Synthesis and biological properties of new constrained CCK-B antagonists: Discrimination of two affinity states of the CCK-B receptor on transfected CHO cells

Article abstract of DOI:10.1021/jm970439a

To improve our knowledge of the bioactive conformation of CCK-B antagonists, we have developed a new series of constrained dipeptoids whose synthesis and biochemical properties are reported here. These compounds, of general structure N(α)-[(2-adamantyloxy

Full text of DOI:10.1021/jm970439a

J . Med. Chem. 1997, 40, 3947 3956
3947
Syn th esis a n d Biologica l P r op er ties of New Con str a in ed CCK-B An ta gon ists:
Discr im in a tion of Tw o Affin ity Sta tes of th e CCK-B Recep tor on Tr a n sfected
CHO Cells
Bruno Bellier, Isabelle McCort-Tranchepain, Bertrand Ducos, Sophie Danascimento, Herve´ Meudal,
Florence Noble, Christiane Garbay, and Bernard P. Roques*
De´partement de Pharmacochimie Mole´culaire et Structurale, U266 INSERM - URA D1500, CNRS,
UFR des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l’Observatoire, 75270 Paris Cedex 06, France
Received J uly 7, 1997X
To improve our knowledge of the bioactive conformation of CCK-B antagonists, we have
developed a new series of constrained dipeptoids whose synthesis and biochemical properties
are reported here. These compounds, of general structure N -[(2-adamantyloxy)carbonyl]- -
methyltryptophanyl-(4-X)-proline, were designed by introducing a cyclization in the structure
of the previously described CCK-B/peptoid antagonist RB 210, N-[N-[(2-adamantyloxy)carbonyl]-
DL- -methyltryptophanyl]-N-(2-phenylethyl)glycine (Blommaert et al. J . Med. Chem. 1993, 36,
2868 2877), by means of a five-membered ring. Structure affinity relationship studies showed
that an R configuration of Trp-C and a cis configuration of the pyrrolidine substituents were
favorable for receptor recognition. The most potent compounds of this new series had similar
affinities for the CCK-B receptor as RB 210 and proved to be far more efficient in inhibiting
inositol phosphate production in CHO cells stably transfected with rat brain CCK-B receptor,
with IC50 values approaching those of the commonly used antagonists L-365,260 and PD-134,-
308. Moreover, binding studies performed using transfected CHO cells showed that two affinity
states of the CCK-B receptor can be discriminated by some of these compounds which also
have different biological profiles and are therefore highly interesting tools for the biochemical
and pharmacological characterization of CCK-B receptor heterogeneity.
In tr od u ction
indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo[3.3.1.13,7]dec-2-
yloxy)carbonyl]amino]propyl]amino]-1-phenylethyl]ami-
no]-4-oxobutanoic acid)10 and were shown to be resistant
to peptidases and endowed with high affinity and
selectivity for CCK-B receptors. In addition, RB 211
appeared to be much more efficient in crossing the
blood brain barrier than L-365,260 and PD-134,30811
and was devoid of the weak CCK-A agonist properties
of dipeptoids.12a,b
Cholecystokinin (CCK), or more precisely its C-
terminal fragment CCK8 (Asp-Tyr(SO3H)-Met-Gly-Trp-
Met-Asp-PheNH2), has been reported to be involved,
through its interaction with the CCK-B receptor, in
analgesia, anxiety, memory processes, and neuropsy-
chiatric disorders.1 Moreover, CCK-B receptor antago-
nists were also shown to suppress panic attacks induced
2
in humans by CCK4 and to potentiate antinociceptive
To improve the properties of PD-134,308, numerous
conformational restrictions were introduced in its struc-
ture. Unfortunately, neither N-terminal cyclization13
nor macrocyclization,14a,b or rigidification of the amide
bond,14c led to any significant positive result. Only a
C-terminal cyclization of PD-134,308 derivatives, by
means of a tetrahydronaphthyl group, has been reported
to increase the affinity for CCK-B receptors15 (Figure
1).
In this study, we made the hypothesis that this latter
approach would also be valid for compounds such as RB
210, in which C-terminal constraints can be easily
introduced. Thus, the â carbon of the phenethyl side
chain of RB 210 was linked to the carbon bearing the
carbonyl function, by means of a methylene bridge. This
resulted in the formation of a proline ring (Figure 1).
The corresponding peptidomimetics 2Adoc- Me-Trp-Pro-
(4-X) provide tools to probe the CCK-B receptor for its
limits of steric tolerance. To evaluate their selectivity
toward CCK-B versus CCK-A receptors, the binding
properties of the new dipeptoids were determined on
both guinea pig brain and pancreatic membranes. The
affinities of the most promising compounds were also
measured on homogenates of CHO cells transfected with
the rat brain CCK-B receptor16 and compared to those
responses induced by exogenous opiates such as mor-
phine3 or by endogenous opioids.4 Such compounds also
exert antidepressant effects in rodents.5 Therefore, the
search for potent and selective CCK-B antagonists,
endowed with good affinities, efficacies, and bioavail-
abilities, remains crucial for further pharmacological
and clinical investigations of such compounds. Recent
binding studies using the CCK-B antagonist L-365,260
((3R)-( )-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-
1,4-benzodiazepin-3-yl)-N′-(3-methylphenyl)urea)6 sug-
gested the possible occurrence of two antagonist binding
subtypes of the CCK-B receptor in rats and guinea pigs,7
which do not seem to be due to receptor structural
heterogeneity, as no experimental evidence has been
found to date to suggest its existence.8
We have previously prepared and studied peptidomi-
metic CCK-B antagonists designated RB 210 and RB
211 (N-[N-[(2-adamantyloxy)carboxyl]-D- -methyltryp-
tophanyl]-N-[2-(4-chlorophenyl)ethyl]glycine).9 These
compounds have smaller chemical structures than the
related dipeptoid PD-134,308 ([R-(R*,R*)]-4-[[2-[[3-(1H-
* To whom correspondence should be addressed. Tel: (33) 01-43-
25-50-45. Fax: (33) 01-43-26-69-18.
X Abstract published in Advance ACS Abstracts, November 1, 1997.
S0022-2623(97)00439-1 CCC: $14.00 © 1997 American Chemical Society

3948 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Bellier et al.
pseudoplanar pyrrolidine ring). Compounds 13c and
14c were obtained by methods reported earlier.18a,b
Because D-trans-hydroxyproline is not commercially
available, compounds 4c 11c were obtained after two
successive stereochemical inversions at the level of the
C4. Thus, a classical Mitsunobu reaction, using Boc-D-
cis-hydroxyproline methyl ester (3a ) yielded benzoate
3z19 whose controlled saponification led to the expected
alcohol 4a , without cleavage of the methyl ester.20
Subsequent Mitsunobu reactions between 4a and dif-
ferent aromatic alcohols yielded substituted prolines
4b 11b with the expected configuration. Deprotection
of these compounds finally provided prolylamines 4c
11c. Following the same scheme, alcohols 1a 3a were
converted into 4-phenoxyproline derivatives 1c 3c. In
these reactions, the inversion of configuration from
trans- to cis-substituted prolines proceeded with a better
yield than the reverse (60% vs 40%) and the purification
was easier.
F igu r e 1. Structures of PD-134,308, RB 210, and RB 211.
Dotted lines show schematically the cyclizations done to obtain
constrained derivatives of PD-134,30813,15 and RB 210 (this
study). Chiral centers created in the new series are shown by
an asterisk.
Preparation of benzyloxy derivatives 15c and 16c does
not involve stereochemical modification and was per-
formed by classical methods starting from Boc-D-4-cis-
hydroxyproline (Scheme 3). Amine 12c was prepared
via homologation by an Arndt-Eistert reaction of N -
Boc-protected phenoxyproline derivative 12a obtained
after saponification of the ester 6b.
Sch em e 1a
N-[(2-Adamantyloxy)carbonyl]-DL- -methyltrypto-
phan, prepared as described previously by Horwell et
al.,10 or its pure D isomer obtained as reported,9,21 was
then coupled to the different prolylamines 1c 16c us-
ing [(benzotriazol-1-yl)oxy]tris(dimethylamino)phospho-
nium hexafluorophosphate (BOP) as a coupling reagent
to give the corresponding amides 1d 16d (Scheme 1).
The reaction time (1 3 days) was dependent on the
nature of the 4-substituent of the proline ring. Com-
pounds 1d 16d were obtained in good yields (75 95%),
in contrast to RB 210 and RB 211 resulting from similar
coupling of Adoc- -methyltryptophan with linear sec-
2
ondary amines (yields ∼ 16%).9 The final dipeptoid
acids 1e 16e and 1f 4f were obtained after saponifica-
tion with 1 N NaOH in dioxane. Esters 1d 4d were
equimolar mixtures of diastereomeric esters, resulting
a
from coupling of racemic Adoc- Me-Trp with enantio-
2
merically pure proline derivatives 1c 4c. Only the
corresponding acids 1e 4e and 1f 4f were separated
by chromatography.
