β-Turned dipeptoids as potent and selective CCK1 receptor antagonists

Article abstract of DOI:10.1021/jm000959x

To improve our knowledge of the bioactive conformation of CCK₁ antagonists, we previously described that replacement of the α-MeTrp residue of dipeptoids with the (2S,5S,11bR)-2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate (IBTM) s

Full text of DOI:10.1021/jm000959x

3770
J . Med. Chem. 2000, 43, 3770-3777
â-Tu r n ed Dip ep toid s a s P oten t a n d Selective CCK1 Recep tor An ta gon ists
Mercedes Mart´ın-Mart´ınez,§ Natalia De la Figuera,§ Miriam Latorre,‡ Rosario Herranz,§
M. Teresa Garc´ıa-Lo´pez,§ Edurne Cenarruzabeitia,‡ J oaqu´ın Del R´ıo,‡ and Rosario Gonza´lez-Mun˜iz*,§
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, 28006 Madrid, Spain, and Departamento de Farmacolog´ıa,
Universidad de Navarra, Irunlarrea 1, 31008 Pamplona, Spain
Received March 30, 2000
To improve our knowledge of the bioactive conformation of CCK1 antagonists, we previously
described that replacement of the R-MeTrp residue of dipeptoids with the (2S,5S,11bR)-2-amino-
3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate (IBTM) skeleton, a probed type II′ â-turn
mimetic, led to restricted analogues (2S,5S,11bR,1′S)- and (2S,5S,11bR,1′R)-2-(benzyloxycar-
bonyl)amino-5-[1′-benzyl-2′-(carboxy)ethyl]carbamoyl-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino-
[8,7-b]indole, 1a ,b, showing high binding affinity and selectivity for CCK1 receptors. In this
report, we describe the synthesis and binding profile of new analogues of compounds 1 designed
to explore the importance of the C-terminal residue and of the type of â-turn on the receptor
binding affinity and selectivity. Structure-affinity relationship studies show that a C-terminal
free carboxylic acid and an S configuration of the Phe and âHph residues are favorable for
CCK1 receptor recognition. Moreover, selectivity for this receptor subtype is critically affected
by the â-turn type. Thus, while compounds 15a and 16a , containing the (2S,5S,11bR)- and
(2R,5R,11bS)-IBTM frameworks, respectively, are both endowed with nanomolar affinity for
CCK1 receptors, restricted dipeptoid derivative 15a , incorporating the type II′ IBTM mimetic,
shows approximately 6-fold higher CCK1 selectivity than analogue 16a , with the type II mimetic.
From these results, we propose that the presence of a â-turn-like conformation within the
peptide backbone of dipeptoids could contribute to their bioactive conformation at the CCK1
receptor subtype. Concerning functional activity, compounds 15a and 16a behave as CCK1
receptor antagonists.
In tr od u ction
discovery of dipeptoids, different constrained analogues
have been prepared in an attempt to establish three-
dimensional structure-activity relationships and to
develop pharmacological agents with improved proper-
ties. Restrictions by NR-CR and NR-NR′ cyclization,23,24
macrocyclization,25,26 and amide bond rigidification27
resulted in dipeptoid analogues with reduced affinity
for CCK2 receptors with respect to the corresponding
acyclic parents. By contrast, conformational restriction
of the C-terminal residue through a Pro ring28 or by
incorporation of a tetrahydronaphthyl group29 have been
reported to maintain or to increase, respectively, the
affinity for CCK2 receptors. However, in both cases the
restricted derivatives showed higher affinity for CCK1
receptors and, therefore, a lower selectivity than their
parents. The enhancement of the affinity for the CCK1
receptor subtype was especially remarkable in con-
strained dipeptoid analogues incorporating dehydro-
and cyclopropylphenylalanine derivatives at the C-
terminus,30 for which conformational studies indicated
the presence of a â-turn within the peptide backbone,
although no preference in â-turn type was observed.31
In a similar way, substitution of the R-methyltryp-
tophan of dipeptoids by dehyrotryptophan led either to
a decrease in the CCK2 selectivity or to CCK1-selective
compounds.32
Cholecystokinin (CCK) is a peptide hormone and
neurotransmitter involved in modulating gastrointes-
tinal and behavioral activities by interacting with
specific receptors.1 Two G-protein-coupled 7TM receptor
subtypes have been characterized for CCK: CCK1
receptors that predominate in the periphery but are also
found in discrete regions of the brain, and CCK2
receptors located mainly in the CNS.2,3 Changes in the
levels of CCK found in tissues and sera of patients with
various disease states, including schizophrenia4 and
eating disorders,5 as well as the effects of CCK deriva-
tives in neuroprotection,6 models of Parkinson’s dis-
ease,7 cancer,8 anxiety,9 and pain,10 indicate the poten-
tial utility of CCK receptor ligands as therapeutic
agents. This potentiality has prompted an intensive
research in this area, and several potent and selective
nonpeptide CCK1 and CCK2 receptor agonists and
antagonists have been reported over the past decade.11-15
Among these compounds, dipeptoids were designed from
structure-activity relationship studies on the endog-
enous CCK2 receptor-selective agonist CCK-4 (H-Trp30-
Met31-Asp32-Phe33-NH2), which revealed the importance
of the aromatic side-chains of Trp and Phe residues as
key binding fragments.16-18 Both CCK1 and CCK2
receptor-selective antagonists were obtained within this
structural family of compounds.19-22 Following the
Taking into account that dehydro amino acids are able
to stabilize â-turn secondary structures, when incorpo-
rated into peptide sequences,33 we decided to investigate
whether a turn-like conformation is responsible for the
enhancement of CCK1 receptor affinity showed by many
* To whom correspondence should be addressed. Tel: 34-91-
5622900. Fax: 34-91-5644853. E-mail: iqmg313@iqm.csic.es.
§ Instituto de Qu´ımica Me´dica.
‡ Universidad de Navarra.
10.1021/jm000959x CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/09/2000

â-Turned Dipeptoids as CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3771
Sch em e 1a
of the restricted dipeptoids. We have recently reported
that replacement of the R-MeTrp residue of dipeptoids
with the (2S,5S,11bR)-2-amino-3-oxohexahydroindolizino-
[8,7-b]indole-5-carboxylate skeleton [(2S,5S,11bR)-IBTM]
led to restricted analogues 1a ,b, showing high binding
affinity and selectivity for CCK1 receptors.34 The (2S,5S,-
11bR)-IBTM skeleton contains the indole side chain of
the Trp residue present in dipeptoids, and as we
previously demonstrated, it is able to mimic a type II′
â-turn conformation.35,36 Therefore, it is plausible that
a turn-like conformation within the peptide backbone
of dipeptoids is favorable for CCK1 receptor recognition.
