Identification of dual Sigma1 receptor modulators/acetylcholinesterase inhibitors with antioxidant and neurotrophic properties, as neuroprotective agents

Article abstract of DOI:10.1016/j.ejmech.2018.09.010

In this manuscript we report on the design, synthesis and evaluation of dual Sigma 1 Receptor (S1R) modulators/Acetylcholinesterase (AChE) inhibitors endowed with antioxidant and neurotrophic properties, potentially able to counteract neurodegeneration. The compounds based on arylalkylaminoketone scaffold integrate the pharmacophoric elements of RRC-33, a S1R modulator developed by us, donepezil, a well-known AChE inhibitor, and curcumin, a natural antioxidant compound with neuroprotective properties. A small library of compounds was synthesized and preliminary in vitro screening performed. Some compounds showed good S1R binding affinity, selectivity towards S2R and N-Methyl-D-Aspartate (NMDA) receptor, AChE relevant inhibiting activity and are potentially able to bypass the BBB, as predicted by the in silico study. For the hits 10 and 20, the antioxidant profile was assessed in SH-SY5Y human neuroblastoma cell lines by evaluating their protective effect against H₂O₂ cytotoxicity and reactive oxygen species (ROS) production. Tested compounds resulted effective in decreasing ROS production, thus ameliorating the cellular survival. Moreover, compounds 10 and 20 showed to be effective in promoting the neurite elongation of Dorsal Root Ganglia (DRG), thus demonstrating a promising neurotrophic activity. Of note, the tested compounds did not show any cytotoxic effect at the concentration assayed. Relying on these encouraging results, both compounds will undergo a structure optimization program for the development of therapeutic candidates for neurodegenerative diseases treatment.

Full text of DOI:10.1016/j.ejmech.2018.09.010

Accepted Manuscript
Identification of dual Sigma1 receptor modulators/acetylcholinesterase inhibitors with
antioxidant and neurotrophic properties, as neuroprotective agents
Marta Rui, Giacomo Rossino, Stefania Coniglio, Stefania Monteleone, Arianna
Scuteri, Alessio Malacrida, Daniela Rossi, Laura Catenacci, Milena Sorrenti, Mayra
Paolillo, Daniela Curti, Letizia Venturini, Dirk Shepmann, Bernhard Wünsch, Klaus R.
Liedl, Guido Cavaletti, Vittorio Pace, Ernest Urban, Simona Collina
PII:
S0223-5234(18)30782-7
10.1016/j.ejmech.2018.09.010
EJMECH 10721
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 June 2018
Revised Date: 3 September 2018
Accepted Date: 4 September 2018
Please cite this article as: M. Rui, G. Rossino, S. Coniglio, S. Monteleone, A. Scuteri, A. Malacrida,
D. Rossi, L. Catenacci, M. Sorrenti, M. Paolillo, D. Curti, L. Venturini, D. Shepmann, B. Wünsch, K.R.
Liedl, G. Cavaletti, V. Pace, E. Urban, S. Collina, Identification of dual Sigma1 receptor modulators/
acetylcholinesterase inhibitors with antioxidant and neurotrophic properties, as neuroprotective agents,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.010.
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

ACCEPTED MANUSCRIPT
Identification of dual Sigma1 receptor modulators/Acetylcholinesterase
inhibitors with antioxidant and neurotrophic properties, as neuroprotective
agents
1
1
1
2,3
4
Marta Rui , Giacomo Rossino , Stefania Coniglio , Stefania Monteleone , Arianna Scuteri ,
4
1
1
1
1
Alessio Malacrida , Daniela Rossi , Laura Catenacci , Milena Sorrenti , Mayra Paolillo , Daniela
5
6
7
7
2
4
Curti , Letizia Venturini , Dirk Shepmann , Bernhard Wünsch , Klaus R. Liedl , Guido Cavaletti ,
8
8
1
Vittorio Pace , Ernest Urban , Simona Collina *
1
Department of Drug Sciences, Medicinal Chemistry, Pharmaceutical Technology and
Pharmacological sections, University of Pavia, Viale Taramelli 6 and 12, 27100 Pavia (Italy).
2
Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 80-82,
6020 Innsbruck (Austria).
3
Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6,
5032 Marburg (Germany).
3
4
School of Medicine and Surgery, University Milano-Bicocca, via Cadore 48, 20900 Monza (Italy).
5
Department of Biology and Biotechnology “L. Spallanzani”, Lab. of Cellular and Molecular
Neuropharmacology, University of Pavia, Via Ferrata 9, 27100 Pavia (Italy).
6
Department of Internal Medicine and Therapeutics, University of Pavia, Via Taramelli 24, 27100
Pavia (Italy).
7
Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster, Correnstrasse 48,
48149 Muenster (Germany).
8
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090 Vienna
(Austria).
1

ACCEPTED MANUSCRIPT
Abstract
In this manuscript we report on the design, synthesis and evaluation of dual Sigma 1 Receptor
S1R) modulators/Acetylcholinesterase (AChE) inhibitors endowed with antioxidant and
(
neurotrophic properties, potentially able to counteract neurodegeneration. The compounds based on
arylalkylaminoketone scaffold integrate the pharmacophoric elements of RRC-33, a S1R modulator
developed by us, donepezil, a well-known AChE inhibitor, and curcumin, a natural antioxidant
compound with neuroprotective properties. A small library of compounds was synthesized and
preliminary in vitro screening performed. Some compounds showed good S1R binding affinity,
selectivity towards S2R and N-Methyl-D-Aspartate (NMDA) receptor, AChE relevant inhibiting
activity and are potentially able to bypass the BBB, as predicted by the in silico study. For the hits
10 and 20, the antioxidant profile was assessed in SH-SY5Y human neuroblastoma cell lines by
evaluating their protective effect against H O cytotoxicity and reactive oxygen species (ROS)
2
2
production. Tested compounds resulted effective in decreasing ROS production, thus ameliorating
the cellular survival. Moreover, compounds 10 and 20 showed to be effective in promoting the
neurite elongation of Dorsal Root Ganglia (DRG), thus demonstrating a promising neurotrophic
activity. Of note, the tested compounds did not show any cytotoxic effect at the concentration
assayed. Relying on these encouraging results, both compounds will undergo a structure
optimization program for the development of therapeutic candidates for neurodegenerative diseases
treatment.
Keywords: Neuroprotective agents, Sigma1 Receptor, acetylcholinesterase inhibitors, antioxidant
properties, neurotrophic properties
2

