Evaluation of novel aryloxyalkyl derivatives of imidazole and 1,2,4-triazole as heme oxygenase-1 (HO-1) inhibitors and their antitumor properties

Article abstract of DOI:10.1016/j.bmc.2013.06.040

A novel series of aryloxyalkyl derivatives of imidazole and 1,2,4-triazole, 17-31, was designed and synthesized as inhibitors of heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). Some of these compounds were found to be good inhibitors of HO-1, in particular those carrying an imidazole moiety as azolyl group and a 3-bromo or 4-iodophenyl as aryl moiety. The most potent compounds 6 and 30 were selected and studied for their antitumor properties in a model of LAMA-84 R cell line overexpressing HO-1 and resistant to imatinib mesylate (IM), a tyrosine-kinase inhibitor used in the treatment of multiple types of cancer, most notably Philadelphia Chromosome positive (Ph⁺) Chronic Myelogenous Leukemia (CML). Results show that both 6 and 30 sensitized LAMA-84 R cell line to antitumor properties of IM.

Full text of DOI:10.1016/j.bmc.2013.06.040

Bioorganic & Medicinal Chemistry 21 (2013) 5145–5153
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of novel aryloxyalkyl derivatives of imidazole
and 1,2,4-triazole as heme oxygenase-1 (HO-1) inhibitors and their
antitumor properties
a
a
a
a
Loredana Salerno a, , Valeria Pittalà , Giuseppe Romeo , Maria N. Modica , Maria A. Siracusa ,
⇑
Claudia Di Giacomo b, Rosaria Acquaviva b, Ignazio Barbagallo b, Daniele Tibullo c, Valeria Sorrenti b
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 Clinical and Molecular Biomedicine, Section of Hematology, Ferrarotto Hospital, University of Catania, via Citelli 6, 95124 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of aryloxyalkyl derivatives of imidazole and 1,2,4-triazole, 17–31, was designed and syn-
thesized as inhibitors of heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). Some of these com-
pounds were found to be good inhibitors of HO-1, in particular those carrying an imidazole moiety as
azolyl group and a 3-bromo or 4-iodophenyl as aryl moiety. The most potent compounds 6 and 30 were
selected and studied for their antitumor properties in a model of LAMA-84 R cell line overexpressing HO-
1 and resistant to imatinib mesylate (IM), a tyrosine-kinase inhibitor used in the treatment of multiple
types of cancer, most notably Philadelphia Chromosome positive (Ph+) Chronic Myelogenous Leukemia
(CML). Results show that both 6 and 30 sensitized LAMA-84 R cell line to antitumor properties of IM.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 10 April 2013
Revised 13 June 2013
Accepted 16 June 2013
Available online 26 June 2013
Keywords:
HO-1inhibitors
Antitumor properties
Imidazole
1,2,4-Triazole
Imatinib
1. Introduction
metabolites produced. In fact, CO and biliverdin, originally viewed
as waste products, have been recently and widely recognized for
Heme oxygenase (HO) is the enzyme which catalyzes the regio-
selective, oxidative degradation of heme, with the simultaneous
release of carbon monoxide (CO), ferrous iron (Fe2+), and biliverdin,
this last further reduced to bilirubin by biliverdin reductase. To
date three enzyme isoforms have been identified: HO-1, HO-2,
and HO-3. HO-1, the most studied isoform also known as heat
shock protein 32 (Hsp32), is an inducible 32-kDa enzyme, predom-
inantly expressed in the liver and spleen, that can be stress-in-
duced by a variety of stimuli such as heavy metals, reactive
oxygen species and, particularly, heat shock. HO-2 is a constitu-
tively expressed 36-kDa protein, widely distributed, with its high
levels in the brain, testes, or endothelial cells. HO-3 is regarded
as a pseudogene of HO-2 and its functional activity is still
uncertain.1
their positive cellular regulatory actions. In particular, CO is in-
volved as a gasotransmitter in antiinflammatory, antiapoptotic,
antiproliferative and vasodilator effects,3 biliverdin and bilirubin
possess antioxidant properties.4
For these reasons, the HO system has generally demonstrated
protective roles towards a variety of stress-induced pathology,
such as diabetes, obesity, atherosclerosis, inflammation, vascular
injury, transplantation, ocular diseases, hypoxia and ischemia;5
consequently, in these pathological conditions an up-regulation
of HO-1 may have therapeutic potential.6,7 However, an up-regula-
tion of the HO system is a double-edge sword and may not exert
exclusively cytoprotective effects, but may contribute to tissue in-
jury under certain pathologic states in which, conversely, inhibi-
tion of HO activity may have therapeutic use.8 A growing body of
evidence suggests that an up-regulation of the HO-1 along with
its catabolism products play a role in the formation, growth, and
metastasis of different tumors including prostate tumor,9 Chronic
Myelogenous Leukemia (CML),10 human renal cell carcinoma,11
and lymphosarcomas.12 It has been reported that in human glio-
mas and melanomas, HO-1 is linked to angiogenesis13,14 and, in
an experimental mouse model, HO-1 accelerates pancreatic cancer
growth by promoting tumor angiogenesis.15
The main physiological role of the HO system is protection of
cells against oxidative stress.2 This effect may be attributed both
to the degradation of the free heme (which is a pro-oxidant agent
since it can catalyze the production of free radicals through Fenton
chemistry) and to the complementary contribution of each of the
⇑
Corresponding author. Tel.: +39 095 7384024.
