5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK1 receptor antagonists: Structure-activity relationship studies on the substituent at N2-position

Article abstract of DOI:10.1021/jm010813d

To establish structure-activity relationships a new series of analogues of the highly potent and selective CCK₁ receptor antagonist (4aS,5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-dioxoperhydropyrido [1,2-c]-pyrimidine (1a) modified at N2-position of the central scaffold has been prepared and evaluated as CCK receptor ligands. With this aim the N2-benzyl group has been replaced by methyl, cyclohexyl, aromatic groups, 1-phenylethyl, and 1-carboxy-2-phenylethyl group. Then, substituents with different electronic and steric properties were introduced into different positions of the phenyl group of analogues 19a and 19b. The results of the CCK receptor binding and in vitro functional activity evaluation suggest the importance of the lipophilic character and an appropriate spatial orientation of the moiety linked at the N2-position of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine template for potent and selective binding and antagonist activity at CCK₁ receptor subtype. The 2-cyclohexyl and (2S)-1-naphthyl derivatives 18a and (2S)-20a have emerged as more potent and selective CCK₁ receptor antagonists than the lead compound 1a. Additionally, the results confirm the (4aS,5R)-stereochemistry at the central bicyclic skeleton as an essential structural requirement for potent binding to this receptor subtype.

Full text of DOI:10.1021/jm010813d

J . Med. Chem. 2001, 44, 2219-2228
2219
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 e-Activity Rela tion sh ip
Stu d ies on th e Su bstitu en t a t N2-P osition
J ose´ M. Bartolome´-Nebreda,§ Rosario Patin˜o-Molina,§ Mercedes Mart´ın-Mart´ınez,§ Isabel Go´mez-Monterrey,§
M. Teresa Garc´ıa-Lo´pez,§ Rosario Gonza´lez-Mun˜iz,§ Edurne Cenarruzabeitia,# Miriam Latorre,#
J oaqu´ın Del R´ıo,# and Rosario Herranz*,§
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, E-28006 Madrid, Spain, and Departamento de Farmacolog´ıa,
Universidad de Navarra, Irunlarrea 1, E-31080 Pamplona, Spain
Received J anuary 10, 2001
To establish structure-activity relationships a new series of analogues of the highly potent
and selective CCK1 receptor antagonist (4aS,5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-
dioxoperhydropyrido[1,2-c]-pyrimidine (1a ) modified at N2-position of the central scaffold has
been prepared and evaluated as CCK receptor ligands. With this aim the N2-benzyl group has
been replaced by methyl, cyclohexyl, aromatic groups, 1-phenylethyl, and 1-carboxy-2-
phenylethyl group. Then, substituents with different electronic and steric properties were
introduced into different positions of the phenyl group of analogues 19a and 19b. The results
of the CCK receptor binding and in vitro functional activity evaluation suggest the importance
of the lipophilic character and an appropriate spatial orientation of the moiety linked at the
N2-position of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine template for potent and selective
binding and antagonist activity at CCK1 receptor subtype. The 2-cyclohexyl and (2S)-1-naphthyl
derivatives 18a and (2S)-20a have emerged as more potent and selective CCK1 receptor
antagonists than the lead compound 1a . Additionally, the results confirm the (4aS,5R)-
stereochemistry at the central bicyclic skeleton as an essential structural requirement for potent
binding to this receptor subtype.
In tr od u ction
shed further light on their functional roles. In this
regard, we reported the design, synthesis,13 and phar-
macological properties14 of the 5-(tryptophyl)amino-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivative 1a (IQM-
Cholecystokinin (CCK) is a regulatory peptide hor-
mone, found predominantly in localized endocrine cells
of the gastrointestinal tract, and a neurotransmitter
present throughout the nervous system.1 In the gas-
trointestinal system CCK regulates motility, pancreatic
enzyme secretion, gastric emptying, and inhibition of
gastric acid secretion.1,2 In the nervous system CCK is
involved in anxiogenesis,1,3,4 satiety,1,5 nociception,1,6
memory and learning processes,3,6-8 and regulation of
dopamine release.1,6 These biological effects are medi-
ated by two specific G protein coupled receptor subtypes,
termed CCK1 and CCK2.6,9
The variety of physiological effects of CCK and its
possible role in some pathological disorders have stimu-
lated research in this area and, over the past decade, a
number of potent and selective non-peptide CCK1 and
CCK2 receptor agonists and antagonists have been
reported.6,10-12 Some of these ligands have been useful
tools for characterizing both CCK receptor subtypes and
for gaining further insight into the functional signifi-
cance of CCK in the periphery and in the central
nervous system (CNS). However, the physiological ef-
fects of CCK mediated by CCK1 or CCK2 receptors are
not completely established.1,6 Therefore, the develop-
ment of CCK receptor antagonists with higher selectiv-
ity for both receptor subtypes is of interest in order to
95,333), which is one of the most selective CCK1 receptor
antagonists described.6 Thus, this compound showed a
CCK1 receptor affinity in the nanomolar range, but it
was devoid of affinity at brain CCK2 receptors.14 In
accordance with this CCK1 receptor affinity, compound
1a was a potent inhibitor of the CCK-8-stimulated
amylase release from isolated pancreatic acini and
blocked the CCK-8-induced hypophagia and hypoloco-
motion in rats.14 Furthermore, despite the predominant
role attributed to CCK2 receptors in the anxiogenic
effects of CCK,3,6 this CCK1 antagonist also showed a
marked anxiolytic-like activity in animal models.14
These results supported the suggestion of some authors
that CCK1 receptors may be involved also in anxio-
genesis.15-17 Previous structure-activity relationship
studies on these 1,3-dioxoperhydropyrido[1,2-c]pyrimi-
dine-based CCK1 antagonists showed the importance of
the 4a,5-trans stereochemistry at the 1,3-dioxoperhy-
* To whom correspondence should be addressed. Tel: 34-91-
5622900. Fax: 34-91-5644853. E-mail: rosario@iqm.csic.es.
§ Instituto de Qu´ımica Me´dica (CSIC).
#
Departamento de Farmacolog´ıa.
10.1021/jm010813d CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/25/2001

2220 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bartolome´-Nebreda et al.
Sch em e 1a
F igu r e 1. NOE relationships observed between the 2,3-
dioxoperhydropyrido[1,2-c]pyrimidine skeleton and the naph-
thyl protons in the DPFGSE-NOE spectra of compounds 20.
Then, sequential N-Boc removal and coupling with Boc-
L-Trp-OH, using BOP as coupling reagent, provided the
(5:1) diastereoisomeric mixtures of 5-(Boc-tryptophyl)-
amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine deriva-
tives 17a ,b-29a ,b, which were chromatographically
resolved in most cases. The 2-methyl derivatives 4 and
17 were prepared using Boc-L-Orn(Z)-OH as starting
material for the synthesis of the piperidine 2, isolating
in this case only the major final diastereoisomer 17b.
The noncommercial 4-pyridylisocyanate was generated
in situ from 4-aminopyridine, by reaction with bis-
(trichloromethyl)carbonate in the presence of Et3N.19
NaH was used as base for the cyclization reaction of
N-carbamoylpiperidines 3, except for the 4-pyridyl and
the 4-nitrophenyl derivatives where, due to the lower
nucleophily of their carbamoyl NH,20,21 this cyclization
was slower than the saponification of the ethyl acetate
group, by hydroxide generated from humidity traces in
the reaction medium, and the respective bicyclic com-
pounds 8 and 15 were not obtained. In both cases, using
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as base, no
saponification was observed, although the cyclization
required longer reaction times (7 h at room temperature
for 15 and 4 days at 60 °C for 8, while in the other cases,
using NaH at room temperature, the reaction was
complete in 30 min).
In the synthesis of the 1-naphthyl derivatives 7, two
atropoisomeric pairs of racemic 1,3-dioxoperhydropy-
rido[1,2-c]pyrimidines (2R*,4a R*,5S*)-7 and (2R*,-
4a S*,5R*)-7 were obtained in a (1:2) ratio, due to the
restricted rotation about the N2-naphthyl bond. After
the coupling with Boc-L-Trp-OH, each one of the two
resulting diastereoisomeric pairs was chromatographi-
cally resolved into (2R)-20a and (2S)-20b and (2S)-20a
and (2R)-20b, respectively, in an a :b ratio of (5:1). We
tried to establish the absolute configuration of these four
diastereoisomers by X-ray diffraction. However, it was
not possible to obtain good crystals for this analysis.
