Structure-activity relationships in a series of orally active γ- hydroxy butenolide endothelin antagonists

Article abstract of DOI:10.1021/jm9606507

The design of potent and selective non-peptide antagonists of endothelin-1 (ET-1) and its related isopeptides are important tools defining the role of ET in human diseases. In this report we will describe the detailed structure-activity relationship (SAR) studies that led to the discovery of a potent series of butenolide ET(A) selective antagonists. Starting from a micromolar screening hit, PD012527, use of Topliss decision tree analysis led to the discovery of the nanomolar ET(A) selective antagonist PD155080. Further structural modifications around the butenolide ring led directly to the subnanomolar ET(A) selective antagonist PD156707, IC₅₀'s = 0.3 (ET(A)) and 780 nM (ET(B)). This series of compounds exhibited functional activity exemplified by PD156707. This derivative inhibited the ET(A) receptor mediated release of arachidonic acid from rabbit renal artery vascular smooth muscle cells with an IC₅₀ = 1.1 nM and also inhibited the ET-1 induced contraction of rabbit femoral artery rings (ET(A) mediated) with a pA₂ = 7.6. PD156707 also displayed in vivo functional activity inhibiting the hemodynamic responses due to exogenous administration of ET-1 in rats in a dose dependent fashion. Evidence for the pH dependence of the open and closed tautomerization forms of PD156707 was demonstrated by an NMR study. X- ray crystallographic analysis of the closed butenolide form of PD156707 shows the benzylic group located on the same side of the butenolide ring as the γ- hydroxyl and the remaining two phenyl groups on the butenolide ring essentially orthogonal to the butenolide ring. Pharmacokinetic parameters for PD156707 in dogs are also presented.

Full text of DOI:10.1021/jm9606507

J . Med. Chem. 1997, 40, 1063-1074
1063
Str u ctu r e-Activity Rela tion sh ip s in a Ser ies of Or a lly Active γ-Hyd r oxy
Bu ten olid e En d oth elin An ta gon ists
William C. Patt,*,† J eremy J . Edmunds,† J oseph T. Repine,† Kent A. Berryman,† Billy R. Reisdorph,† Chet Lee,†
Mark S. Plummer,† Aurash Shahripour,† Stephen J . Haleen,‡ J oan A. Keiser,‡ Mike A. Flynn,‡
Kathleen M. Welch,‡ Elwood E. Reynolds,‡ Ron Rubin,§ Brian Tobias, Hussein Hallak,| and
Annette M. Doherty†
Departments of Medicinal Chemistry, Cardiovascular Therapeutics, Analytical Research, Pharmacokinetics and Drug
Metabolism, and Biomolecular Structure and Drug Design, Parke-Davis Pharmaceutical Research Division,
Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received September 17, 1996X
The design of potent and selective non-peptide antagonists of endothelin-1 (ET-1) and its related
isopeptides are important tools defining the role of ET in human diseases. In this report we
will describe the detailed structure-activity relationship (SAR) studies that led to the discovery
of a potent series of butenolide ETA selective antagonists. Starting from a micromolar screening
hit, PD012527, use of Topliss decision tree analysis led to the discovery of the nanomolar ETA
selective antagonist PD155080. Further structural modifications around the butenolide ring
led directly to the subnanomolar ETA selective antagonist PD156707, IC50’s ) 0.3 (ETA) and
780 nM (ETB). This series of compounds exhibited functional activity exemplified by PD156707.
This derivative inhibited the ETA receptor mediated release of arachidonic acid from rabbit
renal artery vascular smooth muscle cells with an IC50 ) 1.1 nM and also inhibited the ET-1
induced contraction of rabbit femoral artery rings (ETA mediated) with a pA2 ) 7.6. PD156707
also displayed in vivo functional activity inhibiting the hemodynamic responses due to exogenous
administration of ET-1 in rats in a dose dependent fashion. Evidence for the pH dependence
of the open and closed tautomerization forms of PD156707 was demonstrated by an NMR study.
X-ray crystallographic analysis of the closed butenolide form of PD156707 shows the benzylic
group located on the same side of the butenolide ring as the γ-hydroxyl and the remaining two
phenyl groups on the butenolide ring essentially orthogonal to the butenolide ring. Pharma-
cokinetic parameters for PD156707 in dogs are also presented.
In tr od u ction
“ring” portion of the peptide while the C-terminal
hexapeptide chain is identical in all three peptides. The
endothelins are closely related to a series of snake
venom toxins, the sarafotoxins. ET is a potent and
prolonged vasoconstrictor and mitogen in a variety of
cell types. In addition ET has potent renal, pulmonary,
and neuroendocrine actions and has been implicated in
a wide variety of human disease states including is-
chemia,5 cerebral vasospasm,6 stroke,7 renal failure,8
hypertension,9 heart failure,10 pulmonary hyperten-
sion,11 and restenosis.12
Since the 1985 discovery of endothelin (ET),1 as the
peptidic endothelial derived constricting factor, over
2500 papers have been published describing various
aspects of the biochemistry, pharmacology, and physiol-
ogy of ET.2-3 The biochemical pathway leading to the
production of ET has been elucidated. It is derived in
several steps from a 203 amino acid precursor known
as prepro-ET. Prepro-ET is cleaved at two sites by
dibasic pair specific endopeptidases to produce a 39
amino acid peptide known as big ET. Big ET is then
further cleaved by the ET converting enzyme (ECE),
giving rise to the mature peptide ET. The bovine and
mammalian ECE enzymes have recently been cloned
and identified. ECE is a zinc metalloproteinase, and
two different isoforms have been identified, ECE1a and
ECE1b. The two forms of the enzyme differ only in the
size of the N-terminus and in their location. ECE1a is
a cytosolic intracellular enzyme located almost exclu-
sively in the golgi and ECE1b is a membrane bound
extracellular enzyme.
ET exerts its physiological effects by acting upon
specific G protein coupled receptors. Two distinct ET
receptors, ETA and ETB, have been cloned and expressed
in mammalian species.13-16 There is some pharmaco-
logical evidence regarding the existence of ETB receptor
subtypes; however none have been cloned in mam-
malian species.17 A third ET receptor, ETC, has been
isolated and identified in Xenopus dermal melanophores
but has not been described in mammalian tissues.18
The two mammalian ET receptors are defined by their
affinity for the ET isoforms. The ETA receptor exhibits
an affinity order of ET-1 ) ET-2 . ET-3 and Sarafotoxin
6C (S6C). The ETB receptor shows essentially equal
binding affinity for all three ET isoforms and S6C. Both
receptors are widely distributed in both animal and
human tissues. The ETA and/or ETB receptors have
been shown to mediate vasoconstriction in animal
tissues depending upon the species and vascular bed
under study.19-28 Furthermore, the ETB receptor has
The human genome identified for ET was subse-
quently found to encode for three separate isoforms of
the peptide, known as ET-1, ET-2, and ET-3.4 These
isoforms contain several amino acid changes in the
† Department of Medicinal Chemistry.
‡ Department of Cardiovascular Therapeutics.
§ Department of Biomolecular Structure and Drug Design.
Department of Analytical Research.
| Department of Pharmacokinetics and Drug Metabolism.
X Abstract published in Advance ACS Abstracts, March 1, 1997.
S0022-2623(96)00650-4 CCC: $14.00 © 1997 American Chemical Society

1064 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Patt et al.
Sch em e 1a
a
(i) EtOH, NaOH, room temperature, 1.5 h; (ii) KCN, HOAc, reflux, 30 min; (iii) HCl/MeOH or TsOH‚H2O, reflux; (iv) R2PhCHO, Na,
MeOH, reflux; (v) HOAc, reflux; (vi) NaOH.
