Azole endothelin antagonists. 1. A receptor model explains an unusual structure-activity profile

Article abstract of DOI:10.1021/jm950591h

The pseudotetrapeptide FR-139317 is a potent and highly selective antagonist of the endothelin-A (ET(A)) receptor; however, its peptidic nature leads to poor oral absorption characteristics which make it an unlikely drug candidate. In an attempt to improv

Full text of DOI:10.1021/jm950591h

J . Med. Chem. 1996, 39, 957-967
957
Azole En d oth elin An ta gon ists. 1. A Recep tor Mod el Exp la in s a n Un u su a l
Str u ctu r e-Activity P r ofile
Thomas W. von Geldern,* Charles Hutchins,*,† J effrey A. Kester, J inshyun R. Wu-Wong, William Chiou,
Douglas B. Dixon, and Terry J . Opgenorth
Aging and Degenerative Diseases Research and Structural Biology Departments, Pharmaceutical Products Research Division,
Abbott Laboratories, Abbott Park, Illinois 60064
Received August 8, 1995X
The pseudotetrapeptide FR-139317 is a potent and highly selective antagonist of the
endothelin-A (ETA) receptor; however, its peptidic nature leads to poor oral absorption
characteristics which make it an unlikely drug candidate. In an attempt to improve these
properties, we have replaced a portion of the amide bond framework of FR-139317 with a
heterocyclic surrogate. The resultant analogs are also ETA-selective antagonists, but show a
structure-activity profile substantially different from that of the peptidic series, particularly
with regard to the requirements for the side chain group that has been incorporated into the
heterocycle. The nature of the heterocycle itself also has profound effects on the activity of
the compounds. Both of these surprising results can be rationalized through examination of
a 3D model of ET ligand-receptor binding that has previously been developed in our
laboratories.
In tr od u ction
An ta gon ist Design
Endothelin (ET),1 a 21-amino acid bicyclic peptide, is
the most powerful peptidic constrictor of vascular
smooth muscle reported to date, as well as a potent
mitogen. Produced predominantly by endothelial cells,
it acts in both an autocrine and paracrine fashion as a
mediator of vascular function. It has been implicated
as a pathogenic factor for a variety of disease states,2a
including asthma,2b coronary vasospasm and myocardial
infarction,2c pulmonary hypertension,2d restenosis,2e and
atherosclerosis,2f in which excessive vasoconstriction or
smooth muscle proliferation play a role.
Endothelin acts by binding to a family of membrane-
associated, G-protein-coupled receptors (GPCRs).3 Bind-
ing to the ETA receptor subtype, which predominates
in vascular smooth muscle cells, triggers a cascade of
events which leads, via the hydrolysis of inositol phos-
phates, to the observed vasoconstrictive and prolifera-
tive responses. The results of binding to ETB, which is
the major receptor on endothelial cells, are less clearly
understood; while this receptor mediates constriction in
some tissue beds, it has also been linked to the produc-
tion of nitric oxide and to the clearance of endogenous
ET. Because these latter effects might be beneficial in
a number of the diseases described above, it has been
suggested2a that a selective ETA antagonist may provide
some therapeutic advantage over a nonselective agent.
The unique role of endothelial cells as the “gate-
keeper” controlling the exchange of information between
blood and tissue has led to a significant interest in
agents which might modulate their function. In par-
ticular endothelin has attracted attention in the recent
literature; a wide variety of structures exhibiting ET-
antagonist properties have been described,4 including
a number which are both highly potent and well-
absorbed. In this and the following articles we describe
our group’s first attempts to develop a series of orally-
active, ETA-selective antagonists.
Our own interest in the area of endothelin receptor
antagonism began with the report by Ishikawa et al.4a
at Banyu of BQ-123 and its congeners. BQ-123, derived
from the screening lead BE-18257, is a potent and
highly selective blocker of the ETA receptor (IC50 ) 22
nM; ETA/ETB > 800); it was also the first low molecular
weight inhibitor to be disclosed. Chemical modification
studies of this cyclic pentapeptide structure quickly led
both Banyu and Fujisawa to develop the linear pseudot-
etrapeptides BQ-4854b and FR-139317.4c Unfortunately
these early compounds, which have excellent biochemi-
cal properties, are unlikely drug candidates due to their
highly peptidic nature. In fact, we have shown (unpub-
lished results) that when FR-139317 is dosed intraduode-
nally to rats at 10 mg/kg, circulating drug levels are
lower than our 10 ng/mL HPLC detection limit.
We hoped to be able to modify the polyamide backbone
of these peptidic antagonists in such a way as to
maintain receptor affinity while improving physical
properties such as gut transport and plasma stability.
We chose to focus on the linear FR-139317/BQ-485
series as the starting point for modification. Believing
that a dramatic departure from the amide linkage would
be required to achieve our goals, we examined the
heterocycles shown in Scheme 1 as dipeptide mimics.
While the replacement of the CO-N-CR-Câ network
with a five-membered ring is formally an isosteric
substitution, the change results in significant restric-
tions of the flexibility of the dipeptide. This rigidifica-
tion is likely (vide infra) to affect receptor interactions.
Ch em istr y
Our synthetic strategy for preparing the heterocyclic
dipeptide surrogates is modelled on the work of Gordon
and co-workers at Sterling-Winthrop.5 We have pre-
pared mimics of both Leu-Trp and Trp-X dipeptides; the
successful routes are described in Schemes 2 and 3,
respectively. In brief, key intermediate 4 (or 7) arises
from sequential C- and N-acylation of glycine anion
equivalent 3. Because of the instability of the interme-
† Structural Biology Department.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
0022-2623/96/1839-0957$12.00/0 © 1996 American Chemical Society

958 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Sch em e 1. Design Strategy
von Geldern et al.
Sch em e 2. Synthesis of Central Dipeptide Surrogatesa
a
(i) LiHMDS, THF, -78 °C; R1COCl; (ii) HCl/H2O; (iii) Cbz-Leu-OCOOiBu, THF, -20 °C; NMM (dropwise); (iv) PPh3, CCl4, pyr, CH3CN;
(v) Lawesson’s reagent, THF, reflux; (vi) LiOH, H2O, THF (heat); (vii) D-Pal-OEt, EDC, HOBt, NMM, THF, DMF; (viii) H2/10% Pd-C,
EtOH (X ) O) or HBr/HOAc (X ) S); (ix) CDI, Et3N, THF; Pha; (x) LiOH, H2O, THF.
Sch em e 3. Synthesis of Terminal Dipeptide Surrogatesa
a
(i) LiHMDS, THF, -78 °C; R2COCl; (ii) HCl/H2O; (iii) Cbz-D-Trp-OCOOiBu, THF, -20 °C; NMM (dropwise); (iv) NH4OAc, HOAc,
reflux; (v) PPh3, CCl4, pyr, CH3CN; (vi) Lawesson’s reagent, THF, reflux; (vii) H2/10% Pd-C, EtOH (X ) NH, O) or HBr/HOAc (X ) S);
(viii) Pha-Leu-OH, EDC, HOBt, NMM, THF, DMF; (x) LiOH, H2O, THF.
diate amino ketone, care is taken to isolate this material
directly as a hydrochloride salt from the imine hydroly-
sis step; this salt is slowly neutralized in the presence
of a preformed active ester of Cbz-L-Leu (or Cbz-D-Trp)
in order to maximize the production of the desired 4 (or
7). Strategically, compounds 4 and 7 are key to our
efforts. Their rapid assembly from a number of readily-
available starting materials allows the exploration of a
variety of substituents on the heterocyclic ring. Ad-
ditionally, the R-amido ketone moiety allows ready

Azole Endothelin Antagonists. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 959
Ta ble 1. Central Dipeptide Surrogates
compd
X
n
IC50 (ETA), µMa
IC50 (ETB), µMa
formula
solvate
characterization
1a
1b
1c
1d
O
S
O
S
0
0
1
1
>100
>90
68
>100
>100
>100
>100
C33H40N6O5
1.3 TFA
2.0 TFA
1.5 TFA
1.5 TFA
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
C
C
C
33H40N6O4S
34H42N6O5
34H42N6O4S
>90
a
IC50’s calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M.
access from a common intermediate to three different
heterocyclic cores. Thus, for example, reaction of 7 with
an ammonium salt results in cyclization to imidazole
8N; dehydration under Mitsonobu’s conditions leads to
the corresponding oxazole 8O; and treatment with
Lawesson’s thiation reagent provides thiazole 8S.
Assembly of the dipeptide mimics into potential ET
antagonists occurs in straightforward fashion. Leu-Trp
mimics 5 (Scheme 2) are saponified and coupled with
D-2-pyridylalanine ethyl ester (D-Pal-OEt) to give pseudot-
ripeptides 6. The amino terminus of 6 is deprotected
and converted to the corresponding urea; hydrolysis of
the ester provides “internal” heterocyclic analogs 1a -
d . Similarly, deprotection of Trp-X mimic 8 (Scheme
3), followed by coupling with L-leucine perhydroazepinyl
urea (Pha-Leu-OH) and hydrolysis, leads to “terminal”
heterocycles 2a -v.
esters generally occurs smoothly at ambient or slightly
elevated temperatures, imidazoles require significantly
more vigorous conditions to effect this transformation.
The hydrolysis reaction is also impeded by the presence
of bulky substituents at the position adjacent to the
ester. When these factors act in combination (e.g.
compounds 2q and 2t) we find that we are unable to
hydrolyze the ester without a significant loss of stere-
ochemical integrity.
Recep tor Bin d in g P r ofile
Compounds 1a -d and 2a -v were evaluated for their
ability to compete with ET-1 for ETA binding sites in
MMQ cell membranes (a rat pituitary cell line known
to contain ETA receptors) and with ET-3 for ETB sites
in porcine cerebellar membranes. Several points are
apparent from an examination of the IC50 data reported
in Tables 1 and 2. With regard to the Leu-Trp linkage,
the results of Table 1 suggest clearly that our hetero-
In some cases the final saponification step is prob-
lematic. While hydrolysis of the oxazole and thiazole
Ta ble 2. Terminal Dipeptide Surrogates
compd
X
R2
Ph
Ph
Ph
Bn
Bn
Bn
Me
Me
Me
Et
Et
Et
n-Pr
n-Pr
n-Pr
i-Pr
i-Pr
i-Pr
c-Pr
c-Pr
c-Pr
IC50 (ETA), µMa
IC50 (ETB), µMa
formula
solvate
characterization
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2p
2q
2r
NH
O
S
NH
O
S
NH
O
S
NH
O
S
NH
O
S
NH
O
S
NH
O
S
51
7.3
51
8
16
>100
>85
>100
69
90
>90
>100
>100
>100
>100
>80
>100
>100
>100
>100
>100
>60
>100
>100
65
C33H40N6O4
1.25 TFA
NMR, MS, CHN
NMR, MS, HRMS
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, HRMS
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHNb
NMR, MS, CHNc
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, CHN
NMR, MS, HRMS
NMR, MS, HRMS
NMR, MS, CHN
NMR, MS, CHN
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
33H39N5O5
33H39N5O4S
34H42N6O4
34H41N5O5
34H41N5O4S
28H38N6O4
28H37N5O5
28H37N5O4S
29H40N6O4
29H39N5O5
29H39N5O4S
30H42N6O4
30H41N5O5
30H41N5O4S
30H42N6O4
30H41N5O5
30H41N5O4S
30H40N6O4
30H39N5O5
30H39N5O4S
1.0 TFA
1.75 TFA
0.6 TFA
39
0.57
1.43
41
1.5
1.9
41
7.8
7.7
36
0.73
2.5
11
1.5 TFA
0.4 TFA
1.0 TFA
1.6 TFA
0.7 TFA
0.6 TFA
1.2 TFA
0.3 TFA
0.4 TFA
1.6 TFA
0.9 TFA
2s
2t
2u
2v
5.2
3.7
24
0.8 TFA
1.2 TFA
>100
a
b
IC50’s calculated using a mean of at least two measurements (all duplicates) for 11 concentrations from 10-10 to 10-5 M. N calculated
11.69, observed 11.23. c N calculated 11.34, observed 11.92.

