Targeting heme Oxygenase-1 with hybrid compounds to overcome Imatinib resistance in chronic myeloid leukemia cell lines

Article abstract of DOI:10.1016/j.ejmech.2018.09.048

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme and a survival-enhancing factor in a number of cancers. Chronic myeloid leukemia (CML) is a blood cancer caused by pathological kinase activity of the BCR-ABL protein, currently treated with tyrosine kinase inhibitors (TKIs) such as Imatinib (IM). However, resistance to TKIs persists in a number of patients and HO-1 overexpression has been linked with the induction of chemoresistance in CML. With this in mind, in this study, we designed and synthesized the first series of hybrid compounds obtained by combining the structures of IM, as BCR-ABL inhibitor, with imidazole-based HO-1 inhibitors. We found that many hybrids were able to inhibit the enzymatic activity of both targets and to reduce the viability of CML-IM resistant cells, showing that a single molecular entity may reduce the resistance phenomenon.

Full text of DOI:10.1016/j.ejmech.2018.09.048

Accepted Manuscript
Targeting heme Oxygenase-1 with hybrid compounds to overcome Imatinib
resistance in chronic myeloid leukemia cell lines
Valeria Sorrenti, Valeria Pittalà, Giuseppe Romeo, Emanuele Amata, Maria Dichiara,
Agostino Marrazzo, Rita Turnaturi, Orazio Prezzavento, Ignazio Barbagallo, Luca
Vanella, Antonio Rescifina, Giuseppe Floresta, Daniele Tibullo, Francesco Di
Raimondo, Sebastiano Intagliata, Loredana Salerno
PII:
S0223-5234(18)30820-1
10.1016/j.ejmech.2018.09.048
EJMECH 10759
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2018
Revised Date: 14 September 2018
Accepted Date: 15 September 2018
Please cite this article as: V. Sorrenti, V. Pittalà, G. Romeo, E. Amata, M. Dichiara, A. Marrazzo,
R. Turnaturi, O. Prezzavento, I. Barbagallo, L. Vanella, A. Rescifina, G. Floresta, D. Tibullo, F. Di
Raimondo, S. Intagliata, L. Salerno, Targeting heme Oxygenase-1 with hybrid compounds to overcome
Imatinib resistance in chronic myeloid leukemia cell lines, European Journal of Medicinal Chemistry
(2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.048.
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ACCEPTED MANUSCRIPT
Targeting Heme Oxygenase-1 with Hybrid Compounds to Overcome Imatinib Resistance in
Chronic Myeloid Leukemia Cell Lines
a
a,
a
a
a
Valeria Sorrenti , Valeria Pittalà *, Giuseppe Romeo , Emanuele Amata , Maria Dichiara ,
a
a
a
a
a
Agostino Marrazzo , Rita Turnaturi , Orazio Prezzavento , Ignazio Barbagallo , Luca Vanella ,
a
a,
c,
c
Antonio Rescifina , Giuseppe Floresta b, Daniele Tibullo d, Francesco Di Raimondo ,
Sebastiano Intagliata e, Loredana Salerno a,*.
a Department of Drug Sciences, University of Catania, viale A. Doria 6, 95125, Catania, Italy.
b Department of Chemical Sciences, University of Catania, viale A. Doria 6, 95125, Catania, Italia.
c Division of Hematology, A.O.U. Policlinico-OVE, University of Catania, via S. Sofia, 78, 95123,
Catania, Italy.
d Department of Biomedical and Biotechnological Sciences, University of Catania, via S. Sofia, 78,
95123, Catania, Italy.
e Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL,
USA.
