Design of a potent combined pseudopeptide endothelin-a/endothelin-b receptor antagonist, ac-dBhg16-Leu-Asp-IIe-[NMe]IIe-Trp21 (PD 156252): Examination of its pharmacokinetic and spectral properties

Article abstract of DOI:10.1021/jm970161m

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ET(A)/ET(B) receptor antagonists can be developed from the C- terminal hexapeptide of ET (H

Full text of DOI:10.1021/jm970161m

2228
J . Med. Chem. 1997, 40, 2228-2240
Design of a P oten t Com bin ed P seu d op ep tid e En d oth elin -A/En d oth elin -B
Recep tor An ta gon ist, Ac-DBh g16-Leu -Asp -Ile-[NMe]Ile-Tr p 21 (P D 156252):
Exa m in a tion of Its P h a r m a cok in etic a n d Sp ectr a l P r op er ties
Wayne L. Cody,* J ohn X. He, Michael D. Reily, Stephen J . Haleen,† Donnelle M. Walker,† Eric L. Reyner,§
Barbra H. Stewart,§ and Annette M. Doherty
Departments of Chemistry, Vascular and Cardiac Diseases, and Pharmacokinetics & Drug Metabolism, Parke-Davis
Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105
Received March 12, 1997X
The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and
prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor
antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-
Ile20-Trp21), such as Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-
Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic
proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is
thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with
carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By
performing a reduced amide bond and N-methyl amino acid scan, it was discovered that
N-methylation of Ile20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD
156252) that retained full receptor affinity at both endothelin receptor subtypes along with
enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this
bond allows the cis configuration to be readily accessible which greatly alters the preferred
structure of the entire molecule and may be responsible for the observed enhanced metabolic
stability.
In tr od u ction
Endothelin-1 (ET-1; Figure 1) is a potent peptidic
constrictor of vascular smooth muscle that was first
isolated and characterized from the supernatant of
porcine endothelial cells.1-3 ET-1 is one member of a
family of isopeptides that includes ET-2 and ET-34 along
with the structurally and functionally related mouse
vasoactive intestinal contractor (VIC),5 the cardiotoxic
sarafotoxins (SRTXs),6,7 and bibrotoxin.8 All members
of this family possess disulfide bridges between positions
F igu r e 1. Structure of endothelin-1 (ET-1).
1-15 and 3-11, along with a highly conserved C-
terminal hydrophobic hexapeptide, His16-Xxx-Asp-Yyy-
Ile-Trp21 (Xxx ) Leu or Gln, Yyy ) Ile or Val).9-12
Initially, two endothelin receptors were cloned, se-
quenced, and characterized from the bovine and rat
lung, respectively.13,14 The ETA receptor is selective for
ET-1 and ET-2 over ET-3, while the ETB receptor
possesses equal affinity for all of the ET isopeptides.
Subsequently, the corresponding human receptors have
been cloned.15,16 The endothelin receptor subtype popu-
lations (ETA/ETB) are widely distributed in several
tissues and possess different functions dependent upon
the species and location. For example, a highly selective
ETB receptor ligand, sarafotoxin-6c (SRTX-6c),17 has
linked the ETB receptor to vasodilation in the rat aortic
ring,18 while it is functionally linked to vasoconstriction
in several other tissues.19 The existence of additional
ETB receptor subtypes and/or species differences has
also been reported.20-23
antagonists for the ETA and ETB receptors or combined
antagonists for both the ETA and ETB receptors (for
recent reviews, see refs 9-12). This research has
focused on a series of combined ETA/ETB peptidic
antagonists based upon the C-terminal hexapeptide
(His16-Leu-Asp-Ile-Ile-Trp21) of the endothelins. In
designing these antagonists the importance of the
following structural features were considered: (i) the
neutralization of the amine terminus by acetylation
enhanced activity ∼10-fold, (ii) the preference for D-
aromatic amino acids in the 16 position, (iii) the
requirement for a tryptophan of the L-stereochemistry
in the C-terminal 21 position, and (iv) the necessity of
a C-terminal carboxylate.24-26 These observations re-
sulted in the design and preparation of Ac-DDip16-Leu-
Asp-Ile-Ile-Trp21 [compound 2, PD 142893 (Dip ) 3,3-
diphenylalanine)]25,27-29 and Ac-DBhg16-Leu-Asp-Ile-Ile-
Trp21 [PD 145065, compound 3 (Bhg ) 10,11-dihydro-
5H-dibenzo[a,d]cycloheptene-glycine)].30-32 Both of these
compounds exhibited low-nanomolar affinity for the ETA
(58 and 4.0 nM, respectively) and ETB (130 and 30 nM,
respectively) receptors. Likewise, compounds 2 and 3
were able to antagonize ET-1-stimulated vasoconstric-
tion in vitro in the rabbit femoral artery (ETA, pA2 )
6.6 and 6.8, respectively) and SRTX-6c-stimulated vaso-
The identification of these receptors has facilitated
the development of peptidic and nonpeptidic endothelin
* Author to whom correspondence should be addressed. Phone: (313)
996-7729. Fax: (313) 996-3107. E-mail: codyw@aa.wl.com.
† Department of Vascular and Cardiac Diseases.
§ Department of Pharmacokinetics & Drug Metabolism.
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0022-2623(97)00161-1 CCC: $14.00 © 1997 American Chemical Society

Design of a Combined ETA/ ETB Receptor Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2229
constriction in the rabbit pulmonary artery (ETB, pA2
) 6.3 and 7.0, respectively).25,27-32 Unfortunately, both
of these compounds have relatively short half-lives (18.1
( 3.4 and 10.6 ( 2.2 min, respectively) in rat intestinal
perfusate, suggesting limited utility of these compounds
for further in vivo evaluation.33,34
similar X-ray structure that was suggested to be the
bioactive conformation were incorrect using isotope-
edited NMR techniques. The opposite was shown in the
case of rampamycin, in which the solution structure,37
crystal structure38 and receptor-bound conformations
are identical.35
In addition, it has been shown for the
cases of the δ receptor selective opioid antagonist
[DPen2, DPen5]enkephalin and the cyclic octapeptide
known as sandostatin that it is not possible to rely on
only the X-ray crystal or solution NMR structure to
determine the bioactive structure.39,40 In fact, depend-
ing upon the particular situation, either method could
be argued to be more predictive.
In an attempt to further stabilize these compounds
against proteolytic degradation while maintaining re-
ceptor affinity, a reduced amide bond and N-methylated
amino acid scan of compound 2 was performed. These
modifications, in particular the incorporation of N-
methylated amino acids, should have significant effects
on the conformational preferences of the peptide back-
bone. In the case of the N-methylated analogues, the
energy barrier between the cis and trans amide bond
conformers is greatly reduced.
The ability to measure a large number of NMR
observables, particularly nuclear Overhauser effects
(NOEs), and coupling constants in biomolecules has
made possible the determination of several protein
structures in solution.41 It is generally accepted that
these data are more abundant and easier to quantitate
when internal motion is limited as in the case of
medium-sized proteins with extensive secondary and
tertiary structure. A much more difficult problem
exists, however, when NMR is used to measure the
conformational preferences of small peptides in solution.
As is often the case in aqueous solution, linear peptides
exhibit random coil chemical shifts and yield few
structurally relevant NOEs. The lack of NOEs is due
to several factors, including multiple conformations and
dilution of the intramolecular dipolar interactions by
dipolar interactions with solvent. One of the more
useful and informative techniques available is analysis
of the carbon and proton chemical shifts. Since chemical
shifts are sensitive to the peptide backbone angles and
the time scale is much faster than for cross-relaxation
(10-9 versus 10-6 s), chemical shifts can yield informa-
tion on peptides that are undergoing fast exchange
between multiple conformations.42
Analysis of the proton NMR characteristics of com-
pound 15 suggested pronounced conformational prefer-
ences that were dependent upon the solvent, ionization
state, and concentration. For example, in DMSO-d6, the
sodium salt of the peptide adopted a cis peptide bond
between Ile19 and [NMe]Ile20, whereas in aqueous
solution, this peptide bond was trans. In DMSO-d6
solution the structure was characterized by a wide
dispersion in the amide protons with little dispersion
in the methyl protons of Ile and Leu. Similar effects
were observed by modifying the ionization state or
concentration of compound 15 in solution (see below).
By comparison, there were not similar spectral changes
in compound 3 which lacks the [NMe]Ile20 residue.
Herein, this paper will describe the synthesis of a
series of endothelin hexapeptide antagonists based upon
compound 3 that contain reduced and N-methylated
amide bonds along with structure-activity relationships
(SAR) and pharmacokinetic properties. In addition, this
manuscript suggests a potential explanation for the
increased stability observed in rat intestinal perfusate
of compound 15 along with implications to the bioactive
conformation of C-terminal endothelin hexapeptide
antagonists based upon the measurable NMR param-
eters.
In all cases, these modifications, save one, resulted
in significant losses of affinity at one or both of the
endothelin receptor subtypes. In particular, the incor-
poration of [NMe]Ile in the 20 position of compound 2
resulted in an analogue, Ac-DDip16-Leu-Asp-Ile-[NMe]-
Ile-Trp21 (compound 13, PD 149764), that maintained
good affinity at both receptor subtypes. This modifica-
tion was incorporated into the more potent compound
3 template which resulted in an analogue, Ac-DBhg16-
Leu-Asp-Ile-[NMe]Ile-Trp21 (compound 15, PD 156252),
that had high affinity for both receptor subtypes and
greatly enhanced pharmacokinetic properties (stability
in rat intestinal perfusate and cellular permeability).
In an effort to help understand the reasons for the
improved pharmacokinetic properties of compound 15
and, in particular, its conformational preferences with
respect to compound 3, an examination of these ana-
logues by 1H-NMR spectroscopy in solution was per-
1
formed. One powerful H-NMR technique for the con-
formational/structural analysis of macromolecules is
isotope-edited NMR spectroscopy. This strategy is
limited to cases where the soluble macromolecule (in
this case, the receptor) is readily available and either
it or the ligand can be isotopically enriched with 13C and/
or 15N. Unfortunately, in the present scenario this was
not the case, since the ETA and ETB receptor subtypes
were not readily available. A second, more generally
applicable, although less powerful, approach is to de-
termine structural parameters of the receptor ligand
under a variety of conditions (e.g., solvent, temperature,
pH, and the like). If the conformational preferences of
the ligand are preserved under several different condi-
tions, there is a reasonable expectation that such
conformations are the energetically preferred ones.35 If
a compound’s three-dimensional structure can survive
severe environmental changes, it is even more reason-
able to propose that the solution conformation is likely
to represent a biologically relevant conformation.
There are several examples of solvent-dependent
conformational changes in peptides/pseudopeptides. For
example, it is well known from circular dichroism (CD)
and NMR studies that fluorinated alcohols, such as
trifluoroethanol (TFE) and hexafluoro-2-propanol (HFIP),
can induce increased helical structure in linear peptides.
There is also abundant literature available describing
large conformational changes observed for certain cyclic
peptides such as cyclosporin A (CsA) in polar and apolar
solvents.36 In fact, it has been shown that the predicted
conformation of CsA in lipophilic solvents and the
Ch em istr y
P ep tid e Syn th esis, P u r ifica tion , a n d Ch a r a cter -
iza tion . All of the peptide analogues were prepared

