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

Article abstract of DOI:10.1021/jm010898i

To further define the pharmacophore of the potent and selective 5-(tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based CCK₁ receptor antagonists the electronic and topographic properties of the central 1,3-dioxoperhydro-pyrido[1,2-c

Full text of DOI:10.1021/jm010898i

4196
J . Med. Chem. 2001, 44, 4196-4206
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 Cen tr a l 1,3-Dioxop er h yd r op yr id o[1,2-c]p yr im id in e Sca ffold
J ose´ M. Bartolome´-Nebreda,† 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 April 9, 2001
To further define the pharmacophore of the potent and selective 5-(tryptophyl)amino-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine-based CCK1 receptor antagonists the electronic and
topographic properties of the central 1,3-dioxoperhydro-pyrido[1,2-c]pyrimidine scaffold have
been modified. With this aim, the 1- and 3-oxo groups have been replaced by the thioxo- and
deoxi-analogues, and the fused piperidine ring has been contracted to the corresponding
pyrrolidine moiety. The results of the evaluation of the new analogues as CCK receptor ligands,
in rat pancreas and cerebral cortex preparations, showed that, whereas replacement of oxygen
with sulfur is allowed, reduction of the 1- or 3-oxo groups or the contraction of the fused
piperidine ring lead to the complete loss of binding affinity at CCK1 receptors. The thioxo-
analogues 5a , 8a , 12a , and 12b showed functional CCK1 antagonist activity, inhibiting the
CCK-8-stimulated amylase release from pancreatic acinar cells. The 1-thioxo analogue 5a , with
subnanomolar affinity (IC50 ) 0.09 × 10-9 M), was found to be the most potent and selective
compound within the family of 5-(tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-
based CCK1 antagonists.
In tr od u ction
dioxoperhydropyrido[1,2-c]pyrimidine derivative 1a (IQM-
95,333), which is one of the most selective CCK1 receptor
antagonists up to now described.6 Thus, this compound
showed a CCK1 receptor affinity in the nanomolar
range, but it was virtually devoid of affinity at brain
CCK2 receptors.14 In accordance with this CCK1 recep-
tor affinity, compound 1a was a potent inhibitor of
the CCK-8-stimulated amylase release from isolated
pancreatic acini, and blocked the CCK-8-induced hypo-
phagia and hypolocomotion in rats.14 Furthermore,
despite the predominant role attributed to CCK2 recep-
tors 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 sug-
gestion of some authors that CCK1 receptors may be
involved also in anxiogenesis.15-17 From previous struc-
ture-activity relationship studies, the Boc-L-Trp resi-
due and the (4aS,5R)-stereochemistry at the 1,3-dioxo-
perhydropyrido[1,2-c]pyrimidine skeleton emerged as
essential structural requirements for potent CCK1 bind-
ing affinity and subtype receptor selectivity.13,18 These
studies also showed the influence of the lipophilicity and
spatial orientation of the moiety linked at the N2-
position upon the binding profile of this family of CCK1
antagonists.19 Now, to further define the pharmaco-
phore, we have studied the effect of modifying the
electronic and topographic properties of the central 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine scaffold, through
thionation and reduction of the 1- and/or 3-oxo groups,
and the contraction of the fused piperidine to a pyr-
rolidine ring. Because of the lower electronegativity
and higher volume of sulfur with respect to oxygen,20,21
and to the higher polarization of the CdS bond,22,23
thionation produces a decrease in the acceptor hydrogen-
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
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-
* 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.
10.1021/jm010898i CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/18/2001

Structure-Activity Study of CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4197
Sch em e 1a
bonding properties of the 1- and 3-oxo groups. On the
other hand, and differently from oxygen, sulfur could
participate in additional π-π interactions, using empty
3d orbitals.24 The oxo group reduction produces impor-
tant topographic changes due to the transformation of
the plane sp2 hybridization into the tetrahedral sp3, and
also to a loss in the acceptor hydrogen-bonding proper-
ties. In regard to the contraction of the fused piperidine
ring to a pyrrolidine one, this structural modification
affects the topography of the 1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine skeleton, and, therefore, the spatial
arrangement of the groups linked to positions 2 and 5,
both critical for receptor recognition.18,19 As these im-
portant topographic changes could modify the stereo-
chemical requirements for binding at CCK1 and CCK2
receptors, the complete stereochemical space defined by
the eight stereoisomers at the Trp residue and at
positions 4a and 5 of the central skeleton was explored
in this case. The present paper deals with the synthesis
and CCK receptor binding profile of this new series of
derivatives modified at the 1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine scaffold.
Ch em istr y
Similar to the lead compounds 1a and 1b, their ana-
logues 5a ,b and 6a ,b (selectively modified at position 1
of the perhydropyrido[1,2-c]pyrimidine skeleton) were
prepared from the 2,3-trans-3-amino-2-piperidineacetic
derivative 2 [obtained as a (5:1) racemic mixture of
(2S,3R)- and (2R,3S)-isomers from D-Orn(Z)-OH13], as
shown in Scheme 1. This synthetic pathway involved
reaction with benzyl isothiocyanate, followed by in situ
NaH-promoted cyclization of the resulting thiourea to
5-Boc-amino-3-oxo-1-thioxoperhydropyrido[1,2-c]pyrimi-
dine (3). No cyclization through the sulfur was observed
in this reaction.23 Then, sequential N-Boc removal and
coupling with Boc-L-Trp-OH provided a (5:1) diastereo-
isomeric mixture of the 1-thioxo analogues 5a and 5b,
which were chromatographically resolved. Reduction of
3 with nickel boride, generated in situ from NiCl2‚6H2O
and NaBH4 in a (1:1) THF-MeOH mixture,25-28 led to
the 3-oxoperhydropyrido[1,2-c]pyrimidine derivatives 4,
whose N-Boc removal followed by coupling with Boc-L-
Trp-OH, and chromatographic separation yielded the
1-reduced analogues 6a and 6b. On the other hand,
thionation of the 3-oxo-1-thioxoperhydropyrido[1,2-c]-
pyrimidines 3 with Lawesson’s reagent29-31 led to the
1,3-dithioxo derivatives 7, and the following N-Boc
removal and coupling with Boc-L-Trp-OH provided
the corresponding 5-(Boc-tryptophyl)amino derivatives
8a and 8b. Finally, the nickel boride reduction of the
major diastereoisomer 8a led to the corresponding 1,3-
desulfurized analogue 9a .
a
Reagents: (a) Ph-CH2-NCS, THF; (b) NaH, THF; (c) NiCl2‚
6H2O, NaBH4, THF/MeOH; (d) TFA, CH2Cl2; (e) Boc-Trp-OH,
BOP, Et3N, CH2Cl2; (f) Lawesson’s reagent, toluene.
the Lawesson’s reagent led exclusively to the 3-thioxo
analogues 11 in high yield, whose N-Boc removal,
followed by coupling with Boc-L-Trp-OH, yielded the
corresponding (5:1) diastereoisomeric mixture of the
5-(Boc-tryptophyl)amino derivatives 12a and 12b, which
were chromatographically resolved. Then, reduction of
the major diastereoisomer 12a by treatment with nickel
boride, in (1:1) THF/MeOH mixture, provided the de-
sired 3-desulfurized analogue 13a (30%), along with
compound 14a (36%), resulting from a stereospecific
addition of MeOH to the thioxo group, followed by
reductive removal of the mercapto group. A similar
reaction of addition of an alcoholic solvent has been
described in the reductive desulfurization of thiohydan-
toins and thiobarbituric acids with Raney nickel.32
Assignment of the (R)-absolute configuration at position
3 in 14a was made on the basis of the NOE effect
observed between the methoxyl protons and the 4a-H
1
proton in its H NMR NOE difference spectrum, indica-
tive of a biaxial disposition for them (Figure 1).
In comparison with the model compounds 1a and 1b,
the H- and 13C NMR spectra of the thioxo analogues
1
Compounds selectively modified at position 3 were
prepared from a (5:1) (4aS,5R)/(4aR,5S)-racemic mix-
ture of the 5-Boc-amino-1,3-dioxoperhydropyrido[1,2-c]-
pyrimidines13 10 (Scheme 2). First, the reaction with
5, 8, and 12 showed characteristic deshielding in the
chemical shift of the CdS neighboring nuclei. However,
no changes were observed in the H coupling constants
1
and NOE patterns, indicating that important topo-

4198 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Bartolome´-Nebreda et al.
