Development of peptide 3D structure mimetics: Rational design of novel peptoid cholecystokinin receptor antagonists

Article abstract of DOI:10.1021/jm000937a

The two hormones cholecystokinin and gastrin share the same C-terminal sequence of amino acids, namely Gly²⁹-Trp³⁰-Met³¹-Asp³²-Phe³³-NH₂. Nevertheless, this congruence has not precluded usi

Full text of DOI:10.1021/jm000937a

J . Med. Chem. 2000, 43, 3505-3517
3505
Develop m en t of P ep tid e 3D Str u ctu r e Mim etics: Ra tion a l Design of Novel
P ep toid Ch olecystok in in Recep tor An ta gon ists
Caroline M. R. Low,* J ames W. Black, Howard B. Broughton, Ildiko M. Buck, J onathan M. R. Davies,
David J . Dunstone, Robert A. D. Hull, S. Barret Kalindjian, Iain M. McDonald, Michael J . Pether,
Nigel P. Shankley, and Katherine I. M. Steel
J ames Black Foundation, 68 Half Moon Lane, London SE24 9J E, U.K.
Received March 10, 2000
The two hormones cholecystokinin and gastrin share the same C-terminal sequence of amino
acids, namely Gly29-Trp30-Met31-Asp32-Phe33-NH2. Nevertheless, this congruence has not
precluded using this structure to develop selective ligands for either CCK1 or CCK2 receptors.
Manipulation of the hydrophobic residues at positions 31 and 33 gave a series of CCK1 tripeptide
antagonists, typified by N-t-BOC-Trp-2-Nal-Asp-2-(phenyl)ethylamide (pKB 6.8 ( 0.3). Molecular
modeling was used to identify the bioactive conformation of these CCK1-selective compounds
and prompted the design of new peptoid structures. We aimed to maintain the conformation
of the parent series by exploiting patterns of hydrogen-bonding and π-stacking interactions
present in the original molecule, rather than introducing additional covalent bonds. The
prototype, N-(succinyl-D-Asp-2-phenylethylamido)-L-Trp-2-(2-naphthyl)ethylamide, was a potent
and selective CCK1 antagonist (pKB 7.2 ( 0.3). Furthermore, the new series showed patterns
of biological activity that mirrored those of the parent tripeptides. These compounds contain
elements of both peptide primary and secondary structure and represent a novel approach to
designing peptidomimetics. Interesting results were obtained from comparing models of a
representative tripeptide CCK1 antagonist with a conformation of CCK30-33 that others have
proposed to be responsible for its activity at the CCK2 receptor. The results suggest that CCK1
and CCK2 receptors recognize enatiomeric dispositions of the Trp30 indole, Asp32 carboxylic
acid, and C-terminal phenyl groups arrayed about a common backbone configuration. This
“functional chirality” may underpin the mechanism by which these closely related receptor
systems bind CCK30-33 and explain patterns of selectivity observed with optical isomers of a
number of peptoid and nonpeptide ligands.
In tr od u ction
versely, this fragment only has micromolar affinity for
the CCK1 receptor, which requires the sulfated octapep-
tide fragment (CCK-8S) for full activation. This frag-
ment is also common to the structure of the decapeptide
caerulein, found in the skin of amphibia, suggesting that
the mammalian hormones cholecystokinin and gastrin
may share a common evolutionary history.11
Cholecystokinin and gastrin are two closely related
peptide hormones that mediate a range of peripheral
and central biological processes. Cholecystokinin recep-
tors are divided into two subclasses: CCK1 and CCK2
receptors. CCK1 receptors occur centrally in the nucleus
tractus solitarius1 but are mainly located in the periph-
ery in the pancreas,2 gall bladder,3 and colon. CCK2
receptors are the predominant subtype in the brain and
are widely distributed throughout the cortex.4 The CCK2
receptor is also located on the ECL cell of the stomach,
and there is strong evidence to suggest that the central
and peripheral populations of receptors are homoge-
neous, on the basis of their selectivity for a range of
ligands and evidence from molecular hybridization
studies.5 The human CCK1 and CCK2 receptors have
now both been cloned and shown to belong to the family
of G-protein coupled receptors.5-8
CCK1 receptor antagonists have been shown to block
CCK-8-induced contraction of the gallbladder and in-
hibit gastric emptying,12 pancreatic secretion,13 and
satiety.14 On the other hand CCK2 antagonists have
attracted interest on the basis of their ability to inhibit
gastrin-stimulated gastric acid secretion in the periph-
ery.15 A number of central effects have also been noted
for some of these latter compounds, such as anxiolytic
behavior16 and ability to potentiate morphine-induced
analgesia.17
Our aim was to design novel CCK1 and CCK2 antago-
nists using the structure of the C-terminal tetrapeptide,
CCK30-33, as the chemical starting point. The primary
requirement was that the final compounds should be
selective for one receptor type over the other. A further
aim was that their structures had to be sufficiently
different from those of the parent peptides to try to avoid
the recognized problems associated with using peptides
as drugs: namely poor bioavailability, rapid proteolytic
cleavage, and clearance in vivo.
From a chemical point of view, the two hormones need
to be considered together because they share the same
primary sequence of amino acids at their C-termini,
namely Gly-Trp-Met-Asp-Phe-NH2. In fact, only the last
four residues, CCK30-33 (Trp-Met-Asp-Phe-NH2), are
required to elicit a full biological response at the CCK2
receptors in both the periphery9 and the brain.10 Con-
* To whom correspondence should be addressed. Phone: +44
2077378282. Fax: +44 2072749687. E-mail: caroline.low@kcl.ac.uk.
10.1021/jm000937a CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/21/2000

3506 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Sch em e 1. Synthesis of Tetrapeptide 2a
Low et al.
Sch em e 2. Synthesis of Tetrapeptide 3a
a
(a) Ala, NaHCO3, DME, H2O; (b) NHS, DCCI, DME; (c) (i)
TFA, (ii) 39, NaHCO3, DME; (d) H2, Pd/C, EtOH.
Sch em e 3. Synthesis of Compounds 4 and 5a
a
(a) Phe-NH2‚NCl, NaHCO3, DME, H2O; (b) Leu, NaHCO3,
DME, H2O; (c) NHS, DCCI, DME; (d) (i) TFA, (ii) 36, NaHCO3,
DME; (e) H2, Pd/C, EtOH.
Broad cross-screening strategies have led to the
discovery of a number of nonpeptide ligands for these
receptors.18-22 This work has proceeded in parallel with
the alternative strategy based on using the parent
hormone as the initial chemical lead. We have chosen
to use this latter approach and have already reported a
number of compounds designed in this way. These have
included the CCK1-selective sulfonamide 2-NAP,23,24 as
well as a range of nonpeptide CCK2 ligands based on
oxathiazinone,25 dibenzobicyclo[2.2.2]octane (BCO),26,27
and indole skeletons.28 The design of effective antago-
nists for these receptor systems required that we tackled
the twin problems of removing the ability of the
hormone to activate the receptor (efficacy) and biasing
the selectivity of the compounds in favor of one or other
receptor system.
a
(a) Leu, NaHCO3, EtOH, H2O; (b) NHS, DCCI, DME; (c) Ala-
NH2‚HCl, NaHCO3, DME, H2O; (d) (i) TFA, (ii) 42, Et3N, DME;
(e) H2, Pd/C, MeOH; (f) Phe-NH2, DME.
Sch em e 4. Synthesis of Compounds 6-20a
We have not been alone in taking the stucture of the
terminal tetrapeptide as our starting point, and the
Parke-Davis group has described selective CCK1 and
CCK2 ligands designed around the structure of the
dipeptide fragment Trp-Phe.29,30 However, a number of
reports suggest that several examples of these com-
pounds retained the agonist properties of the parent
hormone.31-37 The balance between removing the ability
of a molecule to cause agonism (efficacy) without
disturbing its ability to bind to the receptor is clearly
extremely fine and has been at the heart of our search
for novel and selective antagonists of these two receptor
systems. This paper will focus on our efforts to achieve
this target and will describe the preparation of a
number of peptoids whose structures were derived from
that of BOC-CCK30-33. Our efforts to interpret the
patterns of activity that were obtained in terms of the
3D structure of the molecules led to the design and
synthesis of a second series of peptoids. The aim of this
exercise was to mimic elements of 3D structure identi-
fied in the first series, without the introduction of
additional covalent links. The success of this approach
is reflected in the biological activity of the new series
of compounds - which is directly comparable with that
of the parent series. This new series was optimized to
give a number of potent and selective CCK1 antagonists.
a
(a) (S)-2-Naphthylalanine, NaHCO3, EtOH, H2O; (b) NHS,
DCCI, DME; (c) DME; (d) (i) TFA, (ii) 46, Et3N, DME; (e) H2, Pd/
C, MeOH.
tetrapeptides required to evaluate the contribution of
the amino acid side chains of CCK30-33 were synthesized
using standard methods from commercially available
starting materials (Schemes 1-3). The same methods
were used to prepare compound 6 in which naphthyl-
alanine (Nal) was incorporated in place of Leu31 (Scheme
4). Modification of Phe33 in this latter series was effected
by coupling the appropriately substituted 2-phenylethyl-
amines, or phenylalanines, to the N-hydroxysuccinimide
ester of protected Asp (Scheme 4). The resulting frag-
ments were then coupled to the dipeptide NHS ester
46 and the benzyl protecting group was removed by
hydrogenolysis in the presence of 10% palladium on
charcoal catalyst to give the peptide derivatives 6-20.
Ch em istr y
The compounds described for the first time in this
paper were prepared according to Schemes 1-6. The

