Design, synthesis and SAR analysis of novel selective σ1 ligands (Part 2)

Article abstract of DOI:10.1016/j.bmc.2009.12.039

In order to investigate the molecular features involved in sigma receptors (σ-Rs) binding, new compounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized and their affinity towards σ₁- and σ₂

Full text of DOI:10.1016/j.bmc.2009.12.039

Bioorganic & Medicinal Chemistry 18 (2010) 1204–1212
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and SAR analysis of novel selective r1 ligands (Part 2)
Daniela Rossi a, Mariangela Urbano a, Alice Pedrali a, Massimo Serra a, Daniele Zampieri b,
Maria Grazia Mamolo b, Christian Laggner c, Caterina Zanette d, Chiara Florio d, Dirk Schepmann e,
Bernard Wuensch e, Ornella Azzolina a, Simona Collina a,
*
a Department of Pharmaceutical Chemistry, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
b Department of Pharmaceutical Sciences, University of Trieste, P.le Europa 1, 34127 Trieste, Italy
c Institute of Pharmacy, Department of Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 52c, 6020 Innsbruck, Austria
d Department of Biomedical Sciences (Pharmacology Section), University of Trieste, Via Giorgieri 7, 34127 Trieste, Italy
e Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster, Hittorfstrasse 58-62, 48149 Muenster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to investigate the molecular features involved in sigma receptors (r-Rs) binding, new com-
pounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized
Received 11 November 2009
Revised 10 December 2009
Accepted 11 December 2009
Available online 21 December 2009
and their affinity towards 1- and 2-Rs subtypes was evaluated. The most promising compounds were
r
r
also screened for their affinity at
l-opioid, d-opioid and
j-opioid receptors. Biological results are herein
presented and discussed.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Sigma ligands
Arylalkylaminoalcohols
Arylalkenylamines
Arylalkylamines
Radioligand binding assays
Pd(0) EnCat™ 30NP(en)
1. Introduction
of the NMDA receptor channel complex have been reported to con-
tribute to these effects.2,4 Recently, the
1-Rs agonist PPBP (Fig. 1)
was proved to exert a neuroprotective effect by a mechanism
involving the antiapoptotic protein bcl-2 and sensitive to the r1
Rs antagonist rimcazole.5 Finally, the selective
1-Rs agonist
SA4503 (Fig. 1), was found to significantly suppress hypoxia/hypo-
glycaemia-induced neurotoxicity in rat primary neuronal cultures
and to protect cultured rat retinal neurons from neurotoxicity
induced by glutamate.6 SA4503 is currently undergoing clinical tri-
als for the treatment of depression and of post-stroke motor
dysfunction.
r
The sigma receptors (
named sigma1 ( 1-Rs) and sigma2 (
1-R has been purified and cloned1 and consists of a 26 kDa pro-
tein with no homology to any other known mammalian proteins.
Both -Rs subtypes are expressed in the central nervous system
r
-Rs) system comprises two subtypes,
r
r
2-Rs). Up to now only the
-
r
r
r
(CNS) as well as in peripheral tissues. Although their exact physio-
logical role is not yet completely understood, their involvement in
modulating complex biochemical pathways, as well as several
physiopathological events is well documented.
r
1-Rs have been studied mostly with respect to their functions
Accumulating evidences suggest that both r-Rs subtypes
within the CNS, particularly as putative targets for neuroprotection
regulate cell proliferation and survival. Indeed, cellular transition
from quiescent to proliferative status is associated with an increase
after ischemia.2 There is strong pharmacological evidence which
indicates that
r
1-Rs, at least in part, act as intracellular amplifiers
in
r-Rs expression. Whereas r1-Rs promote cell growth and inhi-
creating a supersensitive state of signal transduction in biochemi-
bit apoptosis,
r
2-Rs activation by selective as well as non-selective
cal pathways in the CNS.3 It was also hypothesized that activation
of
mia in cortical neurons.2 However, the mechanism of neuroprotec-
tion for some 1-Rs ligands has been controversially discussed
because both the 1-Rs and the phencyclidine (PCP) binding site
r
1-Rs can ameliorate Ca2+ dysregulation associated with ische-
r
N
N
OCH3
OCH3
r
N
SA 4503
-Rs ligands.
PPBP
Figure 1. Structures of known
* Corresponding author. Tel.: +39 0382 987379; fax: +39 0382 422975.
E-mail address: simona.collina@unipv.it (S. Collina).
r
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.039

D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
1205
Additionally, useful information concerning the structure and
properties of the ligand-binding site in the 1-Rs was obtained
r
Ar
N
Ar
N
and interesting structure–activity relationships were established
for our novel ligands.
R
R
Arylalkylamines
Arylalkenylamines
In the present paper we address our efforts towards a deeper
evaluation of the influence of the three carbon spacer on
affinity and selectivity, with the final aim to extend the SAR studies
of these chemical classes and also to discover new -Rs ligands
with improved affinity versus 2-Rs subtype. Although an electro-
negative site on the spacer does not seem to have relevance for
conferring affinity towards
1-Rs to our class of compounds,10 this
molecular feature seems to be relevant for 2-Rs binding. Indeed a
2-Rs pharmacophore model recently developed suggested that a
r-Rs
Figure 2. General structures of arylalkenyl- and arylalkylamines.
r
ligands induces growth arrest and cell death in various cell lines.7
Normal cells respond to death stimuli by undergoing apoptosis, the
best characterized form of programmed cell death. In contrast, can-
cer cells frequently escape from spontaneous or therapy-induced
apoptosis due to acquired mutations in the apoptotic machinery.
Therefore, development of novel anticancer drugs able to trigger
alternative death pathways, not dependent on apoptosis-regulat-
r
r
r
r
hydrogen-bond group is involved in
r
2-Rs interaction.11 On the ba-
sis of this observation, new derivatives characterized by three dif-
ferent spacers (alcoholic, olefinic and alkylic spacers, Series I, II and
III compounds, respectively) were designed (Fig. 3). Biphenyl-4-yl
and 6-methoxynaphth-2-yl were selected as aromatic nucleus
while dimethylamine, N-benzyl-N-methylamine and piperidine
were chosen as aminic moieties (Fig. 3).
