Structure-based design of new constrained cyclic agonists of the cholecystokinin CCK-B receptor

Article abstract of DOI:10.1021/jm9603072

New constrained cyclic pseudopeptide cholecystokinin-B (CCK-B) agonists have been designed on the basis of conformational characteristics of the potent and selective CCK-B agonist Boc-Trp-(NMe)Nle-Asp-Phe-NH₂ (K(i) = 0.8 nM, selectivity ratio C

Full text of DOI:10.1021/jm9603072

J . Med. Chem. 1997, 40, 647-658
647
Str u ctu r e-Ba sed Design of New Con str a in ed Cyclic Agon ists of th e
Ch olecystok in in CCK-B Recep tor
Armand G. S. Blommaert,† He´le`ne Dhoˆtel, Bertrand Ducos, Christiane Durieux, Nathalie Goudreau,
Andre´ Bado,‡ Christiane Garbay, and Bernard P. Roques*
De´partement de Pharmacochimie Mole´culaire et Structurale, U266 INSERM-URA D1500 CNRS, Universite´ Rene´ Descartes,
UFR des Sciences Pharmaceutiques et Biologiques 4, Avenue de l’Observatoire, 75270 Paris Cedex 06, France, and Unite´ de
Recherche de Gastro-Ente´rologie, U10 INSERM-Hoˆpital Bichat, 170, Boulevard Ney, 75877 Paris Cedex 18, France
Received April 25, 1996X
New constrained cyclic pseudopeptide cholecystokinin-B (CCK-B) agonists have been designed
on the basis of conformational characteristics of the potent and selective CCK-B agonist Boc-
Trp-(NMe)Nle-Asp-Phe-NH2 (Ki ) 0.8 nM, selectivity ratio CCK-A/CCK-B > 6000) (Goudreau
et al. Biopolymers, 1994, 34, 155-169). These compounds are among the first successful
examples of macrocyclic constrained CCK4 analogs endowed with agonist properties and as
such may be of value for the development of nonpeptide CCK-B agonists. The affinities and
selectivities of these compounds for CCK-B and CCK-A receptors have been determined in
vitro by measuring the displacement of [3H]pCCK8 binding to guinea pig cortex and pancreas
membranes, respectively. The most potent compound, 8b, N-(cycloamido)-R-Me(R)Trp-[(2S)-
2-amino-9-((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH2, has a Ki value of 15 ( 1 nM for guinea
pig cortex membranes with a good CCK-B selectivity ratio (CCK-A/CCK-B ) 147). Furthermore,
8b behaved as a potent and full agonist in a functional assay which measures the stimulation
of inositol phosphate accumulation in CHO cells transfected with the rat CCK-B receptor (EC50
) 7 nM). The in vivo affinity of 8b for mouse brain CCK-B receptors was determined following
intracerebroventricular injection (ID50 ∼ 29 nmol/kg). 8b was also shown to cross the blood-
brain barrier (0.16%), after intravenous administration in mice. 8b also increased gastric acid
secretion measured in anesthetized rats after intravenous injection. Therefore, 8b appears to
be an interesting pharmacological tool and is currently under investigation as a lead for further
development of nonpeptide CCK-B agonists.
In tr od u ction
to hypervigilance effects in rodents and monkeys, sug-
gesting that CCK-B agonists could have interesting
clinical applications.21 Although, several potent and
selective CCK-B antagonists (peptide, pseudopeptide,
and nonpeptide) have been designed, no potent nonpep-
tide agonists are yet available for the CCK-B receptor,
although benzodiazepine derivatives have recently been
reported to behave as CCK-A agonists with affinities
in the micromolar range.22 However, the selectivity of
these compounds versus the CCK-B receptor is poor,
and their agonist or antagonist profile for this receptor
has not been analyzed. It remains therefore an intrigu-
ing and challenging problem to design nonpeptide
compounds able to satisfy the structural requirements
for activation of the CCK-B receptor. An accurate model
of a CCK-B agonist receptor-bound conformation would
be interesting for the rational development of such
compounds. However, the exact three-dimensional
structure of the G-protein-coupled CCK-B receptor is
unknown, and structural information is limited to the
recently determined amino acid sequence23,24 and the
results of site-directed mutagenesis experiments.25-31
The C-terminal octapeptide of cholecystokinin (CCK8
or CCK26-33, H-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-
Phe-NH2) has well-established biological activities in the
periphery and the central nervous system which are
mediated through CCK-A1,2 and CCK-B/gastrin recep-
tors.3,4 In the central nervous system CCK8 is reported
to be involved in motivation and anxiety,5,6 analgesia,7,8
memory processes,9 and neuropsychiatric disorders,10,11
most of these responses being related to CCK-B receptor
or receptor subsite activation.12,13 Accordingly, the
CCK-B receptor constitutes an interesting therapeutic
target. Thus in humans, CCK-B antagonists have been
shown to block panic attacks triggered by CCK4 (Trp-
Met-Asp-Phe-NH2),14 to induce anxiolytic effects,15,16
and to potentiate antinociceptive responses induced by
exogenous opiates such as morphine17 or by endogenous
enkephalins18 in various animal models. Moreover,
CCK-B antagonists exhibit antidepressant-like effects
in rodents.19 The latter results show the existence of a
physiological antagonism between the opioid and CCK
systems modulated by δ opioid and CCK-B receptors.8
In addition, systemic administration of microgram doses
of the potent and selective CCK-B agonist BC 264,20 led
In the absence of this structural information, CCK4
which constitutes the smallest CCK peptide fragment
endowed with a good selectivity for brain over peripheral
receptors, was used, following a deletion approach, to
design the CCK-B peptoid antagonist PD-134,308.32
* To whom correspondence should be sent at: De´partement de
Pharmacochimie Mole´culaire et Structurale U266 INSERM-URA
D1500 CNRS, UFR des Sciences Pharmaceutiques et Biologiques,
Faculte´ de Pharmacie 4, Avenue de l’Observatoire, 75270 Paris Cedex
06, France. Tel. (33)-1-43.25.50.45. Fax (33)-1-43.26.69.18.
† Present address: Ruprecht-Karls-Universita¨t Heidelberg, Phar-
mazeutisch-Chemisches Institut, Im Neuenheimer Feld 364, D-69120
Heidelberg, Germany
Several modifications were made to CCK4 which
increased its CCK-B selectivity, such as the N-terminal
33
protection of the tetrapeptide in Boc-CCK4 or suc-
CCK4 and modifications of the different amino acids
‡ U1O INSERM-Hoˆpital Bichat.
X Abstract published in Advance ACS Abstracts, February 1, 1997.
such as the replacement of Met by Nle and NMeNle.34
S0022-2623(96)00307-X CCC: $14.00 © 1997 American Chemical Society

648 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Blommaert et al.
F igu r e 1. Stereoview of the superposition (heavy atoms only) of the preferential conformation of Boc-Trp-(NMe)Nle-Asp-PheNH2
(normal) and its derived macrocyclic analog 8b (bold).
Finally, restrained analogs of CCK4 containing a dike-
topiperazine nucleus were prepared to provide rigid
templates for the eventual design of peptidomimet-
compounds, bearing bulky N-protected groups, resulted
in affinity increases (by a factor 5 and 35) for the CCK-B
receptor.41
1
ics.35,36 Recent H-NMR and molecular dynamics stud-
A 13-membered heterocyclic ring system appeared to
be suitable for mimicking the conformational and
structural features of the tetrapeptide Boc-Trp-(NMe)-
Nle-Asp-Phe-NH237 (Figure 1). The initial cyclic analog
8 (RB 360) was synthesized by replacing the (NMe)Nle
residue with the non-natural and racemic amino acid
(2R,S)-2-aminononane-1,9-dicarboxylate, H2NCH[(CH2)7-
COOH]COOH, which allowed ring closure by formation
of an amide bond between the carboxylate of its side
chain and the amino group of RMeD-Trp. A further
reduction of the flexibility of the macrocyclic ring
system, in order to probe the structural tolerance of the
receptor binding site, was achieved by introducing an
additional amide bond (compound 16 or RB 370) or a
disulfide bridge (24 or RB 380) into the 13-membered
ring (Schemes 2 and 3), and by changing the size of the
ring (Table 1, compounds 43 and 45).
ies performed in our laboratory indicated that the potent
and CCK-B selective CCK4 agonist, Boc-Trp-(NMe)Nle-
Asp-Phe-NH2 (Ki ) 0.8 nM, selectivity ratio CCK-A/
CCK-B > 6000) adopts an S-shaped conformation
(Figure 1) with a relatively well defined orientation of
the side chains.37 On the basis of these results, we have
already reported the synthesis of novel cyclic CCK-B
agonists involving a diketopiperazine ring in place of
the two N-terminal residues.38
As shown in Figure 1, the side chain of Nle, together
with the N-terminus of tryptophan appeared as good
candidates for another possible cyclization. Thus, cyclic
compounds were designed by molecular modeling in
order to mimic the proposed biologically active confor-
mation of Boc-Trp-(NMe)Nle-Asp-Phe-NH2. These com-
pounds behave as potent and selective CCK-B agonists
able to cross the blood-brain barrier and may be used
for the further design of nonpeptide CCK-B agonists.
