Evaluation of N-substitution in 6,7-benzomorphan compounds

Article abstract of DOI:10.1016/j.bmc.2010.06.005

6,7-Benzomorphan derivatives, exhibiting different μ, δ, and κ receptor selectivity profiles depending on the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic agents. In this work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide spacer for the synthesis of a new series of 5,9-dimethyl-2′-hydroxy-6,7-benzomorphan derivatives (12-22). Data obtained by competition binding assays showed that the μ opioid receptor seems to prefer an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer and less hindered amide. Highly stringent features are required for δ receptor interaction, while an N-acetamide spacer and/or bulkier amide could preferentially lead to κ receptor selectivity. In the propanamide series, compound 12 (named LP1) displayed high μ affinity (K_i = 0.83 nM), good δ affinity (K_i = 29 nM) and low affinity for the κ receptor (K_i = 110 nM), with a selectivity ratio δ/μ and κ/μ of 35.1 and 132.5, respectively. Further, in the adenylyl cyclase assay, LP1 displayed a μ/δ agonist profile, with IC₅₀ values of 4.8 and 12 nM at the μ and δ receptors, respectively. The antinociceptive potency of LP1 in the tail-flick test after sc administration in rat was comparable with the potency of morphine (ED₅₀ = 2.03 and 2.7 mg/kg, respectively), and was totally reversed by naloxone. LP1, possessing a μ/δ agonist profile, could represent a lead in further developing benzomorphan-based ligands with potent in vivo analgesic activity and a reduced tendency to induce side effects.

Full text of DOI:10.1016/j.bmc.2010.06.005

Bioorganic & Medicinal Chemistry 18 (2010) 4975–4982
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of N-substitution in 6,7-benzomorphan compounds
a
a
a
a
Lorella Pasquinucci a, , Orazio Prezzavento , Agostino Marrazzo , Emanuele Amata , Simone Ronsisvalle ,
Zafiroula Georgoussi b, Danai-Dionysia Fourla b, Giovanna M. Scoto c, Carmela Parenti c, Giuseppina Aricò c,
Giuseppe Ronsisvalle a
*
a Department of Pharmaceutical Sciences, Medicinal Chemistry Section, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
b Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of Biology, National Center for Scientific Research ‘‘Demokritos” Ag. Paraskevi 15310, Athens, Greece
c Department of Pharmaceutical Sciences, Pharmacology Section, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
6,7-Benzomorphan derivatives, exhibiting different l, d, and j receptor selectivity profiles depending on
Received 24 February 2010
Revised 27 May 2010
Accepted 4 June 2010
Available online 09 June 2010
the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic agents. In this
work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide
spacer for the synthesis of a new series of 5,9-dimethyl-20-hydroxy-6,7-benzomorphan derivatives
(12–22). Data obtained by competition binding assays showed that the
l opioid receptor seems to prefer
an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer
and less hindered amide. Highly stringent features are required for d receptor interaction, while an
Keywords:
6,7-Benzomorphan derivatives
N-acetamide spacer and/or bulkier amide could preferentially lead to
panamide series, compound 12 (named LP1) displayed high affinity (Ki = 0.83 nM), good d affinity
(Ki = 29 nM) and low affinity for the receptor (Ki = 110 nM), with a selectivity ratio d/ and of
35.1 and 132.5, respectively. Further, in the adenylyl cyclase assay, LP1 displayed a /d agonist profile,
with IC50 values of 4.8 and 12 nM at the and d receptors, respectively. The antinociceptive potency
of LP1 in the tail-flick test after sc administration in rat was comparable with the potency of morphine
(ED50 = 2.03 and 2.7 mg/kg, respectively), and was totally reversed by naloxone. LP1, possessing a /d
j receptor selectivity. In the pro-
l/d opioid agonist
l
cAMP accumulation assay
Tail-flick test
j
l
j/l
l
l
l
agonist profile, could represent a lead in further developing benzomorphan-based ligands with potent
in vivo analgesic activity and a reduced tendency to induce side effects.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
versus the l, d, and j receptors as well as agonist and antagonist
functional activity.6 Variable length N-alkyl chains and the
presence of polar groups or unsaturations in the N-substituent dif-
ferentially affected the pharmacological profile of 5,9-dimethyl-20-
hydroxy-6,7-benzomorphans.7–10 Moreover, a higher potency was
displayed when an N-phenethyl substituent was used instead of
N-methyl, while an N-benzyl substituent produced an inactive
compound.11 In spite of reported data, in the N-substituent the
molecular interactions responsible for the in vitro and in vivo char-
acteristics are not evident, and only a small amount of information
is known about the N-substituent features required to obtain selec-
tivity for a specific opioid receptor subtype.
The opioid receptor system is regarded as one of the most impor-
tant structures in nociception and antinociception. The distribution
pattern of the opioid receptor system is closely related to the circuit
that selectively controls nociceptive transmission.1 Opioid agonists
modulate neurons of this circuit by supporting a pain-inhibiting
state; therefore, the treatment of moderate to severe pain is mainly
based on opioid analgesics.2,3 The opioid system comprises three
classical receptor subtypes:
l, d, and j receptors, all members of
the large G-protein-coupled-receptor family, whose genes are char-
acterized by a high degree of sequence homology.4
Opioid receptors are the target of numerous ligands, including
endogenous or synthetic peptides, opiates and semi-synthetic opi-
oids, and synthetic derivatives with different structural scaffolds.5
In the 6,7-benzomorphan class of ligands, the nature of the N-sub-
stituent is critical for the modulation of affinity and selectivity
An examination of the structures of endogenous opioid ligands
and their synthetic analogs reveals that the Tyr1 and Phe3/Phe4 res-
idues are structural determinants for binding to opioid receptors.
Their distances and reciprocal conformations deeply influence
the affinity and selectivity for the different opioid receptor sub-
types.12–15 Assuming that the tyramine-like moiety of the benzo-
morphan nucleus interacts with opioid receptors in a similar
mode to Tyr1 of endogenous ligands, the introduction of a second
aromatic ring in the N-substituent, and its localization with respect
* Corresponding author. Tel.: +39 095 7384273; fax: +39 095 222239.