Sch em e 2a
Sch em e 3a
a
Reagents: (a) Lawesson’s reagent, toluene; (b) TFA, CH2Cl2;
(c) Boc-Trp-OH, BOP, Et3N, CH2Cl2; (d) NiCl2‚6H2O, NaBH4, THF/
MeOH.
F igu r e 1. Synthesis and C3 configuration assignment of the
3-methoxy derivative 14a .
a
Reagents: (a) CDI, LiCH2CO2Et, THF; (b) H2, Pd(C), EtOH;
(c) NaBH3CN, ZnCl2, EtOH; (d) Ph-CH2-NCO, THF; (e) NaH, THF;
(f) TFA, CH2Cl2; (g) Boc-L-Trp-OH or Boc-D-Trp-OH, BOP, Et3N,
CH2Cl2.
graphic changes have not taken place. Similar to these
compounds, no significant changes in the coupling
constants and NOE patterns for the Boc-Trp residue and
the fused piperidine ring were observed in the deoxy
analogues 6, 9a , and 13a . However, the N9-C1-N2-
C3 planarity in the fused pyrimidine ring is lost because
of the reduction, and, therefore, the spatial disposition
of the N2-benzyl group must change.
Regarding the contraction of the fused piperidine ring
to the pyrrolidine analogue, the construction of the
1,3-dioxoperhydropyrrolo[1,2-c]pyrimidine skeleton was
planned using a synthetic pathway similar to that pre-
viously used for the preparation of 1,3-dioxoperhydro-
pyrido[1,2-c]pyrimidine derivatives. As indicated in
Scheme 3, this route involved synthesis of the appro-
priated pyrrolidines from 4-benzyloxycarbonylamino-2-
tert-butoxycarbonylamino-L-butyric acid (15),33 followed
by reaction with benzyl isocyanate, intramolecular
acylation, and finally N-Boc-removal and coupling
with Boc-Trp-OH. Because in this case we wished to
explore the complete stereochemical diversity, we did
not pay special attention to stereoselectivity. Using the
same reaction conditions that we had used for the
synthesis of Boc-Orn(Z)-OH derived â-keto esters,13,34
the activation of the acid 15 with 1,1′-carbonyldiimida-
zole at room temperature for 2 h, followed by reaction
with LiCH2CO2Et [generated from ethyl acetate and
lithium bis(trimethylsilyl)amide] at -78 °C for 15 min,
led to the required â-keto ester 18 in only a 9% yield,
along with the butyrate derivative 16 (9%), resulting
from transesterification with ethyl acetate, and the
γ-lactam 17 (20%). Then, with the aim of avoiding, or
minimizing, the formation of this γ-lactam, the activa-
tion time and temperature were lowered to 15 min and
0 °C, respectively. In these conditions, the γ-lactam 17
was not detected in the reaction, and the â-ketoester
18 was obtained in 45% yield, along with 12% of
compound 16. The catalytic hydrogenation of 18 in
EtOH, using Pd(C) as catalyst, led to a mixture of the
expected 2,3-trans- and 2,3-cis-pyrrolidines 19a ,b and
19c,d [50%, (10:1)], along with their respective N-ethyl

Structure-Activity Study of CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4199
moiety in a pseudoequatorial disposition. In the case of
the 4a,5-cis-isomers (23c, 23d , 24c, and 24d ), the
coupling constants, and NOE effects observed for the
4a-H with the 5-H and one of the 6-H protons, and for
the 5-NH with the other 6-H, one of the two 7-H and
the 4-H in the same face of the bicyclic ring, suggest
that the fused pyrrolidine ring also adopts a preferred
envelope conformation, but in this case with the C5
below the mean plane of the ring, and the 5-(Boc-
tryptophyl)amino moiety in a pseudoaxial disposition.
These conformations are similar to energy minima
calculated for 5-acetylamino-2-benzyl-1,3-dioxoper-
hydropyrrolo[1,2-c]pyrimidine analogues by molecular
dynamics, followed by energy minimization, using the
Chem3D program.
F igu r e 2. NOE effects and 1H,1H coupling constants used
for the configuration assignment of 1,3-dioxoperhydropyrrolo-
[1,2-c]pyrimidine derivatives.
Biologica l Resu lts a n d Discu ssion
The affinity of the new 5-(Boc-tryptophyl)amino-1,3-
dioxoperhydropyrido[1,2-c]pyrimidine analogues herein
described at CCK1 and CCK2 receptors was determined
by measuring the displacement of [3H]propionyl-CCK-
8 binding to rat pancreatic and cerebral cortex homo-
genates, respectively, as previously described.39 Subse-
quently, those compounds which showed a significant
affinity at CCK1 receptors at concentrations below 10-7
M were tested for their antagonism to CCK-8-stimulated
amylase release from pancreatic acinar cells.40 For
comparative purposes CCK-8 and the model compounds
1a and 1b were also included in the assays. The results
of the evaluation of the thioxo- and deoxi-analogues are
shown in Table 1. Compared with the lead compound
1a , the (4aS,5R)-thioxo-analogues 5a , 8a , and 12a kept
the highly potent and selective binding at the CCK1
versus the CCK2 receptor subtype. It is interesting to
note that the thionation at position 1 led to a 1 order of
magnitude increase in the binding affinity of 5a . This
compound, with a subnanomolar potency (IC50 ) 0.09
nM) is the most potent and selective compound within
the family of 5-(Boc-tryptophyl)amino-1,3-dioxoper-
hydropyrido[1,2-c]pyrimidine-based CCK1 antagonists.
The same thionation in the (4aR,5S)-diastereoisomers
5b and 8b led to a decrease of at least 1 order of
magnitude in the affinity with respect to the model 1b;
the affinity of 12b, however, was slightly increased.
Compounds 5a , 8a , 12a , and 12b antagonized the CCK-
8-stimulated amylase release from pancreatic acinar
cells, although no linear correlation was observed
between the binding affinities and the respective amy-
lase release inhibition potency. The reason for this
discrepancy, particularly marked in compounds 5a and
8a , is unclear. A partial agonist activity does not appear
to account for the results obtained in the amylase assay,
because a much higher concentration of these com-
pounds (10 µM) did not show any intrinsic effect on
amylase release. It should be noted in this regard that
CCK-8 already produces a significant amylase release
at a 10 pM concentration and a maximal release is
generally found with a 0.5 nM concentration of the
peptide.14 In the binding assay, displacement curves
with compounds 5a and 8a produced Hill coefficients
significantly lower than unity (0.38 and 0.52 for 5a and
8a , respectively) suggesting the possibility of an ad-
ditional low-affinity binding site. The Hill coefficient for
the lead compound 1a (0.90) was not, however, different
from unity, indicating a single binding site.
derivatives 20a ,b and 20c,d [40%, (8:1)]. The ratio of
these N-ethyl derivatives, which are are supposed to be
formed by alkylation with acetaldehyde generated from
EtOH, as described in some catalytic hydrogenations
with metallic catalyst,35-37 was not decreased by chang-
ing the time, temperature, or H2 pressure used in the
catalytic hydrogenation. To avoid the formation of
N-ethylpyrrolidine derivatives, the Z protecting group
of 18 was first removed by catalytic hydrogenolysis,
and then, the intramolecular reductive amination was
carried out by reduction with NaBH3CN in the presence
of ZnCl2, to produce a (4:3) 2,3-trans/-cis mixture of
pyrrolidine derivatives 19 in 74% yield. This mixture
was chromatographically resolved into the 1-(N-benzyl)-
carbamoylpyrrolidines 2,3-trans 21a ,b and 2,3-cis 21c,d ,
after its reaction with benzyl isocyanate. Then, NaH-
promoted cyclization, followed by removal of the N-Boc
protecting group, and coupling with Boc-L-Trp-OH or
Boc-D-Trp-OH led, in each case, to a mixture of diastereo-
isomeric 5-(Boc-tryptophyl)amino-1,3-dioxoperhydro-
pyrrolo[1,2-c]pyrimidine derivatives 23a ,b and 24a ,b,
respectively, from the 2,3-trans-pyrrolidines 21a ,b, and
23c,d and 24c,d , from the 2,3-trans-isomers 21c,d . All
these diastereoisomeric mixtures, coming from racem-
ization in the intramolecular reductive amination13
(compounds a 30% of the 4a,5-trans-isomers a and b,
and compounds c 37% of the 4a,5-cis-isomers c and d ),
were chromatographically resolved.
