Synthesis of new anthranilic acid dimer derivatives and their evaluation on CCK receptors

Article abstract of DOI:10.1016/S0014-827X(00)00053-7

We have described previously an innovative bond disconnection strategy of asperlicin, a naturally occurring CCK receptor antagonist, leading to anthranilic acid dimer and tryptophan synthons. We have also demonstrated that when the tryptophan residue is connected to the C- or N-terminal sides of the anthranilic acid dimer, compounds with similar micromolar CCK-A receptor affinities are obtained. In order to investigate the binding effects of different N-terminal substitution, in this paper we describe a new series of anthranilic acid dimer derivatives, characterized by the presence of the tryptophan residue in the C-terminus of the dimer. Among the compounds synthesized, the N-1H-indol-3-propionyl derivative exhibited an improved, at the micromolar range, affinity for the CCK-A receptor in comparison to that of either, the N-unsubstituted derivative and asperlicin. The lead compound emerging from this key step of our investigation represents the new starting point for the development of a new class of CCK-A receptor ligands. (C) 2000 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(00)00053-7

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 369–375
Synthesis of new anthranilic acid dimer derivatives and their
evaluation on CCK receptors
Antonio Varnavas *, Lucia Lassiani, Valentina Valenta
Department of Pharmaceutical Sciences, Uni6ersity of Trieste, P.le Europa 1, 34127 Trieste, Italy
Received 20 February 2000; accepted 7 April 2000
Abstract
We have described previously an innovative bond disconnection strategy of asperlicin, a naturally occurring CCK receptor
antagonist, leading to anthranilic acid dimer and tryptophan synthons. We have also demonstrated that when the tryptophan
residue is connected to the C- or N-terminal sides of the anthranilic acid dimer, compounds with similar micromolar CCK-A
receptor affinities are obtained. In order to investigate the binding effects of different N-terminal substitution, in this paper we
describe a new series of anthranilic acid dimer derivatives, characterized by the presence of the tryptophan residue in the
C-terminus of the dimer. Among the compounds synthesized, the N-1H-indol-3-propionyl derivative exhibited an improved, at the
micromolar range, affinity for the CCK-A receptor in comparison to that of either, the N-unsubstituted derivative and asperlicin.
The lead compound emerging from this key step of our investigation represents the new starting point for the development of a
new class of CCK-A receptor ligands. © 2000 Elsevier Science S.A. All rights reserved.
Keywords: CCK receptor ligands; Anthranilic acid dimer
(gastrointestinal tract) and CCK-B receptors are located
mainly in the CNS [12].
1. Introduction
In view of the importance of CCK in different physio-
pathological processes as irritable bowel syndrome
(IBS), chronic and acute pancreatitis, gastric ulcer, anxi-
ety, panic disorder, eating disorders etc., over the past 10
years, great efforts have been made to develop clinically
useful receptor CCK-ligands [13].
At least two chemical classes (1,4-benzodiazepines,
quinazolinones) of potent non-peptide and selective an-
tagonists for the CCK-A and CCK-B receptors have
been discovered starting from the natural product asper-
licin (Fig. 1) after a sequence of chemical semplifications
and/or modifications [14–17].
Recently, we have proposed anthranilic acid dimer as
a useful template for the development of a new class of
CCK-receptor ligands. The above flexible non-peptide
template derives from asperlicin as a result of an innova-
tive bond disconnection strategy which preserves the
tryptophan residue and the aromatic domains but the
other heteroaromatic bicyclic substructures [18]. More-
over, as in the case of the peptoid derivatives, anthranilic
acid dimer offers the possibility to be derivatized either
at the N-terminal and/or C-terminal site [19,20].
Cholecystokinin (CCK) is a polypeptide hormone
found in the gastrointestinal tract as well as in the brain
[1]. CCK is distributed in various biologically active
molecular forms throughout the peripheral tissues and
central nervous system (CNS), being the C-terminal oc-
tapeptide (CCK-8) the minimal sequence required for
bioactivity [2,3].
The major hormonal effects of CCK are expressed in
the gastrointestinal tract where CCK stimulates the gall
bladder contraction, the pancreatic exocrine secretion
and increases the intestinal motility [4–6].
At the CNS, CCK acts as a neurotransmitter/neuro-
modulator and appears to be involved in anxiety, panic,
depression, nociception and satiety [7–11].
