5-(tryptophylamino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based cholecystokinin receptor antagonists: Reversal of CCK1 receptor subtype selectivity toward CCK2 receptors

Article abstract of DOI:10.1021/jm0498755

With the aim of reversing selectivity or antagonist/agonist functionality in the 5-(tryptophyl-amino)-1,3-dioxoperhydropvrido[1,2-c]pyrimidine-derived potent and highly selective CCK₁ antagonists, a series of 4-benzyl and 4-methyl derivatives h

Full text of DOI:10.1021/jm0498755

5318
J . Med. Chem. 2004, 47, 5318-5329
5-(Tr yp top h yla 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
Ch olecystok in in Recep tor An ta gon ists: Rever sa l of CCK1 Recep tor Su btyp e
Selectivity tow a r d CCK2 Recep tor s
Pilar Mun˜oz-Ruiz,† M. Teresa Garc´ıa-Lo´pez,† Edurne Cenarruzabeitia,‡ J oaqu´ın Del R´ıo,‡ Marlene Dufresne,§
Magali Foucaud,§ Daniel Fourmy,§ and Rosario Herranz*,†
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, E-28006 Madrid, Spain, Departamento de Farmacolog´ıa,
Universidad de Navarra, Irunlarrea 1, E-31080 Pamplona, Spain, and INSERM U531, Institut Louis Bugnard,
CHU Rangueil, Bat. L3, 31403 Toulouse Cedex 4, France
Received February 10, 2004
With the aim of reversing selectivity or antagonist/agonist functionality in the 5-(tryptophyl-
amino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-derived potent and highly selective CCK1
antagonists, a series of 4-benzyl and 4-methyl derivatives have been synthesized. Whereas
the introduction of the benzyl group led, in all cases, to complete loss of the binding affinity,
the incorporation of the methyl group gave a different result depending on the stereochemistry
of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine scaffold. Thus, the introduction of the methyl
group into the (4aS,5R)-diastereoisomers, giving a (4S)-configuration, produced a 3-fold increase
in the CCK1 binding potency and selectivity. However, the same structural manipulation in
the opposite (4aR,5S)-stereochemistry, leading to a (4R,4aR,5S)-configuration, produced reversal
of the selectivity for CCK1 to the CCK2 receptors. The replacement of the Boc group at the
tryptophan moiety by a 2-adamantyloxycarbonyl group also contributed to that reversal. The
resulting compounds displayed moderate CCK2 antagonist activity in rat and human receptors,
and a very small partial agonist effect on the production of inositol phosphate in COS-7 cells
transfected with the wild-type human CCK2 receptor.
In tr od u ction
The cholecystokinin (CCK) family of peptides was
formerly isolated and identified in the gastrointestinal
tract, and later as a neurotransmitter present through-
out the nervous system.1 This family of neuropeptides
include different molecular forms (e.g., CCK-58, CCK-
33, CCK-8) derived from the processing of a 115-amino
acid precursor protein (prepro-CCK), which have the
C-terminal sequence in common,1,2 with CCK-8 being
F igu r e 1.
the minimum sequence for full biological activity.3 In
the gastrointestinal tract CCK is released from endo-
stimulated research in this area and, over the past 15
crine cells, in response to food intake, and regulates
years, a broad assortment of potent and selective
motility, contraction of gallbladder, pancreatic enzyme
nonpeptide CCK1 and CCK2 receptor agonists and
secretion, gastric emptying, and gastric acid secretion.1
In the nervous system CCK is involved in anxio-
genesis,1,4-6 satiety,1,7-9 nociception,1,10 thermoregula-
tion,1,11 and memory and learning processes.4,12,13
Furthermore, the colocalization and interaction of CCK
with other neurotransmitters in some areas of the
central nervous system (CNS),1,14 mainly with dopamine
(DA),15,16 suggests its implication in several neuro-
psychiatric disorders, such as schizophrenia, depression,
and drug addiction.10,15-18 These biological effects are
mediated by two specific G-protein-coupled receptor
subtypes, termed CCK1 and CCK2.1,10
antagonists have been reported.10,19-25 Some of these
ligands have contributed highly to the characterization
and localization of CCK receptor subtypes, as well as
to the study of physiological and pathological actions of
CCK. However, despite the progress in this field, the
complex biological effects of CCK mediated by CCK1 and
CCK2 receptors are not yet fully established.1,10 In this
regard, we have reported the design, synthesis,26 and
pharmacological properties27 of the 5-(tryptophylamino)-
1,3-dioxoperhydropyrido[1,2-c]pyrimidine derivative 1b
(IQM-95,333, Figure 1), prototype of a family of potent
and highly selective CCK1 receptor antagonists, which
include some of the most selective antagonists described
to date.25 This compound showed a CCK1 receptor
affinity in the nanomolar range, but was virtually
devoid of affinity at brain CCK2 receptors.27 In agree-
ment with this CCK1 receptor affinity, compound 1b
was a potent inhibitor of the CCK-8-stimulated amylase
release from isolated pancreatic acini and blocked the
The variety of physiological effects of CCK and its
possible role in certain pathological disorders have
* 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).
‡ Universidad de Navarra.
§ Institut Louis Bugnard.
10.1021/jm0498755 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/04/2004

CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5319
CCK-8-induced hypophagia and hypolocomotion in rats.27
Furthermore, despite the predominant role attributed
to CCK2 receptors in the anxiogenic effects of CCK,4,10
this CCK1 antagonist also showed a marked anxiolytic-
like activity in animal models.27 This result supports
the suggestion of some authors that CCK1 receptors may
be also involved in anxiogenesis.28-30 Structure-activity
relationship studies on these 1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine-based CCK1 receptor antagonists have
shown that the Boc-L-Trp residue, the topography
defined by the (4aS,5R)-1,3-dioxoperhydropyrido[1,2-c]-
pyrimidine scaffold, and the lipophilicity and spatial
orientation of the group attached to the N2 position of
that skeleton are essential structural requirements for
potent and selective binding to CCK1 receptors.26,31-33
We were interested in expanding our assortment of
CCK receptor ligands, reversing the selectivity or the
functionality of our CCK1 highly selective antagonists.
Minor changes in certain groups attached to the core
scaffold or in its stereochemistry have led to intercon-
version of the CCK1/CCK2 receptor subtype selectivity
in most of the known families of CCK receptor ligands.25
There are also several reports which demonstrate the
feasibility of interconverting agonist/antagonist func-
tionality of nonpeptide ligands by minor structural
changes,34 such as, for example, introducing additional
alkyl groups into the structure of an antagonist,35-37 or
changes in the stereochemistry.38 We have approached
our goal of reversing the selectivity or the functionality
of 1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based CCK1
receptor antagonists by introducing additional groups
(Me and CH2Ph) into position 4 of the 1,3-dioxoper-
hydropyrido[1,2-c]pyrimidine skeleton and by bearing
in mind those previous SAR results that pointed out a
decrease in CCK1 receptor affinity and an increase in
that for the CCK2. Taking into account the important
influence of stereochemistry at the tryptophan and 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine domains upon af-
finity and selectivity,26 we have attempted to search
their configurational space as much as possible. Ad-
ditionally, the replacements of the N-Boc group at the
Trp moiety by the 2-adamantyloxycarbonyl group (2-
Adoc) and the benzyl group at the N2 position of the
1,3-dioxoperhydropyrido[1,2-c]pyrimidine scaffold by a
4-dimethylaminophenyl group have been considered, as
both modifications introduced into the diastereoisomer
1a (Figure 1) led independently to a 1 order of magni-
tude increase in the CCK2 receptor affinity of 2a and
3a and to a decrease of more than 1 order of magni-
tude31 or the complete loss of affinity at CCK1 recep-
tors,32 respectively.
Sch em e 1. Retrosynthesis of
4-Substituted-1,3-dioxo-perhydropyrido[1,2-c]pyrimidine
Derivatives
elaboration. These three alternative routes were applied
depending on the substitution and on the required
stereochemistry. Thus, with the aim of facilitating the
obtention of the highest number of stereoisomers, route
A was first attempted. As shown in Scheme 2, for the
synthesis of the 4-benzyl derivatives 8 and 9 from the
â-keto ester 4, this route involved alkylation with benzyl
bromide, using NaH as base at 0 °C. This alkylation
led to the unresolved epimeric mixture of the 2-benzyl
derivatives 5 in a (≈1:1) 1H NMR estimated ratio.
â-Keto ester 4 was obtained from Boc-L-Orn(Z)-OH,
applying a modified method39 of one previously de-
scribed.40 Removal of the benzyloxycarbonyl protecting
group from the 2-benzyl derivatives 5, by catalytic
hydrogenolysis, followed by intramolecular reductive
amination using NaBH3CN in the presence of ZnCl2,
gave a (3:1) mixture of 2,3-trans- and 2,3-cis-disubsti-
tuted piperidine derivatives 6a ,b and 6c,d , which were
chromatographically separated as epimeric mixtures at
the exocyclic stereogenic center in (1.2:1) and (1.4:1)
ratios, respectively. Furthermore, as these reductive
aminations produce racemization at the C2 and C3 of
the piperidine ring in different extent depending on the
substituents and on the reduction conditions,26 both
6a ,b and 6c,d included ≈22% of racemization. Treat-
ment of each one of these mixtures with benzyl iso-
cyanate, followed by in situ cyclization of the respec-
tive urea derivatives, provided the corresponding 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives 7. In-
terestingly, this cyclization took place with total or
partial stereomutation at the exocyclic stereogenic
center, as the (1.2:1) diastereoisomeric mixture 6a ,b
gave exclusively the racemic mixture 7a ,b (80%), with
a (4R*,4aS*,5R*)-relative configuration, while the (1.4:
1) diastereoisomeric mixture 6c,d led to 7c,d and 7e,f,
which were separated in a (7.5:1) ratio. Finally, the
N-Boc removal from the racemic mixtures 7a ,b and
7c,d , followed by coupling with Boc-L- or D-Trp-OH,
using BOP as coupling agent, provided the correspond-
ing (≈3:1) diastereoisomeric mixtures 8a ,b; 8c,d ; and
9a ,b, which were chromatographically resolved.
