Novel imidazole derivatives as heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) inhibitors and their cytotoxic activity in human-derived cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2015.04.003

Heme oxygenase (HO) is a cytoprotective enzyme that can be overexpressed in some pathological conditions, including certain cancers. In this work, novel imidazole derivatives were designed and synthesized as inhibitors of heme oxygenase-1 (HO-1) and heme

Full text of DOI:10.1016/j.ejmech.2015.04.003

European Journal of Medicinal Chemistry 96 (2015) 162e172
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel imidazole derivatives as heme oxygenase-1 (HO-1) and heme
oxygenase-2 (HO-2) inhibitors and their cytotoxic activity in human-
derived cancer cell lines
,
*
,
1
a
a
ꢀ a
Loredana Salerno a , Valeria Pittala , Giuseppe Romeo , Maria N. Modica ,
Agostino Marrazzo a, Maria A. Siracusa a, Valeria Sorrenti b 1, Claudia Di Giacomo b,
,
Luca Vanella b, Neha N. Parayath c, Khaled Greish c
,
d
a Department of Drug Sciences, Section of Medicinal Chemistry, University of Catania, viale A. Doria 6, 95125 Catania, Italy
b Department of Drug Sciences, Section of Biochemistry, University of Catania, viale A. Doria 6, 95125 Catania, Italy
c Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand
d Department of Oncology, Faculty of Medicine, Suez Canal University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Heme oxygenase (HO) is a cytoprotective enzyme that can be overexpressed in some pathological
conditions, including certain cancers. In this work, novel imidazole derivatives were designed and
synthesized as inhibitors of heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). In these com-
pounds the imidazole ring, crucial for the activity, is connected to a hydrophobic group, represented by
aryloxy, benzothiazole, or benzoxazole moieties, by means of alkyl or thioalkyl chains of different length.
Many of the tested compounds were potent and/or selective against one of the two isoforms of HO.
Furthermore, most of the pentyl derivatives showed to be better inhibitors of HO-2 with respect to HO-1,
revealing a critical role of the alkyl chain in discriminating between the two isoenzymes. Compounds
which showed the better profile of HO inhibition were selected and tested to evaluate their cytotoxic
properties in prostate and breast cancer cell lines (DU-145, PC3, LnCap, MDA-MB-231, and MCF-7). In
these assays, aryloxyalkyl derivatives resulted more cytotoxic than benzothiazolethioalkyl ones; in
particular compound 31 was active against all the cell lines tested, confirming the anti-proliferative
properties of HO inhibitors and their potential use in the treatment of specific cancers.
Received 23 January 2015
Received in revised form
2 April 2015
Accepted 4 April 2015
Available online 7 April 2015
Keywords:
Heme oxygenase
HO-1
HO-2
Inhibitors
Anti-proliferative properties
Anti-cancer
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
functional isoforms only: HO-1, which is highly inducible and HO-2,
which is constitutive. HO-1 is a 32 kDa heat shock protein that is
Heme oxygenase (HO) is the rate-limiting enzyme that catalyzes
the metabolism of the pro-oxidant agent heme into equimolar
amounts of carbon monoxide (CO), free iron, and the bile pigment
biliverdin, which in turn is reduced to bilirubin by biliverdin
reductase [1]. Each of these metabolites has regulatory functions:
CO is a cell-signaling molecule with anti-inflammatory, anti-
proliferative, and antiapoptotic effects [2], biliverdin and bilirubin
possess antioxidant properties [3].
expressed in tissues rich in reticuloendothelial cells such as spleen.
In other tissues its expression is induced by numerous stimuli,
including its substrate heme, heavy metals, reactive oxygen species,
hypoxia, NO, ultraviolet radiations, and xenobiotics. HO-2 is a
36 kDa protein constitutively present in brain and testis, but also in
endothelium, distal nephron segment, liver, and gut myenteric
plexus.
The biological functions of HO are mainly associated with a basic
adaptive and defensive response against oxidative and cellular
stress and with a maintaining of cellular homeostasis [5]. Conse-
quently, HO-1 up-regulation might have therapeutic applications in
many oxidative stress-associated diseases, such as diabetes,
obesity, cardiovascular and eye diseases, atherosclerosis, inflam-
mation, and vascular injury. However, over-activity of the HO
Three isoforms of HO have been identified so far, namely HO-1,
HO-2, and HO-3 [4], but enzymatic activity is attributable to two
* Corresponding author.
E-mail address: l.salerno@unict.it (L. Salerno).
These authors contributed equally to the work.
1
http://dx.doi.org/10.1016/j.ejmech.2015.04.003
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
163
system may not exert only cytoprotective effects, but may
contribute to tissue injury under certain unfavorable cases. HO-1
down-regulation or direct inhibition may ameliorate diseases in
which HO over-activity is observed, for example hyper-
bilirubinemia and neonatal jaundice [6], and various bacterial or
fungal infectious diseases [7]. Moreover, an increased expression of
HO-1 has been observed in several cancers, suggesting that one of
the main therapeutic applications of HO-1 inhibitors could be the
treatment of specific cancers. Multiple studies confirm implications
of HO in various tumors [8,9] and the effectiveness of treatment
with HO inhibitors [10,11].
Most of the actual knowledge on HO system is related to HO-1
isoform, whereas spare information is available on HO-2 isoform.
Recent works report significant tumor regression mediated by a
down regulation of both HO-1 and HO-2 system [12]. Therefore,
selective HO-1 or HO-2 inhibitors may provide useful tools for the
elucidation of the physiological roles of HO system and may have
important clinical applications.
reported as HO inhibitors. Their chemistry and the meaningful
structural insights into human HO-1 inhibition have been recently
reviewed [14]. In brief, three key chemical moieties are necessary
for inhibition of HO: an azolyl nucleus, a hydrophobic portion, and a
central alkyl linker. This linker may have different length and may
incorporate a dioxolane ring, a ketone or an alcohol function and
heteroatoms such as sulfur or oxygen. Some representative com-
pounds 1e7, possessing the above-mentioned chemical features,
are depicted in Fig. 1 [15e18].
Selected compounds have been more deeply studied and have
shown to be effective also in intact cells and in in vivo models. For
example, it has been demonstrated that compound 5 inhibits cell
proliferation in vitro and tumor growth in vivo when tested in a
model of hormone-refractory prostate cancer [19].