ACCEPTED MANUSCRIPT
1
. Introduction
The progressive loss of structure and/or function of neurons, within the Central Nervous System
CNS), is the prototypical event leading to neurodegeneration, which is an aspect underpinning a
(
heterogeneous group of disorders [1-2]. The World Health Organization (WHO) recently reported
that neurodegenerative diseases, i.e. Alzheimer’s (AD), Parkinson’s (PD), Amyotrophic Lateral
Sclerosis (ALS) and Multiple Sclerosis (MS), are one of the main causes of death worldwide,
causing the death of 6.8 million people annually (Figure 1) [3-5]. Moreover, the treatment options
of neurodegenerative diseases are meagre, since only few molecules able to attenuate the
devastating outcome and/or to limit the progression of the diseases are available and thus, there is
an urgent need of effective therapies [6]. Neurodegenerative disorders have a multifactorial origin,
being associated with both environmental factors, such as smoking, stress and the use of pesticides,
and specific genetic variations, leading to alteration of several molecular cascades (Figure 1) [7-8].
Dysfunction of neurotransmitter pathways (i.e. acetylcholine or dopamine systems) can sometimes
occur together with abnormal structure and/or accumulation of proteins – i.e. tau protein
hyperphosphorylation, β-amyloid accumulation, further aggravating the pathological condition [9-
12]. An important hallmark of neurodegeneration conditions is an over-production of Reactive
Oxygen Species (ROS) due to alterations of mitochondrial respiratory chain activity [13-14].
3

ACCEPTED MANUSCRIPT
Figure 1. Some aetiological factors that promote multifactorial neurodegenerative diseases.
In our continuing efforts towards the design and development of new molecular entities able to
counteract neurodegeneration, some years ago we designed and characterized a wide series of
Sigma 1 Receptor (S1R) modulators [15]. These compounds may have a great potential as
neuroprotective agents, since under pathological conditions S1R operates as chaperone modulating
different proteins, restoring the calcium homeostasis and controlling the generation of ROS, thus
preventing cellular damage [16-23]. Accordingly, S1R modulators are able to simultaneously
address more than one pathological event and then they represent a good therapeutic opportunity for
treating neurodegenerative disorders. At the same time, it is worth noting that the multifactorial
origin of such disorders prompted the interest of the scientific community toward the so-called
multi-target drugs (MTDs) and along the years, a number of heterogeneous MTDs have been
developed combining drugs already used in therapy with several scaffolds endowed with different
activities [24-27]. With these premises in mind and in the attempt of obtaining molecules able to
modulate S1R, to act as acetylcholinesterase (AChE) inhibitors and endowed with antioxidant
4

ACCEPTED MANUSCRIPT
properties, herein we report on a new series of dual ligands, designed by focussing on RRC-33,
donepezil and curcumin (Cur) (Figure 2), as reference molecules.
In details, RRC-33 is our most promising compound, showing an excellent affinity and selectivity
towards S1R over S2R (Ki S1 = 1.8 ± 0.1 nM; Ki S2 = 45 ± 16 nM) and a high metabolic stability
in several biological matrices [28]. Furthermore, it is able to promote neurite differentiation and
elongation in rat Dorsal Root Ganglia (DRG) experimental model [29]. Donepezil is an AChE
inhibitor able to restore the physiological amount of acetylcholine and it is commonly used ìn
therapy for counteracting the cognitive impairment, a common feature of neurodegenerative
diseases [30-31], while Cur (Figure 2) is an effective ROS scavenging compound [32]. It is the
main yellow-colored pigment of Curcuma longa, historically employed in Ayurvedic medicine
[
33]. Although its numerous drawbacks linked to its solubility, instability in physiological fluids,
and low bioavailability [34], Cur possesses a wide spectrum of action including antioxidant
activities, which may be useful to counteract neurodegenerative diseases [35-37].
Thus, combining the structural features of RRC-33, donepezil and Cur (Figure 2), we designed a
new series of compounds. The newly synthesized compounds were investigated in terms of SRs
affinity and inhibitory activity toward AChE enzyme and their mode of interaction with S1R and
AChE was in silico studied. Furthermore, their in silico ADME parameters have been evaluated to
assess their ability to cross the blood brain barrier (BBB) and reach the central nervous system
(CNS). Lastly, the antioxidant profile and neurotrophic activity of the most interesting derivatives
were evaluated.
5

ACCEPTED MANUSCRIPT
Figure 2. MTD scaffold merges the key functional groups of S1R agonists, AChE inhibitors and curcumin.
2
. Results and discussion
2.1. Chemistry
We prepared a new compound series, characterized by an arylalkylamineketone scaffold with three
elements of structural diversity, i.e. the aromatic ring, the aminic moiety and the length of the linker
between the aromatic and the aminic portions (Figure 2). The conceived arylalkylamineketone
scaffold allowed improving synthetic feasibility and chemical tractability by removal of chiral
center present in the inspiring S1R modulator RRC-33.
Compounds were synthesized by following the general synthetic route outlined in Scheme 1 and
Scheme 2.
6

ACCEPTED MANUSCRIPT
Scheme 1: Weinreb amide formation and nucleophilic substitution. Reagents and reaction conditions: i) K CO ,
2
3
Et O/H O, r.t.; ii) K CO , ACN, r.t.
2
2
2
3
Scheme 2: Lithiated arene formation and quenching with the corresponding Weinreb amide. Reagents and reaction
conditions: iii) t-BuLi, THF, -78 °C iv) Weinreb amide (4a, 5a-c or 6a), -78 °C; v) H O; vi) HCl (1M in Et O), Et O.
2
2
2
A divergent synthesis has been adopted to access the desired products in good yields and in few
steps. Target compounds 7-30 were obtained via Weinreb ketone synthesis from key intermediates
4a, 5a-c and 6a, which in turn were prepared through: i) the Weinreb amide formation and ii) a
nucleophilic substitution (Scheme 1). Specifically, the starting acyl chlorides were easily converted
into the desired β and γ chloro Weinreb amides, using the amine N,O-dimethylhydroxyamine
hydrochloride (NODMHA), in the presence of a basic reagent, K CO . The reactions performed in a
2
3
7