Ch em istr y
The synthesis of the target 4-substituted-1,3-dioxo-
perhydropyrido[1,2-c]pyrimidine derivatives was de-
signed following a similar synthetic scheme to that
previously used for the preparation of 4-unsubstituted
analogues. This methodology involved essentially the
construction of the 1,3-dioxoperhydropyrido[1,2-c]pyr-
imidine scaffold and subsequent coupling of the ap-
propriate N-protected tryptophan residue. As indicated
in the retrosynthetic Scheme 1, the key C-alkylation
step for introducing the additional substituent at posi-
tion 4 of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine
core can be performed at three different stages of its
A similar synthetic scheme for the preparation of
4-methyl-1,3-dioxoperhydropyrido[1,2-c]pyrimidine de-

5320 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Mun˜oz-Ruiz et al.
Sch em e 2a,b
a
Letters are used in compound numeration to indicate different stereoisomers. Thus, a denotes a (4S,4aR,5S)-configuration; b denotes
(4R,4aS,5R)-configuration; c denotes (4R,4aS,5S)-configuration; d denotes (4S,4aR,5R)-configuration; e denotes (4S,4aS,5S)-configuration;
f denotes (4R,4aR,5R)-configuration; g denotes (4R,4aR,5S)-configuration; h denotes (4S,4aS,5R)-configuration. bReagents: (a) CDI, MgCl2,
MeO2CCH2CO2K, THF; (b) NaH, PhCH2Br, THF; (c) H2, 10% Pd(C), MeOH; (d) NaBH3CN, ZnCl2; (e) PhCH2NCO, THF; (f) NaH, THF;
(g) TFA, CH2Cl2; (h) Boc-L- or Boc-D-Trp-OH, BOP, TEA, CH2Cl2.
rivatives was discarded as the starting 2-methyl-â-keto
ester 10 (Scheme 3) was obtained with low yield (39%),
which hampered obtaining the desired 1,3-dioxoper-
hydropyrido[1,2-c]pyrimidine derivatives in acceptable
yields. Therefore, route B was applied, as shown in
Scheme 3, involving akylation of the appropriate N-Z
protected 2-piperidyl acetic acid derivatives 12 with MeI
in THF at -78 °C, using lithium bis(trimethylsilyl)-
amide as base, and in the presence of hexamethylphos-
phoric acid triamide. Under these conditions 2,3-cis-
disubstituted piperidine derivatives 12c,d did not react,
and were recovered unchanged, while raising the reac-
tion temperature from -78 °C to room temperature led
to a complex reaction mixture. However, the (9:1)
racemic mixture of 2,3-trans-disubstituted piperidines
12a ,b led to the methyl derivatives 13a ,b as a single
racemic mixture, whose relative configuration at the
exocyclic center could not be assigned. Removal of the
N-Z protecting group from these 2,3-trans-disubstituted
piperidine derivatives, by catalytic hydrogenolysis, fol-
lowed by treatment with benzyl isocyanate, and in situ
base-promoted intramolecular cyclization of the corre-
sponding urea intermediate, yielded the mixture of 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives 15a ,b
and 15g,h , which was chromatographically resolved in
a (3:1) ratio. This result showed that partial stereo-
mutation at the exocyclic stereogenic center had also
occurred during the urea cyclization. Removal of the
N-Boc protection in the major diastereoisomers 15a ,b,
followed by coupling with Boc-L-Trp-OH and chromato-
graphic resolution, provided the desired compounds 16a
and 16b in a (≈9:1) ratio. N-Boc/N-(2-Adoc) exchange
in the major (4S,4aR,5S)-diastereoisomer 16a , by N-Boc
removal, followed by reaction with 2-adamantyl chloro-
formate, gave the 2-Adoc derivative 17a .
Finally, route C was applied for the preparation of
the 4-methyl-1,3-dioxoperhydropyrido[1,2-c]pyrimidine
derivatives with a 4a,5-cis-relative configuration 16c,d
and 22c,d (Scheme 4), that could not be obtained by the
previous A or B routes, and also for the synthesis of the
2-(dimethylamino)phenyl substituted compounds 23 and
24. This last route involved methylation of the corre-
sponding 4-unsubstituted-1,3-dioxoperhydropyrido[1,2-
c]pyrimidine derivatives 18c,d 26 and 19a ,b,32 respec-
tively, as in route B, by reaction with MeI in THF at
-78 °C, using lithium bis(trimethylsilyl)amide as base,
and in the presence of hexamethylphosphoric acid
triamide, followed by the corresponding N-Boc removal
and coupling with Boc-L- or Boc-D-Trp-OH. Interest-
ingly, the methylation of the 4a,5-cis-compounds 18c,d
was completely stereoselective, giving rise exclusively
to the 4-methyl derivatives with a (4R*,4aS*,5S*)
relative configuration 15c,d , while in the case of the
4a,5-trans-compounds 19a ,b the two possible diastereo-
isomers 21a ,b and 21g,h were obtained in a (≈4:1) ratio.
As mentioned above, and shown in Scheme 4, the N-Boc/
N-(2-Adoc) exchange in the N-Boc derivative 23a pro-
vided the corresponding 2-Adoc analogue 24a . For
biological comparative purposes, the 2-Adoc derivative
20a was similarly prepared from 3a .
The assignment of absolute configuration to the new
4-substituted-1,3-dioxoperhydropyrido[1,2-c]pyrimi-
dine derivatives was done by assuming that, despite the
racemization in the intramolecular reductive amination
steps, the major diastereoisomers maintain the (S)-
configuration of the starting Boc-L-Orn(Z)-OH. There-
fore, the configuration at C5 of the major isomers is (5S).
As shown in Figure 2, the J 4a,5 coupling constant value
was used to assign the relative 4a,5-trans (10-12 Hz)
or 4a,5-cis (0-2 Hz) configuration. With respect to the

CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5321
Sch em e 3a
Sch em e 4a
a
Reagents: (a) NaH, MeI, THF; (b) H2, 10% Pd(C), MeOH; (c)
NaBH3CN, ZnCl2; (d) PhCH2OCOCl, propylene oxide, CH2Cl2; (e)
[(CH3)3Si]2NLi, MeI, HMPA, THF; (f) H2, 10% Pd(C), MeOH; (g)
PhCH2NCO, THF; (h) NaH, THF; (i) TFA, CH2Cl2; (j) Boc-L-Trp-
OH, BOP, TEA, CH2Cl2; (k) 2-adamantyl chloroformate, TEA,
CH2Cl2.
independently of the stereochemistry, none of the 4-ben-
zyl derivatives 8a -d and 9a -b bound at CCK1 or CCK2
receptors at concentrations below 10-5 M. The results
of the 4-methyl derivatives 16, 17, 22-24 and the
[4-(dimethylamino)phenyl]-4-unsubstituted analogue 20a
are shown in Table 1, along with the described affinities
of the 4-unsubtituted compounds 2a ,31 3a , and 3b.32 It
is interesting to note that the introduction of a methyl
group into position 4 of the 1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine skeleton of the prototype 1b led to a
3-fold improvement in the binding potency at CCK1
receptors, providing compound 16b with subnanomolar
affinity and excellent selectivity. This modification in
the model compound having (4aR,5S)-stereochemistry
(1a ) produced a significant increase in the binding
affinity at CCK2 receptors and a higher than 2 orders
of magnitude decrease at CCK1 receptors for the 4-
methyl derivative 16a . Therefore, this modification has
reversed the CCK1 selectivity of compound 1a to the
CCK2 selectivity of compound 16a . A moderate increase
configuration at C4, its assignment was based on the
J 4,4a values and NOE relationships observed in the
DPFGSE-NOE spectra of the new 4-substituted deriva-
tives. On the other hand, the NOE effects observed
between 4a-H, 6-Hax, and 8-Hax protons (not shown in
Figure 1) indicated that in all these derivatives the
fused piperidine ring adopts a preferred chair conforma-
tion with the 4a-H in an axial disposition.
Biologica l Resu lts a n d Discu ssion
The affinities of the new 5-(tryptophylamino)-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives herein
described at rat CCK1 and CCK2 receptors were deter-
mined by measuring the displacement of [3H]propionyl-
CCK-8 binding to rat pancreatic and cerebral cortex
homogenates, respectively, as previously described.41
For comparative purposes, CCK-8, the CCK2 antagonist
PD-135,158,42 and the model compounds 1a and 1b were
also included in the assay. The results showed that,

5322 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Mun˜oz-Ruiz et al.
cerning the 4a,5-cis diastereoisomers 16c, 22c, and 22d ,
the incorporation of the 4-methyl group caused the loss
of the micromolar affinity shown by the 4-unsubsti-
tuted analogues at CCK1 or CCK2 receptors.26 On the
other hand, as in the model compound 1a ,31 the replace-
ment of the Boc group of 3a , 16a , and 23a by the
2-adamantyloxycarbonyl group (2-Adoc) produced a
significant increase in the CCK2 binding potency of 20a ,
17a , and 24a , without affecting the binding at CCK1
receptors. A CCK2 receptor heterogeneity in the rat
cerebral cortex has been previously suggested by means
of the analysis of an exceptionally large number of
competition curves obtained with the CCK2 receptor
antagonist, L-365,260.43 Hill slopes were not however
significantly different from unity in subsequent studies
with other CCK2 receptor ligands when using a much
more reduced data set.44 In the present study, the mean
Hill slope parameter estimates, obtained from the
competition curves for the more potent new CCK2
receptor ligands 17a , 20a , and 24a , were 0.91 ( 0.05,
0.82 ( 0.11 and 0.83 ( 0.10, respectively. These Hill
slopes were not significantly different from unity, sug-
gesting in principle a single binding site.