Furthermore, when we tried to crystallize them from
hot MeOH, partial stereomutation was observed. Due
to these problems, the configuration was tentatively
assigned by 1H NMR, on the basis of the NOEs observed
in the double pulsed field gradient spin-echo NOE
sequence (DPFGSE-NOE) spectra of compounds (2R)-
20a , (2S)-20a , and (2R)-20b. Thus, as shown in Figure
1, weak NOE enhancements were observed between the
8′-H proton of the naphthyl group and the 4-H and 5-H
protons of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine
a
Reagents: (a) R1-NCO, THF; (b) R1-NH2, (CCl3O)2CO, Et3N,
THF; (c) NaH, THF; (d) DBU, THF; (e) TFA, CH2Cl2; (f) Boc-L-
Trp-OH, BOP, Et3N.
dropyrido[1,2-c]pyrimidine skeleton13 and the presence
of the Boc-L-Trp residue13,18 as essential features for
CCK1 binding affinity and subtype receptor selectivity.
Replacement of the acid labile group Boc with the 3,3-
dimethylbutyryl or the tert-butylaminocarbonyl groups
led to acid-stable compounds with enhanced oral bio-
availability.18 Now, to further define the pharmacophore
of this new family of CCK1 antagonists, a structure-
activity relationship study on the nature of the sub-
stituent at N2-position has been carried out. First, we
have explored the replacement of the benzyl group at
this position by methyl, cyclohexyl, aromatic groups (Ph,
naphthyl, and pyridyl), 1-phenylethyl, and the phenyl-
alanine derived 1-carboxy-2-phenylethyl group. Then,
we have studied the effect of introducing substituents
with different electronic and steric properties into
different positions of the N2-phenyl group. These in-
clude the following: a methyl group, electron-withdraw-
ing groups (NO2 and CO2H), and electron-donating
groups [OMe, OAc, OH, and N(Me)2]. The present paper
deals with the synthesis and CCK receptor binding
profile of this new series of 5-(Boc-tryptophyl)amino-
1,3-dioxoperhydropyrido[1,2-c]pyrimidine derivatives.
Ch em istr y
As indicated in Scheme 1, the new 1,3-dioxoperhy-
dropyrido-[1,2-c]pyrimidine derivatives 17a -29a and
17b-29b were prepared applying a synthetic route
similar to that used for the lead compounds 1. In
general, this route involved reaction of the 2,3-trans-3-
amino-2-piperidineacetic acid derivative 2 [obtained as
a (5:1) racemic mixture of (2S,3R)- and (2R,3S)-isomers
from Boc-D-Orn(Z)-OH13] with the corresponding iso-
cyanate, followed by in situ base catalyzed cyclization
of the resulting N-substituted carbamoylpiperidines of
general formula 3 to their respective 5-Boc-amino-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives 4-16.

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2221
Sch em e 2a
Sch em e 3
a
Reagents: (a) 4-(NH2)-Ph-OAc, (CCl3O)2CO, Et3N, THF; (b)
DBU, THF; (c) TFA, CH2Cl2; (d) Boc-L-Trp-OH, BOP, Et3N,
CH2Cl2.
a
Reagents: (a) H2, Pd(C), HCHO, MeOH; (b) TFA, CH2Cl2; (c)
skeleton in diastereoisomers (2S)-20a and (2R)-20b,
while in (2R)-20a the naphthyl 8′-H proton showed
weak NOE with the 4a-H proton.
Boc-L-Trp-OH, BOP, Et3N, CH2Cl2.
Sch em e 4
Similarly, due to restricted rotation, the 2-(2-meth-
ylphenyl)-1,3-dioxoperhydropyrido-[1,2-c]pyrimidines 11
were also obtained as a mixture of atropoisomers at
1
position 2 in a (1.5:1) H NMR estimated ratio. After
N-Boc removing and coupling with Boc-L-Trp-OH, this
mixture was chromatographically resolved in the two
atropoisomers (2R)- and (2S)-24a , and the mixture
(2RS)-24b, in a (a :b ) 5:1) ratio. In this case, the
stereomutation at N2 in each epimeric pair was faster
than in compounds 20, and, after the chromatographic
resolution process, each isomer (2R)- and (2S)-24a was
obtained with, at least, 10% of its corresponding epimer.
This stereomutation was studied in the pair (2R)- and
(2S)-20a , at room temperature and acetonitrile solution,
by RP-HPLC. In these conditions the equilibrium was
reached in 3 days in a (2:3) (2R)/(2S) epimeric ratio. The
absolute configuration at N2 in this epimeric pair was
tentatively assigned on the basis of the NOE observed
in the NOESY spectrum of (2R)-24a between the
methyl protons of the 2-methylphenyl group and the
4-H oriented toward the same face that the Boc-Trp
moiety.
As indicated in Scheme 2, partial deacetylation took
place in the synthesis of the 2-(4-acetoxyphenyl)-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivative 30, ob-
taining a 15% of the phenolic derivative 31. After N-Boc-
removal and coupling with Boc-L-Trp-OH, only the
major diastereoisomers of the tryptophyl derivatives 32a
and 33a could be isolated.
a
Reagents: (a) H-L-Phe-OMe‚HCl, (CCl3O)2CO, Et3N, THF; (b)
NaH, THF; (c) TFA, CH2Cl2; (d) Boc-L-Trp-OH, BOP, Et3N,
CH2Cl2; (e) NaOH, MeOH, H2O.
Compounds incorporating the 3- or 4-(dimethylami-
no)phenyl moiety at position 2 of the perhydropyrido-
[1,2-c]pyrimidine skeleton 36a ,b and 37a ,b were ob-
tained from the corresponding nitro derivatives 14 and
15, by catalytic hydrogenation in the presence of form-
aldehyde, followed by N-Boc-removal and coupling with
Boc-L-Trp-OH (Scheme 3).
Applying a similar synthetic pathway, the reaction
of the 2,3-trans-3-amino-2-piperidineacetic acid deriva-
tive 2 with the hydrochloride of phenylalanine methyl
ester and bis(trichloromethyl)carbonate in the presence
of Et3N gave the corresponding 1-substituted-carbam-
oylpiperidine derivative 38 (Scheme 4). In the following
NaH catalyzed cyclization the ethoxide, generated from
the 2-ethoxycarbonylmethyl group, produced a partial
transesterification of the methyl ester group of 38,
obtaining a (1:1) mixture of methyl and ethyl esters of
the (4aS,5R)-5-(Boc-amino)-1,3-dioxoperhydropyrido-
[1,2-c]pyrimidines 39a and 40a (38%), along with the
ethyl ester of the (4aR,5S)-diastereoisomer 40b (10%).
After N-Boc-removing, and coupling with Boc-L-Trp-OH,
the methyl and ethyl ester mixture 39a and 40a was
chromatographically resolved into the corresponding
Boc-tryptophyl derivatives 41a and 42a , respectively.
The saponification of the methyl ester in compound 41a
gave the 1-carboxy-2-phenylethyl derivative 43a . In the
1H-NMR spectra of compounds 39-43 some signals
appeared duplicated (methyl or ethyl protons, indolic
4-H and phenylalanine R-H), indicating the presence of

2222 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bartolome´-Nebreda et al.
Sch em e 5
These initial results indicated the preference for lipo-
philic groups at the N2-position.
With respect to the introduction of additional sub-
stituents into the N2-phenyl group of 19a this change
led, in general, to slight or moderate decreases in the
CCK1 binding affinity and antagonist potency, indepen-
dently of their electronic and steric properties. Thus,
as an example, the effect of introducing the electron-
withdrawing nitro group (27a and 28a ) was similar to
that of introducing the electron-donating dimethylamino
group (36a and 37a ). The decrease in affinity was higher
when the group was introduced into a meta- than into
a para-position. However, the CCK1 binding profile of
compound 19a was slightly improved when a methyl
group was introduced into ortho-position of the phenyl
group (24a ), or when this group was replaced by a
1-naphthyl group in a (2S)-configuration [(2S)-20a ]. In
both cases the rotation about the N2-aryl bond is
restricted. The difference between both atropoisomers
(2S)-20a and (2R)-20a in binding affinities and antago-
nistic activities (25- and 127-fold, respectively) show the
importance of the aryl group orientation. On the other
hand, it is interesting to note the important decrease
in affinity, by more than 2 orders of magnitude, which
resulted from the replacement of the phenyl group with
the 4-pyridyl in 21a ,b as well as the complete loss of
binding affinity with the introduction of the hydroxy or
carboxy acid groups into the phenyl moiety (33a or
47a ,b).
a
Reagents: (a) H2, Pd (C), EtOH; (b) 3-(NH2)-Ph-CO2H,
(CCl3O)2CO, Et3N, THF; (c) NaH, THF.
two rotamers, probably due to restricted rotation about
the N2-C bond. None of these pairs of rotamers could
be resolved.