Ta ble 1. Intermediate Keto-Esters
also been shown to mediate vasodilatation via release
of endothelin-derived relaxing factor from endothelial
cells.
A large number of peptide and more recently non-
peptide ET antagonists have been described in the
literature. Early small peptide inhibitors included BQ-
5
R1
R2
molecular formula
12329,30 and FR 139317,31 both which are potent ETA
selective antagonists. The hexapeptide antagonists
PD14289332 and PD14506533 with a more balanced ETA/
ETB receptor affinity have also been described. Recently
an ETB selective peptidic antagonist has been described,
BQ-788.34 In addition several non-peptide non-selective
antagonists have appeared in the literature including
Ro 46-2005,35 Ro 47-0203 (Bosentan),36 SB 209670,37
CGS 27830,38 and L-749,329,39 as well as the more ETA
selective antagonists BMS 182874,40 PD156707,41
A-127722,42 TBC-12519,43 and LU127043.44
a
4-Cl
H
4-CH3
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
C18H15ClO5
C18H16O5
C19H18O5
C18H14Cl2O5
C20H20O6
C19H18O6
C18H18O4
C18H17ClO4
C19H20O4
C19H20O5
C18H16Cl2O4
C20H22O6
C20H20O7
C20H20O7
C20H20O7
C21H22O8
C19H16O7
C20H20O5
C19H18O5S
C21H22O6
C25H22O6
C20H18O7
C20H22O6
C20H22O6
C20H22O6
C21H24O7
C21H22O7
C19H18O6
C19H18O6
C19H20O5
b
c
d
e
f
g
h
i
3,4-Cl2
3-CH3,4-OCH3 3,4-OCH2O-
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
2,4-(OCH3)2
3,4-(OCH3)2
3,4,5-(OCH3)3 3,4-OCH2O-
3,4-OCH2O-
4-C2H5
3,4-OCH2O-
H
4-Cl
4-CH3
4-OCH3
3,4-Cl2
j
k
l
3,4-(OCH3)2
3-OCH3,-4,5-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
m
n
o
p
q
r
The discovery of small non-peptide receptor antago-
nists may represent a new group of therapeutic agents
to treat a variety of human diseases. In this paper we
will describe the full structure-activity relationships
of our previously disclosed non-peptide ET antagonist
PD 156707, 1, derived from a compound library screen-
ing lead PD 012527, 2.
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,5-(OCH3)2
2,4-(OCH3)2
2,5-(OCH3)2
3,4,5-(OCH3)3
s
4-SCH3
t
4-O-i-C3H7
4-OCH2Ph
4-CO2CH3
4-OCH3
4-OCH3
4-OCH3
u
v
w
x
y
z
Ch em istr y
Most of the compounds in this report have been
synthesized via the route shown in Scheme 1.45 The
first step involved an aldol condensation of an acetophe-
none with an aldehyde in ethanolic hydroxide, giving
the chalcones 3 in 50-98% yield. The chalcones were
then reacted with cyanide in hot ethoxyethanol contain-
ing acetic acid producing the â-cyano ketones 4 in 40-
90% yields. The cyano group was then hydrolyzed with
either methanolic HCl (saturated) or p-toluenesulfonic
acid hydrate (1 equiv) in methanol at reflux to afford
the keto esters 5a -d d in 30-90% yield (Table 1). The
next series of steps was initiated by reaction of the keto
ester 5 and an aldehyde with sodium methoxide in hot
methanol. One possible mechanism for this series of
reactions is depicted in Figure 1 and involves an aldol
condensation, an elimination, a double bond isomeriza-
tion, and an ester hydrolysis in one pot. Acidification
of the reaction medium with acetic acid afforded cy-
clization to give the readily purified γ-hydroxy buteno-
lides 1-2 and 6-75 (Tables 2-7) in 6-85% yields.
4-OCH3
a a 3-CH3,4-OCH3 3-OCH3,-4,5-OCH2O-
bb 2-OCH3
cc 3-OCH3
d d 4-OCH3
3,4-OCH2O-
3,4-OCH2O-
3-OCH3
These compounds can be converted with a suitable base
to the open chain keto-acid form furnishing stable,
isolable, water soluble salts.
The open form keto-acid salts and the closed form
butenolides exist in a pH dependent equilibrium in
solution, and at physiological pH both forms exist and
the biological activity might reside with either or both
forms (Figure 2). The extent of tautomerization and the
reversibility of tautomerization can be estimated by
examination of the H NMR spectra at various pH*’s.46
PD155080, 8, was studied in 1:1 (DMSO-d6:D2O) at
pH*’s varying from 2.65 to 9.05. At acidic pH* the
compound exists essentially completely in the closed
1

γ-Hydroxy Butenolide Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1065
F igu r e 1. Possible mechanism.
F igu r e 2. Butenolide tautomerization.
F igu r e 3. X-ray structure of 1 (closed form), stereoview.
butenolide form. As the pH* is slowly raised by addition
of 1.0 N NaOD, the compound starts to exhibit proper-
ties associated with the open form keto-acid, and at
basic pH* the compound is essentially all in the open
form. Of particular note is the coupling pattern shown
by the benzylic protons. At acidic pH* the benzylic
protons exhibit an AB quartet pattern consistent with
the ring-closed structure. As the pH* is raised this
pattern coalesces to a singlet, broad at neutral pH*, and
sharp at basic pH* as would be expected with the open
form keto-acid structure. After the pH was basic,
addition of DCl to acidify the solution caused the spectra
to return to its original appearance, proving that the
tautomerization is clean and reversible. As expected,
identical biological results have been obtained with the
salt and the parent in all pharmacological assays.
orthogonal to the butenolide ring (the coordinates are
included as supplementary material).
Compounds 76-79, in Table 8, were synthesized from
the parent butenolides 8 and 1. The two methoxy
analogues, 76 and 78, were synthesized by treating the
parent butenolides with saturated hydrochloric acid in
refluxing methanol, giving the methoxy derivative in
55-72% yield. The reaction can proceed through the
intermediate γ-chloro butenolides, which although un-
stable and reactive, may be isolated by treating the
parent hydroxy butenolide with hydrochloric acid in a
nonprotic solvent such as methylene chloride. The
deshydroxy analogues 77 and 79 were prepared in 70-
85% yield by treatment of the parent with either sodium
borohydride in trifluoroacetic acid at room temperature
or triethylsilane in trifluoroacetic acid at 0 °C.47
The X-ray crystal structure of the closed form of
compound 1, PD156707, has been determined (Figure
3). In the crystal structure the butenolide ring hydroxyl
and the benzylic ring are on the same side of the
butenolide ring and the two phenyl rings are essentially
Biologica l Testin g
Initially our screening assay for ETA receptor affinity
evaluated [125I]ET-1 binding inhibition in rabbit renal
artery vascular smooth muscle cells.48 ETB affinity was

1066 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Ta ble 2. Initial SAR of Butenolide Series, Topliss Tree Approach
Patt et al.
receptor binding affinity, IC50 (nM)a
b
c
compd
R1
R2
R3
ETA
ETB
2
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
4-Cl
H
4-CH3
4-OCH3
3,4-Cl2
3-CH3, 4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
H
H
H
H
H
H
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
3,4-OCH2O-
H
430
600
4900
7.4
400
12
51
27000
30000
>25000
4550
>25000
1750
4-CH3
4-Cl
4-OCH3
3,4-Cl2
3-CH3, 4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
>2500
>2500
1550
>2500
1140
>2500
>2500
>2500
2700
110
1.8
150
2.4
2200
700
570
170
190
4-Cl
4-CH3
3,4-Cl2
4-OCH3
9000
a
IC50 values were measured as described,50 using six concentrations of inhibitor (0.08-2500 nM, depending on inhibitor); n ) 2. b Human
cloned ETA receptor. c Human cloned ETB receptor.
determined similarly in rat cerebellum tissue prepara-
tions.49 More recently compounds have been evaluated
for their affinity toward cloned human ETA and ETB
receptors expressed in Ltk- cells and CHO-K1 cells,
respectively.50 Protocols for these binding assays have
been previously reported.48-50 All data presented in this
report are those obtained using cloned human receptors
unless specifically noted.
response is mediated by both the ETA (60%) and ETB
(40%) receptors.
