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3223
was extracted with 75 mL of diethyl ether; the ether layer was
extracted with 40 mL of water. The combined aqueous phases
were acidified with 7.5 g of acetic acid; the mixture was stirred
until a solid formed. The solid was filtered, washed with
water, and dissolved in dichloromethane. After being dried
with Na2SO4, the solution was concentrated and the residue
crystallized from 1:1 ether-hexanes to get 16.0 g of product,
mp 200-203 °C (90% yield). The crude acid was suspended
in 80 mL of EtOAc and treated with 4.00 g (33.1 mmol) of (S)-
(-)-R-methylbenzylamine. After being heated to dissolve the
acid, 80 mL of ether was added. Scratching with a glass rod
caused the product to crystallize. The solids were filtered and
washed with ether-EtOAc solution to give 8.22 g (81% yield
based on 50% maximum recovery) of salt, mp 165-168 °C.
After one recrystallization, chiral HPLC analysis, using a Regis
Whelk-O column, indicated >99.5% ee. The salt was dissolved
in 500 mL of 36% HCl in ethanol; a white solid forms. The
resultant suspension was warmed for 16 h at 50 °C. After
the suspension was concentrated in vacuo, the residue was
combined with toluene and stirred with potassium bicarbonate
in water for 30 min. The toluene was separated, dried (Na2-
SO4), and concentrated. The residue was chromatographed
on silica gel, eluting with 1:2 hexane-EtOAc to get 6.9 g (99%)
of the resolved amino ester.
Com p ou n d s 9. Compound 8 was dissolved in 1,2-dibro-
moethane (10 mL per 1 g of starting material); diisopropyl-
ethylamine (1 mL per 1 g of starting material) and NaI (100
mg per 1 g of starting material) were added, and the mixture
was stirred at 100 °C for 1 h. Toluene was added, and the
mixture was washed with sodium bicarbonate solution. The
solvents were concentrated, and the resultant black residue
was chromatographed on silica gel, eluting with 4:1 hexanes-
EtOAc to give the N-(2-bromoethyl)pyrrolidine (85-92%). This
compound was combined with the appropriate amine (3.5
equiv) and NaI (10% by weight of bromide) in ethanol (5 mL
per 1 g of bromide) and was heated at 80 °C for 2 h. Toluene
was added, and the mixture was washed with sodium bicar-
ring, are important in optimizing this “balanced” an-
tagonist profile. In particular, the combination of an
N-n-propyl group, an S-alkyl chain between four and
six carbons in length, and a fluorine atom ortho to the
aromatic OCH3 provides compounds with sub-nanomo-
lar affinities for both receptor subtypes, and with ETA/
ETB ratios close to unity. A number of these compounds
also exhibit oral bioavailabilities (in rats) in the 30-
50% range and have relatively long plasma half-lives.
Of these, 15b exhibits the best combination of physical
and biochemical properties.
Our SAR studies suggest that there is no direct
mapping of the N- and S-alkyl groups of the sulfonamide
onto the two amide N-butyl substituents of 1. Instead,
while it is likely that the sulfonamide N-alkyl does share
a hydrophobic domain with one of the amide N-butyls,
the sulfonamide moiety serves to position the longer
S-alkyl into a domain which (unlike the binding site for
the second amide N-butyl) is present in both ETA and
ETB receptor subtypes.
The balanced receptor-binding profile of these potent
and orally-bioavailable compounds complements the
ETA selectivity observed with 1. It remains to be seen
whether this balanced profile will prove to be an
advantage (or disadvantage) in treating diseases in
which endothelin plays a pathogenic role. Studies to
evalute this question are underway in our labs, and will
be reported in due course.
Exp er im en ta l Section
Unless otherwise specified, all solvents and reagents were
obtained from commercial suppliers and used without further
purification. THF was dried over sodium and purified by
distillation. All reactions were performed under nitrogen
atmosphere unless specifically noted. Flash chromatography
was done using silica gel (230-400 mesh) from E. M. Science.
1H-NMR spectra were recorded at 300 MHz; all values are
referenced to tetramethylsilane as internal standard and are
reported as shift (multiplicity, coupling constants, proton
count). Mass spectral analysis is accomplished using fast atom
bombardment (FAB-MS) or direct chemical ionization (DCI-
MS) techniques. All elemental analyses are consistent with
theoretical values to within (0.4% unless indicated. Analyti-
cal HPLC chromatographs are recorded on a Beckman system
using a Vydac “Peptide & Protein” C18 column, eluting with
gradients of acetonitrile in 0.1% aqueous TFA. Melting points
were measured on a Thomas Hoover apparatus and are
uncorrected.
bonate solution, dried (Na2
SO4), and concentrated. More
toluene was added, and removed in vacuo, to get rid of the
primary amine. The residue was dissolved in heptane and
filtered to remove a small amount of insoluble material.
Evaporation of the solvent gave the desired product (86-93%
yield), which was used for the next step without further
purification.
Va r iou s Su lfon yl Ch lor id es. Most of the sulfonyl chlo-
rides employed in this study are commercially available.
Other sulfonyl chlorides were prepared using the procedures
of Roemmele and Rapoport,10 with the exception of 4,4,4-
trifluorobutanesulfonyl chloride, which was synthesized by the
method of Bunyagidi et al.11
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -bu tyl-N-n -bu tan oylam in o)eth yl]pyr r olidin e-
3-ca r boxylic Acid (2). To compound 9 (R1 ) n-Bu, R3
)
Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
EtOAc, ethyl acetate; TFA, trifluoroacetic acid.
OCH3, X ) H) (211 mg, 0.45 mmol) dissolved in 8 mL of 1,2-
dichloroethane was added 0.25 mL of diisopropylethylamine.
The solution was cooled to -40 °C, butyryl chloride (53 mg,
1.1 equiv) was added, the bath was removed, and the mixture
was allowed to warm to ambient temperature and stirred
overnight. Solvents were removed in vacuo; the residue was
taken up in EtOAc, washed sequentially with sodium bicar-
bonate solution, water, and brine, dried (Na2SO4), and con-
centrated in vacuo. The product was purified by flash chro-
matography on silica gel, eluting with a gradient of 1:1
EtOAc-hexane going to EtOAc and finally 10% methanol-
EtOAc. The purified ester was dissolved in 2.5 mL of ethanol;
0.75 mL of a 17% aqueous NaOH solution was added, and the
resultant mixture was stirred at ambient temperature for 3
h. The solvents were evaporated, and the residue was taken
up in water and washed with ether. The aqueous phase was
acidified with 1 N H3PO4 to pH 5 and extracted twice with
ether. The combined organic extracts were washed with brine
and dried over Na2SO4. The solvents were removed in vacuo
to provide 153 mg (61%) of the title compound as a white
foam: 1H NMR (CDCl3, 300 MHz) δ 0.80 (m, 3H), 0.90 (t, J )
8 Hz, 3H), 1.45 (m, 4H), 1.6 (m, 2H), 2.20 (t, J ) 8 Hz, 3H),
2.94 (br m, 2H), 3.10 (br m, 2H), 3.5 (br m, 3H), 3.80 (br m,
Gen er a l Syn th esis of Cor e Com p ou n d s. Eth yl 2-(3-
F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben zod ioxol-5-yl)p yr r o-
lid in e-3-ca r boxyla te a s a Mixtu r e of Tr a n s,Tr a n s a n d
Cis,Tr a n s Dia ster eom er s (8, R3 ) OCH3, X ) F ). This
compound was prepared from ethyl (3-fluoro-4-methoxyben-
zoyl)acetate and 3,4-(methylenedioxy)-2′-nitrostyrene as start-
ing materials according to procedures described in our previous
article.4 The mixture was refluxed in toluene (5 mL/g) and
DBU (1.2 equiv) overnight to convert the cis,cis isomer in the
mixture to trans,trans. Other compounds 8 were prepared in
a similar fashion from the appropriate starting materials.