E-mail address: lpasquin@unict.it (L. Pasquinucci).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.005

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L. Pasquinucci et al. / Bioorg. Med. Chem. 18 (2010) 4975–4982
3. Results and discussion
HO
3.1. Opioid receptor affinity
R1
N
Opioid binding affinities of compounds 12–22 for the l, d, and j
N
R2
n
receptors were determined by radioligand competition analysis
using [3H]DAMGO, [3H]DPDPE, and [3H]U69,593, respectively. These
O
data, in comparison with standard compounds Tyr-
MePhe-Gly-ol (DAMGO), Tyr-c( -Pen-Gly-Phe- -Pen (DPDPE), and
U50,488 are summarized in Table 1. Compounds 12–18, bearing
an N-propanamide spacer, had affinity for the opioid receptor in
the nanomolar range (Kli = 0.83–372 nM). Compound 12 (named
D-Ala-Gly-N-
D
D
12-18
19-22
n = 1
1 = Ph, c-C6H11, Bn, CH2-c-C6H11, t-Bu
R
l
R2 = H, Me, Et
LP1), with a phenyl amide substituent, displayed the highest
l
affinity, similar to that of reference compound DAMGO (Kli = 0.83
and 0.87 nM, respectively). With respect to LP1, all other amide
n = 0
R1 = Ph, c-C6H11
substituents led to reduced
substituent from phenyl (LP1) to cyclohexyl (compound 13) de-
creased
affinity by more than 60-fold (Kli = 56 nM). In com-
pounds 14 and 15, which had a secondary amide with R1 = Me
l affinity. Changing the primary amide
R2 = H, Me, Et
l
Figure 1. Structures of N-substituted 6,7-benzomorphan ligands 12–22.
and Et, respectively,
l affinity decreased as a function of the steric
bulk of the alkyl amide substituents (Kli = 65 and 136 nM, respec-
tively). On the other hand, attempts to increase the distance
between aromatic/alicyclic amide substituents and the benzomor-
to the tyramine moiety, becomes crucial in achieving a determi-
nate pharmacological profile. Therefore, we prepared compounds
12–22 by introducing different functional groups on the N-substi-
tuent of (ꢀ)-(1R,5R,9R)-5,9-dimethyl-20-hydroxy-6,7-benzomor-
phan, such as an aromatic ring and/or alkyl residues linked by an
N-propanamide or N-acetamide spacer (Fig. 1). By having rotatable
groups on the N-substituent, these compounds could differentially
bind opioid receptor subtypes.
phan core (compounds 16 and 17) failed to improve the
(Kli = 105 and Kli = 107 nM, respectively). Finally, the compound
bearing a bulky tert-butyl substituent (18) displayed the lowest
affinity (Kli = 372 nM). Shortening of the spacer in the N-acetamide
compounds (19–22) produced a significant loss of affinity rela-
tive to their N-propanamide homologs (12–15). With respect to
LP1, compound 19 displayed a reduction in Ki values of almost
two orders of magnitude (Kil = 0.83 and Kli = 722 nM, respectively).
LP1 also exhibited good d opioid receptor affinity (Kdi = 29 nM),
while all the other compounds had no d affinity. All compounds
l affinity
l
l
2. Chemistry
The synthesis of the N-benzomorphan compounds started by
separating cis-(ꢀ)-(1R,5R,9R)-N-normetazocine froma racemicmix-
ture, as previously reported.16 Amides 1–8 and 9–11 were prepared
following reported procedures by amine acylation in anhydrous
tetrahydrofuran (THF) at 0 °C with 3-bromopropionyl chloride and
2-chloroacetyl chloride, respectively.17 The target compounds
12–22 were obtained by alkylation of cis-(ꢀ)-(1R,5R,9R)-N-nor-
metazocine with the respective amides 1–11 in N,N-dimethylform-
amide (DMF) at 50 °C in the presence of NaHCO3 (Scheme 1).17
displayed a low affinity for the
tion of compound 15 (Kji = 70 nM). Moreover, the binding data
suggested a preference for the receptor of compounds having a
shorter spacer (20–22) and/or a bulkier amide substituent (15,
21–22), although the affinities were not relevant. In summary,
j receptor, with the partial excep-
j
the
l opioid receptor seems to prefer interaction with the 6,7-ben-
zomorphan ligands having an N-substituent with a propanamide
spacer and aromatic ring. Highly stringent features are required
for d receptor interaction, while an N-acetamide spacer and/or
O
O
a
H
R1
R1
N
X
Cl
X
N
n
n
R2
R2
1-11
X = Cl, Br
b
1-7, 12-18
n = 1
HO
R
R
1 = Ph, c-C6H11, Bn, CH2-c-C6H11, t-Bu
2 = H, Me, Et
R1
N
N
8-11, 19-22
n = 0
R1 = Ph, c-C6H11
R2
n
O
R2 = H, Me, Et
12-22
Scheme 1. The synthetic pathway to compounds 12–22. Reagents and conditions: (a) DMAP, THF, 0 °C, 1 h; (b) cis-(ꢀ)-(1R,5R,9R)-N-normetazocine, NaHCO3, KI, DMF, 50 °C,
4 h.

L. Pasquinucci et al. / Bioorg. Med. Chem. 18 (2010) 4975–4982
4977
Table 1
Opioid receptor binding affinities of compounds 12–22
HO
R1
N
N
R2
n
O
Compound
n
R1
R2
Ki (nM) SEMa,b
Ki ratio
l
d
j
d/
l
j/l
12 (LP1)
13
14
15
16
17
18
19
20
21
22
DAMGO
DPDPE
U50,488
1
1
1
1
1
1
1
0
0
0
0
H
H
Me
Et
H
H
H
H
H
Ph
c-C6H11
Ph
0.83 0.05
29.1
1
110
501 25
261 14
70
237 13
134
422 20
>5000
612 27
339 17
335 17
>5000
>5000
1.14 0.7
6
33.8
>89.2
>76.9
>36.7
>47.6
>46.7
>13.4
>6.9
132.5
8.9
4.0
0.51
2.2
1.2
1.1
>6.9
0.20
0.24
0.29
56
65
136
105
107
3
3
7
6
6
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
Ph
4
Benzyl
CH2-c-C6H11
t-Bu
Ph
c-C6H11
Ph
7
372 18
722 40
2930 161
1370 69
1120 66
0.87 0.6
2120 112
1750 93
>1.7
>3.6
>4.4
Me
Et
Ph
2670 112
2.72 0.9
>5000
a
Values are means SEM of three separate experiments, each carried out in duplicate.