2230 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Cody et al.
Ta ble 1. Binding Affinities (IC50, µM) for Synthetic Analogues of Compounds 2 and 3 at the ETA and ETB Receptor Subtypes
no.
compound
ETA ((SEM)a
ETB ((SEM)b
2c
3d
4
5
6
7
8
9
10
11
12
13h
14
15i
Ac-DDip16-Leu-Asp-Ile-Ile-Trp21
0.058 ( 0.01e
0.130 ( 0.03e
0.015 ( 0.003f
1.3
>0.25
0.020
>0.25
>0.25
8.0
0.60
1.5
Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21
0.0040 ( 0.0004f
Ac-DDip16-[CH2NH]-Leu-Asp-Ile-Ile-Trp21
Ac-DDip16-Leu-[CH2NH]-Asp-Ile-Ile-Trp21
Ac-DDip16-Leu-Asp-[CH2NH]-Ile-Ile-Trp21
Ac-DDip16-Leu-Asp-Ile-[CH2NH]-Ile-Trp21
Ac-DDip16-Leu-Asp-Ile-Ile-[CH2NH]-Trp21
Ac-D[NMe]Dip16-Leu-Asp-Ile-Ile-Trp21
Ac-DDip16-[NMe]Leu-Asp-Ile-Ile-Trp21
Ac-DDip16-Leu-[NMe]Asp-Ile-Ile-Trp21
Ac-DDip16-Leu-Asp-[NMe]Ile-Ile-Trp21
Ac-DDip16-Leu-Asp-Ile-[NMe]Ile-Trp21
Ac-DDip16-Leu-Asp-Ile-Ile-[NMe]Trp21
Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21
2.7
0.26
0.25
0.26
0.88
4.5
0.20
3.2
0.70
0.030
>1.0
0.080
11.0
0.040
3.0
0.0010 ( 0.0003g
a
Binding data in rabbit renal vascular smooth muscle cells ( standard error of the mean where more than one IC50 was determined.
Binding data in rat cerebellar membranes ( standard error of the mean where more than one IC50 was determined. c PD 142893. PD
b
d
145065. e n ) 15 IC50 determinations. f n ) 19 IC50 determinations. n ) 4 IC50 determinations. All other values represent one IC50
g
determination. IC50 values were derived from single competition experiments in which data points were measured in duplicate. Binding
h
i
data were computer-analyzed by nonlinear least squares analysis giving the best fit for a one-site model. PD 149764. PD 156252.
by solid-phase peptide synthetic (SPPS) methodolo-
gies.43,44 The peptide analogues were prepared utilizing
a tert-butyloxycarbonyl (NR-t-Boc) protecting group strat-
egy on a PAM45 (phenylacetoamidomethyl) resin. NR-
t-Boc-protected unusual and N-methyl amino acids were
purchased from commercial sources or prepared utiliz-
ing literature methods.46-48 The reduced amide bonds
were prepared by reductive amination of the corre-
sponding amino aldehyde.49,50 The NR-t-Boc-protected
amino aldehydes were prepared by reduction of the
corresponding N,O-dimethylamide (“Weinreb” amides)
of the amino acid with lithium aluminum hydride or for
aspartic acid by oxidation of the protected amino
alcohol.51 In some cases, N-methyl amino acids were
prepared on the solid support utilizing a temporary
protecting group followed by reductive amination with
formaldehyde or for aspartic acid which was prepared
in solution by reduction of the corresponding oxazoli-
dinone (see the Experimental Section).52,53
were determined to be homogenous by analytical re-
versed-phase HPLC were combined and lyophilized. For
secondary in vitro functional evaluation, the purified
peptides were converted to the corresponding disodium
salt, by treating the fully protonated analogue with 5%
aqueous sodium bicarbonate, followed by solid-phase
extraction on a C18 cartridge, elution with methanol,
concentration under reduced pressure, resuspension in
water, and lyophilization. All final compounds were
analyzed for homogeneity by analytical reversed-phase
HPLC and/or capillary electrophoresis (CE) and char-
acterized for structural integrity by elemental analysis,
electrospray mass spectrometry (FABMS) and proton
nuclear magnetic resonance (1H-NMR) spectroscopy.
Resu lts a n d Discu ssion
It has been well documented that potent ETA and ETB
receptor antagonists can be developed from the C-
terminal hexapeptide of endothelin, such as Ac-DDip16
-
Each NR-t-Boc group was removed with 50% trifluo-
roacetic acid (TFA) in dichloromethane (DCM) and
neutralized with 10% diisopropylethylamine (DIEA) in
DCM prior to incorporation of the next protected amino
acid. All amino acids were single coupled as either their
symmetrical anhydrides or N-hydroxybenzotriazole
(HOBt)-activated esters unless incomplete coupling was
indicated, by the Kaiser test.54 If incomplete incorpora-
tion of the acylating agent was indicated the amino acid
was recoupled until a negative Kasier test was obtained.
After coupling of the last amino acid and NR-amine
deprotection, the peptide was acetylated with an excess
of 1-acetylimidazole in DCM or 10% acetic anhydride
in DCM with a catalytic amount of 4-(dimethylamino)-
pyridine (DMAP). The peptides were simultaneously
deprotected and cleaved from the resin by treatment
with anhydrous liquid hydrogen fluoride (HF) and
anisole (9:1, v/v) at 0 °C for 1 h. The resin was filtered
and washed with diethyl ether, and the crude peptide
was extracted into aqueous solution, concentrated under
reduced pressure, resuspended in water, and lyophi-
lized.
Leu-Asp-Ile-Ile-Trp21 (compound 2, PD 142893)25,27-29
and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (compound 3, PD
145065)30-32 (Table 1). Both compounds showed similar
binding affinities to the ETA (rabbit renal vascular
smooth muscle cells; IC50 ) 58 and 4.0 nM, respectively)
and ETB (rat cerebellar membranes; IC50 ) 130 and 15
nM, respectively) receptors. In addition, in vitro com-
pound 2 was able to block ET-1-stimulated vasocon-
striction in the rabbit femoral artery (ETA) and sarafo-
toxin-6c (SRTX-6c)-stimulated vasoconstriction in the
rabbit pulmonary artery (ETB) with pA2 values of 6.6
and 6.3, respectively.25,27-29 Likewise, compound 3
possessed pA2 values of 6.6 and 6.8 for the ETA and ETB
receptors, respectively.30-32
Both of these compounds had relatively short half-
lives (18.1 ( 3.4 and 10.6 ( 2.2 min, respectively) in
rat intestinal perfusate, thus limiting the utility of these
compounds for further in vivo evaluations.33,34 Each
compound was degraded to a primary metabolite plus
several other fragments under initial rate conditions.
Incubation of compound 3 with a carboxypeptidase
inhibitor from potato tuber reduced the rate of hydroly-
sis by 75%. Given that an aromatic amino acid is at
the C-terminus of the peptide, this provided evidence
of the involvement of carboxypeptidase A which prefers
this substrate configuration.55 In addition, it was shown
by reversed-phase high-performance liquid chromatog-
All crude peptides were purified to homogeneity by
preparative reversed-phase high-performance liquid
chromatography (HPLC) eluting with a linear gradient
of 0.1% aqueous TFA with increasing concentrations of
0.1% TFA in acetonitrile (AcCN). Peptide fractions that

