5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK1 receptor antagonists: Structural modifications at the tryptophan domain

Article abstract of DOI:10.1021/jm991078x

Analogues of the previously reported potent and highly selective CCK₁ receptor antagonist (4aS,5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3- dioxoperhydropyrido-[1,2-c]pyrimidine (2a) were prepared to explore the structural requirements at the Boc-tryptophan domain for CCK₁ receptor affinity. Structural modifications of 2a involved the Trp side chain, its conformational freedom, the Boc group, and the carboxamide bond. Results of the CCK binding and in vitro functional activity evaluation showed three highly strict structural requirements: the type and orientation of the Trp side chain, the H-bonding acceptor carbonyl group of the carboxamide bond, and the presence of the Trp amino protection Boc. Replacement of this acid- labile group with 3,3-dimethylbutyryl or tert-butylaminocarbonyl conferred acid stability to analogues 14a and 15a, which retained a high potency and selectivity in binding to CCK₁ receptors, as well as an in vivo antagonist activity against the acute pancreatitis induced by caerulein in rats. Oral administration of compounds 14a and 15a also produced a lasting antagonism to the hypomotility induced by CCK-8 in mice, suggesting a good bioavailability and metabolic stability.

Full text of DOI:10.1021/jm991078x

J . Med. Chem. 1999, 42, 4659-4668
4659
5-(Tr yp top h yl)a m in o-1,3-d ioxop er h yd r op yr id o[1,2-c]p yr im id in e-Ba sed P oten t
a n d Selective CCK1 Recep tor An ta gon ists: Str u ctu r a l Mod ifica tion s a t th e
Tr yp top h a n Dom a in
J ose´ M. Bartolome´-Nebreda,§ Isabel Go´mez-Monterrey,§ M. Teresa Garc´ıa-Lo´pez,§ Rosario Gonza´lez-Mun˜iz,§
Mercedes Mart´ın-Mart´ınez,§ Santiago Ballaz,‡ Edurne Cenarruzabeitia,‡ Miriam LaTorre,‡ J oaqu´ın Del R´ıo,‡ and
Rosario Herranz*,§
Insituto de Qu´ımica Me´dica (CSIC), J uan de la Cierva 3, E-28006 Madrd, Spain, and Departamento de Farmacolog´ıa,
Universidad de Navarra, Irunlarrea 1, E-31080 Pamplona, Spain
Received May 10, 1999
Analogues of the previously reported potent and highly selective CCK1 receptor antagonist
(4aS,5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-dioxoperhydropyrido-[1,2-c]pyrimidine (2a )
were prepared to explore the structural requirements at the Boc-tryptophan domain for CCK1
receptor affinity. Structural modifications of 2a involved the Trp side chain, its conformational
freedom, the Boc group, and the carboxamide bond. Results of the CCK binding and in vitro
functional activity evaluation showed three highly strict structural requirements: the type
and orientation of the Trp side chain, the H-bonding acceptor carbonyl group of the carboxamide
bond, and the presence of the Trp amino protection Boc. Replacement of this acid-labile group
with 3,3-dimethylbutyryl or tert-butylaminocarbonyl conferred acid stability to analogues 14a
and 15a , which retained a high potency and selectivity in binding to CCK1 receptors, as well
as an in vivo antagonist activity against the acute pancreatitis induced by caerulein in rats.
Oral administration of compounds 14a and 15a also produced a lasting antagonism to the
hypomotility induced by CCK-8 in mice, suggesting a good bioavailability and metabolic
stability.
In tr od u ction
by CCK1 or CCK2 receptors are not completely estab-
lished.1,2 Despite the predominant role suggested for
CCK2 receptors in anxiety and in the control of nocicep-
tion, anxiolytic-like effects and enhancement of mor-
phine analgesia have been reported for the typical CCK1
antagonist Devazepide14,15 (1). It has been suggested16
that these effects could be due to the blockade of CCK2
receptors by Devazepide at high doses, since this
The peptide Cholecystokinin (CCK) displays hormonal
and neurotransmitter activities in the digestive tract
and in the central nervous system (CNS)1 and exerts
its biological effects via at least two G protein coupled
receptor subtypes, termed CCK1 and CCK2.2 CCK1
receptors are mainly found in the digestive tract, where
they are involved in the diverse effects of CCK on the
regulation of digestive processes1 (pancreatic enzyme
secretion, gut motility, and gallbladder contraction).
They are also found in selected CNS areas, where they
regulate dopamine release1-3 and antagonize opioid
analgesia.1,4 Furthermore, CCK1 receptors mediate the
satiety effect of CCK in the CNS and in the peripheral
nervous system1,2,5 and are present in several tumoral
cell lines.2 CCK2 receptors are predominantly found
throughout the CNS2 and are considered to be primarily
involved in anxiety-related conditions,6-9 memory pro-
cesses,1,10 and also in neuropsychiatric disorders.1,11,12
The variety of possible therapeutic utilities for CCK
receptor agonists and antagonists has prompted an
intensive research in this area and, over the past
decade, a number of potent and selective non-peptide
CCK1 and CCK2 receptor antagonists have been re-
ported.13 Some of these antagonists have been useful
tools for characterizing the two CCK receptor subtypes
and for gaining further insight into the functional
significance of CCK in the periphery and in the CNS;
nevertheless, the physiological effects of CCK mediated
compound is not completely devoid of affinity for this
receptor subtype (selectivity for CCK1 over CCK2 varies
from 170-17 to 3000-fold18). Caution has also been
recommended, in general, when attempting to dif-
ferentiate between effects mediated by CCK1 or CCK2
receptors with Devazepide.19 In view of these facts, the
development of CCK receptor antagonists with higher
selectivity for CCK1 over CCK2 receptors is of interest
in order to shed further light on the functional roles of
CCK receptor subtypes. In this regard, we recently
reported the design, synthesis20 and pharmacological
properties21 of the 5-(tryptophyl)amino-1,3-dioxoperhy-
dropyrido-[1,2-c]pyrimidine derivative 2a (IQM-95,333),
a highly potent and selective CCK1 receptor antagonist,
§ Instituto de Qu´ımica Me´dica.
‡ Departamento de Farmacolog´ıa.
10.1021/jm991078x CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/07/1999

4660 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Bartolome´-Nebreda et al.
