Selective endothelin a receptor antagonists. 4. Discovery and structure- activity relationships of stilbene acid and alcohol derivatives

Article abstract of DOI:10.1021/jm970847e

This publication describes the synthesis and optimization of a novel series of stilbene endothelin antagonists. Analysis of the SAR established for previous papers in this series prompted the design and synthesis of (Z)- 4-phenyl-5-(3-benzyloxyphenyl)pent-4-enoic acid 3 which was found to be a moderately active inhibitor of the binding of [¹²⁵I]ET-1 to ET(A) receptors with an IC₅₀ of 6 μM. More interestingly, the intermediate compound (E)-2-phenyl-3-(3-benzyloxyphenyl)propenoic acid 5 was equiactive with 3. Optimization of 5 resulted in the preparation of (E)2-phenyl-3-(2- cyano-5-(thien-3-ylmethoxy))phenylpropenoic acid 18 (RPR111723) which had an IC₅₀ in the binding assay of 80 nM on the ET(A) receptor and a pK(B) of 6.5 in the functional assay, measured on rat aortic strips. Reduction of the acid group of 5 gave the first nonacidic ET(A) antagonist in our series, (E)-2- phenyl-3-(3-benzyloxyphenoxy)prop2-enol 6 with an IC₅₀ of 20 μM. Optimization of 6 resulted in the preparation of 2-(2-methylphenyl)-3-(2- cyano-5-(thien3-ylmethyl)phenyl)prop-2-enol 33 with an IC₅₀ of 300 nM on the ET(A) receptor.

Full text of DOI:10.1021/jm970847e

J . Med. Chem. 1998, 41, 2745-2753
2745
Selective En d oth elin A Recep tor An ta gon ists. 4. Discover y a n d
Str u ctu r e-Activity Rela tion sh ip s of Stilben e Acid a n d Alcoh ol Der iva tives1-3
Peter C. Astles, Thomas J . Brown, Frank Halley,* Caroline M. Handscombe, Neil V. Harris, Clive McCarthy,
Iain M. McLay, Peter Lockey, Tahir Majid, Barry Porter, Alan G. Roach, Christopher Smith, and Roger Walsh
Rhoˆne-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex, RM10 7XS U.K.
Received December 18, 1997
This publication describes the synthesis and optimization of a novel series of stilbene endothelin
antagonists. Analysis of the SAR established for previous papers in this series prompted the
design and synthesis of (Z)-4-phenyl-5-(3-benzyloxyphenyl)pent-4-enoic acid 3 which was found
to be a moderately active inhibitor of the binding of [125I]ET-1 to ETA receptors with an IC50 of
6 µM. More interestingly, the intermediate compound (E)-2-phenyl-3-(3-benzyloxyphenyl)-
propenoic acid 5 was equiactive with 3. Optimization of 5 resulted in the preparation of (E)-
2-phenyl-3-(2-cyano-5-(thien-3-ylmethoxy))phenylpropenoic acid 18 (RPR111723) which had an
IC50 in the binding assay of 80 nM on the ETA receptor and a pKB of 6.5 in the functional
assay, measured on rat aortic strips. Reduction of the acid group of 5 gave the first nonacidic
ETA antagonist in our series, (E)-2-phenyl-3-(3-benzyloxyphenoxy)prop2-enol 6 with an IC50 of
20 µM. Optimization of 6 resulted in the preparation of 2-(2-methylphenyl)-3-(2-cyano-5-(thien-
3-ylmethyl)phenyl)prop-2-enol 33 with an IC50 of 300 nM on the ETA receptor.
In tr od u ction
has been suggested that a selective ETA antagonist may
provide some advantage over a non selective agent.10
In the previous publications (parts 1 and 2 of the present
series),1,2 we described how the first lead compounds
were discovered from a 3D search of the RPR corporate
database using a pharmacophore query derived from the
known ETA antagonists BQ12314 and Shionogi 50-235.15
Optimization of these lead compounds led to the iden-
tification of a new series of nonpeptidic ETA selective
endothelin antagonists, which were described in part
33 and are exemplified by the butanoic acid derivative
1 (Chart 1).
The endothelins comprise a family of closely related
peptides (ET-1, ET-2, and ET-3) whose biological actions
are mediated in mammals by two receptor subtypes
designated ETA and ETB.4 Endothelin-1 (ET-1) is a 21
amino acid peptide with potent vasoconstrictor proper-
ties that was originally identified in conditioned medium
from cultured bovine endothelial cells.5 It has since
been found in different animal and human cells includ-
ing smooth muscle, macrophages, glial cells, and me-
sangial cells.6 Both ETA and ETB receptor subtypes are
also widely distributed in tissues.7 In the vasculature
ETA receptors were first identified on smooth muscle
and were recognized to mediate the ET-1-induced vaso-
constrictor actions whereas ETB receptors were found
on endothelial cells and were responsible for the ET-1
induced vasodilatation. Subsequently it was recognized
that ETB receptors were also distributed to smooth
muscle and were able to mediate contractions.8 The
relative importance of the ETA and ETB vasoconstrictor
receptors varies between tissue and species.9
Ch em istr y
The physical properties of all compounds for which
biological data were available are presented in Tables
1 and 2. The starting benzaldehydes 36, 37, and 38
were made by alkylation of the commercially available
3-hydroxybenzaldehyde and 2-nitro-5-hydroxybenzal-
dehyde, respectively (Scheme 1). Compound 39 was
prepared sequentially by bromination of the 3-hydroxy-
benzaldehyde in dichloromethane16 to give 35 followed
by alkylation, since bromination of the 3-aryloxyben-
zaldehyde (e.g., 37) in the same conditions was known
to give 35 with concomitant debenzylation.17 The cy-
anobenzaldehyde 40 was made by cyanation of the
bromo derivative 39 with copper cyanide in DMF
following a literature procedure for m-bromobenzalde-
hyde.18 Perkin condensation of the appropriate benzal-
dehyde with different arylacetic acids permitted varia-
tion of substitution on the R-phenyl group of the stilbene
derivatives 5, 9-21, 23, and 26 (Scheme 1). Generally
this reaction gave mainly the (E)-R-phenylcinnamic acid.
