Synthesis and biological effects of c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro- Tyr-Ile-Leu) (JMV2012), a new analogue of neurotensin that crosses the blood-brain barrier

Article abstract of DOI:10.1021/jm700925k

The central administration of neurotensin (NT) or of its C-terminal hexapeptide fragment NT(8-13), produces strong analgesic effects in tests evaluating acute pain. The use of NT-derived peptides as pharmaceutical agents to relief severe pain in patients could be of great interest. Unfortunately, peptides do not readily penetrate the blood-brain barrier. We have observed that the cyclic NT(8-13) analogue, c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile- Leu) (JMV2012, compound 1), when peripherally administered to mice produced analgesic and hypothermic effects, suggesting the peptide penetrates the blood-brain barrier and functions effectively like a drug. Moreover, dimeric compounds show increased potency compared to their corresponding monomer. We present the synthesis of the cyclic dimer compound 1 (JMV2012). In mice, compound 1 induced a profound hypothermia and a potent analgesia, even when peripherally administered. Compound 1 appears to be an ideal lead compound for the development of bioactive NT analogues as novel analgesics drugs.

Full text of DOI:10.1021/jm700925k

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J. Med. Chem. 2008, 51, 1610–1616
Synthesis and Biological Effects of c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu)
(JMV2012), a New Analogue of Neurotensin that Crosses the Blood-Brain Barrier
Pierre Bredeloux,† Florine Cavelier,‡ Isabelle Dubuc,† Bertrand Vivet,‡,§ Jean Costentin,† and Jean Martinez*,‡
CNRS FRE 2735, Unité de Neuropsychopharmacologie de la dépression, IFRMP 23, Faculté de Médecine et de Pharmacie, 22 Bd Gambetta,
76183 Rouen cedex, France, and CNRS UMR 5247, IBMM, UniVersités Montpellier 1 & 2, Place E. Bataillon, 34095 Montpellier, France
ReceiVed July 30, 2007
The central administration of neurotensin (NT) or of its C-terminal hexapeptide fragment NT(8-13), produces
strong analgesic effects in tests evaluating acute pain. The use of NT-derived peptides as pharmaceutical
agents to relief severe pain in patients could be of great interest. Unfortunately, peptides do not readily
penetrate the blood-brain barrier. We have observed that the cyclic NT(8-13) analogue, c(Lys-Lys-Pro-
Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012, compound 1), when peripherally administered to mice
produced analgesic and hypothermic effects, suggesting the peptide penetrates the blood-brain barrier and
functions effectively like a drug. Moreover, dimeric compounds show increased potency compared to their
corresponding monomer. We present the synthesis of the cyclic dimer compound 1 (JMV2012). In mice,
compound 1 induced a profound hypothermia and a potent analgesia, even when peripherally administered.
Compound 1 appears to be an ideal lead compound for the development of bioactive NT analogues as novel
analgesics drugs.
Introduction
nociception,13–15 several studies demonstrate that NTS2 recep-
tors are the main receptors involved in NT analgesia, indepen-
dently of the nature of the nociceptive stimulus (thermal,
mechanical, or chemical) used to evaluate nociception.15–22 The
development of NT agonists able to cross the blood-brain
barrier and selective for the NTS2 receptors is of significant
interest as potential novel analgesic agents. In this way, the
C-terminal hexapeptide fragment of NT [NT(8-13)], which
corresponds to the shorter fragment of NT that maintains full
biological activities23 is an obvious lead compound for
development.
Neurotensin (NT,a pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-
Pro-Tyr-Ile-Leu-OH) is a tridecapeptide first isolated by Car-
raway and Leeman (1973)1 from bovine hypothalami. This
peptide, which fulfills the major criteria attributed to a neuro-
modulator/neurotransmitter, exerts a wide range of biological
actions when injected in the central nervous system, including
modulation of dopamine transmissions in the nigro-striatal and
mesocorticolimbic systems,2 hypothermia,3 and analgesia.4 This
last effect is of particular interest because this analgesia was
shown to be more potent than morphine5 and principally
naloxone-insensitive4,6,7 on a molar basis. For this reason, NT
agonists might be useful as nonopioid analgesics from a
therapeutic point of view.
