Potholing of the hydrophobic heme oxygenase-1 western region for the search of potent and selective imidazole-based inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.02.007

Here we report the design, synthesis, and molecular modeling of new potent and selective imidazole-based HO-1 inhibitors in which the imidazole nucleus and the hydrophobic groups are linked by a phenylethanolic spacer. Most of the tested compounds showed

Full text of DOI:10.1016/j.ejmech.2018.02.007

Accepted Manuscript
Potholing of the hydrophobic heme oxygenase-1 western region for the search of
potent and selective imidazole-based inhibitors
Loredana Salerno, Emanuele Amata, Giuseppe Romeo, Agostino Marrazzo, Orazio
Prezzavento, Giuseppe Floresta, Valeria Sorrenti, Ignazio Barbagallo, Antonio
Rescifina, Valeria Pittalà
PII:
S0223-5234(18)30129-6
10.1016/j.ejmech.2018.02.007
EJMECH 10184
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 December 2017
Revised Date: 29 January 2018
Accepted Date: 3 February 2018
Please cite this article as: L. Salerno, E. Amata, G. Romeo, A. Marrazzo, O. Prezzavento, G. Floresta,
V. Sorrenti, I. Barbagallo, A. Rescifina, V. Pittalà, Potholing of the hydrophobic heme oxygenase-1
western region for the search of potent and selective imidazole-based inhibitors, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.007.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Potholing of the hydrophobic heme oxygenase-1 western region for the search of potent and
selective imidazole-based inhibitors
Loredana Salerno a, Emanuele Amata a, Giuseppe Romeo a, Agostino Marrazzo a, Orazio Prezzavento a,
Giuseppe Floresta a,b, Valeria Sorrenti a, Ignazio Barbagallo a, Antonio Rescifina a,*, Valeria Pittalà a,**
a Department of Drug Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
b Department of Chemical Sciences, University of Catania, V.le A. Doria, 95125 Catania, Italy
Corresponding authors:
Antonio Rescifina arescifina@unict.it
Valeria Pittalà vpittala@unict.it
Graphical abstract
Highlights
•
•
•
•
New imidazole-based HO-1 inhibitors have been synthesized
The molecules act as potent HO-1 inhibitors as confirmed by the biological assay
Two molecules show a selectivity >100 toward HO-1 respect to HO-2
In silico studies suggest the binding poses and the ADME-Toxicity profile
Keywords: Heme oxygenase-1 western region, HO-1 imidazole inhibitors, Docking studies, In
silico profiling, ADME-Toxicity.
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Abstract
Here we report the design, synthesis, and molecular modeling of new potent and selective
imidazole-based HO-1 inhibitors in which the imidazole nucleus and the hydrophobic groups are
linked by a phenylethanolic spacer. Most of the tested compounds showed a good inhibitor activity
with IC50 values in the low micromolar range, with two of them (1b and 1j) exhibiting also high
selectivity toward HO-2. These results were obtained by the idea of potholing the entire volume of
the principal hydrophobic western region with an appropriate ligand volume. Molecular modeling
studies showed that these molecules bind to the HO-1 in the consolidated fashion where the
imidazolyl moiety coordinates the heme iron while the aromatic groups are stabilized by
hydrophobic interaction in the western region of the binding pocket. Finally, the synthesized
compounds were analyzed for in silico ADME-Tox properties to establish oral drug-like behavior
and showed satisfactory results.
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1. Introduction
Heme levels are endogenously regulated by the heme oxygenase (HO) enzyme family. Heme
catabolism reaction mediated by HO produces an equimolar amount of Fe2+, carbon monoxide
(CO), and biliverdin (BV) which is readily converted into bilirubin (BR) by biliverdin reductase.
Two major isoforms of HO exist, HO-1 and HO-2. HO-1 (32 kDa), is physiologically expressed at
low levels in the whole human body and more abundantly in spleen and liver. HO-1 is a strongly
inducible isoform and is also referred as heat shock protein 32 (Hsp32). HO-2 (37 kDa) is a non-
inducible constitutive form, mainly found in brain and testis, whose physiological roles have not yet
been fully elucidated [1, 2].
A wide array of stimuli including oxidative, heat shock, heavy metals, UV radiations, and heme
itself are able to upregulate HO-1 which in turn, removing the pro-oxidant heme and forming CO
and BV products, exerts a significant cytoprotective role [3-5]. The HO-1 induction is widely
acknowledged as an adaptive cellular response able to counteract oxidative stress. Moreover, it is
also suggested that both HO-1 and fatty acid binding proteins may have complementary or
synergistic antioxidant effects [6, 7]. HO-1 establishes a protective effect on cells and tissues
against injuries related to pathological conditions as diabetes, obesity, cardiovascular and
pulmonary disorders [3, 8-13]. In this regard, HO-1 inducers may play a role in the management of
the abovementioned diseases [10].
At the same time, the HO-1 inhibition is reported to be beneficial for some pathological conditions
including neonatal jaundice and some type of cancers. Jaundice in newborns is characterized by an
augmented production of BR. The HO-1 inhibition giving a reduction of heme catabolism and BR
formation may be considered a valuable route for neonatal jaundice management [14, 15].
High levels of HO-1 have been frequently reported in different human cancers. Patients showing
HO-1 overexpression are reported to have statistically lower survival rate and poor outcomes [16].
Notwithstanding, the involvement of HO-1 in cancer is not fully clarified. The HO-1 protumoral
action seems to be closely linked to its cytoprotective, antiapoptotic, and angiogenic actions and
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with the modulation of inflammatory and immune responses [16]. Although some papers report a
lower expression of HO-1 in cancerous cells, a larger amount of data clearly show that HO-1 levels
are notably increased in different cancers including multiple myeloma [17], neuroblastoma (NB),
chronic and acute myeloid leukemia [18, 19], breast, prostate, and pancreatic [20]. Moreover, in
most cases where cytotoxicity is connected to reactive oxygen species (ROS), HO-1 overexpression
has been linked with the onset of resistance towards chemo-, radio-, and photodynamic- cancer
therapy [21, 22]. In this perspective, HO-1 inhibitors can be therapeutically useful in cancer
treatment and/or be used as potential enhancers under regimens of chemotherapy, radiotherapy, or
photodynamic therapy. In this contest, the identification of inhibitors endowed with high affinity
and selectivity for HO-1, respect to the constitutive isoform HO-2, may help in better understand
the biological roles of this protein and its implication in several human diseases.