Continuing our study on imidazole-based compounds as enzy-
matic inhibitors [20e22], we recently investigated a series of ary-
loxyalkyl imidazoles as HO-1 and HO-2 inhibitors [17,18]. Among
them, 1-[4-(3-bromophenoxy)butyl]-1H-imidazole 6 and 1-[4-(4-
iodophenoxy)butyl]-1H-imidazole 7 (Fig. 1) showed the highest
inhibitory potency. HO-1 overexpression, together with a variety of
molecular mechanisms, is responsible of resistance to imatinib
In the recent years, a number of imidazole-based compounds
derived from structural modifications of the first described non-
porphyrin HO-inhibitor azalanstat
1 (Fig. 1) [13] have been
Fig. 1. Chemical structures and IC50 values of HO inhibitors 1e7.

164
L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
mesylate (IM) in the treatment of chronic myeloid leukemia (CML)
[23e25]. In our experiments, simultaneous administration of
compounds 6 and 7 with IM in CML cells overexpressing HO-1 and
resistant to IM overcame resistance to IM, sensitizing these cells to
the antitumor effect of IM itself and demonstrating the antitumor
properties of our HO-1 inhibitors.
Based on these premises, in this work we synthesized and tested
novel imidazole-based inhibitors 29e42 (Table 1) to examine two
main chemical modifications of our lead compounds: i) the elon-
gation of the connecting alkyl linker; ii) the replacement of the
hydrophobic aryloxy moieties with bicyclic heterocyclic nuclei such
as benzoxazole, benzothiazole, and 5-chlorobenzothiazole. In light
of the involvement of HO system in the progress of various ma-
lignant tumors and the consequent potential usefulness of HO in-
hibition, we selected compounds showing the better profile as HO
inhibitors, to evaluate their cytotoxic effect against prostate cancer
cells (DU-145, PC3, LnCap) and breast cancer cells (MDA-MB-231,
MCF-7), showing different expression pattern of HO system, and
against non-cancerous canine kidney epithelial cells (MDCK).
starting materials were phenols 8 and 9, in the first step they were
treated with appropriate dibromoalkane in DMSO, in the presence
of KOH, at room temperature, to give aryloxyalkylbromides 15e18
in good yields. When the starting materials were 2-
mercaptobenzoxazole, 2-mercaptobenzothiazole, 2-mercapto-5-
chlorobenzothiazole, 2-benzoxazolone, or 2-benzothiazolone
10e14, the first step was carried in acetone, at room temperature,
with appropriate 1-bromo-u-chloroalkane or dibromoalkane, in
the presence of K2CO3. The intermediates benzoxazole, benzo-
thiazole, or 5-chlorobenzothiazole thioalkylchlorides 19e26, 2-
benzoxazolone or 2-benzothiazolone alkylbromides 27 and 28
were obtained in good yields. In the second step, compounds
15e28 were allowed to react with imidazole or 1,2,4-triazole in
acetonitrile under microwave irradiation, in the presence of trie-
thylamine (TEA) and tetrabutylammoniumbromide (TBAB), at 90 ꢀC
for 30e45 min, to give desired final products 29e42 and 50 in
moderate yields.
Purifications of all new compounds were performed by flash
chromatography using ethyl acetate or mixtures of ethyl acetate
and methanol as eluent. The structures of all new synthesized
compounds were confirmed by analytical data, IR, and 1H NMR
spectra.
2. Results and discussion
2.1. Chemistry
2.2. Structureeactivity relationships
The synthesis of imidazole derivatives 29e42 and 50 followed
the general two-steps pathways illustrated in Scheme 1. When the
In this work, compounds 29e50 were tested to evaluate their
Table 1
Inhibitory potency of compounds 29e50 for HO-1 and HO-2.
Comp.
R
n
X
IC50
HO-1a
42.6 1.2
(
m
M)
HO-2a
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
6
3-Br
3-Br
4-I
4-I
H
H
H
H
H
5
6
5
6
3
4
3
4
5
3
4
5
4
3
3
3
3
4
3
4
3
5
CH
CH
CH
CH
O
O
S
S
S
S
S
S
O
S
N
CH
N
N
0.9 0.01
34
44
42
3
2
1
41
3
0.9 0.02
>100
16.9 2.1
30.9 1.3
0.9 0.01
49
4
74.1 5.1
12.5 2.5
0.8 0.01
11.2 2.1
0.9 0.02
0.9 0.01
0.9 0.02
35.5 1.3
28.8 3.1
>100
44.7 4.1
77.6 5.2
41.7 3.3
33.9 2.5
11.2 0.9
36.3 2.8
0.8 0.01
2.2 0.2b
10 0.5b
1
0.02
39.8 3.1
18.4 1.5
>100
Cl
Cl
Cl
36.3 2.3
17.7 1.2
16.6 1.1
>100b
H
4-NO2
4-NO2
4-NO2
3-Br
3-Br
4-I
>100b
>100b
>100b
N
N
CH
N
>100b
>100b
>100b
3-Br
20
2.1 0.3b
0.01b
1
7
1
a
Data are shown as IC50 values in
Data taken from Ref. [18].
m
M
standard deviation (SD). Values are the mean of triplicate experiments.
b

L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
165
Scheme 1. Reagents and conditions: (a) DMSO, KOH, 1,
u
-dibromoalkane, room temperature, 1 h; (b) acetone, K2CO3, 1-bromo-
u
-chloroalkane or 1,
u
-dibromoalkane, room tem-
perature, 24 h; (c) imidazole or 1,2,4-triazole (to obtain 50), acetonitrile, TEA, TBAB, 90 ꢀC, MW, 30e45 min.
ability to inhibit HO-1 and HO-2 obtained from the microsomal
fractions of rat spleen and rat brain, respectively. The HO-1 and HO-
2 activities were determined by measuring the bilirubin formation
using the difference in absorbance at 464e530 nm, as described in
the experimental section. Compounds 6 and 7 (Fig. 1) were used as
reference substances. Inhibition of enzyme activity is expressed as
imidazole nucleus and a hydrophobic moiety connected by
different spacers; these last can be of different length and can
incorporate various functional groups (Fig. 1). Imidazole ring is
crucial for the binding to the enzyme, since it serves as an anchor by
coordinating with the heme iron of the enzymatic complex; the
hydrophobic moiety stabilizes the complex inhibitor-enzyme
through interactions involving residues lining in hydrophobic
pocket, i.e. Phe33, Met34, Phe37, Val50, Leu54, Leu147, Phe167, and
Phe214. This pocket is very flexible and can accommodate various
hydrophobic groups, such as phenyl, 4-chlorophenyl, adamantly, or
IC50 (mM) and results are summarized in Table 1.