Con for m a tion a l An a lysis
of reference antagonists. Their antagonist potency was
also evaluated by an inositol phosphate assay performed
using the same cells.
1H NMR spectra of several compounds revealed the
presence of two rotamers in a 1:3 ratio corresponding
to a slow N-cis/N-trans isomerization around the proline
amide bond (see Experimental Section for details). The
major form was found to be the N-trans conformer based
on the observation of a strong NOE cross-peak between
the CH3 Trp and H5 Pro in 6e, while the minor rotamer
corresponded to the N-cis form, as confirmed by a NOE
cross-peak between CH3 Trp and H2 Pro. Conforma-
tional analysis was also performed on compound 6e
using a high-temperature-simulated annealing protocol.
Interestingly, among the 100 conformers that were
generated by this method, 30 had a N-cis peptide bond,
which is in agreement with the proportion of N-cis/N-
trans rotamers observed by NMR. Finally, 1H NMR
spectra of 6e in DMSO were recorded over a tempera-
Ch em istr y
All the final compounds listed in Tables 1 3 were
prepared according to Scheme 1, by coupling 2Adoc- Me-
Trp under racemic or pure D form with modified proline
derivatives which were synthesized according to Schemes
2 and 3. The three commercially available hydroxypro-
line isomers provided starting compounds for the ste-
reochemically controlled synthesis of 4-substituted
prolines.17a,b Scheme 2 illustrates the preparation of
several compounds by nucleophilic substitution of pro-
tected cis- and trans-hydroxyproline derivatives (cis and
trans refer to the relative spatial disposition of the
4-substituent and the 2-carboxyl group relative to the

New Constrained CCK-B Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3949
Sch em e 2a
a
(a) Boc2O, NaOH, dioxane; (b) Cs2CO3, MeI, DMF; (c) PPh3, DEAD, PhCO2H, THF; (d) NaOH, 1% MeOH; (e) PPh3, DEAD, PhOH,
THF; (f) CH2Cl2, TFA; (g) (o, p) (X, Y) PhOH, PPh3, DEAD, THF; (h) 1-naphthol, PPh3, DEAD, THF; 5, Ar 1-naphthyl; 6, Ar (p-Cl)Ph;
7, Ar
(p-F)Ph; 8, Ar
(p-I)Ph; 9, Ar
(p-NO2)Ph; 10, Ar
(o,p-Cl2)Ph; 11, Ar
(o,p-F2)Ph.
Sch em e 3a
a
(a) Ag2O, mX-PhCH2OH, DMF; (b) TFA, CH2Cl2; (c) 1 N NaOH, dioxane; (d) 1. iBuCOCl, N-methylmorpholine (NMM), 2. CH2N2,
CH2Cl2, 3. C6H5CO2Ag, NEt3, MeOH.
ture range of 20 80 °C. On the basis of temperature-
dependent changes in the indole NH and OCH (Adoc)
guinea pig cortex and pancreatic membranes, as re-
ported in Tables 1 3.
1
signals, a ∆Gq of 16 kcal mol corresponding to the
The ring closure introduced in the structure of RB 210
led to dipeptoids containing three chiral centers. All
final compounds had lower affinities for CCK-A than
for CCK-B receptors, whatever the stereochemistry of
these centers. Nevertheless, the cyclic constraint in-
troduced reduces the selectivity for CCK-B receptors.
This suggests that reduction of the conformational
freedom may lead to relatively more favorable confor-
mations for CCK-A binding, as compared to the linear
series.
The influence of the stereochemistry on CCK-B recep-
tor recognition was investigated in detail by synthesiz-
ing the eight corresponding stereoisomers of a single
molecule, N -[(2-adamantyloxy)carbonyl]- -methyltryp-
tophanyl-4-phenoxyproline. The results of binding stud-
ies reported in Table 1 clearly indicate that the R(D)
configuration of tryptophan (compounds 1e 4e) is
trans-to-cis isomerization was obtained using the Eyring
relation. The rotational barriers found in Pro-contain-
ing peptides are generally higher (∆Gq G 18 kcal mol 1).
This could be due to a steric hindrance in the planar
N-cis and N-trans forms of 6e.
Resu lts a n d Discu ssion
Str u ctu r e Affin ity Rela tion sh ip s. Conformation-
al restriction of peptides provides a useful approach to
increase receptor potency and selectivity and facilitates
the determination of the bioactive conformation of
peptide ligands.22 Such a strategy was undertaken in
this study by synthesizing cyclic derivatives of the short
peptidomimetic CCK-B antagonist RB 210. The result-
ing compounds 1e 16e and 1f 4f were evaluated for
their potency in inhibiting [3H]pCCK8 binding to both

3950 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Bellier et al.
Ta ble 1. Influence of the Configuration at the Three Chiral
Centers on the Affinity and Selectivity of the New Compounds
optimized with two carbons, the increase or decrease of
this chain length by one carbon resulting in a more than
10-fold loss of affinity.9 This could be related to an
enhanced number of unfavorable conformations, which
seem to be counteracted by the cyclization.
In a further attempt to modify the relative position
of the aromatic ring and the carboxyl group, which is
known to be essential for the activity,9,10 a methylene
was introduced between the ring and the acid group.
Compound 12e (Ki 122 nM) was found to be 5 times
less active than 6e (Ki 24 nM), but its selectivity for
CCK-B receptors was largely improved as a result of
its decreased affinity for CCK-A receptors (Ki 10 µM),
illustrating the even greater sensitivity of the latter to
the position of the carboxyl group (Table 2).
c
c
c
compdb
C2
C4
Ki(CCK-B)a
A/Ba
1e (c)
1f (c)
2e (t)
2f (t)
3e (t)
3f (t)
4e (c)
4f (c)
RB 210
RB 211
R
S
S
S
S
R
R
R
R
S
S
R
R
S
S
R
R
63 3.5
620 188
53.7
10
12
2
12.5
4.3
22.5
26.7
109
173
S
R
S
R
S
R
S
285
9
791 31
98 11
330 67
28
2
91 13
14 0.6
6.1 0.3
A comparison of the properties of both linear (RB 210
and derivatives) and constrained (this study) series of
peptoid antagonists suggests that the phenyl ring is
localized in a putative hydrophobic pocket of the
receptor.23a,b Compounds 14e, 16e, and 5e 11e were
prepared to investigate the requirements for optimal
recognition of this pocket. As shown in Table 3, a
naphthyl group in 5e induced a large loss of affinity,
presumably for reasons of steric hindrance. Introduc-
tion of a chlorine atom on the aromatic ring did not give
any significant improvement for compounds 14e and
16e versus 13e and 15e, respectively. On the contrary,
6e had a 3-fold higher selectivity for the CCK-B receptor
than 4e in the phenoxy series. The availability of
various substituted phenols allowed variations to be
developed in the aromatic ring substitution, which
were more difficult to achieve in the linear series. Table
3 also reports the binding affinities of substituted
4-phenoxyproline-containing dipeptoids. Regarding af-
finities for the CCK-B receptor, only compound 11e,
whose aromatic ring bears a fluorine atom at both ortho
and para positions, showed an improved affinity. This
could result from favorable electron-withdrawing effects,
which would be counteracted by steric interactions in
other compounds, especially 8e. Interestingly, substi-
tution of the aromatic ring by a bulky substituent at
the para position (6e, chlorine; 8e, iodine; 9e, NO2) led
to a 3-fold increase in selectivity for CCK-B versus
CCK-A receptors, probably resulting from more impor-
tant steric requirements at this level for CCK-A recog-
nition.
a
The Ki values (expressed in nM) represent the mean
SEM
of three separate experiments, each performed in triplicate for
determining CCK-B and CCK-A affinities on guinea pig cortex and
pancreas membranes, respectively. Ki for CCK-A receptor is only
expressed as its ratio versus Ki for CCK-B receptor. (c) and (t)
indicate the cis (c) or trans (t) orientation of C2 and C4 substituents
with respect to the mean plane of the proline ring.
refer to the configurations of the tryptophan carbon and carbons
2 and 4 of the proline ring, respectively.
