Synthesis and biological evaluation of new aryl-alkyl(alkenyl)-4-benzylpiperidines, novel Sigma Receptor (SR) modulators, as potential anticancer-agents

Article abstract of DOI:10.1016/j.ejmech.2016.08.067

In the early 2000s, the Sigma Receptor (SR) family was identified as potential “druggable” target in cancer treatment. Indeed, high density of SRs was found in breast, lung, and prostate cancer cells, supporting the idea that SRs could play a role in tumor growth and progression. Moreover, a link between the degree of SR expression and tumor aggressiveness has been postulated, justified by the presence of SRs in high metastatic-potential cancer cells. As a consequence, considerable efforts have been devoted to the development of small molecules endowed with good affinity towards the two SR subtypes (S1R and S2R) with potential anticancer activity. Herein, we report the synthesis and biological profile of aryl-alkyl(alkenyl)-4-benzylpiperidine derivatives - as novel potential anticancer drugs targeting SR. Among them, 3 (RC-106) exhibited a preclinical profile of antitumor efficacy on a panel of cell lines representative of different cancer types (i.e. Paca3, MDA-MB 231) expressing both SRs, and emerged as a hit compound of a new class of SR modulators potentially useful for the treatment of cancer disease.

Full text of DOI:10.1016/j.ejmech.2016.08.067

Accepted Manuscript
Synthesis and biological evaluation of new aryl-alkyl(alkenyl)-4-benzylpiperidines,
novel Sigma Receptor (SR) modulators, as potential anticancer-agent
Marta Rui, Daniela Rossi, Annamaria Marra, Mayra Paolillo, Sergio Schinelli, Daniela
Curti, Anna Tesei, Michela Cortesi, Alice Zamagni, Erik Laurini, Sabrina Pricl, Dirk
Schepmann, Bernhard Wűnsch, Ernst Urban, Vittorio Pace, Simona Collina
PII:
S0223-5234(16)30725-5
10.1016/j.ejmech.2016.08.067
EJMECH 8863
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 July 2016
Revised Date: 29 August 2016
Accepted Date: 30 August 2016
Please cite this article as: M. Rui, D. Rossi, A. Marra, M. Paolillo, S. Schinelli, D. Curti, A. Tesei, M.
Cortesi, A. Zamagni, E. Laurini, S. Pricl, D. Schepmann, B. Wűnsch, E. Urban, V. Pace, S. Collina,
Synthesis and biological evaluation of new aryl-alkyl(alkenyl)-4-benzylpiperidines, novel Sigma Receptor
(SR) modulators, as potential anticancer-agent, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.08.067.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of new aryl-alkyl(alkenyl)-4-
benzylpiperidines, novel Sigma Receptor (SR) modulators, as potential
anticancer-agent.
1
,3#
1#
1
2
2
Marta Rui , Daniela Rossi , Annamaria Marra , Mayra Paolillo , Sergio Schinelli ,
4
5
5
5
6
Daniela Curti , Anna Tesei , Michela Cortesi , Alice Zamagni , Erik Laurini , Sabrina
6
,7
8
8
3
3
Pricl , Dirk Schepmann , Bernhard Wűnsch , Ernst Urban , Vittorio Pace , Simona
1*
Collina
1
Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology
section, University of Pavia, Viale Taramelli 6 and 12, 27100 Pavia (Italy)
2
Department of Drug Sciences, Pharmacology section, University of Pavia, Viale
Taramelli 6 and 12, 27100 Pavia (Italy)
3
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090
Vienna (Austria)
4
Department of Biology and Biotechnology “L. Spallanzani”, Lab. of Cellular and
Molecular Neuropharmacology, University of Pavia, Via Ferrata 9, 27100 Pavia (Italy)
5
Radiobiology and Preclinical Pharmacology Laboratory, Biosciences Laboratory,
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS,
Via P. Maroncelli 40, 47014 Meldola (FC), Italy
6
MOSE – DEA, University of Trieste, Piazzale Europa 1, 34127 Trieste (Italy)
7
National Interuniversity Consortium for Material Science and Technology (INSTM),
Research Unit MOSE-DEA University of Trieste, Trieste, Italy
8
Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster,
Correnstrasse 48, 48149 Muenster (Germany)
*
Correspondence to: Simona Collina, Department of Drug Sciences, Medicinal Chemistry
and Pharmaceutical Technology section, University of Pavia, Viale Taramelli 12, 27100
Pavia (Italy). Phone: (+39)0382-987379, Fax: (+39)0382-422975. E-mail:
simona.collina@unipv.it
#
These Authors equally contributed to this work

ACCEPTED MANUSCRIPT
Abstract
In the early 2000s, the Sigma Receptor (SR) family was identified as potential
druggable” target in cancer treatment. Indeed, high density of SRs was found in breast,
“
lung, and prostate cancer cells, supporting the idea that SRs could play a role in tumor
growth and progression. Moreover, a link between the degree of SR expression and
tumor aggressiveness has been postulated, justified by the presence of SRs in high
metastatic-potential cancer cells. As a consequence, considerable efforts have been
devoted to the development of small molecules endowed with good affinity towards the
two SR subtypes (S1R and S2R) with potential anticancer activity. Herein we report the
synthesis and biological profile of aryl-alkyl(alkenyl)-4-benzylpiperidine derivatives - as
novel potential anticancer drugs targeting SR. Among them, 3 (RC-106) exhibited a
preclinical profile of antitumor efficacy on a panel of cell lines representative of different
cancer types (i.e. Paca3, MDA-MB 231) expressing both SRs, and emerged as a hit
compound of a new class of SRs modulators potentially useful for the treatment of cancer
disease.
Keywords
Sigma Receptor (SR); pan-SR modulators; compound
3
(RC-106); S1R
agonist/antagonist profile; potential anticancer property; apoptotic pathway.
1
. Introduction
Sigma receptors (SRs) are an enigmatic receptor family localized in plasmatic,
mitochondrial and endoplasmic reticulum membranes of several organs including liver,
kidney and brain. Radioligand binding studies and biochemical analyses have shown the
presence of two SR subtypes, Sigma 1 (S1R) and Sigma 2 (S2R) receptors with different
anatomical distribution, distinct physiological and pharmacological profiles [1-4].
It is well known that S1Rs play critical roles in the mammalian nervous system, indeed
their involvement in different neurodegenerative and neuropsychiatric diseases has been
well documented [5-8]. Their ligands can yield both cytoprotective or cytotoxic actions.
In detail, S1R agonists promote neuroprotection, neurite outgrowth, trophic factor
production as well as microglial activation, mitochondrial integrity and reduce production

ACCEPTED MANUSCRIPT
of reactive oxygen species [9,10]. As a consequence, S1R agonists display a high
therapeutic potential for Central Nervous System (CNS) pathologies such as
Amyotrophic Lateral Sclerosis, Multiple Sclerosis, Alzheimer’s disease and Parkinson
disease [5,11,12]. Conversely, S1R antagonists may play a role in neuropathic pain and
anticancer therapy. Overexpression of S1R in high metastatic potential cancer cells,
together with the efficacy of the S1R antagonist Rimcazole (Fig. 1) in inhibiting tumor
cell survival and in promoting apoptosis in breast cancer cells (MCF-7, MDA-MB-231,
MDA-MB-157 and T47D) suggests a link between the degree of S1R expression and
tumor growth and aggressiveness [13]. Despite the evidence supporting the importance of
S1R in cancer, the mechanism of action of S1R antagonists in causing cell death is still
unclear. Currently, it has been hypothesized that the observed apoptototic phenomena are
related to the increase of intracellular calcium levels [14-16].
The S2R subtype is still largely unknown: it has not been cloned yet and its molecular
structure has not been clarified . In the intracellular environment, S2R binding sites are
localized in mitochondria, lysomes, endoplasmic reticulum, and plasma membrane.
Recent studies describe how S2R ligands trigger a cell response which inhibit the activity
of the P-glycoprotein, responsible for the active extrusion of anticancer drugs, leading to
cell death. [17,18]. Moreover, the hypothesis of a correlation between S2R and
Progesterone Receptor Membrane Component 1 (PGRMC1) [19,20] supports the idea
that S2R may exert a critical role in tumorigenesis [18]. Indeed, the over-expression of
PGRMC1 has been associated to tumor stage and to actively proliferating and invasive
cancer cells [21]. It is also relevant that proliferating breast carcinoma cells express S2R
up to ten times more than quiescent cells, and the degree of S2R expression has been
correlated with tumor staging and grading [22-24]. The highest level of S2R has been
detected in pancreatic cancer cell lines (Panc-02, Panc-01, CFPAC-1, AsPC-1) [25]. A
recent study, carried out on mouse breast cancer (EMT-6) and human melanoma (MDA-
MB-435) cell lines, demonstrate that siramesine (Fig. 1), a S2R selective ligand
commonly used as reference compound, can induce cell death (with an EC in both cell
50
lines lower than 10 µM) by three different mechanisms: caspase activation, autophagy,
and impaired cell-cycle progression [26]. In the same work, it has been also demonstrated
that other S2R ligands, i.e. SV119, WC-26 and RHM-138 (Fig. 1), possess a cytotoxic