Finally, in the synthesis of the 3-carboxyphenyl
derivatives 47 (Scheme 5) the construction of the
perhydropyrido[1,2-c]pyrimidine skeleton was posterior
to the coupling with Boc-L-Trp-OH, to avoid the protec-
tion and final deprotection of the carboxyl group. Thus,
(2R*,3S*)-2-ethoxycarbonylmethyl-3-(Boc-tryptophyl)-
aminopiperidine (45a ,b) [obtained from the Boc-L-Trp-
D-Orn(Z)-OH derived â-ketoester 44 as a (2:1) diaste-
reoisomeric mixture of (2S,3R)- and (2R,3S)-isomers,
which could not be resolved22] was used as starting
material for the reaction with 3-carboxyphenyl isocy-
anate, generated in situ from the 3-aminobenzoic acid.
The chromatographic resolution of the diastereoisomeric
pairs 45-47 was not possible.
Finally, the replacement of both prochiral protons of
the benzyl group of the model compound 1a with a
methyl group led to the ten- and 4-fold less potent
compounds 22a and 23a , respectively. The influence of
the configuration at the additional chiral center of these
two compounds shows again the importance of the aryl
group orientation. On the other hand, the phenylalanine
derivatives 41-43 were completely inactive. The ab-
sence of binding affinity in these compounds could be
due to an unfavorable interaction of the additional
alcoxycarbonyl or carboxy groups into the pocket of
accommodation of the N2-substituent within the CCK1
receptor or to an excessive lengthening of the linker
chain between the phenyl group and the central scaffold.
Biologica l Resu lts a n d Discu ssion
As shown in Table 1, the affinity of all new 5-(Boc-
tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimi-
dine derivatives at CCK1 and CCK2 receptors was
determined by measuring the displacement of [3H]-
propionyl-CCK-8 binding to rat pancreatic and cerebral
cortex homogenates, respectively, as previously de-
scribed.23 Subsequently, those compounds which showed
a significant affinity at CCK1 receptors at concentrations
below 10-7 M were tested for their antagonism of the
CCK-8-stimulated amylase release from pancreatic aci-
nar cells.24 For comparative purposes CCK-8 and the
model compounds 1a and 1b were also included in the
assays. From the results, it can be deduced that, in
general, the new N2-modified compounds with a
(4aS,5R)-stereochemistry (compounds a ) keep the high
potency and selectivity in their binding to the CCK1
receptor subtype versus the CCK2 as well as the in vitro
functional antagonistic activity.
The replacement of the N2-benzyl group of the model
compound 1a by a cyclohexyl or phenyl group had no
significant effect on the binding potency and selectivity
of 18a and 19a for CCK1, while in 1b, this change led
to a decrease of an order of magnitude in the affinities
of 18b and 19b. A higher decrease in the CCK1 binding
potency of 1b, of at least 2 orders of magnitude, resulted
when its benzyl group was replaced by a methyl in 17b.
In general, a quite good correlation was observed
between the binding potency of the best compounds to
CCK1 receptors and the values for inhibition of CCK-
8-stimulated amylase release from pancreatic acinar
cells. Like the model compounds 1a and 1b, the new
analogues did not show any intrinsic effect on the
amylase release at a 10 µM concentration.
In conclusion, the biological results herein reported
show that the group linked to the N2-position of the 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine skeleton is an
essential element of the pharmacophore of this CCK1
receptor antagonist family and suggest the importance
of the lipophilic character and spatial orientation of that
group for a potent interaction with the CCK1 receptor.
On the other hand, these results confirm, again, the
(4aS,5R)-stereochemistry at the central scaffold as a
highly strict structural requirement for the selective and
potent binding to this receptor. Ligands 18a , (2S)-20a ,
and 24a , endowed with subnanomolar affinity, have
emerged as more potent and selective CCK1 antagonists
than the lead compound 1a and could serve to fine-tune

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2223
Ta ble 1. Inhibition of [3H]pCCK-8 Specific Binding to Rat Pancreas (CCK1) and Cerebral Cortex Membranes (CCK2) and Inhibition
of Amylase Release from Dispersed Pancreatic Acini
IC50 (nM)a
compd
CCK-8
1a
1b
17b
18a
18b
19a
stereochemistry
R1
CCK1
CCK2
amylase releaseb IC50 (nM)
1.04 ( 0.08
1.59 ( 0.10
22.70 ( 4.0
9162
0.60 ( 0.05
165 ( 36
5.60 ( 0.30
>10000
6153
>10000
>10000
7159
(4aS,5R)
(4aR,5S)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(2R,4aS,5R)
(2S,4aS,5R)
(2R,4aR,5S)
(2S,4aR,5S)
(4aR*,5S*)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aS,5R)
(4aS,5R)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR*,5S*)
CH2-Ph
CH2-Ph
Me
cyclohexyl
cyclohexyl
Ph
0.64 (0.4-0.9)
77 (73-81)
NDc
3.0 (1.8-3.7)
287 (205-325)
3.5 (1.4-10.1)
>1000
1.18 ( 0.72
628.0 ( 28
15.4 ( 7.3
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
1320
19b
Ph
(2R)-20a
(2S)-20a
(2R)-20b
(2S)-20b
21a ,b (5:1)
22a
22b
23a
23b
24a
24b
25a
25b
26a
26b
27a
27b
28a
28b
29a
32a
33a
36a
36b
37a
37b
41a
42a
42b
43a
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
4-pyridyl
93.0 (39-220)
0.73 (0.4-2.1)
NDc
0.59 ( 0.04
>1000
>1000
386 ( 36.2
15.0 ( 1.85
>1000
6.89 ( 1.1
>1000
0.97 ( 0.43
>1000
3.98 ( 2.57
100
2.90 ( 1.2
200
44.0 ( 2.5
>1000
12.0 ( 2.8
NDc
NDc
(R)-CH-(Me)Ph
(R)-CH-(Me)Ph
(S)-CH-(Me)Ph
(S)-CH-(Me)Ph
2-(Me)Ph
12.63 (5.2-23.8)
NDc
112 (55-232)
NDc
1.89 (0.6-3.0)
2-(Me)Ph
3-(Me)Ph
3-(Me)Ph
4-(Me)Ph
4-(Me)Ph
3-(NO2)Ph
3-(NO2)Ph
4-(NO2)Ph
4-(NO2)Ph
4-(OMe)Ph
3-(OAc)Ph
3-(OH)Ph
NDc
10.0 (5.7-24.3)
NDc
>10000
16.6 (9.1-23.4)
2320
NDc
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
2320
>10000
>10000
>10000
>10000
>10000
49.5 (20.8-57.1)
NDc
64.0 (62.6-65.7)
>1000
NDc
15.9 ( 2.9
70.3 ( 4.7
38.1 (34.1-44.3)
NDc
>1000
NDc
3-[N(Me)2]Ph
3-[N(Me)2]Ph
4-[N(Me)2]Ph
4-[N(Me)2]Ph
(S)-CH(CO2Me)-CH2-Ph
(S)-CH(CO2Et)-CH2-Ph
(S)-CH(CO2Et)-CH2-Ph
(S)-CH(CO2H)-CH2-Ph
3-(CO2H)Ph
50.8 ( 15.5
>1000
4.3 ( 1.05
>1000
NDc
NDc
39.8 (32.3-54.5)
NDc
>1000
>1000
>1000
>1000
>1000
NDc
NDc
NDc
NDc
47a ,b (2:1)
NDc
a
Values are the mean or mean( SEM of at least three experiments, performed with seven concentrations of test compounds in triplicate.
b
Inhibition of amylase release stimulated by CCK-8 (0.1 nM) in dispersed pancreatic acini. Data represent the mean of three to six
independent experiments in duplicate (standard errors within (15% of the mean). c ND ) not determined.
mm, 4 µm) column, with a flow rate of 1 mL/min, and using a
interactions with this receptor by further structural
modifications.
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.
