Synthesis, Cytotoxicity Evaluation, and Computational Insights of Novel 1,4-Diazepane-Based Sigma Ligands

Article abstract of DOI:10.1021/acsmedchemlett.9b00524

Among several potential applications, sigma receptor ligands can be used as antipsychotics, antiamnesics, and against other neurodegenerative disorders as well as neuroprotective agents. We present herein a new series of diazepane-containing derivatives as σR ligands obtained by a conformational expansion approach of our previously synthesized piperidine-based compounds. The best results were reached by benzofurane 2c, 3c and quinoline 2d, 3d-substituted diazepane derivatives, which showed the highest σR affinity. The cytotoxic activities of synthesized compounds were evaluated against two cancer cell lines, and the results indicated that none of the compounds induced significant toxicity in these cells. We also evaluated the antioxidant activity by radical scavenging capacity of our best compounds on ABTS and H₂O₂. The results obtained reveal that our new derivatives possess an excellent antioxidant profile and could be protective for the cells. Overall, the benzofurane derivative 2c due to its strong interaction with the active site of the receptor, as confirmed by molecular dynamic simulations, emerged as the optimum compound with high σ1R affinity, low cytotoxicity, and a potent antioxidant activity.

Full text of DOI:10.1021/acsmedchemlett.9b00524

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Letter
Synthesis, Cytotoxicity Evaluation and Computational
Insights of Novel 1,4-Diazepane-Based Sigma Ligands
Daniele Zampieri, Sara Fortuna, Antonella Calabretti, Maurizio Romano, Renzo
Menegazzi, Dirk Schepmann, Bernhard Wünsch, and Maria Grazia Mamolo
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00524 • Publication Date (Web): 16 Dec 2019
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Synthesis, Cytotoxicity Evaluation and Computational Insights of Novel
1,4-Diazepane-Based Sigma Ligands
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Daniele Zampieri†*, Sara Fortuna†, Antonella Calabretti†, Maurizio Romano¥, Renzo Menegazzi¥, Dirk
Schepmann‡, Bernhard Wünsch‡, and Maria Grazia Mamolo†.
†Department of Chemical and Pharmaceutical Sciences, P.le Europa 1-Via Giorgieri 1, University of Trieste, 34127 Trieste,
Italy.
¥Department of Life Sciences, Via Valerio 28/1, University of Trieste, 34127 Trieste, Italy.
‡Institute of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149 Münster, Germany.
KEYWORDS. Sigma receptor, molecular dynamics, binding studies, cytotoxicity, antioxidant.
ABSTRACT: Among several potential applications, sigma receptors ligands can be used as antipsychotics, antiamnesic and against
other neurodegenerative disorders as well as neuroprotective agents. We present herein a new series of diazepane-containing
derivatives as R ligands obtained by a conformational expansion approach of our previously synthesized piperidine-based
compounds. The best results were reached by benzofurane 2c, 3c and quinoline 2d, 3d -substituted diazepane derivatives, which
showed the highest R affinity. The cytotoxic activities of synthesized compounds were evaluated against two cancer cell lines, and
the results indicated that none of the compounds induced significant toxicity in these cells. We also evaluated the antioxidant
activity by radical scavenging capacity of our best compounds on ABTS and H2O2. The results obtained reveal that our new
derivatives possess an excellent antioxidant profile and could be protective for the cells. Overall, the benzofurane derivative 2c due
to its strong interaction with the active site of the receptor, as confirmed by molecular dynamic simulations, emerged as the
optimum compound with high 1R affinity, low cytotoxicity, and a potent antioxidant activity.
The sigma receptors ( σ R) are a class of proteins initially
classified, by Martin and coworkers,1 as a subtype of the
opiate receptors. Further studies revealed them to be a
different receptor class comprising two distinct subtypes: σ1
and σ 2.2-5 The R is a chaperone protein, cloned in 1996
from several tissues including human, consisting of 223 amino
acids6,7 with a MW of 25.3 kDa.8 Crystallized twenty years
later it revealed a trimeric protein organization.9 The σ 1R
subtype is primarily localized to mitochondria-associated ER
membranes (MAM) of neuronal and peripheral cells, such as
cardiac myocytes and hepatocytes. This receptor can also
translocate to the plasma membrane or ER-membrane and
regulate the activity of other proteins by modulating different
ionic channels via an IP3-indipendent mechanism.10,11 The
Rs have neuroprotective and antiamnesic activity,12,13
modulate opioid analgesia14 as well as drug addiction,15 and
their antagonists seem to be effective against the negative
manifestations of schizophrenia without producing
extrapyramidal side effects.16,17 In addition, several studies
suggest a role for R in tumor biology, since its expression
increased in some cancers.18
The 2Rs are overexpressed in many cancer cell lines
including lung cancer,26,27 breast cancer,28 ovarian cancer,29
glioma cancer30 and gastric cancer.31 In this context, since 2R
agonists can induce tumor cell death, they have been proposed
as potential antitumor drugs. On the other hand, the 2Rs are
widely expressed in cerebellum, red nucleus, and substantia
nigra, and are a potential target for the treatment of movement
disorders and of neuroleptic-induced acute dystonia.32 In
addition, 1R antagonists as well as 2R agonists can
modulate neuropathic pain.33,34
In the last decade, our group has synthesized and biologically
evaluated an extensive series of compounds both of
preferential affinity for R and R subtypes. Following up
our studies in this field, we report herein the development of a
new class of sigma ligands designed through the expansion of
the conformational selection paradigm applied to our
previously synthesized piperidine-based R ligands 1 (Figure
1).35
After 40 years from the discovery of Rs,1 in 2017, the 2R
subtype has been purified and identified as transmembrane
protein-97 (TMEM97),19 an endoplasmic-reticulum-resident
transmembrane
molecule
implicated
in
cholesterol
homeostasis due to its association with the lysosomal
transporter NPC1.20,21 The 2R crystal structure is still elusive,
but several pharmacophore models have been proposed.22-25
Figure 1. Conformational expansion approach starting from
previously synthesized sigma ligands 1.