Their synthesis and binding properties are reported in
this study with some pharmacological responses ob-
served with the most potent compounds.
Syn th esis. The cyclic tetrapeptides, listed in Table
1, were synthesized by standard liquid phase peptide
synthesis, following the synthetic pathways summarized
in Schemes 1-4 and Table 2.
Physical and chemical data for all the final com-
pounds and intermediates are also given in Table 2. The
non-natural amino acid 2, methyl[(2R,S)-2-amino-9-
(tert-butyloxycarbonyl)nonanoate, Xaa(OtBu)OMe, was
obtained as a racemate in high yield by simple alkyla-
tion of the benzylidene derivative of glycine methyl ester
with tert-butyl 8-bromooctanoic acid ester as the alky-
lating agent and subsequent acidic hydrolysis of the
imine, according to a procedure described by Stork
et al.42
Resu lts
Molecu la r Design . The aromatic and acidic side
chains of CCK4 are known to play a key role in the
interaction with the CCK-B receptor.32,39,40 In the
proposed biologically “S-shaped” active conformation of
Boc-Trp-(NMe)Nle-Asp-Phe-NH2 (Figure 1), the ali-
phatic side chain of Nle is oriented such that it might
be extended and anchored to the N-terminus of tryp-
tophan by omitting the bulky Boc substituent.
This proposed macrocyclic peptide was built by mo-
lecular modeling, initially preserving S stereochemistry
for the four amino acid residues. The modeling sug-
gested that inversion of the Trp stereochemistry from
S to R was required to preserve the preferred orienta-
tion for the indole residue. This conformation appeared
further stabilized by incorporation of the RMeTrp
residue. The same cyclic peptide with a RMe(L)Trp, in
place of the D isomer, was shown by molecular modeling
to give a much less satisfactory orientation of the Trp
side chain. This is not totally surprising, since changing
LTrp for RMe(L)Trp and RMe(D)Trp, in CCK4-derived
Condensation of 2 (Scheme 1) with Boc-RMe(R)Trp,
which was prepared as described by Horwell et al.,43 led
to intermediate 3 which was subsequently deprotected
by treatment with TFA to give intermediate 4. In-
tramolecular cyclization of 4 was then achieved by
means of amidification with BOP, NaHCO3 using a
dilute (<10-2 M) solution44 of the linear precursor in
DMF. The 13-membered cyclic dipeptide 5 which
1
structure was confirmed by H-NMR spectroscopy, and
mass spectrometry was hence prepared in a 29% yield.
The expected acid derivative 6 was obtained by hydroly-
sis of 5 with an aqueous solution of NaOH, followed by
coupling to the C-terminal dipeptide Asp(OBzl)PheNH2

Agonists of the Cholecystokinin CCK-B Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 649
Ta ble 1. Affinities and Potencies of CCK Ligands in Inhibiting [3H]pCCK8 Specific Binding to Guinea Pig Cortex Membranes,
Pancreatic Membranes, or CHO Cells Transfected with the Rat CCK-B Receptor and in the Stimulation of Inositol Phosphate
Accumulation in CHO Cells
to give 7. Finally, after deprotection of the Asp residue
by catalytic hydrogenolysis, the expected compound 8
was obtained as a mixture of two diastereoisomers
which were separated by semipreparative HPLC to give
the optically pure isomers 8a and 8b (Scheme 1).
The assignment of their configuration has been
proposed for both isomers by using a previously reported
method45 based on the differences in chemical shifts of
side chain protons for dipeptides containing one aro-
matic moiety adjacent to an aliphatic one. For confor-
mational reasons, a closer proximity between the side
chains exists for R,S and S,R isomers in these dipep-
tides, leading the â-CH2 protons of the aliphatic amino
acid with these configurations to be more shielded by
the aromatic moiety than the corresponding protons of
S,S or R,R isomers, in which the side chains are further
apart.45 In the case of 8a and 8b, a simple molecular
model shows that, due to cyclization-induced distorsion,
a closer proximity occurs between the side chains for
R,R isomers of RMeTrp and Xaa, than for R,S isomers.
As the â-CH2 protons of the non-natural amino acid
(Xaa) are more deshielded in 8b than in 8a (respectively
1.50 and 1.35 ppm), we assumed that 8b corresponds
to the R,S,S,S isomer whereas 8a corresponds with the
R,R,S,S isomer.
group of this dipeptide to give the intermediate 11, from
which protecting groups were simultaneously removed
by catalytic hydrogenolysis. The intramolecular cy-
clization was then performed by using BOP in highly
diluted conditions (<10-2 M) to yield 38% of cyclic
dipeptide 13. Saponification of 13 gave the correspond-
ing acid 14 which was coupled to the C-terminal
dipeptide Asp(OBzl)PheNH2 to give 15. An unusually
high rate of epimerization (∼20%) was observed at the
chiral center of the Lys residue following the coupling
step. This can only be explained by the formation of
an oxazolone intermediate,46 presumably favored by
steric and/or electronic effects of the methyl group on
the R-carbon atom of Trp. Chromatographic purification
to homogeneity, however, permitted the pure R,S,S,S
diastereoisomer of 15 to be obtained, which was subse-
quently converted into the final product 16 by catalytic
hydrogenolysis (Scheme 2).
The cyclic peptide 24 was obtained by a stepwise
synthesis as outlined in Scheme 3 using the protecting
groups 4-methylbenzyl (4-MeBzl) and cyclohexyl (cHex)
for the side chains of residues Cys and Asp, respec-
tively. Standard coupling and deprotection procedures
(see the Experimental Section) permitted the linear
tetrapeptide 22 to be obtained, which was further
condensed with S-(carbomethoxysulfenyl)-5-thiopen-
tanoic acid on its N-terminal free amino group using
N,N′-dicyclohexylcarbodiimide (DCC), providing 23 with
a yield of 69%.
The synthesis of the cyclic tetrapeptide 16, outlined
in Scheme 2, also consisted of the condensation of two
dipeptide fragments using the optically pure S isomer
of Lys(Z)OMe instead of the non-natural amino acid 2
used in the synthesis of 8. Condensation with Boc-RMe-
(R)Trp gave 9, which was deprotected by treatment with
TFA to give the dipeptide 10. A monobenzylmalonic
residue was introduced on the N-terminal free amino
S-(carbomethoxysulfenyl)-5-thiopentanoic acid had
been obtained by the action of carbomethoxysulfenyl
chloride (Scm-Cl)47 on tert-butyl 5-thiopentanoic acid
ester and subsequent removal of the tert-butyl ester by

650 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Ta ble 2. Physical and Chemical Data for All Compoundsa
Blommaert et al.
Sch em e 1. Preparation of Compound 8a
scheme;
yield
(%)
TLCc
Rf
compd
procedureb
mp (°C)
oil
oil
175-179
>250
177-187
196-198
177-189
MS (M+)
1
2
3
4
5
6
7
8
I; A
I; B
I; C
I; D
I; E
I; F
I; C
I; G
95
60
64
100
29
100
62
0.90(B)
0.58(D)
0.69(C)
0.21(C)
0.53(C)
0.38(E)
0.52(C)
414
77
8a
8b
9
0.68(E)
0.71(E)
0.71(C)
0.38(C)
0.67(C)
0.33(E)
0.50(C)
0.60(D)
0.81(C)
0.48(E)
0.49(B)
0.46(C)
0.38(B)
0.39(C)
0.59(C)
0.54(C)
0.66(C)
0.62(E)
0.67(D)
0.25(B)
0.30(B)
0.60(C)
0.60(C)
0.60(C)
0.41(C)
0.50(C)
0.55(C)
0.59(C)
0.55(C)
0.50(C)
0.10(C)
0.15(C)
0.30(C)
0.56(C)
194-201
202-211
169-174
145-153
177-181
193-199
167-173
201-209
660
660
II; C
45
100
93
100
38
100
36
96
95
94
62
94
59
85
69
27
55
50
31
57
66
68
100
100
100
95
98
46
100
98
92
29
30
37
81
40
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25d
26
27e
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
II; D
II; H
II; G
II; E
II; F
II; C
II; G
III; H
III; D
III; H
III; D
III; C
III; D
III; I
III; J
IV; K
IV; K
IV; K
IV; C
IV; C
IV; C
IV; G
IV; G
IV; G
IV; C
IV; C
IV; C
IV; D
IV; D
IV; D
IV; C
IV; C
IV; C
IV; J
192-207
195-204
177-178
174-183
182-187
167-179
164-170
175-193
202-213
675
696
165
165
159
167-175
170-179
198
192
187-196
64
138
173-186
152
742
728
756
660
646
674
a
0.71(E)
0.71(E)
0.76(E)
202-211
204-211
203-215
All amino acids have an (S)-configuration unless indicated
otherwise. An asterisk (*) means (R,S)-mixture.