960 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
von Geldern et al.
cycles are poor replacements for the amide bond frame-
work; analogs 1a -d are all essentially inactive. The
data reported in Table 2 indicate that these dipeptide
“mimetics” are also less-than-perfect surrogates for the
C-terminal dipeptide. Studies by Banyu4b have deter-
mined that an aromatic side chain on the C-terminal
amino acid provides optimal activity in the pseudotet-
rapeptide series, and that there is wide latitude in the
variety of substitution which is tolerated at this position.
Compounds 2a -c, containing an aryl side chain con-
sistent with this optimal substitution pattern, show
little affinity for the ETA or ETB receptor. Our attempts
to impart flexibility to the system by adding a methylene
group as a spacer between the heterocycle and the aryl
group (2d -f) met with little success. On the other
hand, when the size of this side chain was reduced
dramatically, leaving just a methyl group, the resultant
imidazole 2g and oxazole 2h improved significantly in
their ability to bind to the ETA receptor. Affinity for
the ETB receptor remains low. To explore this result
further, we prepared a series of analogs having larger
aliphatic substituents on the heterocycle. While an
ethyl group is also tolerated at this position with only
a slight loss of ETA affinity (2j,k ), longer (2m -p ) or
branched (2q-v) chains result in large decreases in
activity.
The nature of the core heterocycle also has a signifi-
cant effect on potency in the binding assay. When the
substituent is sufficiently small, both imidazoles and
oxazoles are active, with the former tending to be
slightly more potent than the latter. The corresponding
thiazoles, however, are less active by several orders of
magnitude. This surprising result likely reflects the
expansion of the azole ring which occurs upon introduc-
tion of the larger sulfur atom. Importantly, none of the
compounds in the series show any significant affinity
for the ETB receptor, suggesting that it may be possible
to develop selective antagonists based upon these azole
lead structures.
tion electron diffraction studies on the bacteriorhodopsin
receptor.7 The seven transmembrane helices, 20-25
residues in length, were identified through hydropho-
bicity analysis8 of the primary human ETA receptor
sequence,9 accounting for the presence of specific resi-
dues which serve as helix initiators or terminators. A
series of highly-conserved sequences (e.g. G97N98 in helix
I, L122AXXD126 in helix II, D182RY184 in helix III, W210
in helix IV, C318WXP321 in helix VI, and N365P366 in helix
VII) facilitate the alignment of ETA with other GPCR.
The essence of our model, and the way in which it
differs from many other models of GPCR binding, is in
the positioning of ligands within the putative binding
domain. It is our hypothesis that, throughout the family
of GPCR and regardless of whether an individual ligand
functions as an agonist or antagonist, all ligands bind
within the cleft formed between helices II, III, VI, and
VII. This binding mode is exemplified in Figure 1 for
the specific case of ET-1 in the ETA receptor. The
disulfide-linked portion of ET-1 remains in the extra-
cellular space, where it may be involved in interactions
with the extracellular loop regions or the N-terminal
tail of the receptor (these regions are not specifically
located in our model); only the C-terminal hexapeptide
makes contact with the interior of the receptor.
A series of receptor-ligand interactions serve to
stabilize the structure as indicated. Lysine166, on helix
III, forms a salt bridge with the carboxylate side chain
of aspartate-18 of ET-1. This lysine residue is almost
unique to the endothelin family of GPCR but is con-
served among ET receptors. An adjacent residue,
glutamine165 on helix III, is involved in a series of
important hydrogen bonds with the main chain of the
native ligand ET-1; specifically, this residue appears to
form good H-bonding interactions with the amide oxy-
gens of the Cys-His and Asp-Ile amide bonds. The
C-terminal carboxylate of ET interacts with the side
chain carboxyl of Asp126 on helix II. Mutagenesis of this
residue has been demonstrated10 to disrupt function
with little effect on the binding properties of the
receptor. We believe that it is a conformational change
in the side chain of this residue upon agonist binding
that serves as the “trigger” to induce the signal trans-
duction cascade. The two isoleucyl residues fit into well-
defined hydrophobic pockets. Isoleucine-19 tucks be-
It is apparent that the flexible backbone of the
Banyu-Fujisawa pseudotetrapeptides allows their C-
terminal side chains to access a portion of the ETA
receptor which is inaccessible to our conformationally
restricted analogs. The difference in steric require-
ments at these formally analogous positions is substan-
tial; while groups as large as indole or naphthyl are
favored in the earlier series,4b only the smallest alkyl
substituents are tolerated in the analogs of the present
study. The steric restrictions around the heterocyclic
ring in compounds 2 are also emphasized by the lack of
activity of the subtly larger thiazoles. To attempt to
understand both of these surprising results, we turned
to our model of the endothelin receptors.
tween helices II and VII into a space defined by Val130
Leu134, Gly354, Leu355, and Ala357 Isoleucine-20 fits
between helices II and III, where it is bounded by Tyr129
,
.
,
Val130, and Val169. The Trp-21 indole is bound between
helices VI and VII in a hydrophobic region which
includes an edge-face interaction with Phe320, and the
Leu-17 side chain fits into a large space between helices
VI and VII near the top of the receptor.
Our model of the conformation of FR-139317 with the
ETA receptor (Figure 2) takes advantage of many of the
same interactions as are described above for ET-1. The
lysine residue on helix III forms a salt bridge with the
terminal carboxylate of the Fujisawa compound; the
adjacent glutamine again provides a lattice of critical
backbone H-bonds, specifically with the urea CdO and
the Leu-Trp amide NH. The leucyl side chain fills the
hydrophobic pocket which is occupied by Ile-19 of ET-
1, and the indole moiety of the D-tryptophyl residue
replaces the Trp-21 indole in the VI-VII “aromatic”
pocket, albeit with a significantly different orientation.
Mod elin g En d oth elin Recep tor -Liga n d
In ter a ction s
A three-dimensional model of the ETA receptor has
been developed by analogy with our earlier work on the
dopamine D1 and D2 receptors.6 Briefly, we began with
the prediction that the receptor will associate with cell
membranes to produce a bundle of seven transmem-
brane hydrophobic helical segments connected by three
intracellular and three extracellular loops. This predic-
tion is in accord with the current understanding of
GPCR tertiary structure, as supported by high-resolu-

Azole Endothelin Antagonists. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 961
F igu r e 1. ET-1 in ETA receptor.
F igu r e 4. Thiazole 2i modeled into the binding pocket. The
methyl group no longer fits into the helix VI pocket; the water
molecule has been displaced by the sulfur atom.
function as an antagonist, as in fact it does. Our model
also provides an explanation for the latitude of substitu-
tion which is permitted at the C-terminal amino acid
in the Banyu-Fujisawa antagonist series. The orienta-
tion of this terminal Câ-Cγ bond positions a γ-substitu-
ent into the large open area at the extracellular inter-
face of the receptor and directly between the four
helices. Such a substituent encounters relatively few
interactions with the receptor, either of a beneficial or
detrimental nature.
Ra tion a lizin g th e Str u ctu r e-Activity P r ofile
Figure 3 provides the corresponding view of our azole
antagonists (specifically imidazole 2g) in the ETA recep-
tor. To position this compound in the binding pocket,
it was first overlayed with our earlier model of FR-
139317 in regions where the two structures are homolo-
gous; the heterocycle was rotated to create a salt bridge
between the terminal carboxylate and Lys166, and the
resultant receptor-ligand system was energy-mini-
mized. Two aspects of this model-development process
are important in evaluating the relevance of any predic-
tive ability it might demonstrate: first, that this model
of azole binding is merely an extension of the model
developed for the Banyu-Fujisawa series; second, that
none of the information generated during our prelimi-
nary structure-activity study has been used in making
this extension.
F igu r e 2. FR-139317 modeled into the II-III-VI-VII bind-
ing pocket, indicating critical ligand-receptor contacts.
The conversion of the Trp-X dipeptide to a heterocyclic
surrogate has only subtle effects on the positioning of
the amino-terminal functionality between the two se-
ries. While there is some shift of the backbone frame-
work toward helices II and III in the azole series,
corresponding minor adjustments in backbone and side
chain torsion angles serve to locate the isobutyl and
indolyl moieties and the perhydroazepinyl substituent
into the same hydrophobic binding sites. The effects of
rigidification on the position of the C-terminal residue
are profound, however. In particular, it is worthwhile
to evaluate these conformational restrictions in light of
the unusual structure-activity profile observed in the
azole antagonist series. The azole 5-methyl substituent,
which is formally analogous to the γ-carbon of the
C-terminal residue of FR-139317, is no longer able to
F igu r e 3. Imidazole 2g modeled into the binding pocket. The
C5-methyl group nests neatly against helix VI; the highlighted
water molecule is within H-bonding distance of the imidazole
NH.
The hydrophobic group of the urea positions itself in a
nondescript hydrophobic cavity near the top of helix VII.
Two consequences of this alignment are worthy of note.
FR-139317 fails to penetrate deeply enough into the II-
II-VI-VII pocket to interact with the side chain of
Asp126; thus, we would predict that the compound would

962 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
von Geldern et al.
fold back into the large open space between the four
helices. Rather, it is forced into direct contact with helix
VI. As a rule such a close interaction would be strongly
disfavored; however, in this case the methyl group finds
a small hydrophobic “nest” comprised of the backbone
and side chain atoms of Leu324 and Ser325 on helix VI.
This hydrophobic pocket not only provides a set of
beneficial contacts which help to stabilize a small alkyl
substituent at this position on the azole ring but also
its size and shape clearly indicate why larger 5-substi-
tutients are not tolerated. Thus, it appears that our
model of the ETA receptor, and of the binding of
compound 2g within that receptor, is capable of “pre-
dicting” this surprising substituent effect with admi-
rable accuracy.
The other significant surprise encountered in our
early structure-activity studies was the dramatic de-
crease in binding which resulted when the imidazole (or
oxazole) heterocyclic core was replaced with a thiazole
(e.g., 2g or 2h f 2i). To explore the effects of such a
modification computationally, we began with the model
developed for imidazole 2g, directly substituted a sulfur
atom for the imidazole NH, and reminimized the result-
ant receptor/ligand system. The results of this analysis
are shown in Figure 4. The expansion of the azole ring
which results from the introduction of the larger sulfur
atom produces two effects, each of which is detrimental.