Sch em e 1a
both in vitro and in vivo. This novel compound showed
a CCK1 receptor affinity similar to that of Devazepide,20
but it was virtually devoid of affinity at brain CCK2
receptors. Interestingly, compound 2a also showed a
marked anxiolytic-like activity in animal models, and
blocked the CCK-8-induced hypophagia and hypoloco-
motion effects after intraperitoneal administration.21
From the first modifications performed on compound 2a ,
the 4a,5-trans stereochemistry of the 1,3-dioxoperhy-
dropyrido[1,2-c]pyrimidine skeleton and the L-configu-
ration of the Boc-Trp residue emerged as essential
features for CCK1 binding affinity and subtype receptor
selectivity.20 Modifications of this residue have been now
performed in order to define which functional groups
are critical for binding to the receptor and to explore
the tolerance of the receptor to conformational con-
straints. These modifications involve the Trp side chain,
its conformational freedom, the Boc group and the
carboxamide bond. Additionally, some of the modifica-
tions herein reported could lead to analogues with
enhanced oral bioavailability and duration of action. The
present paper deals with the synthesis, CCK receptor
binding profile, and in vivo antagonist activity against
the acute pancreatitis induced by caerulein in rats and
against the hypomotility induced by CCK-8 in mice of
this series of Boc-Trp modified analogues of the above
indicated CCK1 receptor antagonists.
Ch em istr y
In an attempt to reduce the molecular weight of the
parent compounds 2, we initially synthesized analogues
7 and 8, in which the Boc-Trp moiety was respectively
replaced by indol-2-yl-carbonyl, the key group in
Devazepide for binding to CCK1 receptors,22 and the
indol-3-yl-acetyl residue.23 Then, analogues 9-12, con-
taining Phe, R-Me-L-Trp, R-Me-D-Trp, and the tetrahy-
dro-â-carbolin-3-yl-carbonyl in place of Trp, were pre-
pared in order to study the influence of the type and
conformational freedom of the side chain upon the
binding affinity at CCK receptors. Compounds 7-12
were prepared following a synthetic route similar to that
used for the synthesis of the model compounds 2, as
indicated in Scheme 1. This route involves sequential
N-Boc removal in the 5-Boc-amino-1,3-dioxoperhydro-
pyrido-[1,2-c]pyrimidine derivatives 6, followed by cou-
pling with the corresponding acid or Boc protected
amino acid derivative. The starting 1,3-dioxoperhydro-
pyrido[1,2-c]pyrimidines 6 were obtained in four steps
from Boc-D-Orn(Z)-OH, as a (4:1) racemic mixture of the
(4aS,5R)- and (4aR,5S)-isomers, or as a (1:4) mixture
from Boc-L-Orn(Z)-OH, as previously described.20 There-
fore, compounds 7 and 8 were obtained as racemic
mixtures, and 9-12 were obtained as diastereoisomeric
mixtures a ,b, which could be chromatographically re-
solved only in the cases of 9 and 12.
a
Reagents: (a) 1,1′-carbonyldiimidazole, LiCH2CO2Et, THF; (b)
H2, Pd(C), EtOH or MeOH; (c) benzyl isocyanate, THF; (d) NaH,
THF; (e) TFA, CH2Cl2; (f) R-OH, BOP, Et3N.
F igu r e 1. Newman projections of the g(-) and g(+) staggered
ratational states of the CR-Câ bond in the Trp residue of
compound 2a , and the corresponding conformations of the
tetrahydro-â-carboline skeleton in 12a .
1
The H NMR data for the lead compound 2a , particu-
larly the Trp J R,â values (2.7 and 5.2 Hz) and the NOE
effect between the NH-Boc and the indole 2-H protons,
suggested a preferred gauche (-) conformation for the
Trp side chain (ø1 ≈ -60)24,25 (Figure 1). We expected
that the tetrahydro-â-carboline skeleton in compound
12a could fix the Trp preferred conformation. However,
hydro-â-carboline 3-H proton and, therefore, a fixed
gauche (+) conformation (ø1 ≈ 60) for the restricted Trp
side chain in this compound.
Taking into account the expected low stability of the
N-Boc protection in the acid medium of the stomach,26
our second objective was to change this protection in
1
the J 3,4 values in the H NMR spectrum of 12a (0 and
6 Hz) indicated an equatorial disposition for the tetra-

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4661
Sch em e 2a
Sch em e 3a
a
Reagents: (a) TFA, CH2Cl2; (b) 3,3-dimethylbutyryl chloride,
Et3N, THF; (c) t-butyl isocyanate, Et3N,THF; (d) 1-adamantylcar-
bonyl chloride, Et3N, THF; (e) 2-adamantyl chloroformate, Et3N,
THF.
order to increase the stability in acid pH, without loss
in the binding affinity. With this aim, the urethane Boc
group was replaced in 2a by its carboxamide and ureido
bioisosters, the 3,3-dimethylbutyryl and tert-butyl-
aminocarbonyl groups, respectively, giving compounds
14a and 15a , and in 2b by the N protecting groups used
in the series of dipeptoid CCK receptor antagonists,27
1-adamantylcarbonyl (1-Adc) and 2-adamantyloxycar-
bonyl (2-Adoc), obtaining analogues 16b and 17b (Scheme
2). A comparative RP HPLC study on the stability of
compounds 2a , 14a and 15a in acid medium showed
that the half-life of 2a in 2 N HCl solution in (1:1) H2O-
MeOH was 17 min, while that of 15a was 11 days, and
the analogue 14a remained unaltered after that time.
Finally, the carboxamide bond between the Trp and
the 1,3-dioxoperhydropyrido-[1,2-c]pyrimidine skeleton
of compound 2a was replaced by the reduced peptide
bond Ψ[CH2NH] in compounds 18a ,b and by the cyano-
methyleneamino Ψ[CH(CN)NH] peptide bond surrogate
in analogues 19-22 (Scheme 3). Both peptide bond
modifications have been used in pseudopeptides to
increase stability toward peptidases and to study if the
replaced peptide bond has a functional role or merely
serves to orient and align the side chains.28-30 The
Ψ[CH2NH] peptide bond surrogate causes an increase
of flexibility in the peptide backbone and a decrease in
the H-bonding properties by loss of the H-bonding
acceptor amide carbonyl group.28,31 In contrast, the Ψ-
[CH(CN)NH] was proposed as peptide bond surrogate
based on the hypothesis that its cyano group keeps
H-bonding acceptor properties and that it could impart
higher backbone rigidity than the Ψ[CH2NH].32,33 On
the other hand, both peptide bond surrogates have the
additional advantage of introducing a basic NH, which
could be protonated, enhancing the solubility in aqueous
systems.28
a
Reagents: (a) TFA, CH2Cl2; (b) Boc-L-Trp-H, Et3N, ZnCl2,
NaBH3CN, MeOH; (c) Boc-L-Trp-H or Z-L-Trp-H, ZnCl2, TMSCN,
MeOH; (d) H2, Pd(C), (Boc)2O, MeOH; (e) H2, Pd(C), MeOH; (f)
bis(trichloromethyl)carbonate, Et3N, CH2Cl2.