Resu lts a n d Discu ssion
The Parke-Davis compound library, of approximately
165 000 compounds, was screened for ETA binding
affinity using the rabbit receptor assay. From a number
of micromolar hits, compound 2 (ETA ) 2.2 µM (rabbit),
430 nM (human) and ETB ) 5.2 µM (rat), 4.5 µM
(human)) was chosen for further development. This
analogue was subsequently found to have modest activ-
ity in our functional assays. Compound 2 inhibited ET-
1-induced arachidonic acid release in rabbit renal
vascular smooth muscle cells with an IC50 of 3.8 µM and
had modest activity in the inhibition of ET-1-induced
vasoconstriction in rabbit femoral artery with a KB of
1.4 × 10-5 M.
Cell-based antagonist activity and potency were mea-
sured using an ET receptor activated arachidonic acid
release assay. Rabbit renal artery vascular smooth
muscle cells and ET-1 (as agonist) were used in the ETA
assay while recombinant human receptors expressed in
CHO-K1 cells and ET-3 (as agonist) were used in the
ETB assay.51,52 Functional antagonism was demon-
strated by ET-1-induced vasoconstriction in rabbit
femoral artery (ETA mediated) or S6C-induced vasocon-
striction in rabbit pulmonary artery (ETB mediated).49,53
Initial attempts to improve the ET receptor binding
affinity of 2 employed the Topliss “decision tree” ap-
proach (Table 2). The Topliss approach takes into
account the electronic, lipophilic, and steric factors for
substitution on a phenyl ring using basic Hansch
principles in a noncomputerized manner.55,56 For op-
timization at the R1 site, we kept the unsubstituted
benzyl at the R2 site and the 3,4-(methylenedioxy)phenyl
moiety at R3, 5-10. From this we discovered compound
8 (PD 155080) with an IC50 of 7.4 nM at the human ETA
receptor.
Optimization at the R2 site via the Topliss tree, 11-
15, was performed with the p-OMe substituent at R1
and the 3,4-(methylenedioxy)phenyl moiety at R3. This
yielded compound 13 with an additional 6-fold increase
in ETA activity, IC50 ) 1.8 nM, versus 8. At both the
R1 and R2 sites the optimal substituents had small -π
values and -σ values.
In vivo antagonism was determined by measuring the
ability of compounds to block the hemodynamic response
to an exogenous ET-1 challenge in rats.41,54 In this
procedure rats were given an oral dose of the antagonist
by gavage followed 15 min later by ganglionic blockade
using mecamylamine. This gave a baseline MABP of
80-90 mmHg, both in antagonist and vehicle-treated
animals. After an additional 15 min the rats were
administered an iv dose of ET-1 (0.3 nM/kg). In vehicle-
treated animals, ET-1 caused a rapid albeit transient
depressor response of approximately 18 mmHg. ET-1
then caused a prolonged pressor response in control
animals with an averaged 44 mmHg peak response 5
min post ET-1 dose. The ETA selective antagonist BQ-
123 failed to block the initial depressor response and
blocked only approximately 60% of the pressor response,
while the nonselective antagonist PD 145065 blocked
100% of both the depressor and pressor responses. This
indicated that in this assay the depressor response is
mediated only by the ETB receptor and the pressor
We also attempted to optimize activity at the R3
position via the Topliss approach keeping optimized
substituents at both R1 and R2, 16-20. While the

γ-Hydroxy Butenolide Endothelin Antagonists
Ta ble 3. Expanded Butenolide SAR at the R1 Site
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1067
receptor binding affinity, IC50 (nM)a
microanalysisd
b
c
compd
R1
ETA
ETB
mol form
21
22
23
24
25
26
27
28
29
30
31
32
2-OCH3
3-OCH
4-C2H5
4-SCH3
4-O-i-C3H7
4-OCH2Ph
4-CO2H
4-CO2CH3
3,4-(OCH3)2
2,4-(OCH3)2
3,4-OCH2O-
3,4,5-(OCH3)3
480
500
630
170
1150
950
>2500
18000
>2500
>2500
11000
5400
>2500
>2500
18000
>2500
2000
C25H20O6‚0.2H2O
C,H,N
C,H,N
C
25H20H6
C26H22O5
C25H20O5S
C27H24O6
C31H24O6
C25H18O7
C26H20O7
C26H22O7
C26H22O7‚0.4H2O
C25H18O7‚0.35H2O
C27H24O8‚0.35H2O
C,H,N
C,H,Ne
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N,H2O(KF)
C,H,N
C,H,N
>25000
400
11
13
19
25
4500
a
b
d
See Table 2. Human cloned ETA receptor. c Human cloned ETB receptor. Analyses for the elements indicated were within 0.4% of
the theoretical values. e Calcd: C, 69.43; H, 4.66. Found: C, 68.57; H, 4.63.
Topliss tree was followed at this site, the activities
achieved in this study were significantly poorer than
with the 3,4-(methylenedioxy)phenyl-substituted com-
pound, 13.
very good potency. The hydrophobic π parameters seem
to be more influential than the electronic σ values with
large negative π values and small negative or positive
σ values being preferred.
These initial studies led quickly to potent and ETA
selective antagonists. All the compounds, 2 and 6-20,
were selective for ETA receptor binding, over ETB
receptor binding, with the majority being >500-fold
selective.
When the substituent was moved to the meta position,
all compounds seemed to be potent regardless of elec-
tronic or hydrophobic characteristics. The 3-carbo-
methoxy compound 40 was potent IC50 ) 8.5 nM, the
3-nitro compound 42 was less potent IC50 ) 70 nM, and
the 3-methoxy derivative 44 was also potent with
nanomolar ETA binding affinity, IC50 ) 3.0 nM. The
addition of a second methoxy substituent gave four
derivatives, 45-48, all with good ETA binding affinity
although not significantly improved over the mono-
methoxy compound 13. In an attempt to link the two
alkoxy substituents together in a ring structure we
synthesized two cyclic 3,4-derivatives, 49 and 50, both
of which were potent compounds. Addition of a third
methoxyl gave some interesting results. The first two
trimethoxy compounds, 51 and 52, were found to be
equally potent to the monomethoxy derivative 13.
However, the 3,4,5-trimethoxy derivative 1 was about
10-fold more potent than any of the previous substitu-
tion patterns, giving an IC50 value of 0.3 nM at the ETA
receptor. Increasing the alkyl portion of the three
alkoxy groups on 1 to ethoxy gave compound 53, which
was even more potent than 1 and somewhat more ETA
selective. The effects of further changes in alkyl size
at this site have been previously reported and are the
subject of a recent publication.57
Having elucidated that electron-donating substituents
seemed to be beneficial to receptor binding we then
started to expand our SAR to further optimize potency.