In the design of novel inhibitors, we took into account previous
SAR and crystallographic studies. These studies have demonstrated
that the key-functional groups required for HO-1 inhibition are an

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L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
biphenyl moieties [15,16,26]. On these basis and taking into account
6 and 7 (Fig. 1) as lead compounds, in this work we designed,
together with benzimidazole derivatives synthesized by Vlahakis
et al. [33], the first potent and selective HO-2 inhibitors described
so far.
synthesized, and tested
derivatives.
a new series of imidazole-based
In the first part of this study, we wanted to examine the influ-
ence of the length of the linker on potency and selectivity against
HO-1 or HO-2. In this regard, we synthesized and tested com-
pounds 29e32, in which the oxybutyl chain of the reference com-
pounds 6 and 7 was replaced with oxypentyl or oxyhexyl chains.
Results show that an elongation of the spacer is detrimental for HO-
1 inhibition whereas it is important to improve the inhibition of
HO-2. In particular, it is possible to observe a decrease of potency of
the oxyhexyl derivatives 30 and 32 for both isoforms, and an in-
crease of potency of oxypentyl derivatives 29 and 31 only for HO-2
when compared to reference compounds 6 and 7. These results
suggest that the length of the linker is crucial for the binding of
these inhibitors with the active site of the enzyme.
In a second step we wanted to investigate the effect of the hy-
drophobic group on HO activity; with this purpose, we synthesized
the imidazole derivatives 33e42 in which the aryl moieties of the
reference compounds 6 and 7 were replaced with benzoxazole,
benzothiazole, 5-chlorobenzothiazole, 2-benzoxazolone, and 2-
benzothiazolone nuclei. These bicyclic heterocycles possess the
chemical features required for the interaction with the hydrophobic
pocket of the enzyme and some of them are part of molecules
showing numerous biological activities, such as anticancer prop-
erties [27e31]. Furthermore, 2-benzoxazolone and 2-
benzothiazolone are regarded as “privileged scaffolds” in medici-
nal chemistry [32].
Results show that benzoxazole (33, 34), 2-benzoxazolone and 2-
benzothiazolone (41, 42) derivatives are poor HO-1 and HO-2 in-
hibitors, demonstrating that these heterocycles give a negative
contribution to the activity. On the other hand, most interesting
compounds belong to the benzothiazole series (35e38); particu-
larly, the thiopropyl derivative 35 results a potent HO-1 inhibitor,
about ten-fold selective for HO-1 over HO-2 whereas the thiobutyl
derivative 36 is potent in inhibiting both isoforms. In addition, the
thiopentyl derivative 37, although less potent than its shorter ho-
mologues 35 and 36, prefers HO-2 with respect to HO-1, confirming
that a pentyl chain is crucial for the binding to HO-2, as observed in
the aryloxyalkyl series 29e32. Furthermore, 5-chlorobenzothiazole
derivatives 38e40 are noteworthy since all of them are potent HO-2
inhibitors and the thiobutyl derivative 39 is the most selective HO-
2 inhibitor among tested compounds.
To the best of our knowledge, very few HO-2 inhibitors, selective
over HO-1, are known so far, with consequent sparse information
on biological and pharmacological roles of the HO-2 isoform.
Among the novel imidazole derivatives described in this work,
some are very good HO-2 inhibitors selective over HO-1, particu-
larly derivatives 29 and 31, belonging to the aryloxyalkyl series, and
compound 39, belonging to the bicyclic series. We had previously
synthesized a series of aryloxyalkyl imidazoles derivatives [18] and
some of them (43e49, Table 1) were not assayed on HO-2 because
they did not inhibit HO-1. In light of the interesting selective HO-2
inhibition observed for compounds 29 and 31, we decided to test
now on HO-2 the previously synthesized aryloxyalkyl imidazoles
43e49. All compounds result poor inhibitors of HO-2, with the
exception of 1-[4-(3-bromophenoxy)butyl]-1H-1,2,4-triazole 48,
2.3. In vitro cytotoxic activity
In light of the HO-1 overexpression in many tumors [34] and on
the basis of the antitumor properties previously observed for
compounds 6 and 7 [18], we selected compounds 6, 7, 31, 35, and
36, showing the better profile as HO inhibitors, for further tests.
Pharmacokinetics and bioavailability may affect the efficacy of a
potential drug as well as pharmacodynamics. With the aim of
predicting pharmacokinetic properties of the above-mentioned
five compounds, we calculated with QikProp 4.3 software [35]
some physicochemical and some ADME-properties. The first
include logarithm of octanolewater partition coefficient (log P) and
logarithm of aqueous solubility (log S), the second include mem-
brane permeability of Caco-2 and MDCK cells (Caucasian colon
adenocarcinoma and MadineDarby canine kidney, respectively),
and % human intestinal absorption (HIA). Values obtained for all
these descriptors (Table 1, supporting material) indicated a good
predicted cell permeation and consequent 100% HIA. On these
premises, these compounds were tested to evaluate their cytotoxic
activity on five cancer cell lines: prostate and breast cancer cells,
both hormone therapy resistant (DU-145, PC3, and MDA-MB-231,
respectively) and sensitive (LnCap and MCF-7, respectively). These
cancer cells show different expression pattern of HO system.
Regarding breast cancer cell lines, moderate levels of HO-1
expression could be observed in MDA-MB-231 cell line [36,37],
whereas concerning to MCF7 cells, conflicting results are reported;
many authors reported moderate levels of HO-1 expression
[36e38] and others reported high levels of HO-1 expression in
MCF7 cells [39]. Consequently, some studies show that HO-1 in-
duction inhibits cell proliferation and invasiveness of breast cancer
cells [38,40,41], other studies affirm that HO-1 may be involved in
breast carcinogenesis and progression and its inhibition may
contribute to reduce cell proliferation [42]. Regarding prostate
cancer cell lines, overexpression of HO-1 has been reported in
LnCap and DU145 cells [19], whereas expression of HO-1 was lower
in PC3 than in LnCap or DU145 cells [43,44].
Cytotoxicity was determined using the sulforhodamine
B
colorimetric assay based on the measurement of cellular protein
content. Results of cytotoxic activity of selected compounds are
described in Figs. 2e4 and in Table 2. As a general comment, ary-
loxyalkyl imidazole derivatives 6, 7, and 31 result more potent than
benzothiazolethioalkyl imidazole derivatives 35 and 36; in partic-
ular, compound 31 is the most interesting compound since has a
good cytotoxicity against all tumor cell lines tested.
Concerning breast cancer, all tested compounds have a good
cytotoxicity in MCF7 cell line, in particular compound 31
(IC50 ¼ 10.98
mM), whereas in MDA-MB-231 cell line, that express
lower levels of HO-1 compared to MCF7 cells, only compounds 6
and 31 maintain a good capacity of inhibiting cell proliferation.
These results might be related to the different levels of HO-1
expression in the two examined cell lines, but also might be due
to the less invasiveness of hormone-responsive MCF7 with respect
to MDA-MB-231 cell line.