IV; J
IV; J
a
1H NMR data and elemental analysis for the final compounds
system, a stereoselective stepwise synthesis (Scheme 4)
was finally performed using the S isomer of the pro-
tected non-natural amino acids 25-27 (ZNH-CH[(CH2)n-
COOtBu]COOH) with n ) 6-8, which were obtained
by an asymmetric alkylation of methyl N-(diphenyl-
methylene)glycinate using the chiral inductor of Op-
polzer.50 This chiral glycine equivalent was prepared
as previously described51 and easily alkylated with tert-
butyl bromoalkanoic acid esters followed by acidic
hydrolysis of the imine with overall yields of around
50%. Subsequent amino protection with a benzyloxy-
carbonyl group and cleavage of the sultam auxiliary
gave the non-natural amino acids 25-27. Stepwise
elongation of the peptide chain by incorporation of
compounds 25-27 and R-Me(R)Trp using classical
coupling and deprotection procedures, followed by cy-
clization and a final deprotection, gave the optical pure
final compounds 43-45 (Scheme 4). Compound 43 was
b
are included in the Experimental Section. Refers to the scheme
in which the synthesis of the compound is outlined; procedures
are detailed in the Experimental Section. c Solvent systems are
indicated in parentheses and explained in the Experimental
Section. [R]20 ) -10.0° (c 1.0, MeOH). e [R]20 ) -7.2° (c 1.0,
d
D
D
MeOH).
the action of TFA (see the Experimental Section). The
Scm group, as described by Hiskey et al.,47 offers the
advantage of acting both as a protecting and as an
activating group, yielding a disulfide when treated with
thiols, with release of the volatile carbonyl sulfide and
methanol. Final deprotection on the cysteine side chain
of 23 using hydrogen fluoride (HF)48 allowed the forma-
tion of a disulfide bridge and consequent intramolecular
cyclization, with simultaneous cleavage of the cyclohexyl
ester49 leading to the final optically pure compound 24
in a 27% yield (Scheme 3). The formation of a signifi-
cant amount (25%) of dimerized product was observed,
as shown by HPLC and mass spectrometry, which is
probably due to the concentrated conditions ((10-1 M)
required for the handling of HF.
1
characterized by H-NMR spectroscopy and mass spec-
trometry and proved to be identical to 8b, supporting
our previously discussed assignment of the two sepa-
rated isomers.
In order to confirm the exact stereochemistry of the
isomers 8a and 8b, and to optimize the size of the ring
Biologica l Eva lu a tion . The apparent affinities of
the synthesized compounds for CCK-A and CCK-B

Agonists of the Cholecystokinin CCK-B Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 651
Sch em e 3. Preparation of Compound 24a
Sch em e 2. Preparation of Compound 16a
a
a
All amino acids have an (S)-configuration unless indicated
All amino acids have an (S)-configuration unless indicated
otherwise. Scm means carbomethoxysulfenyl (SCOOMe).
otherwise.
binding sites were determined by measuring the dis-
placement of [3H]pCCK8 from guinea pig pancreatic and
brain cortex membranes, respectively, as described
previously.20 These data are summarized in Table 1 and
show that most of the compounds designed have a good
CCK-B affinity and selectivity. A comparison of CCK-B
receptor affinities between 8b (Ki ) 15 ( 1 nM), 16 (Ki
) 40 ( 4 nM), and 24 (Ki ) 51 ( 7 nM), which contain
a similar 13-membered cycle, shows that the best
affinity is obtained with the alkyl chain. Thus, incor-
poration of an amide (16) or disulfide bond (24) in the
cycle does not improve the CCK-B affinity, and in the
case of 24, the selectivity has been even decreased (ratio
CCK-A/CCK-B ) 5). This could be due to steric con-
straints imposed on the macrocycle. A comparison of
8b (Ki ) 15 ( 1 nM) and compounds 44 (Ki ) 62 ( 6
nM) and 45 (Ki ) 15 ( 1 nM) suggests that a 13- or
14-membered ring size is optimal for binding to the
CCK-B receptor. In agreement with the molecular
modeling studies, 8b (Figure 1) with a R,S,S,S, config-
uration has a better affinity for the CCK-B receptor than
the R,R,S,S, isomer (8a ).
The apparent in vivo affinity of 8b in mouse brain
was determined by measuring the displacement of [3H]-
pBC 264 as previously described.52 Inhibition of the
specific binding of 10 pmol of [3H]pBC 264 by increasing
concentrations of 8b was studied 15 min after icv
coinjection of both compounds. The apparent ID50 value
of 8b, calculated from the competition curve (not shown),
was 19 µg/kg (29 ( 6.5 nmol/kg). Moreover a significant
inhibition (32 ( 5% and 87 ( 8%) of in vivo [3H]pBC
264 specific binding was observed after iv injection of 5
and 20 mg/kg of 8b, respectively, showing that this
compound is able to enter the mouse brain and to
displace the selective CCK-B agonist [3H]pBC 264 from
its binding sites. The ability of 8b to cross the blood-
brain barrier was estimated to be around 0.16%. This
was calculated, as previously described in detail,52 by
establishing the ratio of the doses, administered either
centrally (7.8 µg/kg) or peripherally (5 mg/kg), which
produced the same percentage of inhibition (32%).
The pharmacological profile of the most potent com-
pounds was investigated by measuring the formation
of inositol phosphates (IP) in CHO cells, stably trans-
fected with the rat brain CCK-B receptor (Table 1,
Figure 2). All the compounds tested behaved as ago-
nists and were able to induce the IP accumulation at
doses inferior to those expected when their CCK-B
affinities are taken into account. Moreover, they were
devoid of antagonist properties at concentrations up to
10-5 M (not shown).
In order to confirm their pharmacological agonist
profile on the CCK-B receptor in vivo, 8b and 24 were
tested for their ability to stimulate the CCK-B/gastrin
receptor by measuring gastric acid output in the anes-
thetized perfused rat stomach. As shown in Figure 3,
iv injected 8b and 24 stimulated gastric acid output in
a dose-dependent manner. On the basis of the ED50
values estimated from the dose-response curves, 8b
(ED50 ) 7.6 ( 1.6 nmol/kg‚h) was almost as potent as
CCK8 (ED50 ) 4 ( 2 nmol/kg‚h), whereas 24 (ED50
)
52 ( 2.3 nmol/kg‚h) was 10-fold less potent than CCK8.

652 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Blommaert et al.
Sch em e 4. Diastereoselective Synthesis of 8b and
Analogsa
F igu r e 2. Stimulation of inositol phosphate accumulation in
CHO cells transfected with the rat CCK-B receptor. Cells were
treated with either CCK8 (b) or compound 8b (0), as described
in the Experimental Section. Results are the mean ( SEM of
three separate measurements of inositol phosphate production,
each in triplicate. EC50 values are given in Table 1.
a
(a) nBuLi, THF, -78 °C; (b) Br(CH2)nCOOtBu, THF/HMPA,
-55 °C to rt; (c) citric acid, H2O/THF; (d) C6H5CH2O2CCl, Et3N,
THF; (e) LiOH, H2O/THF. All amino acids have an (S)-configura-
tion unless indicated otherwise. An asterisk (*) shows n being 7,
6, and 8, respectively.
F igu r e 3. Ability of CCK8 (b), 8b (O) and 24 (9) to increase
the gastric acid output. ED50 values are respectively 4 ( 2,
7.6 ( 1.6, and 52 ( 2.3 nmol/kg‚h for CCK8, 8b, and 24.
residue.54 It is noteworthy that all these CCK-B ago-
nists have a common C-terminal dipeptide: Asp-Phe-
NH2.
