Identification of a potent and selective ρ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells

Article abstract of DOI:10.1016/j.bmc.2011.09.016

Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (ρ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess

Full text of DOI:10.1016/j.bmc.2011.09.016

Bioorganic & Medicinal Chemistry 19 (2011) 6210–6224
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of a potent and selective r1 receptor agonist potentiating
NGF-induced neurite outgrowth in PC12 cells
Daniela Rossi a, Alice Pedrali a, Mariangela Urbano a, Raffaella Gaggeri a, Massimo Serra a,
Leyden Fernández b, Michael Fernández c, Julio Caballero d, Simone Ronsisvalle e, Orazio Prezzavento e,
Dirk Schepmann f, Bernhard Wuensch f, Marco Peviani g, Daniela Curti g, Ornella Azzolina a,
Simona Collina a,
⇑
a Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
b Life Sciences, Computational Genomics Department, Barcelona Supercomputing Center, 08034 Barcelona, Spain
c Department of Bioscience and Bioinformatics, Kyushu Institute of Technology (KIT), 680-4 Kawazu, 820-8502 Iizuka, Japan
d Centro de Bioinformática y Simulación Molecular, Facultad de Ingeniería en Bioinformática, Universidad de Talca, 2 Norte 685, 721 Casilla, Talca, Chile
e Department of Drug Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
f Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster, Hittorfstrasse 58-62, 48149 Muenster, Germany
g Department of Legal Medicine, Forensic and Pharmaco-toxicological Sciences,Lab. of Cellular and Molecular Neuropharmacology, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2011
Revised 5 September 2011
Accepted 8 September 2011
Available online 14 September 2011
Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (r) ligands
based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was
studied. Novel arylalkenylamines generally possess high 1 receptor affinity (Ki values <25 nM) and good
2 selectivity (Ki 2 >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog
(R,S)-33 were observed to be excellent r1 receptor ligands (Ki = 0.70 and 0.86 nM, respectively) and to
display significantly high selectivity over r2 -, and -opioid receptors and phencyclidine (PCP) binding
site of the N-methyl- -aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve
r
r1/
r
r
,
l
j
Keywords:
D
r
Ligands
growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective r1
receptor antagonist BD-1063 totally counteracted this effect, confirming that r1 receptors are involved
in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a r1 agonist profile. As a part
of our work, a threedimensional r1 pharmacophore model was also developed employing GALAHAD
methodology. Only active compounds were used for deriving this model. The model included two hydro-
phobes and a positive nitrogen as relevant features and it was able to discriminate between molecules
with and without affinity toward r1 receptor subtype.
r1 Agonist profile
NGF-induced neurite outgrowth
Neurite elongation
PC12 cells
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
endoplasmic reticulum (ER) at the ER–mitochondria interface,9
where it regulates ER–mitochondrion signaling and ER–nucleus
The sigma (
dine (PCP) binding site. To date, two subtypes of the
have been identified, the r1 and the 2. Concerning the r2 sub-
r
) receptor is unique non-opioid, non-phencycli-
crosstalk.10 The receptor is considered to be an ER chaperone
protein acting as inter-organelle signaling modulator because it
can translocate to the plasma membrane or to other subcellular
compartments under stressful conditions and/or pharmacological
manipulation.11,12 The most prominent action of r1 receptor is
the modulation of voltage-regulated and ligand-gated ion chan-
nels, including Ca2+, K+, Na+, Clꢀ, and NMDA and IP3 receptors.10
Moreover the r1 receptor can interact with ER-resident chaper-
ones like Binding immunoglobulin Protein/78 kDa Glucose-Regu-
lated Protein (BiP/GRP78), acting as sensor of ER-stress caused by
unfolded proteins overload,13 and it can promote cell survival by
modulating the expression of B-cell lymphoma 2 (Bcl-2), the major
antiapoptotic member of the Bcl-2 family.14 Literature evidences
suggest the involvement of the receptor in diseases such as drug
addiction, depression, neurodegeneration, pain-related disorders,
and cancer.1 Recently, a mutation in the r1 receptor gene was
r
receptor
r
type, although it has not yet been cloned, accumulating evidences
suggest the specific involvement of the receptor in the death sig-
naling of cancer cells.1,2 As regards to the
r1 subtype, the receptor
is ubiquitously expressed in mammalian tissues;3 moreover it has
been purified and cloned from several mammalian species as a 223
amino acid protein with 90% identical amino acid sequences across
species.4–7 The macromolecule contains three hydrophobic do-
mains. Two of them are transmembrane-spanning helices with
an extracellular loop in between and an intracellular C terminus.8
The r1 is an intracellular receptor specifically localized in the
⇑
Corresponding author. Tel.: +39 0382987379; fax: +39 0382422975.
E-mail address: simona.collina@unipv.it (S. Collina).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.016

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6211
found to be associated with frontotemporal lobar degeneration
1: Ar = naphth-2-yl, R = CH3
2: Ar = biphen-4-yl, R = CH3
3: Ar = naphth-2-yl, R = CH2C6H5
4: Ar = biphen-4-yl, R = CH2C6H5
(FTLD), the most common cause of dementia under the age of
65 years.15 Interestingly, the r1 receptor has been found impli-
cated in neurite sprouting in vitro suggesting a role for the receptor
in neuroplasticity. In fact, experiments on PC12 cells, an in vitro
model of neuronal differentiation,16 have shown that overexpres-
sion of r1 receptor or exposure to r1 receptor agonists potentiate
neurite outgrowth and elongation induced by growth factors, such
as nerve growth factor (NGF) or epidermal growth factor (EGF).17,18
In line, experiments performed on organotypic spinal cord cultures
have highlighted that exposure to a r1 receptor agonist protects
motoneurons against glutamate-induced excitotoxicity and in-
creases neurite elongation.19
Ar
N
R
Figure 2. Structure of arylalkenylamines (E)-1–4.
O
O
a
+
NHR1R2
NR1R2
5: NR1R2 = N,N-dimethylamine
6: NR1R2 = N-benzyl-N-methylamine
7: NR1R2 = piperidine
8: NR1R2 = 4-benzylpiperidine
9: NR1R2 = morpholine
Therefore, the identification of new and potent ligands, with
high selectivity for the r1 receptor and defined action, will be of
Scheme 1. Synthesis of b-aminoketones 5–9. Reagents and conditions: (a) anhy-
drous toluene, reflux for compounds 5, 6; PEG 400, rt for compounds 7–9.
great interest to better understand the role of
r1 receptor in differ-
ent pathologies. This may pave the way to development of innova-
tive therapeutic strategies addressed to modulate neuronal
protection, plasticity, and regeneration.
and morpholine to but-3-en-2-one in polyethylene glycol 400
(PEG 400) afforded b-aminoketones 8 and 9, respectively (Scheme
1). After an acid–base work up, pure 9 was obtained in good yields;
concerning compound 8, a further purification by filtration on an
alumina pad was required.
The synthesis of arylalkenylamines 10–30 was then carried out
via the nucleophilic addition of the opportune aromatic anion to b-
aminoketones 5–9, followed by direct dehydration under standard
acidic conditions (37% HCl, Scheme 2, method I, see experimental
section).20 A basic work-up allowed the isolation of the desired ole-
finic compounds as free bases. The reaction resulted highly regio-
and stereoselective for all arylalkenylamines, as confirmed by 1H
NMR analysis and NOESY experiments of crude compounds, in
accordance with our previous experience.20
In the last 10 years our research group has conducted extensive
studies aimed at discovering novel
neurodegenerative diseases treatment. In this context, we reported
the design, synthesis and biological evaluation of various 1 recep-
r ligands potentially useful in
r
tor ligands based on both arylalkenyl- and arylalkylaminic scaf-
folds (Fig. 1).20 In that paper we carefully evaluated the influence
of different aromatic portions (naphth-2-yl, naphth-1-yl, biphen-
4-yl, 6-hydroxy-, and 6-methoxy-naphth-2-yl) combined with
two aminic moieties (N,N-dimethylamine and N-benzyl-N-methyl-
amine) on
influence of the three carbon spacer between the aromatic portion
and the aminic moiety on receptor binding by preparing new
r receptors affinity and selectivity. We also studied the
r
derivatives characterized by olefinic, alkylic and alcoholic spacers
(arylalkenyl-, arylalkylamines, and arylalkylaminoalcohols, respec-
tively, Fig. 1).21
4
In the present contribution we extended our SAR studies further
with arylalkenylamines structurally related to (E)-1–4 (Fig. 2);20
particularly, herein we report the synthesis, drug-likeness evalua-
tion, and in vitro profile of new derivatives. For the most active ole-
fin, the corresponding arylalkylamine was studied. Moreover, we
report on a deep investigation of the pharmacological profile of
the most promising compounds by evaluating their effect on
NGF-induced neurite outgrowth and elongation in PC12 cells.
Finally, we report on the development of a r1 pharmacophore
model based on arylalkenyl- and arylalkylamines using GALAHAD
(Genetic Algorithm with Linear Assignment of Hypermolecular
Alignment of Datasets),22 with the final aim of obtaining a model
that could provide a rational hypothetical picture of the chemical
features responsible for the r1 affinity of these compounds.
1
a, b
c, d
Br
Ar
Ar
NR1R2
Ar
NR1R2
3
OH
2
crude 10-30
HCl
NR1R2
e, f
Ar
(E)-10-30 HCl
10: Ar = phenyl
11: Ar = 4-methoxyphenyl
12: Ar = 3-methoxyphenyl
NR1R2
=
N
13: Ar = phenyl
14: Ar = 4-methoxyphenyl
15: Ar = 3-methoxyphenyl
C6H5
N
N
N
2. Results
16: Ar = naphth-2-yl
17: Ar = biphen-4-yl
18: Ar = phenyl
19: Ar = 4-methoxyphenyl
20: Ar = 3-methoxyphenyl
2.1. Chemistry
At first the synthesis of b-aminoketones 5–7 was accomplished
via the Michael addition of N,N-dimethylamine, N-benzyl-N-
methylamine, and piperidine to but-3-en-2-one, essentially
according to the methodologies already described by us (Scheme
1).20,21 Similarly, the Michael addition of both 4-benzylpiperidine
21: Ar = naphth-2-yl
22: Ar = biphen-4-yl
23: Ar = phenyl
24: Ar = 4-methoxyphenyl
25: Ar = 3-methoxyphenyl
C6H5
26: Ar = naphth-2-yl
27: Ar = biphen-4-yl
28: Ar = phenyl
N
O
29: Ar = 4-methoxyphenyl
30: Ar = 3-methoxyphenyl
Ar
NR1R2
Ar
NR1R2
Ar
NR1R2
OH
Arylalkylaminoalcohols
Arylalkenylamines
Arylalkylamines
Scheme 2. Synthesis of arylalkenylamines (E)-10–30 HCl (method I). Reagents and
conditions: (a) t-BuLi, anhydrous Et2O, ꢀ78 °C to rt; (b) ketones 5–9, ꢀ78 °C; (c) 37%
HCl, rt; (d) 1 N NaOH; (e) 37% HCl, rt; (f) crystallization from acetone.
Figure 1. General structures of arylalkenyl-, arylalkylamines, and arylalkylamino-
alcohols.