First, the C5-methyl group is pushed further into the
Leu324-Ser325 pocket on helix VI, such that a beneficial
interaction is no longer possible. This is obviously an
unacceptable situation, and so, upon minimization, the
alkyl group tips out of this pocket, with the backbone
flexing to accommodate the new conformation. Sec-
ondly, the larger sulfur atom protrudes further into the
solvated interior of the II-III-VI-VII pocket, displac-
ing a putative water molecule and disrupting the
internal solvation of the analog. The importance of such
an effect is difficult to predict a priori. To estimate the
magnitude of energy gained or lost through this per-
turbation, we have compared solvation energies for 2g
and 2i as modeled, using three different solvation
parameter sets. The results (see the Experimental
Section for details) suggest that, on average, thiazole
analog 2i may have approximately 1.7 kcal/mol less
solvation stabilization than the corresponding imidazole.
It is clear that a combination of these two effects can
readily account for the difference in binding energy (70×
difference in binding affinity ∼2.6 kcal/mol) we observe
between these two species. Once again, it appears that
the model provides a neat explanation for an apparently
surprising result.
employed in these modeling efforts, the resultant model
not only helps to explain why the two SAR profiles are
different but also accounts for several surprising aspects
of the profile of the new series. This “pseudopredictive”
ability is noteworthy and suggests the possibility that
the model may accurately reflect the details of ET/ET-R
binding.
Exp er im en ta l Section
Unless otherwise specified, all solvents and reagents were
obtained from commercial suppliers and used without further
purification. THF was dried over sodium and purified by
distillation. All reactions were performed under nitrogen
atmosphere unless specifically noted. All final products are
analyzed for purity by analytical HPLC using a 25-cm Vydac
Protein and Peptide C18 column and are >95% pure unless
otherwise stated. 1H-NMR spectra were recorded at 300 MHz;
all values are referenced to tetramethylsilane as internal
standard and are reported as shift (multiplicity, coupling
constants). Mass spectral analysis is accomplished using fast
atom bombardment (FAB-MS) or direct chemical ionization
(DCI-MS) techniques. All elemental analyses are consistent
with theoretical values to within (0.4% unless indicated.
Abbr evia tion s: CDI, 1,1′-carbonyldiimidazole; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DMF, dimethylformamide; EDC,
1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochlo-
ride; HOBt, 1-hydroxybenzotriazole hydrate; LiHMDS, lithium
hexamethyldisilazide; NMM, N-methylmorpholine; Pal, 2-py-
ridylalanine; Pha, hexamethyleneimine (perhydroazepine);
PPh3, triphenylphosphine; pyr, pyridine; TFA, trifluoroacetic
acid; THF, tetrahydrofuran.
Gen er a l Syn th esis of Com p ou n d s 1. Cbz-L-Leu cyl-2-
[(1-m eth ylin d olyl)-3-ca r bon yl]glycin e Eth yl Ester (4, R 1
) 3-[1-m eth ylin dole]). 1-Methylindole-3-carboxylic acid (1.75
g) was suspended in 10 mL of CH2Cl2; 1.05 mL of oxalyl
chloride was added (gas evolves), and the resultant solution
was stirred at room temperature for 90 min. The solvents
were removed in vacuo, and the residue was dried under
vacuum to remove traces of reagent from the crude acid
chloride, which was used without further purification. Simul-
taneously, N-(diphenylmethylene)glycine ethyl ester (2.68 g)
was dissolved in THF (15 mL), and the solution was cooled to
-78 °C. LiHMDS (10 mL, 1 N solution in THF) was added
dropwise, and the resultant yellow slurry was stirred at -78
°C for 30 min. The slurry was transferred via cannula to a
solution of the above acid chloride in THF (10 mL) at -78 °C.
After the addition was complete, the reaction mixture was
allowed to warm to room temperature and stirring was
continued for 2 h. The reaction was then quenched with 2 N
HCl (120 mL) and allowed to stir at room temperature for 1
h. The THF was evaporated, and the resultant aqueous
solution was extracted with EtOAc (2 × 10 mL). The organic
phases were reextracted with 2 N HCl and then discarded;
the combined aqueous phase was concentrated in vacuo. The
resultant slurry was treated with EtOH (20 mL) and filtered.
The filtrate was concentrated in vacuo to give 2-[(1-methylin-
dol-3-yl)carbonyl]glycine ethyl ester hydrochloride as a pinkish
solid which was used without further purification. (Note: this
material may be stored at -20 °C as the salt, but is unstable
as the free base.) Cbz-L-leucine (2.65 g) was dissolved in THF
(15 mL), and the solution was cooled to -20 °C. N-Methyl-
morpholine (1.11 mL, 1.0 equiv) was added, followed by the
dropwise addition of isobutylchloroformate (1.30 mL). After
the addition was complete, the reaction mixture was stirred
for 30 min at -20 °C. The cooling bath was removed; the
above salt was dissolved in DMF (20 mL) and added to the
mixed anhydride. N-Methylmorpholine (1.2 mL) was added
dropwise via syringe pump over a 1 h period. After the
addition was complete, the reaction mixture was allowed to
stir at room temperature for 15 h. The solvents were removed
in vacuo, and the residue was taken up in EtOAc. The organic
layer was washed sequentially with saturated NaHCO3 solu-
tion, 1 N H3PO4, and brine, dried over MgSO4, and evaporated
under reduced pressure to give an orange oil which was
Con clu sion s
We have developed a novel family of antagonists
selective for the ETA receptor by replacing the C-
terminal dipeptide of the known peptidic antagonist FR-
139317 with a heterocyclic dipeptide surrogate. While
this replacement is formally isosteric, the resultant
series exhibits a markedly different structure-activity
profile than was previously reported for the parent
structure. We have simultaneously developed a com-
puter model of the ETA receptor by analogy with our
earlier work in the area of G-protein-coupled receptors
and have applied this model to examine both the
peptidic and peptidomimetic analog structures. Despite
the fact that no structure-activity information was

Azole Endothelin Antagonists. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 963
purified by flash chromatography on silica gel eluting with a
gradient of 1:2 f 1:1 EtOAc/hexane. The title compound was
isolated as a yellowish oil (3.45 g, 68% overall yield).
th ylbu tyl]-5-[[3-(1-m eth ylin d olyl)m eth yl]oxa zol-4-yl]ca r -
bon yl}-D-2-p yr id yla la n in e (1c): 1H NMR (CD3OD, 300 MHz)
δ 0.88 (d, 3H, J ) 7), 0.92 (d, 3H, J ) 7 Hz), 1.47 (m, 4H), 1.64
(m, 5H), 1.74 (m, 2H), 3.36 (m, 4H), 3.47 (dd, 1H, J ) 9, 15
Hz), 3.71 (dd, 1H, J ) 6, 15 Hz), 3.75 (s, 3H), 4.39, (AB, 2H, J
) 15 Hz), 4.97 (dd, 1H, J ) 6, 9 Hz), 5.08 (d, 1H, J ) 7, 9 Hz),
6.99 (ddd, 1H, J ) 1, 7, 8 Hz), 7.01 (s, 1H), 7.16 (ddd, 1H, J )
1, 7, 8 Hz), 7.29 (d, 1H, J ) 8 Hz), 7.45 (d, 1H, J ) 8 Hz), 7.70
(ddd, 1H, J ) 1, 7, 8 Hz), 7.84 (d, 1H, J ) 8 Hz), 8.24 (dt, 1H,
J ) 1, 8 Hz), 8.65 (ddd, 1H, J ) 1, 2, 6 Hz); MS (FAB/NBA)
2-[1(S)-[(Ben zyloxyca r bon yl)a m in o]-3-m eth ylbu tyl]-5-
[3-(1-m eth ylin d olyl)]oxa zole-4-ca r boxylic Acid Eth yl Es-
ter (5O, R1 ) 3-[1-m eth ylin d ole]). Compound 4 (500 mg)
was dissolved in 3 mL of 1:1 CH3CN/pyr; 0.2 mL of CCl4, 0.5
mL of DBU, and 285 mg of PPh3 were added sequentially, and
the resultant solution was allowed to stir at room temperature
for 18 h. The solvents were evaporated under reduced pres-
sure, and the residue was dissolved in EtOAc. The solution
was washed with saturated NaHCO3 solution, 1 N H3PO4, and
brine, dried over MgSO4, and evaporated to give a yellowish
oil which was purified by flash chromatography on silica gel
eluting with 25% EtOAc/hexane to give the title compound
(208 mg, 43% yield).
2-[1(S)-[(Ben zyloxyca r bon yl)a m in o]-3-m eth ylbu tyl]-5-
[3-(1-m eth ylin d olyl)]th ia zole-4-ca r boxylic Acid Eth yl Es-
ter (5S, R1 ) 3-[1-m eth ylin d ole]). Compound 4 above (300
mg) was dissolved in 4 mL of THF; 300 mg of Lawesson’s
reagent were added, and the resultant solution was heated at
80 °C for 4 h. The solvents were evaporated under reduced
pressure, and the residue was dissolved in EtOAc. The
solution was washed with saturated NaHCO3 solution, 1 N
H3PO4, and brine, dried over MgSO4, and evaporated to give
a yellowish oil which was purified by flash chromatography
on silica gel eluting with 25% EtOAc/hexane to give the title
compound (248 mg, 83% yield).
{[2-[1(S)-[(P er h yd r oa zep in -1-ylca r bon yl)a m in o]-3-m e-
th ylbu tyl]-5-[3-(1-m eth ylin d ole)]oxa zol-4-yl]ca r bon yl}-D-
2-p yr id yla la n in e (1a ). Compound 5O described above (100
mg) was dissolved in 2 mL of THF; a solution of 50 mg of LiOH
in 1 mL of water was added, and the resultant mixture was
heated at 80 °C for 6 h. The mixture was acidified with 1 N
H3PO4 and extracted with EtOAc. The organic phase was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude acid was taken up in 4 mL of 1:1 THF/DMF;
50 mg of D-Pal-OEt‚HCl was added, followed by 27 mg of
HOBt, 0.2 mL of NMM, and 39 mg of EDC. The resultant
solution was stirred overnight. The solvents were removed
in vacuo, and the residue was taken up in EtOAc. The organic
layer was washed sequentially with saturated NaHCO3 solu-
tion and brine, dried over Na2SO4, and evaporated under
reduced pressure to give an orange oil which was purified by
flash chromatography on silica gel eluting with a gradient of
2:1 f 3:1 EtOAc/hexane. The product (45 mg) was dissolved
in 4 mL of EtOH; 20 mg of 10% Pd-C was added, and the
mixture was purged with nitrogen. A balloon of hydrogen was
placed on the reaction vessel, and the mixture was stirred at
room temperature for 2 h. The vessel was again purged with
nitrogen, and the mixture was filtered through Celite to
remove the catalyst. The solvents were removed in vacuo, and
the crude product was taken up in 1 mL of THF. Et3N (0.1
mL) was added, followed by 20 mg of CDI; the reaction mixture
was stirred at room temperature for 2 h. Perhydroazepine (0.2
mL) was added, and stirring was continued for 50 h. EtOAc
was added; the reaction mixture was extracted with saturated
NaHCO3 solution, 1 N H3PO4, and brine, dried over MgSO4,
and evaporated to give a yellowish oil. The crude product was
dissolved in 1 mL of THF; a solution of 25 mg of LiOH in 0.5
mL water was added, and the resultant mixture was stirred
at room temperature for 3.5 h. The solution was acidified with
1 N H3PO4, and the product was purified by preparative HPLC
(Vydac µC18) eluting with a 10-70% gradient of CH3CN in
0.1% TFA. The desired fractions were lyophilized to give the
product as a white solid: 21 mg; 1H NMR (CD3OD, 300 MHz)
δ 1.04 (t, 3H, J ) 7), 1.07 (t, 3H, J ) 7), 1.55 (m, 4H), 1.76 (m,
5H), 1.95 (m, 2H), 3.50 (m, 5H), 3.70 (dd, 1H, J ) 6, 15), 3.87
(s, 3H), 5.07 (dd, 1H, J ) 5, 9), 5.18 (d, 1H, J ) 7, 9), 7.18
(ddd, 1H, J ) 1, 7, 8), 7.28 (ddd, 1H, J ) 1, 7, 8), 7.45 (d, 1H,
J ) 8), 7.73 (ddd, 1H, J ) 1, 7, 8), 7.85 (d, 1H, J ) 8), 8.12 (d,
1H, J ) 8), 8.26 (dt, 1H, J ) 1, 8), 8.55 (s, 1H), 8.67 (ddd, 1H,
J ) 1, 2, 6); MS (FAB/NBA) m/ e 601 (M + H)+. Anal. for
C33H40N6O5‚1.3TFA: C, H, N.