In this pharmacological model, the observed effects of
8b and 24 were in agreement with their affinities meas-
ured on CHO cells transfected with the CCK-B receptor
(see Table 1). The less than expected ED50 of CCK8
might reflect its enzymatic degradation or its capacity
to activate CCK-A receptor, thus inducing the release
of somatostatin, which in turn could inhibit gastric acid
secretion.53
Compounds 8b, 16, and 24, which have good affinities
for central CCK receptors, are an interesting series of
cyclic peptidomimetic agonists. To date only a few
CCK4-derived cyclic compounds have been reported,36,38,55
most of them behaving as antagonists. This is the case
for peptoid analogs, such as PD-134,308,32 whose cy-
clization resulted in derivatives with reduced affini-
ties.56 In our compounds, the spatial constraints im-
posed by the formation of a 13-membered ring also
reduced the CCK-B receptor affinity as compared to Boc-
Trp-(NMe)Nle-Asp-Phe-NH2 and even to the nonmethy-
lated Boc-Trp-Nle-Asp-Phe-NH2.37 Nevertheless, the
agonist profile of these molecules is maintained, and 8b
was found to have a similar affinity in vitro as CCK4
(Ki ) 20 nM).57
Discu ssion
The goal of this study was to stabilize the bioactive
conformation of CCK-B agonists in order to aid the
design of non peptide ligands. As shown in Table 1 and
Figures 1-3, cyclic CCK-B agonists have been obtained
using a molecular modeling approach based on the
proposed biologically active conformation of Boc-Trp-
37
(NMe)Nle-Asp-Phe-NH2 (Figure 1). In agreement
with our results, the same type of folded structures have
been reported for several potent agonists derived from
A slight reduction in affinity was observed for 16 and
24, when compared with 8b, suggesting that only a
certain degree of constraint is tolerated by the CCK-B
receptor agonist site. Interestingly, binding to the
CCK4 and containing a [trans-3-propyl-L-proline],35
a
diketopiperazine skeleton,36 or a [(alkylthio)proline31]

Agonists of the Cholecystokinin CCK-B Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 653
CCK-B versus CCK-A receptors was influenced by
changes in the nature of the ring system. A cyclic
moiety with a disulfide bridge in 24 increases CCK-A
receptor recognition (Table 1). This is not surprising
since the nature of amino acid 31 was previously shown
to be important for recognition of central versus periph-
eral receptors. Thus, replacement of the Met31 residue
in Boc-CCK4 with a lysine bearing a phenylurea at the
amino group place resulted in a series of potent and
selective CCK-A agonists exemplified by A-71623.58
Potent CCK-B receptor affinity was restored, but a loss
in selectivity was obtained upon simple substitution of
the lysine amino group.59 On the other hand, introduc-
tion of a Phe31 residue in CCK8 analogs was found to
favor recognition of the CCK-B receptor site.60
definition of the structural and conformational require-
ments for CCK-B receptor recognition and signal trans-
duction.
Exp er im en ta l Section
Com p u ta tion a l P r oced u r es. All calculations were per-
formed on a Silicon Graphics Workstation using the Insight/
Discover Software package (Biosym Technologies Inc.). The
calculations on Boc-Trp-(NMe)Nle-Asp-PheNH2 were per-
formed as previously described.37 Conformational analyses
were performed through high-temperature molecular dynam-
ics. The initial structure of 8 (R,S,S,S isomer) was minimized
using steepest descents and conjugate gradients algorithms
to be subjected afterward to 100 ps of dynamics at 600 K. An
integration time step of 1 fs was used. Molecular geometries
were stored at 1 ps intervals, rendering a set of 100 structures.
All stored structures were then retrieved and cooled down to
300 K by contact with a heat bath using a temperature
coupling constant of 1 ps. The dynamic stimulation continued
for an additional 20 ps under these conditions, and the
instantaneous structures at the end of the annealing phase
were energy minimized using a combination of steepest
descents/conjugate gradients algorithms. The 100 resulting
energy-minimized structures were then clustered according to
conformational similarities (heavy atom superposition) using
the insight/analysis module. Finally the representative con-
former of each family was then compared with the preferential
As listed in Table 1, some of the compounds show
slight differences in binding affinities using either the
CCK-B receptor from guinea pig brain or CHO cells
stably transfected with the rat CCK-B receptor, in
accordance with variations observed in various species.61
In vivo binding experiments on mice, performed using
[3H]pBC 264, gave an ID50 value of (29 ( 6.5 nmol/kg)
for 8b, while in the same conditions an ID50 value of
8.5 ( 0.4 nmol was obtained for CCK8.52 Due to its low
solubility, CCK4 could only be used at a maximal dose
of 10 nmol where it inhibited 40% of the in vivo [3H]-
pBC264 binding. Since CCK8 has an in vitro affinity
higher than that of 8b and CCK4 for the CCK-B receptor
(Table 1), these results suggest that in vivo 8b is
enzymatically much more stable than CCK8 and even
CCK4.
37
conformation of Boc-Trp-(NMe)Nle-Asp-PheNH2 by using a
template-forcing procedure (Insight II User Guide, Version
2.1.0. Copyright 1992, Biosym Technologies Inc., San Diego,
CA). The atoms taken into account for the template-forcing
procedure were matched pairs of atoms which were identical
for both molecules.
Syn t h esis. Commercial chemicals were used without
further purification. When appropriate, solvents were purified
and dried by standard methods before use. All compounds in
Table 2 were dried in vacuo over KOH and silica gel. Flash
column chromatography was performed using Merck silica gel
(230-400 mesh). Merck plates precoated with F254 silica gel
were used for thin layer chromatography with the following
solvent systems (by volume): A, CH2Cl2/MeOH (50/1); B, CH2-
Cl2/MeOH (19/1); C, CH2Cl2/MeOH (9/1); D, AcOEt/CH2Cl2/
MeOH/H2O/AcOH (110/70/30/6/3); E, CH2Cl2/MeOH/H2O/
AcOH (70/30/6/3). Plates were developed with UV, iodine
vapor, ninhydrin, or Ehrlich’s reagent. Melting points were
performed on an Electrothermal melting point apparatus and
are uncorrected. Optical rotations were measured on a Perkin-
Elmer polarimeter (model 241). Mass spectra were recorded
on a quadrupole NERMAG R10-10C apparatus. 1H-NMR
spectra were recorded on a Bruker AC 270-MHz or AM 400-
MHz instrument in DMSO-d6. Chemical shifts were measured
in ppm with HMDS as internal standard. The purity (>98%)
of all final compounds was checked by HPLC (Shimadzu
apparatus) on a 250 × 4.6-mm Kromasil C8-5 µm column with
a mixture of CH3CN and H2O/TFA0.05% as eluent (flow rate 1.2
mL/min, UV detection 214 nm). Elemental analyses, per-
formed by “Service Re´gional de Microanalyses” (Paris, France),
were within (0.4% of the theoretical values unless noted
otherwise.
P r oced u r e A: ter t-Bu tyl 8-Br om oocta n oa te (1). A solu-
tion of 8-bromooctanoic acid (112 mol) was treated with
2-methylpropene (784 mmol) in 20 mL of Et2O and 1.12 mL
of sulfuric acid using the method of McCloskey et al.66 to give
29.8 g of tert-butyl 8-bromooctanoate with a 95% yield: Rf )
0.90 (B); 1H-NMR (DMSO) ∂ 1.15-1.48 (17H, m, (CH2)4 + tBu),
1.70 (2H, m, CH2), 2.10 (2H, t, CH2), 3.45 (2H, t, CH2).
P r oced u r e B: 1-Meth yl-(2R,S)-2-a m in o-9-(ter t)-bu tyl-
oxyca r bon yl)n on a n oa te (2). tert-Butyl 8-bromooctanoate
(16.0 g, 57.3 mmol) was used to alkylate 10.15 g of the
benzylidene derivative of glycine ethyl ester (57.3 mmol) using
lithium diisopropylamine (LDA) and hexamethylphosphora-
mide (HMPA) at -78 °C in dry tetrahydrofuran (THF) as
described by Stork et al.42 After the mixture was stirred
overnight at room temperature, the pH was adjusted to 7 with
1 N HCl, the solvent was removed in vacuo, and 200 mL of
The capacity of 8b to cross the blood-brain barrier,
after iv injection, was evaluated in mice and found to
be 0.16%. This is better than the percentage (0.02%)
found for pBC 264, a highly potent CCK-B agonist (Ki
) 0.17 nM in mouse brain).52 The higher capacity of
8b to cross the blood-brain barrier is illustrated by the
similar dose (20 mg/kg, iv) of 8b and pBC 26452 able to
inhibit 87% of the in vivo binding of [3H]pBC 264.