Synthetic procedures, based on Polymer Assisted Solution Phase
Synthesis (PASPS) methodology, developed for preparing the de-
signed compounds as well as SAR information on newly synthe-
sized compounds are described in this communication.
ing genes, is of great interest.
gets for such drugs.7 Furthermore, the high densities of both
Rs subtypes in many tumour cell lines and tissues suggest their
involvement in cancer biology. Accordingly, -Rs are now com-
monly considered to be tumour biomarkers and ligands with high
affinity towards both -Rs subtypes could have relevant therapeu-
tic and diagnostic applications.8 Actually, many
-Rs ligands are
r2-Rs may represent candidate tar-
r
-
r
r
r
developed nowadays for the detection of tumours at early stages
through imaging techniques (PET).9
Thus, the state of the art suggests that
r-Rs represent exciting
targets for the development of novel neuroprotective agents, for
the in vivo detection of tumours and their metastasis, as well as
for the development of new anticancer drugs. Therapeutic applica-
2. Chemistry
We started our synthetic approach with the synthesis of b-ami-
noketone C, essentially prepared according to the methodology de-
scribed in our previous work, with suitable modifications (Scheme
1).10 The Michael addition of piperidine to but-3-en-2-one was
firstly performed in absolute ethanol and glacial acetic acid (see
Section 6.1.2.1) yielding compound C, for which a purification by
fractional distillation was required. Afterwards, the same reaction
was carried out in PEG 400 (see Section 6.1.2.2), giving the desired
product in a very short time and in higher yield with respect to the
procedure previously described, without any further purification.
The syntheses of racemic 1a–d were then accomplished via
nucleophilic addition of the appropriate arylic anion to the suitable
b-aminoketone, according to the procedure already described by us
(Scheme 1).12 Desired products were obtained in good yields.
Concerning the synthesis of arylalkenylamines 2a–d, prelimin-
ary dehydration experiments of the corresponding arylalkyl-
aminoalcohols under standard acidic conditions (37% HCl or 98%
H2SO4) led to desired olefinic compounds with unsatisfactory
yields and purities. Therefore we considered to take advantage of
the triphenylphosphine–iodine system, recently described for the
regioselective dehydration of tertiary alcohols.13 Moreover, to sim-
plify the reaction work-up, polymer-bound triphenylphosphine
tions of
potent and highly selective
r
-Rs ligands depend on their receptor binding profile:
1-Rs ligands are potential neuropro-
r
tective agents and could represent an innovative pharmacological
approach for the treatment and prevention of neurodegenerative
diseases, on the contrary compounds with high affinity towards
both
r-Rs subtypes and high selectivity against the other receptors
of the CNS are potentially useful in cancer therapy and diagnosis.
In our previous paper we presented the rational drug design,
synthesis and biological evaluation of novel
r-Rs ligands based
on arylalkenyl- and arylalkylaminic scaffolds (Fig. 2) and charac-
terized by three sites of molecular diversity: the aromatic nucleus,
the aminic moiety and the three carbon spacer between these two
structural features.10 In more details, we carefully evaluated the
influence of both the aromatic portion and the aminic moiety on
r
-Rs affinity and selectivity. Concerning the three carbon spacer,
we focused on molecules characterized by either an olefinic (aryl-
alkenylamines) or an alkylic (arylalkylamines) chain (Fig. 2),
according to the
r
1-Rs model developed by us.10 Compounds
with affinity towards
ble selectivity over
r
1-Rs in the nanomolar range and apprecia-
r
2-Rs subtype were identified; moreover,
moderate
r
2-Rs affinity was also observed for some compounds.
R1
R1
R2
R1
R2
Ar
N
Ar
Ar
N
N
OH
Series I
R2
Series II
Series III
Compound
Series II
Ar
R1
R2
Series I
1a
1b
1c
1d
Series III
2a
2b
2c
2d
3a
3b
3c
3d
6-methoxy-naphth-2-yl
6-methoxy-naphth-2-yl
6-methoxy-naphth-2-yl
biphenyl-4-yl
CH3
CH3
CH3
CH2C6H5
(CH2)5
CH3
CH2C6H5
Figure 3. Structures of designed arylalkylaminoalcohols (Series I), arylalkenyl (Series II) and arylalkylamines (Series III).

1206
D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
4
1
R1
R2
3
Ar
N
N
2
d, e
R1
a, b, c
Br
Ar
N
Ar
OH
4
R2
1
(R,S)-1a-d
R1
R2
3
Ar
2
crude 2a-d
f, g
Synthesis of A-C
O
R1
HN
+
R1
N
R2
R2
O
Aminic moiety
HCl
N
R1
dimethylamine
A
Ar
N-benzyl-N-methylamine
B
C
(E)-2a-d HCl
R2
piperidine
Scheme 1. Synthesis of arylalkylaminoalcohols (R/S)-1a–d and arylalkenylamines (E)-2a–d. Reagents and conditions: (a) t-BuLi, anhydrous Et2O, À78 °C to rt; (b) ketones A–
C, À78 °C to 0 °C; (c) H2O rt; (d) I2, TPPP rt; (e) aq 5% NaHSO3 rt; (f) 37% HCl rt; (g) crystallization from acetone.
(TPPP) was employed instead of the conventional reagent (Scheme
1). To the best of our knowledge, the efficacy of the TPPP–iodine
system in the dehydration of tertiary aminoalcohols as well as
the effects of this system on both regioselectivity and stereoselec-
tivity of the reaction have never been studied before.
the (Z) configuration was assigned (see Fig. 4). As regards the ste-
reoselectivity of the reaction, the (E):(Z) isomeric ratio was about
75:25 for all prepared compounds, suggesting that it was not sig-
nificantly affected by the structural properties of both the aromatic
nucleus and the aminic portion of the molecule.
Regarding the regioselective behaviour of the reaction, the
treatment of (R/S)-1a–d with the TPPP–iodine system afforded
exclusively the trisubstituted regioisomers. The 1H NMR analysis
of crude 2a–d revealed only the formation of a C2–C3 double bond;
no signals related to C3–C4 olefinic regioisomers were detectable.
Regarding the configuration of the C2–C3 double bond, 1H NMR
analysis of crude 2a–d showed signals related to both (E) and (Z)
stereoisomers. The main reaction product was the (E) stereoisomer
for all prepared compounds, as confirmed by NOESY experiments.
Indeed, a significant NOE effect corresponding to the interaction
correlating the methyl group on the double bond and the olefinic
hydrogen was evidenced only for the minor stereoisomer, to which
Crude 2a–d were then converted into their respective hydro-
chlorides and successively crystallized from acetone, yielding pure
(E)-2a–d HCl in satisfactory yields.
Finally, racemic 3a–d (Fig. 3) were synthesized by catalytic
reduction of the corresponding arylalkenylamines in hydrogen
atmosphere, applying our already developed protocol with conve-
nient modifications.10 In these experiments Pd(0) EnCat™ 30NP
was used instead of Pd/C (Scheme 2, see Section 6.1.5.1). The
reduction protocol was developed and optimized on compound
(E)-2a and then applied to (E)-2b–d. Among the newly synthesized
compounds, the arylalkylamines 3b and 3d, characterized by an N-
benzyl group, could not be prepared in satisfactory yields using the
CH3
CH3
H
CH3
CH3
N
CH3
H3CO
H
N
H3CO
CH3
N(CH3)2
Olefinic hydrogens
Olefinic methyl groups
major
minor
B
A
Figure 4. NOESY spectra of crude 2a (A) and pure (E)-2a (B). The arrow indicates the NOE effect between the methyl group on the double bond and the olefinic hydrogen of
the minor (Z) stereoisomer.