Exp er im en ta l Section
Gen er al P r ocedu r e for th e Syn th esis of th e (4a R*,5S*)-
5-(ter t-Bu toxyca r bon 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 Der iva tives 4-7, 9-14, a n d 16. Meth od
A. The corresponding isocyanate, methyl isocyanate, cyclohexyl
isocyanate, phenyl isocyanate, 1-naphthyl isocyanate, (1R)-1-
phenylethyl isocyanate, (1S)-1-phenylethyl isocyanate, 2-, 3-,
and 4-methylphenyl isocyanate, 3-nitrophenyl isocyanate, and
4-methoxyphenyl isocyanate (0.39 mmol), was added to a
solution of (2R*,3S*)-3-(tert-butoxycarbonyl)amino-2-(ethoxy-
carbonyl)methylpiperidine25 (2) (100 mg, 0.36 mmol) in dry
THF (2.5 mL). After 1 h of stirring at room temperature, the
reaction mixture was diluted with THF (2.5 mL). Then, NaH
(10 mg of 60% dispersion in mineral oil, 0.39 mmol) was added,
and the stirring was continued for 30 additional min. After-
ward, a 0 °C cooled 1 N HCl solution (25 mL) was added, and
the resulting reaction mixture was extracted with EtOAc (2
× 25 mL). The organic extracts were washed with H2O (25
mL) and brine (25 mL), dried over Na2SO4, and evaporated to
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
with a 0.2 mm layer of silica gel 60 F254, Merck, and prepara-
tive TLC on 20 × 20 cm glass plates coated with a 2 mm layer
of silica gel PF254 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 Varian Gemini-200, Varian INOVA-
300, Varian INOVA-400, or Varian Unity-500 spectrometers,
operating at 200, 300, 400, or 500 MHz, using TMS as
reference. A relaxation time of 1.5 s and a mix time of 600 ms
were used in the NOESY spectra, and 2 s and 500 ms,
respectively, for the DPGSE-NOE spectra. Elemental analyses
were obtained on a CH-O-RAPID apparatus. Analytical RP
HPLC was performed on a Waters Nova-pak C18 (3.9 × 150

2224 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bartolome´-Nebreda et al.
Ta ble 2. Analytical Data of 5-(tert-Butoxycarbonyl)amino-1,3-
dioxoperhydropyrido-[1,2-c]pyrimidines
tography using a (17-66%) gradient of EtOAc in hexane as
eluant to give 15 (123 mg, 85%), whose significant analytical
and spectroscopic data are shown in Tables 2 and 3.
compd
yield (%)
mpa (°C)
formulab
Syn th esis of th e (4a R*,5S*)-2-[4-(Aceth oxy- a n d Hy-
d r oxy)p h e n yl]-5-(t er t -b u t oxyca r b on yl)a m in o-1,3-d i-
oxop er h yd r op yr id o[1,2-c]p yr im id in es 30 a n d 31. These
compounds were prepared as above-mentioned in method B,
from 4-aminophenol acetate [189 mg, 1.25 mmol; obtained from
4-nitrophenol acetate by catalytic hydrogenation in the pres-
ence of 10% Pd(C)26] and (2R*,3S*)-3-(tert-butoxycarbonyl)-
amino-2-(ethoxycarbonyl)-methylpiperidine25 (2) (175 mg, 0.63
mmol). The reaction products were purified by flash chroma-
tography, using a (33-66%) gradient of EtOAc in hexane as
eluant, obtaining the 4-acethoxyphenyl derivative 30 as major
product (79 mg, 37%) with higher Rf and 31 (26 mg, 13%)
in the lower Rf, fraction. The significant analytical and spec-
troscopic data of these compounds are indicated in Tables 2
and 3.
Syn th esis of th e (4a R*,5S*)-5-(ter t-Bu toxyca r bon yl)-
a m in o-2-(3- a n d 4-d im eth yla m in op h en yl)-1,3-d ioxop er -
h yd r op yr id o[1,2-c]p yr im id in es 34 a n d 35. A solution of the
(4aR*,5S*)-5-(tert-butoxycarbonyl)amino-2-(3- or 4-nitrophen-
yl)-1,3-dioxoperhydropyrido-[1,2-c]pyrimidines 14 or 15 (100
mg, 0.25 mmol) in MeOH (25 mL) was hydrogenated in the
presence of 37% aqueous formaldehyde (75 µL, 1 mmol) and
10% Pd(C) (20 mg), at room temperature and 3 atm of H2
pressure, for 2 and 3 days, respectively. Then, the catalyst was
filtered off, washed with MeOH, and the resulting solution was
evaporated to dryness. The residue was purified by flash
chromatography, using a (50-75%) gradient of EtOAc in
hexane as eluant, to give the pure compounds 34 and 35,
whose analytical and spectroscopic data are summarized in
Tables 2 and 3.
Syn th esis of th e (4a R*,5S*)-5-(ter t-Bu toxyca r bon yl)-
a m in o-2-[(1S)-1-(m eth oxyca r bon yl)- a n d (eth oxyca r bo-
n yl)-2-p h en ylet h yl]-1,3-d ioxop er h yd r op yr id o[1,2-c]p y-
r im id in es 39a , 40a , a n d 40b. Bis(trichoromethyl)carbonate
(123 mg, 0.42 mmol) was added to a suspension of H-L-Phe-
OMe‚HCl (226 mg, 1.25 mmol) and TEA (175 µL, 1.25 mmol)
in dry THF (3 mL) cooled at 0 °C. Then, TEA (354 µL, 2.52
mmol) was added dropwise throughout 5 min, continuing the
stirring at 0 °C for 5 additional min. Afterward, a solution of
(2R*,3S*)-3-(tert-butoxycarbonyl)amino-2-(ethoxycarbonyl)-
methylpiperidine25 (2) (175 mg, 0.63 mmol) in dry THF (3 mL)
was added at room temperature, and this mixture was stirred
at room temperature for 1 h. Then, the reaction mixture was
diluted with EtOAc (50 mL), washed successively with H2O
(25 mL) and brine (25 mL), dried over Na2SO4, and evaporated
to dryness. The residue was dissolved into dry THF (9 mL),
and NaH (27 mg of 60% dispersion in mineral oil, 0.61 mmol)
was added to this solution. This reaction mixture was stirred
at room temperature for 30 min, then a 0 °C cooled 1 N HCl
solution (25 mL) was added, and the resulting mixture was
extracted with EtOAc (2 × 25 mL). The organic extracts were
washed with H2O (25 mL) and brine (25 mL), dried over
Na2SO4, and evaporated to dryness. The residue was purified
by preparative TLC, using (1:1) EtOAc-hexane mixture as
eluant, obtaining the unresolved mixture 39a + 40a (136 mg,
46%) as major fraction with higher Rf and 40b (24 mg, 12%)
as lower Rf fraction. Significant analytical and spectroscopic
data of these compounds are summarized in Tables 2 and 3.
Gen er a l P r oced u r e for th e Syn th esis of th e 5-[N-(ter t-
Bu t oxyca r b on yl)-L-t r yp t op h yl]a m in o-1,3-d ioxop er h y-
d r op yr id o[1,2-c]p yr im id in e Der iva tives 17-29, 32, 33, 36,
37, 41, a n d 42. TFA (0.5 mL) was added to a solution of the
corresponding N-Boc protected 1,3-dioxoperhydropyrido[1,2-
c]pyrimidine derivative 4-16, 30, 31, 34, 35, 39, and 40, (0.17
mmol) in dichloromethane (2 mL), and after 30 min at room
temperature, the solvents were evaporated to dryness. The
residue was dissolved in dry dichloromethane (2 mL), and Boc-
L-Trp-OH (65 mg, 0.21 mmol), benzotriazol-1-yloxytris(dim-
ethylamino)phosphonium hexafluorophosphate (BOP, 94 mg,
0.21 mmol), and TEA (54 µL, 0.38 mmol) were added succes-
sively to that solution, and stirring was continued at room
4
5
6
80
78
85
27
48
60
80
71
81
35
48
61
85
40
30
11
46
80
34
10
174-176
foam
210-212
120-122
119-121
foam
syrup
67-69
foam
94-96
209-211c
100-102
90-92
95-97
foam
foam
syrup
foam
C14H23N3O4
C19H31N3O4
C19H25N3O4
C23H27N3O4
C23H27N3O4
C18H24N4O4
C21H29N3O4
C21H29N3O4
C20H27N3O4
C20H27N3O4
C20H27N3O4
C19H24N4O6
C19H24N4O6
C20H27N3O5
C21H27N3O6
C19H25N3O5
C21H30N4O4
C21H30N4O4
(2R*,4a R*,5S*)-7
(2R*,4a S*,5R*)-7
8
9
10
11
12
13
14
15
16
30
31
34
35
39a + 40a (1:1)
40b
syrup
C24H33N3O6
a
b
From EtOAc/hexane. Satisfactory analyses for C, H, N.
c Sublimation.
dryness. Except for the case of the naphthyl derivatives 7, the
residue was purified by flash chromatography using a (33-
50%) gradient of EtOAc in hexane as eluant. The naphthyl
derivatives 7 were purified by preparative TLC using a (1:1)
EtOAc-hexane mixture as eluant. The higher Rf fraction
corresponded to the minor atropoisomeric pair (2R*,4aR*,5S*)-7
(37 mg, 27%), while the lower Rf fraction corresponded to
(2R*,4a S*,5R*)-7 (56 mg, 48%). Significant analytical and
spectroscopic data of these compounds are summarized in
Tables 2 and 3.
