Synthesis and stereochemical structure activity relationships of 1,3- dioxoperhydropyrido[1,2-c]pyrimidine derivatives: Potent and selective cholecystokinin-a receptor antagonists

Article abstract of DOI:10.1021/jm9703247

The synthesis and stereochemical structure-activity relationships of a new class of potent and selective non-peptide cholecystokinin-A (CCK-A) receptor antagonists based on the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine skeleton are described. The most potent member of this series of eight diastereoisomers, (4aS,5R)-2-benzyl-5-[N-[(tert-butoxycarbonyl)-L- tryptophyl]-amino]-1,3-dioxoperhydropyrido[1,2-c]pyrimidine, displays nanomolar CCK-A receptor affinity and higher than 8000-fold potency at the CCK-A than at the CCK-B receptor. As CCK-A antagonist, this compound inhibits the CCK-8-evoked amylase release from pancreatic acinar cells at a low concentration, similar to that of the typical antagonist Devazepide. Highly strict stereochemical requirements for CCK-A receptor binding and selectivity have been found. The L-Trp and the 4a,5-trans disposition of the bicyclic perhydropyrido[1,2-c]pyrimidine are essential for binding potency and selectivity.

Full text of DOI:10.1021/jm9703247

3402
J . Med. Chem. 1997, 40, 3402-3407
Syn th esis a n d Ster eoch em ica l Str u ctu r e-Activity Rela tion sh ip s of
1,3-Dioxop er h yd r op yr id o[1,2-c]p yr im id in e Der iva tives: P oten t a n d Selective
Ch olecystok in in -A Recep tor An ta gon ists
Mercedes Mart´ın-Mart´ınez,§ J ose´ M. Bartolome´-Nebreda,§ Isabel Go´mez-Monterrey,§ Rosario Gonza´lez-Mun˜iz,§
M. Teresa Garc´ıa-Lo´pez,§ Santiago Ballaz,‡ Ana Barber,‡ Ana Fortun˜o,‡ J oaqu´ın Del R´ıo,‡ and Rosario Herranz*,§
Instituto de Qu´ımica Me´dica (CSIC), J uan de la Cierva 3, E-28006 Madrid, Spain, and Departamentos de Farmacolog´ıa y
Fisiologı´a, Universidad de Navarra, Irunlarrea 1, E-31008 Pamplona, Spain
Received May 14, 1997X
The synthesis and stereochemical structure-activity relationships of a new class of potent
and selective non-peptide cholecystokinin-A (CCK-A) receptor antagonists based on the 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine skeleton are described. The most potent member of this
series of eight diastereoisomers, (4aS,5R)-2-benzyl-5-[N-[(tert-butoxycarbonyl)-L-tryptophyl]-
amino]-1,3-dioxoperhydropyrido[1,2-c]pyrimidine, displays nanomolar CCK-A receptor affinity
and higher than 8000-fold potency at the CCK-A than at the CCK-B receptor. As CCK-A
antagonist, this compound inhibits the CCK-8-evoked amylase release from pancreatic acinar
cells at a low concentration, similar to that of the typical antagonist Devazepide. Highly strict
stereochemical requirements for CCK-A receptor binding and selectivity have been found. The
L-Trp and the 4a,5-trans disposition of the bicyclic perhydropyrido[1,2-c]pyrimidine are essential
for binding potency and selectivity.