The biological effects of CCK are mediated by at least
two distinct G-protein-coupled receptors. Both receptors
are present in the periphery and in the CNS, although
CCK-A receptors occur predominantly at the periphery
* 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)00053-7

370
A. Varna6as et al. / Il Farmaco 55 (2000) 369–375
Fig. 1. Structures of lead compounds derived from different discon-
nection approaches of the natural CCK receptors antagonist asper-
licin.
Table 1
Structure of the target compounds
Scheme 1. (a) AcOEt, Et3N; (b) 2-nitrobenzoyl chloride, CH2Cl2,
Et3N; (c) Zn, CH3COOH, CH2Cl2; (d) RCOOH, isobutyl chlorofor-
mate, CH2Cl2, Et3N; (e) RCOCl, CH2Cl2, Et3N; (f) KOH, MeOH.
dimer, via amide bond formation, either directly or by
a short alkyl chain (compounds 1–8). The choice of
these pharmacophoric groups is strongly conditioned
by the hypothesis that the presence of a second
lipophilic pocket on the receptor could involve these
moieties in hydrophobic, charge transfer or, in the case
of the indole, hydrogen-bonding interactions. More-
over, the insertion of a free carboxylic group (com-
pounds 9, 10) was directed to investigate the binding in
the presence of an additional ionic function at the
opposite part of the molecule (N-terminus).
Comp.
R
1
2
3
4
5
6
7
8
9
1H-indol-3-yl-(CH2)2ꢀ
1H-indol-3-yl-CH2ꢀ
1H-indol-2-yl-
C6H5ꢀ
C6H4ꢀp-Cl
C6H5ꢀCH2ꢀ
C6H5ꢀ(CH2)2ꢀ
C6H5ꢀCHꢁCHꢀ
HOOCꢀCH2ꢀ
HOOCꢀ(CH2)2ꢀ
The new compounds, reported in Table 1, were eval-
uated as CCK receptor ligands by determining their
binding affinity for CCK-A and CCK-B receptors.
10
We have also demonstrated that the introduction of
the tryptophan residue, which represents one of the
synthons of the disconnection scheme, either at the N-
or C-terminal site of anthranilic acid dimer, led to
compounds with similar micromolar CCK-A receptor
affinities (IC50:7 mM) [21].
Taking into account the behavior of these anthra-
noyl-anthranilic acid derivatives and trying to optimize
their CCK-A affinity, we have prepared a series of
N-substituted anthranilic acid dimer derivatives holding
the tryptophan residue at the C-terminal site of dimer.
At the present optimization step, the substituents se-
lected to be appended at the N-terminus of the biologi-
cally inert molecular scaffold are the indol-3-yl or
indol-2-yl moieties as well as a phenyl ring linked to the
2. Chemistry
Scheme 1 summarizes the synthetic pathway used to
obtain the compounds listed in Table 1. The reaction of
the tryptophan ethyl ester with isatoic anhydride af-
forded the intermediate 11 in 80% yield. Acylation of
anthranoyl derivative 11 with o-nitrobenzoyl chloride
and subsequent reduction of the nitro group provided
the anthranilic acid dimer derivative 12. Carboxylic
acids were activated by the mixed anhydrides method
or via the acyl chloride formation and subsequently
coupled with the key intermediate 12 to afford com-
pounds 1a–3a, whereas compounds 4a–10a were ob-
tained via commercially available acyl chlorides.

A. Varna6as et al. / Il Farmaco 55 (2000) 369–375
371
Finally, the alkaline hydrolysis of esters 1a–10a af-
forded almost quantitatively the target compounds 1–
10, of which the synthetic data are listed in Tables 2–4.
lar or more potent affinity for CCK-A receptors com-
pared with that of the naturally occurring CCK
antagonist asperlicin (IC50=1.4×10−6 M) [24].
Moreover, the most potent one (compound 1)
showed some sevenfold greater affinity than that of the
N-unsubstituted anthranilic acid dimer derivative com-
pound 0. Compound 2, in which the alkyl chain (ethyl
group) of the substituent of the parent compound 1 was
shortened to a methyl group, exhibited threefold less
potent activity than 1. The introduction of the indol-2-
yl moiety at the N-terminus of compound 0 provided
compound 3 which was comparable to compound 1
with regard to their affinity. Phenyl substituted com-
pounds (compounds 4–8) reversed this effect and dis-
played a decreased potency and selectivity for CCK-A
receptor compared with those bearing the indole ring.