Resolu tion of Com p ou n d 8 (R3 ) OCH3, X ) F ). To
racemic compound 8 (15.0 g, 38.8 mmol), dissolved in 75 mL
of dichloromethane and cooled in an ice bath, was added di-
tert-butyl dicarbonate (9.30 g, 42.7 mmol). After 2 h of stirring
at room temperature, the solution was concentrated in vacuo;
the residue was dissolved in 50 mL of ethanol and treated with
a solution of 3.75 g of NaOH in 19 mL water. The solution
was warmed until homogeneity was achieved. After 2 h of
stirring at room temperature, the solution was concentrated
and redissolved in 200 mL of water. The resultant mixture

3224 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
J ae et al.
2H), 3.82 (s, 3H), 4.30 (br s, 1H), 5.95 (s, 2H), 6.75 (d, J ) 8
Hz, 1H), 6.84 (m, 1H), 6.85 (d, J ) 8 Hz, 2H), 7.04 (d, J ) 1
Hz, 1H), 7.40 (d, J ) 8 Hz, 2H); MS (DCI/NH3) m/e 511 (M +
H)+; HRMS (FAB) calcd for C29H38N2O6 511.2808, found
511.2809; analytical HPLC, 94% pure.
HRMS (FAB) calcd for C28H39N2O7S 547.2478, found 547.2480.
Anal. (C28H38N2O7S‚0.2TFA) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-(et h ylsu lfon yl)a m in o)et h yl]p yr -
r olid in e-3-ca r boxylic a cid (12d ): white solid; mp 70-72 °C;
1H NMR (CDCl3, 300 MHz) δ 0.79 (t, J ) 8 Hz, 3H), 1.28 (t, J
) 7 Hz, 3H), 1.43 (q, J ) 8 Hz, 2H), 2.22-2.30 (m, 1H), 2.71-
2.80 (m, 1H), 2.82-3.10 (m, 6H), 3.18-3.32 (m, 2H), 3.43 (dd,
J ) 3, 9 Hz, 1H), 3.53-3.60 (m, 1H), 3.65 (d, J ) 9 Hz, 1H),
3.80 (s, 3H), 5.96 (s, 2H), 6.73 (d, J ) 7 Hz, 1H), 6.82 (dd, J )
1, 7 Hz, 1H), 6.88 (d, J ) 8 Hz, 2H), 7.00 (d, J ) 1 Hz, 1H),
7.32 (d, J ) 8 Hz, 2H); MS (DCI/NH3) m/e 519 (M + H)+;
HRMS (FAB) calcd for C26H35N2O7S 519.2165, found 519.2165.
Anal. (C26H34N2O7S‚0.3TFA) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-(n -bu tylsu lfon yl)a m in o)eth yl]p yr -
r olid in e-3-ca r boxylic a cid (12e): white solid; mp 65-66 °C;
1H NMR (CDCl3, 300 MHz) δ 0.78 (t, J ) 7 Hz, 3H), 0.92 (t, J
) 7 Hz, 3H), 1.31-1.46 (m, 4H), 1.68 (quintet, J ) 7 Hz, 2H),
2.21-2.32 (m, 1H), 2.70-3.08 (m, 7H), 3.12-3.23 (m, 2H), 3.42
(dd, J ) 2, 9 Hz, 1H), 3.52-3.58 (m, 1H), 3.64 (d, J ) 9 Hz,
1H), 3.80 (s, 3H), 5.96 (s, 2H), 6.72 (d, J ) 7 Hz, 1H), 6.83 (dd,
J ) 1, 7 Hz, 1H), 6.86 (d, J ) 8 Hz, 2H), 7.00 (d, J ) 1 Hz,
1H), 7.32 (d, J ) 8 Hz, 2H); MS (DCI/NH3) m/e 547 (M + H)+.
Anal. (C28H38N2O7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -pr opyl-N-(n -pen tylsu lfon yl)am in o)eth yl]pyr -
r olid in e-3-ca r boxylic a cid (12f): white solid; mp 59-61 °C;
1H NMR (CDCl3, 300 MHz) δ 0.79 (t, J ) 7.5 Hz, 3H), 0.90 (t,
J ) 6 Hz, 3H), 1.26-1.32 (m, 4H), 1.43 (sextet, J ) 7.5 Hz,
2H), 1.67-1.76 (m, 2H), 2.23-2.32 (m, 1H), 2.70-3.08 (m, 7H),
3.15-3.32 (m, 2H), 3.42 (dd, J ) 3, 9 Hz, 1H), 3.52-3.57 (m,
1H), 3.63 (d, J ) 9 Hz, 1H), 3.80 (s, 3H), 5.95 (s, 2H), 6.73 (d,
J ) 7.5 Hz, 1H), 6.83 (dd, J ) 1, 7.5 Hz, 1H), 6.87 (d, J ) 8
Hz, 2H), 7.00 (d, J ) 1 Hz, 1H), 7.32 (d, J ) 8 Hz, 2H); MS
(DCI/NH3) m/e 561 (M + H)+. Anal. (C29H40N2O7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-(n -h exylsu lfon yl)a m in o)eth yl]p yr -
r olid in e-3-ca r boxylic a cid (12g): white solid; mp 59-60 °C;
1H NMR (CDCl3, 300 MHz) δ 0.80 (t, J ) 7.5 Hz, 3H), 0.89 (t,
J ) 7 Hz, 3H), 1.25-1.36 (m, 6H), 1.53 (sextet, J ) 7.5 Hz,
2H), 1.72 (quintet, J ) 7 Hz, 2H), 2.23-2.32 (m, 1H), 2.72-
3.08 (m, 7H), 3.15-3.32 (m, 2H), 3.43 (d, J ) 9 Hz, 1H), 3.55-
3.62 (m, 1H), 3.65 (d, J ) 10 Hz, 1H), 3.80 (s, 3H), 5.96 (s,
2H), 6.74 (d, J ) 7.5 Hz, 1H), 6.82 (d, J ) 7.5 Hz, 1H), 6.87 (d,
J ) 9 Hz, 2H), 7.01 (s, 1H), 7.32 (d, J ) 9 Hz, 2H); MS (DCI/
NH3) m/e 575 (M + H)+. Anal. (C30H42N2O7S‚0.75H2O) C, H,
N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -pr opyl-N-(n -h eptylsu lfon yl)am in o)eth yl]pyr -
r olid in e-3-ca r boxylic a cid (12h ): white solid; mp 58-59 °C;
1H NMR (CDCl3, 300 MHz) δ 0.80 (t, J ) 7.5 Hz, 3H), 0.88 (t,
J ) 7 Hz, 3H), 1.23-1.36 (m, 8H), 1.94 (q, J ) 7.5 Hz, 2H),
1.71 (quintet, J ) 7 Hz, 2H), 2.23-2.32 (m, 1H), 2.70-3.09
(m, 7H), 3.13-3.32 (m, 2H), 3.43 (dd, J ) 3, 9 Hz, 1H), 3.52-
3.58 (m, 1H), 3.65 (d, J ) 9 Hz, 1H), 3.80 (s, 3H), 5.96 (s,
2H), 6.73 (d, J ) 7 Hz, 1H), 6.83 (dd, J ) 1, 7 Hz, 1H), 6.87
(d, J ) 9 Hz, 2H), 7.01 (d, J ) 1 Hz, 1H), 7.32 (d, J ) 9
Hz, 2H); MS (DCI/NH3) m/e 589 M + H)+; HRMS (FAB)
calcd for C31H45N2O7S 589.2947, found 589.2942. Anal.