ACCEPTED MANUSCRIPT
effect in the micromolar range in the aforementioned cancer cell lines [26]. Moreover, the
same compounds are able to inhibit proliferation of pancreatic cancer cells (human lines:
BxPC3, AsPC1, Cfpac, Panc1 and PaCa-2; murine line: Panc02) with an IC ≤ 100 µM
5
0
[25].
Fig. 1. S1R and S2R compounds able to promote antiproliferative effects. Rimcazole (KiS1 = 908.0 ±
9
1
9 nM; KiS2 = 302.0 ± 37 nM) [27]. Siramesine (KiS1 = 17.0 nM; KiS2 = 0.12 nM); SV119 (KiS1 =
417.0 nM; KiS2 = 5.2 nM); WC-26 (KiS1 = 1436.5 ± 166.1 nM; KiS2 = 2.58 ± 0.59 nM); RHM-138
(
KiS1 = 544.0 nM; KiS2 = 12.3 nM); SW43 (KiS1 = 133.0 nM; KiS2 = 19.0 nM) [28].
On the bases of the above findings, we reasoned that both S1R antagonist and S2R
agonists could be useful tools to address novel and more focused cancer treatments.
Hence, SRs could represent an exciting target to develop anticancer drugs with novel
mechanisms of action. Our group has previously prepared and characterized a wide series
of compounds with preferential affinity towards S1R [29a-d]. In an intriguing

ACCEPTED MANUSCRIPT
observation, we documented that the presence of the bulky 4-benzylpiperidine moiety,
while preserving high binding strength for S1R, increases the affinity towards S2R [29c].
Therefore, in the present study we present our efforts aimed at the identification and
characterization of potent SR modulators, able to bind both receptor subtypes.
Specifically, we report herein the preliminary structure-activity relationship (SAR) (Fig.
2
) of aryl-alkyl(alkenyl)-4-benzylpiperidines with the general structure of Fig. 2.
Derivatives 3 and 6 that displayed good affinity toward both receptor subtypes, from now
on called pan-SR ligands, were passed for testing in vitro cytotoxic activity evaluation.
To validate the hypothesis that S1R and S2R modulators could be effective as anticancer
drugs, compound 3 was next screened towards a panel of tumor cell lines representative
of various cancer types, all expressing both sigma receptors. The results of our studies
showed that 3, called by us RC-106, has interesting anticancer activities against prostate,
glioblastoma, pancreas and breast cancer cell lines. The activity against pancreatic PaCa3
cells is of particular interest, being 3 (RC-106) effective against actively proliferating
cells (IC = 42 µM) and against cells with reduced proliferation rate (IC = 7.0 µM,).
5
0
50
The results of this effort are described below.
Fig. 2. SAR exploration. Structural elements subjected to variation are highlighted.
2
. Results
2
.1. Chemistry
The preparation of compounds 1-12 is summarized in Scheme 1. In cases where a chiral
center was present, (semi)preparative chiral chromatography was used to isolate the
stereoisomers.
The key intermediate of the synthetic process is the β-aminoketone A. The synthetic
pathway to obtain A was already described by us and involves a Michael addition of 4-
benzylpiperidine to but-3-en-2-one in absolute ethanol and glacial acetic acid, followed

ACCEPTED MANUSCRIPT
by purification via acid/basic extraction, as reported in our previous work [29c]. The
subsequent lithiation reaction at aryl-bromine in anhydrous Et O at -78 °C with t-butyl
2
lithium, followed by addition/substitution reaction in the presence of the β-aminoketone
2
A and quenching with H O, led to isolate crude compounds. After purification via
acid/basic extraction, 1 and 2 in sufficient amount for the subsequent reaction an suitable
1
13
purity for biological assay were obtained, as confirmed by H-NMR, C-NMR and
UPLC-MS analysis.
Scheme 1. Synthesis of compounds 1-12.
Arylalkylaminoalcohols 1 and 2 were treated with trifluoroacetic anhydride in the
presence of a catalytic amount of cupper triflate, according with a procedure already
experimented by us [30]. In this way, compounds 3 and 4, as (E) stereoisomers C2-C3,
together with low amounts of the olefinic regioisomers C3-C4, were obtained as
1
evidenced by H-NMR. It is worth noting that no signals related to the (Z) stereoisomer,
which represented the minor product using the standard acidic conditions (37% HCl)

ACCEPTED MANUSCRIPT
1
[
29b] were present in the H-NMR of crude products. As a further step, an amount of
arylalkenylamines were subjected to catalytic reduction reaction in hydrogen atmosphere
in the presence of Pd (0)/C 10% (p/p) in absolute EtOH, giving rise to the corresponding
arylalkylamines 5 and 6. Crude 3-6 were purified using alumina (II Brockmann degree)
1
13
column chromatography, yielding pure compounds as confirmed by H-NMR, C-NMR
and UPLC-MS analysis.
In the case of compounds bearing the hydroxyl group at the naphtylic moiety, an
additional step was required, consisting in the protective reaction of the –OH group by t-
butyldimethylsilyl chloride, thus obtaining 7 which was lithiated in anhydrous THF at -78
°
C using an excess of n-butyl lithium. After 20 minutes, the aminoketone A was added to
the C-lithiated intermediate, keeping the temperature below -50 °C for 1.5 h. The reaction
was quenched with saturated aqueous NH Cl and extracted with Et O. The crude product
4
2
was purified by column chromatography giving 8. An amount of this compound was
subjected to an elimination reaction using trifluoroacetic anhydride in the presence of a
catalytic amount of cupper triflate to give the arylalkenylamine 10 [(E) stereoisomer C2-
C3] as main compound, easily isolated by column chromatography. Compounds 8 and 10
were then subjected to the deprotection of –OH-aromatic by drop wise addition of tetra-
N-butylammonium fluoride at 0 °C in argon atmosphere in anhydrous dichloromethane
(DCM), to give 9 and 11, respectively. Lastly, the reduction reaction of 11 using a
catalytic amount of Pd (0) in hydrogen atmosphere gave rise to arylalkylamine 12. With
the exception of compound 11, which was purified by treatment with methanol, pure 9
and 12 were obtained after purification through silica column chromatography. Also in
1
the case of naphthol-derivatives 9 and 11-12, the identities were confirmed by H-NMR,
1
3
C-NMR and UPLC-MS analysis.
All potential SR modulators 1-6, 9 and 11-12 were obtained in a sufficient amount and
with the appropriate degree of purity for the subsequent biological investigations and, in
the case of racemic compound, also for HPLC chiral resolution.