At the R1 position, Table 3, we compared the methoxy
substitution pattern and its effect upon receptor affinity.
The ortho, 21, and meta, 22, methoxy analogues of 8
both suffered about a 50-fold decrease in ETA binding
activity. Next we synthesized the alkyl steric equivalent
of PD155080, (p-Et) 23, and lost significant affinity,
indicating the importance of the heteroatom. Substitut-
ing the methoxy with thiomethoxy (p-SMe), 24, also led
to a decrease in ETA receptor activity. Increasing the
alkyl chain length or bulk on the alkoxy groups at R1
also led to a lowering of binding affinity, 25 and 26.
Replacement of the p-methoxy group with either a
carboxylic acid or an ester group at R1 led to a dramatic
loss of binding affinity, 27 and 28. An additional
electron donating alkoxy group in the R1 ring, 29-31,
gave equipotent derivatives, to the p-OMe derivative 8,
while addition of a third methoxy, 32, was slightly
detrimental to ETA binding affinity.
Exploration of the SAR at the R2 site led to many
highly potent compounds, 1 and 33-60, Tables 4 and
5. While the Topliss approach was accurate in predict-
ing the most active substitution patterns at the R2 site,
the degree of improvement was in general moderate.
Most R2 para-monosubstituted phenyl groups had bind-
ing affinities between 1-50 nM at the ETA receptor, 11-
15 and 33-44. Electron-donating groups such as
p-methoxy, 13, and p-hydroxy, 33, gave potent ana-
logues. However, when the substituent was strongly
electron withdrawing the correlation was less distinct.
The p-nitro derivative 39 had dramatically lower po-
tency whereas the p-methylsulfonyl compound 36 had
The R2 pocket clearly tolerated a wide range of
substitution patterns, and this was extended to explore
some larger aromatic and heteroaromatic substituents
(Table 5). The two naphthylmethyl compounds, 55 and
56, and the two heteroaryl derivatives 57 and 58
retained reasonable ETA binding affinity. Substitution
with even larger aromatic groups such as the biphenyl
derivative 59 and the dibenzosuberone 60 led to sig-
nificant losses of ETA binding affinity. Alkyl substitu-
tion at the R2 site, such as the ethyl compound 61, led
to a loss of ETA potency. When the alkyl substituent
more closely mimicked the benzyl in size and shape,
such as cyclopentylmethyl, cyclohexylmethyl, and cy-

1068 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Ta ble 4. Expanded Butenolide SAR at the R2 Site
Patt et al.
receptor binding affinity, IC50 (nM)a
b
c
compd
R2
ETA
8.0
28
ETB
mol form
microanalysisd
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
1
4-OH
>250
1800
>2500
3000
880
2000
17000
6000
>2500
>25000
>2500
260
C25H20O7‚0.28CH2Cl2
C27H25NO6
C,H,N
4-N(CH3)2
4-SCH3
4-SO2CH3
C,H,Ne
3.9
4.0
1.2
11
800
8.5
C26H22O6S‚0.3H2O
C,H,N,H2O(KF)
C,H,N,H2O(KF)
C,H,N
C,H,N
C,H,N
C26H22O8S‚0.95H2O
4-NHAc
C27H23NO7‚H2O
4-CO2CH3
4-NO2
3-CO2CH3
3-CO2H
C27H22O8
C25H19NO8
C26H20O8‚0.18EtOAc
C26H22O7‚0.28EtOAc
C25H19 O7‚0.4H2O
C26H19F3O6
C26H22O7
C27H24O8‚0.35EtOAc
C27H24O8
C27H24O8‚0.4 H2O
C27H24O8‚0.4H2O
C26H20O8
C,H,N
C,H,N
50
70
43
3-NO2
3-CF3
3-OCH3
C,H,N
C,H,Nf
3.0
11
2.1
1.4
1.7
16
7.5
12
C,H,N
C,H,N
C,H,N
C,H,N,H2O(KF)
C,H,N,H2O(KF)
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
2,3-(OCH3)2
2,5-(OCH3)2
3,4-(OCH3)2
3,5-(OCH3)2
3,4-OCH2O-
3,4-OCH2CH2O-
2,3,4-(OCH3)3
2,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OC2H5)3
3-OCH3,4,5-OCH2O-
2200
1000
950
760
1500
1600
2000
850
780
445
C27H22O8
C28H29O9‚0.25EtOAc
C28H26O9‚0.25EtOAc
C28H26O9
C31H32O8‚0.3EtOAc
C27H22O9
1.8
0.3
0.12
53
54
14
16000
a
b
d
See Table 2. Human cloned ETA receptor. c Human cloned ETB receptor. Analyses for the elements indicated were within 0.4% of
the theoretical values. e Calcd: C, 69.71; H, 5.59; N, 2.23. Found: C, 70.58; H, 5.48; N, 3.05. f Calcd: C, 63.75; H, 3.96. Found: C, 64.47;
H, 3.95.
Ta ble 5. Expanded Butenolide SAR at the R2 Site
receptor binding affinity, IC50 (nM)a
microanalysisd
b
c
compd
R2
ETA
ETB
mol form
55
56
57
58
59
60
61
62
63
64
1-naphthylmethyl
2-naphthylmethyl
3-pyridylmethyl
1-isoquinolylmethyl
4′-phenylbenzyl
dibenzosuberonylmethyl
ethyl
(c-C5H9)CH2
(c-C6H11)CH2
(c-C7H13)CH2
18
14
40
>2500
>2500
>2500
3200
C29H22O6‚0.4H2O
C,H,N,H2O(KF)
C,H,N
C,H,N,H2O(KF)
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C
C
C
C
C
C
29H22O6‚0.35EtOAc
24H19NO6‚0.4H2O
28H21NO6‚0.3H2O
31H24O6‚0.45H2O
34H28O6
10
200
16000
320
10
1900
10000
15000
630
225
1300
20H18O6
C24H24O6
C25H26O6
C26H28O6
13
35
a
b
d
See Table 2. Human cloned ETA receptor. c Human cloned ETB receptor. Analyses for the elements indicated were within 0.4% of
the theoretical values.
cloheptylmethyl (62-64), the ETA binding activity
resembles that seen in 8.
made, compounds 65 and 67-69. The 3,5-dimethoxy
derivative 69 (IC50 of 46 nM, ETA) was the most potent
of the dialkoxy analogues synthesized and was still
5-fold less potent than 8. The 3,4,5-trimethoxy deriva-
tive 70 was synthesized and was only weakly active.
This study indicated that para substituents, larger than
the methylenedioxy moiety, at the R3 site were not well
tolerated, probably due to steric factors. Combining
both the 3,5-dimethoxy and the 3,4-methylenedioxy
moiety gave the 3,4-(methylenedioxy)-5-methoxy ana-
logue 71 with ETA binding affinity, IC50 ) 6.7 nM, equal
to the 3,4-methylenedioxy analogue 8.