Furthermore, no significant differences in CCK-A re-
ceptor affinity were observed between the parent com-
pound 4 and compounds 6 and 7 where the alkyl chain
of the substituent was lengthened. The introduction of
an electron-withdrawing group, such as chloro, at the
4-position of the phenyl ring of the substituent (com-
pound 5) as well as the unsaturation of the alkyl linker
(compound 8) had little effect on receptor affinity. On
the other hand, the introduction of an acidic side chain
(compounds 9 and 10) is detrimental for biological
activity.
3. Biochemistry
Compounds 1–10 (Table 1) were tested for their
ability to displace [3H]-(9)- -364,718 and [3H]-(+)-
L L-
365,260 from their specific binding in rat pancreatic and
guinea pig brain membranes, respectively [22,23].
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 at 10 mM, a
percentage of inhibition (I%) lower than 20% were
considered inactive.
4. Results and discussion
The affinities of the synthesized compounds for the
CCK-A and CCK-B receptors were evaluated by recep-
tor binding assays and are shown in Table 5. In general,
none of the investigated compounds showed apprecia-
ble percentage of inhibition towards CCK-B receptors
at a concentration of 10 mM. By contrast, most of the
tested compounds exhibited binding affinities for the
CCK-A receptors in the micromolar range. Among
these, compounds 1–3 having an indole ring at the
N-terminus of the anthranilic acid dimer showed simi-
These data suggest that for the indole ring containing
substituents an additional binding interaction appears
to be present. The increased affinity observed for these
compounds suggest also that appropriate steric interac-
Table 2
Physicochemical properties of compounds 1–10
Comp.
R
Molecular formula
MW
Crystal solvent a
Rf
M.p. (°C)
1
2
3
4
5
6
7
8
9
3-Indolyl-(CH2)2ꢀ
3-Indolyl-CH2ꢀ
2-Indolyl
Phenyl
4-Chlorophenyl
Benzyl
Phenylethyl
Phenylethenyl
HOOCꢀCH2ꢀ
HOOCꢀ(CH2)2ꢀ
C36H31N5O5
C35H29N5O5
C34H27N5O5
C32H26N4O5
C32H25ClN4O5
C33H28N4O5
C34H30N4O5
C34H28N4O5
C28H24N4O7
C29H26N4O7
611
599
585
546
580.5
560
574
572
528
542
A
A
A
B
C
A
C
C
A
A
0.40 b
0.35 b
0.24 b
0.30 b
0.23 b
0.24 b
0.32 b
0.34 b
0.27 c
0.24 d
190–191
194–196
228–230
175–177
232–234
114–117
169–170
165–168
152–155
193–195
10
a Crystallization solvents: A, MeOH; B, EtOH; C, MeOH–Et2O.
b AcOEt–MeOH: 3/1.
c AcOEt–MeOH: 1/1.
d AcOEt–MeOH: 3/2.

372
A. Varna6as et al. / Il Farmaco 55 (2000) 369–375
Table 3
compounds (compounds 1 and 3) established from this
1H NMR (DMSO-d6) of compounds 1–10 a
key step of our investigation represents the new starting
point for the development of a new class of CCK-A
receptor ligands. Further investigations are needed to
optimize the binding interactions of the N-terminus
substituents of this lead compound in order to confer a
strong affinity for CCK-A receptors. Moreover, it is
reasonable to expect that further improvements in ac-
tivity can be achieved by optimizing the distance and
the spatial arrangements of the N- and C-terminal
pharmacophores of the anthranilic acid dimer. For this
reason additional research to explore this and other
possibilities is in progress.
Comp. l (ppm)
1
2
3
2.73 (t, 2H, ꢀCH2ꢀCOꢀ); 3.00 (t, 2H, ꢀCH2ꢀCH2ꢀCOꢀ);
3.27 (m, 2H, ꢂCHꢀCH2ꢀ); 4.68 (m, 1H, ꢂCHꢀ);
6.85–8.52 (m, 18H, ar); 9.08 (d, 1H, ꢀNHꢀCHꢃ); 10.72
(s, 1H, ꢀNHꢀ ind); 10.77 (s, 1H, ꢀNHꢀ ind); 10.83 (s,
1H, ꢀNHꢀ); 11.96 (s, 1H, ꢀNHꢀ).
