Synthesis of N-terminal substituted anthranilic acid dimer derivatives for evaluation on CCK receptors

Article abstract of DOI:10.1016/S0014-827X(01)01071-0

A series of new N-substituted anthranilic acid dimer derivatives having a C-terminal Phe residue was synthesized and evaluated for their affinity for CCK receptors. These compounds resulted from a blended approach based firstly on the use of an alternative substructure embedded within asperlicin and secondly on the derivatization of this template with substituents chosen considering the C-terminal primary structure of the endogenous ligand. Although these compounds exhibited a regnylogical-type organization similar to that of CCK-4, they are characterized by about 1000-fold greater affinity for CCK-A receptor than the C-terminal tetrapeptide.

Full text of DOI:10.1016/S0014-827X(01)01071-0

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 555–564
Synthesis of N-terminal substituted anthranilic acid dimer
derivatives for evaluation on CCK receptors
Antonio Varnavas a,*, Valentina Valenta a, Federico Berti b, Lucia Lassiani a
a Department of Pharmaceutical Sciences, Uni6ersity of Trieste, P.le Europa 1, 34127 Trieste, Italy
b Department of Chemical Sciences, Uni6ersity of Trieste, Via Giorgieri 1, 34127 Trieste, Italy
Received 15 June 2000; accepted 4 December 2000
Abstract
A series of new N-substituted anthranilic acid dimer derivatives having a C-terminal Phe residue was synthesized and evaluated
for their affinity for CCK receptors. These compounds resulted from a blended approach based firstly on the use of an alternative
substructure embedded within asperlicin and secondly on the derivatization of this template with substituents chosen considering
the C-terminal primary structure of the endogenous ligand. Although these compounds exhibited a regnylogical-type organization
similar to that of CCK-4, they are characterized by about 1000-fold greater affinity for CCK-A receptor than the C-terminal
tetrapeptide. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: CCK receptor ligands; Anthranilic acid dimer
receptor types, CCK-A and CCK-B [5,6]. Both recep-
tors are present in the periphery and in the CNS;
CCK-A receptors are found mostly in the gastrointesti-
nal area, while CCK-B receptors are the predominant
type in the brain [7,8].
These receptor types can be differentiated by their
affinities for CCK-4, CCK-8S, and several receptor
preferring antagonists. The C-terminal tetrapeptide
(CCK-4) preferentially interacts with the central recep-
tor (IC50=3.2×10−8 and 5.0×10−3 M for CCK-B
and CCK-A receptors, respectively) while the sulfated
octapeptide (CCK-8S) binds with nanomolar affinities
at both receptors [9].
Over the past decade potent and selective non-pep-
tide antagonists for CCK-A and CCK-B receptors have
been discovered and suggested to be useful for the
treatment of a variety of diseases such as irritable bowel
syndrome (IBS), chronic and acute pancreatitis, gastric
ulcer, anxiety, panic disorder, schizophrenia, eating dis-
orders [10–12].
The most important strategies adopted for the design
of these antagonists include the discovery of natural
products followed by their chemical manipulation and
the peptoid design approach based on studies of CCK
primary structure. Both strategies include a final step of
1. Introduction
The polypeptide hormone cholecystokinin (CCK)
was discovered in the gastrointestinal tract and subse-
quently in the central nervous system (CNS) [1,2].
Among the different biologically active molecular forms
of CCK (CCK-58, CCK-39, CCK-33, CCK-8, CCK-4),
the sulfated C-terminal octapeptide (CCK-8S), Asp26-
Tyr(SO3H)27 - Met28 - Gly29 - Trp30 - Met31 - Asp32 - Phe33-
NH2, has the same efficacy and potency as the complete
tritriacontapeptide (CCK-33) [3].
CCK is structurally related to gastrin, a gastrointesti-
nal hormone, that acts as mediator of the gastric acid
secretion. These two peptides share an identical C-ter-
minal pentapeptide (Gly-Trp-Met-Asp-Phe-NH2; pen-
tagastrin) which is responsible for all the biological
activities of gastrin [4].
The hormone-like effects of CCK related to the
gastrointestinal functions (such as stimulation of gall
bladder contraction, stimulation of pancreatic enzyme
secretion) as well as its CNS actions as neurotransmit-
ter and neuromodulator are mediated by two distinct
* Correspondence author.
E-mail address: varnavas@univ.trieste.it (A. Varnavas).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01071-0

556
A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
optimization of the obtained non-peptide lead com-
pound performed by chemical modifications.
receptor were obtained [19] and further structural mod-
ifications led to potent and selective CCK-A receptor
antagonists [20].
Thus, the discovery of asperlicin (a natural com-
pound isolated from Aspergillus alliaceus) and its struc-
ture simplification provided two different classes of
CCK-receptor antagonists. The first one, based on the
1,4-benzodiazepine ring system of asperlicin, include
the most important non-peptide CCK receptor antago-
nists since devazepide [3S-(−)-1,3-dihydro-3-(2-indole-
carbonyl-amino)-1-methyl-5-phenyl-2H-1,4-benzo-
Recently, we have proposed a new disconnection
approach of asperlicin in which the rigid polycyclic
system of this natural compound has been opened to
give the flexible anthranilic acid dimer and Trp. In the
first part of our program we demonstrated that the
presence of Trp residue at the N- or C-terminus of
anthranilic acid dimer led to equipotent compounds
with micromolar affinity for CCK-A receptors [21]. In
the subsequent step for the development of this new
class of non-peptide CCK-A receptor ligands our atten-
tion was focused on the independent searching of the
appropriate pharmacophoric groups for the functional-
ization of the N- and C-terminus of this asperlicin-
derived substructure [22]. In particular, we have found
that when the Trp residue is linked to the C-terminus of
the anthranilic acid dimer the highest CCK-A receptor
affinity was achieved with the des-amino Trp at the
N-terminus of the above mentioned dimer [23].
In this paper we wish to report a novel series of
N-terminal substituted anthranilic acid dimer deriva-
tives containing, as in the case of the endogenous
ligands, the Phe residue at the C-terminus of the dimer
(Table 1).
diazepin-2-one] and L-365,260 [(3R-(+)-2,3-dihydro-1-
methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N%-
(3-methylphenyl)-urea] were the first examples of highly
potent, selective, non-peptide antagonists for the CCK-
A and CCK-B receptors, respectively [13,14]. The sec-
ond one, based on quinazolinone nucleus of asperlicin,
represent a class of potent and selective CCK-B recep-
tor antagonists [15,16].