phanal, using NaBH3CN as reducing agent in the
presence of ZnCl2. Only the major isomer 18a could be
isolated from the diastereoisomeric mixture 18a ,b. With
respect to the cyanomethyleneamino analogues 19-22,
these were prepared applying our methodology,34 which
involves reaction of the 1,3-dioxoperhydropyrido[1,2-c]-
pyrimidine derivatives 6a ,b, after N-Boc deprotection,
with Boc-L-tryptophanal or Z-L-tryptophanal and (tri-
methylsilyl)cyanide (TMSCN) in the presence of ZnCl2.
In this process a new chiral center is generated;
therefore, in the reaction with Boc-L-tryptophanal from
the (4:1) racemic mixture 6a ,b, two isomers with a
(4aS,5R)-configuration [19a (31%) and 20a (21%)] were
obtained, and only one (19b (9%)) with the (4aR,5S)-
configuration was obtained. The assignment of the
absolute configuration of the new generated stereogenic
center is usually made through the formation of an
imidazolidin-2-one ring.34 For this purpose it was neces-
sary to remove the Trp amino-protection. However, the
Boc removal was not possible in compounds 19 and 20
due to the extreme susceptibility of the indole ring of
these compounds to oxidative degradation in acid media,
The reduced peptide bond analogues 18a ,b were
obtained from the (4:1) racemic mixture of the 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives 6a ,b
by N-Boc removal, followed by N-alkylation of the
resulting deprotected compounds with Boc-L-trypto-

4662 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Bartolome´-Nebreda et al.
Ta ble 1. Inhibition of the [3H]pCCK-8 Specific Binding to Rat Pancreas (CCK1) and Cerebral Cortex Membranes (CCK2) by
Compounds 7-12
IC50 (nM)a
compd
CCK-8
Devazepide
2a
2b
7a b
8a b
9a
10a b
11a b
12a
stereochemistry
R
CCK1
CCK2
1.04 ( 0.08
0.71 ( 0.05
1.59 ( 0.10
22.7 ( 4.0
>1000
5.60 ( 0.30
696 ( 134
>10000
6153
>10000
>10000
>10000
>10000
>10000
>10000
>10000
(4aS,5R)
(4aR,5S)
Boc-L-Trp
Boc-L-Trp
(4:1) (4aR,5S):(4aS,5R)
(4:1) (4aR,5S):(4aS,5R)
(4aS,5R)
(6:1) (4aS,5R):(4aR,5S)
(6:1) (4aS,5R):(4aR,5S)
(4aS,5R)
In-2-yl-CO
In-3-yl-CH2-CO
Boc-L-Phe
Boc-R-Me-L-Trp
Boc-R-Me-D-Trp
2-Boc-THBC-3-yl-COb
2-Boc-THBC-3-yl-COb
>1000
65.7 ( 22.6
42.4
1236
226
>1000
12b
(4aR,5S)
a
Values are the mean or mean ( SEM of at least three experiments, performed with seven concentrations of test compounds in triplicate.
THBC ) tetrahydro-â-carboline.
b
Ta ble 2. Inhibition of the [3H]pCCK-8 Specific Binding to Rat Pancreas (CCK1) and Cerebral Cortex Membranes (CCK2) by
Compounds 13-22
IC50 (nM)a
compd
stereochemistry
R1
X
CCK1
CCK2
Devazepide
2a
0.71 ( 0.05
1.59 ( 0.10
22.7 ( 4.0
>1000
696 ( 134
>10000
6153
>10000
>10000
>10000
>10000
4390
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aS,5R)
(4aR,5S)
(4aR,5S)
(4aS,5R)
(4aS,5R)
(4aS,5R)
(4aS,5R)
(4aS,5R)
Boc
Boc
H
CO
CO
CO
CO
CO
CO
CO
CO
CH2
[(R)CHCN]
[(S)CHCN]
[(R)CHCN]
[(S)CHCN]
2b
13a b
13bb
14a
H
>1000
tBut-CH2-CO
4.43 ( 0.36
0.91 ( 0.08
486
340
>1000
15a
16b
17b
18a
19a
20a
21a
22a
tBut-HN-CO
1-Adc
2-Adoc
Boc
Boc
Boc
Z
3430
>10000
>10000
>10000
>10000
>10000
7.69 ( 0.86
32.6 ( 5.26
>1000
Z
>1000
a
Values are the mean or mean ( SEM of at least three experiments, performed with seven concentrations of test compounds in triplicate.
Tested as trifluoroacetate salt.
b
even by using different described conditions to minimize
this problem.35 In view of the difficulties in removing
the Boc protection, the Z protected analogues 21a (34%),
22a (17%), and 21b (10%) were prepared similarly.
Then, after Z removal in 21a and 22a by catalytic
hydrogenolysis, both of the resulting deprotected dia-
stereoisomers 23a and 24a were subjected to reaction
with bis(trichloromethyl)carbonate. However, only the
isomer 23a led to the corresponding imidazolidin-2-one
derivative 25a . The absolute configuration at the C5 of
the imidazolidin-2-one ring of this compound, and
therefore at the Ψ[CH(CN)NH] chiral center of 21a and
23a , was established on the basis of its J 4,5 value (5 Hz)
in its 1H NMR spectrum, indicative of a trans disposition
for the 4-H and 5-H imidazolidin-2-one protons.34 After-
ward, the configurational assignment in the Boc ana-
logues was made through the catalytic hydrogenolysis
of 21a in the presence of di(tert-butyl) dicarbonate,
which led directly to the major diastereoisomer 19a . The
low amount obtained of the minor isomers 19b and 21b
did not allow their configuration assignment.