ACCEPTED MANUSCRIPT
mixture of ether and water (in equal amount) were stirred for 24 h. A simple extraction provided
compounds 2-3 in excellent yields. This reaction was not applied for achieving intermediate 1, since
it is commercial available. Accordingly, the Weinreb amides were subjected to a nucleophilic
substitution, exploiting the chlorine as good leaving group. The reaction performed with the
corresponding secondary amine (a-c), needed 24 h and the presence of K CO as base to yield the
2
3
crude α, β and γ amino Weinreb amides 4a, 5a-c and 6a. An acid/base extraction was essential to
obtain the key intermediates, presenting 1 or 2 carbonaceous units, in modest/good yields.
Conversely, 6a required a purification through flash chromatography, since the starting material
conversion into the desired products furnished some side products. Lastly, the smooth bromo-
lithium exchange on the aryl bromide afforded the lithiated arene that, upon quenching with 4a, 5a-
c and 6a, gave the desired ketones 7-30 in good yields, after an adequate purification (Scheme 2).
Specifically, the corresponding aryl bromide were lithiated, in anhydrous THF at -78 °C with t-
butyl lithium. The subsequent addition of compounds 4a, 5a-c and 6a and quenching with H O led
2
to isolate crude compounds, which were purified through flash chromatography. All the potential
MTD molecules 7-30 were obtained in a sufficient amount and with the appropriate degree of
1
13
purity, as confirmed by IR, H-NMR, C-NMR and UPLC-MS analysis, as well as thermal
analysis, for the following biological investigations. Preliminary solid-state characterization by
DSC analysis revealed that all the compounds are crystalline anhydrous phases, with a well-defined
melting point followed by sample decomposition, with the only exception of 28 which crystallized
as solvatomorph.
2.2. Physicochemical and pharmacokinetic predictions
Molecular descriptors were computed to predict six physicochemical properties of each compound:
(a) partition coefficient (ClogP); (b) distribution coefficient at pH 7.4 (ClogD); (c) molecular weight
8

ACCEPTED MANUSCRIPT
(MW); (d) topological polar surface area (TPSA); (e) number of hydrogen-bond donors (HBDs);
and (f) acid dissociation constant (pKa). These parameters are the most representative features to
determine the ADME profile, as well as the ability of a compound in reaching the CNS [38].
According to Wager et al. model, which is a well-established procedure broadly employed in
academic and in industrial settings [39], a score – ranging from 0 to 1 - is attributed to each
property, applying a monotonic decreasing function for ClogP, ClogD, MW, pKa, and HBD,
whereas a hump function for TPSA (Table SI1). The summation of all these values gives access to a
final score (0-6), which can be defined as follows: i) 0-2, compound is unable to cross the BBB; ii)
2-5, compound may reach the CNS; iii) 5-6, compound effectively crosses the BBB [39].
Accordingly, we exploited this model to calculate the potential ability of compounds 7-30 in
reaching the CNS (Table 1). For comparative purposes, we compared the obtained results with
those of RRC-33 and Donepezil (Table 1, Table SI1), for which is known their good CNS
distribution.
The tabulated results show that only four compounds (18 and 21-23) possess a score higher than 5,
whereas the other ones possess values ranging from 2 to 5. Noteworthy, compounds 9, 14, 17, 19,
2
0 and 24 display a good score (> 4). Altogether these values reveal that all molecules 7-30 are able
to reach the CNS. Moreover, reference compounds (R)-RC-33 and Donepezil show good scores
2.57 and 4.41, respectively) and this behaviour is in line with their in vivo CNS distribution, which
was extensively confirmed by experimental data and clinical use.
(
2.3. Binding affinity, enzymatic studies, and structure-activity relationship (SAR) exploration
S1R and S2R binding site affinities of compounds 7-30 were measured through competition
experiments, using radioligands. The assay for S1R was performed using homogenized guinea pig
3
cerebral cortex membranes, in the presence of [ H]-(+)-pentazocine, as a potent and selective S1R
9

ACCEPTED MANUSCRIPT
radioligand. Nonspecific binding values were determined using non-radiolabeled (+)-pentazocine
and haloperidol in excess (see experimental section). Conversely, homogenized rat liver membranes
3
were adopted to evaluate the S2R binding values, employing [ H]-DTG - a nonselective S2R
radioligand - and, non-tritiated (+)-pentazocine to mask the S1R. Moreover, a high concentration of
non-tritiated DTG was used to determine nonspecific binding values. Compounds with high affinity
were tested twice. For compounds with low SR affinity, only one measure was performed. The SR
affinities of compounds 7-30 towards both S1R and S2R are presented in Table 1.
Similar to reference compound RRC-33, almost all compounds possess a weak affinity for S2R, but
only 12 compounds (8, 10, 13-14, 18, 20, 23-28) over 24 present a Ki S1R lower than 50 nM.
Altogether these results allow to draw some SAR considerations: i) the 4-methoxyphenyl ring
seems to play a key role in the interaction with S1R, in fact molecules 8, 14, 18 and 26 belonging to
the three different n series present a good Ki values (27, 11, 8 and 2.9 nM, respectively); ii) a longer
linker guarantees the S1R binding, as it is evident in the 4-benzylpiperidine derivatives (7-16 and
25-30), which follow 1 = 2 < 3 n-scale in the interaction with S1R; iii). the length of the linker can
limit the selectivity, indeed compounds belonging to the n = 3 series present a S2R/S1R ranging
from 1.6 to 6.8 and thus, they may be considered as pan-SR modulators. Lastly,
arylalkylamineketones possess a lower S1R affinity than (R)-RC-33 (Ki S1R = 1.8 nM), this
difference may be associated to the presence of a novel pharmacophoric element, which can limit
the interaction with the molecular target.
At the same time, we extended our study on the N-Methyl-D-Aspartate (NMDA) receptor, an
ionotropic glutamate receptor, constituted by two subunits GluN1 and GluN2. NMDA receptor
plays a relevant role in synaptic plasticity and synapse formation underlying memory and learning.
An alteration of this neurotransmitter pathway causes an accumulation of glutamate in the synaptic
terminations that promotes the GluN2 activation and thus, the long-term signal enhancement
1
0

ACCEPTED MANUSCRIPT
between two neurons. Moreover, the pathological triad, that involves mitochondrial dysfunction,
loss of neuronal structures integrity, and disruption of excitation–transcription coupling, may be
triggered. Accumulating evidence suggests that a negative modulation of this molecular target may
contribute in slowing the progression of neuropathies [40]. Relying on these considerations, we
evaluated the affinity of 7-30 towards GluN2 subunit of NMDA. Competitive binding assays on
membrane extracts of L cells (tk-), stably transfected with a vector containing the genetic
3
information of GluN1a and GluN2B subunits, were performed, using [ H]-ifenprodil, a selective
and potent GluN2 inhibitor radioligand. Compounds with high affinity were tested three times. For
compounds with low NMDA affinity, only one measure was performed. The results are reported in
Table 1 and they show that 7-30 possess a weak affinity towards NMDA receptor, with the
exception of compounds 8-10, 13, 25-29, which show a Ki < 150 nM. These results highlight that
the affinity towards NMDA receptor depends on the length of the spacer, in fact compounds
belonging to the n = 3 series present good affinity towards GluN2 (20-117 nM). Conversely,
molecules with 3 carbonaceous units between the aromatic portion and the aminic moiety are
endowed with a negligible affinity for the ionotropic glutamate receptor, with the exception of
compound 13 (Ki = 39 ± 2.1 nM). Lastly, molecules 7-12 show an intermediate behaviour,
presenting a moderate binding affinity. These results did not discourage us, since the affinity
towards NMDA receptor is not a necessary condition to define our compounds as MTDs. In fact, as
aforementioned S1R can modulate the activity of several client proteins and NMDA receptor is a
molecular target of S1R.
Compounds 7-30 were then tested for defining their potential to inhibit AChE. A
spectrophotometric procedure was adopted, applying suitable modifications to the original Ellman’s
method [41-42]. An initial screening was performed, employing the target compounds at a
concentration of 50 µM. The results of this assay are summarized in Table 1. Noteworthy, 10
1
1