Novel Peptoid CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3507
Sch em e 5. Synthesis of Compounds 23-33a
the atom-centered Gasteiger-Huckel charges used in
SYBYL. We have found that these XED charge descrip-
tions reproduce phenomena such as π-stacking of aro-
matic rings, anomeric effects, and polar atom interac-
tions better than conventional atom-centered charges.
The conditions used for generating sets of conformations
under SYBYL and XED were the same, with the
exception of the charge description used. The second
series of selective CCK1 antagonists (23-33) was mod-
eled exclusively with XED. Full details of the XED
calculations were as follows. All calculations were
carried out at dielectric 4.0, allowing all rotatable bonds,
including the peptide amides, to rotate. The initial set
of conformations was generated using a hard spin
torsional minimizer. Each input conformation was
randomized 250 times (Monte Carlo), twisting each bond
by 10° torsional increments, iterating through all bonds
to 0.001 kcal/mol and storing all conformations within
a 15.0 kcal/mol range of the lowest-energy structure
found. Conformations within 15° on all torsions of any
other were removed. The energies of all structures were
then minimized using a combined parabolic/Fletcher-
Reeves conjugate gradient technique whose accuracy
was set at <0.01 cal/mol. XED charges were then added
to each conformation and the energies of each structure
minimized once again using the same procedure. Con-
formations were collected over a 15.0 kcal/mol energy
range to a maximum of 1000 structures. Thus 100
conformations covering a 9.3 kcal/mol energy range were
identified for the tripeptide 17. The lowest-energy
structures were compared to a model of BOC-CCK30-33
that was created from the published torsion angles
contained in Kolodziej et al.39 (Table 3).
Resu lts a n d Discu ssion
The C-amidated terminal tetrapeptide fragment
CCK30-33 (BOC-Trp-Met-Asp-Phe-NH2) is a potent stimu-
lant of gastric acid secretion as determined by its
behavior in the isolated, lumen-perfused mouse stomach
bioassay.40 We have used this assay on a routine basis
to examine CCK2 receptor ligands as it has proven to
be particularly sensitive in revealing agonist behavior
that would be missed in other tissues.41 Literature
precedent states that the hydrophobic side chain of the
Met31residue does not play an important role in deter-
mining the biological activity of CCK30-33, and it is
widely accepted that this chemically sensitive group can
be replaced by Leu or Nle without detriment to the
pharmacological properties of the molecule.42-44 To
confirm this observation and the suggested role of the
amino acid side chains, we made a series of compounds
in which each residue of the tetrapeptide BOC-Trp-Leu-
Asp-Phe-NH2 was sequentially exchanged with alanine.
This is a recognized method of testing the contribution
of side chain functional groups in a systematic manner
with minimal effect on the conformation of the peptide
backbone.45 This produced an interesting variety of
activities, as summarized in Table 1. The importance
of the carboxylic acid side chain of the Asp residue is
clearly illustrated by the lack of activity of the Ala
analogue 4, when tested at a concentration of 1 × 10-5
M. Furthermore, the bioassay results suggested that it
might be possible to separate the influence of the side
chain functional groups on the ability of the compound
to bind to the CCK2 receptor (affinity) and effect release
Similarly, convergent syntheses of compounds 23-33
were achieved by coupling appropriate L-Trp (49) and
D-Asp (50) fragments (Scheme 5). However, we found
that this reaction proceeded in higher yield if (benzo-
triazol-1-yloxy)trispyrrolidinophosphonium hexafluoro-
phosphate (pyBOP) was employed as the coupling
reagent. Hydrogenolysis of the resulting adducts gave
the compounds described in Table 4.
Molecu la r Mod elin g
The majority of the modeling studies described for the
initial series of tripeptides 6-20 were carried out using
molecular mechanics calculations as implemented in the
commercial package SYBYL (Tripos Associates Inc.).
This included the design of the prototype CCK1 antago-
nist 23 described in the following section. However,
compounds 17, 7, 10, and 12 were also examined using
a modified version of COSMIC equipped with XED
(extended electronic d istribution) charges38 rather than

3508 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Low et al.
Sch em e 6. Design of Prototype CCK1 Antagonist 23 from Tripeptide 7
great a secretory response as BOC-[Leu31]CCK30-33 (1)
when given at a high enough concentration. This sug-
gested that the phenyl ring made a substantial contri-
bution to the way in which the tetrapeptide binds to
the receptor but did not unduly influence the effector
mechanism. Deleting the indole group from the N-
terminal residue (2) produced a loss of both affinity and
efficacy. However, the most significant result was
obtained when the methionine/leucine side chain was
truncated. In this case, it became apparent that com-
pound 3 produced the same level of secretory response
as the peptide in which alanine replaced tryptophan (2)
but had retained a higher level of affinity for the
receptor. This loss of efficacy was surprising in the light
of the early literature precedent,42-44 but it suggested
that elaboration at this point in the peptide structure
might allow us to manipulate this parameter without
detriment to the overall binding.
Ta ble 1. Results of Alanine Scan on the Tetrapeptide
BOC-Trp-Leu-Asp-Phe-NH2: Activity at CCK2 Receptors in the
in Vitro Mouse Stomach Assay40
CCK2
a
no.
compd
pA50
R (%)b
1
2
3
4
5
BOC-Trp-Leu-Asp-Phe-NH2
BOC-Ala -Leu-Asp-Phe-NH2
BOC-Trp-Ala -Asp-Phe-NH2
BOC-Trp-Leu-Ala -Phe-NH2
BOC-Trp-Leu-Asp-Ala -NH2
8.9 ( 0.2
6.0 ( 0.2
7.0 ( 0.2
inactivec
4.0-5.0
100 ( 4
64 ( 9
68 ( 6
>95
a
The pA50 value is the position of half-maximal response of the
agonist curve for the test compound and is the mean of at least
six separate experiments. The errors quoted represent the SEM.
b
R represents the percentage maximal response for the compound
relative to a pentagastrin control and is the mean of at least six
separate experiments. The errors quoted represent the SEM. c The
compound was inactive when tested at a concentration of 1 × 10-5
M.
of gastric acid (efficacy). Removing the phenylalanine
aromatic ring gave a compound (5) with low affinity,
which was nevertheless capable of eliciting almost as
A brief investigation was carried out in which the

Novel Peptoid CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3509
Ta ble 2. CCK1 and CCK2 Data Obtained as a Result of
Modifying the C-Terminal Residue, Phe33, of
BOC-[Nal(2)31]CCK30-33
stomach assay to a level equivalent to that found for
the parent compound BOC-[Leu31]CCK30-33. Unlike
Corringer et al.47 we found that both the mono- and
dimethylamides (Table 2, examples 8 and 9, respec-
tively) showed increased levels of agonist behavior at
CCK2 receptors. Modeling compound 7 showed that the
structure of the calculated global minimum energy
conformation was one in which the three aromatic rings
clustered together in a 3-way π-stack. However, we
cannot equate the level of efficacy observed at CCK2
receptors to the proportion of conformations in which
this arrangement of aromatic groups occurs because
these were not significantly different for the antagonist
6 (30%) and the full agonist 10 (44%) over the energy
range examined. Nevertheless, it is possible that this
small difference is reflected in the modest increase in
CCK1 receptor affinity.
a
b
CCK2
CCK1
pKB
c
no.
X
Y
pKB
6.3 ( 0.2 NS
20 ( 4
R (%)d
6
7
8
9
10
H
H
H
H
H
CONH2
H
CONHMe
CONMe2
5.3 ( 0.2
6.8 ( 0.3
55 ( 13 5.4 ( 0.2
55 ( 6
6.2 ( 0.2
CH2OH
126 ( 26 5.9 ( 0.1
11 4-OMe
12 4-OMe
13 4-Cl
CONH2
H
CONH2
5.6 ( 0.3 NS
6.0 ( 0.2 NS
5.8 ( 0.2
7.2 ( 0.3
74 ( 19 5.9 ( 0.3
60 ( 11 6.3 ( 0.4
14 4-Cl
H
H
H
H
H
H
H
15 2-OMe
16 3-OMe
17 3,4-(OMe)2
18 4-F
19 4-NH2
20 3-CF3
5.2 ( 0.2 NS
5.1 ( 0.3 NS
7.1 ( 0.4
7.2 ( 0.3
6.5 ( 0.3
6.7 ( 0.3
6.5 ( 0.2
6.1 ( 0.3
inactivee
Further progress in reducing the modest levels of
agonism at CCK2 receptors within the series of phenyl-
ethylamides related to 7 was achieved by adding sub-
stituents to the phenyl ring to modify the electron
distribution in this region of the molecule. In general,
it appeared that electron-donating groups were best
suited for this purpose. Thus the 4-methoxyphenyl
analogue 12 was a competitive antagonist, but the
4-chlorophenyl derivative 14 showed increased agonist
activity. The pattern was repeated for the 4-F (18) and
4-NH2 (19) derivatives. This effect was independent of
the presence of a C-terminal amide. Levels of CCK1
affinity were unchanged throughout, and so selectivity
for this class of cholecystokinin receptors began to
emerge as CCK2 activity fell.
29 ( 8
5.9 ( 0.3 NS
inactivee
a
Values determined in the in vitro isolated lumen-perfused
mouse stomach.40 Values determined in the in vitro guinea-pig
b
gallbladder with respect to the shift of the CCK-8S dose-response
curve and are the mean of at least six experiments. c For entries
in this column the values represent pKB ( SEM determined with
respect to the shift of the pentagastrin dose-response curve and
are the mean of at least six separate experiments. For any
compounds that show significant agonism no affinity is stated.d
R
represents the percentage maximal response for the compound
relative to a pentagastrin control and is the mean of at least six
separate experiments. NS, no significant secretory effect observed
when the compound was tested at 1 × 10-5 M. e The compound
was inactive when tested at a concentration of 1 × 10-5 M.
methionine residue of BOC-CCK30-33 was replaced with
a number of other hydrophobic amino acids. This rapidly
led to the identification of the S-3-(2-naphthyl)alanine
(L-2-Nal) derivative 6 (Table 2), which was found to be
a competitive antagonist with modest affinity for both
CCK1 and CCK2 receptors - reinforcing our view that
modification of the residue in position 31 was key to
manipulating efficacy at the CCK2 receptor.
A survey of the literature also showed that the
presence, or absence, of the C-terminal amide of BOC-
CCK30-33 contributes to the observation of an agonist
response. For example, a report some time ago showed
that deletion of the C-terminal amide from the agonist
BOC-[Leu31]CCK30-33 converted the compound from an
agonist to an antagonist, as judged by its behavior in
vitro in the isolated rat stomach assay.46 We have found
that these compounds continue to behave as partial
agonists in the analogous mouse stomach assay -
suggesting that efficacy has been reduced but not
abolished. Also, Corringer et al.47 reported that BOC-
[Phg31,Nal33]CCK30-33 behaved as a full agonist in an
electrophysiological assay on rat hippocampal CCK2
receptors but that the C-terminal dimethylamide was
a competitive antagonist in the same assay. We have
examined the effect of similar modifications on ana-
logues of the L-2-Nal derivative 6.
Molecular modeling showed that the driving force
behind the conformational preferences observed in these
CCK1 antagonists stems from a 3-way π-stacking in-
teraction between the indole, naphthalene, and substi-
tuted phenyl groups. We observe that these compounds
bind preferentially to CCK1 receptors and are antago-
nists rather than agonists at CCK2 receptors. Com-
pounds 7 (Y ) H), 17 (Y ) 3,4-(OMe)2), and 12 (Y )
3-OMe) differ in the number of methoxy substituents
attached to the terminal phenyl group. Careful exami-
nation of the sets of conformations generated with the
XED modeling package showed that there was a steady
increase in the proportion of conformations containing
the 3-way π-stacking interaction (31%, 57%, and 92%,
respectively, within a 3 kcal/mol range of the calculated
global minimum energy conformation). However, nei-
ther the observed increase in affinity for the CCK1
receptor nor differences in the expression of efficacy at
CCK2 receptors can be attributed to this property alone
as only compound 17, of those examined, showed a
single specific biological activity. Instead, we chose to
use this observation to design novel peptoid structures
that maintained the conformation that we believed to
be responsible for the CCK1 activity of this series. Such
compounds should be CCK1 antagonists, as we have no
evidence to suggest that the conformation we have
identified is capable of evoking a response in the
functional GP gallbladder assay. At this point we could
have chosen to introduce covalent bonds to stabilize this
structure but decided against this approach as macro-
cyclization had already been shown to lead to a loss of
affinity for the receptor,47 in addition to making the
In this series, the compound lacking the C-terminal
amide showed low levels of efficacy at CCK2 receptors
but a 30-fold increase in affinity at CCK1, relative to
compound 6. However, replacing the C-terminal amide
with a hydroxymethyl group (10) restored the ability of
the compound to cause acid secretion in the mouse