Syn th esis of (4a R*,5S*)-5-(ter t-Bu toxyca r bon yl)a m in o-
2-(4-p yr id yl)-1,3-d ioxop e r h yd r op yr id o[1,2-c]p yr im i-
d in e (8). Meth od B. Bis(trichoromethyl)carbonate (123 mg,
0.42 mmol) was added to a solution of 4-aminopyridine (118
mg, 1.25 mmol) in dry THF (3 mL) cooled at 0 °C. Then, TEA
(354 µL, 2.52 mmol) was added dropwise throughout 5 min,
continuing the stirring at 0 °C for 5 additional min. Afterward,
a solution of (2R*,3S*)-3-(tert-butoxycarbonyl)amino-2-(ethoxy-
carbonyl)methylpiperidine25 (2) (175 mg, 0.63 mmol) in dry
THF (3 mL) was added at room temperature, and the mixture
was stirred at room temperature for 1 h. Then, the reaction
mixture was diluted with EtOAc (50 mL), washed successively
with H2O (25 mL) and brine (25 mL), dried over Na2SO4,
and evaporated to dryness. The residue was dissolved into
dry THF (7 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (96
µL, 0.69 mmol) was added to this solution. This reaction
mixture was refluxed for 4 days, then a 0 °C cooled 1 N HCl
solution (25 mL) was added, and the resulting mixture was
extracted with EtOAc (2 × 25 mL). The organic extracts were
washed with H2O (25 mL) and brine (25 mL), dried over
Na2SO4, and evaporated to dryness. The residue was purified
by flash chromatography using a (1-4%) gradient of MeOH
in dichloromethane as eluant to give 8 (131 mg, 60%), whose
significant analytical and spectroscopic data are shown in
Tables 2 and 3.
Syn th esis of (4a R*,5S*)-5-(ter t-Bu toxyca r bon yl)a m in o-
2-(4-n itr op h en yl)-1,3-d ioxop er h yd r op yr id o[1,2-c]p yr im i-
d in e (15). Meth od C. 4-Nitrophenyl isocyanate (64 mg, 0.39
mmol) was added to a solution of (2R*,3S*)-3-(tert-butoxycar-
bonyl)amino-2-(ethoxycarbonyl)methylpiperidine25 (2) (100 mg,
0.36 mmol) in dry THF (2.5 mL). After 1 h of stirring at room
temperature, the reaction mixture was diluted with THF (2.5
mL). Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (51 µL, 0.39
mmol) was added, and the stirring was continued for 7
additional h. Afterward, a 0 °C cooled 1 N HCl solution (25
mL) was added, and the resulting mixture was extracted with
EtOAc (2 × 25 mL). The organic extracts were washed with
H2O (25 mL) and brine (25 mL), dried over Na2SO4, and evap-
orated to dryness. The residue was purified by flash chroma-

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2225
Ta ble 3. Significant 1H NMRa Spectroscopic Data of 5-(tert-Butoxycarbonyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidines
compd
4-H
4a-H
5-H
6-H
1.56
7-H
8-H
5-NH
4.52
R1
4
5
2.69
2.65
2.91
2.86
3.10
3.05
3.40
3.38
2.05
1.56
2.69
2.65
4.36
3.20
1.20-1.85 2.07 1.20-1.85
2.23
4.25-4.60 4.25-4.60 4.25-4.60, 2.26,
1.20-1.85
6
2.89
2.97
3.09
3.17
3.22
3.36
3.15
3.28
3.10
3.07
3.50
3.57
3.60
3.58
3.44
3.41
1.72
1.34
1.35
1.31
1.36
1.35
1.41
1.35
1.40
1.26
2.16
2.08
2.07
2.15
2.08
2.07
2.13
2.13
2.11
2.17
1.72
1.56-1.83
2.70
2.70
2.66
2.76
2.65
2.56
4.35
4.35
4.35
4.38
4.33
4.30
4.38
4.37
4.36
4.39
4.58
4.62
4.67
4.47
4.39
4.38
4.51, 4.55
4.48
4.48
4.50
7.10-7.40
7.36, 7.65, 7.92
7.72, 7.67, 7.92
7.14, 8.70
6.09b
(2R*,4a R*,5S*)-7
(2R*,4a S*,5R*)-7
8
9
10
11
12
13
14
3.09
1.67
1.81
1.88
1.81
1.79
2.95
2.77
2.70
3.11
2.93
2.93
1.68
1.66
1.59
1.69
1.66
1.56
1.67
6.07b
2.93, 2.94 3.10, 3.12 3.26 3.53, 3.55
1.83, 1.85 2.73, 2.74
2.10, 2.16c
2.36c
2.91
2.90
2.97
3.10
3.10
3.13
3.18
3.24
3.32
3.52
3.50
3.57
1.85
1.85
1.88
2.72
2.71
2.77
2.37c
7.52, 7.62,
8.07, 8.26
7.48, 8.27
3.82c
15
16
30
31
34
35
2.72
2.90
2.87
2.91
2.94
2.85
2.91
3.10
3.05
3.10
3.11
3.05
3.41
3.26
3.20
3.24
3.25
3.16
2.87
2.93
3.41
3.52
3.49
3.55
3.50
3.47
3.15
2.93
1.48
1.25
1.25
1.27
1.71
1.34
1.93
1.32
1.86
2.13
2.06
2.13
2.12
2.06
1.96
1.94
1.72
2.72
2.72
2.68
2.75
2.76
2.68
2.56
2.44
4.09
4.38
4.33
4.39
4.41
4.33
4.31
4.22
7.10
4.48
4.68
4.57
4.41
4.68
4.31
4.22
1.69
1.64
1.85
1.79
2.28c
1.66-1.86
1.60 1.80
6.69, 6.92
2.94c
1.64-1.69
2.29c
39a + 40a (1:1)
40b
2.68
1.73
5.77, 5.66b
5.60b
2.50
2.79
1.56
1.71
a
b
Spectra registered at 300 MHz in CDCl3, except for 15 which was registered in DMSO-d6. δ corresponding to the 1-H of R1. c δ
corresponding to CH3.
Ta ble 4. Analytical Data of 5-[N-(tert-Butoxycarbonyl)-L-
temperature for 24 h. Afterward, the reaction mixture was
tryptophyl]amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidines
diluted with dichloromethane (25 mL), washed successively
compd
17b
yield
mpa (°C)
formulab
tR (min) (A:B)c
with 10% citric acid (10 mL), 10% NaHCO3 (10 mL), H2O (10
mL), and brine (10 mL), dried over Na2SO4, and evaporated.
The resulting Boc-tryptophyl derivatives were purified and
resolved into diastereoisomers a (4aS,5R) and b (4aR,5S) by
preparative TLC, using 5% EtOAc in ethyl ether (17-26) or
1% MeOH in dichloromethane (27-29, 32, 33, 36, 37, 41, and
42) as eluants. In compounds 17, only the major diastereoi-
somer b was isolated, and the diasteroisomeric resolution was
not possible for the 4-pyridyl derivatives 21. Significant
70
63
16
46
9
174-176
180-182
128-130
143-145
180-182
151-153
148-150
137-139
139-141
foam
C25H33N5O5 8.23 (45:55)d
C30H41N5O5 31.68 (37:63)
18a
18b
19a
19b
(2R)-20a
(2S)-20a
(2R)-20b
(2S)-20b
21a ,b (5:1)
C
C
C
C
C
C
C
C
30H41N5O5 34.40 (37:63)
30H35N5O5 18.20 (33:67)
30H35N5O5 19.67 (33:67)
34H37N5O5 45.93 (33:67)
34H37N5O5 45.67 (33:67)
34H37N5O5 41.87 (33:67)
34H37N5O5 43.33 (33:67)
29H34N6O5 18.22, 18.28
(25:75)
60
65
11
10
67
1
analytical and H NMR spectroscopic data of the resulting Boc-
tryptophyl derivatives are summarized in Tables 4 and 5.
