New piperidine-based derivatives as sigma receptor ligands. Synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.bmcl.2018.08.016

The sigma receptor (σR) family has been considered mysterious for a long time. In fact, the σ2R subtype has been cloned only recently, revealing its identity as TMEM97, a NPC1-binding protein involved in cholesterol biosynthesis and implicated in the pathogenesis of cancer and neurologic disorders. With the aim of developing new chemical entities gifted with σR affinity, herein we report the design and synthesis of new piperidine-based alkylacetamide derivatives with mixed affinity towards both σ1 and σ2R subtypes.

Full text of DOI:10.1016/j.bmcl.2018.08.016

Accepted Manuscript
New piperidine-based derivatives as sigma receptor ligands. Synthesis and
pharmacological evaluation
Daniele Zampieri, Maurizio Romano, Renzo Menegazzi, Maria Grazia Mamolo
PII:
S0960-894X(18)30680-2
https://doi.org/10.1016/j.bmcl.2018.08.016
BMCL 25996
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 June 2018
10 August 2018
13 August 2018
Please cite this article as: Zampieri, D., Romano, M., Menegazzi, R., Mamolo, M.G., New piperidine-based
derivatives as sigma receptor ligands. Synthesis and pharmacological evaluation, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.08.016
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Bioorganic & Medicinal Chemistry Letters
New piperidine-based derivatives as sigma receptor ligands. Synthesis and
pharmacological evaluation.
Daniele Zampieri,a* Maurizio Romano,b Renzo Menegazzi,b Maria Grazia Mamolo.a
a Department of Chemistry and Pharmaceutical Sciences, Piazzale Europa 1, University of Trieste, 34127, Trieste, Italy.
b Department of Life Sciences, Via Valerio 28/1, University of Trieste, 34127, Trieste, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The sigma receptor (R) family has been considered mysterious for a long time. In fact, the
2R subtype has been cloned only recently, revealing its identity as TMEM97, a NPC1-binding
protein involved in cholesterol biosynthesis and implicated in the pathogenesis of cancer and
neurologic disorders. With the aim of developing new chemical entities gifted with R affinity,
herein we report the design and synthesis of new piperidine-based alkylacetamide derivatives with
mixed affinity towards both 1 and 2R subtypes.
Keywords:
Sigma receptors (R)
Alkylacetamide derivatives
Affinity and selectivity
Cytotoxicity
© 2018 Elsevier Ltd. All rights reserved.
Even though more than 40 years have passed since the first
postulation of Martin and coworkers1, the sigma receptor (R)
family and, in particular, the 2R subtype, was considered
enigmatic. Recently, the 2R receptor has been identified with
TMEM97, an endoplasmic-reticulum-resident transmembrane
protein that, with its interactor NPC1, is involved in the
cholesterol metabolism2 and has a role in different cellular
processes, such as cell proliferation and cell signaling.
Moreover, the R are still very attractive because of their
implication in several pathologies, such as cancer and
neurodegenerative disorders. It’s well known that R agonists
promote cytoprotection, while R antagonists and R
agonists induce cytotoxicity. In particular, R are
overexpressed in many cancer cell lines, such as breast
carcinoma3,4, bladder cancer5 and pancreatic cancer cells6.
Different studies confirmed that they are localized in
mitochondria, endoplasmic reticulum, lysosomes and plasma
membrane of human breast cancer cells7,8. Moreover, a
correlation between R and progesterone receptor membrane
component 1 (PGRMC-1), a heme-1 domain protein promoting
tumorigenesis, has been established. Altogether these
observations support the hypothesis that R can exert a pivotal
role in signaling and apoptosis9. Furthermore, a study on human
melanoma cell line, demonstrates that the selective 2R agonist
Siramesine (SRMS), can lead cell death by multiple signaling
pathways such as caspase activation, autophagy (by inhibition
of mTOR kinase) and impaired cell-cycle progression10.
In the last decade, several research groups have been
involved in the field of R ligands, synthesizing and
biologically evaluating extensive series of compounds
exhibiting preferential affinity for R11a-f or R11g-i subtypes.
Moreover, in 2010 our group developed a R pharmacophore
model based on some benzoxazolone derivatives synthesized by
us12 and in 2012 we validate our three-dimensional R
model13,14
.
In our previous works11c,14, we have synthesized some new
acetamide 1 and arylamide 2 derivatives showing high affinity
property towards the 1 receptor, with Ki-values in the low
nanomolar range (Figure 1).
Corresponding author: Tel.: +390405587858; email: dzampieri@units.it