3.10 (m, 2H, ꢀCH2ꢀCHꢃ); 3.74 (s, 2H, ꢀCH2ꢀCOꢀ);
4.65 (m, 1H, ꢀCHꢃ); 6.85–8.50 (m, 18H, ar); 9.15 (d,
1H, ꢀNHꢀCHꢃ); 10.65 (s, 1H, ꢀNHꢀ ind); 10.70 (s,
1H, ꢀNHꢀ ind); 11.50 (s, 1H, ꢀNHꢀ); 12.10 (s, 1H,
ꢀNHꢀ).
3.17 (m, 2H, ꢀCH2ꢀCHꢃ); 4.65 (m, 1H, ꢂCHꢀ);
6.93–8.57 (m, 18H, ar); 8.93 (d, 1H, ꢀNHꢀCHꢃ); 10.80
(s, 1H, ꢀNHꢀ ind); 11.92 (s, 1H, ꢀNHꢀ ind); 11.97 (s,
1H, ꢀNHꢀ); 12.30 (s, 1H, ꢀNHꢀ).
6. Experimental
4
5
6
3.08 (m, 2H, ꢀCH2ꢀ); 4.58 (m, 1H, ꢂCHꢀ); 6.90–8.55
(m, 18H, ar); 8.85 (d, 1H, ꢀNHꢀCHꢃ); 10.75 (s, 1H,
ꢀNHꢀ ind); 11.85 (s, 1H, ꢀNHꢀ); 12.15 (s, 1H, ꢀNHꢀ).
3.24 (m, 2H, ꢀCH2ꢀ); 4.70 (m, 1H, ꢂCHꢀ); 6.95–8.60
(m, 17H, ar); 9.08 (d, 1H, ꢀNHꢀCHꢃ); 10.82 (s, 1H,
ꢀNHꢀ ind); 11.75 (s, 1H, ꢀNHꢀ); 12.18 (s, 1H, ꢀNHꢀ).
3.25 (m, 2H, ꢀCH2ꢀCHꢃ); 3.68 (s, 2H, ꢀCH2ꢀCOꢀ);
4.52 (m, 1H, ꢂCHꢀ); 6.84–8.47 (m, 18H, ar); 8.65 (d,
1H, ꢀNHꢀCHꢃ); 10.71 (s, 1H, ꢀNHꢀ ind); 10.83 (s,
1H, ꢀNHꢀ); 12.15 (s, 1H, ꢀNHꢀ).
6.1. Chemistry
Melting points were determined on a Bu¨chi 510
melting point apparatus and are uncorrected. Proton
(1H NMR, 200 MHz) and carbon (13C NMR, 50 MHz)
nuclear magnetic resonance spectra were recorded on a
Varian-Gemini 2000 Fourier transform spectrometer.
Chemical shifts are reported in l (ppm) relative to
tetramethylsilane (TMS) as internal standard. The fol-
lowing abbreviations are used throughout: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; ar, aromatic
and ind, indole. Spectral data are consistent with as-
signed structures. All reactions were routinely checked
by ascending thin-layer chromatography (TLC) on pre-
coated silica gel plates (60F-254 Merck).
7
8
2.63 (t, 2H, ꢀCH2ꢀCOꢀ); 2.86 (t, 2H, C6H5ꢀCH2ꢀ); 3.17
(m, 2H, ꢀCH2ꢀCHꢃ); 4.68 (m, 1H, ꢂCHꢀ); 6.96–8.60
(m, 18H, ar); 9.05 (d, 1H, ꢀNHꢀCHꢃ); 10.60 (s, 1H,
ꢀNHꢀ ind); 10.82 (s, 1H, ꢀNHꢀ); 11.95 (s, 1H, ꢀNHꢀ).
3.30 (m, 2H, ꢀCH2ꢀ); 4.65 (m, 1H, ꢂCHꢀ); 6.85–8.52
(m, 20H, ar and ꢀCHꢁCHꢀ); 8.95 (d, 1H, ꢀNHꢀCHꢃ);
10.80 (s, 1H, ꢀNHꢀ ind); 10.91 (s, 1H, ꢀNHꢀ); 12.10 (s,
1H, ꢀNHꢀ).
