Sigma receptor ligands carrying a nitric oxide donor nitrate moiety: Synthesis, in silico, and biological evaluation

Full text of DOI:10.1021/acsmedchemlett.9b00661

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Letter
Sigma Receptor Ligands Carrying a Nitric Oxide Donor Nitrate
Moiety: Synthesis, In Silico, and Biological Evaluation
Emanuele Amata,*,∥ Maria Dichiara,∥ Davide Gentile, Agostino Marrazzo, Rita Turnaturi,
Emanuela Arena, Alfonsina La Mantia, Barbara Rita Tomasello, Rosaria Acquaviva, Claudia Di Giacomo,
Antonio Rescifina, and Orazio Prezzavento*
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ABSTRACT: We report the development of molecular hybrids in
which a nitrate group serving as nitric oxide (NO) donor is
covalently joined to σ receptor ligands to give candidates for
double-targeted cancer therapy. The compounds have been
evaluated in radioligand binding assay at both σ receptors and
selected compounds tested for NO release. Compounds 9, 15, 18,
19, and 21 were subjected to MTT test. Compound 15 produced a
significant reduction of MCF-7 and Caco-2 cellular viability with
comparable IC50 as doxorubicin, being also not toxic for fibroblast
HFF-1 cells. Compound 15 has shown a σ1 receptor antagonist/σ2
receptor agonist profile. Two derivatives of compound 15 lacking
the nitrate group did not induce a reduction of MCF-7 cellular
viability, suggesting a potential synergistic effect between the σ receptors and the NO-mediated events. Overall, the combination of
NO donor and σ receptors ligands provided compounds with beneficial effects for the treatment of cancer.
KEYWORDS: Sigma receptors, nitric oxide, nitric oxide donors, cancer, bifunctional ligands
igma (σ) receptors represent a unique receptor class
in several pathological states.11,12 The overexpression of the σ2
receptor has been found in several cancer cells and
proliferating tumors such as lung, colorectal, and breast.13
Due to a 10-fold higher density in proliferating tumor cells
than in quiescent tumor cells, the σ2 receptor also represents a
significant clinical biomarker for determining the proliferative
status of solid tumors.14,15 A vast number of data support the
use of σ1 receptor antagonists and σ2 receptor agonists for the
treatment of cancer due to their antiproliferative effects.16−20
Rimcazole and haloperidol, as well as other σ receptors ligands
endowed with opportune functional profile, have been shown
to inhibit cell proliferation of several cancer cell lines.21,22
Nitric oxide (NO) is a short-lived gas with a dichotomous
role in tumor biology. NO operates as a tumor progressor or
suppressor based on its concentration, duration of exposure,
and cell sensitivity.23 For instance, μM concentrations of NO
result in antitumor effects, while pM−nM concentrations
promote cytoprotective effects. In this view, the modulation of
1
Sinvolved in various biological and pathological conditions.
Two σ receptors subtypes are recognized and termed sigma-1
(σ1) and sigma-2 (σ2), having different structures and
biological functions.2,3 The σ1 receptor is a 25.3 kDa
polypeptide that has been cloned in several species and
recently crystallized revealing a trimeric architecture.4,5 The σ1
receptor has been identified as a ligand-operated chaperone
protein localized in the mitochondria-associated endoplasmic
reticulum (ER) membranes (MAM). At the MAM, the σ1
receptor forms a complex with another chaperone, the
immunoglobulin heavy-chain binding protein (BiP). The σ1
receptor-BiP complex is silent and only activated when cells are
stimulated by σ1 receptor agonists like (+)-pentazocine
[(+)-PTZ] or undergo prolonged stress.6 In contrast,
haloperidol, methamphetamine, and NE-100 can preserve the
σ1 receptor-BiP complex in a silent state.7
The σ2 receptor is a poorly understood protein whose
identification has been controversial. In 2017, Alon and co-
workers identified the σ2 receptor as an endoplasmic
reticulum-resident transmembrane protein (TMEM97) playing
a role in the cholesterol homeostasis and the sterol transporter
Niemann−Pick disease type C1.8 Despite the challenges in
identifying its true identity, σ2 receptor and its ligands, as
determined in radioligand binding assay using [3H]DTG,9,10
have earned a growing scientific interest due to its involvement
Special Issue: In Memory of Maurizio Botta: His
Vision of Medicinal Chemistry
Received: December 29, 2019
Accepted: April 9, 2020
Published: April 9, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00661
© XXXX American Chemical Society
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A

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Letter
a
Scheme 1. Synthetic Strategy for the Preparation of Target Compounds
a
Reagents and conditions: (i) 4-benzylpiperidine, Br[(CH2)1−4]CN, NaI, K2CO3, DMF, 60 °C, overnight; (ii) LiAlH4, THF, rt, N2; (iii) AgNO3,
CH3CN, rt; (iv) EDC, HOBT, CH3CN, rt, 5 h.
