Structure-activity relationships of a novel class of endothelin-A receptor antagonists and discovery of potent and selective receptor antagonist, 2-(benzo[1,3]dioxol-5-yl)-6-isopropyloxy-4- (4-methoxyphenyl)-2H-chromene-3-carboxylic acid (S-1255). 1. Study on structure-activity relationships and basic structure crucial for ETA antagonism

Article abstract of DOI:10.1021/jm010382z

A novel series of endothelin-A (ET_A) selective receptor antagonists having a 2H-chromene skeleton are described. A lead compound, 2-(benzo[1,3] dioxol-5-yl)-2H-chromene-3-carboxylic acid (3), was found by modifications of our own angiotensin II antagonist. A structure-activity relationship (SAR) study of 3 reveals that the structural requirements essential for potent and selective ET_A receptor binding affinity are the m,p-methylenedioxyphenyl, carboxyl, and isopropoxy groups at the 2-, 3-,and 6-positions, respectively, on the (R)-2H-chromene skeleton. The substituent at the 4-position is also important for improving the activity, and various hydrophobic functional groups of 6-9 ? such as liner, branched, and cyclic aliphatic groups, unsubstituted and substituted aryl groups, and even halogen atoms were acceptable. These results suggest that (R)-2-(benzo[1,3]dioxol-5-yl)-6-isopropoxy-2H-chromene-3-carboxylic acid, formula 108, is the crucial basic structure to be recognized by the ETA receptor. The most potent compound is (R)-48 (S-1255), which binds to the ETA receptor with an IC₅₀ value of 0.19 nM and is 630-fold selective for the ET_A receptor than for the ET_B receptor. This compound has 55% oral bioavailability in rats. On the basis of the SAR, the roles of each substituent in the receptor binding are discussed.

Full text of DOI:10.1021/jm010382z

J . Med. Chem. 2002, 45, 2041-2055
2041
Str u ctu r e-Activity Rela tion sh ip s of a Novel Cla ss of En d oth elin -A Recep tor
An ta gon ists a n d Discover y of P oten t a n d Selective Recep tor An ta gon ist,
2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op yloxy-4-(4-m eth oxyp h en yl)-2H-ch r om en e-3-
ca r boxylic Acid (S-1255). 1. Stu d y on Str u ctu r e-Activity Rela tion sh ip s a n d
Ba sic Str u ctu r e Cr u cia l for ETA An ta gon ism
Natsuki Ishizuka,* Ken-ichi Matsumura, Katsunori Sakai, Masafumi Fujimoto, Shin-ichi Mihara, and
Teruo Yamamori
Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, J apan
Received August 10, 2001
A novel series of endothelin-A (ETA) selective receptor antagonists having a 2H-chromene
skeleton are described. A lead compound, 2-(benzo[1,3]dioxol-5-yl)-2H-chromene-3-carboxylic
acid (3), was found by modifications of our own angiotensin II antagonist. A structure-activity
relationship (SAR) study of 3 reveals that the structural requirements essential for potent
and selective ETA receptor binding affinity are the m,p-methylenedioxyphenyl, carboxyl, and
isopropoxy groups at the 2-, 3-, and 6-positions, respectively, on the (R)-2H-chromene skeleton.
The substituent at the 4-position is also important for improving the activity, and various
hydrophobic functional groups of 6-9 Å such as liner, branched, and cyclic aliphatic groups,
unsubstituted and substituted aryl groups, and even halogen atoms were acceptable. These
results suggest that (R)-2-(benzo[1,3]dioxol-5-yl)-6-isopropoxy-2H-chromene-3-carboxylic acid,
formula 108, is the crucial basic structure to be recognized by the ETA receptor. The most
potent compound is (R)-48 (S-1255), which binds to the ETA receptor with an IC50 value of 0.19
nM and is 630-fold selective for the ETA receptor than for the ETB receptor. This compound
has 55% oral bioavailability in rats. On the basis of the SAR, the roles of each substituent in
the receptor binding are discussed.
In tr od u ction
roles of the ETB receptor have not been fully character-
ized. Recently, an ET-3 specific receptor, ETC, was
identified from Xenopus laevis dermal melanophores,10
but the existence of ETC in mammalian tissue is
unknown.
The endothelins (ETs) were isolated from porcine
aortic endothelial cells by Yanagisawa and co-workers
in 1988.1 To date, three isopeptides, ET-1, ET-2, and
ET-3, have been identified.2 They are 21 amino acid
peptides with two disulfide linkages (1-15 and 3-11)
and conserve high homology sequences to each other,
especially where hydrophobic C-terminals are com-
pletely conserved. The mature ETs are synthesized from
big endothelins, of which Trp21-Val22 are cleaved by the
endothelin converting enzyme (ECE).3,4 ET-1 is the most
potent vasoconstrictor peptide known and has a long
duration of action.
ET receptors belong to a seven transmembrane G-
protein-coupled receptor (7TM/GCPR) family,5,6 and two
receptor subtypes, endothelin A (ETA) and endothelin
B (ETB), have been identified.7,8 The ETA receptor binds
to three isopeptides with affinities in the order of ET-1
) ET-2 > ET-3, while the ETB receptor binds equally
to them.6 The ETA receptor is mainly expressed on
vascular smooth muscle cells5 and causes vasoconstric-
tion. On the other hand, the ETB receptor on endothelial
cells causes vasodilatation through the production of
endothelium-derived relaxing factors (EDRF): nitric
oxide (NO) and prostacyclins. The ETB receptor also is
expressed on vascular smooth muscle cells and causes
vasoconstriction as the ETA receptor does.9 However, the
In mammals, ET receptors are distributed in a wide
variety of tissues, and elevated levels of the plasma
concentration of ET-1 were observed in several diseases
such as hypertension,11,12 pulmonary hypertension,13,14
acute myocardial infarction,15 congestive heart fail-
ure,16,17 renal failure,18,19 and atherosclerosis.20 This
observation suggests the relevance of ETs to pathogen-
esis of such diseases. In fact, blocking of ETs using a
monoclonal antibody or ET receptor antagonists in
animal pathological models showed an antihypertensive
activity in hypertension,21,22 reduction of the myocardial
infarct size in acute myocardial ischemia and reperfu-
sion,23,24 reduction in neointimal formation in a carotid
artery balloon angioplasty model,25 improvement of
survival in chronic heart failure,26 and improvement of
the glomerular filtration rate in acute ischemic renal
failure.27 In human clinical trials of chronic heart failure
patients, intravenous administration of a nonpeptidic
antagonist Ro 47-0203 (Bosentan)28,29 reduced their
blood pressure and improved their cardiac perfor-
mance.30 These results strongly indicate that blocking
the ET receptors is a powerful therapeutic strategy in
ameliorating various disease states and ET antagonists
could be promising new therapeutic agents.
* To whom correspondence should be addressed. Tel.: +81(6)6458-
5861. Fax: +81(6)6458-0987. E-mail:natsuki.ishizuka@shionogi.co.jp.
10.1021/jm010382z CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/09/2002

2042 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ishizuka et al.
avairable or synthesized from dihydroxyacetophenone
4 and an alkyl halide, gave 3-formyl derivatives 6.57
Oxidation of the formyl group of 6 with sodium chlorite58
and successive esterification gave esters 8. The Michael
addition of an appropriate Grignard reagent to 8 in the
presence of copper(I) iodide proceeded exclusively at the
2-position to give the key intermediates 9 as a mixture
of keto-enol tautomers, 9a ,b.
Introduction of an alkoxy group at the 4-position was
carried out by the Mitsunobu reaction of intermediates
9 with an appropriate alcohol. Alkaline hydrolysis of the
resulting esters 10 gave 4-alkoxy acids 11-33.
Introduction of an aryl group at the 4-position was
accomplished by reaction of 9 with 2-[N,N′-bis(trifluo-
romethanesulfonyl)amino]pyridine, followed by the Su-
zuki coupling reaction of the resulting triflate 34 and
an appropriate boronic acid. Subsequent alkaline hy-
drolysis provided 4-aryl acids 36-79.
Hydrogenation of intermediates 9 over 10% Pd/C in
ethyl acetate yielded 4-hydroxy esters 80. Dehydration
of 80 with a catalytic amount of p-toluenesulfonic acid
in refluxing toluene afforded 4-unsubstituted esters 81,
which were converted into 4-unsubstituted acids 3 and
82 by alkaline hydrolysis.
The 4-alkyl derivatives, 83-92, shown in Table 4 were
synthesized from 4-methoxy acid 22 (Scheme 2). Thus,
addition of an alkyl Grignard reagent to 22 at low
temperature gave the corresponding 4-alkyl acids 83-
92.
Scheme 3 shows the synthetic routes for 4-chloro,
4-butylthio, and 4-phenoxy derivatives 95, 96, and 98,
respectively. Regioselective alkylation of 2,5-dihydroxy-
acetophenone 4a with 2-bromopropane and successive
condensations of the resulting acetophenone 5a with
piperonal under aqueous basic conditions provided
chromanone 93. Treatment of 93 with phosphorus
oxychloride in dimethylformamide (DMF) caused formy-
lation at the 3-position and chlorination at the 4-position
simultaneously, giving 4-chloro-3-formyl intermediate
94 in good yield.59 Oxidation of the formyl group of 94
with sodium chlorite gave 4-chloro acid 95, which was
further converted into 4-n-butylthio acid 96 by reaction
with sodium butanethiolate.
Addition of sodium p-methoxyphenoxide to 4-chloro-
3-formyl intermediate 94 yielded 4-phenoxy-3-formyl
compound 97. Oxidation of the formyl group of 97 with
sodium chlorite under buffered conditions (pH ) 5)
smoothly proceeded to give 4-phenoxy acid 98 in good
yield.
Modifications of the carboxylic acid at the 3-position
in 4-n-butyl acid 83 and 4-p-anisyl acid 48 are shown
in Scheme 4. Reaction of acid 83 with oxalyl chloride,
followed by treatment with methanol, yielded the cor-
responding 4-n-butyl ester 99. Successive treatment of
acids 83 and 48 with oxalyl chloride and with 28% NH4-
OH afforded corresponding amide derivatives 100 and
101, respectively. Amide 100 was further converted into
tetrazole 102 by reaction with sodium azide and tetra-
chlorosilane.60 3-Acylsulfonamide derivatives 103 and
104 were synthesized by condensation of acids 83 and
48 with methanesulfonamide using the carbodiimide-
4-(dimethylamino)pyridine method.61
F igu r e 1.
Early developments of ET antagonists were initiated
using peptidic compounds. They include BQ-123,31,32
FR139317,33BQ-788,34RES-701-1,35IRL-1038,36PD142893,37
PD145065,38 and TAK-044.39 On the other hand, the
first nonpeptidic antagonist, Ro 46-2005,40 was disclosed
in 1993 by the Roche group. Now, many highly potent
nonpeptidic antagonists such as BMS-182874,41,42 SB-
209670,43,44 PD-156707,45,46 A-127722,47,48 Bosentan,28,29
L-754142,49,50 and A-18208651 are known. Some are
selective antagonists for ETA receptors, and others act
on both ETA and ETB receptors. Although it remains
unclear at present which type of antagonists, selective
ETA or mixed ETA/ETB, is more suitable for the clinical
purposes,52,53 these facts prompted us to develop novel
nonpeptide ET receptor antagonists.
A literature survey at the start of the ET project
revealed that various chemical structures, derived from
both synthetic and natural resources, were reported to
act on ET receptors. Among them, we were very
interested in a series of compounds disclosed in a patent
by the SmithKline-Beecham group.54 These compounds,
for example, 1, were apparently synthesized during the
course of development of angiotensin II (AII) receptor
antagonists because their structures are closely related
to their AII receptor antagonist, Eprosartan55,56 (Figure
1). This prompted us to modify our own AII receptor
antagonists, and compound 2 was one of such starting
materials. Fortunately, we found very weak affinity for
the ETA receptor in compound 3, which was obtained
by the modification of the partial structure of 2.
In this paper, we will describe the discovery of our
lead compound 3 and the structure-activity relation-
ships (SAR) of its derivatives leading to our clinical
candidate (R)-48. Also discussed are the essential roles
of substituents at the 2-, 3-, 4-, and 6-positions of the
2H-chromene skeleton.
Ch em istr y
The 4-alkoxy, 4-aryl, and 4-unsubstituted derivatives,
listed in Tables 1-4 (11-33, 36-79, 3, and 82), were
synthesized via key intermediate 9 as outlined in
Scheme 1. The Vilsmeier-Haack reaction of alkoxy-2-
hydroxyacetophenones 5, which were commercially
Scheme 5 shows the synthesis of 3,4-dihydro com-
pounds 106 and 107 listed in Table 6. Catalytic hydro-