Interestingly, under the same conditions, 20 mg/kg of
CCK4 was unable to modify the binding of [3H]pBC
264.52 Since following iv, ip, and sc administration
CCK4 provokes anxiogenic responses62,63 and is even
able to induce panic attacks when iv administered to
human subjects at doses as low as 10-50 µg,64 these
results suggest that CCK4 might interact with regions
which are not fully shielded by the blood-brain bar-
rier,65 such as the brainstem.
Finally, in two functional assays, our macrocyclic
compounds behaved as full agonists. Their in vivo
potencies for gastric acid induced secretion by stimula-
tion of the rat CCK-B/gastrin receptor correlate well
with their in vitro binding affinities on CHO cells
expressing the rat CCK-B receptor. In the case of the
stimulation of IP accumulation in CHO cells, these
compounds have better efficiencies than would be
expected from their binding affinities to the same cells.
In conclusion, we have shown that, in the case of
CCK-B receptor, it is possible, by rational design of
relatively rigid molecules, to stabilize the bioactive
conformation of CCK4 while conserving agonist proper-
ties. In addition, the new bioactive macrocyclic analogs
of CCK4 exemplified by 8b proved to be relatively easy
to synthesize. Further studies of 8b are being carried
out in the laboratory, aimed at achieving a better

654 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Blommaert et al.
Et2O was added. The organic layer was washed with H2O and
brine, dried with Na2SO4, filtered, and evaporated to yield 21
g of orange oil, which was redissolved in 30 mL of Et2O, and
100 mL of an aqueous 10% citric acid solution was added. This
mixture was stirred for 48 h at room temperature (rt). The
pH of the aqueous layer was adjusted to 12 with NaHCO3,
and it was extracted with EtOAc (3 × 30 mL). The combined
organic layers were washed with H2O and brine, dried with
Na2SO4, filtered, and evaporated to yield 2 (9.9 g, 60%) as a
pale yellow oil: 1H-NMR (DMSO+TFA) ∂ 1.15-1.50 (19H, m,
5 × CH2 + tBu), 1.75 (2H, m, CH2), 2.14 (2H, t, CH2), 3.75
(3H, s, CH3), 4.00 (1H, m, R-CH), 8.30 (3H, s, NH3+).
P r oced u r e C: Boc-rMe(R)Tr p -NHCH[(CH2)7COOtBu ]-
COOCH3 (3). Boc-RMe(R)Trp (1.66 g, 5.2 mmol), which was
prepared as previously described,43 was dissolved in 3 mL of
dimethylformamide (DMF), and at 0 °C were subsequently
added diisopropylethylamine (DIEA, 5.2 mmol) and (benzot-
riazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP, 5.7 mmol). After 30 min of stirring at 0 °C,
the amine 2 was added (1.50 g, 5.2 mmol), plus one additional
equivalent (5.2 mmol) of DIEA, if the amine was used as a
salt. Stirring was continued overnight at rt, the solvent
removed in vacuo, and 50 mL of EtOAc added to the residue.
The organic phase was washed with an aqueous 10% citric
acid solution, H2O, NaHCO3 solution (10%), H2O, and brine,
dried with Na2SO4, filtered, and evaporated. The residual solid
was then further purified by flash column chromatography
(CH2Cl2/EtOAc elution (9/1)) to give 3 (1.97 g, 64%): 1H-NMR
(DMSO) ∂ 1.10-1.65 (33H, m, (CH2)6 + tBu + Boc + R-CH3),
2.10 (2H, t, CH2CO), 3.02-3.40 (2H, m, â-CH2 (Trp)), 3.54 (3H,
s, CH3), 4.08-4.25 (1H, m, R-CH), 6.40 (1H, s, BocNH), 6.85
(1H, t, indole H5), 6.96 (2H, m, indole H2 + H6), 7.25 (1H, d,
indole H7), 7.44 (1H, d, indole H4), 8.30 (1H, m, NH), 10.84
(1H, s, indole NH).
to provide quantitatively 6 (103 mg): 1H-NMR (DMSO) ∂ 1.10-
1.75 (15H, m, (CH2)6 + R-CH3), 1.90-2.15 (2H, m, CH2CO),
2.92, 3.72, 3.20, and 3.40 (2H, d, â-CH2 (Trp)), 4.14 and 4.20
(1H, t, R-CH), 6.85-7.04 (2H, m, indole H5 + H6), 6.95 and
7.74 (1H, s, indole H2), 7.10 and 7.95 (1H, d, NH), 7.28 (1H, d,
indole H7), 7.38 and 7.50 (1H, d, indole H4), 7.68 and 7.82 (1H,
s, NH (Trp)), 10.80 (1H, s, indole NH).
P r oced u r e G: [N-(Cycloa m id o)-rMe(R)Tr p -[(2R,S)-2-
a m in o-9-((cycloa m id o)ca r b on yl)n on a n oyl]]-Asp -P h e -
NH2 (8, RB 360). A solution of the benzyl ester (or carbamate)
7 (100 mg, 0.13 mmol) in 7 mL of MeOH was treated overnight
with 10% palladium on carbon (60 mg/mmol) at room temper-
ature under hydrogen atmosphere. After filtration through
Celite, the filtrate was concentrated to give 8 (68 mg, 77%) as
mixture of two diastereomeric isomers after purification by
flash column chromatography (EtOAc/CH2
Cl2/MeOH/H2O/
AcOH elution (220/70/30/6/3)). Anal. (C35H44N6O7‚0.3H2O) C,
H, N.
Sep a r a tion of th e Dia ster eoisom er s. The diastereoiso-
mers of 8 were separated by semipreparative HPLC (column
330 × 7-mm Kromasil C8; CH3CN/H2O-TFA0.05% ) 72:28
(isocratic); flow 2.5 mL/min) to give the optically pure 8a and
8b, the latter corresponding to the higher retention time, in
TLC.
8a : HPLC (CH3CN/H2OTFA-0.05% ) 35/65) tR ) 11.2 min; 1H-
NMR (DMSO) ∂ 1.06-1.70 (15H, m, (CH2)6 + R-CH3), 1.90-
2.05 (2H, m, CH2CO), 2.18-2.50 (2H, m, â-CH2 (Asp)), 2.74-
3.08 (2H, m, â-CH2 (Phe)); 2.88 and 3.67 (2H, d, â-CH2 (Trp)),
3.94 (1H, m, R-CH (Xaa)), 4.22 (1H, m, R-CH (Phe)), 4.36 (1H,
m, R-CH (Asp)), 6.87 (1H, t, indole H5), 6.91 (1H, s, indole H2),
6.96 (1H, t, indole H6), 7.04-7.22 (8H, m, ArH (Phe) + NH2 +
NH (Xaa)), 7.25 (1H, d, indole H7), 7.32 (1H, d, indole H4), 7.78
(1H, s, NH (Trp)), 8.05 (2H, d, NH (Asp + Phe)), 10.82 (1H, s,
indole NH). Anal. (C35H44N6O7‚0.3H2O) C, H, N.
8b: HPLC (CH3CN/H2OTFA-0.05% ) 35/65) tR ) 12.0 min; 1H-
NMR (DMSO) ∂ 1.06-1.70 (15H, m, (CH2)6 + R-CH3), 1.90-
2.15 (2H, m, CH2CO), 2.33-2.54 (2H, m, â-CH2 (Asp)), 2.72-
3.08 (2H, m, â-CH2 (Phe)), 3.14 and 3.30 (2H, d, â-CH2 (Trp)),
4.15 (1H, m, R-CH (Xaa)), 4.28 (1H, m, R-CH (Phe)), 4.59 (1H,
m, R-CH (Asp)), 6.87 (1H, t, indole H5), 6.96 (1H, t, indole H6),
7.02 (1H, s, indole H2), 7.04-7.22 (7H, m, ArH (Phe) + NH2),
7.25 (1H, d, indole H7), 7.42 (1H, d, indole H4), 7.60 (1H, d,
NH (Xaa)), 7.99 (1H, s, NH (Trp)), 8.18 (2H, d, NH (Asp +
Phe)), 11.02 (1H, s, indole NH). Anal. (C35H44N6O7‚0.5H2O)
C, H, N.