6212
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
Arylalkenylamines 10–30, after purification by either crystalli-
to define nonspecific binding, respectively. The r receptor binding
zation or flash chromatography (when required, see experimental
section), were converted into the corresponding hydrochlorides
and successively crystallized from acetone, yielding desired (E)-
arylalkenylamines in satisfactory yields, with the only exception
of 19 HCl and 29 HCl. In order to improve the reaction yields and
to prepare compounds (E)-19 and (E)-29 in amount suitable for
biological investigation, we changed our synthetic strategy. Thus,
racemic arylalkylaminoalcohols 31 and 32 were synthesized via
nucleophilic addition of the appropriate arylic anion to the oppor-
tune b-aminoketone and isolated in good yields after an acid–base
work up; arylalkenylamines 19 and 29 were then prepared by reg-
ioselective dehydration of the respective arylalkylaminoalcohols
employing the polymer-bound triphenylphosphine (TPPP)-iodine
system (Scheme 3).21 Dehydration reaction of alcoholic intermedi-
ates resulted stereoselective: the main reaction product was the (E)
isomer for both substrates tested, as confirmed by both 1H NMR
analysis of crude products and NOE experiments.21 Crude 19, 29
were then converted into the corresponding hydrochlorides and
crystallized from acetone, providing pure (E)-19 HCl and (E)-29
HCl in satisfactory yields.
affinities, expressed as inhibition constants (Ki), are summarized in
Table 1.
The binding properties of (E)-10–30 HCl were investigated also
for
l-, j-opioid receptors and PCP binding site of NMDA receptors
using, as receptor material, guinea pig brain and pig brain cortex,
respectively. [3H][
D
-Ala2,Me-Phe4,Gly-ol5]enkephalin ([3H]-DAM-
GO, 1 nM,
l
), [3H]-(5R,7R,8b-(ꢀ)-N-methyl-N-[7-(1-pyrrolidinyl)-
1-oxaspiro (4–5) dec-8-yl]-benzeneacetamide) ([3H]-U 69,593,
1 nM,
,b]cyclohepten-5,10-imine ([3H]-MK-801, 2 nM, NMDA) were
employed as specific radioligands. The non-specific binding was
determined with unlabeled naloxone (10 M, ), unlabeled U
69,593 (10 M, ) and unlabeled (+)-MK-801 (10 M, PCP binding
site of NMDA). The residual binding of the radioligand is given at a
concentration of 1 M ( and receptor binding assays) or 10
j
) and [3H]-(5R,10S)-(+)-5-methyl-10,11-dihydro-5I-dibenzo-
[a
l
l
l
j
l
l
l
j
lM
(NMDA receptor binding assays) of tested compounds. When a
significant inhibition of the radioligand was observed, the Ki value
was determined. Results are summarized in Table 1.
2.2.2. NGF-induced neurite outgrowth in PC12 cells
Finally, (R,S)-33 HCl was synthesized by catalytic reduction of
the corresponding arylalkenylamine (E)-17 in hydrogen atmo-
sphere, using Pd(0) EnCat™ 30NP as catalyst (Scheme 4),21 easily
isolated by solid phase extraction (SPE, SCX cartridge) and then
converted into the corresponding hydrochloride.
In a preliminary set of experiments, we assessed the effect of a
known r1 agonist, 2-(4-morpholinethyl)1-phenylcyclohexane-
carboxylate hydrochloride (PRE-084), and an antagonist, 1-[2-
(3,4-dichlorophenyl)ethyl]-4-methylpiperazine
dihydrochloride
(BD-1063), on NGF-induced neurite outgrowth in PC12 cell line.
Cells were treated with NGF (2.5 ng/mL) alone or in the presence
of PRE-084 or BD-1063 for 5 days. NGF alone promoted neurite
sprouting in 18.1% 1.0 of the PC12 cells in culture (n = 8 different
2.2. Biology
2.2.1. Receptor binding studies
experiments); the average neurite length was 100.3 5.0
084 (10 m) significantly increased the percentage of cells with
neurite outgrowth to 27.5% 1.5 (p <0.01) (Fig. 3A) and the length
of neurites to 181.3 11.7 m (p <0.01) (Fig. 3B), whereas it was
ineffective at 2.5 M (Fig. 3). BD-1063 (5 M), co-administered
lm. PRE-
The binding properties of arylalkenylamines (E)-10–30 HCl
l
were investigated for
r receptor subtypes (r1 and r2) expressed
in guinea pig brain membranes. [3H]-(+)-pentazocine (3 nM) was
l
employed for r1 receptor binding assays in the presence of halo-
l
l
peridol (10
l
M) to measure the nonspecific binding. The non selec-
with PRE-084, totally counteracted the effect of PRE-084 on the
percentage of cells with neurite outgrowth (16.0% 2.4, p <0.025)
and significantly decreased the effect of PRE-084 on neurite elon-
tive radioligand [3H]-1,3-di-o-tolylguanidine ([3H]-DTG, 3 nM) was
employed for r2 receptor binding studies in the presence of an
excess of [2S-(2
a,6a
,11R)]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-
gation (140.1 10.8
lm, corresponding to a 23% decrease of neu-
3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol [(+)-SKF10,047,
rite length, p <0.025).
0.4
l
M] and non-tritiated DTG (5
l
M) to mask r1 receptors and
Compounds (E)-17 HCl and (R,S)-33 HCl were then tested in this
experimental setting to characterize their pharmacological profile
and to analyze their effect on NGF-induced neurite outgrowth.
(R,S)-33 HCl was able to increase the percentage of cells with neu-
rite outgrowth starting from 0.01 lM (23.4% 1.8, p <0.05)
(Fig. 4A), whereas at the same concentration no significant re-
a, b, c
Br
d, e
Ar
NR1R2
Ar
NR1R2
crude 19, 29
f, g
Ar
OH
(R,S)-31, 32
sponse was produced by PRE-084. (R,S)-33 HCl also increased neu-
rite elongation starting from 0.25
(Fig. 4B). Co-administration of BD-1063 (5
HCl (0.25 M), totally counteracted the effect of (R,S)-33 HCl on
l
M (130.9 7.2
lm, p <0.01)
lM) with the (R,S)-33
31: Ar = methoxyphenyl, NR1R2 = piperidine
32: Ar = methoxyphenyl, NR1R2 =morpholine
l
HCl
neurite outgrowth (18.6 1.0, p <0.05) and decreased neurite
length of 31% (p <0.01) (data not shown).
Ar
NR1R2
(E)-17 HCl, at the concentrations 0.05, 0.25, and 2.5
significantly enhance NGF-induced neurite outgrowth or elonga-
tion (Fig. 5). Co-administration of (E)-17 HCl (2.5 M) with BD-
1063 (5 M) significantly decreased (p <0.05) the percentage of
lM, did not
(E)-19, 29 HCl
l
Scheme 3. Synthesis of arylalkenylamines (E)-19, 29 HCl (method II). Reagents and
conditions: (a) t-BuLi, anhydrous Et2O, ꢀ78 °C to rt; (b) ketones 7 or 9, ꢀ78 °C; (c)
H2O, rt; (d) I2, TPPP, rt; (e) aq 5% NaHSO3, rt; (f) 37% HCl, rt; (g) crystallization from
acetone.
l
cells with neurite outgrowth.
2.3. Computational study
HCl
N
HCl
N
2.3.1. Drug-likeness studies
The ‘drug-likeness’ of the novel
r receptor ligands was assessed
a, b, c, d
on the basis of their structural properties by applying the Lipinski’s
‘rule of five’ (Table 2).23 Concerning lipophilicity, both clogP and
clogD values were calculated since, given their apparent pKa val-
ues, all molecules are present mainly in the protonated form at
physiological pH (7.4). For all the compounds studied, none of
(E)-17 HCl
(R,S)-33 HCl
Scheme 4. Synthesis of arylalkylamine (R,S)-33. Reagents and conditions: (a) SCX
cartridge; (b) H2, Pd(0) EnCat™ 30NP, abs EtOH, rt; (c) SCX cartridge; (d) 37% HCl, rt.

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6213
Table 1
Affinities of compounds (E)-1–4, 10–30 HCl toward r1,
r
2, NMDA, l-, and j-opioid receptors
Compd
Ki SEM (nM)
r2/
r1
Inhibition of the radioligand
-Opioidd
a
b
r1
r2
l
-Opioidc
j
NMDAe
(E)-1 HClf
(E)-2 HClf
(E)-3 HClf
(E)-4 HClf
(E)-10 HCl
(E)-11 HCl
(E)-12 HCl
(E)-13 HCl
(E)-14 HCl
(E)-15 HCl
(E)-16 HCl
(E)-17 HCl
(E)-18 HCl
(E)-19 HCl
(E)-20 HCl
(E)-21 HCl
(E)-22 HCl
(E)-23 HCl
(E)-24 HCl
(E)-25 HCl
(E)-26 HCl
(E)-27 HCl
(E)-28 HCl
(E)-29 HCl
(E)-30 HCl
(R,S)-33 HCl
19.8 0.3
1.40 0.2
7.88 0.3
6.32 0.3
>1000
148
106
873 12
4
6
7.5
NTg
NT
NT
NT
0%
10%
17%
119h
92h
49%
23%
0%
19%
36%
21%
38%
0%
NT
NT
NT
NT
0%
20%
5%
NT
NT
NT
NT
0%
0%
60%
67%
27%
8%
16%
32%
0%
0%
0%
17%
17%
0%
75.7
110.8
12.0
—
—
—
75.7
4
>1000
>1000
>1000
61.0
155 15
163 11
806 63
>1000
92.0 12
5.25 1.0
10.0 1.8
0.97 0.3
0.86 0.4
459 21
14.0 3.0
91.0 11
23.0 2.6
7.02 0.9
1.07 0.3
4.14 0.3
7.70 1.0
9.60 1.6
11.6 2.0
>1000
5
0.7
438h
197h
301h
44%
6%
29.5
16.3
36.2
129.1
0.4
10.6
3.2
0.7
2.6
5.1
1.7
0.8
23.9
33.3
—
—
—
35.1
9
111 21
191 11
149 13
294 22
16.0 1.1
18.0 1.7
5.49 0.5
7.10 1.2
5.80 1.0
229 32
386 27
>1000
0%
14%
22%
10%
10%
189h
157h
214h
15%
10%
3%
152h
149h
36%
44%
52%
12%
0%
0%
36%
31%
69%
19%
10%
20%
25%
>1000
>1000
0.70 0.3
>1000
>1000
103 10
8%
10%
36%
46%
0%
147.1
Values are means SEM of three experiments.
a
Displacement of 3 nM [3H]-(+)-pentazocine.
b
Displacement of 3 nM [3H]-DTG.
c
Displacement of 1 nM [3H]-DAMGO.
d
Displacement of 1 nM [3H]-U 69593.
e
Displacement of 2 nM [3H]-MK-801.
f
For comparative purposes, affinity data were included.16
g
Not tested.
Ki (nM).
h
Figure 3. Potentiating effect of r1 receptor agonist PRE-084 on NGF (2.5 ng/mL)-induced neurite (A) outgrowth and (B) elongation in PC12 cells. Histograms represent the
mean SEM of at least three different experiments. ⁄⁄, p <0.025; ⁄⁄⁄, p <0.01 versus NGF alone.
the criteria was violated, with the only exception of 22 HCl which
had a logD value above 5. Thus, compounds theoretically should
have good absorption and/or permeability properties.
In detail, 20 pharmacophore models were retained after
GALAHAD run using compounds of the training set. Each of the ob-
tained models represents a different tradeoff among the conflicting
demands of maximizing steric consensus (measured by steric
overlap: SO), maximizing pharmacophore consensus (measured
by pharmacophoric similarity: PhS), and minimizing energy
(measured by strain energy: SE). All the obtained models were
derived from more than seven ligands. In addition, they had Pareto
rank 0; this means no one model is superior to any other. GALA-
HAD models were compared according to Pareto ranking. Small
value of SE and high values of SO and PhS are desired for the best
2.3.2. Pharmacophore modeling studies
A
r1 pharmacophore model was developed by using GALAHAD
(Genetic Algorithm with Linear Assignment of Hypermolecular
Alignment of Datasets).22 GALAHAD models were derived by using
only ten active ligands as a training set [compounds 4, 16, 23–27,
33 herein presented and the structurally related compounds 38
and 43 previously described by us (Fig. 6)].20