In the case of the condensation of benzaldehyde with
phenylacetic acid, some (Z)-R-phenylcinnamic acid 8 was
formed and isolated. In the case of the o-substituted
benzaldehydes (Br, NO2, and CN) the reaction was rapid
and only the (E)-R-phenylcinnamic acid was isolated.19
Endothelin has been implicated in the pathophysiol-
ogy of a large number of diseases. Significantly raised
levels of ET-1 have been detected in the plasma, and
other biological fluids, of patients suffering from conges-
tive heart failure, stable and unstable angina, acute
myocardial infarction, primary pulmonary hypertension,
acute renal insufficiency/failure, shock (septic and car-
diogenic), cerebral ischaemia/stroke (haemorrhagic and
nonhaemorrhagic), asthma, and metastatic prostate
cancer.10,11
The most relevant receptor to target for treatment of
these disease states remains controversial; however the
ETB receptor on endothelium may play a beneficial role
in certain disease states as it is responsible for the
enhanced production of PGI2 and NO induced by ET-
1.12,13 Since these later effects might be beneficial, it
S0022-2623(97)00847-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/18/1998

2746 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
Ch a r t 1
Ta ble 1. Binding Activity of the Stilbene Acids and Related Compounds
compd
R1
R2a
R3
R4
method mp (°C)
formula
analysis IC50 (µM)b
3
5
H
H
Ph
Ph
3-Th
H
H
H
H
CH2CH2CO2H
CO2H
CH2CH2CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
71-74
C24H22O3
175-178 C22H18O3
C, H
C, H
C, H, N
5.3, 4.3
6.0, 6.8
0.5, 0.7
0.6
0.9
0.6
A
7
CN
NO2 Ph
NO2 Ph
NO2 3-Th
NO2 3-Th
NO2 3-Th
NO2 3-Th
NH2 3-Th
111-112 C23H19NO3S‚0.15H2O
9c
A
A
A
A
A
A
Ae
A
A
A
A
A
A
B
221-223 C22H16NNaO5‚3H2O, NaOH C, H, N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
2-F
2-F
2-Cl
2-F, 6-Cl
H
2-F
H
2-CH3
H
3,4-OCH2O- CO2H
2-Cl
157-158 C22H16 FNO5
C, H, N
C, H, N
165-166 C20H14 FNO5S
176-178 C20H14ClNO5S‚1.25H2O
204-205 C20H14ClFNO5S‚1.25H2O
148-150 C20H15NO5S
C, H, Nd 1.3, 1.1
C, H, N
C, H, N
C, H, N
C, H
2
0.5
200-202 C20H16FNO5S
144-147 C20H15BrO3S
125-128 C21H17BrO3S
179-181 C21H15NO3S‚0.2H2O
181-183 C22H15NO5S
185-188 C21H14ClNO3S
155-157 C22H17NO3S‚0.2H2O
176-177 C24H17NO5
147-150 C22H17NO4S
180-182 C17H13NO3
IAf
Br
Br
3-Th
3-Th
3-Th
3-Th
3-Th
3-Th
3,4-OCH2OPh
3-Th
H
CO2H
CO2H
CO2H
0.7
3
C, H
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
0.08, 0.05
0.2, 0.2
0.2, 0.2
0.3, 0.3
1.3
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CONH2
2-CH3
H
3-OCH3
H
A
Ag
B
1.1
IAf
3-Py
3-Th
3-Th
H
226-229 C22H16N2O3‚0.45 CH2Cl2
188-190 C22H17NO4S
169-172 C21H16N2O2S
0.15-0.11
4-OCH3
H
A
IAf
3.1-2.9
a
b
3-Th ) thiophen-3-yl. Inhibition of Et-1 binding to ETA receptor on rat A10 cells (see Experimental Section). c Compound isolated
as the sodium salt. H: calcd 3.79 found 3.28. e Obtained by reduction of compound 11 (see Experimental Section). f IA ) inactive (i.e.,
inhibition <20% at 30 µM). Prepared from phenylacetic acid and compound 44 (Scheme 2).
d
g
The preparation of the stilbene alcohols 6, 28-33 was
achieved in one step by reduction of the corresponding
acid. One to two equivalents of diborane was found to
reduce selectively the acid group in the presence of a
nitro, a cyano, a double bond, and a benzyloxy group,
but a larger excess of reagent resulted in reduction of
the double bond. Diborane in THF is a very efficient
reagent when fresh, but borane-methyl sulfide complex
(BMS) was found to be a more reliable reagent.20 The
amine 15 was prepared by reducing the nitro 11 with
iron in ethanol following a literature procedure.21
Compound 34 was prepared by alkylation of 33 with
methyl iodide. Compound 27 was made from 18 in two
steps, using successively thionyl chloride and ammonia
in DMF (Scheme 1).
Variation of the arylmethyloxy group of the â-aryl was

Selective Endothelin A Receptor Antagonists. 4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2747
Ta ble 2. Binding Activity of the Stilbene Alcohols and Related Compound
compd
R1
R2a
R3
R4
OH
OH
OH
OH
OH
OH
OH
method
mp (°C)
formula
analysis
IC50 (µM)b
6
28
29
30
31
32
33
34
H
Ph
H
Cl
H
H
CH3
H
CH3
CH3
C
C
C
C
C
C
C
Cc
73-74
85-87
92-94
57-59
oil
104-107
99-102
84-86
C22H20O2‚0.2H2O
C20H16ClNO4S
20H17NO4S‚0.5H2O
20H17BrO2S
C20H17NO4S‚0.5H2O
21H17NO2S
C22H19NO2S
C23H21NO2S
C, H
20
0.3, 0.4
1.4
2.1, 2.0
0.6
0.8, 0.6
0.3, 0.3
IAd
NO2
NO2
Br
3-Th
3-Th
3-Th
3-Th
3-Th
3-Th
3-Th
C, H, N
C, H, N
C, H
C
C
Br
C, H
CN
CN
CN
C
C, H, N
C, H, N
C, H, N
OCH3
a
b
3-Th ) thiophen-3-yl. Inhibition of Et-1 binding to ETA receptor on rat A10 cells. c Obtained by methylation of compound 33 (see
d
Experimental Section). IA ) inactive (i.e. inhibition <20% at 30 µM).
Sch em e 1a
a
Reagents: (i) bromine, CH2Cl2, room temperature; (ii) ArCH2Br, NaH, DMF, room temperature; (iii) CuCN, DMF, reflux; (iv)
ArCH2COOH, AcOH, Ac2O, Et3N, reflux; (v) SOCl2, CH2Cl2, room temperature, then ammonia, DMF, room temperature; (vi) iron powder,
NH4Cl, EtOH, reflux; (vii) BMS, THF, room temperature; (viii) NaH, DMF, MeI, room temperature
achieved by an alternative method developed in Scheme
2, in which the cyano derivative was isolated as the
stable acetal 43, hydrolyzed, and condensed with phe-
nylacetic acid to give 24. Demethylation gave compound
45 which was in turn alkylated to provide 22 and 25.
The two chain extended acids 3 and 7 were both
prepared following similar methodologies (Scheme 3)
starting from the two alcohols 6 and 32, respectively.
The starting alcohols were transformed into the bromide
with CBr4 and PPh3 in dichloromethane, following a
literature procedure,22 and reacted with the sodium salt
of dimethyl malonate. The resulting diester was then
hydrolyzed to the bis acid which was in turn decarboxy-
lated by heating (170-190 °C) to give 3 and 7, respec-
tively.
simpler ether derivative 2 and was found to show a
3-fold loss in activity compared to the activity of 2.
Introduction of the cyano and the thiophene groups,
found highly beneficial features in compound 1, into
compound 3 gave compound 7 (Table 1). The resultant
improvement in activity was only a factor of 10, as
compared to 250 for the ether series (comparing com-
pounds 1 and 2). It should be noted that compound 1
also possessed an o-methyl group, which in our experi-
ence conferred a 2.5-fold increase in activity.3 However,
even after making allowance for this it seemed that the
more potent extended stilbene acids were intrinsically
less active than the ether series by more than a log unit.