Endothelin-A Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2043
Sch em e 1a
a
Reagents: (a) Alkyl halide, K2CO3, KI, DMF. (b) POCl3, DMF. (c) NaClO2, H2NSO3H, CH2Cl2, H2O. (d) (i) (COCl)2, CH2Cl2; (ii) CH3OH.
(e) R2MgBr or R2MgCl, CuI, THF. (f) R3OH, diethyl azodicarboxylate, PPh3, THF. (g) 1 M NaOH, THF, CH3OH. (h) 2-[N,N′-
bis(trifluoromathanesulfonyl)amino]pyridine, NaH, DMF. (i) R3B(OH)2, Pd(PPh3)4, 2 M Na2CO3, DME. (j) H2, 10% Pd/C, EtOAc. (k) p-TsOH,
toluene.
Sch em e 2a
synthesized from (R)-22 and n-butyl magnesium chlo-
ride as shown in Scheme 6.
Optical purities of all of the chiral compounds listed
in Table 7 were confirmed to be more than 98% ee by
chiral high-performance liquid chromatography (HPLC)
analysis. In the following discussion, the three rings are
named as the A-, B-, and C-rings as shown in Figure 1.
a
Reagents: (a) RMgBr or RMgCl, THF.
Biologica l Testin g
genation of esters 99 and 35a afforded corresponding
dihydro esters 105, whose all cis configurations were
determined by H nuclear magnetic resonance (NMR)
coupling constants: J 2,3 ) 2-3 Hz and J 3,4 ) 6-7 Hz,
respectively. Alkaline hydrolysis of 105 proceeded with
epimerization at the 3-position to give all trans acids
106 and 107 exclusively. Their stereochemistry was
established on the basis of 1H NMR coupling con-
stants: J 2,3 ) 9-11 Hz and J 3,4 ) 10-12 Hz.
4-p-Anisyl acid 48 was resolved into its enantiomers
(+)-48 and (-)-48 via (1S,2R)-(+)- and (1R,2S)-(-)-
norephedrine salts, respectively. Absolute configurations
of (+)-48 and (-)-48 were assigned to be R and S,
respectively, on the basis of the single-crystal X-ray
structure of the (1S,2R)-(+)-norephedrine salt of (+)-
48 (Figure 2). 4-Unsubstituted acid 82 was resolved into
the enantiomers (-)-82 and (+)-82 via (S)-(-)- and (R)-
(+)-phenylethylamine salts, respectively, whose abso-
lute configurations were determined by comparison of
their CD spectra with the CD spectrum of (R)-48. (R)-
48 and (-)-82 exhibited positive Cotton effects around
240 nm owing to the π-π* transition of the styrene
chromophore. This observation indicates that the ster-
eochemistry of both compounds is the same and thus
the absolute configuration of (-)-82 is R.62
The binding affinities for the rat ETA and pig ETB
receptors of the synthesized compounds were deter-
mined by their inhibition of the [125I]ET-1 binding in
rat aorta smooth muscle cells and the [125I]ET-3 binding
to the cloned pig ETB receptor expressed in COS-7 cells,
respectively.63 The binding studies for human receptors
were performed in a similar fashion, using human ETA
and ETB receptors expressed in Chinese hamster ovary
(CHO) cells.63 The upper limits of the IC50 values
determined for the ETA or ETB receptor bindings were
100 µM. The results are summarized in Tables 1-7.
1
Resu lts a n d Discu ssion
(1) F in d in g of Lea d Com p ou n d . As described in
the Introduction, our initial efforts to find a lead
compound of a novel series of ET receptor antagonists
were focused on various AII antagonists, including 2 and
related analogues. Fortunately, 2H-chromene 3 obtained
by reduction of the 4-carbonyl group of the flavone
substructure of 2 showed very weak binding affinity to
the ETA receptor (IC50 ) 67 µM, Figure 1). The simple
tricyclic structure of 3 offered plenty of room for chemi-
cal modifications to increase activity, and thus, 3 was
selected as our first lead compound.
Surprisingly, introduction of an alkoxy group at the
4-position in 3, which was readily made by treatment
of 9 with an alcohol under Mitsunobu conditions,
dramatically increased the binding affinity for the ETA
receptor (Table 1). As the chain length of the 4-alkoxy
group was increased, the affinity was improved. The
Optical resolution of 4-methoxy acid 22 with (1S,2R)-
(+)-norephedrine gave (+)-22. Because (+)-22 was
converted into (R)-48 by reaction with p-anisylmagne-
sium bromide (Scheme 6), the R-configuration of (+)-
22 was established also. (R)-4-n-Butyl acid, (R)-83, was

2044 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ishizuka et al.
Sch em e 3a
a
Reagents: (a) 2-Bromopropane, K2CO3, CH3CN. (b) Piperonal, 2 M NaOH, MeOH. (c) POCl3, DMF. (d) NaClO2, H2NSO3H, toluene,
H2O. (e) 1-Butanethiol, NaH, THF. (f) 4-Methoxyphenol, NaH, DMF. (g) NaClO2, NaH2PO4, CH3CN, H2O.
Sch em e 4a
a
Reagents: (a) (i) (COCl)2, catalytic DMF, CH2Cl2; (ii) CH3OH. (b) (i) (COCl)2, catalytic DMF, CH2Cl2; (ii) 28% NH4OH. (c) NaN3,
SiCl4, CH3CN. (d) Methanesulfonamide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-(dimethylamino)pyridine, CH2Cl2.
Sch em e 5a
a
Reagents: (a) H2 (500 kPa), 5% Pd/C, AcOH, THF, EtOAc or
H2 (500 kPa), PdCl2, MeOH, CHCl3. (b) 1 M NaOH, THF, MeOH
or 1 M NaOH, DMSO.
F igu r e 2. Single-crystal X-ray structure of (R)-48 as (1S,2R)-
(+)-norephedrine salt. All hydrogens are removed for clarity.
n-butoxy and n-pentoxy derivatives 13 and 14 were
approximately 1000 times more potent than the lead
compound 3.
ate boronic acid, also resulted in dramatic increases in
the binding affinity for the ETA receptor. p-Anisyl and
p-isopropylphenyl groups (39 and 42) were found to be
the most effective substituents, giving the affinities
Introduction of the phenyl or substituted phenyl group
at the 4-position, which was routinely achieved by the
Suzuki coupling reaction of triflate 34 and an appropri-