Table 1. σ1 and σ2 Binding Assays for Compounds 9−20
a
Ki SD (nM)
compd
X
Y
n
m
substitution
σ1
σ2
9
10
11
12
13
14
15
16
17
18
19
20
N
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
2
3
4
5
2
3
4
5
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
meta
meta
meta
meta
para
para
para
para
meta
meta
para
para
49
64 0.7
148
50 0.9
89
145 12
1
330
93
75
142 13
2673 44
230 18
89
70
19 0.2
320
270
2514 24
16
1465 224
18
9
3
2
1
2
250
93
9
2
3
2
24 0.6
19
22 0.8
170
N
N
N
1
4
5
6
haloperidol
(+)-PTZ
DTG
2.5 0.4
4.7 0.7
2
b
ND
1
a
b
Each value is the mean SD of at least two experiments performed in triplicate. Not determined.
NO levels seems to have benefits in the treatment of cancer.
However, NO is a reactive and unstable gas; thus, the use of
NO donors (NODs) is preferred since these agents are stable
chemical compounds able to release NO under specific
chemical or enzymatical conditions. In order to ensure a
high and effective release of cytotoxic amounts of NO, the
choice of the releasing agent is crucial. Among the different
classes of donors, organic nitrates have been reported to
release adequate amounts of NO.16,24
The cytotoxic activity may be carried out by NODs alone, or
they may be conjugated or embedded in chemical compounds
that possess additional antitumoral action for double-targeted
cytotoxic activity.25,26 On the basis of the aforementioned, the
purpose of the present work is the development, biological
evaluation, and molecular modeling studies of a series of
molecular hybrids where the cytotoxic activity of the NOD is
conjugated with that of σ receptors for optimal effect.27
Compounds 9−20 were synthesized according to the steps
illustrated in Scheme 1. Starting from commercially available 4-
benzylpiperidine, intermediates 1−4 were obtained with
opportune nitrile compounds and then converted into the
corresponding amine derivatives by reaction with LiAlH4.6,16
The amine intermediates underwent condensation with
[(nitrooxy)methyl]benzoic acid (5, 6) or para- or meta-
[(nitrooxy)methyl]phenylacetic acid (7, 8) previously pre-
pared via nitration with AgNO3 to give the final compounds
9−16. Compounds 17−20 were obtained through condensa-
tion of 1-benzylpiperidin-4-amine with the same acids 5−8.
The synthesized compounds were evaluated for affinity at
both σ1 and σ2 receptors through radioligand binding assay
(Table 1). Within the series bearing a 4-benzylpiperidine (9−
16), the meta-substituted compound 9 has shown a preferential
σ1 receptor affinity with Ki value of 49 nM, and 330 nM for the
σ2 receptor. The elongation to three-carbon chain (10)
B
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Figure 1. (a) 3D superposition of the best-docked pose for 9 (orange) and 11 (green) bound to the σ1 receptor orthosteric site. The different
lengths of the aliphatic linker reverse the orientation of the ligand inside the receptor. (b) Top-scored docking poses for 9 (orange) and 13
(magenta) bound to the σ1 receptor orthosteric site. The different position of the nitrate group allows better interaction with His154 (green dotted
line).
increased the σ2 receptor affinity by more than 3-fold with Kiσ1
value of 64 nM and Kiσ2 of 93 nM. The increased σ2 receptor
affinity has also been maintained in compound 11 with a Ki
value of 75 nM, although in the four-carbon chain, the σ1
receptor affinity (148 nM) is reduced. Further elongation of
the chain to five carbons takes back σ1 receptor preferential
affinity with a Ki value of 50 nM over the σ2 receptor (Ki value
of 142 nM).