The J 4a,5 values in the 1H NMR spectra of the 1,3-
dioxoperhydropyrrolo[1,2-c]pyrimidine derivatives 23
and 24 (8 Hz in the trans-isomers and 4-4.5 Hz in the
cis-isomers), were not conclusive for the assignment of
the C4a,C5-relative configuration. This assignment was
made on the basis of the NOE effects observed in 1H
NMR NOE difference spectra (Figure 2). Thus, the
NOEs observed in 4a,5-trans-isomers (23a , 23b, 24a ,
and 24b) between the proton 5-H and the 4-Haxial and
7-H protons, located in the same face of the 1,3-
dioxoperhydropyrrolo[1,2-c]pyrimidine skeleton, are in-
dicative of a pseudoaxial disposition for these protons,
as well as the NOEs observed for 5-NH with 4a-H and
one 6-H. These data, along with the coupling constants
for the protons of the bicyclic skeleton, suggest that the
fused pyrrolidine ring adopts a preferred envelope
conformation38 with the C6 projecting below the mean
plane of the ring, and the 5-(Boc-tryptophyl)amino

4200 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Bartolome´-Nebreda et al.
Ta ble 1. Inhibition of [3H]pCCK-8 Specific Binding to CCK Receptors from Rat Pancreas (CCK1) and Cerebral Cortex (CCK2)
Homogenates, and Inhibition of Amylase Release from Dispersed Pancreatic Acini
IC50 (nM)a
amylase releaseb
IC50 (nM)
compd
stereochemistry
X
Y
CCK1
CCK2
CCK-8
1a
1b
5a
5b
6a
6b
8a
8b
1.04 ( 0.08
1.59 ( 0.10
22.7 ( 4.0
0.09 ( 0.06
825
5.60 ( 0.30
>10000
6153
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aS,5R)
(4aR,5S)
(4aS,5R)
(4aS,5R)
CdO
CdO
CdS
CdS
CH2
CdO
CdO
CdO
CdO
CdO
CdO
CdS
CdS
CH2
0.64 (0.4-0.9)
77 (73-81)
33 (16-50)
NDc
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>1000
NDc
CH2
>1000
NDc
CdS
CdS
CH2
CdO
CdO
CdO
CdO
1.34 ( 0.40
62 (49-75)
900
NDc
9a
>1000
NDc
12a
12b
13a
14a
CdS
CdS
CH2
2.83 ( 1.20
13.8 ( 6.6
>1000
4.3 (3.6-22.6)
23 (13-44)
NDc
(R)-CH(OMe)
370
NDc
a
Values are the mean or mean ( SEM of at least three experiments, performed with seven concentrations of test compounds in triplicate.
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.
b
AG. Analytical TLC was performed on aluminum sheets coated
With respect to the reduced analogues 6a , 6b, 9a , and
13a , none of these compounds bound at CCK1 or CCK2
receptors at concentrations below 10-6 M. Compound
14a , resulting from the addition of MeOH at position
3, showed a moderate affinity at CCK1, 2 orders of
magnitude lower than that of the lead compound 1a .
None of the eight diastereoisomers of the 1,3-dioxoper-
hydropyrrolo[1,2-c]pyrimidine derivatives 23 and 24,
resulting from the contraction of the fused piperidine
ring in compounds 1 to a pyrrolidine, showed significant
affinity at CCK1 or CCK2 receptors at concentrations
below 10-6 M. Therefore, whereas some electronic
changes, such as oxygen replacement by sulfur, are
allowed, both topographic changes produced by the
reduction of the 1- and/or 3-oxo groups or by the
contraction of the fused piperidine ring lead to the
complete loss of the binding affinity at CCK1 receptors.
In conclusion, the results reported herein show the
crucial influence of the topography defined by the 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine skeleton, with a
(4aS,5R)-stereochemistry, upon the binding potency at
CCK1 receptors. This influence suggests an essential
role for that skeleton in the appropriate orientation of
the two moieties linked at positions 2 and 5, which, as
previously demonstrated, are the key factors for a good
recognition by the receptor. When these results and
those previously reported13,18,19 are considered, it is
surprising that, although in other families of CCK
receptor ligands it has been possible to reverse receptor
selectivity from CCK1 to CCK2 receptors by means of
small structural modifications or changes in the stere-
ochemistry, none of the structural modifications on the
1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based CCK1
receptor antagonists has led to CCK2 selective ligands.
The high selectivity makes this family of CCK1 receptor
ligands a very specific tool for the study of this receptor
subtype.
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 uncor-
1
rected. H NMR spectra were recorded with Varian Gemini-
200, Varian INOVA-300, and Varian Unity-500 spectrometers,
operating at 200, 300, or 500 MHz, using TMS as reference.
13C NMR spectra were recorded with Varian Gemini-200 or
Varian Unity-500 spectrometers, operating at 50 or 125 MHz.
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.
Syn th esis of (4a R*,5S*)-2-Ben zyl-5-(ter t-bu toxyca r bo-
n yl)a m in o-3-oxo-1-t h ioxop er h yd r op yr id o[1,2-c]p yr im i-
d in e (3). Benzyl isothiocyanate (0.64 mL, 1.99 mmol) was
added to a solution of (2R*,3S*)-3-(tert-butoxycarbonyl)amino-
2-(ethoxycarbonyl)methylpiperidine41 (2) (500 mg, 1.66 mmol)
in dry THF (13 mL). After the reaction mixture was stirred
for 30 min at room temperature, it was diluted with THF (22
mL). Then, NaH (80 mg of 60% dispersion in mineral oil, 1.99
mmol) was added, and the stirring was continued for 15
additional min. Afterward, a 0 °C cooled 1 N HCl solution (50
mL) was added, and the resulting reaction mixture was
extracted with EtOAc (2 × 100 mL). The organic extracts were
washed with H2O (100 mL) and brine (100 mL), dried over
Na2SO4, and evaporated to dryness. The residue was purified
by flash chromatography, using 20% of EtOAc in hexane as
eluant, to give the 3-oxo-1-thioxoperhydropyrido[1,2-c]pyrimi-
dine derivative 3 as a white solid (349 mg, 54%). Significant
analytical and spectroscopic data are summarized in Table 2.
Syn th esis of (4a R*,5S*)-2-Ben zyl-5-(ter t-bu toxyca r bo-
n yl)a m in o-3-oxop er h yd r o-p yr id o[1,2-c]p yr im id in e (4).
NiCl2‚6H2O (323 mg, 1.36 mmol) was added to a solution
of (4aR*,5S*)-2-benzyl-5-(tert-butoxycarbonyl)amino-3-oxo-1-
thioxoperhydropyrido[1,2-c]pyrimidine (3) (67 mg, 0.17 mmol)
in THF/MeOH mixture (1:1, 4 mL). This solution was cooled
at 0 °C, then NaBH4 (154 mg, 4.08 mmol) was added, and the
reaction mixture was stirred for 15 min. Afterward, this
mixture was filtered through Celite diatomaceous earth and
evaporated to dryness. The residue was dissolved in dichloro-
methane (25 mL), and the solution was washed successively
with a saturated solution of NaHCO3 (25 mL), a saturated
solution of ethylenediaminetetraacetic acid (EDTA, 2 × 25
Exp er im en ta l Section
Ch em istr y. All reagents were of commercial quality. Sol-
vents were dried and purified by standard methods. Amino
acid derivatives were obtained from Bachem Feinchemikalien

Structure-Activity Study of CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4201
Ta ble 2. Significant Analytical and Spectroscopic Data of the 2-Benzyl-5-(Boc)aminoperhydropyrido[1,2-c]pyrimidine Derivatives 3, 4,
7, and 11
3
4
7
11
X
Y
CdS
CdO
54
171-173
C20H27N3O3S
CH2
CdO
84
foam
C20H29N3O3
CdS
CdS
63
CdO
CdS
69
139-141
C20H27N3O3S
yield (%)
mp (°C)a
formulac
201-203b
C20H27N3O2S2
1H-RMNd
1-H
3-H
- - - -
- - - -
3.78, 3.85
- - - -
- - - -
- - - -
- - - -
- - - -
4-H
4a-H
5-H
6-H
7-H
8-H
2-CH2
2.87
3.44
3.44
1.57, 2.12
1.81
2.87, 5.31
5.56, 5.71
2.55
2.55
3.46
1.27, 1.82
1.61, 1.65
2.06, 2.76
4.37, 4.73
3.26-3.40
3.26-3.40
3.26-3.40
1.31, 2.07
1.66-1.83
3.06, 5.20
6.23
3.12, 3.59
3.12
3.39
1.34, 2.09
1.60, 1.79
2.67, 4.33
5.56
13C-RMNe
C1
C3
C4
C4a
C5
C6
C7
C8
181.12
164.61
32.89
59.45
51.18
31.07
22.91
53.26
49.59
69.45
167.21
28.18
60.49
50.73
33.67
22.12
47.60
48.66
177.91
197.39
43.48
58.29
51.43
30.90
23.33
54.02
56.89
155.30
201.55
44.51
55.59
52.19
30.99
23.46
44.84
50.51
2-CH2
a
b
d
From EtOAc/hexane. Sublimation. c Satisfactory analyses for C, H, N, and S. Spectra registered at 200 MHz in CDCl3, except for
7 which registered at 500 MHz. e Spectra registered at 50 MHz in CDCl3.