Design of a Combined ETA/ ETB Receptor Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2231
raphy, as well as mass spectrometry, that Ac-DDip16
-
Leu-Asp-Ile-Ile and Ac-DBhg16-Leu-Asp-Ile-Ile were the
primary metabolites of compounds 2 and 3, respec-
tively.33,34
It has been previously shown that N-terminal acety-
lation of these molecules was required for high receptor
affinity; however, modifications of the C-terminus to
protect against exopeptidases, such as carboxypepti-
dases, have not been tolerated.29-31 In addition, mul-
tiple D-amino acid substitutions all led to significant
losses of receptor affinity.56,57 Thus, we have under-
taken a reduced amide and N-methyl amino acid scan
of the C-terminal hexapeptide to explore the structural
features that will maintain receptor affinity while
enhancing stability to enzymatic proteolysis.
F igu r e 2. Structure of compound 15 (PD 156252).
acid chloride of NR-Fmoc-Ile proved to be highly effective
for this coupling (see the Experimental Section for
preparation of compounds 13 and 15).] Also, in vitro
compound 13 was able to block ET-1-stimulated vaso-
constriction in the rabbit femoral artery (ETA) and
SRTX-6c-stimulated vasoconstriction in the rabbit pul-
monary artery (ETB) with pA2 values of 6.6 and 6.3,
respectively.
Red u ced Am id e Bon d Sca n of Com p ou n d 2. The
reduced amide (aminomethylene) analogues (compounds
4-8) were prepared by reductive amination with the
appropriate NR-t-Boc-protected amino aldehyde to the
growing peptide on the solid phase or in solution.49,50
The protected amino aldehydes were prepared by reduc-
tion of the corresponding N,O-dimethylamides (“Wein-
reb” amides) with lithium aluminum hydride or for
aspartic acid by oxidation of the protected amino
alcohol.51 Alternatively, the aminomethylene-contain-
ing dipeptide was prepared, suitably protected, and
coupled directly to the growing peptide (see the Experi-
mental Section). In general, it was observed that the
reduced amide analogues (compounds 4-8; Table 1) had
7-70-fold less affinity for the ETA receptor than the
parent peptide (compound 2). Likewise, 4-fold or more
loss in binding affinity was observed for the ETB
receptor with respect to compound 2. However, com-
pound 6 with the reduced amide bond between Asp18
and Ile19 actually showed 3-fold enhancement of binding
affinity to ETB with respect to compound 2. In spite of
the interesting profile of compound 6, the reduced amide
bond series was not pursued further, since the goal of
this study was to discover a stabilized ETA/ETB receptor
antagonist with high affinity for both receptor subtypes,
thus a profile similar to that of compounds 2 and 3.
Due to the profile of compound 13, the [NMe]Ile20
substitution was incorporated in the more potent Ac-
DBhg16-Leu-Asp-Ile-Ile-Trp21 (compound 3) template to
produce compound 15. Compound 15 showed enhanced
binding affinity to both the rabbit ETA and the rat ETB
receptor subtypes with IC50’s of 1.0 and 40 nM, respec-
tively. Likewise, compound 15 was able to antagonize
ET-1-stimulated vasoconstriction in vitro in the rabbit
femoral artery (ETA, pA2 ) 7.3) and SRTX-6c-stimulated
vasoconstriction in the rabbit pulmonary artery (ETB,
pA2 ) 6.6). Thus, compound 15 represents a highly
potent combined ETA and ETB receptor antagonist
(Figure 2).33
It has been previously reported that a significant
species difference between the binding affinity of ligands
for endothelin receptors can exist, especially in the case
of the ETB receptor. In general, compounds have been
shown to possess significantly lower affinity (150-fold
and greater) for the human cloned ETB receptor than
for the corresponding rat receptor.22,23,29 Compound 15
maintained good binding affinity for both the human
cloned ETA and ETB receptor subtypes (3.0 and 25 nM,
respectively).
N-Meth yl Am in o Acid Sca n of Com p ou n d 2. The
mono-N-methylated analogues (compounds 9-14) were
prepared by incorporation of the NR-t-Boc- or NR-Fmoc-
protected N-methyl amino acid to the growing peptide
on the resin (purchased from commercial sources or
prepared by the method of Freidinger et al.53) or by
reductive amination on the resin with formaldehyde
using a temporary monoprotecting group [4,4′-dimethox-
ylphenylmethyl (Dod)] on the NR-terminal amine (see
the Experimental Section).52 Similar to the reduced
amide bond analogues, most of the N-methylated ana-
logues of compound 2 (compounds 9-12 and 14; Table
1) showed losses in receptor affinity of 7-160-fold.
However, Ac-DDip16-Leu-Asp-Ile-[NMe]Ile-Trp21 (com-
pound 13) maintained high affinity for both the ETA and
ETB receptor subtypes (30 and 80 nM, respectively).33
[Several conditions were explored [N,N′-diisopropylcar-
bodiimide (DIC), DIC/HOBt, (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate (Bop
reagent), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HBTU), and O-(7-azaben-
zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU)] for the coupling of Ile19 to [NMe]Ile20
with little success (<20% incorporation). The use of the
As shown previously, the parent compounds 2 and 3
were unstable in rat intestinal perfusate with half-lives
of 18.1 ( 3.4 and 10.6 ( 2.2 min, respectively.33,34 Large
increases in the half-lives of the [NMe]Ile20 analogues
(PD 149764 and PD 156252) were observed. In fact,
compounds 13 and 15 were approximately 50-fold more
stable than their parent analogues with half-lives in rat
intestinal perfusate of 875 ( 145 and 538 ( 52 min,
respectively (Table 2).
Caco-2 cells are a continuously cultured cell line
derived from human colon adenocarcinoma that spon-
taneously differentiate to resemble the epithelial cells
that line the small intestine and have been widely used
as an in vitro model of intestinal absorption.56-58
Several of the hexapeptide antagonists were examined
in this model: compounds 2, 3, 13, and 15. All of these
compounds are of similar molecular weight and charge
(-2 at physiological pH). The Caco-2 permeabilities of
the hexapeptide ET antagonists ranged from approxi-
mately 2.0 × 10-4 to 6.3 × 10-4 cm/min which, when
compared to standard compounds, suggests a potential
for moderate, but measurable, intestinal absorption in
vivo in the range of 5-10% (Table 2). Compound 2 was
the least permeable, and compound 15 was clearly the

2232 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Cody et al.
Ta ble 2. Stability in Rat Intestinal Perfusate and Permeability in Caco-2 Cell Monolayers for Compounds 2, 3, 13, and 15
no.
compound
permeabilitya (104, cm/min)
stabilityb (t1/2, min)
2c
3d
Ac-DDip16-Leu-Asp-Ile-Ile-Trp21
2.0 ( 0.5
4.7 ( 0.9
2.0 ( 0.8
6.3 ( 2.5
18.1 ( 3.4
10.6 ( 2.2
875 ( 145
538 ( 52
Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21
Ac-DDip16-Leu-Asp-Ile-[NMe]Ile-Trp21
Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21
13e
15f
a
b
Each value represents the mean of three to five cell monolayers. Standard errors are tabulated with the mean values. Values are
the mean of at least three determinations ( standard error of the mean from two perfusate preparations. Leucine aminopeptidase activity
d
ranged from 0.05 to 0.32 nkat/mL in the perfusate preparations. c PD 142893. PD 145065. e PD 149764. f PD 156252.
most permeable. This increase in permeability is likely
due to the enhanced lipophilicity of compound 3. How-
ever, the differences between analogues were small and
the permeabilities were low, as could be anticipated
from the physicochemical properties described above.
In addition, compound 15 was shown to possess in
vivo activity by inhibition of the pressor response to an
ET-1 challenge in a conscious rat model. In particular,
a dose of 10 mg/kg of compound 15 was administered
iv bolus to conscious rats 5 and 30 min prior to an ET-1
challenge (0.30 nM/kg, iv bolus). Compound 15 was
shown to reduce the pressor response to ET-1 by 81%
and 58%, respectively. Thus, compound 15 was effective
as an antagonist in an in vivo model in a time dependent
manner.
NMR Stu d ies of Com p ou n d s 3 a n d 15. The
following discussion involves a description of (1) the 1H-
NMR spectral properties of compounds 3 and 15 under
various conditions including solvent content and ioniza-
tion state and (2) the structural properties of two
different conformations of the sodium salt of compound
15. Under each different set of conditions employed,
rigorous proton NMR assignments were made using
well-established 2D procedures.41 The 1H-NMR spectral
parameters of compound 3 have been previously re-
1
F igu r e 3. 500 MHz H-NMR spectra of compound 3‚Na (A)
ported in dodecylphosphocholine micelles.61 In micelles,
and compound 15‚Na (B). In both cases, the bottom trace is
compound 3 was shown to adopt a standard turn
topology in three closely related families which were
significantly different than the structures observed in
aqueous or DMSO-d6 solution. In addition, it was
shown that compound 3 was in fast chemical exchange
between several closely related conformational states.61
The proton NMR data for the disodium salt and fully
protonated forms of compounds 3 and 15 in aqueous and
DMSO-d6 solution are provided in the Supporting
Information.
the peptide dissolved in 90% H2O/10% D2O with the pH
adjusted to 6.7 and the top trace is the peptide dissolved in
DMSO-d6.
solution of compound 15‚Na in DMSO-d6 caused a
collapse of the dispersion in the amide region and an
increase in dispersion in the methyl region of the
spectrum such that the spectrum becomes similar to the
aqueous spectrum (data not shown). In the converse
experiment, addition of up to 50% (v/v) DMSO-d6 to an
aqueous solution of compound 15‚Na resulted in a slight
increase in the dispersion in the amide protons and a
decrease in the spread of the methyl proton chemical
shifts of leucine and isoleucine. These dramatic differ-
Effect of Solven t on Sp ectr a l Ch a r a cter istics.
1
The 1D H-NMR spectra of the disodium salt forms of
compounds 3 (3‚Na) and 15 (15‚Na) in DMSO-d6 (top
trace) and aqueous (bottom trace) solution are shown
in Figure 3A,B, respectively. In the case of compound
1
ences in the H-NMR spectral properties of compound
1
15‚Na that are solvent dependent are most likely due
to cis ) trans isomerization around the Ile19-[NMe]Ile20
amide bond.
3‚Na, the H-NMR spectrum is slightly different in the
two solvents, with small changes in chemical shift
dispersion in most regions of the spectrum (Figure 3A).
1
1
To further investigate this phenomenon, the H-NMR
However, the H-NMR spectra of compound 15‚Na in
spectrum was qualitatively analyzed for compound
15‚Na in a number of other organic solvents (data not
shown). The spectrum of compound 15‚Na in methanol-
d3 solution was similar to the peptide dissolved in
aqueous solution, whereas the spectrum in DMF-d7 was
very nearly identical to that observed in DMSO-d6. The
peptide dissolved readily in acetone-d6 and sparingly in
AcCN-d3. The resonances were extremely broad in both
solvents, suggesting extensive aggregation.
DMSO-d6 and aqueous solution are significantly differ-
ent from each other (Figure 3B). The DMSO-d6 spec-
trum (top trace) is characterized by significant spectral
dispersion of the amide protons, aspartic acid â protons,
and a clustering of the methyl protons of the leucine
and isoleucine residues. However, the aqueous spec-
trum (bottom trace) is characterized by a large degree
of dispersion in the methyl region, with the γ methyl
protons of Ile19 at exceptionally high field (0.18 ppm).
Other regions of the spectrum are more similar to each
other. Interestingly, addition of 10% (v/v) water to a
Secon d a r y Ch em ica l Sh ifts. It has been well
established that proton and carbon chemical shifts in