ACCEPTED MANUSCRIPT
compounds (9-10, 14-15, 20, 25-27, 29-30) over 24 possess a percentage of inhabitation ≥ 60%,
whereas only five (7-8, 12, 21 and 24) can be considered inactive since they present a percentage of
inhibition < 20%.
In the light of these data, for the most promising compounds, showing at a concentration of 50 µM
an inhibition percentage more than 60%, the IC values were determined. Interestingly, all the
5
0
tested compounds possess an IC lower than 25 µM, with the only exception of 9 (IC = 32.88 ±
5
0
50
1.77 µM) (Table 1). In particular, 27 and 29 result the most promising AChE inhibitors, with an
IC of 7.64 ± 2.07 and 4.28 ± 0.23 µM, respectively.
5
0
From a structural point of view, the most interesting compounds are characterized by the presence
of 4-benzylpiperidine, as aminic portion, with the exception of compound 20 that is characterized
by a piperidine moiety. Moreover, molecules belonging to the n = 3 series (25-27, 29-30) show
greater anti-AChE effect; this inhibitory activity progressively decreases for β- and α-amino
ketones, respectively (9-10, 14-15 and 20). Basing on the obtained results we can conclude that both
the 4-benzylpiperidine portion and the linker length have a crucial role for the anti-AChE activity,
whereas no relation between the aromatic portion and the anti-AChE activity has been evidenced.
Interestingly, RRC-33, the S1R reference compound, demonstrates no inhibition property against
AChE (percentage of inhibition < 5%).
1
2

ACCEPTED MANUSCRIPT
Table 1. Scores related to the potential ability of compounds 7-30 in crossing the BBB. Binding affinities towards
NMDA, S1R and S2R and S1R selectivity (Ki S2R/Ki S1R). AchE inhibition activity and IC50 of the most
promising compounds.
%
of
Ki NMDA
Cmpd
Score
Ki S1R (nM) Ki S2R (nM)
S2R/S1R
inhibition (50
IC50 (µM)
(nM)
µM)
b
b
7
8
9
3.47
3.57
4.04
3.02
3.42
2.97
3.46
4.09
2.75
2.61
4.80
5.30
4.35
4.18
5.48
5.94
5.28
4.91
3.17
3.79
3.74
2.50
75 ± 3.6
27 ± 1.2
131 ± 15
27 ± 1.8
286
2300
2000
1500
802
30.7
74
215
5.65 ± 0.10
8.21 ± 0.06
75.50 ± 0.02
87.40 ± 6.39
48.24 ± 0.02
11.41± 0.04
42.30 ± 0.21
64.10 ± 5.58
85.50 ± 0.02
16 ± 1.2
11 ± 0.4
139 ± 11
>1000
>1000
39 ± 2.1
198
11.5
30
32.82 ± 1.77
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9.13 ± 2.24
b
1400
> 1000
5800
2400
6200
3900
749
4.9
-
b
b
340
16 ± 1.8
11 ± 2.2
211
363
218
29.4
23.4
4.4
256
81
22.02 ± 9.12
>1000
>1000
>1000
1.7 µM
>1000
>1000
>1000
>1000
>1000
723
18.55 ± 7.72
b
167
5
5.04 ± 6.69
a
a
b
b
171
7.9 ± 0.9
2.2 ± 0.7
15 ± 1.1
606
2100
178 ± 12
462
42.07 ± 0.04
45.20 ± 7.07
64.80 ± 0.35
10.75 ± 0.01
30.8
-
13.08 ± 6.31
b
>1000
b
b
b
301
>1000
>1000
-
59.70 ± 2.24
24.62 ± 0.05
12.20 ± 1.72
74.80 ± 3.56
80.01 ± 0.02
9.0 ± 0.7
2.9 ± 0.3
9.0 ± 0.5
31 ± 4.2
17 ± 2.2
24 ± 3.4
-
>1000
-
47 ± 2
5.2
6.8
2.5
2.4
39 ± 1.1
96 ± 2.7
49 ± 1.8
26 ± 0.9
13.07 ± 2.10
12.80 ± 1.79
111 ± 8
43 ± 1.9
58 ± 2.3
71.80 ± 1.60
7.64 ± 2.07
a
a
1
3

ACCEPTED MANUSCRIPT
2
3
9
0
2.73
2.32
2.57
4.41
-
68 ± 1.7
135 ± 11
1.8 ± 0.1
107 ± 10
704
1.6
5.2
25
20 ± 0.7
96 ± 2.6
>1000
-
92.60 ± 0.03
84.10 ± 0.02
4.82 ± 2.15
99.4 ± 0.01
-
4.28 ± 0.23
13.94 ± 2.15
a
RRC-33
Donepezil
Ifenprodil
45 ± 16
c
14.6
-
-
0.012 ± 0.01
-
125 ± 24
98 ± 34
0.8
10 ± 0.7
Values are expressed as mean ± SEM of three experiments. Compounds with high affinity were tested three times. For
compounds with low SR and NMDA affinity (> 150 nM), only one measure was performed.
a
Compounds were not evaluated for solubility issues.
Inhibition % < 60% at a concentration of 50 µM.
Data taken from [43]
b
c
2.4 Molecular dynamic simulations and docking studies
Root mean square deviation (RMSD) values of S1R entire trimeric complex, monomer and binding
pocket indicate that the protein structure is stable during MD simulations (RMSD of Cα atoms
below 2 Å). The S1R structure in complex with 4-IBP was stable and no conformational changes
occurred. The ligand formed H-bonds with the E172 for almost the entire simulation time (above
92%) but only in two binding pockets. In the third one, instead, the interaction was replaced by H-
bonds with water molecules. The most representative cluster (occurring 41% of the simulation time)
slightly differs from the others (average distance of 1.11 Å). However, the second cluster has also
occurrence of 35% and the RMSD between them is 1.34 Å. They differ from the starting
coordinates of 1.53 and 1.56 Å, respectively.
The most representative cluster was used to dock known (training set) and new (test set) S1R
ligands. In particular, the best docking results were obtained with rigid docking without any
constraints (AUC 0.78). However, not all active compounds were docked to the binding pocket: 4
out of 32 agonists were not successful, probably because they bind to a different conformation of
the receptor. Indeed, results did not improve by flexible docking (AUC 0.53), and there is no crystal
structure of the receptor in the active state that could be used to specifically investigate agonists.
1
4