Ta ble 3. Analytical Data of the
2-Benzyl-5-(Boc-Trp)aminoperhydropyrido[1,2-c]pyrimidine
Derivatives 5, 6, 8, 9, and 12-14
compd yield (%) mp (°C)a tR (min) (A:B)b
mL), H2O (25 mL), and brine (25 mL), dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography,
using 33% of EtOAc in hexane as eluant, to give the 3-oxo-
perhydropyrido[1,2-c]pyrimidine derivative 4 as a foam (52 mg,
84%). Significant analytical and spectroscopic data are sum-
marized in Table 2.
formulac
5a
5b
6a
6b
8a
8b
9a
12a
12b
13a
14a
73
9
73
8
39
5
38
52
8
112-114
119-121
99-101
91-92
39.80 (40:60)
37.07 (40:60)
11.67 (33:67)
12.00 (45:55)
17.33 (50:50)
16.87 (50:50)
7.87 (45:55)
43.40 (33:67)
45.00 (33:67)
9.80 (40:60)
5.47 (40:60)
C31H37N5O4S
31H37N5O4S
31H39N5O4
C31H39N5O4
C31H37N5O3S2
C31H37N5O3S2
C31H41N5O3
C31H37N5O4S
C
C
Syn th esis of th e 2-Ben zyl-5-[N-(ter t-bu toxyca r bon yl)-
L-t r yp t op h yl]a m in o-3-oxo-1-t h ioxop er h yd r op yr id o[1,2-
c]p yr im id in e Der iva tives 5a a n d 5b. TFA (0.38 mL) was
added to a solution of (4aR*,5S*)-2-benzyl-5-(tert-butoxycar-
bonyl)amino-3-oxo-1-thioxoperhydropyrido[1,2-c]pyrimidine (3)
(54 mg, 0.14 mmol) in dry dichloromethane (1.5 mL). After 30
min at room temperature, the solvents were evaporated to
dryness. The residue was dissolved in dry dichloromethane
(1.75 mL), and Boc-L-Trp-OH (54 mg, 0.18 mmol), benzotriazol-
1-yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP, 78 mg, 0.18 mmol), and TEA (45 µL, 0.32 mmol) were
added successively to that solution, and stirring was continued
at room temperature for 24 h. The reaction mixture was
diluted with dichloromethane (25 mL), washed successively
with 10% citric acid (10 mL), 10% NaHCO3 (10 mL), H2O (10
mL), and brine (10 mL), before being dried over Na2SO4, and
evaporated. The resulting Boc-tryptophyl derivatives were
purified and resolved into the diastereoisomers 5a (lower Rf)
and 5b (higher Rf) by preparative TLC, using 3% of EtOAc in
diethyl ether as eluant. Significant analytical and spectroscopic
data of these Boc-tryptophyl derivatives are summarized in
Tables 3, 4, and 5.
Syn th esis of th e 2-Ben zyl-5-[N-(ter t-bu toxyca r bon yl)-
L-tr yp top h yl]a m in o-3-oxop er h yd r op yr id o[1,2-c]p yr im i-
d in e Der iva tives 6a a n d 6b. These were prepared from
(4aR*,5S*)-2-benzyl-5-(tert-butoxycarbonyl)amino-3-oxoperhy-
dropyrido[1,2-c]pyrimidine (4) (50 mg, 0.14 mmol) by applying
the same methodology as described above for the preparation
of derivatives 5a and 5b. Significant analytical and spectro-
scopic data of these Boc-tryptophyl derivatives 6a (lower Rf)
and 6b (higher Rf) are summarized in Tables 3, 4, and 5.
Syn t h esis of (4a R*,5S*)-2-Ben zyl-5-(ter t-b u t oxyca r -
bonyl)am in o-1,3-dith ioxoper h ydr opyr ido[1,2-c]pyr im idin e
(7). Lawesson’s reagent (303 mg, 0.75 mmol) was added to a
218-220
107-109
169-171
109-111
113-115
95-97
C
C
31H37N5O4S
31H39N5O4
C32H41N5O5
33
36
111-113
a
b
From EtOAc/ethyl ether. Nova-pak C18, A ) CH3CN, B )
0.05% TFA in H2O. c Satisfactory analyses for C, H, N, and S.
solution of (4aR*,5S*)-2-benzyl-5-(tert-butoxycarbonyl)amino-
3-oxo-1-thioxoperhydropyrido[1,2-c]pyrimidine (3) (195 mg,
0.5 mmol) in toluene (25 mL), and the reaction mixture was
refluxed for 2 h. After evaporation of the solvent, the residue
was purified by flash chromatography using a 20-33% gradi-
ent of EtOAc in hexane as eluant, to give the 1,3-dithioxo-
perhydropyrido[1,2-c]pyrimidine derivative 7 as a yellow solid
(128 mg, 63%). Significant analytical and spectroscopic data
are summarized in Table 2.
Syn th esis of th e 2-Ben zyl-5-[N-(ter t-bu toxyca r bon yl)-
L-tr yptoph yl]am in o-1,3-dith ioxoper h ydr opyr ido[1,2-c]py-
r im id in e Der iva tives 8a a n d 8b. These were prepared from
(4aR*,5S*)-2-benzyl-5-(tert-butoxycarbonyl)amino-1,3-dithioxo-
perhydropyrido[1,2-c]pyrimidine (7) (57 mg, 0.14 mmol) by
applying the same methodology as described above for the
preparation of derivatives 5a and 5b. Significant analytical
and spectroscopic data of these Boc-tryptophyl derivatives
8a (lower Rf) and 8b (higher Rf) are summarized in Tables 3,
4, and 5.
Syn th esis of (4a S,5R)-2-Ben zyl-5-[N-(ter t-bu toxyca r -
b on yl)-L-t r yp t op h yl]-a m in op er h yd r op yr id o[1,2-c]p yr i-
m id in e (9a ). NiCl2‚6H2O (148 mg, 0.62 mmol) was added to

4202 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Bartolome´-Nebreda et al.
Ta ble 4. Significant 1H-NMRa Spectroscopic Data of the 2-Benzyl-5-(Boc-Trp)aminoperhydropyrido[1,2-c]pyrimidine Derivatives 5, 6,
8, 9, and 12-14
(4a,5)
(Trp)
compd config.