On the other hand, the peptoid derivatives resulted
from a strategy based on simplifying the C-terminal
tetrapeptide to the minimal structure with a significant
affinity for the receptor. These studies having estab-
lished that Trp and Phe residues of CCK-4 were both
necessary to obtain micromolar affinity for the CCK-B
receptors, the dipeptide Boc-Trp-Phe-NH2 was the
starting point for the design of the dipeptoid series. For
this dipeptide (Boc-Trp-Phe-NH2) a sycnological inter-
action at the receptor has been suggested, while in the
case of CCK-4 the Trp-Phe-message is non-continuous
(regnylogic-type organization) [17].
Our choice was supported by the observation that
compounds with similar micromolar affinity for the
CCK-A receptors were obtained either in the presence
at the C-terminal site of the anthranilic acid dimer, by
itself inactive, of Trp — suggested by the disconnection
approach of asperlicin — or Phe, a key aminoacid of
the C-terminal sequence of CCK [21].
As a result of a systematic SAR of the N- and
C-terminal sites of the starting dipeptide, characterized
by micromolar affinity for CCK-B receptor (Ki=7.39
0.5×10−5 M [18]), a number of dipeptoids with high
affinity (nanomolar range) and selectivity for CCK-B
2. Chemistry
Table 1
The target compounds summarized in Table 1 were
synthesized by standard procedures according to the
synthetic route depicted in Scheme 1. Construction of
the key intermediate (compound 11) was effected via
ortho-amino benzoylation of the DL-phenylalanine
ethyl ester with isatoic anhydride, to afford compound
10, followed by 2-nitro benzoylation and subsequent
reduction of the nitro group. Derivatives 12 and 13
were synthesized by coupling the intermediate 11 with
the corresponding acid by the mixed-anhydride method
with isobutyl chloroformate and triethylamine, whereas
compounds 14–20 were obtained by condensation of
compound 11 with the corresponding acyl chloride.
Synthetic data of the esters 12–20 are listed in Tables
2–4. The free acids 1–9 were obtained in almost quan-
titative yield by base-catalyzed hydrolysis of the corre-
sponding ethyl esters 12–20; Tables 5–7 give the
physical and spectroscopic properties of compounds
1–9.
Structure of the target compounds
Comp.
R
1
2
3
4
5
6
7
8
9
3-indolylꢀ(CH2)2ꢀ
3-indolylꢀCH2ꢀ
2-indolyl
phenyl
3-methylphenyl
benzyl
4-chlorobenzyl
phenylethenyl-
HOOCꢀ(CH2)2ꢀ

A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
557
Scheme 1.
Table 2
Physicochemical properties of compounds 12–20
b
Comp.
R
Molecular formula
Molecular weight
Yield (%)
Cryst. solvent a
Rf
M.p. (°C)
12
13
14
15
16
17
18
19
20
3-indolylꢀ(CH2)2ꢀ
3-indolylꢀCH2ꢀ
2-indolyl
phenyl
3-methylphenyl
benzyl
4-chlorobenzyl
phenylethenyl-
H5C2OOCꢀ(CH2)2ꢀ
C36H34N4O7
C35H32N4O5
602
588
574
535
549
549
583.5
561
559
70
58
57
56
48
68
60
66
68
A
0.51
0.54
0.69
0.65
0.60
0.65
0.80
0.58
0.66
116–117
166–167
208–210
145–147
123–125
86–87
129–130
177–178
116–117
MeOH
EtOH
MeOH
EtOH
B
A
EtOH
MeOH
C
34H30N4O5
C32H29N3O5
C33H31N3O5
C33H31N3O5
C33H30ClN3O5
C
34H31N3O5
C31H33N3O7
a Crystallizing solvents: A, AcOEt–hexane; B, EtOH 70%.
b AcOEt–hexane, 1:1.
3. Results and discussion
364,718 and [3H]-(+)-
L-365,260 from their specific
binding sites, as previously described [24,25].
The affinities of the synthesized compounds for the
CCK-A and CCK-B receptors were determined, using
rat pancreatic membranes and guinea pig brain mem-
First, the percentage of inhibition (I, %) was deter-
mined at the highest dose of 10 mM and when the I
values were lower than 20%, the compounds were con-
sidered inactive. For the more active ones, the IC50
branes, respectively, by displacement of [3H]-(9)-
L-

558
A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
Table 4
values were then calculated from five-point inhibition
curves by log-probit plots and the reported values are
the geometric means of at least three separate
experiments.
13C NMR (CDCl3) of compounds 12–20
Comp. l (ppm)
12
13
8.79, 15.72, 32.56, 33.62, 48.15, 56.58, 105.66, 109.45,
113.40, 113.83, 115.03, 116.13, 116.51, 117.69, 118.16,
121.41, 121.81, 121.96, 123.26, 123.93, 127.58, 130.15,
130.99, 133.66, 134.91, 162.16, 162.98, 165.86, 166.24
The CCK receptor binding data of N-substituted
anthranilic acid dimer derivatives (compounds 1–9) are
summarized in Table 8.
The present results indicate that indole-containing
derivatives, i.e. compounds 1–3, are the most potent
and selective within this series since they inhibit the
14.19, 35.36, 37.99, 53.51, 61.95, 108.89, 111.08, 118.94,
119.82, 120.44, 121.20, 121.51, 121.87, 122.24, 123.17,
123.40, 123.93, 126.64, 126.95, 127.35, 127.53, 128.65,
129.34, 132.69, 132.79, 135.55, 139.19, 139.91, 167.09,
168.28, 170.66, 171.25
[3H]-(9)-
L-364,718 binding to rat pancreatic mem-
branes at micromolar concentrations and have little or
14
14.25, 38.07, 53.66, 62.03, 103.92, 111.99, 120.09,
120.54, 120.71, 121.31, 122.03, 122.45, 123.29, 123.37,
123.69, 124.77, 126.93, 127.40, 127.54, 127.99, 128.71,
129.42, 131.62, 133.12, 133.25, 135.62, 136.79, 139.35,
140.69, 160.08, 167.83, 168.53, 171.38
Table 3
1H NMR (CDCl3) of compounds 12–20 a
Comp. l (ppm)
15
16
14.27, 38.05, 53.63, 62.02, 120.55, 120.65, 121.64,
121.97, 123.37, 123.67, 126.89, 127.45, 127.53, 128.71,
128.81, 129.41, 131.85, 133.06, 133.24, 135.09, 135.61,
139.35, 140.84, 165.71, 167.92, 168.43, 171.26
12
13
1.25 (t, 3H, ꢀCH3); 2.88 (m, 2H, ꢀCH2ꢀind); 3.25 (m,
4H, ꢀCH2ꢀ, ꢁCHꢀCH2ꢀ); 4.25 (q, 2H, ꢀOꢀCH2ꢀ); 5.06
(m, 1H, ꢀCHꢂ); 6.75 (d, 1H, ꢀNHꢀCHꢂ); 7.05–8.72
(m, 18H, ar); 7.96 (s, 1H, ꢀNHꢀind); 11.25 (s, 1H,
ꢀNHꢀ); 11.85 (s, 1H, ꢀNHꢀ)
14.26, 21.60, 38.04, 53.64, 62.01, 120.52, 120.72, 121.70,
121.90, 123.34, 123.63, 124.33, 126.93, 127.40, 127.48,
128.45, 128.70, 129.40, 132.64, 133.04, 133.19, 135.07,
135.64, 138.61, 139.37, 140.80, 166.12, 167.89, 168.47,
171.11
1.28 (t, 3H, ꢀCH3); 3.22 (m, 2H, ꢀCH2ꢀCHꢂ); 3.92 (s,
2H, ꢀCH2ꢀCOꢀ); 4.23 (q, 2H, ꢀOꢀCH2ꢀ); 5.02 (m, 1H,
ꢀCHꢂ); 6.69 (d, 1H, ꢀNHꢀCHꢂ); 7.03–8.69 (m, 18H,
ar); 8.26 (s, 1H, ꢀNHꢀind); 11.15 (s, 1H, ꢀNHꢀ); 11.65
(s, 1H, ꢀNHꢀ)
17
18
19
14.27, 38.03, 45.79, 53.62, 62.03, 120.43, 120.79, 121.49,
121.77, 123.36, 126.91, 127.18, 127.23, 127.42, 128.71,
128.89, 129.41, 129.67, 132.95, 133.00, 134.71, 135.64,
139.37, 140.20, 167.47, 168.43, 170.04, 171.33.