ACCEPTED MANUSCRIPT
Nevertheless, all active agonists formed ionic interactions with E172 and D126 due to their
positively charged amine group. By contrast, low-ranked ligands lacked of these key residue
contacts. Moreover, further amino acids (W89 and F107) were often involved in protein-ligand
interactions: for instance, these residues were close to the protonated amine and eventually formed
π-cation interactions. Furthermore, Y120 and H154 can form π-π stacking with ligand aromatic
rings.
The docking pose of RRC-33 shows that the positively charged nitrogen atom forms ionic
interactions with E172 and D126, and cation-π interactions with F107 (Figure 3). Moreover,
hydrophobic contacts (not displayed) occur between the aromatic rings and surrounding amino
acids (M93, L95, L105). The test set of new derivatives (7-30) was docked and all molecules
conserved key interactions with the receptor as for the training set. Nevertheless, larger molecules
with four rings (such as 4-biphenyl derivatives 12, 16 and 30) did not fit well into the binding
pocket in comparison with smaller compounds. Indeed, either the biphenyl moiety clashed with
Y206 or the benzyl ring did not have enough space on the opposite side of the pocket. In contrast,
molecules 17, 21 and 22 that have only two rings lacked important hydrophobic contacts resulting
in low binding affinity. In conclusion, compounds with three rings, which conserved H-bond and
hydrophobic interactions, seem to be the most interesting ones. In Figure 3, compounds 10 and 20
are reported by way of example.
All new derivatives were docked also to AChE. Flexible docking on AChE X-ray structure, both
including and excluding water molecules, gave the best results. AUC was 0.71 in both cases,
indicating that the model was able to distinguish between active compounds and decoys. The top-
ranked ligands formed π-π and π-cation interactions with W86, W286, Y337 and Y341 residues of
the binding pocket, as in the donepezil-bound crystal structure (Figure 3). Also, the H-bond with the
backbone of F295 was mostly conserved. In particular, the presence of the carbonyl enhanced the
1
5

ACCEPTED MANUSCRIPT
binding when n was equal to 3 carbon atoms (compounds 25-30), which is the optimal distance to
keep the ionic interaction of the amine and the H-bond to F295. Instead, not all compounds formed
hydrogen bond interactions with the hydroxyl group of Tyr amino acids (compound 20). For
instance, the docking pose of compound 20 showed that the charged amine could fit into a deeper
binding pocket, where it could form interactions with E202 (Figure 3). This was possible only for
unsubstituted piperidine rings. Furthermore, the naphthalene ring (compounds 10 and 29) enhanced
the π-π stacking with W286.
In summary, docking poses suggested that the charged amine plays a key role not only for binding
to negatively charged amino acids, but also to aromatic residues through cation-π interactions. The
binding is strengthened by the presence of aromatic rings, which form hydrophobic and π-π
interactions with both S1R and AChE ligand pockets. Several compounds among our new set of
derivatives showed protein-ligand interaction patterns which are similar to reference compounds
RRC-33 and donepezil, as shown in Figure 3, where the docking poses of 10 and 20 are reported.
1
6

ACCEPTED MANUSCRIPT
Figure 3. Docking poses of reference compounds (RRC-33 and donepezil) and of 10 and 20 on S1R (on the left side)
and AChE (on the right side) binding pockets. Hydrogen atoms are not displayed. Ligands and key residues are shown
as sticks.
2.5. Antioxidant profile and neurotrophic activity
Basing on preliminary biological investigations compounds 8, 10, 13-14, 18-20, 23-28 with either
S1R affinity or both good S1R affinity and relevant AChE inhibitory activity were selected for
further evaluations.
1
7

ACCEPTED MANUSCRIPT
2
.5.1. Free Radical Scavenging (FRS) activity
FRS activity of 8, 10, 13-14, 18-20, 23-28 was determined by 2,2-diphenyl-1-picrylhydrazyl
DPPH) assay, and the results were compared to those of reference molecules RRC-33, Donepezil
(
and Cur. Stock solutions in EtOH of the tested compounds were prepared (5.0 mM). The DPPH
absorbance was spectrophotometrically monitored at 515 nm and at a concentration of 465 µM;
inhibition percentages are reported in Table SI2. These preliminary results demonstrated that
compounds 13-14, 18-20, 23-28, as well as RRC-33 and Donepezil do not display significant
antioxidant activity (values ranging from 2.8% up to 32%). Conversely, compounds 8 and 10 could
be considered promising antioxidant molecules, since they show an FRS activity (> 65%),
comparable with the widely recognised Cur antioxidant activity. Accordingly, only molecules
belonging to the n = 1 series possess intrinsic antioxidant properties, probably due to the methylene
spacer adjacent to both carbonyl and amine moieties which stabilize the radical centre (Scheme 3)
[
44].
Scheme 3. Proposed mechanism for the radical scavenging activity of n = 1 series compounds.
In the light of these data, the IC50 calculation of 8 and 10 was performed at four different
concentrations (ranging from 465 to 45 µM).
1
8

ACCEPTED MANUSCRIPT
2.5.2 Cell viability and 2’,7’-dichlorofluorescein diacetate (DCFDA) assays.
Toxicity of 8, 10, 13-14, 18-20, 23-28 was assessed in SH-SY5Y neuronal cell line, at different
concentrations, ranging from 10 to 50 µM. The cell viability was evaluated through 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay,
after exposing cells to these compounds for 24 h and 48 h. The results are reported in Table 2. The
tested compounds show a dose-dependent effect on SH-SY5Y cell lines (Homo Sapiens bone
marrow neuroblastoma) at different exposure times. Moreover, these values show that almost all
molecules do not exhibit cytotoxic activities, with the exception of compound 23, which decreases
cell viability even at a 10 µM concentration (77%). We speculated that this countertrend could be
due to the presence of morpholine as aminic moiety. Furthermore, compounds belonging to the n =
2
series and presenting a piperidine portion show cytotoxic properties when they are administered
for 48 h at a concentration of 25 µM and 50 µM. Noteworthy, molecules with a 9-atoms linker
between the two aromatic portions do not alter the cell viability even if they are employed at high
doses. Lastly, for comparative purpose, RRC-33, Donepezil and Cur were subjected to MTS test.
Also in this case, the results indicate the lack of toxicity. Considering the results obtained so far
(good BBB score, good S1R affinity, high AChE inhibition percentage) compounds 10, 14 and 20
were selected for a second screening aimed at calculating the IC value, by testing them at 6
5
0
different concentrations (ranging from 1 µM to 100 µM) for 24 and 48 h. Compounds 10 and 20
showed an IC higher than 20 µM (24 h = 52 and 37, respectively; 48 h = 57 and 22, respectively).
5
0
The viability trend is reported in Figure 4.
1
9