Corresponding Authors:
Loredana Salerno; l.salerno@unict.it
Valeria Pittalà; vpittala@unict.it
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Graphical Abstract
Highlights
•
•
•
•
New HO-1/BCR-ABL inhibitors have been synthesized
Many hybrids are able to inhibit the enzymatic activity of both targets
The hybrids overcome tyrosine kinase inhibitors resistance
The hybrids reduce viability of Chronic Myeloid Leukemia Imatinib resistant cells
Keywords: HO-1 inhibitors, Tyrosine kinase inhibitors, BCR-ABL, Imatinib, Chronic myeloid
leukemia
Abstract
Heme oxygenase-1 (HO-1) is a cytoprotective enzyme and a survival-enhancing factor in a number
of cancers. Chronic myeloid leukemia (CML) is a blood cancer caused by pathological kinase
activity of the BCR-ABL protein, currently treated with tyrosine kinase inhibitors (TKIs) such as
Imatinib (IM). However, resistance to TKIs persists in a number of patients and HO-1
overexpression has been linked with the induction of chemoresistance in CML. With this in mind,
in this study, we designed and synthesized the first series of hybrid compounds obtained by
combining the structures of IM, as BCR-ABL inhibitor, with imidazole-based HO-1 inhibitors. We
found that many hybrids were able to inhibit the enzymatic activity of both targets and to reduce the
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viability of CML IM resistant cells, showing that a single molecular entity may reduce the
resistance phenomenon.
1. Introduction
Heme oxygenases (HOs) are enzymes which catalyze the catabolism of heme into free iron, carbon
monoxide (CO), and biliverdin, this last successively reduced to bilirubin [1–3]. The main
consequence of this enzymatic activity is a cyto-protective effect in various stress-related conditions
[4,5]. Three isoforms of HO are present in humans, but only two are enzymatically active: HO-1,
which is inducible, and HO-2, which is constitutive. The latter is ubiquitous and is mainly
responsible for physiological effects. HO-1, also known as heat shock protein 32 (Hsp32), is found
in tissue at low levels with a major presence in the spleen and in the liver, but it may be induced in
many other organs/tissues by a variety of stimuli, such as heat, heme itself, heavy metals, reactive
oxygen species (ROS), hypoxia, nitric oxide (NO), ultraviolet radiations, and xenobiotics.
Therefore, its critical role is the cellular protection against such insults [6–8]. However, an abnormal
HO-1 overexpression may be responsible for pathological conditions; for example, it can modify
the endogenous balance between apoptosis and proliferation, promoting cancer formation and
maintenance [9,10]. Indeed, besides healthy human cells, HO-1 is highly expressed in several solid
tumors as well as hematopoietic neoplasms in which it exerts a critical role as survival molecule,
being involved in their growth, metastasis, angiogenesis, and tolerance to chemo-, radio- and
photodynamic therapies [11–13].
Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy, representing about 15%
of leukemias in adults. It origins from a t(9;22) chromosomal translocation that generates the BCR-
ABL oncogene. The product of this oncogene is the BCR-ABL protein with very high tyrosine
kinase (TK) activity, which in turn leads to an uncontrolled proliferation of myeloid stem cells with
consequent initiation, maintenance and progression of CML [14]. Indeed, blockade of BCR-ABL
protein with tyrosine kinase inhibitors (TKIs) is a well-validated approach for the treatment of CML
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[15]. Imatinib (IM, marketed as Glivec® or Gleevec®) is the first discovered TKI approved by the
FDA in 2001. From that date, it achieved great clinical success becoming the first-line drug in
conventional treatment of CML [16]. Despite this smashing success, its clinical use is often
associated with the emergence of clinical resistance [17,18]. To overcome IM resistance, several
classes of new-generation TKIs have been developed in the last years, and some of them, such as
dasatinib, nilotinib, bosutinib, and ponatinib, are currently approved drugs. Although the
pharmacological use of TKIs has changed CML from a largely incurable disorder to a disease
compatible with a normal lifespan, crucial issues remain to be solved, mainly the permanence of
10–15% of patients resistant to all TKIs and at risk of disease progression. Therefore, new strategies
to improve CML therapy may provide additional therapeutic options for patients in this setting. The
most common mechanism of IM resistance is the result of point mutations in BCR-ABL TK domain
[19]. Besides gene point mutations, IM resistance may be due to amplification of the BCR-ABL
gene with consequent overexpression of BCR-ABL itself [20]. This amplification imposes a
continuous increase of IM dosage with consequent more severe side effects. Moreover, there are
evidences that BCR-ABL protein promotes novel acquired cytogenetic aberrations of other signal
pathways, such as the expression of a number of antiapoptotic proteins, among which HO-1 is one
of the most representative. HO-1 is constitutively expressed in primary CML cells and its
expression is higher in IM-resistant cell lines, such as K562R and LAMA-84R cells, than in IM-
sensitive ones [21,22]. A recent study has provided evidence that silencing HO-1 in the mentioned
cell lines, increased apoptosis and restored IM activity [23]. According to this study, we recently
demonstrated that two novel imidazole-based HO-1 inhibitors 1 (LS0) and 2 (LS1/71) (Figure 1),
were able to restore IM sensitivity in LAMA-84R cells producing growth-inhibitory effects [24].