N
R2
R1
N
O
1
N
R2
R1= H, 4-Cl; R2= Ary
R1
Ary: different aromatic fragments
N
O
N
3 a-o
R1= H, 4-Cl, 2,4-(Cl)2
R2= Alk= Me, Et, Pr, Bu, cyclohexyl
R1
N
O
R2
2
Figure 1. Piperidine-based derivatives 1, 2 and new series 3a-o.
Both the acetamide 1 and arylamide 2 piperidine-based
derivatives were characterized by the presence of chemical
features matching the requirement of a 1 receptor 3D
pharmacophore model developed by our group15. On this basis,
we wanted to verify the importance for the R affinity of some
structural peculiarities of the derivatives, such as the aromatic
pattern on benzyl ring (R1), the central piperidine core as well as
the side chain linked to the amide nitrogen atom. To this aim,
we synthesized a second generation of potential R binders 3a-
o, by replacing the aromatic portion of our original molecules
(R2 of 1 and 2) with different aliphatic fragments and by
including the 2,4-dichloro substitution on the benzyl moiety.
The latter in order to verify if such as substitution could be
important for the selectivity towards R.
The synthetic pathway of the new alkylacetamide derivatives
3a-o (Scheme 1) starts from the commercially available 4-
aminomethylpiperidine and the appropriate alkyl aldehyde in a
typical Shiff reaction. The imine derivatives 4a-e, which were
further alkylated to the piperidine nitrogen atom with the
corresponding substituted benzyl chloride, led to intermediates
5a-o. Subsequently, the Shiff bases were reduced with sodium
borohydride and the corresponding derivatives 6a-o were
acylated with acetyl chloride on the nitrogen atom of the
secondary amine to afford the final alkylacetamide compounds
3a-o.
CH2Cl
CH2NH2
R2
N
N
+
R1-CHO
R2
R1
R1
a
b
N
HN
N
H
4 a-e
5 a-o
N
R1
NaBH4
c
N
R1
CH3-COCl
d
H
R2
R2
N
N
O
6 a-o
3 a-o
R1= Alk= CH3, CH2CH3,(CH2)2CH3, (CH2)3CH3,
cyclohexyl
R2= H, 4-Cl, 2,4-(Cl)2
Scheme 1. Synthetic route for the synthesis of the titled compounds 3a-o. Reagents and conditions: (a) CH2Cl2, 0°C; (b)
Acetone/K2CO3, reflux; (c) MeOH, rt; (d) THF/Et3N, 0°C.
In general, all compounds showed preference for the R
subtype (Table 1) with Ki values ranging from 17.0 to 228 nM
and rather low 2R affinities, except for derivative 3k which,
unexpectedly, showed Ki(2) of 19.6 nM. Intriguingly, the
presence of a linear pentyl chain linked to the acetamide
nitrogen atom affords compounds gifted with the best R
affinity (3d and 3i) and selectivity (3d), whilst a small methyl
group affords compounds with higher Ki(1) values (3a, 3f and
3k), but good 2R affinity (3k). The cyclohexyl moiety (3e, 3j
and 3o), produces compounds gifted with moderate 1R affinity
and devoid of 2R affinity. On the other hand, the presence of a
further chlorine atom on benzene ring of N-benzylpiperidine
moiety, 3k-o, seems to improve the affinity and, particularly, the