Endothelin-A Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2045
Sch em e 6a
effective, giving highly potent derivatives 19 and 48
with the IC50 values of less than 1 nM, which were 100
times more potent than the second lead compounds 13
and 39, respectively. Insertion of a terminal hydroxyl
group into the isopropoxy group led to a slight loss in
the activity (51). Importantly, removal of the oxygen
atom from the isopropoxy group drastically diminished
the activity (52), suggesting that this oxygen atom is
important not only as a spacer but also as a pharma-
cophore that acts as a hydrogen-bonding acceptor at the
receptor active site. In contrast to the 6-position SAR,
introduction of a similar alkoxy group at the 5-, 7-, or
8-position resulted in a significant loss in the activity
(15, 21, 44, 53, and 54). These results indicate that the
alkoxy group at the 6-position, especially the isopropoxy
group, is essential for the potent binding affinity. It is
suggested that the 6-isopropoxy group is properly
recognized by the ETA receptor with its isopropyl part
acting as a hydrophobic region and its oxygen atom
acting as a hydrogen-bonding acceptor.
a
Reagents: (a) (1S,2R)-(+)-Norephedrine, CH3OH. (b) RMgBr
or RMgCl, THF.
Ta ble 1. Substituent Effect at the 4-Position
IC50 (nM)a
Introduction of an alkoxy group at the 6-position also
improved the binding affinity for the ETB receptor, but
the effect was limited (IC50 > 130 nM). As a result, the
selectivity for the ETA receptor became large. The most
significant compounds, 19 and 48, are 1900 and 350
times selective for the ETA receptor over the ETB
receptor, respectively, and thus, they selectively bound
to the ETA receptor.
b
c
compd
R
rETA
pETB
3
11
12
13
14
36
37
38
39
40
41
42
43
H
67000 ( 4000 (2)
>100 000 (1)
OC2H5
O-n-C3H7
O-n-C4H9
O-n-C5H11
C6H5
290 ( 20 (2) 15 000 ( 5000 (2)
160 ( 20 (2)
66 ( 0 (2)
10 000 (1)
4900 (1)
29 ( 7 (2)
1100 ( 290 (2)
>100 000 (1)
31 000 (1)
780 ( 20 (2)
2200 ( 900 (2)
700 (1)
2′-CH3OC6H4
3′-CH3OC6H4
4′-CH3OC6H4
3′,4′-(-OCH2O-)C6H3
4′-CH3C6H4
2800 (1)
1400 (1)
(3) Su bstitu en t Effect a t 2-P osition . Substituent
effects on the 2-phenyl ring were examined by using
compounds shown in Table 3, which had different
substituent patterns at the 4- and 6-positions. As shown
in the table, replacement of the m,p-methylenedioxy
group by another functional group such as an o-, m-, or
p-alkoxy, alkyl, or hydroxyl group consistently dimin-
ished the affinity for the ETA receptor, indicating that
the m,p-methylenedioxy group is exclusively effective
on the 2-phenyl ring.
86 ( 14 (2)
350 (1)
710 ( 160 (2)
6900 ( 1100 (2)
2100 ( 400 (2)
5800 ( 200 (2)
530 ( 50 (2)
83 ( 3 (2)
800 ( 100 (2)
4′-i-C3H7C6H4
4′-ClC6H4
a
A mean IC50 value ( standard error of the mean (SEM) with
b
the number of experiments given in parentheses. Rat receptors.
c Pig receptors.
similar to those of n-butoxy and n-pentoxy groups (13
and 14). The other substituted phenyl derivatives were
less potent than 39 and 42, though considerably more
potent than the lead compound 3.
Because these two types of 4-substituted compounds,
the 4-alkoxy and 4-aryl series, seemed to be equally
promising, compounds 13 and 39 having structurally
distinct 4-substituents were selected as the second lead
compounds for further chemical modifications.
In contrast to the satisfactory improvement in the
binding affinity for the ETA receptor, that for the ETB
receptor was not significant. In the following section,
we will thus focus our discussion on the ETA receptor
binding affinity.
(2) Su bstitu en t Effect on A-Rin g. Substituent
effects on the A-ring for the ET receptor binding affinity
were examined in the second lead compounds 13 and
39 (Table 2). In both series of compounds, introduction
of an alkoxy group at the 6-position gave an equal to
significantly improved affinity (16-20 or 45-51). In
each of these cases, close correlation was observed
between the chain length and the affinity for the ETA
receptor. The optimal number of the carbon atoms is
2-4 (17-20 or 46-49), and with a shorter or longer
group such as methoxy (16 and 45) or benzyloxy (50),
the binding affinity diminishes greatly. Among these
groups, the branched isopropoxy group was especially
Interestingly, some o,p-dimethoxy (57 and 65) and
p-methoxy compounds (58 and 69) retained moderate
affinities. As compared with these compounds, the
affinities of the corresponding o,m-dimethoxy (66), o- or
m-methoxy (67 or 68), and p-alkyl (59) derivatives were
very weak. This observation suggests that the oxygen
atom at the p-position on the 2-phenyl ring plays a
crucial role as a hydrogen-bonding acceptor at the
receptor binding site. Cleavage of the methylenedioxy
bridge (55, 56, and 64) and introduction of a bulkier
alkoxy group at the p-position (70 and 71) also drasti-
cally diminished the affinity, suggesting that the steri-
cally less-hindered and conformationally restricted me-
thylendioxy group is advantageous for forming a
hydrogen bond with the ETA receptor active site.
Interestingly, a combination of the m,p-methylene-
dioxyphenyl group and the n-propyl or n-butyl group
at the 4- and 6-positions, respectively, tended to improve
the affinity for the ETB receptor (62, 63, 65, and 69),
although their binding affinities were still unsatisfac-
tory. This tendency suggests a possibility that ETA/ETB-
balanced antagonists could be found by modifications
of these compounds. In this paper, however, no further
discussion will be made on this subject.

2046 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ta ble 2. Substituent Effect on the A-Ring
Ishizuka et al.
IC50 (nM)a
b
c
compd
R1
R2
rETA
66 ( 0 (2)
3400 (1)
250 (1)
4.2 ( 0.2 (2)
2.0 (1)
pETB
B/Ad
13
15
16
17
18
19
20
21
39
44
45
46
47
48
49
50
51
52
53
54
O-n-C4H9
H
4900 (1)
6000 ( 300 (2)
2300 ( 200 (2)
1600 ( 200 (2)
520 ( 70 (2)
780 (1)
470 ( 90 (2)
2800 ( 200 (2)
1400 (1)
1800 ( 200 (2)
540 ( 20 (2)
400 ( 20 (2)
220 ( 20 (2)
180 (1)
74
1.8
9.2
380
260
1900
160
16
5-O-n-C3H7
6-OCH3
6-OC2H5
6-O-n-C3H7
6-O-i-C3H7
6-O-n-C4H9
7-O-n-C3H7
H
0.41 ( 0.01 (2)
3.0 ( 1.0 (2)
180 ( 40 (2)
86 ( 14 (2)
1200 ( 480 (2)
74 ( 11 (2)
3.8 ( 0.5 (2)
4.4 (1)
0.51 ( 0.04 (2)
17 ( 1 (2)
62 ( 14 (2)
2.8 ( 0.8 (2)
110 ( 40 (2)
970 ( 40 (2)
1600 (1)
4′-CH3OC6H4
16
5-O-n-C3H7
6-OCH3
1.5
7.3
110
50
350
7.6
13
6-OC2H5
6-O-n-C3H7
6-O-i-C3H7
6-O-n-C4H9
6-OCH2C6H5
6-OCH(CH3)CH2OH
6-i-C3H7
130 ( 10 (2)
790 (1)
170 ( 10 (2)
190 (1)
2300 ( 300 (2)
4000 ( 500 (1)
61
1.7
2.4
2.5
7-O-n-C3H7
8-OCH3
d
a-c See footnotes a-c of Table 1. IC50(ETB)/IC50(ETA).
Ta ble 3. Substituent Effect at the 2-Position
IC50 (nM)a
b
c
compd
R1
R2
R3
rETA
pETB
48
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
4′-OCH3
O-i-C3H7
3′,4′-OCH2O-
3′,4′-(OH)2
3′,4′-(OCH3)2
2′,4′-(OCH3)2
4′-OCH3
4′-i-C3H7
3′,4′-OCH2O-
4′-i-C3H7
3′,4′-OCH2O-
4′-CH3O
3′,4′-(OCH3)2
2′,4′-(OCH3)2
2′,5′-(OCH3)2
2′-OCH3
0.51 ( 0.04 (2)
440 ( 20 (2)
170 ( 70 (2)
8.0 ( 2.0 (2)
35 ( 5 (2)
1200 ( 200 (2)
3.2 ( 0.2 (2)
1300 (1)
180 (1)
>100 000 (2)
NTd
770 ( 10 (2)
1300 (1)
5900 (1)
3′,4′-OCH2O-
O-i-C3H7
O-n-C3H7
O-n-C4H9
170 ( 80 (2)
2700 (1)
43 (1)
80 (1)
220 ( 0 (2)
810 ( 140 (2)
18 (1)
160 ( 40 (2)
130 (1)
350 (1)
95 (1)
5200 ( 600 (2)
4800 ( 600 (2)
72 ( 5 (2)
400 ( 50 (2)
35 ( 8 (2)
1600 (1)
760 ( 170 (2)
160 ( 40 (2)
43 ( 7 (2)
3600 (1)
3′-OCH3
4′-OCH3
4′-O-n-C4H9
4′-O-c-C5H9
2200 (1)
d
a-c See footnotes a-c of Table 1. NT ) not tested
(4) Su bstitu en t Effect a t 4-P osition . Preliminary
SAR studies on the substituent effect at the 4-position
(Table 1) indicate that introduction of the structurally
distinct aliphatic and aryl groups were equally effective
for increasing the affinity. To understand why these
distinct groups are acceptable at the 4-position, the
substituent effects at this position were reinvestigated
in detail, fixing the 2- and 6-substituents to the most
promising groups, the m,p-methylenedioxyphenyl and
isopropoxy groups, respectively.
Table 1 reveals that the affinity tends to increase with
increasing chain length of the 4-alkoxy group. To
investigate the optimal length of the 4-substituent,
linear alkoxy groups from methoxy (22) to decyloxy (28)
were systematically introduced into the parent 4-un-
substituted compound 82. The results in Table 4 show