Syn th esis of (4a S,5R)-5-[N-(ter t-Bu toxyca r bon yl)-L-
tr yp top h yl]a m in o-2-[(1S)-1-(ca r boxy)-2-p h en yleth yl]-1,3-
d ioxop er h yd r op yr id o[1,2-c]p yr im id in e (43a ). Sa p on ifi-
ca tion of 41a . NaOH (0.5 N, 1.2 mL, 0.60 mmol) was added
to a solution of (4aS,5R)-5-[N-(tert-butoxycarbonyl)trypto-
phyl]amino-2-[(1S)-1-(methoxycarbonyl)-2-phenylethyl]-1,3-di-
oxoperhydropyrido[1,2-c]pyrimidine (41a ) (36 mg, 60 µmol) in
MeOH (1 mL), and the reaction mixture was stirred a room
temperature for 24 h. Then, it was evaporated to dryness, and
the residue was successively dissolved with EtOAc (15 mL)
and extracted with H2O (10 mL). The aqueous phase was
acidified with 1 N HCl to pH = 2 and extracted with EtOAc
(15 mL). The extract was washed with brine (5 mL), dried
over Na2SO4, and evaporated, to yield 43a (18 mg, 52%),
whose analytical and spectroscopic data are shown in Tables
4 and 5.
Syn th esis of th e Dip ep tid e Boc-L-Tr p -D-Or n -OH.22 Boc-
D-Orn(Z)-OH (5 g, 13.6 mmol) was added slowly to a solution
of thionyl chloride (3.60 mL, 49.5 mmol) in dry MeOH (50 mL)
cooled at -78 °C. Afterward, the reaction mixture was stirred
at room temperature for 24 h. Then saturated NaHCO3
solution was added dropwise until pH = 8, and the MeOH was
evaporated in vacuum. The resulting suspension was diluted
with H2O (100 mL) and extracted with dichloromethane (200
mL). The extract was washed with brine (150 mL), dried over
Na2SO4, and evaporated. The resulting H-D-Orn(Z)-OMe (3.54
g, 13.3 mmol, 98%) was dissolved in dry dichloromethane (150
mL), and Boc-L-Trp-OH (5.15 g, 16.9 mmol), BOP (7.47 g, 16.9
mmol), and TEA (4.27 mL, 16.9 mmol) were added successively
to the solution. After 6 h of stirring at room temperature, the
reaction mixture was washed successively with 10% citric acid
(100 mL), saturated NaHCO3 (100 mL), H2O (100 mL), and
brine (100 mL), dried over Na2SO4, and evaporated. The
residue was purified by flash chromatography, using 50% of
EtOAc in hexane, to give the dipeptide Boc-L-Trp-D-Orn-OMe
22a
22b
23a
54
10
53
11
73
112-114
99-101
116-118
108-110
131-132
C
C
C
C
32H39N5O5 34.13 (37:63)
32H39N5O5 29.80 (37:63)
32H39N5O5 19.53 (40:60)
32H39N5O5 19.00 (40:60)
23b
(2RS)-24a e
C31H37N5O5 25.27, 27.27
(33:67)
C31H37N5O5 29.73, 31.80
(33:67)
C
C
C
(2RS)-24be
15
120-122
25a
25b
26a
26b
27a
27b
28a
28b
29a
32a
33a
36a
36b
37a
37b
41a
42a
42b
43a e
59
10
56
7
131-133
106-108
128-130
31H37N5O5 30.80 (33:67)
31H37N5O5 33.87 (33:67)
31H37N5O5 31.74 (33:67)
234-236f C31H37N5O5 34.94 (33:67)
70
13
68
12
64
75
48
64
12
60
10
44
44
77
54
foam
foam
foam
foam
foam
foam
foam
foam
foam
foam
foam
75-77
foam
94-96
foam
C
C
C
C
C
C
C
C
C
C
C
C
C
C
30H34N6O7 49.53 (30:70)
30H34N6O7 45.56 (30:70)
30H34N6O7 34.78 (33:67)
30H34N6O7 31.44 (33:67)
31H37N5O6 49.53 (30:70)
32H37N5O7 20.94 (25:75)
30H35N5O6 21.89 (28:72)
32H40N6O5 14.17 (28:72)
32H40N6O5 13.00 (28:72)
32H40N6O5 26.00 (25:75)
32H40N6O5 22.83 (25:75)
34H41N5O7 18.20 (40:60)
35H43N5O7 26.47 (40:60)
35H43N5O7 29.13 (40:60)
C33H39N5O7 73.60, 70.60
(26:74)
C
47a ,b (2:1)
41
foam
31H35N5O7 16.20, 17.60
(30:70)
a
b
From ethyl ether/hexane. Satisfactory analyses for C, H, N.
c Nova-pak C18, A ) CH3CN, B ) 0.05% TFA in H2O. µBondapak
d
C18.
e (2:3) atropoisomeric pair. f Sublimation.
(6.09 g, 80%) as white solid: mp ) 137-139 °C; RP HPLC tR
) 23.45 (A:B ) 50:50); 1H NMR (200 MHz, CDCl3) δ 1.09 [m,

2226 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bartolome´-Nebreda et al.
Ta ble 5. Significant 1H NMRa Spectroscopic Data of 5-[N-(tert-Butoxycarbonyl)-L-tryptophyl]amino-1,3-
dioxoperhydropyrido[1,2-c]pyrimidines
compd
4-H
4a-H
5-H
6-H
7-H
8-H
R-H (Trp)
R1
17b
2.48 3.68
2.40 2.52
2.95
2.85
3.49
3.62
1.20-1.70
1.20-1.70
3.68
2.52
4.09
4.28
4.09
4.43
3.33
18a
18b
1.03
1.60
1.60
1.10-1.85, 2.18,
2.42, 4.40
2.12 2.26
2.50
3.57
1.10-1.85
1.10-1.85
2.50
4.24
4.39
1.10-1.85, 2.12,
2.26, 4.49
19a
19b
(2R)-20a
2.63
2.22
2.70 2.80
3.02
2.69
3.20
3.78
3.72
3.89
1.14
1.18
1.19
1.68
1.89
1.75
1.68
1.67
2.63
2.57
2.70
4.31
4.27
4.33
4.48
4.42
4.51
7.13, 7.38, 7.42
7.12, 7.40, 7.45
1.65
1.75
7.38, 7.47, 7.55,
7.89, 7.90
(2S)-20a
(2R)-20b
(2S)-20b
2.75
3.10
2.70
2.65
3.92
3.83
3.74
1.17
1.20
1.16
1.58-1.80
1.90
1.58-1.80
2.64
2.58
2.55
4.32
4.25
4.25
4.50
4.42
4.42
7.56, 7.64. 7.89,
7.24, 7.48
7.32, 7.54, 7.56,
7.59, 7.91, 7.89
7.43, 7.52, 7.53,
7.89, 7.90
2.22 2.47
2.25 2.35
1.57
1.57
1.70
1.68
1.87
21a ,b (5:1) 2.61, 2.43 3.03, 2.97 3.76, 3.65 1.07, 1.23 1.87, 1.54 1.55, 1.54 1.63, 1.54 2.67, 2.62 4.29, 4.11 4.43, 4.87 7.18, 7.15, 8.69, 8.71
22a
22b
23a
23b
2.47
2.16
2.49
2.11
2.91
2.37
2.87
2.48
3.64
3.57
3.66
3.56
3.82
3.71
3.82
3.70
3.82
3.70
3.81
3.73
3.68
3.77
3.73
3.72
3.71
3.78
3.66
3.72
3.68
3.17
3.18
3.29
3.88
3.74
1.12
1.09
1.03
1.22
1.51-1.74
1.89
1.51-1.74
2.47
2.46
2.54
2.42
4.24
4.17
4.24
4.20
4.46
4.41
4.45
4.39
4.49
4.40
4.51
4.40
4.46
4.41
4.45
4.39
4.06
4.42
4.51
4.46
4.45
4.51
4.51
4.51
4.40
4.43
4.43
4.41
4.31
4.09
6.05b
6.02b
1.42
1.66
1.61
1.67
1.61
1.81
1.42
1.50
6.05b
6.03b
(2RS)-24a 2.68, 2.70 3.05, 3.06
(2RS)-24b 2.22, 2.23 2.67, 2.63
1.53-1.78
1.65, 1.73
2.59, 2.57 4.32, 4.33
2.08, 2.29c
2.06, 2.15c
2.38c
1.16, 1.18 1.94, 1.88 1.59, 1.55 1.94, 1.73 2.57, 2.51 4.27, 4.26
25a
25b
26a
26b
27a
27b
28a
28b
29a
32a
33a
36a
36b
37a
37b
41a
42a
42b
43a d
2.64