6214
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
Figure 4. Potentiating effect of
r
1 receptor ligand (R,S)-33 HCl on NGF (2.5 ng/mL)-induced (A) neurite outgrowth and (B) elongation in PC12 cells. Histograms represent the
mean SEM of at least three different experiments. ⁄, p <0.05; ⁄⁄⁄, p <0.01 versus NGF alone.
Figure 5. Effect of
r1 receptor ligand (E)-17 HCl on NGF (2.5 ng/mL)-induced neurite (A) outgrowth and (B) elongation in PC12 cells. Where indicated (BD), BD-1063 (5 lM)
was co-administered with (E)-17 HCl. °, p <0.05 versus (E)-17 HCl without BD-1063.
model. The higher value of SE between all the models is 45.44; and
the lower value is 25.62. In general, SE values were closer to the
maximum for the majority of models; models with low SE value
had the lowest SO and PhS values. SO also had variation between
the minimum (SO = 145.8) and the maximum (SO = 225.4) consid-
ering all the twenty models. Finally, PhS had variation between
19.5 (the minimum) and 30.5 (the maximum). With the intention
to select the best model, we construct a 3D plot to visualize the
Pareto surface as previously described by us.24 The best GALAHAD
model had SE = 39.96, SO = 202.4 and PhS = 26.9 (Fig. 7). It is com-
prised of all the 10 structures, one conformer for each molecule in
the training set, plus a 3D database query derived from features
more or less shared among them. All conformers aligned represent
low-energy conformations of the molecules and it can be seen that
the final alignment shows a satisfactory superimposition of the
pharmacophoric points. In Figure 7, cyan and red spheres indicate
hydrophobes and positive nitrogens, respectively. The best model
includes 3 pharmacophore features: two hydrophobes (H1 and
H2) and one positive nitrogen (N1). The positive nitrogen
represented by N1 indicates that a positive charged atom is a
requirement of 1 ligands. This feature seems to be a requirement
of compounds to have affinity for r1 receptor; the two hydropho-
bic moieties of the pharmacophore schematically represented by
H1 and H2 reflect the need for a hydrophobic structure as the skel-
eton of the r1 ligands with an angular disposition.
We evaluated how well the model reflects r1 binding affinity.
For this, the best model was used as a template in aligning the full
DS including compounds 1–29 and 33 herein presented and the
structurally related compounds 34–44 previously described by us
(Fig. 6).20 We ran a partial least square (PLS) analysis by using
the features of the model as molecular descriptors to predict the
activities of all compounds. The scatter plot of the calculated
versus experimental values of log(1/Ki) using the GALAHAD model
is shown in Figure 8. The affinity log(1/Ki) = ꢀ1.4 was selected as
the threshold value between active and inactive compounds.
According to this graph, the model was able to identify active com-
pounds from the set of molecules which were not used in deriving
the model. Fifteen molecules that do not participate in the
r

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6215
Table 2
Calculated druglike properties of compounds (E)-10–30 HCl and (R,S)-33 HCl
Compound
logP
logDa
pKa
Rotatable bonds
H-Bond acceptors
Molecular weight
(E)-10 HCl
(E)-11 HCl
(E)-12 HCl
(E)-13 HCl
(E)-14 HCl
(E)-15 HCl
(E)-16 HCl
(E)-17 HCl
(E)-18 HCl
(E)-19 HCl
(E)-20 HCl
(E)-21 HCl
(E)-22 HCl
(E)-23 HCl
(E)-24 HCl
(E)-25 HCl
(E)-26 HCl
(E)-27 HCl
(E)-28 HCl
(E)-29 HCl
(E)-30 HCl
(R,S)-33 HCl
2.82
2.67
2.67
4.55
4.39
4.39
4.66
5.32
3.67
3.52
3.52
6.53
7.19
5.54
5.38
5.38
3.59
4.25
2.61
2.45
2.45
5.43
1.50
1.32
1.36
3.26
3.08
3.12
3.07
3.82
2.12
2.04
2.08
4.86
5.60
3.91
3.82
3.86
3.49
4.17
2.51
2.37
2.37
3.00
8.70
8.73
8.68
8.66
8.69
8.64
8.98
8.89
8.94
8.87
8.82
9.05
8.97
8.64
8.95
8.91
6.78
6.69
6.74
6.67
6.67
9.87
3
5
5
5
7
7
3
4
3
5
5
5
6
5
7
7
3
4
3
5
5
5
1
2
2
1
2
2
1
1
1
2
2
1
1
1
2
2
2
2
2
3
3
1
175.27
205.30
205.30
251.37
281.39
281.39
265.39
291.43
215.33
245.36
245.36
355.52
381.55
305.46
335.48
335.48
267.37
293.40
217.31
247.33
247.33
293.45
a
Determined at pH 7.4.
tion of the naphth-2-yl derivative (E)-26 HCl and the biphen-4-yl
derivative (E)-27 HCl, which interact in the low nanomolar range
with the r1 receptor subtype (Ki = 9.60 1.6 nM and 11.6
2.0 nM, respectively). An analogous trend was observed for the
N,N-dimethylamine compound series: only the naphth-2-yl deriv-
ative (E)-1 HCl and the biphen-4-yl derivative (E)-2 HCl signifi-
cantly interact with r1 receptor subtype (Ki = 19.8 0.3 and
1.40 0.2 nM, respectively). Concerning r2 receptor affinity, only
the 4-benzyl-piperidine derivatives (E)-21–25 HCl revealed an
interesting affinity for r2 receptor subtype, having Ki values rang-
ing from 5.49 to 18.0 nM (Fig. 9B).
R
Ar
N
CH3
Ki σ1(nM)
34: Ar = naphth-2-yl
35: Ar = naphth-1-yl
36: Ar = 6-hydroxy-naphth-2-yl
37: Ar = 4-methoxyphenyl
R =
CH3
25
102
229
21.4
Biological results discussed so far clearly evidenced that the
arylalkenylamines considered in this study generally possess high
R
Ar
N
CH3
r1 receptor affinity and good r1
exception of (E)-21–25 HCl, for which good binding affinity for
both receptor subtypes was proved. Therefore, they could repre-
/r2 selectivity, with the only
Ki σ1 (nM)
1.95
47.2
19.0
2.30
1.02
38: Ar = naphth-2-yl
39: Ar = naphth-1-yl
40: Ar = 6-hydroxy-naphth-2-yl
41: Ar = 6-methoxy-napht-2-yl
42: Ar = biphen-4-yl
CH3
R =
r
sent a useful starting point for the next development of
r ligands
potentially helpful in tumor diagnosis.25
Among the considered arylalkenylamines, the most interesting
one in terms of both r1 receptor affinity and selectivity over r2
receptor subtype is the biphen-4-yl derivative (E)-17 HCl
43: Ar = naphth-2-yl
44: Ar = biphen-4-yl
5.4
5.8
CH2C6H5
R =
(Ki = 0.86 0.4 nM, Ki
nucleus as aminic moiety. Thus we prepared the corresponding
arylalkylamine derivative (R,S)-33 HCl: similar 1 receptor affinity
and r1 r2 selectivity were observed (Ki = 0.70 0.3 nM, Kir2
Ki 1 = 147.1).
In order to widen the binding profile of newly synthesized
compounds, their affinity toward - and -opioid receptors and
PCP binding site of the NMDA receptors was also investigated
(Table 1). With regard to - and -opioid receptor binding, novel
r2/Kir1 = 129.1), bearing the piperidine
Figure 6. Chemical structure and r1 binding affinity data of compounds 34–44.
r
/
/
generation of the model have log(1/Ki) values above ꢀ1.4 (Fig. 8).
From them, 40% of the active molecules were correctly predicted.
On the other hand, 12 molecules that do not participate in the gen-
eration of the model, have log(1/Ki) values below ꢀ1.4; none was
predicted with an activity greater than this value.
r
l
j
l
j
compounds generally showed low affinity for both receptor sub-
3. Discussion
types, with the only exception of (E)-13, 14 HCl and (E)-23, 24
HCl (Ki values between 92 and 152 nM at
between 157 and 438 nM at -opioid receptors). Compounds
(E)-15 HCl and (E)-25 HCl showed a moderate inhibition of the
radioligand only in the -opioid receptor binding assays (Ki = 301
l-opioid receptors and
The arylalkenylamines herein presented generally showed
interesting binding properties for r1 receptor subtype (Ki values
<25 nM, Table 1). As evidenced in Figure 9A, all the 4-benzyl-piper-
j
j
idine derivatives (E)-21–25 HCl revealed high affinity for
r
1 recep-
and 214 nM, respectively). Concerning binding studies at the PCP
binding site of the NMDA receptors, none of the tested compounds
displayed a significant binding affinity. Only compounds (E)-12, 13
HCl, and (E)-27 HCl caused an inhibition of the radioligand higher
tor subtype, independently of the aromatic moiety of the molecule
(Ki values ranging from 1.07 nM to 23.0 nM). On the contrary, a
dramatic decrease in r1 receptor affinity was generally evidenced
for morpholine derivatives [Kir1 >1000 nM], with the only excep-
then 50% at the screening concentration of 10 lM.

6216
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
Figure 7. Selected pharmacophore model and molecular alignment of the compounds used to elaborate the model. Cyan and red spheres are represented for hydrophobes
and positive nitrogens, respectively.
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
-3.4 -3.2 -3.0 -2.8 -2.6 -2.4 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4
experimental log (1/Ki)
Figure 8. Scatter plot of the experimental activities versus predicted activities for pharmacophore model (d) predictions for compounds used to elaborate the model (s)
predictions for the remaining compounds. The line in log(1/Ki) = ꢀ1.4 means that this value was selected as the threshold value between active and inactive compounds.
Overall binding results confirmed that (E)-17 HCl and (R,S)-33
HCl are the most promising r1 ligands of this study, displaying
more, cells treatment with SA4503 (1.0
lM) or fluvoxamine
(10
l
M) also potentiates NGF-induced neurite elongation.26
high selectivity toward r2
,
l
-, and
j
-opioid receptors and PCP
To validate this assay, we firstly performed preliminary experi-
ments treating PC12 cells with NGF and with the well known r1
receptor ligands PRE-084 and BD-1063, having agonist and antag-
onist profile, respectively. In agreement with literature results,
NGF promoted neurite sprouting as well as neurite elongation.
The well characterized r1 receptor agonist PRE-084 significantly
increased the percentage of cells with neurite outgrowth and
binding site of the NMDA receptors. Moreover, these compounds
theoretically should have good permeability properties and should
effectively permeate the brain blood barrier, having logP values
higher then 2, as evidenced by drug likeness studies.
Thus, (E)-17 HCl and (R,S)-33 HCl have been selected by us for a
deeper evaluation of their biological profile. In this contest, the
NGF induced-PC12 cells neurite outgrowth and elongation model
represents a valuable tool for drawing agonist/antagonist profile
of selected compounds. Indeed, it has been reported that r1
agonists potentiate the effect of NGF in PC12 cells,17 while r1
receptor antagonists do not exert any relevant influence on
this NGF-induced effect. Particularly, fluvoxamine, 1-[2-(3,4-
dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503),
4-phenyl-1-(4-phenylbutyl) piperi-dine (PPBP), dehydroepian-
drosterone (DHEA)-sulfate26, and donezepil27 significantly increase
the percentage of cells with NGF-induced neurite outgrowth in the
length of neurites only at the concentration of 10
Moreover, the r1 receptor antagonist BD-1063 (5
l
l
M (Fig. 3).
M), when
co-administered with PRE-084, totally counteracted the effect of
the agonist on both neurite outgrowth and elongation.
Concerning our molecules, compound to be highlighted is (R,S)-
33 HCl: it was effective in increasing both neurite outgrowth and
elongation at lower concentration with respect to PRE-084
(0.01 lM vs 10 lM for neurite outgrowth and 0.25 lM vs 2.5 for
neurite elongation) (Figs. 3 and 4). Interestingly, these effects were
totally counteracted by the co-administration of BD-1063,
confirming that r1 receptors are involved in (R,S)-33 HCl modula-
concentration range of 0.1–10
lM. This effect is counteracted by
co-administration of selective r1 receptor antagonists. Further-
tion of the NGF effect in PC12 cells and suggesting the
r1 receptor