Endothelin-A Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2047
Ta ble 4. Substitutent Effect at the 4-Position
the 4-position (92 and 98) showed similar effects on the
binding affinity of 48 giving subnanomolar affinities,
whereas insertion of a longer ethoxy spacer (33) resulted
in a loss in the affinity.
All of these results indicate that the hydrophobic
substituent at the 4-position plays a crucial role for
improving the activity, but its shape is not so important
in the receptor recognition. A predominant factor for
determining the affinity is only the length of the
substituent, and the optimal length is estimated to be
6-9 Å. Presumably, the ETA receptor recognizes only
the length of the 4-substituent but not the shape, and
accordingly, various types of groups are acceptable at
the 4-position.
IC50 (nM)a
b
c
compd
R
rETA
pETB
82
22
23
24
19
25
26
27
28
29
30
31
32
96
83
84
85
86
87
88
89
90
91
95
72
73
74
48
92
98
33
H
330 ( 30 (2)
9.1 ( 0.1 (2)
2.2 (1)
52 000 ( 14 000 (2)
6400 (1)
OCH3
OC2H5
2800 (1)
1300 (1)
780 (1)
O-n-C3H7
O-n-C4H9
1.5 ( 0.2 (2)
0.41 ( 0.01 (2)
0.48 ( 0.03 (2)
0.67 ( 0.13 (2)
4.7 ( 0.3 (3)
130 ( 10 (2)
0.81 ( 0.09 (2)
0.33 ( 0.07 (2)
0.95 (1)
O-n-C5H11
O-n-C6H13
O-n-C7H15
O-n-C10H21
O-i-C3H7
O(CH2)3CHdCH2
O(CH2)5OH
O(CH2)3CN
S-n-C4H9
150 ( 0 (2)
78 ( 5 (2)
410 ( 140 (3)
7600 (1)
One plausible explanation for this observation may
be an induced fit64 of the receptor active site to the
4-substituent, in which ligand-induced conformational
changes of the hydrophobic pocket in the receptor may
take place to accommodate variously shaped 4-substit-
uents. Therefore, in considering the structural diversity
acceptable to the 4-position, this region does not seem
to be essential for specific recognition by the ETA
receptor.
730 (1)
130 ( 30 (2)
880 ( 30 (2)
5800 ( 200 (2)
100 ( 11 (2)
610 ( 70 (2)
150 ( 40 (2)
69 ( 1 (2)
2500 (1)
350 ( 10 (2)
46 ( 2 (2)
480 ( 80 (2)
710 ( 80 (2)
1800 ( 100 (2)
7200 (1)
6.8 ( 1.7 (2)
0.27 ( 0.02 (2)
0.73 ( 0.03 (2)
0.32 ( 0.02 (2)
0.62 ( 0.08 (2)
0.63 ( 0.11 (2)
0.53 ( 0.08 (2)
0.58 ( 0.02 (2)
1.9 ( 0.1 (2)
1.8 ( 0 (2)
n-C4H9
n-C5H11
n-C6H13
i-C3H7
c-C5H9
(5) Su bstitu en t Effect a t 3-P osition . Replacement
of the 3-carboxyl group in the 4-n-butyl acid 83 and 4-p-
anisyl acid 48 by methoxycarbonyl (99 and 35a ) or
carbamoyl (100 and 101) led to a significant loss in the
binding affinity (Table 5). Compounds 102-104, which
contain the tetrazole or acylsulfonamide group as a
carboxyl bioisostere, were more active than the esters
(99 and 35a ) and the amides (100 and 101) but less
potent than the carboxylic acids (83 and 48). Obviously,
the carboxyl group at the 3-position is an essential
functional group. The fact that the acidic compounds
are consistently more active than the nonacidic com-
pounds indicates an important role of an acidic proton
at this region. Thus, the 3-carboxyl group should act as
a hydrogen-bonding donor in the ETA receptor active
site.
CH2-c-C6H11
(CH2)2CH(CH3)2
(CH2)3OCH3
(CH2)4OH
2.3 ( 0.4 (2)
12 ( 1 (2)
Cl
C6H5
3.0 ( 0.2 (2)
3.0 ( 0.7 (2)
1.6 ( 0.6 (2)
0.51 ( 0.04 (2)
0.52 ( 0.19 (2)
0.21 ( 0.03 (2)
3.0 (1)
NTd
2′-CH3OC6H4
3′-CH3OC6H4
4′-CH3OC6H4
4′-CH3OC6H4CH2-
4′-CH3OC6H4O-
4′-CH3OC6H4-
(CH2)2O-
4′-C6H5CH2OC6H4
4′-OHC6H4
4′-i-C3H7C6H4
4′-FC6H4
3500 ( 100 (2)
470 ( 10 (2)
180 (1)
85 (1)
58 ( 1 (2)
200 ( 70 (2)
75
76
77
78
60
6.3 ( 2.0 (2)
4.1 ( 0.1 (2)
4.9 ( 0.2 (2)
8.0 ( 1.0 (2)
3.2 ( 0.2 (2)
600 ( 40 (2)
4800 (1)
750 (1)
NTd
3′,4′-(-OCH2O-)-
170 ( 80 (2)
C6H3
79
5′-methylthio-
phen-2-yl
1.0 ( 0.6 (2)
1800 (1)
(6) Effect of 3,4-Dou ble Bon d . Reduction of the
C3-C4 olefinic bond generally resulted in loss in the
ETA receptor binding affinity (Table 6). The IC50 values
of the 3,4-dihydro compounds 106 and 107 were 77 and
1.3 nM, respectively, which are 2-100 times higher than
those of the corresponding parent compounds. Thus, the
2H-chromene skeleton was found to be the best core
structure in our series of compounds.
a-c See footnote a-c of Table 1. NT ) not tested.
d
a clear correlation between the chain length and the
affinity. The optimal number of the carbon atoms is 4-6
(19, 25, and 26), which corresponds to 6-9 Å in length.
Structurally distinct groups such as linear alkylthio
and alkyl (96 and 83-85), cyclic and branched alkyl
(86-88), as well as different alkoxy (29 and 30), which
have 3-7 carbon atoms, also are similarly effective for
providing subnanomolar affinity. Although substitution
with an oxygen- or nitrogen-containing group at the
terminal position (31, 32, 90, and 91) slightly dimin-
ished the activity, their IC50 values are still respectable.
Interestingly, even chlorine (95) gives moderate ETA
receptor binding affinity with the IC50 value of 12 nM.
The IC50 value of each of the 4-aryl derivatives (72-
79, 48, 92, 98, 33, and 60) is consistently lower by an
order of 102-103 than that of the parent compound 82,
showing an order of affinity similar to the above
4-aliphatic series. Similarly to the results in Table 1,
the p-anisyl group (48) was found to be the optimal
4-aryl substituent. Interestingly, insertion of a meth-
ylene or oxygen spacer between the p-anisyl group and
(7) Effect of Absolu te Con figu r a tion . Relation-
ships between the absolute configuration at the 2-posi-
tion and the binding affinity are summarized in Table
7. R Isomers are more potent than the corresponding
racemates and S isomers and are therefore the eu-
tomers. The most potent compound in our series of
compounds was (R)-48, which bound to the ETA receptor
with the IC50 value of 0.19 nM and with a 630-fold
selectivity over the ETB receptor.
(8) P h ar m acokin etics an d P h ar m acological Stu d-
ies of (R)-48. On the basis of its excellent binding
affinity and selectivity, (R)-48 was chosen for further
evaluation. Compound (R)-48 retained the highly potent
and selective ETA binding affinity against the human
ET receptors (Table 7). The full details of the other
functional and pharmacological studies of (R)-48 had

2048 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ta ble 5. Substitutent Effect at the 3-Position
Ishizuka et al.
d
a-c See footnotes a-c of Table 1. NT ) not tested.
Ta ble 6. Effect of the C3-C4 Double Bond on the Receptor Binding Affinity
d
a-c See footnotes a-c of Table 1. NT ) not tested.
been already reported in our previous report,65 which
demonstrates that this compound is a potent, orally
active and long-lasting ETA receptor antagonist.
curve (from time 0 to infinity) of 65.4 ( 14.7 µg h/mL,
and a total plasma clearance of 1.3 ( 0.3 mL/min/kg.
Orally administrated (R)-48 was rapidly absorbed, and
then, the plasma concentration was kept at a high level
for more than 8 h. A maximum plasma concentration
of 30.6 ( 13.3 µg/mL was achieved at 1.3 ( 0.6 h. Area
under the curve (from time 0 to 48 h) was 214 ( 40 µg
h/mL. The calculated bioavailability was 54.6 ( 10.2%.
Pharmacokinetic studies of (R)-48 were also carried
out. The results of intravenous and oral administration
in rats are shown in Figure 3. Intravenous administra-
tion of (R)-48 gave a terminal-phase half-life of 8.6 (
0.6 h (mean ( standard deviation), an area under the

Endothelin-A Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2049
Ta ble 7. ET Receptors Binding Affinities of the Chiral Compounds
IC50 (nM)a
b
c
compd
82
(R)-82
(S)-82
22
(R)-22
83
(R)-83
48
configuration
R3
rETA
pETB
B/Ad
RS
R
S
RS
R
RS
R
H
H
H
330 ( 30 (2)
240 ( 70 (2)
1600 (1)
52 000 ( 14000 (2) (2)
52 000 (1)
160
220
>60
700
670
840
950
350
630
(440)e
100
>100 000 (1)
OCH3
OCH3
n-C4H9
n-C4H9
4′-CH3OC6H4
4′-CH3OC6H4
9.1 ( 0.1 (2)
6400 (1)
5.2 (1)
3500 ( 1300 (2)
610 ( 70 (2)
400 ( 60 (2)
180 (1)
0.73 ( 0.03 (2)
0.42 ( 0.12 (2)
0.51 ( 0.04 (2)
0.19 ( 0.01 (2)
(0.45 (1))e
RS
R
(R)-48
(S-1255)
(S)-48
120 (1)
(200 (1))e
1500 (1)
S
4′-CH3OC6H4
15 ( 3 (2)
d
a-c See footnotes a-c of Table 1. IC50 (ETB)/IC50 (ETA). e Human ETA and ETB receptors.
F igu r e 4. Crucial basic structure 108 of our ETA receptor
antagonists and its speculated pharmacophore model. H and
A indicate a hydrogen and a hydrogen bonding acceptor on
the receptor active sites, respectively.
F igu r e 3. Pharmacokinetics of (R)-48 in rats.
With these preferable pharmacokinetic and pharmaco-
logical profiles, (R)-48 was selected as our clinical
candidate.
ture 108 (Figure 4) is a crucial basic structure of our
ETA receptor antagonists and is specifically recognized
by the ETA receptor through three hydrogen bonds,
whereas the substituent at the 4-position plays a pivotal
role to help 108 guide into the relevant position of the
receptor active site. This conclusion is illustrated in a
pharmacophore model in Figure 3.
Con clu sion
A unique lead compound 3 was found by screening
our AII receptor antagonists, and the subsequent opti-
mization led us to the discovery of the compounds with
highly potent binding affinity for the ETA receptor, of
which (R)-48 was selected as a clinical candidate.
Extensive chemical modifications of 3 also revealed
the crucial roles of the substituents at the 2-, 3-, 6-, and
4-positions on the (R)-2H-chromene skeleton for binding
affinity. These four substituents are equally indispen-
sable for potent binding affinity, but their roles in the
receptor recognition seem to be significantly different.
Substitutions of the 2-, 3-, and 6-positions with the m,p-
methylenedioxyphenyl group, the carbonyl group, and
the isopropoxy group, respectively, are essential for
improving high potency, and consequently these groups
are recognized by the ETA receptor. They potentially act
as a hydrogen bond acceptor or donor in the receptor
active site and are considered to play predominant roles
in the receptor recognition. In contrast to these posi-
tions, introduction of various aliphatic and aromatic
substituents, 6-9 Å in length, at the 4-position retains
the high potency, thereby suggesting that this region
is not essential for the specific receptor recognition.
These SAR studies led to the conclusion that substruc-
Exp er im en ta l Section
Melting points were determined on a Yanagimoto hot plate
apparatus and are uncorrected. 1H NMR spectra were recorded
on a Varian VXR-300 spectrometer and are reported in parts
per million (δ) relative to tetramethylsilane (TMS) as an
internal standard. Optical rotations were measured in a 10
cm cell on a Perkin-Elmer 241 polarimater. Column chroma-
tography was performed using a column packed with Merck
silica gel 60 (70-230 mesh). Analytical HPLC was performed
on a Shimadzu LC-10AD instrument.
5-Isop r op oxy-2-h yd r oxya cetop h en on e (5a ). A mixture
of 2,5-dihydroxyacetophenone 4a (7.61 g, 50.02 mmol), 2-bro-
mopropane (18.46 g, 150.09 mmol), sodium iodide (1.50 g, 10.01
mmol), powdered potassium carbonate (8.29 g, 59.98 mmol),
and DMF (30 mL) was stirred at 60 °C for 24 h. The mixture
was poured into water and extracted with ethyl acetate. The
combined organic extracts were washed with water and brine,
dried with MgSO4, and concentrated. The residue was purified
by column chromatography on silica gel (ethyl acetate/hexane,
1/19) to give 5a as a yellow oil (4.98 g, 51.3%). 1H NMR
(CDCl3): δ 1.32 (6H, d, J ) 6.1 Hz), 2.60 (3H, s), 4.34-4.52
(1H, m), 6.89-7.23 (3H, m).