m/ e 615 (M + H)+, 637 (M + Na)+. Anal. for C34H42
N6O5‚1.5TFA: C, H, N.
-
{[2-[1(S)-[(P er h yd r oa zep in -1-ylca r bon yl)a m in o]-3-m e-
th ylbu tyl]-5-[3-(1-m eth ylin d olyl)]th ia zol-4-yl]ca r bon yl}-
D-2-p yr id yla la n in e (1b). Compound 5S described above (90
mg) was dissolved in 2 mL of THF; a solution of 50 mg of LiOH
in 1 mL of water was added, and the resultant mixture was
heated at 80 °C for 4 h. The mixture was acidified with 1 N
H3PO4 and extracted with EtOAc. The organic phase was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude acid was taken up in 4 mL of 1:1 THF/DMF;
50 mg of D-Pal-OEt‚HCl was added, followed by 27 mg of
HOBt, 0.2 mL of NMM, and 39 mg of EDC. The resultant
solution was stirred overnight. The solvents were removed
in vacuo, and the residue was taken up in EtOAc. The organic
layer was washed sequentially with saturated NaHCO3 solu-
tion and brine, dried over Na2SO4, and evaporated under
reduced pressure to give an orange oil which was purified by
flash chromatography on silica gel eluting with a gradient of
2:1 f 3:1 EtOAc/hexane. The product (64 mg) was dissolved
in 2 mL of 30% HBr/HOAc and stirred at room temperature
for 2 h. The solvents were removed in vacuo; the residue was
taken up in saturated NaHCO3 solution and extracted with
EtOAc. The organic layer was washed with brine and dried
over Na2SO4. The solvents were removed in vacuo, and the
crude product was taken up in 2 mL of THF. Et3N (0.1 mL)
was added, followed by 25 mg of CDI; the reaction mixture
was stirred at room temperature for 2.5 h. Perhydroazepine
(0.2 mL) was added, and stirring was continued for 15 h.
EtOAc was added; the reaction mixture was extracted with
saturated NaHCO3 solution, 1 N H3PO4, and brine, dried over
Na2SO4, and evaporated to give a yellowish oil. The crude
product was dissolved in 1 mL of THF; a solution of 40 mg of
LiOH in 0.5 mL water was added, and the resultant mixture
was stirred at room temperature for 3 h. The solution was
acidified with 1 N H3PO4, and the product was purified by
preparative HPLC (Vydac µC18) eluting with a 10-70%
gradient of CH3CN in 0.1% TFA. The desired fractions were
lyophilized to give the product as a white solid: 19 mg; 1H
NMR (CD3OD, 300 MHz) δ 1.05 (t, 6H, J ) 7), 1.61 (m, 4H),
1.77 (m, 4H), 1.90 (m, 3H), 3.50 (m, 5H), 3.68 (dd, 1H, J ) 6,
15), 3.83 (s, 3H), 4.98 (dd, 1H, J ) 3, 9), 5.23 (d, 1H, J ) 6, 9),
1
7.13 (ddd, H, J ) 1, 7, 8), 7.26 (ddd, 1H, J ) 1, 7, 8), 7.44 (d,
1H, J ) 10), 7.64 (d, 1H, J ) 9), 7.73 (ddd, 1H, J ) 1, 7, 8),
7.77 (s, 1H), 7.87 (d, 1H, J ) 8), 8.28 (dt, 1H, J ) 1, 8), 8.62
(ddd, 1H, J ) 1, 2, 6); MS (FAB/NBA) m/ e 617 (M + H)+. Anal.
for C33H40N6O4S‚2.0TFA: C, H, N.
Also prepared by a similar procedure was the following.
{[2-[1(S)-[(P er h yd r oa zep in -1-ylca r bon yl)a m in o]-3-m e-
th ylbu tyl]-5-[[3-(1-m eth ylin dolyl)m eth yl]th iazol-4-yl]car -
bon yl}-D-2-p yr id yla la n in e (1d ). 1H NMR (CD3OD, 300
MHz) δ 0.95 (t, 6H, J ) 7 Hz), 1.43 (m, 4H), 1.60 (m, 4H), 1.76
(m, 3H), 3.33 (m, 4H), 3.52 (dd, 1H, J ) 9, 15 Hz), 3.72 (dd,
1H, J ) 6, 15 Hz), 3.75 (s, 3H), 4.58 (s, 2H), 5.06 (dd, 1H, J )
1, 7 Hz), 5.09 (d, 1H, J ) 7 Hz), 6.97 (ddd, 1H, J ) 1, 7, 8 Hz),
7.04 (s, 1H), 7.15 (ddd, 1H, J ) 1, 7, 8 Hz), 7.32 (d, 1H, J ) 10
Hz), 7.35 (d, 1H, J ) 9 Hz), 7.74 (ddd, 1H, J ) 1, 7, 8 Hz),
7.87 (d, 1H, J ) 8 Hz), 8.30 (dt, 1H, J ) 1, 8 Hz), 8.69 (ddd,
1H, J ) 1, 2, 6 Hz); MS (FAB/CH3OH) m/ e 631 (M + H)+, 669
(M + K)+. Anal. Calcd for C34H42N6O4S‚1.5TFA: C, H, N.
Gen er a l Syn th esis of Com p ou n d s 2. Cbz-D-tr yp top h a -
n yl-2-a cetylglycin e Eth yl Ester (7, R2 ) Me). N-(Diphe-
nylmethylene)glycine ethyl ester (30.0 g) was dissolved in THF
(125 mL) and the solution cooled to -78 °C. LiHMDS (100
mL, 1 N solution in THF) was added slowly over 10 min, and
Also prepared by a similar procedure was the following.
{[2-[1(S)-[(P er h yd r oa zep in -1-ylca r bon yl)a m in o]-3-m e-

964 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
von Geldern et al.
the resultant yellow slurry was stirred at -78 °C for 45 min.
The slurry was transferred via cannula to a solution of acetyl
chloride (8.4 mL) in THF (50 mL) at -78 °C. Additional THF
(250 mL) was added to the anion solution to facilitate transfer
to the acetyl chloride solution. After the addition was com-
plete, the reaction mixture was allowed to warm to room
temperature, and stirring was continued for 4 h. The reaction
was then quenched with 2 N HCl (115 mL). The THF was
evaporated, and the resultant aqueous solution was extracted
with EtOAc (2 × 100 mL). The organic phases were discarded,
and the aqueous phase was concentrated in vacuo. The
resultant slurry was treated with EtOH (150 mL) and filtered.
The filtrate was concentrated in vacuo to give 2-acetylglycine
ethyl ester hydrochloride as a yellow solid which was used
without further purification. (Note: this material may be
stored at -20 °C as the salt, but is unstable as the free base.)
Cbz-D-tryptophan (40.6 g) was dissolved in THF (100 mL), and
the solution was cooled to -20 °C. N-Methylmorpholine (13
mL, 1.0 equiv) was added, followed by the dropwise addition
of isobutyl chloroformate (15.6 mL). After the addition was
complete, the reaction mixture was stirred for 30 min at -20
°C. The cooling bath was removed; the above salt was
dissolved in DMF (50 mL) and added to the mixed anhydride.
N-Methylmorpholine (13 mL) was added dropwise via syringe
pump over a 1 h period. After the addition was complete, the
reaction mixture was allowed to stir at room temperature for
1 h. Water (200 mL) was added, and the layers were
separated. The organic layer was washed sequentially with
saturated NaHCO3 solution, 1 N H3PO4, and brine, dried over
MgSO4, and evaporated under reduced pressure to give an
orange oil which was purified by flash chromatography on
silica gel eluting with 15% EtOAc/hexane. The title compound
was isolated as an orange oil (30.7 g, 59% yield overall). 1H
NMR (CDCl3, 300 MHz) δ 1.26 (dt, 3H, J ) 1, 7 Hz), 2.24 (s,
1.5H), 2.30 (s, 1.5H), 3.20 (m, 1H), 3.35 (m, 1H), 4.22 (dq, 2H,
J ) 1, 7 Hz), 4.50 (m, 1H), 5.06 (dd, 1H, J ) 7, 8 Hz), 5.12 (s,
2H), 5.45 (m, 1H), 6.82 (m, 1H), 7.23 (m, 9H), 7.65 (m, 1H),
8.10 (s, 1H); MS (DCI/NH3) m/ e 466 (M + H)+, 483 (M +
NH4)+.
2-{1(R)-[(Ben zyloxyca r bon yl)a m in o]-2-(in d ol-3-yl)eth -
yl}-5-m eth yloxa zole-4-ca r boxylic Acid Eth yl Ester (8O,
X ) O, R2 ) Me). Compound 7 described above (4.44 g) was
dissolved in acetonitrile (15 mL). Pyridine (25 mL), carbon
tetrachloride (2 mL), DBU (2.90 g), and PPh3 (2.75 g) were
added, and the mixture was stirred for 16 h at room temper-
ature. The solvents were evaporated under reduced pressure,
and the residue was dissolved in EtOAc. The solution was
washed with saturated NaHCO3 solution, 1 N H3PO4, and
brine, dried over MgSO4, and evaporated to give an off-white
semisolid which was purified by flash chromatography on silica
gel eluting with 15% EtOAc/hexane to give the title compound
(2.83 g, 66% yield): 1H NMR (CDCl3, 300 MHz) δ 1.37 (t, 3H,
J ) 7 Hz), 2.50 (s, 3H), 3.40 (d, 2H, J ) 8 Hz), 4.37 (q, 2H, J
) 7 Hz), 5.08 (s, 2H), 5.28 (m, 1H), 5.48 (m, 1H), 6.89 (s, 1H),
7.05 (t, 1H, J ) 8 Hz), 7.16 (t, 1H, J ) 8 Hz), 7.35 (m, 7H),
8.06 (s, 1H); MS (DCI/NH3) m/ e 448 (M + H)+, 465 (M +
NH4)+.