3510 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Low et al.
Ta ble 3. Backbone Torsion Angles (deg) for the Tetrapeptide Ac-CCK30-33 (from ref 39) and Calculated Global Minimum of
Tripeptide 17
Trp
Xxx
Asp
Phe
φ4
compd
Xxx
Y
R
ψ1
ω12
φ2
ψ2
φ3
ψ3
Ac-CCK30-33
tripeptide 17
Met
2-Nal
S-CONH2
H
H
169
45
180
-179
-83
-87
93
74
-71
-88
149
56
-118
3,4-(OMe)2
Ta ble 4. CCK1 Data for Novel Peptoids Designed from the 3D
Structure of Peptoid 7
S to R to give 23. Molecular mechanics then showed that
the 3-way interaction of the aromatic nuclei was re-
tained in the low-energy conformations of the new
molecule and that the carboxylic acid of the aspartic acid
residue was oriented in the same region of space as that
found in the parent peptoid 7. This gave a target
molecule that was readily amenable to synthesis and
which appeared to maintain the disposition of the
peptide side chains in space but whose backbone was
substantially different from that of the parent peptide.
The new molecule 23 was a potent CCK1 antagonist
(pKB 7.2 ( 0.3) when tested in vitro in the guinea-pig
gallbladder strip assay. In addition, it was at least 50-
fold selective for this receptor over CCK2, since it did
not produce any significant antagonist effect, or secre-
tory response, when tested at 1 × 10-5 M in the mouse
stomach bioassay. This result merited further investiga-
tion, and a number of analogues of 23 were prepared to
examine the structure-activity relationship (SAR) be-
tween the new series of compounds and those related
to 7.
We have already demonstrated the effect of varying
the nature of the substituent on the terminal phenyl
ring in the parent L-2-Nal series of peptide derivatives
(Table 2). In this case, introduction of electron-with-
drawing groups at either the 3- or 4-position on the
aromatic ring of the new series of compounds (24, 26)
reduced levels of CCK1 activity by a factor of 10 (Table
4). However, the 4-methoxy analogue 25 was of equiva-
lent activity to compound 23 - as observed in the parent
series. The gastrin activity of all of these compounds
remained low, and most were found to be inactive when
tested at a concentration of 3 × 10-5 M in the in vitro
CCK2 bioassay.
We have postulated that the structure of this series
is determined by hydrogen-bonding between elements
of the amide backbone and π-stacking between the three
aromatic moieties. Compounds in which the naphtha-
lene group of compound 25 had been replaced by 3,4-
dichlorophenyl (27) and phenyl (28) showed a sequential
drop in activity, demonstrating the importance of this
group in maintaining the overall topology of the series.
This facet was explored further by introducing an
isopropyl group in place of the 2-naphthalene substitu-
ent in example 29. We were surprised to find that this
compound showed a degree of agonism in the guinea-
pig gall bladder assay that amounted to 44% of the con-
trol value obtained with CCK-8S. In fact, the only other
compound to have shown any agonism (6% of the con-
trol) in this bioassay throughout this investigation was
BOC-[Leu31]CCK30-33, which might be considered to be
the closest relative of compound 29 in the parent series.
Examining a model of the lead compound 23 showed
that the lowest-energy conformation was that in which
CCK1 activity,a
no.
R
X
Y
pKB ( SEM
7.2 ( 0.3
6.0 ( 0.3
4-OMe 6.7 ( 0.2
3-CF3
23
24
25
26
27
28
29
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
(CH2)2
(CH2)2
(CH2)2
(CH2)2
H
4-F
6.0 ( 0.4
3,4-dichlorophenyl (CH2)2
4-OMe 5.9 ( 0.3
phenyl
(CH2)2
(CH2)2
4-OMe 5.5 ( 0.3
iPr
4-OMe 5.4 ( 0.2 (pA50
)
b
30a 2-naphthyl
H
6.2 ( 0.2
30b 2-naphthyl
as above
as above 4-F
as above 4-OMe 6.7 ( 0.2
H
6.5 ( 0.2
6.7 ( 0.1
31
32
2-naphthyl
2-naphthyl
33
2-naphthyl
4-OMe 7.4 ( 0.4
a
Values determined in the in vitro guinea-pig gallbladder with
respect to the shift of the CCK-8S dose-response-curve. pKB
(
SEM values quoted are the mean of at least six experiments. All
compounds were inactive when tested at a concentration of 1 ×
b
10-5 M in the functional CCK2 assay. See text for details.
synthesis more demanding. A prototype molecule was
designed to fulfill these criteria taking the peptide 7 as
the starting point. The aim was to create a structure
that maintained the 3-way π-stack between the indole,
naphthalene, and phenyl groups and incorporated a
carboxylic acid in a position corresponding to that found
in the modeled structure of 7. The design process can
formally be described in the following manner (Scheme
6): First, the tert-butyloxy group from the N-terminus
was deleted in order to free up the valency of the sp2
carbon (*) (21). This was then covalently linked to the
carbonyl of the naphthylalanine residue ([) by two
methylene groups. In addition, the bond between the
R-carbon of the naphthylalanine residue and its adjacent
carbon was broken at this point to avoid the problems
of synthesizing a highly functionalized macrocyclic
system. This gave a structure (22) whose energy was
minimized using molecular mechanics before fitting the
coordinates to those of the original model of 7. This
showed that the overall spatial disposition of the side
chains was retained in the new molecule, with the
exception that the carboxylic acid of the aspartyl residue
was pointing in a different direction. This problem was
overcome by inverting the chirality at the R-carbon from