b
c
, C2, and C4
Ta ble 2. Influence of the Prolyl C2 and C4 Substituents on the
Affinity and Selectivity of New Compounds in the Cis Series
compd
R4
R2
Ki(CCK-B)a
20
32 1.8
A/Ba
13e
14e
15e
16e
4e
Ph
CO2H
CO2H
2
33
Ph-pCl
12.5
68.6
38
22.5
65
OCH2Ph
OCH2Ph-mCl
OPh
OPh-pCl
OPh-pCl
CO2H
CO2H
CO2H
CO2H
24
56
28
24
2
8
2
3
6e
12e
CH2CO2H
122 30
b
a
The Ki values (expressed in nM) represent the mean
SEM
of three separate experiments, each performed in triplicate for
determining CCK-B and CCK-A affinities on guinea pig cortex and
pancreas membranes, respectively. Ki for CCK-A receptor is only
expressed as its ratio versus Ki for CCK-B receptor. Ki(CCK-A)
10 µM.
b
always preferred for CCK-B receptor binding, in agree-
ment with reported data on dipeptoids.10 Regarding the
stereochemistry at C2 and C4 of the proline ring, it may
be inferred that a cis orientation of the substituents
toward the mean plane of the proline ring (both sub-
stituents located on the same side of this plane) is more
favorable. Nevertheless, in compounds with a trans
orientation, 3e, but not 2e, retains a relatively good
CCK-B affinity, showing that a nonoptimal orientation
of the carboxylate does not preclude receptor recogni-
tion. Compound 4e was found to have the best affinity
and gave an indication about the optimal spatial ar-
rangement of the two side chains. This compound was
therefore chosen for further investigation by introduc-
tion of different aromatic moieties in the 4 position.
Modifications of the nature and the length of the chain
between C4 and the phenyl ring in 4e, 13e, and 15e did
not result in significant changes in affinity or selectivity.
Interestingly, this was not the case in the RB 210
(Figure 1) linear series, where the aliphatic chain
between the amide nitrogen and the phenyl ring was
In Vitr o An ta gon ist P r op er ties. To evaluate the
pharmacological profile of the best compounds of this
series (6e 11e), their ability to inhibit CCK8-induced
formation of inositol phosphates (IP) was measured in
CHO cells stably transfected with the rat CCK-B
receptor and compared to that of antagonists L-365,-
260, PD-134,308, and RB 211. Their binding charac-
teristics for the CCK-B receptor were also measured on
membrane preparations of these cells (Table 3). The
affinities determined on CHO cells were generally close
to those obtained using guinea pig brain preparations,
which is in agreement with literature data.24
All the compounds tested behaved as full antagonists,
as they abolished completely IP production triggered by
CCK8 on transfected CHO cells. The inhibitory poten-
cies of all the molecules did not strictly follow the same
order as their affinities measured on the same cell
homogenates. Large variations were even observed in
the ratio: IC50 versus IP production/affinity, which

New Constrained CCK-B Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3951
Ta ble 3. Influence of the Aromatic Group on the Affinity, Selectivity, and Antagonist Activity of the New Compounds: Comparison
with Various CCK-B Antagonists
CHO cells
two-site model, CHO cellsd
Ki %
2
compd
R4
Ki(CCK-B)a
A/Ba
Kib
IC50
Ki
%
1
c
1
2
5e
6e
7e
8e
9e
O-(1-naphthyl)
OPh-pCl
OPh-pF
775 151
2
65
27.4
96.3
80
16.4
22.4
173
24
26.2
3
9
24.1 5.8
19.9
74.9 22
32.7 21
15.3 1.7
18.6 8.4
72
9
5
55.5 12.4
55.8 30
507 120
37.4 8.1
389 158
217 50
OPh-pI
44.8 5.8
26.3 4.8
45.7 5.7
17.6 1.4
6.1 0.3
0.4 0.51
2.0 0.4
OPh-pNO2
OPh-o,pCl2
OPh-o,pF2
0.5
1.9
6.8
32.4
22.7
76.8
75.5
30.4
324.3
67.6
77.3
23.2
10e
11e
RB 211
PD-134,308
L-365,260
14
1
7.3 0.7
15 1.5
30
39
2
7
a
The Ki values (expressed in nM) represent the mean SEM of three separate experiments, each performed in triplicate for determining
CCK-B and CCK-A affinities on guinea pig cortex and pancreas membranes, respectively. Ki for CCK-A receptor is only expressed as its
b
ratio on Ki for CCK-B receptor. Ki values are the mean
SEM of three separate experiments, each performed in triplicate, on the rat
SEM of three separate measurements of
brain CCK-B receptor expressed in CHO cells. c Results (expressed in nM) are the mean
inhibition of inositol phosphate production induced by CCK8 (0.5 nM), each in triplicate (see Experimental Section for details). Results
are the mean of two separate experiments, each in triplcate. Ki values are expressed in nM; %1 and %2 represent the proportions of each
affinity state of the receptor.
d
Biological models used in these various studies are
25 27
guinea pig
and rat brain membranes,7,26 and COS
cells,28 this cell line having been transfected with a
CCK-B receptor gene. In our case, stable transfection
of CHO cells ensures that the receptor panel is homo-
geneous, indicating that the two-site binding mode must
result from the coexistence of two affinity states of a
single receptor, in terms of amino acid sequence. This
also relies on the fact that CCK8 neither binds nor
induces IP production in nontransfected CHO cells
(unpublished results). The lack of CCK8 binding sites
on untransfected CHO cells has already been observed
in a previous study in which the human CCK-B receptor
was transfected.29 In this study, CCK33, CCK8, CCK4,
and gastrin were shown to interact apparently with only
one class of binding sites. We obtained the same results
with these molecules and the transfected rat CCK-B
receptor.
F igu r e 2. Inhibition of [3H]pCCK8 specific binding to a CCK-B
receptor transfected CHO cell membrane preparation by
compound 10e. Each point represents the mean value of
triplicate determinations.
varied from values close to unity (i.e., 8e) to a two-figure
ratio (11e). For example, a comparison of RB 211 with
its constrained equivalent 6e shows that the latter,
although possessing a 2-fold lower affinity, was 3 times
more potent in the IP assay.
In vestiga tion s on th e Occu r r en ce of Tw o Affin -
ity Sta tes for th e CCK-B Recep tor . Binding studies
using CHO cells showed that some compounds, such as
10e and 11e, which are characterized by an additional
substituent at the ortho position of the phenyl ring, and
also compound 9e, bind to the receptor in a way that is
significantly better described by a two-site binding
model (Figure 2). It can be noticed that these three
compounds are the ones bearing the most powerful
electron-withdrawing substituents.
The occurrence of two affinity states of the CCK-B
receptor was reported 10 years ago.25 Several authors
have described CCK-B agonists, apparently capable of
discriminating two26,27 or even three28 binding states,
but only very recently has such a result been obtained
with an antagonist, namely, the benzodiazepine L-365,-
260,7 although, in this case, numerous experiments were
necessary to obtain enough data to reach statistical
significance.
To investigate this problem, the biological profiles of
10e and 11e are of great interest. Indeed, their
antagonist potencies on IP production in CHO cells show
a 10-fold difference, while their mean affinities for the
CCK-B receptor (evaluated from a one-site binding
model) are close to each other (Table 3). This difference
in antagonist behavior is not unique, for a quick
overview of the formely described CCK-B antagonists
L-365,260, PD-134,308, and RB 211 suggests a possible
partition of these molecules according to their biological
properties. On one hand, high-affinity and high-potency
antagonists of IP production would be represented by
L-365,260, PD-134,308, and our new compounds 10e or
7e, whereas on the other hand, RB 211 and 11e or 9e
would be high-affinity and low-potency antagonists
(Table 3).
The two CCK-B receptor affinity states might furnish
a convenient explanation for the discrepancy observed
between the two groups of molecules. In fact, antagonist
activity is linked to a disruption of the corresponding
second-messenger pathway, which is, in our biological
model, phospholipase C (PLC), coupled with the CCK-B
receptor through a G-protein.30 Differential coupling of

3952 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Bellier et al.
Anhydrous solvents were dried over 4 Å molecular sieves
prior to use. Plates were developed with UV, iodine vapor,
ninhydrin, and/or Erlich’s reagent. Melting points of the
crystallized compounds were taken on an Electrothermal
melting point apparatus and are uncorrected. The structure
of all compounds was confirmed by 1H NMR spectroscopy
(Bruker WH 270 MHz) performed in DMSO-d6 (concentrations
ranging 3 5 mM). Chemical shifts were measured in ppm
with HMDSO as internal standard. The purity of compounds
was checked by HPLC (Shimadzu apparatus) on a Kromasil
C8 column (5 µm, 4.6 × 250 mm) or a Kromasil C18 column (5
µm, 4.6 × 250 mm), with a mixture of H2O/TFA0.05% (solvent
A) and CH3CN (solvent B) (flow rate 1.2 mL/min, UV detection
214 nm).