ACCEPTED MANUSCRIPT
2
.2. Chiral resolution
To investigate the relationship between stereochemistry and receptor binding affinity, we
prepared enantiomeric 1-2, 5-6, 9 and 12. On the bases of our previous experience, a
direct chiral HPLC method of enantiomeric separation was applied, and the scaling up of
the process was performed [31a-e]. Baseline separation of racemates was obtained using
cellulose and amylose derived chiral stationary phases (Chiralcel OJ-H, Chiralpack IC
and Chiralpack IA), under different elution conditions, including different mixtures of n-
heptane and polar modifiers (methanol, ethanol or 2-propanol) and alcohols (methanol,
ethanol and 2-propanol). In all cases 0.1 % of diethylamine (DEA) was added to the
mobile phase. Moreover, in the case of the Chiralpak IC, the analysis were carried out
also in the presence of 0.3 % of trifluoroacetic acid (TFA), which improves enantiomer
separations. The optimized analytical methods (Table 1, Fig. SI1, see Supplementary
material) were suitably transferred to the (semi)preparative scale. In detail, enantiomeric
1
, 2, 5 and 9 were resolved by a (semi)preparative Chiralcel OJ-H column, eluting with
ethanol and 0.1 % diethylamine (for the compound 1) or methanol and 0.1 %
diethylamine (for the compounds 2, 5 and 9), whereas compounds 6 and 12 were resolved
on (semi)preparative Chiralpak IA, using methanol and 0.1 % of diethylamine, in all
cases eluting at a flow rate of 2.5 mL/min. The elution conditions applied provided a
quick access to the desired enantiomers (Table 2) with enantiomeric excess over than
9
5%, as evidenced by analytical control of the collected fractions , and in sufficient
amount for preliminary biological assays.
Table 1. Analytical chiral resolution of 1-2, 5-6, 9 and 12.
Compound
Column
Eluent
K1
K2
α
Rs
1
2
5
6
9
Chiralcel OJ-H
Chiralcel OJ-H
Chiralcel OJ-H
Chiralpak IA
Chiralcel OJ-H
Chiralpak IA
A
B
B
B
B
B
1.03 1.63 1.58 3.25
1.24 1.80 1.45 4.05
3.98 4.80 1.21 2.46
0.57 0.81 1.42 3.06
1.10 1.61 1.46 2.62
0.96 1.27 1.32 2.49
1
2
Eluent: A (100% ethanol, 0.1 % diethylamine); B (methanol 100%, 0.1 % diethylamine), flow rate: 1
mL/min; detection UV at 220 (compounds 2 and 6) and at 254 nm (compounds 1, 5, 9 and 12).

ACCEPTED MANUSCRIPT
Table 2. Chiroptical properties of enantiomeric 1-2, 5-6, 9 and 12.
2
0
a
Compound
[α]
D
(c% in MeOH)
ee (%)
96.0
97.0
99.9
98.0
95.0
95.0
99.9
99.9
99.9
99.9
99.9
99.9
K
1
1
2
2
5
5
6
6
9
9
1
1
a
+ 40.5 (0.2)
- 42.3 (0.2)
+ 10.5 (0.6)
- 9.2 (0.6)
1.03
1.63
1.24
1.80
3.98
4.80
0.57
0.81
1.10
1.61
0.96
1.27
b
a
b
a
+ 6.1 (0.2)
- 6.3 (0.2)
b
a
+ 8.2 (0.3)
- 8.3 (0.3)
b
a
+ 24.2 (0.1)
- 24.8 (0.1)
+ 11.8 (0.3)
- 12.0 (0.3)
b
2°
2b
a
Determined by chiral HPLC under the analytical conditions reported in Table 1.
2
.3. SAR studies
The S1R and S2R binding site affinities of the tested compounds were determined in
competition experiments using radioligands. All compounds were tested on guinea pig
brain and rat liver membranes obtained by homogenization, centrifugation, and washing
of the respective tissues. Both S1R and S2R binding site assays were performed with
3
3
[
H]-(+)-pentazocine as radioligand. The S2R binding values were evaluated using [ H]-
DTG was used as radioligand in the S2R assays. Compounds with high affinity were
tested three times. For compounds with low SR affinity, only one measure was
performed. The SR affinities of all compounds towards both S1R and S2R are presented
in Table 3.

ACCEPTED MANUSCRIPT
Table 3. Binding affinities towards S1R and S2R. Values are expressed as mean ± SEM of three
experiments.
Compound
1
Ar
R
K S1 ± SEM K S2 ± SEM
i
i
2-naphtyl
2-naphtyl
2-naphtyl
Phenyl
Phenyl
OH
OH
OH
OH
OH
6.9 ± 2
62.5
1
1
2
2
a
10 ± 2
11 ± 1
9.8 ± 4
27 ± 9
81 ± 35
79 ± 21
57 ± 11
339a
b
a
2
3
4
5
5
b
Phenyl
OH
-
40 ± 4
240a
2-naphtyl
Phenyl
12 ± 5
22 ± 3
47± 13
-
0.7 ± 1
5.6 ± 3
6.0 ± 0.5
a
2-naphtyl
2-naphtyl
H
H
144
a
26± 9
5
6
6
6
9
9
9
b
2-naphtyl
H
6.9 ± 1
2.1 ± 1
2.9 ± 0.4
3.0 ± 0.3
27 ± 5
98
Phenyl
H
6.5 ± 3
8.9 ± 2.1
7.9 ± 1.9
a
Phenyl
H
b
Phenyl
H
a
6-hydroxy naphtyl
6-hydroxy naphtyl
6-hydroxy naphtyl
OH
OH
OH
118
a
70 ± 21
68 ± 8
b
62 ± 4
9.6 ± 3
59 ± 5
35± 2
905a
a
1
1
6-hydroxy naphtyl
6-hydroxy naphtyl
6-hydroxy naphtyl
-
305
a
1
1
1
2
H
H
314
2a
2b
582a
105a
6-hydroxy naphtyl
H
13 ± 4
a
Compounds with high affinity were tested three times. For compounds with low SR affinity (> 100 nM),
only one measure was performed.
All compounds, with the only exception of 9a, 9b and 12, showed an interesting affinity
towards S1R (K S1 ≤ 50 nM) and a good/modest affinity towards S2R, with the exception
i
of compounds 2a, 2b, 9 and 12, which are weak S2R binders. Particularly, all the
arylalkylaminoalcohols 1, 2 and 9 showed a preference affinity towards S1R and the first

ACCEPTED MANUSCRIPT
eluted enantiomers exhibit a slight preferential interaction with the target, in accordance
with our previous work [32]. Conversely, SRs do not show stereoselectivity towards the
enantiomer of arylalkylamines, confirming our previous findings [31a-b]. Of particular
interest are compounds 3 and 6 (racemic and enantiomeric), having a good affinity
towards both S1R and S2R.
To propose a molecular rationale for the experimental affinity of the new 4-
benzylpiperidine derivatives for the S1R, we used our validated three-dimensional model
of the S1R [29d,31a-b,33-35]. We applied a consolidated simulation recipe based on free
energy of binding (∆Gbind) estimation in the framework of the Molecular
Mechanics/PoissonBoltzmann Surface Area (MM/PBSA) computational methodology
(Tables SI1, see Supplementary material) [36]. Taking the naphthalene derivative 3 as a
proof-of-concept, the analysis of the corresponding MD trajectory reveals that 4-benzyl-
piperidine moiety establishes a strong network of polar and hydrophobic interactions with
the receptor. As shown in Fig. 3A and B, the piperidine nitrogen atom is engaged in the
prototypical salt bridge with the carboxylic side chain of Asp126, while the aliphatic
portion of the heterocycle together with the benzyl ring are perfectly encased in the
hydrophobic S1R cavity lined by residues Ile128, Phe133, Tyr173 and Leu186. Finally,
the naphthalene group of 3 performs stabilizing π interactions with the side chains of
Arg119 and Trp121.