To optimize the structural requirements of these IBTM-
containing dipeptoid analogues, we have prepared new
derivatives incorporating L- and D-Phe residues instead
of the corresponding â-homophenylalanine (âHph). To
confirm whether the â II′ is the type of â-turn preferred
for the interaction of these restricted dipeptoids with
the CCK1 receptor, a series of compounds containing the
enantiomeric (2R,5R,11bS)-IBTM skeleton, able to sta-
bilize a type II â-turn, have also been prepared. This
paper describes the synthesis, conformational analysis
by 1H NMR, and CCK receptor binding affinities of this
new family of â-turned restricted dipeptoid analogues.
a
Reagents and conditions: (a) TFA (1 equiv), toluene, rt to
reflux; (b) H2/Pd-C, MeOH; (c) xylene, reflux; (d) 2 N NaOH,
MeOH; (e) TFA, CH2Cl2; (f) ZCl, CH2Cl2, propylene oxide.
carboxylic acid derivative 1035 with H-L- and H-D-Phe-
OMe, respectively, using BOP as the coupling agent. To
prepare compounds 13a ,b, containing the type II â-turn
mimetic (2R,5R,11bS)-IBTM skeleton, two alternate
procedures were undertaken. In the first one, the N-Boc-
protected carboxylic acid derivative 9 was coupled with
H-L-Phe-OMe to provide the conformationally con-
strained dipeptoid 12a , which was then transformed
into the corresponding Z-protected analogue 13a upon
elimination of the Boc group and treatment with benzyl
chloroformate. Compound 13b was prepared by direct
coupling of Z-protected derivative 10 with H-D-Phe-
OMe. Both procedures gave similar final results, and
in both cases, compounds 13a ,b were obtained in 48%
total yield from 7. Condensation of the carboxylic acid
10 with methyl (3S)- and (3R)-amino-4-phenylbutanoate
(H-L- and H-D-âHph-OMe) provided the âHph deriva-
tives 14a ,b, respectively. The C-terminal free carboxylic
acid derivatives 15a ,b, 16a ,b, and 17a ,b were prepared
by saponification with NaOH of the corresponding
methyl esters 11a ,b, 13a ,b, and 14a ,b. It is interesting
to note that while the saponification of the Phe deriva-
tives 11a ,b and 13a ,b proceeded quickly and in very
good yield, a 20-30% of epimerization at C-5 was
observed for the âHph analogues 17a ,b. This epimer-
ization, probably due to the longer reaction times
required for the completion of the reaction, led to lower
yields and difficulties in the purification of the final
compounds. Finally, C-terminal carboxamide deriva-
tives 18a ,b were prepared by ammonolysis of com-
pounds 11a ,b, respectively.
Ch em istr y
The preparation of the (2R,5R,11bS)-2-amino-3-oxo-
hexahydroindolizino[8,7-b]indole-5-carboxylate deriva-
tive 6 was performed by a synthetic route similar to that
described for the corresponding 2S,5S,11bR enanti-
omer,35 but some modifications were introduced in an
attempt to improve overall yield (Scheme 1). The main
differences with the previously described procedure
concern the synthetic steps affording tetrahydro-â-
carboline 4 and the intramolecular lactamization to the
tetracyclic derivative 6. Thus, condensation of the D-Trp
derivative 2 with the D-Asp-derived aldehyde 3 in the
presence of TFA was conducted at room temperature
to afford a diastereoisomeric mixture of the correspond-
ing trans- and cis-tetrahydro-â-carbolines 4 in a 1.5:1
ratio. This mixture was then refluxed in toluene to allow
the complete epimerization of the cis isomer to the
desired trans derivative 437 having the 1S,5R,1′R con-
figuration. Using this method, the yield of the reaction
was improved by 10% with respect to the procedure
involving direct refluxing of the reaction mixture of 2
and 3. The lactamization step did not need previous
esterification as it was done in the initial procedure,
proceeding directly, in very good yield from the car-
boxylic acid derivative 5 obtained by hydrogenation of
compound 4. Removal of the Boc protecting group from
6 followed by treatment with benzyl chloroformate
afforded the corresponding Z-protected derivative 7 in
52% total yield. Finally, saponification of the methyl
ester groups in 6 and 7 furnished the free carboxylic
acid derivatives 8 and 9, respectively.
Con for m a tion a l Stu d ies
The ability of the conformationally restricted dipep-
toid analogues to adopt a â-turn-like conformation in
solution was evaluated by 1H NMR. Independent of the
As indicated in Scheme 2, the Phe-containing deriva-
tives 11a ,b were prepared by condensation of the

3772 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Mart´ın-Martı´nez et al.
Ta ble 2. Inhibition of the [3H]pCCK-8-Specific Binding to Rat
Pancreas (CCK1) and Cerebral Cortex Membranes (CCK2) by
â-Turned Dipeptoids
Sch em e 2a
IC50 (nM)a
config at
2,5,11b,1′
compd
11a
11b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
1a
n
R2
CCK1 CCK2
S,S,R,S
S,S,R,R
R,R,S,S
R,R,S,R
R,R,S,S
R,R,S,R
S,S,R,S
S,S,R,R
R,R,S,S
R,R,S,R
R,R,S,S
R,R,S,R
S,S,R,S
S,S,R,R
S,S,R,S
S,S,R,R
0
0
0
0
1
1
0
0
0
0
1
1
0
0
1
1
OCH3 47.5 ( 11.2 >10000
OCH3 >1000
OCH3 450 ( 50
OCH3 >1000
OCH3 >1000
OCH3 >1000
>10000
5633 ( 338
>10000
>10000
4914 ( 57
>10000
>10000
OH
OH
OH
OH
OH
OH
NH2
NH2
OH
OH
4.7 ( 1.30
54.6 ( 4.8
1.73 ( 0.33 202.0 ( 99.0
>1000
3720 ( 458
97.8 ( 19.6 >10000
>1000
>1000
>1000
>10000
>10000
>10000
88.0 ( 26.2 >10000
7.4 ( 4.1
1b
2700 ( 170
devazepide
0.71 ( 0.05 696 ( 134
a
Values are the mean or mean( SEM of at least three
experiments, performed with seven concentrations of test com-
pounds in duplicate.
the Phe derivatives 11, 13, 15, and 16 were higher (in
absolute value) than those corresponding to this proton
for the âHph analogues 14 and 17, all of them are within
the range expected for a hydrogen-bonded â-turn.