(C31H44N2O7S‚0.3TFA) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -bu tyl-N-(n -p r op ylsu lfon yl)a m in o)eth yl]p yr -
r olid in e-3-ca r boxylic Acid (3). Compound 9 (R1 ) n-Bu,
R3 ) OCH3, X ) H) (200 mg, 0.43 mmol) was dissolved in 5
mL of CH3CN; 110 mg (2 equiv) of N,N-diisopropylethylamine
and 73 mg (1.2 equiv) of 1-propanesulfonyl chloride were added
sequentially, and the resultant solution was allowed to stir at
room temperature for 30 min. The solvent was evaporated
under reduced pressure, and the residue was dissolved in
EtOAc. The solution was washed with sodium bicarbonate
solution, 1 N H3PO4, and brine, dried over Na2SO4, and
evaporated to give a yellowish oil which was purified by flash
chromatography on silica gel, eluting with 40% EtOAc/hexanes
to give 220 mg of product (89%). This ester was dissolved in
5 mL of ethanol, to which was added NaOH (46 mg, 3 equiv)
solution in 2 mL of water. This mixture was stirred for 3 h at
room temperature. The solution was concentrated in vacuo
using a low (<40 °C) bath temperature. Water (10 mL) and
ether (50 mL) were added; the ether layer was extracted with
5 mL of water. The combined aqueous mixture was back-
extracted with ether and then neutralized with acetic acid.
This solution was extracted twice with ether. The acidic
extracts were dried (Na2SO4) and concentrated in vacuo.
EtOAc (1 mL) and ether (1 mL) were added to dissolve the
product, and hexanes was added dropwise to produce a white
solid. The solid was collected and dried in vacuo to give 126
1
mg of 3: mp 64-65 °C; H NMR (CDCl3, 300 MHz) δ 0.83 (t,
J ) 7 Hz, 3H), 0.98 (t, J ) 7 Hz, 3H), 1.12-1.25 (m, 2H), 1.32-
1.41 (m, 2H), 1.75 (sextet, J ) 7 Hz, 2H), 2.23-2.31 (m, 2H),
2.72-3.32 (m, 8H), 3.43 (dd, J ) 3, 9 Hz, 1H), 3.53-3.59 (m,
1H), 3.65 (d, J ) 9 Hz, 1H), 3.80 (s, 3H), 5.95 (s, 2H), 6.73 (d,
J ) 8 Hz, 1H), 6.83 (dd, J ) 1, 8 Hz, 1H), 6.88 (d, J ) 9 Hz,
2H), 7.02 (d, J ) 1 Hz, 1H), 7.33 (d, J ) 9 Hz, 2H); MS (DCI/
NH3) m/e 547 (M + H)+; HRMS (FAB) calcd for C28H39N2O7S
547.2474, found 547.2478; analytical HPLC, 95% pure.
The following compounds are prepared using the procedures
described above for compound 3.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-m eth yl-N-(n -p r op ylsu lfon yl)a m in o)eth yl]p yr -
r olid in e-3-ca r boxylic a cid (12a ): white solid; mp 69-70 °C;
1H NMR (CDCl3, 300 MHz) δ 1.02 (t, J ) 7.5 Hz, 3H), 1.78
(hexad, J ) 7.5 Hz, 2H), 2.18-2.26 (m, 1H), 2.72 (s, 3H), 2.75-
2.95 (m, 5H), 3.13-3.22 (m, 1H), 3.25-3.35 (m, 1H), 3.47-
3.58 (m, 2H), 3.66 (d, J ) 9 Hz, 1H), 3.80 (s, 3H), 5.96 (s, 2H),
6.74 (d, J ) 7.5 Hz, 1H), 6.84 (dd, J ) 3, 7.5 Hz, 1H), 6.87 (d,
J ) 9 Hz, 2H), 7.04 (d, J ) 3 Hz, 1H), 7.43 (d, J ) 9 Hz, 2H);
MS (DCI/NH3) m/e 505 (M + H)+; HRMS (FAB) calcd for
C25H33N2O7S 505.2015, found 505.2008; analytical HPLC, 91%
pure.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-(n -p r op ylsu lfon yl)a m in o)et h yl]-
p yr r olid in e-3-ca r boxylic a cid (12b): white solid; mp 72-
1
73 °C; H NMR (CDCl3, 300 MHz) δ 0.79 (t, J ) 8 Hz, 3H),
0.98 (t, J ) 8 Hz, 3H), 1.43 (sextet, J ) 8 Hz, 2H), 1.75 (sextet,
J ) 8 Hz, 2H), 2.22-2.32 (m, 1H), 2.69-3.32 (m, 9H), 3.42
(dd, J ) 3, 12 Hz, 1H), 3.52-3.58 (m, 1H), 3.64 (d, J ) 12 Hz,
1H), 3.80 (s, 3H), 5.95 (s, 2H), 6.73 (d, J ) 11 Hz, 1H), 6.83
(dd, J ) 1, 11 Hz, 1H), 6.87 (d, J ) 11 Hz, 2H), 7.0 (d, J ) 2
Hz, 1H), 7.32 (d, J ) 11 Hz, 2H); MS (DCI/NH3) m/e 533 (M +
H)+. Anal. (C27H36N2O7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -pr opyl-N-(isobu tylsu lfon yl)am in o)eth yl]pyr -
r olid in e-3-ca r boxylic a cid (12i): white solid; mp 72-73 °C;
1H NMR (CDCl3, 300 MHz) δ 0.82 (t, J ) 7.5 Hz, 3H), 1.04 (d,
J ) 6 Hz, 6H), 1.44 (q, J ) 7.5 Hz, 2H), 2.15-2.33 (m, 2H),
2.57-2.75 (m, 3H), 2.84-3.08 (m, 4H), 3.12-3.21 (m, 1H),
3.23-3.45 (m, 1H), 3.43 (d, J ) 11 Hz, 1H), 3.55-3.62 (m, 1H),
3.66 (d, J ) 9 Hz, 1H), 3.80 (s, 3H), 5.95 (s, 2H), 6.75 (d, J )
9 Hz, 1H), 6.83 (dd, J ) 1, 9 Hz, 1H), 6.87 (d, J ) 9 Hz, 2H),
7.02 (d, J ) 1 Hz, 1H), 7.33 (d, J ) 9 Hz, 2H); MS (DCI/NH3)
m/e 547 M + H)+. Anal. (C28H38N2O7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-isobu tyl-N-(n -pr opylsu lfon yl)am in o)eth yl]pyr -
r olid in e-3-ca r boxylic a cid (12c): white solid; mp 73-74 °C;
1H NMR (CDCl3, 300 MHz) δ 0.80 (d, J ) 6 Hz, 6H), 0.98 (t,
J ) 8 Hz, 3H), 1.62 (sextet, J ) 6 Hz, 1H), 1.74 (sextet, J ) 8
Hz, 2H), 2.23-2.34 (m, 1H), 2.68-2.98 (m, 7H), 3.08-3.18 (m,
1H), 3.26-3.42 (m, 2H), 3.52-3.58 (m, 1H), 3.65 (d, J ) 9 Hz,
1H), 3.80 (s, 3H), 5.90 (s, 2H), 6.74 (d, J ) 8 Hz, 1H), 6.82 (d,
J ) 8 Hz, 1H), 6.86 (d, J ) 8 Hz, 2H), 6.98 (d, J ) 1 Hz, 1H),
7.33 (d, J ) 8 Hz, 2H); MS (DCI/NH3) m/e 547 (M + H)+;
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-((3-ch lor op r op yl)su lfon yl)a m in o)-
eth yl]p yr r olid in e-3-ca r boxylic a cid (12j): white solid; mp
75-76 °C; 1H NMR (CDCl3, 300 MHz) δ 0.80 (t, J ) 7 Hz,

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3225
3H), 1.45 (sextet, J ) 7 Hz, 2H), 2.15-2.31 (m, 3H), 2.70-
2.80 (m, 1H), 2.85-3.10 (m, 6H), 3.23-3.31 (m, 2H), 3.43 (bd,
J ) 9 Hz, 1H), 3.55-3.66 (m, 4H), 3.81 (s, 3H), 5.94 (s, 2H),
6.73 (d, J ) 8 Hz, 1H), 6.82 (d, J ) 8 Hz, 1H), 6.86 (d, J ) 8
Hz, 2H), 7.00 (s, 1H), 7.33 (d, J ) 8 Hz, 2H); MS (DCI/NH3)
m/e 567 (M + H)+. Anal. (C27H35N2O7ClS) C, H, N.