selectivity for 2R subtype, as already pointed out by our
previous research11d
.
Table 1. Affinities and selectivity towards R of derivatives 3a-o.
Ki
Entry
R1
R2
σ2/σ1
σ1 (nMa)
σ2 (nMa)
3a
3b
3c
3d
3e
3f
CH3
C2H5
97.2± 15
36.8± 5
67± 7
422
N.D.
1257
1117
N.D.
N.D.
987
4.3
/
C3H7
H
19
66
/
C4H9
17.0± 4
58.5± 5
228
Cyclohexyl
CH3
/
3g
3h
3i
C2H5
77.4± 11
34.4± 2
31.8± 3
95.3± 7
285
13
40
26
26
0.07
18
24
17
45
C3H7
4-Cl
1377
825
C4H9
3j
Cyclohexyl
CH3
2425
19.6± 4
2583
1837
3204
2343
3k
3l
C2H5
143
3m
3n
3o
C3H7
2,4-(Cl)2
75.9± 18
187
C4H9
Cyclohexyl
51.9± 4
aOnly compounds with highest affinities (<100 nM) were tested in triplicates.
Subsequently, in order to evaluate the toxicity of the novel
1R and 2R ligands, the MTT assay was used to assess the
cytotoxic effect of the two novel 1R (3d) and 2R (3k)
potential lead compounds on human neuroblastoma (SH-SY5Y)
and human pancreas ductal adenocarcinoma cell lines (Panc-1)
after 48 h incubation in the range of 1 to 100 μM. Both these cell
100 and haloperidol) and 2R (agonist: siramesine; antagonist:
AC927) ligands. Accordingly, to their recognized profiles, NE-
100 and haloperidol (1R antagonists) and siramesine2R
agonist) showed a potent cytotoxic effect in both cell lines
(Figure 2). In the SH-SY5Y cell line, the toxicity of the 3k and
3d compounds (concentration: 50 µM) was comparable to that
of siramesine (90%). In addition, in the Panc1 cell line, the
toxicity of the 3k ligand was at least equivalent to that of
haloperidol (66%).
lines have been used in previous studies on sigma receptors.6a,
16-18
The IC50 of 3k (42µM) for Panc-1 cells was similar to that
for SH-SY5Y cells (31μM), while the IC50 of 3d (33 µM) for
Panc-1 cells was approximately three times lower than that for
SH-SY5Y cells (88µM) (Supplementary material).
Importantly, the behavior of the known 1R antagonists and
2R agonists was consistent with the adopted criteria.
Apparently, the 3d sigma-1 derivative exhibits an ambiguous
cytotoxicity profile, since the corresponding values were
comprised between 91% in SH-SY5Y and 32.5% in Panc1 cells
(Figure 2). On the other hand, the cytotoxicity of the 3k sigma-
2 ligand seems to be comparable to that of 2R agonists. This
observation outlines this compound as the most promising
candidate of the series to be exploited as a new pharmacologic
agent for further and more specific investigation on the 2R
biologic activity.
Subsequently, we used the same approach previously
adopted in different studies to carry out a preliminary functional
characterization of the eventual agonist/antagonist activity of
the novel compounds.11e,19-21
Under the assumption that 1R antagonists and 2R agonists
should be cytotoxic, we compared the cytotoxicity of the novel
compounds characterized by the highest 1R and 2R affinity
to that of known 1R (agonist: pentazocine; antagonists: NE-

125
100
75
50
25
0
125
100
75
50
25
0
Figure 2. Cytotoxicity of sigma receptor ligands as obtained from the MTT assay. SH-SY5Y (right chart) and Panc1 (left chart) cells were treated with
different 1R and 2R ligands (50 μM) for 48h. MTT assay was then performed, cytotoxicity of compounds was determined, and data were reported as % of
cytotoxicity vs the control (cells treated with DMSO 1%). The bars represent the mean ± SE from three independent experiments performed in triplicate.
In conclusion, the results of our in vitro investigation confirm
the role of the N-benzylpiperidine core as an excellent scaffold
not only for 1, but also for 2 ligands. At the same time, this
study highlights the following, specific moieties as beneficial to
support -binding: i) the original aromatic fragment linked to
the tertiary nitrogen amide atom (R2 of 1 and 2) seems to be
unnecessary: in fact, its replacement with an alkyl chain does
not affect the R affinity, not only for 1R (3d), but also for
2R subtype (3k); ii) on the other hand, the length of the alkyl
chain can modulate the selectivity towards both receptor
subtypes: a small methyl group leads the selectivity towards
2R, while a longer alkyl chain (eg. pentyl) shifts the selectivity
towards 1R subtype.
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This research did not receive any specific grant from funding
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Graphical Abstract
New piperidine-based derivatives as sigma
ligands. Synthesis and pharmacological
evaluation
Leave this area blank for abstract info.
Daniele Zampieri,a* Maurizio Romano,b Renzo Menegazzi,b Maria Grazia Mamolo.a

Highlights
1. New piperidine-based alkylacetamide derivatives as R ligands were synthesized.
2. Two out of fifteen of new derivatives showed high affinity and selectivity.
3. SAR study was carried out in order to investigate the chemical features.
4. Cytotoxic assay of new derivatives was performed towards 2 cancer cell lines.
5. Compound with the best R affinity/selectivity, resulted to be R agonist.