ACCEPTED MANUSCRIPT
Table 2. Viability test at 24 h and 48 h.
2
4 h
48 h
Cmpd
1
0 µM (%)
25 µM (%)
50 µM (%)
87 ± 0.03
81.3 ± 0.03
49 ± 0.01
56.5 ± 0.05
63 ± 0.01
65.4 ± 0.01
64 ± 0.05
49 ± 0.01
83.3 ± 0.08
87 ± 0.01
84.4 ± 0.01
98 ± 0.01
86 ± 0.07
86 ± 0.04
10 µM (%)
85.5 ± 0.12
89.5 ± 0.02
106 ± 0.17
97.4 ± 0.02
104 ± 0.08
70.3 ± 0.02
95.9 ± 0.03
79 ± 0.04
25 µM (%)
69.3 ± 0.03
77.6 ± 0.04
44 ± 0.04
50.2 ± 0.05
49.4 ± 0.06
42 ± 0.01
49.3 ± 0.04
38 ± 0.06
66 ± 0.03
95 ± 0.05
87 ± 0.04
85.6 ± 0.03
57 ± 0.01
77.1 ± 0.01
50 µM (%)
52.5 ± 0.02
48.4 ± 0.01
36 ± 0.04
8
103 ± 0.03
98.4 ± 0.05
1
0
3
4
8
9
0
3
4
5
6
7
8
107 ± 0.05
93 ± 0.01
104.2 ± 0.04
48 ± 0.01
1
1
1
1
2
2
2
2
2
2
2
99.9 ± 0.01
119 ± 0.02
98.6 ± 0.04
104 ± 0.06
77 ± 0.04
63.9 ± 0.04
86 ± 0.03
44.7 ± 0.01
43.4 ± 0.01
40 ± 0.03
66.4 ± 0.01
95.4 ± 0.01
48 ± 0.01
44.7 ± 0.03
37 ± 0.04
100 ± 0.01
100 ± 0.04
100 ± 0.01
100 ± 0.03
80.2 ± 0.04
100 ± 0.03
73 ± 0.01
100 ± 0.06
96 ± 0.02
62.4 ± 0.01
54 ± 0.02
100 ± 0.02
100 ± 0.04
100 ± 0.01
86 ± 0.01
97.3 ± 0.02
94.8 ± 0.05
77 ± 0.01
61 ± 0.01
65 ± 0.01
61 ± 0.01
RRC-33
Donepezil
Curcumin
100 ± 0.03
84 ± 0.01
57.3 ± 0.01
64.4 ± 0.02
59 ± 0.02
100 ± 0.01
100 ± 0.01
99.3 ± 0.02
76.5 ± 0.01
94.7 ± 0.01
71.5 ± 0.01
94.3 ± 0.01
100 ± 0.01
80.4 ± 0.01
58.7 ± 0.01
Figure 4. Viability trends of compounds 10, 14 and 20, after 24 and 48 h of exposure.
2
0

ACCEPTED MANUSCRIPT
The major intracellular ROS sources are complex I (NADH dehydrogenase ubiquinone-ubiquinone
reductase) and complex III (ubiquinone cytochrome C reductase), both of them take part to the
mitochondrial electron transport chain [45]. A low ROS level plays a crucial role in cellular
pathways. Nevertheless, when the ROS concentration increases and overcomes the cellular
antioxidant machinery, it can induce macromolecular damages (i.e. interaction with the DNA,
proteins and lipids) and compromise the thiol redox circuits, promoting aberrant molecular cascades
[
46]. Considering that oxidative stress, induced by the increase of ROS intracellular concentration,
is a driving issue in neurodegenerative diseases, we evaluated the ability of 10, 14 and 20 and
reference compounds in reducing ROS within SH-SY5Y cells, after an exposure to oxidative
damages mediated by H O . The assay provided the employment of DCFDA reagent, which
2
2
diffuses within the cell and undergoes esterase-mediated de-acethylation. The so-obtained product
is then subjected to oxidation by ROS, giving access to 2’,7’-dichlorofluorescein, a high fluorescent
molecule with adsorption and emission wavelength of 495 and 529 nm, respectively.
The compounds were tested at a concentration of 10 µM in the presence of H O (180 µM) for 24 h.
2
2
In parallel experiments, we evaluated the cell viability by MTS assay, to verify the lack of
cytotoxicity under the same experimental conditions (Figure 5). The results reveal that all
compounds show a safe profile and thus they can be evaluated through fluorescent assay. As
reported in Figure 5, Cur possesses the ability to reduce ROS up to a 0%, whereas donepezil and
RRC-33 show no antioxidant properties. On the basis of our data, 10 and 20 are able to promote
ROS reduction up to 43% and 18% respectively and thus can be considered antioxidant molecules,
whereas 14 resulted ineffective and it was abandoned.
2
1

ACCEPTED MANUSCRIPT
Figure 5. ROS percentage evaluation after administration of compounds 10, 14, 20, Cur, Donepezil and (R)-RC-33.
2.5.3. Neurotrophic activity
Basing on our previous experience, we evaluated the neurotrophic effect of the most interesting
compounds 10 and 20, evaluating the neurite differentiation and elongation by using the
straightforward model of Dorsal Root Ganglia (DRG) [29]. Briefly, DRG explants from E15
Sprague – Dawley rat embryos are able to sprout neurites when cultured with pro-differentiation
agents, such as Nerve Growth Factor (NGF). The neurite elongation is promoted by neurotrophic
drugs and blocked or slowed up by neurotoxic agents. Compounds 10 and 20 were tested at
different concentration, after 24 and 48 hours of exposure (Figure 6 and Figures SI1-SI4).
Interestingly, both compounds showed neurotrophic effect. In particular, best results were obtained
for compound 10 at the concentration of 2.5µM, starting from 24 hours of exposure and leading to
an increase of the neurite length of about 15% with respect to the untreated control DRG. For
compound 20, the concentrations 5µM and 2.5µM resulted to be the most effective, both after 24
hours and after 48 hours of exposure, with an increase of neurite length of about 20% with respect
to control DRG. For both compounds, none concentrations resulted neurotoxic, except at the
concentration 10µM, which showed after 24 hours of exposure an only transient neurotoxic effect,
2
2