2050 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ishizuka et al.
6-Isop r op oxy-4-oxo-4H-ch r om en e-3-ca r ba ld eh yd e (6)
(R1 ) 6-Isop r op oxy). Phosphoryl oxychloride (388 g, 2.54 mol)
was added dropwise to 5a (123 g, 0.634 mol) in DMF (800 mL)
at -30 °C. The mixture was allowed to stand overnight at room
temperature and then poured into ice-water. The resulting
precipitate was collected by filtration, giving 6 (119 g, 81%)
as a dark brown solid. 1H NMR (CDCl3): δ 1.39 (6H, d, J )
6.0 Hz), 4.71 (1H, m), 7.28 (1H, dd, J ) 9.4 and 2.6 Hz), 7.46
(1H, d, J ) 9.4 Hz), 7.64 (1H, d, J ) 2.6 Hz), 8.53 (1H, s),
10.40 (1H, s).
3.94-4.18 (2H, m), 4.40 (1H, m), 5.89 (2H, s), 6.17 (1H, s),
6.65-6.87 (5H, m,), 7.02 (1H, d, J ) 2.8 Hz).
2-(Ben zo[1,3]d ioxol-5-yl)-4-n -b u t yloxy-6-isop r op oxy-
2H-ch r om en e-3-ca r boxylic Acid (19). A mixture of 10 (R1
) 6-isopropoxy, R2 ) m,p-methylenedioxyphenyl, R3 ) n-butyl,
211 mg, 0.479 mmol), 1 M NaOH (2.4 mL), methanol (2.5 mL),
and THF (5 mL) was refluxed for 3 h and then concentrated
under reduced pressure. After water was added, the mixture
was acidified with 1 M HCl and extracted with ether. The
product was recrystallized from diisopropyl ether/acetone to
1
give 19 (183 mg, 90%) as yellow crystals; mp 124-125 °C. H
6-Isopr opoxy-4-oxo-4H-ch r om en e-3-car boxylic Acid (7)
(R1 ) 6-Isop r op oxy). A solution of sodium chlorite (80%, 77
g, 0.684 mol) in water (400 mL) was added dropwise to a
mixture of 6 (R1 ) 6-isopropoxy, 40.0 g, 0.172 mol) in
dichloromethane (860 mL) and NH2SO3H (84.0 g, 0.865 mol)
in water (840 mL) at 0 °C. After 30 min, the organic layer was
separated and the aqueous layer was extracted with chloro-
form. The combined organic phases were washed with water
and brine, dried with MgSO4, and concentrated. Recrystalli-
zation from ethanol afforded 7 (34.8 g, 82%) as yellow crystals;
NMR (CDCl3): δ 1.01 (3H, t, J ) 7.4 Hz), 1.32 (6H, d, J ) 6.0
Hz), 1.52 (2H, m), 1.88 (2H, m), 3.94-4.06 (1H, m), 4.26-4.47
(2H, m), 5.89 (2H, s), 6.27 (1H, s), 6.67 (1H, d, J ) 3.8 Hz),
6.78-6.87 (5H, m). Anal. (C24H26O7) C, H.
Meth yl 2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-4-(tr i-
flu or om eth a n su lfon yloxy)-2H-ch r om en e-3-ca r boxyla te
(34) (R1 ) 6-Isop r op oxy, R2 ) m ,p-Meth ylen ed ioxyp h en -
yl). A solution of 9 (R1 ) 6-isopropoxy, R2 ) m,p-methylene-
dioxyphenyl, 47.5 g, 0.124 mol) in DMF (100 mL) was added
dropwise to sodium hydride (3.60 g, 0.150 mol) in DMF (400
mL) at 0 °C, and the mixture was stirred for 1 h. 2-[N,N′-Bis-
(trifluoromethansulfonyl)amino]pyridine (50.8 g, 0.142 mol)
was added, and the mixture was stirred at 0 °C for 3 h and
then poured into ice-water. Usual workup and purification by
silica gel chromatography (toluene) afforded 34 (63.0 g, 99%)
as pale yellow crystals; mp 88-90 °C. 1H NMR (CDCl3): δ 1.31
(3H, d, J ) 6.4 Hz), 1.31 (3H, d, J ) 6.0 Hz), 3.83 (3H, s), 4.40
(1H, m), 5.92 (2H, s), 6.32 (1H, s), 6.68-6.92 (6H, m). Anal.
(C22H19O9SF3) C, H, S, F.
1
mp 178 °C. H NMR (CDCl3): δ 1.41 (6H, d, J ) 6.0 Hz), 4.72
(1H, m), 7.39 (1H, dd, J ) 9.2 and 3.0 Hz), 7.58 (1H, d, J )
9.2 Hz), 7.62 (1H, d, J ) 3.2 Hz), 8.99 (1H, s), 13.6 (1H, brs).
Meth yl 6-Isop r op oxy-4-oxo-4H-ch r om en e-3-ca r boxyl-
a te (8) (R1 ) 6-Isop r op oxy). Oxalyl chloride (20.4 g, 0.16
mol) was added dropwise to a solution of 7 (R1 ) 6-isopropoxy,
34.8 g, 0.14 mol) and DMF (2.0 mL, 0.026 mol) in dichlo-
romethane (350 mL) at room temperature. After 1 h, methanol
(30 mL) was carefully added, and the mixture was stirred for
15 min. The solvent was removed under reduced pressure, and
the residue was recrystallized from ethanol to give 8 (36.3 g,
Met h yl 2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-4-(4-
m eth oxyph en yl)-2H-ch r om en e-3-car boxylate (35a). A mix-
ture of 34 (R1 ) 6-isopropoxy, R2 ) m,p-methylenedioxyphenyl,
37.5 g, 72.6 mmol), aqueous sodium carbonate (2M, 102 mL,
204 mmol), lithium chloride (9.2 g, 217 mmol), Pd(Ph3P)4 (1.68
g, 1.45 mmol), 4-methoxyphenylboronic acid (12.1 g, 79.6
mmol), and dimethoxyethane (400 mL) was refluxed for 3 h.
After water was added, the mixture was extracted with ether.
The product was purified by column chromatography on silica
gel (ethyl acetate/hexane, 1/4) to give 35a (32.4 g, 94%) as a
1
99%) as colorless needles; mp 124-126 °C. H NMR (CDCl3):
δ 1.37 (6H, d, J ) 6.0 Hz), 3.93 (3H, s), 4.69 (1H, m), 7.24 (1H,
dd, J ) 9.0 and 3.0 Hz), 7.42 (1H, d, J ) 9.0 Hz), 7.63 (1H, d,
J ) 3.0 Hz), 8.66 (1H, s).
Meth yl 2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-4-oxo-
ch r om a n e-3-ca r boxyla te (9) (R1 ) 6-Isop r op oxy, R2
)
m ,p-Meth ylen ed ioxyp h en yl). 1,2-Dibromoethane (634 mg,
3.37 mmol) was added to magnesium turnings (1.72 g, 70.86
mmol) in tetrahydrofuran (THF, 10 mL), and the mixture was
stirred for 10 min at room temperature. A solution of 4-bromo-
1,2-methylenedioxybenzene (13.57 g, 67.49 mmol) in THF (100
mL) was added dropwise, and the mixture was refluxed for 1
h to complete the reaction. The mixture was cooled to room
temperature, and copper(I) iodide (479 mg, 2.52 mmol) was
added. The mixture was stirred for 15 min and then cooled to
0 °C, and then, a solution of 8 (R1 ) 6-isopropoxy, 10.0 g, 38.13
mmol) in THF (100 mL) was added dropwise. After it was
stirred at 0 °C for 1 h, the reaction mixture was poured into
ice-water and 1 M HCl (75 mL) was added. Ether extraction
followed by silica gel chromatography (toluene/acetonitrile, 95/
5) afforded a mixture of keto-enol isomers of 9 (13.6 g, 93%)
1
pale yellow oil. H NMR (CDCl3): δ 1.18 (3H, t, J ) 6.0 Hz),
1.19 (3H, d, J ) 6.0 Hz), 3.48 (3H, s), 3.87 (3H, s), 4.21 (1H,
m), 5.92 (2H, s), 6.18 (1H, s), 6.30 (1H, m), 6.71-7.21 (9H, m).
Anal. (C28H26O7) C, H.
2-(Ben zo[1,3]dioxol-5-yl)-6-isopr opoxy-4-(4-m eth oxyph e-
n yl)-2H-ch r om en e-3-ca r boxylic Acid (48). Acid 48 was
prepared from 35a in a manner similar to that used for the
synthesis of 19 (66%). Yellow crystals; mp 179-181 °C. 1H
NMR (CDCl3): δ 1.15 (3H, d, J ) 6.0 Hz), 1.18 (3H, d, J ) 6.0
Hz), 3.87(3H, s), 4.19 (1H, m), 5.91 (2H, s), 6.15 (1H, s), 6.25-
(1H, m), 6.69-6.74(3H, m), 6.87-6.97 (4H, m), 7.15-7.19(2H,
br). Anal. (C27H24O7) C, H.
2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-2H-ch r om en e-
3-ca r boxylic Acid (82). Ester 9 (R1 ) 6-isopropoxy, R2 ) m,p-
methylenedioxyphenyl, 1.51 g, 3.93 mmol) was hydrogenated
over 10% Pd/C (200 mg) in ethyl acetate (12 mL) for 30 min.
The catalyst was removed by filtration, and the filtrate was
concentrated to give 80 (R1 ) 6-isopropoxy, R2 ) m,p-
methylenedioxyphenyl, 1.10 g, 72%). A solution of 80 and
p-toluenesulfonic acid monohydrate (260 mg, 1.37 mmol) in
toluene (50 mL) was refluxed for 1 h. The reaction mixture
was washed with aqueous NaHCO3 and brine, dried with
MgSO4, and then concentrated. The residue was purified by
silica gel chromatography (ethyl acetate/hexane, 1/4) to afford
81 (R1 ) 6-isopropoxy, R2 ) m,p-methylenedioxyphenyl, 1.00
g, 69%). Ester 81 was hydrolyzed in a similar manner as
described for the synthesis of 19 to give 82 (800 mg, 57% from
9) as yellow crystals; mp 159-161 °C. 1H NMR (CDCl3): δ
1.30 (6H, d, J ) 6.3 Hz), 4.40 (1H, m), 5.90 (2H, s), 6.10 (1H,
s), 6.68-6.85 (6H, m), 7.71 (1H, s). Anal. (C20H18O6) C, H.
1
as a yellow oil. H NMR (CDCl3): δ 1.30 (6H, d, J ) 6.3 Hz,
CH3, enol), 1.32 (6H, d, J ) 6.3 Hz, CH3, keto), 3.68 (3H, s,
CO2CH3, keto), 3.76 (3H, s, CO2CH3, enol), 4.02 (1H, d, J )
12.3 Hz, C(3)H, keto), 4.42-4.50 (1H, m, CH, enol), 4.48-4.56
(1H, m, CH, keto), 5.55 (1H, d, J ) 12.3 Hz, C(2)H, keto), 5.91-
(2H, s, -OCH2O-, enol), 6.00 (2H, s, -OCH2O-, keto), 6.10 (1H,
s, C(2)H, enol), 6.68-7.36 (12H, m, arom, keto and enol).
Met h yl 2-(Ben zo[1,3]d ioxol-5-yl)-4-n -b u t yloxyl-6-iso-
p r op oxy-2H-ch r om en e-3-ca r boxyla te (10) (R1 ) 6-Iso-
p r op oxy, R2 ) m ,p-Meth ylen ed ioxyp h en yl, R3 ) n -Bu tyl).
A solution of diethyl azodicarboxylate (136 mg, 0.780 mmol)
in THF (2 mL) was added dropwise to a stirred solution of 9
(R1 ) 6-isopropoxy, R2 ) m,p-methylenedioxyphenyl, 200 mg,
0.520 mmol), triphenylphosphine (205 mg, 0.780 mmol), and
n-butanol (58 mg, 0.780 mol) in THF (2 mL) at -10 °C. The
stirring was continued for 3 h at -10 °C, and the reaction was
quenched by addition of water. After usual workup, the
product was purified by column chromatography on silica gel
(ethyl acetate/hexane, 1/4), giving 10 (211 mg, 92%) as a pale
2-(Ben zo[1,3]d ioxol-5-yl)-4-n -b u t yl-6-isop r op oxy-2H -
ch r om en e-3-ca r boxylic Acid (83). 4-Methoxy acid 22 was
synthesized as described for the synthesis of 19. A solution of
22 (192 mg, 0.499 mmol) in THF (2.5 mL) was added dropwise
1
yellow oil. H NMR (CDCl3): δ 1.00 (3H, t, J ) 7.4 Hz), 1.30
(6H, d, J ) 6.4 Hz), 1.52 (2H, m), 1.84 (2H, m), 3.76 (3H, s),