Design of a Combined ETA/ ETB Receptor Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2233
peptides and proteins are sensitive to Φ and Ψ angles.62
Thus, the secondary CR and HR chemical shifts for both
forms of compounds 3 and 15 were analyzed. The fully
protonated peptides dissolved in DMSO-d6 show only
small secondary shifts, indicative of highly flexible
conformation properties. For the two analogues in D2O,
a similar secondary shift pattern is observed for com-
pounds 3‚Na and 15‚Na, with the main difference being
a larger negative secondary shift at position 19 in the
N-methylated peptide (compound 15‚Na). In DMSO-
d6, both peptides clearly become more ordered, but their
secondary shifts now differ significantly in both mag-
nitude and sign. Interestingly, the major difference
between the disodium peptides in DMSO-d6 is again at
Ile19, with a ∼5 ppm difference in secondary shift and
a change in sign. Also, the magnitudes of the secondary
shifts for compound 15‚Na are universally higher than
for compound 3‚Na, suggesting a more stable secondary
structure for the methylated peptide.
F igu r e 4. Structures of cis (left) and trans (right) conformers
of compound 15.
compound 15‚Na that does not contain an [NMe]Ile20,
(i.e., compound 3‚Na).
Con for m a t ion a l P r efer en ces for Com p ou n d
15‚Na . Because of the unique spectral features ob-
served for compound 15 (but not compound 3), the
hypothesis that the structure characterized by the
unusually large dispersion of amide proton chemical
shifts was due to the formation of a cis peptide bond
between Ile19 and [NMe]Ile20 was investigated. Methyl-
ation of the amide of Ile20 decreases the energy differ-
ence between the trans and cis configurations, making
both energetically accessible. NOEs between the R
protons of Ile19 and Ile20 or [NMe]Ile20 established that,
indeed, the sodium form of the peptide was 100% cis
(about the Ile19-[NMe]Ile20 peptide bond) in DMSO-d6,
while the lack of NOEs in aqueous solution established
that the peptide bond was 100% trans.
Quantitative analysis of these secondary shifts is
complicated by several factors. First, in our analysis
of Gly-Gly-[NMe]Ile-Gly-Gly, only the trans conformer
was observed in aqueous and DMSO-d6 solutions.
Therefore, the appropriate random coil chemical shift
is not available for the cis conformation of [NMe]Ile.
Hence, the random coil chemical shift for the trans
conformation was used for all peptides. This may be a
reasonable value, since it is known that Pro CR shifts
are not very sensitive to the geometric configuration
about the imide bond. Second, the nearest-neighbor
corrections for the random coil chemical shift of Ile19
(when preceding [NMe]Ile) were taken from work with
proline-containing peptides.63 Here again, the assump-
tion that [NMe]Ile is similar to proline with respect to
secondary shifts may not be valid.
In one 15 mM sample of compound 15‚Na in DMSO-
d6, numerous medium and long range NOEs in a
NOESY spectrum recorded with a mixing time of 400
ms were observed. The sign of the NOEs was consistent
with a correlation time of greater than 1 ns, which was
somewhat surprising for a linear hexapeptide. To
investigate this further, NOESY spectra were obtained
at 80, 120, and 200 ms, respectively. Analysis of the
NOE buildup curves provided clear evidence that many
of the cross-peaks observed in the 400 ms NOESY were
due to spin diffusion. Analysis of the shapes of the
buildup curves suggested that all of the NOE informa-
tion in the 80 ms NOESY was due to direct cross-
relaxation. Sixty-five cross-peak volumes were con-
verted to upper bound distance constraints and used to
generate structures for compound 15‚Na. In addition
to distance constraints, the Φ angles of residues 16, 17,
1
Effect of Ion iza tion Sta te. The H-NMR spectrum
of the fully protonated form of compound 15 in DMSO-
d6 is characterized by poor dispersion with very little
indication of secondary structure. The temperature
coefficients and amide to R proton coupling constants
(3J NHHR, Table 2) that can be measured are consistent
with a conformationally flexible structure. Addition of
20% aqueous sodium hydroxide in approximately equimo-
lar proportional steps initially results in a broadening
of the signals in the amide region, and two sets of
signals could be seen for some of the aliphatic protons.
This is most likely due to cis ) trans isomerization
around the Ile19-[NMe]Ile20 amide bond induced by
deprotonation of the peptide, vide infra. Addition of a
third equivalent of base resulted in a spectrum es-
sentially identical with that of compound 15‚Na. When
a similar titration was performed with aqueous potas-
sium hydroxide or cesium hydroxide, the results were
indistinguishable from those using NaOH.
and 19 were constrained to the range of -75o to -175o,
3
based on J NHHR
.
Using these constraints, 20 structures were generated
by distance geometry/simulated annealing. Nineteen of
these had final DGII optimization errors of <0.1, and
none of these had experimental distance violations of
more than 0.3 Å. Representative structures from these
calculations are shown in Figure 4.
Several structural features that were consistent with
the observed spectral properties were apparent. First,
the close proximity of Leu17 and Ile19 side chains to the
DBhg16 and Trp21 side chains, respectively, could explain
the high-field-shifted methyl protons observed in the
trans conformer. These were also consistent with the
aromatic methyl NOEs observed for compounds 3‚Na
The above results are consistent with the following
hypotheses/observations: Firstly, a distinct conforma-
tion is observed for compound 15‚Na in aprotic anhy-
drous solvents (DMSO-d6, etc.) that is “more ordered”
than the conformation observed in protic solvents or
that of the protonated peptide species in aprotic sol-
vents. Thus, the “ordered” structure is not favored
when the C-terminal and aspartic acid side chain
carboxyl anions are fully or partially neutralized by
protons or efficient solvation. Secondly, this “ordered”
structure is not readily accessible to an analogue of

2234 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Cody et al.
recorded at lower concentrations. Diluting this sample
4-fold resulted in a return to a more normal spectrum
(Figure 5). Some resonances (cf. the Trp indole reso-
nances at 10.6 ppm) suggest a slow exchange between
different species, whereas the amide protons simply
broaden and/or move to higher field at the higher con-
centration. A possible explanation for the concentra-
tion-dependent behavior of the spectrum is a monomer
) multimer equilibrium or an ionic strength/pH effect.
The peak positions of the species that forms at higher
concentrations are similar to those observed for the fully
protonated form of compound 15, suggesting that this
species contains compound 15 in the trans configuration.
Addition of substoichiometric amounts of sodium
hydroxide to a 60 mM solution of the fully protonated
form of compound 15 resulted in a decrease in the
amount of this species with a concomitant increase in
the species identified as the cis configuration of com-
pound 15‚Na. Thus it appears that the second species
observed at higher concentrations may be the trans
configuration of compound 15 and that its relative
abundance may be a function of the effective pH and/or
ionic strength in the DMSO-d6 solution. This is affected
by such difficult to control variables as the extent of
hydration and the exact protonation state of the peptide,
as well as the amount of water present in the solvent.
Con clu sion s
F igu r e 5. Concentration dependence of the 1H-NMR spectrum
of compound 15‚Na in DMSO-d6 at 35 °C. The three concen-
trations shown are 145 mM (top trace), 72 mM (middle trace),
and 36 mM (bottom trace).
Compound 15 represents a constrained analogue of
compound 3 that is a highly potent combined ETA and
ETB receptor antagonist. Compound 15 showed en-
hanced binding affinity to both the rabbit ETA and rat
ETB receptor subtypes with IC50’s of 1.0 and 40 nM,
respectively). Likewise, compound 15 was able to
antagonize ET-1-stimulated vasoconstriction in vitro in
the rabbit femoral artery (ETA, pA2 ) 7.3) and SRTX-
6c-stimulated vasoconstriction in the rabbit pulmonary
artery (ETB, pA2 ) 6.6). In addition, compound 15 was
approximately 50-fold more stable than compound 3 in
rat intestinal perfusate with a half-life greater than 500
min. Since the only difference between compounds 3
and 15 lies in the N-methylation of the amide bond
between Ile19 and Ile20, the enhanced proteolytic stabil-
ity and cellular permeability must be related physically
or conformationally to this modification. In addition,
since N-methylation of an amide bond is known to have
significant effects on the conformational preferences of
the peptide backbone and compound 15 is a highly
potent analogue, it must be assumed that this modifica-
tion has constrained the molecule in a conformation that
is favored for interaction with the ET receptor subtypes.
It is intriguing to speculate that the enhanced stabil-
ity of compound 15 may be a result of the energetically
favored accessibility of the cis amide bond conformer.
From the above 1H-NMR studies it had been shown that
in aqueous solution compound 15‚Na exists exclusively
in the trans amide bond form; however, upon the
addition of DMSO-d6 the cis form became accessible.
The cis form was also shown to depend upon the
ionization state of compound 15. Based upon these
results it was obvious that the energy barrier between
the trans and cis conformers is low and readily obtain-
able; thus it was not possible to predict the preferred
structure of compound 15 under physiological condi-
tions. If the cis conformer was predominant, it can been
and 15‚Na in aqueous solution and for the fully proto-
nated forms of compounds 3 and 15 in DMSO-d6. In
the structures calculated for compound 15‚Na in DMSO-
d6, the three aliphatic side chains were exposed to
solvent and further removed from the aromatic side
chains, consistent with the lack of chemical shift disper-
sion for these species. Second, although the side chain
of Trp for compound 15‚Na was not well defined in the
ensemble of structures, on average it was closer to the
DBhg16 side chain than in the extended structure.
Mutual ring current effects could be invoked to explain
the additional dispersion in the aromatic resonances of
the DBhg16 residue and the unusual chemical shifts of
the Trp21 side chains (compared to Trp in a random coil
peptide) for the sodium salt in DMSO-d6. Third, the
amide protons were internalized in the calculated
structures, consistent with their highly disperse chemi-
cal shifts and relatively small temperature coefficients.
Finally, the C-terminal carboxylate was poised for
hydrogen-bonding interactions with the NH of Ile19 and/
or Asp18 in many of the final structures. The potential
for electrostatic interaction between the C-terminal
carboxylate and amide protons could be a driving force
for cis-peptide bond formation in this peptide dissolved
in an aprotic solvent. Possible structures for the cis and
trans conformers of compound 15‚Na are shown in
Figure 4.
Con cen tr a tion Dep en d en ce of Com p ou n d 15‚Na
in DMSO-d 6 Solu tion . In an attempt to measure the
three bond 13Câ-NH coupling constants, a sample was
prepared by dissolving 72 mg of compound 15‚Na in 0.5
mL of DMSO-d6 (∼150 mM). The resulting spectrum
was more complex than expected based upon spectra