E-mail address: l.salerno@unict.it (L. Salerno).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.040

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L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
In addition, it has been found that expression of HO-1 is in-
Our research group has recently studied a small class of imidaz-
ole-based derivatives behaving as HO-1 inhibitors,48 selected from
previous extensive research conducted in the field of NOS inhibi-
tors.49,50 Among them, 1-[4-(3-bromophenoxy)butyl]-1H-imidaz-
ole (6) emerged for its potency (Fig. 1). In this work, a novel
series of aryloxyalkyl derivatives of imidazole and 1,2,4-triazole
(17–31) was designed, synthesized and tested on HO-1 and HO-2
enzymes obtained from the microsomal fractions of rat spleen
and rat brain, respectively. Taking into account 6 as lead-com-
pound, we evaluated how the variation of the azolyl moiety, the
introduction of different substituents on phenyl ring and the mod-
ification of the length of the connecting oxyalkyl chain, can influ-
ence HO activity.
Moreover, in light of the involvement of HO system in carcino-
genesis, to further establish the utility of HO-1 inhibitors in antitu-
mor therapy it is necessary to explore their pharmacology in
experimental conditions other than broken-cell preparations, such
as the above-mentioned microsomal fractions. Ideally, the range
should span from intact cells to whole organs and animals
in vivo. Herein, we addressed the hypothesis that the more inter-
esting compounds 6 and 30 are effective also in intact cells. These
compounds were selected for studying their antitumor properties
in a model of LAMA-84 R cell line over-expressing HO-1 and resis-
tant to imatinib mesylate (IM), a tyrosine-kinase inhibitor used in
the treatment of multiple types of cancer, most notably of which
includes Philadelphia Chromosome positive (Ph+) CML.51
creased in response to several anticancer treatments, and, by the
means of the protective role of HO-1 in tumor cells, could be
responsible of the development of drug-resistance.16–20 These re-
sults support the idea that HO-1 (particularly its inhibition or
down-regulation), may be a good target in antitumor therapy.
The potential role of HO-1 inhibitors in antitumor therapy has been
demonstrated by the use of the competitive inhibitors metallopor-
phyrins (tin/zinc/chromium protoporphyrins), Mps, in the treat-
ment of some tumors. Mps are heme derivatives in which the
central iron atom of heme is replaced by another element, that
compete with heme for binding to the enzyme. For example,
administration of tin protoporphyrin (SnPP) accentuated the clini-
cal response to 5-aminolevulinic acid-based photodynamic ther-
apy in cultured melanoma tissue21 and had a negative effect on
growth of Kaposi sarcomas.22 Zinc protoporphyrin IX (ZnPPIX) sig-
nificantly suppressed the growth of lung cancer cell lines23 and
augmented the response to radiotherapy in nasopharyngeal
carcinomas.24
To date, the main potential therapeutic application of HO-1
inhibitors belonging to the class of Mps, is the treatment of neona-
tal hyperbilirubinemia, as confirmed by a number of animal and
clinical studies.25–27 However, MPs still have not left the clinical
study stage for their actual applications in human neonates, due
to some limitations in their use, mainly the photosensitizing action
of these compounds.28 Some clinical studies demonstrated that
using a special light with a narrow spectrum (maximum intensity
440–460 nm), photosensitivity of SnMP was a non-issue.29 Never-
theless, since the phototoxicity of MPs appears to be strongly
dependent on the irradiation and spectral quality of the light
source,28–30 the potential use of MPs in diseases different from
hyperbilirubinemia, such as tumors, could be precluded.
2. Chemistry
Compounds 17–31 were obtained as showed in Scheme 1. In
brief, imidazole 7 or 1,2,4-triazole 8 were reacted with appropriate
aryloxyalkybromides 9–16 in acetonitrile under microwave irradi-
ation, in the presence of triethylamine (TEA) and tetra-
butylammoniumbromide (TBAB) as phase transfer catalyst, at
100 °C, for 30–45 min, to give desired final products 17–31. Purifi-
cations were performed by flash chromatography using ethyl ace-
tate or mixtures of ethyl acetate and methanol. The structures of all
new synthesized compounds were confirmed by analytical, IR and
1H NMR spectral data. 1H NMR spectra of triazole derivatives
showed two singlets each corresponding in intensity to one mag-
netically non-equivalent triazole proton, permitting univocally to
assign the structure of regioisomers 1,2,4.
The non-selectivity towards HO-1 and HO-2 isoforms31 and
other heme-utilizing enzymes, such as soluble guanylyl cyclase
(sGC), nitric oxide synthase (NOS), and cytochrome P450
(CYP450),32–34 may represent another problem in the use of MPs.
In fact, although studies have demonstrated that some of MPs
maintain selectivity against HO in vitro if used within a defined
concentration range,33 therapeutic window is narrow and has not
been yet well defined; in addition antitumor activity is reported
only at very high concentrations that also cause inhibition of other
heme-containing enzymes.35
Finally the most of Mps inhibits heme oxygenase activity but
may be also very potent HO-1 inducers in vivo.36,37 Consequently,
in the last few years, in the medicinal chemistry field there has
been an increasing interest in the development of HO-1 inhibitors
structurally distinct from the heme-analogues Mps.
The first discovered non-porphyrin HO-1 inhibitor which acts in
a non-competitive manner with respect to heme, is azalanstat (1,
Fig. 1).38 Starting from the structure of this imidazole-containing
molecule, a number of azole-based compounds has been described
in literature as HO inhibitors. Their chemistry and the meaningful
structural insights into human HO-1 inhibition have been recently
reviewed.39,40 Some representative azole-based HO-1 inhibitors
(2–4) are depicted in Figure 1. On the basis of the results of both
X-ray crystallography studies 41,42 and traditional structure–activ-
ity relationship (SAR) studies,43–46 it is possible to affirm that the
key functional groups which are necessary for enzyme interaction
include an azolyl moiety, a hydrophobic aryl group, and a connect-
ing alkyl chain, usually constituted by four carbons, which incorpo-
rates a dioxolanyl, a hydroxyl or a carbonyl group in the most
interesting substances. Recently, the effect of the introduction of
heteroatoms in the alkyl linker was investigated by compounds
of general formula 5 (Fig. 1);47 interesting results were obtained
with compounds bearing a four or five carbon linker incorporating
oxygen or sulfur as heteroatoms.