The most likely reason for this was the lack of a
hydrogen bond acceptor which was not only present in
2 (ether linker)3 but also in the carbonyl linker series
as in 4.2
Resu lts a n d Discu ssion
Interestingly, during the synthesis of compound 3 we
identified two simple intermediates with unexpectedly
good activities: compounds 5 and 6.
This work was instigated as part of the exploration
of the SAR of the ether series of compounds reported
previously (e.g., 1).3 The objective was to investigate
replacement of the ether with the less labile and more
rigid olefinic linker. The first such compound synthe-
sized (compound 3) was the direct analogue of the
The structure-activity relationships for 5 were first
explored. The activity of this E-isomer 5 (IC50 ) 6 µM)
was compared with the Z-isomer 8 (inactive, i.e., inhibi-

2748 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
Sch em e 2a
a
Reagents: (i) bromine, CH2Cl2, room temperature; (ii) ethylene glycol, toluene, PTSA, reflux; (iii) CuCN, DMF, reflux; (iv) PTSA,
acetone, water, reflux; (v) phenylacetic acid, Et2N, acetic anhydride, reflux; (vi) pyridine hydrochloride, 160 °C; (vii) ArCH2Cl, K2CO3,
acetone, reflux.
Sch em e 3a
a
Reagents: (i) CBr4, PPh3, CH2Cl2, 10 °C; (ii) dimethyl malonate, NaOMe, MeOH, reflux; (iii) NaOH, H2O, EtOH, reflux; (iv) 170-190
°C, neat.
tion <20% at 30 µM) and found to be in full agreement
with the preferred “cisoid” bioactive conformation de-
fined by a receptor model developed and described
previously for the earlier nonstilbene members of this
series3 (e.g., see Figure 1). This model, which in
addition to defining the bioactive conformation also
identifies the position of a critical cationic receptor
group, has proved extremely valuable in the design of
potent analogues such as 1.3 It was used in this work
to compare these stilbene analogues with compounds
derived from earlier series.
can be seen (Figure 1) that 18 fits the receptor site
model very well with the acid-cationic group interaction
at a slightly shallower angle than for the more potent
derivative 1. A rapid study of this readily accessible
stilbene acid series showed that an electron-withdraw-
ing group (which can also act as a H-bond acceptor23
)
at the ortho position of the â-phenyl group was benefi-
cial for activity (Table 1). Indeed reduction of the nitro
group of 11 to the amine 15 abolished the activity
completely. The cyano derivatives showed better activ-
ity than the nitro derivatives (e.g., 18 and 14). This
may be due to steric repulsion in the bioactive confor-
mation between the olefinic proton and the nearby
oxygen of the nitro group. Such an interaction could
Interestingly, the screening of compound 18, which
was a synthetic intermediate for 7, gave a still more
active compound with an IC50 of 80 nM (Table 1). It

Selective Endothelin A Receptor Antagonists. 4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2749
It is noteworthy that the amide compound 27 also
retained some activity (IC50 ) 3 µM) but much less than
the alcohol 32.
Con clu sion
While developing a rigid analogue to the lead com-
pound 1 we identified a new series of small and easily
accessible endothelin antagonists. The most potent
compound in this series was the of (E)-2-phenyl-3-(2-
cyano-5-(thien-3-ylmethoxy))phenylpropenoic acid 18
(RPR111723) with an IC50 of 80 nM which also showed
good functional activity in the rat aorta with a pKB of
6.5 (see the Experimental Section). It is noteworthy
that RPR111723 is comparable in potency to the ETA
selective antagonist BMS-182874 (IC50 ) 150 nM, pKB
) 6.3).24 Compound 18 was selective for ETA receptor
and failed to inhibit binding of ET-1 to ETB receptors
at concentrations up to 30 µM. Although this stilbene
series was not developed further, the observation that
the simple stilbene 18 was 8-fold more active than the
chain extended analogue 7 suggested that a similar
relationship should exist for the more potent ether series
(e.g., 1) and preparation of such short chain compounds
will be the subject of a future publication.
F igu r e 1. Superposition of compounds 1 (in green) and 18
(in cyan) showing the acid groups pointing toward the putative
cationic center (in magenta).
Exp er im en ta l Section
Ch em ica l Meth od s. Melting points were taken on an
Electrothermal melting point apparatus and are uncorrected.
Infrared spectra were recorded in potassium bromide on a
Nicolet 205XB FI spectrometer. Proton NMR spectra were
recorded using a Varian VXR 400 (or a Varian XL 200 when
specified) spectrometer; peak positions are reported in parts
per million relative to internal tetramethylsilane on the δ
cause a rotation about the bond linking the nitroaryl to
the olefinic group, thus disfavoring the bioactive con-
formation.24 The presence of an arylmethyloxy group
on the â-phenyl ring was also important for activity.
Absence of the ring resulted in complete loss of activity
(e.g., the methoxy compound 24). Isosteric replacement
with a thiophene ring was acceptable, but replacement
with an electron rich aromatic group 22 or an electron
deficient aromatic group 25 caused a reduction in
activity. Substitution of the R-phenyl with an ortho
chloro or methyl group led to a 3-fold loss in activity
(compare 18 with 20 and 21). A methoxy group in the
meta position 23 was tolerated, whereas the para
methoxy 26 was inactive.
The compound 6 (IC50 ) 20 µM) was the first nona-
cidic compound with ETA binding activity we had
observed, with an activity only 3-fold lower than the acid
5. Clearly an explanation for this is that while the acid
group interacts with the cationic center through a strong
ionic interaction, the alcohol interaction is through the
acceptance of a weaker H-bonding interaction. However
methylation of the alcohol abolished the activity (com-
pound 34, Table 2) despite the ether, according to
literature values,23 being of similar strength to the
alcohol as a H-bond acceptor. The reason for this was
unclear, but it may be simply that in the conformation
required for receptor interaction the methyl group
infringes the steric constraints of the receptor site.
It is interesting to notice that the SAR seen for the
acid series correlated with the SAR obtained with the
alcohol series with the exception of the effect of the ortho
substituent on the R-phenyl ring. For the acid series
the ortho methyl group decreased activity by a factor
of about 2.5 (compare 18 and 21) while in the alcohol
series the effect is positive by a similar factor (compare
32 and 33), and in this the SAR of the alcohol series
was very similar to that of the earlier published series
related to 1.3
scale. Mass spectra were recorded on
a VG 7070E/250
spectrometer. Microanalyses were performed on a Carlo-Erba
1106 microanalyzer. Where analyses are indicated by the
symbols of the elements, results obtained were within 0.4% of
the theoretical values. All the reactions were performed at
room temperature unless otherwise stated. All organic solu-
tions were dried with magnesium sulfate. Yields are not
optimized.