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6217
Figure 9. r1 (A) and r2 (B) receptor affinity (Ki, nM) of arylalkenylamines (E)-1–4 HCl and (E)-10–30 HCl.
agonist profile. Representative images, in bright field and fluores-
cence microscopy (cells stained with Alexa FluorÒ phalloidin, a
marker of F-actin cytoskeleton) are reported in Figure 10. It has
to be outlined that, at the concentrations showed to potentiate
NGF-induced neurite outgrowth and elongation, (R,S)-33 HCl did
not show detrimental effects on PC12 cells. A MTT based citotoxic-
ity assay, performed after treating spontaneously transformed hu-
man skin (HaCat) cell line with (R,S)-33 HCl for 48 or 72 h,
confirmed these results (see Supplementary data).
As regards to (E)-17 HCl, although it did not significantly en-
hance NGF-induced neurite outgrowth or elongation, the co-
administration of BD-1063 significantly decreased the percentage
of cells with neurite outgrowth (Fig. 5). These findings suggest that
(E)-17 HCl may be a much weaker agonist than (R,S)-33 HCl on
NGF-induced neurite outgrowth and elongation in PC12 cells.
As a part of our work, a threedimensional r1 pharmacophore
model was finally developed employing GALAHAD methodology.
Pharmacophore modeling methods should determine the pattern

6218
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
Figure 10. Representative images of F-actin staining with Alexa FluorÒ phalloidin in PC12 cells treated with (A) (R,S)-33 HCl (0.25
1063 (5 M). Bright field representative images of PC12 cells treated with (C) (R,S)-33 HCl (0.25 M) or (D) (R,S)-33 HCl (0.25 M) and BD-1063 (5 l
l
M) or (B) (R,S)-33 HCl (0.25
M).
lM) and BD-
l
l
l
of active compounds and this pattern should exclude the non-ac-
tive compounds. The quality of a pharmacophore model can be
tested by using it to develop predictions of compounds that were
not used to build the model. A pharmacophore model is good if:
(i) the predictions of the more active compounds that were not
used to built the model have a high value; and (ii) the model
should be able to identify that the less active compounds are not
good ligands of the target.
According to Figure 8 the compounds with experimental log(1/
Ki) above the threshold value ꢀ1.4 are the most active compounds.
Our model fails in the point (i) for 60% of the tested compounds;
this means that our model can lose the detection of active com-
pounds in a virtual screening. On the other hand, the compounds
having experimental log(1/Ki) below ꢀ1.4 were defined as the less
active compounds. All the compounds with log(1/Ki) lower than
ꢀ1.4 were successfully predicted; this means that our model ful-
fills the point (ii).
Basing on binding affinity data, a pharmacophore model was
developed by using GALAHAD. Interestingly, the developed model
was able to discriminate between molecules with and without
affinity toward r1 receptor subtype and thus to correctly predict
r1 receptor affinity of new molecules.
Overall biological investigation suggested that the compound to
be highlighted is (R,S)-33 HCl, showing: (i) an excellent
affinity (Ki = 0.70 0.3 nM); (ii) a high selectivity over r2
-opioid receptors and PCP binding site of the NMDA receptors
r
1 receptor
,
l
-, and
j
and (iii) a r1 receptor agonist profile. Indeed, in our validated
PC12 cell model, it was able to increase both neurite outgrowth
and elongation, resulting more effective than the well character-
ized r1 receptor agonist PRE-084. Moreover, (R,S)-33 HCl is not
toxic for PC12 cells and for HaCaT cells at the concentrations
shown to potentiate NGF-induced neurite outgrowth and elonga-
tion. It has to be outlined that the phospholipase C-c (PLC-c), phos-
phoinositide 3 kinase (PI3K), 38 kDa mitogen activated protein
kinase (p38MAPK), c-Jun N-terminal kinase (JNK), and extracellu-
lar-signal regulated kinase (ERK) signaling pathways have been re-
cently proved to be involved in the potentiation of NGF-induced
neurite outgrowth by r1 receptor agonists in PC12 cells;26 more-
In summary, we built a model that identified the less active
compounds successfully and identified the 40% of the most active
compounds as r1 ligands. Accordingly, our model could be suc-
cessfully employed in a virtual screening study: although some ac-
tive compounds can be neglected, the model rigorously discards
the inactive compounds.
over activation of ERK has been found involved in
r1-agonist med-
iated neuroprotection in primary neuronal cultures exposed to
oxygen-glucose deprivation.27,28 According to these evidences
and based on our biological results, we identified (R,S)-33 HCl as
the optimal candidate for the hit-to-lead process. In particular,
our current efforts are directed toward: (i) isolation of 33 pure
enantiomers, in order to investigate the role of chirality in both
4. Conclusions
In an important extension of previous work, in the present con-
tribution we reported the synthesis, drug-likeness evaluation and
r
receptor binding affinity and efficacy in NGF-induced neurite
outgrowth and elongation; (ii) further characterization of the sig-
naling pathways activated by the novel 1-agonist (R,S)-33 HCl
and the potential neuroprotective effects of this molecule.
in vitro studies of new
scaffold [compounds (E)-10–30 HCl]. In this family, molecules gen-
erally showed interesting binding properties for r1 receptor sub-
type (Ki values <25 nM) and good r1/r2 selectivity (>120).
r ligands based on the arylalkenylaminic
r

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6219
5.1.4. 4-Morpholinobutan-2-one (9)
5. Experimental
5.1. Chemistry
Compound 9 was obtained as a yellow oil. IR (cmꢀ1): 2956–
2853, 2807, 1710, 1457–1447, 1358, 1292–1274, 1137, 1115,
1070. 1H NMR (400 MHz, CDCl3) d: 2.08 (s, 3H), 2.31–2.38 (m,
4H), 2.49–2.59 (m, 4H), 3.56–3.62 (m, 4H). MS m/z [MH+] 157.99.
5.1.1. General remarks
Unless otherwise specified, commercially available reagents
were used as received from the supplier. Solvents were purified
according to the guidelines in Purification of Laboratory Chemi-
cals.29 Pd(0) EnCat™ 30NP (loading = 0.4 mmol/g) and TPPP (load-
ing ꢁ3 mmol/g) were purchased from Sigma–Aldrich. Melting
points were measured on SMP3 Stuart Scientific apparatus and
are uncorrected Analytical TLC were carried out on silica gel pre-
coated glass-backed plates (Fluka Kieselgel 60 F254, Merck) and
visualized by ultra-violet radiation, acidic ammonium molyb-
date(IV), or potassium permanganate. Flash chromatography
was performed with Silica Gel 60 (particle size 230–400 mesh)
purchased from NovaChimica. Bond Elute SCX cartridges were
purchased from Varian. IR spectra were recorded on a Jasco FT/
IR-4100 spectrophotometer; only noteworthy absorptions are gi-
ven. 1H NMR spectra were measured with an AVANCE 400 spec-
trometer Bruker, Germany at rt. Chemical shifts (d) are given in
ppm, coupling constants (J) are in Hertz (Hz) and signals are des-
ignated as follows: (s) singlet, (br s) broad singlet, (d) doublet, (t)
triplet, (q) quartet, and (m) multiplet. TMS was used as internal
standard. MS spectra were recorded on a Finnigan LCQ Fleet sys-
tem (Thermo Finnigan, San Jose, CA, USA), using an ESI source
operating in positive ion mode. The purities of target compounds
were determined on a Jasco HPLC system equipped with a Jasco
autosampler (model AS-2055 plus), a quaternary gradient pump
(model PU-2089 plus), and a multiwavelength detector (model
MD-2010 plus). The HPLC method used was as follows: column
5.1.5. General procedure for the preparation of compounds (E)-
10–30 hydrochlorides (method I)
t-BuLi (10.1 mL, 17.2 mmol, 1.7 M in pentane) was added drop-
wise to
a solution of the appropriate aromatic precursor
(8.58 mmol) in anhydrous diethyl ether (42.9 mL) cooled to
ꢀ78 °C under nitrogen atmosphere, keeping the temperature for
20 min. The reaction mixture was then slowly allowed to warm
to rt. After 1 h stirring, a solution of the appropriate b-aminoketone
(6.86 mmol) in dry diethyl ether (10 mL) was added drop-wise at
ꢀ78 °C. The reaction mixture was slowly allowed to warm to 0 °C
and, after 3 h under stirring, quenched with 37% HCl until pH 2
and stirred overnight.
Reaction work-up for compounds16, 17, 21, 22, 24, and 27: the
organic phase was separated from the aqueous one containing a
white-yellow solid, which was isolated by filtration and dissolved
in water. The aqueous solution was then made alkaline with 1 N
NaOH (pH 10) and extracted with EtOAc. The combined organic
phases were dried over anhydrous Na2SO4 and concentrated in
vacuo, yielding crude16, 17, 21, 22, 24, and 27 as free bases. Com-
pounds 16, 24, and 27 were then converted into the corresponding
hydrochlorides and crystallized from acetone to give pure (E)-16
HCl, (E)-24 HCl, and (E)-27 HCl as white solids. Compounds 17,
21, and 22 were further purified by crystallization from the appro-
priate solvent (mixture of methanol/water, 8:2 v/v for compounds
17 and 22; CH2Cl2 for compound 21) and then converted into the
corresponding hydrochloride, which were crystallized from
acetone affording pure (E)-17 HCl, (E)-21 HCl, and (E)-22 HCl as
white solids.
Reaction work-up for compounds10–15, 18–20, 23, 25, 26, 28–
30: the organic layer was extracted with 10% HCl and the aqueous
phase washed with diethyl ether. The combined acid aqueous
phases were then made alkaline with 1 N NaOH (pH 10) and ex-
tracted with EtOAc. The organic phases were dried over anhydrous
Na2SO4, treated with 37% HCl and crystallized from acetone to give
the desired pure (E) hydrochlorides salts as white solids. For com-
pounds 19 and 29, a further purification by flash chromatography
on silica gel (n-hexane/EtOAc/methanol/7 N NH3 in methanol
9:1:0.5:0.1 v/v/v/v and n-hexane/EtOAc/diethylamine 5:5:0.1 v/v/
v, respectively) was performed prior to salification; crystallization
from acetone furnished the expected pure (E)-19 HCl and 29 HCl as
white solids.
XBridge™ Phenyl, 4.6 mm ꢂ 150 mm, 5
lm; column temperature,
ambient; flow rate, 1 mL/min; gradient elution, 10% methanol in
phosphate buffer (5 mM, pH 7.6) to 90% methanol in phosphate
buffer (5 mM, pH 7.6) in 10 min, followed by isocratic elution,
90% methanol in phosphate buffer (5 mM, pH 7.6) for 10 min.
All of the final compounds had 95% or greater purity. Elemental
analyses (C, H, N) were performed on a Carlo Erba 1106 analyzer
and the analysis results were within 0.4% of the theoretical
values.
5.1.2. General procedure for the preparation of compounds 8, 9
A solution of the appropriate amine (8.71 g and 17.5 g for mor-
pholine and 4-benzylpiperidine, respectively; 100 mmol) and but-
3-en-2-one (10.5 g, 150 mmol) in PEG 400 (250 g) was stirred at rt
for 35 min and then 10% HCl was added until pH 2 was reached.
The aqueous phase was washed with dichloromethane (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 in vacuo to give pure 9 (8.65 g, 55% yield)
and crude 8, which was further purified by filtration on an alumina
pad eluting with ethyl acetate (EtOAc), yielding the desired com-
pound (18.4 g, 75% yield).
5.1.6. (E)-N,N-Dimethyl-3-phenylbut-2-en-1-amine
hydrochloride [(E)-10 HCl]
White solid (0.57 g, 39% yield), mp 199–200 °C. IR (cmꢀ1):
3113b, 2350, 1548, 1515, 1182, 1020, 950, 870, 757, 691. 1H
NMR (400 MHz, CD3OD) d: 2.22 (d, J = 1.2 Hz, 3H), 2.94 (s, 6H),
4.00 (d, J = 7.8 Hz, 2H), 5.88 (qt, J = 1.2, 7.8 Hz, 1H), 7.31–7.43 (m,
3H), 7.48–7.54 (m, 2H). MS m/z [MH+] 176.01. HPLC tR = 12.06 min,
>98.0% pure (k = 241 nm).
5.1.3. 4-(4-Benzylpiperidin-1-yl)butan-2-one (8)
Compound 8 was obtained as a yellow solid: mp 123–124 °C. IR
(cmꢀ1): 3077, 3020, 2925–2902, 2795, 1711, 1603, 1490, 1440,
1377, 1357–1245, 1125, 1108–1056, 947, 748, 701. 1H NMR
(400 MHz, CDCl3) d: 1.27 (dd, J = 3.5, 12.0 Hz, 1H), 1.33 (dd,
J = 3.5, 12.1 Hz, 1H), 1.53 (ttt, J = 3.3, 7.3, 11.0 Hz, 1H), 1.60–1.69
(m, 2H), 1.92 (dt, J = 2.2, 11.8 Hz, 2H), 2.17 (s, 3H), 2.53 (d,
J = 7.1 Hz, 2H), 2.60–2.69 (m, 4H), 2.83–2.91 (m, 2H), 7.11–7.16
(m, 2H), 7.16–7.22 (m, 1H), 7.24–7.31 (m, 2H). MS m/z [MH+]
246.04.
5.1.7. (E)-3-(4-Methoxyphenyl)-N,N-dimethylbut-2-en-1-amine
hydrochloride [(E)-11 HCl]
White solid (0.76 g, 46% yield), mp 200–201 °C. IR (cmꢀ1): 3006,
2573, 2362, 2310, 1511, 1250, 1182, 1029, 954, 835. 1H NMR
(400 MHz, D2O) d: 2.02 (d, J = 1.3 Hz, 3H), 2.78 (s, 6H), 3.73 (s,
3H), 3.82 (d, J = 7.8 Hz, 2H), 5.69 (qt, J = 1.3, 7.8 Hz, 1H), 6.87–
6.94 (m, 2H), 7.37–7.42 (m, 2H). MS m/z [MH+] 206.15. HPLC
tR = 12.06 min, >95.8% pure (k = 251 nm).