D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
1207
H
R1
Ar
+
Ar
N
N
a, b
(R,S)- 3a-d
R2
Ar
HCl
N
6-methoxy-naphth-2-yl
biphenyl-4-yl
(R,S)-3e
(R,S)-3f
R1
Ar
(E)-2a-d HCl
R2
a, c
R1
Ar
N
R2
(R,S)-3b,d
Scheme 2. Synthesis of arylalkylamines (R/S)-3a–f. Reagents and conditions: (a) SCX cartridge, (b) H2, Pd(0) EnCat™ 30NP, abs EtOH, rt (Method I); (c) H2, Pd(0) EnCat™
30NP(en), abs EtOH, rt (Method II).
above described procedure. Complete reduction was achieved for
both compounds, but the concurrent cleavage of the N-benzyl
group was observed. Although both target products 3b and 3d
were isolated from the reaction mixtures by flash chromatography,
the corresponding N-debenzyl-derivatives 3e and 3f (Scheme 2) re-
sulted the main products.
In order to avoid the cleavage of the N-benzyl group and to de-
velop a protocol suitable for the synthesis of 3b and 3d, our strat-
egy consisted in developing a new poisoned catalyst based on
Pd(0) EnCat™ 30NP. Ethylenediamine was used as catalytic poison,
according to the procedure already described by Sajiki et al. for the
Pd/C catalyst.14 The new catalyst will be called by now on Pd(0) En-
Cat™ 30NP(en).
Pd(0) EnCat™ 30NP(en) was firstly experimented on compound
(E)-2a to verify its efficacy in reducing double bonds. 1H NMR anal-
ysis of crude 3a clearly evidenced the effectiveness of Pd(0) En-
Cat™ 30NP(en) in the hydrogenation of the olefinic precursor.
The catalyst was then used for the catalytic hydrogenation of
(E)-2b in hydrogen atmosphere (Scheme 2, see Section 6.1.5.2).
Interestingly, applying our optimized protocol compound 3b was
obtained with good crude purity and satisfactory yield (>45%). 1H
NMR analysis of crude 3b revealed the complete hydrogenation
of the double bond (absence of the signal related to the olefinic
hydrogen) and the presence of one main product in which the N-
benzyl group is still present (Fig. 5). The identity of this product
was then confirmed by both 1H NMR analysis and mass spectros-
copy of pure compound, isolated by solid phase extraction (SPE,
SCX cartridge) followed by flash chromatography.
4. Results and discussion
All novel compounds showed interesting binding affinities for
r
1-Rs subtype (Ki values ranging from 1.29 to 38.8 nM, Table 1)
and poor 2-Rs affinity (Ki values >136 nM, Table 1), with the only
exception of compound 1a which exhibits a negligible affinity for
2-Rs subtype (Ki value >10,000 nM, Table 1). All new molecules
r
r
were mapped onto our 5-points pharmacophore model, which
consisted of four hydrophobic and one positive ionizable fea-
tures.10 Good fit values were obtained for all compounds belonging
to both Series I and Series II. Figure 6 shows compounds (E)-2c and
(S)-3d mapped to pharmacophore model, exemplifying how it
maps the different residues present among our most active com-
pounds. Calculated fit values for the displayed compounds were
3.38 [(E)-2c] and 4.64 [(S)-3d], out of a maximum score of 5.0 (per-
fect fit). The lowest fit values were observed for compounds
belonging to Series I. There the matching algorithm in the software
appears to penalize the proximity of the polar hydroxyl group to
the hydrophobic region, despite the hydroxyl group being well
tolerated.
Contrary to our initial aim, in the present study we identified
novel potent
2-Rs subtypes, suggesting that the structural properties of the
three carbon spacer between the aromatic nucleus and the aminic
moiety do not exert any relevant influence on 1-Rs affinity. On
the contrary, we found that the structural properties of the three
carbon spacer noticeably affect the selectivity over the 2-Rs sub-
type. Indeed Series I and III compounds generally showed selectiv-
ity against 2-Rs subtype higher than compounds belonging to
Series II. Therefore it can be postulated that the alcoholic group
on the spacer is not essential for 1-Rs binding affinity, in accor-
dance with our 1-Rs pharmacophore model, while it appears to
be unfavourable for 2-Rs interaction—at least for certain substitu-
tion patterns—in contrast with the 2-Rs pharmacophore recently
developed for
-tropanyl derivatives.11 Among the most interest-
ing compounds in terms of both 1-Rs affinity and selectivity over
2-Rs subtype, racemic 1b, 1d, 3b and 3d were selected for a dee-
per investigation of their binding profiles. In Table 2 their -opioid,
d-opioid and -opioid receptor affinities are reported. The residual
binding of the radioligand is given at a concentration of 1 M of
r1-Rs ligands with interesting selectivity against
r
r
r
The hydrogenation protocol was then successfully applied to
(E)-2d, providing pure 3d in good yields.
Compounds belonging to both Series I and III, which present
one stereogenic centre, have not been resolved prior to perform
binding studies, therefore they were assayed as racemates.
r
r
r
r
r
a
3. Receptor binding studies
r
r
Sigma receptor binding assays were performed on rat liver
membranes according to the methods of Hellewell et al.,15 slightly
modified as previously described.16 [3H]-(+)-Pentazocine was em-
l
j
l
ployed for
ligand [3H]-DTG (1,3-di-2-tolylguanidine) was employed for
studies in the presence of an excess of non-tritiated (+)-pentazo-
cine (100 nM) to mask 1-Rs. In both assays, the non-specific bind-
ing was defined in the presence of haloperidol (10 M).
In order to determine receptor selectivity the most promising
compounds were screened for their affinity at -opioid, d-opioid
and
-opioid receptors.17
r
1-Rs binding assays, whereas the non-selective radio-
tested compounds. When a significant inhibition of the radioligand
was observed, the Ki value was determined.
r
2-Rs
Novel compounds generally showed low affinity for
and -opioid receptors, with the only exception of 3d (Ki value at
-opioid receptor = 248 nM) and 3b (Ki value at -opioid recep-
l-opioid
r
j
l
l
j
tor = 841 nM). Concerning d-opioid receptor binding assays, only
compounds 1b and 3b showed affinities remarkable high, being
l
j

1208
D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
Table 1
Binding affinities of the prepared compounds towards
r1- and r2-Rs subtypes
Compound
Ki (nM)
a
b
r1
r2
r2/r1
Series I
1a
1b
1c
1d
38.8 4.5
6.04 1.3
10.4 2.4
2.38 0.92
>10,000
>258
156
15
940 148
154 54
215 105
90
Series II
(E)-2a
(E)-2b
(E)-2c
(E)-2d
27.3 2.4
13.7 5.52
4.41 0.60
4.17 1.18
707 111
568 222
136 114
309 116
26
41
31
74
Series III
3a
3b
3c
3d
14.4 1.1
2.31 0.82
1.29 0.23
3.59 0.57
1548 288
169 80
169 105
214 136
108
73
131
60
Values are means SEM of three experiments performed in duplicate.