(Z)-4-P h en yl-5-(3-ben zyloxy)p h en ylp en ten -4-oic Acid
3. Compound 48 (3.5 g, 87 mmol) was heated neat to 170 °C
for 20 min. The melt was then treated with charcoal in hot
cylohexane and filtered. Upon cooling a white solid precipi-
tated which was filtered to give 3 (2.7 g, 87%), having mp 71-
74 °C. 1H NMR (CDCl3) 2.46 (2H, t, J ) 8 Hz, CH2CH2CO2H),
2.84 (2H, t, J ) 8 Hz, CH2CH2CO2H), 4.68 (2H, s, CH2), 6.48
(1H, s, olefin-H), 6.51 (1H, d, J ) 2 Hz, ArH), 6.56 (1H, d, J )
8 Hz, ArH), 6.69 (1H, dt, J ) 2 Hz, J ) 8 Hz, ArH), 7.02 (1H,
t, J ) 8 Hz, ArH), 7.14-7.38 (10H, m, ArH); MS (FAB) m/z
359 [MH]+. Anal. (C24H22O3) C, H.
Meth od A. Gen er a l Meth od for th e P r ep a r a tion of
Com p ou n d s 5, 9-14, 16-21, 23, 24, a n d 26. This procedure
is illustrated for the preparation of (E)-2-phenyl-3-(3-benzyl-
oxy)phenylpropenoic acid 5. 3-Benzyloxybenzaldehyde (49 g,
230 mmol), phenylacetic acid (45.4 g, 340 mmol), acetic
anhydride (115 mL), and triethylamine (32 mL, 230 mmol)
were refluxed for 20 min. The solution was cooled to 90 °C,
water (115 mL) was added to decompose the excess anhydride,
and a solid precipitated which was filtered. Trituration of the
solid with diethyl ether gave a white solid which was filtered
and dried to give 5 (64 g, 85%), having mp 175-178 °C. 1H
NMR (CDCl3) 4.63 (2H, s, CH2), 6.52-7.41 (14H, m, ArH), 7.82
(1H, s, olefin-H); MS (FAB) m/z 331 [MH]+. Anal. (C22H18O3)
C, H.
(Z)-4-P h en yl-5-(2-cya n o-5-(th iop h en -3-ylm eth oxy)p h e-
n yl)p en ten -4-oic Acid 7. Compound 51 (0.85 g, 2 mmol) was
heated neat to 190 °C for 10 min. The melt was treated with
K2CO3 in water (50 mL) and washed with diethyl ether (15

2750 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
mL). The aqueous phase was acidified to pH 1 with concen-
trated aqueous HCl (1 mL), and the mixture was extracted
twice with ethyl acetate (30 mL). The gathered organic
extracts were washed with water, dried, and distilled under
reduced pressure. The resulting gum was triturated with hot
cyclohexane. Upon cooling a white solid precipitated which
was filtered to give 7 (0.47 g, 61%), having mp 111-112 °C.
1H NMR (CDCl3) 2.51 (2H, t, J ) 8 Hz, CH2CH2CO2H), 2.92
(2H, t, J ) 8 Hz, CH2CH2CO2H), 4.53 (2H, s, CH2), 6.38 (1H,
d, J ) 4 Hz, ArH), 6.62 (1H, dd, J ) 4 Hz, J ) 8 Hz, ArH),
6.75 (1H, s, olefin-H), 6.95 (1H, dd, J ) 2 Hz, J ) 4 Hz, ArH),
7.10-7.17 (3H, m, ArH), 7.24-7.36 (4H, m, ArH), 7.45 (1H, d,
the preparation of (Z)-2-phenyl-3-(2-cyano-5-(3-thiophen-3-yl)-
methyloxyphenyl)propenol 32. To a solution of 18 (2.55 g, 7
mmol) in THF (30 mL), under N2, at 0 °C, was added borane
in THF (11 mL, 11 mmol, 1.0 M solution in THF or 5.5 mL,
11 mmol, 2 M solution of BMS in THF), and the solution was
allowed to stir at room temperature for 2 h. The reaction
mixture was then cooled again to 0 °C and quenched sequen-
tially with water (25 mL) and sodium hydroxide (30 mL, 1 N).
The reaction mixture was stirred at room temperature for 30
min and concentrated in vacuo. The residue was dissolved in
ethyl acetate (50 mL), washed twice with water (50 mL), dried,
and evaporated to dryness. The gummy residue was triturated
with pentane to afford 32 (2 g, 83%) as a fawn-colored solid
having mp 104-107 °C. 1H NMR (CDCl3) 1.91 (1H, t, J ) 6
Hz, OH), 4.53 (2H, d, J ) 6 Hz, CH2OH), 4.56 (2H, s, CH2),
6.46 (1H, d, J ) 4 Hz, ArH), 6.75 (1H, dd, J ) 4 Hz, J ) 8 Hz,
ArH), 6.94-6.98 (2H, m, ArH), 7.10-7.47 (7H, m, ArH, olefin-
H), 7.48 (1H, d, J ) 8 Hz, ArH); MS (FAB) m/z 348 [MH]+.
Anal. (C21H17NO2S) C, H, N.
(Z)-2-(2-Met h ylp h en yl)-3-(2-cya n o-5-(t h iop h en -3-yl)-
m eth yloxyp h en yl)p r op en ol Meth yl Eth er 34. To a solu-
tion of 33 (1 g, 2.8 mmol), in DMF (20 mL), was added at room
temperature, under nitrogen, sodium hydride (0.12 g, 3 mmol,
60% dispersion in oil), and the mixture was stirred for 30 min.
Iodomethane (0.22 mL, 3.46 mmol) was added at once, and
the reaction mixture was stirred at room temperature for 3 h.
The solution was then poured into water (200 mL) and
extracted twice with ethyl acetate (50 mL). The gathered
organic phases were washed with water (50 mL) and brine
(25 mL), dried, and distilled under reduced pressure. The
residue was taken up into toluene and purified by flash column
chromatography (eluant, toluene) to give, after trituration with
pentane, 34 (0.22 g, 21%) as a white solid having mp 84-86
°C. 1H NMR (CDCl3) 2.27 (3H, s, CH3), 3.72 (3H, s, OCH3),
3.93 (2H, s, CH2), 5.00 (2H, s, CH2OCH3), 6.07 (1H, s, olefin-
H), 6.75 (1H, dd, J ) 4 Hz, J ) 8 Hz, ArH), 6.84 (1H, d, J )
4 Hz, ArH), 6.87 (1H, d, J ) 8 Hz, ArH), 6.98-7.15 (3H, m,
ArH), 7.25-7.36 (2H, m, ArH), 7.41 (1H, d, J ) 8 Hz, ArH);
MS (EI) m/z 375 [M]+. Anal. (C23H21NO2S‚0.5H2O) C, H, N.
2-Br om o-5-h yd r oxyben za ld eh yd e 35. A solution of bro-
mine (5 mL, 0.1 mol.) in dichloromethane (50 mL) was added
dropwise at room temperature to a solution of 3-hydroxyben-
zaldehyde in dichloromethane (400 mL). The red solution was
stirred for 3 h and the solvent distilled under reduced pressure.