In tr od u ction
Cholecystokinin (CCK) is a gastrointestinal hormone
and neurotransmitter that is found in the digestive tract
and in the central nervous system (CNS).1 While
several endogenous molecular forms of CCK have been
isolated, the C-terminal octapeptide [Asp-Tyr(SO3H)-
Met-Gly-Trp-Met-Asp-Phe-NH2, CCK-8] appears to be
the minimum sequence required for bioactivity.2 Two
CCK receptor subtypes, CCK-A and CCK-B, which
mediate the diverse biological functions of CCK have
been recognized so far.3,4 CCK-A receptors are prima-
rily found in the periphery where they mediate pancre-
atic enzyme secretion, gut motility, and gallbladder
contraction, but they are also found in some discrete
CNS areas.4-6 CCK-A receptors have also been involved
in satiety, gastrointestinal cancer, and neuroprotec-
tion.5,7,8 CCK-B receptors are distributed throughout
the CNS, and they are thought to be involved in the
development of anxiety and in the control of nocicep-
tion.4,9-11
The variety of possible therapeutic utilities for CCK
receptor agonists and antagonists has prompted an
intensive research in this area, and over the past
decade, a number of potent and selective non-peptide
CCK-A and CCK-B receptor antagonists have been
reported.12 Prominent examples are the amino acid
derivatives lorglumide [DL-4-[(3,4-dichlorobenzoyl)amino]-
5-(di-n-pentylamino)-5-oxopentanoic acid] and loxi-
glumide [DL-4-[(3,4-dichlorobenzoyl)amino]-5-[N-(3-meth-
oxypropyl)-N-pentylamino]-5-oxopentanoic acid] as
CCK-A antagonists13 and the 1,4-benzodiazepine CCK-A
and CCK-B antagonists Devazepide14 (MK-329, 1) and
L-365,26015 (2), respectively, both arising from the
manipulation of the natural product asperlicin. All of
these antagonists have served for identifying the two
CCK receptor subtypes and for gaining insight into the
functional significance of CCK in the periphery and in
the CNS; nevertheless, the physiological effects of CCK
mediated by CCK-A or CCK-B receptors remain unclear.
Thus, despite the predominant role suggested for CCK-B
receptors in anxiety and in the control of nociception,
anxiolytic-like effects and enhancement of the morphine
analgesia have been reported for the typical CCK-A
antagonist Devazepide.16,17 As has been suggested,18
these effects could be due to the blockade of the CCK-B
receptors by Devazepide at high doses, since this
compound is not completely devoid of affinity for this
receptor subtype (CCK-B/CCK-A, 17019-300014b). It has
also been indicated that, in general, care should be
taken when attempting to distinguish effects mediated
by CCK-A or CCK-B receptors with Devazepide.20 In
view of these facts, the development of CCK receptor
antagonists with higher selectivity for CCK-A over
CCK-B receptors is desirable in order to shed further
light on the functional roles of CCK receptor subtypes.
In this regard, the pharmacological characteristics of
TP-68021 [(R)-1-[3-[(3-carboxypyridin-2-yl)thio]-2-[(indol-
2-ylcarbonyl)amino]propionyl]-4-(diphenylmethyl)pip-
erazine] and T-063222 [sodium (S)-3-[1-(2-fluorophenyl)-
2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-
2-oxo-1H-indole]propanoate], two novel CCK-A receptor
antagonists with enhanced selectivity relative to
Devazepide, have been recently reported.
As part of our current interest in non-peptide CCK
receptor selective ligands, we directed our efforts toward
the incorporation of conformationally restricted struc-
tures into the sequence of the CCK-B receptor selective
agonist CCK-4 (Trp30-Met31-Asp32-Phe33-NH2) as spacers
between Trp30 and Phe33, key amino acids for interaction
§ Instituto de Qu´ımica Me´dica.
‡ Universidad de Navarra.
X Abstract published in Advance ACS Abstracts, September 15, 1997.
S0022-2623(97)00324-5 CCC: $14.00 © 1997 American Chemical Society

Potent and Selective CCK-A Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3403
Sch em e 1a
with CCK receptors.23 These two amino acids or
structurally related moieties are also commonly found
in various non-peptide CCK-A and CCK-B receptor
antagonists as important fragments for bioactivity.24 In
contrast, a diversity of non-peptide skeletons have
served as core structural motifs of ligands for CCK
receptors.24b,c,25 In spite of this diversity, the presence
of a lactam function is a rather usual feature. These
considerations led us to design a series of CCK-4
restricted analogues in which the Met31-Asp32 fragment
was replaced with a 3-oxoindolizidine ring.26 Among
these bicyclic lactams, compounds 3 and 4 were rela-
tively modest CCK-A and CCK-B receptor antagonists,
respectively. On the basis of this finding, we considered
it of interest to manipulate the 3-oxoindolizidine nucleus
with the main purpose of altering the conformational
properties of compounds 3 and 4, so that the aromatic
side chains could adopt more appropriate orientations
to interact with the corresponding receptor site. This
tactic led us to the 1,3-dioxoperhydropyrido[1,2-c]pyrim-
idine 5a (IQM-95,333), a potent and selective CCK-A
receptor antagonist, both in vitro and in vivo. Very
recently, we described27 the pharmacological properties
of this novel compound, which displays receptor affinity
roughly similar to that of Devazepide, but it is virtually
devoid of affinity at brain CCK-B receptors. Interest-
ingly, it also shows a marked anxiolytic-like activity in
animal models. Here, we report on the synthesis of 5a
and on the importance of the stereochemical structure
for affinity and CCK receptor subtype selectivity of this
compound. To this end, all the possible stereoisomers
(5a -d and 6a -d ) have been prepared and evaluated.