The first reported class of competitive HO-1 inhibitors is represented by the heme analogs
metalloporphyrins (MPs). These compounds, sharing strong structural homologies with heme, are
able to compete with heme itself and to bind other heme-containing enzymes [e.g. nitric oxide
synthase (NOS), soluble guanylyl cyclase (sGC), and cytochromes (CYPs) P450] [23, 24].
Accordingly, the use of MPs to solve the physio-pathological roles of HO-1 may be questionable
and this major limitation, among others, impaired further development of this chemical class.
Other than MPs, only one other class of HO-1 inhibitors deriving from QC-1 (Azalanstat) has been
reported [25]. Later on, structure-activity relationship (SAR) studies around Azalanstat and the X-
ray crystal studies of QC-15, QC-80, QC-82, QC-86, and QC-308 in complex with HO-1 (Table 1)
helped to clarify the essential chemical features required for binding, the binding mode itself, and
solved the issue of off-target activity at other heme-containing enzymes [26-31].
These inhibitors are generally identified by a non-competitive binding mode by preventing Fe2+
oxidation and interfering with O2 binding when heme is located within the HO-1 binding pocket.
Non-competitive HO-1 inhibitors consist of a hydrophobic moiety, a connecting chain of variable
length, and an azole-based structure [32]. The latter is most frequently represented by an imidazole
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ring able to coordinate the Fe2+ atom of heme when heme itself is bound to the HO-1 heme binding
pocket.
Table 1
Chemical structures of selected HO-1 inhibitors, binding regions, and selectivity index (SI).
PDB ID 3HOK, ligand QC-80
Compound
QC-1 (Azalanstat)
QC-15
QC-80
QC-82
SI (HO-2/HO-1)
4.4
>35.7
7.6
>33.3
7.2
PDB ID
—
2DY5
3HOK
3CZY
3K4F
3TGM
HO-1 IC50 (µM)
HO-2 IC50 (µM)
5.3
2.8
2.1
3.0
2.5
0.27
24.4
>100
16
>100
18
QC-86
QC-308
0.46
1.7
Recently,
our
research
group
has
built
a
ligand
database
(HemeOxDB,
http://www.researchdsf.unict.it/hemeoxdb) that incorporates the whole set of published HO-1 and
HO-2 inhibitors, resulting in more than 400 compounds [33]. Among these set, only 32 compounds
(7.4%) showed an HO-1 IC50 <1 µM and, in this subset, only 4 compounds possessed a HO-2/HO-1
ratio >100. On these grounds and motivated by our ongoing interest in developing potent and
selective HO-1 inhibitors [18, 34, 35], here we report the design, synthesis, and molecular modeling
of new HO-1 inhibitors in which the imidazole nucleus and the hydrophobic groups are connected
by a phenylethanolic chain (Table 2). For all the synthesized compounds we describe the inhibitory
activity towards HO-1. For the most interesting compounds (HO-1 IC50 values <1 µM) the HO-2
inhibitory activity has also been determined. Finally, an ADME toxicity in silico prediction was
5

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carried out for the most promising compounds.
2. Results and Discussion
2.1. Design
According to previously described SARs and crystallographic data [31-33], among azole-based HO-
1 inhibitors, the azole moiety, located in the eastern region, serves as an anchor coordinating with
the Fe2+ atom through nitrogen 3 completing the heme hexacoordination. In addition, the enzyme
presents two hydrophobic pockets, one in the proximity region (northeastern) of the heme-binding
pocket, and the other in the distal region (western); this last is substantially constituted by a
principal large cavity and, lateral to this, a small secondary ones (Table 1) [31]. These pockets have
an important role in determining the inhibitor potency by stabilizing the ligand-enzyme binding.
Due to the flexibility of these pockets, hydrophobic groups of different size, such as aryl, biphenyl,
or adamantly moieties, allocated in the western and/or in the northeastern regions of the ligand, can
be accommodated [31, 32]. Moreover, it was ascertained that modifications in the northeastern
region result in a moderate gain in HO-2 inhibitory activity and therefore these are not an efficient
avenue in the development of highly selective HO-1 inhibitors [36-38]. The occupation of the
western secondary small hydrophobic pocket (double-clamp ligands, e.g. QC-308), although brings
to an increase in the HO-1 inhibitory activity, not fulfill the isoform selectivity [30]. Finally, from a
careful review of the literature and from the HemeOxDB inspection, it emerged that compounds
bearing an alcoholic function in the connecting alkyl chain, most of them characterized by a four
methylene chain units, result more active and selective for HO-1 respect to the corresponding
ketone analogues [29, 39, 40].
With this in mind, we decided to focus our attention on the potholing of the principal hydrophobic
heme oxygenase-1 western region for the search of new potent and selective imidazole-based
inhibitors possessing an unadorned imidazole, linked through a phenylethanolic connecting chain to
hydrophobic moieties of variable size, preferentially bearing a halogen residue (compounds 1a‒k,
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Table 2).
These compounds were designed on the basis of the following considerations: i) the analyses of the
pocket cavity volumes near the heme-prosthetic group in the crystal structures of the HO-1 (PDB
ID 1N45) and HO-2 (PDB ID 2QPP) umbound forms revealed a mean difference of about 34 Å3 in
favor of the HO-2 (Table S1); ii) the analogue analyses conducted on five-known corresponding
HO-1/ligand co-crystallized forms (PDB ID 2DY5, 3CZY, 3K4F, 3HOK, and 3TGM) showed that
the volume of the binding pocket is quite flexible and adaptable to that of the ligands and increase
with the size of the enclosed molecule (Table S1); iii) the four most potent and selective HO-1
inhibitors with IC50 <1 µM and HO-2/HO-1 >100 possess a mean Van der Waals volume of 274.34
Å3 with an interval of 239.00–302.87 Å3 (Table S1); iv) inspection of the HemeOxDB for the most
potent and selective HO-2 inhibitors (IC50 <1 µM and HO-1/HO-2 >40) gives out four compounds
with a mean Van der Waals volume of 284.10 Å3 with an interval of 274.37–306.75 Å3 (Table S1).
These observations, taken altogether, highlighted that the volume of the ligands, simplistically,
could be an important factor influencing the selectivity; thus, the larger the cavity of the initial
unbound isoform, the greater the volume of the ligand to achieve the best binding potency and
selectivity. To prove this assumption, the volumes of designed compounds 1a–k encompass the
interval 219.12–329.87 Å3, calculated for the most stable conformer (Table S2).