3.28 (m, 2H, ꢀCH2ꢀCHꢃ); 3.46 (s, 2H, ꢀCH2ꢀCO);
4.74 (m, 1H, ꢂCHꢀ); 6.95–8.57 (m, 13H, ar); 9.10 (d,
1H, ꢀNHꢀCHꢃ); 10.85 (s, 1H, ꢀNHꢀ ind); 10.98 (s,
1H, ꢀNHꢀ); 12.03 (s, 1H, ꢀNHꢀ).
9
6.1.1. N-Anthranoyl-DL-tryptophan ethyl ester (11)
DL-Tryptophan ethyl ester hydrochloride (5.37 g; 20
mmol) was suspended in ethyl acetate (250 ml) and
treated with triethylamine (2.81 ml; 20 mmol) followed
by isatoic anhydride (3.26 g; 20 mmol). The suspension
was refluxed for 2 h, cooled to room temperature (r.t.)
and filtered. The organic phase was shaken in a separa-
tor flask with 1 M NaOH (2×50 ml), water (2×50
ml), dried over anhydrous sodium sulfate and filtered.
The filtrate was evaporated to dryness under reduced
pressure and the resulting oily residue was triturated
with petroleum ether (40–60°; 300 ml). The analytically
pure title compound which precipitated was collected
by filtration, washed with petroleum ether (40–60°) and
dried (5.7 g; 81%).
10
2.54 (t, 2H, ꢀNHCOꢀCH2ꢀ); 2.70 (t, 2H,
ꢀCH2ꢀCOOH); 3.32 (m, 2H, ꢀCH2ꢀCHꢃ); 4.68 (m,
1H, ꢂCHꢀ); 6.95–8.56 (m, 13H, ar); 9.06 (d, 1H,
ꢀNHꢀCHꢃ); 10.71 (s, 1H, ꢀNHꢀ ind); 10.82 (s, 1H,
ꢀNHꢀ); 12.00 (s, 1H, ꢀNHꢀ).
a Abbreviations: ar, aromatic; ind, indole; s, singlet; d, doublet; t,
triplet; m, multiplet.
tions, lipophilicity and hydrogen bond formation of the
N-terminus substituent may be important for fitting
this part of the molecule into a lipophilic pocket on the
receptor.
5. Conclusions
Rf 0.49 (AcOEt–hexane: 1/1); m.p. 98–100°C; 1H
NMR (CDCl3): l 1.24 (t, 3H, ꢀCH3); 3.46 (d, 2H,
ꢂCHꢀCH2ꢀ); 4.16 (q, 2H, ꢀCH2ꢀOꢀ); 5.06 (m, 1H,
ꢂCHꢀ); 5.49 (s, 2H, ꢀNH2); 6.52–7.60 (m, 10H, ar,
ꢀNHꢀ); 8.11 (s, 1H, ꢀNHꢀ ind). 13C NMR (CDCl3): l
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.
We have reported a new series of N-substituted an-
thranilic acid dimer derivatives starting from the lead
compound 0 which derived from a new disconnection
strategy of asperlicin. An increased affinity for CCK-A
receptors was achieved by introducing an indole moiety
at the N-terminus of the starting compound. The lead

A. Varna6as et al. / Il Farmaco 55 (2000) 369–375
373
Table 4
13C NMR (DMSO-d6) of compounds 1–10
Comp. l (ppm)
1
2
20.46, 26.29, 37.66, 53.57, 110.00, 111.10, 111.24, 113.10, 117.90, 117.97, 118.03, 118.17, 120.43, 120.69, 121.65, 122.03, 122.94,
123.34, 123.45, 126.73, 126.86, 127.32, 128.24, 131.97, 132.09, 135.89, 135.99, 138.07, 138.53, 166.05, 168.36, 170.71, 172.82.
34.20, 36.09, 53.95, 109.12, 111.43, 112.05, 113.95, 116.88, 117.87, 118.05, 118.23, 120.35, 120.55, 121.47, 122.10, 123.00, 123.30,
123.80, 126.40, 126.73, 127.25, 128.15, 131.70, 132.23, 135.67, 136.00, 137.95, 138.35, 165.80, 168.15, 169.85, 172.65.
26.44, 54.05, 102.76, 110.31, 111.17, 112.30, 117.97, 118.07, 119.95, 120.65, 120.92, 121.07, 121.43, 121.70, 123.24, 123.34, 123.86,
126.82, 127.06, 127.38, 128.19, 131.33, 132.11, 132.63, 135.89, 136.93, 138.34, 139.05, 159.12, 166.65, 168.13, 172.97.