3. Results and discussion
In this work compounds 17–31 were tested to evaluate their
ability to inhibit HO-1 obtained from the microsomal fractions of
rat spleen. SnPP was used as reference compound. Inhibition of en-
zyme activity is expressed as IC50
(lM) and results are summarized
in Table 1. Compounds which showed IC50 values <100
l
M were
also tested on HO-2 obtained from microsomal fractions of rat
brain.
Structural characterization by X-ray crystallography of azole-
based inhibitors in complex with HO-1, such as compounds 1
and 4 (Fig. 1), showed that the main features for binding to HO-1
included coordination of a nitrogen present in the azolyl moiety
of the inhibitors with the heme iron, and stabilization of the bind-
ing through an interaction between hydrophobic groups of the
inhibitors and a distal hydrophobic pocket in the heme-binding
pocket.41,42 On the basis of these considerations and starting from
the structure of 6,48,52 in this work we designed and synthesized
new aryloxyalkyl derivatives of imidazole and 1,2,4-triazole, 17–
31, which possess the key chemical features needed for enzyme
interaction: (i) an azolyl moiety, represented by imidazole or
1,2,4-triazole, (ii) a hydrophobic group, represented by phenyl or

L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
5147
NH2
S
O2N
N
O
O
N
N
N
O
Cl
azalanstat (1)
HO-1 IC50 = 5.5 µM, HO-2 IC50 = 24.5 µM
2
HO-1 IC50 = 2.5 µM, HO-2 IC50 = >100 µM
N
OH
N
O
N
N
N
I
3
4
HO-1 IC50 = 0.06 µM, HO-2 IC50 = 1.8 µM
HO-1 IC50 = 2.6 µM, HO-2 IC50 = 34 µM
N
Br
O
N
X
( )n
N
Y
N
6
5
X = CH2, O, S
HO-1 IC50 = 2.1 µM, HO-2 IC50 = 2.2 µM
Y = CH2, O, S, NH
n = 1, 2
Figure 1. Chemical structures and HO-inhibition values of compounds 1–6.
N
O
N
( )n
( )n
Br
N
O
X
+
R
X
R
N
H
7,8
9-16
17-31
7, X = CH
8, X = N
17, R = H, n = 1, X = CH
18, R = H, n = 1, X = N
19, R = H, n = 2, X = CH
20, R = H, n = 2, X = N
21, R = 4-NO2, n = 1, X = CH
22, R = 4-NO2, n = 1, X = N
23, R = 4-NO2, n = 2, X = CH
24, R = 4-NO2, n = 2, X = N
9, R = H, n = 1
10, R = H, n = 2
11, R = 4-NO2, n = 1
12, R = 4-NO2, n = 2
13, R = 3-Br, n = 1
14, R = 3-Br, n = 2
15, R = 4-I, n = 1
16, R = 4-I, n = 2
25, R = 3-Br, n = 1, X = CH
26, R = 3-Br, n = 1, X = N
27, R = 3-Br, n = 2, X = N
28, R = 4-I, n = 1, X = CH
29, R = 4-I, n = 1, X = N
30, R = 4-I, n = 2, X = CH
31, R = 4-I, n = 2, X = N
Scheme 1. Reagents and conditions: TEA, TBAB, acetonitrile, 100 °C, 30–45 min.
substituted (4-NO2, 3-Br, 4-I) phenyl, chosen having in mind aryl
moieties present in imidazole derivatives endowed with good or
very good HO inhibitory properties (Fig. 1),45,47 (iii) a linker, repre-
sented by an oxypropyl or oxybutyl chain.
As a general consideration, it is possible to affirm that many of
the tested compounds inhibit HO-1 in the micromolar range even if
with less potency of related 6. In particular, compound 30 pos-
SARs can be discussed: the effect of azolyl moiety is very clear
since imidazole-containing compounds resulted generally more
potent than the corresponding triazole ones (17 vs 18, 19 vs 20,
23 vs 24, 25 vs 26, 6 vs 27 and 30 vs 31). Considering the influence
of the length of the linker chain, compounds containing an oxybu-
tyl linker give the best results with respect to those containing an
oxypropyl chain (19 vs 17, 23 vs 21, 6 vs 25, 30 vs 28, and 31 vs 29).
With regard to the influence of the substituent present in the phe-
nyl group, results show that 4-NO2 is deleterious, 3-Br gives good
results for inhibition of HO-1 but not for selectivity, whereas 4-I
gives the best contribution both in terms of potency on HO-1
and selectivity over HO-2. In order to test the influence on activity
sesses noteworthy HO-1 inhibition (IC50 = 1
tivity towards HO-2 (IC50 = 10 M); this potency, along with that
of 6 (HO-1 IC50 = 2.1 M, HO-2 IC50 = 2.2 M) is comparable to that
of the first discovered HO inhibitor azalanstat (1, Fig. 1, HO-1
IC50 = 5.5 M, HO-2 IC50 = 24.5 M). From the obtained results,
lM) and some selec-
l
l
l
l
l

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L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
Table 1
Table 2
Inhibitory potency of compounds 17–31 against HO-1 and HO-2
HO activity in LAMA-84 R cell lysate
N
Pmol bilirubin/1 h/mg prot.a
% Inhibition
N
O
( )n
Control
103.8 3.5
X
49.5 1.2*
47.1 1.8*
52.3
R
10
10
l
l
M 6
M 30
54.62
a
Compd
R
n
X
IC50 HO-1a
31
>100
IC50 HO-2a
Values are shown from triplicate experiments SD.
