Computer-assisted design, synthesis, binding and cytotoxicity assessments of new 1-(4-(aryl(methyl)amino)butyl)-heterocyclic sigma 1 ligands

Article abstract of DOI:10.1016/j.ejmech.2016.06.001

In this work we applied a blend of computational and synthetic techniques with the aim to design, synthesize, and characterize new σ1 receptor (σ1R) ligands. Starting from the structure of previously reported, high-affinity benzoxazolone-based σ1 ligands, the three-dimensional homology model of the σ1R was exploited for retrieving the molecular determinants to fulfill the optimal pharmacophore requirements. Accordingly, the benzoxazolone moiety was replaced by other heterocyclic scaffolds, the relevant conformational space in the σ1R binding cavity was explored, and the effect on σ1R binding affinity was ultimately assessed. Next, the compounds designed in silico were synthesized, and their affinity and selectivity toward σ₁and σ₂receptors were tested. Finally, a representative series of best σ1R binders were assayed for cytotoxic activity on the SH-SY5Y human neuroblastoma cell line. Specifically, the new 4-phenyloxazolidin-2-one derivatives 2b (i.e., (R)-2b and (S)-2b) emerged as potential leads for further development as σ1R agents, as they were found endowed with the highest σ1R affinity (K_iσ1 values in the range 0.95–9.3?nM), and showed minimal cytotoxic levels exhibited in the selected, cell-based test, in line with a σ1R agonist behavior.

Full text of DOI:10.1016/j.ejmech.2016.06.001

Accepted Manuscript
Computer-assisted design, synthesis, binding and cytotoxicity assessments of new 1-
(4-(aryl(methyl)amino)butyl)-heterocyclic sigma 1 ligands
Daniele Zampieri, Luciano Vio, Maurizio Fermeglia, Sabrina Pricl, Bernhard Wünsch,
Dirk Schepmann, Maurizio Romano, Maria Grazia Mamolo, Erik Laurini
PII:
S0223-5234(16)30478-0
10.1016/j.ejmech.2016.06.001
EJMECH 8662
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 April 2016
Revised Date: 30 May 2016
Accepted Date: 1 June 2016
Please cite this article as: D. Zampieri, L. Vio, M. Fermeglia, S. Pricl, B. Wünsch, D. Schepmann, M.
Romano, M.G. Mamolo, E. Laurini, Computer-assisted design, synthesis, binding and cytotoxicity
assessments of new 1-(4-(aryl(methyl)amino)butyl)-heterocyclic sigma 1 ligands, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.001.
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Graphical Abstract
New 1-(4-(aryl(methyl)amino)butyl)-heterocyclic derivatives were designed and synthesized in
order to evaluate their affinity towards σ receptors by radioligand binding assays. Moreover, in
order to investigate their cytotoxic activity, a functional assay was performed.
Some of these derivatives showed a remarkable affinity and selectivity towards σ1 receptors and
an interesting cytotoxic profile.

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Computer-assisted design, synthesis, binding and cytotoxicity
assessments of new 1-(4-(aryl(methyl)amino)butyl)-heterocyclic sigma
1 ligands.
Daniele Zampieria*, Luciano Vioa†, Maurizio Fermegliab, Sabrina Priclbc*, Bernhard Wünschd,
Dirk Schepmannd, Maurizio Romanoe, Maria Grazia Mamoloa#, Erik Laurinib#
a Department of Chemistry & Pharmaceutical Sciences, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy
b Molecular Simulation Engineering (MOSE) Laboratory, DEA, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy
c
National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-DEA, University of
Trieste, Trieste - Italy
d Department of Pharmaceutical and Medicinal Chemistry, Corrensstrasse 48, University of Münster, 48149 Münster, Germany.
e Department of Life Sciences, Via Valerio 28/1, University of Trieste, 34127 Trieste, Italy
†”This work is dedicated to the fond memory of Prof. Luciano Vio, collegue and friend,
unexpectedly deceased on May 5, 2016.”
Abstract. In this work we applied a blend of computational and synthetic techniques with the
aim to design, synthesize, and characterize new σ1 receptor (σ1R) ligands. Starting from the
structure of previously reported, high-affinity benzoxazolone-based σ1 ligands, the three-
dimensional homology model of the σ1R was exploited for retrieving the molecular
determinants to fulfill the optimal pharmacophore requirements. Accordingly, the
benzoxazolone moiety was replaced by other heterocyclic scaffolds, the relevant
conformational space in the σ1R binding cavity was explored, and the effect on σ1R binding
affinity was ultimately assessed. Next, the compounds designed in silico were synthesized,
and their affinity and selectivity toward σ1 and σ2 receptors were tested. Finally, a
representative series of best σ1R binders were assayed for cytotoxic activity on the SH-SY5Y
human neuroblastoma cell line. Specifically, the new 4-phenyloxazolidin-2-one derivatives 2b
(i.e., (R)-2b and (S)-2b) emerged as potential leads for further development as σ1R agents, as
they were found endowed with the highest σ1R affinity (Kiσ1 values in the range 0.95-9.3
nM), and showed minimal cytotoxic levels exhibited in the selected, cell-based test, in line
with a σ1R agonist behavior.

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Keywords: sigma receptors; binding affinity; cytotoxicity; molecular modeling.
_____________________________________________________________________________________________
*Corresponding author; Phone: +39 040 5587858; e-mail: dzampieri@units.it; Phone: +39 040 5583750; e-mail:
sabrina.pricl@di3.units.it
#These authors contributed equally to this work
Introduction
After their initial misclassification as an opioid receptor subtype [1], sigma receptors (σ-Rs)
actually represent a non-opioid, non-phencyclidine but haloperidol-sensitive receptor family [2].
To date, at least two distinct σ-Rs subtypes - designated as σ1 and σ2 [3] - have been
pharmacologically characterized [4-6]. The amino acid sequence of the σ1 receptor (σ1R)
subtype was determined by purification and cloning from several animal species including man.
Interestingly, this receptor shows no homology with any mammalian protein, whilst it exhibits
significant homology (30%) with sterol C8- C7 isomerase from fungi [7,8]. The σ1R has been
associated with many diseases including stroke, cocaine addiction, pain, cancer, and
neurodegenerative pathologies [9].
On the other hand, the σ2 receptor subtype has not been cloned yet, although its molecular
weight has been determined as approximately 21.5 kDa. [7] It has been proposed that this σ-R
subtype is involved in cellular apoptotic response [10,11], and in the release of Ca2+ through an
IP3-independent manner [12-14].
To date, neither σ1R endogenous ligands have been definitively established [15], nor the role of
all σ1R ligands has been unequivocally established in terms of their agonistic or antagonistic
receptor activity [9]. Ligands displaying preferential affinity for the σ1R subtype - and currently
classified as σ1R agonists - are dextrorotatory benzomorphans such as (+)-pentazocine and (+)-
N-allylnormetazocine (NANM, aka SKF-10,047), whereas haloperidol (categorized as σ1R
antagonist) and 1,3-di-(2-tolyl)guanidine (DTG) exhibit high affinity toward both receptor
subtypes [15]. Given its very low affinity for the σ2 receptors, (+)-pentazocine is now the “gold
standard” molecule used, in its tritiated form, to label σ1 receptors.
In our longstanding research in the field of σ1R ligands design and discovery, in 2009 we
synthesized the series of benzoxazolone derivatives 1 (Figure 1) characterized by high affinity
and selectivity towards σ1receptor subtype [16,17].

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Figure 1. Structure of lead compounds 1a-k
In particular, compounds 1a (H) and 1d (4-Cl) showed the most interesting σ1R affinity within
this molecular series, with Kiσ1 values of 2.6 nM and 7.1 nM and selectivity ratio (Kiσ2/Kiσ1) of
46.2 and 5.1, respectively. Encouraged by these results, we then selected some of these
molecules as training/test set compounds for the construction of a three-dimensional (3D)
pharmacophore model for σ1R binding [17], and the subsequent original development of a σ1R
3D homology model [18], extensively validated in successive works [19-28]. The information
retrieved from the combined application of 3D pharmacophore modeling and molecular
dynamics (MD)-based docking and scoring calculations using the 3D σ1R homology model
allowed us to fully characterize the network of intermolecular interactions responsible for the
potency of compounds 1 as σ1R binders.
In the quest of designing, synthesizing, and testing a second generation of effective σ1R binders,
in this work we exploited this wealth of information at hand and explored the effect of the
replacement of the benzoxazolone moiety in compounds 1 on σ1R affinity. Accordingly, based
upon modeling design and predictions,
a
new series of substituted 1-(4-
(aryl(methyl)amino)butyl)-heterocyclic derivatives 2-11 (Figure 2) were synthesized and tested
for σ1R binding affinity. Finally, the most promising compounds of this new molecular set were
preliminary assessed for their activity profile (agonist/antagonist behavior) in a cell-based assay.