2-{1(R)-[(Ben zyloxyca r bon yl)a m in o]-2-(in d ol-3-yl)eth -
yl}-5-m eth ylth ia zole-4-ca r boxylic Acid Eth yl Ester (8S,
X ) S, R2 ) Me). Compound 7 described above (0.335 g) was
dissolved in THF (5 mL). Lawesson’s reagent (0.45 g) was
added, and the mixture was stirred at reflux for 5 h. The
solvent was evaporated under reduced pressure, and the
residue was taken up in EtOAc (20 mL). The solution was
washed with saturated NaHCO3 solution, 1 N H3PO4, and
brine, dried with MgSO4, and evaporated under reduced
pressure to give a yellow oil which was purified by flash
chromatography eluting with 15% EtOAc/hexane to give the
product as a white solid (155 mg, 46%): 1H NMR (CDCl3, 300
MHz) δ 1.43 (t, 3H, J ) 7 Hz), 2.65 (s, 3H), 3.42 (m, 1H), 3.55
(m, 1H), 4.43 (q, 2H, J ) 7 Hz), 5.09 (s, 2H), 5.38 (m, 1H),
5.63 (br s, 1H), 6.90 (s, 1H), 7.07 (t, 1H, J ) 8 Hz), 7.18 (t, 1H,
J ) 8 Hz), 7.32 (m, 6H), 7.52 (1H, d, J ) 8 Hz), 8.01 (s, 1H);
MS (DCI/NH3) m/ e 464 (M + H)+, 481 (M + NH4)+.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-m eth ylim id a zole-4-ca r boxylic Acid
(2g). Imidazole 8N described above (90 mg) was dissolved in
EtOH (50 mL). The solution was purged of oxygen, 10% Pd/C
(0.5 g) was added, and the mixture was stirred at room
temperature under an atmosphere of hydrogen. After 2 h the
catalyst was removed by filtration, and the solvent was
evaporated in vacuo to give a white solid. This crude material
was dissolved in THF (2 mL). HOBt (30 mg), the above-
described leucine derivative (55 mg), and EDC (42 mg) were
added. N-Methylmorpholine (0.1 mL) and DMF (1 mL) were
added, and the mixture was stirred at room temperature for
18 h. The solvent was evaporated under reduced pressure,
and the residue was taken up in EtOAc. The solution was
washed with saturated NaHCO3 solution, 1 N H3PO4, and
brine, dried over MgSO4, and evaporated in vacuo to give an
orange oil which was purified by flash chromatography on
silica gel eluting with 50% EtOAc/hexane. This material was
dissolved in THF (2 mL), and a solution of LiOH (50 mg) in
H2O (1 mL) was added. The mixture was heated in a Carius
tube at 100 °C for 15 h. The solvents were evaporated under
reduced pressure, and the residue was taken up in 1 N H3-
PO4 (5 mL). The suspension was dissolved in water and
acetonitrile, and the product was purified by preparative
HPLC (Vydac µC18) eluting with a 10-70% gradient of CH3-
CN in 0.1% TFA. The desired fractions were lyophilized to
(P er h yd r oa zep in -1-ylca r bon yl)leu cin e. Leucine benzyl
ester p-toluenesulfonate (7.88 g) was dissolved in THF (40 mL).
Et3N (2.92 mL) was added, and the solution was cooled to 0
°C in an ice bath. Carbonyldiimidazole (3.40 g) was added and
the solution stirred at 0 °C for 1 h. The cooling bath was
removed, and the solution was stirred an additional 1 h at
room temperature. Perhydroazepine (2.68 mL) was added, and
the resultant solution was stirred overnight at room temper-
ature. The reaction mixture was poured onto 400 mL of water;
the mixture was stirred for 30 min, and a white solid was
collected by filtration. The crude ester was dissolved in MeOH
(150 mL), 10% Pd/C (0.9 g) was added, and the mixture was
purged with a stream of nitrogen gas and then placed under
a ballon of hydrogen. After stirring at room temperature for
2 h, the reaction mixture was again purged with nitrogen. The
solvent was removed in vacuo and the residue taken up in
EtOAc and filtered through Celite to remove the catalyst. The
solvent was evaporated in vacuo to give the carboxylic acid as
a white solid (5.06 g, 98%).
2-{(1R)-1-[(Ben zyloxyca r b on yl)a m in o]-2-(in d ol-3-yl)-
eth yl}-5-m eth ylim id a zole-4-ca r boxylic Acid Eth yl Ester
(8N, X ) NH, R2 ) Me). Compound 7 described above (5.0
g) was dissolved in acetic acid (15 mL). Ammonium acetate
(4.0 g) was added, and the mixture was heated at reflux for
16 h. After cooling, the solvent was evaporated under reduced
pressure and the residue was taken up in saturated NaHCO3
solution and extracted with EtOAc. The combined organic
extracts were dried over MgSO4 and concentrated in vacuo.
The resultant orange oil was purified by flash chromatography
on silica gel eluting with 25% EtOAc/hexane to afford 3.60 g
(73%) of the title compound. 1H NMR (CDCl3, 300 MHz) δ
1.31 (t, 3H, J ) 7 Hz), 2.43 (s, 3H), 3.37 (m, 1H), 3.47 (m, 1H),
4.27 (q, 2H, J ) 7 Hz), 5.03 (s, 2H), 5.07 (m, 1H), 5.26 (br s,
1H), 6.82 (s, 1H), 7.04 (t, 1H, J ) 8 Hz), 7.16 (t, 1H, J ) 8 Hz),
7.30 (m, 5H), 7.50 (d, 1H, J ) 8 Hz), 8.05 (s, 1H); MS (DCI/
NH3) m/ e 447 (M + H)+.
1
give the product as a white solid: 40 mg; H NMR (CD3OD,
300 MHz) δ 0.77 (d, 3H, J ) 7 Hz), 0.82 (d, 3H, J ) 7 Hz),
1.30 (m, 2H), 1.52 (m, 5H), 1.66 (m, 4H), 2.52 (s, 3H), 3.4 (m,
6H), 3.59 (dd, 1H, J ) 7, 15 Hz), 4.07 (dd, 1H, J ) 5, 9 Hz),
5.42 (dd, 1H, J ) 6, 8 Hz), 7.01 (ddd, 1H, J ) 1, 7, 8 Hz), 7.11
(ddd, 1H, J ) 1, 7, 8 Hz), 7.12 (s, 1H), 7.36 (d, 1H, J ) 8 Hz),
7.45 (d, 1H, J ) 8 Hz); MS (DCI/NH3) m/ e 523 (M + H)+. Anal.
for C28H38N6O4‚1.5TFA: C, H, N.
The following compounds are prepared using the procedures
described above for compound 2g:
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-p h en ylim id a zole-4-ca r boxylic a cid
1
(2a ): H NMR (CD3OD, 300 MHz) δ 0.80 (d, 3H, J ) 6 Hz),
0.85 (d, 3H, J ) 6 Hz), 1.2-1.45 (m, 10H), 3.2 (m, 4H), 3.43
(dd, 1H, J ) 8, 15 Hz), 3.52 (dd, 1H, J ) 6, 7 Hz), 3.62 (dd,

Azole Endothelin Antagonists. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 965
1H, J ) 6, 15 Hz), 4.13 (t, 1H, J ) 7 Hz), 5.43 (dd, 1H, J ) 6,
8 Hz), 6.96 (ddd, 1H, J ) 1, 7, 8 Hz), 7.10 (ddd, 1H, J ) 2, 7,
8 Hz), 7.14 (s, 1H), 7.36 (ddd, 1H, J ) 1, 2, 8 Hz), 7.45 (m,
4H), 7.69 (dt, 1H, J ) 2, 8 Hz), 7.57 (dd, 1H, J ) 2, 7 Hz); MS
(FAB + NBA) m/ e 585 (M + H)+, 647 (M + Cu)+. Anal. for
C33H40N6O4‚1.25TFA: C, H, N.
at room temperature for 15 h, at which point it had become
homogeneous. The solvents were evaporated under reduced
pressure, and the residue was neutralized with 1 N H3PO4 and
purified by preparative HPLC (Vydac µC18) eluting with a 10-
70% gradient of CH3CN in 0.1% TFA. The desired fractions
were lyophilized to give the product as a white solid: 66 mg;
1H NMR (CD3OD, 300 MHz) δ 0.87 (d, 3H, J ) 7 Hz), 0.88 (d,
3H, J ) 7 Hz), 1.43 (m, 2H), 1.52 (m, 5H), 1.67 (m, 4H), 2.55
(s, 3H), 3.25-3.5 (m, 6H), 4.34 (dd, 1H, J ) 6, 9 Hz), 5.40 (t,
1H, J ) 7 Hz), 6.95 (ddd, 1H, J ) 1, 7, 8 Hz), 6.99 (s, 1H), 7.07
(ddd, 1H, J ) 1, 7, 8 Hz), 7.31 (td, 1H, J ) 1, 8 Hz), 7.37 (d,
1H, J ) 8 Hz); MS (DCI/NH3) m/ e 524 (M + H)+, 541 (M +
NH4)+. Anal. for C28H37N5O5‚0.4TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-ben zylim id a zole-4-ca r boxylic a cid
1
(2d ): H NMR (CD3OD, 300 MHz) δ 0.81 (d, 3H, J ) 6 Hz),
0.85 (d, 3H, J ) 6 Hz), 1.36 (m, 1H), 1.51 (m, 5H), 1.63 (m,
5H), 3.2-3.6 (m, 7H), 4.09 (dd, 1H, J ) 6, 8 Hz), 4.24 (d, 1H,
J ) 15 Hz), 4.36 (d, 1H, J ) 15 Hz), 5.37 (dd, 1H, J ) 7, 8 Hz),
6.94 (ddd, 1H, J ) 1, 7, 8 Hz), 7.04 (s, 1H), 7.1 (d, 2H, J ) 8
Hz), 7.2-7.3 (m, 4H), 7.34 (dd, 2H, J ) 1, 8 Hz). MS (FAB +
G/SG) m/ e 599 (M + H)+. Anal. Calcd for C34H42N6-
O4‚1.75TFA: C, H, N.