Biologica l Resu lts a n d Discu ssion
The affinity of the new 1,3-dioxoperhydropyrido[1,2-
c]pyrimidine derivatives 7-22 at CCK1 and CCK2
receptors was determined by measuring the displace-
ment of [3H]propionyl-CCK-8 binding to rat pancreatic
and cerebral cortex homogenates, respectively, as previ-
ously described.36 The data are depicted in Tables 1 and
2. For comparative purposes CCK-8, Devazepide, and
the model compounds 2a and 2b were also included in
the assay. Like these model compounds, the new 1,3-

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4663
Ta ble 3. Effect of Intraperitoneal Administration of CCK1
Antagonists on the Increase in Plasma Amylase and Lipase
Activities Induced by Caerulein in Ratsa
dioxoperhydropyrido[1,2-c]pyrimidine derivatives 7-22
bound preferentially to the CCK1 receptor subtype. The
replacement of Boc-Trp by the key Devazepide phar-
macophoric group indol-2-yl-carbonyl and by its ana-
logue indol-3-yl-acetyl in compounds 7 and 8, respec-
tively, led to the complete loss of affinity (Table 1). The
phenylalanine analogue 9a showed 1 order of magnitude
lower CCK1 binding potency than the lead compound
2a . In contrast to the good results obtained in the
dipeptoid CCK antagonist series by introducing an
R-methyl substitution into their Trp residue,27,37,38 the
introduction of this substitution into 2a or in its D-Trp
analogue20 to give 10 and 11 led to a decrease of 1 and
3 orders of magnitude, respectively, in the binding
affinity. The conformational restriction introduced by
the tetrahydro-â-carboline system into 12a had a strong
detrimental effect on the binding affinity. This effect
could be due to the aforementioned fact that this
skeleton fixes the gauche (+) Trp side chain conforma-
tion and not the preferred gauche (-) conformation of
the prototype 2a . In regard to the Trp amino protection
(Table 2), it seems necessary for binding to CCK1
receptors, as indicated by the inactivation of compounds
2a and 2b when deprotected to give 13a and 13b. The
replacement of the urethane N-Boc protection of 2a by
its bioisosters, more stable to chemical degradation in
acid media, the 3,3-dimethylbutyryl and tert-butyl-
aminocarbonyl groups in 14a and 15a did not have a
significant effect upon their affinity at CCK receptors.
However, the exchange of this protection in 2b for the
usual27 protections in dipeptoid antagonist 1-adaman-
tylcarbonyl or 2-adamantyloxycarbonyl led to 1 order
of magnitude lower CCK1 binding potency in analogues
16b and 17b. This decrease, along with the fact that
this replacement was the only modification which
produced a slight increase in CCK2 binding affinity,
discouraged the preparation of the corresponding dia-
stereoisomers 16a and 17a . Finally, in relation to the
carboxamide bond (Table 2), its replacement by the
reduced peptide bond in compound 18a led to the
complete loss of activity, while the incorporation of the
[CH(CN)NH] group as a carboxamide surrogate was
translated into a small decrease in the binding affinity
for the (R)-epimer 19a and into 1 order of magnitude
lower potency in the case of the (S)-epimer 20a . As the
main difference between the [CH2NH] and the [CH(CN)-
NH] carboxamide surrogates lies in their H-bonding
properties, rather than in their geometry,31 these data
indicate the importance of the H-bonding acceptor
amide carbonyl or cyano groups for the binding of 2a
or 19a to CCK1 receptors. It also confirms the utility of
the [CH(CN)NH] group as a carboxamide surrogate.31
It is also interesting to note the complete loss of affinity
when the Boc protection of 19a and 20a was exchanged
for the Z protection in 21a and 22a .
plasma amylase plasma lipase
treatment
(IU/mL)
(IU/mL)
vehicle
caerulein
2073 ( 108
15 ( 1
52101 ( 2221
3100 ( 146
125 ( 26**
26 ( 2**
Devazepide (0.1) + caerulein 8793 ( 1353
Devazepide (1) + caerulein
2a (0.1) + caerulein
2a (1) + caerulein
3650 ( 352**
35985 ( 6387
2721 ( 275**
61102 ( 3466
28015 ( 4774** 1140 ( 218**
27103 ( 3539** 1667 ( 224**
5326 ( 434**
1943 ( 505*
112 ( 21**
2956 ( 212
14a (0.1) + caerulein
14a (1) + caerulein
15a (0.1) + caerulein
15a (1) + caerulein
36 ( 5**
a
Pancreatitis induced by four sc injections of caerulein (10 µg/
kg) at hourly intervals. Test compounds (doses in mg/kg ip) given
30 min before the first caerulein injection *p < 0.05, **p < 0.01
vs caerulein (ANOVA followed by Sheffe´’s test).
Ta ble 4. Effect of Oral Administration of CCK1 Antagonists on
the Increase in Plasma Amylase and Lipase Activities Induced
by Caerulein in Ratsa
plasma amylase plasma lipase
treatment
(IU/mL)
(IU/mL)
vehicle
caerulein
Devazepide (0.1) + caerulein
Devazepide (1) + caerulein
2a (0.1) + caerulein
2a (1) + caerulein
14a (0.1) + caerulein
14a (1) + caerulein
15a (0.1) + caerulein
15a (1) + caerulein
3308 ( 285
15 ( 1
39293 ( 3522
7733 ( 1209**
4578 ( 516**
37753 ( 1682
31205 ( 1900
51420 ( 3673
46591 ( 2292
28939 ( 2330
19967 ( 552**
2801 ( 288
871 ( 351**
57 ( 9**
2837 ( 184
1948 ( 232
3089 ( 128
3375 ( 73
2200 ( 164
1062 ( 122**
a
Pancreatitis induced like in Table 3. Test compounds (doses
in mg/kg po) given 60 min before the first caerulein injection *p <
0.05, **p < 0.01 vs caerulein (ANOVA followed by Sheffe´’s test).
All of the tested compounds, at the same dose of 1
mg/kg ip, were able to significantly prevent in the acute
pancreatitis model in rats the enormous rise in plasma
amylase and lipase induced by caerulein. With a 10-
fold lower dose, only Devazepide and 15a showed a
significant antagonism on the rise of both enzyme
activities (Table 3). Since the affinity of 2a and 15a at
CCK1 receptors was approximately identical, it is sup-
posed that the bioavailability of 15a is higher. Such
contention is supported by the results obtained in this
pancreatitis model after oral administration of test
compounds. As can be seen in Table 4, only Devazepide
was effective at the lower dose of 0.1 mg/kg po while
15a , but not 2a , significantly prevented the rise in
enzyme activity at the dose of 1 mg/kg po.