14
15
16
17
18
19
1.26 (t, 3H, ꢀCH3); 3.25 (m, 2H, ꢁCHꢀCH2ꢀ); 4.20 (q,
2H, ꢀOꢀCH2ꢀ); 5.05 (m, 1H, ꢀCHꢂ); 6.75 (d, 1H,
ꢀNHꢀCHꢂ); 7.10–8.85 (m, 18H, ar); 9.23 (s, 1H,
ꢀNHꢀind); 12.05 (s, 1H, ꢀNHꢀ); 12.50 (s, 1H, ꢀNHꢀ)
14.26, 38.03, 45.00, 53.59, 62.03, 120.33, 120.62, 121.36,
121.73, 123.45, 123.63, 126.85, 127.17, 127.43, 128.72,
129.04, 129.40, 131.14, 133.01, 133.20, 135.59, 139.37,
140.16, 167.43, 168.42, 169.47, 171.25
1.29 (t, 3H, ꢀCH3); 3.25 (m, 2H, ꢁCHꢀCH2ꢀ); 4.20 (q,
2H, ꢀOꢀCH2ꢀ); 5.05 (m, 1H, ꢀCHꢂ); 6.70 (d, 1H,
ꢀNHꢀCHꢂ); 7.10–8.90 (m, 18H, ar); 12.00 (s, 1H,
ꢀNHꢀ); 12.30 (s, 1H, ꢀNHꢀ)
14.24, 38.06, 53.59, 62.02, 120.34, 120.72, 121.72,
121.95, 122.32, 123.32, 123.73, 126.91, 127.38, 127.42,
128.14, 128.71, 128.87, 129.40, 129.89, 133.03, 133.20,
134.89, 135.57, 139.30, 140.77, 142.01, 164.45, 167.86,
168.38, 171.23
1.27 (t, 3H, ꢀCH2ꢀCH3); 2.45 (s, 3H, ꢀCH3); 3.22 (m,
2H, ꢁCHꢀCH2ꢀ); 4.20 (q, 2H, ꢀOꢀCH2ꢀ); 5.05 (m,
1H, ꢁCHꢀ); 6.73 (d, 1H, ꢀNHꢀCHꢂ); 7.10–8.90 (m,
17H, ar); 11.98 (s, 1H, ꢀNHꢀ); 12.23 (s, 1H, ꢀNHꢀ)
20
14.16, 29.44, 32.78, 38.02, 53.54, 60.64, 61.93, 120.33,
120.56, 121.50, 121.87, 123.12, 123.58, 126.78, 127.23,
127.35, 128.65, 129.35, 132.98, 135.54, 139.34, 140.35,
167.66, 168.34, 170.15, 171.17, 172.48
1.2 (t, 3H, ꢀCH3); 3.2 (m, 2H, ꢀCH2ꢀ); 3.7 (s, 2H,
ꢀCH2ꢀ); 4.2 (q, 2H, ꢀOꢀCH2ꢀ); 5.0 (m, 1H, ꢁCHꢀ);
6.7 (d, 1H, ꢀNHꢀ); 7.0–8.8 (m, 18H, ar); 11.25 (s, 1H,
ꢀNHꢀ); 11.8 (s, 1H, ꢀNHꢀ)
1.28 (t, 3H, ꢀCH3); 3.25 (m, 2H, ꢁCHꢀCH2ꢀ); 3.72 (s,
2H, ꢀCH2ꢀCOꢀ); 4.22 (q, 2H, ꢀOꢀCH2ꢀ); 5.02 (m, 1H,
ꢁCHꢀ); 6.70 (d, 1H, ꢀNHꢀCHꢂ); 7.10–8.65 (m, 17H,
ar); 11.30 (s, 1H, ꢀNHꢀ); 11.90 (s, 1H, ꢀNHꢀ)
no affinity for CCK-B receptor. The best result was
obtained with compound 1 which has an IC50 of 2.3×
10−6 M, a value about tenfold lower than that of the
N-unsubstituted parent compound (compound 0) cho-
sen as reference.
On the contrary, all the phenyl derivatives, com-
pounds 4–8, exhibit a lower and similar affinity for
both receptors thus indicating a lack of selectivity.
Moreover, the structures considered — in terms of
phenyl ring substitution and/or distance from the an-
thranilic acid dimer template — do not have a substan-
tial effect on binding affinity.
1.26 (t, 3H, ꢀCH3); 3.25 (m, 2H, ꢀCH2ꢀCHꢂ); 4.20 (q,
2H, ꢀOꢀCH2ꢀ); 5.05 (m, 1H, ꢀCHꢂ); 6.60 (d, 1H,
ꢀCOꢀCHꢃ) (J=15.5 Hz); 6.70 (d, 1H, ꢀNHꢀCHꢂ);
7.10–8.85 (m, 19H, ar and ꢃCHꢀPh); 11.60 (s, 1H,
ꢀNHꢀ); 11.95 (s, 1H, ꢀNHꢀ)
20
1.25 (2×t, 6H, 2×ꢀCH3); 2.75 (s, 4H, 2×ꢀCH2ꢀ); 3.2
(m, 2H, ꢀCH2ꢀ); 4.2 (2×q, 4H, 2×ꢀOꢀCH2ꢀ); 5.0 (m,
1H, ꢁCHꢀ); 6.75 (d, 1H, ꢀNHꢀ); 7.0–8.8 (m, 13H, ar);
11.3 (s, 1H, ꢀNHꢀ); 11.9 (s, 1H, ꢀNHꢀ)
a Abbreviations: ar, aromatic; ind, indole; s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet.