Interestingly these effects were obtained only by means of a combination of IM and HO-1
inhibitors, whereas the single molecules were not effective. These studies confirmed that HO-1
over-activity is responsible for IM-resistance and that HO-1 inhibition can be a viable new
anticancer strategy [10,25,26]. Co-treatment with IM and HO-1 inhibitors could find application in
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chronic phase (CP)-CML patients that develop IM resistance and clinically treated with increasing
doses of IM. Synergism between HO-1 inhibitors and IM might decrease IM dosage and side effects
in these patients. Therapeutic application of these findings would require two molecules (IM and
HO-1 inhibitor) co-administration, but combination therapy is generally a difficult and complicated
avenue because it can give rise to drug-drug interactions, as well as a negative patient compliance.
An alternative strategy would be to combine multiple activities within the same compound
providing a superior therapeutic effect and side effect profile, compared to the action of single
molecules. In this respect, conjugation of two biologically active molecules into one hybrid
compound can be beneficial for the treatment of diseases with complex etiologies such as cancer
[27].
On these bases, in this study we designed and synthesized a new series of hybrid compounds,
namely hybrid HO-1/TKI, containing an aryloxyalkylimidazole moiety and an “IM-like portion”
(6a–n, Figure 1), needed for the interaction with HO-1 and BCR-ABL proteins, respectively. In
order to gain insight on the structure activity relationships (SAR), we synthesized various HO-
1/TKIs with the oxybutylimidazole moiety connected at different positions of the benzamide ring
and characterized by the presence of various substituents in the same phenyl ring. We tested all
hybrids to evaluate their capacity of inhibiting the enzymatic activity of both targets and reducing
the viability of IM-resistant and sensitive CML cells. To obtain information on the binding mode of
these HO-1/TKIs towards both proteins and to explain the differences in the experimentally
observed activities, we performed molecular docking studies. Finally, we investigated the
mechanism of action and carried out an in silico prediction of ADME toxicity for the most
promising compounds.
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Figure 1. Chemical structures of IM and parent compounds 1 and 2, and general structure of HO-
1/TKI hybrids 6a–n.
2. Results and Discussion
2.1. Chemistry
The synthesis of HO-1/TK hybrid compounds 6a–n was accomplished in three steps, as outlined in
Scheme 1. In the first step, the commercially available hydroxybenzoic acid methyl esters were
etherified with 1,4-dibromobutane to give corresponding bromobutoxy benzoates 3a–n. In the
second step nucleophilic displacement of the intermediates 3a–n with imidazole in acetonitrile
under microwave irradiation, in the presence of triethylamine (TEA) and tetrabutylammonium
bromide (TBAB), at 95 °C for 30–45 min, gave the desired methyl imidazolylbutoxy benzoates 4a–
n. In the final step, condensation of methyl esters 4a–n with 4-methyl-N3-[4-(pyridin-3-yl)-
pyrimidin-2-yl]1,3-benzenediamine (5) in dry tetrahydrofuran (THF), in the presence of potassium
tert-butoxide (tBuOK), at room temperature, under nitrogen, gave final hybrid compounds 6a–n.