a
Reagents: (a) 1,1′-carbonyldiimidazole, LiCH2CO2Et, THF; (b)
H2, Pd(C), EtOH or MeOH; (c) benzyl isocyanate, THF; (d) NaH,
THF; (e) TFA, CH2Cl2; (f) Boc-L-Trp-OH, BOP, Et3N; (g) Boc-D-
Trp-OH, BOP, Et3N.
constructed from the corresponding 3-amino-2-piper-
idineacetic acid derivatives 9 and 10, which were
prepared from Boc-D-Orn(Z)-OH (7) as previously de-
scribed.28 Reaction of trans-piperidine 9 with benzyl
isocyanate gave the N-benzylcarbamoyl derivative 11,
which after treatment with NaH provided the 1,3-
dioxoperhydropyrido[1,2-c]pyrimidine 13 in 65% overall
yield. Then, sequential N-Boc removal and coupling
with Boc-L- or -D-Trp-OH, using BOP as coupling agent,
led to the diasteroisomeric mixture 5a ,b or 6a ,b in a
4:1 ratio, which was chromatographically resolved to
give 5a and 5b, or 6a and 6b, respectively. In a similar
way, the cis-piperidine 10 led to the 4:1 mixtures 5c,d
and 6c,d , which were separated by semipreparative
RPHPLC. These results indicated that about 20% of
racemization had occurred. As in the synthesis of
8-amino-3-oxoindolizidines 3 and 4 from 4-keto esters
derived from ornithine,29 this racemization could take
place during the formation of the piperidine ring in 9
and 10, due the existence of imine-enamine intermedi-
ates in equilibrium (structures A-C) in the intramo-
lecular reductive amination of 3-keto esters 8.
Ch em istr y
As depicted in Scheme 1, the 1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine skeletons of 5a -d and 6a -d were
In order to clarify this point and to determine the
racemization ratio in 9 and 10, we prepared the Mosher

3404 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Mart´ın-Martı´nez et al.
Sch em e 2a
spectrum of the mixture of piperidines 9 and 10,
previously to their separation, by the decrease in the
integrals of the signals corresponding to the 3-H in both
isomers. Comparison of these results with the ratios
of final 1,3-dioxoperhydropyrido[1,2-c]pyrimidine de-
rivatives 5a :5b and 5c:5d , respectively, measured by
RPHPLC analysis, showed that racemization ratios
resulting from both analyses were similar.
Then, in order to minimize this racemization, we
studied the influence of the reductive amination condi-
tions (solvent, temperature, reducing agent) on the yield
of each stereoisomeric piperidine 9 and 10 and on the
racemization ratio. The results of this study are sum-
marized in Table 1. It is interesting to note that when
the hydrogenation was carried out in MeOD about 45%
of transesterification was observed, but it had no
influence on the yield of the final compounds, because
both ethyl and methyl esters were processed together
for the subsequent construction of the 1,3-dioxoperhy-
dropyrido[1,2-c]pyrimidine nucleus in 13 and 14. The
lowest racemization occurred when the intramolecular
reductive amination was carried out with NaBH3CN/
ZnCl2, after the Z removal by hydrogenolysis. However,
these conditions led to a considerable decrease in the
selectivity for the trans diastereoisomers 9. Minor
isomers 5b,d and 6b,d were obtained as major com-
pounds starting from Boc-L-Orn(Z)-OH.
a
Reagents: (a) benzyl chloroformate, propylene oxide, CH2Cl2;
(b) TFA, CH2Cl2; (c) (R)-(+)-MTPA-OH, BOP, Et3N.