F igu r e 2. NOE relationships and coupling constant values
used for the configuration assignment in 4-substituted-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives.
in the affinity for CCK2 was also observed by the
introduction of the 4-methyl group into the (dimethyl-
amino)phenyl derivative 23a . As the comparison of the
NMR data of the 4-benzyl derivatives 8a ,b with those
of their respective 4-methyl analogues 16a ,b, as well
as with the 4-unsubstituted compounds 1a ,b, did not
show significant conformational differences in the 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine skeleton, the dras-
tic influence of the introduction of a benzyl or a methyl
group into position 4 upon the binding affinity at both
CCK1 and CCK2 receptors seems to indicate the exist-
ence of an additional point of interaction with the
receptor at that position. The complete loss of affinity
resulting from the introduction of the benzyl group could
be due to bad steric contacts with the receptors. Con-
Consistent with its subnanomolar affinity at CCK1
receptors, compound 16b antagonized the CCK-8-
stimulated amylase release from rat pancreatic acinar
cells,45 with an IC50 value of 0.62 ( 0.33 nM. Compounds
that bound to CCK2 receptors at concentrations below
10-6 M were tested for their antagonism of the CCK-
4-induced contractions in isolated longitudinal muscle
myenteric plexus preparations from guinea pig ileum.
In this assay CCK-4 produces a contractile effect by
stimulation of CCK2 receptors.46 As shown in Table 1,
compounds 17a , 20a , and 24a , with submicromolar
affinities at CCK2 receptors, inhibited the CCK-4-
induced contractions with calculated pA2 values of 5.75-
Ta ble 1. Inhibition of the [3H]pCCK-8 Specific Binding to Rat Pancreas (CCK1) and Cerebral Cortex Homogenates (CCK2), and
Inhibition of the CCK-4-Induced Contraction of Isolated Longitudinal Muscle Myenteric Plexus from Guinea-Pig Ileum
IC50 (nM)a
CCK1 CCK2
inhibition of CCK-4 effectb
selectivity:
CCK1/CCK2
c
compd
CCK8
PD-135,158
1a
R1
R2
R3
stereochem.
%
pA2 (CL)d
1.04 ( 0.08 5.60 ( 0.03
1123 ( 23 9.80 ( 0.40
0.19
115
0.004
<10-4
0.1
83.5 ( 6.6 8.10 (7.90-8.30)
Boc-L-Trp
Boc-L-Trp
2-Adoc-L-Trp
Boc-L-Trp
Boc-L-Trp
Boc-L-Trp
Boc-L-Trp
Boc-L-Trp
Boc-D-Trp
Boc-D-Trp
H
H
H
H
H
CH2Ph
CH2Ph
CH2Ph
4-(NMe2)Ph (4aR,5S)
4-(NMe2)Ph (4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
22.7 ( 4.0
1.59 ( 0.10
340
6153
>10000
3430
1b
2a e
3a f
>10000
2320
>10000
3bf
4.30 ( 1.05
<4 × 10-4
2
16a
16b
16c
22c
22d
17a
20a
23a
23g,h
24a
Me CH2Ph
Me CH2Ph
Me CH2Ph
Me CH2Ph
Me CH2Ph
(4S,4aR,5S)
(4R,4aS,5R)
(4R,4aS,5S)
(4R,4aS,5S)
(4S,4aR,5R)
(4S,4aR,5S)
1350 ( 250 700 ( 220
0.47 ( 0.25
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
<5 × 10-5
>10000
>10000
2-Adoc-L-Trp Me CH2Ph
2-Adoc-L-Trp
Boc-L-Trp
181 ( 35
276 ( 36
1265 ( 106
>10000
>55
>36
>8
83.0 ( 6.0 6.62 (6.39-6.81)
61.0 ( 2.0 5.87 (5.52-6.11)
15.3 ( 5.5
H
4-(NMe2)Ph (4aR,5S)
Me 4-(NMe2)Ph (4S,4aR,5S)
Me 4-(NMe2)Ph (4R*,4aR*,5S*)
Boc-L-Trp
2-Adoc-L-Trp Me 4-(NMe2)Ph (4S,4aR,5S)
121 ( 17
>83
75.0 ( 3.8 5.75 (4.97-6.22)
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 CCK-4-induced contraction of isolated longitudinal muscle myenteric plexus preparations from guinea-pig ileum. c Compounds
tested at a fixed 10-5 M concentration. Values are the mean of at least three experiments performed in triplicate. Confidence limits
d
(95%) for pA2 values of four to six experiments. e Reference 31. f Reference 32.

CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5323
Ta ble 2. Binding Affinities and Effects on Inositol Phosphate
Production of 1,3-Dioxoperhydropyrido[1,2-c]pyrimidine
Derivatives 17a , 20a , and 24a on Wild-Type Human CCK2
Receptors Transiently Expressed in COS-7 Cells
stage apparatus and are uncorrected. NMR spectra were
recorded with Varian Gemini 200, Varian INOVA-300, Varian
INOVA-400, and Varian Unity-500 spectrometers, operating
at 200, 300, 400, or 500 MHz for 1H NMR, and at 50, 75, 100,
or 125 MHz for 13C NMR, and using TMS as reference.
Elemental analyses were obtained on a CH-O-RAPID ap-
paratus. Analytical RP 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 phases. Optical rotations were mea-
sured in CHCl3 on a Perkin-Elmer 141 polarimeter.
bindinga
inositol phosphate production
compd
IC50 (nM)
IC50 (nM)b
EC50 (nM)c
(Thr,Nle)-CCK-9 0.96 ( 0.08
1.5 ( 0.7
98 ( 11
17a
20a
24a
723 ( 73
1763 ( 1027
1610 ( 1135 4467 ( 2288
3688 ( 888
517 ( 88
2110 ( 1050
2371 ( 1123
a
Inhibition of specific binding of 125I-BH-(Thr,Nle)-CCK-9 to
Gen er a l P r oced u r e for th e Syn th esis of Meth yl (2RS,
4S )-2-Su b st it u t e d -7-b e n zyloxyca r b on yla m in o-4-(t er t -
bu toxycar bon ylam in o)-3-oxoh eptan oates 5 an d 10. NaH
(60% dispersion in mineral oil, 120 mg, 3 mmol) was added to
a solution of methyl (4S)-7-benzyloxycarbonylamino-4-(tert-
butoxycarbonylamino)-3-oxoheptanoate40 (4) (1.150 g, 2.7 mmol)
in dry THF (40 mL) cooled at 0 °C, and the suspension was
stirred for 20 min at this temperature. Then, the corresponding
alkylating agent, benzyl bromide (0.4 mL, 3.4 mmol) or methyl
iodide (0.22 mL, 3.6 mmol), was added dropwise at 0 °C, and
the stirring was continued at room temperature for 16 h.
Afterward, water (50 mL) was added, and the resulting
reaction mixture was extracted with CH2Cl2 (2 × 150 mL). The
combined organic extracts were washed with water (50 mL),
brine (50 mL), dried over Na2SO4, and evaporated to dryness.
Purification of the crude residue by flash chromatography,
employing a (17-50%) gradient of EtOAc in hexane as eluant,
yielded, in each case, the (1:1) unresolved diastereomeric
mixtures 5 or 10, which could not be resolved.
Met h yl (2RS,4S)-7-Ben zyloxyca r b on yla m in o-4-(ter t-
b u t o x y c a r b o n y la m i n o )-2-p h e n y lm e t h y l-3-o x o h e p -
ta n oa te (5). Syrup (900 mg, 71%). RP HPLC tR ) 19.24 (A:B
) 45:55); 1H NMR (300 MHz, CDCl3) δ 1.12-1.29 (m, 1 H,
6-H), 1.40 (s, 4.5 H, Boc), 1.41 (s, 4.5 H, Boc), 1.39-1.46 (m, 1
H, 5-H), 1.50-1.63 (m, 1 H, 6-H), 1.78 (m, 0.5 H, 5-H), 1.95
(m, 0.5 H, 5-H), 3.0 (m, 1 H, 7-H), 3.14 (m, 3 H, 2-CH2, 7-H),
3.63 (s, 1.5 H, OCH3), 3.66 (s, 1.5 H, OCH3), 4.03 (m, 1 H, 2-H),
4.26 (m, 0.5 H, 4-H), 4.40 (m, 0.5 H, 4-H), 4.75 (m, 1 H, 7-NH),
4.93 (m, 0.5 H, 4-NH), 5.06 [s, 2 H, CH2 (Z)], 5.15 (m, 0.5 H,
4-NH), 7.12-7.37 (m, 10 H, aromatics); 13C NMR (75 MHz,
CDCl3) δ 25.30 and 25.62 (C6), 27.46 and 27.57 (C5), 28.21 [CH3
(Boc)], 33.77 and 34.58 (2-CH2), 40.29 (C7), 52.47 and 52.64
(OCH3), 56.94 and 57.04 (C2), 58.85 (C4), 66.56 [CH2 (Z)], 80.04
[C(CH3)3], 126.70-137.98 (Ph), 155.15, 156.28 and 156.38 [CO
(Boc)] and [CO (Z)], 167.33 and 168.93 (C1), 203.29 and 203.62
(C3). Anal. (C28H36N2O7) C, H, N.
COS-7 cells transfected with wild-type human CCK2 receptors.
Inhibition of (Thr,Nle)-CCK-9-induced IP production. Estimated
b
values, as stimulation could not be totally inhibited at the highest
concentration used (10-4.5 M). c Values were calculated from dose-
response curves of total IP production stimulated by the com-
pounds. Results are expressed as mean ( SEM of three to five
separate experiments.
6.62. None of these compounds showed any intrinsic
contractile effect in the ileum preparations.