Novel Peptoid CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3511
the two carbonyl groups of the succinic acid spacer were
arranged cis to each other, relative to the ethylene unit.
If this were indeed the case, then “locking” the relation-
ship between the two carbonyl groups into this confor-
mation might be expected to increase the affinity of the
resulting compound for the CCK1 receptor by reducing
the overall entropy of the molecule. One way of achiev-
ing this aim was to tie down the conformationally mobile
ethylene group by substituting 1,2-cyclohexanedicar-
boxyl for succinyl. This introduced two new chiral
centers into the molecule, and the compounds were
prepared as racemates from the respective (()-cis- and
(()-trans-dicarboxylic acid anhydrides. However, in
those cases in which the two diastereoisomers were
separated (e.g. 30a ,b) there was little difference in the
activity of the individual diastereoisomers. These two
compounds were equipotent at CCK1 receptors but also
showed low levels of CCK2 activity (pKB ) 5.6). More-
over, their CCK1 activity had not increased relative to
the succinyl derivative 23. Introducing either electron-
withdrawing (31) or electron-donating (32) substituents
in the 4-position of the phenyl ring made no difference
to levels of CCK1 activity in this case. However, chang-
ing the configuration of the cyclohexane group from cis
to trans did lead to a modest increase in activity (33, Y
) 4-OMe: pKB ) 7.4) while maintaining selectivity for
CCK1 receptors. Unfortunately, the compound was
tested as a racemate, and no information is available
for the behavior of the individual enantiomers in this
case. Nevertheless, while this does not represent an
exhaustive study, the behavior of the two series of
peptides/peptoids is remarkably consistent. This sup-
ports the premise that molecular modeling techniques
can be used to design effective 3D mimics of bioactive
conformations without the need to compromise synthetic
accessibility by introducing additional covalent bonds.
F igu r e 1. Conformations of the tetrapeptide Ac-CCK30-33
(taken from ref 39, shown in yellow) and the calculated global
minimum of tripeptide 17 (shown in white). The two structures
were fitted by overlaying the CR atoms of the three amino acid
residues of the peptide backbones (rms ) 0.07). The backbones
of the two molecules are oriented vertically with the N-termini
uppermost. This shows the enantiomeric disposition of the
indole, carboxylic acid, and phenyl groups relative to the
backbone.
Met-Asp-Phe sequence adopt a Z-like bend. The biologi-
cal activities of the compounds described in these
studies were assessed in membrane binding assays, and
hence it is not clear whether the conformation identified
is responsible for the affinity or efficacy of the peptide.
Nevertheless, the authors noted that one consequence
of the molecule adopting this conformation was that the
hydrophobic groups clustered together on one “side” of
the peptide backbone with the hydrophilic side chain
of the Asp32 residue located on the opposite “side”. In
contrast, our model places the Met31 and Asp32 side
chains on the same side of the peptide backbone.
However, we noted that the proposed Z-bend conforma-
tion was highly reminiscent of those low-energy struc-
tures that we had identified for our CCK1 antagonists.
Comparing a model of the 3,4-dimethoxy analogue 17,
a selective CCK1 antagonist, with the structure pro-
Our studies in this area had led us to develop a
molecular model of BOC-CCK30-33 that we used as the
basis for development of our dibenzobicyclo[2.2.2]octane
CCK2 antagonists. This model was originally derived
from fluorescence data that suggested that the indole
and phenyl side chains of the two aromatic amino acids
of H-CCK30-33 were between 5 and 7 Å apart in water.48
The pitch of a 310 helix brings the side chain of every
fourth amino acid into close proximity, and with this
constraint, a number of structures that fulfilled the
experimental criteria were generated using molecular
mechanics.49 Moreover, this arrangement produced
structures of lower energy than those obtained if the
peptide backbone was allowed to adopt an alternative
conformation, such as an R-helix or a simple â-turn.
These structures were in good agreement with energy
calculations on Ac-CCK30-33 previously published by
Pincus et al.50
39
posed for the bioactive conformation of Ac-CCK30-33
allows us to speculate about the origins of this reversal
of selectivity. The overlay, shown in Figure 1, was
generated by fitting the R-carbons of the calculated
global minimum of compound 17 to the equivalent
centers of a model of Ac-CCK30-33, (rms ) 0.07), which
had been generated using the published torsion angles.
In fact, the configuration of the central portion of the
backbone of tripeptide 17 is remarkably similar to the
Z-shaped arrangement proposed for the bioactive con-
formation of Ac-CCK30-33 at CCK2 receptors (Table 3).
In addition, overlaying the two molecules in this way
shows that the side chains of methionine in Ac-CCK30-33
and the (2-naphthyl)alanine side chains of 17 are also
However, more recent studies have led Kolodziej et
al.39 to propose a different structure for the bioactive
conformation of Ac-CCK30-33 at CCK2 receptors. This
research group had also recognized the importance of
the Met31 side chain for CCK2 activity and carried out
a series of studies in which this residue was replaced
with the individual cis and trans isomers of several
4-alkylthioprolines. These studies led them to propose
a 3D structure for the “bioactive” conformation Ac-
CCK30-33 in which the three peptide bonds of the Trp-

3512 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Ch a r t 1. Stereoisomeric Cholecystokinin Ligands
Low et al.
tripeptide 17 are identical. However, the picture ob-
tained from overlaying these two peptides (Figure 1)
strongly suggests that the CCK1 and CCK2 receptors
recognize enantiomeric dispositions of the tryptophan
indole, aspartic acid carboxylate, and terminal phenyl
groups. These results lead us to propose that this
“functional chirality” forms the basis of the mechanism
by which these two closely related receptor systems
select between different conformations of this common
fragment of their parent hormones.
Con clu sion
We have speculated that the CCK1 selectivity of
simple peptide derivatives of CCK30-33 arises from the
disposition of the side chain functional groups and noted
that these mirror the arrangement proposed by Kolodz-
iej et al.39 for the bioactive conformation of Ac-CCK30-33
at CCK2 receptors. In practice, there appear to be two
dominant factors controlling the conformation of these
molecules: namely hydrogen-bonding through the amide
backbone and interaction of the aromatic rings as a
result of hydrophobic collapse. The “functional chirality”
inherent in the arrangement of the amino acid side
chains may underpin the mechanism by which these
closely related receptor systems bind CCK30-33. It may
also explain patterns of selectivity observed with optical
isomers of several series of peptoid and nonpeptide
ligands. We have exploited these features in the design
of a novel series of selective CCK1 antagonists. This led
to the creation of peptidomimetics such as 33 which are
potent CCK1 antagonists (pKB ) 7.4) and are at least
250-fold selective for this receptor over the closely
related CCK2 receptor. Furthermore, no residual efficacy
at CCK2 receptors is evident as we have moved away
from the ”bioactive” conformation of CCK30-33 proposed
by Kolodziej et al.39 This result was achieved without
introducing additional covalent bonds or macrocycliza-
tion. In general, there is a high level of consistency
between the behavior of the original series of peptides
and the new series of compounds. This suggests that
our original proposal concerning the bioactive conforma-
tion of the former was reasonable. In addition, it should
be possible to extend this principle to the design of novel
CCK2 receptor antagonists, using the literature model
Ta ble 5. CCK1/CCK2 Selectivities of Stereoisomeric
Cholecystokinin Receptor Ligands Shown in Chart 1
IC50 (nM)a
compd
CCK1
CCK2
0.15
259
370
19
270
3700
150
2
CCK1/CCK2 selectivity ref
PD 135666
PD 140458
LY288512
LY288513
devazepide
52
25.5
2.8
6400
0.005
100
0.05
0.0009
3375
445
50
0.007
30
30
21
21
51
51
19
19
20500
0.08
8.3
3
53
L-365,260
280
a
IC50 represents the concentration (nM) producing half-
maximal inhibition of specific binding of [125I]Bolton-Hunter
CCK-8 to CCK receptors in the rat pancreas (CCK1) or mouse
cerebral cortex (CCK2). Data taken from the reference specified.
located in similar regions of space relative to the
backbone. However, the other functional groups are
effectively distributed on opposite sides of the peptide
backbones, as if they had been reflected through a
mirror plane running along the plane of the backbone.
Further investigations showed that the same pattern
of behavior was also observed for compounds 7 and 12,
which were examined using the same protocol. In
addition, we have shown that mimicking the arrange-
ment of functional groups, described for the calculated
global minima of these tripeptides, produced the new
series of peptidomimetic CCK1 antagonists related to
23.
39
of Ac-CCK30-33 as a template, although this corollary
remains untested to date.
Exp er im en ta l Section
Gen er a l. Nuclear magnetic resonance spectra were re-
corded on either a Nicolet GE300 or Bruker DRX 300 machine.
Elemental analyses were carried out at the London School of
Pharmacy and all compounds gave analytical results within
(0.4% of the theoretical values. Flash column chromatography
was performed using Merck Kieselgel 60 silica grade 9385.
A number of independent reports have described
opposite CCK1/CCK2 selectivities for enantiomeric pairs
of peptoid,30 benzodiazepine,19,51 and diphenylpyrazoli-
dinone21 cholecystokinin ligands (Chart 1; Table 5). The
structures of these compounds are highly diverse, and
while they cannot be assumed to act at identical sites
on the receptor, there is also no current evidence to the
contrary. Nevertheless, we were intrigued by the pos-
sibility that this stereoselective behavior was a common
theme that also applied to the peptide hormone itself.
The absolute configurations of Ac-CCK30-33 and the
BOC-â-ben zyl-Asp -P h e-NH2 (34). To a solution of phenyl-
alaninamide hydrochloride (0.70 g, 3.5 mmol) and NaHCO3
(0.29 g, 3.5 mmol) in water (5 mL) was added a solution of
BOC-Asp(OBn)-NHS (1.47 g, 3.5 mmol) in DME (10 mL). The
mixture was stirred at room temperature overnight, then
acidified to pH 2 with 1 M HCl. Water (5 mL) was added and
the mixture maintained at 0 °C for 2 h. The resultant white
precipitate was filtered, washed with water, and dried to yield
the title compound (1.43 g, 88%): 1H NMR (DMSO-d6) δ 7.7
(1H, d), 7.4 (1H,s), 7.35 (5H, s), 7.2 (7H, m), 5.0 (2H, s), 4.4
(1H, m), 4.3 (1H, m), 3.0 (1H, m), 2.8 (1H, m), 2.7 (1H, m), 2.5
(1H, m), 1.3 (9H, s).