6220
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
5.1.8. (E)-3-(3-Methoxyphenyl)-N,N-dimethylbut-2-en-1-amine
hydrochloride [(E)-12 HCl]
(400 MHz, D2O) d: 1.24–1.39 (m, 1H), 1.47–1.62 (m, 2H), 1.63–
1.72 (m, 1H), 1.73–1.86 (m, 2H), 2.00 (s, 3H), 2.82 (app. t,
J = 12.0 Hz, 2H), 3.40 (app. d, J = 12.1 Hz, 2H), 3.75 (d, J = 7.8 Hz,
2H), 5.67 (t, J = 7.7 Hz, 1H), 7.21–7.34 (m, 3H), 7.34–7.42 (m, 2H).
MS m/z [MH+] 216.04 HPLC tR = 13.25 min, >98.0% pure
(k = 241 nm).
White solid (1.00 g, 60% yield), mp 156–157 °C. IR (cmꢀ1):
3031b, 2364, 1606, 1574, 1424, 1338, 1213, 1051, 950, 869. 1H
NMR (400 MHz, D2O) d: 2.03 (d, J = 1.4 Hz, 3H), 2.79 (s, 6H), 3.73
(s, 3H), 3.84 (d, J = 7.9 Hz, 2H), 5.73 (qt, J = 1.4, 7.8 Hz, 1H), 6.89
(dd, J = 2.3, 8.0 Hz, 1H), 6.95 (t, J = 2.2 Hz, 1H), 7.04 (app. d,
J = 7.8 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H). MS m/z [MH+] 206.01. HPLC
tR = 12.23 min, >98.0% pure (k = 241 nm).
5.1.15. (E)-1-(3-(3-Methoxyphenyl)but-2-en-1-yl)piperidine
hydrochloride [(E)-20 HCl]
White solid (1.16 g, 60% yield), mp 163–165 °C. IR (cmꢀ1):
2998–3068, 2944, 2834–2856, 2611, 2507, 1600, 1572, 1486,
1427, 1291, 1211, 1158, 1047, 941–958, 878, 827, 780, 697. 1H
NMR (400 MHz, D2O) d: 1.32–1.46 (m, 1H), 1.55–1.70 (m, 2H),
1.70–1.79 (m, 1H), 1.82–1.92 (m, 2H), 2.06 (d, J = 1.3 Hz, 3H),
2.80 (dt, J = 2.7, 12.7 Hz, 2H), 3.48 (app. d, J = 12.1 Hz, 2H), 3.77
(s, 3H), 3.82 (d, J = 7.9 Hz, 2H), 5.75 (qt, J = 1.3, 7.9 Hz, 1H), 6.93
(dd, J = 2.5, 8.1 Hz, 1H), 6.99 (t, J = 2.3 Hz, 1H), 7.10 (d, J = 8.0 Hz,
1H), 7.31 (t, J = 8.0 Hz, 1H). MS m/z [MH+] 246.09. HPLC
tR = 13.39 min, >98.0% pure (k = 241 nm).
5.1.9. (E)-N-Benzyl-N-methyl-3-phenylbut-2-en-1-amine
hydrochloride [(E)-13 HCl]
White solid (0.83 g, 42% yield), mp 180–181 °C. IR (cmꢀ1):
2884b, 2588, 2503, 2317, 1442, 1338, 1212, 1066, 919, 762. 1H
NMR (400 MHz, D2O) d: 1.94 (s, 3H), 2.71 (s, 3H), 3.84 (br s, 2H),
4.23 (br s, 2H), 5.71 (t, J = 7.8 Hz, 1H), 7.23–7.34 (m, 3H), 7.34–
7.43 (m, 7H). MS m/z [MH+] 252.06. HPLC tR = 13.78 min, >98.0%
pure (k = 241 nm).
5.1.10. (E)-N-Benzyl-3-(4-methoxyphenyl)-N-methylbut-2-en-1-
amine hydrochloride [(E)-14 HCl
5.1.16. (E)-4-Benzyl-1-(3-(naphthalen-2-yl)but-2-en-1-
yl)piperidine hydrochloride [(E)-21 HCl]
White solid (1.18 g, 54% yield), mp 172–173 °C. IR (cmꢀ1):
3031b, 2472, 2363, 2309, 1747, 1604, 1511, 1250, 1025, 830. 1H
NMR (400 MHz, D2O) d: 1.93 (d, J = 1.3 Hz, 3H), 2.71 (s, 3H), 3.72
(s, 3H), 3.83 (br s, 2H), 4.23 (br s, 2H), 5.67 (qt, J = 1.3, 7.8 Hz,
1H), 6.86–6.91 (m, 2H), 7.33–7.45 (m, 7H). MS m/z [MH+] 282.11.
HPLC tR = 13.79 min, >98.0% pure (k = 251 nm).
White solid (0.81 g, 30% yield), mp 221–222 °C. IR (cmꢀ1) 2977–
3049, 2926, 2848, 2514, 1597, 1482, 1434–1453, 1157–1287, 1039,
940, 895, 855, 819, 744, 698. 1H NMR (400 MHz, CD3OD) d: 1.41–
1.59 (m, 2H), 1.84–1.99 (m, 3H), 2.28 (d, J = 1.2 Hz, 3H), 2.62 (d,
J = 6.5 Hz, 2H), 3.00 (app. t, J = 12.8 Hz, 2H), 3.61 (app. d,
J = 12.8 Hz, 2H), 3.98 (d, J = 7.7 Hz, 2H), 6.00 (qt, J = 1.2, 7.7 Hz,
1H), 7.14–7.22 (m, 3H), 7.23–7.32 (m, 2H), 7.43–7.52 (m, 2H),
7.63 (dd, J = 1.8, 8.6 Hz, 1H), 7.79–7.90 (m, 3H), 7.93 (s, 1H). MS
m/z [MH+] 356.21. HPLC tR = 16.21 min, >98.0% pure (k = 241 nm).
5.1.11. (E)-N-Benzyl-3-(3-methoxyphenyl)-N-methylbut-2-en-1-
amine hydrochloride [(E)-15 HCl]
White solid (0.96 g, 44% yield), mp 149–150 °C. IR (cmꢀ1):
2949b, 2514, 2363, 2310, 1598, 1425, 1294, 1038, 912, 862. 1H
NMR (400 MHz, D2O) d: 1.93 (d, J = 1.3 Hz, 3H), 2.72 (s, 3H), 3.72
(s, 3H), 3.84 (br d, J = 7.5 Hz, 2H), 4.24 (br s, 2H), 5.71 (qt, J = 1.3,
7.7 Hz, 1H), 6.87 (dd, J = 2.6, 8.2 Hz, 1H), 6.91 (t, J = 2.3 Hz, 1H),
7.00 (app d, J = 8.3 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.34–7.45 (m,
5H). MS m/z [MH+] 282.04. HPLC tR = 13.89 min, >98.0% pure
(k = 241 nm).
5.1.17. (E)-4-Benzyl-1-(3-([1,10-biphen]-4-yl)but-2-en-1-
yl)piperidine hydrochloride [(E)-22 HCl]
White solid (1.38 g, 48% yield), mp 237–240 °C. IR (cmꢀ1):
3085–2979, 2926, 2488, 1641–1580, 1484–1401, 1273–1160,
1038, 943, 835, 765–748, 698. 1H NMR (400 MHz, CD3OD) d:
1.47–1.62 (m, 2H), 1.88–2.02 (m, 3H), 2.23 (d, J = 1.3 Hz, 3H),
2.67 (d, J= 6.7 Hz, 2H), 3.03 (app. t, J = 12.0 Hz, 2H), 3.59 (app. d,
J = 12.0 Hz, 2H), 3.97 (d, J = 7.7 Hz, 2H), 5.94 (qt, J = 1.3, 7.7 Hz,
1H), 7.19–7.24 (m, 3H), 7.27–7.39 (m, 3H), 7.42–7.49 (m, 2H),
7.56–7.61 (m, 2H), 7.62–7.68 (m, 4H). MS m/z [MH+] 382.19. HPLC
tR = 17.17 min, >98.0% pure (k = 271 nm).
5.1.12. (E)-1-(3-(Naphthalen-2-yl)but-2-en-1-yl)piperidine
hydrochloride [(E)-16 HCl]
White solid (1.59 g, 77% yield), mp 209–210 °C. IR (cmꢀ1): 3058,
2931, 2859, 2607, 2484, 2413–2395, 1646–1595, 1439, 1399–
1280, 1160–1078, 1038, 957–944, 896, 850, 818, 741 1H NMR
(400 MHz, D2O) d 1.22–1.40 (m, 1H), 1.46–1.64 (m, 2H), 1.64–
1.74 (m, 1H), 1.74–1.86 (m, 2H), 2.05 (s, 3H), 2.78 (app. t,
J = 12.1 Hz, 2H), 3.38 (app. d, J = 11.5 Hz, 2H), 3.72 (d, J = 7.8 Hz,
2H), 5.76 (t, J = 7.8 Hz, 1H), 7.39–7.47 (m, 2H), 7.50 (dd, J = 1.8,
7.7 Hz, 1H), 7.73–7.86 (m, 4H). MS m/z [MH+] 266.13. HPLC
tR = 14.14 min, >98.0% pure (k = 241 nm).
5.1.18. (E)-4-Benzyl-1-(3-phenylbut-2-en-1-yl)piperidine
hydrochloride [(E)-23 HCl]
White solid (1.06 g, 45% yield), mp 220–222 °C. IR (cmꢀ1):
3055–3025, 2922, 2493, 1650–1598, 1442–1455, 758, 744, 693.
1H NMR (400 MHz, D2O) d: 1.32–1.47 (m, 2H), 1.77–1.89 (m, 3H),
2.06 (d, J = 1.3 Hz, 3H), 2.55 (d, J = 6.7 Hz, 2H), 2.88 (app. t,
J = 12.7 Hz, 2H), 3.51 (app. d, J = 12.8 Hz, 2H), 3.83 (d, J = 7.9 Hz,
2H), 5.75 (qt, J = 1.3, 7.9 Hz, 1H), 7.17–7.25 (m, 3H), 7.27–7.40
(m, 5H), 7.42–7.48 (m, 2H). MS m/z [MH+] 306.17. HPLC
tR = 15.05 min, >95.8% pure (k = 241 nm).
5.1.13. (E)-1-(3-([1,10-Biphen]-4-yl)but-2-en-1-yl)piperidine
hydrochloride [(E)-17 HCl]
White solid (1.24 g, 55% yield), mp 235–236 °C. IR (cmꢀ1): 3038–
2974, 2943, 2612, 2505, 2413, 1651–1514, 1485, 1438, 1400, 1282,
950, 832, 763, 695. 1H NMR (400 MHz, D2O) d: 1.26–1.44 (m, 1H),
1.47–1.67 (m, 2H), 1.67–1.78 (m, 1H), 1.79–1.90 (m, 2H), 2.07 (s,
3H), 2.87 (app. t, J = 12.5 Hz, 2H), 3.45 (app. d, J = 12.0 Hz, 2H), 3.81
(d, J = 7.8 Hz, 2H), 5.79 (t, J = 7.7 Hz, 1H), 7.31–7.38 (m, 1H), 7.39–
7.47 (m, 2H), 7.49–7.55 (m, 2H), 7.58–7.66 (m, 4H). MS m/z [MH+]
292.12. HPLC tR = 14.73 min, >98.0% pure (k = 271 nm).
5.1.19. (E)-4-Benzyl-1-(3-(4-methoxyphenyl)but-2-en-1-
yl)piperidine hydrochloride [(E)-24 HCl]
White solid (1.28 g, 50% yield), mp 190–193 °C. IR (cmꢀ1):
3030–3061, 2837–2973, 2506, 1605, 1511, 1449, 1242, 1027,
821, 751, 702. 1H NMR (400 MHz, D2O) d: 1.30–1.47 (m, 2H),
1.74–1.89 (m, 3H), 2.03 (s, 3H), 2.55 (d, J = 6.4 Hz, 2H), 2.86 (app.
t, J = 12.4 Hz, 2H), 3.49 (app. d, J = 12.6 Hz, 2H), 3.77 (s, 3H), 3.81
(d, J = 7.7 Hz, 2H), 5.70 (t, J = 7.6 Hz, 1H), 6.91–7.00 (m, 2H),
7.16–7.25 (m, 3H), 7.26–7.35 (m, 2H), 7.38–7.46 (m, 2H). MS m/z
[MH+] 336.25. HPLC tR = 15.10 min, >98.0% pure (k = 251 nm).
5.1.14. (E)-1-(3-Phenylbut-2-en-1-yl)piperidine hydrochloride
[(E)-18 HCl]
White solid (0.52 g, 30% yield), mp 235–236 °C. IR (cmꢀ1): 2943,
2609, 2489, 1649, 1434–1454, 947, 846, 758, 691. 1H NMR