The following compounds are prepared using the procedures
described above for compound 2h :
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in dol-3-yl)eth yl}-5-ph en yloxazole-4-car boxylic acid (2b):
1H NMR (CDCl3, 300 MHz) δ 0.84 (d, 3H, J ) 6 Hz), 0.85 (d,
3H, J ) 6 Hz), 1.47 (m, 7H), 1.62 (m, 4H), 3.2-3.4 (m, 5H),
3.47 (d, 2H, J ) 7 Hz), 4.40 (br q, 1H, J ) 8 Hz), 4.87 (br d,
1H, J ) 8 Hz), 5.60 (q, 1H, J ) 8 Hz), 7.02 (d, 1H, J ) 1 Hz),
7.07 (t, 1H, J ) 8 Hz), 7.16 (t, 1H, J ) 8 Hz), 7.33 (d, 1H, J )
8 Hz), 7.40 (m, 4H), 7.53 (d, 1H, J ) 8 Hz), 7.94 (m, 2H), 8.13
(br s, 1H); MS (FAB + NBA) m/ e 586 (M + H)+; HRMS calcd
for C33H40N5O5 586.3029, found 586.3033.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in dol-3-yl)eth yl}-5-ben zyloxazole-4-car boxylic acid (2e):
1H NMR (CDCl3, 300 MHz) δ 0.84 (d, 3H, J ) 6 Hz), 0.85 (d,
3H, J ) 6 Hz), 1.50 (m, 7H), 1.66 (m, 4H), 3.2-3.5 (m, 6H),
4.18 (d, 1H, J ) 15 Hz), 4.32 (d, 1H, J ) 15 Hz), 4.45 (br q,
1H, J ) 8 Hz), 4.94 (br d, 1H, J ) 8 Hz), 5.46 (q, 1H, J ) 8
Hz), 6.70 (d, 1H, J ) 2 Hz), 7.03 (dt, 1H, J ) 1, 8 Hz), 7.14
(dt, 1H, J ) 1, 8 Hz), 7.19 (m, 2H), 7.26 (m, 5H), 7.37 (br s,
1H), 7.46 (d, 1H, J ) 8 Hz), 7.88 (br s, 1H); MS (FAB + NBA)
m/ e 600 (M + H)+, 622 (M + Na)+. Anal. for C34H41N5-
O5‚0.6TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)et h yl}-5-et h ylim id a zole-4-ca r b oxylic a cid
(2j): 1H NMR (CD3OD, 300 MHz) δ 0.81 (d, 3H, J ) 6 Hz),
0.85 (d, 3H, J ) 7 Hz), 1.15 (t, 3H, J ) 8 Hz), 1.3-1.4 (m, 2H),
1.53 (m, 5H), 1.67 (m, 4H), 2.9 (m, 2H), 3.3-3.6 (m, 6H), 4.10
(dd, 1H, J ) 6, 8 Hz), 5.36 (dd, 1H, J ) 7, 8 Hz), 6.98 (ddd,
1H, J ) 1, 7, 8 Hz), 7.10 (ddd, 1H, J ) 1, 7, 8 Hz), 7.12 (s, 1H),
7.36 (d, 2H, J ) 8 Hz); MS (DCI/NH3) m/ e 537 (M + H)+. Anal.
Calcd for C29H40N6O4‚1.6TFA: C, H; N: calcd, 11.69; found,
11.23.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-p r op ylim id a zole-4-ca r boxylic a cid
(2m ): 1H NMR (CD3OD, 300 MHz) δ 0.79 (d, 3H, J ) 6 Hz),
0.84 (d, 3H, J ) 7 Hz), 0.88 (t, 3H, J ) 8 Hz), 1.3-1.4 (m, 2H),
1.53 (m, 5H), 1.60 (m, 2H), 1.68 (m, 4H), 2.94 (m, 2H), 3.3-
3.6 (m, 6H), 4.10 (dd, 1H, J ) 7, 9 Hz), 5.35 (dd, 1H, J ) 7, 8
Hz), 6.97 (ddd, 1H, J ) 1, 7, 8 Hz), 7.07 (s, 1H), 7.10 (ddd, 1H,
J ) 1, 7, 8 Hz), 7.35 (d, 1H, J ) 8 Hz), 7.40 (d, 1H, J ) 8 Hz);
MS (DCI/NH3) m/ e 551 (M + H)+. Anal. for C30H42N6-
O4‚1.2TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in dol-3-yl)eth yl}-5-eth yl-oxazole-4-car boxylic acid (2k):
1H NMR (CD3OD, 300 MHz) δ 0.87 (d, 3H, J ) 7 Hz), 0.88 (d,
3H, J ) 7 Hz), 1.14 (t, 3H, J ) 8 Hz), 1.4 (m, 2H), 1.54 (m,
5H), 1.68 (m, 4H), 2.94 (dq, 2H, J ) 2, 8 Hz), 3.2-3.5 (m, 6H),
4.37 (dd, 1H, J ) 6, 10 Hz), 5.39 (t, 1H, J ) 6 Hz), 6.94 (ddd,
1H, J ) 1, 7, 8 Hz), 6.98 (s, 1H), 7.07 (ddd, 1H, J ) 1, 7, 8 Hz),
7.33 (td, 1H, J ) 1, 8 Hz), 7.34 (dd, 1H, J ) 1, 8 Hz); MS (FAB/
NBA) m/ e 538 (M + H)+. Anal. for C29H39N5O5‚0.7TFA: C,
H; N: calcd 11.34, found 11.92.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in dol-3-yl)eth yl}-5-isopr opylim idazole-4-car boxylic acid
(2q): H NMR (CD3OD, 300 MHz) δ 0.85 (d, 3H, J ) 6 Hz),
0.87 (d, 3H, J ) 6 Hz), 1.10 (d, 3H, J ) 7 Hz), 1.21 (d, 3H, J
) 7 Hz), 1.4-1.5 (m, 2H), 1.52 (m, 5H), 1.68 (m, 4H), 3.3-3.5
(m, 6H), 3.72 (m, 1H), 4.10 (dd, 1H, J ) 6, 10 Hz), 5.29 (t, 1H,
J ) 6 Hz), 6.96 (ddd, 1H, J ) 1, 7, 8 Hz), 7.09 (s, 1H), 7.12
(ddd, 1H, J ) 1, 7, 8 Hz), 7.24 (d, 1H, J ) 8 Hz), 7.35 (td, 1H,
J ) 1, 8 Hz); MS (FAB + NBA) m/ e 551 (M + H)+, 573 (M +
Na)+. Anal. Calcd for C30H42N6O4‚1.6TFA: C, H, N.
1
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)et h yl}-5-p r op yloxa zole-4-ca r b oxylic a cid
(2n ): 1H NMR (CD3OD, 300 MHz) δ 0.85 (d, 3H, J ) 7 Hz),
0.86 (d, 3H, J ) 7 Hz), 0.90 (t, 3H, J ) 8 Hz), 1.4-1.5 (m, 2H),
1.53 (m, 5H), 1.60 (m, 2H), 1.68 (m, 4H), 2.94 (dt, 2H, J ) 2,
8 Hz), 3.3-3.6 (m, 6H), 4.36 (dd, 1H, J ) 6, 10 Hz), 5.38 (t,
1H, J ) 6 Hz), 6.95 (ddd, 1H, J ) 1, 7, 8 Hz), 6.97 (s, 1H), 7.07
(ddd, 1H, J ) 1, 7, 8 Hz), 7.30 (d, 1H, J ) 8 Hz), 7.35 (d, 1H,
J ) 8 Hz); MS (DCI/NH3) m/ e 552 (M + H)+, 569 (M + NH4)+.
Anal. for C30H41N5O5‚0.3TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)et h yl}-5-cyclop r op ylim id a zole-4-ca r b oxy-
lic a cid (2t): H NMR (CD3OD, 300 MHz) δ 0.85 (d, 3H, J )
6 Hz), 0.88 (d, 3H, J ) 6 Hz), 1.08 (d, 3H, J ) 7 Hz), 1.13 (d,
3H, J ) 6 Hz), 1.5-1.8 (m, 11H), 3.3-3.5 (m, 6H), 3.72 (m,
1H), 4.09 (dd, 1H, J ) 6, 10 Hz), 5.38 (t, 1H, J ) 7 Hz), 6.96
(ddd, 1H, J ) 1, 7, 8 Hz), 7.07 (s, 1H), 7.09 (ddd, 1H, J ) 1, 7,
8 Hz), 7.22 (d, 1H, J ) 8 Hz), 7.35 (dd, 1H, J ) 1, 8 Hz); MS
(FAB + NBA) m/ e 549 (M + H)+; HRMS calcd for C30H41N6O4
549.3189, found 549.3210.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)et h yl}-5-m et h yloxa zole-4-ca r b oxylic Acid
(2h ). Oxazole 8O described above (90 mg) was dissolved in
EtOH (5 mL), and 10% Pd/C (50 mg) was added. The mixture
was purged of oxygen and stirred under a balloon of hydrogen
for 5 h. The solvent was removed in vacuo and the residue
taken up in EtOAc and filtered through Celite to remove the
catalyst. The solvent was evaporated to give the amino ester
as a yellow oil. This crude material was dissolved in THF (2
mL). HOBt (30 mg), the above-described leucine derivative
(55 mg), and EDCI (42 mg) were added. N-Methylmorpholine
(0.1 mL) was added, and the resultant solution was stirred at
room temperature for 18 h. The solvent was evaporated under
reduced pressure, and the residue was taken up in EtOAc. This
solution was washed with saturated NaHCO3 solution, 1 N
H3PO4, and brine, dried over Na2SO4, and evaporated in vacuo
to give an orange oil which was purified by flash chromatog-
raphy on silica gel eluting with 50% EtOAc-hexane. To this
material dissolved in THF (2 mL) was added a solution of
LiOH (50 mg) in H2O (1 mL); the resultant mixture was stirred
1
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-isop r op yloxa zole-4-ca r boxylic Acid
1
(2r ): H-NMR (CD3OD, 300 MHz) δ 0.87 (d, 3H, J ) 6 Hz),
0.89 (d, 3H, J ) 6 Hz), 1.15 (d, 3H, J ) 7 Hz), 1.18 (d, 3H, J
) 7 Hz), 1.4-1.5 (m, 2H), 1.52 (m, 5H), 1.68 (m, 4H), 3.3-3.5
(m, 6H), 3.69 (m, 1H), 4.38 (dd, 1H, J ) 6, 10 Hz), 5.37 (t, 1H,
J ) 6 Hz), 6.93 (ddd, 1H, J ) 1, 7, 8 Hz), 6.97 (s, 1H), 7.06
(ddd, 1H, J ) 1, 7, 8 Hz), 7.30 (d, 2H, J ) 9 Hz); MS (DCI/
NH3) m/ e 552 (M + H)+, 569 (M + NH4)+. Anal. for
C30H41N5O5‚0.9TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in dol-3-yl)eth yl}-5-cyclopr opyloxazole-4-car boxylic acid
1
(2u ): H NMR (CD3OD, 300 MHz) δ 0.84 (d, 3H, J ) 6 Hz),
0.86 (d, 3H, J ) 6 Hz), 0.87 (m, 2H), 1.04 (dd, 2H, J ) 3, 9
Hz), 1.4 (m, 2H), 1.53 (m, 5H), 1.68 (m, 4H), 2.65 (m, 1H), 3.3-
3.6 (m, 6H), 4.34 (dd, 1H, J ) 6, 10 Hz), 5.34 (t, 1H, J ) 7
Hz), 6.95 (ddd, 1H, J ) 1, 7, 8 Hz), 6.97 (s, 1H), 7.07 (ddd, 1H,
J ) 1, 7, 8 Hz), 7.32 (d, 1H, J ) 8 Hz), 7.34 (dd, 1H, J ) 1, 8
Hz); MS (DCI/NH3) m/ e 550 (M + H)+, 567 (M + NH4)+. Anal.
for C30H39N5O5‚0.8TFA: C, H, N.