Ta ble 1. Racemization Ratio and Yield of Piperidines 9 and 10
from the 3-Keto Ester 8
overall racemi-
yield zationa
9
10
reductive amination conditions
(%)
(%)
(%) (%)
H2, Pd(C), EtOD, 20 °C, 4 days
H2, Pd(C), EtOD, AcOD, 20 °C, 4 days
H2, Pd(C), MeOD, 20 °C, 3 days
H2, Pd(C), MeOD, 40 °C, 7 h
(1) H2, Pd(C), EtOD, 20 °C, 1 h
(2) NaBH3CN/ZnCl2, 30 min
90
17
90
90
80
20
45
19
10
4
79 11
15
2
72 18
74 10
57 23
a
Racemization ratio determined as 50% of the deuterium
incorporation into position 3 (measured by the decrease in the 3-H
integral in the 1H NMR spectra of 9 and 10) or after the coupling
of 13 and 14 with Boc-L-Trp-OH (measured by the RPHPLC
analysis of the resulting diastereoisomeric mixtures 5a ,b and
6a ,b).
acid derivatives 15a ,b of the ethyl ester of 3-amino-1-
[(benzyloxy)carbonyl]piperidineacetic acid derived from
928 (Scheme 2). However, this diastereoisomeric mix-
ture could not be resolved either by TLC or by RPHPLC,
and its 1H NMR analysis at room temperature in
different solvents [CDCl3, (CD3)2CO, C6D6] showed
broad signals but no duplicity of any of them. It was
necessary to register the spectrum at 80 °C in (CD3)2-
SO to observe the 2-H, 4-H, and OMe signals of 15a
and 15b separately. Taking into account these difficul-
ties in the determination of the racemization ratio by
the Mosher acid derivatives 15a ,b, we studied the
intramolecular reductive amination of the 3-keto ester
8 in deuterated solvent (MeOD or EtOD). In this way,
as the major products, the trans-piperidines 9 should
come only from the hydrogenation of the intermediate
A or C, and the incorporation of deuterium at position
3 into the enamino intermediate B should be twice the
racemization at this position produced in this step. This
Biologica l Resu lts a n d Discu ssion
The affinity of the 1,3-dioxoperhydropyrido[1,2-c]-
pyrimidine derivatives 5a -d and 6a -d at CCK-A and
CCK-B receptors was determined by measuring the
displacement of the [3H]propionyl-CCK-8 binding to rat
pancreatic and brain cortex homogenates, respectively,
as previously described.30 These data are depicted in
Table 2. For comparative purposes the 3-oxoindolizidine
derivatives 3 and 4, CCK-8, and Devazepide were also
included in the assay. In general, these pyrido[1,2-c]-
pyrimidine derivatives showed preference for the CCK-A
versus the CCK-B receptor subtype. The most remark-
able result is that obtained with compound 5a , the best
compound of this series, with a CCK-A receptor affinity
similar to that of Devazepide but with a much higher
selectivity at CCK-A than at CCK-B receptors. Unlike
various precedent series of ligands for CCK receptors,15a,31
stereochemical changes in 5a did not reverse the sense
of selectivity, except for 6d with a very modest affinity
1
deuterium incorporation was measured in the H NMR
Ta ble 2. Inhibition of [3H]pCCK-8 Specific Binding to Rat Pancreas (CCK-A) and Cerebral Cortex Membranes (CCK-B) and
Inhibition of Amylase Release from Dispersed Pancreatic Acini
amylase
releaseb
IC50 (nM) (n)
stereochemistry
4a
Ki (nM)a
compd
CCK-8
Devazepide (1)
3
Trp
5
CCK-A
CCK-B
B/A
0.52 ( 0.04
0.30 ( 0.03
1030
>5000
0.62 ( 0.05
10.6 ( 2
572
2890
298
813
2.8 ( 0.15
342 ( 67
>5000
1560
>5000
2730 ( 273
>5000
2910
>5000
>5000
>5000
1890
5
1140
>4
<0.3
>8000
257
>8
1
>16
>6
25.4 (5)
NDc
ND
21.3 (9)
201 (3)
ND
ND
ND
ND
ND
L
D
L
L
L
L
D
D
D
D
4
5a
5b
5c
5d
6a
6b
6c
6d
S
R
R
S
S
R
R
S
R
S
R
S
R
S
R
S
>5000
>5000
<0.4
ND
a
Values are the mean or mean ( SEM of at least three experiments, performed with seven concentrations of test compounds in triplicate
b
(SEM within (15% of the mean). Inhibition of amylase release stimulated by CCK-8 (0.5 nM) in dispersed pancreatic acini. Data
represent the mean of n independent experiments in duplicate (standard errors within (15% of the mean). c ND ) not determined.