tr a n s,tr a n s-2-[4-(Meth oxym eth oxy)p h en yl]-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-(n -p en tylsu lfon yl)a m i-
n o)eth yl]p yr r olid in e-3-ca r boxylic a cid (13a ): white solid;
mp 57-59 °C; 1H NMR (CDCl3, 300 MHz) δ 0.78 (t, J ) 7 Hz,
3H), 0.90 (t, J ) 7 Hz, 3H), 1.28-1.36 (m, 4H), 1.93 (sextet, J
) 7 Hz, 2H), 1.72 (t, J ) 7 Hz, 2H), 2.20-2.32 (m, 1H), 2.72-
3.10 (m, 7H), 3.18-3.41 (m, 2H), 3.43 (dd, J ) 3, 9 Hz, 1H),
3.48 (s, 3H), 3.52-3.59 (m, 1H), 3.68 (d, J ) 9 Hz, 1H), 5.15
(s, 2H), 5.94 (s, 2H), 6.73 (d, J ) 8 Hz, 1H), 6.82 (dd, J ) 1, 8
Hz, 1H), 6.98-7.02 (m, 3H), 7.32 (d, J ) 9 Hz, 2H); MS (DCI/
NH3) m/e 591 (M + H)+. Anal. (C30H42N2O8S‚0.25H2O) C, H,
N.
tr a n s,tr a n s-2-(4-P r op oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -pr opyl-N-(n -pen tylsu lfon yl)am in o)eth yl]pyr -
r olid in e-3-ca r boxylic a cid (13b): white solid; mp 53-54 °C;
1H NMR (CDCl3, 300 MHz) δ 0.79 (t, J ) 7 Hz, 3H), 0.89 (t, J
) 7 Hz, 3H), 1.03 (t, J ) 7 Hz, 3H), 1.24-1.34 (m, 4H), 1.43
(sextet, J ) 7 Hz, 2H), 1.67-1.75 (m, 2H), 1.80 (sextet, 2H),
2.23-2.33 (m, 1H), 2.72-2.93 (m, 6H), 3.05 (septet, J ) 7 Hz,
2H), 3.15-3.35 (m, 2H), 3.42 (d, J ) 9 Hz, 1H), 3.54-3.62 (m,
1H), 3.67 (d, J ) 9 Hz, 1H), 4.90 (t, J ) 7 Hz, 2H), 5.95 (s,
2H), 6.73 (d, J ) 8 Hz, 1H), 6.85 (d, J ) 8 Hz, 2H), 7.02 (s,
1H), 7.32 (d, J ) 8 Hz, 2H); MS (DCI/NH3) m/e 589 (M + H)+.
Anal. (C31H44N2O7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-((2-m eth oxyeth yl)su lfon yl)a m in o-
)eth yl]p yr r olid in e-3-ca r boxylic a cid (12k ): white solid; mp
62-64 °C; 1H NMR (CDCl3, 300 MHz) δ 0.78 (t, J ) 7 Hz,
3H), 1.42 (sextet, J ) 7 Hz, 2H), 2.23-2.32 (m, 1H), 2.72-
2.79 (m, 1H), 2.86-3.05 (m, 4H), 3.10-3.27 (m, 4H), 3.32 (s,
3H), 3.43 (dd, J ) 3, 9 Hz, 1H), 3.53-3.58 (m, 1H), 3.65 (d, J
) 9 Hz, 1H), 3.69 (t, J ) 6 Hz, 2H), 3.80 (s, 3H), 5.94 (s, 2H),
6.73 (d, J ) 8 Hz, 1H), 6.82 (dd, J ) 1, 8 Hz, 1H), 6.87 (d, J )
8 Hz, 2H), 7.02 (d, J ) 1 Hz, 1H), 7.33 (d, J ) 8 Hz, 2H); MS
(DCI/NH3) m/e 549 (M + H)+. Anal. (C27H36N2O8S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-(ben zylsu lfon yl)a m in o)eth yl]p yr -
r olid in e-3-ca r boxylic a cid (12m ): white solid; mp 88-89 °C;
1H NMR (CDCl3, 300 MHz) δ 0.72 (t, J ) 7 Hz, 3H), 1.32
(sextet, J ) 7 Hz, 2H), 2.06-2.16 (m, 1H), 2.56-2.67 (m, 1H),
2.75-3.10 (m, 6H), 3.30 (dd, J ) 2, 9 Hz, 1H), 3.52 (m, 1H),
3.58 (d, J ) 9 Hz, 1H), 3.78 (s, 3H), 4.09 (d, J ) 12 Hz, 1H),
4.15 (d, J ) 12 Hz, 1H), 5.95 (s, 2H), 6.73 (d, J ) 7 Hz, 1H),
6.80 (dd, J ) 1, 7 Hz, 1H), 6.86 (d, J ) 8 Hz, 2H), 6.97 (d, J )
1 Hz, 1H), 7.27-7.35 (m, 7H); MS (DCI/NH3) m/e 581 (M +
H)+; HRMS (FAB) calcd for C31H37N2O7S 581.2321, found
581.2330. Anal. (C31H36N2O7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-(p h en ylsu lfon yl)a m in o)eth yl]p yr -
r olid in e-3-ca r boxylic a cid (12n ): white solid; mp 89-91 °C;
1H NMR (CDCl3, 300 MHz) δ 0.74 (t, J ) 6 Hz, 3H), 1.33
(sextet, J ) 6 Hz, 2H), 2.20-2.30 (m, 1H), 2.62-2.72 (m, 1H),
2.85-3.05 (m, 4H), 3.12-3.22 (m, 2H), 3.38 (dd, J ) 3, 9 Hz,
1H), 3.49-3.57 (m, 1H), 3.62 (d, J ) 9 Hz, 1H), 3.82 (s, 3H),
5.96 (s, 2H), 6.73 (d, J ) 8 Hz, 1H), 6.84 (dd, J ) 1, 8 Hz, 1H),
6.85 (d, J ) 9 Hz, 2H), 7.02 (d, J ) 1 Hz, 1H), 7.28 (d, J ) 9
Hz, 2H), 7.39-7.54 (m, 3H), 7.70 (d, J ) 7 Hz, 2H); MS (DCI/
NH3) m/e 567 (M + H)+. Anal. (C30H34N2O7S‚0.5H2O) C, H,
N.