With regard to prostate cancer, cytotoxicity of all tested com-
pounds, with the exception of 31, is moderate in PC3 with respect to
LnCap and DU145 cell lines; this result can be related to the
expression of HO-1 which is higher in LnCap and DU145 cells
relative to PC3 cells. Comparing the results obtained in the two
prostatic cell lines overexpressing HO-1, i.e. LnCap and DU145, it
can be observed that LnCap cells are sensitive to the treatment with
with an HO-2 IC50 of 11.22 mM and HO-1 IC50 > 100 mM. This result
prompted us to synthesize the pentyl homologue of 48, i.e 1-[5-(3-
bromophenoxy)pentyl]-1H-1,2,4-triazole 50. This compound is a
very good HO-2 inhibitor with reduced potency for HO-1. This
result confirms the critical role of the pentyl spacer to determine a
different binding with the two isoforms of HO. Moreover, to the
best of our knowledge, compounds 29, 31, 39, 48, and 50 are,
compounds 6, 7, and 31, in particular 7 (IC50 ¼ 16.93
mM), whereas

L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
167
Fig. 2. Cytotoxicity of HO inhibitors against breast cancer cells. A) MDA-MB-231 (triple negative) cells and B) MCF-7 (hormone sensitive) cells were treated with 1.1e200 mM
compounds 6, 7, 31, 35, and 36 for 72 h. Control wells were treated with vehicle only (0.2% DMSO). At the end of treatment Cell number was determined using the sulforhodamine B
assay. Data are expressed as mean SEM (n ¼ 3). Non-linear regression and IC50 values determination was performed using GraphPad Prism 6.
Fig. 3. Cytotoxicity of HO inhibitors against prostate cancer cells. A) DU-145 (hormone resistant) cells B) PC3 (hormone resistant) cells, and C) LnCap (hormone sensitive) cells were
treated with 1.1e200
mM of compounds 6, 7, 31, 35, and 36 for 72 h. Control wells were treated with vehicle only (0.2% DMSO). At the end of treatment Cell number was determined
using the sulforhodamine B assay. Data are expressed as mean SEM (n ¼ 3). Non-linear regression and IC50 values determination was performed using GraphPad Prism 6.

168
L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
tumor regression by decreasing HO-derived CO, mainly involved in
tumor angiogenesis. At the best of our knowledge, compound 31 is
the first example of HO-1 and HO-2 inhibitor more potent on HO-2
isoform, which possesses in vitro cytotoxic effect towards various
kind of tumors.
3. Conclusions
In the present study, imidazole and triazole derivatives 29e50
were investigated as HO-inhibitors. Many of the tested compounds
result interesting both for their potency and/or selectivity against
one of the two isoforms of HO. In particular, among benzothiazole-
containing molecules, thiopropyl derivative 35 is a potent inhibitor
of HO-1, about ten-fold selective for HO-1 over HO-2, and the thi-
obutyl derivative 36 is potent toward both isoforms. Moreover, the
oxypentyl derivatives 29, 31, 48, 50 and the thiobutyl derivative 39,
result potent and selective inhibitors of HO-2 over HO-1, suggesting
their application as pharmacological tools useful to better under-
stand the biological roles of HO-2.
Fig. 4. Cytotoxicity of HO inhibitors against MDCK cells. MDCK (canine kidney
Since HO over-activity is often associated with tumor cells
proliferation, compounds showing the better profile of inhibition
were tested in in vitro models of five tumor cell lines, i.e. DU-145,
PC3, LnCap, MDA-MB-231, and MCF-7, and one non-cancerous
cell line, i.e. MDCK. Results of the present study show that com-
pounds 31, selective inhibitor of HO-2 over HO-1, is the most
interesting derivative among tested compounds.
Moreover, MCF7 and LnCap cells, that represent early stage of
cancer progression, are more sensitive to HO-1/HO-2 inhibition.
Particularly, compound 31, selective for HO-2, is most active in
MCF7 cells expressing moderate levels of HO-1, whereas compound
7, more potent on HO-1, is most active in LnCap cells expressing
high levels of HO-1. All compounds result 2e6 fold selective for
cancer cells with respect to healthy cells.
Based on these in vitro results, each cancer cell line responds
differently to HO-1 or HO-2 inhibitor treatment, suggesting a dif-
ferential expression and distinct roles of the two isoenzymes in
different cancers. These findings strongly suggest that selective
inhibition of HO-1 and/or HO-2 may be an option to enhance the
chemotherapeutic effects of conventional anticancer drugs. In view
of this consideration, it is plausible to speculate that selective in-
hibition of HO-1 or HO-2 in tumor cells utilizing nanotechnology,
may represent a novel strategy for effective anticancer manage-
ment, especially to enhance delivery to the target tumor cells
without affecting normal cells.
epithelial) cells were treated with 1.1e200 mM of compound 6, 7, 31, 35 and 36 for 72 h.
Control wells were treated with vehicle only (0.2% DMSO). At the end of treatment cell
number was determined using the sulforhodamine B assay. Data are expressed as
mean SEM (n ¼ 3). Non-linear regression and IC50 values determination was per-
formed using GraphPad Prism 6.
DU145 cells are sensitive only to compound 31. These effects may
be due to cell type specific response. DU145 cells are considered
more invasive (representative of brain metastasis) and less differ-
entiated as compared to LnCap cells (representative of lymph node
metastasis), therefore the major effect of compounds 6, 7, and 31 in
LnCap cells respect to DU145 cells may be due to the less inva-
siveness of the hormone-responsive LnCap cell line.
Moreover, we the aim of getting more information on the
pharmacodynamic properties of compounds 6, 7, 31, 35, and 36,
they were tested on the non-cancerous cell line MDCK, selected as
example of healthy cells. All compounds are less active against
MDCK cells (Fig. 4 and Table 2), resulting 2e6 fold selective for
cancer cells with respect to healthy cells.
Results obtained in the present study clearly show that each
cancer cell line responds differently to HO-1 or HO-2 inhibitor
treatment. It is believed that intracellular localization of HO iso-
forms may be related to selective functions in different cell types
[45,46] and several studies have implicated intracellular localiza-
tion of HO-1 with different cancers [47]. On the contrary, few re-
ports have associated HO-2 protein expression with tumor
proliferation. Up to date, the role of HO-2 in cancer is almost un-
explored. According to He et al. [48], HO-2 reduction may
contribute to the activation of apoptotic pathway and inhibition of
cell growth. Our previous results evidenced, for the first time, the
ability of ellagic acid to decrease the expression of both HO-1 and
HO-2 and to induce apoptosis in LnCap cells [12]. A down-
regulation of the HO system and in particular of the HO-2 isoen-
zyme, constitutively expressed, could contribute to a significant
4. Experimental procedures
4.1. Chemistry: general methods
Melting points were determined in an Electrothermal IA9200
apparatus with a digital thermometer in glass capillary tubes.