Haloperidol (10 M) was used to define non-specific binding.
l
Series I and Series III compounds were assayed as racemates.
a
Displacement of 1 nM [3H]-(+)-pentazocine.
b
Displacement of 3 nM [3H]-DTG in the presence of (+)-pentazocine (100 nM).
the use of poisoned Pd/C to reduce olefins in the presence of groups
sensitive to hydrogenolytic conditions has been already reported,
this is the first time that Pd(0) EnCat™ 30NP(en) is described and
its efficacy in the selective reduction of double bonds is
demonstrated.
All novel compounds generally showed interesting affinities for
r
r
1-Rs (Ki values ranging from 1.29 to 40 nM) and poor or negligible
2-Rs affinity (Ki values >136 nM). Among the most 1-active
r
compounds, racemic 1d showed a noticeable selectivity towards
2-Rs (factor 90) and also an excellent selectivity with respect to
-opioid, d-opioid and -opioid receptors. Therefore, our current
efforts are directed towards a deeper understanding of the role of
chirality in affecting -Rs ligand binding by resolving the racemic
mixtures of 1d and at evaluating the -Rs affinities of pure enan-
tiomers. Finally, we intend to address our attention to clarify the
r
l
j
r
r
influence of a hydrogen-bond donor group on
applying a molecular modelling approach.
r2-Rs interaction
6. Experimental
6.1. Chemistry
6.1.1. General
All reagents and solvents were purchased from commercial
suppliers and used without any further purification. Anhydrous
solvents were obtained according to standard procedures. Pd(0)
EnCat™ 30NP (loading = 0.4 mmol/g) was purchased from Sigma–
Aldrich. Melting points were determined in open capillaries on
SMP3 Stuart Scientific apparatus and are uncorrected. NOESY
NMR spectra were recorded on Bruker AVANCE 400 MHz spec-
trometer using tetramethylsilane as internal standard (d = 0) and
the chemical shift values are expressed in ppm (d). IR spectra were
recorded on a Jasco FT/IR-4100 spectrophotometer; only notewor-
thy absorptions are given. Reaction courses and the purity of com-
pounds were checked by thin layer chromatography (TLC) on silica
gel (Kieselgel 60 F254, Merck) pre-coated glass-backed plates pur-
chased from Fluka and the chromatograms were detected by UV
radiations, potassium permanganate and acidic ammonium
molybdate(IV). Flash chromatography was performed with Silica
Gel 60 (particle size 230–400 mesh) purchased from Nova Chimica.
Bond Elut SCX cartridges were purchased from Varian. MS spectra
Figure 5. 1H NMR spectra of (E)-2b (A), crude and pure 3b (B and C, respectively).
the Ki values 41 and 171 nM, respectively. Interestingly, binding re-
sults clearly evidenced that compound 1d (Ki
2 = 215 nM) display high selectivity towards
and -opioid receptors.
r
1 = 2.38 nM, Ki
r
l-opioid, d-opioid
j
5. Conclusion
Three different series of
r-Rs ligands were synthesized apply-
ing PASPS methodology whenever it was possible. In this context
a new catalyst suitable for the selective reduction of olefinic bonds
in the presence of an N-benzyl group was developed using Pd(0)
EnCat™ 30NP and ethylenediamine as catalytic poison. Although

D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
1209
Figure 6. Compounds (E)-2c (left) and (S)-3d (right) mapped to our
r1-Rs pharmacophore model. Cyan spheres: hydrophobic features, red sphere: positive ionizable feature.
Table 2
with water (30 mL); after an acid–base work-up, the combined or-
ganic phases were evaporated under vacuum. Crude products were
purified by flash chromatography, affording the desired
compounds.
Affinities of racemic 1b, 1d, 3b and 3d towards opioid receptors
Compound
l
([3H]-DAMGO)
d ([3H]-DPDPE)
j
([3H]-U 69593)
1b
1d
3b
3d
32%a
41 nM
31%a
30%a
45%a
30%a
32%a
171 nM
841 nM
6.1.3.1. (R/S)-4-(Dimethylamino)-2-(6-methoxynaphth-2-yl)bu-
tan-2-ol [(R/S)-1a]. Flash chromatography mobile phase: hex-
ane–ethyl acetate–7 N NH3 in methanol (80:20:1.5). Yield: 69%;
white solid; mp 98–100 °C. IR (cmÀ1): 3183, 2969, 2948, 2821,
2779, 1604, 1461, 1261, 1201, 1030, 849, 750. 1H NMR
(400 MHz, DMSO-d6) d: 1.48 (s, 3H, CH3–C), 1.88–1.96 (m, 2H, C–
CH2), 1.98–2.10 (m, 7H, HCH–N + N(CH3)2), 2.14–2.25 (m, 1H,
HCH–N), 3.86 (s, 3H, OCH3), 5.93 (br s, 1H, OH, exchanges with
D2O), 7.13 (dd, 1H, J = 2.4 Hz, J = 8.9 Hz, aromatic), 7.28 (d, 1H,
J = 2.2 Hz, aromatic), 7.52 (dd, 1H, J = 1.1 Hz, J = 8.5 Hz, aromatic),
7.75 (d, 1H, J = 8.6 Hz, aromatic), 7.80 (d, 1H, J = 8.9 Hz, aromatic),
7.87 (s, 1H, aromatic). MS: m/z 274.18 [MH+].
248 nM
0%a
15%a
a
Percent inhibition of the radioligand at a concentration of 1
compound.
lM of the test
were recorded on a Waters Micromass ZQ using an APCI ionization
source operating in positive ion mode. Elemental analyses (C, H, N)
were performed on a Carlo Erba 1106 analyzer and the analysis re-
sults were within 0.4% of the theoretical values.
6.1.2. 4-Piperidin-1-yl-butan-2-one (C)
6.1.2.1. Method a. But-3-en-2-one (30 mL, 370 mmol) was added
dropwise to a stirred solution of piperidine (40.6 mL, 410 mmol)
and glacial acetic acid (1 mL) in absolute ethanol (63 mL). Reaction
mixture was stirred at room temperature for 3 h; the solvent was
then removed under reduced pressure, affording crude b-aminok-
etone C (brown oil), which was purified by fractional distillation,
yielding the desired product as a yellow oil (yield 46%).