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Figure 2. Structure of new sigma ligands 2-11a,b
2. Results and discussion
2.1 Computer-Aided Design of new 1-(4-(aryl(methyl)amino)butyl)- heterocyclic derivatives
Previous simulations performed on compounds 1a and 1d in complex with the 3D homology
model of the σ1R [18,19] led to the identification of a specific map of interaction for each
functional group of these molecules, and to the recognition of the protein residues mainly
involved in ligand binding. Yet, a detailed quantification of the interaction energies and the
derivation of the relevant interaction spectra for these compounds – instrumental to complete the
information set required for the design of new derivatives based on 1 - were not carried out at
that time. Thus, the optimized structure of the benzoxazolone derivatives 1a and 1d were re-
docked in the binding pocket of the 3D σ1R model, and the corresponding protein/ligand free
energy of binding (∆Gbind) were calculated via the MM/PBSA (Molecular Mechanics/Poisson-
Boltzmann Surface Area) approach [29], yielding values in agreement with the previous report
[17], as expected (∆Gbind= -10.59 kcal/mol for 1a, and -10.68 kcal/mol for 1d, respectively,
Table SI1). Taking compound 1d as a proof-of-concept, the analysis of the equilibrated portion

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of the respective MD trajectory (Figure 3A) revealed in detail the qualitative pattern of the
interactions between this compound and the σ1R, i.e., the engagement of the basic amine
nitrogen of 1d in a persistent salt bridge with the COO- group of D126, and a stable hydrogen
bond between the oxygen atom of the benzoxazolone ring of 1d and the –NH group of the
peptidic bond linking residues T151 and V152 of the receptor. Moreover, the MD simulation
accounts for the presence of stabilizing π−π interactions between the p-chlorobenzyl ring of 1d
and the side chains of the protein amino acids W121 and R119 (see also Figure SI1 in the
supplementary data). Finally, the aromatic fragment of the benzoxazolone moiety and the butyl
linker chain of 1d are nestled in an hydrophobic pocket lined by the side chains of the receptor
residues I128, F133, Y173 and L182, with the further stabilizing contribution of E172 (Figure
3A).
Figure 3. (A) Details of compound 1d in the binding pocket of the σ1R. Compound 1d is depicted as atom-colored
sticks-and-balls (C, gray, N, blue, O, red, Cl, green). The side chains of σ1R residues mainly interacting with 1d
are highlighted as colored sticks (R119 and W121, cyan; D126, red; I128, F133, E172, Y173 and L182, magenta;
T151 and V152, green) and labeled. The salt bridge and the hydrogen bond are shown as black broken lines.
Hydrogen atoms, water molecules, ions, and counterions are omitted for clarity. (B) Per-residue binding free energy
decomposition of the selected amino acids for the σ1R in complex with 1d.
A quantification of the single contribution of each identified receptor residue to ligand binding
was further carried out through a per-residue binding free energy deconvolution (PRBFED) of
the enthalpic term (∆Hbind,res) of the binding free energy, as shown in Figure 3B. The PRBFED
analysis confirmed that the network of favorable enthalpic interactions is substantially afforded
by the above mentioned σ1R residues. In particular, the network of hydrophobic interactions
involving the alkyl and aromatic fragments of 1d and the receptor cavity generated by residues
I128, F133, E172, Y173 and L182, considerably concurs in stabilizing receptor/ligand binding,

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with an overall contribution of -6.34 kcal/mol. Contextually, the π interactions (π-cation and π-
π, respectively) between the N-p-chlorobenzyl ring of 1d and the side chains of R119 and W121
produce an overall favorable contribution of -2.73 kcal/mol. Noteworthy, the polar interactions
afforded by the receptor/ligand permanent salt bridge and hydrogen bond provide a favorable
contribution to σ1R/1d binding of -5.46 kcal/mol.
On the basis of these results, we next proceeded with the design of new 1-(4-
(aryl(methyl)amino)butyl)- heterocyclic derivatives potentially endowed with comparable, if not
enhanced, σ1R affinity. To the purpose, we reasoned that the N-benzyl or the N-p-chlorobenzyl
amine and the butyl spacer, with their corresponding interactions, were essential for effective
σ1R binding. Thus, we decided to replace the benzoxazolone moiety with others, correlated,
heterocyclic scaffolds, in order to explore the conformational space within the relevant binding
cavity and to gain further information on the relevance of the specific intermolecular
interactions. Accordingly, the following new molecular structures were selected to address
specific requirements: (i) the 4-phenyloxazolidin-2-one ((R)-2b and (S)-2a,b) and 5-phenyl-
1,3,4-oxadiazol-2(3H)-one (3a,b) derivatives were chosen as non-condensed analogues of the
benzoxazolone
moiety;
(ii)
the
dihydroquinolin-2(1H)-one
(4a,b),
2H-
benzo[b][1,4]oxazin3(4H)-one (5a,b), 2H-benzo[b][1,4]thiazin-3(4H)-one (6a,b), and 4,5-
dihydro-1H-benzo[b]azepin-2(3H)-one (7a,b) substituents were selected to analyze the effect of
an increased steric hindrance within the receptor cavity; (iii) the isoindoline-1,3-dione (8a,b) and
indolin-2,3-dione (9a,b) rings were chosen as they are provided with two oxygen atoms, able to
potentially perform mutual hydrogen bonds as acceptors with the receptors counterpart; and
finally iv) the piperidin-2-one (10a,b) and the indole (11a,b) scaffolds were selected to study the
role of the two peculiar pharmacophore features of the benzoxazolone substituent, i.e., its
aromatic part and the hydrogen bond acceptor atom, respectively. In total, a set of 22 new
compounds (2-11) was designed.
The same docking/MM-PBSA scoring computational procedure described for compounds 1a
and 1d was next applied to the optimized structures of 2-11 in complex with the σ1R (Table S1).
Figure 4 shows the results of the MM/PBSA analysis, from which a direct structure-affinity
correlation can be made.

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Figure 4. In silico estimated free energies of binding (∆Gbind) for the newly designed set of 22 σ1R ligands in
complex with receptor. The ∆Gbind of the benzoxazolone derivatives 1a and 1d are also shown for comparison, the
dotted red line serving as a guide.
As seen in Figure 4, computational data predict that the replacement of the benzoxazolone
moiety with a non-constrained analogous such as the 4-phenyloxazolidin-2-one group in
derivatives (R)-2a,b and (S)-2a,b could slightly increase the affinity of these compounds for the
receptor, derivative (R)-2b exhibiting the best ∆Gbind value of -11.72 kcal/mol. Furthermore, in
agreement with previous observations [21,23,28], simulations indicate that the eventual presence
of a stereo center does not substantially affect the interactions of the relevant compound with the
receptor, since all the phenyloxazolidinones are provided with comparable σ1R affinity (Figures
4 and 5, Table SI1). Application of the PRBFED analysis further confirmed the interpretation of
the overall free energy of binding values: all σ1R residues involved in ligand binding establish
the same qualitative interactions with the ligands, and afford a favorable ∆Hbind,res contribution
comparable with those detected with lead compounds 1a and 1d (Figure 5B).

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Figure 5. (A) Superposition of equilibrated MD snapshots of the σ1R in complex with 1d (light sea green) and (R)-
2b (firebrick). Hydrogen atoms, water molecules, ions, and counterions are omitted for clarity. (B) Comparison of
the per-residue binding enthalpy decomposition ∆Hbind,res for compounds 1d, (R)-2b and (S)-2b in complex with the
σ1R.
On the other hand, the ∆Gbind values predicted for compounds 4-7 point to a small decrease of
σ1R binding capability for these molecules compared to the benzoxazolone derivatives 1a and
1d, with free energy of binding values in the range -9.84 − -10.43 kcal/mol (Figure 4, Table
SI1). In fact, the presence of the amidic group in each scaffold allow them to perform the
fundamental hydrogen bond with the receptor via their acceptor oxygen, while the aromatic
fragment of the corresponding heterocyclic moiety can exploit stabilizing interactions while
nicely encased within the σ1R hydrophobic cavity.
The results obtained with the remaining derivatives 8-11 deserved a more specific interpretation.
From a computational perspective, the lack of one of the important pharmacophore requirements
in the piperidin-2-one (10a,b) and the indole (11a,b) derivatives leads to a plummet in the
corresponding affinity for the σ1R, with a loss in ∆Gbind of about 4 and 3 kcal/mol, respectively.
The absence of the aromatic fragment in the piperidinone derivatives 10a,b not only affects the
hydrophobic interactions with the σ1R residues I128, F133, E172, Y173, and L182, but also, via
a sort of domino effect, negatively reflects on the bound ligand conformation, with a consequent
drastic reduction of the entire interaction spectrum, as testified in Figure 6. The same behavior is
reproduced by the indole derivatives 11a,b, for which not only the contributions of T151 and
V152 are zero, as expected, but also the rest of the σ1R residues involved in ligand binding
diminish their favorable contribution to binding enthalpy.