Spontaneous locomotor activity of mice was signifi-
cantly reduced by CCK-8, 10 µg/kg, by approximately
60% (Figure 2). All of the tested compounds were able
to significantly reverse the hypomotility when given ip
at the dose of 0.1 mg/kg (not shown). The time course
of the antagonism to CCK-8 was studied after oral
administration of a 10-fold higher dose, 1 mg/kg, which
did not produce on its own any significant effect on the
locomotion. While the antagonism to the hypomotility
was significant with all four compounds given 1 or 2 h
before CCK-8, only Devazepide significantly antago-
nized the hypomotility when given 4 h before CCK
(Figure 2). Nevertheless, at this time point the distance
travelled by mice receiving CCK-8 or the combined
treatment CCK-8 + 2a was almost identical (Figure 2A),
Compounds with the higher affinity at CCK1 recep-
tors, 14a and 15a , were tested in vivo for their protec-
tive effect on the experimental acute pancreatitis in-
duced by caerulein in rats, which is known to be
sensitive to CCK1 antagonists,39 and for their antago-
nism to the hypomotility induced by CCK-8 in mice,
which is also selectively reversed by antagonists at this
CCK receptor subtype.40 The model compound 2a , as
well as Devazepide, were comparatively included in
these studies.

4664 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Bartolome´-Nebreda et al.
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
Gemini-200,Varian XL-300, or Unity-500 spectrometer, oper-
ating at 200, 300, or 500 MHz, using TMS as reference. 13C
NMR spectra were recorded with the Varian Gemini-200
spectrometer, operating at 50 MHz. Elemental analyses were
obtained on a CH-O-RAPID apparatus. Analytical HPLC was
performed on a Waters Nova-pak C18 (3.9 × 150 mm, 4 µm)
column, with a flow rate of 1 mL/min, and using a tunable
UV detector set at 214 nm. Mixtures of CH3CN (solvent A)
and 0.05% TFA in H2O (solvent B) were used as mobile phase.
Gen er a l P r oced u r e for th e Syn th esis of th e 1,3-Dioxo-
p er h yd r op yr id o[1,2-c]p yr im id in e Der iva tives 7-12. TFA
(0.5 mL) was added to a solution of (4aR*,5S*)-2-benzyl-5-
[(tert-butoxycarbonyl)amino]-1,3-dioxoperhydropyrido[1,2-c]py-
rimidine [6a ,b, obtained as a (1:4) racemic mixture of 6a and
6b, from Boc-L-Orn(Z)-OH for 7a ,b and 8a ,b, and as a (4:1)
mixture from Boc-D-Orn(Z)-OH for 9-12] (75 mg, 0.2 mmol)
in CH2Cl2 (1 mL); after 1 h at room temperature, the solvents
were evaporated to dryness. The residue was dissolved in dry
CH2Cl2 (5 mL), and the corresponding acid (indol-3-yl-acetic
acid, indol-2-yl-carboxylic acid, Boc-L-Phe-OH, Boc-R-Me-L-Trp-
OH, Boc-R-Me-D-Trp-OH, or 2-Boc-tetrahydro-â-carbolin-3-yl-
carboxylic acid) (0.22 mmol), (benzotriazolyloxy)-tris(dimeth-
ylamino)phosphonium hexafluorophosphate (BOP; 97 mg, 0.22
mmol), and triethylamine (63 µL, 0.45 mmol) were added
successively to that solution; stirring was continued at room
temperature for 12 h. Afterward, the reaction mixture was
diluted with CH2Cl2 (20 mL), and the resulting solution was
washed successively with 10% citric acid (2 × 10 mL),
saturated NaHCO3 (2 × 10 mL), H2O (2 × 10 mL), and brine
(10 mL), dried over Na2SO4, and evaporated. Flash chroma-
tography of the residue with 20-50% gradients of EtOAc in
hexane yielded in each case: the (1:4) racemic mixtures 7a b
(68%) and 8a b (71%), the (6:1) diastereoisomeric mixtures
10a b (90%) and 11a b (94%), which could not be resolved, and
the (4:1) diasteroisomeric mixtures 9a b and 12a b. Preparative
TLC of 9a b with (9:1) hexane-EtOAc yielded isolated 9a
(85%), while 12a b was resolved by preparative TLC with
(9:1) ethyl ether-EtOAc into 12a (49%) and 12b (7%). The
most significant analytical and spectroscopic data of these
compounds are summarized in Table 5.
F igu r e 2. Effect of the CCK1 antagonists devazepide (1) and
2a (A), 14a and 15a (B) on the hypomotility induced by CCK-8
in mice. Antagonists (1 mg/kg po) were given at the times
indicated before CCK-8 (10 µg/kg). Spontaneous locomotor
activity (mean ( SEM of 8-12 animals) was measured 5 min
after CCK-8 for 30 min *p < 0.05 **p < 0.01 vs control; +p <
0.05, ++p < 0.01 vs CCK-8 (ANOVA followed by Scheffe´’s test).
whereas a certain trend toward an antagonism of the
CCK-8 effect by either compound 14a or 15a was
observed in Figure 2B, suggesting a slightly higher oral
bioavailability in mice of compounds 14a and 15a as
compared to compound 2a .
Syn th esis of (4a S,5R)- a n d (4a R,5S)-2-Ben zyl-5-(L-tr yp -
t op h yl)a m in o-1,3-d ioxop e r h yd r op yr id o[1,2-c]p yr im i-
d in e Tr iflu or oa ceta te (13a a n d 13b). TFA (0.5 mL) was
added to a solution of (4aS,5R)- or (4aR,5S)-2-benzyl-5-(Boc-
L-tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimi-
dine (2a or 2b) (112 mg, 0.2 mmol) in CH2Cl2 (1 mL); after 1
h at room temperature, the solvents were evaporated to
dryness. Flash chromatography of the residue with 1-5%
gradient of MeOH in CH2Cl2 yielded the corresponding N
deprotected tryptophyl derivative 13a or 13b, respectively,
whose significant analytical and spectroscopic data are sum-
marized in Table 6.