Several receptors are involved in NT biological activity. So
far, three NT receptors have been cloned and designated NTS1,
NTS2, and NTS3. NTS18 and NTS29,10 belong to the family of
G protein coupled receptors, while NTS3,11 constituted by a
single transmembrane domain, corresponds to the gp95/sorti-
lin.12 Although it was recently shown that stimulation of the
NTS1 receptor was required for the full expression of NT
analgesia when a thermal stimulus was used to induce
Thus, peptide NT1 or “Eisai peptide”,24–26 a modified
analogue of NT(8-13), was the first agonist able to induce
biological activity, such as analgesia, hypothermia, and hypolo-
comotion, after systemic administration in animals.27,28 More
recently, the group of Richelson was interested in peptidase-
resistant NT agonists able to cross the blood-brain barrier and
presenting high binding affinity for the NTS1 receptor. Among
the different peptides they developed, NT69L was the most
studied. This peptide exhibits high affinity for the neurotensin
receptor (0.82 nM for the rat NTS1; 1.55 nM for the human
NTS1; 2.1 nM for the human NTS2 receptor) and induces potent
analgesia and hypothermia after intraperitoneal injection in
rats.29
* To whom correspondence should be addressed: Phone: +33 (0)4 67
14 38 44. Fax: +33 (0)4 67 14 48 66. E-mail: martinez@univ-montp1.fr.
A different strategy to obtain agonist that cross the blood-brain
barrier is the development of cyclic analogues. A few years ago,
a structure-activity relationship study revealed that a cyclic
analogue (JMV1193) comprising the minimal effective NT
fragment was able to cross the blood-brain barrier.30 This cyclic
compound has the sequence c(Lys-Lys-Pro-Tyr-Ile-Leu) and
displays an affinity in the 0.3 µM range for mouse brain NT
receptors. The i.v. administration of this compound induces the
same hypothermic and analgesic effects as icv injection.
However, like NT and NT(8-13), compound JMV1193 was
inactivated by the endopeptidase neprilysin. It has been shown
that dimeric compounds were usually able to show increased
potency compared to their corresponding monomer.31 We then
decided to synthesize the dimeric compound of Lys-Lys-Pro-
†
CNRS FRE 2735.
CNRS UMR 5247.
Present address: Sanofi-aventis, recherche & développement, 16 rue
‡
§
d’Ankara, 67080 Strasbourg, France.
a Abbreviations: Boc, NR-tert-butyloxycarbonyl; DCM, dichloromethane;
DE35; dose that brings the mean body temperature to 35 °C over 4 h; DIEA,
diisopropylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide;
ED50, effective dose 50; Fmoc, 9-fluorenylmethyloxycarbonyl; HBTU,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
HOBt, N-hydroxybenzotriazole; icv., intracerebroventricular; i.v., intrave-
nous; NT, neurotensin; NTS1, neurotensin receptor type 1; NTS2 neuro-
tensin receptor type 2; NTS3, neurotensin receptor type 3; per os, oral route
of administration; s.c., subcutaneous; tBu, tert-butyl; TEA, triethylamine;
TFA, trifluoroacetic acid. The symbols and abbreviations are in accordance
with recommendations of the IUPAC-IUB Joint Commission on Biochemi-
cal Nomenclature and Symbolism for Amino Acids and Peptides. Biochem
J. 1984, 219, 345–373.
10.1021/jm700925k CCC: $40.75
2008 American Chemical Society
Published on Web 03/06/2008

Synthesis of JMV2012, an Analogue of Neurotensin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1611
Scheme 1. Synthetic Procedure for Cyclic Peptide Compound 1
Table 1. Optimization of the Cyclization Reaction
Tyr-Ile-Leu, maintaining the cyclic character of this molecule.
We synthesized compound JMV2012 (compound 1), c(Lys-Lys-
Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu). The cyclization con-
ditions were modulated to favor dimerization starting from the
hexapeptide Lys-Lys-Pro-Tyr-Ile-Leu.
Initial studies performed in vitro showed that this peptide
was able to bind to both human NTS1 and NTS2 receptors with
a similar affinity close to 150 ( 100 nM. Under the same
experimental conditions, NT was found to bind to the human
NTS1 receptor with an affinity of 0.16 nM and to the human
NTS2 receptor with an affinity of 1.10 nM. Furthermore, it has
been shown that NT(8-13) binds to the rat and human NTS1
receptor with an affinity of 0.16 and 0.14 nM, respectively.32
Nevertheless, we decided to evaluate the in vivo activity
(hypothermia and analgesia) of compound 1 after various routes
of administration in mice.