A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
559
4. Molecular modeling
Compound 9, characterized by an aliphatic side
chain having a second free carboxyl group, was inactive
on both receptors.
It is interesting to note that the present data, with
respect to the CCK-A receptor, are closely related to
those already observed for the same receptor with
anthranilic acid dimer derivatives having a C-terminal
Trp residue [23].
The geometry of compound 1 depends on the ar-
rangement of the central anthranoyl anthranilic acid
moiety: both the oxygens O1 and O2 are hydrogen
bonded to the amide protons in the ortho position H1
and H2, as can be deduced from the interatomic dis-
,
tances of 1.74 and 1.72 A, respectively (Fig. 1). The
system is not planar, being the plane containing one
phenyl ring of the dimer puckered by 42° with respect
to the other. The phenylalanine and the indolepropi-
onic groups lie on the same side of the structure with a
distance between the indole and the phenyl rings of
Hence, it may be inferred that the C-terminal
aminoacid (Phe or Trp) do not significantly modulate
the activity since almost equipotent compounds were
obtained. Moreover, the indole moiety appears to im-
part the best CCK-A receptor binding affinity and can
therefore be considered the optimum N-terminus group
in these series of compounds.
Finally, we have also observed that the most active
compound (compound 1) resembles some structural
features of CCK-4 having these two ‘tetrapeptides’ the
same N- and C-terminal aminoacid side chains and
differing for the presence of the anthranilic acid dimer
instead of the Met-Asp dipeptide, respectively. For this
reason, we hypothesize that compound 1 and CCK-4
could have a similar regnylogical-type organization al-
though characterized by quite different affinities for the
pancreatic receptor (IC50=2.3×10−6 M for com-
pound 1 versus 5.0×10−3 M for CCK-4 [9]).
With an aim to assess this fact we have undertaken a
molecular modeling study of compound 1 and CCK-4
structures.
,
about 10 A (Fig. 2).
The low-energy conformation determined for com-
pound 1 appeared to have some common features with
that described for CCK-4 [26]. Therefore, a conforma-
tional analysis was performed also on CCK-4 in order
to compare the geometries of these two structures par-
ticularly with respect to the spatial arrangement of the
aromatic domains of the N- and C-terminal sites previ-
ously shown to be crucial for receptor recognition.
The optimized geometry of CCK-4 obtained by our
conformational search was quite similar to the absolute
minimum already reported [26]. The values of the b
and c dihedral angles for the central residues of me-
thionine and aspartate are localized in the a-helix inter-
val and the side chains of tryptophane and
phenylalanine are found on the same side of the pep-
,
tide, at an average distance of 10.6 A (Fig. 3). In this
Table 5
Physicochemical properties of compounds 1–9 a
b
Comp.
R
Molecular formula
Molecular weight
Rf
M.p. (°C)
1
2
3
4
5
6
7
8
9
3-indolylꢀ(CH2)2ꢀ
3-indolylꢀCH2ꢀ
2-indolyl
phenyl
3-methylphenyl
benzyl
4-chlorobenzyl
phenylethenyl-
HOOCꢀ(CH2)2ꢀ
C34H30N4O7
C33H28N4O5
C32H26N4O5
C30H25N3O5
C31H27N3O5
C31H27N3O5
C31H26ClN3O5
C32H27N3O5
C27H25N3O7
574
560
546
507
521
521
555.5
533
503
0.40
0.50
0.54
0.74
0.69
0.31
0.46
0.50
0.25
208–210
210–211
269–270
158–160
110–112
146–147
210–212
163–165
182–184
a Compounds 1–9 were crystallized from methanol.
b AcOEt–EtOH, 1:1.

560
A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
Table 6
In both cases the aromatic rings of the N- and C-termi-
nal sites approach each other, thus indicating a similar
regnylogical-type organization.
1H NMR (DMSO-d6) of compounds 1–9 a
Comp.
l (ppm)
1
2.69 (t, 2H, ꢀCH2ꢀind); 2.99 (t, 2H, ꢀCH2ꢀCH2ꢀ);
3.15 (m, 2H, ꢁCHꢀCH2ꢀ); 4.64 (m, 1H, ꢀCHꢂ);
6.90–8.50 (m, 18H, ar); 9.05 (d, 1H, ꢀNHꢀCHꢂ);
10.70 (s, 1H, ꢀNHꢀ ind); 10.78 (s, 1H, ꢀNHꢀ);
11.85 (s, 1H, ꢀNHꢀ)
5. Conclusions
In conclusion, we have shown that compounds with
micromolar affinity for CCK-A receptor could be ob-
tained by anchoring two appropriate pharmacophoric
groups on the anthranilic acid dimer template. The best
results were obtained with the N- and C-terminal aro-
matic rings already found in the likewise spatial ori-
2
3
3.66 (m, 2H, ꢀCH2ꢀCHꢂ); 3.76 (s, 2H,
ꢀCH2ꢀCOꢀ); 4.68 (m, 1H, ꢀCHꢂ); 6.8–8.35 (m,
18H, ar); 9.15 (d, 1H, ꢀNHꢀCHꢂ); 10.72 (s, 1H,
ꢀNHꢀ ind); 11.05 (s, 1H, ꢀNHꢀ); 11.75 (s, 1H,
ꢀNHꢀ)
3.10 (m, 2H, ꢀCH2ꢀ); 4.70 (m, 1H, ꢀCHꢂ);
7.05–8.65 (m, 18H, ar); 9.15 (d, 1H, ꢀNHꢀCHꢂ);
11.95 (s, 2H, ꢀNHꢀ and ꢀNHꢀ ind); 12.10 (s, 1H,
ꢀNHꢀ)
Table 7
13C NMR (DMSO-d6) of compounds 1–9
Comp. l (ppm)
4
5