2.26
2.61
2.27
2.64
2.34
2.78 3.01
2.34
2.60
2.61
2.56
2.59
2.31
2.60
2.30
2.36
2.34
2.35
3.04
2.69
3.06
2.65
3.12
2.79
3.40
2.79
3.02
2.98
2.88
3.01
2.65
2.95
2.54
2.79
2.80
2.72
4.37
3.45
1.15
1.17
1.13
1.19
1.16
1.19
1.21
1.20
1.10
1.09
1.05
1.14
1.26
1.10
1.14
1.12
1.08
1.11
0.90
1.39
1.69
1.87
1.67
1.88
1.83
1.84
1.68
1.90
1.67
1.60
1.55
1.67
1.89
1.56
1.88
1.71
1.70
1.61
1.25
1.45
1.69
1.67
1.66
2.64
2.64
2.61
2.56
2.64
2.64
2.73
2.66
2.60
2.61
2.56
2.59
2.57
2.60
2.54
2.58
2.45
2.36
2.75
2.59
4.33
4.26
4.31
4.26
4.30
4.27
4.06
4.28
4.31
4.29
4.27
4.31
4.30
4.29
4.28
4.14
4.15
4.13
3.77
4.09
1.59
1.56
1.69
1.68
2.37c
2.37c
2.37c
7.49, 7.62, 8.26
7.51, 7.60, 8.05, 8.26
7.48, 8.26
8.28, 8.31
3.81c
1.59
1.52
1.59
1.75
1.68
1.75
1.67
1.60
1.55
1.67
2.29c
6.75, 6.91
2.94c
1.61
1.59
1.77
1.73
2.95c
1.56
2.95c
2.96c
1.42-1.60
1.35-1.61
5.63b
5.61b
1.61
5.47b
2.63
1.25, 1.50 1.40, 1.67
4.42, 4.50b
7.92, 7.68, 7.32
47a ,b (2:1) 2.59 3.97
1.60
1.74
a
Spectra registered at 300, 400 or 500 MHz in CDCl3 except for 28 and 47a ,b, which were registered in DMS0-d6, and 35a which was
registered in (CD3)2CO. δ corresponding to the 1-H of R1. c δ corresponding to CH3. Data of the (1:1) atropoisomeric mixture assigned
in the TOCSY spectrum.
b
d
2H, 4-H (Orn)], 1.37-1.59 [m, 2H, 3-H (Orn)], 1.48 (s, 9H, Boc),
3.05 [m, 2H, 5-H (Orn)], 3.13 [dd, 1H, J ) 9 and 14 Hz, 3-H
(Trp)], 3.34 [dd, 1H, J ) 5 and 14 Hz, 3-H (Trp)], 3.67 (s, 3H,
OMe), 4.51 [m, 2H, 2-H (Orn) and 2-H (Trp)], 4.82 [t, 1H, J )
6 Hz, 5-NH (Orn)], 5.18 [s, 2H, CH2 (Z)], 5.28 [bs, 1H, 2-NH
(Trp)], 6.04 [d, 1H, 2-NH (Orn)], 7.02 [d, 1H, J ) 2 Hz, 2-H
(In)], 7.11-7.25 [m, 2H, 5-H and 6-H (In)], 7.31-7.44 [m, 6H,
7-H (In) and Ph (Z)], 7.72 [d, 1H, J ) 7.5 Hz, 4-H (In)], 8.46
[s, 1H, 1-H (In)]. Anal. (C30H38N4O7) C, H, N. This dipeptide
methyl ester (5.84 g, 10.3 mmol) was dissolved into MeOH (75
mL), and 1 N NaOH (24 mL) and H2O (8 mL) were added to
the solution. After 15 min of stirring at room temperature,
the MeOH was evaporated, and the resulting solution was
diluted with H2O (150 mL) and washed with EtOAc (100 mL).
The aqueous phase was acidified to pH = 5, by dropwise
addition of 1 N HCl, and extracted with EtOAc (2 × 150 mL).
The organic extracts were dried over Na2SO4 and evaporated
to dryness to yield the dipeptide Boc-L-Trp-D-Orn-OH (5.69
g, 100%) as a syrup, which was used without further purifica-
tion for the following step in the synthetic pathway. RP HPLC
tR ) 21.45 (A:B ) 50:50); 1H NMR [200 MHz, (CD3)2CO] δ
1.24-1.50 [m, 2H, 4-H (Orn)], 1.35 (s, 9H, Boc), 1.52-1.85 [m,
2H, 3-H (Orn)], 3.11 [m, 3H, 5-H (Orn) and 3-H (Trp)], 3.27
[dd, 1H, J ) 6.5 and 14 Hz, 3-H (Trp)], 4.43 [m, 2H, 2-H (Orn)
and 2-H (Trp)], 5.06 [s, 2H, CH2 (Z)], 6.02 [bs, 1H, 2-NH (Trp)],
6.31 [bs, 1H, 2-NH (Orn)], 7.00-7.12 [m, 2H, 5-H and 6-H (In)],
7.21 [d, 1H, J ) 2 Hz, 2-H (In)], 7.31 [m, 7H, 2-NH (Orn), 7-H
(In) and Ph (Z)], 7.65 [d, 1H, J ) 7 Hz, 4-H (In)], 10.05 [s, 1H,
1-H (In)].
Syn th esis of Eth yl (4RS)-7-Ben zyloxyca r bon yla m in o-
4-[N-(ter t-b u t oxyca r b on yl)-L-t r yp t op h yl]a m in o-3-oxo-
h ep ta n oa te (44).22 1,1-Dicarbonyldiimidazole (2.01 g, 12.4
mmol) was added to a solution of Boc-L-Trp-D-Orn-OH (5.69
g, 10.3 mmol) in dry THF (95 mL), and the mixture was stirred
at room temperature for 2 h. Then, this reaction mixture was
added dropwise, and under argon atmosphere, to a -78 °C
cooled solution of the lithium enolate of ethyl acetate [obtained
by dropwise addition of dry EtOAc (5.23 mL, 53.56 mmol),
under argon atmosphere, to a 1 M solution of lithium bis-
(trimethylsilyl)amide in hexane (53.56 mL, 53.56 mmol) cooled
to -78 °C, followed by 15 min of stirring]. After 25 min of
stirring at this temperature, 1 N HCl was added until pH =
7, and the resulting mixture was extracted with EtOAc (2 ×
150 mL). The organic extracts were successively washed with
saturated NaHCO3 (200 mL), H2O (200 mL), and brine (200
mL), dried over Na2SO4, and evaporated to dryness. The
residue was purified by flash chromatography using 25% of
EtOAc in hexane as eluant, to yield the title compound (3.69
g, 63%) as a white solid. A 10% of epimerization at the C2 of
1
ornithine was observed in the H NMR spectrum. Therefore,
this Boc-L-Trp-D-Orn-OH derived â-ketoester 44 was obtained
as a (9:1) (4S/4R)-epimeric pair, which could not be resolved.
RP HPLC tR ) 24.93 (A:B ) 50:50); 1H NMR (200 MHz, CDCl3)
δ 0.99 (m 2H, 6-H), 1.19 and 1.21 (2 t, 3 H, Et), 1.32-1.57 (m,
2H, 5-H), 1.39 and 1.43 (2 s, 9 H, Boc), 3.11 and 3.13 [2 d, 1
H, 3-H (Trp)], 3.29 and 3.39 [2 d, 1 H, 3-H (Trp)], 3.34 and
3.37 (2 s, 2 H, 2-H), 4.11 (c, 2 H, Et), 4.42 [m, 2 H, 2-H and
2-H (Trp)], 4.85 (m, 1 H, 7-NH), 5.13 [s, 2 H, CH2 (Z)], 5.22 [d,

Selective CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2227
1H, 2-NH (Trp)], 5.82 and 6.29 (2 d, 1 H, 4-NH), 6.97 [s, 1 H,
2-H (In)], 7.06-7.24 [m, 2 H, 5-H and 6-H (In)], 7.25-7.34 [m,
6 H, 7-H (In) and Ph (Z)], 7.64 [d, 1 H, 4-H (In)], 8.59 [s, 1 H,
1-H (In)]. Anal. (C33H42N4O8) 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.23 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 50 000g. 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 100 000g. 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 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.