In mice, compound 1 was able to induce a profound
hypothermia and an NTS2-dependent analgesia in the acetic
acid-induced writhing and hot plate test after icv injection.
Consequently, compound 1 was further characterized with
respect to its ability to cross the blood-brain barrier. For
this purpose, we studied its ability to trigger hypothermia
and analgesia observed with the help of acetic acid-induced
writhing test after various routes of administration such as
the intravenous (i.v.), subcutaneous (s.c.), and oral routes
(per os) in mice.
coupling
reagent
concentration
(linear peptide)
reaction
time
yieldsa
(mono/dimer)
base
HBTU (1.5 TEA (10
equiv) equiv)
HBTU (1.5 TEA (10
equiv) equiv)
1 mmol/L
1 h 30 min
87/0%
100 mmol/L
1 h 30 min
0/85%
a Yields after purification on preparative HPLC.
carbonyl (Fmoc) protection was used as temporary protection
of the N-terminal amino groups, and NR-tert-butyloxycarbonyl
(Boc) and tert-butyl (tBu) were used for the side-chain protec-
tions. Couplings of protected amino acids were carried out with
a solution of HBTU/HOBt reagents.
The use of the 2-chlorotrityle resin, as well as of mild
conditions (0.5% trifluoroacetic acid (TFA) in DCM) for
cleaving the peptide from the resin, allowed the release of the
protected peptide from the resin. Cyclization of the resulting
protected linear hexapeptide was achieved using O-(benzotria-
zol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HBTU) and triethylamine (TEA) in dimethylformamide. We
modulated the cyclization conditions to favor dimerization. The
linear precursor concentration was used at a high concentration
to obtain the desired cyclic peptide 1 (Table 1).
Finally, the protected cyclic analogue was deprotected with
TFA in the presence of anisole as scavenger. The free cyclic
analogue 1 was purified by preparative reverse-phase HPLC on
a C18 column, and its structure was confirmed by ESI mass
spectrometry. To confirm the structure of this compound, we
prepared the corresponding linear dodecapeptide stepwise on
the same solid support, followed by cyclization, deprotection,
and purification. The cyclic compound obtained by this way
Results
Chemistry. The linear hexapeptide precursor was synthesized
by solid-phase using the 2-chlorotrityl chloride resin preloaded
with proline residue (Scheme 1). The 9-fluorenylmethyloxy-

1612 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Bredeloux et al.
This hypothermia was significant from the dose of 100 ng at
30 min postinjection (p ) 0.006 vs saline). At the highest tested
dose (3 µg), a profound hypothermia occurred with a maximal
effect at 90 min postinjection (p < 0.001 vs saline). This
hypothermia was prolonged because at 4 h postinjection the
body temperature of mice treated with compound 1 (3 µg)
remained significantly lower than that of the saline controls (p
< 0.001). To integrate the intensity of the hypothermia and its
duration into one parameter, the mean body temperature over
4 h was calculated as the area under the time-response curve
for each mouse over a period of 240 min (4 h) divided by 240
min (Figure 1B). From this dose–response curve, we determined
that the ED35 for compound 1 (dose of compound 1 that brings
the mean body temperature to 35 °C over 4 h) was 300 ng.
Moreover, this hypothermia was not potentiated by the mixed
multipeptidase inhibitor JMV390-1 (data not shown), indicating
that compound 1 is resistant to the major NN/NT degrading
enzymes, such as aminopeptidase N and endopeptidases 24.11,
24.15, and 24.16.33,34
Nociception. In the hot plate test, compound 1 increased dose-
dependently, between 300 ng and 3 µg, latencies of both paw
licking and jumping (F(3,27) ) 9.976; p < 0.001 for the paw
licking latency, and F(3,24) ) 7.297; p ) 0.001 for the jump
latencies; Figure 2A,B). This result indicates that compound 1
triggers analgesia by icv administration in mice. Furthermore,
the NTS2 receptor antagonist levocabastine (1350 pmol), which
prevents the increase in jump latency produced by neurotensin
after icv administration,18 also prevented the increase in jump
latency induced by compound 1 (300 ng; interaction [levo-
cabastine × compound 1]: F(1,22) ) 5.201; p ) 0.035). As it
was already demonstrated for neurotensin,18 these data clearly
established that NTS2 receptors are involved in the analgesic
effect developed by compound 1 (Figure 2C).