tr a n s,tr a n s-2-(3,4-Dim eth oxyph en yl)-4-(1,3-ben zodioxol-
5-yl)-1-[2-(N-n -p r op yl-N-(n -p en tylsu lfon yl)a m in o)eth yl]-
p yr r olid in e-3-ca r boxylic a cid (13c): white solid; mp 75-
1
86 °C; H NMR (CD3OD, 300 MHz) δ 0.75 (t, J ) 7 Hz, 3H),
0.82 (t, J ) 7 Hz, 3H), 1.32-1.43 (m, 6H), 1.65-1.77 (m, 2H),
3.0-3.09 (m, 4H), 3.23-3.27 (m, 2H), 3.44 (t, J ) 6 Hz, 1H),
3.47-3.56 (m, 2H), 3.78 (d, J ) 9 Hz, 1H), 3.83-3.93 (m, 1H),
3.87 (s, 3H), 3.92 (s, 3H), 4.63 (d, J ) 13 Hz, 1H), 5.97 (s, 2H),
6.82 (d, J ) 7 Hz, 1H), 6.93 (d, J ) 7 Hz, 1H), 7.06 (d, J ) 7
Hz, 1H), 7.08 (d, J ) 3 Hz, 2H), 7.16 (dd, J ) 3, 7 Hz, 1H),
7.27 (d, J ) 3 Hz, 1H); MS (DCI/NH3) m/e 591 (M + H)+;
HRMS (FAB) calcd for C30H43N2O8S 591.2740, found 591.2725.
Anal. (C30H42N2O8S‚0.6TFA) C, H, N.
tr a n s,tr a n s-2-(Ben zod ioxa n -6yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -pr opyl-N-(n -pen tylsu lfon yl)am in o)eth yl]pyr -
r olid in e-3-ca r boxylic a cid (13d ): white solid; mp 68-69
1
°C; H NMR (CDCl3, 300 MHz) δ 0.82 (t, J ) 7 Hz, 3H), 0.89
(t, J ) 6 Hz, 3H), 1.28-1.37 (m, 4H), 1.45 (quintet, J ) 7 Hz,
2H), 1.73 (m, 2H), 2.28 (m, 1H), 2.65-2.95 (m, 5H), 3.0-3.12
(m, 2H), 3.15-3.32 (br, 2H), 3.41 (m, 1H), 3.50-3.64 (m, 2H),
4.25 (s, 4H), 5.95 (s, 2H), 6.73 (d, J ) 7 Hz, 1H), 6.81-6.98
(m, 5H); MS (DCI/NH3) m/e 589 (M + H)+; HRMS (FAB)
calcd for C30H41N2O8S 589.2594, found 589.2573. Anal.
(C30H40N2O8S‚1.0H2O) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-((4-ch lor op h en yl)su lfon yl)a m in o)-
eth yl]p yr r olid in e-3-ca r boxylic a cid (12o): white solid; mp
105-106 °C; 1H NMR (CD3OD, 300 MHz) δ 0.72 (t, J ) 7 Hz,
3H), 1.34 (sextet, J ) 7 Hz, 2H), 2.56-2.62 (m, 1H), 2.78-
2.86 (m, 1H), 2.96-3.03 (m, 3H), 3.13-3.26 (m, 3H), 3.51 (dd,
J ) 5, 9 Hz, 1H), 3.62-3.68 (m, 1H), 3.80 (s, 3H), 3.94 (d, J )
9 Hz, 1H), 5.92 (s, 2H), 6.75 (d, J ) 8 Hz, 1H), 6.84 (dd, J )
2, 8 Hz, 1H), 6.94 (d, J ) 8 Hz, 2H), 6.98 (d, J ) 2 Hz, 1H),
7.36 (d, J ) 8 Hz, 2H), 7.49 (d, J ) 8 Hz, 2H), 7.68 (d, J ) 8
Hz, 2H); MS (DCI/NH3) m/e 601 (M + H)+. Anal. (C30H33N2-
ClO7S) C, H, N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-((4-m eth ylp h en yl)su lfon yl)a m in o-
)eth yl]p yr r olid in e-3-ca r boxylic a cid (12p ): white solid; mp
78-79 °C; 1H NMR (CDCl3, 300 MHz) δ 0.73 (t, J ) 7 Hz,
3H), 1.33 (sextet, J ) 7 Hz, 2H), 2.20-2.30 (m, 1H), 2.40 (s,
3H), 2.61-2.72 (m, 1H), 2.83-3.05 (m, 4H), 3.08-3.19 (m, 2H),
3.48 (dd, J ) 3, 9 Hz, 1H), 3.49-3.57 (m, 1H), 3.62 (d, J ) 9
Hz, 1H), 3.81 (s, 3H), 5.95 (s, 2H), 6.73 (d, J ) 8 Hz, 1H), 6.82
(d, J ) 8 Hz, 1H), 6.87 (d, J ) 8 Hz, 2H), 7.00 (s, 1H), 7.21 (d,
J ) 8 Hz, 2H), 7.29 (d, J ) 8 Hz, 2H), 7.57 (d, J ) 8 Hz, 2H);
MS (DCI/NH3) m/e 581 (M + H)+. Anal. (C31H36N2O7S) C, H,
N.
tr a n s,tr a n s-2-(4-Meth oxyp h en yl)-4-(1,3-ben zod ioxol-5-
yl)-1-[2-(N-n -p r op yl-N-((4-m eth oxyp h en yl)su lfon yl)a m i-
n o)eth yl]p yr r olid in e-3-ca r boxylic a cid (12q): white solid;
mp 96-97 °C; 1H NMR (CDCl3, 300 MHz) δ 0.73 (t, J ) 7 Hz,
3H), 1.34 (sextet, J ) 7 Hz, 2H), 2.20-2.30 (m, 1H), 2.62-
2.71 (m, 1H), 2.82-3.03 (m, 4H), 3.08-3.18 (m, 2H), 3.38 (dd,
J ) 3, 9 Hz, 1H), 3.48-3.56 (m, 1H), 3.62 (d, J ) 9 Hz, 1H),
3.81 (s, 3H), 3.86 (s, 3H), 5.95 (s, 2H), 6.73 (d, J ) 8 Hz, 1H),
6.81-6.89 (m, 5H), 7.01 (d, J ) 1 Hz, 1H), 7.28 (d, J ) 8 Hz,
2H), 7.62 (d, J ) 8 Hz, 2H); MS (DCI/NH3) m/e 597 (M + H)+.