Am yla se Relea se. Dispersed rat pancreatic acini were
prepared by using a modification of the technique of J ensen
et al.24 The rat was decapitated, and the pancreas was carefully
cleaned. Tissue was injected with 1 mL of a solution of
collagenase (type V, Sigma) at a concentration of 1 mg/mL (in
distilled water) and subjected to the digestion step consisting
in two 6 min incubations at 37 °C and washing three times
the tissue in buffer A (composition in mM: NaCl 140, KCl 4.87.
MgCl2 1.13, CaCl2 1.10, Glucose 10 and Hepes 10, pH ) 7.4)
after each incubation. The tissue obtained after the last
incubation was dispersed with the aid of a Pasteur pipet, and
the homogenate was centrifuged twice (100 g, 1 min, 4 °C).
The final pellet was resuspended in 100 mL of buffer B (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-glutaric acid 5 mM, fumaric acid 5 mM, pyruvic acid 5 mM,
SBTI 0.01%, bovine serum albumin 1%, essential amino acid
mixture 1%, and essential vitamin mixture 1%). Amylase
release was measured using the procedure of Peikin et al.27
Samples (2 mL) of 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 deter-
mined using the Amyl Kit Reagent (Boeringher 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 of amylase release elicited by a fixed CCK-8
concentration (0.1 nM) produced by the assayed compounds
was calculated according to the formula
Syn th esis of th e (2R*,3S*)- a n d (2R*,3R*)-3-[N-(ter t-
Bu toxyca r bon yl)-L-tr yp top h yl]a m in o-2-(eth oxyca r bon -
yl)m eth ylp ip er id in es 45.22 A solution of the (9:1) epimeric
mixture of â-ketoester 44 (400 mg, 0.64 mmol) in EtOH (40
mL) was hydrogenated at room temperature and 3 atm of H2
pressure, in the presence of 10% Pd (C) (40 mg) for 7 days.
Afterward, the catalyst was filtered off and washed with
EtOH (10 mL), and the resulting solution was evaporated to
dryness. The residue was purified by preparative TLC using
4% of MeOH in dichloromethane as eluant to isolate two
fractions. The lower Rf and major fraction (90 mg, 30%)
corresponded to a (2:1) epimeric mixture of the 2,3-trans-
piperidines 45a ,b, and the higher Rf fraction corresponded to
the (2:1) epimeric mixture of the 2,3-cis-piperidines 45c,d (44
mg, 14%).
(2R*,3S*)-3-[N-(ter t-Bu toxycar bon yl)-L-tr yptoph yl]am i-
n o-2-(eth oxycar bon yl)m eth ylpiper idin e (45a,b). RP HPLC
1
tR ) 18.89 (A:B ) 25:75); H NMR (300 MHz, CDCl3) δ 0.85
and 1.05 (m, 1 H, 4-H), 1.22 and 1.25 (t, 3 H, Et), 1.44 (s, 9 H,
Boc), 1.55 (m, 2 H, 5-H), 1.81 and 2.03 (m, 1 H, 4-H), 2.20 and
2.43 (m, 2 H, 2-CH2), 2.45 (m, 1 H, 2-H), 2.45 and 2.91 (m, 2
H, 6-H), 3.13 and 3.30 [m, 2 H, 3-H (Trp)], 3.51 (m, 1 H, 3-H),
4.07 and 4.08 (c, 2 H, Et), 4.36 [dd, 1H, 2-H (Trp)], 5.18 [bs, 1
H, 2-NH (Trp)], 5.59 and 5.70 (d, 1 H, 3-NH), 7.05 [d, 1 H, J
) 2 Hz, 2-H (In)], 7.08-7.22 [m, 2 H, 5-H and 6-H (In)], 7.35
[d, 1 H, J ) 7.5 Hz, 7-H (In)], 7.66 [d, 1 H, J ) 7.5 Hz, 4-H
(In)], 8.27 [bs, 1 H, 1-H (In)]. Anal. (C25H36N4O5) C, H, N.
(2R*,3R*)-3-[N-(ter t-Bu toxycar bon yl)-L-tr yptoph yl]am i-
n o-2-(eth oxycar bon yl)m eth ylpiper idin e (45c,d). RP HPLC
1
tR ) 27.72 (A:B ) 25:75); H NMR (300 MHz, CDCl3) δ 0.84
and 0.90 (m, 1 H, 5-H), 1.07 (m, 1 H, 4-H), 1.23 and 1.24 (t, 3
H, Et), 1.43 (s, 9 H, Boc), 1.32 (m, 1 H, 4-H), 2.00 and 2.45 (m,
1 H, 2-CH2), 2.45 and 2.54 (m, 4 H, 6-H and 2-CH2), 2.90 (m,
1 H, 2-H), 3.15 and 3.33 [m, 2 H, 3-H (Trp)], 3.83 and 3.92 (m,
1 H, 3-H), 4.09 and 4.14 (c, 2 H, Et), 4.43 [m, 1H, 2-H (Trp)],
5.19 [bs, 1 H, 2-NH (Trp)], 6.32 and 6.54 (bs and d, 1 H, 3-NH),
7.05 [d, 1 H, J ) 2 Hz, 2-H (In)], 7.21-7.10 [m, 2 H, 5-H and
6-H (In)], 7.34 [d, 1 H, J ) 7.5 Hz, 7-H (In)], 7.68 [d, 1 H, J )
7.5 Hz, 4-H (In)], 8.15 [bs, 1 H, 1-H (In)]. Anal. (C25H36N4O5)
C, H, N.
Syn th esis of (4a R*,5S*)-5-[N-(ter t-Bu toxyca r bon yl)-
tr yp top h yl]a m in o-2-(3-ca r boxy)p h en yl-1,3-d ioxop er h y-
d r op yr id o[1,2-c]p yr im id in e (47a ,b). Bis(trichoromethyl)-
carbonate (96 mg, 0.32 mmol) was added to a 0 °C cooled
solution of 3-aminobenzoic acid (163 mg, 1.3 mmol). Afterward,
TEA (0.60 mL, 4.28 mmol) was added dropwise throughout 5
min. After 5 min of stirring at 0 °C, a solution of (2R*,3S*)-
3-[N-(tert-butoxycarbonyl)-L-tryptophyl]amino-2-(ethoxycarbo-
nyl)methylpiperidine (45a ,b) (200 mg, 0.43 mmol) in dry THF
(3 mL) was added at room temperature, and the mixture was
stirred at room temperature for 16 h. Then, the reaction
mixture was diluted with EtOAc (50 mL), washed successively
with H2O (25 mL), 10% citric acid (25 mL) and brine (25 mL),
dried over Na2SO4, and evaporated to dryness. The residue
was purified by flash chromatography to give the piperidine
derivative 46a ,b (60 mg, 0.1 mmol, 23%) as a syrup, which
was dissolved into dry THF (2 mL). Then, NaH (12 mg of 60%
dispersion in mineral oil, 0.30 mmol) was added to this
solution. This reaction mixture was stirred at room temper-
ature for 30 min, then a 0 °C cooled 1 N HCl solution (25 mL)
was added, and the resulting mixture was extracted with
EtOAc (2 × 25 mL). The organic extracts were washed with
H2O (25 mL) and brine (25 mL), dried over Na2SO4, and
evaporated to dryness. The residue was purified by flash
chromatography using 10-50% gradient of MeOH in dichlo-
romethane as eluant, to yield the (2:1) diastereoisomeric
mixture of 1,3-dioxoperhydropyrido[1,2-c]pyrimidines 47a ,b
(40 mg, 67%), which could not be resolved. The significant
analytical and spectroscopic data of these compounds are
shown in Tables 4 and 5.
% I ) [(SCCK-SC) - (ST-SC)/(SCCK-SC)] × 100
where SC was control release (vehicle), SCCK the release elicited
by CCK-8, and ST the release elicited by CCK-8 in the presence
of increasing drug concentrations. Linear regression analysis
was used in order to estimate the IC50 values of the compounds
on the dose response curves.
Ack n ow led gm en t. This work has been supported
by the CICYT (SAF 97-0030), Fundacio´n La Caixa (97/
022) and Consejeria de Educacio´n y Cultura de la
Comunidad de Madrid (CAM-08.5/0006/1998).
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(3) Dauge´, V.; Le´na, I. CCK in Anxiety and Cognitive Processes.
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