Novel Peptoid CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3513
BOC-Ala -Leu (35). To a solution of leucine (0.66 g, 5 mmol)
and NaHCO3 (0.42 g. 5 mmol) in water (10 mL) was added a
solution of BOC-Ala-NHS (1.14 g, 4 mmol) in DME and the
solution was stirred at room temperature for 3 h. The mixture
was acidified to pH 2 with 1 M HCl and a further 10 mL water
was added. On evaporation of the DME, an oil separated which
was extracted into ethyl acetate, dried and evaporated to give
the title compound (1.16 g, 96%): 1H NMR (DMSO-d6) δ 7.9
(1H, d), 6.8 (1H, d), 4.2 (1H, m), 4.0 (1H, m), 1.6 (1H, m), 1.5
(2H, m), 1.3 (9H, s), 1.1 (3H, d), 0.9 (3H, d), 0.8 (3H, d).
BOC-Ala -Leu N-Hyd r oxysu ccin im id e Ester (36). To a
solution of 35 (1.14 g, 3.8 mmol) in dry DME (20 mL) were
added N-hydroxysuccinimide (0.44 g, 3.8 mmol) and DCCI
(0.78 g, 3.8 mmol) and the mixture stirred at 5 °C overnight.
The precipitated dicyclohexylurea was removed by filtration
and the filtrate evaporated to give 1.59 g of crude product,
which was used without further purification: 1H NMR (DMSO-
d6) δ 8.4 (1H, d), 6.9 (1H, d), 4.6 (1H, m), 4.0 (1h, m), 2.8 (4H,
s), 1.8-1.5 (3H, m), 1.3 (9H, s), 0.9 (3H, d), 0.8 (3H, d).
(DMSO-d6) δ 8.25 (1H, d), 8.0 (1H, d), 7.8 (1H, d), 7.6 (1H, d),
7.4-6.9 (12H, m), 6.85 (1H, d), 5.0 (2H, s), 4.6 (1H, m), 4.3
(1H, m), 4.2 (2H, m), 3.2-2.6 (6H, m), 1.3 (9H, s), 1.1 (3H, m).
BOC-Tr p -Ala -Asp -P h e-NH 2 (3). The benzyl ester 40 (0.30
g, 0.4 mmol) was dissolved in MeOH (30 mL) and stirred with
10% palladium-on-carbon (30 mg) under an atmosphere of
hydrogen for 4 h. The catalyst was removed by filtration and
washed with hot MeOH. The filtrate was evaporated and the
residue crystallized from aqueous MeOH to afford 0.16 g (64%)
white solid: mp 213-215 °C; [R]20 ) -21.2° (c 0.85, DMSO);
D
1H NMR (DMSO-d6) δ 8.2 (1H, d), 8.0 (1H, d), 7.8 (1H, d), 7.6
(1H, d), 7.3-6.9 (11H, m), 6.8 (1H, d), 4.5-4.0 (4H, m), 3.0
(2H, m), 2.8 (2H, m), 2.7-2.3 (2H, m), 1.2 (9H, s), 1.1 (3H, m).
Anal. (C32H40N6O8‚0.5H2O) C, H, N.
BOC-Tr p -Leu -H (41). BOC-Trp-NHS was coupled to leu-
cine using the method described for 38: 1H NMR (DMSO-d6)
δ 10.8 (1H, s), 7.05 (1H, d), 7.6 (1H, d), 7.3 (1H, d), 7.1-6.9
(3H, m), 6.7 (1H, d), 4.2 (2H, m), 3.0 (2H, m), 1.6 (1H, br s),
1.5 (2H, br s), 1.3 (9H, s), 0.8 (6H, m).
BOC-Tr p -Leu -NHS (42). Prepared from 41 using the
method described for 39: 1H NMR (DMSO-d6) δ 10.8 (1H, s),
8.6 (1H, d), 6.9-7.1 (3H, m), 6.8 (1H, d), 4.7 (1H, m), 4.2 (1H,
m), 3.0 (2H, m), 2.8 (4H, s), 1.6 (1H, m), 1.5 (2H, m), 1.3 (9H,
s), 0.9 (6H, dd).
BOC-Asp (OBn )-Ala -NH2 (43). Prepared according to the
method given for 34 except that Ala-NH2 hydrochloride was
used in place of Phe-NH2 hydrochloride: yield (65%); 1H NMR
(DMSO-d6) δ 7.8 (1H, s), 7.3 (6H, s), 7.2 (1H, d), 7.0 (1H, s),
5.0 (2H, s), 4.3 (1H, m), 4.1 (1H, m), 2.3 (2H, m), 1.3 (9H, s),
1.1 (3H, d).
BOC-Ala -Leu -Asp (OBn )-P h e-NH2 (37). Benzyl ester 34
(0.35 g, 0.75 mmol) was deprotected on stirring in trifluoro-
acetic acid (3 mL) for 1 h. The solvent was evaporated and
the residue dissolved in DME (6 mL). NEt3 (0.3 mL, 2.2 mmol)
and NHS ester 36 (0.33 g, 0.75 mmol) were added and the
mixture stirred at room temperature overnight. Water (10 mL)
was added and the mixture stirred at 0 °C for a further 1 h.
The resultant precipitate was filtered, washed with cold water,
and dried. Recrystallization from ethanol/water gave 0.17 g
(35%) of product: 1H NMR (DMSO-d6) δ 8.2 (1H, d), 7.8 (2H,
m), 7.3 (5H, s), 7.1 (5H, m), 7.0 (1H, d), 5.0 (2H, s), 4.5 (1H,
m), 4.3 (1H, m), 4.2 (1H, m), 3.9 (1H, m), 3.0 (2H, m), 2.8 (2H,
m), 1.6 (1H, m), 1.3 (11H, s), 1.1 (3H, m), 0.8 (6H, m).
BOC-Ala -Leu -Asp -P h e-NH2 (2). The benzyl ester 37 (0.24
g, 0.37 mmol) was dissolved in MeOH (30 mL) and stirred with
10% palladium-on-carbon (20 mg) under an atmosphere of
hydrogen for 4 h. The catalyst was removed by filtration
through a pad of Celite and the solvent evaporated. The
residue was recrystallized from aqueous ethanol to yield the
title compound (0.14 g, 68%): [R]20D ) -46.5° (c 0.86, MeOH);
1H NMR (DMSO-d6) δ 8.2 (1H, d), 7.8 (2H, m), 7.2 (7H, m),
7.0 (1H, t), 4.4 (1H, m), 4.3 (2H, m), 4.0 (1H, m), 3.0 (1H, m),
2.8 (1H, m), 2.6 (1H, m), 2.4 (1H, m), 1.6 (1H, m), 1.4 (11H,
m), 1.1 (3H, m), 0.8 (6H, m). Anal. (C27H41N5O8) C, H, N.
BOC-Tr p -Ala -H (38). To a solution of alanine (0.18 g, 2
mmol) and NaHCO3 (0.34 g, 4 mmol) in water (5 mL) was
added a suspension of BOC-Trp-NHS (0.8 g, 2 mmol) in ethanol
(6 mL). The mixture was stirred at room-temperature over-
night, the ethanol was evaporated and the residue was
acidified to pH 2 with 1 M HCl. The separated oil was
extracted into ethyl acetate, dried and evaporated to give 0.76
g crude product that was used without further purification:
1H NMR (DMSO-d6) δ 8.2 (1H, d), 7.6 (1H, d), 7.3 (1H, d), 7.1
(1H, s), 7.0 (2H, m), 6.7 (1H, d), 4.2 (2H, m), 3.1 (1H, m), 2.8
(1H, m), 1.25 (9H, s), 1.1 (3H, d).
BOC-Tr p -Ala -NHS (39). To a solution of dipeptide 38 (0.75
g, 2 mmol) in dry DME (10 mL) were added N-hydroxysuc-
cinimide (0.23 g, 2 mmol) and DCCI (0.41 g, 2 mmol), and the
mixture stirred at 5 °C overnight. The precipitated dicyclo-
hexylurea was removed by filtration and the filtrate evapo-
rated. The crude product (0.98 g) was dissolved in a 1:1
mixture of CH2Cl2 and EtOAc (20 mL) and filtered through
silica to afford 0.61 g (67%) of the title compound: 1H NMR
(DMSO-d6) δ 10.7 (1H, s), 8.7 (1H, d), 7.6 (1H, d), 7.3 (1H, d),
7.1 (1H, s), 7.0 (2H, m), 6.7 (1H, d), 4.7 (1H, m), 4.2 (1H, m),
3.1 (1H, m), 2.8 (5H, m), 1.5 (3H, d), 1.25 (9H, s).
BOC-Tr p -Leu -Asp (OBn )-Ala -NH2 (44). Benzyl ester 43
was deprotected and coupled to NHS ester 42 following the
method described for 40. The product was recrystallized from
1
aqueous MeOH: yield 61%; H NMR (DMSO-d6) δ 10.8 (1H,
s), 8.4 (1H, d), 7.9 (1H, d), 7.8 (1H, d), 7.6 (1H, d), 7.3 (6H, m),
7.0 (5H, m), 5.0 (2H, s), 4.6 (1H, m), 4.3 (1H, m), 4.1 (2H, m),
2.8 (4H, m), 1.6 (1H, br s), 1.65 (2H, m), 1.6 (2H, m), 1.3 (9H,
s), 1.2 (3H, d), 0.8 (6H, m).
BOC-Tr p -Leu -Asp -Ala -NH2 (5). Using the method de-
scribed for compound 2, benzyl ester 44 was hydrogenolysed
to give the title compound: yield 96%; [R]20 ) -50.9° (c 0.31,
D
MeOH); 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.3 (1H, d), 7.9
(1H, d), 7.8 (1H, d), 7.6 (1H, d), 7.3 (1H, d), 7.2 (1H, s), 7.0
(4H, m), 6.8 (1H, d), 4.5 (1H, m), 4.3 (1H, m), 4.1 (2H, m), 3.1-
2.6 (4H, m), 1.6 (1H, m), 1.4 (2H, m), 1.25 (9H, s), 1.2 (3H, d),
0.8 (6H, m). Anal. (C29H42N6O8) C, H, N.
BOC-Tr p -Leu -Ala -P h e-NH2 (4). Dipeptide 45 was pre-
pared from BOC-Ala-NHS and Phe-NH2‚hydrochloride, ac-
cording to the method given for 34, and then coupled to 42:
yield 72%; 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.0 (1H, d), 7.9
(1H, d), 7.8 (1H, d), 7.6 (1H, d), 7.2 (11H, m), 6.8 (1H, d), 4.3
(2H, m), 4.2 (2H, m), 3.1-2.7 (4H, m), 1.6-1.0 (15H, m), 0.8
(6H, m). Anal. (C34H46N6O6‚0.5H2O) C, H, N.
BOC-Tr p -3-(2-n a p h th yl)a la n in e-NHS (46). Using the
method described for 35, BOC-Trp-NHS was coupled to L-3-
(2-naphthyl)alanine. The NHS ester 46 was then prepared
using the method described for 36: yield 98%; 1H NMR
(DMSO-d6) δ 10.7 (1H, s), 8.8 (1H, d), 7.8 (4H, m), 7.5 (4H,
m), 7.3 (1H, m), 7.0 (3H, m), 6.7 (1H, d), 5.1 (1H, m), 4.2 (1H,
m), 2.8 (4H, s), 2.6-3.3 (4H, m), 1.2 (9H, s).
BOC-Tr p -L-3-(2-n a p h th yl)a la n yl-Asp -P h e-NH2 (6). Ben-
zyl ester 34 was deprotected using trifluoroacetic acid and
coupled to NHS ester 46 following the method described for
37. The product was recrystallized from EtOH-H2O: yield
1
65%; H NMR (DMSO-d6) δ 10.7 (1H, s), 8.5 (1H, d), 8.0 (2H,
d), 7.8 (4H, m), 7.5-6.8 (20H, m), 5.0 (2H, s), 4.6 (2H, m), 4.4
BOC-Tr p -Ala -Asp (OBn )-P h e-NH2 (40). Benzyl ester 34
(0.47 g, 1.0 mmol) was deprotected by stirring in trifluoroacetic
acid (3 mL) for 1h. The solvent was evaporated and the residue
was dissolved in DME (6 mL). NEt3 (0.4 mL, 3 mmol) followed
by 39 were added and the mixture stirred at room temperature
overnight. Water (10 mL) was added and the mixture was
stirred at 0 °C for a further 1 h. The resultant precipitate was
filtered, washed with cold water, and dried. Recrystallization
from ethanol/water gave 0.40 g (55%) of product: 1H NMR
(1H, m), 4.1 (1H, m), 3.2-2.5 (8H, m), 1.2 (9H, s).
The resulting benzyl ester (0.35 g, 0.4 mmol) was then
dissolved in MeOH (40 mL) and AcOH (0.5 mL) and a catalytic
amount of 10% palladium-on-carbon added. The mixture was
stirred under an atmosphere of hydrogen overnight. The
catalyst was removed by filtration and the filtrate evaporated
to give the title compound (0.28 g, 90%). An analytically pure
sample of 6 was obtained by recrystallization from EtOH-