Optical rotations were measured on a Perkin-Elmer pola-
rimeter (model 241). Mass spectra were recorded on a quad-
rupole NERMAG R10-10C apparatus or a double-focusing VG
70-250 SEQ instrument. Elemental analyses were performed
by Service Re´gional de Microanalyse (Universite´ Pierre et
Marie Curie, Paris, France).
the CCK-B receptor to G-proteins illustrated by differ-
ences in the sensitivity of the binding of agonists to the
addition of Gpp(NH)p, a nonhydrolyzable analogue of
GTP, has been suggested by several authors to explain
the presence of two affinity states.27,31
This hypothesis can explain the two-site binding
models obtained here, provided that differential cou-
pling of the receptor results in different conformations
at the antagonist binding region of the receptor. More-
over, IP assays start with incubation with CCK8 (0.5
nM). Therefore, if there is an equilibrium between pre-
and postcoupled states of the receptor, it will be shifted
in favor of the precoupled state. Thus, the inhibitory
potency of antagonists in this experiment would merely
result from their ability to bind to the putative pre-
coupled state of the receptor, whatever their “mean”
affinity, as determined from one-site binding models.
In any case, the hypotheses evoked by Harper et al.,7
such as different posttranslational modifications of
CCK-B receptors, remain valid here, but if they can
justify two-site binding models, they fail to explain the
large variations observed in antagonist activity on the
PLC pathway. Be that as it may, further biological
experiments are necessary to confirm any of the hy-
potheses described here.
Gen er a l Meth od A. Phenol (4.5 mmol), substituted phenol
(4.5 mmol), 1-naphthol (4.5 mmol), or benzoic acid (4.5 mmol)
and triphenylphosphine (4.5 mmol) were added to the alcohol
(3 mmol) in THF (10 mL). To this cooled (0 °C) solution was
added dropwise diethyl azodicarboxylate (4.5 mmol) over a
period of 1 h. After standing overnight at room temperature,
the mixture was concentrated and the residue was suspended
in Et2O (13 mL). The mixture was filtered, the filtrate was
concentrated, and the residue was chromatographed.
P r oced u r e B. Benzoate (8.3 mmol) was added to a solution
of 1% NaOH in MeOH (225 mL), and the resulting mixture
was stirred at room temperature for 30 min. After quenching
of the reaction with AcOH (2.3 mL) to avoid saponification of
the methyl ester, evaporation of the MeOH, addition of water,
and workup (EtOAc), the residue was chromatographed on
silica gel.
Gen er a l Meth od C. At 0 °C the carbamate (5 mmol) in
CH2Cl2 (10 mL) and TFA (10 mL) was stirred for 1 h and at
room temperature for an additional 2 h. After evaporation of
the solvents, the residue was either lyophilized and triturated
from Et2O or purified by chromatography.
Gen er a l Meth od D. Silver(I) oxide (10 mmol) and benzyl
bromide or m-Cl-benzyl bromide (10 mmol) were added to a
solution of the hydroxy acid (3 mmol) in DMF (6 mL). After
being stirred for 24 h at room temperature, the suspension
was filtered, the filtrate evaporated, and the residue subjected
again to the same procedure, before being purified by flash
chromatography on silica gel.
Con clu sion
The introduction of a bridging methylene unit in the
structure of RB 211 preserved the affinity for the CCK-B
receptor, indicating that unfavorable steric interaction
between the receptor and the proline ring did not occur.
Optimization of the configurational requirements for
receptor recognition led to compounds containing two
D-modified amino acids, the second being a proline
bearing an aromatic substituent on C4, with a cis
configuration toward the carboxylate. Reduction of the
conformational freedom, and therefore of the unfavor-
able entropy factor, unexpectedly did not improve the
affinity of parent RB 211, but only its antagonist
activity. Moreover, these new molecules should possess
enhanced stability toward enzymatic and acid degrada-
tion and increased lipophilicity, as compared to the
parent dipeptoids RB 210 and PD-134,308. This is
expected to facilitate blood brain barrier penetration,
which is crucial for pharmacological studies of these new
antagonists, and is currently under evaluation in our
laboratory. Last, the discrimination of two affinity
states of CCK-B receptors by some of the new com-
pounds described here is suggested by biochemical
studies and is now the object of further investigation.
Gen er a l Meth od E. To the ester (0.5 mmol) in dioxane (6
mL) was added 1 N NaOH (0.55 mmol) at 0 °C. After 5 h at
room temperature the solvent was evaporated. After addition
of 1 N HCl until pH 2 and workup (EtOAc), the residue was
chromatographed on silica gel (typical yield: 85%).
P r oced u r e F . To a stirred solution of the acid (3.68 mmol)
and N-methylmorpholine (3.68 mmol) in anhydrous THF (8
mL) at 20 °C was added dropwise isobutyl chloroformate
(3.68 mmol). After 20 min, the N-methylmorpholine hydro-
chlororide was filtered off, and a solution of diazomethane (15
mmol) in CH2Cl2 (45 mL) was added to the filtrate at 10 °C.
The cooled solution was stirred for 2 days at 4 °C. The
solvents were removed in vacuo to give the intermediate diazo
ketone, which was not further characterized. To a stirred
solution of this pale yellow oil in dry MeOH (7 mL) was added
a solution of silver benzoate (0.48 mmol) in Et3N (7.36 mmol)
causing evolution of nitrogen. When gas evolution had ceased,
the resulting tan solution was stirred for a further 10 min.
The solution was then treated with charcoal and filtered, and
the solvents were removed in vacuo to give a residue which
was dissolved in EtOAc (50 mL), worked up (water, brine),
and purified by flash chromatography.
Exp er im en ta l Section
Ch em istr y. D-cis-Hyp-OH was purchased from Aldrich
(France). Boc-L-trans-Hyp-OMe, Boc-L-cis-Hyp-OMe, Boc-L-
trans-Hyp(Bzl)-OH, and DL- -methyltryptophan were from
Bachem (Switzerland). BOP was from Novabiochem. Solvents
were from SDS (France). PD-134,308 was synthesized as
previously described.10
Flash column chromatography was performed using Merck
silica gel (230 400 mesh). TLC was performed on precoated
silica gel plates (60F-254, 0.2 mm thick; Merck) with the
following solvent systems (v/v): A, CH2Cl2 MeOH AcOH (9:
1:0.5); B, CH2Cl2 MeOH (95:5); C, CH2Cl2 MeOH (100:2); D,
CH2Cl2; E, cyclohexane EtOAc (9:1); F, cyclohexane EtOAc
(4:1); G, CHCl3 MeOH H2O AcOH EtOAc (7:3:0.6:0.3:5.5);
H, CH2Cl2 EtOAc (9:1). I, CH2Cl2 EtOAc (8:2).
Gen er a l Meth od G. To a solution of the amine (1.24 mmol)
in dry DMF (440 µL) was added N -[(2-adamantyloxy)carbo-
nyl]- -methyl-(RS)-tryptophan (0.62 mmol), DIEA (2.48 or 3.72
mmol when the amine was used as salt), and, at 0 °C, BOP
(0.69 mmol). After 1 h at this temperature, the solution was
allowed to warm to room temperature and stirred further for

New Constrained CCK-B Antagonists
3 days. The solvent was then evaporated, and EtOAc (20
mL) was added to the residue. The organic phase was washed
successively with 10% aqueous citric solution (2 × 15 mL),
water (1 × 15 mL), 10% aqueous NaHCO3 solution (2 × 15
mL), water (1 × 15 mL), and brine (1 × 15 mL). The organic
solution was dried over Na2SO4 and filtered. Evaporation of
the solvent gave a residue which was chromatographed on
silica gel.
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3953
1
2.68 (1H, m, H3), 3.55 (3H, s, OCH3), 3.64 (2H, m, 2H5), 5.32
(1H, m, H4), 6.95 7.98 (7H, m, Ar).
TF A H-D-cis-Hyp (P h -p Cl)-OMe (6c): obtained from 6b
according to general method C; yield 85%, after flash chro-
matography using CH2Cl2 MeOH (95:5) as eluent; Rf 0.37-
(B); mp 198 200 °C; 1H NMR (DMSO) δ 2.33 (1H, dd, H3),
2.55 (1H, m, H3), 3.46 (2H, m, 2H5), 3.66 (3H, s, OCH3), 4.63
(1H, dd, H2), 5.12 (1H, m, H4), 6.86 (2H, dd, Ar), 7.31 (2H, dd,
Ar); FAB/MS (MH ) 256.