Elemental analyses for C, H, N, S were within 0.4% of theoretical
values and were performed on a Carlo Erba Elemental Analyzer
Table 2
IC50 values for HO inhibitor compounds in hormone resistant and hormone responsive breast, prostate cancer cells, and in non-cancerous canine kidney epithelial cells.
Comp
IC50 (m
M)a
MDA-MB-231
MCF-7
DU145
PC3
LnCap
MDCK
6
7
31
35
36
27.46 0.17
55.06 2.77
27.44 0.23
55.12 0.99
55.07 0.48
27.57 0.06
27.69 0.08
10.98 0.03
27.51 0.05
27.46 0.06
46.56 0.52
54.91 0.82
27.68 0.22
54.84 0.55
82.23 0.26
54.87 0.53
82.49 0.61
27.61 0.21
82.64 0.98
82.51 0.12
27.61 0.20
16.93 0.07
27.79 0.18
55.03 0.91
54.73 0.80
82.41 0.01
102.6 0.88
55.08 0.04
82.41 0.05
58.46 3.45
a
Data are shown as IC50 values in
m
M
standard deviation (SD). Values are the mean of triplicate experiments.

L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
169
Mod.1108 apparatus. The IR spectra were recorded in KBr disks on a
Perkin Elmer 1600 series FT-IR spectrometer. 1H NMR spectra were
determined with a Varian Inova Unity 200 (200 MHz) instrument in
4.3.2. 2-[(3-Chloropropyl)thio]-5-chlorobenzothiazole (24)
Flash chromatography on silica gel with cyclohexane/ethyl ac-
etate (9.8:0.2, v:v) gave a white solid; mp 50.0e51.0 ꢀC; yield 72%;
IR (KBr) cmꢂ1: 2961, 1584, 1546, 1453, 1425, 1247, 1082; 1H NMR
DMSO-d6 solution. Chemical shifts are in
d values (ppm) using
tetramethylsilane as the internal standard; coupling constants (J)
are given in Hz. Signal multiplicities are characterized as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), br (broad signal).
All the synthesized compounds were checked by TLC on Merck
plates (Kieselgel 60 F254) and spots were visualized under the UV
(DMSO-d6) d 8.08e8.04 (m, 1H, aromatic), 7.95e7.94 (m, 1H, aro-
matic), 7.45e7.40 (m, 1H, aromatic), 3.79 (t, J ¼ 6.4 Hz, 2H, CH2Cl),
3.48 (t, J ¼ 6.8 Hz, 2H, SCH2), 2.30e2.17 (m, 2H, CH2CH2CH2). Anal.
C10H9Cl2NS2 (C, H, N, S).
light (
l
¼ 254 and 366 nm). Preparative column chromatography
4.3.3. 2-[(4-Chlorobutyl)thio]-5-chlorobenzothiazole (25)
Recrystallization by petroleum ether 40e60 ꢀC gave a white
was performed using Merck silica-gel 60 (230e400 mesh). Micro-
wave irradiation experiments were carried out with a CEM
Discover instrument using closed Pyrex glass tubes (ca. 10 mL) with
Teflon-coated septa. All chemicals and solvents were reagent grade
and were purchased from commercial sources. Analytical and
spectral data of unknown compounds are following reported.
solid; mp 49.0e50.0 ꢀC; yield 69%; IR (KBr) cmꢂ1: 3089, 2953, 1544,
1425, 1321, 1007, 812; 1H NMR (DMSO-d6)
d 8.07e8.03 (m, 1H, ar-
omatic), 7.94e7.93 (m, 1H, aromatic), 7.45e7.39 (m, 1H, aromatic),
3.72 (t, J ¼ 6.2 Hz, 2H, CH2Cl), 3.41 (t, J ¼ 6.8 Hz, 2H, SCH2),
1.95e1.86 (m, 4H, CH2CH2CH2CH2). Anal. C11H11Cl2NS2 (C, H, N, S).
4.2. General procedure for the synthesis of aryloxyalkyl bromides
15e18
4.3.4. 2-[(5-Chloropentyl)thio]-5-chlorobenzothiazole (26)
Flash chromatography on silica gel with cyclohexane/ethyl ac-
etate (9.8:0.2, v:v) gave an orange oil; yield 85%; IR (KBr) cmꢂ1
:
3066, 2937, 1547, 1455, 1428, 1296, 1067, 996; 1H NMR (DMSO-d6)
8.07e8.03 (m, 1H, aromatic), 7.93e7.92 (m, 1H, aromatic),
Intermediates 15e18 were prepared following literature pro-
cedures [49]. To a DMSO solution (10 mL) was added powdered
KOH (20 mmol). After stirring for 5 min, 3-bromo or 4-iodophenol
d
7.45e7.39 (m, 1H, aromatic), 3.65 (t, J ¼ 6.6 Hz, 2H, CH2Cl), 3.38 (t,
J ¼ 7.2 Hz, 2H, SCH2), 1.90e1.68 (m, 4H, CH2CH2CH2CH2CH2),
1.62e1.46 (m, 2H, CH2CH2CH2). Anal. C12H13Cl2NS2 (C, H, N, S).
was added (5 mmol), followed by appropriate 1,u-dibromoalkanes
(10 mmol). After stirring for 1 h, the reaction mixture was poured
into water and extracted with CH2Cl2 (3 ꢁ 30 mL). The combined
organic extracts were washed with brine (2 ꢁ 20 mL) and dried
over anhydrous sodium sulfate. After rotary evaporation, oil resi-
dues were used without any further purification for the next step.
For analytical purpose, crude 17 was purified by flash
chromatography.
4.4. General procedure for the synthesis of imidazole derivatives
29e42, 50
A literature method was employed, with modifications [51]. To a
suspension of the appropriate intermediates 15e28 (5 mmol) in
acetonitrile (3 mL), imidazole or 1,2,4 triazole (7.5 mmol), TEA
(5 mmol), and a catalytic amount of TBAB (0.1 g) were added. The
mixture and a magnetic bar was sealed in a Pyrex test tube (ca.
10 mL) and heated at 90 ꢀC under microwaves irradiation for
30e45 min (run time 2 min, microwave max power 150 W, max
pressure 150 Psi). The solvent was eliminated in vacuo to give a
residue which was suspended in water; the mixture was alkalinized
with NaOH 0.1 N and extracted with ethyl acetate (3 ꢁ 50 mL). The
combined extracts were washed with water, dried on anhydrous
sodium sulfate, and evaporated to obtain a residue which was pu-
rified by flash column chromatography on silica gel using ethyl
acetate or mixtures of ethyl acetate/methanol as eluent.