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In addition to the spacer replacement, we opted to expand the subtype plays important roles in synaptic transmission and
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new series of compounds by using various aromatic
fragments, including heterocycles, both monocycles and
bicycles, linked to the amide carbonyl group. Moreover, in
order to verify the influence on the sigma affinity of the
substitution on benzyl moiety, we decided to retain the
unsubstituted phenyl ring, present in many 1R ligands and
the 2,4-dimethyl substituted phenyl ring, typical of several
2R ligands.36 The synthesis of our new diazepane-based
derivatives 2a-g and 3a-g is depicted in Scheme 1.
plasticity, learning, memory and other physiological and
pathological processes.37,38 Hence, antagonist of GluN2b
subunit are of interest as neuroprotective drugs for various
CNS disorders. The radioligand used in the competition assay
was [3H]-labeled Ifenprodil, a prototypical allosteric inhibitor
of GluN2b subunit (Supporting Information).
For compounds with affinity value higher than 100 nM, only
one measure was performed. The R, R and GluN2b
affinities of compounds 2a-g and 3a-g are presented in Table
1.
9
The synthetic route of our new series of compounds
(Supporting information) was carried out by treating the
appropriate, commercially available, 1-benzyl-1,4-diazepane,
which was made to react with the appropriate aroyl chloride to
give the corresponding first subseries 2a-g. These compounds
did not need further purification after the classical work-up.
The 2,4-dimethyl derivatives 3a-g were obtained in three
reaction steps, starting from 1-Boc-1,4-diazepane and the
corresponding aroyl chloride to provide the acylated
intermediates 4a-g. The cleavage of protecting N-Boc group
with TFA, led to the intermediates 5a-g which were
subsequently N-alkylated, with a direct reductive amination
using 2,4-dimetylbenzaldheyde and NaCNBH3, to give the
final subseries 3a-g. These compounds were purified by
DCVC technique.
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Table 1. Affinities of compounds 2a-g and 3a-g towards 1,
2 and GluN2b receptors.
Ki (nM) a
Cmpd
R
Ar
GluN2b
1
2
2a
2b
333
560
1 %
147
297
0 %
2c
8.0 ± 0.6
19 ± 3.2
47 ± 15
47 ± 19
1.6 M
1.5 M
Scheme 1. Synthesis of compounds 2a-g and 3a-ga
2d
H
2e
2f
267
495
187
0 %
9 %
41 ± 12
2g
3a
116
690
0 %
0 %
0 %
3.9 M
3b
3c
336
3 %
1.8 M
20 ± 5
28 ± 11
59 ± 12
3d
3e
29±10
254
35±4
108
116
2,4(CH3)2
2 M
3f
70 ± 7
849
102
79 ± 19
294
aa) DCM, Et3N, 0°C; b) DCM, TFA, rt; c) DCM, 2,4-
dimethylbenzaldheyde, NaCNBH3 rt.
3g
3.9 M
The 1R and 2R 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 (di-o-tolylguanidine) as radioligand in the 2R
assay. Compounds 2a-g and 3a-g were tested against 1R and
2R of animal origin, prepared from guinea pig brain and rat
liver membranes by homogenization, centrifugation and
washing of the respective tissues. We also performed a
competition experiment towards GluN2b subunit containing
NMDA receptors in a radioligand binding assay. This receptor
Ifp
Hal
-
-
-
-
-
-
125 ± 24
6.3 ± 1.6
89 ± 29
98 ± 34
78 ± 2.3
57 ± 18
10 ± 7.0
ntb
DTG
nt
aOnly compounds with highest affinities (<100 nM) were
tested in triplicates. For low-affinity compounds, the
competition curves were recorded only once (single value),
whereas the inhibition of the radioligand binding (shown as
%) was assayed at a test compound concentration of 1 μ M;
bnot tested.