The solid was dissolved into water (250 mL) with potassium
carbonate (2 equiv). The greenish suspension was filtered and
the solution cautiously acidified to pH 1 with concentrated
aqueous HCl. A solid separated which was filtered and
recrystallized from acetic acid to give 35 as a white solid (9 g,
45%) having mp 131-132 °C (lit.16 mp ) 133 °C). 1H NMR
(CDCl3) 6.99 (1H, dd, J ) 4 Hz, J ) 8 Hz, ArH), 7.38 (1H, d,
J ) 4 Hz, ArH), 7.44 (1H, d, J ) 8 Hz, ArH), 9.51 (1H, s, OH),
10.26 (1H, s, ArCHO).
3-Ben zyloxyben za ld eh yd e 36. To a solution of 3-hy-
droxybenzaldehyde (10 g, 82 mmol) in DMF (250 mL) was
added potassium tert-butoxide (9.2 g, 82 mmol) at room
temperature under N2. The mixture was stirred 15 min and
the benzyl bromide (14 g, 82 mmol) added at once. The
mixture was stirred for 3 h at room temperature and the
solvent distilled under reduced pressure. The residue was
partitioned between water and ethyl acetate, the phases were
separated, and the organics were concentrated. The residue
was purified by distillation (bp 180 °C, 0.1 mbar) to give 36
(14.8 g, 80%) as a colorless oil which solidified on standing
mp 52-54 °C (lit.26 mp 54 °C). 1H NMR (CDCl3) 5.13 (2H, s,
CH2) 7.23-7.51 (9H, m, ArH) 9.98 (1H, s, CHO).
J ) 8 Hz, ArH); MS (FAB) m/z 390 [MH]+ . Anal. (C23H19
NO3S‚0.15H2O) C, H, N.
-
(Z)-2-P h en yl3-(3-ben zyloxy)p h en ylp r op en oic Acid 8.
The filtrates of the preparation of compound 5 were concen-
trated and columned on silica gel (eluant CH2CL2/MeOH, 95/
5) to give compound 8 (1.5 g, 2%) as a gum. 1H NMR (CDCl3)
5.06 (2H, s, CH2), 6.99-7.51 (15H, m, ArH, olefin-H); MS (FAB)
m/z 331 [MH]+. Anal. (C22H18O3) C, H.
(E)-2-(2-F lu or op h en yl)-3-(2-a m in o-5-(3-th iop h en -3-yl)-
m eth yloxy)p r op en oic Acid 15. Compound 11 (2 g, 5 mmol),
iron powder (2 g, slight excess), and ammonium chloride (1.5
g, 28 mmol), in aqueous methanol (50 mL, 50/50), were
refluxed for 90 min. The mixture was filtered through Celite
and the filter washed with hot MeOH. The gathered filtrate
was basified and the compound extracted with ethyl acetate
to give 15 (0.7 g, 37%) as an off-white solid, having mp 200-
202 °C. 1H NMR (DMSO-d6) 4.40 (2H, s, CH2), 5.10 (2H, bs,
NH2), 6.08 (1H, d, J ) 4 Hz, ArH), 6.60-6.68 (2H, m, ArH),
6.92 (1H, dd, J ) 2 Hz, J ) 4 Hz, thiophen-H), 7.10-7.51 (6H,
m, ArH) 7.85 (1H, s, olefin-H). Anal. (C20H16FNO3S) C, H,
N.
Meth od B. Gen er a l Meth od for th e P r ep a r a tion of
Com p ou n d s 22 a n d 25. This procedure is illustrated for the
preparation of (E)-2-phenyl-3-(2-cyano-5-(pyridin-3-ylmeth-
oxy)phenyl)propenoic acid 25. Compound 45 (0.44 g, 1.7
mmol), 3-picolyl chloride hydrochloride (0.44 g, 1.7 mmol),
potassium carbonate (1.5 g, 10.9 mmol), potassium iodide (0.01
g, cat.) and tetrabutylammonium bromide (2 mg, cat.), in
methyl ethyl ketone (25 mL) was refluxed for 20 h. The
mixture was cooled to room temperature, and the precipitated
inorganics were filtered. The filtrate was evaporated under
reduced pressure and the residue taken up into ethanol (25
mL) and treated with 1 N sodium hydroxide solution (2 mL)
at reflux for 2 h. The solvent was distilled under reduced
pressure and the residue dissolved into water (75 mL), washed
with diethyl ether, and acidified with 1 N HCl to pH 5. A solid
was obtained which was filtered and recrystallized from
2-ethoxyethanol to give 25 as a white solid (0.21 g, 35%) having
mp 230-232 °C. 1H NMR (DMSO-d6) 4.72 (2H, s, CH2) 6.51
(1H, d, J ) 4 Hz, ArH), 7.06 (1H, dd, J ) 4 Hz, J ) 8 Hz,
ArH), 7.13-7.20 (2H, m, ArH), 7.32-7.44 (4H, m, ArH), 7.63-
7.70 (1H, m, ArH), 7.85 (1H, d, J ) 8 Hz, ArH), 7.79 (1H, s,
olefin-H) 8.47-8.58 (2H, m, ArH); MS (FAB) m/z 357 [MH]+.
Anal. (C22H16N2O3, 0.45 mol CH2Cl2) C, H, N.
(E )-2-P h e n yl-3-(2-cya n o-5-(3-t h iop h e n -3-yl)m e t h yl-
oxy)p h en ylp r op en a m id e 27. Compound 18 (1 g, 2.8 mmol)
in dichloromethane (20 mL) was treated with thionyl chloride
(0.4 mL) at reflux for 1 h. The solvent was distilled under
reduced pressure and the residue taken up in DMF (10 mL)
and treated with concentrated aqueous NH4OH (1 mL) for 1
h. The mixture was concentrated in vacuo and the residue
taken up in ethyl acetate and washed with water. Distillation
of the solvent gave an off white solid which was recrystallized
from 2-propanol to give 27 (0.26 g, 25%) as a white solid having
mp 169-172 °C. 1H NMR (DMSO-d6) 4.75 (2H, s, CH2), 6.44
(1H, d, J ) 4 Hz, ArH), 6.92-7.03 (2H, m, ArH), 7.10-7.18
(3H, m, ArH) 7.36-7.43 (3H, m, ArH), 7.51-7.57 (2H, m, ArH,
olefinic-H), 7.53 (1H, d, J ) 8 Hz, ArH); MS (FAB) m/z 361
[MH]+. Anal. (C21H16NO2S) C, H, N.
5-Ben zyloxy-2-n itr oben za ld eh yd e 37. Following the
same procedure as above, but replacing the 3-hydroxybenzal-
dehyde with 5-hydroxy-2-nitrobenzaldehyde, compound 37 was
obtained in 69% yield as a yellow oil, after purification by
filtration through silica (eluant, CH2Cl2). 1H NMR (CDCl3)
5.21 (2H, s, CH2), 7.21 (1H, dd, J ) 4 Hz, J ) 8 Hz, ArH),
7.32-7.45 (6H, m, ArH), 8.16 (1H, d, J ) 8 Hz, ArH), 10.52
(1H, s, CHO).