P r oced u r e H : (Ben zyl)m a lon yl-rMe(R )Tr p -Lys(Z)-
OMe (11). To a cold solution (0 °C) of the carboxylic acid,
monobenzylmalonate (352 mg, 1.81 mmol), which was pre-
pared as described by Strube67 in 25 mL of CH2Cl2, were
subsequently added DCC (2.0 mmol), Et3N (2.0 mmol), and
the amine 10 (1 g, 1.81 mmol), plus one additional equivalent
(1.81 mmol) of Et3N if the amine was used as a salt. The
mixture was stirred at 0 °C for 1 h and at rt overnight. After
filtration of the N,N′-dicyclohexylurea (DCU), the solvent was
removed in vacuo and 30 mL of EtOAc was added. The organic
layer was washed with an aqueous 10% citric acid solution,
H2O, saturated NaHCO3 solution, H2O, and brine, dried with
Na2SO4, filtered, and evaporated to yield 11 (1.12 g, 93%): 1H-
NMR (DMSO) ∂ 1.10-1.70 (9H, m, (CH2)3 + R-CH3), 2.88 (2H,
m, â-CH2 (Lys)), 3.18-3.30 (4H, m, â-CH2 (Trp) + COCH2CO),
3.54 (3H, s, OCH3), 4.08 (1H, m, R-CH), 4.92 (2H, s, CH2 (Bzl)),
5.08 (2H, s, CH2 (Bzl)), 6.86 (1H, t, indole H5), 6.96 (1H, t,
indole H6), 7.05 (1H, d, indole H2), 7.15 (1H, t, NH (Lys)), 7.20-
7.38 (11H, m, ArH (Bzl) + indole H7), 7.42 (1H, d, indole H4),
7.82 (1H, d, NH (Lys), 7.94 (1H, s, NH (Trp), 10.84 (1H, s,
indole NH).
P r oced u r e D: TF A‚Me(R )Tr p -NH CH [(CH 2)7COOH ]-
COOCH3 (4). The carbamate 3 (1.9g, 3.2 mmol), dissolved in
a mixture of CH2Cl2 (10 mL), trifluoroacetic acid (TFA, 10 mL),
and anisole (1 mL), was treated for 1 h at 0 °C and an
additional 2 h at rt. After evaporation and trituration with
dry Et2O, the residual solid was dried to provide quantitatively
4 (1.76 g): 1H-NMR (DMSO) ∂ 1.10-1.80 (15H, m, (CH2)6 +
R-CH3), 2.10 (2H, m, CH2CO), 3.15-3.30 (2H, m, â-CH2 (Trp)),
3.55 and 3.60 (3H, s, CH3), 4.14 and 4.40 (1H, m, R-CH), 6.95
(1H, t, indole H5), 7.04 (1H, t, indole H6), 7.12 and 7.20 (1H,
d, indole H2), 7.30 (1H, d, indole H7), 7.58 and 7.65 (1H, d,
indole H4), 7.94 and 8.00 (3H, s, NH3+), 8.58 and 8.68 (1H, d,
NH), 11.02 and 11.08 (1H, s, indole NH).
P r oced u r e E : N-(Cycloa m in o)-rMe(R )Tr p -[1-m et h yl-
(2R,S)-2-a m in o-9-((cycloa m id o)ca r bon yl)n on a n oa te] (5).
To a solution at 0 °C of the linear precursor 4 (1.5 g, 2.75 mmol)
in 550 mL of DMF were subsequently added NaHCO3 (8.25
mmol) and BOP (3.03 mmol). The mixture was stirred for 30
min at 0 °C and for 5 h at rt. After evaporation, 50 mL of
EtOAc was added to the residue. The organic phase was
washed with an aqueous 10% citric acid solution, H2O,
NaHCO3 solution (10%), H2O, and brine, dried with Na2SO4,
filtered, and evaporated. The residual solid was then further
purified by flash column chromatography (CH2Cl2/MeOH
elution (50/1)) and provided 5 (325 mg, 29%): 1H-NMR
(DMSO) ∂ 1.10-1.70 (15H, m, (CH2)6 + R-CH3), 1.88-2.10 (2H,
m, CH2CO), 2.90, 3.18, 3.24, and 3.68 (2H, d, â-CH2 (Trp)),
3.54 and 3.60 (3H, s, CH3), 4.14 and 4.22 (1H, t, R-CH), 6.82-
7.00 (2H, m, indole H5 + H6), 6.90 and 7.10 (1H, s, indole H2),
7.24 (1H, d, indole H7), 7.26 and 7.98 (1H, d, NH), 7.32 and
7.42 (1H, d, indole H4), 7.64 and 7.80 (1H, s, NH (Trp)), 10.78
and 10.82 (1H, s, indole NH).
P r oced u r e F : N-(Cycloa m id o)-rMe(R )Tr p -[(2R,S)-2-
a m in o-9-((cycloa m id o)ca r bon yl)n on a n oic a cid ] (6). To
a cold (0 °C) solution of the ester 5 (107 mg, 0.26 mmol) in 5
mL of MeOH was added 1 N NaOH (0.77 mL, 3 equiv). The
reaction mixture was stirred overnight at rt, the solvents were
evaporated, and H2O was added. The pH was adjusted to 3
with an aqueous 10% citric acid solution and the product
extracted with EtOAc (3 × 20 mL). The combined organic
layers were washed with an aqueous 10% citric acid solution,
H2O, and brine, dried with Na2SO4, filtered, and evaporated
[Cycloa m id o[m a lon yl-rMe(R)Tr p -Lys]]-Asp -P h e-NH2
(16, RB 370). 16 (42 mg) was obtained in 96% yield according
to procedure G from 50 mg of 15 (0.065 mmol) after purification
by flash column chromatography (EtOAc/CH2Cl2/MeOH/H2O/
AcOH elution (220/70/30/6/3)): 1H-NMR (DMSO) ∂ 1.10-1.65
(9H, m, (CH2)3 + R-CH3), 2.30-2.64 (2H, m, â-CH2 (Asp)),
2.75-3.56 (8H, m, â-CH2 (Trp + Phe) + COCH2CO + CH2
(Lys)), 4.00 (1H, t, R-CH (Lys)), 4.28 (1H, m, R-CH (Phe)), 4.54
(1H, m, R-CH (Asp)), 6.88 (1H, t, indole H5), 6.96 (1H, m, indole
H6), 7.04-7.28 (9H, m, ArH (Phe) + indole H2, H7 + amide

Agonists of the Cholecystokinin CCK-B Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 655
NH + NH (Lys)), 7.40 (1H, d, indole H4), 7.48 (1H, s, amide
NH), 8.04 (1H, d, NH (Lys)), 8.15 (2H, m, NH (Phe + Asp)),
8.36 (1H, s, NH (Trp)), 11.00 (1H, s, indole NH). Anal.
(C34H41N7O8) C, H, N.
15.2 mmol) was added at -78 °C. The mixture was stirred
for 1 h while being warmed to -55 °C. Afterward tert-butyl
8-bromooctanoate (6.7 g, 25.3 mmol) and HMPA (12.0 mL, 68
mmol) in 40 mL of THF were added, and stirring was
continued at rt for 16 h. The pH was adjusted to 6 with a
solution of acetic acid in THF (1 mL/4 mL). Et2O (200 mL)
was added, and the organic phase was washed successively
with saturated solutions of NH4Cl, NaHCO3, water, and brine,
dried over Na2SO4, and evaporated to dryness. The residue
was redissolved in a solution of aqueous 10% citric acid and
THF (70 mL/70 mL). After the mixture was stirred for 48 h
at rt, the solvents were evaporated, and a mixture of H2O and
Et2O was added. The aqueous layer was basified with
NaHCO3 (pH ) 8) and extracted with EtOAc. The organic
phase was then washed with H2O and brine, dried over Na2-
SO4, and evaporated to dryness. The residue was further
purified by chromatography using CH2Cl2/MeOH (100/2) elu-
tion to yield 2.88 g of a yellow oil (50%): ((-)-10,2-bornane)-
sultam (2S)-2-amino-9-(tert-butyloxycarbonyl)nonanoate: Rf )
0.50 (D); 1H-NMR (DMSO) ∂ 0.88 (3H, s, CH3), 0.98 (3H, s,
CH3), 1.10-1.90 (31H, m, CH2 + CH + tBu + NH2), 2.08 (2H,
t, CH2), 3.58-3.80 (3H, m, R-CH + CH2).