6.1.3.2. (R/S)-4-(Benzyl(methyl)amino)-2-(6-methoxynaphth-2-
yl)butan-2-ol [(R/S)-1b]. Flash chromatography mobile phase:
hexane–ethyl acetate–7 N NH3 in methanol (90:10:1.5). Yield:
81%; yellow oil. IR (cmÀ1): 3190, 3026, 2965, 2930, 2836, 2799,
1632, 1604, 1262, 1202, 734, 697. 1H NMR (400 MHz, DMSO-d6)
d: 1.48 (s, 3H, CH3–C), 1.98–2.06 (m, 5H, C–CH2 + N–CH3), 2.18–
2.28 (m, 1H, HCH–N), 2.33–2.44 (m, 1H, HCH–N), 3.29 (d, 1H,
J = 13.1 Hz, HCH-Ph), 3.42 (d, 1H, J = 13.1 Hz, HCH-Ph), 3.86 (s,
3H, OCH3), 5.84 (br s, 1H, OH, exchanges with D2O), 7.13 (dd, 1H,
J = 2.5 Hz, J = 8.9 Hz, aromatic), 7.18–7.31 (m, 6H, aromatic), 7.52
(dd, 1H, J = 1.8 Hz, J = 8.6 Hz, aromatic), 7.73 (d, 1H, J = 8.6 Hz, aro-
matic), 7.78 (d, 1H, J = 8.9 Hz, aromatic), 7.86 (d, 1H, J = 1.9 Hz, aro-
matic). MS: m/z 350.21 [MH+].
6.1.2.2. Method b. A mixture of piperidine (0.1 mL, 1 mmol), but-
3-en-2-one (0.12 mL, 1.5 mmol) and PEG 400 (2.5 g) was stirred at
room temperature for 35 min and then 10% HCl was added until pH
2 was reached. The aqueous phase was washed with CH2Cl2, made
alkaline with 1 N NaOH solution (pH 10) and extracted with
CH2Cl2. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated under reduced pressure affording the de-
sired product as yellow oil (yield 62%).
6.1.3.3. (R/S)-2-(6-Methoxynaphth-2-yl)-4-(piperidin-1-yl)butan-
2-ol [(R/S)-1c]. Flash chromatography mobile phase: hexane–
ethyl acetate–7 N NH3 in methanol (98:2:2). Yield: 46%; white
solid; mp 79–86.5 °C. IR (cmÀ1): 3126, 2965, 2932, 2828, 2789,
1632, 1604, 1481, 1262, 1204, 1121, 851, 808. 1H NMR
(400 MHz, DMSO-d6) d: 1.28–1.39 (m, 2H, N(CH2CH2)2CH2), 1.40–
1.54 (m, 7H, CH3–C + N(CH2CH2)2CH2), 1.87–1.98 (m, 2H, C–CH2),
2.05–2.20 (m, 4H, CH2–N + N(CHHCH2)2CH2), 2.24–2.40 (br, 2H,
N(CHHCH2)2CH2), 3.86 (s, 3H, OCH3), 6.20 (br s, 1H, OH, exchanges
with D2O), 7.12 (dd, 1H, J = 2.5 Hz, J = 8.9 Hz, aromatic), 7.27 (d, 1H,
J = 2.4 Hz, aromatic), 7.51 (dd, 1H, J = 1.7 Hz, J = 8.5 Hz, aromatic),
7.74 (d, 1H, J = 8.6 Hz, aromatic), 7.79 (d, 1H, J = 8.9 Hz, aromatic),
7.87 (s, 1H, J = 1.8 Hz, aromatic). MS: m/z 314.23 [MH+].
bp6 mmHg 103 °C. IR (cmÀ1): 2932, 2852, 2763–2797, 1711,
1442, 1376, 1354, 1301, 1226, 1165, 1152, 1117, 1038, 862, 773–
752. 1H NMR (400 MHz, CDCl3) d: 1.39 [m, 2H, N(CH2CH2)2CH2],
1.54 [m, 4H, N(CH2CH2)2CH2], 2.12 (s, 3H, CH3CO), 2.33 [br s, 4Y,
N(CH2CH2)2CH2], 2.58 (m, 4H, COCH2CH2N). MS: m/z 156.01 [MH+].
6.1.3. General procedure for the preparation of
arylalkylaminoalcohols (R/S)-1a–d
t-BuLi (25 mmol, 1.7 M in pentane) was added dropwise to a
solution of the appropriate aromatic precursor (12.5 mmol) in
anhydrous diethyl ether (50 mL) cooled to À78 °C, under nitrogen
atmosphere, keeping the temperature for 20 min. The reaction
mixture was then slowly allowed to warm to room temperature.
Stirring was continued for 1 h and a solution of the appropriate
b-aminoketone (10 mmol) in anhydrous diethyl ether (15 mL)
was then added dropwise at À78 °C. The reaction mixture was
slowly allowed to warm to 0 °C, stirred for 3 h and then quenched
6.1.3.4. (R/S)-4-(Benzyl(methyl)amino)-2-(biphenyl-4-yl)butan-
2-ol [(R/S)-1d]. Flash chromatography mobile phase: hexane–
ethyl acetate–7 N NH3 in methanol (80:20:1.5). Yield: 84%; yellow
oil. IR (cmÀ1): 3186, 3026, 2967, 2923, 2841, 2798, 1736, 1599,

1210
D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
1484, 1452, 1075, 1005, 837, 765, 732. 1H NMR (400 MHz, DMSO-
d6) d: 1.42 (s, 3H, CH3–C), 1.94–2.00 (m, 2H, C–CH2), 2.06 (s, 3H, N–
CH3), 2.22–2.31 (m, 1H, HCH–N), 2.36–2.46 (m, 1H, HCH–N), 3.33
(d, 1H, J = 12.7 Hz, HCH-Ph), 3.45 (d, 1H, J = 13.0 Hz, HCH-Ph),
5.83 (br s, 1H, OH, exchanges with D2O), 7.20–7.27 (m, 3H, aro-
matic), 7.28–7.39 (m, 3H, aromatic), 7.43–7.52 (m, 4H, aromatic),
7.54–7.61 (m, 2H, aromatic), 7.63–7.69 (m, 2H, aromatic). MS: m/
z 346.17 [MH+].
7.51–7.56 (m, 2H, aromatic), 7.57–7.67 (m, 4H, aromatic). MS: m/
z 328.17 [MH+].
6.1.5. General procedure for the preparation of arylalkylamines
(R/S)-3a–f
6.1.5.1. Method I. Before use, Pd(0) EnCat™ 30NP (supplied as a
water wet solid with water content 45% w/w) was washed thor-
oughly with absolute ethanol to remove water. Pre-washed Pd(0)
EnCat™ 30NP (0.20 equiv) was added to a stirred solution of the
appropriate arylalkenylamine as free base (0.14 mmol) in absolute
ethanol (11 mL) and the reaction mixture was left at room temper-
ature in hydrogen atmosphere (balloon) for 30 h. The catalyst was
then filtered off and washed with absolute ethanol; the filtrates
were loaded on SCX cartridge and eluted with 1 M NH3 in metha-
nol, the organic phases were finally dried in vacuo. In this way,
pure 3a and 3c were obtained as yellow oils. Compounds 3b and
3d–f were isolated from crude residue by flash chromatography.