The binding affinities and effects of compounds 17a ,
20a , and 24a were also studied in COS-7 cells trans-
fected with wild-type human CCK2 receptors. As shown
in Table 2, the binding affinities were 1 order of
magnitude lower than the respective affinities observed
in rat cerebral cortex homogenates. The Hill coefficients
were also close to unity. The species-specific differences
in receptor structure and the use of a distinct radio-
ligand ([3H]propionyl-CCK-8 and 125I-BH-(Thr,Nle)-
CCK-9) may account for the discrepancies in the affinity
values from the two CCK2 receptor binding assays. The
compounds inhibited the (Thr,Nle)-CCK-9-induced pro-
duction of inositol phosphate with potencies in close
agreement with their affinity values. However, they are
not pure antagonists, as they also showed a small par-
tial agonist activity with EC50 values also in the same
micromolar range as the binding affinities, and efficacies
in the stimulation of inositol phosphate production lower
than 15% of the maximum stimulation produced by a
10-7 M concentration of (Thr,Nle)-CCK-9.
In conclusion, the introduction of a methyl group into
position C4 of the 5-(Boc-tryptophylamino)-1,3-dioxo-
perhydropyrido[1,2-c]pyrimidine-based CCK1 antago-
nists has increased the binding potency and selectivity
for CCK1 receptors in the (4aS,5R)-diastereoisomers,
while in the (4aR,5S)-isomers the same structural
modification, along with the replacement of the Boc
group by the 2-adamantyloxycarbonyl group, has led to
the reversal of the CCK1 receptor subtype selectivity
toward the CCK2. Despite their low potency, these are
the first CCK2 selective antagonists in this series of 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivatives and
may be good starting structures for obtaining potent and
selective CCK2 antagonists by further structure ma-
nipulation.
Met h yl (2RS,4S)-7-Ben zyloxyca r b on yla m in o-4-(ter t-
bu toxyca r bon yla m in o)-2-m eth yl-3-oxoh ep ta n oa te (10).
1
Syrup (400 mg, 39%). RP HPLC tR ) 6.08 (A:B ) 45:55); H
NMR (300 MHz, CDCl3) δ 1.29 (d, 1.5 H, J ) 7 Hz, 2-CH3),
1.33 (d, 1.5 H, J ) 7 Hz, 2-CH3), 1.40 (s, 4.5 H, Boc), 1.41 (s,
4.5 H, Boc), 1.46-1.55 (m, 2 H, 6-H), 1.77-1.89 (m, 2 H, 5-H),
3.19 (m, 2 H, 7-H), 3.67 (s, 1.5 H, OCH3), 3.69 (s, 1.5 H, OCH3),
3.74 (q, 1 H, J ) 7 Hz, 2-H), 4.44 (m, 1 H, 4-H), 4.91 (m, 1 H,
7-NH), 5.06 [s, 2 H, CH2 (Z)] 5.11 (m, 1 H, 4-NH), 7.27-7.34
(m, 5 H, aromatics); 13C NMR (50 MHz, CDCl3) δ 12.64 and
13.21 (2-CH3), 25.80 and 25.93 (C6), 27.91 [CH3 (Boc)], 28.26
and 28.60 (C5), 40.46 (C7), 48.94 and 49.56 (C2), 52.36 and 52.48
(OCH3), 58.14 and 58.67 (C4), 66.63 [CH2 (Z)], 80.14 [C(CH3)3],
128.03, 128.46 and 136.60 (Ph), 155.40 and 156.45 [CO (Boc)]
and [CO (Z)], 170.41 (C1), 204.65 (C3). Anal. (C22H32N2O7) C,
H, N.
Exp er im en ta l Section
Syn th esis of th e 3-(ter t-Bu toxyca r bon yla m in o)-2-(1-
m eth oxyca r bon yl-2-p h en yleth yl)p ip er id in es 6. A solution
of methyl (2RS,4S)-7-benzyloxycarbonylamino-4-(tert-butoxy-
carbonylamino)-2-phenylmethyl-3-oxoheptanoate (5) (769 mg,
1.5 mmol) in MeOH (100 mL) was hydrogenated, at room
temperature and 1 atm of H2 pressure, in the presence of 10%
Pd (C) (80 mg) for 2 h. After filtration of the catalyst,
NaBH3CN (190 mg, 3 mmol) and ZnCl2 (216 mg, 1.76 mmol)
were added and the resulting mixture stirred at room tem-
perature for 1 h. Then, the solvent was evaporated, and the
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). Preparative
radial chromatography was performed on 20 cm diameter glass
plates coated with a 1-mm layer of silica gel 60 PF254 (Merck).
Silica gel 60 (230-400 mesh) (Merck) was used for flash
chromatography. Melting points were taken on a micro hot

5324 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Mun˜oz-Ruiz et al.
Ta ble 3. Significant Analytical and Spectroscopic Data of 5-(tert-Butoxycarbonyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine
Derivatives
7a ,b
CH2Ph
CH2Ph
7c,d
CH2Ph
CH2Ph
7e,f
CH2Ph
CH2Ph
15a ,b
15g,h
15c,d
21a ,b
21g,h
R2
R3
Me
CH2Ph
Me
CH2Ph
Me
CH2Ph
Me
4-(NMe)2Ph
Me
4-(NMe)2Ph
stereochem (4R*,4aS*,5R*) (4R*,4aS*,5S*) (4R*,4aR*,5R*) (4R*,4aS*,5R*) (4R*,4aR*,5S*) (4R*,4aS*,5S*) (4R*,4aS*,5R*) (4R*,4aR*,5S*)
formulaa
yield (%)
mp (°C)b
tR (A:B)c
C27H33N3O4
80
syrup
C27H33N3O4
60
120-122
6.99 (50:50)
C27H33N3O4
8
150-157
9.55 (50:50)
C21H29N3O4
60
71-73
C21H29N3O4
19
syrup
C21H29N3O4
86
foam
C22H32N4O4
56
224-225
3.17 (30:70)
C22H32N4O4
13
syrup
7.81 (50:50)
4.76 (45:55)
5.82 (45:55)
6.00 (40:60)
4.03 (30:70)
1H NMRd
4-H
4a-H
5-H
5-NH
6-H
7-H
3.05
2.83
3.30
4.04
2.00, 1.23
1.59
3.08-3.17
3.19
3.77
4.61
1.65-1.75
1.53-1.57
3.13-3.16
3.16
4.13
4.44
1.51, 1.71
1.47-1.50
2.96
2.94-2.98
3.28
2.90
3.08
3.66
4.33
2.73
3.13
4.01
4.69
3.03-3.09
3.03-3.09
3.46
3.01
3.24
3.65-3.79
4.45
1.19-1.32, 2.05
1.54-1.86
4.53
4.50
1.42-1.49, 2.08 1.19, 2.05
1.56-1.68
1.56-1.70, 1.92 2.14
1.50, 1.80
2.67, 4.17
1.30
4.84, 4.97
3.5
1.56-1.70
4.46, 4.70
1.30
4.90, 4.96
8.0
1.64-1.73
2.64-2.72, 4.43 2.73, 4.21
1.45
2.93
0
8-H
2.45-2.59, 4.38 2.53-2.68, 4.45 2.57-2.66, 4.40 2.63, 4.42
3.07, 2.74
5.05, 4.84
0
R2e
3.05, 2.91
4.95
2
0
2.95, 3.48
4.89
0
0
1.31
4.90-5.03
0
8
1.41
2.94
3
R3f
J 4,4a (Hz)
J 4a,5 (Hz)
12
10
1
10
10
13C NMRg
C1
C3
C4
C4a
C5
C6
C7
C8
R2e
R3f
C1
154.90
170.32
42.87
58.76
49.58
32.18
24.12
46.51
37.14
44.04
154.90
155.34
170.57
43.32
57.72
48.71
29.89
20.12
45.66
36.81
44.11
155.34
138.29
170.10
43.26
57.75
45.02
30.61
20.32
46.22
29.77
44.40
138.29
155.88
171.53
35.89
63.82
50.26
32.11
24.20
46.60
18.09
43.85
155.88
154.68
172.91
37.55
57.75
47.19
30.94
22.61
44.00
11.37
44.15
154.68
155.25
171.39
37.25
60.64
46.69
29.52
19.44
45.24
14.44
44.40
155.25
155.31
172.31
36.42
63.74
50.71
32.52
24.45
46.97
18.58
40.86
155.31
155.07
173.56
37.69
57.57
47.25
30.88
22.61
43.95
11.58
40.47
155.07
a
b
Satisfactory analyses for C, H, N. From EtOAc/hexane. c Novapak C18 (3.9 × 150 mm, 4 µm), using mixtures of A ) CH3CN and B
) 0.05% TFA in H2O. Measured in CDCl3 at 300 MHz, except for 7a ,b, 15a ,b, and 15c,d measured at 400 MHz. e CH3 except for
d
compounds 7, where it refers to the CH2 of CH2Ph. f The CH2 of R3 except for compounds 21, where it refers to NMe2. Measured in
g
CDCl3 at 75 MHz, except for 15a ,b and 15c,d , which were measured at 100 MHz.
1
resulting residue was treated with water (50 mL). Afterward,
1 N NaOH solution was added dropwise until pH ) 9, and
the aqueous phase was extracted with CH2Cl2 (2 × 100 mL).
The combined organic extracts were washed with brine (100
mL) and dried over Na2SO4, and the solvent was evaporated
to dryness. The resulting residue was purified by flash
chromatography, employing a (1-9%) gradient of MeOH in
CH2Cl2 as eluant, yielding the (1.4:1) unresolved diastereo-
meric mixture of the 2,3-cis piperidines 6c,d (higher Rf, 19%)
and the (1.2:1) unresolved diastereomeric mixture of the 2,3-
trans piperidines 6a ,b (lower Rf, 60%).