Ki values were obtained as [3H]DAMGO displacement for the receptor, [3H]DPDPE displacement for the d receptor, and [3H]U69,593 displacement for the
b
l
j
receptor.
bulkier amide substituents could preferentially lead to
interactions.
j
receptor
of cAMP accumulation were performed in HEK293 cells stably
expressing the d opioid receptor. LP1 from 100 pM to 10
lM
The most promising compound, LP1, was further evaluated for its
ability to inhibit adenylyl cyclase in HEK293 cells stably expressing
revealed a dose-dependent inhibition of cAMP accumulation, with
an IC50 of 12 nM and an efficacy of 83%. d-Selective agonist DPDPE
displayed a lower IC50 value of 0.14 nM, with a maximal inhibition
of 69% (Fig. 3). LP1 was approximately a 100-fold less potent than
DPDPE at inhibiting cAMP accumulation. This data was unexpected
compared to the binding values at the d receptor, with LP1 having
only a 10-fold lower affinity with respect to DPDPE. Naloxone at
either the
l or d opioid receptor, and by the tail-flick test in male
Sprague–Dawley rats in order to characterize its in vivo antinocicep-
tive effects.
3.2. Adenylyl cyclase-mediated effects
10
HEK293 cells stably expressing both
not shown). These results suggest that LP1 acts in vitro as a potent
opioid agonist with a lower d agonist activity.
l
M completely reversed LP1-mediated cAMP inhibition in
Opioid receptors typically signal through coupling to Gi/Go pro-
teins to inhibit adenylyl cyclase.18 To examine the functional role
of LP1 in opioid receptor signaling, the ability of LP1 to affect for-
skolin-stimulated adenylyl cyclase activity in HEK293 cells stably
l
or d opioid receptors (data
l
expressing the
are reported in Table 2. Treatment of HEK293 cells stably express-
ing the opioid receptor with LP1 from 10 pM to 10 M revealed a
l or the d opioid receptor was tested and the results
l
l
dose-dependent inhibition of cAMP accumulation, with an IC50 of
4.8 nM and an efficacy of 73%. The efficacy of LP1 at inhibiting
adenylyl cyclase was similar to that displayed by the
l selective
agonist DAMGO (IC50 = 3.18 nM; Imax = 73%) (Fig. 2). To elucidate
the effect of LP1 at the d opioid receptor, similar measurements
Table 2
Measurements of adenylyl cyclase
Compound
IC50 (nM) SDa,b
Imax SDc
l
d
l
d
LP1
DAMGO
DPDPE
4.8 0.5
3.18 0.3
ND
12 1.2
NDd
0.14 0.03
73 3.8
73 0.3
ND
83 1.2
ND
69 0.7
a
Agonist properties of compounds in the inhibition of forskolin-stimulated cAMP
accumulation by and d opioid receptors. The inhibition of cAMP accumulation was
Figure 2. The effect of LP1 on cAMP accumulation by the
HEK293 cells stably expressing the
opioid receptor were labeled with [3H]adenine
(1.5
Ci/mL), and generation of [3H]cAMP was measured in response to treatment
with 50 M forskolin. Forskolin-stimulated cAMP accumulation was also measured
in the presence of various concentrations of the opioid receptor agonist DAMGO
(h). The data are presented as cAMP accumulation (percentage of maximum) and
represent the average SD of triplicate determinations from four independent
experiments.
l opioid receptor (j).
l
l
measured as described in Section 5.
l
b
IC50 value is the concentration of the compound needed to produce half max-
l
imal inhibition, with all values presented as the average SD of triplicate deter-
minations from four independent experiments.
l
c
ICmax value is the maximal percent inhibition obtained with the compound.
ND, not determined.
d

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L. Pasquinucci et al. / Bioorg. Med. Chem. 18 (2010) 4975–4982
Figure 4. Naloxone (3 mg/kg sc) pre-treatment and post-treatment antagonizes the
LP1 (4 mg/kg sc) analgesic response. The arrow indicates the administration of
naloxone at the peak of the LP1 effect. Data are expressed as means SE (n = 8–10).
Differences were considered significant when p < 0.05.
Figure 3. The effect of LP1 on cAMP accumulation by the d opioid receptor (d).
HEK293 cells stably expressing the d opioid receptor were labeled as described in
Figure 2. Forskolin-stimulated cAMP accumulation was also measured in the
presence of various concentrations of the d opioid receptor agonist DPDPE (s). Data
are presented as cAMP accumulation (percentage of maximum) and represent the
average SD of triplicate determinations from five different experiments.
18, the optimal opioid binding affinities at both
l and d receptors
occurred with LP1, which has an N-propanamide spacer and a phe-
nyl amide substituent. All the structural modifications evaluated
Table 3
reduced the
all attempted modulations induced a complete loss of affinity.
Higher receptor selectivity was present in compounds with an
l affinity compared to LP1, while on the d receptor,
ED50 values of LP1 and morphine in the tail-flick test
a
Compound
ED50 mg/kg sc (C.L.95%)
j
N-acetamide spacer and/or bulkier amide substituent.
LP1 (2–5 mg/kg sc)
Morphine (1–10 mg/kg sc)
2.03 (1.53–2.53)
2.7 (2.56–2.94)
The in vivo tail-flick test, performed to assess the pharmacolog-
ical properties of LP1, showed that LP1 is a potent analgesic and
that its effect is opioid receptor-mediated. This evidence, together
with measurements of cAMP accumulation, suggests that LP1 pos-
a
ED50 value is the dose of the compound needed to produce half maximal effect.
Confidence limits 95% are given in brackets.
sesses a peculiar mixed
l/d agonist profile. The relevance of simul-
3.3. Tail-flick test
taneous and d agonist activation to produce analgesia with
l
reduced tolerance is well established, and is important in produc-
ing useful drugs for chronic pain treatment.19 Clinically used opi-
oids exert their pain relief action mainly through the activation
The antinociceptive effect induced by subcutaneous (sc) admin-
istration of LP1 using the tail-flick test in rats was evaluated. LP1
produced a dose-dependent analgesic effect in the dose-range of
2–5 mg/kg. Table 3 shows the ED50 value of LP1 with its confidence
limits (C.L.95%) compared to the ED50 value of morphine. LP1, at a
dose of 4 mg/kg sc, significantly increased the nociceptive latency,
demonstrating a clear analgesic effect compared to the group of
of the
l receptor. However, their prolonged administration pro-
duces tolerance to the analgesic effects, requiring escalating doses
that are associated with side effects such as respiratory depression,
constipation, and physical dependence, which limit their therapeu-
tic potential.20,21 In view of these findings, the mixed
l/d agonist
*
rats treated with saline ( p < 0.05 vs saline-treated rats). The cut-
LP1 could be useful as an analgesic agent for chronic pain treat-
ment. Further, in vivo experiments are in progress to evaluate its
tolerance-inducing capability.
off latency of 10 s, established as the maximal analgesic threshold,
was reached 20 min after injection, and lasted until 80 min after
treatment. Pretreatment with naloxone (3 mg/kg sc, 30 min prior
to LP1) prevented the analgesic effect induced by LP1 at a dose
of 4 mg/kg sc (Fig. 4). The administration of naloxone at the peak
of the LP1 effect caused a fast decrease of tail-flick latencies (TFLs),
as indicated by the arrow in Figure 4. All of these data established
that the LP1 analgesic effect was comparable to morphine and was
opioid receptor-mediated.