3.07 (m, 2H, ꢀCH2ꢀ); 4.67 (m, 1H, ꢀCHꢂ);
7.06–8.55 (m, 18H, ar); 9.13 (d, 1H, ꢀNHꢀCHꢂ);
11.82 (s, 1H, ꢀNHꢀ); 12.05 (s, 1H, ꢀNHꢀ)
1
2
3
20.49, 36.09, 37.65, 53.96, 111.12, 113.12, 117.98,
118.04, 120.63, 120.70, 121.72, 122.03, 122.99, 123.36,
123.52, 126.13, 126.74, 127.38, 127.89, 128.08, 128.84,
132.03, 135.99, 137.76, 138.00, 138.41, 166.00, 168.18,
170.73, 172.51
2.40 (s, 3H, ꢀCH3); 3.05 (m, 2H, ꢀCH2ꢀ); 4.68 (m,
1H, ꢀCHꢂ); 7.11–8.51 (m, 17H, ar); 9.14 (d, 1H,
ꢀNHꢀCHꢂ); 11.74 (s, 1H, ꢀNHꢀ); 12.04 (s, 1H,
ꢀNHꢀ)
34.36, 36.05, 53.86, 107.12, 111.20, 118.10, 118.37,
120.51, 120.70, 120.89, 121.02, 122.44, 123.05, 123.13,
124.58, 126.17, 126.90, 126.94, 127.90, 128.84, 132.06,
136.16, 137.70, 138.13, 138.33, 165.71, 168.22, 169.97,
172.46
6
7
3.07 (m, 2H, ꢀCH2ꢀ); 3.67 (s, 2H, ꢀCH2ꢀ); 4.68 (m,
1H, ꢁCHꢀ); 7.1–8.5 (m, 18H, ar); 9.11 (d, 1H,
ꢀNHꢀ); 10.7 (s, 1H, ꢀNHꢀ); 11.8 (s, 1H, ꢀNHꢀ)
3.08 (m, 2H, ꢁCHꢀCH2ꢀ); 3.68 (s, 2H,
ꢀCH2ꢀCOꢀ); 4.67 (m, 1H, ꢀCHꢂ); 7.10–8.42 (m,
17H, ar); 9.08 (d, 1H, ꢀNHꢀCHꢂ); 10.65 (s, 1H,
ꢀNHꢀ); 11.79 (s, 1H, ꢀNHꢀ)
36.05, 53.85, 102.74, 112.30, 119.97, 120.87, 120.99,
121.05, 121.39, 121.71, 123.33, 123.89, 126.16, 126.81,
127.40, 127.90, 128.19, 128.85, 131.28, 132.21, 132.70,
136.92, 137.67, 138.19, 139.03, 159.12, 166.62, 168.27,
172.44
8
9
3.08 (m, 2H, ꢀCH2ꢀ); 4.68 (m, 1H, ꢀCH2ꢀCHꢂ);
6.90 (d, 1H, ꢀCOꢀCHꢃ) (J=15.6 Hz); 7.02–8.51
(m, 19H, ar and ꢃCHꢀPh); 9.13 (d, 1H,
ꢀNHꢀCHꢂ); 10.88 (s, 1H, ꢀNHꢀ); 11.87 (s, 1H,
ꢀNHꢀ)
4
5
36.03, 53.85, 120.76, 120.89, 121.37, 122.06, 123.27,
123.60, 126.17, 126.87, 127.41, 127.91, 128.16, 128.69,
128.84, 131.85, 132.17, 132.57, 134.29, 137.67, 138.19,
138.98, 164.53, 166.53, 168.24, 172.46
3.1 (m, 2H, ꢀCH2ꢀ); 3.3 (m, 4H, 2×ꢀCH2ꢀ); 4.7
(m, 1H, ꢁCHꢀ); 6.9–8.6 (m, 13H, ar); 9.0 (d, 1H,
ꢀNHꢀ); 11.4 (s, 1H, ꢀNHꢀ); 12.0 (s, 1H, ꢀNHꢀ)
20.82, 36.00, 53.84, 120.75, 120.84, 121.53, 122.38,
123.27, 123.64, 123.78, 126.18, 127.42, 127.67, 127.92,
128.14, 128.55, 128.84, 132.17, 132.45, 132.59, 134.30,
137.65, 138.05, 138.18, 138.81, 164.74, 166.47, 168.24,
172.46
a Abbreviations: ar, aromatic; ind, indole; s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet.
6
7
8
9
36.03, 43.68, 53.82, 120.49, 120.65, 121.76, 123.02,
123.60, 123.80, 126.18, 126.48, 127.30, 127.92, 128.09,
128.16, 128.84, 129.18, 131.92, 132.05, 135.01, 137.68,
138.37, 165.81, 168.22, 169.12, 172.46
case, the Phe side chain is relatively close to the pyrrole
,
ring of Trp (ca. 8 A) whereas in compound 1 the indole
ring offers its phenyl ring towards the Phe moiety.
Finally, a geometrical comparison was made by an
rms overlay between compound 1 and CCK-4. This
superimposition was performed matching 11 atom pairs
(the five atoms of the pyrrole ring of the Trp and the
six aromatic carbons of the Phe) and the rms error in
36.00, 42.65, 53.82, 120.31, 120.51, 121.85, 122.94,
123.75, 124.21, 126.18, 127.37, 127.93, 128.07, 128.84,
131.16, 131.90, 132.05, 133.89, 137.41, 137.70, 138.39,
165.69, 168.22, 168.80, 172.48
36.02, 53.82, 120.29, 120.68, 121.89, 122.07, 123.15,
123.62, 123.78, 126.18, 127.31, 127.55, 127.77, 128.14,
128.72, 128.85, 129.35, 129.74, 132.05, 133.20, 134.29,
135.57, 138.30, 140.82, 163.47, 166.05, 168.23, 172.44
,
the overlay resulted to be 1.07 A (Fig. 4).
It can be seen that compound 1 exhibits an extensive
overlap with CCK-4, the main difference regarding the
tetrapeptide backbone as compared with the anthranilic
acid dimer scaffold. In fact, the three peptide bonds of
the CCK-4 sequence form a S-like bend whereas com-
pound 1 presents a staggered U-shaped conformation.
28.57, 31.48, 36.02, 53.81, 120.62, 120.71, 121.52,
123.07, 123.30, 126.18, 127.37, 127.92, 128.11, 128.89,
132.07, 137.68, 138.06, 138.35, 166.02, 168.21, 169.94,
172.44, 173.42

A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
561
Table 8
CCK receptors binding data
Comp.
R
H
CCK-A (%) a
CCK-B (%) b
0
1
2
3
4
5
6
7
8
9
(26×10−6 M) c
IN d
30
28
25
35
43
28
43
1H-indol-3-ylꢀ(CH2)2ꢀCOꢀ
1H-indol-3-ylꢀCH2ꢀCOꢀ
1H-indol-2-ylꢀCOꢀ
C6H5ꢀCOꢀ
C6H4-m-CH3ꢀCOꢀ
C6H5ꢀCH2ꢀCOꢀ
C6H4-p-ClꢀCH2ꢀCOꢀ
C6H5ꢀCHꢃCHꢀCOꢀ
HOOCꢀ(CH2)2ꢀCOꢀ
(2.3×10−6 M) c
(6.8×10−6 M) c
(3.8×10−6 M) c
30
35
38
40
43
37
IN d
IN d
a Percentage of inhibition at 10 mM of [3H]-(9)-
L
-364,718 binding in rat pancreatic membranes.
b Percentage of inhibition at 10 mM of [3H]-(+)-
L-365,260 in guinea pig brain membranes.
c IC50 (mM) given as the mean of at least three independent determinations. The maximum standard error was always less than 20% of the
geometric mean.
d Inactive: percentage of inhibition less than 20% at 10 mM.
ented sequence of CCK-4 as well as in the dipeptoid
series otherwise characterized by a sycnological-type
organization.