In conclusion, the results herein described show the
strong structural requirements at the tryptophan do-
main for CCK1 receptor binding of this new family of
highly selective CCK1 antagonists based on the struc-
ture of 5-(tryptophyl)amino-1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine. Thus, both the indol of the Trp side
chain and its orientation are important structural
features, and the presence of the H-bonding acceptor
carbonyl group of the carboxamide bond and the Boc
group or some surrogate, such as the 3,3-dimethylbu-
tyryl or the tert-butylaminocarbonyl, is essential. Com-
pounds 14a and 15a , which incorporate these surro-
gates, with much higher chemical stability in acid media
than the lead compound 2a , showed similar CCK
receptor in vitro activity to that of this prototype. These
changes enhanced or tended to enhance their oral
bioavailability. This improvement in the in vivo CCK1
receptor antagonism profile was particularly significant
in the case of the tert-butylaminocarbonyl derivative
15a .
Gen er a l P r oced u r e for th e Rep la cem en t of th e N-Boc
P r otectin g Gr ou p of 2a a n d 2b. Syn th esis of Com p ou n d s
14a -17b. TFA (0.5 mL) was added to a solution of (4aS,5R)-
or (4aR,5S)-2-benzyl-5-(Boc-L-tryptophyl)amino-1,3-dioxoper-
hydropyrido[1,2-c]pyrimidine (2a or 2b) (112 mg, 0.2 mmol)
in CH2Cl2 (1 mL); after 1 h at room temperature, the solvents
were evaporated to dryness. The residue was dissolved in dry
THF (1 mL), and triethylamine (56 µL, 0.4 mmol) was added
to that solution, which was stirred for 10 min. Then, the
corresponding acylating agent (3,3-dimethylbutyryl chloride,
tert-butyl isocyanate, 1-adamantylcarbonyl chloride, or 2-ada-
mantyl chloroformate) was added (0.25 mmol), stirring was
continued at room temperature for 1.5 h, and the reaction
mixture was evaporated to dryness. The resulting residue was
purified by preparative TLC with (1:2) EtOAc-ethyl ether in
the tryptophyl derivatives 14a and 15a , and by flash chroma-
tography in the case of 16b and 17b , using a (33-50%)
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 Feinchemikalien
AG. Analytical TLC was performed on aluminum sheets coated

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4665
Ta ble 5. Significant Analytical and Spectroscopic Data of Compounds 7-12
7a b
8a b
9a
10a b
11a b
12a
12b
R
In-2-yl-CO
In-3-yl-CH2-CO Boc-L-Phe
(4aR*,5S*) (4aS,5R)
Boc-R-Me-L-Trp Boc-R-Me-D-Trp 2-Boc-THBC-3-yl-COa 2-Boc-THBC-3-yl-COa
stereochemistry (4aR*,5S*)
(4aR*,5S*)
(4aR*,5S*)
C32H39N5O5
94
(4aS,5R)
C32H37N5O5
49
(4aR,5S)
C32H37N5O5
7
formulab
yield (%)
mp (°C)
tR (A:B)c
1H NMRd
R (R)e
C24H24N4O3 C25H26N4O3 C29H36N4O5 C32H39N5O5
71
210-212
13.16 (45:55) 9.36 (45:55)
68
85
90
91-92
168-170
46.54 (33:67) 42.00 (33:67)
128-130
37.93 (37:63)
139-141
36.13 (37:63)
46.27 (33:67)
3.64
4.15
1.53, 1.55
4.96
2.54, 2.76
1.52, 1.54
5.02
2.57, 2.69
2.57, 2.54
2.87, 2.95
3.69
5.16f
4.88, 4.96
2.34
5.20f
4.92
2.39, 2.49
2-CH2
4-H
4.93
2.76, 2.91
4.80, 4.90
2.61
4.92, 4.98
2.56, 2.72
4a-H
5-H
3.22
3.94
2.70
2.92
2.89, 2.96
3.71
3.07
3.64
3.00
3.62
3.64
3.61
5-NH
6-H
5.88
1.20, 2.09
5.47
1.00, 1.81
5.59
1.08, 1.66
6.18, 6.35
1.25, 1.88
1.25, 1.73
1.59, 1.73
2.60, 4.33
2.60, 4.31
6.16, 6.35
1.18, 1.81
1.25, 1.87
1.44-1.59
2.56, 4.30
2.56, 4.33
5.83
1.30, 1.94
5.76
1.40, 1.88
7-H
8-H
1.45-1.90
2.65, 4.34
1.40-1.65
2.38, 4.18
1.50
2.51, 4.18
1.56, 1.76
2.61, 4.32
1.56, 1.73
2.60, 4.32
a
b
d
THBC ) tetrahydro-â-carboline. Satisfactory analyses for C, H, N. c A ) CH3CN; B ) 0.05% TFA in H2O. In CDCl3, measured at
300 MHz. e H, except for 10a b and 11a b where it is CH3. J 3,4 in the tetrahydro-â-carboline skeleton 0 and 6 Hz.
f
Ta ble 6. Significant Analytical and Spectroscopic Data of Compounds 13-17
13a
13b
14a
15a
16b
1-Adc
(4aR,5S)
C37H43N5O4
83
17b
2-Adoc
(4aR,5S)
C37H43N5O5
59
R1
H
H
tBut-CH2-CO
(4aS,5R)
C32H39N5O4
80
tBut-NH-CO
(4aS,5R)
C31H38N6O4
86
stereochemistry
formulaa
yield (%)
mp (°C)
tR (A:B)c
1H NMRd
R-H (Trp)
NH-R1
2-CH2
4-H
(4aS,5R)
C28H30F3N5O5
89
(4aR,5S)
C28H30F3N5O5
92
b
b
foam
5.33 (33:67)
foam
14.34 (30:70)
138-140
132-134
117-119
137-139
35.13 (40:60)
57.67 (33:67)
31.20 (33:67)
23.67 (33:67)
4.29
4.19
4.66
5.99
4.89, 4.98
2.51, 2.69
2.86
4.53
5.51
4.84, 4.96
2.52, 2.64
2.70
4.71
5.74
4.91, 4.97
2.23
2.51
4.48
5.32
4.91, 4.98
2.16
2.54
4.82, 4.93
2.75, 2.90
3.27
4.87, 4.97
2.64, 2.78
3.42
4a-H
5-H
3.52
3.76
3.54
3.57
3.57
3.59
5-NH
8.27
7.32
5.98
5.51
5.74
5.38
6-H
7-H
8-H
1.20
1.56
2.62, 4.12
1.48-1.89
1.48-1.89
2.71, 4.25
0.97, 1.59
1.43
2.50, 4.26
0.98, 1.50
1.50
2.46, 4.21
1.14, 1.87
1.48-1.84
2.50, 4.24
1.17, 1.89
1.41-1.84
2.49, 4.24
a
b
d
Satisfactory analyses for C, H, N. As trifluoroacetate salt. c A ) CH3CN; B ) 0.05% TFA in H2O. Measured at 300 MHz in CDCl3,
except for 13a and 13b which were registered in (CD3)2CO.
gradient of EtOAc in hexane as eluant. Significant analytical
and spectroscopic data of these compounds are summarized
in Table 6.