Compound 1 was also tested in the writhing test. This test is
more sensitive than the hot plate test. In the writhing test,
compound 1, injected 20 min before testing, reduced dose-
dependently the number of writhes produced by acetic acid
injection in mice (F(3,37) ) 9.740; p < 0.001) from the dose
of 30 ng (p ) 0.001 vs saline; Figure 3). In this experiment,
the ED50 was below this dose of 30 ng.
This test was then used for the evaluation of the analgesic
activity of compound 1 by the other route of administration.
EffectsofCompound1onBodyTemperatureandNocicep-
tion in Mice after i.v. Administration. Considering that
compound 1 induced hypothermia and analgesia after icv
administration, we have evaluated its ability to produce hypo-
thermia and analgesia after i.v. administration.
Figure 1. Time course and dose–response relationships for the
hypothermic effect of icv injected compound 1 in the mouse. (A) Body
temperature as a function of time after icv injections of saline or
compound 1 at the indicated doses. (B) Mean body temperature over
4 h after icv injection of compound 1 at various doses. M ( SEM
from eight mice per group; (a) p < 0.05, (b) p < 0.01, and (c) p <
0.001 vs saline controls.
With regard to hypothermia, the two-way repeated measures
analysis of variance performed on the results of this experience
indicates a significant interaction between the different doses
of i.v. administered compound 1 and the different times of the
measure (F(12,114) ) 19.236; p < 0.001), thus, compound 1,
i.v. administered at the dose of 1 and 3 mg/kg, induced a dose
and time-dependent hypothermia that lasted at least 60 min, with
a maximal effect at 30 min postinjection (Figure 4).
Compound 1 produced a strong analgesic effect by i.v.
administration (F(3,42) ) 7.914; p < 0.001) in the writhing
test from the dose of 0.3 mg/kg (p ) 0.042 vs saline).
Furthermore, at the dose of 1 mg/kg, compound 1 was close to
abolish writhing in mice (p ) 0.001 vs saline; Figure 5A).
Finally, at the dose of 0.3 mg/kg, the two-way analysis of
variance indicates that compound 1, by i.v. administration,
induced a dose- and time-dependent analgesia (F(3,78) ) 4.529;
p ) 0.006). This analgesia was still evident for 60 min (Figure
has the same chemical and physical properties than compound
1 prepared previously.
The biological profile of the cyclic peptide was then evaluated
by its ability to induce analgesic and hypothermic activities after
various systemic routes of administration.
Biological Results. Effects of Compound 1 on Body Tem-
perature and Nociception in Mice after icv Administration. In
a first series of experiments, compound 1 was injected icv for
studying its ability to trigger hypothermia and analgesia in mice.
Hypothermia. The two-way repeated measures analysis of
variance performed on the results of this experience indicates a
statistically significant interaction between the different doses
of compound 1 icv administered and the different time of the
measure (F(35238) ) 16.699; p < 0.001). Thus, compound 1
icv administered between the dose of 30 ng and 3 µg reduced
dose and time-dependent mice body temperature (Figure 1A).

Synthesis of JMV2012, an Analogue of Neurotensin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1613
Figure 3. Dose–response relationships for the analgesic effect of icv
injected compound 1 in the writhing test. Mice were icv injected with
compound 1 at various indicated doses 15 min before receiving acetic
acid solution 0.5% (i.p.). Mice were then placed individually in large
beakers and the stretches were counted over a 5 min period 5 min after
the acetic acid solution injection. M ( SEM from 8 to 14 mice per
group; (b) p < 0.01 and (c) p < 0.001 vs saline controls.
Figure 4. Time course for the hypothermic effect of i.v. injected
compound 1 at various doses in the mouse. M ( SEM from 6 to 8
mice per group; (c) p < 0.001 vs saline controls.
compound 1 to induce hypothermia and analgesia in mice was
also tested after s.c. administration.
Compound 1, s.c. administered, produced a time- and dose-
dependent hypothermia (interaction [dose × time]: F(24,272)
) 7.647; p < 0.001) with a maximal effect reached for each
tested dose at 30 min postinjection (Figure 6A). This hypoth-
ermic effect lasted between 30 and 45 min for the doses of 1
and 3 mg/kg, whereas at the highest tested dose of 10 mg/kg,
this hypothermia was more pronounced and still significant for
more than 3 h (Figure 6A).
In the writhing test, compound 1, between the doses of 1
and 10 mg/kg, reduces dose-dependently the number of writhes
(F(3,22) ) 16.465; p < 0.001) at the dose of 3 mg/kg (p <
0.001 vs saline; Figure 6B).