At the R3 site, Table 6, the 3,4-methylenedioxy moiety
was optimal in the initial set of analogues 13. The 3,4-
dimethoxy compound 65 was 10-fold less active than the
3,4-methylenedioxy 8 at this site. Comparison of the
3-methoxy, 66, and the 4-methoxy, 20, derivatives
showed that the former was preferred by at least 2-fold
(20 contains a 4-methoxy substituent at the R2 position,
which is about 4 fold more potent than an R2 unsubsti-
tuted compound, e.g. 66). To explore alkoxy substitution
patterns further, a series of dialkoxy analogues were

γ-Hydroxy Butenolide Endothelin Antagonists
Ta ble 6. Expanded Butenolide SAR at the R3 Site
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1069
receptor binding affinity, IC50 (nM)a
b
c
compd
R3
ETA
ETB
mol form
C26H24O6
C25H22O5
C26H24O6
C26H24O6‚0.6EtOAc
C26H24O6
C27H26O7‚0.15H2O
C26H22O7‚0.5H2O
microanalysisd
65
66
67
68
69
70
71
3,4-(OCH3)2
3-OCH3
70
90
300
240
46
3900
12000
25000
>2500
5200
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N,H2O(KF)
C,H,N
2,4-(OCH3)2
2,5-(OCH3)2
3,5-(OCH3)2
3,4,5-(OCH3)3
3-OCH3, 4,5-OCH2O-
1300
1600
640
6.7
a
b
d
See Table 2. Human cloned ETA receptor. c Human cloned ETB receptor. Analyses for the elements indicated were within 0.4% of
the theoretical values.
Ta ble 7. Combination of Best R1, R2, and R3 Groups
receptor binding affinity, IC50 (nM)a
b
c
compd
R1
4-OCH3
4-OCH3
R2
R3
ETA
ETB
mol form
C26H22O7
C29H28O10‚0.4H2O
C30H30O10
C29H28O9‚0.1EtOAc
C27H24O7‚0.5H2O
microanalysisd
71
72
73
74
75
H
3-OCH3,4,5-OCH2O-
6.7
0.5
0.9
0.5
8.2
640
260
180
240
1410
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
3,4,5-(OCH3)3 3-OCH3,4,5-OCH2O-
3-CH3,4-OCH3 3,4,5-(OCH3)3 3-OCH3,4,5-OCH2O-
3-CH3,4-OCH3 3,4,5-(OCH3)3 3,4-OCH2O-
3-CH3,4-OCH3
H
3-OCH3,4,5-OCH2O-
a
b
d
See Table 2. Human cloned ETA receptor. c Human cloned ETB receptor. Analyses for the elements indicated were within 0.4% of
the theoretical values.
Ta ble 8. Expanded SAR at the Butenolide Ring γ-Site
receptor binding affinity, IC50 (nM)a
b
c
compd
R2
R4
OH
OCH3
H
OH
OCH3
H
ETA
7.4
300
700
0.3
ETB
mol form
microanalysisd
8
76
77
1
78
79
H
H
H
4550
>25000
>2500
780
>2500
>25000
C26H22O6‚0.7H2O
29H28O9‚0.2H2O
C,H,N,H2O(KF)
C,H,N
C
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
600
30
C29H28O7
C28H26O8
C,H,N
C,H,N
a
b
d
See Table 2. Human cloned ETA receptor. c Human cloned ETB receptor. Analyses for the elements indicated were within 0.4% of
the theoretical values.
The five compounds in Table 7, 71-75, exemplify
combination of the best groups from all three sites, R1,
R2, and R3. As expected, these derivatives are all
extremely potent and selective for the ETA receptor,
170-520-fold selective.
The results tabulated in Table 8 demonstrate the
importance of the ring hydroxyl in retaining potency.
Conversion of 8 and 1 to their corresponding methyl
ethers, 76 and 78, led to a loss of potency. Similarly,
removal of the hydroxy group to give the corresponding
methines, 77 and 79, also led to loss in binding affinity.
These analogues, by design, cannot tautomerize to form
the open chain keto-acids. Whether it is the loss of
the hydroxy and any hydrogen-bonding interactions, it
forms with the receptor, or the elimination of the open
chain form that is responsible for the loss of potency is
being examined.58
Many of the compounds were then examined in the
functional assay for antagonist activity. In general as
receptor binding potency improved we saw a corre-
sponding improvement in the functional activity. Table
9 includes data for selected butenolides and their open

1070 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Ta ble 9. Functional Data for Selected Butenolide ET Antagonists
Patt et al.
inhibition of ET-1 induced
inhibition of agonist-induced
arachidonic acid release, IC50(nM)a
contraction in arterial rings, KB(M)b
c
d
e
f
compd
ETA
ETB
ETA
ETB
2
3800
49
ia
22000
8200
120
1.4 × 10-5 (n ) 2)
5.6 × 10-7 (n ) 2)
7.8 × 10-8 (n ) 1)
1.9 × 10-8 (n ) 2)
2.9 × 10-8 (pA2 ) 7.6)g
8.5 × 10-8 (n ) 1)
2.2 × 10-8 (n ) 1)
2.5 × 10-8 (n ) 1)
2.8 × 10-8 (n ) 1)
ia at 10-5
M
8, choline salt
37, K salt
1
1, Na salt
71, Na salt
72, Na salt
73, Na salt
74, Na salt
2.6 × 10-5
4.9
1.1
1.1
nt
nt
130
3.4 × 10-5
10
15700
5100
68
nt
nt
nt
nt
0.34
0.78
0.98
1900
See Table 2. IC50 values were measured as described,51-52 using six concentrations of inhibitor (0.01-100 nM, depending on inhibitor);
a
b
n ) 3. c ET-1 used as the agonist, rabbit renal artery vascular smooth muscle cells. ET-3 used as the agonist, human cloned receptors/
d
CHO-K1 cells. e ET-1 used as the agonist, rabbit femoral artery. f S6C used as the agonist, rabbit pulmonary artery. Schild regression
g
analysis was run to give pA2 value.
Ta ble 10. In Vivo Effects of Orally Dosed Butenolide ET
Antagonists
compd
N
MABP response (% ET-1)a
8, choline salt
1, Na salt
71, Na salt
72, Na salt
73, Na salt
74, Na salt
4
5
6
6
6
6
80 ( 9
50 ( 8
76 ( 7
52 ( 9
86 ( 12
53 ( 9
a
Effect of antagonists at 10 mg/kg on MABP in anesthetized,
ganglionic-blocked rats versus an ET-1 (0.3 nmol/kg, iv) baseline.
F igu r e 4. Dose-response curve of the inhibition (1 and 8) of
the pressor response due to ET-1 (0.3 nmol/kg, iv) in anesthe-
sized, ganglionic-blocked rats. The antagonists were admin-
istered at doses from 0.3 to 100 mg/kg (po) by oral gavage.
chain keto-acid salt forms. The data, as expected,
shows a very good correlation between the tautomeric
forms. The initial screening hit 2 inhibited ET-1-
induced arachidonic acid release in rabbit renal vascular
smooth muscle cells with an IC50 of 3.8 µM. Compound
8 (ETA ) 7.4 nM, ETB ) 4500 nM) with an approximate
100-fold increase in binding affinity over 2 inhibited ET-
1-induced arachidonic acid release with an IC50 of 150
nM and inhibited ET-1-induced vasoconstriction with
a KB of 5.6 × 10-7 M. Compounds with subnanomolar
ETA receptor binding affinity showed corresponding
increases in functional activity. Compound 1 inhibited
ET-1-induced arachidonic acid release with an IC50 of
1.1 nM and inhibited ET-1-induced vasoconstriction
with a KB of 2.9 × 10-8 M (pA2 of 7.6). This compound
further has ETB functional activity with an IC50 of 120
nM in the ET-3-induced arachidonic acid release and a
KB of 3.4 × 10-5 M in the S6C-induced vasoconstriction
in rabbit pulmonary artery. The most potent ETA
selective antagonist in this study, functionally (overall),
is compound 72. Compound 72 inhibited ET-1-induced
arachidonic acid release with an IC50 of 0.34 nM and
inhibited ET-1-induced vasoconstriction with a KB of 2.2
× 10-8 M. This compound also had modest ETB
functional activity with an IC50 of 5.1 µM for inhibition
of ET-3-induced arachidonic acid release.