4.2.1. 5-(4-Iodophenoxy)pentyl bromide (17)
Flash chromatography on silica gel with cyclohexane/ethyl ac-
etate (9.5:0.5, v:v) gave a white solid; mp 90.0e91.5 ꢀC; yield 80%;
IR (KBr) cmꢂ1: 2940, 1585, 1486, 1284, 1243, 1174, 817; 1H NMR
(DMSO-d6)
d 7.61e7.53 (m, 2H, aromatic), 6.81e6.73 (m, 2H, aro-
matic), 3.93 (t, J ¼ 6.4 Hz, 2H, OCH2), 3.55 (t, J ¼ 6.4 Hz, 2H, CH2Br),
1.92e1.65 (m, 4H, CH2CH2CH2CH2CH2), 1.57e1.42 (m, 2H,
CH2CH2CH2). Anal. C11H14BrIO (C, H, N).
4.3. General procedure for the synthesis of benzoxazole,
benzothiazole, or 5-chlorobenzothiazole thioalkylchlorides 19e26
and 2-benzoxazolone or 2-benzothiazolone alkylbromides 27, 28
By use of this procedure, the subsequent new compounds were
obtained:
Intermediates 19e26 were prepared following literature pro-
cedures, with slight modifications [50]. A solution of 10e14
(6.6 mmol) in acetone (20 mL) was mixed with K2CO3 (10 mmol)
and stirred at room temperature for 10 min. After addition of the
4.4.1. 1-[5-(3-Bromophenoxy)pentyl]-1H-imidazole (29)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a colorless oil; yield 18%; IR (KBr) cmꢂ1: 3108, 2940,
2868, 1588, 1508, 1468, 1294, 1228,1077; 1H NMR (DMSO-d6)
d 7.61
appropriate 1-bromo-u-chloroalkane or 1,u-dibromoalkane
(7.3 mmol), the mixture was stirred for further 24 h. Inorganic
materials was removed by filtration, then the solvent was removed
in vacuum to obtain a residue which was used without any further
purification for the next step. For analytical purpose, crude 23e26
were purified by flash chromatography or by recrystallization.
(s, 1H, imidazole), 7.27e7.08 (m, 3H aromatic þ 1H imidazole),
6.95e6.87 (m, 1H aromatic þ 1H imidazole), 4.00e9.93 (m, 4H,
OCH2 þ CH2N), 1.83e1.64 (m, 4H, CH2CH2CH2CH2CH2), 1.40e1.28
(m, 2H, CH2CH2CH2CH2CH2). Anal. C14H17BrN2O (C, H, N).
4.4.2. 1-[6-(3-Bromophenoxy)hexyl]-1H-imidazole (30)
4.3.1. 2-[(5-Chloropentyl)thio]benzothiazole (23)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a light yellow oil; yield 19%; IR (KBr) cmꢂ1: 3107,
2937, 2860, 1588, 1572, 1486, 1284, 1228, 1077; 1H NMR (DMSO-d6)
Flash chromatography on silica gel with cyclohexane/ethyl ac-
etate (9:1, v:v) gave an orange oil; yield 75%; IR (KBr) cmꢂ1: 3061,
2937, 1456, 1427, 1309, 1239, 995, 756. 1H NMR (DMSO-d6)
d
7.60 (s, 1H, imidazole), 7.27e7.08 (m, 3H aromatic þ 1H imid-
d
8.03e7.98 (m, 1H, aromatic), 7.88e7.83 (m, 1H, aromatic),
azole), 6.95e6.87 (m, 1H aromatic þ1H imidazole), 3.98e3.91 (m,
4H, OCH2 þ CH2N), 1.75e1.61 (m, 4H, CH2CH2CH2CH2CH2CH2),
1.48e1.24 (m, 4H, CH2CH2CH2CH2CH2CH2). Anal. C15H19BrN2O (C, H,
N).
7.05e7.32 (m, 2H, aromatic), 3.64 (t, J ¼ 6.6 Hz, 2H, CH2Cl), 3.38 (t,
J ¼ 7.0 Hz, 2H, SCH2), 1.87e1.67 (m, 4H, CH2CH2CH2CH2CH2),
1.62e1.46 (m, 2H, CH2CH2CH2). Anal.C12H14ClNS2 (C, H, N, S).

170
L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
4.4.3. 1-[5-(4-Iodophenoxy)pentyl]-1H-imidazole (31)
(t, J ¼ 7.2 Hz, 2H, SCH2), 1.85e1.67 (m, 4H, CH2CH2CH2CH2CH2),
1.43e1.31 (m, 2H, CH2CH2CH2CH2CH2). Anal. C15H17N3S2 (C, H, N, S).
Flash chromatography on silica gel with ethyl acetate/methanol
(9.5:0.5, v:v) gave a white solid; mp 90.9e92.9 ꢀC; yield 15%; IR
(KBr) cmꢂ1: 3096, 2933, 2861, 1585, 1510, 1487, 1286, 1234, 1176,
4.4.10. 2-[[3-(1H-Imidazol-1-yl]propyl]thio]-5-chlorobenzothiazole
(38)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a light orange oil; yield 23%; IR (KBr) cmꢂ1: 3108,
2936, 1507, 1455, 1428, 1230, 1078, 1003, 908; 1H NMR (DMSO-d6)
1076; 1H NMR (DMSO-d6)
d
7.61e7.52 (m, 2H aromatic þ 1H
imidazole), 7.15 (br s, 1H, imidazole), 6.86 (br s, 1H, imidazole),
6.79e6.72 (m, 2H aromatic), 3.99e3.88 (m, 4H, OCH2 þ CH2N),
1.78e1.63 (m, 4H, CH2CH2CH2CH2CH2), 1.39e1.27 (m, 2H,
CH2CH2CH2CH2CH2). Anal. C14H17IN2O (C, H, N).
d
8.07e8.02 (m, 1H aromatic), 7.92e7.91 (m, 1H aromatic), 7.67 (s,
1H, imidazole), 7.45e7.39 (m, 1H, aromatic), 7.23 (s, 1H, imidazole),
6.91 (s, 1H, imidazole), 4.13 (t, J ¼ 7.0 Hz, 2H, CH2N), 3.25 (t,
J ¼ 7.6 Hz, 2H, SCH2), 2.30e2.16 (m, 2H, CH2CH2CH2). Anal.