Design of a Combined ETA/ ETB Receptor Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2235
reversed-phase HPLC analysis was carried out on a Vydac
column (218TP54) (0.46 × 25.0 cm, 5 mM particle size). The
analytical HPLC system used was the same as that described
in detail above for peptide purification. The mobile phase
utilized for the analytical HPLC analysis was 80% A:20% B
to 14% A:86% B [0.1% aqueous TFA (A):0.1% TFA in AcCN
(B)]; linear gradient was over 22 min at 1.5 mL/min (λ ) 214
and 280 nm) on a Vydac 218TP54 column.
Chemical ionization mass spectra (CIMS) were acquired
with a Fisons VG Trio-2A quadrupole mass spectrometer using
1% ammonia in methane as the reagent gas. Fast atom
bombardment mass spectra (FABMS) were measured with a
seen from Figure 4 that C-terminal carboxylate would
be buried and less accessible to carboxypeptidase activ-
ity, accounting for the increased stability of compound
15 in rat intestinal perfusate. In the same regard, it is
possible that methylation of the p-1 amino acid of a
carboxypeptidase substrate may lead to an analogue
that does not fit properly into the enzyme, either
sterically or simply by disruption of a critical hydrogen-
bonding interaction. Resolution of the exact reasons for
the increased stability will require additional studies,
which are not the focus of this manuscript. Such studies
could include replacement of the Ile19-[NMe]Ile20 amide
bond with peptidomimetic isosteres (cis alkenes and the
like) that would lock the molecule into the cis configu-
ration.
It is also quite intriguing that the simple modification
of N-methylating a single amide bond of a hexapeptide
can have such profound effects on the intrinsic stability
of the molecule in rat intestinal perfusate without
concomitant effects on cellular permeability. In this
regard, it should become common practice in the devel-
opment of second-generation peptidomimetic compounds
designed from a peptide lead for extended in vitro and
in vivo evaluations to perform a reduced amide bond
and N-methylated amino acid scan. As can be seen from
above, this can impart desirable physicochemical prop-
erties into the molecule as well as provide insights into
the understanding of the bioactive conformation.
VG analytical 7070E/HF mass spectrometer in either
a
thioglycerol or 3-nitrobenzyl alcohol matrix using xenon as the
target gas. Electrospray mass spectra (ESMS) were obtained
on either a Finnigan TSQ70 or Fisons VG Trio 2000 quadru-
pole mass spectrometer using 50:50 water:methanol made 1%
in acetic acid as the solvent. Routine 1H-NMR spectra were
measured with a Varian Gemini 2300 or Varian Unity 400
instrument using tetramethylsilane as an external standard
in chloroform or dimethyl sulfoxide (CDCl3 or DMSO-d6;
Cambridge Isotope Laboratories).
[14C]PEG-4000 was purchased from New England Nuclear,
DuPont (NEN). Reference compounds that were used to
establish the correlation between fractions absorbed in humans
and transport across CACO-2 cell monolayers were either
obtained from Sigma Chemical Co., with the radiolabeled form
of the compounds purchased from NEN (D-mannitol, hydro-
cortisone, phenytoin, and L-phenylalanine), or synthesized at
Parke-Davis (gabapentin and cefdinir). Modified MES buffer
was prepared from MES [2-(N-morpholino)ethanesulfonic
acid], NaCl, and KCl (Sigma Chemical Co.). D-Glucose and
human serum albumin were also purchased from Sigma.
Anesthetics used included Ketaset (ketamine HCl injection,
USP, 100 mg/mL; Aveco Co., Inc.), Rompun (xylazine, 20 mg/
mL; Miles Inc.), and sodium pentobarbital (64.8 mg/mL;
Anthony Products) for all animal surgery.
Gen er a l Str a tegy: 1. P ep tid e Syn th esis. All of the
peptides were synthesized by solid-phase peptide synthetic
techniques on an ABI Model 430A or 431A peptide synthesizer.
The peptide analogues were prepared using an NR-t-Boc
protection scheme with NR-t-Boc-Trp-PAM resin and the
aspartic acid side chain carboxylate protected as the benzyl
ester. Individual NR-t-Boc amino acids were coupled via DCC
in DMF. Deprotection of the NR-t-Boc group was achieved with
50% TFA in DCM, and removal of the NR-Fmoc group was
accomplished with 20% piperidine in DCM. N-Terminal
acetylation was carried out on the resin in DCM with an excess
of 1-acetylimidazole (20-fold) or 10% acetic anhydride in DCM
with a catalytic amount of DMAP. The resin was then washed
in turn with DMF, MeOH, and DCM (2 × each) and dried
under reduced pressure. The peptides were then deprotected
and cleaved from the resin using anhydrous liquid HF:anisole
(9:1). The resin was washed with anhydrous ethyl ether, and
the crude peptide was extracted from the resin with AcCN:
water (1:1) with 0.1% TFA, concentrated under reduced
pressure, and lyophilized. (See ref 27 for the detailed solid-
phase synthesis of compound 2, which is representative of all
solid-phase syntheses in this report.)
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Orthogonally protected NR-t-Boc
amino acids, NR-Fmoc amino acids, and NR-t-Boc-Trp-PAM
resins were purchased from either Advanced Chemtech, Ap-
plied Biosystems Inc., Bachem California, Bachem Bioscience,
Novabiochem, Peninsula Laboratories, Inc., or Synthetech Inc.
All amino acids were of the L-configuration unless otherwise
noted.
TFA was purchased from Halocarbon. N,N′-Dicyclohexyl-
carbodiimide (DCC), DIEA, and HOBt were purchased from
Applied Biosystems Inc. (ABI). N,N-Dimethylformamide
(DMF), DCM, and NMP were purchased from Burdick &
J ackson and were of reagent grade or better. HPLC grade
solvents (AcCN and water) were obtained from Burdick and
J ackson, EM Science, or Mallinckrondt. HF was purchased
from Matheson Gas Products. All of the other reagents for
chemical synthesis were purchased from Aldrich Chemical Co.,
Inc. or similar suppliers. Tris(hydroxymethyl)aminomethane
(Trizma), ethylenediaminetetraacetate (EDTA), and N-(2-
hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES)
were purchased from Sigma Chemical Co. Phenylmethane-
sulfonyl fluoride (PMSF) and bacitracin were purchased from
Boehringer Mannheim Biochemicals. Bovine serum albumin
(BSA) was purchased from Miles Inc., Diagnostics Division.
[
125I]ET-1 (2000 Ci/mmol) was purchased from New England
Nuclear, DuPont and [3H]arachidonic acid (218 Ci/mmol) was
purchased from Amersham.
2. P ep tid e P u r ifica tion . Crude peptides were dissolved
in a mixture of aqueous 0.1% TFA and AcCN (exact ratio
depending on the solubility of the peptide) and then purified
by preparative reversed-phase HPLC (see above). Peptide
fractions determined to be pure by analytical HPLC were
combined, concentrated under reduced pressure, and lyophi-
lized.
The peptides were prepared on an ABI Model 430A or 431A
peptide synthesizer using software version 1.40. For NR-t-Boc
syntheses, the HF cleavages were performed on an Immuno-
Dynamics Inc. Model 2A/2B HF Apparatus. High-pressure
liquid chromatographs were obtained on a Waters HPLC
system from Millipore Corp. equipped with a Model 600E
system controller, a Model 600 solvent delivery system, a
Model 490 variable wavelength detector operating at 214 and
280 nm, and a Bio-Rad Laboratories Model AS-100 autosam-
pler. Vydac analytical and preparative C18 HPLC columns
were purchased from The Nest Group. Preparative reversed-
phase HPLC was performed using a C18 preparative scale
Vydac column (218TP1022) (2.2 × 25.0 cm, 10-20 mM particle
size) eluting with a linear gradient of 0.1% aqueous TFA with
increasing concentrations of AcCN at 15 mL/min. Analytical
3. P ep tid e Hom ogen eity a n d Ch a r a cter iza tion . Pep-
tides were assessed for homogeneity by analytical reversed-
phase analytical HPLC. The peptides were characterized by
1
ESMS or FABMS and H-NMR spectroscopy. All final pepti-
1
domimetic compounds (4-15) provided an H-NMR spectrum
that was consistent with the desired structure; however, due
to the complexity of the resulting spectrum individual assign-
ments are not provided.
P r ep a r a t ion of Ac-DDip -Ψ[CH 2NH ]-Leu -Asp -Ile-Ile-
Tr p (4). P r ep a r a tion of Nr-t-Boc-DDip -N(CH3)OCH3. To

