C-terminal anthranoyl-anthranilic acid derivatives and their evaluation on CCK receptors

Article abstract of DOI:10.1016/S0014-827X(00)00043-4

A series of C-terminal anthranoyl-anthranilic acid derivatives arising from a strict bond disconnection approach of asperlicin were synthesized and examined for their CCK receptor affinities. These compounds represent the second step of our investigation

Full text of DOI:10.1016/S0014-827X(00)00043-4

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 293–302
C-terminal anthranoyl–anthranilic acid derivatives and their
evaluation on CCK receptors
Antonio Varnavas *, Lucia Lassiani, Elena Luxich, Valentina Valenta
Department of Pharmaceutical Sciences, Uni6ersity of Trieste, P. le Europa 1, I-34127 Trieste, Italy
Received 10 February 1999; accepted 1 September 1999
Abstract
A series of C-terminal anthranoyl–anthranilic acid derivatives arising from a strict bond disconnection approach of asperlicin
were synthesized and examined for their CCK receptor affinities. These compounds represent the second step of our investigation
directed toward the search for alternative substructures of asperlicin as a starting point for the development of a new class of CCK
ligands. The obtained micromolar affinities for CCK-A rather than CCK-B receptor confirm that the anthranilic acid dimer
represents a useful template for the development of selective CCK-A receptor ligands. © 2000 Elsevier Science S.A. All rights
reserved.
Keywords: CCK ligands; Anthranilic acid dimer
1. Introduction
classes of potent and selective antagonists have been
described [11–17].
Cholecystokinin (CCK) is an endogenous peptide
that occurs in both the gastrointestinal tract and central
nervous system (CNS) [1]. Among its different molecu-
lar forms the C-terminal octapeptide (CCK-8) repre-
sents the minimum sequence required for bioactivity [2].
The entire range of the biological functions of CCK
appears to be mediated by two receptors subtypes,
CCK-A and CCK-B [3,4]. CCK-A receptors predomi-
nate in the periphery and mediate the hormone-like
CCK activity (stimulation of gall bladder contraction
and pancreatic enzyme secretion), but were also found
in CNS [5,6].
CCK-B receptors are widely distributed in CNS and
are thought to be involved in the control of anxiety and
nociception [7–10].
The therapeutic potential of CCK ligands in treating
gastrointestinal CCK-related disorders and in patho-
physiological situations concerning different CCK-
modulated central neural pathways, has stimulated an
extensive research in this area and up to now manifold
Among these, non-peptide derivatives deserve a par-
ticular mention in consideration of their favourable in
vivo stability. Many classes of these ligands could be
related to substructures recognised in the molecule of
asperlicin, the first non-peptide selective antagonist for
CCK-A receptors isolated from Aspergillus alliaceus
[13].
As part of our interest in non-peptide CCK receptor
ligands, we have directed our efforts toward the search
for alternative substructures of asperlicin as a starting
point for the development of a new class of ligands.
We recently described an innovative bond disconnec-
tion of asperlicin, summarized in Fig. 1, showing that
the structure of this natural product may be viewed as
the result of intramolecular cyclization of the an-
thranilic acid dimer and tryptophan [18].
The derivatization of the N-terminal part of the
anthranilic acid dimer, per se inactive, led to com-
pounds with micromolar affinities for the CCK-A re-
ceptor subtype.
In this paper we wish to report the second and
obvious step of our investigation concerning the synthe-
sis and CCK receptor binding of C-substituted anthra-
noyl–anthranilic acid derivatives (Table 1).
* Corresponding author. Tel.: +39-040-676 7861; fax: +39-040-
525 72.
E-mail address: varnavas@univ.trieste.it (A. Varnavas).
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00043-4

294
A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
Fig. 1. Disconnection bond strategy. Path A: cut C followed by cut A both on amidinic group. Path B: cut C (amidinic group) followed by cut
B (CꢀN amide). The preliminary bond disconnection of the fused leucine residue and FGI (dehydration) on the indolenine moiety is common to
path A and B.
In consideration of the synthons obtained from the
disconnection scheme, the substituents chosen are es-
sentially amino acids of the C-terminus of CCK and
already shown to be crucial for the interaction at the
receptors.
1, 2 and 8 gave the corresponding amides 21–23 (route
e).
3. Biochemistry
Compounds 1–25 (Table 1) were tested for their
ability to displace [3H]-(9)- -364,718 and [3H]-(+)-
L L-
2. Chemistry
365,260 from their specific binding in rat pancreatic and
guinea pig brain membranes, respectively [19,20].
The IC50 values were calculated from five-point inhi-
bition curves by log–probit plots and the reported
values are the geometric means of at least three sepa-
rate experiments. Compounds exhibiting a percentage
of inhibition (I%) at 30 mM less than 20% were consid-
ered inactive.
The synthesis of the C-substituted antranoyl–an-
thranilic acid derivatives 1–25 was accomplished by
standard procedures as depicted in Scheme 1.
Briefly, the o-aminobenzoyl derivatives (compounds
1a–10a and 24a–25a) were obtained in almost quanti-
tative yield by reacting isatoic anhydride with the C-
protected or with the decarboxylated (Trp and Phe)
DL-amino acids, respectively (route a).
A further one-pot o-aminobenzoylation reaction per-
formed as above on these intermediates (1a–10a and
24a–25a) was excluded because of either steric hin-
drance and poor nucleophilicity toward isatoic
anhydride.
Hence, we accomplished an o-nitrobenzoylation
(route b) followed by reduction (route c) to obtain
compounds 1–10 and 24–25.
4. Results and discussion
The reported C-substituted anthranoyl–anthranilic
acid derivatives showed preferences for the CCK-A
receptor resulting inactive against CCK-B. Further-
more, as previously observed for the N-substituted
anthranoyl–anthranilic acid derivatives, only com-
pounds with a free C-terminal carboxylic group exhib-
ited receptor affinity [18]. In fact the corresponding
esters (compounds 1–10), as well as the amides (com-
The subsequent alkaline hydrolysis of the esters 1–10
afforded almost quantitatively the target compounds
11–20 (route d) and similarly the ammonolysis of esters

A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
295
Table 1
Moreover and in support of our initial choice, inside
this series, derivatives (11, 12, 19) having amino acids
of the C-terminal tetrapeptide of CCK (i.e. Trp-Met-
Asp-Phe-NH2) exhibited the highest degree of affinity
with the only exception of compound 20.