19%). RP HPLC tR ) 3.80 (A:B ) 40:60); H NMR (200 MHz,
CDCl3) δ 1.39 (s, 2.97 H, Boc), 1.46 (s, 6.03 H, Boc), 1.49-1.88
(m, 4 H, 4-H and 5-H), 2.59-3.08 (m, 7 H, 2-CH, 2-H, 6-H,
1-NH and CH2Ph), 3.45 (s, 3 H, OCH3,), 3.69 (d, 0.33 H, J )
11 Hz, 3-H), 4.02 (d, 0.67 H, J ) 11 Hz, 3-H), 5.45 (m, 1 H,
3-NH), 7.06-7.20 (m, 5 H, aromatics); 13C NMR (50 MHz,
CDCl3) δ 20.86 (C5), 28.39 [CH3 (Boc)], 28.42 (C4), 30.49 and
34.95 (CH2Ph), 45.35 (C3), 47.06 (C3 and C6), 50.34 and 51.18
(2-CH), 51.37 (OCH3), 61.00 and 61.32 (C2), 78.74 and 79.09
[C(CH3)3], 126.29-138.81 (Ph), 155.17 and 155.46 [CO (Boc)],
174.00 and 174.97 [CO (ester)]. Anal. (C20H30N2O4) C, H, N.
(2R*,3S*)-3-(ter t-Bu toxyca r bon yla m in o)-2-(1-m eth oxy-
ca r bon yl-2-p h en yleth yl)p ip er id in es (6a ,b). Syrup (326 mg,
Gen er a l P r oced u r e for th e Syn th esis of th e 2,4-Di-
ben zyl-5-(ter t-bu toxycar bon ylam in o)-1,3-dioxoper h ydr o-
p yr id o[1,2-c]p yr im id in es 7. Benzyl isocyanate (64 µL, 0.5
mmol) was slowly added to a solution of the corresponding
diastereoisomeric mixture of 3-(tert-butoxycarbonylamino)-2-
(1-methoxycarbonyl-2-phenylethyl)piperidines 6a ,b or 6c,d
(182 mg, 0.5 mmol) in dry THF (8 mL). After 1 h of stirring at
room temperature, the reaction mixture was diluted with THF
(8 mL). Then, NaH (60% dispersion, 24 mg, 0.6 mmol) was
added, and the stirring was continued for an additional 3 h.
Afterward, the reaction mixture was poured into 1 N HCl
solution (25 mL) cooled to 0 °C, and the mixture was extracted
with EtOAc (2 × 50 mL). The combined organic extracts were
washed with brine (25 mL) and dried over Na2SO4, and the
solvent was evaporated to dryness. The resulting residue was
purified either by flash chromatography, employing 25% of
EtOAc in hexane as eluant, in the case of the 1,3-dioxoper-
hydropyrido[1,2-c]pyrimidine derivative 7a ,b (186 mg, 80%),
1
60%). RP HPLC tR ) 3.59 (A:B ) 40:60); H NMR (300 MHz,
CDCl3) δ 1.17 (m, 1 H, 4-Hax), 1.43 (s, 6.3 H, Boc), 1.48 (s, 2.7
H, Boc), 1.43-1.50 (m, 1 H, 5-H), 1.60-1.69 (m, 1 H, 5-H),
1.89 (s, 1 H, 1-NH), 2.01 (m, 0.7 H, 4-Hec), 2.09 (m, 0.3 H, 4-Hec),
2.37-2.61 (m, 2 H, 2-H and 6-Hax), 3.02 (m, 4 H, 2-CH, 6-Hec
and CH2Ph), 3.44 (m, 0.7 H, 3-H), 3.68 (m, 0.3 H, 3-H), 3.51
(s, 2.1 H, OCH3,) 3.62 (s, 0.9 H, OCH3,), 4.31 (d, 0.7 H, J ) 10
Hz, 3-NH), 4.42 (d, 0.3 H, J ) 10 Hz, 3-NH), 7.18-7.36 (m, 5
H, aromatics); 13C NMR (50 MHz, CDCl3) δ 26.03 and 27.02
(C5), 28.73 [CH3 (Boc)], 32.46, 32.75, 33.53 (C4 and CH2Ph),
35.47 (CH2Ph), 45.94 (C6), 48.21 (2-CH), 50.60 (C3), 46.50, 49.00
and 49.53 (C6, 2-CH, C3), 51.86 (OCH3), 61.70 and 64.23 (C2),
79.55 [C(CH3)3], 126.37, 128.72, 128.49, 129.41 (Ph), 140.03
[C(Ph)], 155.47 and 155.68 [CO (Boc)], 174.95 [CO (ester)].
Anal. (C20H30N2O4) C, H, N.
(2R*,3R*)-3-(ter t-Bu toxyca r bon yla m in o)-2-(1-m eth oxy-
ca r bon yl-2-p h en yleth yl)p ip er id in es (6c,d ). Syrup (106 mg,

CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5325
Ta ble 4. Analytical Data of the New 5-[N-(tert-
Butoxycarbonyl)tryptophyl-1,3-dioxoperhydropyrido[1,2-c]
Pyrimidine Derivatives
) 17 Hz, 2-CH2), 3.02 (m, 1 H, J ) 11 Hz, 6-Hec) 3.31 (dq, 1 H,
J ) 10 and 4 Hz, 3-H), 3.70 (s, 3 H, OCH3,), 4.39 (d, 1 H, J )
10 Hz, 3-NH); 13C NMR (50 MHz, CDCl3) δ 25.79 (C5), 28.34
[CH3 (Boc)], 32.44 (C4), 37.68 (2-CH2), 45.78 (C6), 51.57 and
51.84 (C2 and C3), 58.97 (OCH3), 79.34 [C(CH3)3], 155.34 [CO
(Boc)], 173.20 [CO (ester)]. Anal. (C13H24N2O4) C, H, N.
(2R*,3R*)-3-(ter t-b u t oxyca r b on yla m in o)-2-(m et h oxy-
ca r bon ylm eth yl)p ip er id in e (11c,d ). White solid (128 mg,
24%). Mp 44-46 °C (CH2Cl2/MeOH); 1H NMR (200 MHz,
CDCl3) δ 1.44 (s, 9 H, Boc), 1.52-1.64 (m, 3 H, 4-H and 5-H),
1.86 (m, 1 H, 4-H), 1.99 (s, 1 H, 1-NH), 2.31 (dd, 1 H, J ) 17
and 9 Hz, 2-CH2), 2.44 (dd, 1 H, J ) 17 and 4 Hz, 2-CH2), 2.67
(m, 1 H, 6-Hax), 2.97 (m, 1 H, 6-Hec), 3.04 (m, 1 H, 2-H), 3.69
(s, 4 H, 3-H and OCH3,), 5.35 (d, 1 H, J ) 9 Hz, 3-NH); 13C
NMR (50 MHz, CDCl3) δ 20.60 (C5), 28.32 [CH3 (Boc)], 30.14
(C4), 37.56 (2-CH2), 40.16 (C6), 40.73 (C3) 51.61 (C2), 55.98
(OCH3), 78.89 [C(CH3)3], 155.54 [CO (Boc)], 172.98 [CO (ester)].
Anal. (C13H24N2O4) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of th e 1-(Ben zyl-
oxyca r bon yl)-3-(ter t-bu toxyca r bon yla m in o)-2-(m eth oxy-
ca r bon ylm eth yl)p ip er id in es 12. Benzyl chloroformate (0.54
mL, 3.80 mmol) was slowly added to a stirred solution of the
corresponding 3-(tert-butoxycarbonylamino)-2-(methoxycar-
bonylmethyl)piperidine 11a ,b or 11c,d (523 mg, 1.90 mmol)
and propylene oxide (2.02 mL, 28.80 mmol) in CH2Cl2 (10 mL)
at 0 °C, and the stirring was continued for 20 h. Evaporation
of the solvent to dryness gave a residue which was purified
by flash chromatography, using a (17-50%) gradient of EtOAc
in hexane, to give the 2,3-trans- and 2,3-cis-disubstituted
piperidines 12a ,b and 12c,d , respectively.
(2R *,3S*)-1-(Be n zyloxyca r b on yl)-3-(t er t -b u t oxyca r -
b on yla m in o)-2-(m e t h oxyca r b on ylm e t h yl)p ip e r id in e s
(12a ,b). Syrup (717 mg, 92%); RP HPLC tR ) 4.30 (A:B )
50:50); 1H NMR (300 MHz, CDCl3) δ 1.41 (s, 9 H, Boc), 1.55-
1.75 (m, 4 H, 4-H and 5-H), 2.53 (dd, 1 H, J ) 14 and 6 Hz,
2-CH2), 2.67 (dd, 1 H, J ) 14 and 9 Hz, 2-CH2), 2.91 (m, 1 H,
6-Hax), 3.55 (s, 3 H, OCH3,), 3.71 (m, 1 H, 3-H) 4.10 (m, 1 H,
6-Hec), 4.72 (t, 1 H, J ) 7 Hz, 2-H), 4.89 (d, 1 H, J ) 7 Hz,
3-NH), 5.12 [s, 2 H, CH2(Z)], 7.26-7.36 (m, 5 H, aromatics);
13C NMR (75 MHz, CDCl3) 19.59 and 23.36 (C4 and C5), 28.30
[CH3 (Boc)], 34.71 (2-CH2), 38.45 (C6), 47.41 (C2), 51.81 (C3),
52.90 (OCH3), 67.34 [CH2(Z)], 79.54 [C(CH3)3], 127.69, 127.95,
128.45 and 136.41 (Ph), 154.81 [CO (Boc) and CO (Z)], 170.60
[CO (ester)]. Anal. (C21H30N2O6) C, H, N.
(2R*,3R*)-1-(Ben zyloxycar bon yl)-3-(ter t-bu toxycar bon -
ylam in o)-2-(m eth oxycar bon ylm eth yl)piper idin es (12c,d).