Potent and Selective CCK-A Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3405
500 spectrometer, operating at 200 or 500 MHz, using TMS
as reference. Elemental analyses were obtained on a CH-O-
RAPID apparatus. Analytical RPHPLC was performed on a
Waters Nova-pak C18 (3.9 × 150 mm, 4 µm) column, with a
flow rate of 1 mL/min, using a tunable UV detector set at 214
nm. Mixtures of CH3CN (solvent A) and 0.05% TFA in H2O
(solvent B) were used as mobile phase. Semipreparative
RPHPLC was performed on a Waters Nova-pak C18 (25 × 100
mm, 6 µm) cartridge, with a flow rate of 6.5 mL/min of 40:60
CH3CN and 0.05% TFA in H2O.
for CCK-B receptors. However, a dramatic influence of
the stereochemical structure on the binding potency and
selectivity is observed. Thus, the replacement of the
L-Trp residue of compounds 5a -d by D-Trp in 6a -d led
to a 2-500-fold decrease in the CCK-A binding affinity.
Concerning the stereochemistry of the bicyclic skeleton,
compounds 5a,b, having a 4a,5-trans disposition, showed
from 2 to 3 orders of magnitude higher CCK-A binding
potency than their corresponding 4a,5-cis diastereoiso-
mers 5c,d . What is clear is that the 5R-configured
diastereoisomers 5a ,c have higher affinity and selectiv-
ity for CCK-A receptors than their 4S-configured coun-
terpart 5b,d , respectively. It is also clear that replace-
ment of the 3-oxoindolizidine skeleton in compound 3
with a 1,3-dioxoperhydropyrido[1,2-c]pyrimidine tem-
plate, having the same stereochemistry, produced the
analogue 5b and an important increase in CCK-A
affinity (≈200-fold). This considerable improvement in
affinity could be attributed to a better spatial disposition
of the pharmacophoric 2-benzyl and L-Trp groups.
However, additional interactions of the 1,3-dioxoperhy-
dropyrido[1,2-c]pyrimidine ring with the CCK-A binding
sites can not be discarded.
The best compounds of this series, 5a ,b, were tested
for their antagonism of the CCK-8-stimulated amylase
release from pancreatic acinar cells.32 In accordance
with the binding results, compound 5a showed an
antagonist potency similar to that of the well-known
CCK-A antagonist Devazepide (Table 2), while the
diastereoisomer 5b was approximately 1 order of mag-
nitude less potent. Compounds 5a ,b did not show any
intrinsic effect on amylase release at a 1 µM concentra-
tion and, like all other compounds of the series, were
not able to induce at a 10 µM concentration a contractile
response in the isolated longitudinal muscle-myenteric
plexus preparation from guinea pig ileum, which is
known to be sensitive to both CCK-A and CCK-B
receptor agonists.33
In conclusion, the use of the 1,3-dioxoperhydropyrido-
[1,2-c]pyrimidine as template for appending the aro-
matic side chains of CCK-4 has led to potent and highly
selective CCK-A receptor ligands with antagonist activ-
ity. Compound 5a is one of the most selective CCK-A
antagonists reported to date and remains the best
compound of this series. Besides its selectivity, the most
important feature of this potent and novel CCK-A
antagonist is its lack of structural resemblance to those
previously reported and its independence in genesis.