ACCEPTED MANUSCRIPT
being disappeared after 48 hours. The present results demonstrated the pro-differentiation properties
of compounds 10 and 20, thus confirming their neurotrophic effect.
Figure 6. Evaluation of Neurotrophic activity of compounds 10 and 20, after 24h and 48h of exposure.
3
. Conclusion
In the present paper we discovered dual S1R modulators/AChE inhibitors endowed with antioxidant
and neurotrophic properties. The adopted medicinal chemistry approach led to a novel series of
compounds sharing the pharmacophoric requirements for interacting with both S1R and AChE, as
confirmed by the molecular modelling study. Within the whole series, three compounds resulted
endowed with considerable S1R affinity, good selectivity over S2R and AChE inhibition activity.
2
3

ACCEPTED MANUSCRIPT
Their effect on neuroblastoma cell (SH-SY5Y) survival was evaluated and the ROS damage was
mimicked by exposing SH-SY5Y cells to H O . The results highlighted that only compounds 10
2
2
and 20 decrease ROS production, thus ameliorating the cellular survival and therefore, they were
selected for further experimental investigations. To this purpose, their effect on neurite outgrowth in
DRG in vitro model was assessed and their neurotrophic role confirmed. Of note, the tested
compounds did not show any cytotoxic effect at the concentration assayed.
Overall, our hit compounds 10 and 20 combine in the same molecule the S1R modulation and the
AchE inhibition, together with cell damage protection and neurotrophic properties. It is important to
underline that RRC-33 has no anti-AChE effect, whereas both RRC-33 and donepezil show no
antioxidant properties in our cellular model of ROS damage. Importantly, both compounds maintain
the high affinity toward S1R.
Both 10 and 20 compounds will undergo a structure optimization program for the development of
therapeutic candidates for neurodegenerative diseases treatment.
4
. Materials and Methods
4.1. General remarks
Reagents and solvents for synthesis were obtained from Aldrich (Italy). Solvents were purified
according to the guidelines in Purification of Laboratory Chemicals. Thermal characterization was
performed by using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)
e
e
on a Mettler Star System equipped with a DSC 821 and TGA-DSC1 cell, respectively. Samples
were weighed and placed in sealed aluminum (DSC) or alumina (TGA) pans. The samples were
heated from 30 °C to 300 °C with a scanning rate of 10 °C/min under nitrogen atmosphere. The
instruments were previously calibrated with Indium as standard reference. For FT-IR analysis a
Spectrum One Perkin Elmer spectrophotometer equipped with a MIRacle™ ATR device was used.
-1
-1
The IR spectra were scanned over wavenumber range of 4000-650 cm with a resolution of 4 cm .
2
4

ACCEPTED MANUSCRIPT
Analytical thin-layer-chromatography (TLC) was carried out on silica gel precoated glass-backed
plates (Fluka Kieselgel 60 F254, 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 Sigma Aldrich). Proton
nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500, 400 and 200
spectrometers operating at 500 MHz, 400 MHz and 200 MHz, respectively. Proton chemical shifts
(δ) are reported in ppm with the solvent reference relative to tetramethylsilane (TMS) employed as
the internal standard (CDCl , δ = 7.26 ppm). The following abbreviations are used to describe spin
3
multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad signal, dd =
1
3
doublet-doublet, td = triplet-doublet. The coupling constant values are reported in Hz. C NMR
spectra were recorded on a 500 MHz and 400 MHz spectrometers operating at 125 MHz, 100 MHz
and 50 MHz, respectively, with complete proton decoupling. Carbon chemical shifts (δ) are
reported in ppm relative to TMS with the respective solvent resonance as the internal standard
(
CDCl , δ = 77.23 ppm).
3
UPLC-UV-ESI/MS analyses 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 Phenyl (ABP) (50 x 2.1 mm, 1.7 µm) or
ACQUITY BEH Shield (ABS) (100 x 2.1 mm, 1.7 µm) columns, 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 1.5 minutes, return to the initial conditions in 0.2 minutes) at a flow rate of 0.5 mL min-
1. All of the final compounds had 95% or greater purity.
4.2. General procedure for the preparation of compounds 2-3
2
5

ACCEPTED MANUSCRIPT
To an aqueous solution of K CO (2.0 equiv.) was added Et O and N,O-dimethylhydroxyamine
2
3
2
hydrochloride (1.5 equiv.). The resulting mixture was cooled at 0 °C and after 2 minutes the
corresponding acyl chloride (1.0 equiv.) was added dropwise. The reaction was stirred overnight
until the room temperature was reached. The reaction mixture was extracted with Et O (2 x 5 mL)
2
and washed with water (5 mL) and brine (10 mL). The organic phase was dried (anhydrous
Na SO ), filtered and, after removal of the solvent under reduced pressure, the pure compounds 2-3
2
4
were obtained.
3-chloro-N-methoxy-N-methylpropanamide (2) By following the General Procedure, starting from
3
-chloropropanoyl chloride (127 mg, 1.00 mmol, 1.0 equiv), K CO (276 mg, 2.0 mmol, 2.0 equiv),
2
3
N,O-dimethylhydroxyamine hydrochloride (146 mg, 1.5 mmol, 1.5 equiv), H O (3 mL) and Et O (3
2
2
mL), the desired product was obtained in a quantitative amount (> 99.9 %) (151 mg) as a bright
1
yellow oil. H NMR (500 MHz, CDCl ) δ: 3.80 (t, J = 6.9 Hz, 2H, CH CH Cl), 3.70 (s, 3H,
3
2
2
13
NOCH ), 3.19 (s, 3H, NCH ), 2.91 (t, J = 6.7 Hz, 2H, CH CH CO). C NMR (125 MHz, CDCl ) δ:
3
3
2
2
3
170.8, 61.4, 39.2, 35.0, 32.0.
4-chloro-N-methoxy-N-methylbutanamide (3) By following the General Procedure, starting from 4-
chlorobutanoyl chloride (141 mg, 1.00 mmol, 1.0 equiv), K CO (276 mg, 2.0 mmol, 2.0 equiv),
2
3
N,O-dimethylhydroxyamine hydrochloride (146 mg, 1.5 mmol, 1.5 equiv), H O (3 mL) and Et O (3
2
2
1
mL), the desired product was obtained in 92% (152 mg) as a bright yellow oil. H NMR (500 MHz,
CDCl ) δ: 3.70 (s, 3H, NOCH ), 3.63 (t, J = 6.4 Hz, 2H, CH CH Cl), 3.18 (s, 3H, NCH ), 2.62 (t, J
3
3
2
2
3
13
=
6.7 Hz, 2H, CH CH CO), 2.11 (m, J = 6.9 Hz, 2H, CH CH CH ). C NMR (125 MHz, CDCl ) δ:
2 2 2 2 2 3
173.3, 61.3, 44.7 32.1, 28.6, 27.1.
4.3. General procedure for the preparation of compounds 4a, 5a-c and 6a
2
6