5. Experimental
5.1. General
All commercial chemicals were used as received from Aldrich
Chemical Co. unless otherwise specified. cis-( )-N-Normetazocine
was obtained from Fabbrica Italiana Sintetici. Melting points were
determined in open capillary tubes with a Büchi 530 apparatus and
are uncorrected. Analytical thin-layer chromatography was
performed on silica gel 60 F254 aluminum sheets (Merck) with fluo-
rescent indicator. Components were visualized by UV light (k = 254
and 366 nm) and iodine vapor. Flash column chromatography was
performed on Merck silica gel 60 (230–400 mesh). Optical rota-
tions were determined in MeOH solution with a Perkin-Elmer
241 polarimeter. Infrared spectra were recorded on a 1600 FT-IR
Perkin-Elmer instrument. 1H and 13C NMR spectra were routinely
recorded at a constant temperature of 27 °C on a Varian Inova-200
spectrometer in CDCl3 solution; chemical shifts d are expressed in
4. Conclusion
In this study, our attention was focused on the evaluation of
N-substitution in 6,7-benzomorphan compounds to extend the
body of information about this structural region in the opioid
receptor interaction. Collectively, the Ki values obtained from com-
petition binding assays confirmed that the N-substituent nature
influences the
variable way. We observed that an N-propanamide chain (com-
pounds 12–18) allows an improved affinity with respect to an
N-acetamide chain (compounds 19–22). Among compounds 12–
l, d, and j receptor affinity and selectivity in a very
l

L. Pasquinucci et al. / Bioorg. Med. Chem. 18 (2010) 4975–4982
4979
ppm with reference to TMS as an internal standard. The data are
reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet), coupling con-
stants (Hz) and number of protons. Unless otherwise specified,
all final compounds were purified to P95% purity, as determined
by elemental analysis using a Carlo Erba analyzer. Receptor radio-
ligands [3H]DAMGO, [3H]DPDPE, and [3H]U69,593 were purchased
from Perkin-Elmer Life Sciences. Some of the propanamide and
acetamide compounds are known, although we report the experi-
mental procedures and spectral characterization since they were
prepared by different synthetic routes.
C10H12BrNO: C, 49.61; H, 5.00; N, 5.79. Found: C, 49.78; H, 5.54;
N, 5.67.
5.2.1.6. 3-Bromo-N-(cyclohexylmethyl)propanamide (6). Col-
orless oil (53%); IR (neat, cmꢀ1): 1640; 1H NMR (CDCl3): d 5.88
(s, 1H, exchangeable in D2O), 3.65 (t, J = 6.6 Hz, 2H), 3.15–3.13
(m, 2H), 2.76 (t, J = 6.6 Hz, 2H), 1.76–1.63 (m, 5H), 133–1.11 (m,
3H), 1.57–1.39 (m, 1H), 1.01–0.84 (m, 2H). 13C NMR (CDCl3): d
169.63, 45.86, 39.84, 37.81, 30.73, 27.75, 26.29, 25.73. Anal. Calcd
for C10H18BrNO: C, 48.40; H, 7.31; N, 5.64. Found: C, 48.61; H, 7.44;
N, 5.32.
5.2.1.7. 3-Bromo-N-(tert-butyl)propanamide (7). Colorless oil
(40%); IR (neat, cmꢀ1): 1648; 1H NMR (CDCl3): d 5.40 (s, 1H,
exchangeable in D2O), 3.63 (t, J = 6.6 Hz, 2H), 2.67 (t, J = 6.6 Hz,
2H), 1.38 (s, 9H). 13C NMR (CDCl3): d 168.23, 51.61, 40.42, 28.71,
27.90. Anal. Calcd for C7H14BrNO: C, 40.40; H, 6.78; N, 6.73. Found:
C, 40.44; H, 6.69; N, 6.65.
5.2. Compounds synthesis
5.2.1. General procedure for the preparation of 3-bromo-N-
substituted- and 3-bromo-N,N-disubstituted propanamide
derivatives (1–7)
To a solution of 3-bromopropionyl chloride (5.14 g, 3.02 mL,
30 mmol) in THF (10 mL) cooled at 0 °C and kept under vigorous
stirring, a solution of the appropriate amine (20 mmol) and
4-(dimethylamino)pyridine (DMAP) (1.15 g, 9.4 mmol) in THF
(20 mL) was added dropwise. After 1 h, the reaction mixture was
quenched with H2O and extracted with CHCl3. The organic phase
was washed with a saturated aqueous solution of NaHCO3, dried
over anhydrous Na2SO4 and evaporated. The crude product was
purified by flash chromatography on silica gel using cyclohexane
and ethyl acetate (AcOEt) as solvents.
5.2.2. General procedure for the preparation of 2-chloro-N-
substituted- and 2-chloro-N,N-disubstituted acetamide
derivatives (8–11)
By employing the previously-described procedure for the pro-
panamide derivatives, the following compounds were also
prepared.
5.2.2.1. 2-Chloro-N-phenylacetamide (8). White solid (96%);
mp: 136–138 °C; IR (KBr, cmꢀ1): 1669; 1H NMR (CDCl3): d 8.44
(s, 1H, exchangeable in D2O), 7.55–7.54 (m, 2H), 7.50–7.27 (m,
2H), 7.16–7.09 (m, 1H), 4.16 (s, 2H). 13C NMR (CDCl3): d 164.00,
136.86, 129.02, 125.09, 120.10, 42.91. Anal. Calcd for C8H8ClNO:
C, 56.65; H, 4.75; N, 8.26. Found: C, 56.74; H, 4.91; N, 8.17.
5.2.1.1. 3-Bromo-N-phenylpropanamide (1). White solid (80%);
mp 123–124 °C; IR (KBr, cmꢀ1): 1658; 1H NMR (CDCl3): d 7.91 (s,
1H, exchangeable in D2O), 7.54–7.50 (m, 2H), 7.37–7.26 (m, 2H),
7.16–7.09 (m, 1H), 3.71 (t, J = 6.6 Hz, 2H), 2.94 (t, J = 6.6 Hz, 2H).