3514 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Low et al.
1
H2O: [R]20 ) -21.0° (c 1.0, DMSO); H NMR (DMSO-d6) δ
for 7, except that 2-(4-chlorophenyl)ethylamine was used in
place of 2-phenylethylamine: 1H NMR (DMSO-d6) δ 10.8 (1H,
s), 8.4 (1H, d), 8.0 (1H, d), 7.8 (5H, m), 7.3 (7H, m), 7.2 (2H,
m), 7.0 (2H, m), 6.9 (2H, m), 4.6 (1H, m), 4.5 (1H, m), 4.2 (1H,
m), 3.2 (4H, m), 2.8 (6H, m), 1.2 (9H, s). Anal. (C41H44ClN5O7)
C, H, N.
D
10.7 (1H, s), 8.5 (1H, d), 8.0 (2H, m), 7.7 (4H, m), 7.5-6.9 (15H,
m), 6.8 (1H, d), 4.7 (1H, m), 4.5 (1H, m), 4.4 (1H, m) 4.1 (1H,
m), 3.2-2.6 (8H, m) 1.2 (9H, s). Anal. (C42H46N6O8‚2.8H2O‚
1.0EtOH) C, H, N.
BOC-Asp (OBn )-2-p h en yleth yla m id e (47). A solution of
BOC-Asp(OBn)-NHS (1.0 g, 2.4 mmol) and 2-phenylethylamine
(0.3 mL, 2.4 mmol) in dry DME (15 mL) was stirred at room
temperature overnight. The mixture was poured into water
(50 mL) and the resultant precipitate was filtered, washed with
water and dried to yield the title compound (0.88 g, 84%): 1H
NMR (DMSO-d6) δ 7.9 (1H, t), 7.4-7.2 (10H, m), 7.1 (1H, d),
5.1 (2H, s), 4.3 (1H, m), 3.3 (2H, m), 2.7 (2H, m), 2.6 (2H, m),
1.4 (9H, s).
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(2-m eth oxyph en -
yl)eth yla m id e (15). Prepared following the methods de-
scribed for 7, except that 2-(2-methoxyphenyl)ethylamine was
used in place of 2-phenylethylamine: [R]20 ) -35.5° (c 0.45,
D
MeOH); 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4 (1H, d), 8.0
(1H, d), 7.8-6.7 (18H, m), 4.6 (1H, m), 4.5 (1H, m), 4.1 (1H,
m), 3.7 (3H, s), 3.1 (2H, m), 3.1-2.4 (8H, m), 1.2 (9H, s). Anal.
(C42H47N5O8‚0.8H2O) C, H, N.
BOC-Tr p -L-3-(2-n a p h t h yl)a la n yl-Asp -2-p h en ylet h yla -
m id e (7). Benzyl ester 47 was deprotected using trifluoroacetic
acid and coupled to NHS ester 46 in DME containing NEt3
following the method described for 37. The crude product was
purified by column chromatography then hydrogenolysed
following the procedure used for the preparation of example
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(3-m eth oxyph en -
yl)eth yla m id e (16). Prepared following the methods de-
scribed for 7, except that 2-(3-methoxyphenyl)ethylamine was
used in place of 2-phenylethylamine: [R]20 ) -30.6° (c 0.49,
D
MeOH); 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4 (1H, d), 8.0
(1H, d), 7.9-6.7 (18H, m), 4.6 (1H, m), 4.5 (1H, m), 4.1 (1H,
m), 3.7 (3H, s), 3.5-2.6 (10H, m), 1.2 (9H, s). Anal. (C42H47N5O8‚
0.8H2O) C, H, N.
2: [R]20 ) -8.9° (c 0.9, DMSO); H NMR (DMSO-d6) δ 10.9
1
D
(1H, s), 8.4 (1H, m), 8.1 (2H, m), 7.9-6.8 (17H, m), 6.7 (1H,
m), 4.6 (1H, m), 4.5 (1H, m), 4.1 (1H, m), 3.5-2.3 (10H, m),
1.2 (9H, s). Anal. (C41H45N5O7) C, H, N.
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-P h e-NHMe (8). Pre-
pared following the methods described for 7, except that Phe-
BOC-Tr p -L-3-(2-n a p h th yl)a la n yl-Asp -2-(3,4-d im eth oxy-
p h en yl)eth yla m id e (17). Prepared following the methods
described for 7, except that 2-(3,4-dimethoxyphenyl)ethylamine
was used in place of 2-phenylethylamine: [R]20 ) -42.0° (c
D
NHMe was used in place of 2-phenylethylamine: [R]20
)
1
0.45, MeOH); H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4 (1H, d),
D
-70.0° (c 0.21, EtOH); 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4
(1H, d), 8.0 (2H, m), 7.7 (4H, m), 7.4 (4H, m), 7.2 (7H, m), 6.9
(4H, m), 4.6 (1H, m), 4.5 (1H, m), 4.3 (1H, m), 4.0 (1H, m), 2.9
(8H, m), 2.5 (3H, d), 1.2 (9H, s). Anal. (C43H47N6O8) C, H, N.
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-P h e-NMe2 (9). Pre-
pared following the methods described for 7, except that Phe-
NMe2was used in place of 2-phenylethylamine: [R]20D ) -37.7°
8.0 (1H, d), 7.9-6.5 (17H, m), 4.6 (1H, m), 4.5 (1H, m), 4.1
(1H, m), 3.7 (3H, s), 3.6 (3H, s), 3.5-2.4 (10H, m), 1.2 (9H, s).
Anal. (C43H49N5O9) C, H, N.
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(4-flu or oph en yl)-
eth yla m id e (18). Prepared following the methods described
for 7, except that 2-(4-fluorophenyl)ethylamine was used in
place of 2-phenylethylamine: [R]20D ) -38.7° (c 0.98, MeOH);
1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4 (1H, d), 8.0 (1H, d),
7.9-6.7 (18H, m), 4.6 (1H, m), 4.5 (1H, m), 4.1 (1H, m), 3.2-
2.5 (10H, m), 1.2 (9H, s). Anal. (C41H44FN5O7) C, H, N.
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(4-am in oph en yl)-
eth yla m id e (19). Prepared following the methods described
for 7, except that 2-(4-aminophenyl)ethylamine was used in
place of 2-phenylethylamine: [R]20D ) -20.4° (c 0.49, MeOH);
1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4 (1H, d), 8.0 (1H, d),
7.9-6.8 (14H, m), 6.8 (2H, m), 6.4 (2H, m), 4.6 (1H, m), 4.5
(1H, m), 4.1 (1H, m), 3.3-2.3 (10H, m), 1.2 (9H, s). Anal.
(C41H46N6O7‚0.7EtOAc) C, H, N.
1
(c 0.37, EtOH); H NMR (DMSO-d6) δ 10.7 (1H, s), 8.4 (1H,
d), 8.0 (1H, d), 7.9 (1H, d), 7.7 (4H, m), 7.4 (3H, m), 7.2 (7H,
m), 6.9 (4H, m), 4.8 (1H, m), 4.6 (1H, m), 4.5 (1H, m), 4.1 (1H,
m), 3.0 (6H, m), 2.7 (6H, d), 2.6 (2H, m), 1.2 (9H, s). Anal.
(C44H45N6O8‚2.5H2O) C, H, N.
BOC-Tr p -L-3-(2-n a p h t h yl)a la n yl-Asp -p h en yla la n in ol
(10). Prepared following the methods described for 7, except
that L-phenylalaninol was used in place of 2-phenylethyl-
amine: [R]20 ) -60.0° (c 0.20, EtOH); 1H NMR (DMSO-d6) δ
D
10.7 (1H, s), 8.4 (1H, d), 7.9 (1H, d), 7.7 (5H, m), 7.2 (10H, m),
7.0 (2H, m), 6.9 (2H, m), 4.6 (1H, m), 4.5 (1H, m), 4.1 (1H, m),
3.8 (1H, m), 3.2 (2H, m), 2.8 (8H, m), 1.2 (9H, s). Anal.
(C42H47N5O8) C, H, N.
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(3-tr iflu or om eth
ylp h en yl)eth yla m id e (20). Prepared following the methods
described for 7, except that 2-(3-trifluoromethylphenyl)ethy-
BOC-Tr p -L-3-(2-n a p h th yl)a la n yl-Asp -(4-m eth oxy)P h e-
NH2 (11). Prepared following the methods described for 7,
except that L-(4-methoxy)Phe-NH2 was used in place of 2-phen-
lamine was used in place of 2-phenylethylamine: [R]20
)
D
-40.3° (c 0.77, MeOH); 1H NMR (DMSO-d6) δ 10.8 (1H, s),
8.4 (1H, d), 8.0 (1H, d), 7.9-6.8 (20H, m), 4.6 (1H, m), 4.5 (1H,
m), 4.1 (1H, m), 3.4-2.5 (10H, m), 1.2 (9H, s). Anal.
(C42H44F3N5O7‚0.5H2O) C, H, N.
ylethylamine: [R]20 ) -45.7° (c 0.33, EtOH); 1H NMR
D
(DMSO-d6) δ 10.7 (1H, s), 8.5 (1H, d), 7.9 (2H, m), 7.8 (4H,
m), 7.4 (6H, m), 7.0 (6H, m), 6.8 (3H, m), 4.6 (1H, m), 4.5 (1H,
m), 4.3 (1H, m), 4.1 (1H, m), 3.5 (3H, m), 2.8 (8H, m), 1.2 (9H,
s). Anal. (C43H48N6O9‚1.0H2O) C, H, N.
BOC-Tr p -2-(2-n a p h th yl)eth yla m id e (48). BOC-Trp-NHS
(1.20 g, 3.0 mmol) was added to a solution of 2-(2-naphthyl)-
ethylamine (0.51 g, 3.0 mmol) in dry DME. The mixture was
stirred at room temperature for 2 h and partitioned between
1 M HCl (100 mL) and EtOAc (50 mL). The aqueous phase
was then washed with a further portion of EtOAc and the
combined organic layers washed with H2O (3 × 50 mL), dried
(MgSO4) and concentrated in vacuo. The crude product was
purified by flash column chromatography, on silica gel with
2% MeOH-CH2Cl2, to give the title compound as a white foam
(1.30 g 95%): 1H NMR (CDCl3) δ 7.95 (1H, s), 6.9-7.8 (12H,
m), 5.7 (1H, bs), 5.15 (1H, bs), 4.4 (1H, m, CH), 3.45 (2H, m),
3.3 (1H, m), 2.7 (1H, m), 2.7 (2H, m), 1.4 (9H, s).
N-Su ccin yl-Tr p -2-(2-n a p h th yl)eth yla m id e (49). Com-
pound 48 (0.5 g, 1.1 mmol) was treated with trifluoroacetic
acid (5 mL) and the mixture stirred for 40 min at room
temperature. The solvent was then removed in vacuo and the
crude salt taken up in dry THF. The solution was then treated
with NEt3 (0.4 mL) followed by succinic anhydride (0.12 g, 1.2
mmol) and a catalytic amount of 4-(dimethylamino)pyridine.
The reaction mixture was stirred at room temperature for 18
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(4-m eth oxyph en -
yl)eth yla m id e (12). Prepared following the methods de-
scribed for 7, except that 2-(4-methoxyphenyl)ethylamine was
used in place of 2-phenylethylamine: [R]20 ) -30.6° (c 0.46,
D
EtOH); 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.4 (1H, d), 8.0 (1H,
d), 7.7 (5H, m), 7.4 (5H, m), 7.0 (4H, m), 6.9 (2H, m), 6.8 (2H,
m), 4.6 (1H, m), 4.4 (1H, m), 4.1 (1H, m), 3.6 (3H, s), 3.1 (2H,
m), 2.8 (6H, m), 2.6 (2H, m), 1.2 (9H, s). Anal. (C42H47N5O8‚
0.5H2O) C, H, N.
BOC-Tr p -L-3-(2-n a p h th yl)a la n yl-Asp -L-(4-ch lor o)P h e-
NH2 (13). Prepared following the methods described for 7,
except that L-(4-chloro)Phe-NH2 was used in place of 2-phen-
ylethylamine: [R]20D ) -7.8° (c 0.13, DMF); 1H NMR (DMSO-
d6) δ 10.8 (1H, s), 8.5 (1H, d), 8.0 (2H, m), 7.8 (4H, m), 7.3
(11H, m), 7.0 (2H, m), 6.9 (1H, m), 6.8 (1H, d), 4.7 (1H, m), 4.6
(1H, m), 4.4 (1H, m), 4.1 (1H, m), 2.8 (8H, m), 1.2 (9H, s). Anal.
(C42H45ClN6O8) C, H, N.
BOC-Tr p-L-3-(2-n aph th yl)alan yl-Asp-2-(4-ch lor oph en yl)-
eth yla m id e (14). Prepared following the methods described