4.4.4. 1-[6-(4-Iodophenoxy)hexyl]-1H-imidazole (32)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a white solid; mp 85.6e87.3 ꢀC; yield 21%; IR (KBr)
cmꢂ1: 3103, 2940, 2855, 1586, 1488, 1284, 1244, 1101, 811; 1H NMR
C13H12ClN3S2 (C, H, N, S).
(DMSO-d6)
d
7.60e7.54 (m, 2H aromatic þ 1H imidazole), 7.15 (br s,
4.4.11. 2-[[4-(1H-Imidazol-1-yl)butyl]thio]-5-chlorobenzothiazole
(39)
1H, imidazole), 6.86 (br s, 1H, imidazole), 6.80e6.72 (m, 2H aro-
matic), 3.97e3.87 (m, 4H, OCH2 þ CH2N), 1.78e1.60 (m, 4H,
Flash chromatography on silica gel with ethyl acetate/methanol
(9.5:0.5, v:v) gave a light yellow solid; mp 72.4e73.1 ꢀC; yield 21%;
IR (KBr) cmꢂ1: 3090, 2838, 1546, 1508, 1427, 1289, 1240, 1070, 804;
CH2CH2CH2CH2CH2CH2),
1.47e1.21
(m,
4H,
CH2CH2CH2CH2CH2CH2). Anal. C15H19IN2O (C, H, N).
1H NMR (DMSO-d6)
d 8.06e8.02 (m, 1H aromatic), 7.94e7.93 (m, 1H
4.4.5. 2-[[3-(1H-Imidazol-1-yl)propyl]thio]benzoxazole (33)
aromatic), 7.65 (br s, 1H, imidazole), 7.44e7.39 (m, 1H, aromatic),
7.18 (br s, 1H, imidazole), 6.88 (br s, 1H, imidazole), 4.02 (t,
J ¼ 6.6 Hz, 2H, CH2N), 3.37 (t, J ¼ 7.4 Hz, 2H, SCH2), 1.90e1.63 (m,
4H, CH2CH2CH2CH2). Anal. C14H14ClN3S2 (C, H, N, S).
Flash chromatography on silica gel with ethyl acetate/methanol
(9.5:0.5, v:v) gave a yellow oil; yield 22%; IR (KBr) cmꢂ1: 3108, 2938,
1600, 1501, 1453, 1237, 1131, 1096, 744; 1H NMR (DMSO-d6)
d
7.68e7.60 (m, 2H aromatic þ 1H imidazole), 7.38e7.29 (m, 2H
aromatic), 7.23 (br s, 1H, imidazole), 6.91 (br s, 1H, imidazole), 4.13
(t, J ¼ 6.8 Hz, 2H, CH2N), 3.26 (t, J ¼ 7.2 Hz, 2H, SCH2), 2.32e2.18 (m,
2H, CH2CH2CH2). Anal. C13H13N3OS (C, H, N, S).
4.4.12. 2-[[5-(1H-Imidazol-1-yl)pentyl]thio]-5-chlorobenzothiazole
(40)
Flash chromatography on silica gel with ethyl acetate gave a
colorless oil; yield 17%; IR (KBr) cmꢂ1: 3107, 2935, 2859, 1507, 1454,
4.4.6. 2-[[4-(1H-Imidazol-1-yl)butyl]thio]benzoxazole (34)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a colorless oil; yield 20%; IR (KBr) cmꢂ1: 3109, 2940,
1427, 1229, 1068, 1001; 1H NMR (DMSO-d6)
d 8.07e8.02 (m, 1H
aromatic), 7.93e7.92 (m, 1H aromatic), 7.61 (s, 1H, imidazole),
7.44e7.39 (m, 1H, aromatic), 7.15 (s, 1H, imidazole), 6.87 (s, 1H,
imidazole), 3.96 (t, J ¼ 7.0 Hz, 2H, CH2N), 3.35 (t, J ¼ 7.6 Hz, 2H,
SCH2), 1.86e1.67 (m, 4H, CH2CH2CH2CH2CH2), 1.42e1.31 (m, 2H,
CH2CH2CH2). Anal. C15H16ClN3S2 (C, H, N, S).
1499, 1453 1237, 1131, 1096, 744; 1H NMR (DMSO-d6)
d 7.68e7.59
(m, 2H aromatic þ 1H imidazole), 7.37e7.26 (m, 2H aromatic), 7.17
(br s, 1H, imidazole), 6.86 (br s, 1H, imidazole), 4.01 (t, J ¼ 6.8 Hz,
2H, CH2N), 3.39 (t, J ¼ 7.2 Hz, 2H, SCH2), 1.90e1.67 (m, 4H,
CH2CH2CH2CH2). Anal. C14H15N3OS (C, H, N, S).
4.4.13. 3-[4-(1H-Imidazol-1-yl)butyl]-2(3H)-benzoxazolone (41)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a white solid; mp 90.1e91.9 ꢀC; yield 20%; IR (KBr)
cmꢂ1: 3133, 2943, 1767, 1611, 1498, 1351, 1247, 1078, 755; 1H NMR
4.4.7. 2-[[3-(1H-Imidazol-1-yl)propyl]thio]benzothiazole (35)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a light brown oil; yield 23%; IR (KBr) cmꢂ1: 3109,
2935, 1507, 1456, 1427, 1232, 1078, 997, 757; 1H NMR (DMSO-d6)
(DMSO-d6)
d
7.60 (s, 1H, aromatic), 7.36e7.08 (m, 4H aromatic þ 1H
imidazole), 6.86 (br s, 1H, imidazole), 3.98 (t, J ¼ 6.6 Hz, 2H, CH2N)
3.82 (t, J ¼ 7.0 Hz, 2H, CONCH2), 1.78e1.56 (m, 4H, CH2CH2CH2CH2).
Anal. C14H15N3O2 (C, H, N).
d
8.03e7.99 (m, 1H aromatic), 7.98e7.93 (m, 1H aromatic), 7.68 (s,
1H, imidazole), 7.51e7.32 (m, 2H, aromatic), 7.23 (br s, 1H, imid-
azole), 6.92 (br s, 1H, imidazole), 4.13 (t, J ¼ 6.8 Hz, 2H, CH2N), 3.29
(t, J ¼ 7.2 Hz, 2H, SCH2), 2.30e2.16 (m, 2H, CH2CH2CH2). Anal.
4.4.14. 3-[3-(1H-Imidazol-1-yl)propyl]-2(3H)-benzothiazolone
(42)
C
13H13N3S2 (C, H, N, S).