13C NMR (CDCl3): d 168.33, 137.36, 128.95, 124.69, 120.31, 40.38,
27.13. Anal. Calcd for C9H10BrNO: C, 47.39; H, 4.42; N, 6.14. Found:
C, 47.55; H, 4.67; N, 6.10.
5.2.2.2. 2-Chloro-N-cyclohexylacetamide (9). White solid (87%);
mp: 113–115 °C; IR (KBr, cmꢀ1): 1648; 1H NMR (CDCl3): d 6.40 (s,
1H, exchangeable in D2O), 3.96 (s, 2H), 3.76–3.67 (m, 1H), 1.88–
1.82 (m, 2H), 1.72–1.52 (m, 3H), 1.42–1.04 (m, 5H). 13C NMR
(CDCl3): d 164.77, 48.57, 42.66, 32.70, 25.33, 24.63. Anal. Calcd
for C8H14ClNO: C, 54.70; H, 8.03; N, 7.97. Found: C, 54.85; H,
8.21; N, 7.78.
5.2.1.2. 3-Bromo-N-cyclohexylpropanamide (2). White solid
(46%); mp 98–100 °C IR (KBr, cmꢀ1): 1640; 1H NMR (CDCl3): d
5.47 (s, 1H, exchangeable in D2O), 3.90–3.77 (m, 1H), 3.65 (t,
J = 6.6 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 1.96–1.90 (m, 2H), 1.75–
1.46 (m, 3H), 1.42–1.12 (m, 5H). 13C NMR (CDCl3): d 168.57,
48.37, 39.85, 32.97, 27.78, 25.36, 24.71. Anal. Calcd for C9H16BrNO:
C, 46.17; H, 6.89; N, 5.98. Found: C, 46.32; H, 6.95; N, 5.92.
5.2.2.3. 2-Chloro-N-methyl-N-phenylacetamide (10). White so-
lid (90%); mp: 77–79 °C; IR (KBr, cmꢀ1): 1680; 1H NMR (CDCl3): d
7.47–7.42 (m, 3H), 7.28–7.23 (m, 2H), 3.86 (s, 2H), 3.32 (s, 3H).
13C NMR (CDCl3): d 166.16, 142.57, 129.98, 128.49, 126.96, 41.45,
37.89. Anal. Calcd for C9H10ClNO: C, 58.86; H, 5.49; N, 7.63. Found:
C, 58.92; H, 5.55; N, 7.54.
5.2.1.3. 3-Bromo-N-methyl-N-phenylpropanamide (3). Color-
less oil (79%); IR (neat, cmꢀ1): 1659; 1H NMR (CDCl3): d 7.50–
7.33 (m, 3H), 7.26–7.18 (m, 2H), 3.60 (t, J = 6.8 Hz, 2H), 3.30 (s,
3H), 2.65 (t, J = 6.8 Hz, 2H). 13C NMR (CDCl3): d 169.99, 143.28,
129.91, 128.10, 127.35, 37.32, 37.07, 27.47. Anal. Calcd for
5.2.2.4. 2-Chloro-N-ethyl-N-phenylacetamide (11). Colorless oil
(93%); IR (neat, cmꢀ1): 1669; 1H NMR (CDCl3): d 7.51–7.41 (m,
3H), 7.28–7.21 (m, 2H), 3.81 (s, 2H), 3.79 (q, J = 7.2 Hz, 2H), 1.15
(t, J = 7.2 Hz, 3H). 13C NMR (CDCl3): d 165.59, 140.84, 129.91,
128.61, 128.11, 44.81, 41.91, 12.68. Anal. Calcd for C10H12ClNO:
C, 60.76; H, 6.12; N, 7.09. Found: C, 60.82; H, 6.21; N, 7.00.
C10H12BrNO: C, 49.61; H, 5.00; N, 5.79. Found: C, 49.75; H, 5.21;
N, 5.63.
5.2.1.4. 3-Bromo-N-ethyl-N-phenylpropanamide (4). Colorless
oil (70%); IR (neat, cmꢀ1): 1658; 1H NMR (CDCl3): d 7.50–7.34
(m, 3H), 7.20–7.13 (m, 2H), 3.78 (q, J = 7.2 Hz, 2H), 3.59 (t,
J = 7.0 Hz, 2H), 2.60 (t, J = 7.0 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H). 13C
NMR (CDCl3): d 170.04, 141.53, 129.80, 128.42, 128.19, 44.09,
37.35, 27.59, 27.95. Anal. Calcd for C11H14BrNO: C, 51.58; H,
5.51; N, 5.47. Found: C, 51.71; H, 5.53; N, 5.35.
5.2.3. General procedure for the preparation of N-substituted cis
-(ꢀ)-N-normetazocine derivatives (12–22)
A
mixture of cis-(ꢀ)-(1R,5R,9R)-N-normetazocine (500 mg,
2.3 mmol), the appropriate amide (1–11, 3.45 mmol), NaHCO3
(289.83 mg, 3.45 mmol) and a catalytic amount of KI was stirred in
DMF (10 mL) at 50 °C for 4 h. After cooling, the reaction mixture
was diluted with AcOEt (200 mL) and H2O (30 mL). The organic layer
was separated, washed with brine, dried over anhydrous Na2SO4,
and evaporated in vacuo. The residue was purified by flash chroma-
tography using CHCl3 and CH3OH as solvents. Compounds 12–22
were recrystallized from absolute ethanol to give white solids.