White solid (681 mg, 86%). Mp 111-113 °C (hexane/EtOAc);
RP HPLC tR ) 4.44 (A:B ) 50:50); 1H NMR (300 MHz, CDCl3)
δ 1.43 (s, 9 H, Boc), 1.50-1.80 (m, 4 H, 4-H and 5-H), 2.49
(dd, 1 H, J ) 14 and 9 Hz, 2-CH2), 2.56 (dd, 1 H, J ) 14 and
6 Hz, 2-CH2), 2.79 (dt, 1 H, J ) 14 and 2 Hz, 6-Hax), 3.55 (s, 3
H, OCH3,), 3.75 (m, 1 H, 3-H) 4.06 (d, 1 H, J ) 14, 6-Hec) 4.47
(bs, 1 H, 3-NH), 5.09 [d, 1 H, J ) 13 Hz, CH2(Z)], 5.18 [d, 1 H,
J ) 13 Hz, CH2(Z)], 5.09-5.18 (m, 1 H, 2-H), 7.26-7.39 (m, 5
H, aromatics); 13C NMR (50 MHz, CDCl3) 24.43 and 25.51 (C4
and C5), 28.30 [CH3 (Boc)], 31.18 (2-CH2), 38.28 (C6), 49.96 (C2),
51.40 (OCH3), 51.79 (C3), 67.27 [CH2(Z)], 79.95 [C(CH3)3],
127.82, 128.40, and 136.77 (Ph), 154.69 and 155.19 [CO (Boc)
and CO (Z)], 171.65 [CO (ester)]. Anal. (C21H30N2O6) C, H, N.
Syn th esis of (2R*,3S*)-1-(Ben zyloxyca r bon yl)-3-(ter t-
b u t oxyca r b on yla m in o)-2-[1-(m et h oxyca r b on yl)et h yl]-
p ip er id in es 13a ,b. A solution of (2R*,3S*)-1-(N-benzyloxy-
carbonyl)-3-(tert-butoxycarbonylamino)-2-(methoxycarbonyl-
methyl)piperidine (12a ,b) (406 mg, 1 mmol) in dry THF (7 mL)
was added dropwise to a stirred solution of lithium bis-
(trimethylsilyl)amide (1 M solution in THF, 2.0 mL, 2 mmol)
in THF (5 mL) at - 78 °C, and the stirring was continued for
45 min at the same temperature. Afterward, a solution of
methyl iodide (123 µL, 2 mmol) and hexamethylphosphoramide
(100 µL, 0.58 mmol) in dry THF (5 mL) was added dropwise
at - 78 °C. After the reaction mixture was stirred at this
temperature for further 4 h, the resulting solution was then
treated with 10% NH4Cl solution (50 mL) and extracted with
diethyl ether (2 × 50 mL). The combined organic extracts were
yield
(%)
mp
compd
(°C)a
formulab
tR(min) (A:B)c
8a
8b
8c
8d
70
23
60
16
71
19
66
7
80
3
56
81
3
118-120 C38H43N5O5 9.90 (50:50)
111-113
100-102
95-97
C
C
C
38H43N5O5 10.91 (50:50)
38H43N5O5 9.68 (50:50)
38H43N5O5 10.96 (50:50)
9a d
9be
16a
16b
16c
16d
17a
111-113 C38H43N5O5 10.39 (50:50)
118-120 C38H43N5O5 9.13 (50:50)
125-127
108-110
113-116
foam
C
C
C
C
32H39N5O5 7.00 (45:55)
32H39N5O5 7.31 (45:55)
32H39N5O5 10.45 (40:60)
32H39N5O5 11.89 (40:60)
140-143 C38H45N5O5 20.30 (45:55)
foam C32H39N5O5 10.29 (40:60)
113-116 C32H39N5O5 11.92 (40:60)
22cf
22d g
20a
23a
23b
54
82
9
170-172
foam
foam
foam
172-174
C
C
C
C
C
38H46N6O5 7.71(40:60)
33H42N6O5 9.60 (30:70)
33H42N6O5 10.19 (30:70)
33H42N6O5 9.41, 7.87 (30:70)
39H48N6O5 10.05 (40:60)
23e,f (e:f, 9:1) 62
24a
40
a
From EtOAc/hexane, except for 20a and 24a from CH2Cl2/
MeOH. Satisfactory analyses for C, H and N. c Novapak C18 (3.9
b
× 150 mm, 4 µm), using mixtures of, A ) CH3CN and B ) 0.05%
d
TFA in H2O. Enantiomer of 8b. e Enantiomer of 8a . f Enantiomer
g
of 16d . Enantiomer of 16c.
or by radial chromatography, using 17% of EtOAc in hexane
as eluant, for 7c,d (166 mg, 60%) and 7e,f (22 mg, 8%), whose
significant analytical and spectroscopic data are summarized
in Table 3.
Gen er a l P r oced u r e for th e Syn th esis of th e 2,4-Di-
ben zyl-5-[N-(ter t-bu toxyca r bon yl)tr yp top h yla m in o]-1,3-
dioxoper h ydr opyr ido[1,2-c]pyr im idin e Der ivatives 8a-d
a n d 9a ,b. TFA (0.5 mL) was added dropwise to a stirred
solution of the corresponding 2,4-dibenzyl-5-(tert-butoxycar-
bonylamino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine 7a,b and
7c,d (84 mg, 0.18 mmol) in CH2Cl2 (2 mL), and the stirring
was continued for 45 min at room temperature. Evaporation
of the solvent to dryness gave a residue which was dissolved
in dry CH2Cl2 (3 mL). Then, Boc-L- or -D-Trp-OH (66 mg, 0.26
mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP, 95 mg, 0.26 mmol), and TEA (50
µL, 0.40 mmol) were added successively to that solution, and
the stirring was continued at room temperature for 18 h. The
solvent was evaporated to dryness, and the residue was
dissolved in EtOAc (25 mL). The resulting solution was washed
successively with 10% citric acid (10 mL), 10% NaHCO3 (10
mL), water (10 mL), and brine (20 mL), dried over Na2SO4,
and the solvent was evaporated. The resulting diastereo-
isomeric pairs of Boc-tryptophyl derivatives 8a ,b, 8c,d , and
9a ,b were purified and resolved by flash chromatography using
a (10-50%) gradient of EtOAc in hexane as eluant. Significant
analytical and spectroscopic data of these compounds are
summarized in Tables 4-6.
Syn th esis of th e 3-(ter t-Bu toxycar bon ylam in o)-2-(m eth -
oxyca r bon ylm eth yl)p ip er id in es 11. These compounds were
prepared from methyl (4S)-7-benzyloxycarbonylamino-4-(tert-
butoxycarbonylamino)-3-oxoheptanoate (4) (844 mg, 2 mmol),
by applying the same methodology above mentioned for the
synthesis of analogue piperidines 6. The resulting diastereo-
isomeric mixture was resolved by flash chromatography, using
a (1-9%) gradient of MeOH in CH2Cl2 as eluant, into the 2,3-
cis-disubstituted-piperidine 11c,d (higher Rf, 128 mg, 24%) and
the 2,3-trans-disubstituted piperidine 11a ,b (lower Rf, 284 mg,
52%) as (≈9:1) racemic mixtures.
(2R*,3S*)-3-(ter t-Bu t oxyca r b on yla m in o)-2-(m et h oxy-
ca r bon ylm eth yl)p ip er id in e (11a ,b). White solid (284 mg,
52%). Mp 81-83 °C (CH2Cl2/MeOH); 1H NMR (200 MHz,
CDCl3) δ 1.25 (dq, 1 H, J ) 11 and 4 Hz, 4-Hax), 1.45 (s, 9 H,
Boc), 1.56-1.76 (m, 2 H, 5-H), 1.94 (s, 1 H, 1-NH), 2.02 (m, 1
H, 4-Hec), 2.38 (dd, 1 H, J ) 17 and 9 Hz, 2-CH2), 2.58 (dt, 1
H, J ) 11 and 3 Hz, 6-Hax), 2.70 (m, 1 H, 2-H), 2.73 (d, 1 H, J

5326 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Mun˜oz-Ruiz et al.
Ta ble 5. Significant 1H NMRa Spectroscopic Data of 5-(Boc- and 2-Adoc-Trp)-Amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine
Derivatives
R-H
compd
4-H
2.33
4a-H
2.43
5-H
3.60
6-H
1.89
1.01-1.05
0.94, 1.81
7-H
1.51
8-H
R2b
R3c
(Trp) J 4,4a J 4a,5
8a d
2.04-2.36
4.32
2.35-2.43
4.36
2.51, 2.79 5.05, 4.98 4.09
2.51, 2.85 5.00, 5.08 4.48
0
0
11.5
11
2
8be
8c
2.74
2.37
2.78
2.10
2.35-2.43 3.71
1.52
2.95
3.06
2.53
4.16
4.02
3.56
1.37-1.41
1.63
1.25
1.30-1.41
1.21, 1.85
1.37-1.41 2.37-2.46
2.81, 3.17 4.93
4.36
8
4.19
2.44, 4.25
8d
0.86
1.22-1.27
1.51
2.90, 3.00 4.84, 4.88 4.31
5
2
16a
16b
16cf
16d g
2.43-2.54
4.32
2.49-2.57
4.35
1.02
1.17
1.07
1.18
4.91, 4.98 4.34
4.90, 5.00 4.42
4.85, 4.97 4.54
4.82, 4.92 4.34
4.85, 4.92 4.36
0
11
11
2
2.49-2.57 2.70
1.68-1.76 2.84
3.55-3.61 1.14-1.54
1.46
0
4.19
1.34-1.41
1.72
1.04
2.46, 4.12
11
10
0
1.34-1.41
1.22-1.40 2.47
2.41
2.96
4.17-4.22 1.22-1.42
2
4.17-4.22
2.41, 4.27
2.47
4.22-4.12
2.-8-2.59
4.31
17a
20a
1.95
2.50
3.50
3.64
1.16, 1.95
1.20,1.82
1.59
154-1.67
0.96
-
11
9
2.14-2.24 2.57
2.93
2.93
2.97
4.43
4.26
4.49
9.5
23a
1.98
2.62
3.69
1.20-1.27
1.90
1.03-1-87 1.63-1.68 2.61, 4.39
1.08, 1.87
1.24
1.62
1.08
0
11
23b
23g,h
24a
2.56
1.96
2.82
2.57
3.84
3.90
3.67
1.28
1.28
1.08
0
h
0
11
11
10
1.46, 1.81
1.56
2.61, 4.12
2.51, 4.30
2.95, 2.94 4.47
2.94 4.37
1.97-2.01 2.62
1.97-2.01
a
b
Spectra registered in CDCl3 at 400 MHz except for 8a -c, 16a -d , and 24g,h registered at 500 MHz. The CH3 of R2, except for
compounds 8, where it is the CH2. c The CH2 of R3, except for 20a , 23, and 23a , where it refers to NMe2. The same data for its enantiomer
d
9b. e The same data for its enantiomer 9a . f The same data for its enantiomer 22d . The same data for its enantiomer 22c. The 4-H
g
h
and 4a-H signals did not have enough resolution to measure this coupling constant.