Anal. (C31H36N2O8S) C, H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-(n -p en tylsu lfon yl)a m i-
n o)eth yl]p yr r olid in e-3-ca r boxylic a cid (13e): white solid;
1
mp 66-68 °C; H NMR (CDCl3, 300 MHz) δ 0.81 (t, J ) 7.5
Hz, 3H), 0.89 (t, J ) 7 Hz, 3H), 1.26-1.35 (m, 4H), 1.45 (sextet,
J ) 7.5 Hz, 2H), 1.68-1.76 (m, 2H), 2.25-2.33 (m, 1H), 2.72-
2.92 (m, 5H), 2.97-3.12 (m, 2H), 3.16-3.33 (m, 2H), 3.43 (dd,
J ) 3, 9 Hz, 1H), 3.53-3.60 (m, 1H), 3.66 (d, J ) 10 Hz, 1H),
3.88 (s, 3H), 5.95 (s, 2H), 6.74 (d, J ) 8 Hz, 1H), 6.82 (dd, J )
1, 8 Hz, 1H), 6.92 (t, J ) 8 Hz, 1H), 6.97 (d, J ) 1 Hz, 1H),
7.12 (d, J ) 8 Hz, 1H), 7.18 (dd, J ) 1, 12 Hz, 1H); MS (DCI/
NH3) m/e 579 (M + H)+. Anal. (C29H39N2FO7S‚0.25H2O) C,
H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-eth oxyp h en yl)-4-(1,3-ben zo-
d ioxol-5-yl)-1-[2-(N-n -p r op yl-N-(n -p en tylsu lfon yl)a m in o)-
eth yl]p yr r olid in e-3-ca r boxylic a cid (13f): white solid; mp
65-66 °C; 1H NMR (CDCl3, 300 MHz) δ 0.82 (t, J ) 7 Hz,
3H), 0.90 (t, J ) 7 Hz, 3H), 1.26-1.36 (m, 4H), 1.41-1.52 (m,
5H), 1.73 (quintet, J ) 7 Hz, 2H), 2.23-2.33 (m, 1H), 2.69-
2.96 (m, 5H), 2.97-3.12 (m, 2H), 3.16-3.37 (m, 2H), 3.43 (d,
J ) 9 Hz, 1H), 3.52-3.59 (m, 1H), 3.66 (d, J ) 9 Hz, 1H), 4.08
(q, J ) 7 Hz, 2H), 5.95 (s, 2H), 6.74 (d, J ) 8 Hz, 1H), 6.82 (d,
J ) 8 Hz, 1H), 6.92 (t, J ) 8 Hz, 1H), 6.97 (s, 1H), 7.07 (d, J
) 8 Hz, 1H), 7.15 (d, J ) 12 Hz, 1H); MS (DCI/NH3) m/e 593
(M + H)+. Anal. (C30H41N2O7FS‚0.25H2O) C, H, N.
tr a n s,tr a n s-2-(3,4-Diflu or op h en yl)-4-(1,3-ben zod ioxol-
5-yl)-1-[2-(N-n -p r op yl-N-(n -p en tylsu lfon yl)a m in o)eth yl]-
p yr r olid in e-3-ca r boxylic a cid (13g): white solid; mp 71-

3226 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
J ae et al.
1
72 °C; H NMR (CDCl3, 300 MHz) δ 0.82 (t, J ) 7 Hz, 3H),
3.67 (d, J ) 9 Hz, 1H), 3.88 (s, 3H), 5.97 (s, 2H), 6.73 (d, J )
7 Hz, 1H), 6.82 (dd, J ) 1, 7 Hz, 1H), 6.92 (t, J ) 7 Hz, 1H),
6.96 (d, J ) 1 Hz, 1H), 7.10 (d, J ) 9 Hz, 1H), 7.17 (dd, J ) 1,
12 Hz, 1H); MS (DCI/NH3) m/e 619 (M + H)+; HRMS (FAB)
calcd for C28H35N2O7F4S1 619.2101, found 619.2125. Anal.
(C28H34N2O7F4S) C, H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-(ben zylsu lfon yl)a m in o-
)eth yl]p yr r olid in e-3-ca r boxylic a cid (14g): white solid; mp
80-81 °C; 1H NMR (CDCl3, 300 MHz) δ 0.73 (t, J ) 7 Hz,
3H), 1.34 (sextet, J ) 7 Hz, 2H), 2.12 (m, 1H), 2.60 (m, 1H),
2.76-3.08 (m, 6H), 3.28 (dd, J ) 3, 9 Hz, 1H), 3.48 (m, 1H),
3.58 (d, J ) 9 Hz, 1H), 3.87 (s, 3H), 4.13 (dd, J ) 9, 22 Hz,
2H), 5.95 (s, 2H), 6.72 (d, J ) 7 Hz, 1H), 6.77 (dd, J ) 1, 7 Hz,
1H), 6.88 (t, J ) 9 Hz, 1H), 6.92 (d, J ) 1 Hz, 1H), 7.05 (d, J
) 9 Hz, 1H), 7.12 (dd, J ) 1, 12 Hz, 1H), 7.32 (m, 5H); MS
(DCI/NH3) m/e 599 (M + H)+. Anal. (C31H35N2O7FS‚0.5H2O)
C, H, N.
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 of [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., Cambridge, 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.
0.90 (t, J ) 7 Hz, 3H), 1.25-1.38 (m, 4H), 1.46 (sextet, J ) 7
Hz, 2H), 1.74 (quintet, J ) 7 Hz, 2H), 2.26-2.36 (m, 1H), 2.72-
2.95 (m, 5H), 2.98-3.12 (m, 2H), 3.15-3.34 (m, 2H), 3.45 (dd,
J ) 3, 9 Hz, 1H), 3.53-3.60 (m, 1H), 3.71 (d, J ) 9 Hz, 1H),
5.96 (s, 2H), 6.75 (d, J ) 9 Hz, 1H), 3.82 (dd, J ) 2, 9 Hz, 1H),
5.96 (d, J ) 2 Hz, 1H), 7.09-7.18 (m, 2H), 7.23-7.34 (m, 1H);
MS (CDI/NH3) m/e 567 (M + H)+. Anal. (C28H36N2F2O6S) H,
N; C: calcd, 59.36; found, 59.92.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zodioxol-5-yl)-1-[2-(N-n -pr opyl-N-(n -bu tylsu lfon yl)am in o)-
eth yl]p yr r olid in e-3-ca r boxylic a cid (14a ): white solid; mp
1
65-66 °C; H NMR (CDCl3, 300 MHz) δ 0.82 (t, J ) 7.5 Hz,
3H), 0.92 (t, J ) 7.5 Hz, 3H), 1.34-1.52 (m, 4H), 1.72 (quintet,
J ) 7.5 Hz, 2H), 2.25-2.35 (m, 1H), 2.72-2.94 (m, 5H), 2.97-
3.12 (m, 2H), 3.19-3.46 (m, 2H), 3.44 (d, J ) 9 Hz, 1H), 3.53-
3.60 (m, 1H), 3.67 (d, J ) 9 Hz, 1H), 3.89 (s, 3H), 5.95 (s, 2H),
6.74 (d, J ) 8 Hz, 1H), 6.82 (d, J ) 8 Hz, 1H), 6.92 (t, J ) 9
Hz, 1H), 6.97 (s, 1H), 7.12 (d, J ) 9 Hz, 1H), 7.18 (d, J ) 12
Hz, 1H); MS (DCI/NH3) m/e 565 (M + H)+. Anal. (C28H37N2-
FO7S) C, H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-(n -h exylsu lfon yl)a m i-
n o)eth yl]p yr r olid in e-3-ca r boxylic a cid (14b): white solid;
mp 63-65 °C; 1H NMR (CDCl3, 300 MHz) δ 0.82 (t, J ) 7 Hz,
3H), 0.88 (t, J ) 6 Hz, 3H), 1.23-1.47 (m, 6H), 1.44 (sextet, J
) 7 Hz, 2H), 1.71 (quintet, J ) 6 Hz, 2H), 2.24-2.34 (m, 1H),
2.70-2.93 (m, 5H), 2.96-3.12 (m, 2H), 3.15-3.35 (m, 2H), 3.43
(dd, J ) 3, 9 Hz, 1H), 3.52-3.59 (m, 1H), 3.66 (d, J ) 9 Hz,
1H), 3.87 (s, 3H), 5.95 (s, 2H), 6.74 (d, J ) 8 Hz, 1H), 6.82 (d,