However, the ∆δ/∆T values for the 1′-NH amide protons
of compounds 18a ,b were slightly higher than those
required for conformational stabilization through an
intramolecular hydrogen bond, suggesting that the
presence of a C-terminal carboxamide function disrupts
to some extent the â-turn-like conformation.
a
Reagents and conditions: (a) H-Xaa-OMe, BOP, Et3N, CH2Cl2;
(b) 2 N NaOH, MeOH; (c) NH3, MeOH; (d) TFA, CH2Cl2; (e) ZCl,
CH2Cl2, propylene oxide.
Biologica l Resu lts a n d Discu ssion
The affinity of all the new dipeptoid analogues at
CCK1 and CCK2 receptors was determined by measur-
ing the displacement of [3H]propionyl-CCK-8 binding to
rat pancreatic and cerebral cortex homogenates, respec-
tively, as previously described.39 These data are depicted
in Table 2. For comparative purposes, binding affinities
of model compounds 1a ,b, and the well-known CCK1
antagonist devazepide,40 were also included in Table 2.
The results show that compounds with a free car-
boxylic acid at the C-terminus have better affinity for
CCK1 receptors than the corresponding methyl ester
derivatives (15a vs 11a , 15b vs 11b, 16a vs 13a , 17a
vs 14a ). These results are in agreement with previous
findings regarding SAR of dipeptoids.20 A more surpris-
ing result is the lack of binding affinity of the C-terminal
carboxamide derivative 18a , for which it could be
expected to have binding values in the same order of
magnitude than those found for the methyl ester
analogue 11a .20 One possible explanation for this fact
comes from the conformational study in solution, above-
mentioned, that seems to indicate that compound 18a
is not able to stabilize a â-turn conformation.
Ta ble 1. Temperature Coefficients for Amide Protons
∆δ/∆T (10-3 ppm/K)a
∆δ/∆T (10-3 ppm/K)a
compd
2-NH
1′-NH
compd
2-NH
1′-NH
11a (13b)
11b (13a )
14a
14b
15a (16b)
-4.3
-4.3
-5.7
-4.8
-5.3
-2.9
-2.8
-1.9
-1.8
-2.8
15b (16a )
17a (1b)
17b (1a )
18a
-5.3
-5.0
-5.3
-5.3
-5.2
-2.7
-1.7
-1.8
-3.2
-4.0
18b
a
Determined by least-squares linear regresion analysis from
measurements over the temperature range 30-60 °C (seven
points), DMSO-d6.
solvent used, the spectra of all compounds exhibited no
coupling between the downfield H-6 proton and the
vicinal H-5 proton of the IBTM moiety (see Table 3,
Experimental Section). This agrees with an axial dis-
position of the 5-carboxylate group, as required for the
adoption of â-turn conformations in IBTM-containing
analogues.35,36
It has been described that small absolute values for
the temperature coefficient (∆δ/∆T e 3 × 10-3 ppm/K)
of amide protons indicate their protection from solvent
exchange either by involvement in an intramolecular
hydrogen bond or by inaccessibility to solvent, whereas
values higher than 4 × 10-3 ppm/K indicate exposure
to the solvent.38 As can be seen in Table 1, the ∆δ/∆T
value for the 2-NH amide proton, in all the cases, was
indicative of the accessibility of these proton to bulk
solvent. Although these values for the 1′-NH proton of
In the type II′ â-turned dipeptoids 1a ,b and 15a ,b,
the interchange of âHph with Phe was well-tolerated
at the CCK1 receptor, but the influence of the configu-
ration of these residues on the binding potency differed.
Thus, while (R)-âHph derivative 1b showed 1 order of
magnitude higher affinity than its S-configured ana-
logue 1a , in the Phe analogues, this increase was shown

â-Turned Dipeptoids as CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3773
Ta ble 3. Selected 1H NMR (300 MHz) Chemical Shifts and Coupling Constants of â-Turned Dipeptoids
δ (ppm)
compd
H-1
H-2
2-NH
H-5
5-CONH
H-6
H-11b
H-1′
H-2′
R1
R2
3.61
J 5,6 (Hz)
11a (13b)a
11a (13b)b
11b (13a )a
11b (13a )b
12a a
2.15
2.26
2.03, 2.28
2.34
2.53
2.33
2.19, 2.40
2.31
2.23, 2.36
2.31
2.25
2.10
2.34
2.34
2.45
2.29
2.45
2.23
2.10, 2.26
2.35
3.87
4.04
3.76
4.04
4.04
3.99
3.89
3.99
3.93
3.95
4.03
4.10
4.04
4.02
4.06
3.97
4.06
4.03
4.09
4.04
3.66
6.16
8.14
5.90
8.19
8.05
8.26
5.94
8.21
6.08
7.72
8.10
7.99
8.19
8.31
5.61
8.20
6.70
8.11
6.54
8.14
5.83
5.21
4.99
5.05
4.97
5.04
4.88
5.09
4.92
5.15
4.99
4.95
5.05
4.95
4.90
4.90
4.91
5.03
5.02
5.20
4.98
5.12
7.60
8.17
7.78
8.21
d
7.71
6.98
7.69
7.42
d
8.00
d
8.12
7.86
d
2.85, 3.60
2.74, 3.31
2.93, 3.68
2.78, 3.21
3.02, 3.61
2.75, 3.35
2.85, 3.68
2.78, 3.35
2.91, 3.70
2.70, 3.49
2.71, 3.34
2.84, 3.61
2.77, 3.20
2.72, 3.32
2.85, 3.65
2.72, 3.35
2.90, 3.65
2.70, 3.35
2.85, 3.66
2.78, 3.11
2.91, 3.53
4.55
4.60
5.35
5.25
5.39
5.09
5.09
4.85
4.70
4.61
4.57
5.46
5.29
5.15
5.27
4.83
4.92
4.41
3.86
5.36
4.89
4.82
4.41
4.71
4.41
4.65
4.19
4.39
4.24
4.56
4.61
4.37
4.60
4.33
4.19
4.42
4.23
4.48
4.41
4.74
4.36
4.34
3.06, 3.20
2.94, 3.07
2.93, 3.10
2.90, 3.06
2.83, 3.20
2.68
2.40
2.78
2.54
3.09
2.91, 3.10
3.14
2.88, 3.08
2.72
2.51
2.72
2.54
2.85, 3.07
3.21
2.78, 2.98
2.91
5.07
5.11
5.02
5.07
1.46
5.04
4.95
5.13
5.15
5.07
5.11
5.14
5.07
5.04
5.04
5.10
5.21
5.15
5.18
5.07
4.83
7.3, 0.0
6.9, 0.0
7.3, 0.0
7.3, 0.0
7.1, 0.0
7.3, 0.0
7.7, 0.0
7.1, 0.0
7.1, 0.0
7.4, 0.0
7.2, 0.0
7.5, 0.0
7.0, 0.0
7.2, 0.0
7.3, 0.0
7.3, 0.0
7.3, 0.0
7.0, 0.0
6.5, 0.0
7.2, 0.0
6.0, 0.0
3.54
3.54
3.51
3.53
3.26
3.14
3.48
3.58
14a b
14a c
14bb
14bc
15a (16b)a
15a (16b)b
15b (16a )a
15b (16a )b
17a b
17a c
17bb
7.66
d
7.85
7.56
7.98
7.99
17bc
18a b
d
18a c
6.14, 6.62
d
5.58, 5.81
18bb
18bc
1.72, 1.98
a
b
d
Registered in (CD3)2CO. Registered in DMSO-d6. c Registered in CDCl3. Within Ar protons.
