Synthesis and biological characterisation of [3H]BBL454, a new CCK2 selective radiolabelled agonist displaying original pharmacological properties

Article abstract of DOI:10.1016/j.bmcl.2003.11.001

[³H]BBL454, a new CCK₂ selective tritiated agonist was prepared via the reductive tritiation of a 5-aminopentyn-1-yl moiety introduced on the N-terminal end of a pentapeptide derivative of cholecystokinin. The binding properties of this labelled compound were determined on CHO cells transfected with the rat CCK₂ receptor. [³H]BBL454 is able to discriminate two affinity states of the CCK₂ receptor a supplementary indication of its validity for further exploring the heterogeneity of this receptor.

Full text of DOI:10.1016/j.bmcl.2003.11.001

Bioorganic & Medicinal Chemistry Letters 14 (2004) 369–372
Synthesis and biological characterisation of [3H]BBL454, a new
CCK2 selective radiolabelled agonist displaying original
pharmacological properties
Bruno Bellier,a,y Christophe Dugave,b Frederic Etivant,a Roger Genet,b
Veronique Gigouxa and Christiane Garbaya,*
a
´
Laboratoire de Pharmacochimie Moleculaire et Structurale, FRE CNRS 2463-INSERM U266,
UFR des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’Observatoire, 75270 Paris Cedex 06, France
b
´ ´ ´
Departement d’Ingenierie et d’Etude desProte ines, Batiment 152, Centre d’EtudesNucle airesde Saclay,
´
91191 Gif Sur Yvette Cedex, France
ˆ
Received 9April 2003; revised 17 July 2003; accepted 3 November 2003
Abstract—[3H]BBL454, a new CCK2 selective tritiated agonist was prepared via the reductive tritiation of a 5-aminopentyn-1-yl
moiety introduced on the N-terminal end of a pentapeptide derivative of cholecystokinin. The binding properties of this labelled
compound were determined on CHO cells transfected with the rat CCK2 receptor. [3H]BBL454 is able to discriminate two affinity
states of the CCK2 receptor a supplementary indication of its validity for further exploring the heterogeneity of this receptor.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
cesses. On the other hand, the heptapeptide BC264 [Boc
-Tyr(SO3H)-gNle-mGly-Trp-NMeNle-Asp-Phe-NH2,
Table 1]5 was the only available CCK2B agonist for a
decade; this widely studied compound is non-anxio-
genic,6 increases the exploratory behaviour of rats,7 sti-
mulates dopamine release in vivo8 and reinforces
working memory.9 Recently, a new series of BC264-
derived pentapeptides10 was developed by a rational
process, in which the lead compound RB400 (HO2C-
CH2-CO-Trp-NMeNle-Asp-Phe-NH2, Table 1), and
some of its derivatives were shown to share the CCK2B
pharmacology.11 In particular, the most potent com-
The peptide cholecystokinin (CCK-8, Asp-Tyr(SO3H)-
Met-Gly-Trp-Met-Asp-Phe-NH2), both
a
neuro-
transmitter and a hormone, is involved in several
physiological processes including regulation of appetite
and gastrointestinal functions, anxiety, cognitive pro-
cesses and analgesia.1,2 All these effects result from the
interaction of cholecystokinin with two seven trans-
membrane receptors termed CCK1 and CCK2 (formerly
CCK-A and CCK-B respectively), the latter being
widely distributed throughout the brain.2 Although no
evidence for a structural heterogeneity of the CCK2
receptors has been obtained to date,3 numerous data
suggest that CCK2 agonists can be separated into two
classes, regarding their pharmacological properties in
vivo.4 On the one hand, most CCK2 agonists including
the endogenous peptides CCK-8 and CCK-4 are asso-
ciated with the so-called ‘CCK2A’ profile (formerly
‘CCK-B10), characterised by anxiogenic properties and
a negative or non-measurable effect on memory pro-
pound,
BBL454
[H-(CH2)5-NHCO-CH2CO-Trp-
NMeNle-Asp-Phe-NH2] shares the favourable proper-
ties of BC264 (increase of working memory, for
instance), but at doses 10–100 times lower (as low as
0.03 mg/kg after ip injection in the rat), and remains
active in a large range of doses11 (0.03–300 mg/kg in the
open-field test). In contrast, BC264, the former CCK2B
compound, is not satisfying from this point of view, as it
is only active in a small concentration range9 (i.e., in a
two-trial memory test, BC264 is only active at 3 mg/kg,
but not at any dose lower nor higher).
* Corresponding author at present address: Laboratoire de Pharma-
cochimie Moleculaire et Cellulaire, FRE CNRS 2718-INSERM
U266, UFR Biomedicale, 45, rue des Saints-Peres, 75270 Paris Cedex
06, France. Tel.: +33-1-4286-4080; fax: +33-1-4286-4082; e-mail:
christiane.garbay@univ-paris5.fr
Unfortunately, numerous experiments in several recep-
tor preparations (cortical membranes from diverse spe-
cies or CHO cells stably transfected with the rat CCK2
receptor) have been so far unsuccessful in the aim of
establishing a biochemical discrepancy between CCK2A
y
Present address: Section Analyse Chimique, Centre d’Etudes du
Bouchet, BP3, 91710 Vert Le Petit, France.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.001