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with both compounds through van der Waals interactions are
circled in red. Thr 181 (circled in green) form a hydrophobic
interaction with compounds 2a, while a hydrogen bond with
compound 2c.
From the obtained data we can summarize the following: i) the
2
bulky diazepane spacer retained, or even improved, the R
affinity to both 1 and 2, with respect to piperidine ring; ii)
only bicycle derivatives displayed moderate to high affinity
towards both R subtypes, while the corresponding
monocycles analogues were weak inhibitors or avoiding of R
affinity; iii) the best results against 1R was reached by
benzofurane derivative 2c, while its 2,4-dimethyl substituted
analogue 3c gave the best pan-affinity with Ki values of 8.0
and 28 nM towards both R subtypes and also the best
GluN2b inhibition value of 59 nM; iv) the 2,4-dimethyl
substitution on benzyl moiety derivatives, improves the 2
over 1 affinity of the bicycle derivatives, as well as the
affinity towards GluN2b subunit receptor.
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To confirm the docking result and understand the different
behavior of the two compounds we ran 250 ns of molecular
dynamics (MD) simulation of the complexes in water solvent.
The ligand topologies were built with ATB.39 The topologies
were validated as the molecular mechanics minimized
structure of compound 2a had root mean square deviation
(RMSD) of 0.01007 nm with respect to the semi-empirical
minimized structure, while for compound 2c the same RMSD
was 0.0082 nm.
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The trajectories, scored with the Autodock Vina scoring
function,40 showed
a constant binding score for both
compounds (Figure 3a,b). For both systems the protein
backbone RMSD diverged along the dynamics up to 0.6 nm
(Figure 3c,d), as expected by simulating only a monomer of
the extracellular domain. The ligands RMSD with respect to
the fixed protein backbone, below 0.4 nm for compound 2c
(Figure 3c), revealed a major conformational change for
compound 2a (RMSD > 1.0 nm, Figure 3d). These variations
were not reflected in the protein backbone radius of gyration
constant at 1.6 nm for both systems (Figure 3e,f). Larger
protein rearrangements peaks at residues 190-200 for both
systems, as observed in the protein backbone root mean square
fluctuation (RMSF > 0.5 nm, Figure 3g,h). These are part of
the helical structure delimitating the pocket but not directly
interacting with the ligands (Figure 3i,j and insets in Figure 2).
Along the whole simulated time ligands were not observed to
leave the pocket with compound 2c maintaining its position
but rotating its benzene ring (Figure 3i) and compound 2a
changing position by both flipping its orientation and further
sliding inside the pocket (Figure 3j), a movement not
associated with a gain in binding energy as calculated by
Autodock Vina score (Figure 3b).
To get insight the interaction of our compounds into the 1R
active site, we performed a computational assessment of the
best 1R ligand of the series, 2c, in comparison with its
monocycle analogue 2a.
We prepared the 1R following our previously procedure36
(Supporting information) and we docked compounds 2a and
2c to the target by following the same protocol.
The comparison between the optimum pose obtained for each
compound (Figure 2) suggests that compound 2c slides further
into the pocket than 2a pushing its benzene ring to interact
with Trp164 and Phe133, closed at the bottom by Tyr206, and
forming an H-bond with Thr181. Moreover, compound 2c
optimum pose is predicted to be in touch through 17
hydrophobic interactions with target residues and a hydrogen
bond with Thr181 (inset in Figure 2a). Also compound 2a
interacts with the target with 17 hydrophobic interactions
including Thr181 and shares 14 of those interactions with
compound 2c (inset in Figure 2b).
Figure 3. Molecular dynamics simulations analysis for compound
2c (left column) and 2a (right column): (a,b) Autodock Vina
score, (c,d) protein backbone RMSD (green) and ligands RMSD
in the protein backbone frame (blue), (e,f) protein backbone
radius of gyration,(g,h) protein backbone RMSF with values
above 0.25 nm highlighted in magenta. All values measured with
respect to the starting configuration correspond to the minimized
optimum pose identified by docking. Simulation snapshots taken
at the lowest Autodock Vina score (circled in panels a,b) for (i)
compound 2c and (j) compound 2a. Protein residues with RMSF
Figure 2. 3D putative of (a) compound 2c and (b) compound 2a
in the optimum AutoDock pose. Protein residues interacting with
both compounds by van der Waals interactions are highlighted in
red, Thr 181 in green, other interacting residues (Trp 164, Phe
133, Tyr 206 in panel (a) and Thr202, Val84, Ile124 in panel (b))
are white. In the insets: schematic diagrams of the interaction
between the receptor and the respective compounds in the same
optimum AutoDock pose. Protein residues interacting with the
compounds by van der Waals interactions are highlighted in red,
while hydrogen bonds are indicated by green dotted lines. The
hydrogen bonds distances are also indicated. Residues interacting
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that 1R agonists promote cell survival,41,42 these results
support the hypothesis that compounds 2c, 2d, 3c and 3d, can
above 0.25 nm are highlighted (magenta). The starting ligand
configuration is also indicated (white).
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be included in this category.