by compound 15a , having the 1′S configuration, with
respect to the 1′R isomer 15b. The importance of the
1′S configuration of the Phe moiety in these type-II′
â-turned dipeptoids for efficient interaction with CCK1
receptors is also evidenced from comparing the binding
affinity of methyl ester 11a to that of 11b. It is worth
noting the high affinity and selectivity of compound 15a
for CCK1 receptors. Thus, this Phe derivative displayed
nanomolar CCK1 receptor affinity similar to that of the
best homologue 1b, but on the contrary to this homo-
logue, compound 15a was unable to bind to CCK2
receptors at 10-5 M concentrations. This result, sup-
porting previous findings in which homologation of the
phenylalanine carboxylic acid group led to a remarkable
increase in CCK2 affinity but not to an appreciable
change in CCK1 receptor binding,20 suggests that the
through-bond distance of the carboxylic acid group from
the phenethyl backbone is important for the interaction
of 1b with CCK2 receptors.
With regard to the type of â-turn, some interesting
conclusions may be drawn from the binding affinities
of dipeptoids 13, 14, 16, and 17, incorporating the type
II mimetic (2R,5R,11bS)-IBTM. In general, the best
interaction with CCK1 receptors was found for those
derivatives having an S-configured Phe or âHph resi-
due, but none of the analogues with the R configuration
were able to recognize this receptor subtype. While in
the â II′-turned dipeptoids the Phe derivative 15a and
its âHph analogue 1b bind to CCK1 receptor with
nanomolar affinity, in the type II â-turned series the
âHph derivative 17a was unable to reach the nanomolar
affinity showed by the corresponding Phe analogue 16a .
Moreover, compound 16a showed about a 20-fold de-
crease in CCK2/CCK1 selectivity when compared to the
type II′-constrained dipeptoid analogue 15a . In connec-
tion with this, many of the type II-restricted dipeptoids
(13a , 14b, and 16b) were able to bind to the CCK2
receptors in the micromolar range. This tendency to
bind to CCK2 receptors was found to be independent of
the ability of these derivatives to recognize the CCK1
receptors. These facts indicate that, while both the
(2S,5S,11bR)- and (2R,5R,11bS)-IBTM skeletons are
able to facilitate the correct orientation of the pharma-
cophoric side chains to interact with the CCK1 receptor,
the conformational constraint imposed by the type II′
â-turn mimetic is not tolerated at all by the CCK2
receptor binding site. This clear difference in confor-
mational requirements for the interaction with CCK1
and CCK2 receptors could be helpful to design con-
strained ligands with high selectivity for the CCK1
receptors.
The best compounds in this series, 15a and 16a , were
tested for their ability to inhibit the CCK-8-stimulated
amylase release from pancreatic acinar cells.41 In ac-
cordance with the binding results, these compounds
were able to antagonize the amylase release with IC50
values of 6.0 (2.6-13.7) and 1.2 (0.6-2.2) nM for 15a
and 16a , respectively. These compounds did not show
any intrinsic effect on the amylase release at a 1 µM
concentration; therefore, they behave as CCK1 antago-
nists. The antagonist potency of compounds 15a and
16a was comparable to that found for the model CCK1
antagonist devazepide in the same assay [IC50 ) 2.5
(2.1-2.7) nM)].
In conclusion, we have prepared compounds 15a and
16a , two highly constrained dipeptoid analogues con-
taining the (2S,5S,11bR)- and (2R,5R,11bS)-IBTM frame-
works, which are endowed with high binding affinity
for CCK1 receptors. As these frameworks are probed
type II′ and type II â-turn mimetics, respectively, we
propose that the presence of a â-turn-like conformation
within the backbone of dipeptoids could contribute to
their bioactive conformation at the CCK1 receptor
subtype. Moreover, we have shown that the type of
â-turn is critical for maintaining good selectivity for the
CCK1 receptor. Thus, restricted dipeptoid analogue 15a ,
containing the type II′ IBTM mimetic, is apparently
devoid of affinity for CCK2 receptors and is therefore a
new potent and selective CCK1 receptor antagonist.
Exp er im en ta l Section
Ch em istr y. All reagents were of commercial quality. Sol-
vents were dried and purified by standard methods. Amino
acid derivatives were obtained from Bachem AG. Analytical

3774 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Mart´ın-Martı´nez et al.
TLC was performed on aluminum sheets coated with a 0.2-
mm layer of silica gel 60 F254, Merck. Silica gel 60 (230-400
mesh), Merck, was used for flash chromatography. Melting
points were taken on a micro hot stage apparatus and are
uncorrected. 1H NMR spectra were recorded with a Varian XL-
300, operating at 300 MHz, using TMS as reference. Temper-
ature coefficients were obtained from least-squares fits to data
of 30, 35, 40, 45, 50, 55, and 60 °C in DMSO-d6. 13C NMR
spectra were recorded with a Varian Gemini-200 operating at
50 MHz. Elemental analyses were obtained on a CH-O-RAPID
apparatus. Optical rotations were determined with a Perkin-
Elmer 141 polarimeter. Analytical HPLC was performed on a
Waters Nova-pak C18 column (3.9 × 150 mm, 4 µm) with a
flow rate of 1 mL/min, and using a tuneable UV detector set
at 214 nm. Mixtures of CH3CN (solvent A) and 0.05% TFA in
H2O (solvent B) were used as mobile phase. Compounds 2 and
10 were prepared as described.35 H-L-âHph-OMe and H-D-
âHph-OMe were synthesized from Z-L- and Z-D-Phe-OH,
respectively, following a described procedure.42
(1S,3R,2′R)-1-[2′-(ter t-Bu t oxyca r b on yl)a m in o-2′-ca r -
boxyeth yl]-3-m eth oxyca r bon yl-1,2,3,4-ter ta h yd r o-â-ca r -
bolin e (5). Compound 4 (3 g, 5.02 mmol) was dissolved in
MeOH (100 mL) and hydrogenated at room temperature and
30 psi of pressure for 2h in the presence of 10% Pd-C (0.6 g).