370
B. Bellier et al. / Bioorg. Med. Chem. Lett. 14 (2004) 369–372
Table 1. Essential biological data of reference CCK2 agonists20
Compd
Ki (CCK2)
Ki (CCK1)
Pharmacology
CCK-8
Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-PheNH2
Mixed CCK1/CCK2A agonist
0.6 nM
0.7 nM
BC264
Boc-Tyr(SO3H)-gNle-mGly-Trp-NMeNle-Asp-PheNH2
0.31 nM
0.75 nM
2.6 nM
96 nM
>3 mM
>5 mM
3.64 mM
CCK2B agonist
CCK2B agonist
CCK2A agonist
CCK2B agonist
RB400
HO2C-CH2-CO-Trp-NMeNle-Asp-Phe-NH2
PJC-42
(CH3)3C-OCO-Trp-NMeNle-Asp-Phe-NH2
BBL454
H-(CH2)5NHCOCH2CO-Trp-NMeNle-Asp-PheNH2
2.66 nM
and CCK2B agonists. However, binding studies using
CCK2 agonists or antagonists12 support the hypothesis
that the pool of CCK2 receptors could be constituted of
several affinity states. Additional data also show that
multiple transduction systems are associated with CCK2
receptors.13
precursors by tritium gas. In our case, the presence of
both N-methylnorleucine and phenylalanine in the
sequence of BBL454 allowed us to apply this strategy,
but this necessitated the preparation of unnatural,
enantiomerically pure aminoacids, whose reported
synthesis gave poor yields.14,15 Thus, an unsaturated N-
terminal analogue of BBL454 appeared as much more
promising, since its reduction would lead to
[3H]BBL454 without any structural modification but the
substitution of hydrogen atoms by their isotopes.
Therefore, the design of more ‘selective’ CCK2A or
CCK2B compounds appeared as mandatory to further
study the relevance of CCK2A/CCK2B discrimination in
vitro. CCK2B selectivity is a difficult parameter to
determine, but we hypothesised that the width of the
concentration range of activity of CCK2B agonists in
vivo could be an indication of this parameter. BC264,
which is generally only active at a single dose in vivo
would in this case be classified as a poorly selective
CCK2B agonist, whereas BBL454, having a large range
of activity, could be considered highly CCK2B-selective.
Thus, a radiolabelled analogue of BBL454 was of great
interest, all the more since desirable pharmacological
effects displayed by BBL454 remain associated with
some unexplained issues. In particular, why is BBL454
100 times more active than BC264 in the open-field test
using rats while its affinity is 10 times lower on rat
CCK2 receptors? Variations in the bioavailability of the
compounds, which a radiolabelled analogue could help
to determine, might be a convenient explanation for
these observations. This prompted us into the design of
a labelled derivative of BBL454.
The key step in the chosen strategy was thus the pre-
paration of any unsaturated five-carbon-length linear
primary amine. Obviously, the more insaturated bonds
that are present in the resulting precursor, the higher the
specific activity that should be obtained by catalytic tri-
tiation. Thus, 1-aminopentynes were chosen as potential
intermediates.
Preliminary attempts to convert commercially available
3-pentyn-1-ol into the corresponding amine failed,
probably due to the susceptibility of intermediate acti-
vated alcohols to elimination and/or nucleophilic
substitution, even by triethylamine. Therefore, a chloro-
pentyne was found to be preferable than a pentynol as
starting material. Thus, 5-chloropent-1-yne (3) was
converted in quantitative yields to N-(pent-4-yn-1-yl)-
phthalimide (4) by nucleophilic substitution using
sodium iodide and potassium phthalimide; the resulting
compound was purified by recrystallization as pre-
viously described.16 Surprisingly, hydrazinolysis under
the conditions described by the same authors afforded a
complex mixture of compounds, from which the desired
1-amino-pent-4-yne could neither be isolated as such,
nor as the hydrochloride salt. Alternative attempts at
deprotection of the phthalimide group in acidic (HCl/
AcOH) medium led to deprotected compounds showing
addition of HCl to the triple bond.
2. Strategy and synthesis
The extensive structure–affinity relationships obtained
with derivatives of RB40010 and the in vivo study of
several representative compounds has proved that slight
structural differences could lead to a change of the
pharmacological profile from CCK2A to CCK2B. Thus,
it seemed crucial for our purposes that the introduction
of a radioisotope should modify as little as possible the
structure of BBL454. Previous works on tritiated CCK
agonists had used the unsaturated analogue of norleu-
cine l-2-amino-4-hexynoic acid14 or the phenylalanine
Therefore, milder deprotection conditions were used
involving reduction of the phtalimide by sodium bor-
ohydride in a water/isopropyl alcohol mixture followed
by treatment with diluted acetic acid.17 Purification, as
published,17 of the compound by ion-exchange chro-
matography was avoided because of the suspected high
precursor
l-3(30,40,50-tribromophenyl)alanine15
to
introduce labels by means of catalytic reduction of the