N-P r otection of th e Su lta m In ter m ed ia te. A solution
of 5.6 g of ((-)-10,2-bornane)sultam (2S)-2-amino-9-(tert-
butyloxycarbonyl)nonanoate (12.3 mmol) in 115 mL of THF
was treated at 0 °C with 1.62 g of benzyl chloroformate (13.5
mmol) and 2.36 g of Et3N (13.5 mmol). The mixture is stirred
at rt for 2 h. After filtration and evaporation to dryness, the
residue was redissolved in EtOAc and the organic phase
washed with an aqueous 10% citric acid solution, H2O, a
saturated solution of NaHCO3, H2O, and brine, dried over Na2-
SO4, and evaporated to dryness to give 8.21 g of a colorless oil
(99%): ((-)-10,2-bornane)sultam N-(benzyloxycarbonyl)-(2S)-
2-amino-9-(tert-butyloxycarbonyl)nonanoate: Rf ) 0.28 (hex-
ane/acetone ) 8/2); 1H-NMR (DMSO) ∂ 0.88 (3H, s, CH3), 0.98
(3H, s, CH3), 1.10-1.90 (29H, m, CH2 + CH + tBu), 2.08 (2H,
t, CH2), 3.54-3.80 (2H, m, CH2), 4.50 (1H, m, R-CH), 4.98 (2H,
s, CH2), 7.30 (5H, m, ArH), 7.54 (1H, d, NH).
Syn th esis of 25. A solution of 8.2 g of ((-)-10,2-bornane)-
sultam N-(benzyloxycarbonyl)-(2S)-2-amino-9-(tert-butyloxy-
carbonyl)nonanoate (13.9 mmol) in 40 mL of THF was treated
with 36 mL of 0.5 N LiOH (18.1 mmol) at rt for 4 h. After
evaporation to dryness, EtOAc was added and the organic
phase was washed with an aqueous 10% solution of citric acid,
H2O, and brine. Then the organic phase was dried over
sodium sulfate and evaporated to dryness. The resulting
residue was purified by chromatography using CH2Cl2/MeOH
(100/2) elution to give 25 (2.66 g, 55%) as a colorless oil:
N-(benzyloxycarbonyl)-(2S)-2-amino-9-(tert-butyloxycarbonyl)-
nonanoic acid: 1H-NMR (DMSO) ∂ 1.10-1.60 (21H, m, CH2 +
tBu), 2.08 (2H, t, CH2), 3.85 (1H, m, R-CH), 4.98 (2H, s, CH2),
7.28 (5H, m, ArH), 7.48 (1H, d, NH).
[N-(Cycloa m id o)-rMe(R)Tr p -[(2S)-2-a m in o-9-((cycloa -
m id o)ca r bon yl)n on a n oyl]]-Asp -P h e-NH2 (43). 40 (35 mg,
0.047 mmol) was treated at 0 °C with 15 mL of anhydrous HF
and 0.4 mL of anisole during 75 min following procedure J .
After removal of the HF by vacuum distillation, the residue
was precipitated and washed with Et2O. Purification by
semipreparative HPLC (column 330 × 7-mm Kromasil C8; CH3-
CN/H2O-TFA0.05% ) 72:28 (isocratic); flow 2.5 mL/min) gave
25 mg of the final product 43 (81%) showing the same physical
characteristics as does 8b.
P r oced u r e I: [S-(Ca r bom eth oxysu lfen yl)-5-th iop en -
t a n oyl]-rMe(R )Tr p -Cys(4-MeBzl)-Asp (OcH ex)-P h e-NH 2
(23). Syn th esis of HS(CH2)4COOtBu . A solution of 5.0 g
of Br(CH2)4COOtBu (21 mmol) in 10 mL of EtOH was treated
with 1.6 g of thiourea (21 mmol) following a procedure
described by Urquhart et al.68 The reaction mixture was
refluxed for 3 h, a solution of 1.25 g of NaOH in 5 mL of H2O
was then added, and stirring was continued for another 2 h
at reflux temperature. After evaporation of the EtOH, ether
(30 mL) was added. The organic phase was separated, washed
with brine, dried on Na2SO4, filtered, and evaporated to
dryness to give 2.91 g of pale yellow oil (73%): HS(CH2)4-
1
COOtBu; Rf ) 0.90 (C); H-NMR (DMSO) ∂ 1.30 (9H, s, tBu),
1.50 (4H, m, CH2), 2.10-2.45 (5H, m, CH2 + SH).
Syn th esis of CH3OOC-S-S-(CH2)4COOtBu . To a cold
(0 °C) solution of HS(CH2)4COOtBu (1.5 g, 7.9 mmol) in 50
mL of MeOH was added at once 1.0 g of carbomethoxysulfenyl
chloride (Scm-Cl, 7.9 mmol).47 The reaction mixture was
stirred for 3 h at rt, the solvent was then removed in vacuo,
and the residue was purified by flash column chromatography
(100% CH2Cl2 elution) to give 2.36 g of a colorless oil (95%):
CH3OOC-S-S-(CH2)4COOtBu; Rf ) 0.75 (A); 1H-NMR
(DMSO) ∂ 1.35 (9H, s, tBu), 1.52 (4H, m, CH2), 2.25 (2H, t,
CH2), 2.75 (2H, t, CH2), 3.80 (3H, s, CH3).
Syn th esis of CH3OOC-S-S-(CH2)4COOH. A 600 mg
sample of CH3OOC-S-S-(CH2)4COOtBu (1.9 mmol) was
stirred at 0 °C with a mixture of TFA/CH2Cl2 (1 mL/2 mL) for
2 h and at rt, and then solvents were evaporated to give in
quantative yield a colorless oil: CH3OOC-S-S-(CH2)4COOH;
Rf ) 0.38 (C); 1H-NMR (DMSO) ∂ 1.50 (4H, m, CH2), 2.15 (2H,
t, CH2), 2.75 (2H, t, CH2), 3.78 (3H, s, CH3).
Syn th esis of 23. To a cold solution (0 °C) of 22 (1.23 g,
1.39 mmol) in 5 mL of DMF were subsequently added DIEA
(0.27 mL, 1.53 mmol), DCC (316 mg, 1.53 mmol), and CH3-
OOC-S-S-(CH2)4COOH (344 mg, 1.53 mmol). The mixture
was stirred at 0 °C for 1 h, and after the mixture was warmed
to rt, stirring was continued overnight. After filtration of the
DCU, the solvent was removed in vacuo and 50 mL of EtOAc
were added. The organic layer was washed with an aqueous
10% citric acid solution, H2O, saturated NaHCO3 solution,
H2O, and brine, dried with Na2SO4, filtered, and evaporated.
The residual solid was further purified by flash column
chromatography (CH2Cl2/MeOH elution (100/3)) to yield 936
mg of 23 (69%) as a white solid: 1H-NMR (DMSO) ∂ 1.10-
1.75 (21H, m, cHex + (CH2)4 + R-CH3), 2.20 (3H, s, CH3), 2.55-
3.55 (8H, m, â-CH2), 3.60 (2H, s, CH2(Bzl)), 3.80 (3H, s, OCH3),
4.20-4.65 (5H, m, R-CH + OCH), 6.85-7.55 (17H, m, ArH +
NH2 + NH), 8.08 (1H, d, NH), 8.20 (2H, m, NH), 10.90 (1H, s,
indole NH).
P r oced u r e J : [Cyclo-S,S-[(5-th iop en ta n oyl)-rMe(R)-
Tr p -Cys]]-Asp -P h e-NH2 (24, RB 380). 23 (800 mg, 0.82
mmol) was treated at 0 °C with 15 mL of anhydrous hydrogen
fluoride (HF) in the presence of 0.8 mL of anisole during 75
min following the procedure of Sakakibara et al.48 After
removal of the HF by vacuum distillation, the residue was
precipitated and washed with Et2O. Purification by flash
chromatography (CH2Cl2/MeOH/AcOH elution (18/2/1)) gave
24 (1.55 mg, 27%) as a white solid: 1H-NMR (DMSO) ∂ 1.25
(3H, s, R-CH3), 1.54-1.75 (4H, m, CH2), 2.10-2.24 (2H, m,
CH2), 2.35-3.45 (10H, m, â-CH2 + CH2), 4.30 (2H, m, R-CH),
4.48 (1H, m, R-CH), 6.90 (1H, t, indole H5), 6.98 (1H, m, indole
H6), 7.03 (1H, s, indole H2), 7.05-7.23 (7H, m, ArH (Phe) +
NH2), 7.28 (1H, d, indole H7), 7.40 (1H, d, indole H4), 7.84 (1H,
s, NH), 7.92 (1H, d, NH), 8.05 (1H, s, NH (Trp)), 8.25 (1H, d,
NH), 10.88 (1H, s, indole NH). Anal. (C33H40N6O7S2) C, H;
N: calcd, 12.06; found, 12.67.