Endothelin-A Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2051
to n-butylmagnesium chloride (2.0 M solution in ether, 1.3 mL,
2.60 mmol) at -35 °C. After it was stirred for 1 h at -35 °C
and at 0 °C for 2 h, the mixture was poured into acetic acid
(0.15 mL, 2.64 mmol) in ice-water and extracted with ethyl
acetate. The product was purified by column chromatography
on silica gel (ethyl acetate/hexane, gradient from 1/4 to 1/2) to
with ether and purification by silica gel chromatography
(toluene/ethyl acetate, 9/1), the product was recrystallized from
diisopropyl ether/acetone to give 97 (480 mg, 78%) as yellow
1
crystals; mp 94-95 °C. H NMR: δ 1.05 (3H, d, J ) 6.0 Hz),
1.19 (3H, d, J ) 6.0 Hz), 3.75 (3H, s), 4.14-4.23 (1H, m), 5.89
(2H, s), 6.32 (1H, s), 6.69 (2H, d, J ) 8.1 Hz), 6.80-6.88 (6H,
m), 6.98 (2H, d, J ) 9.0 Hz), 10.09 (1H, s). Anal. (C27H24O7) C,
H.
1
give 83 (161 mg, 79%) as yellow crystals; mp 140-141 °C. H
NMR (CDCl3): δ 0.98 (3H, t, J ) 6.8 Hz), 1.30 (3H, d, J ) 6.0
Hz), 1.31 (3H, d, J ) 6.0 Hz), 1.38-1.74 (4H, m), 3.08 (2H,
m), 4.40 (1H, m), 5.89 (2H, s), 6.13 (1H, s), 6.66 (1H, d, J )
7.8 Hz), 6.73-6.85 (3H, m), 6.74 (1H, d, J ) 7.8 Hz), 6.96 (1H,
d, J ) 2.2 Hz). Anal. (C24H26O6) C, H.
2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-4-(4-m et h oxy-
p h en oxy)-2H-ch r om en e-3-ca r boxylic Acid (98). A solution
of sodium chlorite (80%, 130 mg, 1.15 mmol) in water (1 mL)
was added to a mixture of aldehyde 97 (100 mg, 0.217 mmol)
in dimethyl sulfoxide (DMSO, 7 mL) and NaH2PO4‚2H2O (64
mg, 0.412 mmol) in water (1 mL) at room temperature. After
it was stirred for 20 h, the reaction mixture was diluted with
water, extracted with ethyl acetate, and recrystallized from
ethyl acetate/hexane to afford 98 (148 mg, 82%) as yellow
2-(Be n zo[1,3]d ioxol-5-yl)-6-isop r op oxych r om a n -4-
on e (93). A solution of 5a (219 g, 1.13 mol), piperonal (170 g,
1.13 mol), and 2 M NaOH (1.13 L) in methanol (570 mL) was
stirred at room temperature for 3 days. The resulting precipi-
tate was collected to give 93 as yellow crystals (336 g, 91%);
mp 125-126 °C. 1H NMR (CDCl3): δ 1.33 (6H, d, J ) 6.1 Hz),
2.76-3.11 (2H, m), 4.44-4.62 (1H, m), 5.34 (1H, dd, J ) 3.2
and 13.0 Hz), 5.99 (2H, s), 6.81-7.36 (6H, m).
1
crystals; mp 188-190 °C. H NMR (CDCl3 + CD3OD): δ 1.06
(3H, d, J ) 6.0 Hz), 1.19 (3H, d, J ) 6.0 Hz), 3.75 (3H, s),
4.11-4.29 (1H, m), 5.92 (2H, s), 6.30 (1H, s), 6.69-6.97 (10H,
m). Anal. (C27H24O8) C, H.
2-(Ben zo[1,3]d ioxol-5-yl)-4-ch lor o-6-isop r op oxy-2H -
ch r om en e-3-ca r ba ld eh yd e (94). Phosphoryl oxychloride
(6.09 g, 39.7 mmol) was added to DMF (12 mL) at 0 °C. After
30 min, a solution of 93 (5.19 g, 15.9 mmol) in DMF (20 mL)
was added dropwise thereto at 0 °C, and then, the mixture
was stirred at room temperature for 2 days. The reaction
mixture was poured into ice-water and extracted with ethyl
acetate. The product was purified by silica gel chromatography
(toluene) followed by recrystallization (diisopropyl ether/
acetone) to afford 94 (4.83 g, 82%) as yellow crystals; mp 126-
Meth yl 2-(Ben zo[1,3]dioxol-5-yl)-4-n -bu tyl-6-isopr opoxy-
2H-ch r om en e-3-ca r boxyla te (99). Oxalyl chloride (78 µL,
0.877 mmol) was added dropwise to a solution of 83 (300 mg,
0.731 mmol) and DMF (15 µL) in CH2Cl2 (2.5 mL) at room
temperature. After it was stirred at room temperature for 20
min, methanol (2 mL) was added. After 5 min, water was
added and the mixture was extracted with ethyl acetate. Usual
workup followed by column chromatography on silica gel (ethyl
acetate/hexane, 1/5) gave 99 (190 mg, 83%) as a yellow oil. 1H
NMR (CDCl3): δ 0.99 (3H, t, J ) 7.4 Hz), 1.30 (3H, d, J ) 6.0
Hz), 1.31 (3H, t, J ) 6.0 Hz), 1.43-1.69 (4H, m), 2.96-3.08
(2H, m), 3.72 (3H, s), 4.26-4.44 (1H, m), 5.90 (2H, s), 6.11 (1H,
s), 6.65-6.82 (5H, m), 6.95 (1H, d, J ) 2.4 Hz). Anal. (C25H28O6‚
0.15H2O) C, H.
1
127 °C. H NMR (CDCl3): δ 1.33 (6H, d, J ) 6.0 Hz), 4.38-
4.57 (1H, m), 5.90 (2H, s), 6.23 (1H, s), 6.60-7.26 (6H, m),
10.26 (1H, s).
2-(Ben zo[1,3]d ioxol-5-yl)-4-ch lor o-6-isop r op oxy-2H -
ch r om en e-3-ca r boxylic Acid (95). A solution of 94 (92.0 g,
247 mmol) in toluene (1.38 L) was added to NH2SO3H (57.6 g,
593 mmol) in water (346 mL). After the mixture was cooled to
0 °C, a solution of sodium chlorite (80%, 56.0 g, 495 mmol) in
water (346 mL) was added dropwise, and the mixture was
stirred for 20 min. Aqueous solution of sodium sulfite (31.0 g
in 100 mL water, 246 mmol) and aqueous NaOH (40.0 g in
700 mL water) were successively added to the reaction
mixture. The aqueous layer was separated and acidified with
37% HCl. The resulting precipitate was collected by filtration
to give 95 (87.5 g, 91%) as yellow crystals; mp 177 °C. 1H NMR
(CDCl3) δ: 1.31 (6H, d, J ) 6.0 Hz), 4.45 (1H, m), 5.90 (2H, s),
6.24 (1H, s), 6.66-6.85 (5H, m), 7.23-7.26 (1H, m). Anal.
(C20H17ClO6) C, H, Cl.
2-(Ben zo[1,3]d ioxol-5-yl)-4-n -b u t yl-6-isop r op oxy-2H -
ch r om en e-3-ca r boxa m id e (100). Oxalyl chloride (78 µL,
0.877 mmol) was added dropwise to a solution of 83 (300 mg,
0.731 mmol) and DMF (15 µL) in CH2Cl2 (3 mL) at room
temperature. After it was stirred at room temperature for 1
h, the mixture was mixed with 28% NH4OH (3 mL) and then
extracted with ethyl acetate. Usual workup and column
chromatography on silica gel (chloroform/acetonitrile, 95/5)
followed by recrystallization from isopropyl ether/acetone gave
1
100 (231 mg, 77%) as colorless crystals; mp 115-116 °C. H
NMR (CDCl3): δ 0.96 (3H, d, J ) 7.2 Hz), 1.31 (3H, d, J ) 6.3
Hz), 1.32 (3H, d, J ) 6.3 Hz), 1.42-1.63 (4H, m), 2.70-2.88
(2H, m), 4.35-4.49 (1H, m), 5.40 (2H, br), 5.83 (1H, s), 5.92
(2H, s), 6.66-6.73 (3H, m), 6.86-6.88 (3H, m). Anal. (C24H27
NO5) C, H, N.
-
2-(Ben zo[1,3]d ioxol-5-yl)-4-bu tylsu lfa n yl-6-isop r op oxy-
2H-ch r om en e-3-ca r boxylic Acid (96). A solution of 1-bu-
tanethiol (108 mg, 1.20 mmol) in THF (1 mL) was added to
sodium hydride (60%, 84 mg, 2.10 mmol) in THF (1 mL) at
room temperature. The mixture was stirred for 30 min, and
then, a solution of 95 (382 mg, 0.982 mmol) in THF (6 mL)
was added at 0 °C. After it was stirred for 30 min at 0 °C, the
reaction was quenched by addition of acetic acid (0.2 mL) and
the mixture was extracted with ethyl acetate. The usual
workup and purification by silica gel column chromatography
(ethyl acetate/hexane, gradient from 1/9 to 1/4), followed by
recrystallization from 2-propanol/hexane afforded 96 (33 mg,
2-(Ben zo[1,3]d ioxol-5-yl)-4-n -b u t yl-6-isop r op oxy-2H -
ch r om en e-3-tetr a zole (102). A mixture of 100 (100 mg, 0.244
mmol), sodium azide (238 mg, 3.66 mmol), tetrachlorosilane
(140 µL, 1.22 mmol), and acetonitrile (4 mL) was refluxed for
16 h. The reaction was quenched with 2 M Na2CO3, and the
mixture was extracted with ethyl acetate. Usual workup and
purification by column chromatography on silica gel (chloroform/
acetonitrile, 95/5) afforded 102 (65 mg, 61%) as a light brown
1
oil. H NMR (CDCl3): δ 0.96 (3H, t, J ) 7.3 Hz), 1.33 (3H, d,
J ) 6.0 Hz), 1.34 (3H, d, J ) 6.0 Hz), 1.42-1.55 (2H, m), 1.62-
1.73 (2H, m), 2.93-3.10(2H, m), 4.40-4.48 (1H, m), 5.90 (2H,
s), 6.15 (1H, s), 6.66-6.85 (5H, m), 6.98-6.99 (1H, m). Anal.
(C24H26N4O4‚0.3H2O) C, H, N.
1
15%) as yellow crystals; mp 138-142 °C. H NMR (CDCl3): δ
0.88 (3H, t, J ) 7.2 Hz), 1.17-1.69 (4H, m), 1.31 (3H, d, J )
6.0, Hz), 1.32 (3H, d, J ) 6.0 Hz), 2.83 (2H, t, J ) 7.4 Hz),
4.44 (1H, m), 5.89 (2H, s), 6.37 (1H, s), 6.64 (1H, d, J ) 6.0
Hz), 6.72 (1H, dd, J ) 0.6 and 1.6 Hz), 6.73-6.87 (3H, m),
7.27 (1H, m). Anal. (C24H26O6S) C, H, S.
N-[2-(Ben zo[1,3]d ioxol-5-yl)-4-n -b u t yl-6-isop r op oxy-
2H-ch r om en e-3-ca r bon yl]m eth a n esu lfon a m id e (103). A
solution of 83 (100 mg, 0.244 mmol), methanesulfonamide (25
mg, 0.268 mmol), 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide hydrochloride (51 mg, 0.268 mmol), and 4-(dimethyl-
amino)pyridine (33 mg, 0.268 mmol) in THF (2 mL) and CH2-
Cl2 (4 mL) was stirred for 16 h. Water was added, and the
mixture was extracted with ethyl acetate. The product was
purified by column chromatography on silica gel (ethyl acetate/
hexane, 1/1) to give 103 (65 mg, 55%) as a yellow oil. 1H NMR
(CDCl3): δ 0.94 (3H, t, J ) 7.1 Hz), 1.30 (3H, d, J ) 6.0 Hz),
2-(Benzo[1,3]dioxol-5-yl)-6-isopropoxy-4-(4-methoxyphen-
oxy)-2H -ch r om en e-3-ca r b a ld eh yd e (97). A solution of
4-methoxyphenol (366 mg, 2.95 mmol) in THF (4 mL) was
added dropwise to sodium hydride (60%, 134 mg, 3.35 mmol)
in THF (2 mL) at room temperature. After it was stirred for 1
h, a solution of 94 (500 mg, 1.34 mmol) in THF (5 mL) was
added dropwise. The mixture was stirred for 6 h at room
temperature and then poured into ice-water. After extraction