Table 2
General structure of compounds 1a‒k.
Compound
R1
3-Br
3-Ph
4-I
4-Ph
4-(4′-ClC6H4)
4-PhO
1a
1b
1c
1d
1e
1f
4-PhCH2
4-PhCH2O
4-(4-ClC6H4)CH2O
4-(4-BrC6H4)CH2O
4-(4-IC6H4)CH2O
1g
1h
1i
1j
1k
2.2. Synthesis
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Compounds 1a–k have been synthesized according to the general pathway illustrated in Scheme 1.
Intermediates 3a–i were synthesized by nucleophilic displacement of the commercially available 1-
substituted bromomethyl ketones 2a–i using an excess of imidazole. The 1-substituted
bromomethyl ketones 2j,k were obtained by bromination [41], in the glacial acetic acid, of the
corresponding commercially available ketones and used as it to synthesize compounds 3j,k as
reported above. The imidazole ketones 3a–k, were then reduced with NaBH4 affording the final
compounds 1a–k in high yield.
Scheme 1. Reagents and conditions: (i) imidazole, K2CO3, dry DMF, room temperature, 2 h, then water; (ii) NaBH4,
CH3OH, 2 h, reflux, then HCl 1N, 0.5 h, reflux.
2.3. HO inhibition and structure-activity relationships (SARs)
For inhibitory activity assay, HO-1 and HO-2 have been extracted from rat spleen and rat brain
microsomal fractions, respectively. Enzyme activity inhibition is expressed as IC50 (µM) and results
are outlined in Table 3, utilizing Azalanstat as reference compound. All the novel synthesized
derivatives exhibited good inhibitory potency towards HO-1. Only compounds which showed HO-1
IC50 values <1 µM, were tested on HO-2. Above all, compounds 1a, 1b, 1f, 1h, and 1j stand out for
their potency on HO-1 (IC50 <1 µM), and a couple of them showed a remarkable selectivity towards
HO-2.
Table 3
Inhibitory potency and selectivity index (SI) of compounds 1a–k towards HO-1 and HO-2.
IC50 (µM)a
HO-2
Compound
SI (HO-2/HO-1)
HO-1
0.40 ±0.01
0.90 ±0.08
14.80 ±0.8
11.70 ±0.3
26.90 ±0.8
0.90 ±0.1
26.90 ±1.1
0.50 ±0.01
13.10 ±0.9
32.00 ±2.2
>100
80
>111
—
—
—
11.6
—
23.4
—
1a
1b
1c
1d
1e
1f
1g
1h
1i
NDb
NDb
NDb
10.50 ±0.2
NDb
11.70 ±0.9
NDb
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0.95 ±0.02 ACCEPTED MANUSCRIPT
>100
>105
—
4.6
1j
1k
Azalanstat
24.50 ±1.0
5.30 ±0.4
NDb
24.40 ±0.8
a Data are shown as IC50 values in µM ± standard deviation (SD). Values are the mean of triplicate experiments.
b ND = not determined. The HO-2 activity was determined only for those compounds with an HO-1 IC50<1 µM.
Compound 1a, possessing a bromine residue at the 3-position of the phenyl ring, demonstrated to be
more active (IC50 = 0.40 µM) than compound 1c bearing an iodine at the 4-position (HO-1 IC50 =
14.80 µM). The bromo-derivate 1a gave good results also in terms of HO-2/HO-1 selectivity,
although this ratio was not satisfactory when compared to HO-1 inhibitors reported so far [38-40].
When the second phenyl ring is directly linked to the first one, as in the plain or 4-chlorosubstituted
biphenyl derivatives 1d and 1e, we observed a decrease of HO-1 activity (HO-1 IC50 = 11.70 µM
and IC50 = 26.90 µM, respectively), compared with 1a. Conversely, when the second phenyl ring is
linked at the 3-position, as in compound 1b, the inhibitory activity reach an IC50 value of 0.95 µM
coupled with an optimal selectivity towards the HO-2 isoform (IC50 > 100 µM).
When the second phenyl ring is separated by a methylene bridge, as for compound 1g, a similar
trend with respect to compounds 1d and 1e was observed, being 1g HO-1 IC50 = 26.90 µM.
Conversely, compound 1f, possessing a diphenyl ether residue, showed a 30-fold increase in
inhibitory potency (HO-1 IC50 = 0.90 µM). Unfortunately, this compound also significantly
inhibited HO-2. The elongation of the spacer between the two phenyl rings afforded compounds
1h,k. The potency of these four compounds was strongly influenced by the substituent of the
second phenyl ring, being the unsubstituted 1h and the 4-iodophenyl substituted 1k, respectively,
the best and the worse HO-1 inhibitors in this subset. In term of selectivity, compound 1j
demonstrated good potency towards HO-1 (IC50 = 0.95 µM), and remarkable selectivity against
HO-2 (IC50 >100 µM).
Interestingly, the most potent and selective compound 1b has a calculated Van der Waals volume of
275.12 Å3 that is the nearest to the mean value of 274.34 Å3 calculated on the best four potent and
selective compounds (Table S2), according to the postulated assumption on the relation between
cavity volumes and potency/selectivity.
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2.4. Docking studies
After the publication of various imidazole derivatives in complex with HO-1 (ID PDB, Table 1),
different docking studies were successfully performed for understanding the ligand-protein
interactions [34, 42]. In order to figure out the key interactions of our new compounds, we
investigated the binding modes of 1a–k using the crystal structure of HO-1 complexed with QC-80
(ID PDB 3HOK) in order to explore the largest binding pocket and, at the same time to exclude the
involvement of the northeastern cavity. Docking was performed using AutoDock [43] and the setup
was done with the YASARA molecular modeling program [44].
To validate the docking model, and to ascertain that the choice of the largest pocket does not
excessively invalidate the results with the less bulky ligands, we docked the Azalanstat and the five
co-crystallized ligands (QC-15, QC-80, QC-82, QC-86, and QC-308) in the binding pocket of the
3HOK structure. The results reported in Table 4 show that the calculated binding potencies are in
good agreement with the experimental ones. Moreover, the docked pose of the original ligand QC-
80 shows high analogy to the corresponding crystallographic structure with an RMSD value of 0.75
Å.