X
Y
1-H
- - - -
- - - -
3.61, 3.75 - - - -
3.43, 3.63 - - - -
3-H
- - - -
- - - -
4-H
4a-H
5-H
6-H
7-H
1.64
1.72
8-H
2-CH2
R-H
5a
5b
6a
6b
8a
8b
9a
12a
12b
13a
14a
(S,R) CdS
(R,S) CdS
(S,R) CH2
(R,S) CH2
(S,R) CdS
(R,S) CdS
(S,R) CH2
(S,R) CdO
(R,S) CdO
(S,R) CdO
CdO
CdO
CdO
CdO
CdS
CdS
CH2
CdS
CdS
CH2
2.56, 2.66
2.34
2.16, 2.39
1.71, 2.17
2.63, 3.02
2.71, 3.01
3.18
2.85
2.16
1.59
2.73
2.64
3.69 1.18, 1.64
3.67 1.27, 1.84
3.73 1.00, 1.57
3.56 1.14, 1.59
2.90, 5,22 5.52, 5.71 4.42
2.85, 5.16 5.51, 5.73 4.40
1.30-1.76 1.91, 2.67 4.36, 4.69 4.44
1.31-1.50 1.87, 2.59 4.37, 4.56 4.29
- - - -
- - - -
- - - -
- - - -
3.76 1.27, 1.64-1.73 1.64-1.73 2.98, 5.17 6.23, 6.32 4.46
3.61 1.15-1.84
1.46, 1.65 2.59, 4.26 5.54, 5.60 4.38
1.37, 1.64 1.90, 2.84 3.45, 3.55 4.36
1.55-1.70 2.56, 4.28 5.55, 5.59 4.45
1.55-1.74 2.92, 5.11 6.21, 6.31 4.38
2.49, 3.57 1.82, 2.62 1.37-1.64 1.37-1.64 3.67 1.37-1.64
- - - -
- - - -
- - - -
- - - -
- - - -
2.90, 3.25
2.92
2.90
3.07
2.74
2.85
3.63 1.06, 1.70
3.71 1.27, 1.83
2.92, 3.19 1.64
4.20 (3.18b) 1.60, 1.94
3.16 0.87, 1.44-1.58 1.44-1.58 2.37, 4.54 4.46, 4.54 4.33
3.56 1.47-1.53
(S,R) CdO (R)-CH(OMe) - - - -
1.47-1.53 2.85, 4.47 4.00, 5.25 4.34
a
b
Spectra registered in CDCl3 at 300 MHz, except for 6a , 9a , 12a , and 14a which registered at 500 MHz. OMe.
Ta ble 5. Significant 13C-NMRa Spectroscopic Data of the 2-Benzyl-5-(Boc-Trp)aminoperhydropyrido[1,2-c]pyrimidine Derivatives 5, 6,
8, 9, and 12-14
(4a,5)
compd config.
X
Y
C1
C3
C4
C4a
C5
C6
C7
C8
2-CH2 Trp CR
5a
5b
6a
6b
8a
8b
9a
12a
12b
13a
14a
(S,R)
(R,S)
(S,R)
(R,S)
(S,R)
(R,S)
(S,R)
(S,R)
(R,S)
(S,R)
(S,R)
CdS
CdS
CH2
CH2
CdS
CdS
CH2
CdO
CdO
CdO
CdO
CdO
CdO
CdO
CdS
CdS
CH2
CdS
CdS
CH2
180.87
181.94
63.39
164.63
165.44
167.24
167.24
197.30
197.67
51.46
201.32
201.52
44.03
32.03 59.24 49.24 30.69 22.73 53.46
33.03 59.13 50.10 31.15 23.28 53.59
28.01 59.53 49.09 29.69 21.90 49.05
32.46 58.98 49.04 29.68 21.80 48.07
42.66 57.30 49.63 30.19 22.83 53.50
43.00 57.16 49.82 30.15 22.85 53.33
26.92 65.78 50.38 30.99 23.39 51.99
43.68 54.65 50.22 30.20 23.03 44.49
43.57 54.47 50.42 30.29 22.93 44.41
24.51 59.57 49.83 31.49 24.51 42.35
49.61
50.23
47.65
47.71
56.37
56.61
59.45
50.22
50.27
51.48
50.44
56.08
56.03
55.93
55.81
56.19
53.33
55.61
56.28
55.66
55.73
55.48
69.33
177.70
177.25
77.16
150.77
150.65
155.97
CdO (R)-CH(OMe) 149.89 83.78 (55.57b) 30.66 54.45 40.99 30.10 23.68 42.77
a
b
Spectra registered in CDCl3 at 50 MHz. OMe.
a solution of (4aS,5R)-2-benzyl-5-[N-(tert-butoxycarbonyl)-L-
tryptophyl]amino-1,3-dithioxoperhydropyrido[1,2-c]pyrimi-
dine (8a ) (23 mg, 0.04 mmol) in a THF/MeOH mixture (1:1, 3
mL). This solution was cooled at 0 °C, then NaBH4 (73 mg,
1.92 mmol) was added, and the reaction mixture was stirred
for 15 min at this temperature. Afterward, this mixture was
worked up as indicated for the synthesis of compound 4,
yielding the perhydropyrido[1,2-c]pyrimidine derivative 9a (8
mg, 38%) as a yellow solid, whose more significant analytical
and spectroscopic data are summarized in Tables 3, 4, and 5.
Syn th esis of (4a R*,5S*)-2-Ben zyl-5-(ter t-bu toxyca r bo-
n yl)a m in o-1-oxo-3-t h ioxop er h yd r op yr id o[1,2-c]p yr im i-
d in e (11). 2,4-Bis(4-methoxyphenyl)-1,3,2,4-dithiaphosphe-
tane (Lawesson’s reagent, 303 mg, 0.75 mmol) was added to a
solution of (4aR*,5S*)-2-benzyl-5-(tert-butoxycarbonyl)amino-
1,3-dioxoperhydropyrido[1,2-c]pyrimidine13 (10) (188 mg, 0.5
mmol) in toluene (25 mL), and the reaction mixture was
refluxed for 2 h. After evaporation of the solvent, the residue
was purified by flash chromatography, using a 20-33%
gradient of EtOAc in hexane as eluant, to give the 1-oxo-3-
thioxoperhydropyrido[1,2-c]pyrimidine derivative 11 as a pale
yellow solid (134 mg, 69%). Significant analytical and spec-
troscopic data are summarized in Table 2.
derivatives 12a (lower Rf) and 12b (higher Rf) are summarized
in Tables 3, 4, and 5.
Syn th esis of (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-1-oxop er h yd r op yr id o[1,2-c]-
p yr im id in e (13a ) a n d (3R,4a S,5R)-2-Ben zyl-5-[N-(ter t-
b u t oxyca r bon yl)-L-t r yp t op h yl]a m in o-3-m et h oxy-1-oxo-
p er h yd r op yr id o[1,2-c]p yr im id in e (14a ). NiCl2‚6H2O (83
mg, 0.35 mmol) was added to a solution of (4aS,5R)-2-benzyl-
5-[N-(tert-butoxycarbonyl)-L-tryptophyl]amino-1-oxo-3-thioxo-
perhydropyrido[1,2-c]pyrimidine (12a ) (23 mg, 0.04 mmol) in
THF/MeOH mixture (1:1, 1.5 mL). This solution was cooled
at 0 °C, then NaBH4 (40 mg, 1.04 mmol) was added, and the
reaction mixture was stirred for 15 min at this temperature.
This mixture was filtered through Celite diatomaceous earth
and evaporated to dryness. The residue was dissolved in
dichloromethane (25 mL), and the solution was washed
successively with a saturated solution of NaHCO3 (15 mL), a
saturated solution of EDTA (2 × 15 mL), H2O (15 mL), and
brine (15 mL), then dried over Na2SO4, and evaporated. The
residue was purified by preparative TLC, using 33% of EtOAc
in hexane as eluant, yielding the 1-oxoperhydropyrido[1,2-c]-
pyrimidine derivative 13a (lower Rf, 8 mg, 33%) and the
3-methoxy-1-oxoperhydropyrido[1,2-c]pyrimidine derivative 14a
(higher Rf, 9 mg, 36%). Significant analytical and spectroscopic
data of these Boc-tryptophyl derivatives are summarized in
Tables 3, 4, and 5.
Syn th esis of th e 2-Ben zyl-5-[N-(ter t-bu toxyca r bon yl)-
L-t r yp t op h yl]a m in o-1-oxo-3-t h ioxop er h yd r op yr id o[1,2-
c]p yr im id in e Der iva tives 12a a n d 12b. These were pre-
pared from (4aR*,5S*)-2-benzyl-5-(tert-butoxycarbonyl)amino-
1-oxo-3-thioxoperhydropyrido[1,2-c]pyrimidine (11) (54 mg,
0.14 mmol) by applying the same methodology described above
for the preparation of derivatives 5a and 5b. Significant
analytical and spectroscopic data of these Boc-tryptophyl
Syn t h esis of E t h yl (4S)-6-(Ben zyloxyca r b on yl)a m i-
n o-4-(ter t-b u t oxyca r b on yl)a m in o-3-oxoh exa n oa t e (18).