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Scheme 1. Synthesis of HO-1/TKI hybrids 6a–n. Reagents and conditions: (i) method A, 1,4-
dibromobutane, acetone, K2CO3, 90 °C under microwaves irradiation; method B, 1,4-
dibromobutane, dimethylsulfoxide, KOH, room temperature; method C, 1,4-dibromobutane,
acetonitrile, K2CO3, reflux. (ii) Imidazole, acetonitrile, TEA, TBAB, 95 °C, under microwaves
irradiation. (iii) intermediate 5, dry THF, tBuOK, room temperature, under nitrogen.
2.2. Biological evaluation
Designed and synthesized compounds 6a–n are hybrid compounds bearing the pharmacophoric
portions for inhibition of HO-1 and TK, respectively. Since the structure of these hybrids is very
close to that of IM, but rather different with respect to HO-1 inhibitors 1 and 2 (Figure 1), a crucial
challenge was to demonstrate whether HO-1 inhibition was maintained. Thus, as a first step, we
tested the ability of all compounds of inhibiting HO-1 enzymatic activity. We focused our attention
towards HO-1 since only this inducible isoform is involved in tumorigenesis and in tumor
progression, and is extremely overexpressed in cancer such as CML. HO-1 was obtained from the
microsomal fractions of rat spleen, and its activity was determined by measuring the bilirubin
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formation using the difference in absorbance at 464–530 nm, as described in the experimental
section [28]. Compounds 1 and 2 (Figure 1), and azalanstat (Table 1, Figure S1, supporting
information, SI) were used as reference substances. Azalanstat is the first discovered imidazole-
based HO-1 inhibitor, representing the lead-compound for SAR and crystallographic studies in the
field of HO-1 inhibitors [29–32]. Despite its potential hepatotoxicity at high doses, imidazole
nucleus is the central core in many commercially available drugs, such as antimycotic agents, and
continues to be widely used in the development of new potential pharmacologically active
compounds [33–35]. One of the most critical issues in the clinical use of azole based HO-1
inhibitors is the non-selectivity towards other heme-containing enzymes, such as nitric oxide
synthase, soluble guanylate cyclase, and cytochrome P450. For this reason azalanstat and some of
its congeners were tested on above-mentioned hemoproteins, and showed a good profile of
selectivity for HO-1 [36]. HO-1 inhibition by 6a–n is expressed as IC50 (µM) and results are
summarized in Table 1. It is evident that all hybrids inhibited HO-1 at micromolar concentrations,
with IC50 values ranging from 0.9 to 74 µM, and in sub-micromolar range for three of them, 6f, 6g,
and 6m. Inhibition potency of these last compounds is comparable to or higher than those of the
parent compounds 1 and 2, and better than that of azalanstat. Additionally, compound 5
(representing the “IM-like portion” of the hybrids) as well as IM, were not active on HO-1,
confirming that HO-1 inhibition is due to the imidazole portion of the molecules. SAR studies
revealed that the position of the oxybutylimidazole moiety OR2 strongly influenced HO-1
inhibition. The best results were obtained when this moiety was in the 2- position of the benzamide
ring with respect to the 3- and, above all, the 4-substituted compounds (6a vs 6b and 6c, or 6d and
6e vs 6l). Indeed, hybrids 6f and 6g were the most potent compounds in this series. The only
exception to this rule was given by derivative 6m which, despite being a 4-substituted compound,
inhibited HO-1 in submicromolar concentration. Regarding the effect of the substituent R1,
generally unsubstituted compounds were less potent than those carrying a substituent; among
selected substituents, bromine generally gave the best contribution (6f, 6i, and 6m). Taken together,
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these results confirm that these compounds behave as imidazole-based HO-1 inhibitors, although
the different chemical structure with respect to the parent compounds 1 and 2.