5.2.1.5. N-Benzyl-3-bromopropanamide (5). Colorless oil (70%);
IR (neat, cmꢀ1): 1639; 1H NMR (CDCl3): d 7.32–7.25 (m, 5H), 6.30
(s, 1H, exchangeable in D2O), 4.42 (d, 2H), 3.61 (t, J = 6.6 Hz, 2H),
2.75 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3):
d
169.60, 137.80,
128.64, 127.71, 127.49, 43.63, 39.41, 27.48. Anal. Calcd for

4980
L. Pasquinucci et al. / Bioorg. Med. Chem. 18 (2010) 4975–4982
5.2.3.1. 3-[(2R,6R,11R)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetra-
hydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropana-
(m, 1H), 6.76–6.75 (m, 1H), 6.68–6.63 (m, 1H), 3.15–3.09 (m,
2H), 3.08–2.57 (m, 6H), 2.43–2.40 (m, 2H), 2.19–2.05 (m, 1H),
1.77–1.73 (m, 7H), 1.43–1.18 (m, 4H), 1.35 (s, 3H), 1.12–0.85 (m,
3H), 0.87 (d, J = 5 Hz, 3H). 13C NMR (CDCl3): d 172.99, 154.68,
142.45, 128.15, 127.15, 113.25, 112.30, 57.59, 50.62, 45.40, 44.58,
42.02, 41.93, 37.88, 36.31, 31.74, 30.96, 26.35, 25.85, 25.37,
23.33, 14.01. Anal. Calcd for C24H36N2O2: C, 74.96; H, 9.44; N,
7.28. Found: C, 75.03; H, 9.51; N, 7.47.
mide (12). White solid (86%); mp: 173–174 °C; ½a D20
ꢀ50° (c 1.0,
ꢁ
MeOH); 1H NMR (CDCl3): d 11.27 (s, 1H), 7.58–7.54 (m, 2H),
7.38–7.26 (m, 2H), 7.13–7.10 (m, 1H), 6.97–6.93 (m, 1H), 6.78–
6.77 (m, 1H), 6.69–6.63 (m, 1H), 3.07–2.53 (m, 6H), 2.51–2.49
(m, 2H), 2.27–2.13 (m, 1H), 1.97–1.77 (m, 2H), 1.40 (s, 3H), 1.50–
1.25 (m, 1H), 0.89 (d, J = 5 Hz, 3H). 13C NMR (CDCl3): d 170.67,
154.95, 142.08, 138.53, 129.00, 128.32, 126.41, 123.88, 119.75,
113.51, 112.39, 57.79, 50.31, 44.73, 41.40, 36.14, 32.52, 29.51,
25.21, 23.60, 14.01. Anal. Calcd for C23H28N2O2: C, 75.79; H, 7.74;
N, 7.69. Found: C, 75.44; H, 7.56; N, 7.55.
5.2.3.7. N-(tert-Butyl)-3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-
1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]propan-
amide (18). White solid (67%); mp: 157–159 °C; ½a D20
ꢀ50° (c 1.0,
ꢁ
MeOH); 1H NMR (CDCl3): d 7.28 (s, 1H), 6.93–6.89 (m, 1H), 6.79–
6.74 (m, 2H), 3.34–3.20 (m, 3H), 3.00–2.90 (m, 3H), 2.80–2.75
(m, 2H), 2.43–2.30 (m, 2H), 2.20–2.07 (m, 1H), 1.36, (s, 12H),
1.46–1.25 (m, 1H), 0.86 (d, J = 5 Hz, 3H). 13C NMR (CDCl3): d
169.88, 155.79, 140.86, 128.55, 124.14, 114.26, 112.47, 59.46,
51.50, 50.60, 45.98, 39.53, 38.98, 35.55, 32.49, 28.68, 24.61,
23.77, 13.68. Anal. Calcd for C21H32N2O2: C, 73.22; H, 9.36; N,
8.13. Found: C, 73.11; H, 9.43; N, 8.31.
5.2.3.2. N-Cyclohexyl-3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-
1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]propan-
amide (13). White solid (73%); mp: 152-154 °C; ½a D20
ꢀ52° (c 1.0,
ꢁ
MeOH); 1H NMR (CDCl3): d 7.49 (s, 1H), 6.97–6.92 (m, 1H), 6.73–
6.69 (m, 2H), 3.86–3.73 (m, 1H), 3.33–2.88 (m, 6H), 2.85–2.79 (m,
2H), 2.50–2.07 (m, 3H), 1.91–1.86 (m, 2H), 1.69–1.58 (m, 3H), 1.38
(s, 3H), 1.48–1.17 (m, 6H), 0.88 (d, J = 5 Hz, 3H). 13C NMR (CDCl3):
d 169.83, 155.68, 140.97, 128.49, 124.35, 114.15, 112.44, 59.23,
50.59, 48.39, 45.85, 39.27, 35.56, 32.85, 32.79, 31.76, 25.42, 24.73,
24.63, 23.63, 13.67. Anal. Calcd for C23H34N2O2: C, 74.56; H, 9.25;
N, 7.56. Found: C, 74.88; H, 9.04; N, 7.54.
5.2.3.8. 2-[(2R,6R,11R)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetra-
hydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylacet-
amide (19). White solid (92%); mp: 186–188 °C; ½a D20
ꢀ64° (c 1.0,
ꢁ
MeOH); 1H NMR (CDCl3): d 9.52 (s, 1H), 7.62–7.57 (m, 2H), 7.40–
7.26 (m, 2H), 7.16–7.08 (m, 1H), 6.97–6.93 (m, 1H), 6.76–6.75
(m, 1H), 6.68–6.63 (m, 1H), 3.37 (d, J = 17 Hz, 1H), 3.12 (d,
J = 17 Hz, 1H), 2.91–2.81 (m, 3H), 2.51–2.26 (m, 2H), 1.98–1.72
(m, 2H), 1.41–1.40 (m, 1H), 1.37 (s, 3H), 0.86 (d, J = 5 Hz, 3H). 13C
NMR (CDCl3): d 169.43, 154.34, 142.64, 137.54, 129.05, 128.33,
127.66, 124.24, 119.42, 113.25, 112.34, 59.46, 59.05, 46.31, 42.35,
42.24, 35.89, 26.08, 25.36, 14.04. Anal. Calcd for C22H26N2O2: C,
75.40; H, 7.48; N, 7.99. Found: C, 75.32; H, 7.12; N, 8.06.
5.2.3.3. 3-[(2R,6R,11R)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetra-
hydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-methyl-N-phenyl-
propanamide (14). White solid (82%); mp: 169–171 °C; ½a D20
ꢀ56°
ꢁ
(c 1.0, MeOH); 1H NMR (CDCl3): d 7.44–7.31 (m, 3H), 7.17–7.14 (m,
2H), 6.89–6.85 (m, 1H), 6.68–6.56 (m, 2H), 3.26 (s, 3H), 2.99–2.53
(m, 5H), 2.36–2.32 (m, 3H), 2.05–1.87 (m, 1H), 1.86–1.67 (m, 2H),
1.27 (s, 3H), 1.21–1.17 (m, 1H), 0.78 (d, J = 5 Hz, 3H). 13C NMR
(CDCl3): d 172.04, 154.57, 143.66, 142.73, 129.81, 128.10, 127.88,
127.18, 126.40, 113.18, 112.23, 58.27, 51.10, 45.65, 41.44, 40.92,
37.39, 36.09, 32.25, 25.23, 23.58, 14.00. Anal. Calcd for C24H30N2O2:
C, 76.16; H, 7.99; N, 7.40. Found: C, 76.45; H, 8.09; N, 7.65.