Meth od C. Gen er a l Meth od for th e P r ep a r a tion of
Com p ou n d s 6 a n d 28-33. This procedure is illustrated for

Selective Endothelin A Receptor Antagonists. 4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2751
5-(3-Th ioph en -3-yl)-m eth yloxy-2-n itr oben zaldeh yde 38.
To a solution of 5-hydroxy-2-nitrobenzaldehyde (6 g, 36 mmol)
in DMF (150 mL) was added potassium tert-butoxide (4.3 g,
38 mmol) at room temperature under N2. The mixture was
stirred 15 min and the 3-(chloromethyl)-thiophene3 (5 g, 38
mmol) added dropwise over 3 min. The mixture was stirred
for 6 h at room temperature and the solvent distilled under
reduced pressure. The residue was partitioned between water
and ethyl acetate, the phases were separated, and the organics
were concentrated. The residue was purified by filtration
through silica (eluant CH2Cl2) to give 38 (3.6 g, 38%) as a
yellow oil. 1H NMR (CDCl3) 5.22 (2H, s, CH2), 7.14 (1H, dd, J
) 2 Hz, J ) 4 Hz, thiophen-H), 7.21 (1H, dd, J ) 4 Hz, J ) 8
Hz, ArH), 7.36-7.43 (3H, m, aromatic-H), 8.16 (1H, d, J ) 8
Hz, ArH), 10.48 (1H, s, CHO).
2-B r o m o -5-(3-t h io p h e n -3-y l-m e t h y lo x y )b e n za ld e -
h yd e 39. To a solution of 2-bromo-5-hydroxy-benzaldehyde
35 (12.5 g, 62 mmol) in DMF (150 mL) was added potassium
tert-butoxide (7.3 g, 65 mmol) at room temperature under N2.
The mixture was stirred 15 min and the 3-(chloromethyl)-
thiophene3 (8.7 g, 65 mmol) added dropwise over 3 min. The
mixture was stirred for 6 h at room temperature and left to
stand overnight. The solvent was distilled under reduced
pressure, the residue was partitioned between water and ethyl
acetate, the phases were separated, and the organics were
concentrated to give 39 (18 g, 100%) as a crude oil. 1H NMR
(CDCl3) 5.10 (2H, s, CH2), 7.09 (1H, dd, J ) 4 Hz, J ) 8 Hz,
ArH), 7.14 (1H, dd, J ) 2 Hz, J ) 4 Hz, thiophen-H), 7.36-
7.43 (2H, m, thiophen-H), 7.49 (1H, d, J ) 4 Hz, ArH), 7.53
(1H, d, J ) 8 Hz, ArH), 10.30 (1H, s, CHO).
2-Cyan o-5-(3-th ioph en -3-yl)m eth yloxyben zaldeh yde 40.
A stirred solution of compound 39 (5 g, 17 mmol) in DMF (100
mL) under N2 was treated with copper(I) cyanide (3 g, 34
mmol). The resulting mixture was refluxed for 90 min. The
cooled reaction mixture was added to water (500 mL) and
extracted twice with ethyl acetate (250 mL). The combined
organic extracts were washed twice with water (100 mL),
dried, filtered, and evaporated to dryness. The residual oil
was taken up into dichloromethane and purified by filtration
through silica. Distillation of the eluant gave compound 40
(2 g, 48%) as an amber oil which solidified on standing to give
a waxy solid. 1H NMR (CDCl3) 5.21 (2H, s, CH2), 7.14 (1H,
dd, J ) 2 Hz, J ) 4 Hz, thiophen-H), 7.28 (1H, dd, J ) 4 Hz,
J ) 8 Hz, ArH), 7.36-7.41 (2H, m, thiophen-H), 7.58 (1H, d,
J ) 4 Hz, ArH), 7.75 (1H, d, J ) 8 Hz, ArH), 10.33 (1H, s,
CHO).
mixture was then poured into aqueous ammonia (made from
concentrated aqueous ammonia, 200 mL, and water, 200 mL)
and extracted twice with ethyl acetate (200 mL). The organic
phase was in turn washed with ammonia until the blue
coloration faded. The organics were washed with water, dried,
and distilled under reduced pressure to give 43 as a brown oil
(40 g, 100% crude). 1H NMR (CDCl3) 3.88 (3H, s, CH3), 4.06-
4.24 (4H, m, OCH2CH2O), 5.86 (1H, s, CH), 6.94 (1H, dd, J )
4 Hz, J ) 8 Hz, ArH), 7.12 (1H, d, J ) 4 Hz, ArH), 7.62 (1H,
d, J ) 8 Hz, ArH).
2-Cya n o-5-m eth oxyben za ld eh yd e 44. A solution of 43
(10 g, 43 mmol) in aqueous acetic acid (50%, 80 mL) was heated
to 100 °C for 2 h. Water (100 mL) was then added and the
solution left to cool to 10 °C in ice. The solid formed was
isolated by filtration and purified by recrystallization from
toluene to give 44 (3.9 g, 55%) as a white solid having mp 105-
109 °C. 1H NMR (CDCl3) 3.94 (3H, s, CH3), 7.22 (1H, dd, J )
4 Hz, J ) 8 Hz, ArH), 7.51 (1H, d, J ) 4 Hz, ArH), 7.74 (1H,
d, J ) 8 Hz, ArH), 10.33 (1H, s, CHO).
(E)-2-P h en yl-3-(2-cya n o-5-h yd r oxy)p h en ylp r op en oic
Acid 45. A mixture of 24 (1.4 g, 5 mmol) and pyridine
hydrochloride (14 g, 10 equiv w/w) was heated at 160 °C for
17 h. The cooled reaction mixture was partitioned between
ethyl acetate (40 mL) and 1 N HCl (40 mL). The aqueous layer
was washed with ethyl acetate, and the gathered organic
extracts were washed with water and dried. Distillation of
the solvent gave a residue which was purified by column
chromatography (eluant CH2Cl2/MeOH, 19/1) to give 45 as a
waxy solid. 1H NMR (DMSO-d6) 6.26 (1H, d, J ) 4 Hz, ArH),
6.77 (1H, dd, J ) 4 Hz, J ) 8 Hz, ArH), 7.10-7.16 (2H, m,
ArH), 7.31-7.36 (3H, m, ArH), 7.13 (1H, d, J ) 8 Hz, ArH),
7.84 (1H, s, olefin-H); MS (FAB) m/z 366 [MH]+. Anal.
(C16H11NO3) C, H, N.
(E)-4-P h en yl-5-(3-b en zyloxy)p h en yl-1-b r om op r op -2-
en e 46. To a solution of 6 (11.75 g, 35 mmol) in dichlo-
romethane (275 mL), cooled to 10 °C, was added carbon
tetrabromide (15.4 g, 46 mmol) and triphenylphosphine (14.5
g, 55 mmol). The solution was stirred at that temperature
for 1 h and filtered through a pad of silica with dichlo-
romethane. Distillation of the filtrate gave 46 (13 g, 100%)
as a crude oil. 1H NMR (CDCl3) 4.38 (2H, s, CH2Br), 4.59 (2H,
s, CH2), 6.55 (1H, t, J ) 2 Hz, ArH), 6.53 (1H, d, J ) 8 Hz,
ArH), 6.75 (1H, dt, J ) 2 Hz, J ) 8 Hz, ArH), 6.78 (1H, s,
olefin-H), 7.05 (1H, t, J ) 8 Hz, ArH), 7.26-7.72 (10H, m,
ArH).