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6221
5.1.20. (E)-4-Benzyl-1-(3-(3-methoxyphenyl)but-2-en-1-
yl)piperidine hydrochloride [(E)-25 HCl]
with diethyl ether; the aqueous phase was then made alkaline with
1 N NaOH (pH 10) and extracted with EtOAc. The combined organic
phases were dried over anhydrous Na2SO4 and evaporated under
reduced pressure affording pure (R,S)-31 and crude (R,S)-32, which
was further purified by flash column chromatography on silica gel
(n-hexane/EtOAc/diethylamine 5:5:0.1 v/v/v).
The alcohols (R,S)-31 or (R,S)-32 (0.53 g, 2.0 mmol) dissolved in
CH2Cl2 (10 mL) were then added to a freshly prepared solution of
TPPP (1.00 g, 3.0 mmol) and iodine (0.76 g, 3.0 mmol) in CH2Cl2
(24 mL). The mixture was further stirred at rt for 48 h. Aqueous
5% NaHSO3 was added and after 10 min under stirring the mixture
was filtered through a pad of CeliteÒ. The organic phase was
washed with 1 N NaOH, dried over anhydrous Na2SO4 and concen-
trated under reduced pressure yielding crude 19 and 29, which
were converted into the corresponding hydrochlorides and crystal-
lized from acetone, providing pure (E)-19 HCl and (E)-29 HCl.
White solid (1.15 g, 45% yield), mp 169–173 °C. IR (cmꢀ1):
3079–3024, 2998–2919, 2490, 1576, 1454–1432, 1287, 1210,
1046, 776, 743, 698, 692. 1H NMR (400 MHz, D2O) d: 1.32–1.47
(m, 2H), 1.74–1.90 (m, 3H), 2.05 (s, 3H), 2.55 (d, J = 6.7 Hz, 2H),
2.87 (app. t, J = 12.6 Hz, 2H), 3.50 (app. d, J = 11.8 Hz, 2H), 3.78 (s,
3H), 3.82 (d, J = 7.9 Hz, 2H), 6.75 (t, J = 7.8 Hz, 1H), 6.93 (dd,
J = 2.5, 8.2 Hz, 1H), 6.97–7.01 (m, 1H), 7.03–7.09 (m, 1H), 7.16–
7.24 (m, 3H), 7.26–7.35 (m, 3H). MS m/z [MH+] 336.15. HPLC
tR = 15.23 min, >98.0% pure (k = 241 nm).
5.1.21. (E)-4-(3-(Naphthalen-2-yl)but-2-en-1-yl)morpholine
hydrochloride [(E)-26 HCl]
White solid (1.19 g, 57% yield), mp 221–222 °C. IR (cmꢀ1): 3056,
2939–2976, 2453–2526, 1639, 1399–1440, 1123, 1081, 961, 812,
740. 1H NMR (400 MHz, D2O) d: 2.13 (d, J = 1.2 Hz, 3H), 2.99–3.57
(br, 4H), 3.60–4.19 (br, 4H), 3.88 (d, J = 7.9 Hz, 2H), 5.83 (qt,
J = 1.2, 8.0 Hz, 1H), 7.46–7.51 (m, 2H), 7.56 (dd, J = 1.8, 8.7 Hz,
1H), 7.80–7.87 (m, 3H), 7.88 (d, J = 1.2 Hz, 1H). MS m/z [MH+]
268.01. HPLC tR = 13.16 min, >98.0% pure (k = 241 nm).
5.1.26. (R,S)-2-(4-Methoxyphenyl)-4-(piperidin-1-yl)butan-2-ol
[(R,S)-31]
Yellow oil (1.17 g, 65% yield). IR (cmꢀ1): 3187, 2930, 2805–
2850, 1609, 1508, 1244, 1035, 829. 1H NMR (400 MHz, DMSO) d:
1.26–1.37 (m, 2H), 1.32 (s, 3H), 1.38–1.50 (m, 4H), 1.71–1.89 (m,
2H), 2.05–2.17 (m, 4H), 2.20–2.38 (br, 2H), 3.69 (s, 3H), 5.92–
6.20 (br s, 1H, exchanges with D2O), 6.82 (d, J = 8.7 Hz, 2H), 7.29
(d, J = 8.7 Hz, 2H). MS m/z [MH+] 264.08.
5.1.22. (E)-4-(3-([1,10-Biphen]-4-yl)but-2-en-1-yl)morpholine
hydrochloride [(E)-27 HCl]
White solid (0.95 g, 42% yield), mp 244–252 °C. IR (cmꢀ1):
2975–3075, 2865, 2519, 2401–2445, 1516–1644, 1485, 1443,
1402, 1260, 1121, 1079, 959, 827, 762, 693. 1H NMR (400 MHz,
D2O) d: 2.06 (s, 3H), 3.05–3.19 (m, 2H), 3.36–3.50 (m, 2H), 3.60–
3.76 (m, 2H), 3.89 (d, J = 8.0 Hz, 2H), 3.95–4.06 (m, 2H), 5.77 (t,
J = 7.9 Hz, 1H), 7.28–7.35 (m, 1H), 7.37–7.44 (m, 2H), 7.47–7.53
(m, 2H), 7.56–7.64 (m, 4H). MS m/z [MH+] 294.12. HPLC
tR = 13.64 min, >98.0% pure (k = 271 nm).
5.1.27. (R,S)-2-(4-Methoxyphenyl)-4-morpholinobutan-2-ol
[(R,S)-32]
Yellow oil (1.00 g, 55% yield). IR (cmꢀ1): 3154b, 2955, 2817,
1609, 1508, 1243, 1115, 1031, 830. 1H NMR (400 MHz, DMSO) d:
1.40 (s, 3H), 1.73–1.81 (m, 2H), 2.14–2.26 (m, 2H), 2.28–2.37 (m,
4H), 3.51–3.63 (m, 4H), 3.77 (s, 3H), 5.74–5.88 (br s, 1H, exchanges
with D2O), 6.82 (d, J = 7.3 Hz, 2H), 7.35 (d, J = 7.3 Hz, 2H). MS m/z
[MH+] 266.04.
5.1.23. (E)-4-(3-Phenylbut-2-en-1-yl)morpholine hydrochloride
[(E)-28 HCl]
White solid (0.84 g, 48% yield), mp 203–212 °C. IR (cmꢀ1): 3050,
2871–2983, 2476–2538, 1437, 1258, 1125, 1082, 959, 763. 1H NMR
(400 MHz, D2O) d: 2.04 (s, 3H), 3.02–3.44 (br, 4H), 3.63–4.01 (br,
4H), 3.86 (d, J = 7.9 Hz, 2H), 5.71 (t, J = 7.8 Hz, 1H), 7.23–7.36 (m,
3H), 7.37–7.46 (m, 2H). MS m/z [MH+] 218.05. HPLC tR = 12.25 min,
>98.0% pure (k = 241 nm).
5.1.28. (E)-1-(3-(4-Methoxyphenyl)but-2-en-1-yl)piperidine
hydrochloride [(E)-19 HCl]
White solid (0.27 g, 48% yield, method II), mp 210–212 °C. IR
(cmꢀ1): 2934, 2614, 2520, 1607, 1509, 1464, 1454, 1287, 1244,
1173, 1033, 958, 830, 817. 1H NMR (400 MHz, D2O) d: 1.31–1.44
(m, 1H), 1.53–1.68 (m, 2H), 1.69–1.78 (m, 1H), 1.80–1.91 (m,
2H), 2.04 (s, 3H), 2.88 (dt, J = 2.5, 12.6 Hz, 2H), 3.46 (app. d, 2H),
3.76 (s, 3H), 3.80 (d, J = 8.0 Hz, 2H), 5.71 (t, J = 7.9 Hz, 1H), 6.94
(d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H). MS m/z [MH+] 246.01.
HPLC tR = 13.27 min, >98.0% pure (k = 251 nm).
5.1.24. (E)-4-(3-(3-Methoxyphenyl)but-2-en-1-yl)morpholine
hydrochloride [(E)-30 HCl]
White solid (0.76 g, 39% yield), mp 158–163 °C. IR (cmꢀ1): 3021,
2834–2999, 2454–2469, 1580, 1444–1464, 1213, 1125, 1080,
1032, 962, 865, 840, 783, 709, 689. 1H NMR (400 MHz D2O) d:
2.01 (d, J = 1.1 Hz, 3H), 2.99–3.45 (br, 4H), 3.56–3.95 (br, 4H),
3.71 (s, 3H), 3.85 (d, J = 7.9 Hz, 2H), 5.69 (qt, J = 1.2, 7.9 Hz, 1H),
6.87 (dd, J = 2.2, 8.2 Hz, 1H), 6.93 (t, J = 2.1 Hz, 1H), 7.00 (d,
J = 7.9 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H). MS m/z [MH+] 248.02. HPLC
tR = 12.29 min, >98.0% pure (k = 241 nm).
5.1.29. (E)-4-(3-(4-Methoxyphenyl)but-2-en-1-yl)morpholine
hydrochloride [(E)-29 HCl]
White solid (0.30 g, 53% yield, method II), mp 209–210 °C. IR
(cmꢀ1): 3000–3032, 2934, 2866, 2433, 1512, 1244, 1128, 1026,
970, 827. 1H NMR (400 MHz, D2O) d: 2.00 (d, J = 1.2 Hz, 3H),
3.09–3.33 (br, 4H), 3.71 (s, 3H), 3.73–3.94 (br, 4H), 3.82 (d,
J = 8.0 Hz, 2H), 5.66 (qt, J = 1.4, 8.0 Hz, 1H), 6.88 (d, J = 8.8 Hz,
2H), 7.37 (d, J = 8.8 Hz, 2H). MS m/z [MH+] 248.02. HPLC
tR = 12.25 min, >98.0% pure (k = 251 nm).
5.1.25. Preparation of compounds (E)-19, 29 hydrochlorides
(method II)
t-BuLi (10.1 mL, 17.2 mmol, 1.7 M in pentane) was added drop-
wise to a solution of the 1-bromo-4-methoxy-benzene (1.60 g,
8.58 mmol) in anhydrous diethyl ether (42.9 mL) cooled to
ꢀ78 °C under nitrogen atmosphere, keeping the temperature for
20 min. The reaction mixture was then slowly allowed to warm
to rt. After 1 h under stirring a solution of the appropriate b-ami-
noketone (1.06 g and 1.08 g for compounds 7 and 9, respectively;
6.86 mmol) in dry diethyl ether (10 mL) was then added drop-wise
at ꢀ78 °C. The reaction mixture was slowly allowed to warm to
0 °C, stirred for 3 h and then quenched with water (15 mL). The
mixture was added with 5% DL-tartaric acid until pH 4 and washed
5.1.30. Preparation of (R,S)-1-(3-([1,10-biphen]-4-yl)butyl)piper-
idine [(R,S)-33]
Prior to use, Pd(0) EnCat™ 30NP (supplied as a water wet solid
with water content 45% w/w) was washed thoroughly with abso-
lute ethanol to remove water. Pre-washed Pd(0) EnCat™ 30NP
(0.21 g, 0.2 equiv) was added to a stirred solution of (E)-17 as free
base (0.12 g, 0.41 mmol) in absolute ethanol (21 mL) and the reac-
tion mixture was left at rt in hydrogen atmosphere (balloon) for
22 h. The catalyst was then filtered off and washed with absolute
ethanol; the filtrate was loaded on SCX cartridge and eluted with