Boc-D-tr a n s-Hyp (Bz)-OMe (3z): obtained from Boc-D-cis-
Hyp-OMe (3a ) according to general method A; yield 100%,
after flash chromatography with CH2Cl2 MeOH (100:2) as
TF A H-D-cis-Hyp (P h -p F )-OMe (7c): obtained from 7b
according to general method C; yield 100%, without further
eluent; Rf 0.20(C); mp 91 92 °C; [ ]18
42.8° (c 0.5, CHCl3).
1
D
purification; Rf 0.51(B); H NMR (DMSO) δ 2.37 (1H, m, H3),
Anal. (C18H23NO6) C, H, N.
2.57 (1H, m, H3), 3.48 (2H, m, 2H5), 3.69 (3H, s, OCH3), 4.53
(1H, m, H2), 5.09 (1H, brs, H4), 6.84 (2H, dd, Ar), 7.06 (2H, dd,
Ar).
Boc-L-tr a n s-Hyp (P h )-OMe (2b) a n d Boc-D-tr a n s-Hyp -
(P h )-OMe (3b): obtained respectively from Boc-L-cis-Hyp-OMe
(2a ) and 3a according to general method A; yield 40%, after
flash chromatography with CH2Cl2 as eluent; Rf 0.16(D); mp
TF A H-D-cis-Hyp (P h -p I)-OMe (8c): obtained from 8b
according to general method C; yield 100%, without further
101 °C; 2b [ ]18
37.7° (c 0.5, CHCl3); 3b [ ]18
38.3°
1
D
D
purification; Rf 0.6(B); H NMR (DMSO) δ 2.40 (1H, m, H3),
(c 0.2, CHCl3); 1H NMR (DMSO) δ 1.29 and 1.32 (9H, s, (CH3)3),
2.16 (1H, m, H3), 2.40 (1H, m, H3), 3.44 3.67 (5H, m, OCH3
2H5), 4.25 (1H, q, H2), 4.98 (1H, brs, H4), 6.90 (3H, m, Ar),
7.26 (2H, dd, Ar). Anal. (C17H23NO5) C, H, N.
2.62 (1H, m, H3), 3.51 (2H, m, 2H5), 3.75 (3H, s, OCH3), 4.69
(1H, m, H2), 5.17 (1H, m, H4), 6.74 (2H, dd, Ar), 7.63 (2H, dd,
Ar).
TF A H-D-cis-Hyp (P h -p NO2)-OMe (9c): obtained from 9b
according to general method C; yield 93%, after precipitation
in Et2O; Rf 0.35(B); 1H NMR (DMSO) δ 2.45 (1H, m, H3), 2.71
(1H, m, H3), 3.59 (2H, m, 2H5), 3.78 (3H, s, OCH3), 4.73 (1H,
m, H2), 5.36 (1H, brs, H4), 7.14 (2H, dd, Ar), 8.22 (2H, dd, Ar).
TF A H-D-cis-Hyp (P h -o,p Cl2)-OMe (10c): obtained from
10b according to general method C; yield 100%, without
further purification; Rf 0.55(B); 1H NMR (DMSO) δ 2.50 (1H,
m, H3), 2.64 (1H, m, H3), 3.62 (2H, m, 2H5), 3.77 (3H, s, OCH3),
4.72 (1H, m, H2), 5.22 (1H, m, H4), 7.19 (1H, d, Ar), 7.38 (1H,
d, Ar), 7.54 (1H, s, Ar).
TF A H-D-cis-Hyp (P h -o,p F 2)-OMe (11c): obtained from
11b according to general method C; yield 100%, without
further purification; Rf 0.3(B); 1H NMR (DMSO) δ 2.4 (2H, m,
2H3), 3.48 (2H, m, 2H5), 3.72 (3H, s, OCH3), 4.67 (1H, m, H2),
5.12 (1H, brs, H4), 6.87 7.3 (3H, m, Ar).
Boc-D-cis-Hyp (P h -p Cl)-OH (12a ): obtained from 6b ac-
cording to general method E without further purification; yield
90%; Rf 0.10(H); 1H NMR (DMSO) δ 1.29 and 1.32 (9H, s,
(CH3)3), 2.09 (1H, m, H3), 2.50 (1H, m, H3), 3.32 (1H, d, H5),
3.65 (1H, m, H5), 4.20 (1H, m, H2), 4.91 (1H, brs, H4), 6.82
and 7.26 (4H, d, Ar). Anal. (C16H20NO5Cl) C, H, N.
Boc-D-cis-â-h om o-Hyp(P h -pCl)-OMe (12b): obtained from
12a according to procedure F; yield 70%, after flash chroma-
tography with cyclohexane EtOAc (4:1) as eluent; Rf 0.21(F);
amorphous solid. Anal. (C18H24NO5Cl) C, H, N.
Boc-L-cis-Hyp (P h )-OMe (1b) a n d Boc-D-cis-Hyp (P h )-
OMe (4b): obtained respectively from Boc-L-trans-Hyp-OMe
(1a ) and Boc-D-trans-Hyp-OMe (4a ), according to general
method A; yield 60%, after flash chromatography with CH2-
Cl2 and then CH2Cl2 MeOH (100:2) as eluent; Rf 0.16(D), 0.35-
(C); 1b [ ]18
32.6° (c 0.5, CHCl3); 4b [ ]18
33.4° (c
D
D
0.6, CHCl3); 1H NMR (DMSO) δ 1.30 and 1.35 (9H, s, (CH3)3),
2.13 (1H, m, H3), 2.51 (1H, m, H3), 3.36 (1H, m, H5), 3.53
3.70 (4H, m, OCH3 H5), 4.34 (1H, m, H2), 4.99 (1H, m, H4),
6.70 (2H, m, Ar), 6.80 (1H, dd, Ar), 7.24 (2H, dd, Ar). Anal.
(C17H23NO5) C, H, N.
Boc-D-cis-Hyp (1-n a p h th yl)-OMe (5b): obtained from 4a
according to general method A; yield 52%, after flash chro-
matography with cyclohexane EtOAc (9:1, 8:2) as eluent; Rf
0.36(F); mp 103 105 °C. Anal. (C21H25NO5) C, H, N.
Boc-D-cis-Hyp (P h -p Cl)-OMe (6b): obtained from 4a ac-
cording to general method A; yield 78%, after flash chroma-
tography with cyclohexane EtOAc (9:1, 8:2) as eluent; Rf
0.15(E), 0.31 (F); mp 67 68 °C; [ ]
Anal. (C17H22NO5Cl) C, H, N.
30.2° (c 0.6, CHCl3).
Boc-D-cis-Hyp (P h -p F )-OMe (7b): obtained from 4a ac-
cording to general method A; yield 79%, after chromatography
with cyclohexane EtOAc (9:1, 8:2) as eluent; Rf 0.22(F); mp
64 66 °C. Anal. (C17H22NO5F) C, H, N.
Boc-D-cis-Hyp (P h -p I)-OMe (8b): obtained from 4a accord-
ing to general method A; yield 70%, after chromatography with
cyclohexane EtOAc (9:1, 8:2) as eluent; Rf 0.41(F); mp 70
72 °C. Anal. (C17H22NO5I) C, H, N.
Boc-D-cis-Hyp (P h -p NO2)-OMe (9b): obtained from 4a
according to general method A; yield 79%, after chromatog-
raphy with cyclohexane EtOAc (9:1, 8:2) as eluent; Rf 0.22(F);
mp 64 66 °C. Anal. (C17H22NO5F) C, H, N.
Boc-D-cis-Hyp (P h -o,p Cl2)-OMe (10b): obtained from 4a
according to general method A; yield 74%, after chromatog-
raphy with cyclohexane EtOAc (9:1, 8:2) as eluent; Rf 0.36(F);
mp 52 54 °C. Anal. (C17H21NO5Cl2) C, H, N.
TF A H-D-cis-â-h om o-Hyp (P h -p Cl)-OMe (12c): obtained
from 12b according to general method C; yield 90%, after
precipitation in Et2O; Rf 0.17(B); mp 87 °C; FAB/MS (MH )
270.
TF A H-D-cis-Hyp (Bzl)-OBzl (15c). Boc-D-cis-Hyp(Bzl)-
OBzl was obtained from Boc-D-cis-Hyp-OH according to gen-
eral method D; yield 65%, after flash chromatography with
CH2Cl2 and then CH2Cl2 MeOH (100:2) as eluent, as an oil;
Rf 0.30(C). 15c was obtained from the preceding compound
according to general method C; yield 85%, after chromatog-
raphy using CH2Cl2 MeOH (95:5) as eluent; Rf 0.19(B); mp
93 °C; 1H NMR (DMSO TFA) δ 2.40 (2H, m, 2H3), 3.30 (1H,
m, H5), 3.46 (1H, dd, H5), 4.26 (1H, m, H2), 4.40 (2H, dd,
OCH2Φ), 4.65 (1H, brt, H4), 5.16 (2H, s, CO2CH2Φ), 7.10 7.40
(10H, m, Ar).