Significant versus untreated control cells: p <0.001
*
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
6
H
H
H
H
4-NO2
4-NO2
4-NO2
4-NO2
3-Br
3-Br
3-Br
4-I
4-I
4-I
4-I
2-Br
4-Br
3-Br
1
1
2
2
1
1
2
2
1
1
2
1
1
2
2
2
2
2
CH
N
CH
N
CH
N
CH
N
CH
N
N
CH
N
CH
N
2
>100
NT
21
92
1
5
30
27
NT
NT
21
NT
1
2
Results reported in Table 2 evidence that compounds 6 and 30
at 10 M concentration are able to inhibit HO activity in LAMA-84
>100
>100
l
R cell line respect to untreated cells. Therefore they are effective
not only in broken-cell preparation, such as the above-described
microsomal fractions, but also in intact cells, suggesting that they
are able to cross the cellular membrane and that could be active
in vivo.
Moreover we evaluated if the HO-1 inhibition induced by com-
pounds 6 and 30 was able to overcome IM resistance. This hypoth-
esis is confirmed by data reported in Figure 2. Results evidence that
the single treatment with IM or compound 6 or compound 30 does
not modify percentage of cell survival whereas treatment of LAMA-
84 R cell line with compounds 6 or 30 plus IM is able to reduce per-
centage of cell survival.
56
2
1
>100
69
3
>100
NT
NT
>100
>100
>100
NT
75
1
38
53
31
5
0.01
1
3
1
90
4
10 0.5
70
24
75
3
3
4
CH
CH
CH
2.1 0.3
0.58 0.03
2.2 0.2
0.36 0.01
SnPP
a
Data are shown as IC50 values in
mean of triplicate experiments.
l
M
standard deviation (SD). Values are the
According to these results, Fluorescence Activated Cell Sorting
(FACS) analysis reveals that only compounds 6 or 30 (10
lM) plus
IM (1 M) treatment, but not the single treatment with IM or com-
pound 6 or 30, induces decrease of S and G2/M phases with in-
creased percentage of apoptotic cells (Fig. 3).
l
of the position of substituent in the phenyl ring, in this work we
also evaluated previously synthesized derivatives 32 and 33,52 car-
rying a 2- and 4-bromophenyl as aryl moieties, respectively. Both
compounds result less potent than corresponding 3-bromo analog
6, towards both HO-1 and HO-2.
In our previous study62 we reported that HO-1 mRNA levels
were significantly higher in LAMA-84 resistant cells when com-
pared to non-resistant clones. In the present paper we measured
HO-1 and HO-2 mRNA levels in LAMA-84 resistant cells treated
and untreated with compounds 6 or 30. It is interesting to evidence
that compounds 6 or 30 are able to decrease HO-1 mRNA levels
whereas they don’t modify HO-2 mRNA levels (Fig. 4). Thus, com-
pounds 6 and 30, contrarily to Mps and other HO-1 inhibitors, do
not behave as very potent HO-1 inducers but they are able both
to down-regulate HO-1 expression and to inhibit HO-1/HO-2
activity.
Compounds 6 and 30, showing the highest potency against
HO-1, 30 being also 10-fold selective for HO-1 versus HO-2, were
selected to further biological studies, in order to explore the poten-
tial role of HO-1 inhibitors in antitumor therapy. Moreover we
studied the mechanisms involved in their antitumor properties.
It has been reported that different human cancers express high
levels of HO-1, which may provide them a growth advantage.9–15,53
Therefore, inhibition of HO-1 might lead to reduced tumor growth
in vitro and in vivo.21–23,35,54,55 Recent studies demonstrated that
induction of HO-1 made leukemia and colon cancer cells resistant
to merocyanine and pyrrolidine dithiocarbamate, two experimen-
tally used chemotherapeutic drugs.56,57 Berberat et al.15 reported
that high HO-1 levels in pancreatic cancer cells may, at least partly,
be responsible for their resistance to anticancer therapy.
Datta et al. reported that down-regulation of HO-1 expression
lead to cellular apoptosis.63 According to these data, compounds
6 or 30 plus IM treatment lead to cellular apoptosis.
Moreover we reported that HO-1 siRNA abolished IM resistance
in LAMA-84 R cell population, restoring apoptotic effects of IM.62
CML is a stem cell disease in which BCR/ABL 51 promotes the sur-
vival of leukemic cells. Identification of IM, a tyrosine-kinase inhib-
itor used in the treatment of multiple cancers, and the subsequent
findings that this compound displays growth inhibitory and pro-
apoptotic effects in Bcr–Abl+ cells, has tremendously modified
CML treatment, but the initial striking efficacy of this drug has been
overshadowed by the development of clinical resistance.58–60
Although the majority of Ph+ CML patients benefit from IM treat-
ment, a substantial number of patients are either initially refractory
to treatment (primary resistance) or develop resistance during the
course of treatment (secondary or acquired resistance).61
Our previous study62 showed that HO-1 might be involved in the
resistance mechanisms of Bcr–Abl+ cells, contributing to their fail-
ure in undergoing apoptosis in the presence of IM. In fact, HO-1
expression resulted higher in IM-resistant K562 and LAMA cell
lines, than in IM-sensitive ones. Moreover, in these resistant cells,
the combining of siRNA to IM treatment overcome IM resistance.
In the present paper we evaluated the effects of compounds 6
and 30 in a model of LAMA-84 R cell line overexpressing HO-1
and resistant to IM.
Figure 2. Effects of 1 lM IM alone, or 10 lM 6 alone, or 10 lM 30 alone, or a
combination of drugs. Survival analyzed by ATP lite assay. Values are shown from
triplicate experiments SD. ⁄Significant versus untreated control cells and versus
1 lM IM alone, or 10 lM 6 alone, or 10 lM 30 alone treated cells: p <0.05.