2236 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Cody et al.
a solution of NR-t-Boc-DDip (3.0 g, 8.8 mmol) in DMF (20 mL)
were added HCl‚HN(CH3)OCH3 (0.88 g, 8.8 mmol), and DIEA
(3.0 mL, 17.2 mmol) followed by BOP reagent (3.9 g, 8.8 mmol).
The reaction was allowed to continue for 2 h. The reaction
mixture was then concentrated to dryness. The residue was
taken up with ethyl acetate (EtOAc; 50 mL), washed with
saturated aqueous Na2CO3 (2 × 50 mL), water, and 1 M
aqueous KHSO4 (2 × 50 mL), dried over MgSO4, and concen-
trated under reduced pressure to yield a white foam (3.3 g,
88%): 1H-NMR (CDCl3) δ 1.31 (s, 9H), 2.94 (s, 3H), 3.61 (s,
3H), 4.32 (d, 1H), 4.99 (d, 1H), 5.68 (t, 1H), 7.27 (m, 10H);
CIMS (m/ z)+ calcd 384.2, found 385 (M + H).
(s, 9H), 1.48 (m, 4H), 2.95 (m, 5H), 3.88 (m, 1H), 4.22 (m, 3H),
4.54 (m, 1H), 7.32 (m, 2H), 7.63 (m, 2H), 7.88 (m, 2H); FABMS
(m/ z)+ calcd 510.2, found 511 (M + H).
P r ep a r a t ion of Ac-DDip -Leu -Ψ[CH 2NH ]-Asp -Ile-Ile-
Tr p (5). The peptide synthesis, cleavage from the resin, and
deprotection were performed as described in the general
procedure. The crude peptide was purified by reversed-phase
HPLC to afford 33 mg of the title compound: HPLC tR ) 14.6
min (>96%); ESMS (m/ z)+ calcd 910.1, found 910.4 (M).
P r ep a r a t ion of Ac-DDip -Leu -Asp -Ψ[CH 2NH ]-Ile-Ile-
Tr p (6). P r ep a r a tion of Nr-t-Boc-Asp (OBzl)-CH2OH. To
a solution of NR-t-Boc-Asp(OBzl) (6.3 g, 20 mmol) in dry THF
(20 mL) at 0 °C under N2 was added 1 M BH3‚THF (40 mL,
40 mmol) dropwise over a period of 2 h. The solution was
stirred for an additional 2 h at 0 °C, and 20 mL of HOAc was
added. The reaction mixture was evaporated under reduced
pressure, dissolved in EtOAc (100 mL), washed with 10%
aqueous NaHCO3, water, 1 N HCl, and brine (2 × 50 mL,
each), dried with MgSO4, filtered, and concentrated under
reduced pressure to an oil (5.5 g, 90%): 1H-NMR (CDCl3) δ
1.45 (s, 9H), 2.15 (d, 2H), 2.20 (br, 1H), 3.65 (d, 2H), 4.01 (m,
1H), 5.12 (s, 2H), 5.33 (d, 1H), 7.45 (m, 5H); CIMS (m/ z)+ calcd
309.4, found 310 (M), 210 (M - Boc).
P r ep a r a tion of Nr-t-Boc-DDip -CHO. To a solution of NR-
t-Boc-DDip-N(CH3)OCH3 (3.3 g, 8.5 mmol) in dry THF (50 mL)
at 0 °C, was added, in portions, lithium aluminum hydride
(0.43 g, 11.1 mmol). The reaction was allowed to continue for
30 min at 0 °C and was quenched by adding 1 M aqueous
KHSO4 (20 mL). The organic phase was separated and the
aqueous phase extracted with EtOAc (50 mL). The combined
organic layer was washed with brine, dried with MgSO4, and
concentrated under reduced pressure to give a colorless oil
which crystallized on standing at room temperature (2.75, g
99%): 1H-NMR (CDCl3) δ 1.39 (s, 9H), 4.50 (d, 1H), 4.88 (d,
1H), 5.07 (t, 1H), 7.28 (m, 10H), 9.61 (s, 1H); CIMS (m/ z)+
calcd 325.4, found 326 (M + H).
P r ep a r a t ion of Ac-DDip -Ψ[CH 2NH ]-Leu -Asp -Ile-Ile-
Tr p (4). The synthesis of NR-t-Boc-Leu-Asp-Ile-Ile-Trp-PAM
resin was performed as described in the general procedure on
a 1.0 mmol scale. NR-t-Boc-Leu-Asp-Ile-Ile-Trp-PAM resin was
treated with 50% TFA in DCM (20 mL) for 30 min at room
temperature and washed successively with DCM (3 × 20 mL),
10% DIEA in DCM (20 mL), DMF (3 × 20 mL), and 5% acetic
acid in DMF (2 × 20 mL). NR-t-Boc-DDip-CHO (1.2 g, 3.0
mmol) was added followed by 1% acetic acid in DMF (20 mL)
and 1 M NaBH3CN‚THF (3.3 mL, 3.3 mmol). The mixture
was shaken at room temperature for 3 h. The resin was then
washed in turn with DMF, MeOH, and DCM (2 × 20 mL, each)
and dried under reduced pressure. The peptide was depro-
tected and cleaved from the resin using anhydrous HF as
described in the general procedure. The crude peptide was
purified by reversed-phase HPLC to afford 230 mg of the title
compound: HPLC tR ) 12.5 min (>99%); ESMS (m/ z)+ calcd
910.1, found 910.6 (M).
P r ep a r a t ion of Ac-DDip -Leu -Ψ[CH 2NH ]-Asp -Ile-Ile-
Tr p (5). P r ep a r a t ion of Nr-Ben zyloxyca r b on yl(Cb z)-
Leu -Ψ[CH2NH]-Asp(OBu t)-OBzl. NR-Cbz-Leu-CHO was pre-
pared in two steps from NR-Cbz-Leu as described for NR-t-Boc-
DDip-CHO (30.8 g, 99%): 1H-NMR (CDCl3) δ 0.95 (m, 6H), 1.74
(m, 3H), 4.38 (m, 1H), 5.12 (s, 2H), 5.38 (m, 1H), 7.34 (s, 5H),
9.60 (s, 1H); CIMS (m/ z)+ calcd 249.3, found 250 (M + H).
NR-Cbz-Leu-CHO (14.5 g, 58 mmol) was dissolved in dry
MeOH (500 mL) and treated with H-Asp(OBut)-OBzl‚HCl (16.2
g, 58.0 mmol) followed by NaBH3CN (3.77 g, 60.1 mmol) and
acetic acid (HOAc; 4.0 mL, 66.5 mmol). The reaction was
allowed to continue overnight and concentrated under reduced
pressure to dryness. The residue was taken up with EtOAc
(200 mL); the organic layer was washed with saturated
NaHCO3 and brine, dried with MgSO4, and concentrated under
reduced pressure to an oil (15.1 g, 52%): 1H-NMR (CDCl3) δ
0.89 (m, 6H), 1.28 (m, 1H), 1.40 (s, 9H), 1.52-2.51 (m, 4H),
2.75 (m, 4H), 3.75 (m, 2H), 5.08 (s, 2H), 5.17 (s, 2H), 7.32 (m,
10H); CIMS (m/ z)+ calcd 512.3, found 513 (M + H).
P r ep a r a tion of Nr-t-Boc-Asp (OBzl)-CHO. To a solution
of NR-t-Boc-Asp(OBzl)-CH2OH (2.0 g, 9.7 mmol) in DCM was
added pyridinium dichromate (5.7 g, 15.0 mmol). The solution
was allowed to stir at room temperature overnight. The
reaction mixture was filtered through a Celite pad, and the
filtrate was evaporated to dryness under reduced pressure to
an oil (1.5 g, 75%). This oil was used without further
purification.
P r ep a r a t ion of Ac-DDip -Leu -Asp -Ψ[CH 2NH ]-Ile-Ile-
Tr p (6). The peptide synthesis, cleavage from the resin, and
deprotection were performed as described in the general
procedure. The crude peptide was purified by reversed-phase
HPLC to afford 43 mg of the title compound: HPLC tR ) 15.9
min (>97%); ESMS (m/ z)+ calcd 910.1, found 910.5 (M).
P r ep a r a t ion of Ac-DDip -Leu -Asp -Ile-Ψ[CH 2NH ]-Ile-
Tr p (7). P r ep a r a tion of Nr-Cbz-Ile-Ψ[CH2NH]-Ile-OMe.
NR-Cbz-Ile-CHO was prepared in two steps from NR-Cbz-Ile
as described for NR-t-Boc-DDip-CHO (8.3 g, 64%). NR-Cbz-Ile-
CHO was dissolved in dry MeOH (250 mL) and treated with
Ile-OMe‚HCl (4.8 g, 33.4 mmol) followed by NaBH3CN (2.8 g,
44.3 mmol) and acetic acid (2.0 mL, 33.3 mmol). The reaction
mixture was allowed to stir overnight and concentrated under
reduced pressure to dryness. The residue was dissolved in
EtOAc (100 mL), washed with saturated aqueous NaHCO3 and
brine (2 × 50 mL, each), dried with Na2SO4, filtered, and
concentrated under reduced pressure to an oil which crystal-
lized upon standing (9.35 g, 74%): 1H-NMR (CDCl3) δ 0.88
(m, 12H), 1.45 (m, 7H), 2.42 (m, 1H), 2.78 (m, 1H), 3.02 (d,
1H), 3.59 (m, 1H), 3.69 (s, 3H), 4.88 (m, 1H), 5.10 (s, 2H), 7.35
(s, 5H); CIMS (m/ z)+ calcd 378.4, found 379 (M + H).
P r ep a r a tion of Nr-Cbz-Ile-Ψ[CH2NH]-Ile. To a solution
of NR-Cbz-Ile-Ψ[CH2NH]-Ile-OMe (4.35 g, 11.5 mmol) in p-
dioxane (45 mL) was added 1 M aqueous LiOH (12.5 mL, 12.5
mmol). The reaction mixture was stirred at room temperature
for 4 days. The solvent was removed under reduced pressure,
and the residue was dissolved in water (10 mL). The resulting
solution was treated with 2 N HCl (23 mL). The precipitate
which formed was collected and dried (3.1 g, 74%): 1H-NMR
(DMSO-d6) δ 1.35 (m, 6H), 2.52 (m,1H), 2.78 (m, 1H), 2.99 (d,
2H), 5.02 (s, 1H), 7.22 (d, 1H), 7.35 (s, 5H); CIMS (m/ z)+ calcd
364.4, found 365 (M + H).
P r ep a r a tion of Nr-F m oc-Leu -Ψ[CH 2NH]-Asp (OBu t ).
NR-Cbz-Leu-Ψ[CH2NH]-Asp(OBut)-OBzl was dissolved in MeOH
(200 mL), treated with 20% Pd/C (2.0 g), and placed under a
hydrogen atmosphere at 50 psi (2 h, room temperature). The
reaction mixture was filtered through a Celite pad, and solvent
was evaporated under reduced pressure to provide a white
solid (6.0 g, 71%) which was suspended in a mixture of
p-dioxane:water (1:1, 200 mL). The solution was treated with
triethylamine (7.35 mL, 52.8 mmol) followed by 9-fluorenyl-
methyloxycarbonyl-N-hydroxysuccinimide (Fmoc-OSu, 7.0 g,
20 mmol). The solution was stirred at room temperature
overnight and treated with 1 M aqueous KHSO4 (52.0 mL),
and the precipitate was collected by filtration (6.4 g, 55%
yield): 1H-NMR (DMSO-d6) δ 0.82 (m, 6H), 1.18 (m, 1H), 1.40
P r ep a r a tion of Nr-F m oc-Ile-Ψ[CH2NH]-Ile. NR-Cbz-Ile-
Ψ[CH2NH]-Ile was dissolved in MeOH (50 mL) and hydroge-
nated in the presence of 20% Pd/C (0.30 g) for 2 h. The
reaction mixture was filtered, and the solvent was concen-
trated under reduced pressure to a white powder (1.8 g) which
was suspended in a mixture of water:p-dioxane (1:1, 70 mL).
The solution was treated with triethylamine (2.7 mL, 19.5
mmol) followed by Fmoc-OSu (2.6 g, 7.7 mmol). The reaction
mixture was stirred at room temperature overnight, and
treated with an aqueous solution of KHSO4 (2.7 g, 19.5 mmol
in 100 mL). The precipitate was collected, triturated with