Boc-D-cis-Hyp (P h -o,p F 2)-OMe (11b): obtained from 4a
according to general method A; yield 82%, after chromatog-
raphy with cyclohexane EtOAc (9:1, 8:2) as eluent; Rf 0.24(F).
Anal. (C17H21NO5F2) C, H, N.
TF A H-L-tr a n s-Hyp (P h )-OMe (2c) a n d TF A H-D-tr a n s-
Hyp (P h )-OMe (3c): obtained respectively from 2b and 3b
according to general method C; yield 85%, after flash chro-
matography using CH2Cl2 MeOH (95:5) as eluent; Rf 0.36-
(B); mp 191 °C: FAB/MS (MH ) 222.
TF A H-D-cis-Hyp (Bzl-m Cl)-OBzl-m Cl (16c). Boc-D-cis-
Hyp(Bzl-mCl)-OBzl-mCl was obtained from Boc-D-cis-Hyp-OH
according to general method D; yield 50%, after flash chro-
matography with CH2Cl2 MeOH (100:2) as eluent, as an oil;
1
Rf 0.17(D); H NMR (DMSO) δ 1.20 and 1.35 (9H, s, (CH3)3),
H-L-cis-Hyp (P h )-OMe (1c) a n d H-D-cis-Hyp (P h )-OMe
(4c): obtained respectively from 1b and 4b according to general
method C; yield 85%, after flash chromatography using CH2-
Cl2 MeOH (95:5) as eluent; Rf 0.35(B); mp 66 67 °C. Anal.
(C12H15NO3) C, H, N.
2.0 2.21 (1H, m, H3), 2.38 (1H, m, H3), 3.18 3.58 (2H, m, 2H5),
4.28 4.50 and 4.87 5.17 (6H, m, 2(OCH2) H2 H4), 7.10
7.41 (8H, m, Ar). 16c was obtained as an oil from the
preceding compound according to general method C; yield 85%;
Rf 0.25(B); 1H NMR (DMSO TFA) δ 2.46 (2H, m, 2H3), 3.35
(1H, m, H5), 3.51 (1H, m, H5), 4.31 (1H, brs, H2), 4.43 (2H, dd,
OCH2Φ), 4.72 (1H, m, H4), 5.20 (2H, s, CO2CH2Φ), 7.12 7.56
(8H, m, Ar); FAB/MS (MH ) 380.
TF A H-D-cis-Hyp (1-n a p h th yl)-OMe (5c): obtained from
5b according to general method C; yield 100%, without further
1
purification; Rf 0.66(B); H NMR (DMSO) δ 2.55 (1H, m, H3),

3954 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Bellier et al.
2Ad oc- -Me-DL-Tr p -L-cis-H yp (P h )-OMe (1d ), 2Ad oc- -
Me-DL-Tr p -D-cis-Hyp (P h )-OMe (4d ), 2Ad oc- -Me-D-Tr p -L-
tr a n s-H yp (P h )-OMe (2d ), 2Ad oc- -Me-DL-Tr p -D-tr a n s-
Hyp (P h )-OMe (3d ): according to general method G (2 days),
1d 4d were obtained from 1c 4c, respectively; yield 85%. 1d
and 4d FAB/MS (MH ) 600; HPLC, tR 9.60 and 8.45 min, 70%
B. 2d and 3d FAB/MS (MH ) 600; HPLC, tR 9.25 and 10.25
min, 70% B.
Hyp (P h )-OH (3e), 2Ad oc- -Me-D-Tr p -D-tr a n s-Hyp (P h )-OH
(3f): according to general method E, 1e,f 4e,f were obtained
from 1d 4d , respectively. 1e and 4f mp 180 182 °C; 1H NMR
(DMSO
2H3), 2.80 3.60 (2H, m, 2Hâ), 3.70 4.20 (2H, m, 2H5), 4.35
4.72 (2H, m, H2 OCH), 4.96 and 5.07 (1H, brs, H4), 6.60
7.42 (11H, m, Ar NH), 10.85 (1H, brs, NHind). 1f and 4e mp
161 164 °C; 1H NMR (DMSO
TFA) δ 1.0 2.50 (19H, m,
adamantyl CH3 2H3), 2.90 3.90 (4H, m, 2Hâ 2H5),
4.38 (1H, m, H2), 4.64 and 4.84 (1H, brs, OCH), 4.73 and 4.95
), 6.70 7.40 (11H, m, Ar NH), 10.84 (1H, brs,
TFA) δ 1.0 2.50 (19H, m, adamantyl
CH3
2Ad oc- Me-D-Tr p -D-cis-Hyp (1-n a p h th yl)-OMe (5d ): ac-
cording to general method G (3 days), 5d was obtained from
1
5c; yield 76%; Rf(H) 0.21; H NMR (DMSO) δ 1.4 2.6 (19H,
(1H, brs, H4
1
CH3
adamantyl
2H3), 3.05 and 3.12 (1H, d, Hâ), 3.20
NHind). 2e and 3f mp 240 244 °C; H NMR (DMSO) δ 1.04
2.60 (19H, m, adamantyl
2Hâ), 3.65 5.10 (5H, m, 2H5
3.41 (1H, d, Hâ), 3.47 and 3.49 (3H, s, OCH3), 3.70 4.00 (2H,
m, 2H5), 4.56 (1H, m, H2), 4.67 and 4.82 (1H, brs, OCH), 5.12
CH3 2H3), 2.90 3.60 (2H, m,
H2 OCH H4), 6.70 7.50
and 5.30 (1H, brs, H4), 6.8 8.02 (13H, m, Ar
and 10.91 (1H, brs, NH ind).
NH), 10.88
(11H, m, Ar NH), 10.88 (1H, brs, NHind). 2f and 3e mp 140
141 °C; 1H NMR (DMSO
TFA) δ 1.10 2.45 (19H, m,
adamantyl CH3 2H3), 3.03 and 3.13 (1H, d, Hâ), 3.50
and 3.55 (1H, d, Hâ), 3.84 4.18 (2H, m, 2H5), 4.37 (1H, m, H2),
4.62 and 4.83 (1H, brs, OCH), 5.08 (1H, brs, H4), 6.80 7.50
2Ad oc- -Me-D-Tr p -D-cis-Hyp (P h -p Cl)-OMe (6d ): accord-
ing to general method G (2 days), 6d was obtained from 6c;
yield 85%; FAB/MS (MH ) 634; HPLC, tR 12.0 min, 70% B.
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -p F )-OMe (7d ): according
to general method G (3 days), 7d was obtained from 7c; yield
(11H, m, Ar
NH), 10.90 (1H, s, NHind).
2Ad oc- Me-D-Tr p -D-cis-Hyp (1-n a p h th yl)-OH (5e): ac-
cording to general method E, 5e was obtained from 5d ; 1H
NMR (DMSO TFA) δ 1.18 and 1.22 (3H, s, CH3), 1.25 2.6
1
85%; Rf(I) 0.38; H NMR (DMSO) δ 1.14 2.5 (19H, CH3
adamantyl 2H3), 3.05 (1H, d, Hâ), 3.44 (1H, d, Hâ), 3.56 and
3.58 (3H, s, OCH3), 3.55 3.85 (2H, m, 2H5), 4.49 (1H, m, H2),
4.65 and 4.82 (1H, brs, OCH), 4.84 and 5.04 (1H, brs, H4),
(16H, m, adamantyl
2H3), 3.05 and 3.12 (1H, d, Hâ), 3.45
(1H, d, Hâ), 3.70 4.10 (2H, m, H5), 4.45 (1H, m, H2), 4.61 and
4.85 (1H, brs, OCH), 5.04 and 5.23 (1H, brs, H4), 6.78 8.12
6.72 7.40 (10H, Ar
NH), 10.87 (1H, brs, NH ind).
(13H, m, Ar
MS ((M Na) ) 658.3.
NH), 10.84 and 10.87 (1H, brs, NH ind); ES/
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -p I)-OMe (8d ): according
to general method G (3 days), 8d was obtained from 8c; yield
88%; Rf(H) 0.25; H NMR (DMSO) δ 1.12 2.5 (19H, CH3
1
2Ad oc- -Me-D-Tr p -D-cis-Hyp (P h -p Cl)-OH (6e): according
to general method E, 6e was obtained from 6d ; mp 167 169
°C; 1H NMR (DMSO TFA) δ 1.08 2.45 (19H, m, adamantyl
CH3 2H3), 3.10 (1H, m, Hâ), 3.45 (1H, m, Hâ), 3.60 3.90
(2H, m, 2H5), 4.41 (1H, m, H2), 4.66 and 4.86 (1H, brs, OCH),
adamantyl 2H3), 3.08 (1H, d, Hâ), 3.43 (1H, d, Hâ), 3.52 and
3.53 (3H, s, OCH3), 3.60 3.85 (2H, m, 2H5), 4.45 (1H, m, H2),
4.65 and 4.83 (1H, brs, OCH), 4.83 and 5.03 (1H, brs, H4),
6.60 7.55 (10H, Ar NH), 10.87 and 10.90 (1H, brs, NH ind).