After filtration of the catalyst the solvent was evaporated to
provide 2.05 g (98%) of the title compound as a foam. 1H NMR
(300 MHz, DMSO-d6): δ 11.17 (s, 1H, NHi), 7.49-7.02 (m, 5H,
In and 2′-NH), 4.80 (m, 1H, H-2′), 3.79 (s, 3H, CO2CH3), 3.60
(m, 2H, H-1 and H-3), 3.33 (dd, 1H, H-4, J ) 15.2, 5.1), 3.04
(dd, 1H, H-4, J ) 15.2, 9.5), 2.39 (m, 2H, H-1′), 1.40 [s, 9H,
CH3 (Boc)]. Anal. (C21H27N3O6) C, H, N.
(2R,5R,11b S)-2-(ter t-Bu t oxyca r b on yl)a m in o-5-m et h -
oxycar bon yl-3-oxo-2,3,5,6,11,11b-h exah ydr o-1H-in dolizin o-
[8,7-b]in d ole (6). A solution of tetrahydro-â-carboline 5 (1.5
g, 3.58 mmol) in xylene (50 mL) was refluxed for 2 h. After
evaporation of the solvent the resulting residue was purified
on a silica gel column using 30% of EtOAc in hexane as eluent,
to provide 1.34 g (93%) of the title compound as a white solid.
mp 118-120 °C (EtOAc/hexane). HPLC: tR ) 34.76 min
Boc-D-Asp (H)-OBzl (3). To a solution of Boc-D-Asp-OBzl
(12 g, 33.6 mmol) in dry THF (25 mL), at -15 °C, were added
NMM (3.7 mL, 33.6 mmol) and isobutyl chloroformate (4.35
mL, 33.6 mmol) and stirred 10 min at that temperature. The
salts were filtered and washed with THF (2 × 120 mL). To
the resulting organic solution, cooled to -10 °C, was added a
solution of NaBH4 (1.88 g, 50.4 mmol) in H2O (17 mL) and
stirring continued until evolution of H2 ceased. Then, H2O (600
mL) and EtOAc (800 mL) were added and the phases sepa-
rated. The organic layer was successively washed with 10%
citric acid, 10% NaHCO3 and brine, dried over Na2SO4 and
evaporated. The resulting residue was purified on a silica gel
column using 20% EtOAc in hexane, to give 8.63 g (83%) of
benzyl (2R)-(tert-butoxycarbonyl)amino-4-hydroxybutanoate.
This compound (8.63 g, 27.9 mmol) in CH2Cl2 (30 mL) was
slowly added to a solution previously prepared as follows:
Oxalyl chloride (2.64 mL, 30.7 mmol) in CH2Cl2 (15 mL) was
treated at -60 °C with DMSO (4.75 mL, 67 mmol) in CH2Cl2
(15 mL) and stirred 10 min at that temperature. The reaction
mixture was stirred at -60 °C for 30 min, treated with TEA
(19.8 mL, 143 mmol) and then allowed to warm to room
temperature. H2O (60 mL) was added and the phases were
separated. The organic layer was dried over Na2SO4 and
evaporated leaving a residue which was purified on a silica
gel column using 15% EtOAc in hexane as eluent. The title
1
(A:B ) 28:72). [R]D -73.0° (c 4 in CHCl3). H NMR (300 MHz,
DMSO-d6): δ 11.08 (s, 1H, NHi), 7.56 (d, 1H, 2-NH, J ) 8.4),
7.43-6.93 (m, 4H, In), 5.18 (m, 2H, H-5 and H-11b), 4.08 (m,
1H, H-2), 3.62 (s, 3H, CO2CH3), 3.21 (d, 1H, H-6, J ) 15.8),
3.00 (ddd, 1H, H-6, J ) 15.8, 7.1, 2.0), 2.34 (m, 2H, H-1), 1.40
[s, 9H, CH3 (Boc)]. 13C NMR (50 MHz, DMSO-d6): δ 171.80,
170.75, 155.17 (CO), 136.11, 133.03, 126.25, 121.33, 118.79,
117.94, 111.26, 103.99 (Ar), 78.27 [C (Boc)], 52.42 (OCH3),
50.91 (C-2), 49.92 (C-5), 49.60 (C-11b), 31.73 (C-1), 28.20 [CH3
(Boc)], 22.85 (C-6). Anal. (C21H25N3O5) C, H, N.
(2R,5R,11bS)-2-(Ben zyloxyca r bon yl)a m in o-5-m eth oxy-
ca r bon yl-3-oxo-2,3,5,6,11,11b-h exa h yd r o-1H-in d olizin o-
[8,7-b]in d ole (7). A solution of compound 6 (1.33 g, 3.32 mmol)
in CH2Cl2 (4 mL) was treated at room temperature with TFA
(2 mL, 26 mmol). After stirring at that temperature for 1h the
reaction mixture was evaporated to dryness. The resulting
residue was dissolved in CH2Cl2 (10 mL), cooled to 0 °C, and
treated successively with TEA (1.4 mL, 6.64 mmol) and benzyl
chloroformate (0.95 mL, 6.64 mmol). After stirring overnight
at room temperature the solvent was evaporated, the residue
was extracted with EtOAc and washed with H2O and brine.
The organic extract was separated, dried over Na2SO4 and
evaporated. The resulting residue was purified on a silica gel
column using 30% of EtOAc in hexane as eluent, to provide
0.63 g (52%) of the title compound as a white solid. Mp: 109-
111 °C (EtOAc). HPLC: tR ) 10.49 min (A:B ) 40:60). 1H NMR
(300 MHz, DMSO-d6): δ 11.11 (s, 1H, NHi), 8.05 (d, 1H, 2-NH,
J ) 8.4), 7.42-6.95 [m, 9H, In and C6H5 (Z)], 5.21 (m, 2H, H-5
and H-11b), 5.05 [s, 2H, CH2 (Z)], 4.16 (m, 1H, H-2), 3.61 (s,
3H, CO2CH3), 3.23 (d, 1H, H-6, J ) 15.6), 2.97 (ddd, 1H, H-6,
J ) 15.6, 7.1, 2.0), 2.38 (m, 2H, H-1). 13C NMR (50 MHz,
DMSO-d6): δ 171.38, 170.75, 156.20 (CO), 136.26-105.79 (14
C, Ar), 67.22 [CH2 (Z)], 52.42 (OCH3), 51.57 (C-2), 50.11 (C-5),
49.91 (C-11b), 31.80 (C-1), 23.05 (C-6). Anal. (C24H23N3O5) C,
H, N.
compound (6.8 g, 84%) was obtained as a syrup. HPLC: tR
)
1
10.53 min (A:B ) 47:35). H NMR (300 MHz, CDCl3): δ 9.71
(s, 1H, CHO), 7.35 (m, 5H, Ph), 5.42 (d, 1H, R-NH, J ) 8.1),
5.17 (m, 2H, OCH2), 4.70 (m, 1H, R-H), 3.06 (m, 2H, â-H), 1.42
[s, 9H, CH3 (Boc)]. Anal. (C16H21NO5) C, H, N.