[N-(Cycloa m id o)-rMe(R)Tr p -[(2S)-2-a m in o-8-((cycloa -
m id o)ca r bon yl)octa n oyl]]-Asp -P h e-NH2 (44). Compound
44 was obtained in the same way as 43: 1H NMR (DMSO) ∂
1.06-1.65 (13H, m, (CH2)5 + R-CH3), 1.95-2.10 (2H, m, CH2-
CO), 2.60-2.80 (2H, m, â-CH2 (Asp)), 2.75-3.00 (2H, m, â-CH2
(Phe)), 3.20 and 3.41 (2H, d, â-CH2 (Trp)), 4.10 (1H, m, R-CH
(Xaa)), 4.35 (1H, m, R-CH (Phe)), 4.50 (1H, m, R-CH (Asp)),
6.87 (1H, t, indole H5), 6.96 (1H, t, indole H6), 7.02 (1H, s,
indole H2), 7.04-7.22 (7H, m, ArH (Phe) + NH2), 7.25 (1H, d,
indole H7), 7.42 (1H, d, indole H4), 7.55 (1H, d, NH (Phe)), 7.80
(1H, d, NH (Xaa)), 7.95 (1H, s, NH (Trp)), 8.25 (1H, d, NH
(Asp)), 10.80 (1H, s, indole NH). Anal. (C34H42N6O7) C, H, N.
[N-(Cycloa m id o)-rMe(R)Tr p -[(2S)-2-a m in o-10-((cycloa -
m id o)ca r bon yl)-d eca n oyl]]-Asp -P h e-NH2 (45). Compound
P r oced u r e K: N-(Ben zyloxyca r bon yl)-(2S)-2-a m in o-9-
(ter t-bu tyloxyca r bon yl)n on a n oic Acid (25). Syn th esis of
th e Su lta m In ter m ed ia te. According to J osien et al.,51 ((-
)-10,2-bornane)sultam N-(diphenylmethylene)glycinate (the
chiral inductor of Oppolzer50) (5.51 g, 12.6 mmol) was solubi-
lized in 50 mL of THF, and 9.47 mL of nBuLi (1.6 M/hexane,

656 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Blommaert et al.
45 was obtained in the same way as 43: 1H NMR (DMSO) ∂
1.10-1.65 (17H, m, (CH2)7 + R-CH3), 2.10 (2H, m, CH2CO),
2.40-3.40 (6H, m, â-CH2 (Asp + Phe + Trp)), 4.10-4.60 (3H,
m, R-CH (Asp + Phe + Xaa)), 6.90 (1H, t, indole H5), 6.95 (1H,
t, indole H6), 7.10-7.20 (8H, m, indole H2 + ArH (Phe) + NH2),
7.25 (1H, d, indole H7), 7.40 (1H, d, NH (Phe)), 7.60 (1H, d,
NH (Xaa)), 7.90 (1H, s, NH (Trp)), 8.20 (1H, d, NH), 10.85 (1H,
s, indole NH). Anal. (C36H46N6O7‚0.3H2O) C, H, N.
formate. Total [3H]inositol phosphates were then eluated 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 percent-
age of the maximal response induced by the compounds.
In Vivo Bin d in g Assa ys. The experiments with icv and
iv injections were performed as previously described.52 Briefly,
mice were killed by cervical dislocation 15 min after icv
injection of [3H]pBC 264 (10 pmol, 1 µCi) alone or with 8b,
and their brains were quickly removed. A delay of 15 min was
chosen, as previous studies showed that specific binding of [3H]-
pBC 264 is maximum at this time.52 The brain (minus
cerebellum) was homogenized in 10 mL of cold 50 mM Tris-
HCl buffer containing 0.02% bacitracin. Aliquots (0.15 mL)
of the homogenate were immediately filtered through What-
man GF/B glass filters and rinsed twice with 5 mL of cold
buffer. Free radioactivity was calculated as the difference
between total radioactivity and radioactivity retained on the
filters, which was considered as bound radioactivity. 8b was
prepared as a stock solution in ethanol/Chremophor EL/H2O
(1/1/8) and diluted in saline before icv coinjection with [3H]-
pBC 264 or iv administration at different concentrations 5 min
prior to [3H]pBC 264.
In Vitr o Bin d in g Assa ys. [3H]pCCK8 (specific activity 60
Ci/mmol) was purchased from Amersham. Brain cortex and
pancreas homogenates were prepared from guinea pig as
previously described.20 Protein concentration was estimated
using the Pierce bicinchoninic acid protein assay reagent with
bovin serum albumin as a standard. 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 of bacitracin for 60 min
in the presence of brain membranes (0.6 mg of protein per
tube) or in 10 mM Pipes-HCl buffer (pH 6.5), 30 mM MgCl2,
0.2 mg/mL of bacitracin, and 0.2 mg/mL of soybean trypsin
inhibitor for 120 min in the presence of pancreatic membranes
(0.2 mg of protein per tube). [3H]pCCK8 was incubated with
brain membranes at 0.2 nM and with pancreatic membranes
at 0.1 nM in the presence of varying concentrations of the
competitor. Nonspecific binding was determined in the pres-
ence of 1 µM CCK8. Incubation was terminated by rapid
filtration through Whatman GF/B glass-fiber filters precoated
with buffer containing 0.1% bovine serum albumin. The filters
were rinsed with 2 × 5 mL of ice-cold buffer and dried and
the radioactivity counted. Ki values were calculated using the
Cheng-Prusoff equation.
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 Na 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.70 Two 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.
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 of
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
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 containing 0.02% of bacitracin. The
membranes were aliquoted and frozen at -80 °C. The binding
assays were performed as with brain membranes with 40 µg
of protein/tube and 0.4 nM of [3H]pCCK8.
In ositol P h osp h a te Assa ys. CHO cells stably expressing
wild-type CCK-B rat receptor were assayed for agonists
stimulated membrane phosphoinositides hydrolysis essentially
as previously described.70 Cells were plated in 24-well micro-
titer plates. Before confluency cells were grown in the pres-
ence of 1 µCi/mL myo-[γ-3H]inositol for 16 h at 37 °C. Cells
were treated with 10 mM LiCl for 30 min at 37 °C, and various
concentrations of agonists 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 200 µL of ice-cold 67% methanol and 300
µL of 0.125% Triton. The cells were scraped, the wells were
then rinsed with 200 µL of ice-cold 67% methanol, and the
suspension was subjected to chloroform extraction. Then 0.5
mL of the aqueous phase was added to 4.5 mL of water. The
column of 0.5 mL anion exchange resin (Dowex AG1 × 8 200-
400 mesh from BioRad) was washed with 1 mL of distilled
water followed by 5 mL of 5 mM sodium borate/60 mM sodium
Ga str ic Acid Secr etion . Experiments were performed
using male Sprague-Dawley rats weighting 300 ( 25 g, fasted
for 18 h with free access to water.71 The technical aspects of
the operation have been described by Ghosh and Schild72 and
modified by Lai.73 Briefly, the rats were anesthetized by
intramuscular injection of urethane (0.6-0.7 mL of 25%
solution per 100 g of body weight). A polyethylene catheter
introduced in the oesophagus and passed to the cardia was
connected to a peristaltic pump (Minipuls 2, Gilson Medical
Electronics) to deliver a solution of 0.9% NaCl at a constant
rate of 1.0 mL/min. This perfusate was collected through
another catheter placed through the pylorus and secured with
a ligature.
The tests started after stabilization of the gastric perfusion,
which was achieved usually between 30 and 60 min after
completion of the surgical preparation. During this period,
an intravenous infusion of physiological saline at 2.4 mL/min
(perfusor Braun, Roucaire) was made into the dorsal vein of
the penis. Stimulation by the tested compounds was obtained
by continuous infusion through the same route diluted in
saline as described.37 Gastric secretion was collected every
15 min, and the H+ levels were evaluated by titrating the
entire sample with 0.01 N NaOH to pH 7. The results are
expressed as a percentage (mean of the two experimental
points preceding administration of the antagonist) of gastric
acid output induced by each compound. Statistical evaluations
were performed using Student’s t-test.
Ack n ow led gm en t. The authors would like to thank
C. Dupuis for expert manuscript drafting and Dr. A.
Beaumont for stylistic revision. The work was sup-
ported by Funds from the “Institut National de la Sante´
et de la Recherche Me´dicale”, the “Centre National de
la Recherche Scientifique”, and the “University Rene´
Descartes”.
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