2052 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ishizuka et al.
1.31 (3H, d, J ) 6.0 Hz), 1.40-1.50 (2H, m), 1.54-1.64 (2H,
m), 2.77-2.97 (2H, m), 3.04 (3H, s), 4.36-4.44 (1H, m), 5.87
(2H, s), 5.91 (1H, s), 6.65-6.71 (3H, m), 6.82-6.89 (3H, m).
Anal. (C25H29NO7S‚1.0H2O) C, H, N, S.
(R)-83. Acid (R)-83 was synthesized from (R)-22 in a manner
similar to that described for the synthesis of the racemate
23
(89%, 100% ee); mp 148-149 °C. [R]D +61.3 (c 1.01, MeOH).
1H NMR (CDCl3): δ 0.98 (3H, t, J ) 7.2 Hz), 1.30 (3H, d, J )
6.0 Hz), 1.31 (3H, d, J ) 6.0 Hz), 1.38-1.73 (4H, m), 2.92-
3.22 (2H, m), 4.40 (1H, m), 5.89 (2H, s), 6.13 (1H, s), 6.66 (1H,
d, J ) 7.8 Hz), 6.70-6.86 (4H, m), 6.96 (1H, d, J ) 2.2 Hz).
Anal. (C24H26O6) C, H. The enantiomeric purity was deter-
mined by HPLC: column, CHIRALCEL OJ -R (4.6 mm × 150
Met h yl 2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-4-(4-
m eth oxyp h en yl)-ch r om a n e-3-ca r boxyla te (105b). A mix-
ture of 35a (1.50 g, 3.15 mmol), palladium chloride (2.01 g,
11.34 mmol), methanol (30 mL), and CH2Cl2 (10 mL) was
stirred under 500 kPa of hydrogen pressure for 72 h. The
catalyst was removed by filtration, and the filtrate was
concentrated under reduced pressure. The precipitate was
triturated with isopropyl ether to give 105b (667 mg, 45%).
1H NMR (CDCl3): δ 1.21 (3H, d, J ) 6.2 Hz), 1.24 (3H, d, J )
6.2 Hz), 3.21 (3H, s), 3.27 (1H, dd, J ) 2.6 and 6.5 Hz), 3.80
(3H, s), 4.24-4.33 (1H, m), 4.63 (1H, d, J ) 6.5 Hz), 5.27 (1H,
d, J ) 2.6 Hz), 5.96 (2H, s), 6.47-6.48 (1H, m), 6.74-6.95 (7H,
m), 7.14 (2H, d, J ) 8.7 Hz).
2-(Ben zo[1,3]d ioxol-5-yl)-6-isop r op oxy-4-(4-m et h oxy-
p h en yl)-ch r om a n e-3-ca r boxylic Acid (107). Acid 107 (53%
yield) was synthesized from 105b in a manner similar to that
used for synthesis of 19. Colorless crystals; mp 272 °C (dec).
1H NMR (CDCl3 + CD3OD): 1.17 (3H, d, J ) 6.0 Hz), 1.22
(3H, d, J ) 6.3 Hz), 3.09 (1H, dd, J ) 9.9 and 11.4 Hz), 3.80
(3H, s), 4.26 (1H, m), 4.50 (1H, d, J ) 11.4 Hz), 5.05 (1H, d, J
) 9.9 Hz), 5.96 (2H, s), 6.30 (1H, br), 6.70-7.00 (7H, m), 7.13
(2H, d, J ) 8.4 Hz). Anal. (C27H26O7‚0.1H2O) C, H.
mm, DAICEL); solvent, acetonitrile/0.5 M NaClO4-HClO4
)
60/40; flow rate, 1.0 mL/min; detection, 286 nm; tR ) 10.1 min.
Sin gle-Cr ysta l X-r a y An a lysis of (1S,2R)-(+)-Nor ep h e-
d r in e Sa lt of (R)-48. Colorless prismatic crystals of the salt
36H37O8 were grown from methanol solution. A single crystal
C
with approximate dimensions 0.20 mm × 0.30 mm × 0.35 mm
was picked up and used for the X-ray data collection. X-ray
diffraction measurements were performed at 295 K on a
Rigaku AFC7R diffractometer using a graphite-monochro-
mated Cu KR radiation (λ ) 1.541 78 Å) and a rotating anode
generator. Cell constants were obtained by least-squares
refinement using the setting angles of 25 carefully centered
reflections in the range of 45° < 2θ < 50°. Crystal data are as
follows: space group P212121, a ) 20.432(1) Å, b ) 23.575(1)
Å, c ) 6.628(5) Å, V ) 3192.7(5) Å3, Z ) 4, Dcal ) 1.272 g/cm3.
The 3491 reflections were collected (2θ × 140.2°) ω/2θ mode
with ω scan width is (1.73 + 0.30tan θ)° and ω scan speed is
16.0°/min. The linear absorption coefficient, µ, for Cu KR
radiation is 7.37 cm-1. The data were corrected for Lorentz
and polarization effects. The structure was solved by direct
methods66 and expanded using Fourier techniques. The non-
hydrogen atoms were refined anisotropically. Hydrogen atoms
in the OH group were refined isotropically, while the rest were
included in fixed positions. The final cycle of full-matrix least-
squares refinement was based on 3461 observed reflections [I
> 0.00σ(I)] and 447 variable parameters. Final R and weighted
R values were 0.087 and 0.139, respectively. The maximum
and minimum peaks on the final difference Fourier map
corresponded to 0.40 and -0.26 e/Å3, respectively. All calcula-
tions were performed using the teXsan67 crystallographic
software package of Molecular Structure Corporation. Atom
coordinates, thermal parameters, bond lengths, and angles are
available as Supporting Information.
P r ep a r a tion of Ch ir a l Com p ou n d s (R)-48 a n d (S)-48.
A solution of 48 (20.0 g, 43.4 mmol) and (1S,2R)-(+)-norephe-
drine (6.56 g, 43.4 mmol) in acetonitrile (40 mL) was allowed
to stand overnight at room temperature. The precipitate was
recrystallized successively from 2-propanol and ethanol to give
the salt (7.8 g, 30%, 99% ee). A solution of the salt in water
was treated with 1 M HCl (12.8 mL) and extracted with ethyl
acetate. The acid was recrystallized from 2-propanol to give
(R)-48 (5.03 g, 25%, 100% ee) as pale yellow crystals; mp 170-
171 °C. [R]D24 +178.8 (c 1.00, CH3OH). 1H NMR(CDCl3): δ 1.15
(3H, d, J ) 5.8 Hz), 1.18 (3H, d, J ) 5.8 Hz), 3.88 (3H, s), 4.19
(1H, m), 5.91 (2H, s), 6.16 (1H, s), 6.25 (1H, m), 6.69-6.74 (3H,
m), 6.90-6.97 (4H, m), 7.16-7.19 (2H, m). Anal. (C27H24O7)
C, H. The enantiomeric purity of (R)-48 was determined by
HPLC: column, CHIRALCEL OJ -R (4.6 mm × 150 mm,
DAICEL); solvent, acetonitrile/H2O/trifluoroaceti acid ) 68/
31/0.1; flow rate, 0.5 mL/min, detection, 286 nm: tR ) 6.1 min.
In a similar manner, (S)-48 was obtained via the (1R,2S)-
Ra t ETA Recep tor s Bin d in g Assa y. The rat aorta smooth
muscular cells were cultured on 48 well culture plates. After
3-5 days, the cluster medium was aspirated, and the cells
were washed twice with ice-cold HEPES-buffered Hank’s
solution (20 mM, pH 7.4). The cells in each well were incubated
with 12.5 pM [125I]ET-1 in HEPES-buffered Hank’s solution
(0.3 mL) containing PMSF (0.1 mM), protinin (10 µg/mL),
leupeptin (10 µg/mL), pepstatin A (10 µg/mL), bacitracin (250
µg/mL), and soybean trypsin inhibitor (10 µg/mL) in the
absence or presence of a test compound at varying concentra-
tions. Equilibrium binding studies were performed at 37 °C
for 60 min. The incubation was terminated by rapid removal
of the incubation medium and addition of ice-cold HEPES-
buffered Hank’s solution (0.25 mL). The free ligand was
removed by washing the intact attached cells two times with
ice-cold HEPES-buffered Hank’s solution. The cells were
dissolved in 0.1 M NaOH and transferred to a test tube, and
then the radioactivity was counted. The nonspecific binding
was determined in the presence of 10-7 M ET-1 and was about
5-10% of the total binding.
24
(-)-norephedrine salt (28%, 100% ee); mp 170-171 °C. [R]D
-177.3 (c 1.00, CH3OH). Anal. (C27H24O7) C, H. HPLC: tR
)
12.4 min; 100%ee.
(R)-22. Acid (R)-22 was obtained in a manner similar to that
used for preparation of (R)-48, except that acetonitrile was
24
replaced by methanol (37%, 99.7% ee); mp 136-137 °C. [R]D
1
+42.6 (c 1.00, CH3OH). H NMR (CDCl3): δ 1.30 (3H, d, J )
6.0 Hz), 1.31 (3H, d, J ) 6.0 Hz), 4.01 (3H, s), 4.43(1H, m),
5.89 (2H, s), 6.25 (1H, s), 6.65-6.92 (6H, m). Anal. (C21H20O7)
C, H. The enantiomeric purity was determined by HPLC:
column, CHIRALCEL OJ -R (4.6 mm × 150 mm, DAICEL);
solvent, acetonitrile/0.2 M H3PO4-KH2PO4 ) 50/50; flow rate,
0.5 mL/min; detection, 286 nm; tR ) 10.1 min.
(R)-82 a n d (S)-82. Acid 82 (800 mg, 2.26 mmol) was treated
with (R)-(+)-phenylethylamine (274 mg, 2.26 mmol) in metha-
nol. The precipitate was collected and recrystallized repeatedly
from methanol to give the salt (99.5% ee). Acid hydrolysis and
recrystallization from ethanol gave (S)-82 (200 mg, 25%,
98%ee) as yellow needles; mp 151.5-152 °C. [R]D28 +6.6 (c 1.01,
CH3OH). 1H NMR (CDCl3): δ 1.30 (6H, d, J ) 6.3 Hz), 4.40
(1H, m), 5.90 (2H, s), 6.10 (1H, s), 6.68-6.85 (6H, m), 7.71 (1H,
s). Anal. (C20H18O6) C, H. The enantiomeric purity was
determined by HPLC: column, CHIRALCEL OJ -R (4.6 mm
× 150 mm, DAICEL); solvent, acetonitrile/H2O/trifluoroaceti
acid ) 70/30/0.1; flow rate, 0.5 mL/min; detection, 286 nm; tR
) 5.5 min.
P ig ETB Recep tor s Bin d in g Assa y. COS cells translated
with cDNA encoding porcine ETB type receptor (90 µM, 10-3
-
10-4 cells in Hank’s solution containing 20 mM HEPES buffer
and protease inhibitors) was incubated with [125I]ET-3 (25 pM,
10 µL) and a test solution (1 µL) at various concentrations at
37 °C for 60 min. The incubation was terminated by filtering
through a Whatman GF/C glass fiber filter (presoaked in 1%
polyethylene imine), which was washed four times with Tris-
HCl (2.5 mL, 50 mM, pH 7.4), and the radioactivity was
counted. Specific binding was calculated by subtracting the
nonspecific binding obtained in the presence of 0.1 µM ET-3.
In a similar manner, (R)-82 was obtained via the (S)-(-)-
28
phenylethylamine salt (23%, 99% ee); mp 151-152 °C. [R]D
-7.3 (c 1.01, CH3OH). Anal. (C20H18O6) C, H. HPLC: tR ) 7.1
min.