B. Bellier et al. / Bioorg. Med. Chem. Lett. 14 (2004) 369–372
371
volatility of the expected amine, which would have
made it difficult to isolate from the elution solvent.
Thus, the final reaction mixture was acidified with
hydrochloric acid, filtered and precipitated in ether to
separate inorganic and organic boron salts. Smooth
evaporation of the ethereal solution provided the amine
5 only contaminated with remaining boron salts which
could not be completely eliminated (thus leading to an
apparent overall yield exceeding 100%), but which did
not compromise the following steps of synthesis. Next, 5
was coupled with the pentapeptide analogue 6 (obtained
by condensation of mono-tert-butyl malonate with the
tetrapeptide Trp-NMeNle-Asp(OBzl)Phe-NH2 followed
by deprotection of the tert-butyl ester with tri-
fluoroacetic acid) yielding the unsaturated precursor of
BBL454, 7. Catalytic hydrogenation at atmospheric
pressure of 7 in methanol, with palladium oxide as cat-
alyst, gave BBL454 (1)18 in 100% raw yield (without
purification); tritiation of 7 under the same conditions
led to [3H]BBL454 (2) with an estimated specific activity
of 50 Ci/mmol (Scheme 1).
[3H]BBL454 were assayed on intact CHO cells following
a protocol described previously.19 Cold saturation
experiments conducted with BBL454 competing with
[3H]BBL454 proved that [3H]BBL454 binds to two sites
in CHO cells, in a specific and saturable way. Scatchard
analysis of these results reveals a high affinity and low
populated
binding
site
(KD1=0.71Æ0.17 nM,
Bmax1=120Æ16 fmol/mg of protein) and a low affinity
and high populated binding site (KD2=194Æ76 nM,
Bmax2=4.41Æ0.71 pmol/mg of protein). A representa-
tive Scatchard diagram of these experiments is presented
in Figure 1. That BBL454 is able to discriminate two
affinity states of the CCK2 receptor is a supplementary
indication as to its validity for further exploring the
heterogeneity of these receptors. Therefore, competition
experiments using this new radiolabelled agonist and
various standard CCK2 ligands are now under progress.
Owing to its remarkable pharmacological properties in
vivo, the CCK2B agonist BBL454 emerges as a valuable
tool for studying the functional heterogeneity of CCK2
receptors, and merely as a major starting point for the
research of compounds exerting stimulant and memory
reinforcing. Its tritiated analogue will now be used
to assay the bioavailability of BBL454 in vivo, and to
determine, via autoradiographic studies, the location of
its sites of action. Its is also firmly hoped that this
3. Biochemical characterization
Cold BBL454 was assayed following standard proto-
cols10 by competition of [3H]pCCK-8 from membranes
of CHO cells transfected with CCK2 receptors
(Ki=2.7Æ0.3 nM), and proved to behave as a full ago-
nist of inositol phosphate production (EC50=2Æ0.3 nM)
and arachidonic acid liberation (EC50=6.9Æ0.8 nM) by
intact transfected CHO cells (data not shown). Then,
the binding properties of the new radiolabelled agonist
radioligand will be useful for designing
a high
throughput biochemical test for discriminating easily
and rapidly CCK2A from CCK2B agonists; its capacity
to discriminate between two affinity states of the
receptor is a first indication that it may be useful for
such analyses.
Scheme 1. Synthesis of [3H]BBL454.