6222
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
1 N NH3 in methanol; the organic phase was finally evaporated in
vacuo to give pure (R,S)-33 as free base (yellow oil, 0.072 g, 60%
yield). IR (cmꢀ1): 3055, 3027, 2929, 2851, 2799, 2762, 1600,
1486, 1450, 1154, 1119.835, 764. 1H NMR (CDCl3) d 1.31 (d,
J = 7.0 Hz, 3H), 1.39–1.49 (m, 2H), 1.55–1.64 (m, 4H), 1.79–1.92
(m, 2H), 2.21 (ddd, J = 5.8, 9.9, 12.1 Hz, 1H), 2.29–2.46 (br, 4H),
2.31 (ddd, J = 6.2, 9.8, 12.1 Hz, 1H), 2.78 (sextet, J = 7.2 Hz, 1H),
7.28 (d, J = 8.1 Hz, 2H), 7.32–7.38 (m, 1H), 7.42–7.48 (m, 2H),
7.54 (d, J = 8.2 Hz, 2H), 7.59–7.64 (m, 2H). MS m/z [MH+] 294.26.
HPLC tR = 14.82 min, >95.7% pure (k = 241 nm).
for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of
buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23.500ꢂg
(20 min, 4 °C). This procedure was repeated twice. The final pellet
was resuspended in 5-6 volumes of buffer, the protein concentra-
tion was determined according to the method of Bradford using
bovine serum albumin as standard and subsequently the prepara-
tion was frozen (ꢀ80 °C) in 1.5 mL portions containing about
1.5 mg protein/mL.
5.2.2.3.
with the radioligand [3H]-DAMGO (51 Ci/mmol, Perkin Elmer LAS).
The thawed membrane preparation (about 75 g of the protein)
l-Opioid receptor binding assay. The test was performed
5.2. Biology
l
was incubated with various concentrations of test compounds,
1 nM [3H]-DAMGO and TRIS-MgCl2-PMSF-buffer (50 mM, 8 mM
5.2.1. Sigma receptor binding assays
Crude membranes were prepared as previously described.30,31
The protein concentration of the membrane preparations was
determined by the Lowry method and the membranes were stored
MgCl2, 400 lM PMSF, pH 7.4) in a total volume of 200 lL for
150 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
at ꢀ80 °C until use.
r1 Binding affinity was determined incubating
membrane aliquots (400
lL, 500 lg protein) at 37 °C for 150 min,
with 3 nM [3H]-(+)-pentazocine (45 Ci/mmol) and nine different
concentrations of test ligand in assay buffer (50 mM Tris–HCl, pH
determined with 10
GO is 0.57 nM.
lM unlabeled naloxone. The Kd-value of DAM-
7.4) to a final volume of 1 mL. Haloperidol at 10
measure the nonspecific binding. For the r2 binding assays the
membranes (300 L, 360 g protein) were incubated at rt for
120 min with 3 nM [3H]DTG (31 Ci/mM) in the presence of (+)-
SKF10,047 (0.4 M) and nine different concentrations of test com-
lM was used to
l
l
5.2.2.4.
with the radioligand [3H]-U 69,593 (55 Ci/mmol, Amersham, Little
Chalfont, UK). The thawed membrane preparation (about 75 g of
j-Opioid receptor binding assay. The test was performed
l
l
pound. Incubation was carried out in 50 mM Tris–HCl (pH 8.0) to a
final volume of 0.5 mL. Nonspecific binding was evaluated in the
presence of 5 lM DTG. After incubation the samples were filtered
through Whatman GF/B glass fiber filters, presoaked in a 0.5% poly-
ethyleneimine solution. The filters were washed two times each,
with 4 mL of suitable ice-cold buffer and the radioactivity was
counted in 4 mL of ‘Ultima Gold MV’ in a 1414 Winspectral Perkin-
Elmer Wallac liquid scintillation counter. Ki values for the tested
compounds were calculated using the EBDALIGAND program 34
purchased from Elsevier/Biosoft.
the protein) was incubated with various concentrations of test
compounds, 1 nM [3H]-U 69,593, and TRIS–MgCl2-buffer (50 mM,
8 mM MgCl2, pH 7.4) in a total volume of 200 lL for 150 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 determined with
10 lM unlabeled U 69,593. The Kd-value of U 69,593 is 0.69 nM.
5.2.3. Binding studies at the PCP binding site of the NMDA
receptor
5.2.2. Opioid receptor binding assays
5.2.2.1. Materials and general procedures. The guinea pig brains
were commercially available (Harlan-Winkelmann, Borchen, Ger-
many). Homogenizer: Elvehjem Potter (B. Braun Biotech Interna-
tional, 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 temperature 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 (Perkin Elmer). The so-
lid scintillator was melted on the filtermat at a temperature of
95 °C for 5 min. After solidifying of the scintillator at rt, the scintil-
lation was measured using a MicroBeta Trilux scintillation analyzer
(Perkin Elmer). The overall counting efficiency was 20%. All exper-
iments were carried out in triplicates using standard 96-well-mul-
tiplates (Diagonal, Muenster, Germany). The IC50 values were
determined in competition experiments with at least six concen-
trations of the test compounds and were calculated with the pro-
gram GraphPad PrismÒ 3.0 (GraphPad Software, San Diego, CA,
USA) by non-linear regression analysis. The Ki values were calcu-
lated according to the formula of Cheng and Prusoff.
5.2.3.1. Preparation of the receptor material. Fresh pig brain
cortex was homogenized with the potter (500–800 rpm, 10 up-
and-down strokes) in 6 volumes of cold 0.32 M sucrose. The sus-
pension was centrifuged at 1200g for 10 min at 4 °C. The superna-
tant was separated and centrifuged at 23,500g for 20 min at 4 °C.
The pellet was resuspended in buffer [5 mM Tris–acetate with
1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5] and centri-
fuged again at 31,000g (20 min, 4 °C). This procedure was repeated
twice. The final pellet was resuspended in buffer, the protein con-
centration was determined according to the method of Bradford32
using bovine serum albumin as standard, and subsequently the
preparation was frozen (ꢀ83 °C) in 1.5 mL portions containing
about 0.8 mg protein/mL.
5.2.3.2. Performance of the assay. The test was performed with
the radioligand [3H]-(+)-MK-801 (22.0 Ci/mmol; Perkin–Elmer).
The thawed membrane preparation (about 100 lg of the protein)
was incubated with various concentrations of test compounds,
2 nM [3H]-(+)-MK-801, and TRIS/EDTA-buffer (5 mM/1 mM pH
7.5) in a total volume of 200 lL for 150 min at rt. The incubation
was terminated by rapid filtration through the presoaked filter-
mats using the cell harvester. After washing each well five times
with 300 lL of water, the filtermats were dried at 95 °C. Subse-
5.2.2.2. Preparation of the tissue. 5 guinea pig brains (l- and j-
quently, the solid scintillator was placed on the filtermat and
melted at 95 °C. After 5 min, the solid scintillator was allowed to
solidify at rt. The bound radioactivity trapped on the filters was
counted in the scintillation analyzer. The non-specific binding
opioid receptor assay) were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M su-
crose. The suspension was centrifuged at 1200ꢂg for 10 min at
4 °C. The supernatant was separated and centrifuged at 23.500ꢂg