At the same time, the para substitution of the nitrate group
gives compounds with generally lower affinity with respect to
both receptor subtypes. It is observed an improvement of the
σ2 receptor affinity with the linker elongation. Indeed,
compound 13 has a Kiσ2 of 2673 nM, while compound 16
has a Kiσ2 of 70 nM. In terms of σ1 receptor affinity,
compounds 9−16 show a mixed behavior with affinity ranging
from 89 nM for compound 13 to 249 nM for compound 15.
Compounds bearing the 1-benzylpiperidin-4-amine (17−20)
have shown worthy σ1 receptor affinities and a general lower
affinity at the σ2 receptor. In particular, compounds 17−19
have a σ1 receptor affinity in the low double digits nM range
(Kiσ1 of 24, 19, and 22 nM, respectively), while only
compound 17 shows σ2 receptor affinity in the same range
(Kiσ2 of 19 nM). The other compounds show a lower affinity
to the σ2 receptor, which leads to a >10 times selectivity with
respect to the σ1 receptor.
protonated piperidine ring is rather characteristic, being close
to the carboxyl function of Glu172. The docking studies
conducted upon the σ2 receptor were performed by using its
homology model previously built by us.28 In this case, the salt
bridge interactions between the ligands and the residues Asp29
and Asp56 were considered. In the 4-benzylpiperidine series
with the nitrate group in the meta position (9−12), compound
9 showed a preferential affinity for the σ1 receptor with a Ki
value of 49 nM.
Compound 9 is placed inside the receptor site with the
piperidinium proton engaged in a salt bridge interaction with
Glu172 and a hydrogen bond with N−H amide. The nitrate
group is positioned on the opposite side helices α4 and α5,
showing polar interactions with the residues Asp126 and
His154. The optimal position of the piperidine ring favors the
π−π stacking of the hydrogen in position 4 of the piperidine
system with the residue Tyr103. The benzyl group is involved
in hydrophobic interactions with the Leu182 and Met93
residues. The elongation to the four-carbon chain (11)
decreases the affinity for the σ1 receptor while increasing the
affinity for the σ2 receptor. Indeed, derivative 11 has an
inverted orientation with respect to derivative 9, with the
nitrate group toward the helix α5 to allow the accommodation
of the aliphatic chain within the receptor pocket (Figure 1a).
The para substitution of the nitrate group (13−16)
decreases the affinity with respect to the ligands substituted
in the meta position. This is probably due to a lower cation−π
interaction with the His154 residue (Figure 1b). An elongation
of the aliphatic portion of the ligands leads to a better affinity
for the σ2 receptor. The most similar compounds in the series
(11 and 16) show comparable interactions within the σ2
receptor pocket. Both form the salt bridge with the residue
Asp56 while the benzyl portion in position 4 to the piperidine
ring shows aliphatic interactions with the residues Leu70,
Met108, Pro113, and Val146. The nitrate group establishes
electrostatic interactions with the Arg36 residue, discriminating
against the different affinity between the different isomers. A
detailed description of the interactions with σ2 receptor is
reported in the Molecular Docking section in the Supporting
Information.
A docking study was conducted to identify and evaluate the
key molecular interactions involved in the receptor/ligand
recognition. The crystal structure of the σ1 receptor PD144418
(PDB code 5HK1)4 reveals an occluded and elongated binding
cavity in a similar β-barrel fashion, with the highly conserved
Glu172 amino acid residue located near the center of the
cavity, forming a salt bridge with the ligands. Since all
compounds possess a tertiary amine, we performed the study
considering the N-protonated structures (pH = 7.4). There-
fore, the formation of the salt bridge was used as a filter for the
docking poses. The calculated free binding energies (ΔG) and
Ki values to the catalytic site of the σ1 receptor for compounds
9−20 and haloperidol are reported in Table S1.
Active analysis of the site showed that the orientation
changes according to the length of the ligands and the
protonated N-position. Moreover, the position of the
C
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Compound 18 showed the preeminent affinity to the σ1
receptor. On this compound, a molecular dynamics (MD)
simulation was performed (Supporting Information). An
elongation of the aliphatic portion of the ligands leads to a
better affinity for σ2 receptor.
Once the affinity profiles of the synthesized compounds at σ
receptors were evaluated, we determined the ability of these
compounds to release NO. Nitrite content was measured by
the Griess method incubating the compounds (100 μM), at 37
°C, in Tris-HCl buffer for 30 min (Figure 2).