5.2.3.9. N-Cyclohexyl-2-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-
1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]acet-
amide (20). White solid (76%); mp: 156–158 °C; ½a D20
ꢀ65° (c 1.0,
ꢁ
5.2.3.4. 3-[(2R,6R,11R)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetra-
hydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-ethyl-N-phenyl-
MeOH); 1H NMR (CDCl3): d 7.48 (d, 1H), 6.92–6.88 (m, 1H), 6.74–
6.60 (m, 2H), 3.83–3.78 (m, 1H), 3.21 (d, J = 16.6 Hz, 1H), 2.97 (d,
J = 16.6 Hz, 1H), 2.97–2.74 (m, 4H), 2.36–2.10 (m, 2H), 2.03–1.65
(m, 6H), 1.36 (s, 3H), 1.49–1.26 (m, 6H), 0.83 (d, J = 5 Hz, 3H). 13C
NMR (CDCl3): d 170.34, 154.51, 142.57, 128.17, 127.53, 113.26,
112.34, 58.86, 58.79, 47.26, 46.13, 42.27, 42.08, 35.92, 32.91,
25.71, 25.48, 25.33, 24.55, 14.16. Anal. Calcd for C22H32N2O2: C,
74.12; H, 9.05; N, 7.86. Found: C, 74.33; H, 9.01; N, 7.95.
propanamide (15). White solid (78%); mp: 156–158 °C; ½a D20
ꢁ
ꢀ58° (c 1.0, MeOH); 1H NMR (CDCl3): d 7.45–7.33 (m, 3H), 7.17–
7.12 (m, 2H), 6.86–6.67 (m, 1H), 6.61–6.57 (m, 2H), 3.73 (q,
J = 7.0 Hz, 2H), 2.90–2.56 (m, 5H), 2.31–2.16 (m, 3H), 2.05–1.78
(m, 3H), 1.28 (s, 3H), 1.21–1.18 (m, 1H), 1.11 (t, J = 7.0 Hz, 3H),
0.77 (d, J = 5 Hz, 3H). 13C NMR (CDCl3): d 171.04, 154.89, 142.29,
141.76, 129.85, 128.27, 128.17, 125.99, 113.45, 112.31, 58.50,
50.93, 45.80, 44.25, 40.95, 40.43, 35.96, 32.12, 29.68, 25.09,
23.60, 13.94, 12.98. Anal. Calcd for C25H32N2O2: C, 76.50; H, 8.22;
N, 7.14. Found: C, 76.40; H, 8.54; N, 7.34.
5.2.3.10. 2-[(2R,6R,11R)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetra-
hydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-methyl-N-phen-
ylacetamide (21). White solid (90%); mp: 166–168 °C; ½a D20
ꢀ63°
ꢁ
(c 1.0, MeOH); 1H NMR (CDCl3): d 7.46–7.30 (m, 3H), 7.27–7.21 (m,
2H), 6.82–6.78 (m, 1H), 6.70–6.68 (m, 1H), 6.61–6.56 (m, 1H), 3.28
(s, 3H), 3.96 (d, J = 14.8 Hz, 1H), 3.14 (d, J = 14.8 Hz, 1H), 2.62–2.54
(m, 3H), 2.44–2.38 (m, 1H), 2.05–1.93 (m, 1H), 1.79–1.64 (m, 2H),
1.26 (s, 3H), 1.21–1.14 (m, 1H), 0.72 (d, J = 5 Hz, 3H). 13C NMR
(CDCl3): d 170.71, 154.34, 143.75, 143.11, 129.41, 127.93, 127.83,
127.60, 127.18, 112.85, 112.24, 57.65, 57.01, 45.86, 41.82, 41.18,
37.65, 35.90, 25.31, 23.94, 13.93. Anal. Calcd for C23H28N2O2: C,
75.79; H, 7.74; N, 7.69. Found: C, 75.67; H, 7.66; N, 7.58.
5.2.3.5. N-Benzyl-3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,
5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]propana-
mide (16). White solid (84%); mp: 162–164 °C; ½a D20
ꢀ50° (c 1.0,
ꢁ
MeOH); 1H NMR (CDCl3): d 9.21 (s, 1H), 7.39–7.26 (m, 5H), 6.89–
6.85 (m, 1H), 6.69–6.60 (m, 2H), 4.43 (q, J = 14.4 Hz, 1H), 4.40 (q,
J = 14.4 Hz, 1H), 2.83–2.66 (m, 5H), 2.59–2.44 (m, 3H), 2.12–1.99
(m, 1H), 1.54–1.21 (m, 3H), 1.18 (s, 3H), 0.69 (d, J = 5 Hz, 3H). 13C
NMR (CDCl3): d 172.53, 154.73, 142.37, 138.29, 128.69, 128.14,
128.05, 127.47, 126.85, 113.25, 112.28, 57.70, 50.43, 44.39, 43.63,
41.44, 41.13, 36.02, 31.59, 25.02, 23.39, 13.89. Anal. Calcd for
5.2.3.11. 2-[(2R,6R,11R)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetra-
hydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-ethyl-N-phenyl-
C24H30N2O2: C, 76.16; H, 7.99; N, 7.40. Found: C, 76.32; H, 7.67; N, 7.54.
acetamide (22). White solid (94%); mp: 152–154 °C; ½a D20
ꢀ62° (c
ꢁ
5.2.3.6. N-(Cyclohexylmethyl)-3-[(2R,6R,11R)-8-hydroxy-6,11-
dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-
1.0, MeOH); 1H NMR (CDCl3): d 7.47–7.35 (m, 3H), 7.22–7.17 (m,
2H), 6.82–6.78 (m, 1H), 6.68–6.67 (m, 1H), 6.60–6.55 (m, 1H),
3.74 (q, J = 7.0 Hz, 2H), 3.09 (d, J = 14.8 Hz, 1H), 2.90 (d,
J = 14.8 Hz, 1H), 2.60–2.53 (m, 3H), 2.44–2.38 (m, 1H), 2.02–1.91
yl]propanamide (17). White solid (77%); mp: 163–164 °C; ½a D20
ꢁ
ꢀ56° (c 1.0, MeOH); 1H NMR (CDCl3): d 8.76 (s, 1H), 6.94–6.89

L. Pasquinucci et al. / Bioorg. Med. Chem. 18 (2010) 4975–4982
4981
(m, 1H), 1.79–1.65 (m, 2H), 1.26 (s, 3H), 1.21–1.18 (m, 1H), 1.11 (t,
J = 7.0 Hz, 3H), 0.72 (d, J = 5 Hz, 3H). 13C NMR (CDCl3): d 170.04,
154.18, 143.23, 142.00, 129.35, 128.39, 128.08, 127.99, 127.76,
112.83, 112.24, 57.68, 57.35, 45.88, 44.41, 41.88, 41.21, 35.95,
25.36, 23.99, 13.95, 12.99. Anal. Calcd for C24H30N2O2: C, 76.16;
H, 7.99; N, 7.40. Found: C, 76.34; H, 8.13; N, 7.55.
the data was performed using Origin 7.5 software (OriginLab Corpo-
ration, Northampton, USA).