Structure of the target compounds
Another structural feature that strongly affects the
receptor affinity concerns the presence of a carboxylic
group since either derivatization (esters and amides) or
decarboxylation (compounds 24 and 25) led to inactive
compounds. These facts could be ascribed to the lack of
specific ionic interactions at the receptor (esters, amides
and decarboxylated derivatives) and/or to unfavourable
steric interactions between the pharmacophoric group
and receptor site arising from a modified spatial ar-
rangement as a result of decarboxylation.
Comp.
R
R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀH
ꢀCOOC2H5
ꢀCOOC2H5
ꢀCOOC2H5
ꢀCOOC2H5
ꢀCOOC2H5
ꢀCOOC2H5
ꢀCOOCH3
ꢀCOOCH3
ꢀCOOC2H5
ꢀCOOC2H5
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀCOOH
ꢀC6H5
ꢀCH(CH3)2
ꢀCH2ꢀCH(CH3)2
ꢀ(CH2)2ꢀCH3
ꢀ(CH2)3ꢀCH3
ꢀ(CH2)2ꢀSꢀCH3
ꢀCH2ꢀCOOC2H5
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀH
5. Conclusions
In this paper we have reported the synthesis and
biological evaluation of compounds representing the
second step of anthranoyl–anthranilic acid derivatiza-
tion according to the disconnection approach of asper-
licin we proposed.
It appears that the N- as well as the C-substitution of
anthranilic acid dimer, employed as template for step-
wise optimization, with different amino acids, led to
compounds that prefer CCK-A receptor with affinity of
the same order of magnitude of asperlicin (IC50=1.4
mM [21]), the starting point of our investigation.
Further investigation demonstrating a higher affinity
for the same receptor of compounds obtained by the
simultaneous substitution of this template will be dis-
cussed in forthcoming papers and additional studies are
in progress to explore the SAR of the central nucleus.
ꢀC6H5
ꢀCH(CH3)2
ꢀCH2ꢀCH(CH3)2
ꢀ(CH2)2ꢀCH3
ꢀ(CH2)3ꢀCH3
ꢀ(CH2)2ꢀSꢀCH3
ꢀCH2ꢀCOOH
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀ(CH2)3ꢀCH3
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀCOOH
ꢀCONH2
ꢀCONH2
ꢀCONH2
ꢀH
ꢀH
pounds 21–23) resulted inactive. The CCK-A receptor
binding data of compounds 11–20 are summarized in
Table 11.
Compound 11, a tryptophan derivative, showed the
greater affinity inside this series. It is interesting to note
that a similar binding behaviour was observed with the
Trp residue connected at the N-terminus of the an-
thranilic acid dimer [18]. This fact well agrees with our
disconnection strategy applied to asperlicin nucleus.
The observed binding data suggest a strong depen-
dence of the affinity on the side chain of the amino
acids employed. In particular nor-Val, nor-Leu, Met
and Phe (compounds 17–19 and 12) exhibited similar
affinity and are characterized by a linear aliphatic side
chain or by the presence of a benzyl group. On the
other hand, the presence of branched aliphatic chains
(compounds 15, 16), the shortening of the Phe residue
(compound 14) and particularly the absence of any
residue (compound 13) dramatically reduce the affinity.
As a result of this analysis it can be pointed out that
the amino acid side chain represents a pharmacophoric
group involved in receptor recognition.
6. Experimental
6.1. Chemistry
Melting points were determined on a Bu¨chi 510
melting point apparatus (Bu¨chi, Flawil, Switzerland)
and are uncorrected. Ascending thin-layer chromatog-
raphy (TLC) was performed on precoated silica gel
plates (60F-254 Merck) by using UV light to visualize
the chromatograms. Proton (1H NMR, 200 MHz) and
carbon (13C NMR, 50 MHz) nuclear magnetic reso-
nance spectra were recorded on a Varian-Gemini 200
Fourier Transform spectrometer. Chemical shifts are
reported as l (ppm) relative to tetramethylsilane (TMS)
as internal standard and the signals are described as
s=singlet, d=doublet, dd=double doublet, t=
triplet, q=quartet, b=broad and m=multiplet. Spec-
tral data are consistent with assigned structures.

296
A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
Scheme 1. (a) AcOEt, Et3N; (b) CH2Cl2, Et3N; (c) Zn, CH3COOH, CH2Cl2; (d) KOH, MeOH; (e) NH3(gas), MeOH.
Physicochemical properties and spectroscopic (1H
6.1.1. General procedure for the preparation of the
anthranilamides 1a–10a and 24a–25a
and 13C NMR) data of compounds 1a–10a are pre-
sented in Tables 2–4, respectively.
A suspension of 20 mmol of the corresponding two-
substituted ethyl amine (phenylethylamine and
tryptamine) or of the appropriate hydrochloride salt of
the C-protected DL-amino acid 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 down to room temperature (r.t.) and filtered.
The organic phase was thoroughly washed with a 1 M
NaOH aqueous solution (2×50 ml), water (2×50 ml),
dried over anhydrous sodium sulphate and concen-
trated in vacuo. Trituration with petroleum ether (40–
70°C) afforded the analytically pure title compounds.
6.1.1.1. N-anthranoyltryptamine (24a). Compound 24a
was prepared according to the general procedure de-
scribed earlier in 83% yield; m.p. 159–161°C; Rf 0.43
1
(1:1 AcOEt–hexane); H NMR (DHSO): l 2.96, t-2H
(ꢀCH2ꢀ); 3.53, q-2H (ꢀCH2ꢀNHꢀ); 6.45, s-2H (ꢀNH2);
6.48–8.40, m-10H (arom., ꢀNHꢀ), 10.84, s-1H (ꢀNHꢀ,
indole). 13C NMR (DHSO): l 25, 111, 112, 114, 115,
116, 118, 118.1, 121, 122, 127, 131, 136, 149, 169.