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B. Bellier et al. / Bioorg. Med. Chem. Lett. 14 (2004) 369–372
Figure 1. Saturation of BBL454 on CHO cells and corresponding Scatchard diagram.
12. Reviewed in: Bellier, B.; Gigoux, V.; Garbay, C. Curr.
Med. Chem. CNSA 2001, 1, 209.
Acknowledgements
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Million, E.; Garbay, C.; Roques, B. P.; Noble, F. J. Neu-
rochem. 1999, 73, 281.
The authors wish to acknowledge Michel Vidal for
numerous helpful technical suggestions and support.
14. Sasaki, N. A.; Funakoshi, S.; Potier, P.; Morgat, J. L.;
Genet, R.; Gacel, G.; Charpentier, B.; Roques, B. P. J.
Labelled Compd. Radiopharm. 1985, 22, 1123.
15. Charpentier, B.; Roques, B. P.; Roy, J.; Morgat, J. L. J.
Labelled Compd. Radiopharm. 1989, 27, 387.
16. L’abbe, G.; Leurs, S.; Dehaen, W. Tetrahedron 1993, 49,
4439.
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TFA) (d ppm/HMDS; two rotamers due to the NMeNle
residue): 0.6–1.7 (m, –(CH2)3CH3 Nle and CONHCH2–
ðCH2Þ –H) 2.38–3.1 (m, 10H, COCH2CONHCH2(CH2)4–,
CH2b 4Trp, CH2b Asp, CH2b Phe), 2.77 and 3.03 (s, 3H,
NCH3 Nle), 4.19(m, a Nle), 4.72 (m, a Nle), 4.31 (m, 1H,
a Phe), 4.40 and 4.48 (m, 1H, a Asp), 4.90 (m, 1H, a Trp),
6.85–7.16 (m) and 7.22 (d) and 7.45 (m) and 7.75 (d),
(13H, 5 Ar Trp+5 Ar Phe+amide NH Phe+CONH2
Phe), 7.78 (d) and 7.88 (d) (1H, NHðCH2Þ5ꢀH) 7.90 (d)
and 8.13 (d) (1H, amide NH Asp), 8.41 (d) and 8.83 (d)
(1H, amide NH Trp) 10.68 and 10.78 (s, 1H, indole NH).
HPLC: tR=23.4 min [gradient 10–90% B in 30 min (sol-
vent systems A: H2O + 0.1% TFA, B: 70% acetonitrile,
30% H2O, 0.09% TFA), column: C18 Vydac (5 m, 4.6 ꢁ
150 mm), 1 mL/min, l=210 nm].
19. Gigoux, V.; Escrieut, C.; Fehrentz, J. A.; Poirot, S.; Mai-
gret, B.; Moroder, L.; Gully, D.; Martinez, J.; Vaysse, N.;
Fourmy, D. J. Biol. Chem. 1999, 274, 20457.
20. Affinities for CCK1 and CCK2 receptors were determined
respectively on guinea-pig pancreatic membranes and on
membranes of CHO cells stably transfected with the rat
CCK2 receptor, by competition with 1 nM [3H]pCCK-8.
Results are the mean of at least three separate experi-
ments, each performed in triplicate.