L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
5149
Figure 5. Intracellular oxidants in LAMA-84 R cells untreated and treated with
M IM alone, or 1 M IM + 10 M 6, or 1 M IM + 10 M 30. Values are shown
from triplicate experiments SD. ⁄Significant versus untreated control cells and
versus 1 M IM alone treated cells: p <0.05.
1
l
l
l
l
l
l
4. Conclusions
In the present study, a novel series of aryloxyalkyl derivatives of
imidazole and 1,2,4-triazole, 17–31, was investigated as HO-inhib-
itors. Best results were obtained for imidazole derivatives carrying
an oxybutyl chain as linker. Particularly, 1-[4-(3-bromophen-
oxy)butyl]-1H-imidazole 6 and 1-[4-(4-iodophenoxy)butyl]-1H-
imidazole 30 showed the highest inhibitory potency against
HO-1; 30 resulted moderately selective for HO-1 versus HO-2.
This effect was confirmed also in LAMA-84 R cell line, demon-
strating the capacity of these compounds to be active also in intact
cells. Moreover compounds 6 and 30 besides to inhibit HO-1/HO-2
activity are able to down-regulate HO-1 expression and in this con-
dition, in LAMA-84 R cell line, IM can cause an increase of ROS lev-
els with consequent cell apoptosis. Therefore, simultaneous
administration of 6 or 30 with IM sensitized LAMA-84 R cell line
to IM.
Figure 3. Cell cycle distribution of survival cells (Panel A) and apoptotic cells (Panel
B) analyzed by FACS. Values are shown from triplicate experiments SD. ⁄Signif-
icant versus untreated control cells and versus 1
10 M 30 alone treated cells: p <0.001.
lM IM alone, or 10 lM 6 alone, or
l
In conclusion, we suggest that specific HO-1 activity decrease
mediated by HO-1 inhibitors, such as 6 and 30, in combination
with conventional radiotherapy and chemotherapy may open up
new perspectives in the treatment of CML.
5. Experimental procedures
5.1. Chemistry
Melting points were determined in an Electrothermal IA9200
apparatus with a digital thermometer in glass capillary tubes. Ele-
mental analyses for C, H, N were within 0.4% of theoretical values
and were performed on a Carlo Erba Elemental Analyzer Mod.1108
apparatus. The IR spectra were recorded in KBr disks on a Perkin
Elmer 1600 series FT-IR spectrometer. 1H NMR spectra were deter-
mined with a Varian Inova Unity 200 (200 MHz), instrument in
DMSO-d6 solution. Chemical shifts are in d values (ppm) using tet-
ramethylsilane 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
light (k = 254 and 366 nm). Preparative column chromatography
was performed using Merck silica-gel 60 (230–400 mesh). All
chemicals and solvents were reagent grade and were purchased
from commercial sources. Aryloxyalkylbromides 11, 13–16 were
synthesized following described procedures64 with slight modifi-
cations, in brief: corresponding phenols (5 mmol) were reacted
Figure 4. Effect of 10 lM 6 and 10 lM 30 on mRNA expression analyzed by Real
Time PCR. Values are shown from triplicate experiments SD. ⁄Significant versus
untreated control cells: p <0.05.
Since apoptotic effect of IM may be mediated by ROS formation,62
here we measured the levels of intracellular oxidants in LAMA-84 R
cells untreated and treated with IM alone, or IM combined with 6
or 30. Figure 5 shows that co-administration of IM and both com-
pounds increases ROS levels. This effect may be related to in-
creased percentage of apoptotic cells mediated by compounds 6
or 30 plus IM treatment. Therefore, the inhibition of HO-1 may al-
low the IM mediated generation of ROS and the elevated ROS levels
may facilitate tumor killing.
Overall results reported in the present paper demonstrate that
compounds 6 or 30 plus IM treatment can down-regulate HO-1
expression and, in this condition, IM can cause an increase of
ROS levels with consequent tumor cell apoptosis.

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L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
with 1,3-dibromopropane or 1,4-dibromobutane (10 mmol) and
potassium carbonate (10 mmol) in acetone (3 mL), under micro-
waves irradiation in a sealed vial at 90 °C, 200 W, 150 Psi, for
30 min. The solvent was removed in vacuum to give a residue
which was suspended in water and extracted with dichlorometh-
ane (3 ꢀ 50 mL); the combined extracts were washed with water,
dried, and evaporated to obtain a residue which was used without
any further purification. Analytical and spectral data of previous
5.2.6. 1-[3-(4-Nitrophenoxy)propyl]-1H-1,2,4-triazole (22)
The title compound was isolated as white solid; yield 25%; mp
115–117 °C; IR (KBr) cmꢁ1: 3136, 2968, 1592, 1502, 1330, 1262; 1H
NMR (DMSO-d6) d 8.54 (s, 1H, triazole), 8.25–8.17 (m, 2H, aro-
matic), 7.98 (s, 1H, triazole), 7.16–7.08 (m, 2H, aromatic), 4.37 (t,
J = 6.8 Hz, 2H, OCH2), 4.12 (t, J = 6.0 Hz, 2H, NCH2), 2.34–2.21 (m,
2H, CH2CH2CH2). Anal. C11H12N4O3 (C,H,N).