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a thick colorless oil; yield 24%; IR (KBr) cmꢂ1: 3110,
2944, 1669, 1588, 1509, 1472, 1327, 1230, 749; 1H NMR (DMSO-d6)
4.4.8. 2-[[4-(1H-Imidazol-1-yl)butyl]thio]benzothiazole (36)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a light yellow solid; mp 85.0e86.0 ꢀC; yield 27%; IR
(KBr) cmꢂ1: 3096, 2946, 1511, 1453, 1369, 1225, 1081, 1005, 754; 1H
d
7.60 (s, 1H, aromatic), 7.36e7.08 (m, 4H aromatic þ 1H imidazole),
6.86 (br s, 1H, imidazole), 3.98 (t, J ¼ 6.6 Hz, 2H, CH2N) 3.82 (t,
J ¼ 7.0 Hz, 2H, CONCH2), 1.78e1.56 (m, 2H, CH2CH2CH2). Anal.
NMR (DMSO-d6)
d 8.02e7.98 (m, 1H aromatic), 7.87e7.83 (m, 1H
aromatic), 7.63 (s, 1H, imidazole), 7.50e7.32 (m, 2H, aromatic), 7.17
(br s, 1H, imidazole), 6.86 (br s, 1H, imidazole), 4.01 (t, J ¼ 6.8 Hz,
2H, CH2N), 3.37 (t, J ¼ 7.2 Hz, 2H, SCH2), 1.90e1.66 (m, 4H,
CH2CH2CH2CH2). Anal. C14H15N3S2 (C, H, N, S).
C
13H13N3OS (C, H, N, S).
4.4.15. 1-[5-(3-Bromophenoxy)pentyl]-1H-1,2,4-triazole (50)
Flash chromatography on silica gel with ethyl acetate gave a
colorless oil; yield 25%; IR (KBr) cmꢂ1: 3118, 2944, 2869, 1589, 1572,
4.4.9. 2-[[5-(1H-Imidazol-1-yl)pentyl]thio]benzothiazole (37)
Flash chromatography on silica gel with ethyl acetate/methanol
(9:1, v:v) gave a pale orange oil; yield 20%; IR (KBr) cmꢂ1: 3107,
2935, 1507, 1456, 1426, 1309, 1232, 996, 758; 1H NMR (DMSO-d6)
1469, 1273, 1228, 1014; 1H NMR (DMSO-d6)
d 8.41 (s, 1H, triazole),
7.95 (s, 1H, triazole), 7.26e7.18 (m, 1H aromatic), 7.12e7.07 (m, 2H,
aromatic), 6.96e6.88 (m, 1H, aromatic), 4.19 (t, J ¼ 6.8 Hz, 2H,
d
8.02e7.98 (m, 1H aromatic), 7.86e7.82 (m, 1H aromatic), 7.60 (s,
OCH2), 3.95 (t,
J
¼
6.4 Hz, 2H, CH2N), 1.86e1.64 (m, 4H,
1H, imidazole), 7.50e7.31 (m, 2H, aromatic), 7.15 (br s, 1H, imid-
azole), 6.86 (br s, 1H, imidazole), 3.95 (t, J ¼ 7.0 Hz, 2H, CH2N), 3.37
CH2CH2CH2CH2CH2), 1.40e1.28 (m, 2H, CH2CH2CH2CH2CH2). Anal.
13H16BrN3O (C, H, N).
C

L. Salerno et al. / European Journal of Medicinal Chemistry 96 (2015) 162e172
171
4.5. Biology
4.5.4. Cell cultures
MDA-MB-231 cells (hormone resistant), MCF-7 (hormone sen-
sitive), PC3 cells (hormone resistant), DU145 cells (hormone
resistant), LnCap cells (hormone sensitive), and MDCK (non-
cancerous canine kidney epithelial cells), were maintained in
complete growth media (DMEM/Ham's F12 supplemented with
10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin,
100 units/mL of streptomycin, and 2.2 g/L of NaHCO3) at 37 ꢀC in a
humidified atmosphere of 5% CO2. For all procedures, cells were
harvested using TrypLE Express (Life Technologies, Auckland, New
Zealand).
4.5.1. Preparation of spleen and brain microsomal fractions
HO-1 and HO-2 were obtained, respectively, from rat spleen and
brain as the microsomal fraction prepared by differential centrifu-
gation; the dominance of HO-1 protein in the rat spleen and of HO-
2 in the rat brain has been well documented [33]. These particular
microsomal preparations were selected in order to use the most
native (i.e., closest to in vivo) forms of HO-1 and HO-2. Spleen and
brain (SpragueeDawley rats) microsomal fractions were prepared
according to the procedure outlined by Ryter et al. [4]. The exper-
iments reported in the present paper complied with current Italian
law and met the guidelines of the Institutional Animal Care and Use
Committee of University of Catania (Italy). The experiments were
performed in male SpragueeDawley 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 and HO-2 activities, each rat was
sacrificed and their spleen and brain were excised and weighed. A
homogenate (15%, w/v) of spleens and brains 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 PottereElvehjem
homogenizing system with a Teflon pestle. The microsomal fraction
of rat spleen and brain 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 100,000 g pellet
(microsomes) was resuspended in 100 mM potassium phosphate
buffer, pH 7.8, containing 2 mM MgCl2 with a PottereElvehjem
homogenizing system. The rat spleen and brain microsomal frac-
tions were divided into equal aliquots, placed into microcentrifuge
tubes, and stored at ꢂ80 ꢀC for up to 2 months. Protein concen-
tration of the microsomal fraction was determined by Lowry
method [52].
4.5.5. In vitro cytotoxicity of HO inhibitors against breast and
prostate cancer cell lines and against non-cancerous kidney
epithelial cells
To test the cytotoxic effect of HO inhibitors, cells were seeded
into 96-well plates and incubated for 24 h. This was followed by
treatment with HO inhibitors at the concentrations indicated for
72 h. Following the incubation, cells were fixed using 10% trichloro-
acetic acid (TCA). Cytotoxicity was determined using the sulfo-
rhodamine B assay as previously described [53]. The concentration
required to decrease the cell number by 50% (IC50) was determined
by non-linear regression using Graphpad prism 6 software. Treat-
ments were performed in triplicate and data represents mean of
three independent experiments.
Acknowledgments
This work was supported by grants from the Italian MIUR and
the University of Catania.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.003.
4.5.2. Preparation of biliverdin reductase
Liver cytosol has been used as a source of biliverdin reductase.
Rat liver was perfused through the hepatic portal vein with cold
0.9% NaCl, then it was cut and flushed with 2 ꢁ 20 mL of ice cold
PBS to remove all of the blood. Liver tissue was homogenized in 3
volumes of 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 g
supernatant was saved and then stored in small amounts at ꢂ80 ꢀC
after its protein concentration was measured.
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