26.57, 54.47, 110.61, 111.09, 117.94, 118.03, 120.54, 121.02, 121.36, 122.10, 123.00, 123.25, 123.60, 126.60, 126.86, 127.23, 127.42,
128.02, 128.70, 131.82, 131.94, 132.50, 134.35, 135.83, 138.33, 139.02, 164.54, 166.57, 167.61, 173.19.
3
4
5
26.33, 53.58, 110.02, 111.23, 117.88, 118.16, 120.66, 120.74, 120.84, 121.70, 122.78, 123.16, 123.40, 123.88, 126.91, 127.45, 128.30,
128.75, 128.84, 132.13, 132.42, 133.15, 135.93, 136.65, 138.38, 138.56, 163.62, 166.44, 168.31, 172.75.
6
26.66, 43.90, 55.21, 110.95, 111.15, 117.74, 118.16, 120.35, 120.50, 121.16, 121.53, 122.92, 123.09, 123.28, 123.43, 126.46, 127.17,
127.55, 127.75, 128.15, 129.16, 131.54, 131.84, 134.96, 135.77, 138.10, 138.46, 165.92, 167.37, 169.06, 173.95.
26.33, 30.46, 38.12, 53.56, 110.03, 111.23, 117.89, 118.17, 120.58, 120.74, 121.74, 122.96, 123.39, 123.54, 125.71, 126.91, 127.31,
127.98, 128.04, 128.23, 131.91, 132.04, 135.93, 137.88, 138.51, 141.00, 166.04, 168.33, 170.17, 172.75.
7
8
26.41, 53.87, 110.23, 111.18, 117.95, 118.09, 120.68, 120.87, 120.96, 121.82, 122.17, 123.10, 123.33, 123.58, 127.04, 127.27, 127.86,
128.17, 128.69, 129.70, 131.93, 132.01, 134.33, 135.90, 138.27, 138.40, 140.79, 163.48, 166.13, 168.16, 172.90.
26.31, 53.56, 110.04, 111.21, 117.90, 118.15, 120.62, 120.72, 121.77, 123.02, 123.37, 123.70, 126.92, 127.24, 128.22, 131.91, 132.01,
135.91, 137.59, 138.45, 164.55, 165.83, 168.29, 168.79, 172.76.
9
10
26.30, 28.57, 31.48, 53.56, 110.03, 111.22, 117.90, 118.16, 120.52, 120.74, 121.47, 122.95, 123.27, 123.39, 126.88, 127.31, 128.24,
132.08, 135.88, 138.10, 138.48, 166.07, 168.32, 169.92, 172.80, 173.39.
6.1.2. N-(N-Anthranoyl)anthranoyl-DL-tryptophan ethyl
ester (12)
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.
A solution of N-anthranoyl-DL-tryptophan ethyl es-
ter (11) (17.52 g; 50 mmol) and triethylamine (7.03 ml;
50 mmol) in dry dichloromethane (250 ml) was cooled
at 0°C and stirred during the dropwise addition of a
solution of 2-nitrobenzoyl chloride (9.25 g; 50 mmol) in
dry dichloromethane (60 ml). After stirring for 2 h at
r.t. the reaction mixture was washed in succession with
1 M NaOH (2×30 ml) and water (2×30 ml). The
organic layer was dried over anhydrous sodium sulfate
and filtered. The filtrate was evaporated under reduced
pressure to give the crude nitro derivative, which was
used immediately without further purification in the
next step of the synthesis. The nitro derivative was
dissolved in dichloromethane (100 ml) 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) and
water (2×50 ml), dried over anhydrous sodium sulfate
and evaporated. Crystallization from 90% ethanol af-
forded the analytically pure compound 12 (16.2 g;
69%).