(1S,3R,2′R)-2-Ben zyl-1-[2′-b en zyloxyca r b on yl-2′-(ter t-
bu toxyca r bon yl)a m in oeth yl]-3-m eth oxyca r bon yl-1,2,3,4-
tetr a h yd r o-â-ca r bolin e (4). To a solution of aldehyde 3 (3.42
g, 11.09 mmol) and Bzl-D-Trp-OMe (2; 3.42 g, 11.09 mmol) in
toluene (34 mL) was added TFA (0.85 mL, 11.09 mmol) at room
temperature. After stirring overnight at that temperature, the
mixture was refluxed for 1 h. The reaction mixture was allowed
to warm to room temperature, neutralized with 10% NaHCO3
and evaporated to dryness. The resulting residue was ex-
tracted with EtOAc, washed with H2O and the organic phase
was separated, dried over Na2SO4 and evaporated. The residue
was purified on a silica gel column, using 15% of EtOAc in
hexane as eluent, to give 4.54 g (68%) of the title compound
as a syrup. HPLC: tR ) 7.19 min (A:B ) 70:30). [R]D -23.0° (c
0.4 in CHCl3). 1H NMR (300 MHz, CDCl3): δ 9.35 (s, 1H, NHi),
7.58-7.08 [m, 14H, In and C6H5 (Bzl)], 5.24 (d, 1H, 2′-NH,
J ) 7.3), 5.10 [m, 1H, CH2 (Bzl)], 4.41 (m, 1H, H-2′), 4.10 (dd,
1H, H-3, J ) 10.1, 5.9), 3.82 (m, 5H, CO2CH3, 2-CH2, H-1),
3.44 (d, 1H, 2-CH2, J ) 13.5), 3.08 (m, 2H, H-4), 2.19 (m, 2H,
H-1′), 1.35 [s, 9H, CH3 (Boc)]. 13C NMR (50 MHz, CDCl3): δ
173.06, 171.71, 156.12 (CO), 138.89-106.85 (20C, Ar), 80.32
[C (Boc)], 67.37 [CH2 (Bzl)], 57.12 (OCH3), 52.93 (2-CH2), 52.75
(C-2′), 52.69 (C-3), 52.25 (C-1), 38.64 (C-1′), 28.17 [CH3 (Boc)],
20.19 (C-4). Anal. (C35H39N3O6) C, H, N.
(2R,5R,11bS)-2-(Ben zyloxyca r bon yl)a m in o-5-ca r boxy-
3-oxo-2,3,5,6,11,11b -h exa h yd r o-1H -in d olizin o[8,7-b]in -
d ole (8). A solution of compound 6 (0.6 g, 1.38 mmol) in MeOH
(15 mL) was treated with 2 N NaOH (1.05 mL, 2.1 mmol) and
the mixture was stirred at room temperature for 18 h. After
evaporation of the MeOH the remaining aqueous mixture was
diluted with H2O (10 mL), acidified with 1 N HCl to pH 3,
and extracted with EtOAc. The extract was dried (Na2SO4) and
evaporated. The residue was purified on a silica gel column
using 15% of MeOH in CH2Cl2 to provide 0.55 g (96%) of the
title compound as a white solid. Mp: 135-137 °C. HPLC:
1
tR ) 16.23 min (A:B ) 30:70). H NMR (300 MHz, DMSO-d6):
δ 11.08 (s, 1H, NHi), 8.03 (d, 1H, 2-NH, J ) 8.4), 7.41-6.92
[m, 9H, In and C6H5 (Z)], 5.25 (m, 1H, H-11b), 5.08 (d, 1H,
H-5, J ) 7.2), 5.04 [s, 2H, CH2 (Z)], 4.15 (m, 1H, H-2), 3.23 (d,
1H, H-6, J ) 15.8), 2.92 (ddd, 1H, H-6, J ) 15.8, 7.2, 1.6),
2.37 (m, 2H, H-1). 13C NMR (50 MHz, DMSO-d6): δ 172.12,
171.64, 156.02 (CO), 137.05-104.38 (14 C, Ar), 65.75 [CH2 (Z)],

â-Turned Dipeptoids as CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3775
51.60 (C-2), 50.16 (C-5), 49.89 (C-11b), 31.76 (C-1), 23.29 (C-
6). Anal. (C23H21N3O5) C, H, N.
at room temperature the solvent was evaporated, the residue
was extracted with EtOAc and washed with H2O and brine.
The organic extract was separated, dried over Na2SO4 and
evaporated. The resulting residue was purified on a silica gel
column using 60% of EtOAc in hexane as eluent, to provide
0.19 g (62%) of the title compound as a foam. HPLC: tR ) 21.82
min (A:B ) 40:60). Anal. (C33H32N4O6) C, H, N.
Gen er a l P r oced u r e for Rem ova l of th e C-Ter m in a l
P r otectin g Gr ou p . A solution of the corresponding 1′-
methoxycarbonyl derivative (0.17 mmol) in MeOH (3 mL) was
treated with 2 N NaOH (0.13 mL, 0.25 mmol) and the mixture
was stirred at room temperature for 5-18 h. After evaporation
of the MeOH the remaining aqueous mixture was diluted with
H2O (5 mL), acidified with 1 N HCl to pH 3, and extracted
with EtOAc. The extract was dried (Na2SO4) and evaporated.
The residue was purified on a silica gel column using a 15%
of MeOH in CH2Cl2.
(2R,5R,11bS)-2-(ter t-Bu toxycar bon yl)am in o-5-car boxy-
3-oxo-2,3,5,6,11,11b -h exa h yd r o-1H -in d olizin o[8,7-b]in -
d ole (9). This compound 0.62 g (93%) was obtained from 6
(0.67 g, 1.7 mmol) following the above-described procedure for
the preparation of compound 8. White solid. Mp: 141-144 °C
1
(EtOAc). HPLC: tR ) 10.39 min (A:B ) 30:70). H NMR (300
MHz, DMSO-d6): δ 10.97 (s, 1H, NHi), 7.40-6.93 (m, 5H, 2-NH
and In), 5.34 (m, 1H, H-11b), 4.80 (d, 1H, H-5, J ) 7.4), 4.03
(m, 1H, H-2), 3.30 (d, 1H, H-6, J ) 15.2), 2.82 (dd, 1H, H-6,
J ) 15.2, 7.4), 2.32 (m, 2H, H-1), 1.41 [s, 9H, CH3 (Boc)]. 13C
NMR (50 MHz, DMSO-d6): δ 172.56, 172.38, 155.75 (CO),
136.66, 133.73, 126.87, 121.83, 119.34, 118.42, 111.80, 104.85
(Ar), 78.83 [C (Boc)], 51.60 (C-2), 50.64 (C-5), 50.18 (C-11b),
32.22 (C-1), 28.73 [CH3 (Boc)], 23.66 (C-6). Anal. (C20H23N3O5)
C, H, N.