J ) 8 Hz, 1H), 6.42 (t, J ) 8 Hz, 1H), 6.96 (s, 1H), 7.12 (d, J
) 9 Hz, 1H), 7.17 (d, J ) 12 Hz, 1H); MS (DCI/NH3) m/e 593
(M + H)+. Anal. (C30H41N2O7FS) C, H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-(isobu tylsu lfon yl)a m i-
n o)eth yl]p yr r olid in e-3-ca r boxylic a cid (14c): white solid;
mp 77-78 °C; 1H NMR (CDCl3, 300 MHz) δ 0.83 (t, J ) 7 Hz,
3H), 1.06 (d, J ) 6 Hz, 6H), 1.45 (q, J ) 7 Hz, 2H), 2.20 (septet,
J ) 6 Hz, 1H), 2.26-2.36 (m, 1H), 2.62-2.78 (m, 3H), 2.85-
2.95 (m, 2H), 2.97-3.10 (m, 2H), 3.15-3.35 (m, 2H), 3.43 (dd,
J ) 3, 9 Hz, 1H), 3.53-3.62 (m, 1H), 3.66 (d, J ) 9 Hz, 1H),
3.88 (s, 3H), 5.95 (s, 2H), 6.74 (d, J ) 8 Hz, 1H), 6.82 (dd, J )
2, 8 Hz, 1H), 6.92 (t, J ) 8 Hz, 1H), 6.97 (d, J ) 2 Hz, 1H),
7.12 (d, J ) 9 Hz, 1H), 7.18 (dd, J ) 2, 12 Hz, 1H); MS (DCI/
NH3) m/e 565 (M + H)+. Anal. (C28H37N2O7FS‚0.25H2O) C,
H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-((3-m eth yl-1-bu tyl)su l-
fon yl)a m in o)eth yl]p yr r olid in e-3-ca r boxylic a cid (14d ):
1
white solid; mp 65-67 °C; H NMR (CDCl3, 300 MHz) δ 0.82
(t, J ) 7 Hz, 3H), 0.88 (d, J ) 5 Hz, 6H), 1.46 (sextet, J ) 7
Hz, 2H), 1.56-1.64 (m, 3H), 2.24-2.33 (m, 1H), 2.68-2.93 (m,
5H), 2.98-3.12 (m, 2H), 3.15-3.35 (m, 2H), 3.43 (dd, J ) 3, 9
Hz, 1H), 3.52-3.58 (m, 1H), 3.65 (d, J ) 12 Hz, 1H), 3.87 (s,
3H), 5.95 (s, 2H), 6.73 (d, J ) 8 Hz, 1H), 6.82 (dd, J ) 2, 8 Hz,
1H), 6.92 (t, J ) 8 Hz, 1H), 6.97 (d, J ) 2 Hz, 1H), 7.10 (d, J
) 9 Hz, 1H), 7.16 (dd, J ) 2, 12 Hz, 1H); MS (DCI/NH3) m/e
579 (M + H)+. Anal. (C29H39N2FO7S) C, H, N.
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 as described by
Berridge.12 The upper aqueous phase (1 mL) was retained,
and a small portion (100 µL) was counted. The rest of the
aqueous sample was analyzed by batch chromatography using
anion-exchange resin AG1-X8 (Bio-Rad).
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-((3-ch lor op r op yl)su lfo-
n yl)am in o)eth yl]pyr r olidin e-3-car boxylic acid (14e): white
1
foam; H NMR (CD3OD, 300 MHz) δ 0.80 (t, J ) 7 Hz, 3H),
1.47 (bd hex, J ) 8 Hz, 2H), 2.15 (pen, J ) 7 Hz, 2H), 2.32 (m,
1H), 2.7-3.2 (m, 9H), 3.46 (dd, J ) 4, 10 Hz, 1H), 3.57 (m,
1H), 3.64 (t, J ) 6 Hz, 2H), 3.67 (d, J ) 9 Hz, 1H), 3.86 (s,
3H), 5.92 (s, 2H), 6.74 (d, J ) 8 Hz, 1H), 6.84 (dd, J ) 2, 8 Hz,
1H), 6.96 (d, J ) 2 Hz, 1H), 7.06 (t, J ) 9 Hz, 1H), 7.18 (m,
2H); MS (DCI/NH3) m/e 585 (M + H; Cl35)+, 587 (M + H; Cl37)+.
Anal. (C27H34N2O7ClFS) C, H, N.
tr a n s,tr a n s-2-(3-F lu or o-4-m eth oxyp h en yl)-4-(1,3-ben -
zod ioxol-5-yl)-1-[2-(N-n -p r op yl-N-((3,3,3-tr iflu or op r op yl)-
su lfon yl)a m in o)eth yl]p yr r olidin e-3-ca r boxylic a cid (14f):
ETB. Chinese hamster ovary cells (CHO) permanently
transfected with the human ETB receptor were grown to
confluence in 24-well tissue culture plates and labeled with
5µCi/well of [3H]myoinositol in F-12 media + 10%FBS + 1xP/
S/F. The adherent cells were washed gently with PBS and
then incubated in 200 µL of buffer A containing protease
1
white solid; mp 72-73 °C; H NMR (CDCl3, 300 MHz) δ 0.82
(t, J ) 7 Hz, 3H), 1.46 (sextet, J ) 7 Hz, 2H), 2.03 (quintet, J
) 7 Hz, 2H), 2.16-2.32 (m, 3H), 2.74 (m, 1H), 2.82-3.13 (m,
6H), 3.26 (m, 1H), 3.43 (dd, J ) 3, 9 Hz, 1H), 3.56 (m, 1H),

Pyrrolidine-3-carboxylic Acids as Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3227
(5) Opgenorth, T. J .; Adler, A. L.; Calzadilla, S. V.; Chiou, W. J .;
Dayton, B. D.; Dixon, D. B.; Gehrke, L. J .; Hernandez, L.;
Magnuson, S. R.; Marsh, K. C.; Novosad, E. I.; von Geldern, T.
W.; Wessale, J . L.; Winn, M.; Wu-Wong, J . R. Pharmacological
Characterization of A-127722: An Orally Active and Highly
Potent ETA-Selective Receptor Antagonist. J . Pharmacol. Exp.
Ther. 1996, 276, 473-481.
(6) Tasker, A. S.; Sorensen, B. K.; J ae, H.-S.; Winn, M.; von Geldern,
T. W.; Dixon, D. B.; Chiou, W. J .; Dayton, B. D.; Calzadilla, S.
V.; Hernandez, L.; Marsh, K. C.; Wu-Wong, J . R.; Opgenorth,
T. J . Potent and Selective non-Benzodioxole-Containing Endot-
helin-A Receptor Antagonists. J . Med. Chem. 1997, 40, 322-
330.
inhibitors and 10 mM LiCl for 60 min at 37 °C in a CO2
incubator. Test compounds were then added followed by the
addition of 1 nM ET-1 and incubated for 30 min at 37 °C. The
cells were then solubilized by the addition of 50 µL of 1 N
NaOH and then neutralized by the addition of 50 µL of 1 N
HCl. The solubilized cell suspension was transfered to glass
tubes and extracted by the addition of 1.5 mL of 1:2 (v/v)
chloroform-methanol. Total inositol phosphates were ex-
tracted and analyzed by batch chromatography on anion-
exchange resin as above. All IC50 values are calculated using
an average of at least two separate determinations.