ACCEPTED MANUSCRIPT
Fig. 3. (A) 2D schematic representation of the identified interactions between the S1R 3D model and 3. (B)
Zoomed view of SR1 in complex with 3. The compound is in atom colored sticks-and-balls (C, gray; N,
blue). Hydrogen atoms, water molecules, ions, and counterions are not shown for clarity. A dashed black
line highlights the salt bridge between the S1R D126 side chain and the piperidine nitrogen atom of 3. (C)
Per-residue binding enthalpy (∆Hbind,res) decomposition for the S1R/3 complex. Only SR1 amino acids from
positions 100 to 200 – most relevant to ligand binding - are shown for clarity.
For each compound, a quantitative analysis of ligand/protein interactions was next
performed via a per-residue deconvolution of the enthalpic contribution to binding (Fig.
SI3, see Supplementary material). Taking again compound 3 as reference, Fig. 3C shows
the resulting interaction spectrum. Substantially, the hydrophobic interactions of 3 with
the side chains of residues Ile128, Phe133, Tyr173, and Leu186 contribute an overall
stabilization term to binding equal to -3.25 kcal/mol, while the permanent salt bridge
between the N-atom of 3 and the side chain of Asp126 (average dynamic length (ADL) =
4
.09 ± 0.04 Å) reflects in a stabilizing contribution of -1.87 kcal/mol. Finally, the
important π/π and π/cation interactions (established between the naphthyl ring of the
molecule and the indole ring of Trp121 and the cationic side chain of Arg119,
respectively) result in a strong enthalpic stabilization of -2.01kcal/mol.

ACCEPTED MANUSCRIPT
To sum up, compounds 3 and both racemic and enantiomeric 6 revealed good affinity
towards S1R, in agreement with the molecular modelling studies, together with a S2R
affinity and therefore they have been selected for a deeper biological investigation.
2
.4. Quantification of SRs expression in cancer cell lines
As stated in the introduction section, an appropriate modulation of both receptors could
have a synergic effect in inducing tumor cell death. Therefore, the first step of our in
depth biological investigation consisted in testing a panel of cancer cell lines
representative of different human solid tumors (Table 4) for SRs expression [37-40]. In
details, we determined the expression levels of mRNA of S1R and PGRMC1, considered
as the S2R binding site, by Real Time RT- Polymerase Chain Reaction (RT-PCR).
Table 4. Tumor cell lines expressing both SR selected for this study
Cell line
Capan-2
Paca3
Origin
Tumor source
Primary tumor
Primary tumor
Metastatic site
Primary tumor
Metastatic site
Metastatic site
Metastatic site
Primary tumor
Morphology
Epithelial
Epithelial
Epithelial
Epithelial
Epithelial
Epithelial
Epithelial
Epithelial
Pancreas
Pancreas
Pancreas
Breast
CFPaC-1
SUM 159
MDA-MB 231
PC3
Breast
Prostate
Prostate
Glioblastoma
LNCaP
U87
The expression values of S1R and PGRMC-1 in CFPAC-1 line are very similar as
evidenced by western blot and related densitometric analysis showed in supplematary
data (Fig. SI4) and for this reason were used as reference values (arbitrary set equal to 1)
for Real Time RT-PCR evaluation of S1R or PGRMC-1 expression in the panel of cell
line investigated. As shown in Fig. 4 the highest S1R mRNA expression levels were
found in MDA-MB 231, LNCaP and PaCa3 cell lines, whereas PGRMC1 mRNA was
found to be highly expressed in PC3, CAPAN-2, and PaCa3 cell lines, respectively.

ACCEPTED MANUSCRIPT
Fig. 4. Relative Quantification (RQ) of the target genes SR1 and PGRMC1 RNA expression. The
mRNA levels were normalized to the endogenous reference genes GAPDH and HPRT, and quantified
respect to the value of S1R or PGRMC-1 found in CFPaC-1 cell line that was arbitrary set equal to 1
(
RQ=1). Values are the mean ± SD of three independent experiments.
2
.5. Preliminary biological evaluation of 3 and 6
We perform a preliminary assessment of the anticancer potential of compounds 3 and 6,
showing a good affinity towards SRs, on PaCa3 cells that express both SRs at high level,
using siramesine, a well known commercial S2R agonist, and NE100, a S1R antagonist,
as reference compounds [26,41]. The effect of compounds 3, 6, siramesine and NE100 on
cell viability was evaluated by the MTS assay. Cell lines grown in a 10% serum-
containing medium were exposed to increasing concentrations of compounds (0.1 µM -
1
00 µM) for 24 hours. Compound 3 showed an interesting cytotoxic activity, comparable
to siramesine, whereas compounds 6 and NE100 exhibited a poor cytotoxic effect (Fig.
). Since fetal bovine serum (FBS) is enriched in a variety of growth factors and
5
neurosteroids that may interfere and/or mask SR binding sites, the effect of compounds 3
and 6 on PaCa3 was evaluated also in serum-free medium. Interestingly, the decrease of
cell viability induced by 3 was enhanced by starvation conditions (IC50 = 49.8 ± 4.1 µM
and IC50 = 7.0 ± 0.2 µM, respectively). A similar effect was observed for siramesine (IC50
=
45.4 ± 2.0 µM in complete medium and IC = 6.0 ± 0.3 µM, in starvation condition).
50
On the contrary, starvation conditions are irrelevant for the cytotoxic properties of 6 and
NE100.

ACCEPTED MANUSCRIPT
According to this data, compound 3, by now on called RC-106, was selected for further
investigation.
Fig. 5. Effect of different SR modulators. The cells were exposed to compounds NE100, Siramesine, 3
(
RC-106) and 6, for 24 hours in the presence or in the absence of 10% FBS. The viability of the cells was
determined by MTS assay (mean ± SD of 3 independent experiments).
2
.6. S1R agonist/antagonist profile of compound 3 (RC-106)
Considering that the anticancer activity of a S1R ligands is related to their antagonist
profile [13], we investigated the profile of 3 (RC-106) by assessing its in vitro ability to
modulate NGF-induced neurite outgrowth in PC12 cells. Indeed, S1R agonists are known
to potentiate NGF-induced neurite outgrowth when used in the low micromolar
concentration range [29c,31a]. In detail, PC12 cells were incubated in a medium
containing 0.5% FBS plus NGF (2.5 ng/ml), in the presence of increasing concentrations
of 3 (RC-106) (0-10µM) for 96 h. Subsequently, cells were fixed and those displaying a
neurite longer than the diameter of the cell body were counted. Neurite outgrowth was
not affected by the addition of 3 (RC-106) up to 1 µM concentration; on the other hand,
starting from 2.5 µM neurite outgrowth was completely inhibited (Fig. SI3, see
Supplementary material), thus suggesting a S1R antagonist profile. To confirm this
hypothesis, competition assays were performed. Specifically, PC12 cells were incubated
with the standard S1R agonist PRE-084 (5, 10 and 25µM) in the absence/presence of 3
(RC-106) at 0.25 or 2.5 µM. At 0.25 µM concentration, compound 3 (RC-106)
significantly antagonized the effect of PRE-084 (10 and 25 µM) on NGF-induced neurite
outgrowth (Fig. 6). At 2.5 µM concentration, 3 (RC-106) completely blocked NGF-

ACCEPTED MANUSCRIPT
induced neurite sprouting, even in the presence of PRE-084 (results not shown). The
results confirm that 3 (RC-106) is a S1R antagonist.
Fig. 6. Assay of NGF–induced neurite outgrowth in PC12 cells. Effect of PRE-084 alone or in
combination with 3 (RC-106), at 0.25 µM. Histograms represent the mean±sem of at least 5 different
experiments performed in triplicate. **= p<0.005; ***=p<0.0008; ****=p<0.000004 vs control (0 PRE-
§
0
84). °°=p<0.007; °°°=p<0.00004 vs PRE-084 10µM and = p<0.004 vs PRE-084 25µM.
2
.7. Effect of 3 (RC-106) on cell viability
The cytotoxic acititvity of the pan-SR modulator 3 (RC-106) was evaluated by the MTS
assay on the panel of cancer cell lines (LNCaP, PC3, U87, Paca3, Capan-2, MDA MB
2
31, SUM 159) expressing both S1R and S2R. Briefly, cell lines grown in a 10% serum-
containing medium were exposed to increasing concentrations of 3 (RC-106) (0.1 µM -
00 µM) for 24 hours (Fig. 7). Compound 3 (RC-106) induced a decrease of cell viability
in all cell lines starting at 25 µM, with IC values ranging from 50 µM to 64 µM. The
1
5
0
IC50 values did not vary significantly by increasing the incubation time up to 48 h, except
for the Paca3 cell line, whose IC value markedly decreased after 48 h incubation (28.73
5
0
±
4.6, data not shown).