Design of a Combined ETA/ ETB Receptor Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2237
EtOAc:hexane (1:4), and dried under reduced pressure to a
white solid (2.0 g, 51%): CIMS (m/ z)+ calcd 452.1, found 453
(M + H).
P r ep a r a tion of Ac-[NMe]DDip -Leu -Asp -Ile-Ile-Tr p (9).
H-[NMe]DDip-Leu-Asp(OBzl)-Ile-Ile-Trp was cleaved from the
resin and deprotected using anhydrous HF:anisole (9:1, 0 °C,
60 min). The crude peptide was purified by reversed-phase
HPLC and lyophilized. The purified peptide was dissolved in
90% HOAc (20 mL) and treated with acetic anhydride (2 mL)
for 2 h. Evaporation followed by lyophilization afforded 40
mg of the title compound: HPLC tR ) 18.5 min (97%); ESMS
(m/ z)+ calcd 938.1, found 937.6 (M).
P r ep a r a t ion of Ac-DDip -Leu -Asp -Ile-Ψ[CH 2NH ]-Ile-
Tr p (7). The peptide synthesis, cleavage from the resin, and
deprotection were performed as described in the general
procedure. The crude peptide was purified by reversed-phase
HPLC to afford 190 mg of the title compound: HPLC tR ) 14.5
min (>98%); FABMS (m/ z)+ calcd 910.1, found 910.7 (M).
P r ep a r a t ion of Ac-DDip -Leu -Asp -Ile-Ile-Ψ[CH 2NH ]-
Tr p (8). P r ep a r a tion of Nr-t-Boc-Ile-N(CH3)OCH3. To a
solution of NR-t-Boc-Ile (10 g, 43.2 mmol) in DMF (20 mL) were
added HCl‚HN(CH3)OCH3 (4.4 g, 45.1 mmol) and DIEA (15.7
mL, 90.1 mmol) followed by BOP reagent (9.1 g, 43.2 mmol).
The reaction mixture was stirred for 2 h. The reaction mixture
was then concentrated under reduced pressure to dryness. The
residue was taken up with EtOAc (100 mL), washed with
saturated aqueous Na2CO3, water, and 1 M aqueous KHSO4
(2 × 50 mL, each), dried with MgSO4, and concentrated under
reduced pressure to give a pale yellow oil (10.1 g, 91%): 1H-
NMR (CDCl3) δ 0.95 (m, 6H), 1.38 (s, 9H), 1.50 (m, 2H), 1.74
(m, 1H), 3.21 (s, 3H), 3.80 (s, 3H), 4.80 (m, 1H), 5.33 (d, 1H);
CIMS (m/ z)+ calcd 274.1, found 274 (M).
P r ep a r a tion of Ac-DDip -[NMe]Leu -Asp -Ile-Ile-Tr p (10).
The peptide synthesis, cleavage from the resin, and deprotec-
tion were performed as described in the general procedure.
NR-t-Boc-[NMe]Leu was obtained from commercial sources.
The crude peptide was purified by reversed-phase HPLC to
afford 18.0 mg of the title compound: HPLC tR ) 17.5 min
(>98%); ESMS (m/ z)+ calcd 938.1, found 939.1 (M + H).
P r ep a r a tion of Ac-DDip -Leu -[NMe]Asp -Ile-Ile-Tr p (11).
P r ep a r a tion of Nr-F m oc-[NMe]Asp (OBzl). To a suspen-
sion of NR-Fmoc-Asp(OBzl) (3.0 g, 9.3 mmol) and paraformal-
dehyde (2.0 g) in toluene (100 mL) was added p-toluenesulfonic
acid (0.2 g). The mixture was refluxed with azeotropic water
removal for 30 min. The reaction mixture was then cooled to
room temperature, washed with saturated aqueous NaHCO3
and brine (2 × 50 mL, each), dried with MgSO4, filtered, and
concentrated under reduced pressure (oil, 2.8 g) which was
then treated with a mixture of CHCl3 (50 mL), TFA (40 mL),
and Et3SiH (4.3 mL, 27.0 mmol) at room temperature for 40
h. The reaction mixture was evaporated under reduced
pressure to dryness. The residue was dissolved in EtOAc (50
mL), washed with saturated aqueous NaHCO3, water, 1 N
HCl, and brine (2 × 50 mL, each), filtered, dried with MgSO4,
and concentrated under reduced pressure to a foam. Crystal-
lization from EtOAc/hexane yielded a white powder (2.5 g,
80%): 1H-NMR (CDCl3) δ 2.85 (m, 1H), 2.90 (s, 3H), 3.11 (m,
2H), 4.25 (m, 2H), 5.12 (m, 2H), 7.25 (m, 7H), 7.55 (m, 3H),
P r ep a r a tion of Nr-t-Boc-Ile-CHO. To a solution of NR-
t-Boc-Ile-N(CH3)OCH3 (10.0 g, 38.9 mmol) in dry THF (200
mL) at 0 °C was added, in portions, lithium aluminum hydride
(1.7 g, 44.8 mmol). The reaction was stirred for 30 min at 0
°C and quenched by the addition of 1 M aqueous KHSO4 (100
mL). The organic phase was separated and the aqueous phase
extracted with EtOAc (100 mL). The combined organic phases
was washed with brine (1 × 50 mL), dried with MgSO4,
filtered, and concentrated under reduced pressure to a colorless
oil (6.6 g 87%): 1H-NMR (CDCl3) δ 0.96 (m, 6H), 1.36 (s, 9H),
1.38-1.84 (m, 3H), 4.38 (m, 1H), 5.38 (d, 1H), 9.60 (s, 1H);
CIMS (m/ z)+ calcd 215.3, found 216 (M + H).
1
P r epar ation of Nr-t-Boc-Ile-Ψ[CH2NH]-Tr p-P AM-Resin .
NR-t-Boc-Trp-PAM resin (1.0 mmol total) was treated with 50%
TFA in DCM (20 mL) for 30 min at room temperature; the
resin was washed successively with DCM (3 × 20 mL), 10%
DIEA in DCM (20 mL), DMF (3 × 20 mL), and 5% HOAc in
DMF (2 × 20 mL). NR-t-Boc-Ile-CHO (2.5 mmol) was added
followed by 1% acetic acid in DMF (20 mL) and NaBH3CN (2.5
mmol). The mixture was shaken at room temperature for 3
h. The resin was washed with DMF, MeOH, and DCM (3 ×
20 mL, each) and dried under reduced pressure.
P r ep a r a t ion of Ac-DDip -Leu -Asp -Ile-Ile-Ψ[CH 2NH ]-
Tr p (8). The peptide synthesis, cleavage from the resin, and
deprotection were performed as described in the general
procedure. The crude peptide was purified by reversed-phase
HPLC to afford 30 mg of the title compound: HPLC tR ) 14.5
min (>98%); FABMS (m/ z)+ calcd 910.1, found 910.7 (M).
P r ep a r a tion of Ac-[NMe]DDip -Leu -Asp -Ile-Ile-Tr p (9).
P r ep a r a tion of Nr-4,4′-Dim eth oxylp h en ylm eth yl(Dod )-
DDip -Leu -Asp (OBzl)-Ile-Ile-Tr p -P AM R esin . NR-t-Boc-
DDip-Leu-Asp(OBzl)-Ile-Ile-Trp-PAM resin (0.5 mmol) was
prepared as described in the general procedure. NR-t-Boc-
DDip-Leu-Asp(OBzl)-Ile-Ile-Trp-PAM resin (0.5 mmol) was
treated with 50% TFA/DCM for 30 min in a manual shaker
and washed with DCM (3 × 20 mL), 10% DIEA/DCM (20 mL),
and DCM (2 × 20 mL). Dod-Cl (0.20 g, 0.76 mmol) in DCM
(20 mL) was added followed by DIEA (0.5 mL). The reaction
was allowed to proceed for 1 h. The resin was drained washed
with DCM and DMF (3 × 20 mL, each), and dried under
reduced pressure.
P r ep a r a tion of H-[NMe]DDip -Leu -Asp (OBzl)-Ile-Ile-
Tr p -P AM R esin . The formaldehyde in DMF solution was
prepared as follows: To a 38% aqueous formaldehyde solution
(20 mL) was added to DMF (180 mL) followed by MgSO4 (50
g). The solution was stirred for 1 h under N2 and filtered. The
filtrate (20 mL) was added to NR-Dod-DDip-Leu-Asp(OBzl)-Ile-
Ile-Trp-PAM resin followed by HOAc (0.30 mL) and 1 M
NaBH3CN in DMF (1.25 mL, 1.25 mmol). The mixture was
shaken for 30 min and the procedure repeated. The resin was
then treated with 50%TFA/DCM (2 × 20 mL, 20 min each),
washed in turn with DCM, 10% DIEA/DCM, and DMF (3 ×
20 mL, each), and dried under reduced pressure.
7.78 (m, 3H) [In the H-NMR of this compound a doubling of
the resonances was observed, presumably due to rotational
isomerization about the urethane bond. In this case the
chemical shift of the major isomer is reported (probably the
trans conformer), but the total integration of both resonances
is reported.] FABMS (m/ z)+ calcd 459.5, found 459.1 (M).
P r ep a r a tion of Ac-DDip -Leu -[NMe]Asp -Ile-Ile-Tr p (11).
The peptide synthesis, cleavage from the resin, and deprotec-
tion were performed as described in the general procedure.
The crude peptide was purified by reversed-phase HPLC to
afford 25.8 mg of the title compound: HPLC tR ) 15.0 min
(>99%); ESMS (m/ z)+ calcd 938.1, found 939.4 (M + H), 960.1
(M + Na).
P r ep a r a tion of Ac-DDip -Leu -Asp -[NMe]Ile-Ile-Tr p (12).
The peptide synthesis, cleavage from the resin, and deprotec-
tion were performed as described in the general procedure.
NR-t-Boc-[NMe]Ile was obtained from commercial sources. The
crude peptide was purified by reversed-phase HPLC to afford
58.5 mg of the title compound: HPLC tR ) 18.5 min (97%);
ESMS (m/ z)+ calcd 938.1, found 938.1 (M), 960.7 (M + Na).
P r ep a r a tion of Ac-DDip -Leu -Asp -Ile-[NMe]Ile-Tr p (13).
P r ep a r a tion of Nr-F m oc-Ile-COCl. To a suspension of NR-
Fmoc-Ile (10.0 g, 28.3 mmol) in DCM (100 mL) was added
oxalyl chloride (2.9 mL, 33.0 mmol) dropwise at 0 °C. The
reaction mixture was stirred for 1 h at 0 °C and warmed to
room temperature. The reaction mixture was stirred at room
temperature for an additional 2 h and evaporated under
reduced pressure to an oil (8.5 g, 81%): 1H-NMR (CDCl3) δ
0.85 (t, 3H), 1.05 (d, 3H), 1.18 (m, 1H), 1.45 (m, 1H), 2.12 (m,
2H), 4.25 (t, 1H), 4.50 (m, 2H), 5.25 (d, 1H), 7.25 (t, 2 H), 7.38
(t, 2H), 7.55 (m, 2H), 7.74 (d, 2H) [In the 1H-NMR for this
compound a doubling of the resonances was observed, presum-
ably due to rotational isomerization about the urethane bond.
In this case the chemical shift of the major isomer is reported
(probably the trans conformer), but the total integration of both
resonances is reported.] CIMS (m/ z)+ calcd 370.2, found 369
(M + H).
P r ep a r a tion of Nr-F m oc-Ile-[NMe]Ile-Tr p -P AM Resin .
NR-t-Boc-[NMe]Ile-Trp-PAM resin (0.5 mmol) was prepared as
described in the general procedure. NR-t-Boc-[NMe]Ile was
obtained from commercial sources. NR-t-Boc-[NMe]Ile-Trp-