6.1.4. General procedure for the preparation of
arylalkenylamines (E)-2a–d
Triphenylphosphine polymer-bound (TPPP) (1.5 mmol) was
added to a solution of iodine (1.5 mmol) in CH2Cl2 (12 mL) and
the mixture was stirred at room temperature for 10 min. A solution
of the appropriate alcohol (1.0 mmol) in CH2Cl2 (5 mL) was then
added and the mixture was further stirred at room temperature.
Aqueous 5% NaHSO3 was added and the mixture was stirred for
10 min and then filtered through Celite. The organic phase was
washed with 1 M NaOH, dried over anhydrous Na2SO4 and concen-
trated under reduced pressure yielding crude 2a–d, which were
converted into the corresponding hydrochlorides and crystallized
from acetone, providing pure (E)-2a–d HCl as white solids. All com-
pounds were characterized as free bases.
6.1.5.2. Method II: procedure for the preparation of the aryl-
alkylamines (R/S)-3b, 3d. 6.1.5.2.1. Preparation of Pd(0) EnCat™
30NP(en): Pd(0) EnCat™ 30NP (240 mg, 0.096 mmol of Pd) was
stirred with ethylenediamine (67.2 mL of 0.1 M solution in metha-
nol, 6.72 mmol) at room temperature for 48 h and then the catalyst
was filtered off, washed with methanol and diethyl ether, dried un-
der a vacuum pump for 48 h and stored under nitrogen.
6.1.4.1. (E)-3-(6-Methoxynaphth-2-yl)-N,N-dimethylbut-2-en-1-
amine [(E)-2a]. Yield: 53%; white solid; mp 87.4–88.4 °C. IR
(cmÀ1): 3008, 2956, 2631, 2590, 2487, 1625, 1598, 1482, 1247,
1210, 1030, 851. 1H NMR (400 MHz, CDCl3) d: 2.18 (s, 3H, CH3–
C@CH), 2.35 (s, 6H, N(CH3)2), 3.18 (d, 2H, J = 6.7 Hz, CH2–N), 3.94
(s, 3H, OCH3), 6.00–6.07 (m, 1H, CH3–C@CH), 7.10–7.19 (m, 2H,
aromatic), 7.60 (dd, 1H, J = 1.7 Hz, J = 8.6 Hz, aromatic), 7.70 (d,
1H, J = 8.7 Hz, aromatic), 7.74 (d, 1H, J = 8.7 Hz, aromatic), 7.77
(d, 1H, J = 1.8 Hz, aromatic). MS: m/z 256.11 [MH+].
6.1.5.2.2. Preparation of compounds (R/S)-3b, 3d: Pd(0) EnCat™
30NP(en) (0.3 equiv) was added to a stirred solution of the appro-
priate arylalkenylamine as free base (0.14 mmol) in absolute etha-
nol (11 mL). The reaction mixture was stirred at room temperature
in hydrogen atmosphere (balloon) for 24 h. The catalyst was then
filtered off and washed with absolute ethanol; the filtrate was
loaded on SCX cartridge and eluted with 1 M NH3 in methanol
and the organic phase was finally dried in vacuo. Desired com-
pounds were finally isolated by flash chromatography.
6.1.4.2. (E)-N-Benzyl-3-(6-methoxynaphth-2-yl)-N-methylbut-2-
en-1-amine [(E)-2b]. Yield: 30%; yellow solid; mp 61.2–63.4 °C.
IR (cmÀ1): 3059, 2955, 2561, 2501, 2409, 1912, 1624, 1599, 1483,
1458, 1206, 1024, 854, 749, 700. 1H NMR (400 MHz, CDCl3) d:
2.16 (s, 3H, CH3–C@CH), 2.32 (s, 3H, N–CH3), 3.29 (d, 2H,
J = 6.7 Hz, CH2–N), 3.62 (s, 2H, CH2-Ph), 3.95 (s, 3H, OCH3), 6.07–
6.13 (m, 1H, CH3–C@CH), 7.11–7.18 (m, 2H, aromatic), 7.26–7.32
(m, 1H, aromatic), 7.33–7.41 (m, 4H, aromatic), 7.60 (dd, 1H,
J = 1.7 Hz, J = 8.6 Hz, aromatic), 7.70 (d, 1H, J = 8.7 Hz, aromatic),
7.74 (d, 1H, J = 8.7 Hz, aromatic), 7.76 (d, 1H, J = 1.8 Hz, aromatic).
MS: m/z 332.24 [MH+].
6.1.5.3. (R/S)-3-(6-Methoxynaphth-2-yl)-N,N-dimethylbutan-1-
amine [(R/S)-3a]. Yield: 68%; yellow oil. IR (cmÀ1): 3053, 2954,
2856, 2813, 2762, 1604, 1459, 1262, 1030, 849, 805. 1H NMR
(400 MHz, CDCl3) d: 1.35 (d, 3H, J = 6.9 Hz, CH3–C), 1.86–2.00 (m,
2H, CH–CH2), 2.19–2.48 (m, 2H, CH2–N), 2.30 (s, 6H, N–(CH3)2),
2.83–2.97 (m, 1H, CH3–CH), 3.93 (s, 3H, OCH3), 7.10–7.20 (m, 2H,
aromatic), 7.34 (dd, 1H, J = 1.6 Hz, J = 8.4 Hz, aromatic), 7.56 (s,
1H, aromatic), 7.68–7.73 (m, 2H, aromatic). MS: m/z 258.05 [MH+].
6.1.5.4. (R/S)-N-Benzyl-3-(6-methoxynaphth-2-yl)-N-methylbutan-
1-amine [(R/S)-3b]. Flash chromatography mobile phase: hexane–
ethyl acetate–7 N NH3 in methanol (80:20:1.5). Yield: 46%; yellow
oil. IR (cmÀ1): 2952, 2925, 2836, 2784, 1633, 1604, 1452, 1263,
1030, 849, 734, 697. 1H NMR (400 MHz, CDCl3) d: 1.33 (d, 3H,
J = 6.9 Hz, CH3–C), 1.82–1.99 (m, 2H, CH–CH2), 2.16 (s, 3H, N–
CH3), 2.24–2.34 (m, 1H, HCH–N), 2.35–2.45 (m, 1H, HCH–N),
2.89–3.02 (m, 1H, CH3–CH), 3.45 (AB system, 2H, J = 13.0 Hz,
CH2-Ph), 3.94 (s, 3H, OCH3), 7.11–7.18 (m, 2H, aromatic), 7.22–
7.39 (m, 6H, aromatic), 7.55 (s, 1H, aromatic), 7.66–7.74 (m, 2H,
aromatic). MS: m/z 334.28 [MH+].