(Z)-Met h yl 4-P h en yl-5-(3-b en zyloxy)p h en yl-2-m et h -
oxyca r bon ylp en ten -4-oa te 47. To a solution of sodium
methoxide (2 g, 37 mmol) in anhydrous methanol (200 mL)
was added dimethyl malonate (5.1 g, 38.5 mmol) at room
temperature. After 15 min, compound 46 (13 g, 34 mmol) was
added in solution into methanol (75 mL). The reaction mixture
was refluxed for 1 h and the solvent distilled under reduced
pressure. The residue was then partitioned between ethyl
acetate (150 mL) and 1 N HCl (50 mL). The aqueous layer
was washed with ethyl acetate (50 mL), and the gathered
organic layers were washed in turn with water, dried, and
distilled under reduced pressure. Purification of the residue
with flash column chromatography (eluant pentane/ethyl
acetate, 3/1) gave 47 (10.8 g, 73%) as an amber oil. 1H NMR
(CDCl3) 3.10 (2H, d, J ) 8 Hz, CH2CH), 3.46 (1H, t, J ) 8 Hz,
CH2CH), 3.70 (6H, s, CH3), 4.68 (2H, s, CH2), 6.48 (2H, m, ArH,
olefin-H), 6.53 (1H, d, J ) 8 Hz, ArH), 6.75 (1H, dt, J ) 2 Hz,
J ) 8 Hz, ArH), 7.02 (1H, t, J ) 8 Hz, ArH), 7.13-7.48 (10H,
m, ArH).
(Z)-4-P h en yl-5-(3-ben zyloxy)p h en yl-2-ca r boxyp en ten -
4-oic Acid 48. A mixture of 47 (7 g, 16.3 mmol), sodium
hydroxide (2.25 g, 56 mmol), methanol (150 mL), and water
(100 mL) was heated at reflux for 3 h. The solvent was
distilled under reduced pressure and the residue dissolved into
water (100 mL) and acidified to pH 1 with concentrated
aqueous HCl. The precipitate was extracted twice with ethyl
acetate (100 mL), and the gathered organic extracts were
washed with water, dried, and distilled under reduced pres-
sure. The residue was triturated with cyclohexane to give 48
2-Br om o-5-m eth oxyben za ld eh yd e 41. To a solution of
3-methoxybenzaldehyde (334 g, 2.45 mol) in dichloromethane
(3 L), maintained at 0 °C, was added a solution of bromine
(393 g, 2.45mol) in dichloromethane (500 mL) dropwise over
2.5 h. After addition, the reaction mixture was allowed to
reach room temperature overnight. The solvent was then
distilled under reduced pressure and the residue recrystallized
from cyclohexane to give 41 as a brown solid (405 g, 76%)
having mp 73-76 °C (lit.27 mp 75-76 °C). 1H NMR (CDCl3)
3.85 (3H, s, CH3), 7.04 (1H, dd, J ) 4 Hz, J ) 8 Hz, ArH), 7.42
(1H, d, J ) 4 Hz, ArH), 7.53 (1H, d, J ) 8 Hz, ArH), 10.31
(1H, s, CHO).
2-(2-Br om o-5-m eth oxy-p h en yl)d ioxola n e 42. A mixture
of 41 (305 g, 1.4 mol), ethylene glycol (260 g, 4.2 mol), and
p-toluenesulfonic acid (5 g) in toluene (2.4 L) was refluxed with
a Dean-Stark for 3 h. The solvent was then distilled under
reduced pressure and the residue partitioned between ethyl
acetate and water. The aqueous layer was washed twice with
ethyl acetate, and the gathered organic phase was washed in
turn with water, dried over magnesium sulfate, and distilled
under reduced pressure to give 42 (375 g, 100% crude) as a
brown oil. 1H NMR (CDCl3) 3.81 (3H, s, CH3), 4.05-4.18 (4H,
m, OCH2CH2O), 6.03 (1H, s, CH), 6.79 (1H, dd, J ) 4 Hz, J )
8 Hz, ArH), 7.15 (1H, d, J ) 4 Hz, ArH), 7.43 (1H, d, J ) 8
Hz, ArH).
2-(2-Cya n o-5-m eth oxy-p h en yl)d ioxola n e 43. A mixture
of 42 (53 g, 0.2 mol) and cuprous cyanide (17.8 g, 0.2 mol) in
dimethylformamide (50 mL) was refluxed for 1 h. The cooled

2752 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Astles et al.
1
(3.5 g, 53%) as a white solid having mp 112-116 °C dec. H
mL), pepstatin A (10 µg/mL), leupeptin (10 µg/mL), and phenyl
methenyl sulfonyl fluoride (0.1 mM). After homogenization
using a glass/Teflon manual homogenizer, the samples were
centrifuged at 4 °C for 17 min at 1000g, and the resulting
supernatants were retained. This material was centrifuged
at 40000g for 35 min at 4 °C, the pellets were resuspended in
50 mM Tris buffer (pH 7.4), and the protein content was
determined. Aliquots of 100 µL were snap frozen using a
mixture of methanol and solid carbon dioxide and stored at
-20 °C until required. Samples were diluted as necessary
with 50 mM Tris buffer pH 7.4 containing 0.1%w/v bovine
serum albumin for use in the assay.
NMR (CDCl3) 3.13 (2H, d, J ) 8 Hz, CH2CH), 3.50 (1H, t, J )
8 Hz, CH2CH), 4.66 (2H, s, CH2), 6.48-6.55 (3H, m, ArH,
olefin-H), 6.69 (1H, dt, J ) 2 Hz, J ) 8 Hz, ArH), 6.98 (1H, t,
J ) 8 Hz, ArH), 7.12-7.37 (10H, m, ArH); MS (FAB) m/z 403
[MH]+. Anal. (C25H22O5, 0.3 C6H12) C, H.
(E)-4-P h en yl-5-(2-cyan o-5-(th ioph en -3-yl)ph en yl)-1-br o-
m op r op -2-en e 49. To a solution of 32 (2.65 g, 7 mmol), in
dichloromethane (75 mL), cooled to 10 °C, were added carbon
tetrabromide (3.3 g, 9.9 mmol) and triphenylphosphine (3 g,
11.3 mmol). The reaction mixture were stirred at this tem-
perature for 1 h and allowed to reach room temperature
overnight. The solution was then filtered on a pad of silica
(dichloromethane) and the solvent distilled under reduced
pressure to give 49 (2.8 g, 98%) as an amber oil. 1H NMR
(CDCl3) 4.38 (2H, s, CH2Br), 4.53 (2H, s, CH2), 6.48 (1H, d, J
) 2 Hz, ArH), 6.53 (1H, dd, J ) 2 Hz, J ) 8 Hz, ArH), 6.75-
7.50 (10H, m, olefin-H, ArH).