The MM/PBSA analysis confirmed that the van der Waals
interactions were the major liable for binding the compounds,
a contribution that is more relevant for compound 2c which
also benefits of a less unfavorable polar solvation energy than
that calculated for compound 2a (Figure 4a,b). This result is
reflected by the single amino acid contribution to the binding
energy. Indeed, for compound 2a several amino acids opposed
to the binding with Arg119 and Glu172 contribution larger
than 1 kcal/mol, followed by Gln135, Asp126, and His154.
Instead, compound 2c is synergistically kept bound to its
interacting site by several residues (Figure 4d). More in details
(Figure 4e,f) there are seven residues (namely Leu105, Ile124,
Phe107, Trp89, Val84, Leu182, and Tyr103) contributing to
the binding energy with more than 0.9 kcal/mol with the major
contributing force to be ascribed to van der Waals forces and
hydrophobic interactions.
Motivated by these results, we evaluated the in vitro
antioxidant activity of the same compounds tested in the
aforementioned cytotoxic assay. We tested the ability to
scavenge ABTS derived radicals and H2O2 oxidant. Ascorbic
acid and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid) were used as standard antioxidants in
comparison test. The assayed compounds potently inhibited
ABTS radicals and H2O2, compared to the standards (Table 2).
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Table 2. In vitro antioxidant activity of compounds 2c, 2d,
3c and 3d.
IC50 (g/mL)a
Cmpd
ABTS
H2O2
2c
12.71 ± 0.25
14.26 ± 0.15
10.05 ± 0.09
9.43 ± 0.11
12.75 ± 0.12
18.73 ± 0.26
15.89 ± 0.18
20.35 ± 0.27
18.56 ± 0.31
17.44 ± 0.18
19.27 ± 0.54
20.38 ± 0.19
2d
3c
3d
Ascorbic Acid
Trolox
aAll measurements were performed in triplicate
Among the series, the dimethyl substituted compounds 3c and
3d exhibited a significant radical scavenging capacity on both
ABTS and H2O2 with values of 10.05 and 18.56 M/mL for 3c
and 9.43 and 17.44 M/mL for 3d, lower than compared
standards, ascorbic acid and Trolox (12.75, 19.27 M/mL and
18.73, 20.38 M/mL, respectively).
Furthermore, in order to evaluate their drug likeness and the
potential ability to cross the BBB, the compounds 2a-g and
3a-g were also in-silico scored for their physiochemical and
pharmacokinetic parameters (ADME) by using the extended
version of Lipinski’s rule of five. All the compounds were
found to be BBB permeant and none of them violate any
Lipinski’s RO5 (Supporting information).
Figure 4. MMPBSA analysis: energetic contributions to the
receptor binding of (a) compound 2c, and (b) compound 2a, and
amino acids contribution to the total binding energy of (c)
compound 2a, and (d) compound 2c with highlighted the amino
acids opposing to the binding with predicted energy larger than
0.3 kcal/mol; (e) details of the contributions of each amino acid
with binding energy larger than 0.9 kcal/mol for compound 2c; (f)
snapshot of the complex 1/2c with highlighted amino acids
(shades of red) contributing to the binding energy with more than
± 0.9 kcal/mol. All data averaged over the last 150 ns of the
molecular dynamics trajectory.
Overall the larger ring keeps the ligand fixed in its position,
which is more accessible to Thr181 for H-bonding. A larger
number of contacts deep into the pocket further inhibited the
molecular rearrangement inside the protein pocket.
In conclusion, we have synthesized a new series of ring-
expanded diazepane-based compounds. The new series
showed enhanced affinity than its original counterpart35
towards both R subtypes and, among the series, the
benzofurane derivative 2c showed the best 1R affinity and
molecular dynamic simulations confirmed a strong interaction
with the active site of the receptor. The benzofurane and
quinoline derivatives 2c, 3c and 2d, 3d, displayed the best Ki
values and a safe profile towards two human cell lines.
Altogether these data, along with the documented radical
scavenging and cell survival promoting activities, support the
interest for further studies aiming at evaluating the potential
neuroprotective activity of this novel series of 1R ligands.
The effects of this new set of 1R ligands on cell health were
evaluated by testing the cytotoxic response of the human
pancreatic carcinoma (PANC1) and human neuroblastoma
(SH-SY5Y) cell lines, selected because they express high
levels of 1R.18 To this aim, we selected the most interesting
compounds (2c, 2d, 3c and 3d) and tested their potential
toxicity by MTT assay (Supporting information, Table S1 and
Table S2). The experiments revealed that none of our
diazepane-containing
derivatives
showed
significant
cytotoxicity at different concentrations, with the exception of
compound 3d which exhibited a moderate toxicity toward
PANC1 cells, but only at 100 M concentration (viability of
51%). Interestingly, compounds 2c and 2d, which exhibited
the best 1R affinities (Ki = 8 and 19 nM, respectively),
resulted to be the less toxic (viability: 127 and 196% at 50
M; 98 and 127% at 100 M, in SH-SY5Y and PANC1,
respectively). Therefore, considering the general consensus
ASSOCIATED CONTENT
Supporting Information
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distribution of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine
The Supporting Information is available free of charge on the
(DTG) binding sites in guinea pig brain: a comparative study, Brain
Res. 1992, 581, 33–38.