52
synthesized 1-[4-(3-bromophenoxy)butyl]-1H-imidazole (6)
5.2.7. 1-[4-(4-Nitrophenoxy)butyl]-1H-Imidazole (23)
The title compound was isolated as yellow solid; yield 27%; mp
67–69 °C; IR (KBr) cmꢁ1: 3087, 2938, 1591, 1500, 1331, 1269; 1H
NMR (DMSO-d6) d 8.24–8.16 (m, 2H, aromatic), 7.65 (s, 1H, imidaz-
ole), 7.20–7.09 (m, 2H aromatic + 1H imidazole), 6.89 (br s, 1H,
imidazole), 4.12 (t, J = 6.4 Hz, 2H, OCH2), 4.04 (t, J = 6.8 Hz, 2H,
NCH2), 1.94–1.61 (m, 4H, CH2CH2CH2CH2). Anal. C13H15N3O3
(C,H,N).
are: colorless oil; yield 32%; IR (Neat) cmꢁ1: 3112, 2913, 1589,
1488, 1228, 780; 1H NMR (CDCl3) d 7.49 (br s, 1H, imidazole),
7.18–7.02 (m, 3H aromatic + 1H imidazole), 6.93 (br s, 1H, imidaz-
ole), 6.83–6.77 (m, 1H, aromatic), 4.06–3.91 (m, 4H, OCH2 + NCH2),
2.06–1.94 (m, 2H, OCH2CH2), 1.83–1.73 (m, 2H, NCH2CH2). Anal.
C13H15 BrN2O (C,H,N). Analytical and spectral data of all unknown
compounds are following reported.
5.2. General procedure for the synthesis of aryloxyalkyl
derivatives of imidazole or 1,2,4-triazole (17–31)
5.2.8. 1-[4-(4-Nitrophenoxy)butyl]-1H-1,2,4-triazole (24)
The title compound was isolated as white solid; yield 28%; mp
90–91 °C; IR (KBr) cmꢁ1: 3114, 2942, 1590, 1500, 1335, 1254; 1H
NMR (DMSO-d6) d 8.55 (s, 1H, triazole), 8.23–8.16 (m, 2H, aro-
matic), 7.98 (s, 1H, triazole), 7.18–7.09 (m, 2H, aromatic), 4.26 (t,
J = 6.8 Hz, 2H, OCH2), 4.14 (t, J = 6.2 Hz, 2H, NCH2), 1.99–1.88 (m,
2H, OCH2CH2), 1.77–1.67 (m, 2H, NCH2CH2). Anal. C12H14N4O3
(C,H,N).
A
solution of the appropriate aryloxyalkylbromides 9–16
(5 mmol) commercially available or prepared as above-described,
imidazole 7 or 1,2,4-triazole 8 (7.5 mmol), TEA (5 mmol) and cata-
lytic amount of TBAB (0.1 g) in acetonitrile (3 mL) was heated un-
der microwaves irradiation in a sealed vial at 100 °C, 200 W, 150
Psi, for 30–45 min. The solvent was removed in vacuo to give a res-
idue which was suspended in water, alkalinized with NaOH 0.1 N
and extracted with dichloromethane (3 ꢀ 50 mL); the combinated
extracts were washed with water, dried, and evaporated to obtain
a residue which was purified by flash column chromatography on
silica gel using ethyl acetate or ethyl acetate/methanol 9:1 as elu-
ent. By use of this procedure, the subsequent compounds were
obtained:
5.2.9. 1-[3-(3-Bromophenoxy)propyl]-1H-imidazole (25)
The title compound was isolated as colorless oil; yield 21%; IR
(Neat) cmꢁ1: 3110, 2938, 1588, 1468, 1284, 1228; 1H NMR
(DMSO-d6) d 7.62 (s, 1H, imidazole), 7.28–7.10 (m, 3H aromatic +
1H imidazole), 6.97–6.88 (m, 1H aromatic + 1H imidazole), 4.12
(t, J = 7.0 Hz, 2H, OCH2), 3.91 (t, J = 6.0 Hz, 2H, NCH2), 2.21–2.08
(m, 2H, CH2CH2CH2). Anal. C12H13BrN2O (C,H,N).
5.2.1. 1-(3-Phenoxypropyl)-1H-imidazole (17)
5.2.10. 1-[3-(3-Bromophenoxy)propyl]-1H-1,2,4-triazole (26)
The title compound was isolated as pale yellow oil; yield 20%; IR
(Neat) cmꢁ1: 3117, 2947, 1589, 1468, 1274, 1228; 1H NMR (DMSO-
d6) d 8.53 (s, 1H, triazole), 7.97 (s, 1H, triazole), 7.28–6.90 (m, 4H,
aromatic), 4.34 (t, J = 6.8 Hz, 2H, OCH2), 3.97 (t, J = 6.0 Hz, 2H,
NCH2), 2.28–2.15 (m, 2H, CH2CH2CH2). Anal. C11H12BrN3O (C,H,N).
Analytical and spectral data were in agreement with Ref. 65.
5.2.2. 1-(3-Phenoxypropyl)-1H-1,2,4-triazole (18)
The title compound was isolated as colourless oil; yield 28%; IR
(Neat) cmꢁ1: 3117, 2948, 1600, 1497, 1244, 757; 1H NMR (DMSO-
d6) d 8.53 (s, 1H, triazole), 7.98 (s, 1H, triazole), 7.32–7.24 (m, 2H,
aromatic), 6.96–6.89 (m, 3H, aromatic), 4.36 (t, J = 6.8 Hz, 2H,
OCH2), 3.94 (t, J = 6.2 Hz, 2H, NCH2), 2.26–2.19 (m, 2H, CH2CH2-
CH2). Anal. C11H13N3O (C,H,N).
5.2.11. 1-[4-(3-Bromophenoxy)butyl]-1H-1,2,4-triazole (27)
The title compound was isolated as pale yellow oil; yield 23%;
IR (Neat) cmꢁ1: 3117, 2946, 1589, 1469, 1228, 680; 1H NMR
(DMSO-d6) d 8.52 (s, 1H, triazole), 7.97 (s, 1H, triazole), 7.27–6.90
(m, 4H, aromatic), 4.24 (t, J = 6.8 Hz, 2H, OCH2), 3.99 (t, J = 6.4 Hz,
2H, NCH2), 1.99–1.85 (m, 2H, OCH2CH2), 1.71–1.60 (m, 2H, NCH2-
CH2). Anal. C12H14 BrN3O (C,H,N).