6.1.4.3. (E)-1-(3-(6-Methoxynaphth-2-yl)but-2-enyl)piperidine
[(E)-2c]. Yield: 50%; pale yellow solid; mp 91.2–93.1 °C. IR
(cmÀ1): 2935, 2507, 1628, 1600, 1482, 1455, 1203, 1028, 844,
810. 1H NMR (400 MHz, CDCl3) d: 1.42–1.55 (m, 2H, N(CH2CH2)2-
CH2), 1.61–1.71 (m, 4H, N(CH2CH2)2CH2), 2.17 (s, 3H, CH3–C@CH),
2.42–2.62 (m, 4H, N(CH2CH2)2CH2), 3.23 (d, 2H, J = 6.7 Hz, CH2–
N), 3.94 (s, 3H, OCH3), 6.04–6.11 (m, 1H, CH3–C@CH), 7.10–7.18
(m, 2H, aromatic), 7.58–7.64 (m, 1H, aromatic), 7.70 (d, 1H,
J = 8.7 Hz, aromatic), 7.73 (d, 1H, J = 8.7 Hz, aromatic), 7.77 (d,
1H, J = 1.2 Hz, aromatic). MS: m/z 296.21 [MH+].
6.1.5.5. (R/S)-1-(3-(6-Methoxynaphth-2-yl)butyl)piperidine [(R/S)-
3c]. Yield: 63%; yellow oil. IR (cmÀ1): 3053, 2928, 2850, 2761,
2058, 1904, 1633, 1604, 1263, 1158, 1032, 848. 1H NMR
(400 MHz, CDCl3) d: 1.34 (d, 3H, J = 6.9 Hz, CH3–CH), 1.38–1.48
(m, 2H, N(CH2CH2)2CH2), 1.54–1.64 (m, 4H, N(CH2CH2)2CH2),
1.84–1.98 (m, 2H, CH–CH2), 2.13–2.24 (m, 1H, HCH–N), 2.25–
2.45 (m, 5H, HCH–N + N(CH2CH2)2CH2), 2.80–2.94 (m, 1H, CH3–
CH), 3.94 (s, 3H, OCH3), 7.11–7.18 (m, 2H, aromatic), 7.34 (dd,
1H, J = 1.6 Hz, J = 8.4 Hz, aromatic), 7.56 (s, 1H, aromatic),
7.67–7.73 (m, 2H, aromatic). MS: m/z 298.27 [MH+].
6.1.4.4. (E)-N-Benzyl-3-(biphenyl-4-yl)-N-methylbut-2-en-1-amine
[(E)-2d]. Yield: 49%; pale yellow solid; mp 67.2–68.3 °C. IR
(cmÀ1): 3032, 2978, 2537, 2488, 2388, 1644, 1486, 1469, 1420,
765, 696. 1H NMR (400 MHz, CDCl3) d: 2.11 (s, 3H, CH3–C@CH),
2.32 (s, 3H, N–CH3), 3.27 (d, 2H, J = 6.7 Hz, CH2–N), 3.61 (s, 2H,
CH2-Ph), 6.02–6.10 (m, 1H, CH3–C@CH), 7.25–7.32 (m, 1H, aro-
matic), 7.33–7.42 (m, 5H, aromatic), 7.43–7.50 (m, 2H, aromatic),

D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
1211
6.1.5.6. (R/S)-N-Benzyl-3-(biphenyl-4-yl)-N-methylbutan-1-am-
ine [(R/S)-3d]. Flash chromatography mobile phase: hexane–
ethyl acetate–7 N NH3 in methanol (80:20:1.0). Yield: 49%; yellow
oil. IR (cmÀ1): 3059, 3025, 2922, 2851, 2783, 1484, 1451, 1026,
1006, 835, 731, 695. 1H NMR (400 MHz, CDCl3) d: 1.30 (d, 3H,
J = 6.9 Hz, CH3–C), 1.79–1.94 (m, 2H, CH–CH2), 2.19 (s, 3H, N–
CH3), 2.27–2.46 (m, 2H, CH2–N), 2.82–2.93 (m, 1H, CH3–CH), 3.48
(s, 2H, CH2-Ph), 7.23–7.39 (m, 8H, aromatic), 7.42–7.49 (m, 2H, aro-
matic), 7.51–7.56 (m, 2H, aromatic), 7.59–7.64 (m, 2H, aromatic).
MS: m/z 330.24 [MH+].
6.2.2. Opioid receptor radioligand binding assays
6.2.2.1. Materials and general procedures. The guinea pig
brains and rat brains were commercially available (Harlan-Winkel-
mann, Borchen, Germany). Homogenizer: Elvehjem Potter (B.
Braun Biotech International, Melsungen, Germany). Centrifuge:
High-speed cooling centrifuge model Sorvall RC-5C plus (Thermo
Fisher Scientific, Langenselbold, Germany). Filter: Printed Filtermat
Typ A and B (Perkin–Elmer LAS, Rodgau-Jügesheim, Germany),
presoaked in 0.5% aqueous polyethylenimine for 2 h at room tem-
perature before use. The filtration was carried out with a MicroBeta
FilterMate-96 Harvester (Perkin–Elmer). The scintillation analysis
was performed using Meltilex (Typ A or B) solid scintillator (Per-
kin–Elmer). The solid scintillator was melted on the filtermat at a
temperature of 95 °C for 5 min. After solidifying of the scintillator
at room temperature, the scintillation was measured using a
MicroBeta Trilux scintillation analyzer (Perkin–Elmer). The overall
counting efficiency was 20%. All experiments were carried out in
triplicates using standard 96-well-multiplates (Diagonal, Muen-
ster, Germany). The IC50 values were determined in competition
experiments with at least six concentrations of the test compounds
6.1.5.7. (R/S)-3-(6-Methoxynaphth-2-yl)-N-methylbutan-1-am-
ine [(R/S)-3e]. Flash chromatography mobile phase: ethyl ace-
tate–methanol–7 N NH3 in methanol (70:30:2). Yield: 35%; yellow
oil. IR (cmÀ1): 2953, 2922, 2851, 2789, 1604, 1262, 1029, 849, 807.
1H NMR (400 MHz, DMSO-d6) d: 1.26 (d, 3H, J = 6.9 Hz, CH3–C),
1.68–1.79 (m, 2H, CH–CH2), 2.20 (s, 3H, N–CH3), 2.25–2.43 (m,
2H, CH2–N), 2.84–2.96 (m, 1H, CH3–CH), 3.27–3.32 (br s, 1H, NH,
exchanges with D2O), 3.86 (s, 3H, OCH3), 7.10–7.15 (m, 1H, aro-
matic), 7.25–7.29 (m, 1H, aromatic), 7.34–7.39 (m, 1H, aromatic),
7.61 (s, 1H, aromatic), 7.72–7.78 (m, 2H, aromatic). MS: m/z
244.08 [MH+].