Endothelin-A Receptor Antagonists
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Hu m a n ETA Recep tor s Bin d in g Assa y. Membranes of
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[
125I]ET-3 was replaced by [125I]-ET1.
Hu m a n ETB Recep tor s Bin d in g Assa y. Membranes of
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P h a r m a cok in etic Stu d ies in Ra ts. On the 2 days before
the experiment, the right jugular vein was cannulated with
polyethylene tubing for venous blood collection under light
anesthesia with ethyl ether. Antagonist was dissolved in a
mixture of 10% v/v N,N-dimethylacetamide, 50% v/v poly-
(ethylene glycol) 400, and 40% v/v saline. The solution was
administrated intravenously to nonfasted rats at a dose of 5
mg/2 mL/kg. Venous blood was collected at certain intervals
after dosing.
Antagonist suspended in 0.5% hydroxypropyl cellulose
(HPC-SL) solution was administrated orally to nonfasted rats
at a dose of 30 mg/2 mL/kg. Venous blood was collected at
certain intervals after dosing.
The concentration of antagonist in plasma was analyzed by
HPLC method. Briefly, 100 µL of plasma was mixed with 200
µL of acetonitrile, following by shaking and centrifuging. The
supernatant was analyzed.
The maximum plasma concentration (Cmax) was defined as
the highest value actually recorded, and the time to reach Cmax
was measured accordingly. The area under the curve was
calculated by the trapezoidal rule. Pharamcokinetic param-
eters were calculated using the nonlinear least-squares regres-
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Ack n ow led gm en t. The authors are grateful to Dr.
T. Konoike and K. Oda, Manufacturing Technology R
& D Laboratories, and T. Yorifuji, S. Shinomoto, Y. Ide,
T. Oya, M. Nishiuchi, and H. Kinoshita, Discovery
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1
Su p p or tin g In for m a tion Ava ila ble: Melting points, H
NMR spectra, and elemental analysis for the assayed com-
pounds, X-ray crystallographic data for the (1S,2R)-(+)-
norephedrine salt of (R)-48, and CD spectra of (R)-48, (R)-82,
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