ACCEPTED MANUSCRIPT
Fig. 7. Effect of 3 (RC-106) on cell viability was evaluated on a panel of cancer cell lines with
different histotypes. Cells were exposed to the drug for 24 hours in a 10% FBS containing medium. Cell
viability was determined by the MTS assay (average of three independent experiments ± SD).
The effect of 3 (RC-106) on U87, Capan-2 and LNCaP cancer cells in the absence of
serum-induced cell cycle stimulation was also evaluated. Interestingly, in all the cell lines
treated with 3 (RC-106) in serum-free conditions, a marked decrease of cell survival at
low compound 3 (RC-106) concentrations compared to cells treated in FBS containing

ACCEPTED MANUSCRIPT
medium, was shown, as evidenced by the low IC values (9.6 -10.5 µM) (Fig. 8). This
5
0
trend is similar to that already evidenced on PaCa3 cells.
Fig. 8. MTS assay in serum-free conditions. After a 24 hours starvation, cell lines were treated for 24
hours with the indicated concentrations of 3 (RC-106). Data are expresses as percent of control values ±
standard deviation.
2
.8. Study of 3 (RC-106) apoptotic pathway through capase 3 activation
Lastly, to evaluate whether the observed decrease of cell viability under both conditions
was due to apoptosis, TUNEL and Annexin V stainings (analyzed by FACS) and caspase
3
activation (western blot, WB) were performed on PaCa3 cells, the tumor line displaying
also the highest PGRMC1 mRNA levels. Moreover, according to Zeng C. et al. [42] we
adopted caspase 3 assay in order to clarify the S2R agonist/antagonist profile of 3 (RC-
1
06).
The TUNEL assay showed that in serum containing medium a significant induction of
apoptosis after 24 h exposure to 3 (RC-106) could be observed only at 25 µM
concentration (Fig. 9A). This result is supported by the detection of the cleaved form of
caspase 3 by WB analysis (Fig. 9C) and by the Annexin V assay. In these FACS
experiments, a significant increase of early and late apoptotic cells (43.1% ± 3.4 and
3
4.1% ± 8.2, respectively) at 25 µM compound 3 (RC-106) was detected (Fig. 9B).

ACCEPTED MANUSCRIPT
Fig. 9. Apoptosis and apoptotic-related markers analysis in Paca3 cells grown in 10% FBS complete
medium. (A) TUNEL assay. Percentage of apoptotic cells after 24 h exposure to compound 3 (RC-106) at
1
0, 25 and 50 µM. *p<0.05. (B). Cytofluorimetric (FACS) analysis of apoptosis by Annexin V test. Cells
were exposed for 24 h to compound 3 (RC-106) (25 µM). Q1 area represents viable cells; Q2 early-
apoptotic cells; Q3 late-apoptotic cells; Q4 necrotic cells. The images are representative of three
experiments. (C) WB analysis of apoptotic-related marker after 24 h exposure to 3 (RC-106) at 25 µM.
Images are representative of two independent experiments.
When the same experiments were repeated under serum-free conditions (Fig. 10), a
significant increase of apoptotic cells was detected by FACS analysis. In particular, at 10
µM, 3 (RC-106) induces significant apoptosis in PaCa3 cells (early apoptotic cells = 10.9
%
± 0.0 and late apoptotic cells = 17.2 % ± 0.6). The apoptosis induction was further
confirmed by WB detection of cleaved caspase 3, after exposure of the cells at the same 3
RC-106) concentration.
(
The apoptotic effect of compound 3 (RC-106) is caspase-dependent, as evidenced by WB
assay. Therefore, through this functional assay, we can conclude that 3 (RC-106) is a S2R
agonist.

ACCEPTED MANUSCRIPT
Fig. 10. Apoptosis and apoptotic-related markers analysis in Paca3 cells.
Paca3 cells were exposed to 24 h starvation-condition and to 10 µM compound 3 (RC-106) and the
percentage of apoptotic cells was compared in untreated cells (UTR).(A) TUNEL assay: the values are the
mean±SD of 3 individual experiments. *p<0.05 (B) Representative images of FACS analysis of apoptosis
by Annexin V test. (C) WB analysis of apoptotic-related markers. Images are representative of two
independent experiments.
3
. Discussion
On the basis of recent literature evidences, we hypothesized that pan- SR modulators can
evoke anticancer activity. However, the design of new such compounds represents a
major challenge, since no structural information is currently available on S2R, which
could enable the adoption of effective techniques such as e.g., computer-aided drug
design. Notwithstanding these difficulties, and with this new goal in mind, we capitalized
our previous work [29c], according to which the presence of a bulky aminic portion
seemed to be an important feature in favoring ligand binding to both receptors. Therefore,
we designed a new molecular series of aryl-alkyl(alkenyl) 4-benzylamines. The chemical
strategy to obtain compounds 1-6, 9 and 11-12 followed a divergent synthesis, based on
the initial preparation of the common β-aminoketone intermediate A, easily obtainable

ACCEPTED MANUSCRIPT
via Michael chemistry (Scheme 1) [29c]. Accordingly, the smooth bromo-lithium
exchange on the appropriate aryl bromide afforded the lithiated arene that, upon
quenching with β-aminoketone A, gave the tertiary alcohols 1-2 in high yields. The
subsequent dehydration with trifluoroacetic anhydride under Cu(OTf)
2
catalysis
conditions [30] afforded a mixture of olefinic regioisomers C3-C4 and the E stereoisomer
C2-C3. After purification, olefins 3 and 4 were isolated and finally hydrogenated to
access the desired amines 5 and 6. The same strategy was applied for accessing
hydroxylated compounds 9 and 11-12. However, the protection of the aromatic alcohols
as TBS ethers (and their corresponding removal) was required to avoid interference with
the lithiation step. Concerning the synthetic pathway of 9 and 11-12, two additional
observations are worth at this point: i) the lithiation of the protected TBS-bromonaphtols
with n-BuLi in THF performed better than the t-BuLi/Et O based method, and ii) keeping
2
temperature below -50 °C after quenching with β-aminoketone A improved the efficiency
of the alcohol synthesis. In the case of racemic compounds, a (semi)preparative chiral
high performance liquid chromatography (HPLC) resolution process was performed and
the pure enantiomers obtained in amounts sufficient to support a preliminary biological
investigation. All compounds showed – in silico and in vitro - very high/good affinity for
the S1R, with K S1 values in the range 0.7 – 120 nM (Tables 1, SI1 and SI2, see
i
Supplementary material). Molecular modeling revealed that the main molecular
requirements for high S1R affinity (i.e., the instauration of the prototypical salt bridge
involving the ligand basic N atom and the carboxylic side chain of Asp126, the
encasement of an aromatic portion of the ligand within the hydrophobic S1R cavity lined
by residues Ile128, Phe133, Tyr173 and Leu186, and the generation of a set of further
stabilizing ligand/receptor π interactions) were all satisfied by the present series of
compounds.
Keeping in mind that the purpose of the work was the identification of dual S1R and S2R
ligands, compounds 3, called by us RC-106, and 6 have been selected for a preliminary
investigation of their cytotoxic properties, being the molecules in the full series endowed
i
with a good affinity towards both receptor subtypes [3 (RC-106) K S1 = 12.0 ± 5.0 nM;
K S2 = 22.0 ± 3.0 nM; 6 K S1 = 2.1 ± 1.0; K S2 = 6.5 ± 3.0]. The preliminary biological
i
i
i
evaluation of 3 (RC-106) and 6 were carried out using PaCa3 cell line (MTS assay)