D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
6223
was determined with 10
l
M (+)-MK-801. The Kd value of the radi-
(Sigma–Aldrich) to the flask (i.e., 1 mL in a T25 cm2 flask). Then,
complete growth medium was added and the cells were pelletted
at 250g ꢂ 5 min. HaCaT cells were resuspended in medium with
10% FBS and plated 5000 cells per well in 96-well plates. After
24 h, cells were treated with compound (R,S)-33 HCl in the concen-
tration range 1 ꢂ 10ꢀ8 to 1 ꢂ 10ꢀ5 and incubated for 48 or 72 h at
37 °C, 5% CO2 in media with 5% FBS (N = 6) or without serum
(N = 6). A MTT based citotoxicity test (CellTiter 96Ò AQueous One
Solution Cell Proliferation Assay, Promega) was performed and
the optical density was read in a microplate reader (BioTekÒ
Instruments, Inc.).
oligand [3H]-(+)-MK-801 is 2.26 nM.
5.2.4. NGF neurite outgrowth in PC12 cells
5.2.4.1. Cell culture. PC12 cells were cultured at 37 °C, 5% CO2
with RPMI medium supplemented with 5% heat-inactivated fetal
bovine serum (FBS), 10% heat-inactivated horse serum (HS),
200 mM glutamine, and 1% penicillin/streptomycin. The medium
was changed two or three times a week. When NGF with or with-
out the compounds to be tested, had to be added, cells were de-
tached from the culture dishes, centrifuged at 150g for 5 min,
and plated at 0.25 ꢂ 104 cells/cm2 onto 19 mm-wide glass cover-
slips coated with poly-D-lysine in 12-well tissue culture plates.
5.2.4.5. Statistical analysis. Data are expressed as the
mean standard error of the mean (SEM). Statistical analysis was
performed by two-way analysis of variance (ANOVA) and post
hoc Bonferroni/Dunn test. Values of p less than 0.05 were consid-
ered statistically significant.
Twenty-four hours after plating, the medium was replaced with
RPMI medium containing 0.5% HS, 200 mM glutamine, 1% penicil-
lin/streptomycin and with NGF (2.5 ng/mL) with or without drugs.
Compounds (R,S)-33 HCl and (E)-17 HCl were dissolved in DMSO
(1 mg/100
genic H2O to 1 mM solution and added to the cell medium to reach
the selected final concentrations. The well characterized 1 recep-
lL and 1 mg/180 lL, respectively), diluted with apyro-
5.3. Computational study
r
tor agonist PRE-084 and antagonist BD-1063 at 10 mM stock solu-
tions were diluted in apyrogenic H2O to 1 mM solutions and then
added to the cell medium. In some experiments, BD-1063 was
co-administered with (R,S)-33 HCl, (E)-17 HCl or PRE-084 at a final
5.3.1. Drug-like properties
Drug-like properties such as logP, pKa, and logD at pH 7.4 were
calculated using MARVIN.33 For logP calculations, MARVIN used a
predefined pool of fragments34 and the extended group contribu-
tion approach.35 pKa values were calculated from empirically
calculated partial charges, and hydrogen bonds were also parame-
terized and taken into account within the calculation. First, MAR-
VIN assigns ionization sites to the molecule. Then, it generates all
microspecies and calculates partial charge distribution of micro-
species. After that, it calculates the micro ionization constant pKa
for microspecies. Finally, it calculates the ratio of microspecies
and macro ionization constant pKa. From values of logP and pKa,
logD was determined at desired pH.
concentration of 5 lM.
5.2.4.2. Quantification of neurite outgrowth and length. Five
days after incubation with NGF (2.5 ng/mL) with or without
(R,S)-33 HCl and (E)-17 HCl, PC12 cells, grown on glass coverslips
were fixed at rt for 15 min in phosphate-buffered saline (PBS) con-
taining 4% (wt/vol) paraformaldehyde. Morphometric analysis was
performed on digitized images of fixed cells taken under phase-
contrast illumination with an inverted microscope (Optika) linked
to a digital camera or with a laser scanning confocal system (TCP-
SP2, Leica). Images of six fields per coverslip were taken at 10ꢂ or
40ꢂ magnification, with an average of 200–20 cells per field. At
least three independent experiments were performed for each con-
dition. Neurite outgrowth and elongation was scored by measuring
the percentage of differentiated cells bearing at least one neurite
with a length equal to the cell body diameter and following the
length of the neurite from the cell body to the tip. The cell counting
and neurite length measurements were performed in a blinded
manner by two independent examiners using Image Tool software.
5.3.2. Pharmacophore modeling
The molecules reported in this work and the ones reported in
our previous work20 were sketched by using the SYBYL program.36
They were optimized and their activities were collected as log(1/
Ki) values. Pharmacophore modeling was achieved by using the
GALAHAD method. Pharmacophore models consist of a group of
features that are positioned relative to each other in coordinate
space as points surrounded by a sphere of tolerance. Each sphere
represents the region in space that should be occupied by a certain
chemical functionality capable of the kind of interaction specified
by the feature type. GALAHAD is able to identify a set of ligand con-
formations that have an optimal combination of low strain energy
(SE), steric overlap (SO), and pharmacophoric similarity (PhS). GAL-
AHAD conducts the model building process into two stages: (1) GA
search in the internal coordinates (torsional space) and (2) align-
ment of the conformers produced in cartesian space by a linear
assignment routine.37 GALAHAD uses a true multi-objective (MO)
function in which each term (SE, SO, and PhS) is considered inde-
pendently.38 This MO functions are employed for three different
purposes: to assess reproductive fitness, to select which candidates
should survive to the next generation, and to rank models after
Cartesian alignment of their constituent ligand conformers. The
three MO functions constitute a multi-objective triage (MOTriage)
approach, which make use of the Pareto rank for each individual
model.39
5.2.4.3. Immunocytochemistry. PC12 cells were fixed with 4%
paraformaldehyde, washed three times with PBS and then perme-
abilized for 5 min with PBS containing 0.2% (wt/vol) Triton X-100.
The cells were washed with PBS and blocked with 1.5% bovine ser-
um albumin (BSA) in PBS for 1 h. For F-actin visualization, the cells
were incubated for 30 min with Alexa FluorÒ 633 phalloidin, a
high-affinity F-actin probe (Molecular Probes, Invitrogen), diluted
in 1% BSA in PBS (1 U/200
ll). At the end of the incubation, cells
were counterstained with Hoechst 33342 (1
lM) and washed once
in distilled water. The coverslips were mounted with Mowiol/Dab-
co on glass slides. All procedures were performed at rt. Alexa
FluorÒ 633 phalloidin staining was viewed with an Olympus
CX31 microscopy equipped with FRAEN AFTER fluorescence UV
and RED cassettes and with a Scion CFW-1312M digital camera.
Image J software was utilized for image acquisition.
Ten of the most active r1 ligands were selected to generate
pharmacophore models. GALAHAD was run for 100 generations
with a population size of 60 and a tournament pool size of 250. De-
fault values were used for other settings. Only models with more
than 8 ligands with contribution to the consensus feature were
considered. Between the selected models, the one with the best
5.2.4.4. Citotoxicity test. In vitro spontaneously transformed
keratinocytes from histologically normal human skin (HaCaT) were
purchased from CLS (Cell Lines Service, D69214 Eppelhein, Ger-
many) and cultivated in DMEM/high glucose; 2 mM glutamine;
Pen/Strep 1%; FBS 10%. Cells were dissociated using an appropriate
volume of pre-warmed TrypLE™ Select cell dissociation reagent

6224
D. Rossi et al. / Bioorg. Med. Chem. 19 (2011) 6210–6224
15. Luty, A. A.; Kwok, J. B.; Dobson-Stone, C.; Loy, C. T.; Coupland, K. G.; Karlstrom,
H.; Sobow, T.; Tchorzewska, J.; Maruszak, A.; Barcikowska, M.; Panegyres, P. K.;
Zekanowski, C.; Brooks, W. S.; Williams, K. L.; Blair, I. P.; Mather, K. A.; Sachdev,
P. S.; Halliday, G. M.; Schofield, P. R. Ann. Neurol. 2010, 68, 639.
16. Sassone-Corsi, P.; Der, C. J.; Verma, I. M. Mol. Cell. Biol. 1989, 8, 3174.
17. Takebayashi, M.; Hayashi, T.; Su, T. P. J. Pharmacol. Exp. Ther. 2002, 303, 1227.
18. Takebayashi, M.; Hayashi, T.; Su, T. P. Synapse 2004, 53, 90.
19. Guzman-Lenis, M. S.; Navarro, X.; Casas, C. Neuroscience 2009, 162, 31.
20. Collina, S.; Loddo, G.; Urbano, M.; Linati, L.; Callegari, A.; Ortuso, F.; Alcaro, S.;
Laggner, C.; Langer, T.; Prezzavento, O.; Ronsisvalle, G.; Azzolina, O. Bioorg.
Med. Chem. 2007, 15, 771.
21. Rossi, D.; Urbano, M.; Pedrali, A.; Serra, M.; Zampieri, D.; Mamolo, M. G.;
Laggner, C.; Zanette, C.; Florio, C.; Schepmann, D.; Wuensch, B.; Azzolina, O.;
Collina, S. Bioorg. Med. Chem. 2010, 18, 1204.
22. Richmond, N. J.; Abrams, C. A.; Wolohan, P. R. N.; Abrahamian, E.; Willett, P.;
Clark, R. D. Aided Mol. Des. 2006, 20, 567.
SE, SO, and PhS values based on Pareto ranking was selected as the
best model.
Acknowledgments
The authors are indebted to M. Ceriani and E. Martegani, Dept.
Biotechnology and Biosciences, University of Milano-Bicocca, Mi-
lan and to F. Leoni, Italfarmaco SpA, Cinisello Balsamo (MI) for
the gift of different batches of PC12 cells. We acknowledge espe-
cially Paola Moro, Department of Drug Sciences, University of Pavi-
a, for the technical support.
23. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46, 3.
Supplementary data
24. Caballero, J. J. Mol. Graph. Model. 2010, 29, 363.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.016.
25. Mégalizzi, V.; Mathieu, V.; Mijatovic, T.; Gailly, P.; Debeir, O.; De Neve, N.; Van
Damme, M.; Bontempi, G.; Haibe-Kains, B.; Decaestecker, C.; Kondo, Y.; Kiss, R.;
Lefranc, F. Neoplasia 2007, 9, 358.
26. Nishimura, T.; Ishima, T.; Iyo, M.; Hashimoto, K. PLoS One 2008, 3, e2558.
27. Ishima, T.; Nishimura, T.; Iyo, M.; Hashimoto, K. Prog. Neuro-Psychopharmacol.
Biol. Psychiatry 2008, 32, 1656.
28. Tan, F.; Guio-Aguilar, P. L.; Downes, C.; Zhang, M.; O’Donovan, L.; Callaway, J.
K.; Crack, P. J. Neuropharmacology 2010, 59, 416.
References and notes
1. Maurice, T.; Su, T. P. Pharmacol. Ther. 2009, 124, 195.
2. Crawford, K. W.; Bowen, W. D. Cancer Res. 2002, 62, 313.
3. Mavlyutov, T. A.; Epstein, M. L.; Andersen, K. A.; Ziskind-Conhaim, L.; Ruoho, A.
E. Neuroscience 2010, 167, 247.
29. Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals, 3th ed.;
Pergamon Press: Oxford, 1988.
4. Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H. G.; Striessnig, J.; Kempner,
E.; Glossmann, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8072.
5. Kekuda, R.; Prasad, P. D.; Fei, Y. J.; Leibach, F. H.; Ganapathy, V. Biochem.
Biophys. Res. Commun. 1996, 229, 553.
30. Matsumoto, R. R.; Bowen, W. D.; Tom, M. A.; Vo, V. N.; Truong, D. D.; De Costa,
B. R. Eur. J. Pharmacol. 1995, 280, 301.
31. Mach, R. H.; Smith, C. R.; Childers, S. R. Life Sci. 1995, 57.
32. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
6. Seth, P.; Leibach, F. H.; Ganapathy, V. Biochem. Biophys. Res. Commun. 1997, 241,
535.
7. Seth, P.; Fei, Y. J.; Li, H. W.; Huang, W.; Leibach, F. H.; Ganapathy, V. J.
Neurochem. 1998, 70, 922.
8. Toussaint, M.; Debreu-Fontaine, M. A.; Maurice, T.; Melnyk, P. Med. Chem. 2010,
6, 355.
33. Kashiwagi, H.; McDunn, J. E.; Simon, P. O., Jr.; Goedegebuure, P. S.; Xu, J.; Jones,
L.; Chang, K.; Johnston, F.; Trinkaus, K.; Hotchkiss, R. S.; Mach, R. H.; Hawkins,
W. G. Mol. Cancer 2007, 6, 48. doi:10.1186/1476-4598-6-48.
34. Kashiwagi, H.; McDunn, J. E.; Simon, P. O., Jr.; Goedegebuure, P. S.;
Vangveravong, S.; Chang, K.; Hotchkiss, R. S.; Mach, R. H.; Hawkins, W. G. J.
Transl. Med. 2009, 7, 24. doi:10.1186/1479-5876-7-24.
9. Hayashi, T.; Rizzuto, R.; Hajnoczky, G.; Su, T. P. Trends Cell Biol. 2009, 19, 81.
10. Su, T. P.; Hayashi, T.; Maurice, T.; Buch, S.; Ruoho, A. E. Trends Pharmacol. Sci.
2010, 31, 57.
11. Morin-Surun, M. P.; Collin, T.; Denavit-Saubie, M.; Baulieu, E. E.; Monnet, F. P.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8196.
35. Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.; Niso, M.; Simone, G. M.;
Perrone, R.; Paradiso, A. Mol. Cancer Ther. 2006, 5, 1807.
36. SYBYL, version 7.3; Tripos Inc.: 1699 South Hanley Rd., St. Louis, MO 63144, USA,
2006.
37. Richmond, N. J.; Willett, P.; Clark, R. D. J. Mol. Graph. Model. 2004, 23, 199.
38. Gillet, V. J.; Khatib, W.; Willett, P.; Fleming, P. J.; Green, D. V. S. J. Chem. Inf.
Comput. Sci. 2002, 42, 375.
12. Hayashi, T.; Su, T. P. J. Pharmacol. Exp. Ther. 2003, 306, 726.
13. Hayashi, T.; Su, T. P. Cell 2007, 131, 596.
14. Meunier, J.; Hayashi, T. J. Pharmacol. Exp. Ther. 2010, 332, 388.
39. Clark, R.; Abrahamian, E. J. Comput. Aided Mol. Des. 2009, 23, 765.