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Figure 6. (A) Superposition of equilibrated MD snapshots of the σ1R in complex with 1d (light sea green) and 10b
(hot pink). Hydrogen atoms, water molecules, ions and counterions are omitted for clarity. (B) Comparison of the
per residue binding enthalpy decomposition ∆Hbind,res for compounds 1d, 10b and 11b in complex with σ1R.
More interesting are the computational results obtained with isoindoline-1,3-dione (8a,b) and
indolin-2,3-dione (9a,b) derivatives. Although the structures of these compounds are provided
with all optimal pharmacophore requirements for σ1R binding, the insertion of a further
hydrogen acceptor group in the heterocyclic scaffold substantially seems to compromise the
affinity of these molecules for the σ1R. In fact, as the inspection of the relevant MD trajectory
reveals, to maintain the interaction with the –NH group of the backbone between T151 and V152
both the hetero-aromatic groups pay the cost of a configuration rearrangement within the binding
cavity; this, in turn, impair the rest of the favorable interactions with the other σ1R residues
involved in binding, as shown in Figure 7. The resulting binding conformations assumed by
compounds 8a,b and 9a,b do not allow them to satisfactorily snug their aromatic fragment
within the receptor binding cavity, the consequent reorganization of the N,N-arylmethyl amine
moiety preventing this group from exploiting optimal interactions with residues R119, W121,
and D126.

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Figure 7. (A) Superposition of equilibrated MD snapshots of the σ1R in complex with 1d (light sea green) and 8b
(sienna). Hydrogen atoms, water molecules, ions and counterions are omitted for clarity. (B) Comparison of the per-
residue binding enthalpy decomposition ∆Hbind,res for compounds 1d, 8b, and 9b in complex with σ1R.
2.2 Chemistry
All derivatives 2-11 were synthesized starting from the corresponding intermediate (Schemes 1
and 2), according to the synthetic pathway reported in Scheme 3 (Table SI2) The synthetic
pathway of each derivative started from the relevant commercially available scaffolds: 3,4-
dihydroquinolin-2(1H)-one (16), 2H-benzo[b][1,4]-oxazin-3(4H)-one (17), 2H-benzo[b][1,4]-
thiazin-3(4H)-one (18), potassium phtalimide (19), indolin-2,3-dione (20), piperidin-2-one (21)
and indole (22), respectively. The series 2a,b was obtained as racemate form (2a,b), as well as
enantiomerically pure R (R-2a,b) and S (S-2a,b) isomers, starting from pure phenylglycine (R/S,
R and S). Compounds 3a,b were obtained from the intermediate 5-phenyl-1,3,4-oxadiazol-
2(3H)-one 14 resulting from cyclization of benzoic acid hydrazide with triphosgene. Series 7a,b
was obtained following Schmidt cyclization starting from 1-tetralone and sodium azide in conc.
H2SO4. The N-methyl-1-phenylmethanamine is commercially available, while the 1-(4-
chlorophenyl)-N-methylmethanamine was obtained by reaction from 4-chlorobenzyl chloride
and methylamine solution.

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Scheme 1. Synthesis of intermediate 12-22 (phenylglycine, benzoic acid hydrazide, triphosgene, 1-tetralone and
compounds 16-22 are commercially available). Reagents and conditions: a) NaBH4, I2, THF/100°C, MeOH, rt
then KOH 20% 4h; b) (EtO2)CO 130°C; c) H2O/Tol. 0°C; d) H2SO4 conc., NaN3, Toluene 0°C 0.5h, rt overnight.

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Scheme 2. Synthesis of intermediate 23-32. Reagents and conditions: a) K2CO3/KI (cat.), ACN, 100°C, 6-12h.
Scheme 3. Synthesis of final compounds 2-11a,b. Reagents and conditions: a) K2CO3, ACN, 100°C, 6-12h.

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2.3 Receptor binding studies
σ1R and σ2R affinities of the test compounds were determined in competition experiments by
radiometric assays, using [3H]-(+)-Pentazocine as radioligand for the 1R assay, and [3H]-DTG
as radioligand in the 2R assay. Compounds 2-11a,b were tested against σ1R and σ2R of animal
σ
σ
origin prepared from guinea pig brain and rat liver, respectively.
In principle, the receptor binding studies on σ1R confirmed the structure-affinity relationship
predicted by the computational approach, as testified from the strict correlation between the
calculated free energies of binding and the corresponding Kiσ1 values listed in Table 1 (R2=
0.84). In fact, in agreement with MM-PBSA calculations, the 4-phenyloxazolidin-2-one
derivatives (2a,b, (R)-2a,b and (S)-2a,b), and the 5-phenyl-1,3,4-oxadiazol-2(3H)-one (3a,b)
compounds are experimentally ranked as the best σ1R binders, with a Kiσ1 values in the range
0.95-25 nM. Also, these compounds are provided with a good selectivity against the σ2R
subtype, for which they show affinity values at least one order of magnitude lower with respect
to the σ1R counterpart. The slight decrease in σ1R affinity predicted by modeling for
compounds 4-7 as a consequence of the replacement of the benzoxazolone moiety with a larger
heterocyclic group is confirmed by the corresponding experimental data (Table 1). Interestingly,
however, these molecules preserve a good selectivity profile against the σ2 subtype.
The experimental analysis carried out for the remaining new derivatives 8-11 confirmed that the
absence of the fundamental heterocyclic groups dramatically affects receptor/ligand binding, the
corresponding Kiσ1 spanning the high nanomolar - micromolar concentration range.
It is interesting to note that, although the tested Kiσ2 were very weak, all compounds show a
similar behavior against the σ2R subtype, with affinity values all of the order of hundreds of
nanomolar. The σ2 receptor is even more enigmatic than σ1R, and the information about its
binding site for the ligands is exceedingly scares and fragmented; therefore any computational
structure-affinity relationship derivation can be barely attempted, if at all. Anyway, on the basis
of these results, we are be tempted to speculate that the σ2R region in which the heterocyclic
scaffold is likely to be nestled might be characterized by higher adaptability with respect to σ1R,
thereby accommodating the presence of different chemical moieties without requiring major
energetical penalties. If verified, this information could be exploited in the future studies to
investigate and rationalize the molecular determinants for the σ2R binding.

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Kiσ1
(nM)
Kiσ2
(nM)
Cpd
2a
Ar
R
Kiσ2/Kiσ1
H
2.7±0.9
89±19
32
Cl
H
0.95±0.13
7.7±0.7
93±17
>3000
98
2b
>390
(R)-2a
(R)-2b
(S)-2a
Cl
H
2.9±0.6
25±5
603
190
208
7.6
Cl
H
9.3±0.8
5.2±1.9
317
110
8.1
17
(S)-2b
3a
Cl
H
18.7±1.3
16±6
315
222
16.8
13.9
3b
4a
Cl
H
33±9
30±8
239
274
7.2
9.1
4b
5a
Cl
H
16±3
119
105
7.4
21
5b
6a
5.0±1
Cl
H
13±3
9.0±3
18±2
844
169
254
412
621
13
28
6b
7a
7b
8a
Cl
H
23
0.73
Cl
>3000
42±24
<0.014
8b