Many of the open chain salt forms of the compounds
in Table 9 were further examined for their ability after
oral administration, to inhibit the increase in mean
arterial blood pressure (MABP) due to administration
of exogenous ET-1 to conscious ganglionic blocked rats
(Table 10). Compound 8 at 10 mg/kg shows a 20%
reduction in the peak pressor response to ET-1. The
more potent compounds 1, 72, and 74 show, at 10 mg/
kg, the maximal effect seen with an ETA selective
antagonist in this screen, approximately 50% reductions
in peak pressor response. Figure 4 shows graphically
the dose response curves for 8 and 1 in this assay.
Ta ble 11. ETA Receptor Selectivity
receptor binding
affinity, IC50 (nM)a
ET receptor
selectivity ETB/ETA
b
c
compd
ETA
ETB
1
8
53
72
74
0.3
7.4
0.12
0.5
780
4550
445
260
240
2600
615
3700
520
0.5
480
a
b
See Table 2. Human cloned ETA receptor. c Human cloned
ETB receptor.
Compound 8 is essentially inactive at 3 mg/kg and
maximally effective at 30 mg/kg. Compound 1, the more
potent derivative, shows moderate activity at a 1 mg/
kg dose and maximal effect at the 10 mg/kg dose.
A selection of compounds in Table 11 demonstrate
that, as a class, the γ-hydroxy butenolides are extremely
ETA potent and selective antagonists. All of the buteno-
lide compounds, Tables 2-8, were ETA selective, and
of those in Table 11, compounds 1 and 53 were greater
than 2500-fold selective and had sub-nanomolar ETA
receptor binding affinity. Additionally compound 1 was
tested by Panlabs against a battery of 28 receptor
binding and seven enzyme inhibition assays and was
found to be inactive against all (at 10 µM) except the
endothelin receptors.59
The pharmacokinetics parameters for compound 1,
initially reported in rats41 have now been characterized
in dogs (Table 12). In a three-way crossover study
design, four male beagle dogs were dosed with a single
2.5 mg/kg intravenous dose to fed animals, or 5 mg/kg
oral capsule to fed or fasted animals. Serial heparinized
plasma samples were collected up to 24 h postdose.
Plasma concentrations were measured by a specific
HPLC assay.

γ-Hydroxy Butenolide Endothelin Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1071
Ta ble 12. Mean PD156707 Pharmacokinetic Parameters in
Dogs (n ) 4)a
to TMS. Mass spectra (MS) were recorded on a Varian VG
7070 spectrometer at nominal 5000 resolution or a Finnigan
MAT 900Q spectrometer. Microanalyses were determined by
the Parke-Davis Pharmaceutical Analytical Department or
under contract with Robertson Analytical Services.
parameter
intravenous
oral fasted
oral fed
dose (mg/kg)
2.5
0.62
5.0
1.2
1.52
0.69
2.0
5.0
1.9
0.48
2.5
0.59
20
t1/2 (h)b
Gen er a l Exa m p le of Syn th esis of Com p ou n d s 5a -s
a n d 5w -ff. 3-(1,3-Ben zod ioxol-5-yl)-1-(4-m eth oxyp h e-
n yl)p r op -2-en -1-on e (3f). 4-methoxyacetophenone (200 g,
1.33 mol) was dissolved in absolute ethanol (300 mL), and the
solution was treated with piperonal (200 g, 1.33 mol). The
solution was then treated with 10% aqueous NaOH (41 mL)
and the mixture stirred at room temperature for 1.5 h and
then allowed to precipitate. The yellow solid was collected by
filtration and washed with ethanol:water (1:1) (600 mL) and
dried in vacuo overnight. The solid was used as in the next
step: 360.15 g (96%); 1H NMR (CDCl3) δ 3.89 (s, 3H), 6.02 (s,
1H), 6.84 (s, 2H), 6.97 (d, 2H), 7.16 (d, 2H), 7.38 (d, 1H), 7.72
(s, 1H), 8.02 (s, 1H); MS (EI+): 310 Da; mp 127-131 °C.
2-(1,3-Ben zod ioxol-5-yl)-1-(4-m eth oxyp h en yl)-4-oxobu -
tyr on itr ile (4f). A solution of 3f (360.15 g, 1.28 mol) in freshly
distilled 2-ethoxyethanol (1 L) and acetic acid (91.9 g, 1.53 mol)
at 105 °C was treated in portions with a solution of KCN (124.4
g, 1.91 mol) in water (217 mL). The mixture was stirred at
105 °C for 30 min and allowed to cool to room temperature.
The mixture was allowed to stand overnight to precipitate. The
solid was collected by filtration, washed with methanol:water
(1:1) (1.6 L), and dried under high vacuum. This gave 338.8
c
Cmax
tmax
d
AUC (0-∞)e
F (%)f
1.5
67
a
b
Following a single intravenous or oral dose. Apparent ter-
minal phase elimination half-life; arithmetic mean. c Maximum
observed concentration (µg/mL). Time to reach Cmax (h). e Area
d
under the concentration-time curve from time zero to infinity (µg
at h/mL). f Absolute oral bioavailability.
Following intravenous administration, PD 156707, 1,
plasma concentrations declined in a biexponential man-
ner. PD 156707 was rapidly absorbed following oral
dosing. Food appeared to slow oral absorption and
decrease oral bioavailability: fasted 66%, fed 20%. The
mean plasma terminal elimination half-life was longer
following oral (1.9 h) than intravenous dosing (0.62 h).
Con clu sion
1
Starting from the modestly active compound library
screening hit, 2, we were able to develop, using the
structured Topliss tree approach, a number of potent
nanomolar ETA selective ET antagonists. Further lead
optimization via examination of the electron and struc-
tural requirements led to a series of sub-nanomolar
affinity ETA antagonists, represented by 1, with oral
activity against an exogenous ET-1 challenge. Com-
pounds 1 and 8 are being evaluated in a number of
preclinical disease models, and efficacy in stroke for 1
has been demonstrated.7
g of a yellow/brown solid (85%); H NMR (CDCl3) δ 3.43 (m,
1H), 3.62 (m, 1H), 3.87 (s, 3H), 4.48 (m, 1H), 5.97 (s, 2H), 6.79
(d, 1H), 6.88 (m, 2H), 6.93 (m, 2H), 7.90 (d, 2H); MS (EI+) 310
Da; mp 90-92 °C. Anal. (C18H15NO4‚0.2H2O) C, H, N.
2-(1,3-Ben zod ioxol-5-yl)-1-(4-m eth oxyp h en yl)-4-oxobu -
tyr ic Acid Meth yl Ester (5f). A suspension of 4f and
p-toluenesulfonic acid hydrate (208.3 g, 1.10 mol) in methanol
(1 L) and dioxane (0.5 L) was heated to reflux overnight under
nitrogen. The mixture was cooled to room temperature and
the precipitate collected by filtration, washed with methanol,
and dried to give 354.3 g of a white solid: 1H NMR (CDCl3) δ
3.20 (d of d, 1H), 3.70 (s, 3H), 3.82 (d of d, 1H), 3.86 (s, 3H),
4.18 (d of d, 1H), 5.95 (s, 2H), 6.80 (m, 3H), 6.92 (d, 2H), 7.94
(d, 2H); MS (EI+) 343 Da; mp 113-114°C. Anal.