Effects of Compound 1 on Nociception after Administra-
tion per os in Mice. After oral administration, compound 1
administered at the dose of 30 mg/kg reduced significantly the
number of writhes induced by the i.p. injection of an acetic
acid solution (Figure 7).
Figure 2. Dose–response relationships for the analgesic effect of icv
injected compound 1 in the hot plate test. Mice were icv injected with
compound 1 at various indicated doses 20 min before being placed on
the hot plate at 55 °C. (A) Dose–response relationship for the paw
licking latency. (B) Dose–response relationship for the jump latency.
M ( SEM from 6 to 9 mice per group; (a) p < 0.05, (b) p < 0.01, and
(c) p < 0.001 vs saline controls. (C) Effect of icv injected levocabastine
on the increase in jump latency produced by icv injected compound 1.
Mice were icv injected with saline, compound 1 (300 ng), levocabastine
(1350 pmol), or with a coadministration of compound 1 (300 ng) and
levocabastine (1350 pmol) 20 min before being placed on the hot plate
at 55 °C. M ( SEM from 6 to 8 mice per group; (a) p < 0.05 vs saline
controls; **p < 0.01 vs compound 1 alone.
Conclusion
This paper presents the synthesis and the pharmacological
evaluation of a new cyclic dimeric NT derivative. This
compound, c(Tyr-Ile-Leu-Lys-Lys-pro-Tyr-Ile-Leu-Lys-Lys-
Pro) (1), exhibited analgesic and hypothermic activities after
icv., i.v., s.c., and per os administration. These data suggest
that this peptide, owing to its cyclic structure, resists to the NT
degrading enzymes and crosses the blood-brain barrier. More-
over, the ability of levocabastine to inhibit the analgesic activity
5B). These data indicate that compound 1 was able to cross the
blood-brain barrier.
Effects of Compound 1 on Body Temperature and
Nociception after s.c. Administration in Mice. The ability of

1614 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Bredeloux et al.
Figure 5. Dose–response and time-course relationships for the
analgesic effect of i.v. injected compound 1 in the writhing test. (A)
Dose–response relationships for the analgesic effect of i.v. injected
compound 1 in the writhing test. Mice were i.v. injected with compound
1 at various indicated doses 15 min before receiving acetic acid solution
0.5% (i.p.). Mice were then placed individually in large beakers and
the stretches were counted over a 5 min period 5 min after the acetic
acid solution injection. (B) Time-course for the analgesic effect of i.v.
injected compound 1 (0.3 mg/kg) in the writhing test. M ( SEM from
8 to 18 mice per group; (a) p < 0.05, (b) p < 0.01, and (c) p < 0.001
vs saline controls.
Figure 6. Time-course for the hypothermic effect of s.c. injected
compound 1 and dose–response relationship for the analgesic effect of
s.c. injected compound 1 in the writhing test. (A) Body temperature as
a function of time after s.c. injections of saline or compound 1 at the
indicated doses. M ( SEM from 8 to 10 mice per group. (B)
Dose–response relationships for the analgesic effect of s.c. injected
compound 1 in the writhing test. Mice were s.c. injected with compound
1 at various indicated doses 15 min before receiving acetic acid solution
0.5% (i.p.). Mice were then placed individually in large beakers and
the stretches were counted over a 5 min period 5 min after the acetic
acid solution injection. M ( SEM from 6 to 8 mice per group; (a) p <
0.05, (b) p < 0.01, and (c) p < 0.001 vs saline controls.
of compound 1 in the hot plate test also suggests that compound
1 behaves like an agonist of the NTS2 receptors. However,
compound 1 probably triggers other NT receptors because it
produces hypothermia in mice, a NT effect independent of the
NTS2 receptors stimulation. In conclusion, compound 1 appears
at this stage to be an ideal lead compound for the development
of bioactive cyclic dimer more selective for the NTS2 receptors
as novel analgesic drugs.
Preparative HPLC was performed on a Waters Delta-Prep 4000
chromatography equipped with a Waters 486 UV detector with
detection at 214 nm, using a Delta-Pak C18 column (40 × 100
mm, 15 µm, 100 Å) at a flow rate of 50 mL/min of a binary eluent
system of A/B (A: H2O, TFA 0.1%; B: CH3CN, TFA 0.1%).