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H
519
237
0.46
9a
Cl
H
385
173
0.45
n.d.
9b
>3000
>3000
10a
Cl
H
770
310
252
252
0.33
0.81
0.27
10b
11a
11b
Cl
4400
1200
Haloperidol
DTG
-
-
-
-
6.6±0.9
71±8
78±2.0
54±8
11.8
0.8
Table 1. σ1R and σ2R affinities of the synthesized compounds 2-11a,b. All compounds with highest affinities
(<100 nM) were tested in triplicates. n.d.: not determined. The last row shows the correlation (R2=0.84) between the
predicted values ∆Gbind and the corresponding experimental ꢀGbind,exp, calculated using the following relationship:
ꢀGbind,exp = -RT ln(1/Kiσ1).
2.4 Functional characterization
With the aim of performing an investigation of the cytotoxic activity of this new series of σ1R
ligands, and attempting a preliminary functional characterization of their eventual
agonist/antagonist profile, we compared the toxic effects of a set of novel compounds obtained
on the SH-SY5Y neuroblastoma cell line with those measured for NE-100 and haloperidol (two
commonly accepted σ1R antagonist) and pentazocine (the gold standard σ1R agonist) (Figure
8). Although SH-SY5Y cells seem to express σ1R at moderate level [30], previous works used
this human cell line of neuronal origin for σ1R studies [31-33] and obtained results overlapping
with those obtained with guinea pig brain subcellular fraction [34].
To the purpose, we followed the approach originally proposed by Zeng et al. for σ2R ligands
[35], based on the MTT assay. The underlying assumption for performing these tests is that σ1R

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agonists should promote cytoprotection via target activation, while antagonists should be
generally endowed with an opposite (i.e., cytotoxic) effect [36]. Specifically, we tested those
new compounds characterized by the highest σ1R affinity (that is, 2b (R)-2b, (S)-2b, 3a, and
6a), while the lead compounds 1a and 1d were also tested for comparison.
Accordingly to its recognized antagonist profile, NE-100 has a potent cytotoxic effect, as clearly
shown in Figure 9, where the effect exerted by the best σ1R binders is also shown as % of NE-
100 cytotoxicity at 50 µM (100%).
Considering the criteria adopted, the behavior of pentazocine and haloperidol in SH-SY5Y cells
is consistent with an activity of agonist and antagonist compounds, respectively. Actually,
haloperidol showed a cytotoxic effect (99%) at 50 µM strictly comparable with NE-100, while
pentazocine exhibited a negligible cytotoxicity (4.4%) at the same concentration. The
benzoxazolone derivatives 1a and 1d displayed an ambiguous cytotoxicity profile, since their
corresponding values are comprised between 50% and 75%. Clearly, further investigations are
required in this respect. On the other hand, the newly designed derivatives 2b (R)-2b, (S)-2b, 3a,
and 6a all exhibit a plausible σ1R agonist profile, seemingly in line with the low cytotoxic
activity detected for pentazocine. In particular, the 4-phenyloxazolidin-2-one derivatives, both as
pure enantiomers (R)-2b, (S)-2b and in their racemic form 2b, demonstrate very low cytotoxic
effects (< 15%), strictly comparable to the prototypical σ1R agonist, as inferred from Figure 9.
For the record, these compounds are also characterized by the best σ1R affinity along the new
series of σ1R designed and tested in the present work (Table 1).
Figure 8. Test compounds used for functional assay to define σ1R agonist/antagonist profile:

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Figure 9. Cytotoxicity of σ1R ligands as obtained from the MTT assay. SH-SY5Y cells were treated with different
σ1R ligands (50 ꢁM) for 48h. MTT assay was then performed, cytotoxicity of compounds was determined, and data
were reported as % of NE-100 cytotoxicity at 50 µM (100%). The bars represent the mean ± SD from three
independent experiments performed in triplicate.
3. Conclusions
On the basis of the molecular information retrieved by the in silico analysis of the binding mode
of the two lead benzoxazolone derivatives 1a and 1d, both endowed with high σ1R affinity
(Kiσ1 = 2.6 and 7.1 nM, respectively), we designed and synthesized the new series of derivatives
2-11a,b, with a twofold aim of (i) dissecting the effect of the replacement of the benzoxazolone
moiety on sigma 1 receptor affinity, and (ii) preliminarily evaluating the behavior of these
compounds as σ1R agonists or antagonists. All new molecules maintain the 1-(4-
(aryl(methyl)amino)butyl)- spacer characterizing the compound series 1, whereas the
benzoxazolone moiety, linked to the butyl chain, has been replaced with others, correlated,
heterocyclic scaffolds to obtain the corresponding 4-phenyloxazolidin-2-one (2a,b, (R)-2a,b and
(S)2a,b), 5-phenyl-1,3,4-oxadiazol-2(3H)-one (3a,b), dihydroquinolin-2(1H)-one (4a,b), 2H-
benzo[b][1,4]oxazin3(4H)-one (5a,b), 2H-benzo[b][1,4]thiazin-3(4H)-one (6a,b) and 4,5-

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dihydro-1H-benzo[b]azepin-2(3H)-one (7a,b), isoindoline-1,3-dione (8a,b), indolin-2,3-dione
(9a,b), piperidin-2-one (10a,b) and indole (11a,b) derivatives.
The results achieved by scoring the affinity of these molecules for the σ1R via their molecular
dynamics simulations in complex with our 3D σ1R model allowed us to predict that, from the
perspective of receptor binding ability, modifications of the benzoxazolone moiety are well
tolerated only if the new molecular scaffolds preserve the peculiar molecular determinants for
the optimal encasement in the relevant σ1R binding cavity. In fact, the new derivatives 2-7, in
which the aromatic substituent and the hydrogen bond acceptor atom are preserved, display a
binding capacity comparable to, or even slightly higher (as in the case of the derivatives 4-
phenyloxazolidin-2-one 2a,b) to that of the lead compounds 1a and 1d. Conversely, when one of
the pharmacophore requirements is missing (as in the piperidone (10a,b) or in the indole (11a,b)
derivatives), the favorable interactions established between the σ1R and the ligand significantly
decreases, with effects extending, in a domino effect, beyond the receptor hydrophobic cavity
directly involved in ligand binding. Finally, it is interesting to highlight that the addition of a
supplementary element, i.e. an additional hydrogen bond acceptor (e.g., isoindolindiones 8a,b
and indolindiones 9a,b), leads to a substantial impairment of the resulting compound σ1R
binding capability, by virtue of a suboptimal conformation of the aromatic fragment nestled in
the receptor binding cavity.
All computer-based predictions were substantiated by the experimental characterization of the
σ1R affinity of all new compounds through radioligand binding assays of the Ki values against
both sigma receptor subtypes. The analysis confirmed the quality of the in silico prediction with
an outstanding agreement between the calculated σ1R affinity and the experimental inhibitory
concentrations. Moreover, the experiments revealed a clear preference of the new derivative 2-7
for the σ1R with a good selectivity profile against the σ2 receptor subtypes.
Finally, the MTT viability assay on human neuroblastoma cells (SH-SY5Y) was performed in
order to test the cytotoxic effects of the novel compounds. These data were also useful for a
preliminary evaluation of the agonist/antagonist behavior on a selection of the best σ1R binder
in the new series. According to these tests, the new 4-phenyloxazolidin-2-one derivatives 2b,
(R)-2b and (S)-2b demonstrated a very low level of cytotoxicity, comparable with that obtained
with the σ1R prototypical agonist pentazocine. This property, coupled with their remarkable
σ1R affinities (Kiσ1 values in the low nanomolar range), outlines these compounds as the most

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promising candidates of the series to exploit them as new pharmacologic agents for further and
more specific investigation on the σ1R biologic activity.
4. Experimental
4.1 Computational details
The optimized structure of all compounds was docked into the putative binding pockets for the
sigma1 receptor by applying a consolidated procedure [18-28] with AutoDock 4.2 [37]. For each
compound, only the molecular conformation satisfying the combined criteria of having the lowest
(i.e., more favorable) Autodock energy and belonging to a highly populated cluster was selected
to carry out for further modeling. The ligand/receptor complexes obtained from the docking
procedure was further refined in Amber 14 [38] using the quenched molecular dynamics (QMD)
method as previously described [18-28]. According to QMD, the best energy configuration of
each complex resulting from this step was subsequently solvated by a cubic box of TIP3P [39]
water molecules extending at least 10 Å in each direction from the solute. The system was
neutralized and the solution ionic strength was adjusted to the physiological value of 0.15 M by
adding the required amounts of Na+ and Cl- ions. Each solvated system was relaxed by 500 steps
of steepest descent followed by 500 other conjugate-gradient minimization steps and then
gradually heated to a target temperature of 300 K in intervals of 50 ps of NVT MD, using a
Verlet integration time step of 1.0 fs. The Langevin thermostat was used to control temperature,
with a collision frequency of 2.0 ps-1 The protein was restrained with a force constant of 2.0
kcal/(mol Å), and all simulations were carried out with periodic boundary conditions.
Subsequently, the density of the system was equilibrated via MD runs in the isothermal-isobaric
(NPT) ensemble, with isotropic position scaling and a pressure relaxation time of 1.0 ps, for 50 ps
with a time step of 1 fs. All restraints on the protein atoms were then removed, and each system
was further equilibrated using NPT MD runs at 300 K, with a pressure relaxation time of 2.0 ps.
Three equilibration steps were performed, each 2 ns long and with a time step of 2.0 fs. To check
the system stability, the fluctuations of the rmsd of the simulated position of the backbone atoms
of the receptor with respect to those of the initial protein were monitored. All chemophysical
parameters and rmsd values showed very low fluctuations at the end of the equilibration process,
indicating that the systems reached a true equilibrium condition. The equilibration phase was