(2S,5S,11bR,1′S)-2-(Ben zyloxycar bon yl)am in o-5-(1′-car -
b oxy-2′-p h e n yle t h yl)ca r b a m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (15a ). Yield: 83%
(from 11a ). White foam. [R]D 20.9° (c 0.01 in MeOH). HPLC:
tR ) 7.73 min (A:B ) 40:60). Anal. (C32H30N4O6) C, H, N.
(2S,5S,11b R,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
ca r boxy-2′-p h en ylet h yl)ca r ba m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (15b). Yield: 75%
(from 11b). White foam. HPLC: tR ) 8.73 min (A:B ) 40:60).
Anal. (C32H30N4O6) C, H, N.
(2R,5R,11b S,1′S)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
ca r boxy-2′-p h en ylet h yl)ca r ba m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (16a ). Yield: 93%
(from 13a ). White foam. HPLC: tR ) 8.70 min (A:B ) 40:60).
Anal. (C32H30N4O6) C, H, N.
(2R,5R,11b S,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
ca r boxy-2′-p h en ylet h yl)ca r ba m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (16b). Yield: 87%
(from 13b). White foam. [R]D -20.1° (c 0.01 in MeOH). HPLC:
tR ) 7.71 min (A:B ) 40:60). Anal. (C32H30N4O6) C, H, N.
(2R,5R,11b S,1′S)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
b en zyl-2′-ca r b oxyet h yl)ca r b a m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (17a ). Yield: 60%
(from 14a ). White solid. Mp: 210-212 °C. HPLC: tR ) 9.73
min (A:B ) 40:60). Anal. (C33H32N4O6) C, H, N.
(2R,5R,11b S,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
b en zyl-2′-ca r b oxyet h yl)ca r b a m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (17b). Yield: 40%
(from 14b). White foam. HPLC: tR ) 8.95 min (A:B ) 40:60).
Anal. (C33H32N4O6) C, H, N.
Gen er a l P r oced u r e for Cou p lin g of 5-Ca r boxyh exa h y-
d r oin d olizin o[8,7-b]in d oles w ith P h e Der iva tives. A solu-
tion of the 5-carboxyhexahydroindolizino[8,7-b]indole 8, 9 or
10 (0.54 mmol) and the corresponding Phe or âHph derivative
(0.54 mmol) in dry CH2Cl2 (14 mL) was successively treated
with BOP (0.24 g, 0.54 mmol) and TEA (0.15 mL, 1.08 mmol)
at room temperature. After stirring overnight the solvent was
evaporated, the residue was dissolved in EtOAc and washed
with citric acid (10%), NaHCO3 (10%)and brine. The organic
layer was dried (Na2SO4) and evaporated leaving a residue
which was purified on a silica gel column as specified in each
case.
(2S,5S,11b R,1′S)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
m eth oxycar bon yl-2′-ph en yleth yl)car bam oyl-3-oxo-2,3,5,6,-
11,11b-h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (11a ). Yield:
73% (from 10 and H-L-Phe-OMe). Eluent: 40% of EtOAc
in hexane. White foam. [R]D -2.5° (c 0.01 in MeOH). HPLC:
tR ) 18.73 min (A:B ) 40:60). Anal. (C33H32N4O6) C, H, N.
(2S,5S,11b R,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
m eth oxycar bon yl-2′-ph en yleth yl)car bam oyl-3-oxo-2,3,5,6,-
11,11b-h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (11b). Yield:
63% (from 10 and H-D-Phe-OMe). Eluent: 40% of EtOAc in
hexane. White foam. HPLC: tR ) 21.80 min (A:B ) 40:60).
Anal. (C33H32N4O6) C, H, N.
(2R,5R,11b S,1′S)-2-(ter t-Bu t oxyca r b on yl)a m in o-5-(1′-
m eth oxycar bon yl-2′-ph en yleth yl)car bam oyl-3-oxo-2,3,5,6,-
11,11b-h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (12a ). Yield:
84% (from 9 and H-L-Phe-OMe). Eluent: 50% of EtOAc in
hexane. White foam. HPLC: tR ) 13.20 min (A:B ) 40:60).
Anal. (C30H34N4O6) C, H, N.
(2R,5R,11b S,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
m eth oxycar bon yl-2′-ph en yleth yl)car bam oyl-3-oxo-2,3,5,6,-
11,11b-h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (13b). Yield:
94% (from 8 and H-D-Phe-OMe). Eluent: 40% of EtOAc in
Gen er a l P r oced u r e for P r ep a r a tion of C-Ter m in a l
Ca r ba m oyl Der iva tives. A solution of the corresponding 1′-
methoxycarbonyl derivative (0.1 g, 0.17 mmol) in saturated
NH3 in MeOH was stirred overnight at room temperature.
After evaporation to dryness, the resulting residue was puri-
fied on a silica gel column using a 3% MeOH in CH2Cl2.
(2S,5S,11bR,1′S)-2-(Ben zyloxycar bon yl)am in o-5-(1′-car -
b a m oyl-2′-p h en ylet h yl)ca r b a m oyl-3-oxo-2,3,5,6,11,11b -
h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (18a ). Yield: 89%
(from 11a ). White foam. HPLC: tR ) 5.27 min (A:B ) 40:60).
Anal. (C32H31N5O5) C, H, N.
hexane. White foam. [R]D 2.4° (c 0.01 in MeOH). HPLC: tR
18.73 min (A:B ) 40:60). Anal. (C33H32N4O6) C, H, N.
)
(2R,5R,11b S,1′S)-2-(Ben zyloxyca r b on yl)a m in o-5-[1′-
b e n zyl-2′-(m e t h oxyca r b on yl)e t h yl]ca r b a m oyl-3-oxo-
2,3,5,6,11,11b-h exah ydr o-1H-in dolizin o[8,7-b]in dole (14a).
Yield: 65% (from 8 and H-L-âHph-OMe). Eluent: 40% of
EtOAc in hexane. White foam. HPLC: tR ) 9.93 min (A:B )
45:55). Anal. (C34H34N4O6) C, H, N.