5.2.3. 1-(4-Phenoxybutyl)-1H-imidazole (19)
Analytical and spectral data were in agreement with Ref. 65.
5.2.4. 1-(4-Phenoxybutyl)-1H-1,2,4-triazole (20)
5.2.12. 1-[3-(4-Iodophenoxy)propyl]-1H-imidazole (28)
The title compound was isolated as white solid; yield 23%; mp
87–88 °C; IR (KBr) cmꢁ1: 3092, 2944, 1584, 1487, 1229, 824; 1H
NMR (DMSO-d6) d 7.62–7.54 (m, 2H, aromatic + 1H imidazole),
7.18 (br s, 1H, imidazole), 6.88 (br s, 1H, imidazole), 6.81–6.75
(m, 2H aromatic), 4.11 (t, J = 7.0 Hz, 2H, OCH2), 3.87 (t, J = 6.2 Hz,
2H, NCH2), 2.21–2.11 (m, 2H, CH2CH2CH2). Anal. C12H13IN2O
(C,H,N).
The title compound was isolated as white solid; yield 25%; mp
48–49 °C; IR (KBr) cmꢁ1: 3101, 2947, 1602, 1499, 1255, 755; 1H
NMR (DMSO-d6) d 8.55 (s, 1H, triazole), 7.98 (s, 1H, triazole),
7.32–7.24 (m, 2H, aromatic), 6.95–6.88 (m, 3H, aromatic), 4.25 (t,
J = 6.8 Hz, 2H, OCH2), 3.96 (t, J = 6.2 Hz, 2H, NCH2), 1.98–1.90 (m,
2H, OCH2CH2), 1.70–1.62 (m, 2H, NCH2CH2). Anal. C12H15N3O
(C,H,N).
5.2.5. 1-[3-(4-Nitrophenoxy)propyl]-1H-Imidazole (21)
5.2.13. 1-[3-(4-Iodophenoxy)propyl]-1H-1,2,4-triazole (29)
The title compound was isolated as white solid; yield 21%; mp
77–79 °C; IR (KBr) cmꢁ1: 3103, 2944, 1585, 1508, 1487, 1236; 1H
NMR (DMSO-d6) d 8.51 (s, 1H, triazole), 7.97 (s, 1H, triazole),
7.62–7.54 (m, 2H, aromatic), 6.80–6.72 (m, 2H, aromatic), 4.34 (t,
J = 6.8 Hz, 2H, OCH2), 3.93 (t, J = 6.0 Hz, 2H, NCH2), 2.28–2.15 (m,
2H, CH2CH2CH2). Anal. C12H12IN3O (C,H,N).
The title compound was isolated as light yellow solid; yield
34%; mp 76–78 °C. IR (KBr) cmꢁ1: 3103, 2950, 1593, 1504, 1339,
1258; 1H NMR (DMSO-d6) d 8.22–8.17 (m, 2H, aromatic), 7.63 (s,
1H, imidazole), 7.20–7.09 (m, 2H aromatic + 1H imidazole), 6.89
(br s, 1H, imidazole), 4.15 (t, J = 7.0 Hz, 2H, OCH2), 4.05 (t,
J = 6.2 Hz, 2H, NCH2), 2.27–2.14 (m, 2H, CH2CH2CH2). Anal.
C12H13N3O3 (C,H,N).

L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
5151
5.2.14. 1-[4-(4-Iodophenoxy)butyl]-1H-imidazole (30)
6.3. Measurement of HO-1 and HO-2 enzymatic activities in
The title compound was isolated as white solid; yield 16%; mp
90–92 °C; IR (KBr) cmꢁ1: 3112, 2942, 1586, 1488, 1468, 1254; 1H
NMR (DMSO-d6) d 7.62–7.53 (m, 2H, aromatic + 1H imidazole),
7.17 (br s, 1H, imidazole), 6.88 (br s, 1H, imidazole), 6.80–6.73
(m, 2H, aromatic), 4.04–3.90 (m, 4H, OCH2 + NCH2), 1.90–1.76
(m, 2H, OCH2CH2), 1.68–1.58 (m, 2H, NCH2CH2). Anal. C13H15IN2O3
(C,H,N).
microsomal fraction of rat spleen and brain
The HO-1 and HO-2 activities were determined by measuring
the bilirubin formation using the difference in absorbance at
464–530 nm as described by Ryter et al.1 Reaction mixtures (500
l
L) consisted of 20 mM 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,
10 L of DMSO (or the same volume of DMSO solution of test com-
pounds to a final concentration of 100, 10, and 1 M). Incubations
l
5.2.15. 1-[4-(4-Iodophenoxy)butyl]-1H-1,2,4-triazole (31)
The title compound was isolated as pale yellow solid; yield 19%;
mp 57–58 °C; IR (KBr) cmꢁ1: 3104, 2913, 1585, 1511, 1487, 1247:
1H NMR (DMSO-d6) d 8.52 (s, 1H, triazole), 7.96 (s, 1H, triazole),
7.61–7.53 (m, 2H, aromatic), 6.81–6.73 (m, 2H, aromatic), 4.23 (t,
J = 6.8 Hz, 2H, OCH2), 3.95 (t, J = 6.4 Hz, 2H, NCH2), 1.98–1.84 (m,
2H, OCH2CH2), 1.67–1.60 (m, 2H, NCH2CH2). Anal. C12H14 IN3O
(C,H,N).
l
l
were carried out for 60 min at 37 °C in a circulating water bath in
the dark. Reactions were stopped by adding 1 volume of chloro-
form. After recovering the chloroform phase, the amount of biliru-
bin formed was measured with a double-beam spectrophotometer
as OD464–530 nm (extinction coefficient, 40 mM/cmꢁ1 for bilirubin).