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followed by a data production run consisting of 40 ns of MD simulations in the canonical (NVT)
ensemble. Only the last 20 ns of each equilibrated MD trajectory were considered for statistical
data collections. A total of 1000 trajectory snapshots were analyzed the each ligand/receptor
complex. The binding free energy, ∆Gbind, between the ligands and the sigma1 receptor was
estimated by resorting to the MM/PBSA approach implemented in Amber 14. According to this
well validated methodology [18-28], the free energy was calculated for each molecular species
(complex, receptor, and ligand), and the binding free energy was computed as the difference:
∆Gbind = Gcomplex – (Greceptor + Gligand) = ∆EMM + ∆Gsol - T∆S
in which ∆EMM represents the molecular mechanics energy, ∆Gsol includes the solvation free
energy and T∆S is the conformational entropy upon ligand binding. The per residue binding free
energy decomposition was performed exploiting the MD trajectory of each given
compound/complex, with the aim of identifying the key residues involved in the ligand/receptor
interaction. This analysis was carried out using the MM/GBSA approach [40], and was based on
the same snapshots used in the binding free energy calculation. All simulations were carried out
using the Pmemd modules of Amber 14, running on our Mose25 CPU/GPU calculation cluster.
4.2 Chemistry
Commercially available chemicals were of reagents grade and used as received. Column
chromatography was performed on Silica Gel 60 (230-400 mesh, Merk) and reaction courses and
product mixtures were routinely monitored by thin-layer chromatography (TLC) on silica gel
precoated F254 Merck plates. Melting points were determined with a Büchi 510 capillary
apparatus or a Stuart SMP300, and are uncorrected. Infrared spectra were recorded on a Perkin
Elmer RXI spectrophotometer in nujol mulls. Proton nuclear magnetic resonance (1H-NMR)
spectra were determined on a Varian Gemini 200 MHz , a Jeol 400 MHz and a Varian Inova 500
MHz; chemical shifts are reported as δ (ppm) in CDCl3 solution (0.05% v/v TMS). ESI-MS
spectra were obtained on a Bruker Daltonics Esquire 4000 spectrometer by infusion of a solution
of the sample in MeOH.

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4.2.1 Synthesis of (R,S)4-phenyloxazolidin-2-one 13 [41,42]
To a solution of 2.5 g (66.0 mmol) of NaBH4 in 125 ml of THF at 0°C, 8.4 g of I2 (33.0 mmol)
dissolved in the minimum amount of THF, were slowly added. When the solution turns white,
5.0 g of (R,S)-phenylglycine were added. The mixture was heated under reflux for 18h under
TLC monitoring (CH2Cl2/EtOH 9:1). The solution was allowed to cool then added of 4 ml of
MeOH until it turns clear. The solvents were removed under reduced pressure.
The solid residue has been taken with 60 ml of a KOH 20% acq. solution and allowed to stir for
4h at room temperature. CH2Cl2 was added and the organic phase was separated, washed with
water, dried with Na2SO4 and concentrated in vacuum to afford 4.3g (95%) of (R,S)-
phenylglycinol 12 as a light-yellow oil.
1H NMR (CDCl3-TMS) ppm (
δ): 2.84 (broad s., 3H, NH2 + OH); 3.46 (m, 1Ha, CH2); 3.56 (m,
1Hb, CH2); 3.87 (m, 1H, CH); 7.20 (m, 5H, arom.). MS: m/z 138 [MH+].
In the same manner pure (R)-phenylglycinol (R)-12 and pure (S)-phenylglycinol (S)-12 were
obtained, starting from the corresponding R- and S-phenylglicine.
A dry 250 ml three-necked round bottom flask equipped with a thermometer and a 10 cm
vigreux column with a distillation head, was charged with 4.3 g of the (R,S)-phenylglycinol
(31.4 mmol) obtained and added of 9.3 g of diethyl carbonate (79.0 mmol) and 0.43 g of K2CO3
(3.10 mmol). The mixture was heated carefully to 130-140°C and the ethanol was distils as it
formed. The oily residue was cooled and added by 50 ml of CH2Cl2 to facilitate the filtration of
the remaining potassium carbonate. The organic phase was then washed with a satured solution
of NaHCO3, separated and dried over anhydrous Na2SO4, filtrate and evaporated in vacuo. The
residue was crystallized from AcOEt/Etp (1:3) to afford 4.3 g (26.4 mmol, 85%) of 13 as a white
solid; mp 134-136°C.
1H-NMR (CDCl3/TMS)
δ: 4.22 (dd 1H, CH2 oxad., J=8.6; 7.0 Hz); 4.77 (dd, 1H, CH oxad., J=
8.6; 8.6 Hz); 4.98 (dd, 1H, CH2 oxad., J= 8.6; 7.0 Hz); 5.40 (broad s., 1H, NH oxad.
disappearing on D2O); 7.40 (m, 5H, arom.). MS: m/z 164 [MH+].
In an analogous way the pure isomers (R)-4-phenyloxazolidin-2-one (R)-13 and (S)-4-
phenyloxazolidin-2-one (S)-13 were obtained:

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4.2.2. (R)-4-phenyloxazolidin-2-one (R)-13
White solid; m.p.: 126-128°C; Yield (%): 85. 1H-NMR (CDCl3/TMS)
δ: 4.22 (dd 1H, CH2 oxad.,
J= 8.6; 7.0 Hz); 4.77 (dd, 1H, CH oxad., J= 8.6; 8.6 Hz); 4.98 (dd, 1H, CH2 oxad., J= 8.6; 7.0
Hz); 5.40 (broad s., 1H, NH oxad. disappearing on D2O); 7.40 (m, 5H, arom.). MS: m/z 164
[MH+].
4.2.3. (S)-4-phenyloxazolidin-2-one (S)-13
1
White solid; m.p.: 124-126°C; Yield (%): 77. H-NMR (CDCl3/TMS)
δ: 4.22 (dd 1H, CH2
oxad., J= 8.6; 7.0 Hz); 4.77 (dd, 1H, CH oxad., J= 8.6; 8.6 Hz); 4.98 (dd, 1H, CH2 oxad., J= 8.6;
7.0 Hz); 5.40 (broad s., 1H, NH oxad. disappearing on D2O); 7.40 (m, 5H, arom.). MS: m/z 164
[MH+].
4.2.4. 5-Phenyl-1,3,4-oxadiazol-2(3H)-one 14 [43]
To an ice-bath solution of benzoic acid hydrazide (2.5 g, 18.0 mmol) in 20 ml of water a solution
of triphosgene (5.34 g, 18.0 mmol) in 20 ml of toluene was added dropwise. A white solid was
formed which dissolved by addiction of water. The organic phase was eliminated and the water
was neutralized with an aqueous solution of NaOH (10%). The white solid formed was filter off,
wash with distilled water and recrystallized from abs. EtOH to afford 1.4 g of 14 as a white
solid.
Yield (%) 48; m.p.= 132-136°C; I.R.: (nujol, cm-1): 1751, 3257. 1H-NMR (CDCl3/TMS): δ 7.51
(m, 3H arom.), 7.89 (d, 2H arom.), 10.0 (broad s., 1H, NH disappearing on deuteration). MS:
m/z 163 [MH+].
4.2.5. 4,5-Dihydro-1H-benzo[b]]azepin-2(3H)-one 15 [44]
3.00 g (21.0 mmol) of 1-tetralone were dissolved in 120 ml of toluene and cooled with an ice
bath at 0°C, under stirring. Sodium azide (2.67 g, 41.0 mmol) was added in one portion and,
subsequently, 10.2 ml of H2SO4 conc. were added drop by drop, over a period of 30 minutes.
The solution was left to stir overnight, then 82 ml of distilled water were slowly added and the
organic phase was separated, dried over anhydrous Na2SO4, filtrate and evaporated in vacuo.
The solid residue was crystallized from CH2Cl2/n-hexane (1:1) to afford 2.20 g (14.0 mmol,
76%) of 15 as a white crystalline solid.
1
Melting point: 138-139°C. H-NMR (CDCl3/TMS): δ 2.36 (dq, 2H, H4-,4’ CH2 benzoaz .); 2.49
(q, 2H, H3-3’ CH2 benzoaz.); 2.93 (t, 2H, H5-5’ benzoaz.); 7.09 (d, 1H, H6 arom.); 7.29 (t, 1H, H7
arom.); 7.37 (m, 2H, H8 e H9 arom.); 7.60 (broad s., 1H, NH-CO disappearing on deuteration).