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3
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ACS Publications website.
(5)
Bouchard, P.; Quirion, R. [3H]1,3-di(2-tolyl)guanidine and
Detailed experimental, synthetic procedures and characterization
of compounds, additional computational details, pharmacology
and cytotoxicity assays, antioxidant activity, drug likeness
prediction (PDF).
[3H](+)pentazocine binding sites in the rat brain: autoradiographic
visualization of the putative sigma1
sigma2 receptor subtypes,
Neuroscience 1997, 76, 467–477.
(6)
Hanner, M.; Moebius, F.F.; Flandorfer, A.; Knaus, H.G.;
Striessnig, J.; Kempner, E.; Glossmann, H. Purification, molecular
cloning, and expression of the mammalian sigma1-binding site. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 8072-8077.
AUTHOR INFORMATION
9
(7)
Kekuda, R.; Prasad, P.D.; Fei, Y.J.; Leibach, F.H.;
Corresponding Author
Ganapathy, V. Cloning and functional expression of the human type 1
sigma receptor (hSigmaR1). Biochem. Biophys. Res. Commun. 1996,
229, 553−558.
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* Daniele Zampieri; tel. +390405587858; dzampieri@units.it
Author Contributions
(8)
Hellewell, S.B.; Bruce, A.; Feinstein, G.; Orringer, J.;
Williams, W.; Bowen, W.D. Rat liver and kidney contain high
densities of 1 and 2 receptors: characterization by ligand binding and
photoaffinity labeling, Eur. J. Pharmacol., Mol. Pharmacol. Sect.
1994, 268, 9−18.
DZ synthesized and characterized the compounds and wrote the
manuscript; SF generated the computational results and analysis;
AC performed the antioxidant assay; MR and RM tested the
cytotoxicity of the compounds and wrote the manuscript; DS and
BW performed the binding assay; DZ and MGM conceived the
project. The manuscript was written through contributions of all
authors. All authors have given approval to the final version of the
manuscript.
(9)
Schmidt, H.R.; Zheng, S.; Gurpinar, E.; Koehl, A.;
Manglik, A.; Kruse, A.C. Crystal structure of the human 1 receptor,
Nature 2016, 532, 527−530.
(10)
of sigma-1 receptors, Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 491-
496.
Hayashy, T.; Su, T.-P. Regulating ankyrin dynamics: Roles
(11)
Wu, Z.; Bowen, W.D. Role of sigma-1 receptor C-terminal
Notes
segment in inositol 1,4,5-trisphosphate receptor activation:
constitutive enhancement of calcium signaling in MCF-7 tumor cells,
J. Biol. Chem. 2008, 283, 28198-28215.
The authors declare no competing financial interest.
(12)
Cobos, E.J.; Entrena, J.M.; Nieto, F.R.; Cendan, C.M.;
ACKNOWLEDGMENT
Pozo, E.D. Pharmacology and therapeutic potential of sigma-1
ligands, Curr. Neuropharmocol. 2008, 6, 344-366.].
(13)
amnesic potentials of sigma (sigma) receptor ligands, Prog.
Neuropsychopharmacol. Biol. Psychiatry. 1997, 21, 69-102.
(14)
G.W.; Enhanced kappa-opioid receptor-mediated analgesia by
antisense targeting the sigma1 receptor, Eur. J. Pharmacol. 1997,
331, (1997) R5-6.
We thank Dr. Fabio Hollan (University of Trieste, DSCF) for the
MS data. We acknowledge the CINECA Awards N.
HP10CKYM0P, 2019, for the availability of high performance
computing resources and support.
Maurice, T.; Lockhart, B.P. Neuroprotective and anti-
King, M.; Pan, Y.X.; Mei, J.; Chang, A.; Xu, J.; Pasternak,
ABBREVIATIONS
1R and 2R, sigma-1 and sigma-2 receptor; ABTS, 2,2'-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid; ADME, absorption
distribution metabolism excretion; ATB, automated topology
builder; BBB, blood brain barrier; Boc, tert-butyloxycarbonyl;
CNS, central nervous system; DCM, dichloromethane; DCVC,
direct column vacuum chromatography; DTG, di-o-
tolylguanidine; Et3N, triethylamine; ER, endoplasmatic reticulum;
DMEM, Dulbecco’s Modified Eagle’s Medium; MAM,
mitochondrion-associated membrane; MD, molecular dynamics;
MMPBSA, molecular mechanics Poisson-Boltzmann surface
area; MW, molecular weight; NMDA, N-methyl-D-aspartic acid;
NPC1, Niemann-Pick cholesterol transporter type1; RMSD, root
mean square deviation; RMSF root mean squared fluctuation;
RO5, rule of five; TLC, thin-layer chromatography; TFA,
trifluoroacetic acid; TMEM97, transmembrane protein-97;
(15)
McCracken, K.A.; Bowen, W.D.; Walker, F.O.; De Costa,
B.; Matsumoto, R.R.; Two novel sigma receptor ligands, BD1047 and
LR172, attenuate cocaine-induced toxicity and locomotor activity,
Eur. J. Pharmacol. 1999, 370, 225-232.