METHOD A: 1,1′-Carbonyldiimidazole (CDI, 1.40 g, 8.62
mmol) was added to a solution of (2S)-4-(benzyloxycarbonyl)-
amino-2-(tert-butoxycarbonyl)aminobutyric acid33 (15) (2.53 g,

Structure-Activity Study of CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4203
7.18 mmol) in dry THF (20 mL), and the solution was stirred
at room temperature for 2 h. Then this solution was added,
under argon atmosphere, to a -78 °C cooled solution of LiCH2-
CO2Et [obtained by slow addition of dry EtOAc (3.1 mL, 31.6
mmol) to 1 M solution of lithium bis(trimethylsilyl)amide in
hexane (31.6 mL, 31.6 mmol)], and stirring was continued for
20 min at this temperature. Afterward, 1 N HCl was slowly
added until pH 7 was reached, and the mixture was extracted
with EtOAc (2 × 100 mL). The organic extracts were washed
successively with saturated NaHCO3 (150 mL), H2O (150 mL),
and brine (150 mL), dried over Na2SO4, and evaporated to
dryness. The residue was purified by flash chromatography,
using 33% of ethyl ether in hexane as eluant, to give, from
higher to lower Rf, ethyl (2S)-4-(benzyloxycarbonyl)amino-2-
(tert-butoxycarbonyl)aminobutyrate (16) (265 mg, 10%), the
â-ketoester 18 (265 mg, 9%), and the 2-oxopyrrolidine 17 (480
mg, 20%). METHOD B: Similar to method A, but the initial
activation with CDI was carried out at 0 °C for 15 min. In
this way, only ethyl (2S)-4-(benzyloxycarbonyl)amino-2-(tert-
butoxycarbonyl)aminobutyrate (16) (322 mg, 12%) and the
â-ketoester 18 (1.34 g, 45%) were obtained.
1H NMR (500 MHz, CDCl3) 2,3-trans-20a ,b: δ 1.01 and 1.20
[2t, 3H, CH3 (Et)], 1.37 (s, 9H, Boc), 1.54 (m, 1H, 4-H), 2.11-
2.21 [m, 2H, 4-H and CH2 (1-Et)], 2.30 (dd, 1H, J ) 9 and 17.5
Hz, 5-H), 2.41 and 2.49 (2m, 2H, 2-CH2), 2.57 (m, 1H, 2-H),
2.71 [m, 1H, CH2 (1-Et)], 3.00 (m, 1H, 5-H), 3.82 (m, 1H, 3-H),
4.08 [c, 2H, CH2 (OEt)], 4.74 (s, 1H, 3-NH). 2,3-cis-20c,d : δ
0.99 and 1.20 [2t, 3H, CH3 (Et)], 1.37 (s, 9H, Boc), 1.54 (m,
1H, 4-H), 2.11-2.21[m, 2H, 4-H and CH2 (1-Et)], 2.30 (m, 1H,
5-H), 2.41 and 2.49 (2m, 2H, 2-CH2), 2.57 (m, 1H, 2-H), 2.71
[m, 1H, CH2 (1-Et)], 3.11 (m, 1H, 5-H), 3.82 (m, 1H, 3-H), 4.08
[c, 2H, CH2 (OEt)], 5.09 (d, 1H, J ) 8 Hz, 3-NH). Anal.
(C15H28N204) C, H, N.
Syn th esis of th e 3-(ter t-Bu toxyca r bon yl)a m in o-2-
(eth oxyca r bon yl)m eth ylp yr r olid in es 19. METHOD B: A
solution of ethyl (4S)-6-(benzyloxycarbonyl)amino-4-(tert-bu-
toxycarbonyl)amino-3-oxo-hexanoate (18) (475 mg, 1.16 mmol)
in EtOH (50 mL) was hydrogenated, at room temperature and
1 atm of H2 pressure, in the presence of 10% Pd(C) (50 mg)
for 1 h. The catalyst was filtered off, washed with EtOH (5
mL), and NaBH3CN (602 mg, 9.28 mmol) and ZnCl2 (76 mg,
0.58 mmol) were added to the solution. After the solution was
stirred at room temperature for 48 h, the solvent was evapo-
rated to dryness, and the residue was dissolved in EtOAc (50
mL). The resulting solution was successively washed with H2O
(25 mL), 0.1 N HCl (25 mL), saturated NaHCO3 (25 mL), and
brine (25 mL), dried over Na2SO4, and evaporated to dryness.
The residue was purified by flash chromatography, using 4%
of MeOH in CH2Cl2 as eluant, to yield a 4:3 mixture of 2,3-
trans- and 2,3-cis-3-(tert-butoxycarbonyl)amino-2-(ethoxycar-
bonyl)methylpyrrolidines 19 (236 mg, 74%).
Syn th esis of th e 1-(Ben zylcar bam oyl)-3-(ter t-bu toxycar -
bon yl)a m in o-2-(eth oxyca r bon yl)m eth ylp yr r olid in es 21.
Benzyl isocyanate (90 µL, 0.86 mmol) was added to a solution
of the 4:3 mixture of 2,3-trans- and 2,3-cis-3-(tert-butoxycar-
bonyl)amino-2-(ethoxycarbonyl)methylpyrrolidines 19 (154 mg,
0.56) in dry THF (3 mL), and this reaction mixture was stirred
at room temperature for 4 h. EtOAc (25 mL) was added, and
the resulting solution was successively washed with a 10%
solution of citric acid (15 mL), saturated NaHCO3 (15 mL),
H2O (15 mL), and brine, dried over Na2SO4, and evaporated
to dryness. The residue was purified by preparative TLC, using
2% of MeOH in CH2Cl2 as eluant, to yield the 2,3-trans-
pyrrolidines 21a ,b (higher Rf, 100 mg, 43%) and the 2,3-cis-
pyrrolidines 21c,d (lower Rf, 70 mg, 31%).
2,3-tr a n s-1-(Ben zylcar bam oyl)-3-(ter t-bu toxycar bon yl)-
a m in o-2-(et h oxyca r b on yl)m et h ylp yr r olid in es (21a ,b ).
Foam. 1H NMR (200 MHz, CDCl3) δ 1.23 [t, 3H, J ) 7 Hz,
CH3 (Et)], 1.43 (s, 9H, Boc), 1.85 (m, 1H, 4-H), 2.16 (dddd, 1H,
J ) 3.5, 6, 9, and 13 Hz, 4-H), 2.51 (dd, 1H, J ) 6 and 16 Hz,
2-CH2), 2.70 (dd, 1H, J ) 6.5 and 16 Hz, 2-CH2), 3.35 (ddd,
1H, J ) 3.5, 9.5, and 10 Hz, 5-H), 3.62 (dd, 1H, J ) 9 and 10
Hz, 5-H), 3.97 (m, 1H, 3-H), 4.10 [c, 2H, J ) 7 Hz, CH2 (Et)],
4.12 (m, 1H, 2-H), 4.40 and 4.42 (2s, 2H, CH2-Ph), 4.74 (d, 1H,
J ) 5.5 Hz, 3-NH), 5.47 (br s, 1H, 1-CONH), 7.30 (m, 5H, Ph).
Anal. (C21H31N305) C, H, N.
Eth yl (2S)-4-(Ben zyloxycar bon yl)am in o-2-(ter t-bu toxy-
1
ca r bon yl)a m in obu tyr a te (16). Syrup. H NMR (200 MHz,
CDCl3) δ 1.27 [t, 3H, J ) 7 Hz, CH3 (Et)], 1.45 (s, 9H, Boc),
1.79 and 2.22 (2m, 2H, 3-H), 3.30 and 3.65 (2m, 2H, 4-H), 4.18
[c, 2H, J ) 7 Hz, CH2 (Et)], 4.29 (m, 1H, 2-H), 5.11 [s, 2H,
CH2(Z)], 5.57 (m, 1H, 2-NH), 5.91 (s, 1H, 4-NH), 7.34 [m, 5H,
Ph (Z)]. Anal. (C19H28N206) C, H, N.