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Compounds 16-22 are commercially available.
4.2.6 General synthesis of intermediate 23-32
4.2.6.1 (R,S) 3-(4-bromobutyl)-4-phenyloxazolidin-2-one 23
To a solution of 1.6 g (10.0 mmol) of (R,S) 4-phenyloxazolin-2-one (13), 3,4 g (25.0 mmol) of
K2CO3 and a catalytic amount of KI in acetonitrile, 5.4 g (25.0 mmol) of 1,4-dibromobutane
were slowly added drop by drop stirring at rt. The mixture was then heated under reflux for 6-
12h, monitored by TLC (CH2Cl2/EtOH 9:1). The hot solution was filtered and evaporated under
reduced pressure. The yellow oil residue of (R,S) 3-(4-bromobutyl)-4-phenyloxazolidin-2-one
23 was used without further purification.
Yield: 96%; I.R.: (nujol, cm-1): 1735; 1H-NMR (CDCl3/TMS): δ 1.42 (m, 2H, N-
CH2CH2CH2CH2Br); 1.60 (m, 2H, N-CH2CH2CH2CH2Br); 2.65 (m, 1H, H , N-CH2(CH2)3Br);
3.20 (m, 3H, H , N-CH2(CH2)3Br + N-(CH2)3CH2Br); 3.97 (dd, 1H, CH2 oxad., J= 8.8; 6.6 Hz);
4.46 (dd 1H, CH oxad., J= 8.8; 8.8 Hz); 4.62 (dd, 1H, CH2 oxad., J= 8.8; 6.6 Hz).
In the same way the enantiomerically pure compounds (R)-23, (S)-23 and compounds 24-32
were obtained.
4.2.6.2 (R) 3-(4-bromobutyl)-4-phenyloxazolidin-2-one (R)-23
1
Trituration with petroleum ether (40°-70°) give a yellow oil; yield: 57%; H-NMR: (CDCl3-
TMS) δ: 1.29 (m, 2H, N-CH2CH2CH2CH2Br); 1.50 (m, 2H, N-CH2CH2CH2CH2Br); 2.54 (m,
1H, H , N-CH2(CH2)3Br); 3.10 (m, 3H, H , N-CH2(CH2)3Br + N-(CH2)3CH2Br); 3.80 (m, 1H,
CH2 oxad.); 4.35 (m 1H, CH oxad.); 4.62 (m, 1H, CH2 oxad.).
4.2.6.3 (S) 3-(4-bromobutyl)-4-phenyloxazolidin-2-one (S)-23
1
Trituration with petroleum ether (40°-70°) give a yellow oil; yield: 40%; H-NMR: (CDCl3-
TMS), : 1.29 (m, 2H, N-CH2CH2CH2CH2Br); 1.50 (m, 2H, N-CH2CH2CH2CH2Br); 2.54 (m,
1H, H , N-CH2(CH2)3Br); 3.10 (m, 3H, H , N-CH2(CH2)3Br + N-(CH2)3CH2Br); 3.80 (m, 1H,
CH2 oxad.); 4.35 (m 1H, CH oxad.); 4.62 (m, 1H, CH2 oxad.).
4.2.6.4 3-(4-bromobutyl)-5-Phenyl-1,3,4-oxadiazol-2(3H)-one 24
1
White solid (upon cooling overnight), yield: 53%; m.p. 60-66 °C; H-NMR: (CDCl3/TMS) δ:
1.97-2.00 (m, 4H, N-CH2CH2CH2CH2Br), 3.48 (m, 2H, N CH2CH2CH2CH2Br), 3.83 (m, 2H,
N-CH2CH2CH2CH2Br), 7.48 (m, 3H, Ph), 7.85 (m, 2H, Ph).

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4.2.6.5 1-(4-bromobutyl)-3,4-dihydroquinolin-2(1H)-one 25
1
Chromatography column (AcOEt-EtOH 95:5); yellow oil; yield: 92%; H-NMR: (CDCl3/TMS)
δ: 1.75-1.80 (m, 2H, NCH2CH2CH2CH2Br), 1.95-2.00 (m, 2H, NCH2CH2CH2CH2Br), 2.60 (t,
2H, CH2CH2CO dihydroq., J= 8 Hz) 2.85 (t, 2H, CH2CH2CO dihydroq., J= 8 Hz), 3.40 (t, 2H,
N-CH2CH2CH2CH2Br), 3.93 (t, 2H, NCH2CH2CH2CH2Br), 6.96-7.23 (m, 4H arom.).
4.2.6.6 4-(4-bromobutyl)-2H-benzo[b][1,4]oxazin-3(4H)-one 26
1
Yellow oil; yield: 92%; H-NMR: (CDCl3/TMS) δ: 1.77-1.90 (m, 4H, N-CH2CH2CH2CH2Br);
3.39 (t, 2H, N-(CH2)3CH2Br); 3.90 (m, 2H, N-CH2(CH2)3Br); 4,51 (m, 2H, H3-3’ CH2 benzox.);
6,94 (m, 4H, arom.).
4.2.6.7 4-(4-bromobutyl)-2H-benzo[b][1,4]thiazin-3(4H)-one 27
1
Yellow oil; yield: 87%; H-NMR: (CDCl3/TMS) δ: 1.75-1.85 (m, 4H, N-CH2CH2CH2CH2Br);
3.32 (t, 2H, N-(CH2)3CH2Br); 3.40 (s, 2H, H3-3’ CH2 benzothiaz.); 4.00 (t, 2H, N-CH2(CH2)3Br);
6,99-7.34 (m, 4H, arom.).
4.2.6.8 1-(4-bromobutyl)-2,3,4,5-tetrahydro-1H-benzo[b]]azepin-2(3H)-one 28
1
Orange oil; yield: 76%; H-NMR: (CDCl3/TMS) δ: 1.53-1.72 (m, 4H, N-CH2CH2CH2CH2Br);
1.90-2.10 (m, 4H, H3-3’-H4-4’ 2xCH2 benzoaz.); 2.58 (m, 2H, H5-5’ CH2 benzoaz.); 3.22 (t, 2H, N-
(CH2)3CH2Br); 3.76 (m, 2H, N-CH2(CH2)3Br); 7.03-7.17 (m, 4H, arom.).
4.2.6.9 2-(4-bromobutyl)isoindoline-1,3-dione 29
This compound has been synthesized starting from potassium phtalimide (1 eq.) and 1,4-
dibromobutane (2.5 eq.) by refluxing the mixture in acetone for 6 hours. The hot solution was
filtered off from inorganic salts then concentrated in vacuo and recrystallized from n-hexane to
afford a white solid.
1
Yield: 80%; m.p. 78-82 °C; H-NMR (CDCl3/TMS) δ: 1.85 (m, 4H, N-CH2CH2CH2CH2Br),
3.43 (t, 2H, N-CH2CH2CH2CH2Br), 3.71 (t, 2H, N-CH2CH2CH2CH2Br), 7.72 (m, 2H, H4, H7),
7.82 (m, 2H, H5, H6).
4.2.6.10 1-(4-bromobutyl)indoline-2,3-dione 30
To a solution (1eq.) of isoindolin-1,3-dione in DMF, 1.2eq. of NaH (95%) was added at 0°C and
the mixture was stirred for 30 minutes. Then 2.5eq. of 1,4-dibromobutane was slowly added and
the mixture was allowed to stir at room temperature overnight. The solution was poured into ice-
cold water and extracted three times with diethyl ether which was collected, dried over
anhydrous Na2SO4, filtered and evaporated. The residue was purified by chromatography
column (100% CH2Cl2).