(16)
Modell, S.; Nober, D.; Holzbach, R. Efficacy and safety of
an opiate sigma-receptor antagonist (SL 82.0715) in schizophrenic
patients with negative symptoms: an open dose-range study,
Pharmacopsychiatry 1996, 29, 63-66.
(17)
Huber, M.T.; Gotthardt, U.; Schreiber, W.; Krieg, J.C.
Efficacy and safety of the sigma receptor ligand EMD 57445
(panamesine) in patients with schizophrenia: an open clinical trial,
Pharmacopsychiatry 1999, 32, 68-72.
(18)
Kim, F.J.; Maher, C.M. Sigma1 Pharmacology in the
Context of Cancer. Handbook of Experimental Pharmacology:
Springer, 2017, pp 237-308.
(19)
S.F.; Kruse, A.C. Identification of the gene that codes for the 2
receptor, Proc. Nat. Ac. Sc. 2017, 114, 7160–7165.
(20)
T.; Wirkner, U.; Erfle, H.; Muckenthaler, M.; Pepperkok, R.; Runz,
H. Identification of cholesterol-regulating genes by targeted RNAi
screening, Cell. Metab. 2009, 10, 63-75.
Alon, A.; Schmidt, H.R.; Wood, M.D.; Sahn, J.J.; Martin,
REFERENCES
(1)
Martin, W.R.; Eades, C.E.; Thompson, J.A.; Huppler, R.E.;
Bartz, F.; Kern, L.; Erz, D.; Zhu, M.; Gilbert, D.; Meinhof,
Gilbert, P.E. The effects of morphine- and nalorphine- like drugs in
the nondependent and morphine-dependent chronic spinal dog. J.
Pharmacol. Exp. Ther. 1976, 197, 517-532.
(2)
Su, T.-P. Evidence for sigma opioid receptor: binding of
(21)
Riad, A.; Zeng, C.; Weng, C.C.; Winters, H.; Xu, K.;
Sigma-2
[3H]SKF-10047 to etorphine-inaccessible sites in guinea-pig brain. J.
Pharmacol. Exp. Ther. 1982, 223, 284–290.
Makvandi, M.; Metz, T.; Carlin, S.; Mach, R.H.
Receptor/TMEM97 and PGRMC-1 Increase the Rate of
Internalization of LDL by LDL Receptor through the formation of a
Ternary Complex, Sc. Rep. 2018, 8, 16845.
(3)
Quirion, R.; Bowen, W.D.; Itzhak, Y.; Junien, J.L.;
Musacchio, J.M.; Rothman, R.B.; Su, T.-P.; Tam S.W.; Taylor, D.P.
A proposal for the classification of sigma binding sites Trends
Pharmacol. Sci. 1992, 13, 85–86.
(22)
Cratteri, P.; Romanelli, M.N.; Cruciani, G.; Bonaccini, C.;
Melani, F. Grind-derived pharmacopore model for a series of alfa-
trophanyl derivative ligands of the sigma-2 receptor, J. Comuput.
Aided Mol. Des. 2004, 18, 361-374.
(4)
Walker, J.M.; Bowen, W.D.; Goldstein, S.R.; Roberts,
A.H.; Patrick, S.L.; Hohmann, A.G.; DeCosta, B. Autoradiographic
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 10
(23)
Laurini, E.; Zampieri, D.; Mamolo, M.G.; Vio, L.; Zanette,
(42)
Hayashi, T.; Su, T.P., Sigma-1 receptor chaperones at the
C.; Florio, C.; Posocco, P.; Fermeglia, M.; Pricl, S.
A
3D-
ER-mitochondrion interface regulate Ca(2+) signaling and cell
survival. Cell, 2007, 131, 596-610.
1
2
3
4
Pharmacophore model for 2 receptors based on a series of
substituted benzo[d]oxazol-2(3H)-one derivatives. Bioorg. Med.
Chem. Lett. 2010, 20, 2954-2957.
(24)
Rhoades, D.J.; Kinder, D.H.; Mahfouz, T.M.
A
5
6
7
8
Comprehensive Ligand Based Mapping of the 2 Receptor Binding
Pocket. Med. Chem. 2014, 10, 98-121.
(25)
Foresta, G.; Rescifina, A.; Marrazzo A.; Dichiara, M.;
Pistarà, V.; Pittalà, V.; Prezzavento, O.; Amata, E. Hyphenated 3D-
QSAR statistical model-scaffold hopping analysis for the
identification of potentially potent and selective sigma-2 receptor
ligands. Eur. J. Med. Chem. 2017, 139, 884-891.