ACCEPTED MANUSCRIPT
To a solution of Weinreb amide (1.0 equiv.) in ACN, the corresponding amine (1.0 equiv.) and
K CO (1.5 equiv.) were added. The mixture was stirred overnight, at room temperature. After
2
3
removal of the solvent under reduced pressure, the crude was extracted with DCM (3 x 5 mL) and
washed with water (5 mL) and brine (10 mL). In the case of compound 4a, this work-up was
sufficient to obtain the pure compound. Conversely, an acid (pH = 3-4) / base (pH = 8-9) work-up
was required for 5a-c and 6a, the combined organic phases were dried (anhydrous Na SO ), filtered
2
4
and, evaporated under vacuum to get the desired compounds. The crude compound 6a was further
purified through flash chromatography (silica gel) to afford pure compound 6a.
2-(4-benzylpiperidin-1-yl)-N-methoxy-N-methylacetamide (4a) By following the General Procedure,
starting from 2-chloro-N-methoxy-N-methylacetamide (138 mg, 1.00 mmol, 1.0 equiv), K CO3
2
(207 mg, 1.5 mmol, 1.5 equiv), 4-benzylpiperidine (175 mg, 176 mL 1.0 mmol, 1.0 equiv) and
ACN (7 mL), the desired product was obtained in quantitative amount (276 mg) as a bright yellow
1
oil. H NMR (500 MHz, CDCl ) δ: 7.27 (m, 2H, Ar), 7.18 (t, 1H, J = 7.5 Hz, Ar), 7.14 (d, 2H, J =
3
7
6
4
6
.3 Hz, Ar), 3.69 (s, 3H, NOCH ), 3.29 (s, 2H, CH N), 3.16 (s, 3H, NCH ), 2.96 (d, 2H, Pip-2, Pip-
3 2 3
), 2.53 (d, 2H, CH Pip-4), 2.07 (t, 2H, Pip-2, Pip-6), 1.62 (d, 2H, Pip-3, Pip-5), 1.51 (m, 1H, Pip-
2
1
3
), 1.40 (q, 2H, Pip-3, Pip-5). C NMR (125 MHz, CDCl ) δ: 171.4, 140.5, 129.1, 128.1, 125.7,
3
1.3, 58.5, 54.1, 43.1, 37.5, 32.1, 32.0.
3-(4-benzylpiperidin-1-yl)-N-methoxy-N-methylpropanamide (5a) By following the General
Procedure, starting from 3-chloro-N-methoxy-N-methylpropanamide (152 mg, 1.00 mmol, 1.0
equiv), K CO (207 mg, 1.5 mmol, 1.5 equiv), 4-benzylpiperidine (175 mg, 176 mL, 1.0 mmol, 1.0
2
3
equiv) and ACN (7 mL), the desired product was obtained in quantitative amount (290 mg) as a
1
bright yellow oil. H NMR (500 MHz, CDCl ) δ: 7.27 (m, 2H, Ar), 7.18 (t, 1H, J = 7.4 Hz, Ar),
3
2
7

ACCEPTED MANUSCRIPT
7
2
2
1
.13 (d, 2H, J = 7.4 Hz, Ar), 3.68 (s, 3H, NOCH ), 3.16 (s, 3H, NCH ), 2.95 (d, 2H, Pip-2, Pip-6),
3
3
.72-2.70 (m, 4H, CH N, CH CH N), 2.53 (d, 2H, CH Pip-4), 2.00 (t, 2H, Pip-2, Pip-6), 1.65 (d,
2
2
2
2
1
3
H, Pip-3, Pip-5), 1.54 (m, 1H, Pip-4), 1.36 (q, 2H, Pip-3, Pip-5). C NMR (125 MHz, CDCl ) δ:
3
73.1, 140.5, 129.1, 128.1, 125.8, 61.3, 53.8, 53.6, 43.0, 37.6, 37.1, 32.1, 29.5.
N-methoxy-N-methyl-3-(piperidin-1-yl)propanamide (5b) By following the General Procedure,
starting from 3-chloro-N-methoxy-N-methylpropanamide (152 mg, 1.00 mmol, 1.0 equiv), K CO3
2
(207 mg, 1.5 mmol, 1.5 equiv), piperidine (85 mg, 99 mL, 1.0 mmol, 1.0 equiv) and ACN (7 mL),
1
the desired product was obtained in quantitative amount (200 mg) as a bright yellow oil. H NMR
(
400 MHz, CDCl ) δ: 3.76 (s, 3H, NOCH ), 3.24 (s, 3H, NCH ), 2.76 (s, 4H, CH N, CH CH N),
3 3 3 2 2 2
1
3
2
1
.52 (t, 4H, Pip-2, Pip-6), 1.70-1.56 (m, 6H, Pip-3, Pip-4, Pip-5). C NMR (100 MHz, CDCl ) δ:
3
75.2, 61.3, 54.5, 54.2, 29.7, 25.9, 24.2.
N-methoxy-N-methyl-3-morpholinopropanamide (5c) By following the General Procedure, starting
from 3-chloro-N-methoxy-N-methylpropanamide (152 mg, 1.00 mmol, 1.0 equiv), K CO (207 mg,
2
3
1.5 mmol, 1.5 equiv), morpholine (87 mg, 87 mL, 1.0 mmol, 1.0 equiv) and ACN (7 mL), the
1
desired product was obtained in 46 % (93 mg) as a bright yellow oil. H NMR (400 MHz, CDCl )
3
δ: 3.78-3.73 (m, 4H, NCH CH O), 3.73 (s, 3H, NOCH ), 3.22 (s, 3H, NCH ), 2.81-2.63 (m, 4H,
2
2
3
3
1
3
CH CH N, CH CH N), 2.56-2.51 (t, 4H, NCH CH O). C NMR (100 MHz, CDCl ) δ: 175.3, 66.9,
2
2
2
2
2
2
3
61.3, 53.9, 53.6, 32.2, 29.4.
4-(4-benzylpiperidin-1-yl)-N-methoxy-N-methylbutanamide (6a) By following the General
Procedure, starting from 4-chloro-N-methoxy-N-methylbutanamide (166 mg, 1.00 mmol, 1.0
equiv), K CO (207 mg, 1.5 mmol, 1.5 equiv), 4-benzylpiperidine (175 mg, 176 mL 1.0 mmol, 1.0
2
3
2
8