Novel Peptoid CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3515
h before removal of the solvent in vacuo. The residue was then
dissolved in CH2Cl2 (40 mL), washed with 1 M HCl (2 × 30
mL) and H2O (30 mL) and the organic phase dried (MgSO4),
and concentrated to give the crude product which was used
directly in the next stage (0.47 g, 94%): 1H NMR (DMSO-d6)
δ 12.1 (1H, bs), 10.8 (1H, s), 8.0-6.9 (14H, m), 4.4 (1H, m),
3.6-2.2 (10H, m).
BOC-D-Asp (OBn )-2-p h en yleth yla m id e (50). 2-Phenyl-
ethylamine (0.89 mL, 7.1 mmol) was added to BOC-D-Asp-
(OBn)-H (2.3 g, 7.0 mmol) and DCCI (1.5 g, 7.5 mmol) in
anhydrous CH2Cl2 (50 mL) at -10 °C under argon. The
reaction mixture was stirred at -10 °C for 45 min, than at 4
°C for 64 h, then filtered and evaporated to dryness. The
residue was dissolved in EtOAc, filtered and evaporated to
dryness yielding a yellow oil. Trituration with ether gave a
white amorphous solid (1.2 g). The concentrated liquors were
chromatographed on silica gel with acetone-toluene eluant
yielding a further 0.6 g of product (total yield 61%): 1H NMR
(DMSO-d6) δ 7.85 (1H, t), 7.3 (5H, s), 7.2 (5H, m), 7.1 (1H, t),
5.05 (2H, s), 4.25 (1H, q), 3.2 (2H,m), 2.6 (4H, m), 1.3 (9H, s).
N-[Su ccin yl-D-Asp -2-(3,4-d ich lor op h en yl)eth yla m id o]-
Tr p -2-p h en yleth yla m id e (27). The compound was prepared
as in example 23, except that 2-(3,4-dichlorophenyl)ethylamine
was used in place of 2-(2-naphthyl)ethylamine and 2-(4-
methoxyphenyl)ethylamine was used in place of 2-phenylethyl-
1
amine: [R]20 ) +18.9° (c 0.74, DMF); H NMR (DMSO-d6) δ
D
10.8 (1H, s), 8.2 (2H, dd), 7.95 (2H, dt), 7.5 (1H, d), 7.3 (3H,
m), 7.1 (4H, m), 7.0 (1H, t), 6.8 (2H, d), 4.4 (2H, m), 3.7 (3H,
s), 3.2 (4H, m), 3.1 (2H, m), 2.8 (2H, m), 2.6 (4H, m), 2.4 (4H,
m). Anal. (C36H39Cl2N5O7‚4.0H2O) C, H, N.
N-[Su ccin yl-D-Asp -2-(4-m et h oxyp h en yl)et h yla m id o]-
Tr p -2-p h en yleth yla m id e (28). The compound was prepared
as in example 23, except that 2-phenylethylamine was used
in place of 2-(2-naphthyl)ethylamine and 2-(4-methoxyphenyl)-
ethylamine was used in place of 2-phenylethylamine: [R]20
D
1
) 17.7° (c 0.8, DMF); H NMR (DMSO-d6) δ 10.8 (1H, s), 8.2
(2H, dd), 8.0 (2H, dt), 7.55 (1H, s), 7.3 (1H, d), 7.2 (2H, d), 7.1
(7H, m), 7.0 (1H, t), 6.8 (2H, d), 4.45 (2H, m), 3.7 (3H, s), 3.2
(8H, m), 2.6 (4H, t), 2.4 (4H, m). Anal. (C36H41N5O7‚0.6CH2-
Cl2) C, H, N.
N-(Su ccin yl-D-Asp -2-p h en yleth yla m id o)-Tr p -(2-m eth -
yl)p r op yla m id e (29). The compound was prepared as in
example 23, except that 2-methylpropylamine was used in
place of 2-(2-naphthyl)ethylamine and 2-(4-methoxyphenyl)-
N-(Su ccin yl-D-Asp (OBn )-2-p h en ylet h yla m id o)-Tr p -2-
(2-n a p h th yl)eth yla m id e (51). Benzyl ester 50 (0.25 g, 0.6
mmol) was treated with trifluoroacetic acid (3 mL) and the
mixture stirred at room temperature for 1 h before removal of
the solvent in vacuo. The residue was then dissolved in CH2-
ethylamine was used in place of 2-phenylethylamine: [R]20
D
1
i
) +21.4° (c 0.7, MeOH); H NMR (DMSO-d6) δ 10.8 (1H, s),
Cl2 (5 mL) and treated sequentially with Pr2NEt (0.3 mL, 1.7
8.2 (2H, dd), 7.9 (2H, q), 7.6 (1H, d), 7.3 (1H, d), 7.1 (5H, m),
6.8 (2H, d), 4.4 (2H, t), 3.7 (3H, s), 3.2 (4H, m), 2.9 (2H, m),
2.5 (4H, m), 2.4 (2H, m), 1.6 (1H, m), 0.8 (6H, dd). Anal.
(C32H41N5O7) C, H, N.
mmol), 49 (0.27 g, 0.6 mmol) and (benzotriazol-1-yloxy)-
trispyrrolidinophosphonium hexafluorophosphate (pyBOP; 0.31
g, 0.6 mmol). The mixture was stirred for 1 h at room
temperature and the solvent removed in vacuo. The residue
was then taken up in EtOAc (30 mL) and washed with 5%
KHSO4 (3 × 20 mL), NaHCO3 (20 mL) and brine (20 mL). The
organic phase was then dried (MgSO4) and evaporated to give
the crude product which was purified by flash column chro-
matography on silica gel in 5% MeOH-CH2Cl2 to give the title
compound as a white solid (0.16 g, 37%): 1H NMR (DMSO-d6)
δ 10.8 (1H, s), 8.2 (1H, d), 8.1 (1H, d), 8.0 (1H, t), 7.9 (1H, t),
7.8 (2H, m), 7.6 (1H, s), 7.5-6.8 (19H, m), 5.0 (2H, s), 4.6 (1H,
m), 4.4 (1H, m), 3.4-2.2 (16H, m).
N-(1,2-cis-Cycloh exan edicar boxyl-D-Asp-2-ph en yleth yl-
a m id o)-Tr p -2-(2-n a p h th yl)eth yla m id e (30a ,b). The com-
pounds were prepared as in example 23, except that (()-1,2-
cis-cyclohexanedicarboxylic anhydride was used in place of
succinic anhydride. The two diastereoisomeric benzyl esters
were separated by flash column chromatography on silica gel,
using toluene/EtOAc (1:1), before being hydrogenated sepa-
rately to give examples 30a ,b. 30a : [R]20 ) +8.2° (c 0.73,
D
MeOH); 1H NMR (DMSO-d6) δ 10.8 (1H s), 8.1 (1H, t), 8.0 (3H,
m), 7.8 (3H, m), 7.6 (1H. s), 7.5-6.9 (13H, m), 4.5 (1H, m), 4.4
(1H, m), 3.4-2.5 (12H, m), 1.8-1.2 (10H, m). Anal. (C43H47N5O6)
C, H, N. 30b: [R]20D ) +5.3° (c 0.76, MeOH); 1H NMR (DMSO-
d6) δ 10.8 (1H, s), 8.1-7.7 (6H, m), 7.6 (1H, s), 7.45 (4H, m),
7.4-6.9 (10H, m), 4.4 (2H, m), 3.5-2.4 (12H, m), 2.2-1.2 (10H,
m). Anal. (C43H47N5O6‚3.5H2O) C, H, N.
N-(Su ccin yl-D-Asp-2-ph en yleth ylam ido)-Tr p-2-(2-n aph -
th yl)eth yla m id e (23). 51 (0.23 g, 0.3 mmol) was dissolved
in MeOH (50 mL) with warming. The solution was then cooled
to room temperature and stirred under 1 atm of hydrogen in
the presence of a catalytic amount of 10% palladium on
charcoal for 18 h. The mixture was then filtered through Celite,
and the filtrate concentrated in vacuo to give the title
N-[(()-1,2-cis-Cycloh exa n ed ica r boxyl-D-Asp -2-(4-flu o-
r oph en yl)eth ylam ido]-Tr p-2-(2-n aph th yl)eth ylam ide (31).
The compound was prepared as in example 23, except that
(()-1,2-cis-cyclohexanedicarboxylic anhydride was used in
place of succinic anhydride and 2-(4-fluorophenyl)ethylamine
was used in place of 2-phenylethylamine: 1H NMR (DMSO-
d6) δ 10.8 (1H, s), 8.4-6.9 (20H, m), 4.4 (2H, m), 3.5-2.2 (12H,
m), 2.0-1.2 (10H, m). Anal. (C43H46FN5O6‚2.2H2O) C, H, N.
N-[(()-1,2-cis-Cycloh exa n ed ica r boxyl-D-Asp -2-(4-m eth -
oxyp h en yl)et h yla m id o]-Tr p -2-(2-n a p h t h yl)et h yla m id e
(32). The compound was prepared as in example 23, except
that (()-1,2-cis-cyclohexanedicarboxylic anhydride was used
in place of succinic anhydride and 4-methoxyphenylethylamine
was used in place of 2-phenylethylamine: 1H NMR (DMSO-
d6) δ 10.8 (1H, s), 8.2 (3H, m), 7.8 (3H, m) 7.7 (1H, s), 7.4 (5H,
m), 7.1 (5H, m), 6.8 (2H, m), 4.5 (1H, m), 4.4 (1H, m), 4.1 (1H,
m), 3.7 (3H, s), 3.2-2.3 (11H, m), 1.9-1.1 (11H, m). Anal.
(C44H49N5O7‚2.0H2O‚0.8CH2Cl2) C, H, N.
N-[(()-1,2-tr a n s-Cycloh exa n ed ica r b oxyl-D-Asp -2-(4-
m eth oxyp h en yl)eth yla m id o]-Tr p -2-(2-n a p h th yl)eth yla -
m id e (33). The compound was prepared as in example 23,
except that (()-1,2-trans-cyclohexanedicarboxylic anhydride
was used in place of succinic anhydride and 4-methoxyphe-
nylethylamine was used in place of 2-phenylethylamine: 1H
NMR (DMSO-d6) δ 10.8 (1H, s), 8.2 (2H, m), 7.8-6.7 (18H,
m), 4.5-4.35 (2H, m), 3.7 (3H, s), 3.2-2.4 (12H, m), 1.9-1.3
(10H, m). Anal. (C44H49N5O7‚4.0H2O‚0.3CH2Cl2) C, H, N.
Biological Meth ods. Mater ials: BOC-pentagastrin (Sigma)
was dissolved in DMF to a concentration of 40 mM serially
compound as a white solid (0.15 g, 74%): [R]20 ) +19.0° (c
D
1
1.0, DMSO); H NMR (DMSO-d6) δ 10.8 (1H, s), 8.2 (2H, m),
8.05 (1H, t), 7.9 (1H, t), 7.8 (3H, m), 7.6 (1H, s), 7.4 (3H, m),
7.35-6.9 (10H, m); 4.45 (2H, m), 3.4-2.2 (16H, m). Anal.
(C39H41N5O6‚0.65H2O) C, H, N.
N-[Su ccin yl-D-Asp -2-(4-flu or op h en yl)eth yla m id o]-Tr p -
2-(2-n a p h th yl)eth yla m id e (24). The compound was prepared
as in example 23, except that 2-(4-fluorophenyl)ethylamine
was used in place of 2-phenylethylamine: [R]20 ) +15.0° (c
D
0.83, DMSO); 1H NMR (DMSO-d6) δ 10.8 (1H, s), 8.2 (2H, m),
8.1 (1H, t), 8.0 (1H, t), 7.8 (3H, m), 7.6 (1H, s), 7.5 (3H, m), 7.3
(2H, m), 7.1 (2H, m), 7.0 (4H, m), 6.9 (1H, m), 4.45 (2H, m),
3.4-2.3 (16H, m). Anal. (C39H40FN5O6) C, H, N.
N-[Su ccin yl-D-Asp -2-(4-m et h oxyp h en yl)et h yla m id o]-
Tr p -2-(2-n a p h th yl)eth yla m id e (25). The compound was
prepared as in example 23, except that 2-(4-methoxyphenyl)-
ethylamine was used in place of 2-phenylethylamine: [R]20
D
) +15° (c 1.0, DMF); 1H NMR (DMSO-d6) δ 10.8 (1H,s), 8.2-
7.9 (4H, m), 7.8-6.7 (16H, m), 4.5 (2H, m), 3.7 (3H, s), 3.4-
2.2 (16H, m). Anal. (C40H43N5O7) C, H, N.
N-[Su ccin yl-D-Asp -2-(3-t r iflu or om et h ylp h en yl)et h yl-
a m id o]-Tr p -2-(2-n a p h th yl)eth yla m id e (26). The compound
was prepared as in example 23, except that 2-(3-trifluoro-
methylphenyl)ethylamine was used in place of 2-phenylethyl-
amine: [R]20 ) +13.0° (c 1.0, DMSO); 1H NMR (DMSO-d6) δ
D
10.8 (1H, s), 8.1-7.95 (4H, m), 7.9 (3H, m), 7.6 (1H, s), 7.5-
7.3 (9H, m), 7.1-6.9 (3H, m), 4.45 (2H, m), 3.4-2.2 (16H, m).
Anal. (C40H40F3N5O6‚1.5H2O) C, H, N.