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21
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23
24
25
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27
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42
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44
45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
(26)
Han K.Y.; Gu, X.; Wang, H.R.; Liu, D.; Lv, F.Z.; Li, J.N.
Overexpression of MAC30 is associated with poor clinical outcome in
human non-small cell lung cancer, Tumour Biol. 2013, 34, 821-825.
(27)
Y.; Sheng, Y.L. Prognostic value of MAC30 expression in human
pure squamous cell carcinomas of the lung, Asian Pac. J. Cancer
Prev. 2016, 17, 2705-2710.
Ding, H.; Gui, X.H.; Lin, X.B.; Chen, R.H.; Cai, H.R.; Fen,
(28)
Xiao, M.; Li, H.; Yang, S.; Huang, Y.; Jia, S.; Wang, H.;
Wang, J.; Li, Z. Expression of MAC30 protein is related to survival
and clinicopathological variables in breast cancer, J. Surg. Oncol.
2013, 107, 456-462.
(29)
Yang, S.; Li, H.; Liu, Y.; Ning, X.; Meng, F.; Xiao, M.;
Wang, D.; Lou, G.; Zhang, Y. Elevated expression of MAC30
predicts lymph node metastasis and unfavourable prognosis in
patients with epithelial ovarian cancer, Med. Oncol. 2013, 30, 324.
(30)
Qiu, G.; Sun, W.; Zou, Y.; Cai, Z.; Wang, P.; Lin, X.;
Huang, J.; Jiang, L.; Ding, X.; Hu, G.RNA interference against
TMEM97 inhibits cell proliferation, migration, and invasion in
glioma cells, Tumour Biol. 2015, 36, 8231-8238.
(31)
Xu, X.Y.; Zhang, L.J.; Yu, Y.Q.; Zhang, X.T.; Huang,
W.J.; Nie, X.C.; Song, G.Q. Downregulated MAC30 expression
inhibits proliferation and mobility of human gastric cancer cells, Cell.
Physiol. Biochem. 2014, 33, 1359-1368.
(32)
Rousseaux C.G.; Greene, S.F., Sigma receptors [σRs]:
biology in normal and diseased states. J. Recept. Signal Transduct
Res. 2016, 36, 327–388.
(33)
1 receptor: a new therapeutic target for pain. Eur. J. Pharmacol. 2013,
716, 78−93.
(34)
Zamanillo, D.; Romero, L.; Merlos, M.; Vela, J.M., Sigma
Sahn, J.J.; Mejia, G.L.; Ray, P.R.; Martin, S.F.; Price, T.J.,
2 Receptor/Tmem97 Agonists Produce Long Lasting
Sigma
Antineuropathic Pain Effects in Mice”, ACS Chem. Neurosci. 2017, 8,
1801-1811.
(35)
M.; Pricl, S.; Mamolo, M.G. Synthesis and receptor binding studies of
some new arylcarboxamide derivative as sigma-1 ligands. Bioorg.
Med. Chem. Lett. 2014, 24, 1021-1025.
Zampieri, D.; Laurini, E.; Vio, L.; Golob, S.; Fermeglia,
(36)
Zampieri, D.; Fortuna, S.; Calabretti, A.; Romano, M.;
Menegazzi, R.; Schepmann, D.; Wünsch, B.; Collina, S.; Zanon, D.;
Mamolo, M.G. Discovery of new potent dual sigma receptor/GluN2b
ligands with antioxidant property as neuroprotective agents. Eur. J.
Med. Chem. 2019, 180, 268-282.
(37) Riedel, G.; Platt, B.; Micheau, J. Glutamate receptor
function in learning and memory. Behav. Brain Res. 2003, 140, 1-47.
(38)
Paoletti, P..; Bellone, C.; Zhou, Q. NMDA receptor subunit
diversity: impact on receptor properties, synaptic plasticity and
disease. Nat. Rev. Neurosci. 2013, 14, 383-400.
(39)
Nair, P.C.; Oostenbrink, C.; Mark A.E. An Automated Force Field
Topology Builder (ATB) and Repository: Version 1.0. J. Chem.
Theory and Comput. 2011, 7, 4026-4037.
Malde, A.K., Zuo, L.; Breeze, M.; Stroet, M.; Poger, D.;
(40)
Trott, O.; Olson A.J., AutoDock Vina: improving the speed
and accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J. Comput. Chem. 2010, 31, 455-
461.
(41)
Maurice, T.; Su, T.P., The pharmacology of sigma-1
receptors, Pharmacol. Ther. 2009 124, 195-206.
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Ar
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Ar
N
O
N
R
R
N
N
Et
O
2 a-g (R = H); 3 a-g (R = 2,4(CH3)2
Scheme 1. Synthesis of compounds 2a-g and 3a-ga
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Kiσ1 = 13.6 - 373 nM
Kiσ2 = 270 - >1000 nM
Ar = Furyl (a), thiophenyl (b), benzofuranyl (c), quinolinyl
(d), pyridin-4-yl (e), naphtalen-1-yl (f), phenyl (g).