Table 1. Inhibition potency of hybrids 6a–n towards HO-1.
HO-1 IC50 (µM) ± SDa
28.02 ± 0.8
38.54 ± 1.2
66.23 ± 2
Compound
6a
6b
6c
R1
H
H
H
OR2 position
2
3
4
4-I
5-I
2
2
2
2
2
3
3
3
4
4
4
—
—
—
—
—
15.13 ± 0.2
38.87 ± 1.1
0.95 ± 0.01
0.95 ± 0.02
61.56 ± 2.1
28.65 ± 1.8
15.14 ± 0.9
4.03 ± 0.1
74.89 ± 2.1
0.92 ± 0.01
56.61 ± 2.3
> 100
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
5
IM
1
4-Br
5-CH3
4-OCH3
5-Br
4-OCH3
4-NO2
3-I
3-Br
3-OCH3
—
—
—
—
—
> 100
2.10 ± 0.3
1.00 ± 0.05
6 ± 1c
2
Azalanstatb
aEach value is the mean ± SD of three determinations; bFigure S1; cValue from reference [29]
Subsequently, we evaluated if compounds 6a–n were able to reduce the viability of CML cells. We
used both IM-resistant (K562R) and IM-sensitive (K562S) cell lines. The K562R cells were
selected because they did not have the two most common mutations in BCR–ABL gene, i.e. T315I
and P-binding phosphate loop (P-loop) mutations [37]. In these cells, resistance mechanism is
mainly due to BCR-ABL gene amplification and HO-1 over-expression, as already experimentally
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reported by Tibullo et al. [23]. Results, reported in Table 2, were determined by a XTT assay as
described in experimental section and are expressed as IC50. IM and HO-1 inhibitors 1 and 2 were
used as reference compounds.
Table 2: Viability of resistant and sensitive CML cell lines in the presence of hybrid compounds
6a–n. K562R and K562S were incubated for 24 h with various concentrations of compounds 6a–n
and cell viability was assessed using the XTT assay kit.
IC50 (µΜ)a
Compounds
K562R
K562S
83.42 ± 2.2
63.40 ± 1.8
99.38 ± 2.3
51.82 ± 1.2
81.70 ± 1.7
44.58 ± 2.1
48.57 ± 1.4
46.56 ± 0.9
19.14 ± 0.3
18.74 ± 0.7
47.62 ± 1.1
52.39 ± 1.5
31.76 ± 0.8
27.10 ± 0.7
81.70 ± 1.7
84.38 ± 1.3
98.82 ± 1.8
27.33 ± 1.2
25.56 ± 0.9
47.50 ± 1.3
74.57 ± 2.1
49.89 ± 1.2
57.41 ± 2.2
66.50 ± 0.9
66.09 ± 0.7
59.48 ± 1.0
58.87 ± 1.3
6.50 ± 0.2
10.11 ± 0.3
25.02 ± 0.8
32.47 ± 0.4
12.00 ± 0.2
29.51 ± 1.0
68.30 ± 0.9
69.09 ± 0.6
1.00 ± 0.09
—
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
1
2
IM
IM + 1
IM + 2
—
aEach value is the mean ± SD of three determinations.
With regard to resistant cells, many hybrids were more effective with respect to single molecules,
IM or imidazole-based HO-1 inhibitors 1 and 2 (IC50 = 98, 81, 84 µΜ, respectively). Specifically,
some hybrids were 2 to 5-fold more potent than IM (IC50 = 98 µΜ), particularly 6i, 6j, and 6n (IC50
= 19, 18, and 27, respectively). Notably, 6i and 6j were slightly more effective also with respect to
the IM and 1 or 2 co-administration. This result confirms that the combination of inhibitory
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properties against HO-1 and BCR-ABL in the same hybrid molecule has the same effect of IM and
HO-1 inhibitors co-administration [24] with the advantage of avoiding the potential side-effects of a
combination therapy. Due to HO-1 over-expression in K562R cells, it is possible to affirm that the
introduction of a moiety that inhibits HO-1 in the structure of IM to give hybrids 6a–n is critical to
reduce the K562R cells viability, and contributes positively in overcoming IM-resistance. However,
since the most active compounds are not those with the highest inhibitory potency towards HO-1, a
contribution to the antiproliferative activity may be due to the IM-portion of the molecule or to off-
target effects.
On the contrary, all compounds were less active than IM in sensitive cells. This result may be
related to HO-1 activity too; indeed, inhibition of HO-1 by hybrids 6a–n does not give a positive
contribution since this enzyme is not over-expressed in K562S cells [23]. Compounds 6i and 6j
were the most active also in these cells, particularly 6i which showed a potency close to IM (6i IC50
= 6 µΜ, ΙΜ IC50 1 µΜ). Consequently, 6i may be considered as a lead compound for further
development also for the treatment of IM-sensitive cells.
Since our attention was mainly devoted to the involvement of HO-1 in the phenomena of
chemoresistance, we focused our attention on K562R resistant cells and determined the inhibitory
activity of compounds 6–n towards BCR-ABL protein. For this purpose, we measured the amount
of phosphorylated CrKL (pCrKL) by immunoblot analysis (Figure S2, SI). CrKL is a BCR-ABL
substrate whose phosphorylation reflects the level of TK activity, and which is commonly used to
test BCR-ABL activity in vivo [38]. Results are summarized in Figure 2 and are expressed as
pCrKL/CrKL ratio in K562R cells treated with 10 µM of IM or 10 µM of tested compounds,
compared to the control. We selected 10 µM dose since this was, on average, the concentration at
which almost all hybrids were able to inhibit HO-1. IM at the concentration of 1 µM (data not
shown) and 10 µM in resistant cells was not able to inhibit phosphorylation of CrkL, whereas in
sensitive cells it was effective already at 1 µM (Table 2). Of interest, many hybrids, particularly 6f,
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6i, 6j, 6k, 6l, 6m, and 6n were more effective than IM itself in inhibiting the phosphorylation of
CrkL, and consequently, in reducing TK activity of BCR-ABL protein. Differently to HO-1,
hybrids with the highest TK inhibitory ability carry the oxybutylimidazole moiety at the 3- or 4-
position of the benzamide ring, instead of the 2-position. This is obviously due to different SARs
for the two different targets. The noteworthy result is that some hybrid compounds were able to
interact with both HO-1 and BCR-ABL at micromolar concentrations, confirming that it is possible
to inhibit both targets with one single molecule. Simultaneous HO-1 and BCR-ABL inhibition may
have therapeutic application when IM-resistance is caused by BCR-ABL gene amplification, such
as in this K562R cells, with downstream overexpression of BCR-ABL and HO-1 proteins. This is
particularly true in CP-CML patients clinically treated with increasing doses of IM, as they
consequently suffer more severe side effects. HO-1/TK hybrids may represent an alternative to
higher IM doses, especially if their action is selective towards leukemic cells.
Figure 2. pCrKL expression in K562R cells treated with 10 µM IM and 10 µM hybrid compounds
6a–n. Protein detection was carried out using immunoblot analysis and densitometric evaluation of
protein were obtained from four experiments performed in triplicate. Data, expressed as
pCrKL/CrKL ratio, represent the mean ± SD. Significant vs untreated control (CTRL): *p<0.005.
Lymphocytes collected from healthy volunteers were selected as an example of non-cancerous
cells. The viability of these lymphocytes was tested in the presence of the most interesting
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compounds (6i, 6j, 6k, 6m, and 6n). Since tested compounds, at 10 µM, did not alter lymphocytes
viability, they can be considered non-toxic for healthy cells (Figure S17, SI).