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1
Orange oil; yield: 85%; m.p. 76-80 °C; I.R.: (nujol, cm-1): 1610, 1737; H-NMR (CDCl3/TMS)
δ: 1.91 (m, 4H, NCH2CH2CH2CH2Br), 3.46 (t, 2H, NCH2CH2CH2CH2Br), 3.76 (t, 2H,
NCH2CH2CH2CH2Br), 6.92 (d, 1H, H7, indol.), 7.12 (t, 1H, H5, indol.), 7.59 (m, 2H, H4, H6
indol.).
4.2.6.11 1-(4-bromobutyl)piperidin-2-one 31
The synthesis of this intermediate started from 1 eq. of piperidin-2-one dissolved in ACN, 5%
mol of TBAB and 1.5eq. of CsCO3 as base. The reaction mixture was allowed to stir overnight at
room temperature then the solvent was evaporated under reduced pressure. The residue was
dissolved with CH2Cl2 and washed with water 3 times. The oily residue was purified by
chromatography column on silica gel (eluent: AcOEt/EtOH 95-5).
1
Yield: 15%; H-NMR (CDCl3-TMS),
δ: 1.54-1.77 (m, 8H, (4) CH2CH2CH2CH2 and (4)
NCH2CH2CH2CH2CO pip.), 2.24 (m, 2H, NCH2CH2CH2CH2CO pip.), 3.13-3.30 (m, 4H, (2)
CH2CH2CH2CH2N and (2) NCH2CH2CH2CH2CO pip.), 3.35 (m, 2H, CH2CH2CH2CH2Br).
4.2.6.12 1-(4-bromobutyl)indole 32
The synthesis of this compound was carried out with the same procedure described above for the
corresponding indolin-2,3-dione derivative (30), with the addiction of a catalytic amount (5%
mol) of TBAB into the reaction mixture. The product was purified by chromatography column
on silica gel using petroleum ether (40°-70°) as eluent to afford a light yellow oil.
1
Yield: 23%; H-NMR (CDCl3/TMS)
δ
: 1.85 (m, 2H, NCH2CH2CH2CH2Br), 2.01 (m, 2H,
NCH2CH2CH2CH2Br), 3.37 (t, 2H, NCH2CH2CH2CH2Br), 4.17 (t, 2H, NCH2CH2CH2CH2Br),
6.50 (d, 1H, H3, indol., J= 2.8 Hz), 7.09 (t, 1H, H2, indol., J= 2.8 Hz), 7.11 (t, 1H, H5, indol.),
7.21 (t, 1H, H6, indol.), 7.34 (d, 1H, H4 indol.), 7.63 (d, 1H, H7 indol.).
4.2.7 General synthesis of final compounds 2-11a,b
4.2.7.1 (R,S) 3-(4-Benzyl-(methyl)-amino)-butyl)-4-phenyl-oxazolidin-2-one 2a
A solution of 0.17 g (1.40 mmol) of N-benzylmethylamine, 0.20 g (1.40 mmol) of K2CO3 and
0.40 g of (R,S)-3(4-bromobutyl)-5-phenyloxazolidin-2-one 23 in ACN was allowed to stirring
under reflux for 6-12 h and monitored by TLC until the reaction was completed. The hot

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solution was filtered and concentrated in vacuo to afford 0.29 g (85.0 mmol) of a
chromatography pure yellow oil 2a.
1
I.R.: (nujol, cm-1) 1754 (C=O); H-NMR: (CDCl3-TMS; 200 MHz), δ: 1.40-1.60 (m, 4H,
CH2CH2CH2CH2); 2.14 (s, 3H, NCH3); 2.32 (t, 2H, (CH2)3CH2NCH3); 2.80 (m, 1H, Ha,
NCH2(CH3)3NCH3); 3.43 (m, 3H, Hb, NCH2(CH2)3NCH3 + (2) N(CH3)CH2Ph); 4.11 (dd, 1H,
CH2 oxad.; J= 8.8; 6.6 Hz); 4.60 (t, 1H, CH oxad. J= 8.8; 8.8 Hz); 4.77 (dd, 1H, CH2 oxad. J=
8.8; 6.6 Hz); 7.20-7.40 (m, 10H, arom). MS: m/z 339 [MH+].
The 1-(4-chlorophenyl)-N-methylmethanamine was synthesized starting from 4-chlorobenzyl
chloride (14.0 mmol) and 20 ml of ethanolic solution of methylamine (33%) under reflux for 2h.
The mixture was distilled under vacuum to eliminate the excess of methylamine and washed
with a satured NaHCO3 solution to afford a yellow oil (56%) that was used without further
purification.
The 1-(2,4-dimethylphenyl)-N-methylmethanamine was synthesized in analogous way.
4.2.7.2 (R,S) 3-(4-((4-Chlorobenzyl)-(methyl)-amino)-butyl)-4-phenyl-oxazolidin-2-one 2b.
Starting from 1-(4-chlorophenyl)-N-methylmethanamine and (R,S)-3(4-bromobutyl)-5-
phenyloxazolidin-2-one 23 following the same procedure described above, 70% of 2b was
obtained as a yellow pure oil.
1
I.R.: (nujol, cm-1) 1754 (C=O). H-NMR: (CDCl3-TMS; 200 MHz) δ: 1.30-1.50 (m, 4H,
CH2CH2CH2CH2); 2.11 (s, 3H, NCH3); 2.30 (m, 1H, (CH2)3CH2NCH3); 2.80 (m, 1H,
(CH2)3CH2NCH3); 3.38 (s, 2H, NCH2(CH2)3NCH3); 3.45 (s, 2H, N(CH3)CH2Ph); 4.10 (dd, 1H,
CH2 oxad.; J= 8.0; 6.0 Hz); 4.59 (t, 1H, CH oxad.; J= 8.0; 8.0 Hz); 4.78 (dd, 1H, CH2 oxad.; J=
8.0; 6.0 Hz); 7.20-7.40 (m, 9H, arom). MS: m/z 371 [MH+], 373 [MH++2].
Following the same procedure described above, compounds (R)-2a,b, (S)-2a,b and 3-11a,b were
obtained.
4.2.7.3 (R) 3-(4-Benzyl-(methyl)-amino)-butyl)-4-phenyl-oxazolidin-2-one (R)-2a
1
Yellow oil; yield: 83%; I.R.: (nujol, cm-1) 1754 (C=O). H-NMR: (CDCl3-TMS; 400 MHz) δ:
1.20-1.40 (m, 4H, CH2CH2CH2CH2); 1.99 (s, 3H, NCH3); 2.19 (t, 2H, (CH2)3CH2NCH3); 2.65
(m, 1H, N-CH2(CH3)3NCH3);3.30 (m, 3H, N(CH3)CH2Ph + 1H N-CH2(CH2)3NCH3); 3.93 (m,