Table 4
Docking results for Azalanstat and co-crystallized ligands.
Compound
Azalanstat
QC-15
QC-80
QC-82
∆GB (kcal/mol)
–7.45
–7.59
–7.04
–7.55
Ki calcd (µM)
IC50 exp (µM) HO-1
3.4
2.7
6.8
2.9
13.7
1.1
5.3
4.0
2.1
3.0
2.5
0.27
QC-86
QC-308
–6.63
–8.11
Since our compounds present a stereocenter (at the C1 ethanolic position, Table 2), we performed a
preliminary docking study on both stereoisomers of the most potent compound 1a to verify the
eventual existence of an eudysmic ratio; the obtained results highlighted only a very small
difference in the calculated binding energy in favor of the (S)-enantiomer (0.02 kcal/mol), because
the hydroxylic moiety occupying the hydrophobic region not establish any hydrogen bond (Fig. S1).
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Moreover, the superimposition of the binding pose of QC-80 ligand, possessing the 1,3-dioxolane
ring, to the binding poses of the two enantiomers of compound 1a, shows that the oxygen atoms of
the two enantiomers occupy the same three-dimensional space as those of the dioxolane moiety
without any possibility to establish an opportune non-bonding interaction (Fig. S2), so justifying
the possible absence of an eudysmic ratio. Thus, once validated the model, we focused the attention
only on the (S)-enantiomer of compounds 1a–k docking them into the active site (encompassing all
the hydrophobic cavities) of HO-1. The results are collected in Table 5 and the docking poses are
shown in Fig. S3.
All docked compounds correctly present the ferrous iron of heme coordinated by the nitrogen atom
of the imidazole ring (eastern pocket). By means of this coordination binding, Fe2+ is protected
from oxidation by disruption of an ordered solvent structure involving the critical Asp140
hydrogen-bond network (Tyr58, Tyr114, Arg136, and Asn210) and consequent displacement of
water residues needed for catalysis.
Table 5
Docking results for molecules (S)-1a–k.
Compound
Ki calcd. (µM)
2.8
0.5
9.5
12.0
23.2
1.0
Exp. IC50 (µM) HO-1
0.40
0.90
14.80
11.70
26.90
0.90
∆GB calcd. (kcal/mol)
–7.58
–8.64
–6.85
–6.71
1a
1b
1c
1d
1e
1f
–6.32
–8.16
–6.75
–8.69
–6.93
–8.48
11.2
0.4
8.3
26.90
0.50
13.10
0.95
1g
1h
1i
0.6
1j
–6.16
30.4
24.50
1k
In the docked structures, the substituted phenylethanolic linker of our inhibitors is always located in
the western region of the binding pocket, whereas the northeastern pocket remains free. The
docking poses for the most potent and selective inhibitors 1a,b,j are reported in Fig. 1.
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Fig. 1. Docked poses of compounds 1a (orange), 1b (pink), and 1j (turquoise). Left: binding pocket full surface. Right:
highlighted sidechains of the amino acids involved in the key interactions.
In particular, compound 1a results as the most potent compound of the series, with a selectivity of
80 toward HO-2; the docking pose of 1a revealed interactions similar to the classical HO-1
inhibitors, with the phenyl-connecting group in the principal western region pocket (Leu54, Phe167,
Val50 and Arg 136) and the bromine pointing to the external portion inside the western region (Fig.
S1). On the other hand, the second most selective compound 1j, with a selectivity index HO-2/HO-
1 >105, due to its length and volume, showed a slight difference in the calculated pose inside the
protein with the former compound. In this case, the connecting phenyl group is in the western
region of the binding site, but the secondary aromatic group (4-bromophenyl) is pointing to the
external surface of the protein (Fig. S4). However, with the docking experiments it was verified that
the secondary aromatic group is not located deep inside the western pocket, neither in this case and
nor for compounds 1e and 1g–k. Also, the use of the co-crystallized form with QC-80, allow us to
exclude even a pose with this secondary aromatic moiety inside the northeastern region. Finally, the
most selective compound 1b also shows a binding arrangement similar to the previous compounds.
The aromatic linker portion is located at the bottom of the western cavity, while the phenyl in
position 3 points to the outside of the cavity (Fig. S5).
2.5. ADME and toxicity risk assessment
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To verify if the designed compounds showed a good pharmacokinetic profile and no adverse side
effects (ADME-Toxicity), we conducted an in silico study for the compounds with the highest
activity and selectivity 1a,b,j. These results are reported in Tables 5 and 6 together with that of the
best four compounds present in the HemOxDB.
The in silico ADME results (Table 5) clearly show that the new synthesized compounds should
exhibit a good oral availability [human intestinal absorption (HIA) >70% and Caco-2 cell
permeability >4) but with a strong plasma protein binding (PPB >90%), with the exception of 1a,
penalizing their diffusion and transport across cell membranes. Interestingly, compounds 1a,b,j are
supposed to discreetly permeate the blood-brain barrier (BBB penetration <1) making them, and
especially 1a, potential candidates for neuroblastoma therapy. Moreover, all these imidazole
derivatives resulted non-mutagen, non-tumorigenic, non-irritant and without negative effects on the
reproductive system (Table 6). Finally, all the three new synthesized compounds possess a positive
value of drug-likeness, stating that the molecule predominantly contains fragments which are
frequently present in commercial drugs, and a drug-score, that encompass the contributions of
partition coefficient, solubility, molecular weight, drug-likeness and the four toxicity risk, higher
than that of the four best compounds so far reported.
Table 5
Selected in silico ADME profiling of most selective compounds in HemOxDB and 1a,b,ja.
Absorption
HIA (%) In vitro Caco-2 cell permeability
(nm s–1)
Distribution
Compound
In vitro PPB In vivo BBB penetration
(%)
(Cbrain/Cblood)
HemOxDB2
HemOxDB18
HemOxDB16 100.00
96.83
98.48
45.99
57.26
57.55
48.64
29.63
41.25
47.67
98.72
84.58
88.16
77.57
65.33
94.03
90.94
0.28
0.42
0.47
1.49
0.72
0.26
0.10
HemOxDB20
98.69
96.07
96.22
96.58
1a
1b
1j
a The properties related to ADME were predicted using PreADMET web-based application (http://preadmet.bmdrc.kr).
Table 6
In silico toxicity parameters of most selective compounds in HemOxDB and 1a,b,ja.