2Adoc- Me-D-Tr p -D-cis-Hyp(P h -pNO2)-OMe (9d ): accord-
ing to general method G (3 days), 9d was obtained from 9c;
4.86 and 5.01 (1H, brs, H4), 6.72 7.44 (10H, m, Ar
10.88 (1H, brs, NHind).
NH),
1
yield 95%; Rf(H) 0.21; H NMR (DMSO) δ 1.1 2.6 (19H, m,
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -p F )-OH (7e): according
to general method E, 7e was obtained from 7d ; 1H NMR
(DMSO TFA) δ 1.13 and 1.22 (3H, s, CH3), 1.3 2.5 (16H,
m, adamantyl 2H3), 3.05 (1H, d, Hâ), 3.42 (1H, d, Hâ), 3.58
3.82 (2H, m, H5), 4.37 (1H, m, H2), 4.65 and 4.90 (1H, brs, H4),
4.67 and 4.79 (1H, brs, OCH), 6.72 7.40 (10H, m, Ar NH),
10.78 (1H, brs, NH ind); ES/MS ((M Na) ) 626.3.
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -p I)-OH (8e): according to
general method E, 8e was obtained from 8d ; 1H NMR (DMSO
TFA) δ 1.12 and 1.17 (3H, s, CH3), 1.35 2.6 (16H,
adamantyl 2H3), 3.02 and 3.09 (1H, d, Hâ), 3.40 (1H, d, Hâ),
3.55 3.80 (2H, m, 2H5), 4.63 and 4.82 (1H, brs, OCH), 4.68
CH3
adamantyl
2H5), 3.01 3.12 (2H, m, Hâ), 3.5 3.8
(5H, m, OCH3 2H5), 4.44 5.32 (3H, OCH H4 H2), 6.82
7.70 (7H, m, Ar), 8.15 (2H, m, Ar), 10.88 (1H, brs, NH ind).
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -o,p Cl2)-OMe (10d ): ac-
cording to general method G (3 days), 10d was obtained from
10c; yield 94%; Rf(H) 0.25; 1H NMR (DMSO) δ 1.18 2.6 (19H,
CH3
adamantyl
2H3), 3.05 and 3.12 (1H, d, Hâ), 3.43
(1H, d, Hâ), 3.55 and 3.57 (3H, s, OCH3), 3.60 3.83 (2H, m,
2H5), 4.50 (1H, m, H2), 4.57 and 4.84 (1H, brs, OCH), 4.84 and
5.11 (1H, brs, H4), 6.8 7.5 (9H, Ar NH), 10.90 (1H, brs, NH
ind).
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -o,p F 2)-OMe (11d ): ac-
cording to general method G (3 days), 11d was obtained from
11c; yield 93%; Rf(H) 0.13; 1H NMR (DMSO) δ 1.1 2.6 (19H,
CH3 adamantyl 2H3), 3.0 3.5 (1H, d, Hâ), 3.55 and 3.56
(3H, s, OCH3), 3.6 3.85 (2H, m, 2H5), 4.5 (1H, m, H2), 4.62
and 4.85 (1H, brs, OCH), 4.7 and 5.04 (1H, brs, H4), 6.7 7.45
and 4.96 (1H, brs, H4), 6.55 7.55 (10H, m, Ar
and 10.87 (1H, brs, NH ind); ES/MS ((M Na) ) 734.2.
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -p NO2)-OH (9e): accord-
ing to general method E, 9e was obtained from 9d ; H NMR
NH), 10.85
1
(DMSO
TFA) δ 1.08 2.6 (19H, m, CH3
adamantyl
2H3), 3.03 and 3.11 (1H, d, Hâ), 3.41 (1H, d, Hâ), 3.72 (2H, m,
2H5), 4.36 (1H, m, H2), 4.63 and 4.83 (1H, brs, OCH), 4.95 and
5.20 (1H, brs, H4), 6.7 8.2 (10H, m, Ar NH), 10.83 and 10.87
(1H, brs, NH ind); ES/MS ((M Na) ) 653.2.
(9H, Ar
NH), 10.88 (1H, brs, NH ind).
2Adoc- -Me-D-Tr p-D-cis-â-h om o-Hyp(P h -pCl)-OMe (12d):
according to general method G (2 days), 12d was obtained from
12c; yield 82%; FAB/MS (MH ) 648; tR 26.50 min, 60% B.
2Ad oc- -Me-D-Tr p -D-cis-P r o(4-P h )-OMe (13d ): according
to general method G (3 days), 13d was obtained from H-D-cis-
Pro(4-Ph-pCl)-OMe (13c); yield 78%; FAB/MS (MH ) 584;
HPLC, tR 9.35, min, 70% B.
2Ad oc- -Me-D-Tr p -D-cis-P r o(4-P h -p Cl)-OMe (14d ): ac-
cording to general method G (3 days), 14d was obtained from
14c; yield 85%; FAB/MS (MH ) 618; HPLC tR 9.50 min, 70%
B.
2Ad oc- Me-D-Tr p -D-cis-H yp (P h -o,p Cl2)-OH (10e): ac-
cording to general method E, 10e was obtained from 10d ; H
1
NMR (DMSO TFA) δ 1.18 and 1.73 (3H, s, CH3), 1.3 2.6
(16H, m, adamantyl
2H3), 3.00 and 3.08 (1H, d, Hâ), 3.39
(1H, d, Hâ), 3.55 (1H, m, H5), 3.8 (1H, m, H5), 4.35 and 4.40
(1H, m, H2), 4.60 and 4.82 (1H, brs, OCH), 4.64 and 4.97 (1H,
brs, H4
), 6.78 7.50 (10H, m, Ar
NH), 10.83 and 10.86 (1H,
brs, NH ind); ES/MS (35Cl, 35Cl (M Na) , and 35Cl,37Cl
) 676.2 and 678.2.
(M
Na)
2Adoc- -Me-DL-Tr p-D-cis-Hyp(Bzl)-OBzl (15d) an d Adoc-
2Ad oc- Me-D-Tr p -D-cis-Hyp (P h -o,p F 2)-OH (11e): accord-
ing to general method E, 11e was obtained from 11d ; 1H NMR
(DMSO TFA) δ 1.15 and 1.21 (3H, s, CH3), 1.3 2.6 (16H,
m, adamantyl 2H3), 3.03 and 3.12 (1H, d, Hâ), 3.45 (1H, d,
Hâ), 3.72 (1H, m, 2H5), 4.40 (1H, m, H2), 4.63 and 4.84 (1H,
2
-Me-DL-Tr p -D-cis-Hyp (Bzl-m Cl)-OBzl-m Cl (16d ): accord-
ing to general method G (1 day), 15d and 16d were obtained
from 15c and 16c with 85% and 80% yields, respectively. 15d
FAB/MS (MH ) 690; HPLC tR 14.80 and 12.05 min, 80% B.
16d FAB/MS (MH ) 758; HPLC tR 11.2 and 9.2 min, 80% B.
2Ad oc- -Me-D-Tr p -L-cis-Hyp (P h )-OH (1e), 2Ad oc- -Me-
L-Tr p-L-cis-Hyp(P h )-OH (1f), 2Adoc- -Me-D-Tr p-D-cis-Hyp-
(P h )-OH (4e), 2Ad oc- -Me-L-Tr p -D-cis-H yp (P h )-OH (4f),
2Ad oc- -Me-D-Tr p -L-tr a n s-Hyp (P h )-OH (2e), 2Ad oc- -Me-
L-Tr p-L-tr a n s-Hyp(P h )-OH (2f), 2Adoc- -Me-L-Tr p-D-tr a n s-
brs, OCH), 4.67 and 4.93 (1H, m, H4), 6.8 7.4 (m, Ar
10.88 (1H, brs, NH ind); ES/MS ((M Na) ) 644.3.