Ta ble 6. Significant 13C NMRa Spectroscopic Data of 5-(Boc- and 2-Adoc-Trp)-Amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine
Derivatives
compd
C1
C3
C4
C4a
C5
C6
C7
C8
R2 b
R3 c
CR (Trp)
8a d
8be
8c
155.49
155.78
153.37
152.66
151.97
152.29
151.98
154.59
154.35
150.23
152.30
150.25
154.96
152.35
171.45
171.17
171.47
171.84
171.65
171.94
171.74
170.97
171.61
171.44
171.79
171.44
173.92
171.43
41.61
42.16
42.88
43.81
34.50
35.63
32.09
36.30
36.77
33.46
35.09
35.30
36.57
32.40
58.22
58.59
56.00
56.71
34.50
63.83
63.07
59.01
59.39
54.28
62.84
63.24
56.92
62.82
47.46
47.38
45.18
46.42
63.07
48.60
48.40
43.83
44.24
50.55
48.47
48.44
45.62
48.63
31.40
32.23
28.94
29.13
31.63
31.94
31.70
28.80
28.62
30.63
31.75
29.67
30.28
31.95
23.72
24.03
18.95
19.29
23.83
24.17
23.86
18.27
18.47
23.06
23.88
23.94
22.42
23.86
46.56
46.82
45.04
45.41
46.54
46.85
46.63
44.56
44.79
44.48
46.66
46.72
43.77
46.71
36.66
37.18
33.47
35.51
17.94
18.19
17.97
12.02
12.96
-
44.06
44.38
44.28
44.10
43.78
44.08
43.83
44.42
44.39
40.51
40.54
40.59
40.53
40.53
55.82
56.82
54.68
55.78
55.80
56.24
55.81
54.71
55.98
55.82
55.63
56.02
55.40
55.99
8d
16a
16b
16cf
16d g
17a
20a
23a
23b
23g,h
24a
18.15
18.22
11.53
18.21
a
b
Spectra registered in CDCl3 at 75 MHz except for 8c, 20a , and 23g,h , which were registered at 100 MHz. CH3 except for compounds
8 and 9, where it is the CH2 of CH2Ph. c The CH2 of CH2Ph, except for compounds 22 and 23a where it refers to NMe2. The same data
d
for its enantiomer 9b. e The same data for its enantiomer 9a . f The same data for its enantiomer 22d . The same data for its enantiomer
g
22c.
washed successively with water (50 mL) and brine (50 mL)
and dried over Na2SO4. Evaporation of the solvent to dryness
gave a residue which was purified by flash chromatography,
using a (10-50%) gradient of EtOAc in hexane as eluant, to
give the title compounds 13a ,b (syrup, 391 mg, 93%) as a
single racemic mixture. RP HPLC tR ) 8.27 (A:B ) 45:55); 1H
NMR (300 MHz, DMSO, 80 °C) δ 0.95 (d, 1 H, J ) 7 Hz, CH3),
1.37 (s, 9 H, Boc), 1.37-1.57 (m, 2 H, 4-H) 1.82 (m, 2 H, 5-H),
2.73 (m, 1 H, 6-Hax), 2.92 (q, 0.5 H, J ) 7 Hz, 2-CH), 2.95 (q,
0.5 H, J ) 7 Hz, 2-CH), 3.48 (m, 1 H, 3-H), 3.66 (s, 3 H, OCH3,),
3.95 (m, 1 H, J ) 13 Hz, 6-Hec), 4.30 (d, 1 H, J ) 11 Hz, 2-H),
5.10 [s, 2 H, CH2(Z)], 6.47 (ws, 1 H, 3-NH), 7.30-7.37 (m, 5 H,
aromatics). 13C NMR (75 MHz, CDCl3) 14.16 (Me) 19.40 and
19.68 (C5) 23.42 (C4), 28.32 [CH3 (Boc)], 38.52 (C6), 39.11 and
39.35 (2-CH), 46.28 and 46.86 (C3), 52.13 (OCH3), 57.80 and
57.73 (C2), 67.37 [CH2(Z)], 79.98 [C(CH3)3], 127.73, 127.98,
128.49 and 136.40 (Ph), 154.61 and 156.28 [CO (Boc) and CO
(Z)], 174.49 [CO (ester)]. Anal. (C22H32N2O6) C, H, N.
Syn th esis of (2R*,3S*)-3-(ter t-Bu toxyca r bon yla m in o)-
2-[1-(m eth oxyca r bon yl)eth yl]p ip er id in es 14a ,b. A solu-
tion of (2R*,3S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl-
amino)-2-(1-(methoxycarbonyl)ethyl)piperidine (13a ,b) (350
mg, 0.83 mmol) in MeOH (30 mL) was hydrogenated at room
temperature and 1 atm of H2 pressure in the presence of 10%
Pd(C) (35 mg) for 30 min. Afterward, the catalyst was filtered
off and washed with MeOH (2 × 3 mL), and the filtrate was

CCK Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5327
evaporated to dryness. The resulting residue was purified by
flash chromatography, using a (1-4%) gradient of MeOH in
CH2Cl2 as eluant The title compound 14a ,b was obtained as
a syrup which solidified on standing as a white solid (197 mg,
83%). Mp 61-62 °C (CH2Cl2/MeOH); 1H NMR δ 1.07-1.27 (m,
3 H, 4-Hax), 1.18 (d, 3 H, J ) 7 Hz, CH3), 1.40 (s, 9 H, Boc),
1.48-1.64 (m, 2 H, 5-H), 1.67 (s, 1 H, 1-NH), 2.03 (m, 1 H,
4-Hec), 2.50 (dq, 1 H, J ) 12 and 3 Hz, 6-Hax), 2.64 (dd, 1 H, J
) 10 and 3 Hz, 2-H), 2.76 (m, 1 H, 2-CH), 2.98 (m, 1 H, J ) 12
Hz, 6-Hec) 3.38 (dq, 1 H, J ) 10 and 4 Hz, 3-H), 3.61 (s, 3 H,
OCH3,), 4.30 (d, 1 H, J ) 10 Hz, 3-NH). 13C NMR (50 MHz,
CDCl3) 10.76 (Me) 25.91 (C5) 28.34 [CH3 (Boc)], 33.00 (C4),
39.92 (2-CH), 46.02 (C6), 49.45 (C3), 51.80 (OCH3), 62.90 (C2),
79.30 [C(CH3)3], 155.24 [CO (Boc)], 176.72 [CO (ester)]. Anal.
(C14H26N2O4) C, H, N.
mL) at - 78 °C, and the stirring was continued for 45 min at
the same temperature. Afterward, a solution of methyl iodide
(92 µL, 1.5 mmol) and hexamethylphosphoramide (75 µL, 0.44
mmol) in dry THF (4 mL) was added dropwise at -78 °C. After
the reaction mixture was stirred at this temperature for
further 4 h, the resulting solution was then treated with 10%
NH4Cl solution (50 mL) and extracted with diethyl ether (2 ×
50 mL). The combined organic extracts were washed succes-
sively with water (50 mL) and brine (50 mL) and dried over
Na2SO4. Evaporation of the solvent to dryness gave a residue
which was purified by flash chromatography, using a (20-
50%) gradient of EtOAc in hexane as eluant, to give the title
compounds 15c,d , 21a ,b, and 21g,h as racemic mixtures,
whose significant analytical and spectroscopic data are sum-
marized in Table 3.
Syn th esis of th e 2-Ben zyl-5-(ter t-bu toxyca r bon yla m i-
n o)-4-m e t h yl-1,3-d ioxo-p e r h yd r op yr id o[1,2-c]p yr im i-
d in e Der iva tives 15. These compounds were prepared from
the (2R*,3S*)-3-(tert-butoxycarbonylamino)-2-(1-(methoxycar-
bonyl)ethyl)piperidines 14a ,b (150 mg, 0.52 mmol), by applying
the same methodology as described above for the preparation
of analogues 7. The resulting diastereoisomeric mixture of
15a ,b and 15g,h was purified and resolved by flash chroma-
tography, employing 25% of EtOAc in hexane as eluant,
followed by preparative radial chromatography, employing a
(0.2-1%) gradient of MeOH in CH2Cl2 as eluant, to give the
racemic mixtures 15g,h (higher Rf, 38 mg, 19%) and 15a ,b
(lower Rf, 120 mg, 60%). Significant analytical and spectro-
scopic data of these compounds are summarized in Table 3.
Syn th esis of (4S,4a R,5S)- a n d (4R,4a S,5R)-2-Ben zyl-5-
[[N-(ter t-bu toxyca r bon yl)-L-tr yp top h yl]a m in o]-4-m eth yl-
1,3-d ioxop er h yd r op yr id o[1,2-c]p yr im id in es 16a a n d 16b.