The presence of such pharmacophoric groups joined
together by means of a spacer can therefore be consid-
ered as important elements for CCK receptor recogni-
tion and we are continuing our efforts to search for new
templates that are able to support these groups with the
correct spatial arrangement.
6. Experimental
6.1. Chemistry
All chemicals and solvents used in syntheses were
reagent-grade products and were used without addi-
Fig. 1. Stable conformation of compound 1.
Fig. 2. Stereo view of the lowest energy conformer of compound 1.

562
A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
Fig. 3. Stereo view of the lowest energy conformer of Ac-CCK-4.
tional purification. Melting points were determined on
a Bu¨chi 510 melting point apparatus (Bu¨chi, Flawil,
Switzerland) and are uncorrected. Ascending thin-layer
chromatography (TLC) was performed on precoated
silica gel plates (60F-254 Merck) using UV light to
visualize the chromatograms. Proton (1H NMR, 200
MHz) and carbon (13C NMR, 50 MHz) NMR spectra
were recorded on a Varian-Gemini 2000 Fourier Trans-
form spectrometer. Chemical shifts are reported in
parts per million (ppm, l units) relative to tetramethyl-
silane (TMS) as internal standard. Splitting patterns are
designated as s, singlet; d, doublet; dd, double doublet;
t, triplet; q, quartet; and m, multiplet. Spectral data are
consistent with the assigned structures.
washed in succession with 1 M NaOH (2×30 ml) and
water (2×30 ml). The dried (sodium sulfate) organic
phase was concentrated to give the crude nitro deriva-
tive, which was used without further purification. The
residue was taken up with 100 ml of dichloromethane
and treated with 10 g of zinc dust. The resulting
suspension was cooled at 0°C and, within 10 min, 12 ml
of glacial acetic acid was added dropwise with stirring.
The mixture was stirred for 1 h at r.t. and then filtered.
The organic phase was washed with 1 M NaOH (2×50
ml), water (2×50 ml), dried over sodium sulfate, and
evaporated. Crystallization from 90% ethanol afforded
the analytically pure compound 11 in 71% yield.
1
Rf 0.73 (AcOEt–hexane, 1:1); m.p. 137°C; H NMR
(CDCl3): l 1.28 (t, 3H, ꢀCH3); 3.23 (m, 2H, ꢀCH2ꢀ);
4.24 (q, 2H, ꢀOꢀCH2ꢀ); 5.03 (m, 1H, ꢀCHꢂ); 5.75 (s,
2H, ꢀNH2); 6.68–8.67 (m, 14H, arom and ꢀNHꢀ);
11.62 (s, 1H, ꢀNHꢀ). 13C NMR (CDCl3): l 14.14,
37.97, 53.48, 61.83, 115.68, 116.92, 117.39, 120.29,
121.65, 122.71, 126.68, 127.28, 127.80, 128.60, 129.33,
132.73, 135.58, 139.93, 149.71, 167.94, 168.47, 171.22.
6.1.1. N-Anthranoyl-DL-phenylalanine ethyl ester (10)
A suspension of DL-phenylalanine ethyl ester hydro-
chloride (4.59 g, 20 mmol) in 500 ml of ethyl acetate
was treated with triethylamine (2.81 ml, 20 mmol)
followed by isatoic anhydride (3.26 g, 20 mmol). The
resulting mixture was refluxed under stirring for 2 h,
cooled to room temperature (r.t.) and filtered. The
organic phase was thoroughly washed with 1 M NaOH
(2×50 ml), water (2×50 ml), dried over anhydrous
sodium sulfate and concentrated in vacuo. Trituration
with petroleum ether (40–70°C) afforded the analyti-
cally pure title compound in 79% yield.
6.1.3. General procedure for the synthesis of
N-[N-(substituted)anthranoyl]
anthranoyl-DL-phenylalanine ethyl ester deri6ati6es
(12 and 13)
A solution of 10 mmol of the corresponding acid in
100 ml of dry dichloromethane cooled at −10°C was
treated with triethylamine (1.40 ml, 10 mmol) followed
by isobutyl chloroformate (1.31 ml, 10 mmol). The
resulting mixture was stirred at −10°C for 20 min and
Rf 0.69 (AcOEt–hexane, 1:1); m.p. 83–84°C; 1H
NMR (CDCl3): l 1.24 (t, 3H, ꢀCH3); 3.21 (m, 2H,
ꢀCH2ꢀCHꢂ); 4.18 (q, 2H, ꢀCH2ꢀOꢀ); 4.97 (m, 1H,
ꢁCHꢀ); 5.45 (s, 2H, ꢀNH2); 6.52 (d, 1H, ꢀNHꢀ);
6.61–7.28 (m, 9H, arom). 13C NMR (CDCl3): l 14.19,
38.05, 53.20, 61.63, 115.41, 116.69, 117.29, 127.15,
127.43, 128.60, 129.43, 132.59, 136.04, 148.84, 168.66,
171.75.
6.1.2. N-(N-Anthranoyl) anthranoyl-DL-phenylalanine
ethyl ester (11)
A solution of N-anthranoyl-DL-phenylalanine ethyl
ester (10) (6.24 g, 20 mmol) in 100 ml of dry
dichloromethane cooled at 0°C was treated with tri-
ethylamine (2.81 ml, 20 mmol) followed by 2-nitro-ben-
zoyl chloride (3.70 g, 20 mmol). The resulting mixture
was stirred at r.t. for 2 h. The reaction mixture was
Fig. 4. Superimposition (heavy atoms only) of the preferential confor-
mation of compound 1 (red) and Ac-CCK-4 (gray).