Mutagenic
none
Tumorigenic
none
Reproductive Effects
Irritant
none
low
none
none
none
none
Drug-Likeness
3.05
Drug-Score
0.77
HemOxDB2
HemOxDB18
HemOxDB16
HemOxDB20
1a
none
high
none
none
none
none
none
none
none
none
none
none
none
none
4.07
0.63
–2.33
3.05
4.84
0.42
0.72
0.51
0.92
none
none
0.89
1b
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none
none
none
none
2.65
0.79
1j
a The properties related to toxicity were predicted using DataWarrior software [45].
3. Conclusion
In this study, we report the design, synthesis and biological properties of a series of imidazole-based
derivatives 1a‒k. All of the obtained compounds were tested for their inhibitory activity in HO-1
and those which showed a HO-1 IC50 inhibitory activity <1 µM were tested on HO-2. Above all,
compounds 1a, 1b, 1f, 1h, and 1j, presented an IC50 <1 µM on HO-1 and 1b,j showed a >100
selectivity toward HO-2, according to the design predictions. Modeling studies of these imidazole-
based inhibitors proved that in all of the molecules the imidazole interacts with the iron of the heme
group; moreover, all hydrophobic groups reside only in the principal western pocket and the most
potent and selective compounds fill it almost completely. Moreover, in silico prediction of the
ADME-Tox profiles for the most potent and selective compounds 1a,b,j highlighted that they
exhibit a good oral availability, a strong plasma protein binding, with the exception of 1a, and a
discrete capacity to permeate the BBB making them, and especially 1a, potential candidates for
neuroblastoma therapy. Moreover, all these imidazole derivatives resulted non-mutagen, non-
tumorigenic, non-irritant and without negative effects on the reproductive system (Table 6). Finally,
all the three new synthesized compounds possess an elevated value of drug-score, higher than that
of the four best compounds so far reported, suggesting them as the new lead candidates for further
studies.
4. Experimental section
4.1. Chemistry
Melting points were determined in an IA9200 Electrothermal apparatus equipped with a digital
thermometer in glass capillary tubes and are uncorrected. Infrared spectra were recorded on a
Perkin Elmer 281 FTIR spectrometer in KBr disks or NaCl crystal windows. Elemental analyses for
C, H, N, and S were performed on a Carlo Erba Elemental Analyzer Mod. 1108 apparatus. Analyses
14

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indicated by the symbols of the elements or functions were within ±0.4% of the theoretical values.
1H NMR spectra were recorded on a Varian Unity Inova 200 or 500 MHz spectrometer in DMSO-
d6 solution. Chemical shifts are given in d values (ppm), using tetramethylsilane (TMS) 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). All the synthesized
compounds were tested for purity on TLC (aluminum sheet coated with silica gel 60 F254, Merck)
and visualized by UV (λ = 254 and 366 nm). Purification of synthesized compounds by column
chromatography was performed using silica gel 60 (Merck). All chemicals and solvents were
reagent grade and were purchased from commercial vendors.
4.1.1. General procedure for the synthesis of 2-bromo-1-{4-[(4-bromobenzyl)oxy]phenyl}ethanone
and 2-bromo-1-{4-[(4-iodobenzyl)oxy]phenyl}ethanone (2j,k)
The appropriate commercially available 1-{4-[(4-bromobenzyl)oxy]phenyl}ethanone or 1-{4-[(4-
iodobenzyl)oxy]phenyl}ethanone (5 mmol) was dissolved in 4 mL of glacial acetic acid, bromine (5
mmol) was added dropwise and the reaction mixture was stirred at 22 °C for 1 h. The obtained
reaction mixture was dropped in saturated NaHCO3, extracted with EtOAc (3 × 30 mL). The
combined organic layers were dried over Na2SO4, filtered and evaporated. The obtained crude 2j,k
were used such as.
4.1.2. General procedure for the synthesis of 1-substituted-2-(1H-imidazol-1-yl)ethanones (3a–k)
The appropriate, 2-bromo-1-substituted ethanone (2a–k, 5 mmol), was dissolved in anhydrous
DMF (15 mL) and added drop-wise to a previously prepared suspension of imidazole (15 mmol)
and K2CO3 (15 mmol) in anhydrous DMF (30 mL). The obtained reaction mixture was left stirring
for 2 h; then, water was added and the resulting suspension was filtered. The crude filtrate was
purified by column chromatography using a Biotage® chromatographic system with Biotage®
SNAP KP-Sil flash chromatography cartridges and DCM/methanol (98:2) as eluent. Analytical and
spectral data are reported only for unknown compounds 3e and 3i–k. Compounds 3a–d, and 3f–h
were previously reported in the literature [27, 40, 46].
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4.1.2.1. 1-(4'-chlorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethanone (3e)
The title compound was obtained as a pure white solid (49%): mp 189–191 °C; IR (KBr, selected
lines) cm–1 3102, 2921, 1696, 1686, 1602, 1487, 1392, 1235, 1097, 991, 813, 663; NMR (200 MHz,
DMSO-d6): 8.18–8.05 (m, 2H, aromatic), 7.95–7.78 (m, 4H, aromatic), 7.62–7.53 (m, 2H + 1H,
aromatic + imidazole), 7.14 (s, 1H, imidazole), 6.94 (s, 1H, imidazole), 5.78 (s, 2H, CH2). Anal.
(C17H13ClN2O) C, H, N.
4.1.2.2. 1-{4-[(4-chlorobenzyl)oxy]phenyl}-2-(1H-imidazol-1-yl)ethanone (3i)
The title compound was obtained as a pure white solid (52%): mp 118–119 °C; IR (KBr, selected
1
lines) cm–1 3130, 1690, 1600, 1508, 1235, 1176, 1086, 1015, 990, 810, 609; H NMR (200 MHz,
DMSO-d6): 8.06–7.96 (m, 2H, aromatic), 7.58 (s, 1H, imidazole), 7.50–7.42 (s, 4H, aromatic),
7.20–7.15 (m, 2H, aromatic), 7.10 (s, 1H, imidazole), 6.91 (s, 1H, imidazole), 5.67 (s, 2H, CH2N),
5.25 (s, 2H, CH2O). Anal. (C18H15ClN2O) C, H, N.