6.1.1.2. N-anthranoyl-2-phenylethylamine (25a). The
compound was obtained in 82% yield as an oil. Rf 0.65

A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
297
Table 2
Physicochemical properties of compounds 1a–10a
a
Comp.
R
R2
Molecular formula
MW
Yield (%)
Rf
M.p. (°C)
1a
2a
3a
4a
5a
6a
7a
8a
9a
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀH
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀCH3
ꢀCH3
ꢀC2H5
ꢀC2H5
C20H21N3O3
C18H20N2O3
C11H14N2O3
C17H18N2O3
C14H20N2O3
C15H22N2O3
C13H18N2O3
C14H20N2O3
C14H20N2O3S
C15H20N2O5
351
312
222
298
264
278
250
264
296
308
81
79
66
42
65
75
65
25
70
59
0.49
0.69
0.52
0.72
0.70
0.73
0.61
0.64
0.63
0.56
98–100
83–84
70–72
81–83
48
49–50
87
55
ꢀC6H5
ꢀCH(CH3)2
ꢀCH2ꢀCH(CH3)2
ꢀ(CH2)2ꢀCH3
ꢀ(CH2)3ꢀCH3
ꢀ(CH2)2ꢀSꢀCH3
ꢀCH2ꢀCOOC2H5
53–55
89–90
10a
a 1:1 AcOEt–hexane.
1
(1:1 AcOEt–hexane); H NMR (CDCl3): l 2.91, t-2H
(ꢀCH2ꢀ); 3.65, q-2H (ꢀCH2ꢀNHꢀ); 5.4, s-2H (ꢀNH2);
6.19, m-1H (ꢀNHꢀ); 6.55–7.37, m-9H (arom.). 13C
NMR (CDCl3): l 36, 41, 116.6, 117, 126.6, 127, 128.7,
129, 132, 139, 149, 169.
Table 3
1H NMR (CDCl3) of compounds 1a–10a
Comp. l (ppm)
1a
2a
1.24, t-3H (ꢀCH3); 3.46, d-2H (ꢀCH2ꢀ); 4.16, q-2H
(ꢀCH2ꢀOꢀ); 5.06, m-1H (ꢁCHꢀ); 5.49, s-2H (ꢀNH2);
6.52–7.60, m-10H (arom. ꢀNHꢀ); 8.11, s-1H (ꢀNHꢀ,
indole).
1.24, t-3H (ꢀCH3); 3.21, m-2H (ꢀCH2ꢀCHꢂ); 4.18,
q-2H (ꢀCH2ꢀOꢀ); 4.97, q-1H (ꢁCHꢀ); 5.45, s-2H
(ꢀNH2); 6.52, d-1H (ꢀNHꢀ); 6.61–7.28, m-9H (arom.).
1.26, t-3H (ꢀCH3); 4.18, m-4H (ꢀCH2ꢀOꢀ, ꢀCH2ꢀ);
5.41, s-2H (ꢀNH2); 6.57–7.40, m-5H (arom. ꢀNHꢀ).
1.23, t-3H (ꢀCH3); 4.20, q-2H (ꢀCH2ꢀ); 5.03, s-2H
(ꢀNH2); 5.70, d-1H (ꢀCHꢂ); 6.64, m-2H (ꢀNHꢀ, H
arom.); 7.10–7.43, m-8H (arom.).
0.99, d-6H (ꢀCHꢀ(CH3)2); 1.30, t-3H (ꢀCH3); 2.25,
m-1H (ꢀCHꢀ(CH3)2); 4.22, q-2H (ꢀOꢀCH2ꢀ); 4.70,
m-1H (ꢀNHꢀCHꢂ); 5.12, s-2H (ꢀNH2); 6.57, d-1H
(ꢀNHꢀCHꢂ); 6.67–7.44, m-4H (arom.).
0.97, d-6H (ꢁCHꢀCH3); 1.27, t-3H (ꢀCH2ꢀCH3); 1.67,
m-3H (ꢁCHꢀCH2ꢀ); 4.19, q-2H (ꢀOꢀCH2ꢀ); 4.76,
m-3H (ꢀCHꢀNHꢀ, ꢀNH2); 6.52, d-1H (ꢀNHꢀ);
6.59–7.40. m-4H (arom.).
0.94, t-3H (ꢀCH3); 1.42, m-2H (ꢀCH2ꢀCH3); 1.87,
m-2H (ꢁCHꢀCH2ꢀ); 3.76, s-3H (ꢀOꢀCH3); 4.75, m-1H
(ꢀCHꢂ); 5.29, s-2H (ꢀNH2); 6.57, d-1H (ꢀNHꢀCHꢂ);
6.68–7.41, m-4H (arom.).
0.88, t-3H (ꢀCH3); 1.33, m-4H (ꢀ(CH2)2ꢀCH3); 1.85,
m-2H (ꢁCHꢀCH2ꢀ); 3.76, s-3H (ꢀOꢀCH3); 4.74, m-1H
(ꢀCHꢂ); 5.40, s-2H (ꢀNH2); 6.56, d-1H (ꢀNHꢀCHꢂ);
6.68–7.41, m-4H (arom.).
1.30, t-3H (ꢀCH2ꢀCH3); 2.10, s-3H (ꢀSꢀCH3); 2.07,
m-2H (ꢁCHꢀCH2); 2.57, t-2H (ꢀCH2ꢀSꢀ); 4.23, q-2H
(ꢀCH2ꢀCH3), 4.83, m-3H (ꢀCHꢂ, ꢀNH2); 6.61–7.42,
m-5H (arom. ꢀNHꢀ).
1.24, t-3H (ꢀCH3); 1.28, t-3H (ꢀCH3); 3.00, m-2H
(ꢀCH2ꢀ); 4.13, q-2H (ꢀOꢀCH2ꢀ); 4.24, q-2H
(ꢀOꢀCH2ꢀ); 4.76, s-2H (ꢀNH2); 4.97, m-1H (ꢀCHꢂ);
6.62, d-1H (ꢁCHꢀNHꢀ); 6.68–7.42, m-4H (arom.).