μM for compound 9 and 26 μM for compound 15. None of
the synthesized compounds, nor compound 21, were
demonstrated to be toxic for the human fibroblasts HFF-1 at
the maximal tested concentration. Compounds 18 and 19 have
been shown to be nontoxic for the three evaluated cell lines
(IC50 > 100 μM), probably due to the lower rate of NO release
and to an unsuitable functional profile at σ receptors.
In order to determinate the precise mechanism for the
reduction of cellular viability, compound 15 (IC50 concen-
tration) was evaluated in combination with the σ1 receptor
agonist (+)-PTZ (1 μM) and the σ2 receptor antagonist 1-
phenethylpiperidine (AC927, 1 μM) by MTT.30
Incubation of compound 15 with (+)-PTZ or AC927 wholly
restored the loss of cell viability induced by 15 alone (Figure
3). This outlines a σ1 and σ2 receptors involvement in the
observed cellular events and a σ1 receptor antagonist/σ2
receptor agonist functional profile for compound 15.
Figure 2. Total nitrite content measured using Griess reagent.
For this assay, we selected the compounds based on their
affinity profile against σ receptors. In particular, we evaluated
compounds 9, 11, 15, and 17−19, having shown good affinity
at both receptors or prevalence for one receptor subtype.
Compounds 9 and 15 were able to release a significant amount
of NO in the μM range (9, 13.0 0.5 μM; 15, 13.0 0.4
μM). The amount of NO released by compound 18 was 1.3
0.2 μM, while compound 19 produced 6.3
0.3 μM NO.
Negligible NO amounts were detected for compounds 11 and
17 (data not shown).
Figure 3. Effects of compound 15 in combination with the selective
σ1 receptor agonist (+)-PTZ and σ2 receptor antagonist AC927 on
MCF-7 and Caco-2 viability by MTT test.
After having obtained the desired chemical tools, we
evaluated their activity in the appropriate cell lines. We
evaluated those compounds that, in previous experiments, have
been demonstrated to possess the desired profile (compounds
9, 15, 18, and 19). Two cancer cell lines were selected, Caco-2
and MCF-7 cells, for their expression of both σ receptors.
Although MCF-7 cells are reported to exclusively express the
σ2 subtypes, a Western blot analysis has shown the presence of
the σ1 isoform in our in-house cell line (Figure S4).29 The
toxicity against human fibroblast HFF-1 cells was also
evaluated. Doxorubicin (21) was used as the standard
cytotoxic compound. Results showed a reduction of cellular
viability on both Caco-2 and MCF-7 cell lines for compounds
9 and 15 (Table 2). These compounds were those with a
higher rate of NO release. Measured IC50 values were better
with respect to compound 21 for MCF-7 cells, with IC50 of 36
To dig into the dual mechanism of the prepared compounds,
we tested fragments 5 and 22a,b and synthesized two
derivatives of compound 15 lacking the nitrate function,
compounds 23a and 23b, as negative control (Table 3 and
Scheme S1).31,32
The fragments 5 and 22a,b have shown no significant
viability reduction on MCF-7 and no affinity for both σ
receptors. Compound 23a showed a loss of affinity at both σ
receptors, while compound 23b retained a similar affinity
profile at both σ receptors with respect to compound 9 or 15.
When evaluated on the MCF-7 cell line, compound 23a
induced lower than 50% viability reduction at 100 μM, while
compound 23b had an IC50 of 87 μM. Overall compound 23b,
while maintaining a similar profile against σ receptors, showed
a lower ability in reducing MCF-7 viability, thus sustaining a
possible synergistic effect between σ receptors and the NO-
mediated events.
In conclusion, this contribution reports the development of
novel hybrid compounds able to release NO and to bind σ
receptors as candidates for double-targeted cancer therapy.
The compounds have been evaluated for their affinity at σ
receptors and ability to release NO. Four compounds showed
the desired profile with compounds 9 and 15 also able to
induce a marked loss of viability in MCF-7 and Caco-2 cell
lines while not being toxic for healthy human fibroblast HFF-1.
Table 2. MTT Test on MCF-7, Caco-2, and HFF-1 for
Compounds 21, 9, 15, 18, and 19
a
IC50 SD (μM)
compd
MCF-7
Caco-2
HFF-1
b
21
9
15
18
19
44 0.3
36 0.2
26 0.4
21 0.3
59 0.5
28 0.2
>100
>100
>100
>100
b
b
b
b
b
>100
>100
b
b
b
>100
>100
>100
a
Each value is the mean SD of at least two experiments performed
b
in quadruplicate. Cell viability reduction lower than 50% at 100 μM.