4.1.2.3. 1-{4-[(4-bromobenzyl)oxy]phenyl}-2-(1H-imidazol-1-yl)ethanone (3j)
The title compound was obtained as pure off-white solid (35%): mp 115–116 °C; IR (KBr, selected
1
lines) cm–1 3114, 1691, 1678, 1599, 1498, 1235, 1178, 1029, 992, 904, 807; H NMR (200 MHz,
DMSO-d6): 8.07–7.95 (m, 2H, aromatic), 7.68–7.57 (s, 2H + 1H, aromatic + imidazole), 7.47–7.36
(m, 2H, aromatic), 7.20–7.15 (m, 2H, aromatic), 7.11 (s, 1H, imidazole), 6.92 (s, 1H, imidazole),
5.67 (s, 2H, CH2N), 5.23 (s, 2H, CH2O). Anal. (C18H15BrN2O) C, H, N.
4.1.2.4. 2-(1H-imidazol-1-yl)-1-{4-[(4-iodobenzyl)oxy]phenyl}ethanone (3k)
The title compound was obtained as pure off-white solid (33%): mp 195–196 °C; IR (KBr, selected
1
lines) cm–1 3132, 3106, 1694, 1600, 1500, 1232, 1174, 1086, 1011, 989, 826,660; H NMR (200
MHz, DMSO-d6): 8.15–7.63 (m, 4H, aromatic), 7.61 (s, 1H, imidazole), 7.18–7.00 (m, 4H + 1H,
aromatic + imidazole), 6.93 (s, 1H, imidazole), 5.68 (s, 2H, CH2N), 5.22 (s, 2H, CH2O). Anal.
(C18H15IN2O) C, H, N.
4.1.3. General procedure for the synthesis of 1-(substituted)-2-(1H-imidazol-1-yl)ethanoles 1a–k
A mixture of the appropriate imidazole-ketone (3a–k, 2.0 mmol) and NaBH4 (2.2 mmol) in
16

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anhydrous methanol (10 mL) was refluxed for 2 h; then it was evaporated to dryness, added with
deionized water (40 mL), acidified with HCl 2N and heated to 110 °C for 0.5 h. After cooling to
room temperature, the reaction mixture was treated with NaOH 0.5N up to a pH of 8–9 and the
obtained suspension was filtered, washed repeatedly with water to neutrality, and dried. Using this
procedure, the following compounds were obtained.
4.1.3.1. 1-(3-bromophenyl)-2-(1H-imidazol-1-yl)ethanol (1a)
Recrystallization from EtOH gave the title compound as a pure light yellow solid (72%): mp 109–
1
111 °C; IR (KBr, selected lines) cm–1 3122, 1567, 1508, 1418, 1233, 1105, 1065, 920, 750; H
NMR (500 MHz, DMSO-d6): 7.54 (bs, 1H, imidazole), 7.50–7.48 (m, 1H, aromatic), 7.36–7.27 (m,
2H + 1H, aromatic), 7.11 (s, 1H, imidazole), 6.83 (s, 1H, imidazole), 5.83 (d, J = 5 Hz, 1H, OH),
4.88–4.80 (m, 1H, CH), 4.15 (dd, J = 14.5 Hz, J = 4 Hz, 1H, CHACHB), 4.04 (dd, J = 8 Hz, J = 14
Hz, 1H, CHACHB); 13C NMR (125 MHz, DMSO-d6) 145.42, 137.69, 130.25, 130.09, 128.79,
127.72, 125.035, 121.47, 120.00, 71.30, 53.18. Anal. (C11H11BrN2O) C, H, N.
4.1.3.2. 1-(biphenyl-3-yl)-2-(1H-imidazol-1-yl)ethanol (1b)
Recrystallization from EtOH gave the title compound as a pure light brown sticky oil (90%): IR
(KBr, selected lines) cm–1 3117, 1512, 1484, 1390, 1233, 1080, 1003, 814, 747, 649; 1H NMR (500
MHz, DMSO-d6): 7.65–7.35 (m, 9H + 1H, aromatic + imidazole), 7.15 (s, 1H, imidazole), 6.85 (s,
1H, imidazole), 5.77 (bs 1H, OH), 4.93–4.88 (m, 1H, CH), 4.21 (dd, J = 14 Hz, J = 4 Hz, 1H,
CHACHB), 4.10 (dd, J = 8 Hz, J = 14 Hz, 1H, CHACHB). 13C NMR (125 MHz, DMSO-d6) 143.29,
140.20, 139.94, 128.85, 128.65, 127.69, 127.39, 126.67, 125.63, 125.04, 124.45, 120.06, 72.05,
53.51. Anal. (C17H16N2O) C, H, N.
4.1.3.3. 2-(1H-imidazol-1-yl)-1-(4-iodophenyl)ethanol (1c)
Recrystallization from EtOH gave the title compound as a pure off-white solid (88%): mp 182–183
°C; IR (KBr, selected lines) cm–1 3116, 2933, 1512, 1484, 1399, 1280, 1231, 1073, 1102, 1073,
1
1006, 918, 818, 738; H NMR (500 MHz, DMSO-d6): 7.70–7.67 (m, 2H, aromatic), 7.46 (s, 1H,
imidazole), 7.18–7.08 (m, 2H + 1H, aromatic + imidazole), 6.82 (s, 1H, imidazole), 5.78 (d, J = 4
17

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Hz, 1H, OH), 4.82–4.78 (m, 1H, CH), 4.12 (dd, J = 14 Hz, J = 4 Hz, 1H, CHACHB), 4.02 (dd, J = 8
13
Hz, J = 14 Hz, 1H, CHACHB); C NMR (125 MHz, DMSO-d6) 142.38, 136.74, 128.37, 127.74,
120.00, 93.18, 71.43, 53.17. Anal. (C11H11IN2O) C, H, N.
4.1.3.4. 1-(biphenyl-4-yl)-2-(1H-imidazol-1-yl)ethanol (1d)
Recrystallization from EtOH gave the title compound as a pure off-white solid (80%): mp 182–183
°C (183–184, lit. [46]); IR (KBr, selected lines) cm–1 3188, 3110, 1511, 1488, 1282, 1234, 1075,
1
833, 753; H NMR (500 MHz, DMSO-d6): 7.69–7.61 (m, 4H, aromatic) 7.53 (s, 1H, imidazole),
7.49–7.41 (m, 5H, aromatic), 7.16 (s, 1H, imidazole), 6.85 (s, 1H, imidazole), 5.76 (d, J = 4.2 Hz,
1H, OH), 4.88–4.85 (m, 1H, CH), 4.19 (dd, J = 14 Hz, J = 4 Hz, 1H, CHACHB), 4.08 (dd, J = 8 Hz,
J = 14 Hz, 1H, CHACHB), 13C NMR (125 MHz, DMSO-d6) 141.84, 139.85, 139.13, 137.72, 128.89,
127.74, 127.35, 126.63, 126.56, 126.33, 120.03, 71.82, 53.43. Anal. (C17H16N2O) C, H, N.