NH),
2Ad oc- -Me-D-Tr p -D-cis-â-h om o-Hyp (P h -p Cl)-OH (12e):
according to general method E, 12e was obtained from 12d ;
mp 149 151 °C; FAB/MS (MH ) 634; HPLC tR 17.00, min, 60%
1
B; H NMR (DMSO) δ 1.0 2.62 (19H, m, adamantyl
CH3

New Constrained CCK-B Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3955
2H3), 2.75 3.90 (9H, m, 2HâPro
4.03 and 4.13 (1H, m, H2), 4.53 and 4.60 (1H, s, OCH), 4.78
(1H, brs, NHind).
2HâTrp
OCH3
2H5),
and centrifuged under the same conditions. The final pellet
was homogenized at 4 °C in 5 mL of Tris-HCl buffer (pH 7.4)
with 5 mM MgCl2 and bacitracin. The membranes were
aliquoted and frozen at 80 °C. Protein concentration was
estimated using the Pierce bicinchoninic acid protein assay
reagent with bovine serum albumin as a standard. The
binding assays were performed in 50 mM Tris-HCl, pH 7.4, 5
mM MgCl2, and 0.2 mg/mL bacitracin as previously described.23b
Each assay contained the membrane preparation (around 40
µg of protein) and [3H]pCCK8 (0.4 nM) in a final volume of 1
mL. The nonspecific binding was determined in the presence
of 1 µM CCK8.
In ositol P h osp h a te Assa ys. CHO cells stably expressing
wild-type CCK-B rat receptor were assayed for agonist (and/
or antagonist)-stimulated IP hydrolysis essentially as previ-
ously described.29,33 Cells were plated in 24-well microtiter
plates. Before confluency cells were grown in the presence of
1 µCi/mL myo-[2-3H]inositol for 16 h, at 37 °C. Cells were
treated with 10 mM LiCl for 30 min at 37 °C and with 0.5 nM
CCK8 for 5 min, and various concentrations of antagonists
were then added to the cells. After 45 min at 37 °C, the
incubation medium was removed and the cells were washed
twice with 1 mL of PBS. The reaction was stopped by adding
400 µL of ice-cold 67% methanol and 300 µL of 0.125% Triton.
The cells were scraped, and the suspension was subjected to
chloroform extraction; 0.5 mL of the aqueous phase was added
to 4.5 mL of water. The solution was then loaded onto a 0.5-
mL column of AG1-X8 Dowex anion-exchange resin. The
column was washed with 1 mL of distilled water followed by
5 mL of 5 mM sodium borate/60 mM sodium formate. Total
[3H]inositol phosphates were then eluted into scintillation vials
with 5 mL of 1 M ammonium formate/0.1 M formic acid.
Scintillation mixture was then added and the radioactivity
counted. The data are expressed as the percentage of the
maximal response induced by 0.5 nM of CCK8.
2Ad oc- -Me-D-Tr p -D-cis-P r o(4-P h )-OH (13e): according
to general method E, 13e was obtained from 13d ; mp 171
174 °C; 1H NMR (DMSO
TFA) δ 1.10 2.12 (17H, m,
CH3), 2.38 (1H, m, H3), 2.61 (1H, m, H3), 2.85
3.51 (4H, m, 2Hâ 2H5), 4.01 4.40 (2H, m, H4 H2), 4.68
adamantyl
and 4.82 (1H, brs, OCH), 6.80 7.44 (11H, m, Ar NH), 10.86
(1H, brs, NHind).
2Ad oc- -Me-D-Tr p -D-cis-P r o(4-P h -p Cl)-OH (14e): accord-
ing to general method E, 14e was obtained from 14d ; mp 165
168 °C; 1H NMR (DMSO
TFA) δ 1.20 2.31 (17H, m,
adamantyl
m, 2Hâ
CH3), 2.34 2.80 (2H, m, 2H3), 3.0 3.60 (4H,
2H5), 4.07 4.40 (2H, m, H4
H2), 4.73 and 4.89
NH), 10.92 (1H, s,
(1H, brs, OCH), 6.88 7.50 (10H, m, Ar
NHind).
2Ad oc- -Me-D-Tr p -D-cis-Hyp (Bzl)-OH (15e) a n d 2Ad oc-
-Me-D-Tr p -D-cis-Hyp (Bzl-m Cl)-OH (16e): according to gen-
eral method E, 15e was obtained from 15d and 16e from 16d .
15e mp 229 231 °C; 1H NMR (DMSO
TFA) δ 1.05 2.35
(19H, m, adamantyl
3.36 3.58 (2H, m, Hâ
CH3 2H3), 3.0 and 3.10 (1H, d, Hâ),
H5), 3.71 (1H, d, H5), 3.86 and 4.13
(1H, s, H4), 4.32 (3H, dd, H2 OCH2), 4.62 and 4.81 (1H, brs,
OCH), 6.78 7.40 (11H, m, Ar NH), 10.83 (1H, brs, NHind).
16e mp 154 157 °C; 1H NMR (DMSO
TFA) δ 1.05 2.35
(19H, m, adamantyl CH3 2H3), 3.06 and 3.14 (1H, d, Hâ),
3.40 3.80 (3H, m, Hâ 2H5), 3.94 and 4.12 (1H, brs, H4), 4.40
(3H, m, H2 OCH2), 4.69 and 4.89 (1H, brs, OCH), 6.83 7.46
(10H, m, Ar
NH), 10.90 (1H, s, NHind).
Bin d in g Assa ys. [3H]pCCK8 (specific activity 60 Ci/mmol)
was purchased from Amersham. Incubations (final volume 1
mL) were carried out at 25 °C in 50 mM Tris-HCl buffer (pH
7.4), 5 mM MgCl2, and 0.2 mg/mL bacitracin for 60 min in the
presence of brain membranes (0.6 mg of protein/tube) or in 10
mM Pipes-HCl buffer (pH 6.5), 30 mM MgCl2, 0.2 mg/mL
bacitracin, and 0.2 mg/mL soybean trypsin inhibitor for 120
min in the presence of pancreatic membranes (0.2 mg of
protein/tube).
Str u ctu r a l An a lyses by NMR. The N-cis and N-trans
isomers of the proline compounds were established by measur-
ing the relative intensities of at least three different signals.
Assignment of the peaks to each rotamer was established by
NOESY experiments (200 ms of mixing time) using a Bru¨ker
AMX600 spectrometer operating at 600 MHz for proton. The
rotational barrier (∆Gq) around the proline bond was measured
by evaluation of the line widths of three signals at various
temperatures.
[3H]pCCK8 was incubated at a concentration of 0.2 nM with
brain membranes and 0.1 nM with pancreatic membranes, in
the presence of varying concentrations of the competitor.
Nonspecific binding was determined in the presence of 1 µM
CCK8. Incubation was terminated by rapid filtration through
Whatman GF/B glass-fiber filters precoated with buffer con-
taining 0.1% bovine serum albumin. The filters were rinsed
with 2 × 5 mL of ice-cold buffer and dried, and the radioactiv-
ity was counted. Ki values were calculated using the Cheng
Prusoff equation. Moreover, when the Hill slope values of the
curves was significantly less than unity, the data were
reanalyzed with a two-site model. The displacement curves
Ack n ow led gm en t. The authors are grateful to C.
Dupuis for expert manuscript drafting and to N. Guil-
laume for technical assistance. We acknowledge Dr.
Beaumont for stylistic revisions.
better fitted (p
model were considered significant.
0.05) by a two-site model than a one-site
Refer en ces
Cell Gr ow th , Tr a n sfection , a n d Tr a n sfor m a n t Cell
Selection . Chinese hamster ovary (CHO) cells were grown
in HAM-F12 medium containing 10% fetal calf serum, 50 µg/
mL gentamycin, and 1 mM sodium pyruvate, in 5% CO2 at 37
°C. One day before transfection, cells were plated at a density
of 3 × 105 cells/9-cm diameter tissue culture dish. Cells were
transfected with 15 µg of the pcDNA3/RKB vector using the
calcium phosphate method;32 2 days after the transfection, cells
were grown in the presence of 0.4 mg/mL G 418. After 3
weeks, growing clones of cells resistant to G 418 were observed.
A pure cell line was obtained by cloning by the limit dilution
method. CHO cells transfected with the human CCK-B
receptor have already been described.29
P r ep a r a tion of CHO Mem br a n es a n d Liga n d Bin d in g
Assa ys. Cells were plated at a density of 1 × 106 cells/15-cm
diameter tissue culture dish in the presence of 0.4 mg/mL G
418. At confluency, cells were rinsed with cold phosphate-
buffered saline (PBS), scraped from the tissue culture dish,
and resuspended in PBS. The cells were centrifuged at 4 °C
for 5 min at 2000 rpm. The pellet was homogenized at 4 °C
in 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl2
and centrifuged at 4 °C for 35 min at 100000g. The resulting
pellet was rehomogenized in a large excess of ice-cold buffer
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