966 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
von Geldern et al.
1
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-m eth ylth ia zole-4-ca r boxylic Acid
(2i). Thiazole 8S described above (72 mg, 0.15 mmol) was
dissolved in 2 mL of 30% HBr in HOAc and stirred at ambient
temperature for 3 h. The solvents were removed in vacuo; the
residue was taken up in saturated sodium bicarbonate solution
and extracted with EtOAc. The combined organic extracts
were washed with brine and dried over Na2SO4. The solvents
were removed in vacuo, and the crude product was dissolved
in THF (2 mL). HOBt (30 mg), the above-described leucine
derivative (55 mg), and EDCI (42 mg) were added. N-
Methylmorpholine (0.1 mL) was added, and the mixture was
stirred at room temperature for 18 h. The solvent was
evaporated under reduced pressure and the residue taken up
in EtOAc. The solution was washed with saturated NaHCO3
solution, 1 N H3PO4, and brine, dried with MgSO4, and
evaporated in vacuo to give an orange oil which was purified
by flash chromatography on silica gel eluting with 50% EtOAc/
hexane. This material was dissolved in THF (2 mL), a solution
of LiOH (50 mg) in H2O (1 mL) was added, and the mixture
was warmed at 80 °C for 10 h. The solvents were evaporated
under reduced pressure; the residue was neutralized with 1
N H3PO4 and purified by preparative HPLC (Vydac µC18)
eluting with a 10-70% gradient of CH3CN in 0.1% TFA. The
desired fractions were lyophilized to give the product as a
white solid: 54 mg; 1H NMR (CD3OD, 300 MHz) δ 0.80 (d,
3H, J ) 8 Hz), 0.82 (d, 3H, J ) 8 Hz), 1.25-1.45 (m, 3H), 1.51
(m, 4H), 1.68 (m, 5H), 2.68 (s, 3H), 3.2-3.45 (m, 6H), 4.29 (dd,
1H, J ) 6, 8 Hz), 5.50 (dd, 1H, J ) 6, 8 Hz), 6.97 (dt, 1H, J )
1, 7 Hz), 7.04 (s, 1H), 7.08 (dt, 1H, J ) 1, 7 Hz), 7.31 (d, 1H,
J ) 8 Hz), 7.52 (d, 1H, J ) 8 Hz); MS (DCI/NH3) m/ e 540 (M
+ H)+, 557 (M + NH4)+. Anal. for C28H37N5SO4‚1.0TFA: C,
H, N.
(2s): H NMR (CD3OD, 300 MHz) δ 0.83 (d, 3H, J ) 7 Hz),
0.84 (d, 3H, J ) 7 Hz), 1.24 (d, 3H, J ) 8 Hz), 1.25 (d, 3H, J
) 8 Hz), 1.3-1.4 (m, 2H), 1.53 (m, 5H), 1.68 (m, 4H), 2.94 (m,
2H), 3.3-3.5 (m, 6H), 4.09 (septet, 1H, J ) 7 Hz), 4.30 (dd,
1H, J ) 6, 10 Hz), 5.49 (dd, 1H, J ) 6, 8 Hz), 6.94 (ddd, 1H,
J ) 1, 7, 8 Hz), 7.07 (s, 1H), 7.08 (ddd, 1H, J ) 1, 7, 8 Hz),
7.30 (d, 1H, J ) 8 Hz), 7.40 (dd, 1H, J ) 1, 8 Hz); MS (DCI/
NH3) m/ e 568 (M + H)+; HRMS calcd for C30H42N5O4S
568.2958, found 568.2958 .
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)et h yl}-5-cyclop r op ylt h ia zole-4-ca r b oxylic
a cid (2v): H NMR (CD3OD, 300 MHz) δ 0.63 (m, 2H), 0.90
(d, 3H, J ) 6 Hz), 0.91 (d, 3H, J ) 6 Hz), 1.20 (m, 2H), 1.2-
1.4 (m, 2H), 1.51 (m, 5H), 1.67 (m, 4H), 2.96 (m, 1H), 3.3-3.6
(m, 6H), 4.28 (dd, 1H, J ) 6, 10 Hz), 5.44 (dd, 1H, J ) 6, 8
Hz), 6.94 (ddd, 1H, J ) 1, 7, 8 Hz), 7.05 (s, 1H), 7.08 (ddd, 1H,
J ) 1, 7, 8 Hz), 7.32 (d, 1H, J ) 8 Hz), 7.46 (dd, 1H, J ) 1, 8
Hz); MS (DCI/NH3) m/ e 566 (M + H)+. Anal. for C30H39N5-
O4S‚1.2TFA: C, H, N.
1
Recep tor Bin d in g Assa ys. All samples were kept at 4
°C throughout the process of membrane isolation. MMQ cells
(prolactin secreting rat pituitary cells known to contain ETA
receptors) or porcine cerebellar tissues (known to contain ETB
receptors) are homogenized in 25 mL of 10 mM Hepes (pH
7.4) containing 0.25 M sucrose and a protease inhibitor cocktail
[50 mM EDTA , 0.1 mM PMSF, and 5 µg/mL Pepstatin A, and
0.025% Bacitracin] using a microultrasonic cell disruptor
(Kontes). The mixture was centrifuged at 1000g for 10 min.
The supernatant was collected and centrifuged at 60000g for
60 min. The precipitate was resuspended in 20 mM Tris, pH
7.4, containing protease inhibitor cocktail and centrifuged
again. The final membrane pellet was resuspended in 20 mM
Tris, pH 7.4, containing protease inhibitors and stored at -80
°C until used. Protein content was determined by the Bio-
Rad dye-binding protein assay.
The following compounds are prepared using the procedures
described above for compound 2i:
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-p h en yl-th ia zole-4-ca r boxylic a cid
(2c): 1H NMR (CDCl3, 300 MHz) δ 0.86 (d, 3H, J ) 6 Hz),
0.87 (d, 3H, J ) 6 Hz), 1.45 (m, 6H), 1.62 (m, 5H), 3.30 (m,
5H), 3.37 (dd, 1H, J ) 7, 15 Hz), 3.48 (dd, 1H, J ) 7 , 15 Hz),
4.32 (br q, 1H, J ) 8 Hz), 4.70 (br d, 1H, J ) 8 Hz), 5.60 (br
q, 1H, J ) 8 Hz), 7.06 (d, 1H, J ) 2 Hz), 7.12 (dt, 1H, J ) 1,
8 Hz), 7.20 (dt, 1H, J ) 1, 8 Hz), 7.36 (m, 5H), 7.47 (m, 2H),
7.58 (d, 1H, J ) 8 Hz), 8.12 (br s, 1H); MS (DCI/NH3) m/ e 602
(M + H)+. Anal. for C33H39N5O4S‚1.0TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in dol-3-yl)eth yl}-5-ben zylth iazole-4-car boxylic acid (2f):
1H NMR (CDCl3, 300 MHz) δ 0.86 (d, 3H, J ) 6 Hz), 0.87 (d,
3H, J ) 6 Hz), 1.48 (m, 6H), 1.63 (m, 5H), 3.24 (m, 4H), 3.34
(m, 3H), 4.32 (br q, 1H, J ) 8 Hz), 4.52 (s, 1H), 4.57 (br d, 1H,
J ) 8 Hz), 5.50 (br q, 1H, J ) 8 Hz), 6.93 (d, 1H, J ) 2 Hz),
7.07 (dt, 1H, J ) 1, 8 Hz), 7.18 (m, 2H), 7.26 (m, 5H), 7.34 (dd,
1H, J ) 7, 8 Hz), 7.50 (d, 1H, J ) 8 Hz), 8.03 (br s, 1H); MS
(FAB + NBA) m/ e 616 (M + H)+, 638 (M + Na)+, 678 (M +
Cu)+; HRMS calcd for C34H42N5O4S: 616.2958, found: 616.2943.
Binding assays were performed in 96-well microtiter plates
pretreated with 0.1% BSA. Membranes were diluted ∼100-
fold in buffer B (20 mM Tris, 100 mM NaCl, 10 mM MgCl2,
pH 7.4, with 0.2% BSA, 0.1 mM PMSF, 5 µg/mL Pepstatin A,
0.025% bacitracin, and 50 mM EDTA) to a final concentration
of 0.2 mg/mL of protein. In competition binding studies,
membranes (0.02 mg) were incubated with 0.1 nM of [125I]ET-
1 (for ETA assay in MMQ) or [125I]ET-3 (for ETB assay in
porcine cerebellum) in buffer B (final volume: 0.2 mL) in the
presence of increasing concentrations of the test compound for
3 h at 25 °C. After incubation, unbound ligands were
separated from bound ligands by a vacuum filtration method
using glass-fiber filter strips in PHD cell harvesters (Cam-
bridge Technology, Inc., MA), washing the filter strips three
times with saline (1 mL). Nonspecific binding was determined
in the presence of 1 µM ET-1. IC50 values are calculated using
an average of at least two separate determinations.
Recep tor Mod elin g Stu d ies. Model development and
refinement studies were performed using the InsightII model-
ing software package from Biosym Technologies, Inc. Energy
minimizations and molecular dynamics calculations were
performed using the Discover module and were accomplished
using a modified version of the CVFF parameter set. Solvent
water molecules were incorporated into the model by applying
the SOAK subroutine in InsightII to the modeled receptor in
the absence of ligand. All water molecules containing oxygen
atoms within 2.0 Å of a non-hydrogen atom of the ligand were
then removed. The receptor and ligand were held fixed during
a short minimization (100 steps of conjugate gradient) and
molecular dynamics (2 ps at 300 K) to allow the water
molecules to adjust position. The entire complex was then
subjected to a short minimization (100 steps of conjugate
gradient) and molecular dynamics (2 ps at 300 K holding the
backbone of the receptor fixed) procedure to relieve major
stresses. Solvation calculations were performed using three
different solvation models as implemented in the Solvation
module of Insight. All three models11-13 use solvation param-
eters corresponding to CFF91 force field atom types. The
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-eth ylth ia zole-4-ca r boxylic a cid (2l):
1H NMR (CD3OD, 300 MHz) δ 0.82 (d, 3H, J ) 7 Hz), 0.83 (d,
3H, J ) 7 Hz), 1.24 (t, 3H, J ) 8 Hz), 1.4 (m, 2H), 1.52 (m,
5H), 1.67 (m, 4H), 3.18 (q, 2H, J ) 8 Hz), 3.2-3.5 (m, 6H),
4.30 (dd, 1H, J ) 6, 10 Hz), 5.49 (dd, 1H, J ) 6, 8 Hz), 6.94
(ddd, 1H, J ) 1, 7, 8 Hz), 7.04 (s, 1H), 7.07 (ddd, 1H, J ) 1, 7,
8 Hz), 7.32 (d, 1H, J ) 8 Hz), 7.47 (dd, 1H, J ) 1, 8 Hz); MS
(DCI/NH3) m/ e 554 (M + H)+, 551 (M + NH4)+. Anal. for
C29H39N5O4S‚0.6TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)et h yl}-5-p r op ylt h ia zole-4-ca r b oxylic a cid
1
(2p ): H NMR (CD3OD, 300 MHz) δ 0.82 (d, 3H, J ) 7 Hz),
0.83 (d, 3H, J ) 7 Hz), 0.93 (t, 3H, J ) 8 Hz), 1.4 (m, 2H), 1.52
(m, 5H), 1.67 (m, 6H), 3.14 (t, 2H, J ) 8 Hz), 3.2-3.5 (m, 6H),
4.30 (dd, 1H, J ) 6, 10 Hz), 5.50 (dd, 1H, J ) 6, 8 Hz), 6.95
(ddd, 1H, J ) 1, 7, 8 Hz), 7.04 (s, 1H), 7.06 (ddd, 1H, J ) 1, 7,
8 Hz), 7.31 (d, 1H, J ) 8 Hz), 7.46 (dd, 1H, J ) 1, 8 Hz); MS
(DCI/NH3) m/ e 568 (M + H)+. Anal. for C30H41N5O4-
S‚0.4TFA: C, H, N.