4.1.3.5. 1-(4'-chlorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethanol (1e)
Recrystallization from EtOH gave the title compound as a pure white solid (82%): mp 232–233 °C;
1
IR (KBr, selected lines) cm–1 3116, 1513, 1485, 1391, 1233, 1099, 1080, 1003,814, 649; H NMR
(500 MHz, DMSO-d6): 7.76–7.63 (m, 4H, aromatic), 7.55–7.41(m, 4H + 1H, aromatic +
imidazole), 7.16 (s, 1H, imidazole), 6.85 (s, 1H, imidazole), 5.76 (d, J = 4.5 Hz, 1H, OH), 4.18 (dd,
13
J = 14 Hz, J = 4 Hz, 1H, CHACHB), 4.07 (dd, J = 8 Hz, J = 14 Hz, 1H, CHACHB); C NMR (125
MHz, DMSO-d6) 142.23, 138.63, 137.74, 132.21, 128.83, 128.30, 127.77, 126.69, 126.27, 71.74,
53.40. Anal. (C17H15ClN2O) C, H, N.
4.1.3.6. 2-(1H-imidazol-1-yl)-1-(4-phenoxyphenyl)ethanol (1f)
Recrystallization from EtOH gave the title compound as a pure white solid (85%): mp 159–160 °C;
IR (KBr, selected lines) cm–1 3116, 1590, 1506, 1488, 1240, 1167, 1082, 1073, 834, 694; 1H NMR
(500 MHz, DMSO-d6): 7.55–7.45 (m, 1H, imidazole), 7.40–7.30 (m, 4H, aromatic), 7.17–7.08 (m,
1H + 1H, aromatic + imidazole), 7.00–6.95 (m, 4H, aromatic), 6.83 (s, 1H, imidazole), 5.72 (d, J =
4.5 Hz, 1H, OH), 4.81 (m, 1H, CH), 4.13 (dd, J = 14 Hz, J = 4 Hz, 1H, CHACHB), 4.04 (dd, J = 8
13
Hz, J = 14 Hz, 1H, CHACHB). C NMR (125 MHz, DMSO-d6) 156.78, 155.73, 137.78, 129.98,
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127.66, 123.30, 119.98, 118.38, 118.32, 71.56. Anal. (C17H16N2O2) C, H, N.
4.1.3.7. 1-(4-benzylphenyl)-2-(1H-imidazol-1-yl)ethanol (1g)
Recrystallization from EtOH gave the title compound as a pure off-white solid (90%): mp 159–160
°C; IR (KBr, selected lines) cm–1 3118, 2921, 1510, 1494, 1417, 1235, 1079, 1017, 920, 733, 697;
1H NMR (500 MHz, DMSO-d6): 7.49 (s, 1H, imidazole), 7.35–7.02 (m, 9H, aromatic), 6.83 (s, 1H,
imidazole), 6.84 (s, 1H, imidazole), 5.64 (bs, 1H, OH), 4.78–4.73 (m, 1H, CH), 4.10 (dd, J = 14 Hz,
J = 4 Hz, 1H, CHACHB), 4.00 (dd, J = 8 Hz, J = 14 Hz, 1H, CHACHB), 3.92 (s, 2H, CH2); 13C NMR
(125 MHz, DMSO-d6) 141.29, 140.30, 128.60, 128.39, 128.36, 127.70, 126.12, 125.89, 71.91,
53.51. Anal. (C18H18N2O) C, H, N.
4.1.3.8. 1-[4-(benzyloxy)phenyl]-2-(1H-imidazol-1-yl)ethanol (1h)
Recrystallization from EtOH gave the title compound as a pure white solid (92%): mp 145–146 °C;
IR (KBr, selected lines) cm–1 3115, 2886, 1612, 1509, 1454, 1255, 1238, 1171, 1076, 1045, 823,
1
731; H NMR (500 MHz, DMSO-d6): 7.50–7.29 (m, 5H + 1H, aromatic + imidazole), 7.26–7. –23
(m, 2H, aromatic), 7.09 (s, 1H, imidazole), 6.99–6.92 (m, 2H, aromatic), 6.82 (s, 1H, imidazole),
5.58 (bs, 1H, OH), 4.79–4.67 (m, 1H, CH), 4.09 (dd, J = 14 Hz, J = 4 Hz, 1H, CHACHB), 4.01 (dd,
J = 8 Hz, J = 14 Hz, 1H, CHACHB); 13C NMR (125 MHz, DMSO-d6) 157.58, 137.10, 134.86,
128.37, 127.73, 127.60, 127.17, 114.34, 71.62, 69.12, 53.54. Anal. (C18H18N2O2) C, H, N.
4.1.3.9. 1-{4-[(4-chlorobenzyl)oxy]phenyl}-2-(1H-imidazol-1-yl)ethanol (1i)
The obtained crude material was purified by column chromatography using a Biotage®
chromatographic system with Biotage® SNAP KP-Sil flash chromatography cartridges and
DCM/methanol (98:2) as eluent, to afford the title compound as pure off-white solid (65%): mp
197–198 °C; IR (KBr, selected lines) cm–1 3115, 1611, 1510, 1490, 1231, 1175, 1081, 1052, 1013,
1
822, 661; H NMR (500 MHz, DMSO-d6): 7.50–7.41 (m, 4H + 1H, aromatic + imidazole), 7.28–
7.21 (m, 2H, aromatic), 7.09 (s, 1H, imidazole), 6.99–6.92 (m, 2H, aromatic), 6.82 (s, 1H,
imidazole), 5.58 (d, J = 4 Hz, 1H, OH), 5.09 (s, 2H, CH2), 4.78–4.71 (m, 1H, CH), 4.09 (dd, J = 14
13
Hz, J = 4 Hz, 1H, CHACHB), 4.01 (dd, J = 8 Hz, J = 14 Hz, 1H, CHACHB); C NMR (125 MHz,
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DMSO-d6) 157.36, 137.63, 136.17, 135.01, 132.29, 129.39, 128.36, 127.66, 127.18, 119.95, 114.36,
71.60, 71.50, 68.27, 53.52. Anal. (C18H17ClN2O) C, H, N.