Table 5
CCK receptors binding data
Comp. R
CCK-A (%) a
1×10−5
CCK-B (%) b
1×10−5
M
M
0
1
2
3
4
5
6
7
8
H
(7.0×10−6 M) c IN d
1H-indol-3-yl-(CH2)2ꢀCOꢀ (1.1×10−6 M) c IN d
1H-indol-3-yl-CH2ꢀCOꢀ
1H-indol-2-ylꢀCOꢀ
C6H5ꢀCOꢀ
C6H4ꢀp-ClꢀCOꢀ
C6H5ꢀCH2ꢀCOꢀ
C6H5ꢀ(CH2)2ꢀCOꢀ
C6H5ꢀCHꢁCHꢀCOꢀ
HOOCꢀCH2ꢀCOꢀ
HOOCꢀ(CH2)2ꢀCOꢀ
(3.2×10−6 M) c IN d
(1.8×10−6 M) c IN d
60
40
75
65
58
35
IN d
45
53
38
9
10
44
IN d
IN d
IN d
1
Rf 0.45 (AcOEt–hexane: 1/1); m.p. 150°C; H NMR
a Percentage of inhibition at 10 mM of [3H]-(9)-
L
-364,718 binding
(CDCl3):
l
1.25 (t, 3H, ꢀCH3); 3.43 (m, 2H,
in rat pancreatic membranes.
b Percentage of inhibition at 10 mM of [3H]-(+)-
pig brain membranes.
L-365,260 in guinea
ꢂCHꢀCH2ꢀ); 4.17 (q, 2H, ꢀOꢀCH2ꢀ); 5.08 (m, 1H,
ꢀCHꢃ); 5.76 (s, 2H, ꢀNH2); 6.68–7.69 (m, 12H, ar);
8.30 (s, 1H, ꢀNHꢀ ind); 8.65 (d, 1H, ar); 11.69 (s, 1H,
ꢀNHꢀ). 13C NMR (CDCl3): l 14.19, 27.65, 53.53,
61.86, 109.76, 111.42, 115.97, 117.10, 117.51, 118.53,
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.

374
A. Varna6as et al. / Il Farmaco 55 (2000) 369–375
6.1.3. General procedure for the synthesis of
6.2. Biological e6aluation
N-[N-(substituted)anthranoyl] anthranoyl-DL-tryptophan
ethyl ester deri6ati6es (1a–2a)
6.2.1. General
A solution of 10 mmol of the corresponding acid in
dry dichloromethane (100 ml) cooled at −10°C was
treated with triethylamine (1.4 ml; 10 mmol) followed
by isobutyl chloroformate (1.31 ml; 10 mmol). The
resulting mixture was stirred at −10°C for 20 min and
treated dropwise with a solution of compound 12 (4.7
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 and the organic phase was washed with di-
luted aqueous sodium hydroxide solution and water.
The organic layer was dried over anhydrous sodium
sulfate and evaporated under reduced pressure. The
residue was used without further purification in the
next step of the synthesis.
Male rats (Wistar) and male guinea pigs (Hartley)
were obtained from Charles River, Calco, Como
(Italy). [3H]-(9)- -364,718 and [3H]-(+)-
L L-365,260
were purchased from NEN Research products (Brus-
sels) 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 described previously
[26]. 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 as described previously [22]. 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)
6.1.4. General procedure for the synthesis of
and a solution of various concentrations of the com-
pounds 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
(Whatman) filters and rinsed four times with 4 ml of
cooled Tris buffer (50 mM, pH 7.4). Non-specific bind-
N-[N-(substituted)anthranoyl] anthranoyl-DL-tryptophan
ethyl ester deri6ati6es (3a–10a)
A solution of 10 mmol of the corresponding acyl
chloride (the indole-2-carbonyl chloride was prepared
according to the procedure described by Kermack [25])
in 30 ml of dry dichloromethane was added gradually
to a solution of compound 12 (4.7 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 dichlomethane and washed in succession with
0.1 N sodium hydroxide solution, water, 0.1 N hy-
drochloric acid and water. The dried (sodium sulfate)
organic phase was evaporated under reduced pressure
to give a crude residue, which was used without further
purification in the next step of the synthesis.
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.
CCK-B receptor affinities were determined by dis-
placement of [3H]-(+)-
L-365,260 from guinea pig cere-
bral cortex membranes as described previously by
Chang et al. [23]. 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]-(+)-
6.1.5. General procedure for the preparation of
compounds 1–10
A mixture of 5 mmol of the corresponding ester
(compounds 1a–10a) and potassium hydroxide (0.56 g;
10 mmol) in methanol (50 ml) was warmed gently 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 combined organic extracts
were dried over sodium sulfate and evaporated to give
the corresponding title compound. Crystallization from
the appropriate solvent afforded the analytically pure
derivatives 1–10 (Table 2).
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 total.
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