(2R,5R,11b S,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-[1′-
b e n zyl-2′-(m e t h oxyca r b on yl)e t h yl]ca r b a m oyl-3-oxo-
2,3,5,6,11,11b-h exah ydr o-1H-in dolizin o[8,7-b]in dole (14b).
Yield: 87% (from 8 and H-D-âHph-OMe). Eluent: 60% of
EtOAc in hexane. White foam. HPLC: tR ) 15.95 min (A:B )
40:60). Anal. (C34H34N4O6) C, H, N.
(2R,5R,11b S,1′S)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
m eth oxycar bon yl-2′-ph en yleth yl)car bam oyl-3-oxo-2,3,5,6,-
11,11b-h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (13a ). A so-
lution of compound 12a (0.29 g, 0.54 mmol) in CH2Cl2 (1 mL)
was treated with TFA (0.33 mL, 4.3 mmol) and stirred at room
temperature for 1 h. After evaporation to dryness, the residue
was dissolved in CH2Cl2 (5 mL), cooled to 0 °C, and treated
successively with TEA (0.19 mL, 1.35 mmol) and benzyl
chloroformate (0.12 mL, 0.81 mmol). After stirring overnight
(2S,5S,11b R,1′R)-2-(Ben zyloxyca r b on yl)a m in o-5-(1′-
ca r b a m oyl-2′-p h en ylet h yl)ca r b a m oyl-3-oxo-2,3,5,6,11,-
11b-h exa h yd r o-1H-in d olizin o[8,7-b]in d ole (18b). Yield:
92% (from 11b). White solid. Mp: 139-141 °C. HPLC: tR
6.20 min (A:B ) 40:60). Anal. (C32H31N5O5) C, H, N.
)
Bin d in g Assa ys. CCK1 and CCK2 receptor binding assays
were performed using rat pancreas and cerebral cortex homo-
genates, respectively, according to the method previously
described.39 Briefly, rat pancreas tissue was carefully cleaned
and homogenized in Pipes HCl buffer, pH 6.5, containing 30
mM MgCl2 (15 mL/g of wet tissue) and the homogenate was
then centrifuged twice at 4 °C for 10 min at 50000g. For
displacement assays, pancreatic membranes (0.2 mg protein/
tube) were incubated with 0.5 nM [3H]pCCK-8 in Pipes HCl
buffer, pH 6.5, containing MgCl2 (30 mM), bacitracin (0.2 mg/
mL) and soybean trypsin inhibitor (SBTI, 0.2 mg/mL), for 120

3776 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Mart´ın-Martı´nez et al.
(6) Katsuura, G.; Shinohara, S.; Shintaku, H.; Eigyo, M.; Mat-
sushita, A. Protective Effect of CCK-8 and Ceruletide on
Glutamate-Induced Neuronal Cell Death in Rat Neurone Cul-
tures: Possible Involvement of CCK-B Receptors. Neurosci. Lett.
1991, 132, 159-162.
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8S Inhibits L-Dopa-Induced Dyskinesias in Parkinsonian Squir-
rel Monkeys. Neurology 1990, 40, 717-718.
(8) Guo, Y.-S.; Towsend, C. M. Gastrointestinal Hormones and
Gastrointestinal Cancer Growth. Contemp. Endocrinol. 1999, 8,
189-214.
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Neurosci. Biobehav. Rev. 1998, 22, 815-825.
(10) Roques, B. P.; Noble, F. Association of Enkephalin Catabolism
min at 25 °C. Rat brain cortex was homogenized in 50 mM
Tris-HCl buffer pH 7.4 containing 5 mM MgCl2 (20 mL/g of
wet tissue) and the homogenate was centrifuged twice at 4 °C
for 35 min at 100000g. Brain membranes (0.45 mg protein/
tube) were incubated with 1nM [3H]pCCK-8 in 50 mM Tris-
HCl buffer, pH 7.4, containing MgCl2 (5 mM) and bacitracin
(0.2 mg/mL) for 60 min at 25 °C. Final incubation volume was
0.5 mL in both cases. Non specific binding was determined
using CCK-8 (1 µM) as the cold displacer. The inhibition
constants (Ki) were calculated using the equation of Cheng-
Prusoff from the displacement curves analyzed with the
receptor fit competition LUNDON program.
Inhibitors and CCK-B Antagonists:
a Potential Use in the
Am yla se Relea se. Dispersed rat pancreatic acini were
prepared by using a modification of the technique of J ensen
et al.41 The rat was decapitated and the pancreas was carefully
cleaned. Tissue was injected three times with 5 mL of a
solution of collagenase (Worthinton) at a concentration of 70
U/mL (in mix buffer) and subjected to the digestion step
consisting in three 10-min incubations at 37 °C in atmosphere
of pure O2 and agitation (200 cycles/min). The tissue was
washed two times tin mix buffer (composition: NaCl 98 mM,
KCl 6 mM, NaH2PO4 2.5 mM, CaCl2 0.5 mM, theophylline 5
mM, glucose 11.4 mM, L-glutamine 2 mM, L-glutamic acid 5
mM, fumaric acid 5 mM, piruvic acid 5 mM, SBTI 0.01%,
essential amino acid mixture 1%, essential vitamin mixture
1%, pH ) 7.4) after each incubation. The tissue obtained after
the last incubation was transferred to stop buffer (4% BSA)
and shook vigorously for 10 min. The homogenate was
centrifuged twice (100g, 1 min, 4 °C); the pellet obtained in
the first centrifugation was resuspended in wash buffer (0.2%
BSA) and the final pellet was resuspended in incubation buffer
(1% BSA). Cells were allowed to rest for 30 min and then they
were centrifuged (100g, 1 min, 4 °C) and resuspended again
in incubation buffer.
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Amylase release was measured using the procedure of
Peikin et al.43 Samples (2 mL) of the acini suspension were
placed in plastic tubes and incubated for 30 min at 37 °C in
atmosphere of pure O2 with agitation (70 cycles/min). Amylase
activity was determined using the amyl kit reagent (Boering-
her Mannheim). Release (S) was calculated as the percentage
of the amylase activity in the acini that was released into
extracellular medium during the incubation period. The
percentage of inhibition by drugs of amylase release elicited
by a fixed CCK-8 concentration (0.1 nM) was calculated
according to the formula: % I ) [(SCCK - SC) - (ST - SC)/
(SCCK - SC)] × 100, where SC is control release (vehicle), SCCK
is release elicited by CCK-8, and ST is release in the presence
of increasing drug concentrations. IC50 values were calculated
for the drug tested.
Ack n ow led gm en t. We thank the Comisio´n Inter-
ministerial de Ciencia y Tecnolog´ıa (SAF 97 0030),
Fundacio´n La Caixa (97/022), and Comunidad Au-
to´noma de Madrid (08.5/0006/1998) for financial sup-
port.
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