4.1.3.10. 1-{4-[(4-bromobenzyl)oxy]phenyl}-2-(1H-imidazol-1-yl)ethanol (1j)
The obtained crude material was purified by column chromatography using a Biotage®
chromatographic system with Biotage® SNAP KP-Sil flash chromatography cartridges and
DCM/methanol (98:2) as eluant, to afford the title compound as a pure white solid (61%): mp 205–
206 °C; IR (KBr, selected lines) cm–1 3117, 1610, 1509, 1489, 1230, 1174, 1071, 1010, 821, 661;
1H NMR (500 MHz, DMSO-d6): 7.60–7.57 (m, 2H, aromatic), 7.46 (s, 1H, imidazole), 7.41–7.38
(m, 2H, aromatic), 7.28-7.20 (m, 2H, aromatic), 7.08 (s, 1H, imidazole), 6.98–6.92 (m, 2H,
aromatic), 6.81 (s, 1H, imidazole), 5.58 (d, J = 4.5 Hz, 1H, OH), 5.07 (s, 2H, CH2), 4.08 (dd, J = 14
13
Hz, J = 4 Hz, 1H, CHACHB), 4.00 (dd, J = 8 Hz, J = 14 Hz, 1H, CHACHB). C NMR (125 MHz,
DMSO-d6) 157.34, 137.63, 135.02, 131.28, 129.69, 127.67, 127.18, 120.81, 119.94, 114.36, 71.60,
68.30, 53.51. Anal. (C18H17BrN2O) C, H, N.
4.1.3.11. 1-{4-[(4-iodobenzyl)oxy]phenyl}-2-(1H-imidazol-1-yl)ethanol (1k)
The obtained crude material was purified by column chromatography using a Biotage®
chromatographic system with Biotage® SNAP KP-Sil flash chromatography cartridges and
DCM/methanol (98:2) as eluant, to afford the title compound as a pure white solid (89%): mp 197–
198 °C; IR (KBr, selected lines) cm–1 3116, 1610, 1508, 1458, 1231, 1077, 1008, 918, 802, 660; 1H
NMR (500 MHz, DMSO-d6): 7.78–7.72 (m, 2H, aromatic), 7.47 (s, 1H, imidazole), 7.28–7.22 (m,
4H, aromatic), 7.08 (s, 1H, imidazole), 6.95–6.91 (m, 2H, aromatic), 6.82 (s, 1H, imidazole), 5.58
(d, J = 3.5 Hz, 1H, OH), 5.06 (s, 2H, CH2), 4.78–4.70 (m, 1H, CH), 4.09 (dd, J = 14 Hz, J = 4 Hz,
1H, CHACHB), 4.00 (dd, J = 8 Hz, J = 14 Hz, 1H, CHACHB); 13C NMR (125 MHz, DMSO-d6)
157.35, 137.14, 136.97, 134.99, 128.76, 127.64, 127.18, 119.96, 114.37, 93.73, 71.60, 68.42, 53.53.
Anal. (C18H17IN2O) C, H, N.
4.2. Biology
4.2.1. Preparation of spleen and brain microsomal fractions
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HO-1 and HO-2 were obtained, respectively, from rat spleen and brain as the microsomal fraction
prepared by differential centrifugation; the dominance of HO-1 protein in the rat spleen and of HO-
2 in the rat brain has been well documented [47-50]. 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 (Sprague-Dawley rats) microsomal fractions were prepared according to the procedure
outlined by Ryter et al. [51]. 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 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 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 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 divided 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 [52].
4.2.2. Preparation of biliverdin reductase
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 a 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 minutes at 4 °C. The supernatant was decanted and centrifuged at 100,000g for 1 h
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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.
4.2.3. Measurement of HO-1 and HO-2 enzymatic activities in 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 to 530 nm as described by Ryter et al. [51]. Reaction mixtures (500
µL) consisted of 20 mM Tris-HCl, pH 7.4, (1 mg/mL) microsomal extract, 0.5–2.0 mg/mL
biliverdin reductase, 1 mM NADPH, 2 mM glucose 6-phosphate (G6P), 1 U G6P dehydrogenase,
25 µM hemin, 10 µL of DMSO (or the same volume of DMSO solution of test compounds to a final
concentration of 100, 10, and 1 µM). Incubations 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 chloroform. After
recovering the chloroform phase, the amount of bilirubin formed was measured with a double-beam
spectrophotometer as OD464-530 nm (extinction coefficient, 40 mM/cm−1 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.
4.3. Docking
4.3.1. Preparation of ligands
The 3D structures of ligands were built using Gabedit (2.4.8) software [53] and all geometries were
fully optimized, in the same software, with the semi-empirical PM6 Hamiltonian [54] implemented
in MOPAC2016 (17.130W) [55].
4.3.2. Docking protocol
Macromolecules and ligands, were prepared within YASARA; the point charges were initially
assigned according to the AMBER14 force field [56], and then damped to mimic the less polar
Gasteiger charges used to optimize the AutoDock scoring function. Fine docking was performed by
applying the Lamarckian genetic algorithm (LGA) implemented in AutoDock. The ligand-centered
maps were generated by the program AutoGrid with a spacing of 0.375 Å and dimensions that
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encompass all atoms extending 5 Å from the surface of the ligand. All of the parameters were
inserted at their default settings. In the docking tab, the macromolecule and ligand are selected, and
GA parameters are set as ga_runs = 100, ga_pop_size = 150, ga_num_evals = 20000000,
ga_num_generations = 27000, ga_elitism = 1, ga_mutation_rate = 0.02, ga_crossover_rate = 0.8,
ga_crossover_mode = two points, ga_cauchy_alpha = 0.0, ga_cauchy_beta = 1.0, number of
generations for picking worst individual = 10.
From the crystal structures of the HO-1/QC-xx complex, we retained only the chain B and the
prosthetic-heme group. Because no water molecules are directly involved in complex stabilization
they were not considered in the docking process. All protein amino acidic residues were kept rigid
whereas all single bonds of ligands were treated as full flexible.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://doi.org/
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