0.1 mmol), and NaBH3CN (16 mg, 0.21 mmol) were added
successively, and stirring at room temperature was continued
for 15 h. Afterward, the reaction mixture was filtered and
evaporated to dryness. The residue was dissolved in EtOAc
(50 mL), and the resulting solution was washed successively
with 0.1 N HCl (2 × 25 mL), saturated NaHCO3 (2 × 25 mL),
H2O (2 × 25 mL), and brine (25 mL), dried over Na2SO4, and
evaporated. Flash chromatography of the residue with 4% of
EtOAc in ethyl ether allowed the isolation of the major
diastereoisomer 18a , whose significant analytical and spec-
troscopic data are shown in Table 7.
Syn th esis of (4aS,5R)-2-Ben zyl-5-[(2S)-2-N-(ter t-bu toxy-
ca r b on yl)a m in o-3-(in d ol-3-yl)-p r op yl]a m in o-1,3-d ioxo-
p er h yd r op yr id o[1,2-c]p yr im id in e (18a ). TFA (0.5 mL) was
added to a solution of (4aR*,5S*)-2-benzyl-5-[(tert-butoxycar-
bonyl)amino]-1,3-dioxoperhydropyrido[1,2-c]pyrimidine [6a ,b,
obtained as a (9:1)20 racemic mixture of 6a and 6b] (75 mg,
0.2 mmol) in CH2Cl2 (1 mL); after 1 h at room temperature,
the solvents were evaporated to dryness. The residue was
dissolved in MeOH (1.5 mL), and 4 Å molecular sieves and
triethylamine (28 µL, 0.2 mmol) were added; after 15 min of
stirring, Boc-L-tryptophanal (58 mg, 0.2 mmol), ZnCl2 (14 mg,
Syn th esis of th e Cya n om eth ylen ea m in o Ca r boxa m id e
Su r r oga te Con ta in in g Com p ou n d s 19-22. TFA (1 mL) was
added to a solution of (4aR*,5S*)-2-benzyl-5-[(tert-butoxycar-

4666 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Bartolome´-Nebreda et al.
Ta ble 7. Significant Analytical and Spectroscopic Data of Compounds 18-22
18a
19a
19b
20a
21a
21b
22a
R1
Boc
Boc
Boc
Boc
Z
Z
Z
X
CH2
[(R)CHCN]
(4aS,5R)
C32H38N6O4
31
99-101
17.40 (45:55)
[CHCN]
(4aR,5S)
C32H38N6O4
9
102-104
16.53 (45:55)
[(S)CHCN]
(4aS,5R)
C32H38N6O4
21
106-108
20.13 (45:55)
[(R)CHCN]
(4aS,5R)
C35H36N6O4
34
68-70
20.13 (45:55)
[CHCN]
(4aR,5S)
C35H36N6O4
10
foam
20.33 (45:55)
[(S)CHCN]
(4aS,5R)
C35H36N6O4
17
67-69
20.20 (45:55)
stereochemistry
formulaa
yield (%)
mp (°C)
tR (A:B)b
1H NMRc
1′-Η
(4aS,5R)
C31H39N5O4
62
80-82
31.27 (40:60)
2.46, 2.98
3.96
4.60
4.96
2.70, 3.02
2.98
3.57
4.22
4.74
4.95
2.60, 2.93
2.86
3.64
4.26
4.80
4.95
2.47, 2.87
2.87
3.79
4.27
4.86
4.96, 4.90
2.50, 2.92
2.93
3.49
4.17
5.07
4.87
2.43, 2.76
2.61
3.64
4.28
5.03
4.93
2.33, 2.80
2.80
3.81
4.34
5.20
4.97, 4.90
2.45, 2.84
2.84
2′-H
NH-R1
2-CH2
4-H
4a-H
5-H
2.15
2.23
2.21
2.40
2.09
2.24
2.37
5-NH
6-H
7-H
2.17
1.69
1.71
1.72
1.69
1.62
1.68
1.10, 2.15
1.72, 1.47
2.63, 4.31
1.12, 2.40
1.44, 1.70
2.53, 4.26
1.06, 2.21
1.39, 1.73
2.53, 4.27
1.12, 1.95
1.44, 1.69
2.60, 4.30
1.03, 2.27
1.38, 1.65
2.28, 4.21
1.16, 2.24
1.25-1.76
2.56, 4.28
0.98, 1.93
1.36, 1.66
2.54, 4.27
8-H
13CNMRd
C1′
50.60
53.17
53.94
52.93
54.34
54.75
53.94
CN
120.10
118.48
117.77
120.18
118.53
117.82
a
b
d
Satisfactory analyses for C, H, N. A ) CH3CN; B ) 0.05% TFA in H2O. c In CDCl3, registered at 300 MHz. In CDCl3, measured
at 50 MHz.
1
bonyl)amino]-1,3-dioxoperhydropyrido[1,2-c]pyrimidine [6a ,b,
obtained as a (9:1)20 racemic mixture of 6a and 6b] (160 mg,
0.43 mmol) in CH2Cl2 (2 mL); after 1 h at room temperature,
the solvents were evaporated to dryness. The residue was
dissolved in MeOH (3 mL), to this solution triethylamine (67
µL, 0.47 mmol) was added, and after 15 min of stirring, the
reaction mixture was cooled at -20 °C. Boc- or Z-L-tryptopha-
nal (1.29 mmol), ZnCl2 (59 mg, 0.43 mmol), and TMSCN (0.161
µL, 1.29 mmol) were added successively, and stirring was
continued, first at -20 °C for 1 h and then at 0 °C for 15 h.
Afterward, the reaction mixture was evaporated to dryness.
The residue was dissolved in EtOAc (50 mL), and the resulting
solution was washed successively with H2O (2 × 25 mL) and
brine (25 mL), dried over Na2SO4, and evaporated. Preparative
TLC of the residue using 1% of MeOH in CH2Cl2 yielded in
each case, in decreasing order of Rf, the Boc protected
compounds 19a , 19b, and 20a , and the Z protected analogues
21a , 21b, and 22a , whose significant analytical and spectro-
scopic data are summarized in Table 7.