(3S)-1-Ben zyloxyca r bon yl-3-(ter t-bu toxyca r bon yl)a m i-
n o-2-oxop yr r olid in e (17). White solid. Mp 112-114 °C
(EtOAc/hexane). 1H NMR (200 MHz, CDCl3) δ 1.45 (s, 9H, Boc),
1.89 and 2.63 (2m, 2H, 4-H), 3.60 and 3.91 (2m, 2H, 5-H), 4.27
(m, 1H, 2-H), 5.08 (m, 1H, 3-NH), 5.29 [s, 2H, CH2 (Z)], 7.32-
7.50 [m, 5H, Ph (Z)]. Anal. (C17H22N205) C, H, N.
Eth yl (4S)-6-(Ben zyloxycar bon yl)am in o-4-(ter t-bu toxy-
ca r bon yl)a m in o-3-oxoh exa n oa te (18). White solid. Mp 69-
1
71 °C (ethyl ether). H NMR (200 MHz, CDCl3) δ 1.27 [t, 3H,
J ) 7 Hz, CH3 (Et)], 1.44 (s, 9H, Boc), 1.61 and 2.10 (2m, 2H,
5-H), 3.07 and 3.47 (2m, 2H, 6-H), 3.53 (s, 2H, 2-H), 4.17 [c,
2H, J ) 7 Hz, CH2 (Et)], 4.33 (m, 1H, 4-H), 5.11 [s, 2H, CH2
(Z)], 5.47 (m, 2H, 3-NH and 5-NH), 7.25-7.38 [m, 5H, Ph (Z)].
Anal. (C21H30N207) C, H, N.
Syn th esis of th e 3-(ter t-Bu toxyca r bon yl)a m in o-2-
(eth oxyca r bon yl)m eth ylp yr r olidin es 19 a n d Th eir 1-Eth -
yl Der iva tives 20. METHOD A: A solution of ethyl (4S)-6-
(benzyloxycarbonyl)amino-4-(tert-butoxycarbonyl)amino-3-oxo-
hexanoate (18) (256 mg, 0.62 mmol) in EtOH (25 mL) was
hydrogenated, at room temperature and 3 atm of H2 pressure,
in the presence of 10% Pd(C) (100 mg) for 24 h. Afterward,
the catalyst was filtered off, washed with EtOH (10 mL), and
the solution was evaporated to dryness. The residue was
purified by flash chromatography, using 3% of MeOH in CH2-
Cl2 as eluant. In this way, the (8:1) mixture of 2,3-trans- and
2,3-cis-3-(tert-butoxycarbonyl)amino-2-(ethoxycarbonyl)methyl-
1-ethylpyrrolidines 20 (higher Rf, 87 mg, 44%) was separated
from the (10:1) mixture of 2,3-trans- and 2,3-cis-3-(tert-butoxy-
carbonyl)amino-2-(ethoxycarbonyl)methylpyrrolidines 19 (lower
Rf, 84 mg, 50%). Neither of these two mixtures could be
resolved.
2,3-cis-1-(Ben zylca r b a m oyl)-3-(ter t-b u t oxyca r b on yl)-
a m in o-2-(et h oxyca r b on yl)m et h ylp yr r olid in es (21c,d ).
Foam. 1H NMR (200 MHz, CDCl3) δ 1.21 [t, 3H, J ) 7 Hz,
CH3 (Et)], 1.44 (s, 9H, Boc), 1.75 (ddd, 1H, J ) 9, 9.5, and 12
Hz, 4-H), 2.21 (dddd, 1H, J ) 2, 7, 7.5, and 12 Hz, 4-H), 2.46
(dd, 1H, J ) 4 and 15.5 Hz, 2-CH2), 2.64 (dd, 1H, J ) 6 and
15.5 Hz, 2-CH2), 3.35 (ddd, 1H, J ) 2, 9, and 10 Hz, 5-H), 3.47
(ddd, 1H, J ) 7.5, 9.5, and 10 Hz, 5-H), 4.08 [c, 2H, J ) 7 Hz,
CH2 (Et)], 4.22 (m, 1H, 3-H), 4.40 and 4.42 (2s, 2H, CH2-Ph),
4.43 (m, 1H, 2-H), 4.93 (br s, 1H, 3-NH), 5.23 (br s, 1H,
1-CONH), 7.28 (m, 5H, Ph). Anal. (C21H31N305) C, H, N.
Syn t h esis of t h e 2-Ben zyl-5-(ter t-b u t oxyca r b on yl)-
a m in o-1,3-d ioxop er h yd r op yr r olo[1,2-c]p yr im id in e De-
r iva tives 22. NaH (14 mg of 60% dispersed in mineral oil,
0.36 mmol) was added to a solution of the corresponding 2,3-
trans- and 2,3-cis-1-(benzylcarbamoyl)-3-(tert-butoxycarbonyl)-
amino-2-(ethoxycarbonyl)methylpyrrolidines 21a ,b or 21c,d
(114 mg, 0.24 mmol) in dry THF (6 mL), and the mixture was
stirred at room temperature for 15 min. The mixture was
2,3-tr a n s- a n d 2,3-cis-3-(ter t-Bu toxyca r bon yl)a m in o-2-
1
(eth oxyca r bon yl)m eth ylp yr r olid in es 19. Syrup. H NMR
(500 MHz, CDCl3) 2,3-Trans-19a ,b: δ 1.26 [t, 3H, CH3 (Et)],
1.44 (s, 9H, Boc), 1.61 (ddd, 1H, J ) 7, 12.5 and 13.5 Hz, 4-H),
2.24 (ddd, 1H, J ) 7, 8 and 13.5 Hz, 4-H), 2.36 (dd, 1H, J )
9.5 and 16 Hz, 2-CH2), 2.58 (dd, 1H, J ) 3.5 and 16 Hz, 2-CH2),
2.89 (s, 1H, 1-H), 3.06 (m, 2H, 5-H), 3.17 (m, 1H, 2-H), 3.41
(m, 1H, 3-H), 4.14 [c, 2H, CH2 (Et)], 4.16 (d, 1H, J ) 7 Hz,
3-NH). 2,3-Cis-19c,d : δ 1.19 [t, 3H, CH3 (Et)], 1.44 (s, 9H, Boc),
1.61 (m, 1H, 4-H) 2.24 (m, 1H, 4-H), 2.32 (m, 1H, 2-CH2), 2.69
(dd, 1H, J ) 2.5 and 16 Hz, 2-CH2), 2.81 and 3.06 (2m, 2H,
5-H), 3.11 (m, 1H, 2-H), 3.34 (m, 1H, 3-H), 4.07 (c, 2H, CH2
(Et)], 4.94 (d, 1H, J ) 8 Hz, 3-NH). Anal. (C13H24N204) C, H, N.
2,3-Tr a n s- a n d 2,3-Cis-3-(ter t-bu toxyca r bon yl)a m in o-
2-(eth oxyca r bon yl)m eth yl-1-eth ylp yr r olid in es 20. Foam.

4204 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Bartolome´-Nebreda et al.
Ta ble 6. Significant Analytical and Spectroscopic Data of the 2-Benzyl-5-(Boc-Trp)amino-1,3-dioxoperhydropyrrolo[1,2-c]pyrimidine
Derivatives 23 and 24
23a (24b)a
23b (24a )a
23c (24d )a
23d (24c)a
(4a,5) config.