A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
563
treated dropwise with a solution of compound 11 (4.31
g, 10 mmol) in 50 ml of dry dichloromethane. After the
addition was complete, the reaction was stirred at r.t.
for 1 h and then refluxed for 3 h. The solvents were
evaporated, the residue was dissolved in dichloro-
methane; the organic layer was washed with diluted
aqueous sodium hydroxide solution and with water,
dried (sodium sulfate), and evaporated to dryness. The
residue was purified by crystallization to yield com-
pounds 12 and 13 (Table 2).
as previously described [24]. Briefly, samples of 0.4 ml
containing 0.8 mg of wet tissue (:7.2 mg/ml protein)
were incubated for 30 min at 37°C in the presence of
[3H]-
L-364,718 (0.2 nM final concentration) and a solu-
tion of various concentrations of the compounds to be
tested. The buffer used for binding assay was 50 mM
Tris–HCl (pH 7.4 at 37°C), 5 mM MgCl2, 5 mM
dithiothreitol, 2 mg/ml of bovine serum albumin and
0.14 mg/ml bacitracin. The samples were then filtered
under reduced pressure using glass fiber GF/B (What-
man) filters and rinsed four times with 4 ml of cooled
Tris buffer (50 mM, pH 7.4). Non-specific binding was
6.1.4. General procedure for the synthesis of
N-[N-(substituted)anthranoyl]
anthranoyl-DL-phenylalanine ethyl ester deri6ati6es
(14–20)
determined in the presence of (R,S)- -364,718 (0.3 mM
L
final concentration) and was always less than 10% of
the total binding.
A solution of 10 mmol of the corresponding acyl
chloride (the indole-2-carbonyl chloride was prepared
according to the procedure described by Kermack et al.
[27]) in 30 ml of dry dichloromethane was gradually
added to a solution of compound 11 (4.31 g, 10 mmol)
in the same solvent (50 ml). The pH of the reaction was
adjusted to 9.5 with triethylamine and the solution
stirred for 2 h. The reaction mixture was diluted with
100 ml of dichloromethane and washed in succession
with 0.1 N NaOH, water, 0.1 N HCl and water. The
dried (sodium sulfate) organic phase was rotary evapo-
rated and the residue was purified by crystallization to
yield compounds 14–20 (Table 2).
CCK-B receptor affinities were determined by dis-
placement of [3H]-(+)-
L-365,260 from guinea pig cere-
bral cortex membranes as previously described by
Chang et al. [25]. The buffer used was 10 mM HEPES,
5 mM MgCl2, 1 mM EGTA, 130 mM NaCl and 0.25
mg/ml bacitracin, pH 6.5 and the glass fiber filters were
GF/C (Whatman). Briefly, displacement experiments
were performed by incubation of 0.5 ml of brain mem-
branes corresponding to 6.2 mg of wet tissue (200 mg/ml
protein) for 30 min at 25°C in the presence of [3H]-(+)-
L
-365,260 (1 nM final concentration) plus various con-
centrations of the compounds to be tested. Non-specific
binding was determined in the presence of (R,S)-
L
-
365,260 (2 mM final concentration). Specific binding
was defined as the radioactivity after subtracting non-
specific binding and was 45% of the total.
6.1.5. General procedure for the synthesis of compounds
1–9
A mixture of 5 mmol of the corresponding ethyl ester
(compounds 12–20) in methanol (50 ml) and in the
presence of potassium hydroxide (0.56 g, 10 mmol) was
gently warmed for 4 h. The solvent was removed under
reduced pressure and the residue was taken up with
water. After cooling, the solution was adjusted to pH
2–3 with diluted HCl to obtain the precipitation of the
acid (compounds 1–9, Table 5).
6.3. Computational procedures
All calculations were carried out on the form of the
tetrapeptide (CCK-4) with the NH2-terminal amino
group blocked by an acetyl group.
The minimum energy conformations of compound 1
and CCK-4 were obtained from a conformational
search carried out with a modification of the weighted
random Monte Carlo search algorithm of Chang et al.
6.2. Biological e6aluation
[29],
Simulations).
implemented
in
Conformer
(Princeton
6.2.1. General
Male rats (Wistar) and male guinea pigs (Hartley)
were obtained from Charles River, Calco, Como
The dihedral angles O1ꢀC2ꢀC3ꢀN1, C3ꢀN1ꢀC4ꢀO2,
C5ꢀC6ꢀN2ꢀH2, and the two h, i torsional angles at
phenylalanine and indolepropionic acid were selected
for the generation of the starting geometries of com-
pound 1, while all the b and c angles of the peptide
backbone were used for the starting geometries of
CCK-4.
The initial geometries obtained from the algorithm
were optimized first with the MM2 forcefield, and the
searches were considered complete when either 1000
initial conformations in a row were rejected as similar
to previously found conformations, or the number of
consecutive minimized conformations which are proved
(Italy). [3H]-(9)- -364,718 and [3H]-(+)-
L L-365,260
were purchased from NEN Research Products (Brux-
elles) with specific activities of 87 and 75.3 Ci/mmol,
respectively. (R,S)-L-364,718 and (R,S)-L-365,260 were
synthesized in our laboratory as previously described
[28]. Radioactivity was counted with 4 ml of Aquassure
(NEN) high performance LSC cocktail in a Packard
TRI-CARB 300 liquid scintillator.
6.2.2. Binding studies
All experiments were performed in triplicate. CCK-A
receptor binding of [3H]-(9)-
L-364,718 was performed

564
A. Varna6as et al. / Il Farmaco 56 (2001) 555–564
[15] M.J. Yu, K.J. Thrasher, J.R. McCowan, N.R. Mason, L.G.
Mendelsohn, Quinazolinone cholecystokinin-B receptor ligands,
J. Med. Chem. 34 (1991) 1505–1508.
[16] J.K. Padia, M. Field, J. Hinton, K. Meecham, J. Pablo, R.
Pinnock, B.D. Roth, L. Singh, N. Suman-Chauhan, B.K.
Trivedi, L. Webdale, Novel nonpeptide CCK-B antagonists:
design and development of quinazolinone derivatives as potent,
selective, and orally active CCK-B antagonists, J. Med. Chem.
41 (1998) 1042–1049.
[17] D.C. Horwell, B. Birchmore, P.R. Boden, M. Higginbottom, Y.
Ping Ho, J. Hughes, J.C. Hunter, R.S. Richardson, a-Methyl
tryptophanylphenyl-alanine and their arylethylamine ‘‘dipep-
toid’’ analogues of the tetrapeptide cholecystokinin (30–33),
Eur. J. Med. Chem. 25 (1990) 53–60.
[18] D.C. Horwell, A. Beeby, C.R. Clark, J. Hughes, Synthesis and
binding affinities of analogues of cholecystokinin-(30–33) as
probes for central nervous system cholecystokinin receptors, J.
Med. Chem. 30 (1987) 729–732.
to be the same as the conformations previously found
reach 2500. Two minimized conformations were consid-
ered different if the steric energies differ by more than
0.5 kcal/mol or a torsional angle by torsional angle
comparison shows that one or more torsional angles
differ by more than 0.1 rad.