Syn th esis of (4a S,5R)-2-Ben zyl-5-[(4S,5R)-5-cya n o-4-
(in d ol-3-yl)m eth yl-2-oxoim id a zolid in -1-yl]-1,3-d ioxop er -
h yd r op yr id o[1,2-c]p yr im id in e (25a ). A solution of (4aS,5R)-
2-benzyl-5-[(1R,2S)-2-N-(benzyloxycarbonyl)amino-1-cyano-
3-(indol-3-yl)-propyl]amino-1,3-dioxoperhydropyrido[1,2-c]py-
rimidine (21a ) (60 mg, 0.1 mmol) in 10-5 N HCl in MeOH (20
mL) was hydrogenated at room temperature and 40 psi of
pressure for 4 days, in the presence of 10% Pd(C) (28 mg). After
filtering off the catalyst, the solvent was evaporated, and the
resulting residue was dissolved in dry CH2Cl2 (6 mL). Tri-
ethylamine (28 µL, 0.2 mmol) was added to the resulting
solution, and the mixture was stirred at room temperature for
15 min. Then, bis(trichloromethyl)carbonate (6 mg, 0.05 mmol)
and triethylamine (17 µL, 0.12 mmol) were added at 0 °C, and
the stirring was continued at this temperature for 12 h.
Afterward, the reaction mixture was diluted with CH2Cl2 (25
mL), washed with water (10 mL) and brine (10 mL), and dried
over Na2SO4. Removal of the solvent and preparative TLC of
the residue, using 6% of MeOH in CH2Cl2 as eluant, gave the
2-oxoimidazolidine derivative 25a as a foam (13 mg, 26%): RP
HPLC tR ) 5.73 (A:B ) 40:60); H NMR (300 MHz, CDCl3) δ
1.54 (m, 1H, 7-H), 1.80 (m, 3H, 6-H and 7-H), 2.63 (m, 1H,
4-H), 2.67 (m, 1H, 8-H), 2.94 (dd, 1H, 4-H, J ) 6 and 17 Hz),
3.34 and 3.36 [2dd, 2H, 4-CH2 (2-oxoimidazolidine), J ) 6 and
15 Hz], 3.37 (m, 1H, 5-H), 3.79 (m, 1H, 4a-H), 4.16 [d, 1H,
5-H (2-oxoimidazolidine), J ) 5 Hz], 4.25 [m, 1H, 4-H (2-
oxoimidazolidine)], 4.35 (m, 1H, 8-H), 4.94 and 5.00 (2d, 2H,
2-CH2, J ) 14 Hz), 7.13-7.54 (m, 10H, aromatics), 8.27 [s,
1H, 1-H (In)]; 13C NMR (125 MHz, CDCl3) δ 23.77 (C7), 27.10
(C6), 30.75 [4-CH2 (2-oxoimidazolidine)], 33.59 (C4), 44.14 (2-
CH2), 44.96 (C8), 49.14 [C5 (2-oxoimidazolidine)], 52.72 (C4a),
55.47 [C5, and C4 (2-oxoimidazolidine)], 108.41 [C3 (In)], 111.75
[C7 (In)], 117.28 (CN), 117.95 [C4 (In)], 120.39 [C5 (In)], 122.91
[C4 (Ph)], 123.18 [C2 (In)], 126.97 [C3a (In)], 127.36 [C6 (In)],
128.40 and 128.46 [C2 and C3 (Ph)], 136.33 [C7a (In)], 137.59
[C1 (Ph)], 153.53 (C1), 158.19 [C1 (2-oxoimidazolidine)], 167.38
(C3). Anal. (C28H28N6O3) 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 described by
Dauge´ et al,36 with minor modifications. 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 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 1 nM
[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.
Nonspecific binding was determined using 1 µM CCK-8 as the
cold displacer. The inhibition constants (Ki) were calculated

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4667
Pharmacol. 1990, 99, 138P. (c) Hendrie, C. A.; Neill, J . C.;
Shepherd, J . K.; Dourish, C. T. The Effects of CCK-A and CCK-B
Antagonists on Activity in the Black/White Exploration Model
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using the equation of Cheng and Prusoff from the displacement
curves analyzed with the receptor fit competition LUNDON
program.
Ca er u lein -In d u ced Acu te P a n cr ea titis in Ra ts. Acute
pancreatitis was induced in male Wistar rats (190-210 g)
deprived of food for 24 h by four subcutaneous injections of
caerulein (10 µg/kg) at hourly intervals as described.41 CCK
antagonists were administered by intraperitoneal or oral route
(0.25 mL/100 g) 30 or 60 min, respectively, before the first
caerulein injection. Three hours after the last caerulein
injection, blood was collected and the pancreas was removed.
Amylase and lipase activities in plasma were measured by
means of commercially available kits (Boehringer Mannheim).
Sp on ta n eou s Locom otor Activity. Hypomotility was
induced in mice by ip injection of CCK-8 (10 µg/kg) 5 min before
recording the locomotion for 30 min.40 Test compounds (0.1
mL/10 g) were given ip 30 min before CCK or po at different
times before CCK. Locomotion was measured by placing the
mice in a black wooden open-topped box (65 × 65 × 45 cm3)
and recording the distance travelled in centimeters by using
a digital Videomex-V-system (Columbus Inst.) working with
the appropriate computer program.
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Characterization of a New and Extremely Potent and Selective
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rey, I.; Gonza´lez-Mun˜iz, R.; Garc´ıa-Lo´pez, M. T.; Ballaz, S.;
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M.; Go´mez-Monterrey, I.; Herranz, R.; Gonza´lez-Mun˜iz, R.;
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a Highly Selective CCKA Receptor Antagonist with Anxiolytic-
like Activity in Animal Models. Br. J . Pharmacol. 1997, 121,
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Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.;
Anderson, P. S.; Chang, R. S. L.; Lotti, V. J .; Cerino, D. J .; Chen,
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Ack n ow led gm en t. This work has been supported
by the CICYT (SAF 97-0030) and Fundacio´n La Caixa
(97/022). The contribution of J ose´ M. Bartolome´-Ne-
breda to this work was awarded with the 1997 J anssen-
Cilag award of the Spanish Society of Therapeutic
Chemistry for Young Researchers.
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