Trp
S,R (R,S)
L (D)
R,S (S,R)
L (D)
R,R (S,S)
L (D)
S,S (R,R)
L (D)
yield (%)
12 (28)
C30H35N5O5
19.20
26 (18)
C30H35N5O5
16.53
27 (50)
C30H35N5O5
17.80
48 (17)
C30H35N5O5
14.53
formulab
c
tR
1H-RMNd
4-H
2.41, 2.68
3.08 (8)
4.03
2.23, 2.37
2.39 (8)
3.94
2.09, 2.53
3.68 (4.5)
4.42
1.60, 2.30
3.47 (4.5)
4.33
4a-H [J 4a-5 (Hz)]
5-H
6-H
7-H
2-CH2
5-NH
1.33, 1.95
3.45, 3.53
4.85, 4.91
5.85
1.38, 2.06
3.37
4.88
1.35, 1.94
3.18, 3.48
4.82, 4.88
5.92
1.56, 1.93
2.76, 3.37
4.75, 4.95
5.56
5.69
(Trp) R-H
4.40
4.46
4.42
4.64
13C-RMNe
C1
C3
C4
C4a
C5
C6
C7
2-CH2
(Trp) CR
151.46
168.53
36.50
55.52
54.89
28.63
43.32
43.59
55.85
151.31
168.70
36.37
56.04
54.37
28.72
43.24
43.47
54.91
151.74
169.15
32.86
54.74
51.22
28.93
43.46
43.64
55.60
151.37
169.36
32.86
54.74
51.38
28.87
42.92
43.68
54.49
a
b
Foam. Satisfactory analyses for C, H, and N. c Nova-pak C18, using (35:65) mixture of CH3CN and 0.05% TFA in H2O as mobile
phase. Registered in CDCl3 at 500 MHz. e Registered in CDCl3 at 125 MHz.
d
added to a 0 °C cooled 1 N solution of HCl (50 mL), and the
resulting solution was extracted with EtOAc (2 × 25 mL). The
organic extracts were washed with brine (15 mL), dried over
Na2SO4, and evaporated to dryness. The residue was purified
by flash chromatography, using 50% of EtOAc in hexane as
eluant, to yield, in each case, the (4aR*,5S*)- and (4aR*,5R*)-
1,3-dioxoperhydropyrrolo[1,2-c]pyrimidine derivatives 22a ,b
and 22c,d , respectively.
23 a n d 24. From the (4aR*,5S*)- and (4aR*,5R*)-2-benzyl-5-
(tert-butoxycarbonyl)amino-1,3-dioxoperhydropyrrolo[1,2-c]py-
rimidines 22a ,b and 22c,d (50 mg, 0.14 mmol), applying the
same methodology as described for the synthesis of the Boc-
tryptophyl derivatives 5a and 5b. Each resulting diastereoiso-
meric mixture 23a ,b, 23c,d , 24a ,b, and 24c,d , was resolved
by preparative TLC, using 2% of MeOH in CH2Cl2 as eluant.
Significant analytical and spectroscopic data of these Boc-
tryptophyl derivatives are summarized in Table 6.
(4a R*,5S*)-2-Ben zyl-5-(ter t-bu toxyca r bon yl)a m in o-1,3-
d ioxop er h yd r op yr r olo[1,2-c]p yr im id in es (22a ,b). Foam
(100 mg, 98%). 1H NMR (500 MHz, CDCl3) δ 1.44 (s, 9H, Boc),
1.73 (ddd, 1H, J ) 9.5, 10, and 12.5 Hz, 6-H), 2.32 (dddd, 1H,
J ) 2, 7, 7.5, and 12.5 Hz, 6-H), 2.57 (dd, 1H, J ) 13 and 16
Hz, 4-H), 3.02 (dd, 1H, J ) 4 and 16 Hz, 4-H), 3.37 (ddd, 1H,
J ) 4, 8.5, and 13 Hz, 4a-H), 3.54 (ddd, 1H, J ) 7.5, 10, and
11.5 Hz, 7-H), 3.67 (ddd, 1H, J ) 3, 5 and 11.5 Hz, 7-H), 3.94
(m, 1H, 5-H), 4.66 (br s, 1H, 5-NH), 4.89 and 4.94 (2d, 2H, J
) 15 Hz, 2-CH2), 7.19-7.39 (m, 5H, Ph). 13C NMR (50 MHz,
CDCl3) δ 28.23 [CH3 (Boc)], 29.06 (C6), 36.84 (C4), 43.20 and
43.55 (C7 and 2-CH2), 56.40 and 56.45 (C4a and C5), 80.28
[C(CH3)3], 127.27, 128.30, 128.56, 137.67 (Ph), 151.57 (C1),
155.12 [CO(Boc)], 168.65 (C3). Anal. (C19H25N304) C, H, N.
(4a R*,5R*)-2-Ben zyl-5-(ter t-bu toxyca r bon yl)a m in o-1,3-
d ioxop er h yd r op yr r olo[1,2-c]p yr im id in es (22a ,b). Foam
Bin d in g Assa ys. CCK1 and CCK2 receptor binding assays
were performed using rat pancreas and cerebral cortex homog-
enates respectively, according to the method described by
Dauge´ et al39 with minor modifications. Briefly, rat pancreas
tissue was carefully cleaned and homogenized in Pipes HCl
buffer, pH 6.5, containing 30 mM MgCl2 (15 mL/g of wet tissue)
and the homogenate was then centrifuged twice at 4 °C for 10
min at 50000g. For displacement assays, pancreatic mem-
branes (0.2 mg protein/tube) were incubated with 0.5 nM [3H]-
pCCK-8 in Pipes HCl buffer, pH 6.5, containing MgCl2 (30
mM), bacitracin (0.2 mg/mL), and soybean trypsin inhibitor
(SBTI, 0.2 mg/mL), for 120 min at 25 °C. Rat brain cortex was
homogenized in 50 mM Tris-HCl buffer pH 7.4 containing 5
mM MgCl2 (20 mL/g of wet tissue), and the homogenate was
centrifuged twice at 4 °C for 35 min at 100000g. Brain
membranes (0.45 mg protein/tube) were incubated with 1 nM
[3H]pCCK-8 in 50 mM Tris-HCl buffer, pH 7.4, containing
MgCl2 (5 mM) and bacitracin (0.2 mg/mL) for 60 min at 25 °C.
Final incubation volume was 0.5 mL in both cases. Nonspecific
binding was determined using CCK-8 1 µM as the cold
displacer. The inhibition constants (Ki) were calculated, using
the equation of Cheng and Prusoff, from the displacement
curves analyzed with the receptor fit competition LUNDON
program.
1
(42 mg, 42%). H NMR (500 MHz, CDCl3) δ 1.43 (s, 9H, Boc),
1.92 (dddd, 1H, J ) 2, 2.5, 7, and 13.5 Hz, 6-H), 2.19 (dddd,
1H, J ) 6, 9.5, 10, and 13.5 Hz, 6-H), 2.49 (dd, 1H, J ) 14 and
16 Hz, 4-H), 2.78 (dd, 1H, J ) 4 and 16 Hz, 4-H), 3.56 (ddd,
1H, J ) 7, 9.5, and 10 Hz, 7-H), 3.66 (dt, 1H, J ) 2.5 and 9.5
Hz, 7-H), 3.78 (dt, 1H, J ) 4 and 14 Hz, 4a-H), 4.33 (ddd, 1H,
J ) 2, 4, and 6 Hz, 5-H), 4.74 (d, 1H, J ) 8 Hz, 5-NH), 4.89
and 4.94 (2d, 2H, J ) 14 Hz, 2-CH2), 7.21-7.40 (m, 5H, Ph).
13C NMR (50 MHz, CDCl3) δ 28.25 [CH3 (Boc)], 29.61 (C6),
33.04 (C4), 43.55 and 43.71 (C7 and 2-CH2), 52.71 and 55.30
(C4a and C5), 80.48 [C(CH3)3], 127.33, 128.31, 128.80, 137.69
(Ph), 151.90 (C1), 155.123 [CO(Boc)], 169.31 (C3). Anal.
(C19H25N304) C, H, N.
Am yla se Relea se. Dispersed rat pancreatic acini were
prepared by using a modification of the technique of J ensen
et al.40 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
of two 6-min incubations at 37 °C, and washing the tissue three
Gen er a l P r oced u r e for th e Syn th esis of th e 2-Ben zyl-
5-[N-(ter t-bu toxyca r bon yl)-L- a n d -D-tr yp top h yl]a m in o-
1,3-d ioxop er h yd r op yr r olo[1,2-c]p yr im id in e Der iva tives

Structure-Activity Study of CCK1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4205
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times 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 (100g, 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.42 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 determined
using the Amyl Kit Reagent (Boehringer 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
% I ) [(SCCK - SC) - (ST - SC)/(SCCK - SC)] × 100
where SC is the control release (vehicle), SCCK is the release
elicited by CCK-8, and ST is the release elicited by CCK-8 in
the presence of increasing drug concentrations. Linear regres-
sion analysis was used in order to estimate the IC50 values of
the compounds on the dose response curves.
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Ack n ow led gm en t. This work has been supported
by CICYT (SAF 97-0030 and SAF 2000-0147) and
Fundacio´n La Caixa (97/022).
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