One unit of the enzyme was defined as the amount of enzyme cat-
alyzing the formation of 1 nmol of bilirubin/mg protein/h.
6. Biology
6.4. Cell cultures and treatments
6.1. Preparation of spleen and brain microsomal fractions
Ph+ cell lines LAMA-84 R, were cultured in RPMI-1640 (Sigma,
Milan, Italy) with 10% Fetal Bovine Serum (FBS) and 1% penicil-
lin–streptomycin at a final concentration of 15 ꢀ 104 cells/mL
and were incubated at 37 °C in 5% CO2. Cells were incubated for
HO-1 and HO-2 were obtained, respectively, from rat spleen
and brain as the microsomal fraction prepared by differential cen-
trifugation; the dominance of HO-1 protein in the rat spleen and of
HO-2 in the rat brain has been well documented.47,66–68 These par-
ticular 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 (Sprague–Dawley rats) microsomal fractions were pre-
pared according to the procedure outlined by Ryter et al.1 The
experiments 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 experi-
ments 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 and HO-2 activi-
ties, each rat was sacrificed and their spleen and brain were ex-
cised and weighed. A homogenate (15%, w/v) of spleens and
brains pooled from four rats was prepared in ice-cold HO-homog-
enizing buffer (50 mM Tris buffer, pH 7.4, containing 0.25 M su-
crose) using a Potter–Elvehjem homogenizing system with a
Teflon pestle. The microsomal fraction of rat spleen and brain
homogenate was obtained by centrifugation at 10,000g for
20 min at 4 °C, followed by centrifugation of the supernatant at
100,000g for 60 min at 4 °C. The 100,000g pellet (microsomes)
was resuspended in 100 mM potassium phosphate buffer, pH 7.8,
containing 2 mM MgCl2 with a Potter–Elvehjem homogenizing
system. The rat spleen and brain microsomal fractions were di-
vided into equal aliquots, placed into microcentrifuge tubes, and
stored at ꢁ80 °C for up to 2 months. Protein concentration of the
microsomal fraction was determined by Lowry method.48
24 h either with IM 1
lM alone, or 10 lM compound 6 alone, or
10 M compound 30 alone, or a combination of drugs.
l
6.5. ATP-lite1step assay for cell survival
After drug treatment, the viability of cells was evaluated by the
ATP-lite1step assay (PerkinElmer, Monza, Italy), as described by
the manufacturer. Briefly, cells were plated onto 96-well micro-
plates in 100
lL growth medium and 100 lL of the reconstituted
reagent was added to each well. Cells were incubated at 37 °C for
20 min at 700 rpm using an orbital microplate shaker. Lumines-
cence was measured using a Victor3 (PerkinElmer). Drug-resis-
tance was calculated as the percentage of viable cells in the
treated suspension culture in comparison to the untreated one.
6.6. Cell cycle analysis
Cells were washed and resuspended in cold 80% ethanol to a fi-
nal concentration of 0.5 ꢀ 106 cells/mL for 1 h at 4 °C. The ethanol-
fixed cells were centrifuged to remove ethanol and the pellet was
resuspended in propidium iodide staining reagent (0.1% triton X-
100, 0.1 mM EDTA, 0.05 mg/mL RNase A and 50 lg/mL propidium
iodide). Cells were stored in the dark at room temperature for
about 3 h. Cells were then analyzed with a FACS flow cytometer
(FC500 Beckman coulter; Beckman Coulter S.p.a., Milan, Italy)
and processed by the ModFit program. The treated cells were eval-
uated by FACS analysis for identifying the cells at different stages
of the cell cycle.
6.2. Preparation of biliverdin reductase
6.7. Real-time PCR quantification
Liver cytosol has been used as a source of biliverdin reductase
(BVR). 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 10,000g,
for 20 min at 4 °C. Supernatant was decanted and centrifuged at
100,000g for 1 h at 4 °C to sediment the microsomes. The
100,000g supernatant was saved and then stored in small amounts
at ꢁ80 °C after its protein concentration was measured.
Expression of HO-1/HO-2 and GAPDH were evaluated by real-
time PCR. Cultured cell layers in different conditions were rinsed
with cold PBS and immediately lysed using Trizol Reagent (Qia-
gen). Total RNA was isolated, treated with RNase-free DNase I,
and quantified by UV spectrophotometry. For RT-PCR analysis of
mRNA expression, 1.0 lg of total RNA (in 20 lL reaction volume)
was reverse-transcribed using reverse transcriptase (Roche Diag-
nostic) and oligo-dT primers in a standard reaction. The resultant
cDNA was then used as the template for PCR amplification. The
quantitative real-time polymerase chain reaction (qRT-PCR) was

5152
L. Salerno et al. / Bioorg. Med. Chem. 21 (2013) 5145–5153
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manufacturer. A value of 10 ng of reverse transcribed RNA samples
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line
Total HO activity in the cell lysate was determined by measur-
ing the bilirubin formation using the difference in absorbance at
464–530 nm as described by Ryter et al.1
Reaction mixtures (500
lL) consisted of 20 mM Tris–HCl, pH
7.4, (1 mg/mL) cell lysate, 0.5–2 mg/mL biliverdin reductase,
1 mM NADPH, 2 mM glucose 6-phosphate (G6P), 1 U G6P dehydro-
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probe 20,70-dichlorofluorescein diacetate (DCFH-DA), as previously
described.69 The fluorescence [corresponding to the oxidised radi-
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6.10. Statistical analyses
The data are presented as means SD for 4 experiments in trip-
licate. One-way variance analysis and Student’s t-test were used
where appropriate; p <0.05 was regarded as significant.
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Acknowledgments
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This work was supported by grants from the Italian MIUR and
the University of Catania.
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