O
Figure 1. Conformational expansion approach starting from
previously synthesized sigma ligands 1.
N
NH
N
N
a)
Ar
(74-97%)
2a-g
O
a)
(91-99%)
b)
Boc
Boc
N
NH
N
N
Ar
(65-91%)
O
4a-g
H3C
O
N
N
c)
(29-40%)
Ar
HN
N
Ar
5a-g
3a-g
H3C
Ar = Furyl, thiophenyl, benzofuranyl, quinolinyl, pyridin-4-yl, naphtalen-1-yl,
phenyl.
aa) DCM, Et3N, 0°C; b) DCM, TFA, rt; c) DCM, 2,4-
dimethylbenzaldheyde, NaCNBH3 rt.
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Table 1. Affinities of compounds 2a-g and 3a-g towards
s
1,
s
2 and GluN2b receptors.
Ar
O
N
R
N
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Ki (nM) a
Cmpd
R
Ar
s
1
s
2
GluN2b
O
2a
2b
333
147
560
297
1 %
S
0 %
O
2c
8.0 ± 0.6
19 ± 3.2
47 ± 15
47 ± 19
1.6 µM
1.5 µM
N
2d
H
2e
2f
267
495
187
0 %
9 %
N
41 ± 12
2g
3a
116
690
0 %
0 %
0 %
O
3.9 µM
S
3b
3c
336
3 %
1.8 µM
O
20 ± 5
28 ± 11
59 ± 12
N
3d
3e
29±10
254
35±4
108
116
2,4(CH3)2
2 µM
N
3f
70 ± 7
102
79 ± 19
294
3g
849
3.9 µM
Ifp
Hal
-
-
-
-
-
-
125 ± 24
98 ± 34
10 ± 7.0
ntb
6.3 ± 1.6 78 ± 2.3
89 ± 29 57 ± 18
DTG
nt
aOnly compounds with highest affinities (<100 nM) were
tested in triplicates. For low-affinity compounds, the competition
curves were recorded only once (single value), whereas the
inhibition of the radioligand binding (shown as %) was assayed
at a test compound concentration of 1 µM; bnot tested.
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Figure 4. MMPBSA analysis: energetic contributions to the
receptor binding of (a) compound 2c, and (b) compound 2a, and
amino acids contribution to the total binding energy of (c)
compound 2a, and (d) compound 2c with highlighted the amino
acids opposing to the binding with predicted energy larger than
0.3 kcal/mol; (e) details of the contributions of each amino acid
with binding energy larger than 0.9 kcal/mol for compound 2c;
(f) snapshot of the complex s1/2c with highlighted amino acids
(shades of red) contributing to the binding energy with more than
± 0.9 kcal/mol. All data averaged over the last 150 ns of the
molecular dynamics trajectory.
Figure 2. 3D putative of (a) compound 2c and (b) compound 2a
in the optimum AutoDock pose. Protein residues interacting with
both compounds by van der Waals interactions are highlighted in
red, Thr 181 in green, other interacting residues (Trp 164, Phe
133, Tyr 206 in panel (a) and Thr202, Val84, Ile124 in panel (b))
are white. In the insets: schematic diagrams of the interaction
between the receptor and the respective compounds in the same
optimum AutoDock pose. Protein residues interacting with the
compounds by van der Waals interactions are highlighted in red,
while hydrogen bonds are indicated by green dotted lines. The
hydrogen bonds distances are also indicated. Residues
interacting with both compounds through van der Waals
interactions are circled in red. Thr 181 (circled in green) form a
hydrophobic interaction with compounds 2a, while a hydrogen
bond with compound 2c.
Table 2. In vitro antioxidant activity of compounds 2c, 2d,
3c and 3d.
IC50 (µg/mL)a
Cmpd
ABTS
H2O2
2c
12.71 ± 0.25
14.26 ± 0.15
10.05 ± 0.09
9.43 ± 0.11
12.75 ± 0.12
18.73 ± 0.26
15.89 ± 0.18
20.35 ± 0.27
18.56 ± 0.31
17.44 ± 0.18
19.27 ± 0.54
20.38 ± 0.19
2d
3c
3d
Ascorbic Acid
Trolox
aAll measurements were performed in triplicate
Figure 3. Molecular dynamics simulations analysis for
compound 2c (left column) and 2a (right column): (a,b)
Autodock Vina score, (c,d) protein backbone RMSD (green) and
ligands RMSD in the protein backbone frame (blue), (e,f) protein
backbone radius of gyration,(g,h) protein backbone RMSF with
values above 0.25 nm highlighted in magenta. All values
measured with respect to the starting configuration correspond to
the minimized optimum pose identified by docking. Simulation
snapshots taken at the lowest Autodock Vina score (circled in
panels a,b) for (i) compound 2c and (j) compound 2a. Protein
residues with RMSF above 0.25 nm are highlighted (magenta).
The starting ligand configuration is also indicated (white).
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