ACCEPTED MANUSCRIPT
considering the high level of expression of both SRs. For comparative purposes, the
effects of siramesine (S2R agonist) and NE100 (S1R antagonist) were also evaluated.
Compounds 3 (RC-106) and siramesine showed an interesting antiproliferative activity,
both in complete medium and in starvation conditions. Conversely, compound 6 and
NE100 showed poor cytotoxic properties and therefore 6 were discarded.
To in depth characterize 3 (RC-106) from a functional point of view, we assessed its S1R
agonist/antagonist profile on NGF-induced neuronal differentiation in PC12 cells model
[43]. Indeed, it has been reported that S1R agonists, such as (+)-pentazocine, imipramine,
fluvoxamine, PRE-084 and RC-33, among the others, potentiate NGF-induced neurite
outgrowth in PC12 cells, and that selective S1R antagonist (such as NE-100 and
BD1063) significantly attenuate the efficacy of S1R agonists both in the same in vitro
assay [29c,43-47]. Our results (Fig. 6) clearly show that compound 3 (RC-106) has a S1R
antagonist profile.
We then investigated the cytotoxic activity of 3 (RC-106) on a panel of tumor cell lines
representative of various cancer types all expressing both SRs. In particular, with regard
to S2R, we evaluated PGRMC1 mRNA by RT-PCR as equivalent to S2R expression,
even if the actual identity of S2R is still controversial [20,40-54]. When we tested the
effect of 3 (RC-106) on actively proliferating tumor cell lines, we observed significant
cytotoxicity at concentrations starting from 10 µM in all the cell lines under
investigation. Interestingly, 3 (RC-106) showed cytotoxic effect in all the cell lines tested
under low proliferation conditions induced by serum deprivation. In particular, our data
indicated that a short term starvation (24 hours) enhances the cytotoxic effect of 3 (RC-
1
06), as evidenced by the low IC values detected and by the low dose of 3 (RC-106)
50
needed to trigger apoptotic mechanism under these conditions. This result is also in
agreement with recent data from pre-clinical models showing that short term starvation
may be able to potentiate the effectiveness of chemotherapy and radiotherapy [55-59].
Several clinical trials are currently studying the effect of fasting or fasting-mimic diets in
patients undergoing chemotherapy (NCT01304251, NCT01175837, NCT00936364,
NCT01175837, NCT01802346, NCT02126449). Importantly, the apoptotic effect of 3
(RC-106) is caspase-mediated both in normal and in starvation conditions, evidencing the
S2R the agonist profile of 3 (RC-106).

ACCEPTED MANUSCRIPT
To sum up, we identified a S1R antagonist and a S2R agonist exerting an interesting
cytotoxic action toward a panel of tumor cell lines (pancreas, breast, prostate and
glioblastoma) both in complete medium and under starvation conditions. Our data also
suggest that the observed decrease of cell viability is due to an apoptotic process
triggered by 3 (RC-106).
4
. Conclusion
There is increasing evidence that both S1R and S2R play a significant role in cancer
biology, therefore modulator of both SR subtypes could be of high interest for developing
novel anti-cancer drugs. In this scenario, we report the design and synthesis of a
compound series targeting both SR subtypes with high affinity. Among these, we
identified 3 (RC-106), a compound able to induce a strong cytotoxic effect in a wide
panel of cancer cell lines, all expressing SRs, both actively proliferating and in low
proliferation rate in response to serum deprivation. The antitumor properties of 3 (RC-
1
06) have been observed in all cell lines independently from tumor histotype. In
particular, in pancreatic PaCa3 cells, the cell line expressing the highest levels of both
receptors, 3 (RC-106) acts as pro-apoptotic drug, inducing a fast triggering of cell death
program.
To sum up, 3 (RC-106) exhibited a promising cytotoxic activity on a panel of cancer cell
lines of different tumors, representative of various cancer expressing both SRs. This
compound could meet the requirements of new-generation drugs to enter into the so-
called basket trials, consisting in treating several neoplastic diseases, all characterized by
the same molecular alterations, in this case represented by high expression of S1R or S2R
or both [60]. Lastly, it has to be underlined that S1R antagonists can be used for
alleviating chronic pain, especially in conditions such as neuropathic pain, a pathologic
condition that frequently occurs in cancer patients [61-63]. Accordingly, the
identification of new, potent pan-sigma receptor modulators will be of great interest, to
develop antitumor and analgesic drugs, representing an innovative pharmacological
approach for the treatment of cancer patients with advanced disease. Therefore, 3 (RC-
1
06) represents the hit compound of a new class of dual-action ligands targeting S1R and
S2R potentially useful for the treatment of cancer disease. Moreover, the evaluation of

ACCEPTED MANUSCRIPT
the antinociceptive properties of 3 (RC-106) is under investigation and will be reported in
due course.
5
. Experimental section
5
.1. General remarks
Reagents and solvents for synthesis were obtained from Aldrich (Italy). Solvents were
purified according to the guidelines in Purification of Laboratory Chemicals. Melting
points were measured on SMP3 Stuart Scientific apparatus and are uncorrected.
Analytical thin-layer-chromatography (TLC) was carried out on silica gel precoated
glass-backed plates (Fluka Kieselgel 60 F254, Merck) and on aluminiumoxid precoated
aluminium-backed plates (DC-Alufolien Aluminiumoxid 60 F254 neutral, Merck);
visualized by ultra-violet (UV) radiation, acidic ammonium molybdate (IV), or potassium
permanganate. Flash chromatography (FC) was performed with Silica Gel 60 (particle
size 230–400 mesh, purchased from Nova Chimica) and neutral aluminium oxide
(
particle size 0.05-0.15 mm, purchased from Fluka). Proton nuclear magnetic resonance
NMR) spectra were recorded on a Bruker Avance 500 spectrometer operating at 500
(
MHz. Proton chemical shifts (
δ
) are reported in ppm with the solvent reference relative to
= 7.26 ppm). The
tetramethylsilane (TMS) employed as the internal standard (CDCl3,
δ
following abbreviations are used to describe spin multiplicity: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, br = broad signal, dd = doublet-doublet, td = triplet-
1
3
doublet. The coupling constant values are reported in Hz. C-NMR spectra were
recorded on a 500 MHz spectrometer, with complete proton decoupling. Carbon chemical
shifts (
δ
) are reported in ppm relative to TMS with the respective solvent resonance as
= 77.23 ppm).
the internal standard (CDCl , δ
3
UPLC-UV-ESI/MS analysis were carried out on a Acuity UPLC Waters LCQ FLEET
system using an ESI source operating in positive ion mode, controlled by ACQUIDITY
PDA and 4 MICRO (Waters). Analyses were run on a ACQUITY BEH C18 (50 x 2.1
mm, 1.7 ꢀm) column, at room temperature, with gradient elution (solvent A: water
containing 0.1% of formic acid; solvent B: methanol containing 0.1% of formic acid;
gradient: 10% B in A to 100% B in 3 minutes, followed by isocratic elution 100% B for