These compounds were prepared from the (≈9:1) racemic
mixture of (4R*,4aS*,5R*)-2-benzyl-5-(tert-butoxycarbonyl-
amino)-4-methyl-1,3-dioxoperhydropyrido[1,2-c]pyrimidines
15a ,b (97 mg, 0.25 mmol), applying the same procedure as
described for the preparation of analogues 8. Significant
analytical and spectroscopic data of these Boc-tryptophyl
derivatives 16a (higher Rf, 94 mg, 66%) and 16b (lower Rf, 10
mg, 7%) are summarized in Tables 4-6.
Gen er a l P r oced u r e for th e Syn th esis of (4S,4a R,5S)-
5-[[N-(2-Ad a m a n t yloxyca r b on yl)-L-t r yp t op 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 Der iva tives
17a , 20a , a n d 24a . TFA (0.2 mL) was added to a stirred
solution of the corresponding (4S,4aR,5S)-5-[[N-(tert-butoxy-
carbonyl)-L-tryptophyl]amino]-1,3-dioxoperhydropyrido[1,2-c]-
pyrimidine 16a , 3a , or 23a (0.10 mmol) in CH2Cl2 (2 mL). After
4 h of stirring at room temperature, the solvent was evaporated
to dryness, and the resulting residue was dissolved in dry THF
(2 mL). Then, dry triethylamine (28 µL, 0.20 mmol) was added
to the resulting solution, and the reaction mixture was stirred
for 10 min. Afterward, a solution of 2-adamantyl chloroformate
[0.30 mmol, prepared from 2-adamantanol (50 mg, 0.33 mmol)
as previously described47 in THF (2 mL) was added, and the
stirring was continued for further 18 h. After removal of the
solvent to dryness under reduced pressure, water (5 mL) was
added, and the suspension was extracted with CH2Cl2 (2 × 10
mL). The combined organic extracts were washed with brine
(10 mL) and dried over Na2SO4, and the solvent was evapo-
rated to dryness. The crude residue was purified by prepara-
tive radial chromatography, using a (25-50%) gradient of
EtOAc in hexane (17a ) or (1-10%) gradient of MeOH in
CH2Cl2 (20a and 24a ) as eluants, yielding the corresponding
5-N-[(2-adamantyloxycarbonyl)-L-tryptophyl]amino derivatives
17a , 20a , and 24a , whose analytical and spectroscopic data
are summarized in Tables 4-6.
Gen er a l P r oced u r e for th e Syn th esis of th e 5-[[N-(ter t-
bu toxyca r bon yl)-L- a n d D-tr yp top h yl]a m in o]-4-m eth yl-
1,3-d ioxop er h yd r op yr id o[1,2-c]p yr im id in e Der iva tives
16c,d 22c,d , a n d 23. These compounds were prepared from
the appropriate 5-(tert-butoxycarbonylamino)-4-methyl-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine derivative 15c,d , 21a ,b,
or 21g,h (0.2 mmol), by applying the same procedure as above
indicated for the synthesis of the analogues 8 and 9. The (9:1)
diastereoisomeric mixture 23g,h , resulting from 21g,h , could
not be resolved. Significant analytical and spectroscopic data
of the title compounds are summarized in Tables 4-6.
Biological Meth ods. Mater ials. [3H]Propionyl-CCK-8 (spe-
cific activity, 60-80 mCi mmol-1) was from Amersham Bio-
sciences. CCK-8 and CCK-4 were from Sigma-Aldrich. PD-
135,158 was a gift from Parke Davis. Amylase kit reagent was
from Boeringher Mannheim. (Thr, Nle)-CCK-9 was synthe-
sized by Luis Moroder (Max Planck Institut fur Biochimie,
Munchen, Germany). 125INa was from Amersham Biosciences.
(Thr, Nle)CCK-9 was conjugated with Bolton-Hunter reagent,
purified and radioiodinated as described previously by Fourmy
et al.48 The specific activity of radioiodinated peptide was
1600-2000 Ci/mmol. Myo-2-[3H]inositol was from Amersham
Biosciences.
Ra t CCK1 a n d CCK2 Recep tor Bin d in g Assa ys. CCK1
and CCK2 receptor binding assays were performed using rat
pancreas and cerebral cortex homogenates, respectively, ac-
cording to the method described by Dauge´ et al.,41 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]propionyl-CCK-8 in
PIPES HCl buffer, pH 6.5, containing MgCl2 (30 mM), baci-
tracin (0.2 mg/mL) and soybean trypsin inhibitor (SBTI, 0.2
mg/mL), for 120 min at 25 °C. Rat brain cortex was homog-
enized 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. IC50 values were calculated from the displace-
ment curves analyzed with GraphPad Prism software.49
Am yla se Relea se. Dispersed rat pancreatic acini were
prepared by using a modification of the technique of J ensen
et al.45 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
Gen er a l P r oced u r e for th e Syn th esis of th e 5-(ter t-
Bu t oxyca r b on yla m in o)-4-m e t h yl-1,3-d ioxop e r h yd r o-
p yr id o[1,2-c]p yr im id in e Der iva t ives 15c,d , 21a ,b , a n d
21g,h . A solution of the corresponding 5-(tert-butoxycarbonyl-
amino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine derivative
18c,d 27 or 19a ,b32 (0.75 mmol) in dry THF (5 mL) was added
dropwise to a stirred solution of lithium bis(trimethylsilyl)-
amide (1 M solution in THF, 1.5 mL, 1.5 mmol) in THF (5

5328 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Mun˜oz-Ruiz et al.
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.50
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:
presence of 2 µCi/ml myo-2-[3H] inositol and incubated over-
night in 24-well dishes.
Wild -Typ e Hu m a n CCK2 Recep tor Bin d in g Assa y.
Approximately 24 h after the transfer of transfected cells to
24-well plates, the cells were washed with phosphate-buffered
saline pH 6.95, 0.1% BSA and then incubated for 60 min at
37 °C in 0.5 mL Dulbelcco’s Modified Eagle’s Medium, 0.1%
BSA with either 71 pM 125I-BH-(Thr, Nle)CCK-9 in the
presence or the absence of competing compound. The cells were
washed twice with phosphate-buffered saline pH 6.95 contain-
ing 2% BSA, and cell-associated radioligand was collected with
0.1 N NaOH added to each well. The radioactivity was directly
counted in a gamma counter (Auto-Gamma, Packard, Downers
Grove, IL) or added to scintillant and counted for the tritiated
radioligand. Nonspecific binding was always less than 10% of
total binding.
In ositol P h osp h a te Assa ys. Approximately 24 h after the
transfer to 24-well plates and following overnight incuba-
tion in complete medium containing 2 µCi/mL of myo-2-
[3H]inositol, the transfected cells were washed with Dubelcco’s
Modified Eagle’s Medium and then incubated for 30 min in 2
mL/well Dubelcco’s Modified Eagle’s Medium containing 20
mM LiCl at 37 °C. The cells were washed with PI buffer at
pH 7.45: phosphate-buffered saline containing 135 mM NaCl,
20 mM HEPES, 2 mM CaCl2, 1.2 mM MgSO4, 1 mM EGTA,
10 mM LiCl, 11.1 mM glucose, and 0.5% BSA. The cells were
then incubated for 60 min at 37 °C in 0.5 mL PI buffer with
or without ligands at various concentrations. The reaction was
stopped by adding 1 mL methanol/chlorhydric acid to each
well, and the content was transferred to a column (Dowex AG
1-X8, formate form, Bio-Rad, Hercules, CA) for the extraction
of inositol phosphates. The columns were washed twice with
5 mL of distilled water and twice more with 2 mL of 5 mM
sodium tetraborate/60 mM sodium formate. The content of
each column was eluted by addition of 2.5 mL of 1 M
ammonium formate/100 mM formic acid. Samples of the eluted
fraction (0.5 mL) were added to scintillant, and â-radioactivity
was counted.
% 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.
Isola t ed Lon git u d in a l Mu scle-Myen t er ic P lexu s
(LMMP ) P r ep a r a tion fr om Gu in ea -P ig Ileu m . Guinea-pigs
were killed and bled. The ileum was excised approximately
10 cm from ileo-caecal junction, and longitudinal muscle strips
with the myenteric plexus (LMMP) attached were prepared.51
LMMP strips were suspended in a 10 mL organ bath contain-
ing Krebs bicarbonate solution (composition in mM: NaCl
118.2, KCl 4.6, CaCl2‚2H2O 1.6, MgSO4 1.2, KH2PO4 1.2,
NaHCO3 24.8 and glucose 1.0) maintained at 37 °C and
aerated with 95% O2/5% CO2. Tissues were equilibrated for
30 min at 0.5 mg applied force and then field-stimulated (1
Hz, 1 ms, 10-15 V) for 30 min. The strips were subsequently
stimulated by KCl (40 mM) to obtain a maximal contractile
response. The preparation was then washed with Krebs
bicarbonate solution and equilibrated for 20 min period before
performing the different experiments. After control responses
to KCl had been obtained, noncumulative concentration-
response curves (CRC) to CCK4 were obtained by stepwise
increases in concentration every 10 min; the preceding con-
centration was washed out, and the tissue was exposed to the
peptide for a period of 2 min. CRC for each peptide were
calculated as percentages of the initial KCl contraction, and
EC50 values were determined. In studies with drugs, each strip
was used to record two CRC: the first for the agonist alone
and the second for the agonist in the presence of the antago-
nist, each strip serving as its own control. Antagonists were
allowed to preequilibrate for 30 min prior to addition of the
agonist. The effect of antagonists was expressed as percentage
of inhibition of maximal response obtained with the agonist
alone in the same tissue, and pA2 values were calculated
according to the following equation,52
Ack n ow led gm en t. This work has been supported
by CICYT (SAF2000-0147).
Su p p or tin g In for m a tion Ava ila ble: Table of combustion
analysis data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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