D
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Davide Gentile − Department of Drug Sciences, Chemistry
Section, University of Catania, 95125 Catania, Italy
Agostino Marrazzo − Department of Drug Sciences, Medicinal
Chemistry Section, University of Catania, 95125 Catania,
Italy; orcid.org/0000-0002-8728-8857
Table 3. Binding Assays and MTT Viability Test on MCF-7
for Compounds 22a,b, 23a,b, and 5
Rita Turnaturi − Department of Drug Sciences, Medicinal
Chemistry Section, University of Catania, 95125 Catania,
Italy; orcid.org/0000-0002-5895-7820
Emanuela Arena − Department of Drug Sciences, Medicinal
Chemistry Section, University of Catania, 95125 Catania, Italy
Alfonsina La Mantia − Department of Drug Sciences,
Biochemistry Section, University of Catania, 95125 Catania,
Italy
Barbara Rita Tomasello − Department of Drug Sciences,
Biochemistry Section, University of Catania, 95125 Catania,
Italy
a
b
Ki SD (nM)
IC50 SD (μM)
compd
σ1
σ2
MCF-7
c
c
c
c
d
5
>10000
>10000
>10000
>10000
>10000
>10000
>100
>100
>100
>100
c
c
d
22a
22b
23a
23b
d
d
711 127
62
2600 730
127 16
3
87 0.3
Rosaria Acquaviva − Department of Drug Sciences,
Biochemistry Section, University of Catania, 95125 Catania,
Italy
Claudia Di Giacomo − Department of Drug Sciences,
Biochemistry Section, University of Catania, 95125 Catania,
Italy
a
Each value is the mean SD of at least two experiments performed
b
in triplicate. Each value is the mean SD of at least two experiments
performed in quadruplicate. Radioligand displacement lower than
50% at 10 μM. Cell viability reduction lower than 50% at 100 μM.
c
d
Antonio Rescifina − Department of Drug Sciences, Chemistry
Section, University of Catania, 95125 Catania, Italy;
orcid.org/0000-0001-5039-2151
In cellular experiments involving the use of selective σ
receptors agonist and antagonist, compound 15 has shown a σ1
receptor antagonist/σ2 receptor agonist functional profile. The
elimination of the nitrate function of compound 15 as in
compounds 23a,b determined the loss of its ability to reduce
MCF-7 viability sustaining a possible synergistic effect between
the σ receptors and the NO-mediated events. Molecular
docking studies have shown that the length of the aliphatic
linker has an essential role in the orientation of the ligands
inside the receptor pocket and for σ1/σ2 selectivity. The nitrate
group stabilizes the ligand/receptor complex to cation−π
interaction with His154 residue in the σ1 receptor, also
confirmed by the MD simulation, and with Phe71 and Pro113
residues in the σ2 receptor. Overall, the combination of NO
donor and σ receptors ligands provided compounds with
potential beneficial effects in the treatment of neoplastic
disorders.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00661
Author Contributions
∥E.A. and M.D. contributed equally.
Funding
̀
This work was financially supported by Universita degli Studi
di Catania, “Piano per la Ricerca 2016−2018”, Grants
57722172105 and 57722172114.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Prof. Giuseppina Imme, Dr. Roberto
Catalano, and Nunzio Giudice from the Department of Physics
and Astronomy, University of Catania, for technical and
instrumental support of the Beckman LS6500 liquid
scintillation counter.
■
̀
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00661.
■
sı
ABBREVIATIONS
General synthetic methods and spectral data of final
compounds, procedures for in vitro biological assays,
and computational methods (PDF)
■
σ, sigma; σ1, sigma-1; σ2, sigma-2; ER, endoplasmic reticulum;
[3H]-DTG, [3H]-1,3 di-o-tolylguanidine; NO, nitric oxide;
NOD, nitric oxide donor; AC927, 1-phenetylpiperidine
AUTHOR INFORMATION
Corresponding Authors
■
REFERENCES
■
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3396392909; Email: prezzave@unict.it
Authors
Maria Dichiara − Department of Drug Sciences, Medicinal
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was originally published ASAP on April 15, 2020.
Due to a production error, additional corrections were needed
to Tables 2 and 3. The corrected version was reposted on April
15, 2020.
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https://dx.doi.org/10.1021/acsmedchemlett.9b00661
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX