Combination of heme oxygenase-1 inhibition and sigma receptor modulation for anticancer activity

Article abstract of DOI:10.3390/molecules26133860

Cancer is a multifactorial disease that may be tackled by targeting different signaling pathways. Heme oxygenase-1 (HO-1) and sigma receptors (σRs) are both overexpressed in different human cancers, including prostate and brain, contributing to the cancer

Full text of DOI:10.3390/molecules26133860

molecules
Communication
Combination of Heme Oxygenase-1 Inhibition and Sigma
Receptor Modulation for Anticancer Activity
Giuseppe Romeo 1 , Valeria Ciaffaglione 1,
*
, Emanuele Amata 1 , Maria Dichiara 1, Loredana Calabrese 1,
Luca Vanella 1 , Valeria Sorrenti 1 , Salvo Grosso 1, Agata Grazia D’Amico 1, Velia D’Agata 2
,
Sebastiano Intagliata 1,
*
and Loredana Salerno 1
1
Department of Drug and Health Sciences, University of Catania, viale A. Doria 6, 95125 Catania, Italy;
gromeo@unict.it (G.R.); eamata@unict.it (E.A.); mariadic.md@gmail.com (M.D.);
loredanacalabrese94@gmail.com (L.C.); lvanella@unict.it (L.V.); sorrenti@unict.it (V.S.);
salvogrosso@outlook.it (S.G.); agata.damico@unict.it (A.G.D.); l.salerno@unict.it (L.S.)
Department of Biomedical and Biotechnological Sciences, University of Catania, 95100 Catania, Italy;
vdagata@unict.it
2
*
Correspondence: valeria.ciaffaglione@phd.unict.it (V.C.); s.intagliata@unict.it (S.I.);
Tel.: +39-095-738-4006 (V.C.); +39-095-738-4053 (S.I.)
Abstract: Cancer is a multifactorial disease that may be tackled by targeting different signaling
pathways. Heme oxygenase-1 (HO-1) and sigma receptors ( Rs) are both overexpressed in different
human cancers, including prostate and brain, contributing to the cancer spreading. In the present
study, we investigated whether HO-1 inhibitors and R ligands, as well a combination of the two,
σ
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
σ
ꢀꢁꢂꢃꢄꢅꢆ
may influence DU145 human prostate and U87MG human glioblastoma cancer cells proliferation.
In addition, we synthesized, characterized, and tested a small series of novel hybrid compounds
Citation: Romeo, G.; Ciaffaglione, V.;
Amata, E.; Dichiara, M.; Calabrese, L.;
Vanella, L.; Sorrenti, V.; Grosso, S.;
D’Amico, A.G.; D’Agata, V.; et al.
Combination of Heme
(HO-1/
σ
Rs)
1–
4
containing the chemical features needed for HO-1 inhibition and
σR modulation.
Herein, we report for the first time that targeting simultaneously HO-1 and
σ
R proteins may be
a good strategy to achieve increased antiproliferative activity against DU145 and U87MG cells,
with respect to the mono administration of the parent compounds. The obtained outcomes provide
Oxygenase-1 Inhibition and Sigma
Receptor Modulation for Anticancer
Activity. Molecules 2021, 26, 3860.
https://doi.org/10.3390/
an initial proof of concept useful to further optimize the structure of HO-1/σRs hybrids to develop
novel potential anticancer agents.
molecules26133860
Keywords: heme oxygenase; HO-1 inhibitors; sigma receptors;
σR ligands; DU145; anticancer
activity; combination therapy; hybrid compounds
Academic Editor:
Diego Muñoz-Torrero
Received: 14 May 2021
Accepted: 22 June 2021
Published: 24 June 2021
1. Introduction
Despite a large number of molecules approved as anticancer drugs, cancer remains a
serious cause of death worldwide [ ]. Moreover, most antineoplastic agents currently in
clinical practice have developed severe side effects along with multidrug resistance [ ].
Therefore, the identification of new molecules, acting at classical or innovative molecular
targets, which may improve or restore the effects of anticancer drugs, is of general interest.
Cancer can be considered, in all respects, a multi-genic disease. Cancer initiation and
1
2,3
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
progression depend on more than one receptor or signaling pathway [
multi-targeted therapies may be advantageous over mono-therapy [ ]. To date, diseases
with complex etiologies such as cancer are often treated with “drug-cocktails” combining
two or more molecules acting at different molecular targets, to optimize clinical response [ ].
4], suggesting that
5
6
Copyright:
© 2021 by the authors.
Combination therapy is generally associated with some drawbacks, such as the risk of
metabolic interactions between drugs, and low patient compliance, due to multiple intakes
during the day [7]. In order to overcome these negative aspects, an emerging strategy in
medicinal chemistry is the search for novel bioactive compounds which combine in one
molecule multi-target properties, called multi-target ligands or hybrid compounds [8,9].
Among the numerous pathways involved in the formation, growth, and survival of
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cancer cells, the heme oxygenase (HO) system and sigma receptors (σRs) play pivotal
Molecules 2021, 26, 3860. https://doi.org/10.3390/molecules26133860
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3860
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roles [10
,11]. HO is the enzyme responsible for the oxidative catabolism of the pro-oxidant
heme into free iron, carbon monoxide and biliverdin, subsequently reduced to bilirubin [12].
Two main isoforms catalyze this reaction in humans: HO-1 and HO-2. HO-2 is constitutive,
ubiquitous, and mainly responsible for physiological effects [13]. HO-1 is inducible and
expressed at basal levels only in the spleen and the liver, but it may be induced in many
other organs and tissues by various stimuli, including heat, heme itself, heavy metals,
ROS, and xenobiotics [14]. It has been widely acknowledged that HO-1 is a cytoprotective
enzyme particularly active against oxidative stress [15,16]. However, HO-1 also exerts pro-
tective effects towards cancer cells, and aberrant high levels of HO-1 have been frequently
reported in different human cancers, including prostate, lung, and pancreatic cancer, neu-
roblastoma, and chronic and acute myeloid leukemia. In addition, HO-1 overexpression
has been associated with drug-resistance towards commonly used cancer therapies [17–19]
.
Thus, it is conceivable that HO-1 inhibitors can be useful in anticancer treatment either alone
or in combination with chemotherapy, radiotherapy, or photodynamic therapy, according
to recent literature reports [20–22].
σ
Rs were earlier discovered as a subclass of opioid receptors [23]. Based on the
current knowledge, Rs are considered non-opioid, non-GPCR transmembrane proteins
σ
expressed mainly in the endoplasmic reticulum (ER) membrane, classified into two sub-
types: σ1R and σ2R/TMEM97. From their discovery, these receptors have attracted the
attention of pharmacologists and medicinal chemists due to their pleiotropic functions
on mitochondrial metabolism, apoptosis, ion channels modulation, lipid transport and
metabolism regulation, neuritogenesis, mediation of Ca2+ release, and interplays with G
protein-coupled receptors (GPCRs) [24]. As a consequence, these receptors are involved
in several pathological conditions, including SNC disorders (neuropathic pain, depres-
sion, Alzheimer’s, Parkinson’s) [25
prostate, breast, colorectal cancer, glioblastoma) [27
might have a critical role in cancer growth, cell proliferation, and tumor aggressiveness [29].
In recent years, many selective or mixed R ligands with potential biological effects have
been developed [30 33]. It is generally accepted that, for anticancer activity, antagonism at
,
26], and expressed in many types of cancer cells (e.g.,
,28]. Indeed, both the σ1R and the σ2
R
σ
–
σ1R or agonism at σ2R are preferable [34,35].
On these premises, this work aimed to evaluate whether HO-1 inhibitors and σR
ligands, as well as a combination of the two, may counteract cancer cell proliferation. In this
regard, we selected DU145 and U87MG cells as representative cell lines for humane prostate
cancer and glioblastoma, respectively, in which both HO-1 and
Among HO-1 inhibitors, which we sourced from our library of compounds [40], we chose
LS/0 LS4/28, and LS6/42 as lead molecules, since it has been previously demonstrated
that they are endowed with a very good HO inhibition profile (Figure 1), as well as
antitumor properties [21 41 44]. Regarding R ligands, we selected haloperidol (Figure 1)
as an example of mixed R ligand with anticancer activity against some tumors [45
σRs are involved [36–39].
,
,
–
σ
σ
]
and benzylpiperazine derivatives SI1/13 and RFB/13, which emerged from our recent
studies as very potent σ1R ligands, and very selective over the σ2R (Figure 1) [46]. Then,
we synthesized new HO-1/σRs hybrid compounds 1–4, which contain the structural
requirements for interacting with both targets i.e., an azole-based moiety and a hydrophobic
group connected by a central linker for HO-1, and a basic cyclic amine such as piperazine
linked to two different hydrophobic moieties for σRs (Figure 2) [41,47]. The novel hybrids
were tested to evaluate their capacity to inhibit HO-1, their affinity for σ1R and σ2R,
and their cytotoxicity against DU145 and U87MG cancer cell lines.

Molecules 2021, 26, 3860
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Figure 1. Structures of selected HO-1 inhibitors and
σ
R ligands and their potency against respec-
tive targets.
Figure 2. Structural requirements for both targets in HO-1/
colors. For HO-1 inhibitors: azole nucleus (orange), hydrophobic group (blue), and central linker
(magenta). For R ligands: first hydrophobic portion HYD1 (blue), second hydrophobic portion
σRs hybrids 1–4 are depicted in different
σ
HYD2 (red), and basic nitrogen (green).
2. Results and Discussions
2.1. Chemistry
The synthesis of compounds
Scheme 1. The first step was the formation of the amides 9–
1
–
4
was accomplished in three steps, as shown in
12 obtained by means of
a reaction between benzylpiperazine and a carboxylic acid derivative , activated using
1,10-carbonyldiimidazole (CDI). The following step was the etherification of
12 with
1,4–dibromobutane in acetonitrile under reflux and in presence of K2CO3, to give the
corresponding bromobutoxy amides 13 16. The final hybrids were obtained through
16 with imidazole in THF, using sodium
5–8
9–
–
1–4
nucleophilic displacement of intermediates 13
–
hydride (NaH) as base.

Molecules 2021, 26, 3860
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Scheme 1. Reagents and conditions: (i) benzylpiperazine, CDI, THF, 0 °C 10 min, room temperature,
8 h; (ii) 1,4-dibromobutane, acetonitrile, K2CO3, reflux, 6 h; (iii) imidazole, THF, NaH, reflux, 9 h.
2.2. Biological Activity
2.2.1. HO-1 Inhibition
The new HO-1/σRs hybrids 1–4 were tested to evaluate their HO-1 inhibitory activity.
The enzyme was obtained from the microsomal fractions of rat spleen. Determination of
HO-1 activity was performed by measuring the bilirubin formation using the difference
in absorbance at 464–530 nm, as reported in the experimental section. Compounds LS/0
LS4/28 LS6/42 and azalanstat were used as reference substances. The inhibitory potency
is expressed as IC50 M) and results are shown in Table S1. We focused our attention
,
,
(
µ
on HO-1, since only this inducible isoform is involved in tumorigenesis and in tumor
progression [48].
Structure–activity relationship (SAR) studies performed so far have evidenced that
the pharmacophore for HO-1 inhibition contains three main critical portions: (i) an azole
nucleus, preferably an imidazole, (ii) a hydrophobic moiety, and (iii) a central linker
connecting the imidazole and the hydrophobic groups (Figure 2) [14]. Each chemical
portion interacts with a specific amino acid located in three principal regions of the enzyme,
near to the catalytic site which binds the substrate heme, and known as the eastern,
western, and central regions, respectively, as described in crystallographic studies [49].
Many studies have demonstrated that the western region, responsible for binding with
the hydrophobic portions of the inhibitors, is very flexible and can allocate both simple
aromatic moieties, such as the phenyl group of reference compound LS/0, but also hindered,
heteroaromatic, and ramified aryl moieties [50
we thought to increase molecular complexity by introducing a further moiety, i.e., benzyl
piperazine, as this feature is necessary to target Rs. To find multiple contact points with
,51]. Then, using LS/0 as a lead compound,
σ
the protein, we connected the benzylpiperazine with the phenoxybutylimidazole through
a chain containing from 1 to 4 carbon atoms, then allowing free rotation, and, consequently,
many possible different conformations. Unfortunately, all new hybrids are less potent
than reference substances. It is likely that the western region of the enzyme, formed
only by hydrophobic aminoacids, does not tolerate the presence of polar amino group,
such as the N-atom belonging to the benzylpiperazine moiety present in hybrids, charged at
physiological pH. Among hybrids 1–4, compound 4, containing the longest chain between
benzylpiperazine and phenoxybutylimidazole, is the most potent compound among the
series (Table S1), suggesting that a major distance of the piperazine ring allows a better
binding of the first hydrophobic portion inside the western region of the enzyme.
2.2.2. σRs Binding Properties
All new synthesized HO-1/
σ1R and the σ2R, through radioligand binding assay. Compounds
σ
hybrids
1
–
4
were evaluated for affinity at both the
were designed
1–4

Molecules 2021, 26, 3860
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taking into account pharmacophoric features for
σ
Rs affinity. Specifically,
σ
R ligands
require two hydrophobic moieties located at an appropriate distance from a basic nitrogen
(HYD1, HYD2, and basic N, Figure 2), in order to properly allocate into the R binding
σ
site [52]. In our σ1R lead compounds, SI1/13 and RFB/13, the benzyl group linked to
piperazine represents HYD2, the N-4 of piperazine is the basic nitrogen, and the other
phenyl ring acts as HYD1. These moieties were maintained in our HO-1/
; while an imidazole linked by an oxybutyl chain was further added with the aim of
also targeting the HO-1 enzyme. Moreover, the choice of the benzylpiperazine moiety as
R pharmacophoric portion was further supported by the recent discovery of our highly
potent and selective σ1R antagonist SI1/13 46]. Unexpectedly, none of the novel hybrids
showed Rs affinity (Table S1), suggesting that the additional oxybutyl chain and the distal
imidazole, with respect to SI1/13 and RFB/13, are not tolerated by both Rs. According
σRs hybrids
1–4
σ
[
σ
σ
to Glennon’s pharmacophoric σ1R model [52], HYD1 can tolerate bulky groups; however,
the presence of a flexible 4-(imidazolyl)butoxy group as a substituent might interfere
through a steric hindrance in establishing essential hydrophobic interaction between the
phenyl ring (located at the HYB1) and key amino acid residues inside the binding pockets.
2.2.3. Cytotoxicity against DU145 and U87MG Cell Lines
In some types of human prostate and brain cancers, both HO-1 and
σRs are overex-
pressed, influencing cancer cell proliferation. Consequently, R ligands and HO-1 inhibitors
σ
may have anticancer activity. Literature data highlight the potential antiproliferative activ-
ity of various σ1R ligands in highly diffusive glioblastoma and prostate cancer cells, likely
by interfering with the progression of cell cycle and decreasing the migration of cancer
cells [11
,28]. Likewise, HO-1 inhibition hinders cancer progression, mainly by decreasing
CO-mediated angiogenesis and disrupting the antioxidant HO-1 activity [22
,
36 53]. In this
,
study, we selected DU145 cells as representative for prostate cancer [37], and U87MG as
cancer cells of glioblastoma [39], to evaluate whether inhibition of HO-1 and modulation
of σRs may be effective for anticancer activity. Cytotoxicity of all new and previous synthe-
sized compounds were evaluated in vitro via MTT assay. The cell viability of DU145 and
U87-MG cells was assessed after 72 h of continuous treatment with all tested compounds,
at the concentrations of 1, 10, and 50 µM.
Firstly, we tested the cytotoxicity against both cell lines of the
RFB/13, and HO-1 inhibitors LS/0 LS4/28, and LS6/42 (Figure 3, panels A and B). Among
R ligands we also included haloperidol, an antipsychotic drug endowed with antitumor
properties due to its affinity for both σ1R and σ2R. The results in Figure 3A show that
both R ligands and HO-1 inhibitors were able to reduce DU145 cells proliferation with a
different range of potency. In particular, haloperidol, the R1 ligand SI1/13, and the HO-
1 inhibitor LS6/42 were the most potent, since at 10 M they reduced the cell viability of
σR1 ligands, SI1/13 and
,
σ
σ
σ
µ
about 50%. The same trend was observed in U87MG glioblastoma cells. In fact, as displayed
in Figure 3B, SI1/13 showed high efficacy in downregulation of cell viability, similarly to
haloperidol, at all concentrations tested, whereas RFB/13 and LS/0 were able to significantly
reduce cell viability only at 50 µM. LS4/28 did not affect U87MG cell viability; on the
contrary, LS6/42 was the most efficacious since they reduced cellular viability by about 50%
at 10 µM.

Molecules 2021, 26, 3860
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Control
1 M
(A)
DU145
150
10 M
50 M
100
50
0
***
***
***
***
***
***
Haloperidol
SI1/13
RFB/13
LS/0
LS4/28
LS6/42
Control
1 µM
(B)
U87MG
10 µM
50 µM
150
100
50
***
***
***
***
***
0
Haloperidol
SI1/13
RFB/13
LS/0
LS4/28
LS6/42
Figure 3. Effect of
and LS6/42 treatments on cell viability of DU145 (panel
by MTT assay at the doses of 1, 10 and 50
experiments and values are expressed as percentage of control (% of control). Data represent
means SEM. *** p < 0.001 vs. control as determined by One-way ANOVA followed by Tukey’s
multiple comparison test.
σ
R ligands haloperidol, SI1/13 and RFB/13 and of HO-1 inhibitors LS/0
,
LS4/28
A
) and U87MG (panel B) cell lines, assessed
µM. Results are representative of at least three independent
±
Since HO-1 and
whether a simultaneous treatment with
advantage with respect to single compounds. In this regard, we combined 10
haloperidol, SI1/13, or RFB/13 with the same amount of LS/0 LS4/28, or LS6/42. Results
are shown in Figure 4A for DU145 cells and in Figure 4B for U87MG cells, respectively.
Combination of the R ligand SI1/13 with HO-1 inhibitors, in particular LS6/42, was note-
worthy in DU145 cells; in fact, 10 M of SI1/13 plus 10 M of LS6/42 reduced cell viability
of about 75% with respect to the 50% effects showed by the single compounds. The effect of
R ligands and HO-1 inhibitors co-administration was noteworthy in U87MG cells, where
σ
Rs are both involved in examined cancers, we wanted to evaluate
R ligands and HO-1 inhibitors may have some
M of
σ
µ
,
σ
µ
µ
σ
all the combinations afforded to reduced cell proliferation with respect to that obtained
with single compounds. Specifically, the viability was significantly reduced for compound
RFB/13 only when combined with LS4/28 or LS6/42, whereas the antiproliferative action
of haloperidol and SI1/13 was increased by the addition of all the tested HO-1 inhibitors.
The most efficacious combinations were haloperidol plus LS6/42 and SI1/13 plus LS6/42.

Molecules 2021, 26, 3860
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DU145
(A)
150
100
50
***
##
***
***
###
0
l
l
l
l
0
8
L
2
0
8
2
4
0
/
8
2
4
3
/
3
1
o
/
o
o
r
2
4
2
2
o
r
r
1
/
/
/
/
/
/
S
S
S
t
t
t
/
/
d
4
l
6
4
6
4
6
i
L
l
L
3
L
3
n
n
1
n
B
r
I
S
S
S
S
S
S
o
o
o
e
+
+
F
+
3
S
L
L
L
L
L
C
l
C
C
p
R
3
+
+
+
I
+
+
+
o
o
1
1
/
l
/
d
i
3
3
a
1
o
o
B
r
I
1
1
1
1
H
/
/
/
/
d
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e
F
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i
r
S
1
I
1
B
B
r
p
R
e
e
F
F
o
S
S
l
p
p
R
R
a
o
o
l
l
H
a
a
H
H
U87MG
(B)
150
100
50
***
###
***
###
###
0
l
l
l
l
0
8
2
0
8
2
0
/
8
2
3
1
/
3
o
/
o
o
r
2
/
4
2
/
4
2
4
/
o
r
r
/
/
1
/
/
S
S
S
t
t
t
/
d
4
l
6
4
6
4
6
i
L
l
L
3
L
3
n
n
1
n
B
r
I
S
S
L
S
S
S
S
o
o
o
e
+
+
3
F
+
S
L
L
L
L
L
C
l
C
C
p
R
3
+
+
+
I
+
+
+
o
o
1
1
/
l
/
d
i
3
3
a
1
o
o
B
r
I
1
1
1
1
/
H
/
/
/
d
d
e
F
i
i
r
S
1
I
1
B
B
r
p
R
e
e
F
F
o
S
S
l
p
p
R
R
a
o
o
l
l
H
a
a
H
H
Figure 4. Effects of the combination of
1 inhibitors LS/0 LS4/28 and LS6/42 treatments on cell viability of DU145 (panel
(panel ) cell lines, assessed by MTT at the doses of 10 M, and compared to the effect obtained
with R ligands alone at the same dose. Results are representative of at least three independent
experiments and values are expressed as percentage of control (% of control). Data represent
means R ligand as determined by
SEM. *** p < 0.001 vs. control, ## p < 0.001 and ### p < 0.001 vs.
one-way ANOVA followed by Tukey’s multiple comparison test.
σ
R ligands haloperidol, SI1/13 and RFB/13 and of HO-
,
A
) and U87MG
B
µ
σ
±
σ
Finally, we tested the viability of DU145 and U87MG cancer cells in the presence
of all new HO-1/ Rs hybrids . Results showed in Figure 5A evidence that the new
compounds were able to influence cell proliferation of DU145 cell line only at high
concentrations. Glioblastoma U87MG cancer cells became more sensitive after the treatment
with hybrids . In fact, as showed in Figure 5B, compounds and reduced U87MG
cell viability at all concentrations, especially at 50 M, compared to control group; instead,
compound 3 showed less efficacy than the control at 1 µM.
σ
1–4
1–4
1–4
1,
2
4
µ

Molecules 2021, 26, 3860
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Control
1 M
DU145
(A)
150
100
50
10 M
50 M
**
**
**
***
0
1
2
3
4
Control
1 µM
U87MG
(B)
10 µM
50 µM
150
100
50
***
**
***
***
***
0
1
2
3
4
Figure 5. Effect of HO-1/
(panel ) cell lines, assessed by MTT assay at the doses of 1, 10 and 50
of at least three independent experiments and values are expressed as percentage of control (% of
control). Data represent means SEM. ** p < 0.01, *** p < 0.001 vs. control as determined by one-way
σ
R hybrids
1–
4
treatments on cell viability of DU145 (panel
A) and U87MG
B
µM. Results are representative
±
ANOVA followed by Tukey’s multiple comparison test.
The low cytotoxicity against DU145 cells and the moderate antiproliferative activity
towards U87MG cells of HO-1/
both HO-1 and Rs proteins showed by the same compounds
aging reduction in the viability of both cancer cells was obtained after co-administration of
HO-1 inhibitors and R ligands parent molecules, confirming that simultaneous inhibition
σ
Rs hybrids
1–
4
correlate well to the low potency towards
σ
1–4. Nevertheless, an encour-
σ
of HO-1 and modulation of σRs may be a valuable target for anticancer activity.
3. Materials and Methods
3.1. Chemistry
Melting points were determined by using an Electrothermal IA9200 apparatus contain-
ing a digital thermometer. Determinations were achieved after introducing glass capillary
1
tubes, filled with analytes, inside the apparatus, and are uncorrected. H NMR and 13
C
NMR spectra were recorded on Varian Inova Unity (200 MHz) spectrometers in DMSO-d6
or CDCl3 solution. Chemical shifts are given in values to two digits after the decimal
δ
point in part per million (ppm), using tetramethylsilane (TMS) as the internal standard;
coupling constants (J) are given in Hz. Signal multiplicities are indicated with the fol-
lowing abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br
(broad signal). The IR spectra were recorded in KBr disks or Neat, on a Perkin Elmer
1600 series FT-IR spectrometer. Reactions were monitored by thin-layer chromatography
(TLC), carried out on Merck plates (Kieselgel 60 F254), using UV light (254 and 366 nm) for

Molecules 2021, 26, 3860
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visualization and developed using iodine chamber. Flash column chromatography was
performed on Merck silica gel 60 0.040–0.063 mm (230–400 mesh). Reagents, solvents and
starting materials were purchased from commercial suppliers.
The synthetic procedures and characterization of intermediates
the Supplementary Materials.
9–16 are described in
General procedure for the synthesis of (1H-imidazol-1-yl)butoxy phenyl ketones (1–4
).
NaH (2.54 mmol) was added to a solution of 1H-imidazole (1.52 mmol) in anhydrous THF
(12 mL) under nitrogen. After 15 min, the appropriate bromobutoxy phenyl derivative
( –16), previously solubilized in THF (12 mL), was added and the reaction mixture was
13
left stirring for 9 h under reflux. The solvent was evaporated under vacuum, then water
(100 mL) was added to the resulting residue and extracted with ethyl acetate (3 50 mL).
×
The organic layer was washed with a basic solution (NaOH 0.5N 20 mL), brine (50 mL),
dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was
purified by flash column chromatography using ethyl acetate/methanol (9.5/ 0.5).
(4-(4-(1H-imidazol-1-yl)butoxy)phenyl)(4-benzylpiperazin-1-yl)methanone (1). Colorless
oil: yield 96,39 %. IR (KBr, selected lines) cm−1: 3402, 2940, 1657, 1610, 1512, 1461, 1300, 1176,
1
1026, 842. H NMR (200 MHz, DMSO-d6):
δ 7.64 (s, 1H, imidazole), 7.38–7.21 (m, 5H + 2H,
aromatic), 7.19 (s, 1H, imidazole), 6.99–6.90 (m, 2H, aromatic), 6.89 (s, 1H, imidazole),
4.07–3.95 (m, 2H + 2H, O-CH2-CH2-CH2-CH2-N), 3.55–3.39 (m, 2H + 4H, Ar-CH2-N +
piperazine), 2.42–2.31 (m, 4H, piperazine), 1.93–1.71 (m, 2H, O-CH2-CH2-CH2-CH2-N),
1.71–1.58 (m, 2H, O-CH2-CH2-CH2-CH2-N). 13C NMR (50 MHz, CDCl3):
δ 170.2, 159.9,
137.4, 129.4, 129.2, 128.8, 128.7, 128.4, 128.1, 127.4, 118.8, 114.1, 67.2, 62.9, 53.1, 50.4, 46.8,
28.0, 26.2. Anal. Calcd. for (C25H30N4O2): C, 71.74; H, 7.23; N, 13.39. Found: C, 71.56; H,
7.21; N, 13.42.
2-(4-(4-(1H-imidazol-1-yl)butoxy)phenyl)-1-(4-benzylpiperazin-1-yl)ethan-1-one (2). Color-
less oil: yield 72,95 %. IR (KBr, selected lines) cm−1: 2939, 2810, 1640, 1512, 1452, 1244, 1178,
1
1000, 742. H NMR (200 MHz, CDCl3):
δ 7.53 (s, 1H, imidazole), 7.29 (s, 5H, aromatic), 7.18–
7.02 (m, 3H, aromatic), 6.94 (s, 1H, imidazole), 6.81 (d, J = 8.5 Hz, 2H, aromatic + imidazole),
4.06–3.91 (m, 4H, O-CH2-CH2-CH2-CH2-N + O-CH2-CH2-CH2-CH2-N), 3.64–3.62 (m, 4H,
CO-CH2-Ar + Ar-CH2-N), 3.47–3.41 (m, 4H, piperazine), 2.40 (t, J = 10 Hz, 2H, piperazine),
2.27 (t, J = 8 Hz, 2H, piperazine), 2.02–1.91 (m, 2H, O-CH2-CH2-CH2-CH2-N), 1.82–1.72
(m, 2H, O-CH2-CH2-CH2-CH2-N). 13C NMR (50 MHz, CDCl3):
δ 169.8, 157.5, 137.5, 129.7,
129.3, 129.1, 128.7, 128.3, 127.3, 127.3, 118.8, 114.6, 67.1, 62.8, 52.9, 52.7, 46.8, 46.0, 41.8, 40.0,
28.1, 26.3. Anal. Calcd. for (C26H32N4O2): C, 72.19; H, 7.46; N, 12.95. Found: C, 71.98; H,
7.44; N, 12.99.
3-(4-(4-(1H-imidazol-1-yl)butoxy)phenyl)-1-(4-benzylpiperazin-1-yl)propan-1-one (3). Col-
orless oil: yield 60,49 %. IR (KBr, selected lines) cm−1: 3456, 2942, 1631, 1513, 1443, 1242,
1
825. H NMR (200 MHz, DMSO-d6):
δ 7.66 (s, 1H, imidazole), 7.33–7.23 (m, 5H, aromatic),
7.19 (s, 1H, imidazole), 7.12 (d, J = 8.6 Hz, 2H, aromatic), 6.89 (s, 1H, imidazole), 6.81 (d, J =
8.6 Hz, 2H, aromatic), 4.02 (t, J = 7.0 Hz, 2H, O-CH2-CH2-CH2-CH2-N), 3.95 (t, J = 6.2 Hz,
2H, O-CH2-CH2-CH2-CH2-N), 3.45–3.35 (m, 2H + 4H, Ar-CH2-N + piperazine), 2.72 (t, J
= 6.8 Hz, 2H, CO-CH2-CH2-Ar), 2.59–2.51 (m, 2H, CO-CH2-CH2-Ar), 2.27–2.51 (m, 4H,
piperazine), 1.89–1.76 (m, 2H, O-CH2-CH2-CH2-CH2-N), 1.69–1.56 (m, 2H, O-CH2-CH2-
CH2-CH2-N). 13C NMR (50 MHz, CDCl3):
δ 170.7, 157.2, 137.5, 133.6, 129.5, 129.2, 128.7,
128.4, 127.4, 118.9, 118.9, 114.5, 67.2, 62.9, 53.0, 52.8, 46.9, 45.6, 41.6, 35.4, 30.7, 28.2, 26.4.
Anal. Calcd. for (C27H34N4O2): C, 72.62; H, 7.67; N, 12.55. Found: C, 72.53; H, 7.66;
N, 12.58.
4-(4-(4-(1H-imidazol-1-yl)butoxy)phenyl)-1-(4-benzylpiperazin-1-yl)butan-1-one (4). Or-
ange oil: yield 85 %. IR (KBr, selected lines) cm−1: 3430, 2926, 1631, 1512, 1443, 1241,
1
1028, 999, 832, 744. H NMR (200 MHz, CDCl3)
δ 7.67 (s, 1H, imidazole), 7.39–7.28 (m,
4H + 1H, aromatic + imidazole), 7.10 (d, J = 8.2 Hz, 3H, aromatic), 6.98 (s, 1H, imidazole),
6.81 (d, J = 8.4 Hz, 2H, aromatic), 4.08 (t, J = 7.2 Hz, 2H, O-CH2-CH2-CH2-CH2-N), 3.97

Molecules 2021, 26, 3860
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(t, J = 5.8 Hz, 2H, O-CH2-CH2-CH2-CH2-N), 3.74–3.62 (m, 2H, piperazine), 3.57 (s, 2H,
Ar-CH2-N), 3.49–3.37 (m, 2H, piperazine), 2.62 (t, J = 7.5 Hz, 2H, piperazine), 2.52–2.37
(m, 4H, CO-CH2-CH2-CH2-Ar), 2.31 (t, J = 7.5 Hz, 2H, piperazine), 2.00–1.86 (m, 2H + 2H,
O-CH2-CH2-CH2-CH2-N + CO-CH2-CH2-CH2-Ar), 1.88–1.69 (m, 2H, O-CH2-CH2-CH2-
CH2-N). 13C NMR (50 MHz, CDCl3):
δ 171.3, 157.0, 137.3, 134.0, 129.5, 129.4, 129.2, 128.7,
128.4, 127.4, 114.3, 67.1, 62.9, 53.1, 52.8, 47.0, 45.5, 41.5, 34.5, 32.4, 28.1, 27.0, 26.3. Anal.
Calcd. for (C28H36N4O2): C, 73.01; H, 7.88; N, 12.16. Found: C, 72.91; H, 7.87; N, 12.20.
3.2. Biology
3.2.1. Preparation of Spleen Microsomal Fractions
Microsomal fractions obtained from rat spleen were used as sources of HO-1. Mi-
crosomal preparations obtained by differential centrifugation, were selected in order to
use the most native (i.e., closest to in vivo) forms of HO-1. The experiments reported
in the present paper complied with current Italian law and met the guidelines of the In-
stitutional Animal Care and Use Committee of Ministry of Health (Directorate General
for Animal Health and Veterinary Medicines) (Italy). The experiments were performed
in male Sprague–Dawley albino rats (150 g body weight and age 45 d). They had free
access to water and were kept at room temperature with a natural photo-period (12 h
light, 12 h dark cycle). For measuring HO-1 activities, each rat was sacrificed and their
spleens were excised and weighed. A homogenate (15%, w/v) of spleens pooled from
four rats was prepared in ice-cold HO-homogenizing buffer (50 mM Tris buffer, pH 7.4,
containing 0.25 M sucrose) using a Potter–Elvehjem homogenizing system with a Teflon
pestle. The microsomal fraction of rat spleen homogenate was obtained by centrifugation at
◦
10,000
×
g for 20 min at 4 C, followed by centrifugation of the supernatant at 100,000
×
g for
◦
60 min at 4 C. The 100,000g pellet (microsomes) was resuspended in an 100 mM potassium
phosphate buffer, pH 7.8, containing 2 mM MgCl2 with a Potter–Elvehjem homogenizing
system. The rat spleen microsomal fractions were divided into equal aliquots, placed into
microcentrifuge tubes, and stored at −80 ◦C for up to 2 months.
3.2.2. Preparation of Biliverdin Reductase
Biliverdin reductase was obtained from liver cytosol. Rat liver was perfused through
the hepatic portal vein with cold 0.9% NaCl, then it was cut and flushed with 2
of ice-cold PBS to remove all of the blood. Liver tissue was homogenized in 3 volumes of
×
20 mL
solution containing 1.15% KCl w/v and Tris buffer 20 mM, pH 7.8 on ice. Homogenates
◦
were centrifuged at 10,000
×
g, for 20 min at 4 C. Supernatant was decanted and centrifuged
◦
at 100,000
×
g for 1 h at 4 C to sediment the microsomes. The 100,000
was saved and then stored in small amounts at
was measured.
×
g supernatant
◦
−
80 C after its protein concentration
3.2.3. Measurement of HO-1 Enzymatic Activity in Microsomal Fraction of Rat Spleen
The HO-1 activities were determined by measuring the bilirubin formation using the
difference in absorbance at 464–530 nm. Reaction mixtures (500
Tris–HCl, pH 7.4, (1 mg/mL) microsomal extract, 0.5–2 mg/mL biliverdin reductase,
1 mM NADPH, 2 mM glucose 6-phosphate (G6P), 1 U G6P dehydrogenase, 25 M hemin,
and 10 L of DMSO (or the same volume of DMSO solution of test compounds to a final
concentration of 100, 10, and 1
µL) contained 20 mM
µ
µ
◦
M). Samples were incubated for 60 min at 37 C in a
µ
circulating water bath in the dark. Reactions were stopped by adding the same volume of
chloroform. After recovering the chloroform phase, the amount of bilirubin formed was
measured with a double-beam spectrophotometer as OD464–530 nm (extinction coefficient,
−1
40 mM/cm for bilirubin). One unit of the enzyme was defined as the amount of enzyme
catalyzing the formation of 1 nmol of bilirubin/mg protein/h.

Molecules 2021, 26, 3860
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3.2.4. Radioligand Binding Assay
Brain and liver homogenates for σ1R and σ2R binding assays were prepared from
male Dunkin–Hartley guinea pigs and Sprague–Dawley rats, respectively, (ENVIGO RMS
S.R.L., Udine, Italy) as previously reported [54]. In vitro σ1R ligand binding assays were
carried out in Tris buffer (50 mM, pH 7.4) for 150 min at 37 ◦C. The thawed membrane
preparation of guinea pig brain cortex was incubated with increasing concentrations of
test compounds and [3H](+)-pentazocine (2 nM) in a final volume of 0.5 mL. Unlabeled
(+)-pentazocine (10 µM) was used to measure non-specific binding. Bound and free
radioligand were separated by fast filtration under reduced pressure using a Millipore
filter apparatus through Whatman GF 6 glass fiber filters, which were presoaked in a 0.5%
poly(ethyleneimine) water solution. Each filter paper was rinsed three times with ice-cold
Tris buffer (50 mM, pH 7.4), dried at rt, and incubated overnight with scintillation fluid
into pony vials. The bound radioactivity has been determined using a liquid scintillation
counter (Beckman LS 6500) [55]. In vitro σ2R ligand binding assays were carried out in
Tris buffer (50 mM, pH 8.0) for 120 min at rt. The thawed membrane preparation of
rat liver was incubated with increasing concentrations of test compounds and [3H]DTG
(2 nM) in the presence of (+)-pentazocine (5
µM) as σ1R masking agent in a final volume
of 0.5 mL. Non-specific binding was evaluated with unlabeled DTG (10
µM). Bound and
free radioligand were separated by fast filtration under reduced pressure using a Millipore
filter apparatus through Whatman GF 6 glass fiber filters, which were presoaked in a 0.5%
poly(ethyleneimine) water solution. Each filter paper was rinsed three times with ice-cold
Tris buffer (10 mM, pH 8), dried at rt, and incubated overnight with scintillation fluid into
pony vials. The bound radioactivity was determined using a liquid scintillation counter
(Beckman LS 6500) [56]. The Ki-values were calculated with the program GraphPad Prism®
7.0 (GraphPad Software, San Diego, CA, USA). The Ki-values are given as mean value
from at least two independent experiments performed in duplicate.
±
SD
3.2.5. Cell Cultures
Two lines of cancer cells were used to conduct our investigations. In particular,
we used the human glioblastoma cell line U87MG (ATCCC number #HTB-14) and the
prostate cancer cell line DU145 (ATCC HTB-81). These cell lines were obtained from the
American Type Culture Collection (ATCC, Rockville, Md., USA). Cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of heat-inactivated
fetal bovine serum (FBS), 100 U/mL penicillin and 100-µg/mL streptomycin (Sigma-
Aldrich, Steinheim, Germany) and incubated at 37 ◦C in a humidified atmosphere with
5% CO2.
3.2.6. In Vitro Cytotoxicity of HO-1 Inhibitors, σR Ligands, and HO-1/σR Hybrids
1–4 against DU145 and U87MG Cancer Cell Lines
The cytotoxicity of HO-1 inhibitors and
as well as novel HO-1/ R hybrids compounds were evaluated. The effect on cell viability
was assessed by performing MTT assay. Cells were seeded into 96-well plates at a density
of 7.0 L of culture medium. The day after, cells were treated
103 cells/well in 100
with each molecule at three different concentrations (1 M, 10 M and 50 M) for 72 h.
σR ligands previous synthesized compounds
σ
×
µ
µ
µ
µ
Following treatments, 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) (Sigma-Aldrich) was added to each well and incubated for 4 h at 37 ◦C.
Finally, dimethyl-sulfoxide (DMSO) was used to dissolve formazan salts and absorbance
was measured at 450 nm in a microplate reader (Biotek Synergy-HT). Six replicate wells
were used for each group.
4. Conclusions
Due to its complex etiology, cancer may be counteracted by targeting different biolog-
ical pathways. In this paper, we investigated whether the inhibition of HO-1 enzymatic
activity and simultaneous modulation of the σR functions may have some advantages in

Molecules 2021, 26, 3860
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reducing the proliferation of DU145 human prostate and U87MG glioblastoma cancer cell
lines. In this regard, compounds alone, a combination of two compounds, i.e.,
haloperidol, SI1/13 or RFB/13 plus HO-1 inhibitors LS/0 LS4/28 or LS6/42, and HO-1/
hybrid compounds were evaluated. Although hybrids showed only moderate
antiproliferative activity against glioblastoma cells, we proved for the first time that simul-
taneously targeting HO-1 and R proteins reduces DU145 and U87MG cell proliferation to
a major extent concerning the effect achieved with single compounds. The obtained results
σ
R ligands
,
σ
Rs
1–
4
1–4
σ
serve as an initial proof of concept, useful for optimizing HO-1/
develop novel potential anticancer agents.
σRs hybrids’ structure to
Supplementary Materials: The following are available online at: Synthesis of intermediates
9–12.
Synthesis of intermediates 13 16. Table S1: HO-1 inhibition and binding properties of hybrids 1–4
–
1
and reference compounds. Table S2: Elemental analysis data for compounds
NMR spectra of intermediates
1
–
4. Figures S1–S8: H
1
9–
16. Figures S9-S16: H NMR and 13C NMR spectra of compounds
1–4. Figure S17: Viability of MDA-MB 231 cell line, References.
Author Contributions: G.R., L.S., V.S. and S.I. designed the study and the experiments; G.R., L.C.
and V.C. synthesized, purified and characterized the compounds; G.R., V.C. and S.I. performed
and analyzed 1H and 13C-NMR spectra; V.S. and L.V. performed HO-1 inhibition experiments; E.A.
and M.D. performed the radioligand binding assay; A.G.D. and S.G. performed cancer cell lines
experiments; G.R., L.S., V.S., V.D., E.A. and A.G.D. provided reagents, materials, and analysis tools;
L.S. and G.R. prepared the original draft. All authors revised the draft. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by (1) PON R&I funds 2014–2020 (CUP: E66C18001320007,
AIM1872330, activity 1); (2) Programma Ricerca di Ateneo UNICT 2020-22 linea 2; (3) Project autho-
rized by the Ministry of Health (Directorate General for Animal Health and Veterinary Medicines)
“Dosing of enzymatic activities in rat microsomes” (2018–2022; project code 02769.N.VLY).
Institutional Review Board Statement: All animal experiments were performed based on the rules
and regulations and policy of the University of Catania and approved by the Italian Ministry of
Health (Directorate General for Animal Health and Veterinary Medicines, 2018–2022; project code
02769.N.VLY).
Conflicts of Interest: The authors declare no conflict of interest.
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