6.1.2. General procedure for the preparation of the
N-(N-anthranoyl) anthranilamides 1–10
A solution of 20 mmol of the corresponding an-
thranilamides (1a–10a) 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
washed in succession with a 1 M NaOH (2×30 ml)
and water (2×30 ml). The dried (sodium sulphate)
organic phase was concentrated to give the crude nitro
derivative, 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 sulphate, and evaporated. Crystallization from
the appropriate solvent afforded the analytically pure
3a
4a
5a
6a
7a
8a
1
derivatives 1–10 (Table 5). Spectroscopic H and 13C
9a
NMR data are presented in Tables
6 and 7,
respectively.
N-(N-anthranoyl)anthranoyltryptamine (24) and N-
(N-anthranoyl)anthranoyl-2-phenylethylamine (25)
were prepared according to the method described ear-
lier. Crystallization from methanol afforded the pure
title compounds in 54 and 48% yield, respectively.
10a

298
A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
Table 4
6.1.2.2. N-(N-anthranoyl)anthranoyl-2-phenylethylamine
13C NMR (CDCl3) of compounds 1a–10a
(25). Rf 0.69 (1:1 AcOEt–hexane); m.p. 158°C; 1H
NMR (CDCl3): l 2.93, t-2H (ꢀCH2ꢀC6H5); 3.70, q-2H
(ꢀNHꢀCH2ꢀ); 5.74, s-2H (ꢀNH2); 6.37, t-1H
(ꢀNHꢀCH2); 6.67–8.62, m-13H (arom.); 11.74, s-1H
(ꢀNHꢀ). 13C NMR (CDCl3): l 35.55, 41.07, 115.73,
117.08, 117.45, 121.13, 121.75, 122.70, 126.46, 126.76,
127.88, 128.80, 132.36, 132.80, 138.56, 139.71, 149.74,
168.07, 169.14.
Comp. l (ppm)
1a
2a
14.14, 27.71, 53.26, 61.61, 110.08, 111.32, 115.49,
116.67, 117.27, 118.69, 119.51, 119.66, 122.17, 122.24,
122.92, 127.60, 132.53, 148.79, 168.96.
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.
3a
4a
14, 42, 62, 115, 116, 117, 128, 133, 149, 169, 170.
14, 57, 62, 116.6, 117.3, 127.2, 127.6, 128.5, 129, 133,
137, 149, 168, 171.
14.28, 17.99, 19.05, 31.61, 57.11, 61.37, 115.83, 116.72,
117.30, 127.42, 132.52, 148.73, 169.02, 172.22.
14.20, 22.15, 22.88, 25.03, 41.85, 50.93, 61.42, 115.86,
116.65, 117.29, 127.44, 132.52, 148.77, 168.93, 173.34.
13.75, 18.73, 34.72, 52.14, 52.41, 115.45, 116.67, 117.34,
127.45, 132.58, 148.81, 168.92, 173.40.
13.89, 22.38, 27.49, 32.35, 52.28, 52.39, 115.43, 116.62,
117.31, 127.44, 132.56, 148.86, 168.86, 173.36.
14, 16, 30, 32, 52, 62, 115, 116.7, 117, 133, 149, 169,
172.
14.14 (2C), 36.49, 48.74, 61.10, 61.96, 115.19, 116.74,
117.29, 127.65, 132.70, 148.88, 170.96, 171.17 (2C).
6.1.3. General procedure for the preparation of com-
pounds 11–20
5a
6a
A mixture of 5 mmol of the corresponding ester
(compounds 1–10) 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 taken up with water.
After cooling, the solution was adjusted to pH 2–3
with diluted HCl. The resulting milky white suspension
was extracted with AcOEt (2×50 ml), and the com-
bined organic extracts were dried over sodium sulphate
and rotoevaporated to give the corresponding title com-
pound. Crystallization from the appropriate solvent
afforded the analytically pure derivatives 11–20 (Table
8).
7a
8a
9a
10a
6.1.2.1. N-(N-anthranoyl)anthranoyltryptamine (24). Rf
0.53 (1:1 AcOEt–hexane); m.p. 169–170°C; H NMR
1
1
Spectroscopic H and 13C NMR data are presented
(CDCl3): l 3.07, t-2H (ꢀCH2ꢀ, indole); 3.69, q-2H
(ꢀNHꢀCH2); 6.24, s-2H (ꢀNH2); 6.65–8.66, m-13H
(arom.); 8.42, t-1H (ꢀNHꢀCH2ꢀ); 10.28, s-1H (ꢀNHꢀ,
indole); 12.22, s-1H (ꢀNHꢀ). 13C NMR (CDCl3): l
24.78, 40.26, 11.19, 111.87, 114.74, 115.78, 117.05,
118.14, 118.28, 120.44, 120.72, 120.96, 121.89, 122.13,
127.14, 127.31, 127.65, 131.49, 132.25, 136.23, 139.53,
149.97, 167.46, 168.84.
in Tables 9 and 10, respectively. The CCK-A receptor
binding data of compounds 11–20 are summarized in
Table 11.
6.1.4. General procedure for the preparation of
compounds 21–23
Amides 21–23 were prepared from the corresponding
esters (compounds 1, 2 and 8, respectively). A suspen-
Table 5
Physicochemical properties of compounds 1–10
b
Comp.
R
R2
Molecular formula
MW
Yield (%)
Crystal solvent a
Rf
M.p. (°C)
1
2
3
4
5
6
7
8
9
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀH
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀC2H5
ꢀCH3
ꢀCH3
ꢀC2H5
ꢀC2H5
C27H26N4O4
C25H25N3O4
C18H19N3O4
C24H23N3O4
C21H25N3O4
C22H27N3O4
C20H23N3O4
C21H25N3O4
C21H25N3O4S
C22H25N3O6
470
431
341
417
383
397
369
383
415
427
69
71
36
46
32
48
18
56
28
42
A
A
B
B
B
C
B
B
B
C
0.45
0.73
0.51
0.71
0.63
0.66
0.64
0.70
0.58
0.56
150
137
128–129
148–150
118–120
95–96
118–119
113–114
115–116
89–90
ꢀC6H5
ꢀCH(CH3)2
ꢀCH2ꢀCH(CH3)2
ꢀ(CH2)2ꢀCH3
ꢀ(CH2)3ꢀCH3
ꢀ(CH2)2ꢀSꢀCH3
ꢀCH2ꢀCOOC2H5
10
a Crystallizing solvents: A=EtOH 90%; B=MeOH; C=EtOH 50%.
b 1:1 AcOEt–hexane.

A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
299
Table 7
Table 6
13C NMR (CDCl3) of compounds 1–10
1H NMR (CDCl3) of compounds 1–10
Comp. l (ppm)
Comp. l (ppm)
1
2
14.19, 27.65, 53.53, 61.86, 109.76, 111.42, 115.97,
1
1.25, t-3H (ꢀCH3); 3.43, m-2H (ꢀCH2ꢀ); 4.17, q-2H
117.10, 117.51, 118.53, 119.75, 120.51, 121.67, 122.33,
122.77, 122.92, 126.97, 127.61, 127.86, 132.67, 132.85,
136.14, 139.80, 149.68, 168.00, 168.70, 171.68.
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.
14.19, 41.85, 61.84, 115.70, 116.97, 117.47, 120.02,
121.64, 122.75, 126.97, 127.81, 132.83, 139.98, 149.74,
168.05, 169.19, 169.68.
14.06, 56.84, 62.26, 115.68, 116.91, 117.39, 120.13,
121.73, 122.64, 122.72, 126.95, 127.26, 127.91, 128.72,
128.99, 129.08, 132.74, 132.91, 136.25, 140.05, 149.73,
168.02, 168.42, 170.66.
14.26, 18.00, 18.99, 31.66, 57.38, 61.59, 115.70, 116.93,
117.44, 120.72, 121.75, 122.75, 126.78, 127.81, 132.75,
139.93, 149.77, 167.99, 168.97, 171.75.
14.20, 22.12, 22.89, 25.08, 41.77, 51.23, 61.64, 115.71,
116.90, 117.42, 120.53, 121.71, 122.71, 126.77, 127.86,
132.75, 139.96, 149.78, 168.01, 168.86, 172.81.
13.72, 18.66, 34.65, 52.40, 52.55, 115.71, 116.95, 117.44,
120.43, 121.75, 122.72, 126.81, 127.83, 132.76, 140.00,
149.77, 168.01, 168.79, 172.86.
13.88, 22.36, 27.40, 32.30, 52.54, 115.70, 116.96, 117.44,
120.46, 121.75, 122.74, 126.82, 127.83, 132.78, 139.98,
149.76, 168.01, 168.77, 172.85.
14.22, 15.06, 30.12, 31.45, 52.16, 62.00, 115.69, 116.93,
117.45, 120.13, 121.73, 122.76, 126.88, 127.81, 132.78,
132.92, 140.11, 149.78, 168.01, 168.90, 171.70.
14.14, 36.24, 48.97, 61.26, 62.18, 115.71, 116.91, 117.45,
119.98, 121.66, 122.77, 127.01, 127.78, 132.77, 132.96,
140.18, 149.79, 168.00, 168.70, 170.42, 171.12.
(ꢀOꢀCH2); 5.08, m-1H (ꢀCHꢂ); 5.76, s-2H (ꢀNH2);
6.68–7.69, m-12H (arom.); 8.30, s-1H (ꢀNHꢀ, indole);
8.65, d-1H (arom.); 11.69, sꢀ1H (ꢀNHꢀ).
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-13H (arom.); 11.62, s-1H (ꢀNHꢀ).
1.29, t-3H (ꢀCH3); 4.17, d-2H (ꢀCH2ꢀ); 4.25, q-2H
(ꢀOꢀCH2ꢀ); 5.74, s-2H (ꢀNH2); 6.66–6.77, m-2H
(arom.); 6.95, t-1H (ꢀNHꢀCH2); 7.06–8.66, m-6H
(arom.); 11.71, s-1H (ꢀNHꢀ).
2
3
3
4
4
5
1.24, t-3H (ꢀCH3); 4.24, q-2H (ꢀOꢀCH2ꢀ); 5.60, d-1H
(ꢀCHꢂ); 5.75, s-2H (ꢀNH2); 6.66–8.76, m-14H (arom.
and ꢀNHꢀCHꢂ); 11.60, s-1H (ꢀNHꢀ).
1.00, dd-6H (ꢁCHꢀ(CH3)2); 1.30, t-3H (ꢀCH2ꢀCH3);
2.27, m-1H (ꢁCHꢀ(CH3)2); 4.23, q-2H (ꢀOꢀCH2ꢀ);
4.75, m-1H (ꢁCHꢀNHꢀ); 5.75, s-2H (ꢀNH2);
6.66–6.77, m-2H (arom.); 6.82, d-1H (ꢁCHꢀNHꢀ);
7.08–8.68, m-6H (arom.); 11.63, s-1H (ꢀNHꢀ).
1.00, dd-6H (ꢁCHꢀ(CH3)2); 1.29, t-3H (ꢀCH2ꢀCH3);
1.73, m-3H (ꢀCH2ꢀCH(CH3)2); 4.22, q-2H (ꢀOꢀCH2ꢀ);
4.83, m-1H (ꢁCHꢀNHꢀ); 5.75, s-2H (ꢀNH2);
6.67–8.68, m-9H (arom. and ꢀNHꢀCHꢂ); 11.65, s-1H
(ꢀNHꢀ).
0.95, t-3H (ꢀCH3); 1.40, m-2H (ꢀCH2ꢀCH3); 1.82,
m-2H (ꢁCHꢀCH2ꢀ); 3.77, s-3H (ꢀOꢀCH3); 4.81, m-1H
(ꢁCHꢀNHꢀ); 5.75, s-2H (ꢀNH2); 6.67–6.77, m-2H
(arom.); 6.81, d-1H (ꢀNHꢀCHꢂ); 7.07–8.68, m-6H
(arom.); 11.66, s-1H (ꢀNHꢀ).
0.88, t-3H (ꢀCH3); 1.33, m-4H (ꢀ(CH2)2ꢀCH3); 1.81,
m-2H (ꢁCHꢀCH2ꢀ); 3.78, s-3H (ꢀOꢀCH3); 4.80, m-1H
(ꢁCHꢀNHꢀ); 5.75, s-2H (ꢀNH2); 6.66–6.77, m-2H
(arom.); 6.81, d-1H (ꢀNHꢀCHꢂ); 7.07–8.68, m-6H
(arom.); 11.66, s-1H (ꢀNHꢀ).
1.30, t-3H (ꢀCH3); 2.09, s-3H (ꢀSꢀCH3); 2.26, m-2H
(ꢁCHꢀCH2ꢀ); 2.59, t-2H (ꢀCH2ꢀSꢀ); 4.24, q-2H
(ꢀOꢀCH2); 4.89, m-1H (ꢀCHꢂ); 5.75, s-2H (ꢀNH2);
6.66–8.68, m-9H (arom. and ꢀNHꢀCHꢂ); 11.72, s-1H
(ꢀNHꢀ).
5
6
6
7
7
8
9
8
10
9
(ꢀCHꢂ); 6.50, s-2H (ꢀNH2); 6.66–8.53, m-13H
(arom.); 7.69, s-2H (ꢀCOꢀNH2); 8.82, d-1H
(ꢀNHꢀCHꢂ); 10.79, s-1H (ꢀNHꢀ, indole); 11.95, s-1H
(ꢀNHꢀ). 13C NMR (DMSO-d6): l 27.15, 53.91, 110.34,
111.14, 113.88, 115.02, 116.81, 118.04, 118.29, 120.03,
120.20, 120.71, 122.08, 123.40, 126.99, 127.40, 128.27,
131.91, 132.37, 135.87, 139.19, 150.19, 167.01, 168.38,
173.10.
10
1.25, t-3H (ꢀCH3); 1.28, t-3H (ꢀCH3); 3.00, m-2H
(ꢀCH2ꢀ); 4.15, q-2H (ꢀOꢀCH2); 4.25, q-2H (ꢀOꢀCH2ꢀ);
5.02, m-1H (ꢁCHꢀ); 5.75, s-2H (ꢀNH2); 6.67–7.27,
m-4H (arom.); 7.38, d-1H (ꢀNHꢀCHꢂ); 7.49–8.69,
m-4H (arom.); 11.74, s-1H (ꢀNHꢀ).
6.1.4.2. N-(N-anthranoyl)anthranoylphenylalaninamide
(22). Rf 0.54 (3:1 AcOEt–hexane); m.p. 256°C; 1H
NMR (DMSO-d6): l 3.15, m-2H (ꢀCH2ꢀ); 4.73, m-1H
(ꢀCHꢂ); 6.58, s-2H (ꢀNH2); 6.63–8.52, m-13H
(arom.); 7.68, s-2H (ꢀCOꢀNH2); 8.91, d-1H
(ꢀNHꢀCHꢂ); 11.79, s-1H (ꢀNHꢀ). 13C NMR
(DMSO-d6): l 36.96, 54.40, 113.84, 114.93, 116.80,
120.01, 120.27, 122.09, 126.03, 127.02, 127.80, 128.23,
128.88, 131.83, 132.37, 138.18, 139.04, 150.24, 166.95,
168.35, 172.60.
sion of 5 mmol of the corresponding ester in methanol
(50 ml) was stirred and cooled at 0°C while a
continuous stream of ammonia gas was bubbled into
the reaction flask. After 2 h introduction of the
ammonia was discontinued and the solution was
allowed to warm up to r.t. and left to stand overnight.
The solvent was removed in vacuo and the dry residue
was crystallized from methanol to give the analytically
pure target compounds.
6.1.4.1. N-(N-anthranoyl)anthranoyltryptophanamide
(21). Rf 0.23 (3:1 AcOEt–hexane); m.p. 248–249°C; H
NMR (DMSO-d6): l 3.26, m-2H (ꢀCH2ꢀ); 4.75, m-1H
6.1.4.3. N-(N-anthranoyl)anthranoylnorleucinamide (23).
Rf 0.44 (3:1 AcOEt–hexane); m.p. 210–211°C; 1H
NMR (DMSO-d6): l 0.85, t-3H (ꢀCH3); 1.31, m-4H
1

300
A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
Table 8
Physicochemical properties of compounds 11–20
Comp.
R
Molecular formula
MW
Crystal solvent a
Rf
M.p. (°C)
11
12
13
14
15
16
17
18
19
20
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀH
C25H22N4O4
C23H21N3O4
C16H15N3O4
C22H19N3O4
C19H21N3O4
C20H23N3O4
C19H21N3O4
C20H23N3O4
C19H21N3O4S
C18H17N3O6
442
403
313
389
355
369
355
369
387
371
A
A
A
A
B
0.26 b
0.54 b
0.34 c
0.52 c
0.65 c
0.69 c
0.66 b
0.52 b
0.37 b
0.63 d
217–219
176–177
198–199
244
199
177
211
204
210–211
210
ꢀC6H5
ꢀCH(CH3)2
ꢀCH2ꢀCH(CH3)2
ꢀ(CH2)2ꢀCH3
ꢀ(CH2)3ꢀCH3
ꢀ(CH2)2ꢀSꢀCH3
ꢀCH2ꢀCOOH
B
A
A
A
A
a Crystallizing solvents: A=MeOH; B=EtOH 95%.
b 3:1 AcOEt–MeOH.
c 2:1 AcOEt–MeOH.
d 1:2 AcOEt–MeOH.
Table 9
((ꢀCH2)2ꢀCH3); 1.77, m-2H (ꢁCHꢀCH2ꢀ); 4.39, m-1H
(ꢁCHꢀ); 6.58, s-2H (ꢀNH2); 6.65–8.54, m-8H (arom.);
7.07, s-2H (ꢀCOꢀNH2); 8.73, d-1H (ꢀNHꢀCHꢂ);
11.96, s-1H (ꢀNHꢀ). 13C NMR (DMSO-d6): l 13.67,
21.64, 27.79, 30.97, 53.08, 113.83, 114.95, 116.84,
120.27, 120.69, 122.24, 127.03, 128.50, 131.79, 132.39,
139.01, 150.28, 167.04, 168.49, 173.30.
1H NMR (DMSO-d6) of compounds 11–20
Comp. l (ppm)
11
12
13
3.36, m-2H (ꢀCH2ꢀ); 4.75, m-1H (ꢁCHꢀ); 6.53–8.65,
m-15H (arom. and ꢀNH2); 9.04, d-1H (ꢀNHꢀCHꢂ);
10.83, s-1H (ꢀNHꢀ, indole); 11.95, s-1H (ꢀNHꢀ).
3.21, m-2H (ꢀCH2ꢀ); 4.70, m-1H (ꢁCHꢀ); 6.57–8.56,
m-15H (arom. and ꢀNH2); 9.10, d-1H (ꢀNHꢀCHꢂ);
11.80, s-1H (ꢀNHꢀ).
3.97, d-2H (ꢀCH2ꢀ); 6.59–8.62, m-10H (arom. and
ꢀNH2); 9.22, t-1H (ꢀNHꢀCH2ꢀ); 12.15, s-1H (ꢀNHꢀ);
12.80, b-1H (ꢀOH).
5.62, d-1H (ꢀCHꢂ); 6.58–8.52, m-13H (arom. and
ꢀNH2); 9.40, d-1H (ꢀNHꢀCHꢂ); 11.78, s-1H (ꢀNHꢀ).
1.00, d-6H (ꢀCHꢀ(CH3)2); 2.23, m-1H (ꢁCHꢀ(CH3)2);
4.31, m-1H (ꢀNHꢀCHꢂ); 6.58–8.53, m-10H (arom. and
ꢀNH2); 8.83, d-1H (ꢀNHꢀCHꢂ); 11.76, s-1H (ꢀNHꢀ);
12.80, b-1H (ꢀOH).
6.2. Biochemistry
6.2.1. General
Male rats (Wistar) and male guinea pigs (Hartley)
were obtained from Charles River, Calco, Como
14
15
(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,
16
17
18
19
20
0.91, d-6H (ꢀCHꢀ(CH3)2); 1.75, m-2H (ꢀCH2ꢀ); 4.50,
m-1H (ꢀNHꢀCHꢂ); 6.58–8.57, m-10H (arom. and
ꢀNH2); 8.99, d-1H (ꢀNHꢀCHꢂ); 11.94, s-1H (ꢀNHꢀ);
12.75, b-1H (ꢀOH).
0.90, t-3H (ꢀCH3); 1.41, m-2H (ꢀCH2ꢀCH3); 1.80,
m-2H (ꢁCHꢀCH2ꢀ); 4.40, m-1H (ꢁCHꢀ); 6.59–8.55,
m-10H (arom. and ꢀNH2); 8.97, d-1H (ꢀNHꢀCHꢂ);
11.91, s-1H (ꢀNHꢀ).
0.87, t-3H (ꢀCH3); 1.35, m-4H ((ꢀCH2)ꢀCH3); 1.83,
m-2H (ꢁCHꢀCH2ꢀ); 4.40, m-1H (ꢁCHꢀ); 6.59–8.57,
m-10H (arom. and ꢀNH2); 8.98, d-1H (ꢀNHꢀCHꢂ);
11.93, s-1H (ꢀNHꢀ).
2.06, s-3H (ꢀCH3); 2.11, m-2H (ꢁCHꢀCH2); 2.62,
m-2H (ꢀSꢀCH3); 4.57, m-1H (ꢁCHꢀ); 6.59–8.54,
m-10H (arom. and ꢀNH2); 9.03, d-1H (ꢀNHꢀCHꢂ);
11.93, s-1H (ꢀNHꢀ).
respectively. (R,S)-L-364,718 and (R,S)-L-365,260 were
synthesized in our laboratory as previously described
[22]. Radioactivity was counted with 4 ml of Aquassure
(NEN) high performance LSC cocktail in a Packard
TRI-CARB 300 liquid scintillator.
6.2.2. Radioligand binding assays
All experiments were performed in triplicate.
CCK-A receptor affinities were determined by dis-
placement of [3H]-(9)-
L-364,718 from rat pancreas
membranes as previously described [19]. Briefly, sam-
ples 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)
2.84, m-2H (ꢀCH2); 4.79, m-1H (ꢁCHꢀ); 6.60–8.60,
m-10H (arom. and ꢀNH2); 9.12, d-1H (ꢀNHꢀCHꢂ);
and a solution of various concentrations of the com-
pounds to be tested. The buffer used for binding assay
12.00, s-1H (ꢀNHꢀ); 12.70, b-2H (ꢀOH).
.

A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
301
Table 10
Table 11
CCK receptors binding data
13C NMR (DMSO-d6) of compounds 11–20
Comp.
l (ppm)
11
26.28, 53.64, 110.13, 111.31, 113.77, 115.09, 116.91,
117.96, 118.25, 119.68, 120.06, 120.83, 122.16,
123.44, 126.98, 128.31, 132.15, 132.49, 135.97,
139.35, 150.36, 167.04, 168.69, 172.88.
36.00, 53.89, 113.71, 115.02, 116.85, 119.84, 120.05,
122.17, 126.19, 126.95, 127.94, 128.10, 128.83,
132.03, 132.45, 137.78, 139.13, 150.30, 166.95,
168.51, 172.49.
a
Comp.
R
CCK-A (IC50)
12
11
12
13
14
15
16
17
18
19
20
ꢀCH2ꢀ3-indolyl
ꢀCH2ꢀC6H5
ꢀH
7.0
26
IN c
24 b
IN c
30 b
26
13
14
41.08, 113.77, 115.02, 116.87, 119.43, 120.17, 122.26,
126.97, 127.97, 132.18, 132.45, 139.47, 150.31,
167.05, 168.90, 170.63.
56.71, 113.75, 115.01, 116.85, 120.27, 120.34, 122.27,
127.01, 127.82, 128.04, 128.22, 128.85, 132.06,
132.45, 136.27, 138.98, 150.28, 167.03, 168.42,
171.31.
18.51, 19.06, 29.15, 58.21, 113.62, 114.99, 116.87,
120.26, 120.58, 122.26, 126.93, 128.72, 131.91,
132.45, 138.84, 150.30, 166.86, 168.93, 172.43.
20.92, 22.76, 24.42, 38.95, 50.76, 113.71, 115.00,
116.89, 120.18, 120.23, 122.25, 126.95, 128.38,
132.01, 132.46, 139.10, 150.34, 167.02, 168.77,
173.51.
13.25, 18.88, 32.15, 52.25, 113.77, 115.09, 116.88,
120.28, 120.42, 122.33, 126.96, 128.39, 132.01,
132.49, 138.99, 150.24, 167.03, 168.83, 173.23.
13.58, 21.53, 27.80, 29.87, 52.47, 113.74, 115.03,
116.88, 120.27, 120.50, 122.28, 126.96, 128.41,
131.99, 132.46, 139.05, 150.30, 167.03, 168.81,
173.20.
ꢀC6H5
ꢀCH(CH3)2
ꢀCH2ꢀCH(CH3)2
ꢀ(CH2)2ꢀCH3
ꢀ(CH2)3ꢀCH3
ꢀ(CH2)2ꢀSꢀCH3
ꢀCH2ꢀCOOH
24
22
IN c
15
16
a 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.
b Percentage of inhibition at 30 mM of [3H]-(9)-
L-364,718 binding
in rat pancreatic membranes.
c Inactive: the test compound inhibited [3H]-(9)-
L-364,718 specific
17
18
binding in rat pancreatic membranes by less than 20% at 30 mM.
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 total.
19
20
14.36, 29.75, 29.90, 51.47, 113.72, 115.02, 116.87,
120.07, 120.20, 122.25, 126.97, 128.41, 132.08,
132.46, 139.12, 150.31, 167.02, 168.95, 172.82.
34.69, 48.51, 113.10, 114.44, 116.26, 119.03, 119.54,
121.60, 126.35, 127.53, 131.56, 131.85, 138.69,
149.70, 166.40, 167.78, 170.84, 171.28.
References
[1] J.A. Williams, Biomed. Res. 3 (1982) 107–121.
[2] J.N. Crawley, R.L. Corwin, Peptides 15 (1994) 731–755.
[3] R.B. Innis, S.H. Snyder, Proc. Natl. Acad. Sci. USA 77 (1980)
6917–6921.
[4] C.T. Dourish, D.R. Hill, Trends Pharmacol. Sci. 8 (1987) 207–
208.
[5] M. D’Amato, F. Makovec, L.C. Rovati, Drug News Perspect. 7
(1994) 87–95.
[6] D.R. Hill, L. Singh, P. Boden, R. Pinnock, G.N. Woodruff, J.
Hughes, in: C.T. Dourish, S.J. Cooper, S.D. Iversen, L.L. Iversen
(Eds.), Multiple Cholecystokinin Receptors in the CNS, Oxford
University Press, London, 1992, pp. 57–76.
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
(Whatman) filters and rinsed four times with 4 ml of
cooled Tris buffer (50 mM, pH 7.4). Non-specific bind-
ing was determined in the presence of (R,S)-L-364,718
(0.3 mM final concentration) and was always less than
10% of the total binding.
[7] A. Rex, C.A. Marsden, H. Fink, Neurosci. Lett. 228 (1997)
79–82.
Central CCK-B receptor affinities were determined
by displacement of [3H]-(+)-
L-365,260 from guinea pig
[8] S. Ballaz, A. Barber, A. Fortuno, J. Del Rio, M. Martin-Mar-
tinez, I. Gomez-Monterrey, R. Herranz, R. Gonzalez-Muniz,
M.T. Garcia-Lopez, Br. J. Pharmacol. 121 (1997) 759–767.
[9] L. Singh, R.J. Oles, M.J. Field, P. Atwal, G.N. Woodruff, J.C.
Hunter, Br. J. Pharmacol. 118 (1996) 1317–1325.
[10] M.O. Urban, D.J. Smith, G.F. Gebhart, J. Pharmacol. Exp.
Ther. 278 (1996) 90–96.
[11] F. Makovec, Drugs Future 18 (1993) 919–931.
[12] F. Makovec, W. Peris, S. Frigerio, R. Giovanetti, O. Letari, L.
Mennuni, L. Revel, J. Med. Chem. 39 (1996) 135–142.
[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, Proc.
Natl. Acad. Sci. USA 83 (1986) 4918–4922.
cerebral cortex membranes as previously described by
Chang et al. [20]. 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 compound to be tested. Non-specific
.

302
A. Varna6as et al. / Il Farmaco 55 (2000) 293–302
[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, J. Med.
Chem. 32 (1989) 13–16.
[15] M.J. Yu, K.J. Trasher, J.R. McCowan, N.R. Mason, L.G.
Mendelson, J. Med. Chem. 34 (1991) 1505–1508.
[16] 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, J. Med. Chem. 36 (1993) 552–565.
[17] M.J. Drysdale, M.C. Pritchard, D.C. Horwell, J. Med. Chem.
35 (1992) 2573–2581.
[19] R.S.L. Chang, V.J. Lotti, T.B. Chen, K.A. Kunkel, Mol. Phar-
macol. 30 (1986) 212–217.
[20] R.S.L. Chang, T.B. Chen, M.G. Bock, R.M. Freidinger, R.
Chen, A. Rosegay, V.J. Lotti, Mol. Pharmacol. 35 (1989) 803–
808.
[21] M.G. Bock, R.M. Di Pardo, K.E. Rittle, B.E. Evans,
R.M. Freidinger, D.F. Veber, R.S.L. Chang, T.B. Chen,
M.E. Keegan, V.J. Lotti, J. Med. Chem. 29 (1986) 1941–
1945.
[22] M.G. Bock, R.M. Di Pardo, B.E. Evans, K.E. Rittle, D.F.
Veber, R.M. Freidinger, J. Hirshfield, J.P. Springer, J. Org.
Chem. 52 (1987) 3232–3239.
[18] A. Varnavas, L. Lassiani, E. Luxich, M. Zacchigna, Pharmazie
51 (1996) 697–700.
.