Synthesis of Protected Linear Peptide. The linear hexapeptide
was synthezised by the solid-phase method on a Perkin-Elmer
ABI433A automatic synthesizer on a 0.25 mmol scale with Pro-
preloaded 2-chlorotrityl chloride resin (loading 0.56 mmol/g, 446
mg). The coupling reagent was a 0.45 M solution of HBTU/HOBt
(2 mL). Four times excess of amino acid was used in coupling (1
mmol). Deprotection cycles were carried out in piperidine/DMF
(20/80, 5 mL) and monitored by conductimetry. Elongation was
performed by single 30 min couplings in DMF (15 mL) with DIEA
(2 M, 1 mL) as base. Washings were carried out with DMF (3 ×
15 mL) and DCM (1 × 15 mL). Final cleavage was carried out
with 0.5% TFA in DCM for 15 min (10 mL). The resin was washed
extensively with DCM, then dried in vacuo, dissolved in an
acetonitrile-water mixture (5/5, 10 mL), and freeze-dried. The side
chain protected intermediate peptide was obtained in 89% yield
(247 mg of TFA salt). tR ) 18.4 min (20–50% B, 30 min, C18).
ES-MS [M + H]+ 1017.4.
Experimental Section
The starting 2-chlorotrityl chloride resin preloaded with proline
residue was purchased from Novabiochem; Fmoc-amino acids were
obtained from Bachem.
HBTU, HOBt, DIEA, TEA, and piperidine were purchased from
Aldrich. Water was obtained from Milli-Q plus system (Millipore),
and acetonitrile and trifluoroacetic acid (TFA) were obtained from
Merck. Mass spectra were obtained by electron spray ionization
(ESI-MS) on a Micromass Platform II quadrupole mass spectrom-
eter (Micromass) fitted with an electrospray source coupled with
an HPLC Waters. HPLC runs were performed on Waters equipment,
using columns packed with Nucleosil 100 Å, 5 µm, C18 particles,
unless otherwise stated. The analytical column (250 × 4.6 mm)
operated at 1 mL/min, with a photodiode array detector 996,
wavelength 214 nm. Solvent A consisted of 0.1% TFA in H2O,
and solvent B consisted of 0.1% TFA in acetonitrile. After
lyophilisation, the product was stored at -20 °C.
Cyclization of Protected Peptide. The resulting protected linear
hexapeptide (220 mg, 0.22 mmol)) was allowed to cyclize for 90
min at high concentration in DMF (2.2 mL), with HBTU (125 mg,

Synthesis of JMV2012, an Analogue of Neurotensin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1615
performed with homogenates freshly prepared from COS 7 cells
(0.01 mg protein per tube) expressing either the human NTS1 or
NTS2. Competition experiments were carried out in 250 µL of 50
mM Tris/HCl buffer, pH 7.5, containing 0.2% BSA and 1 mM
o-phenanthroline. Homogenates, 10 µg of protein from cells, were
incubated for 30 min at 25 °C with increasing concentrations of
compound 1 and 125I-Tyr3-NT (100 Ci/mmol, 0.05 nM). Total,
specific, and nonspecific binding were determined by using the
filtration method, as described previously. It was checked that
membranes from untransfected COS cells were devoid of specific
125I-Tyr3-NT binding.
Analgesic Tests. Hot Plate Test. This test was derived from
that of Eddy and Leimbach (1953).36 A plastic cylinder (height )
20 cm, diameter ) 14 cm) was used to confine the mouse to the
heated surface of the plate. The plate was heated to a temperature
of 55 ( 0.5 °C, using a thermoregulated water circulating pump.
For each animal, the paw licking and the jump latencies were
determined. To avoid injury, mice that did not respond within 240 s
were removed from the hot plate. Each mouse was tested only once
and sacrificed immediately thereafter. This protocol was approved
by the Regional Ethical Committee for Animal Research (Nor-
mandy) with the following number: N/12-04-04-14 (hot plate test).
Writhing Test. This test was derived from that of Sigmund et
al. (1957).37 Mice received intraperitoneally (i.p.) a 0.5% acetic
acid solution in a volume of 10 mL/kg. They were then placed
individually in large beakers. The stretches were counted over a 5
min period from the fifth minute after the acetic acid solution
injection. A stretch was characterized by an elongation of the body,
the development of tension in the abdominal muscles, and the
extension of the forelimbs. This protocol was approved by the
Regional Ethical Committee for Animal Research (Normandy) with
the following number: N/01-05-04-20 (writhing test).