5.3.2. Antinociception
Male Sprague–Dawley rats (Morini, S. Polo d’Enza, RE, Italy)
weighing 180–200 g were used. The animals were kept at a con-
stant room temperature (25 1 °C) under a 12:12 h light and dark
cycle, with free access to food and water. Each rat was used for
only one experiment. The antinociceptive response was evaluated
by recording the latency with the radiant heat tail-flick test.28
Briefly, it consisted of an irradiation of the lower third of tail with
an IR source (Ugo Basile, Comerio, Italy). The day before the exper-
iment, rats were habituated to the procedure for measuring the
nociception threshold. The basal pre-drug latency was established
between 3 and 4 s, and was calculated as the average of the first
three measurements, which were performed at 5 min intervals. A
cut-off latency of 10 s was established to minimize damage to
the tail. Post-treatment tail flick latencies (TFLs) were determined
at 20, 40, 60, 80, and 100 min after sc injection. The behavioral
tests were conducted by researchers blinded to the treatment
group. Data were expressed as means SE of the values recorded
in animals of the same group. Intergroup comparisons were as-
sessed using an initial two-way analysis of variance (ANOVA) fol-
lowed by Duncan’s multiple range post-hoc test. Differences
were considered significant when *p < 0.05.
5.3. Radioligand binding assay
Membranes were prepared from male Hartley guinea pigs (250–
300 g) and Sprague–Dawley rats (200–250 g) (Morini, S. Polo d’Enza,
RE, Italy). Binding to the
l and d opioid receptors was carried out on
rat membranes obtained from whole brain minus the cerebellum, as
previously reported.22 The
l binding assay was carried out as re-
ported by Furness et al.23 Briefly, membranes (1 mg/mL) were incu-
bated in 50 mM Tris–HCl, pH 7.4, containing 100 mM choline
chloride, 3 mM MnCl2, 100 nM DPDPE to eliminate binding to d
opioid receptors and a protease inhibitor cocktail (100
lg/mL baci-
tracin, 10 g/mL bestatin, 4 g/mL leupeptin, and 2 g/mL chymo-
l
l
l
statin). Incubation proceeded for 2 h at 25 °C with 2 nM
[3H]DAMGO. Nonspecific binding was defined in the presence of
1 lM unlabeled DAMGO. The d binding assay was performed with
2 nM [3H]DPDPE and rat brain membranes as reported above. Ali-
quots of the homogenates (1 mg/mL) were incubated under the
same conditions with the exception of DAMGO in place of DPDPE
to eliminate binding to the
was defined in the presence of 20
ing assay was performed on membranes obtained from guinea pig
cerebella, prepared as previously described.6 Aliquots of homoge-
nates (1 mg/mL) were incubated in 50 mM Tris–HCl, pH 7.4, con-
taining a protease inhibitor cocktail, 100 nM DPDPE and 100 nM
l
opioid receptor. Nonspecific binding
M unlabeled DPDPE. The bind-
The ED50 values were calculated from linear regression of the
dose–effect functions, and means of the 95% confidence interval
were obtained. Linear regression was determined using the Graph-
Pad Prism program (GraphPad, San Diego, CA, USA). Experimental
procedures were approved by the local ethical committee I.A.C.U.C.
(Institutional Animal Care and Use Committee) and were con-
ducted in accordance with International Guidelines as well as
European Communities Council Directive and National Regulations
(EEC Council 86/609 and DL 116/92).
l
j
DAMGO, with the latter two to eliminate binding to d and
l opioid
receptors, respectively. Incubation proceeded for 1 h at 25 °C with
2 nM [3H]U69,593. Nonspecific binding was defined in the presence
of 10 lM U50,488. The incubation was terminated by rapid filtration
through Whatman GF/C glass filters presoaked in 0.1% polyethyl-
eneimine solution for 1 h. The filters were washed twice with
4 mL of ice-cold 50 mM Tris–HCl buffer. After overnight incubation
in 4 mL of Filter Count Cocktail (Packard), radioactivity retained on
the filters was measured by liquid scintillation spectrometry using a
1414 Winspectral Perkin-Elmer Wallac. Competition inhibition con-
stant (Ki) values were calculated with the EBDA/LIGAND program
(Elsevier/Biosoft).24 All values are presented as means SEM of
three separate experiments, each carried out in duplicate.
Acknowledgments
This work was supported by a FP6 European Grant (LSHC-CT-
2006-037733) and by the Italian MIUR (COFIN and FIRB Programs).
The authors gratefully acknowledge Fabbrica Italiana Sintetici
(Italy) for providing cis-( )-N-normetazocine.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.005.
5.3.1. cAMP accumulation assay
5.3.1.1. Cell culture
. HEK293 cells stably expressing either the
EE-tagged or d opioid receptor were grown in Dulbecco’s modi-
l
MOL files
fied Eagle’s medium (DMEM) supplemented with 10% fetal calf ser-
um, 2 mM glutamine, 100 U/mL penicillin, and 0.1 mg/mL
streptomycin under 5% CO2 at 37 °C.25,26
The following ZIP file contains the MOL files of the most impor-
tant compounds referred to in this article.
5.3.1.2. Measurements of cAMP accumulation. Measurements
of adenylyl cyclase activitywere performed as described by Merkou-
References and notes
ris et al.27 Briefly, HEK293 cells stably expressing the
l
or the d opi-
oid receptor were seeded in 12-well plates and incubated for 24 h in
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Ci/well). The generation of
[3H]cAMP was assessed in response to treatment of the cells with
various concentrations of the appropriate ligand (1 pM–10 V)
using 50 V forskolin at 37 °C for 30 min. Results were calculated
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