(C19H18O6‚0.2H2O) C, H, N.
Exp er im en ta l Section
P h a r m a cok in etics Meth od s. PD156707 was isolated
from dog plasma by reverse-phase solid phase extraction.
Liquid chromatographic separation was achieved on a C18,
Spherex 5µ (3.2 × 150 mm), Phenomenex reverse phase
column. The mobile phase consisted of 40% acetonitrile/60%
ammonium acetate buffer (50 mM, pH 3.5). Column effluent
was monitored by a fluorescence detector with an excitation
wavelength of 312 nm and emission of 430 nm. Assay limit
of quantitation ranged from 10 to 5000 ng/mL. Plasma
samples contained more than 5000 ng/mL PD156707 and were
diluted with blank dog plasma and reanalyzed. Minimum
limit of quantitation for PD156707 was 10 ng/mL from a 200
µL dog plasma sample aliquot.
Non sta n d a r d In ter m ed ia tes. 3-(1,3-Ben zod ioxol-5-yl)-
1-(4-(m eth ylth io)p h en yl)p r op -2-en -1-on e (3s). From 4′-
(methylthio)acetophenone (15.5 g, 93.3 mmol) and piperonal
(14 g, 93.3 mmol) in the same manner as 3f was synthesized
the chalcone 3s. This gave 27 g (97%) of a yellow solid; 1H
NMR (CDCl3) δ 2.54 (s, 3H), 6.03 (s, 2H), 6.83-7.96 (m, 9H);
MS (CI+) 298 Da; mp 135-137 °C. Anal. (C17H14O3S) C, H,
N.
2-(1,3-Ben zod ioxol-5-yl)-1-(4-(m eth ylth io)p h en yl)-4-ox-
obu tyr ic Acid Eth yl Ester (5s). Triphenyl orthothioformate
(6.8 g, 20 mmol) was treated with n-BuLi (2.1 M, 10 mL) at
-78 °C under nitrogen and stirred for 30 min. To this was
added the chalcone 3s (5.4 g, 18.1 mmol) in THF (100 mL),
and the mixture was stirred cold for another 30 min. The
mixture warmed to room temperature and stirred for 30 min.
The solution was treated with NH4Cl (saturated) (5 mL),
passed a short packed silica gel column (200 g), and evaporated
to dryness. The crude product was recrystallized in ethyl
acetate/ether to give the ortho thioester as a white solid, 7.2 g
Pharmacokinetic parameter values were estimated by non-
compartmental analysis of individual dog plasma PD156707
concentration-time data. Maximum PD156707 plasma con-
centrations (Cmax) and times for these to occur (tmax) were
recorded as observed. Apparent terminal elimination-rate
constants (λz) were calculated by linear regression of the log-
linear terminal phase of concentration-time profiles. Appar-
ent terminal elimination half-life values were calculated from
elimination-rate constants as t1/2 ) 0.69/λz. Area under
plasma concentration-time curve AUC(0-∞) values were
calculated from time zero to time of last detectable concentra-
tion using the trapezoidal rule and were extrapolated to
infinity. Absolute oral bioavailability F% ) (doseiv × AUC-
(0-∞)po/dosepo × AUC(0-∞)iv) × 100.
1
(62%), which was identified by H NMR and used as is. The
ortho thioester (3.7 g, 5.8 mmol), HgCl2 (7.8 g, 28.7 mmol),
and HgO (2.5 g, 11.5 mmol) were refluxed in 95% ethanol (150
mL) for 7 h under nitrogen. The mixture was then filtered
and the filtrate diluted with water (75 mL) and extracted with
methylene chloride (2 × 100 mL). The combined extracts were
washed successively with 1 N HCl (200 mL) and brine (200
mL) and then dried over MgSO4. Evaporation of the solvent
gave 1.05 g (49%) of the ester: 1H NMR (CDCl3) δ 1.25 (m,
3H), 2.53 (s, 3H), 3.20 (m, 1H), 3.83 (m, 1H), 4.07-4.21 (m,
4H), 5.95 (s, 2H), 6.75-7.90 (m, 7H). Anal. (C20H20O5S) C,
H, N (not run).
Gen er a l Ch em ica l P r oced u r es. All reagents were either
purchased from common commercial sources or synthesized
according to literature references using commercial sources.
Melting points were determined on a Thomas-Hoover capillary
melting point apparatus and are reported uncorrected. Proton
NMR (1H NMR) were obtained on Bruker AC-200 or AM-400
or Varian Unity 400 MHz spectrometers and are referenced
3-(1,3-Ben zod ioxol-5-yl)-1-(4-h yd r oxyp h en yl)p r op -2-
en -1-on e (3ee). From 4′-hydroxyacetophenone (9.1 g, 69
mmol) and piperonal (10 g, 67 mmol) in the same manner as

1072 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Patt et al.
3f was synthesized 16.5 g (91%) of the chalcone; 1H NMR
(CDCl3) δ 6.10 (s, 2H), 6.87-8.09 (m, 9H), 10.38 (br s, 1H);
MS (CI+) 268 Da; mp 204-205 °C. Anal. (C16H12O4) C, H, N.
2-(1,3-Ben zod ioxol-5-yl)-1-(4-isop r op oxyp h en yl)-4-ox-
obu tyr on itr ile (4t). The chalcone 3ee (5.35 g, 19.7 mmol)
was dissolved in DMF (100 mL) and treated with K2CO3 (3 g,
20 mmol) and isopropyl bromide (3 g, 24 mmol). The mixture
warmed to 100 °C and stirred for 3 h. The mixture filtered
and the filtrate evaporated in vacuo to give the crude solid.
The material was purified by flash chromatography (1000 g
of flash silica gel, chloroform:ethyl acetate (200:1)). Evapora-
tion of the appropriate fractions gave 4.8 g (79%) of the
chalcone. From this chalcone (4.0 g, 12.9 mmol) in the same
manner as 4f was isolated the nitrile: 3.85 g (89%); 1H NMR
(CDCl3) δ 1.37 (m, 6H), 3.51 (m, 2H), 4.49 (m, 1H), 4.65 (m,
1H), 5.99 (d, 2H), 6.78-8.03 (m, 7H); MS (CI+) 338 Da. Anal.
(C20H19NO4) C, H, N (not run).
2-(1,3-Ben zod ioxol-5-yl)-1-(4-isop r op oxyp h en yl)-4-ox-
obu tyr ic Acid Meth yl Ester (5t). From the nitrile 4t (3.0
g, 8.9 mmol) in the same manner as 4f was synthesized the
ester 4t, giving 2.4 g (73%): 1H NMR (CDCl3) δ 1.37 (d, 6H),
3.42-4.50 (m, 3H), 3.64 (s, 3H), 4.66 (m, 1H), 6.01 (s, 2H),
6.665-7.55 (m, 7H); MS (CI+) 371 Da. Anal. (C21H22O2) C,
H, N (not run).
3-Ben zo[1,3]-d ioxo-5-yl-1-(4-cya n op h en yl)p r op -2-en -1-
on e (3ff). From 4′-cyanoacetophenone (9.7 g, 67 mmol) and
piperonal (10 g, 67 mmol) was synthesized 3ff in the same
manner as 3f. This gave 13.5 g (73%): 1H NMR (CDCl3) δ
6.05 (s, 2H), 6.85-8.08 (m, 9H); MS (CI+) 277 Da. Anal.
(C17H11NO3) C, H, N (not run).