The geometries obtained from the search were refined
by a semiempirical calculation using the AM1 hamilto-
nian as implemented in MOPAC-93 [30] (RHF, SCF
convergence 1×10−5; the geometry optimization was
carried out with the Polak–Ribiere algorithm at a
convergence limit of 1×10−4 kcal/A mol).
,
References
[19] J. Hughes, P. Boden, B. Costall, A. Domeney, E. Kelly, D.C.
Horwell, J.C. Hunter, R.D. Pinnock, G.N. Woodruff, Develop-
ment of a class of selective cholecystokinin type B receptor
antagonists having potent anxiolytic activity, Proc. Natl. Acad.
Sci. USA 87 (1990) 6728–6732.
[20] P.R. Boden, M. Higginbottom, D.R. Hill, D.C. Horwell, J.
Hughes, D.C. Rees, E. Roberts, L. Singh, N. Suman-Chauhan,
G.N. Woodruff, Cholecystokinin dipeptoid antagonists: design,
synthesis, and anxiolytic profile of some novel CCK-A and
CCK-B selective and ‘‘mixed’’ CCK-A/CCK-B antagonists, J.
Med. Chem. 36 (1993) 552–565.
[21] A. Varnavas, L. Lassiani, E. Luxich, M. Zacchigna, Anthranoyl-
anthranilic acid: a template for the development of a new class of
cholecystokinin receptor ligands, Pharmazie 51 (1996) 697–700.
[22] A. Varnavas, L. Lassiani, E. Luxich, V. Valenta, C-terminal
anthranoyl-anthranilic acid derivatives and their evaluation on
CCK receptors, Farmaco 55 (2000) 293–302.
[23] A. Varnavas, L. Lassiani, V. Valenta, Synthesis of new an-
thranilic acid dimer derivatives and their evaluation on CCK
receptors, Farmaco 55 (2000) 369–375.
[1] V. Mutt, J.E. Jorpes, Hormonal polypeptides of the upper
intestine, Biochem. J. 125 (1971) 57P–58P.
[2] L.I. Larsson, J.F. Rehfeld, Localization and molecular hetero-
geneity of cholecystokinin in the central and peripheral nervous
systems, Brain Res. 165 (1979) 201–218.
[3] M.L. Villanueva, S.M. Collins, R.T. Jensen, J.D. Gardner,
Structural requirements for action of cholecystokinin on enzyme
secretion from pancreatic acini, Am. J. Physiol. 242 (1982)
G416–G422.
[4] G.F. Stening, M.I. Grossman, Gastrin-related peptides as stimu-
lants of pancreatic and gastric secretion, Am. J. Physiol. 217
(1969) 262–266.
[5] R.B. Innis, S.H. Snyder, Distinct cholecystokinin receptors in
brain and pancreas, Proc. Natl. Acad. Sci. USA 77 (1980)
6917–6921.
[6] C.T. Dourish, D.R. Hill, Classification and function of CCK
receptors, Trends Pharmacol. Sci. 8 (1987) 207–209.
[7] A. Saito, H. Sankaran, I.D. Goldfine, J.A. Williams, Cholecys-
tokinin receptors in the brain: characterization and distribution,
Science 208 (1980) 1155–1156.
[8] T.H. Moran, P.H. Robinson, M.S. Goldrich, P.R. McHugh,
Two brain receptors: implications for behavioral actions, Brain
Res. 362 (1986) 175–179.
[9] F. Makovec, M. Bani, R. Chiste`, L. Revel, L.C. Rovati, L.A.
Rovati, Differentiation of central and peripheral cholecystokinin
receptors by new glutaramic acid derivatives with cholecys-
tokinin — antagonistic activity, Arzneim.-Forsch./Drug Res. 36
(1986) 98–102.
[10] H. Ito, H. Sogabe, Y. Satoh, CCK-A receptor antagonists,
Drugs Future 20 (1995) 587–599.
[11] L. Revel, F. Makovec, Update on nonpeptide CCK-B receptor
antagonists, Drugs Future 23 (1998) 751–766.
[12] J.G. Wettstein, L. Bueno, J.L. Junien, CCK antagonists: phar-
macology and therapeutic interest, Pharmacol. Ther. 62 (1994)
267–282.
[13] B.E. Evans, M.G. Bock, K.E. Rittle, R.M. Di Pardo, W.L.
Whitter, D.F. Veber, P.S. Anderson, R.M. Freidinger, Design of
potent, orally effective, nonpeptidal antagonists of the peptide
hormone cholecystokinin, Proc. Natl. Acad. Sci. USA 83 (1986)
4918–4922.
[24] R.S.L. Chang, V.J. Lotti, T.B. Chen, K.A. Kunkel, Characteri-
zation of the binding of [3H]-(9)-
L-364,718: a new potent,
nonpeptide cholecystokinin antagonist radioligand selective for
peripheral receptors, Mol. Pharmacol. 30 (1986) 212–217.
[25] R.S. Chang, T.B. Chen, M.G. Bock, R.M. Freidinger, R. Chen,
A. Rosegay, V.J. Lotti, Characterization of the binding of
[3H]-
L-365,260: a new potent and selective brain cholecystokinin
(CCK-B) and gastrin receptor antagonist radioligand, Mol.
Pharmacol. 35 (1989) 803–808.
[26] S.A. Kolodziej, G.V. Nikiforovich, R. Skeean, M.F. Lignon, J.
Martinez, G.R. Marshall, Ac-[3- and 4-alkylthioproline31]-CCK4
analogs: synthesis and implications for the CCK-B receptor-
bound conformation, J. Med. Chem. 38 (1995) 137–149.
[27] W.O. Kermack, W.H. Perkin, R. Robinson, Harmin and harma-
line. Part V. The synthesis of norharman, J. Chem. Soc. 119
(1921) 1602–1642.
[28] M.G. Bock, R.M. Di Pardo, B.E. Evans, K.E. Rittle, D.F.
Veber, R.M. Freidinger, J. Hirshfield, J.P. Springer, Synthesis
and resolution of 3-amino-1,3-dihydro-5-phenyl-2H-1,4-benzodi-
azepin-2-ones, J. Org. Chem. 52 (1987) 3232–3239.
[29] R. Chang, W.C. Guida, W.C. Still, An internal coordinate
Monte Carlo method for searching conformational space, J. Am.
Chem. Soc. 111 (1989) 4379–4386.
[14] M.G. Bock, R.M. Di Pardo, B.E. Evans, K.E. Rittle, W.L.
Whitter, D.F. Veber, P.S. Anderson, R.M. Freidinger, Benzodi-
azepine gastrin and brain cholecystokinin receptor ligands:
L-
365,260, J. Med. Chem. 32 (1989) 13–16.
[30] J.J.P. Stewart, MOPAC-93.02, Fujitsu Ltd, Tokyo, Japan, 1994.
.