3516 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Low et al.
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diluted to 4 mM in DMF and subsequently in water to 0.4 µM.
CCK-8S (Genosys) was diluted in water to a concentration of
2 mM and subsequently serially diluted in water to 20 nM.
P r otocols: At least eight preparations were used simulta-
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throughout for the allocation of experimental treatments to
each organ bath. Test compound or vehicle was incubated for
1 h before a single cumulative E/[A] curve was obtained. In
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baths did not exceed 700 µL in a single experiment.
Gu in ea -p ig ga ll bla d d er m u scle str ip CCK1 bioa ssa y:
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described previously.51 In brief, four longitudinal strips of
smooth muscle were dissected from each gall bladder taken
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produced and the preparations were washed twice during this
period. Force was continuously recorded with an isometric
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Gastric acid secretion was measured in isolated, lumen-
perfused mouse stomachs, prepared essentially as described
previously.40 Male mice (Charles River CD1, 22-26 g, 18 h
fasted, water ad libitum) were used. After sacrifice, the
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esophagus was ligated at the level of the cardiac sphincter and
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region, a cannula ligated tightly into the incision, and the
contents of the stomach flushed through with mucosal solution
to remove any remaining food. The stomach was placed in an
organ bath containing 40 mL of buffered serosal solution. The
serosal solution was maintained at 37 ( 1 °C and gassed
vigorously with 95% O2 and 5% CO2. The stomachs were
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improved the signal-to-noise ratio in preliminary experiments
when BOC-pentagastrin was used as the agonist. The prepa-
rations were allowed to stabilize for 90 min before the addition
of drugs to the serosal solution in the organ bath. Agonist-
stimulated responses were expressed as the change in the pH
of the lumen-perfusate from the basal pH immediately prior
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were considered to be significant.
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