Therefore, this compound may be a useful pharmaco-
logical tool to investigate the functional role of CCK-A
receptors. Modifications of the two structural domains
on the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine core of
5a , namely, Boc-L-Trp and the 2-benzyl group, which
could lead to improve oral bioavailability and duration
of action, are in progress.
(2R*,3S*)-1-(N-Ben zylca r b a m oyl)-3-[(ter t-b u t oxyca r -
bon yl)am in o]-2-[(eth oxycar bon yl)m eth yl]piper idin e (11).
Benzyl isocyanate (0.62 mL, 5 mmol) was added to a solution
of (2R*,3S*)-3-[(tert-butoxycarbonyl)amino]-2-[(ethoxycarbon-
yl)methyl]piperidine (9) [obtained as a 4:1 mixture of 2S,3R-
and 2R,3S-stereoisomers from Boc-D-Orn(Z)-OH as previously
described28] (1.145 g, 4 mmol) in dry THF (30 mL). After 1 h
of stirring at room temperature, the solvent was evaporated,
and the residue was purified by flash chromatography, using
a 17-25% gradient of AcOEt in hexane as eluant, to give 11,
with 20% of its enantiomer, as a foam (1.443 g, 86%): 1H NMR
(200 MHz, CDCl3) δ 1.12 [t, 3H, CH3(Et)], 1.25 (m, 1H, 5-H),
1.34 (s, 9H, Boc), 1.56 (m, 3H, 4-H, 5-H), 2.36 (dd, 1H, J )
10.3, 16.5 Hz, 2-CH2), 2.64 (m, 1H, 6-H), 2.81 (dd, 1H, J )
3.7, 16.5 Hz, 2-CH2), 3.52 (m, 1H, 3-H), 4.00 [q, 2H, CH2 (Et)],
4.12 (m, 1H, 6-H), 4.26 [m, 2H, CH2 (Bzl)], 4.39 (m, 1H, 2-H),
5.06 (d, 1H, J ) 7.3 Hz, 3-NH), 5.89 (br s, 1H, 1-CONH), 7.14-
7.22 (m, 5H, aromatics). Anal. (C22H33N3O5) C, H, N.
(2R*,3R*)-1-(N-Ben zylca r b a m oyl)-3-[(ter t-b u t oxyca r -
bon yl)am in o]-2-[(eth oxycar bon yl)m eth yl]piper idin e (12).
As above indicated for the synthesis of 11, this compound was
obtained from (2R*,3R*)-3-[(tert-butoxycarbonyl)amino]-2-
[(ethoxycarbonyl)methyl]piperidine (10),28 as a foam (1.175 g,
70%): 1H NMR (200 MHz, CDCl3) δ 1.12 [t, 3H, CH3 (Et)],
1.24 (m, 1H, 4-H), 1.34 (s, 9H, Boc), 1.59 (m, 3H, 4-H, 5-H),
2.50 (m, 3H, 6-H, 2-CH2), 3.58 (m, 1H, 3-H), 4.00 [q, 2H, CH2
(Et)], 4.18 (m, 1H, 6-H), 4.29 [m, 2H, CH2 (Bzl)], 4.50 (d, 1H,
J ) 6.5 Hz, 3-NH), 4.61 (m, 1H, 2-H), 5.83 (br s, 1H, 1-CONH),
7.15-7.25 (m, 5H, aromatics). Anal. (C22H33N3O5) C, H, N.