Ò
and were calculated with the program GRAPHPAD PRISM 3.0 (Graph-
Pad Software, San Diego, CA, USA) by non-linear regression analy-
sis. The Ki values were calculated according to the formula of
Cheng and Prusoff.
6.1.5.8. (R/S)-3-(Biphenyl-4-yl)-N-methylbutan-1-amine [(R/S)-
3f]. Flash chromatography mobile phase: ethyl acetate–metha-
nol–7 N NH3 in methanol (70:30:2). Yield: 38%; white solid; mp
178–180 °C. IR (cmÀ1): 3295, 3025, 2956, 2923, 2868, 2776,
2461, 1597, 1484, 1349, 1006, 763, 696. 1H NMR (400 MHz,
DMSO-d6) d: 1.25 (d, 3H, J = 6.9 Hz, CH3–C), 1.84–1.98 (m, 2H,
CH–CH2), 2.48 (s, 3H, N–CH3), 2.59–2.70 (m, 1H, HCH–N), 2.75–
2.92 (m, 2H, CH3–CH + HCH–N), 7.32–7.39 (m, 3H, aromatic),
7.42–7.50 (m, 2H, aromatic), 7.59–7.69 (m, 4H, aromatic), 8.46–
8.90 (br s, 1H, NH, exchanges with D2O). MS: m/z 240.10
[MH+].
6.2.2.2. Preparation of the tissue. Five guinea pig brains (
l- and
j-opioid receptor assay) or six rat brains (d opioid receptor assay)
were homogenized with the potter (500–800 rpm, 10 up-and-down
strokes) in six volumes of cold 0.32 M sucrose. The suspension was
centrifuged at 1200g for 10 min at 4 °C. The supernatant was sepa-
rated and centrifuged at 23,500g for 20 min at 4 °C. The pellet was
resuspended in 5-6 volumes of buffer (50 mM Tris, pH 7.4) and cen-
trifuged again at 23,500g (20 min, 4 °C). This procedure was re-
peated twice. The final pellet was resuspended in 5-6 volumes of
buffer, the protein concentration was determined according to the
method of Bradford using bovine serum albumin as standard, and
subsequently the preparation was frozen (À80 °C) in 1.5 mL por-
tions containing about 1.5 mg protein/mL.
6.2. Pharmacology
6.2.1. Sigma radioligand binding assays
Binding assays were performed on rat liver membranes as pre-
6.2.2.3.
j-Opioid receptor binding assay. The test was per-
viously described.16 For
r1-Rs assay 250 lg of rat liver homoge-
formed with the radioligand [3H]-U-69593 (55 Ci/mmol, Amer-
sham, Little Chalfont, UK). The thawed membrane preparation
nate was incubated for 120 min at 37 °C with 1 nM [3H]-(+)-
pentazocine (Perkin–Elmer, specific activity 34.9 Ci/mmol) in
50 mM Tris–HCl, pH 8.0, 0.5 mL final volume. Non-specific binding
(about 75
tions of test compounds, 1 nM [3H]-U-69593, and Tris–MgCl2-buf-
fer (50 mM, 8 mM MgCl2, pH 7.4) in a total volume of 200 L for
lg of the protein) was incubated with various concentra-
was defined in the presence of 10 lM haloperidol. The reaction
l
was stopped by vacuum filtration through GF/B glass-fibre filters
presoaked with 0.5% polyethylenimine, followed by rapid washing
with 2 mL ice-cold buffer. Filters were placed in 3 mL scintillation
cocktail and the radioactivity determined by liquid scintillation
counting.
120 min at 37 °C. The incubation was terminated by rapid filtration
through the presoaked filtermats using a cell harvester. After
washing each well five times with 300 lL of water, the filtermats
were dried at 95 °C. The bound radioactivity trapped on the filters
was counted as described above. The non-specific binding was
For
r2-Rs assay, 150 lg of rat liver homogenate were incubated
determined with 10
69593 is 0.69 nM.
lM unlabelled U-69593. The Kd-value of U-
for 120 min at room temperature with 3 nM [3H]-DTG (Perkin–El-
mer, specific activity 58.1 Ci/mmol) in 50 mM Tris–HCl, pH 8.0,
0.5 mL final volume. (+)-pentazocine (100 nM) and haloperidol
6.2.2.4.
formed with the radioligand [3H]-DAMGO (51 Ci/mmol, Perkin–
Elmer LAS). The thawed membrane preparation (about 75 g of
l-Opioid receptor binding assay. The test was per-
(10
lM) were used to mask r1-Rs and to define non-specific bind-
ing, respectively.
l
Competition studies were done using at least 11 different con-
centrations of the compound under investigation prepared as
10 mM stock solutions in 100% DMSO and diluted with buffer on
the day of the experiment. The maximal DMSO final concentration
in the incubation tubes was 0.1%.
IC50 values and Hill’s coefficients nH were calculated by non-lin-
ear regression using a four parameters curve-fitting algorithm of
the SigmaPlot software, and are the means SEM of three separate
determinations performed in duplicate. The corresponding Ki val-
ues were obtained by means of the Cheng–Prusoff equation.
the protein) was incubated with various concentrations of test
compounds, 3 nM [3H]-DAMGO, and Tris–MgCl2–PMSF-buffer
(50 mM, 8 mM MgCl2, 400
200 L for 150 min at 37 °C. The incubation was terminated by ra-
pid filtration through the presoaked filtermats using a cell har-
vester. After washing each well five times with 300 L of water,
lM PMSF, pH 7.4) in a total volume of
l
l
the filtermats were dried at 95 °C. The bound radioactivity trapped
on the filters was counted as described above. The non-specific
binding was determined with 10 lM unlabelled naloxone. The
Kd-value of DAMGO is 0.57 nM.

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D. Rossi et al. / Bioorg. Med. Chem. 18 (2010) 1204–1212
6.2.2.5. d-Opioid receptor binding assay. The test was per-
Supplementary data
formed with the radioligand [3H]-DPDPE (40 Ci/mmol, Perkin–
Elmer LAS). The thawed membrane preparation (about 75 lg of
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.12.039.
the protein) was incubated with various concentrations of test
compounds, 1 nM [3H]-DPDPE, and Tris–MgCl2–PMSF-buffer
(50 mM, 8 mM MgCl2, 400
200 L for 150 min at 37 °C. The incubation was terminated by ra-
pid filtration through the presoaked filtermats using a cell har-
vester. After washing each well five times with 300 L of water,
the filtermats were dried at 95 °C. The bound radioactivity trapped
on the filters was counted as described above. The non-specific
lM PMSF, pH 7.4) in a total volume of
References and notes
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Acknowledgements
This work was supported by Grant from Ministero dell’Istruzi-
one, dell’Università e della Ricerca, Italy (MIUR).
Special thanks to Sigma–Aldrich, in the person of Claudio
Costantini, for providing Pd(0) EnCat™ 30NP.