ACCEPTED MANUSCRIPT
1H, CH oxad.; J= 12.0; 8.0 Hz); 4.45 (t, 1H, CH2 oxad. J= 12.0; 12.0 Hz); 4.65 (m, 1H, CH2
oxad. J= 12.0; 8.0 Hz); 7.15-7.25 (m, 10H, arom). MS: m/z 339 [MH+].
4.2.7.4 (R) 3-(4-((4-Chlorobenzyl)-(methyl)-amino)-butyl)-4-phenyl-oxazolidin-2-one (R)-2b
1
Yellow oil; yield: 82%; I.R.: (nujol, cm-1) 1754 (C=O). H-NMR: (CDCl3-TMS; 400
MHz) δ: 1.30-1.50 (m, 4H, CH2CH2CH2CH2); 2.06 (s, 3H, NCH3); 2.24 (m, 1H,
(CH2)3CH2NCH3); 2.75 (m, 1H, (CH2)CH2NCH3); 3.34 (s, 2H, N(CH3)CH2Ph); 3.42 (m,
2H, NCH2(CH2)3NCH3); 4.05 (m, 1H, CH2 oxad.; J= 12.0; 8.0 Hz); 4.55 (t, 1H, CH
oxad.; J= 12.0; 12.0 Hz); 4.74 (t, 1H, CH2 oxad.; J= 12.0; 8.0 Hz); 7.13-7.43 (m, 9H,
arom). MS: m/z 371 [MH+], 373 [MH++2].
4.2.7.5 (S) 3-(4-Benzyl-(methyl)-amino)-butyl)-4-phenyl-oxazolidin-2-one (S)-2a
1
Yellow oil; yield: 91%; I.R.: (nujol, cm-1) 1754 (C=O). H-NMR: (CDCl3-TMS; 500 MHz) δ:
1.40-1.50 (m, 4H, CH2CH2CH2CH2); 2.11 (s, 3H, NCH3); 2.30 (t, 2H, (CH2)3CH2NCH3); 2.77
(m, 1H, NCH2(CH3)3NCH3); 3.41 (m, 3H, N(CH3)CH2Ph + 1H N-CH2(CH2)3NCH3); 4.08 (dd,
1H, CH2 oxad.; J= 10.0; 5.0 Hz); 4.57 (t, 1H, CH oxad. J= 10.0; 5.0 Hz); 4.76 (dd, 1H, CH2
oxad. J= 10.0; 5.0 Hz); 7.25-7.40 (m, 10H, arom). MS: m/z 339 [MH+].
4.2.7.6 (L) 3-(4-((4-Chlorobenzyl)-(methyl)-amino)-butyl)-4-phenyl-oxazolidin-2-one (S)-2b
1
Yellow oil; yield: 83%; I.R.: (nujol, cm-1) 1754 (C=O). H-NMR: (CDCl3-TMS; 500 MHz) δ:
1.30-1.45 (m, 4H, CH2CH2CH2CH2); 2.09 (s, 3H, NCH3); 2.27 (m, 1H, (CH2)3CH2NCH3); 2.77
(m, 1H, (CH2)3CH2NCH3); 3.36 (s, 2H, N(CH3)CH2Ph); 3.40 (m, 2H, NCH2(CH2)3NCH3); 4.06
(dd, 1H, CH2 oxad.; J= 10.0; 5.0 Hz); 4.57 (t, 1H, CH oxad.; J= 10.0; 5.0 Hz); 4.76 (dd, 1H, CH2
oxad.; J= 10.0; 10.0 Hz); 7.20-7.40 (m, 9H, arom). MS: m/z 371 [MH+], 373 [MH++2].
4.2.7.7 3-(4-(Benzyl(methyl)amino)butyl)-5-phenyl-1,3,4-oxadiazol-2(3H)-one 3a
1
Yellow oil; yield: 91%; I.R.: (nujol, cm-1) 1787 (C=O). H-NMR: (CDCl3-TMS; 200 MHz) δ:
1.58-1.83 (m, 4H, CH2CH2CH2CH2); 2.17 (s, 3H, NCH3); 2.43 (t, 2H, (CH2)3CH2NCH3); 3.47
(s, 2H, N(CH3)CH2Ph); 3.76 (t, 2H, NCH2(CH3)3NCH3); 7.28-7.82 (m, 10H, arom). MS: m/z
338 [MH+].
4.2.7.8 3-(4-((4-Chlorobenzyl)(methyl)amino)butyl)-5-phenyl-1,3,4-oxadiazol-2(3H)-one 3b
1
Yellow oil; yield: 47%; I.R.: (nujol, cm-1) 1775 (C=O). H-NMR: (CDCl3-TMS; 200 MHz) δ:
1.90-2.00 (m, 4H, CH2CH2CH2CH2); 2.15 (s, 3H, NCH3); 3.43 (m, 4H,
(CH2)3CH2N(CH3)CH2Ph); 3.82 (t, 2H, NCH2(CH3)3NCH3); 7.26-7.83 (m, 9H, arom). MS: m/z
371 [MH+], 373 [MH++2].

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4.2.7.13 1-(4-(Benzyl(methyl)amino)butyl)-3,4-dihydroquinolin-2(1H)-one 4a
1
Yellow oil; yield: 80%; I.R.: (nujol, cm-1) 1634 (C=O). H-NMR: (CDCl3-TMS; 200 MHz) δ:
1.50-1.70 (m, 4H, CH2CH2CH2CH2); 2.00 (s, 3H, N-CH3); 2.27 (t, 2H, N-CH2CH2CH2CH2);
2.49 (t, 2H, CH2CH2-CO dihydroq., J= 8.0 Hz); 2.71 (t, 2H, CH2CH2-CO dihydroq., J= 8.0 Hz);
3.30 (s, 2H, PhCH2N); 3.77 (t, 2H, N-CH2(CH2)3-N-CH3); 6.80-7.20 (m, 9H, arom). MS: m/z
323 [MH+].
4.2.7.14 1-(4-((4-Chlorobenzyl)(methyl)amino)butyl)-3,4-dihydroquinolin-2(1H)-one 4b
1
Yellow oil; yield: 73%; I.R.: (nujol, cm-1) 1679 (C=O). H-NMR: (CDCl3-TMS; 200 MHz) δ:
1.55-1.68 (m, 4H, CH2CH2CH2CH2); 2.24 (s, 3H, N-CH3); 2.54 (t, 2H, (CH2)3-CH2-N-CH3);
2.64 (t, 2H, CH2CH2-CO chin., J= 8 Hz); 2.88 (t, 2H, CH2CH2-CO chin., J= 8 Hz); 3.55 (s, 2H,
PhCH2N); 3.95 (t, 2H, N-CH2CH2CH2CH2-NCO); 6.90-7.30 (m, 8H, arom). MS: m/z 357
[MH+], 359 [MH++2].
4.2.7.15 2-(4-Benzyl(methyl)amino)butyl)-2H-benzo[b][1,4]oxazin-3(4H)-one 5a
1
Yellow oil; yield: 88%; I.R.: (nujol, cm-1) 1695 (C=O). H-NMR: (CDCl3-TMS; 400 MHz) δ:
1.50-1.60 (m, 4H, CH2CH2CH2CH2); 2.05 (s, 3H, NCH3); 2.29 (m, 2H, (CH2)3CH2NCH3); 3.33
(s, 2H, NCH2Ph); 3.79 (m, 2H, CH2(CH2)3NCH3); 4,40 (s, 2H, H3-3’ CH2 benzox.); 6,85 (m, 4H,
arom. benzox.); 7.16 (m, 5H, arom. Ph). MS: m/z 325 [MH+].
4.2.7.16 2-(4-((4-Chlorobenzyl)(methyl)amino)butyl)-2H-benzo[b][1,4]oxazin-3(4H)-one 5b
1
Yellow oil; yield: 88%; I.R.: (nujol, cm-1) 1682 (C=O). H-NMR: (CDCl3-TMS; 400 MHz) δ:
1.46-1.60 (m, 4H, CH2CH2CH2CH2); 2.04 (s, 3H, NCH3); 2.30 (m, 1H, (CH2)3CH2NCH3); 3.33
(s, 3H, NCH2Ph e (CH2)3CH2NCH3); 3.78 (m, 2H, CH2(CH2)3NCH3); 4,41 (s, 2H, H3-3’ CH2
benzox.); 6,85 (m, 4H, arom. benzox.); 7.12 (m, 4H, arom. Ph). MS: m/z 359 [MH+], 361
[MH++2].
4.2.7.17 2-(4-Benzyl(methyl)amino)butyl)-2H-benzo[b][1,4]thiazin-3(4H)-one 6a
1
Yellow oil; yield: 38%; I.R.: (nujol, cm-1) 1674 (C=O). H-NMR: (CDCl3-TMS; 400 MHz) δ:
1.49-1.59 (m, 4H, CH2CH2CH2CH2); 2.09 (s, 3H, NCH3); 2.33 (m, 1H, (CH2)3CH2NCH3);
3.21-3.24 (m, 3H, NCH2Ph e 1H (CH2)3CH2NCH3); 3.40 (s, 2H, H3-3’ CH2 benzothiaz.); 3.91
(m, 2H, -CH2(CH2)3N-CH3); 6,90-7.20 (m, 9H, arom. benzothiaz. and Ph). MS: m/z 341 [MH+];
4.2.7.18 2-(4-((4-Chlorobenzyl)(methyl)amino)butyl)-2H-benzo[b][1,4]thiazin-3(4H)-one 6b
1
Yellow oil; yield: 52%; I.R.: (nujol, cm-1) 1651 (C=O). H-NMR: (CDCl3-TMS; 400 MHz) δ:
1.42-1.54 (m, 4H, CH2CH2CH2CH2); 2.02 (s, 3H, NCH3); 2.27 (m, 1H, (CH2)3CH2NCH3);
3.17-3.20 (m, 3H, NCH2Ph e 1H (CH2)3CH2NCH3); 3.32 (s, 2H, H3-3’ CH2 benzothiaz.); 3.87