(4a R*,5S*)-2-Ben zyl-5-[(ter t-b u t oxyca r b on yl)a m in o]-
1,3-dioxoper h ydr opyr ido[1,2-c]pyr im idin e (13). NaH (108
mg of dispersion in mineral oil, 2.7 mmol) was added to a
solution of the piperidine 11 (1.133 g, 2.7 mmol) in dry THF
(50 mL). After the mixture stirred for 3 h at room tempera-
ture, H2O (100 mL) was added, and the reaction mixture was
extracted with AcOEt (2 × 200 mL). Then, the organic phase
was washed with 0.1 N HCl (100 mL) and brine (100 mL),
dried over Na2SO4, and evaporated. The residue was purified
by flash chromatography, using a 0.1-1% gradient of acetone
in CH2Cl2 as eluant, to give 13 as a foam (766 mg, 76%): 1H
NMR (200 MHz, CDCl3) δ 1.33 (m, 1H, 6-H), 1.44 (s, 9H, Boc),
1.55 and 1.78 (2m, 2H, 7-H), 2.08 (m, 1H, 6-H), 2.64 (m, 1H,
8-H), 2.76 (dd, 1H, J ) 9, 17 Hz, 4-H), 2.96 (dd, 1H, J ) 5, 17
Hz, 4-H), 3.07 (m, 1H, 4a-H), 3.41 (m, 1H, 5-H), 4.38 (m, 2H,
8-H, 5-NH), 4.98 (s, 2H, 2-CH2), 7.14-7.23 (m, 5H, aromatics).
Anal. (C20H27N3O4) C, H, N.
(4a R*,5R*)-2-Ben zyl-5-[(ter t-b u t oxyca r b on yl)a m in o]-
1,3-dioxoper h ydr opyr ido[1,2-c]pyr im idin e (14). This com-
pound was obtained as a foam in 48% yield, from the piperidine
12 as above indicated for the synthesis of 13: 1H NMR (200
MHz, CDCl3) δ 1.44 (s, 9H, Boc), 1.62 (m, 3H, 6-H, 7-H), 1.84
(m, 1H, 6-H), 2.64 (m, 1H, 8-H), 2.73 (m, 2H, 4-H), 3.45 (m,
1H, 4a-H), 3.78 (m, 1H, 5-H), 4.33 (m, 1H, 8-H), 4.68 (d, 1H,
J ) 10 Hz, 5-NH), 4.91 and 4.82 (2d, 2H, J ) 15 Hz, 2-CH2),
7.17-7.33 (m, 5H, aromatics). Anal. (C20H27N3O4) C, H, N.
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)tr yp top h yl]a m in o]-1,3-d ioxop er h yd r o-
p yr id o[1,2-c]p yr im id in e Der iva tives 5a -d a n d 6a -d .
TFA (3 mL) was added to a solution of the corresponding
N-Boc-protected 1,3-dioxoperhydropyrido[1,2-c]pyrimidine de-
rivative 13 or 14 (373 mg, 1 mmol) in CH2Cl2 (6 mL); after 4
h at room temperature, the solvents were evaporated to
dryness. The residue was dissolved in dry CH2Cl2 (25 mL),
and Boc-L- or -D-Trp-OH (365 mg, 1.2 mmol), (benzotriazolyl-
oxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP; 549 mg, 1.2 mmol), and triethylamine (32 mL, 2.2 mmol)
Exp er im en ta l Section
Ch em istr y. All reagents were of commercial quality.
Solvents 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. Silica
gel 60 (230-400 mesh), Merck, was used for flash chromatog-
raphy. Melting points were taken on a micro hot stage
apparatus and are uncorrected. Optical rotations were deter-
mined with a Perkin-Elmer-241 MC polarimeter. 1H NMR
spectra were recorded with a Varian Gemini-200 or Unity-

3406 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Mart´ın-Martı´nez et al.
Ta ble 3. Significant 1H NMR Spectroscopic Dataa of the
1,3-Dioxoperhydropyrido[1,2-c]pyrimidine Derivatives 5a -d
and 6a -d
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 of protein/tube) were
incubated with 1 nM [3H]pCCK-8 in 50 mM Tris-HCl buffer,
pH 7.4, containing MgCl2 (5 mM) and bacitracin (0.2 mg/mL)
for 60 min at 25 °C. Final incubation volume was 0.5 mL in
both cases. Nonspecific binding was determined using 1 µM
CCK-8 as the cold displacer. The inhibition constants (Ki) were
calculated using the equation of Cheng and Prusoff from the
displacement curves analyzed with the Receptor Fit Competi-
tion LUNDON program.