2-(1,3-Ben zod ioxol-5-yl)-1-(4-cya n op h en yl)-4-oxobu ty-
r on itr ile (4ff). From the chalcone 3ff (12.5 g, 45.8 mmol) in
the same manner as 4f was synthesized the nitrile 4ff, giving
8.6 g (62%): 1H NMR (CDCl3) δ 3.59 (m, 2H), 4.46 (m, 1H),
5.99 (s, 2H), 6.78-8.03 (m, 7H); MS (CI+) 305 Da. Anal.
(C18H12N2O2) C, H, N (not run).
lyophilized. This gave 3.65 g (88%) of a white lyophilate: 1H
NMR (D2O) δ 3.21 (s, 2H), 3.54 (s, 3H), 3.55 (s, 6H), 3.83 (s,
3H), 5.94 (s, 2H), 6.03 (s, 2H), 6.85-6.90 (m, 4H), 6.98 (d, 1H),
7.66 (d, 2H); MS (CI+) 505 Da. Anal. (C28H25O9Na‚0.8H2O)
C, H, N, H2O (KF), Na.
Gen er a l Syn th esis of γ-Meth oxy Bu ten olid es 76 a n d
78. 3-(1,3-Ben zodioxol-5-yl)-4-ben zyl-5-m eth oxy-5-(4-m eth -
oxyp h en yl)-5H-fu r a n -2-on e (76). A solution of 8 (205 mg,
0.5 mmol) in methanol (15 mL) was saturated with HCl gas.
The solution warmed to 50 °C and stirred for 18 h. The cooled
solution was evaporated in vacuo to give a crude product. The
crude was purified by flash chromatography (120 g of flash
silica gel, hexane:ethyl acetate (3:1)). Evaporation of the
appropriate fractions gave 119 mg (55%) as a colorless solid:
1H NMR (CDCl3) δ 3.26 (s, 3H), 3.76 (q, 2H), 3.79 (s, 3H), 5.96
(s, 2H), 6.77-6.83 (m, 5H), 6.94-6.96 (m, 2H), 7.05-7.09 (m,
3H), 7.34 (d, 2H); MS (EI+) 430 Da. Anal. (C26H22O6‚0.7H2O)
C, H, N, H2O (KF).
Syn th esis of Desh ydr oxy Bu ten olides 77 an d 79. 3-(1,3-
Ben zod ioxol-5-yl)-4-ben zyl-5-(4-m et h oxyp h en yl)-5H-fu -
r a n -2-on e (77). In trifluoroacetic acid (8 mL), at 0 °C, under
a nitrogen stream was added in parts a mixture of sodium
borohydride (378 mg, 10 mmol) and 8 (416 mg, 1.0 mmol). The
resultant deep green solution was stirred for 5 min and
evaporated free of TFA. The residue was then carefully
quenched with water (20 mL). The aqueous solution was
extracted with ethyl acetate (25 mL) and the organic phase
washed with brine (25 mL). The organic phase then dried over
MgSO4 and evaporated in vacuo to give the crude product. The
crude material was purified by flash chromatography (70 g of
flash silica gel, 5% ethyl acetate:methylene chloride). The
appropriate fractions were evaporated in vacuo to give 282 mg
1
(70%) as an off-white solid: H NMR (CDCl3) δ 3.68 (q, 2H),
3.83 (s, 3H), 5.59 (s, 1H), 6.01 (s, 2H), 6.88-6.92 (m, 3H), 6.96-
6.98 (m, 2H), 7.03-7.09 (m, 4H), 7.26-7.29 (m, 2H); MS (CI+)
400 Da. Anal. (C25H20O5‚0.2H2O) C, H, N.
3-(1,3-Ben zod ioxol-5-yl)-4-(3,4,5-tr im eth oxyben zyl)-5-
(4-m eth oxyp h en yl)-5H-fu r a n -2-on e (79). In TFA (6.5 mL)
frozen at -78 °C was added 53 (2.53 g, 5.0 mmol). The deep
green solution was treated with triethylsilane (7.0 mL) and
stirred for 1 h. The mixture was warmed to reflux for 3 h
and stripped in vacuo to give an oil. The oil was triturated
with petroleum ether and decanted. The insoluble residue was
dissolved in ethyl acetate, washed successively with saturated
NaHCO3 and brine, and then dried over MgSO4. The solvents
were evaporated, and the resultant solid was recrystallized
from ether. The solid dried in vacuo to give 2.08 g (85%) of
79: 1H NMR (CDCl3) δ 3.63 (d of d, 2H), 3.75 (s, 6H), 4.02 (s,
6H), 5.63 (s, 1H), 6.02-6.03 (m, 2H), 6.10 (s, 2H), 6.89-6.92
(m, 3H), 7.05-7.09 (m, 4H); MS (CI+) 400 Da. Anal. (C28H26O8)
C, H, N.
2-(1,3-Ben zod ioxol-5-yl)-1-(4-(ca r boxym eth yl)p h en yl)-
4-oxobu tyr ic Acid Meth yl Ester (5v). The nitrile 4ff (6.2
g, 20.3 mmol) in methanol (50 mL) was saturated with HCl
gas and warmed to 45 °C until no trace of starting material
remained. The solution was cooled and treated with water
(100 mL). The resultant solid was collected, washed with
methanol:water (8:2) (50 mL), and dried in vacuo. This gave
the diester: 6.1 g (81%); 1H NMR (CDCl3) δ 3.25 (m, 1H), 3.71
(s, 3H), 3.90 (m, 1H), 3.96 (s, 3H), 4.21 (m, 1H), 5.96 (m, 2H),
6.75-8.16 (m, 7H); MS (CI+) 370 Da. Anal. (C20H18O7) C, H,
N (not run).
Gen er a l Exa m p le for th e Syn th esis of Com p ou n d s 1,
2, a n d 6-75). 3-Ben zo[1,3]d ioxol-5-yl-5-h yd r oxy-5-(4-
m eth oxyp h en yl)-4-(3,4,5-tr im eth oxyben zyl)-5H-fu r a n -2-
on e (1). To methanol (6 mL) was added sodium metal (57
mg, 2.5 mmol), and the mixture was stirred to dissolve. To
this was added 5f (0.822 g, 2.4 mmol) followed by 3,4,5-
trimethoxybenzaldehyde (0.50 g, 2.5 mmol). The mixture was
warmed to reflux for 16 h. The solution was treated with
acetic acid (1.5 mL) and refluxed an additional 6 h. The
solvents were evaporated in vacuo to give a pasty residue. This
was partitioned between ethyl acetate (20 mL) and water (20
mL). The organic phase was quickly separated, dried over
MgSO4, and evaporated to dryness. The crude product was
purified by flash chromatography (150 g of flash silica gel, 20%
ethyl acetate:methylene chloride). The product was isolated
by evaporation of the appropriate fractions to give 0.719 g
(59%) as a white foam: 1H NMR (CDCl3) δ 3.64 (s, 6H), 3.69
(m, 2H), 3.74 (s, 3H), 3.80 (s, 3H), 5.97 (s, 2H), 6.01 (s, 2H),
6.79-6.98 (m, 5H), 7.39 (d, 2H); MS (CI+) 507 Da; mp 73-75
°C. Anal. (C28H26O9) C, H, N.
Ack n ow led gm en t. We thank Dr. Gary McClusky
and colleagues for providing the spectroscopic and
analytical data.
Su p p or tin g In for m a tion Ava ila ble: Spectral and gen-
eral experimental information for all compounds which have
either not previously been published or not been specifically
mentioned in the preceding experimental, and X-ray coordi-
nates for compound 1 (51 pages). Ordering information is
given on any current masthead page.
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