ACCEPTED MANUSCRIPT
-1
1
.5 minutes, return to the initial conditions in 0.2 minutes) at a flow rate of 0.5 mL min .
All of the final compounds had 95% or greater purity.
Chiral HPLC runs were conducted on a Jasco (Cremella, LC, Italy) HPLC system
consisting of PU-1580 pump, 851-AS auto-sampler, and MD-1510 Photo Diode Array
(PDA) detector. Experimental data were acquired and processed by Jasco Borwin PDA
and Borwin Chromatograph Software. Solvents used for chiral chromatography were
HPLC grade and supplied by Carlo Erba (Milan, Italy). Chiral analytical columns:
Chiralcel OJ-H (4.6 mm diameter x 150 mm length, dp 5 ꢀm), Chiralpack IC (4.6 mm
diameter x 250 mm length, dp 5ꢀm) and Chiralpack IA (4.6 mm diameter x 250 mm
length, dp 5 ꢀm). Analytes were detected photometrically at 220 and 254 nm. Unless
otherwise specified, sample solutions were prepared dissolving analytes at 1 mg/mL in
ethanol and filtered through 0.45 ꢀm PTFE membranes before analysis. The injection
volume was 10 ꢀL. Detection was performed at 220 and 254 nm. The retention factor
R 0 0 R
(k) was calculated using the equation k = (t - t )/ t , where t is the retention time and
t0 the dead time (t was considered to be equal to the peak of the solvent front and was
0
taken from each particular run). The enantioselectivity (α) and the resolution factor (Rs)
were calculated as follows: α= k / k and Rs = 2 (t - t ) / (w + w ) where t and t
2
1
R2
R1
1
2
R2
R1
are the retention times of the second and the first eluted enantiomers, and w and w are
1
2
the corresponding base peak widths. All HPLC analyses were performed at room
temperature.
The best conditions found by the screening protocol were applied to a semi-preparative
scale-up. The enantiomers of 1, 2, 5 and 9 were then completely resolved by a (semi)-
preparative process using a Chiralcel OJ-H column (10 mm diameter × 250 mm length, 5
ꢀm), eluting with ethanol (for the compound 2) or methanol (for the compounds 3, 5 and
8
) at RT with a flow rate of 2.5 mL/min. Compounds 6 and 12 were resolved on
Chiralpak IA (10 mm diameter × 250 mm length, 5 ꢀm) using MeOH at a flow rate of 2.5
mL/min as eluent. The eluate was fractioned according to the UV profile (detection
preformed at 220 and 254 nm). The fractions obtained containing the enantiomers were
evaporated at reduced pressure. Analytical control of collected fractions was performed
using the analytical columns.

ACCEPTED MANUSCRIPT
Optical rotation values of enantiomeric compounds were measured on a Jasco
photoelectric polarimeter DIP 1000 using a 0.5 dm cell and a sodium and mercury lamp
-2
-1
(
λ = 589 nm, 435 nm); sample concentration values are given in 10 g mL (Table 2)
5
.2. General procedure for the preparation of compounds 1 and 2
Under nitrogen atmosphere, tert-BuLi (2.5 equiv, 1.7 M in pentane) was added dropwise
to a -78 °C cooled solution of the appropriate aryl bromide (1.25 equiv) in anhydrous
diethyl ether (20 mL). After 20 minutes the reaction was slowly allowed to reach room
temperature. The stirring was continued for 1 additional hour and a solution of 4-(4-
benzyl-piperidin-1-yl)-butan-2-one (1.0 equiv) in anhydrous diethyl ether (6 mL) was
then added dropwise at -78 °C. The reaction mixture was slowly warm to 0 °C, stirred for
3
h and then quenched with water (12 mL); after an acid (pH = 3-4) / base (pH = 8-9)
work-up, the combined organic phases were evaporated under vacuum to get the desired
compounds.
5
.2.1. 4-(4-benzylpiperidin-1-yl)-2-(naphthalen-2-yl)butan-2-ol, [1]: Yield: 44%; white
-1
1
solid; mp: 105-107 °C; IR (cm ): 3434, 2918, 1653, 1438, 1156, 1112, 820; H-NMR
500 MHz) (CDCl ) δ (ppm): 8.01 (s, 1H), 7.86-7.81 (m, 3H), 7.47-7.45 (m, 3H), 7.28 (t,
(
3
J = 7.8 Hz, 2H), 7.19 (t, J = 7.0 Hz, 1H), 7.13 (d, J = 7.1 Hz, 2H), 3.19 (d, 1H), 2.54-2.50
(
(
(
m, 3H), 2.31 (m, 1H), 2.22-2.14 (m, 2H), 1.92 (d, 1H), 1.84 (m, 1H), 1.76 (m, 1H), 1.68
1
3
m, 1H), 1.60 (m, 1H), 1.57 (s, 3H), 1.50 (m, 1H), 1.31 (m, 2H); C-NMR (500 MHz)
CDCl ) δ (ppm): 146.3, 140.5,133.3, 132.0, 129.1, 128.2, 128.1, 127.6, 127.4, 125.8,
3
1
25.4, 123.8, 123.6, 75.7, 55.1, 54.8, 52.6, 43.1, 37.8, 37.4, 32.6, 32.1, 31.4; UHPLC-
+
R
ESI-MS: t = 2.03, > 99.9% pure (λ = 225 nm), m/z = 374 [M + H]
5
.2.1.1. (+)-4-(4-benzylpiperidin-1-yl)-2-(naphthalen-2-yl)butan-2-ol, [(+)-1]: White
2
0
solid; [α]D = +40.5 (c 0.2, CH OH). The IR and NMR spectra are identical to that of 1.
3
HPLC: t = 8.5 min, ee 96.0%.
R
5
.2.1.2. (-)-4-(4-benzylpiperidin-1-yl)-2-(naphthalen-2-yl)butan-2-ol, [(-)-1]: White
2
0
solid; [α]D = -42.3 (c 0.2, CH OH). The IR and NMR spectra are identical to that of 1.
3
R
HPLC: t = 11.1 min, ee 97.0%.

ACCEPTED MANUSCRIPT
5
.2.2. 4-(4-benzylpiperidin-1-yl)-2-phenylbutan-2-ol, [2]: Yield: 77%; white solid; mp:
-1
1
9
0.9-93 °C; IR (cm ): 3184, 3125, 1602, 1369, 1343, 1156, 846, 699; H-NMR (500
) δ (ppm): 7.45 (d, J = 8.9 Hz, 2H), 7.33 (t, J = 8.1 Hz, 2H), 7.27 (t, J = 7.4
Hz, 2H), 7.21-7.19 (m, 2H), 7.13 (d, J = 7.0 Hz, 2H), 3.15 (d, 1H), 2.54 (m, 1H), 2.52 (m,
MHz) (CDCl
3
2
1
H) 2.30 (m, 1H), 2.22 (m, 1H), 2.07 (m, 1H), 1.86 (m, 1H), 1.80 (m, 1H) 1.75 (m, 1H),
1
3
.67 (m, 1H) 1.60 (m, 1H), 1.50 (m, 1H), 1.49 (s, 3H) 1.31 (m, 2H); C-NMR (500
MHz) (CDCl ) δ (ppm): 148.9, 140.5, 129.1, 127.9, 128.2, 126.0, 125.8, 125.0, 75.5,
3
5
R
5.1, 54.8, 52.6, 43.1, 37.8, 37.7, 32.6, 32.1, 31.4; UHPLC-ESI-MS: t = 1.75, > 97%
+
pure (λ = 210 nm), m/z = 324 [M + H]
2
0
5
.2.2.1. (+)-4-(4-benzylpiperidin-1-yl)-2-phenylbutan-2-ol, [(+)-2]: Yellow oil; [α]D =
+
10.5 (c 0.6, CH OH). The IR and NMR spectra are identical to that of 2. HPLC: t = 3.4
3 R
min, ee 99.9%.
2
0
5
.2.2.2. (-)-4-(4-benzylpiperidin-1-yl)-2-phenylbutan-2-ol, [(-)-2]: Yellow oil; [α]D = -
.2 (c 0.6, CH OH). The IR and NMR spectra are identical to that of 2. HPLC: t = 4.2
9
3
R
min, ee 98.0%.
5
.3. General procedure for the preparation of compounds 3 and 4
Under argon atmosphere trifluoroacetic anhydride (2.0 equiv) was added dropwise to a
solution of the tertiary alcohol (1.0 equiv) and copper triflate (2 mol %) in anhydrous
dichloromethane (5 mL) cooled to 0 °C. After stirring the reaction a solution of
NaHCO3(aq) (5 %) was added. The phases were separated and the organic phase was dried
2 4
over anhydrous Na SO and concentrated under reduced pressure. The obtained crude
was purified by alumina (II Brockmann degree) column chromatography (9 n-hexane – 1
ethyl acetate).
5
.3.1. (E)-4-benzyl-1-[3-(naphthalen-2-yl)but-2-en-1-yl]piperidine, [3]: Yield: 50%,
-1
yellow solid; mp: 221-222 °C; IR (cm ): 3049-2977, 2926, 2848, 2514, 1597, 1482,
1
1
453-1434, 1287-1157, 1039, 940, 895, 855, 819, 744, 689; H-NMR (500 MHz)
3
(CDCl ) δ (ppm): 7.82-7.80 (m, 3H). 7.78 (d, J = 9.0 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H),