(Z)-Meth yl 4-P h en yl-5-(2-cya n o-5-(th iop h en -3-ylm eth -
oxy)p h en yl)-2-m eth oxyca r bon ylp en ten -4-oa te 50. To a
solution of sodium methoxide (0.48 g, 8.9 mmol) in anhydrous
methanol (32 mL) was added dimethyl malonate (1.3 g, 9.6
mmol) at room temperature. After 15 min, compound 49 (2.8
g, 6.8 mmol) was added in solution into methanol (20 mL).
The reaction mixture was refluxed for 5 h and the solvent
distilled under reduced pressure. The residue was then
partitioned between ethyl acetate (50 mL) and 1 N HCl (10
mL). The aqueous layer was washed with ethyl acetate (20
mL), and the gathered organic layers were washed in turn with
water, dried, and distilled under reduced pressure. Purifica-
tion of the residue with flash column chromatography (eluant
pentane/ethyl acetate, 3/1) gave 50 (1.5 g, 49%) as a pale yellow
gum. 1H NMR (CDCl3) 3.19 (2H, d, J ) 8 Hz, CH2CH), 3.47
(1H, t, J ) 8 Hz, CH2CH), 3.75 (6H, s, CH3), 4.51 (2H, s, CH2),
6.34 (1H, d, J ) 4 Hz, ArH), 6.71-6.77 (2H, m, olefin-H, ArH),
6.95 (1H, dd, J ) 2 Hz, J ) 4 Hz, ArH), 7.10-7.37 (7H, m,
ArH), 7.45 (1H, d, J ) 8 Hz, ArH).
Bin d in g Assa y. Binding assays were performed in Milli-
pore 0.22 µm 96 well Multiscreen plates and consisted of 20
pM [125I]ET-1, test compound or vehicle, and A10 cells or
cerebellum protein in a final volume of 250 µL. Nonspecific
binding was measured using 500 nM unlabeled ET-1. [125I]-
ET-1 was prepared in 50 mM Tris buffer pH 7.4 containing
0.1% w/v BSA, and test compounds were prepared in the same,
supplemented with dimethyl sulfoxide at 5% v/v final assay
concentration. Reactions were started by the addition of cells
or cerebellum protein and allowed to proceed for 2 h at 37 °C
before being terminated by vacuum filtration. The filters were
then washed and collected for γ-counting.
Da ta An a lysis. Data from binding assays was corrected
for nonspecific binding and expressed as a percentage of total
[
125I]ET-1 binding in the presence of vehicle. IC50 values were
obtained by curve fitting. In a minority of cases, duplicate
IC50 values were not obtained; the results of IC50 measure-
ments were confirmed by retesting a single concentration
selected to fall on the concentration response curve. Com-
pounds are referred to IA (inactive) when the inhibition is
inferior to 20% at 30 µM.
F u n ction a l Assa y. Male Sprague-Dawley rats were
sacrificed, the aorta removed, de-endothelialized and placed
in organ baths containing oxygenated Krebs at 37 °C. The
tissues were equilibrated under 2 g tension and exposed to
phenylephrine (30 nM). A maximum contractile response to
3 µM phenylephrine was then measured. Tissues were
incubated for 30 min with vehicle (DMSO) or antagonist in
the presence of protease inhibitors leupeptin (1 µM), thiorphan
(1 µM), bestatin (1 µM), bacitracin (750 units l-1), and captopril
(1 µM). A cumulative concentration response curve to ET-1
(0.01-1000 nM) in 0.1% BSA was generated. All results are
expressed as percentage of maximal PE response and pKB
values calculated.
(Z)-4-P h en yl-5-(2-cya n o-5-(th iop h en -3-ylm eth oxy)p h e-
n yl)-2-ca r boxyp en ten -4-oic Acid 51. A mixture of 50 (1.5
g, 3.3 mmol), sodium hydroxide (0.33 g, 8.3 mmol), methanol
(50 mL), and water (10 mL) was heated at reflux for 5 h. The
solvent was distilled under reduced pressure and the residue
dissolved into water (150 mL) and acidified to pH 1 with
concentrated aqueous HCl. The precipitate was extracted
twice with ethyl acetate (75 mL), and the gathered organic
extracts were washed with water, dried, and distilled under
reduced pressure. The residue was triturated with dichlo-
romethane and recrystallized from toluene/ethyl acetate to give
51 (0.7 g, 51%) as a white solid having mp 164-168 °C dec.
1H NMR (DMSO-d6) 3.02 (2H, d, J ) 8 Hz, CH2CH), 3.17 (1H,
t, J ) 8 Hz, CH2CH), 4.21 (2H, s, CH2), 6.35 (1H, d, J ) 4 Hz,
ArH), 6.68 (1H, s, olefin-H), 6.90 (1H, dd, J ) 4 Hz, J ) 8 Hz,
ArH), 7.01 (1H, dd, J ) 2 Hz, J ) 4 Hz, ArH), 7.12-7.18 (2H,
m, ArH), 7.29-7.43 (4H, m, ArH), 7.53 (1H, m, ArH), 7.64 (1H,
Molecu la r Mod elin g. All structures were initially created
using Concord 3D builder (distributed by Tripos Inc., 1699
Hanley Road, Suite 303, St. Louis, MO 63144). The remain-
ing modeling was carried out within Chem-X (developed and
distributed by Chemical Design Ltd., Roundway House, Crom-
well Park, Chipping Norton, Oxfordshire, OX7 5SR, UK).
Charges were first set using the Gasteiger method, and
conformational analysis and energy calculations were carried
out using the default Chem-X force field.
d, J ) 8 Hz, ArH); MS (FAB) m/z 434 [MH]+. Anal. (C24H19
NO5S‚1.25H2O) C, H, N.
-
P r ep a r a tion of ETA Recep tor s. A10 cells, a rat aortic
smooth muscle cell line (ATCC number CRL-1476), were grown
to confluence in Dulbecco’s modified Eagle’s medium contain-
ing 10% v/v foetal bovine serum. Two days after the final
medium change cells were scraped from the base of the flask
and centrifuged at 1000g for 10 min at 4 °C. The resulting
pellets were washed twice in 50 mM Hepes buffer pH 7.3
containing calcium chloride (1 mM) and magnesium chloride
(5 mM) and resuspended at a density of 140000 cells/mL in
50 mM Hepes buffer pH 7.3. Aliquots of 5 mL were snap
frozen using a mixture of methanol and solid carbon dioxide
and stored at -20 °C until required. For use in the binding
assay, cells were diluted to the required density with 50 mM
Hepes buffer pH 7.3.
Ack n ow led gm en t. The authors thank the following
people for their technical contributions to the work
described in this paper: Imtiaz Ahmed, J anet Archer,
Clare Howels, David Neighbor and Barry Wyman for
the synthesis; J effrey Philips, Andrea Stoppard, and
Melanie Wong for the binding data; Michael Podmore
and Mark Vine for spectroscopy; and Anne Stevens for
microanalysis.
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