proton
5a or 6b
5b or 6a
5c or 6d
5d or 6c
R-Trp
2-CH2
4-H
4a-H
5-NH
5-H
4.43
4.91 4.97
2.53
2.83
5.67
4.81
4.91 4.96
2.18
2.48
5.45
4.39
4.82 4.90
2.39 2.53
3.67
5.98
4.03
4.59
4.79 5.01
1.92 2.35
3.20
5.62
3.94
3.62
3.58
6-H
7-H
8-H
1.01 1.66
1.55
2.53 4.27
1.06 1.82
1.50 1.67
2.48 4.24
1.39
0.93 1.39
2.53 4.18
1.40 1.64
0.45 1.40
2.44 3.94
Am yla se Relea se. Dispersed rat pancreatic acini were
prepared by using a modification of the technique of J ensen
et al.32 The rat was decapitated,and the pancreas was care-
fully 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 (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.34 Samples (2
mL) of acini suspension were placed in plastic tubes and
incubated for 30 min at 37 °C in an 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 (0.5 nM) produced by the assayed compounds
was calculated according to the formula:
a
Registered in CDCl3 at 500 MHz.
were added successively to that solution; stirring was contin-
ued at room temperature for 12 h. Afterwards, the solvent
was evaporated, the residue was dissolved in AcOEt (25 mL),
and the resulting solution was washed successively with 10%
citric acid (10 mL), 10% NaHCO3 (10 mL), H2O (10 mL), and
brine (10 mL), dried over Na2SO4, and evaporated. Flash
chromatography of the residue with a 20-50% gradient of
AcOEt in hexane yielded, in each case, the isolated trans-
(4aS,5R)- and (4aR,5S)-1,3-dioxoperhydropyrido[1,2-c]pyrimi-
dine derivatives 5a , 5b and 6a , 6b, respectively, and the
diastereoisomeric cis mixtures 5c,d and 6c,d . These mixtures
were resolved by semipreparative HPLC as above mentioned.
The 1H NMR spectroscopic data of all these compounds are
summarized in Table 3.
(4a S,5R)-2-Ben zyl-5-[N-[(ter t-bu toxyca r bon yl)-L-tr 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 i-
d in e (5a ): white solid (70% from 13); mp 119-121 °C; [R]26
D
) -31.8° (c 1.00, CHCl3); RPHPLC tR ) 50.87 min (33:67).
Anal. (C31H37N5O5) C, H, N.
(4a R,5S)-2-Ben zyl-5-[N-[(ter t-bu toxyca r bon yl)-L-tr 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 i-
d in e (5b): white solid (17% from 13); mp 174-176 °C; [R]26
D
) +52.6° (c 1.00, CHCl3); RPHPLC tR ) 46.87 min (33:67).
Anal. (C31H37N5O5) C, H, N.
(4a R,5R)-2-Ben zyl-5-[N-[(ter t-bu toxyca r bon yl)-L-tr 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 i-
d in e (5c): white solid (60% from 14); mp 116-118 °C; HPLC
tR ) 41.93 min (35:65). Anal. (C31H37N5O5) C, H, N.
(4a S,5S)-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]-1,3-d ioxop er h yd r op yr id o[1,2-c]p yr im i-
d in e (5d ): white solid (14% from 14); mp 112-114 °C; HPLC
tR ) 37.07 min (35:65). Anal. (C31H37N5O5) C, H, N.
(4a S,5R)-2-Ben zyl-5-[N-[(ter t-bu toxyca r bon yl)-D-tr 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 i-
% I ) [(SCCK - SC) - (ST - SC)/(SCCK - SC)] × 100
where SC is control release (vehicle), SCCK is release elicited
by CCK-8, and ST is release 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.
d in e (6a ): enantiomer of 5b (74% from 13); [R]26 ) -51.8°
(c 1.00, CHCl3). Anal. (C31H37N5O5) C, H, N.
Ack n ow led gm en t. This work has been supported
by the CICYT (SAF 94-0705) and Comunidad de Madrid
(AE 0004/95).
D
(4a R,5S)-2-Ben zyl-5-[N-[(ter t-bu toxyca r bon yl)-D-tr 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 i-
d in e (6b): enantiomer of 5a (18% from 13); [R]26 ) +31.4°
D
Refer en ces
(c 1.00, CHCl3). Anal. (C31H37N5O5) C, H, N.
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