2-{1(R)-[(P er h yd r oa zep in -1-ylca r bon yl)leu cyla m in o]-
2-(in d ol-3-yl)eth yl}-5-isop r op ylth ia zole-4-ca r boxylic a cid

Azole Endothelin Antagonists. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 967
Antagonist, 97-139. J . Pharmacol. Exp. Ther. 1994, 268, 1122-
1128. (e) Stein, P. D.; Hunt, T. J .; Floyd, D. M.; Moreland, S.;
Dickinson, K. E. J .; Mitchell, C.; Liu, E. C.-K.; Webb, M. L.;
Murugesan, N.; Dickey, J .; McMullen, D.; Zhang, R.; Lee, V. G.;
Serafino, R.; Delaney, C.; Schaeffer, T. R.; Kozlowski, M. The
Discovery of Sulfonamide Endothelin Antagonists and the
Development of the Orally Active ETA Antagonist 5-(Dimethy-
lamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfona-
mide. J . Med. Chem. 1994, 37, 329-331. (f) Clozel, M.; Breu,
V.; Gray, G. A.; Kalina, B.; Loffler, B. M.; Burri, K.; Cassal, J .
M.; Hirth, G.; Muuler, M.; Neidhart, W.; Ramuz, H. Pharmaco-
logical Characterization of Bosentan, a New Potent Orally Active
Non-peptide Endothelin Receptor Antagonist. J . Pharmacol.
Exp. Ther. 1994, 270, 228-235. (g) Ohlstein, E. H.; Nambi, P.;
Douglas, S. A.; Edwards, R. M.; Gellai, M.; Lago, M. A.; Leber,
J . D.; Cousins, R. D.; Gao, A.; Frazee, J . S.; Peishoff, C. E.; Bean,
J . W.; Eggleston, D. S.; Elshourbagy, N. A.; Kumar, C.; Lee, J .
A.; Yu, T.-L.; Louden, C.; Brooks, D. P.; Weinstock, J .; Feuer-
stein, G.; Poste, G.; Ruffolo, R. R., J r.; Gleason, J . G.; Elliott, J .
D. SB 209670, a Rationally Designed Potent Nonpeptide En-
dothelin Receptor Antagonist. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 8052-8056. (h) Greenlee, W. J .; Walsh, T. F.; Pettibone, D.
J .; Tata, J . R.; Rivero, R. A.; Williams, D. L.; Bagley, S. W.;
Dhanoa, D. S.; Chakravarty, P. K.; Fitch, K. J .; Broten, T. P.;
Kevin, N. J . Phenoxyphenylacetic Acid Derivatives. WO 94/
21590, 1994. (i) Doherty, A. M.; Patt, W. C.; Edmunds, J . J .;
Berryman, K.; Reisdorph, B. R.; Plummer, M.; Shahripour, A.;
Lee, C.; Cheng, X.-M.; Walker, D. M.; Haleen, S.; Keiser, J . A.;
Flynn, M. A.; Welch, K. M.; Hallak, H. O.; Taylor, D. G.;
Reynolds, E. E. Discovery of a Novel Series of Orally Active Non-
Peptide Endothelin-A (ETA) Receptor-Selective Antagonists. J .
Med. Chem. 1995, 38, 1259-1263.
results of these calculations are summarized below.
Wesson-
Eisenberg12
(Kyte-Doolittle8
parameterization)
Wesson-
Eisenberg12
(Sharp13 para-
meterization)
Eisenberg-
McLachlan11
imidazole 2g 10.33 kcal/mol
-16.05 kcal/mol
-14.03
2.02
-10.80 kcal/mol
-8.47
2.33
thiazole 2i
∆E
11.05
0.72
Ack n ow led gm en t. The authors thank the Abbott
Analytical Department for assistance in acquiring H-
NMR and mass spectra.
1
Refer en ces
(1) Yanagisawa, M.; Kurihara, H.; Kimura, S.; Tomobe, Y.; Koba-
yashi, M.; Mitsui, Y.; Yazaki, Y.; Goto, K.; Masaki, T. A Novel
Potent Vasoconstrictor Peptide Produced by Vascular Endothe-
lial Cells. Nature 1988, 332, 411-415.
(2) (a) Opgenorth, T. J . Endothelin Receptor Antagonism. Adv.
Pharmacol. 1995, in press. (b) Mattoli, S.; Soloperto, M.; Marini,
M.; Fasoli, A. Levels of Endothelin in the Bronchoalveolar
Lavage Fluid of Patients with Symptomatic Asthma and Revers-
ible Airflow Obstruction. J . Allergy Clin. Immunol. 1991, 88,
376-384. (c) Golfman, L.; Hata, T.; Beamish, R.; Dhall, N. Role
of Endothelin in Heart Function and Disease. Can. J . Cardiol.
1993, 9, 635-653. (d) Li, H.; Elton, T.; Chen, Y.; Oparil, S.
Increased Endothelin Receptor Gene Expression in Hypoxic Rat
Lung. Am. J . Physiol. 1994, 266, L553-L560. (e) Ohlstein, E.;
Douglas, S. Endothelin-1 Modulates Vascular Smooth Muscle
Structure and Vasomotion: Implications in Cardiovascular
Pathology. Drug Dev. Res. 1993, 29, 108-128. (f) Alberts, G.;
Peifley, K.; J ohns, A.; Kleha, J .; Winkles, J . Constitutive
Endothelin-1 Overexpression Promotes Smooth Muscle Cell
Proliferation via an External Autocrine Loop. J . Biol. Chem.
1994, 269, 10112-10118.
(5) Gordon, T. D.; Singh, J .; Hansen, P. E.; Morgan, B. A. Synthetic
Approaches to the ‘Azole’ Peptide Mimetics. Tetrahedron Lett.
1993, 34, 1901-1904.
(6) Hutchins, C. Three-dimensional Models of the D1 and D2
Dopamine Receptors. Endocrine J . 1994, 2, 7-23.
(7) Henderson, R.; Baldwin, J .; Ceska, T. H.; Zemlin, F.; Beckmann,
E.; Downing, K. Model for the Structure of Bacteriorhodopsin
Based on High-resolution Electron Cryo-microscopy. J . Mol. Biol.
1990, 213, 899-929.
(3) Sakurai, T.; Yanagisawa, M.; Masaki, T. Molecular characteriza-
tion of endothelin receptors. Trends Pharmacol. Sci. 1992, 13,
103-108. Masaki, T.; Vane, J . R.; Vanhoutte, P. M.; V. Inter-
national Union of Pharmacology Nomenclature of Endothelin
Receptors. Pharmacol. Rev. 1994, 46, 137-142.
(8) Kyte, J .; Doolittle, R. F. A Simple Method for Displaying the
Hydropathic Character of a Protein. J . Mol. Biol. 1982, 157,
105-132.
(4) (a) Ishikawa, K.; Fukami, T.; Nagase, T.; Fujita, K.; Hayama,
T.; Niiyama, K.; Mase, T.; Ihara, M.; Yano, M. Cyclic Pentapep-
tide Endothelin Antagonists with High ETA Selectivity. Potency-
and Solubility-Enhancing Modifications. J . Med. Chem. 1992,
35, 2139-2142. (b) Itoh, S.; Sasaki, T.; Ide, K.; Ishikawa, K.;
Nishikibe, M.; Yano, M. A Novel Endothelin ETA Receptor
Antagonist, BQ-485, and its Preventive Effect on Experimental
Cerebral Vasospasm in Dogs. Biochem. Biophys. Res. Commun.
1993, 195, 969-975. Ishikawa, K.; Fukami, T.; Hayama, T.;
Niiyama, K.; Nagase, T.; Mase, T.; Fujita, K.; Ihara, M.; Ikemoto,
F.; Yano, M. Endothelin antagonistic peptide derivatives. EP
460679, 1991. (c) Aramori, I.; Nirei, H.; Shoubo, M.; Sogabe, K.;
Nakamura, K.; Kojo, H.; Notsu, Y.; Ono, T.; Nakanishi, S.
Subtype Selectivity of a Novel Endothelin Antagonist FR-139317
for the Two Endothelin Receptors in Transfected Chinese
Hamster Ovary Cells. Mol. Pharmacol. 1993, 43, 127-131.
Hemmi, K.; Neya, M.; Fukami, N.; Hashimoto, M.; Tanaka, H.;
Kayakiri, N. New Endothelin Antagonists and Their Prepara-
tion. WO 93/10144, 1993. (d) Mihara, S. I.; Nakajima, S.;
Matumura, S; Kohnoike, T.; Fujimoto, M. Pharmacological
Characterization of a Potent Nonpeptide Endothelin Receptor
(9) Elshourbagy, N. A.; Korman, D. R.; Wu, H.-L.; Sylvester, P. R.;
Lee, J . A.; Nuthalaganti, P.; Bergsma, D. O.; Kumar, C. S.;
Nambi, P. Molecular Characterization and Regulation of the
Human Endothelin Receptors. J . Biol. Chem. 1993, 268, 3873-
3879.
(10) Rose, P. M.; Krystek, S. R., J r.; Patel, P. S.; Liu, E. C. K.; Lynch,
J . S.; Lach, D. A.; Fisher, S. M.; Webb, M. L. Aspartate Mutation
Distinguishes ETA but not ETB Receptor Subtype-selective
Ligand Binding While Abolishing Phospholipase C Activation
in Both Receptors. FEBS Lett. 1995, 361, 243-249.
(11) Eisenberg, D.; McLachlan, A. D. Solvation Energy in Protein
Folding and Binding. Nature 1986, 319, 199-203.
(12) (a) Wesson, L.; Eisenberg, D. Atomic Solvation Parameters
Applied to Molecular Dynamics of Proteins in Solution. Protein
Sci. 1992, 1, 227-235.
(13) Sharp, K. A.; Nicholls, A.; Friedman, R.; Honig, B. Extracting
Hydrophobic Free Energies from Experimental Data: Relation-
ship to Protein Folding and Theoretical Models. Biochemistry
1991, 30, 9686-9697.
J M950591H