Body Temperature. Colonic temperature was measured with a
thermistor probe (Thermalert TH-5, Physitemp, NJ, U.S.A.) intro-
duced to a depth of 2 cm into the rectum. This protocol was
approved by the Regional Ethical Committee for Animal Research
(Normandy) with the following number: N/16-04-04-18 (measure-
ment of colonic temperature in rat and mouse).
Statistical Analysis. The data were expressed as means ( SEM.
Results were analyzed using one-way analysis of variance or two-
way analysis of variance, when appropriate, followed by Student
Newman Keul’s comparisons. Comparisons between two groups
were analyzed by Student’s t-test. For the study of the hypothermic
effect of compound 1, statistical analysis was performed using a
two-way repeated measures followed by a Student Newman Keul’s
test. A p-value of <0.05 was considered significant, and statistical
analyses were performed with SigmaStat (SPSS Inc., Chicago, IL,
U.S.A.).
Figure 7. Analgesic effect of compound 1 administered at the dose
of 30 mg/kg per os in the writhing test. Mice were treated with
compound 1 15 min before receiving acetic acid solution 0.5% (i.p.).
Mice were then placed individually in large beakers and the stretches
were counted over a 5 min period 5 min after the acetic acid solution
injection. M ( SEM from 10 mice per group; (a) p < 0.05 vs saline
controls.
0.33 mmol) as coupling reagent and TEA (300 µl, 2.20 mmol) as
base (Table 1). After concentration under reduced pressure, the
residue was dissolved in ethyl acetate (6 mL) and then successively
washed (3 × 1.5 mL) with 1 M aqueous solution of KHO4S,
saturated NaCl aqueous solution, saturated NaHCO3 aqueous
solution, and water. The ethyl acetate phase was dried over MgSO4,
filtered off, then concentrated under reduced pressure to afford a
colorless oil, which precipitated from ether/hexane (1/5) to afford
a white solid (92% yield, 195 mg).
Preparation of Free Cyclic Peptide. The dried residue from
the cyclization reaction (150 mg, 0.15 mmol) was dissolved in a
mixture TFA/anisole (8/2, 2 mL) and stirred for 30 min. The
reaction mixture was evaporated under reduced pressure and then
coevaporated several times with hexane to remove residual TFA.
Addition of a solution ether/hexane (1/5) allowed the peptide to
precipitate as a TFA salt. The afforded solid was dried under
vacuum and then purified on preparative HPLC.
These conditions afforded the expected cyclic dimer (1) in 85%
yield, after purification. tR ) 18.1 min (20–50% B, 30 min, C18).
ES-MS [M + H]+ 1485.9.
Animals. Male Swiss albino mice (CD1, Charles River,
L’Arbresle, France) weighing 20–22 g were obtained at least one
week before the beginning of the experiments. The animals were
housed in a room maintained at a constant temperature (21 ( 1
°C), with a regular light cycle (light on between 7 a.m. and 7 p.m.).
Food and water were freely available, except at the time of testing.
This study was performed in accordance with the guidelines
published in the NIH Guide for the Care and Use of Laboratory
Animals (National Institutes of Health No. 85-23, revised 1985),
and the principles were presented in the European Communities
Council Directive of 24 November 1986 (86/609/EEC).
Drugs and Solutions. c(Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu-
Lys-Lys-Pro) 1 was dissolved in saline. Levocabastine (a generous
gift from Janssen-Cilag laboratory, Beerse, Belgium) was dissolved
in dimethylsulfoxide (DMSO) and cremophor EL and then diluted
with saline to a final concentration of 5% DMSO and 5%
cremophor.
Compound 1 was injected in mice intravenously (i.v.), subcu-
taneously (s.c.), and by a gastric canula (per os) in a volume of 10
mL/kg. The intracerebroventricular injections (icv.) were performed
according to the method of Haley and Mc Cormick (1957)35 in a
volume of 10 µL/mouse. These protocols were approved by the
Regional Ethical Committee for Animal Research (Normandy) with
the following numbers: N/10-04-04-12 (intracerebroventricular
injection in mouse) and N/04-05-04-23 (per os injection).
Acknowledgment. This work was supported by a grant from
the Société Française de Pharmacologie et de Thérapeutique.
Supporting Information Available: Mass spectrometry and
HPLC tracing for compound 1. This material is available free of
charge via the Internet at the http://pubs.acs.org.
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