P h a r m a cok in etic An a lysis. The pharmacokinetic behav-
ior of compound 16h was evaluated in male Sprague-Dawley
rats. Briefly, the test compound was prepared as a 10 mg/
mL solution in an ethanol-propylene glycol-D5W (20:30:50,
by volume) vehicle containing 1 molar equiv of sodium
hydroxide. Groups of rats (n ) 4 per group) received either a
10 mg/kg (1 mL/kg) intravenous dose administered as a slow
bolus in the jugular vein or a 10 mg/kg (1 mL/kg) oral dose
administered by gavage. Heparinized blood samples (∼0.4 mL/
sample) were obtained from a tail vein of each rat 0.1 (iv only),
0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 9, and 12 h after dosing. The
samples were analyzed by reverse phase HPLC following
liquid-liquid extraction from the plasma. Initial estimates
of the pharmacokinetic parameters (e.g. the maximum con-
centration Cmax) for NONLIN8413 were obtained with the
program CSTRIP.14 Area under the curve (AUC) values were
calculated by the trapezoidal rule over the time course of the
study. The terminal-phase rate constant (â) was utilized in
the extrapolation of the AUC from 12 h to infinity to provide
an AUC0-∞ value, and in the calculation of T1/2 values.
Assuming dose proportionality and correcting for the differ-
ences in dosing, a comparison of the AUC following oral dosing
with that obtained following an intravenous dose provided an
estimate of the bioavailability (F).
(7) Opgenorth, T. J . Endothelin Receptor Antagonism. Adv. Phar-
macol. 1995, 33, 1-65.
(8) For example: (a) Clozel, M.; Breu, V.; Gray, G. A.; Kalina, B.;
Loffler, B. M.; Burri, K.; Cassal, J . M.; Hirth, G.; Muuler, M.;
Neidhart, W.; Ramuz, H. Pharmacological Characterization of
Bosentan, a New Potent Orally Active Non-peptide Endothelin
Receptor Antagonist. J . Pharmacol. Exp. Ther. 1994, 270, 228-
235. (b) Ikeda, S.; Awane, Y.; Kusumoto, K.; Wakimasu, M.;
Watanabe, T.; Fujino, M. A New Endothelin Receptor Antago-
nist, TAK-044, Shows Long-Lasting Inhibition of Both ETA- and
ETB-Mediated Blood Pressure Responses in Rats. J . Pharmacol.
Exp. Ther. 1994, 270, 728-733. (c) (1) Ohlstein, E. H.; Nambi,
P.; Lago, A.; Hay, D. W. P.; Beck, G.; Fong, K.-L.; Eddy, E. P.;
Smith, P.; Ellens, H.; Elliott, J . D. Nonpeptide Endothelin
Receptor Antagonists. VI: Pharmacological Characterization of
SB 217242,
A Potent and Highly Bioavailable Endothelin
Receptor Antagonist. J . Pharmacol. Exp. Ther. 1996, 276, 609-
617. (2) Ohlstein, E. H.; Nambi, P.; Douglas, S. A.; Edwards, R.
M.; Gellai, M.; Lago, M. A.; Leber, J . D.; Cousins, R. D.; Gao,
A.; Frazee, J . S.; Peishoff, C. E.; Bean, J . W.; Eggleston, D. S.;
Elshourbagy, N. A.; Kumar, C.; Lee, J . A.; Yu, T.-L.; Louden,
C.; Brooks, D. P.; Weinstock, J .; Feuerstein, G.; Poste, G.; Ruffolo,
R. R., J r.; Gleason, J . G.; Elliott, J . D. SB 209670, a Rationally
Designed Potent Nonpeptide Endothelin Receptor Antagonist.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8052-8056. (d) Williams,
D. L., J r.; Murphy, K. L.; Nolan, N. A.; O’Brien, J . A.; Pettibone,
D. J .; Kivlighn, S. D.; Krause, S. M.; Lis, E. V., J r.; Zingaro, G.
J .; Gabel, R. A.; Clayton, F. C.; Siegl, P. K. S.; Zhang, K.; Naue,
J .; Vyas, K.; Walsh, T. F.; Fitch, K. J .; Chakravarty, P. K.;
Greenlee, W. J .; Clineschmidt, B. V. Pharmacology of L-754,-
142, a Highly Potent, Orally Active, Nonpeptidyl Endothelin
Antagonist. J . Pharmacol. Exp. Ther. 1995, 275, 1518-1526.
(9) Wu-Wong. J . R.; Chiou, W. J .; Hoffman, D. J .; Winn, M.; von
Geldern, T. W.; Opgenorth, T. J . Endothelins and Endothelin
Receptor Antagonists: Binding to Plasma Proteins. Life Sci.
1996, 58, 1839-1847.
(10) Roemmele, R. C.; Rapoport, H. Removal of N-Arylsulfonyl
Groups from Hydroxy R-Amino Acids. J . Org. Chem. 1988, 53,
2367-2371.
(11) Bunyagidi, C.; Piotrowaska, H.; Aldridge, M. Synthesis of 2,2,2-
Trifluoroethanesulfonic Acid. J . Org. Chem. 1981, 46, 3335-
3336.
(12) Berridge, M. J .; Downes, C. P.; Hanley, M. R. Lithium Amplifies
Agonist Dependent Phosphatidylinositol Responses in Brain and
Salivary Glands. Biochem. J . 1982, 206, 587-595.
(13) Statistical Consultants, Inc. PCNONLIN and NONLIN84: Soft-
ware for the Statistical Analysis of Nonlinear Models. Am. Stat.
1986, 40, 52.
Ack n ow led gm en t. The authors thank the Abbott
1
Analytical Department for assistance in acquiring H-
NMR and mass spectra.
Refer en ces
(1) von Geldern, T. W.; Hutchins, C.; Kester, J . A.; Wu-Wong, J .
R.; Chiou, W.; Dixon, D. B.; Opgenorth, T. J . Azole Endothelin
Antagonists. I. A Receptor Model Explains an Unusual Structure-
Activity Profile. J . Med. Chem. 1996, 39, 957-967.
(2) von Geldern, T. W.; Kester, J . A.; Bal, R.; Wu-Wong, J . R.; Chiou,
W.; Dixon, D. B.; Opgenorth, T. J . Azole Endothelin Antagonists.
II. Structure-Activity Studies. J . Med. Chem. 1996, 39, 968-
981.
(3) von Geldern, T. W.; Hoffman, D. J .; Kester, J . A.; Nellans, H.
N.; Dayton, B. D.; Calzadilla, S. V.; Marsh, K. C.; Hernandez,
L.; Chiou, W.; Dixon, D. B.; Wu-Wong, J . R.; Opgenorth, T. J .
Azole Endothelin Antagonists. III. Using ∆logP as a Tool to
Improve Absorption. J . Med. Chem. 1996, 39, 982-991.
(4) Winn, M.; von Geldern, T. W.; Opgenorth, T. J .; J ae, H.-S.;
Tasker, A. S.; Boyd, S. A.; Kester, J . A.; Mantei, R. A.; Bal, R.;
Sorensen, B. K.; Wu-Wong, J . R.; Chiou, W.; Dixon, D. B.;
Novosad, E. I.; Hernandez, L.; Marsh, K. C. 2,4-Diarylpyrroli-
dine-3-carboxylic Acids--Potent ETA Selective Endothelin Recep-
tor Antagonists. 1. Discovery of A-127722. J . Med. Chem. 1996,
39, 1039-1048.
(14) Sedman, A. J .; Wagner, J . G. CSTRIP - A FORTRAN Computer
Program for Obtaining Initial Polyexponential Estimates. J .
Pharm. Sci. 1976, 65, 1006-1010.
J M970101G