ACCEPTED MANUSCRIPT
7
2
.46-7.44 (m, 2H), 7.29 (t, J = 7.4 Hz, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.16 (d, J = 7.6 Hz,
H), 6.09 (t, J = 6.6 Hz, 1H), 3.22 (d, J = 6.5 Hz, 2H), 3.03 (d, 2H) 2.57 (d, J = 6.9 Hz, 2
1
3
H), 2.16 (s, 3H), 1.98 (t, 2H), 1.68 (d, 2H), 1.57 (m, 1H), 1.38 (m, 2 H); C-NMR (500
MHz) (CDCl
3
) δ (ppm): 140.7, 140.4, 136.9, 133.4, 132.5, 129.1, 128.1, 128.0, 127.6,
1
27.5, 126.0, 125.7, 125.6, 124.2, 124.1, 57.2, 54.1, 43.2, 37.9, 32.2, 16.1; UHPLC-ESI-
+
MS: t
R
= 2.20, >98% pure (λ = 245 nm), m/z = 356 [M + H]
5
.3.2. (E)-4-benzyl-1-(3-phenylbut-2-en-1-yl)piperidine, [4]: Yield: 42%; yellow solid;
-1
mp: 220-222 °C; IR (cm ): 3085-2979, 2926, 2488, 1641-1580, 1484-1401, 1273-1160,
1
1
2
5
2
038, 943, 835, 765-748, 689; H-NMR (500 MHz) (CDCl ) δ (ppm): 7.40 (d, J = 7.9,
3
H), 7.31-7.28 (m, 4H); 7.24 (m, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 7.3 Hz, 2H),
.92 (t, J = 6.4 Hz, 1H), 3.16 (d, J = 6.5 Hz, 2H), 2.99 (d, 4H), 2.55 (d, J = 6.8 Hz, 2H),
1
3
.05 (s, 3H), 1.95 (t, 2H), 1.66 (d, 2H), 1.54 (m, 1H), 1.36 (m, 2H); C-NMR (500 MHz)
) δ (ppm): 143.3, 140.7, 137.1, 129.1, 128.2, 128.1, 126.9, 125.7, 125.6, 124.9,
(CDCl
3
5
7.0, 54.0, 43.2, 37.6, 32.1, 16.1; UHPLC-ESI-MS: t = 1.97, > 98% pure (λ = 205 nm),
R
+
m/z = 306 [M + H]
5
.4. General procedure for the preparation of compound 5 and 6.
To a solution of olefin (1.0 equiv) in absolute ethanol (10 mL) was added a catalytic
amount of Pd (0) / C 10% (p/p, 0.06 equiv). The suspension was stirred vigorously under
hydrogen atmosphere (1 atm). The reaction mixture was then filtered through Celite,
using dichloromethane as solvent. The crude was purified by alumina (II Brockmann
degree) column chromatography (9 n-hexane – 1 ethyl acetate).
5
.4.1. 4-benzyl-1-[3-(naphthalen-2-yl)butyl]piperidine, [5]: Yield: 56%, yellow oil; IR
-
1
1
(
cm ): 3025, 2924, 2508, 1631, 1602, 1542, 1496, 1453; H-NMR (500 MHz) (CDCl
ppm): 7.79-7.77 (t, J = 8.7 Hz, 3H), 7.59 (s, 1H), 7.45-7.42 (m, 2H), 7.32 (d, J = 8.8 Hz,
H), 7.25 (m, 2H), 7.17 (m, 1H), 7.08 (m, 2H), 3.30 (broad peak, 2H), 2.90 (m, 1H), 2.76
m, 1H), 2.55 (d, J = 7.4 Hz, 2H), 2.46 (m, 1H), 2.27-2.13 (m, 4H), 1.82-1.71 (m, 4H),
3
) δ
(
1
(
1
3
1
1
.59 (m, 1H), 1.36 (d, J = 6.5 Hz, 3H); C-NMR (500 MHz) (CDCl ) δ (ppm): 142.4,
3
39.3, 133.4, 132.3, 128.9, 128.5, 128.3, 127.6, 127.5, 126.1, 125.5, 125.3, 124.8, 56.1,

ACCEPTED MANUSCRIPT
5
R
2.4, 41.9, 38.3, 36.5, 31.8, 29.1, 22.7; UHPLC-ESI-MS: t = 2.17, > 97% pure (λ = 220
+
nm), m/z = 358 [M + H]
2
0
5
D
.4.1.1. (+)-4-benzyl-1-(3-(naphthalen-2-yl)butyl)piperidine, [(+)-5]: Yellow oil; [α] =
+
6.1 (c 0.2, CH OH). The IR and NMR spectra are identical to that of 5. HPLC: t = 7.7
3 R
min, ee 95.0%.
2
0
5
.4.1.2. (-)-4-benzyl-1-(3-(naphthalen-2-yl)butyl)piperidine, [(-)-5]: Yellow oil; [α]D = -
.3 (c 0.2, CH OH). The IR and NMR spectra are identical to that of 5. HPLC: t = 9.0
6
3
R
min, ee 95.0%.
-1
5
.4.2. 4-benzyl-1-(3-phenylbutyl)piperidine, [6]: Yield: 47%, yellow oil; IR (cm ): 3682,
1
3
019, 2929, 2856, 2434, 2400, 1230; H-NMR (500 MHz) (CDCl ) δ (ppm): 7.28-7-27
3
(m, 4H), 7.17 (m, 4H), 7.13 (d, J = 7.0 Hz, 2H), 2.86 (broad peak, 2H), 2.71 (m, 1H),
2
1
.52 (d, J = 6.6 Hz, 2H), 2.26 (m, 1H), 2.14 (m, 1H), 1.87-1.72 (m, 4H), 1.61 (d, 2H),
1
3
.49 (m, 1H), 1.29 (m, 2H), 1.24 (d, J = 7.3 Hz, 3H); C-NMR (500 MHz) (CDCl ) δ
3
(ppm): 147.3, 140.7, 129.1, 128.3, 128.1, 126.9, 125.9, 125.7, 57.3, 54.1, 53.9, 43.2, 38.4,
3
7.9, 35.4, 32.1, 22.6; UHPLC-ESI-MS: t = 1.92, > 97% pure (λ = 200 nm), m/z = 308
R
+
[M + H]
2
0
5
0
9
5
.4.2.1. (+)-4-benzyl-1-(3-phenylbutyl)piperidine, [(+)-6]: Yellow oil; [α]D = +8.2 (c
.3, CH
9.9%.
3 R
OH). The IR and NMR spectra are identical to that of 6. HPLC: t = 3.7 min, ee
20
.4.2.2. (-)-4-benzyl-1-(3-phenylbutyl)piperidine, [(-)-6]: Yellow oil; [α]D = -8.3 (c 0.3,
OH). The IR and NMR spectra are identical to that of 6. HPLC: t = 5.3 min, ee
CH
3
R
9
9.9%.
5
.5. (6-bromonaphthalen-2-yloxy)-tert-butyldimethtyl-silane, [7]
Under argon atmosphere 6-bromo-2-naphthol (1.0 equiv), imidazole (1.0 equiv) and tert-
butyldimethylsilyl chloride (1.2 equiv) were solubilized in anhydrous dimethylformamide
(20 mL). After stirring the solution overnight, the reaction mixture was extracted by