2238 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Cody et al.
PAM resin was treated with 50% TFA/DCM (20 mL) for 30
min and washed with DCM (3 × 20 mL), 10% DIEA/DCM (20
mL), and DCM (3 × 20 mL). NR-Fmoc-Ile-COCl (0.6 g, 1.6
mmol) in DCM (20 mL) was added followed by DIEA (2.7 mL,
1.55 mmol). The reaction mixture was shaken for 2 h, and
the procedure was repeated. The peptide resin was then
washed with DCM (3 × 20 mL) and dried under reduced
pressure.
P r ep a r a tion of Ac-DDip -Leu -Asp -Ile-[NMe]Ile-Tr p (13).
The peptide synthesis, cleavage from the resin, and deprotec-
tion were performed as described in the general procedure.
The crude peptide was purified by reversed-phase HPLC to
afford 70.1 mg of the title compound: HPLC tR ) 17.3 min
(98%); ESMS (m/ z)+ calcd 938.1, found 939.1 (M + H).
P r ep a r a tion of Ac-DDip -Leu -Asp -Ile-Ile-[NMe]Tr p (14).
P r ep a r a tion of Nr-Dod -Tr p -P AM Resin . NR-t-Boc-Trp-
PAM resin (0.5 mmol) was treated with 50% TFA/DCM for 30
min in a manual shaker and washed with DCM (3 × 20 mL),
10% DIEA/DCM (20 mL), and DCM (2 × 20 mL). Dod-Cl (0.20
g, 0.76 mmol) in DCM (20 mL) was added followed by DIEA
(0.5 mL). The reaction was allowed to proceed for 1 h. The
resin was drained, washed with DCM and DMF (3 × 20 mL,
each), and dried under reduced pressure.
P r ep a r a tion of H-[NMe]Tr p -P AM Resin . The formal-
dehyde in DMF solution was prepared as follows: To a 38%
aqueous formaldehyde solution (20 mL) was added DMF (180
mL) followed by MgSO4 (50 g). The solution was stirred for 1
h and filtered. The filtrate (20 mL) was added to NR-Dod-Trp-
PAM resin followed by HOAc (0.30 mL) and 1 M NaBH3CN
in DMF (1.25 mL, 1.25 mmol). The mixture was shaken for
30 min and the procedure repeated. The resin was then
treated with 50% TFA/DCM (2 × 20 mL, 20 min each), washed
in turn with DCM, 10% DIEA/DCM, and DMF (3 × 20 mL,
each), and dried under reduced pressure.
P r ep a r a tion of Ac-DDip -Leu -Asp -Ile-Ile-[NMe]Tr p (14).
The peptide synthesis was performed as described in the
general procedure. After the coupling of NR-t-Boc-DDip, the
peptide resin was treated with 50% TFA/DCM (30 min). and
the peptide was deprotected and cleaved from the resin using
anhydrous HF:anisole (9:1, 0 °C, 60 min). The crude peptide
was purified by reversed-phase HPLC and lyophilized. The
purified peptide was dissolved in 90% acetic acid (20 mL) and
treated with acetic anhydride (2 mL) for 2 h. Evaporation
followed by lyophilization afforded 40 mg of the title com-
pound: HPLC tR ) 16.7 min (97%); ESMS (m/ z)+ calcd 938.1,
found 937.6 (M).
bolus). Compound 15 was administered at a dose of 10 mg/
kg (iv, bolus) 5 and 30 min prior to the ET-1 challenge.
Nu clea r Ma gn etic Reson a n ce Stu d ies. 1H-NMR spectra
were recorded on a Bruker AMX 500 spectrometer using
samples typically 5-10 mM dissolved in deuterated solvents
obtained from Cambridge Isotope Laboratories. Two-dimen-
sional total correlation spectroscopy (TOCSY),64 double-
quantum-filtered correlation spectroscopy (dqf-COSY),65 ro-
tating frame nuclear Overhauser spectroscopy (ROESY),66,67
and nuclear Overhauser spectroscopy (NOESY)68 were ac-
quired as 1024 × 512 matrices and were processed using
UXNMR (Bruker Instruments). Quadrature detection in the
t1 dimension was achieved by the time proportional phase
incrementation (TPPI)69 method in 2D spectra. Prior to zero-
filling to 1K in the second dimension and Fourier transforma-
tion, cos2 weighting functions were applied to the time domain
data in both dimensions. Finally, the baselines in the 2D time
domain matrices were flattened using a polynomial fitting
routine. NOE data were converted to distance constraints as
previously described.70 NOESY cross-peaks for which volume
buildup curves (80-400 ms) suggested contributions from spin
diffusion were excluded from the analysis. Coupling constants
were measured directly from resolution-enhanced 1D spectra
and were used as converted to dihedral constraints where
warranted. Amide temperature coefficients were measured
relative to an internal reference [3-(trimethylsilyl)propionic
acid-d4 sodium salt (TSP) for aqueous samples, the residual
solvent resonance in organic solvents] for at least three
different temperatures. Structures were generated using the
distance geometry/simulated annealing program DGII (Biosym
Technologies) followed by constrained energy minimization
using a previously described protocol.70
Secondary shifts were calculated by subtracting the random
coil chemical shift from the observed chemical shift, and all
observed chemical shifts were referenced directly or indirectly
to TSP protons at 0.0 ppm.71 Carbon-13 random coil chemical
shifts for natural amino acids were obtained from the ap-
propriate literature references for peptides dissolved in D2O71
and DMSO.72 To correct the DMSO solution random coil
chemical shifts for referencing relative to TSP protons, 3.34
ppm was added to the reported values. For unnatural
residues, random coil shifts were measured in the peptides
Gly-Gly-Xxx-Gly-Gly, where Xxx ) DBhg and [NMe]Ile as
previously described.71 The random coil chemical shifts used
to calculate secondary shifts are provided in the Supporting
Information. For the disodium salt peptides, 1.5 ppm was
added to the random coil chemical shift of the R carbon of Asp18
to compensate for differences in ionization state from the
literature values. This correction factor was derived from the
reported shifts for Asp carbons in the neutral and anionic
forms of Asp-containing peptides in D2O.72 For Ile19 in com-
pound 15, 2.4 ppm was subtracted from the random coil
chemical shift of the Ile R carbon to compensate for its
attachment to a tertiary amide group.63 This correction factor
was derived from the reported shifts for Ile in the peptides
Gly-Gly-Ile-Ala-Gly-Gly and Gly-Gly-Ile-Pro-Gly-Gly.72
P r epar ation of Ac-DBh g-Leu -Asp-Ile-[NMe]Ile-Tr p (15).
The peptide synthesis, cleavage from the resin, and deprotec-
tion were performed as described for Ac-DDip-Leu-Asp-Ile-
[NMe]Ile-Trp (compound 13). The crude peptide was purified
by reversed-phase HPLC to afford 530 mg of the title com-
pound: HPLC tR ) 18.0 min (>97%); ESMS (m/ z)+ calcd 964.1,
found 963.4 (M).
En d oth elin Recep tor Bin d in g Assa y P r otocol. The
receptor binding assay protocols using rabbit renal vascular
smooth muscle cells (ETA), rat cerebellar membranes (ETB),
or the corresponding cloned human ETA and ETB receptors
have been previously described.29
Ack n ow led gm en t. The authors would like to thank
Dana DeJ ohn J oseph Loo, Tracy Stevenson, and Brian
Tobias for analytical and spectral data; Michael Flynn
and Kathleen Welch for pharmacological data; Vlad
Beylin, Huaigu Chen, and Om Goel for the preparation
of DDip and DBhg; and J ames Kaltenbronn and Bill
Reisdorph for the preparation of protected intermedi-
ates.
In Vitr o Con tr a ctility Stu d ies. The experimental pro-
tocols using vascular rings of rat femoral artery (ETA) or rabbit
pulmonary artery (ETB) have been previously described.29
Ca co-2 Cell Tr a n sp or t a n d Sta bility in Ra t In testin a l
P er fu sa te Exp er im en ts. These studies were performed as
previously described.34
Con sciou s R a t Du r a t ion of Act ion St u d y w it h P D
156252. Nonfasted rats (350-500 g) were anesthetized with
methoxyflurane by inhalation and instrumented with a jugular
cannula (PE50) for iv administration of test agents and with
a carotid artery cannula (PE50) for arterial blood pressure
measurements. Rats were attached to a swivel for freedom
of movement, and food and water were available ad libidum.
Prior to the experiment, the animals were allowed to recover
from the anesthesia for 60 min. Following recovery the rats
were ganglionically blocked with mecamylamine‚HCl (1.25 mg/
kg, iv) 20 min prior to the ET-1 challenge (0.30 nM/kg, iv,
Su p p or tin g In for m a tion Ava ila ble: Tables containing
1
selected H-NMR data for compounds 3 and 15 and the random
coil chemical shifts used to calculate secondary shifts and
figures illustrating the secondary CR/HR chemical shifts of
compounds 3 and 15 and the titration of compound 15 (7
pages). Ordering information is given on any current mast-
head page.

Design of a Combined ETA/ ETB Receptor Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2239
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