Subsequently, we studied the mechanism of cell death. As we previously reported [24], HO-1
inhibitors 1 and 2 plus IM treatment lead to cellular apoptosis. This data, according to Datta et al.
[39], demonstrated that down-regulation of HO-1 expression induced cellular apoptosis. In order to
evaluate whether the cellular death was due to an apoptotic cell death, we determined the apoptosis
profile of the most active compounds (6i, 6j, 6k, 6m, and 6n) using a cytofluorimetric assay.
Results are shown in Figures 3 and 4. All the tested compounds were able to increase the number of
apoptotic dead cells to a greater extent than IM. In particular, compounds 6i, 6j, and 6n highly
reduced the viability of resistant cells increasing the number of cells in late and early apoptosis.
Among them, hybrids 6i and 6j were the most potent, then they were selected for more in-depth
biological studies.
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Figure 3. Representative apoptotic profile of K562R cells untreated and treated with 10 µM IM and
10 µM 6i, 6j, 6k, 6m, 6n hybrid compounds. Each profile shows the percentage of live, apoptotic
and dead cells.
Figure 4. Percentage of apoptotic K562R cells untreated and treated with 10 µM IM and 10 µM 6i,
6j, 6k, 6m, 6n hybrid compounds. Values represent the means ± SD of four experiments performed
in triplicate. Significant vs untreated control (CTRL): *p<0.005.
In particular, we desired to determine whether HO-1 inhibition was maintained in intact cells too.
At this purpose, we measured HO-1 enzymatic activity in K562R cells treated with 10 µM IM, 1, 2
imidazole compounds, and 10 µM hybrids 6i and 6j, with respect to untreated cells. Results,
described in Figure 5, show that both compounds reduced the HO-1 activity in the lysates obtained
after treatment of intact cells, thus behaving as HO-1 inhibitors. Therefore, as parent compounds 1
and 2, they are effective not only in broken-cell preparations but also in intact cells, suggesting that
they are able to cross the cellular membrane and might have a potential in vivo application.
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Figure 5. HO-1 activity in K562R cell lysate obtained after 24 h cell treatment in absence and in
presence of 10 µM IM, 1 and 2 imidazole compounds, 6i and 6j hybrid compounds. HO-1 activity
was determined by measuring the bilirubin formation using the difference in absorbance at 464 to
530 nm as described in the experimental section. Values represent the means ± SD of four
experiments performed in triplicate. Significant vs untreated control cells: *p<0.001.
It is generally recognized that, in some specific cases, HO-1 inhibition may induce HO-1 expression
in vivo, giving rise to opposite effects to the expected results. This is particularly true for
metalloporphyrins (Mps), which are HO-1 inhibitors structurally related to heme and which
consequently act as competitive HO-1 inhibitors [40]. HO-1 induction, together with other side-
effects of Mps, limit their clinical use. Therefore, in the present paper, in order to exclude a
potential indirect HO-1 induction, we measured HO-1 and HO-2 mRNA levels in K562R cells
treated and untreated with IM, 1 and 2 reference compounds, and with hybrid compounds 6i and 6j
(Figure 6). Results revealed that compounds 6i and 6j markedly decreased mRNA levels of HO-1,
whereas HO-2 mRNA levels were not modified. In contrast, IM gave opposite effects since
enhanced HO-1 mRNA expression. These results confirmed what had been observed in our
previous paper about the combination of IM and the parent HO-1 inhibitors, 1 and 2 [24]. Thus,
compounds 6i and 6j, contrarily to Mps and other HO-1 inhibitors, do not behave as HO-1 inducers
but they are both able to down-regulate HO-1 expression and to inhibit HO-1 activity.
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Figure 6. Effect of 10 µM IM, 1 and 2 imidazole compounds, 10 µM 6i and 10 µM 6j on mRNA
expression analyzed by Real-Time PCR. Values represent the means ± SD of four experiments
performed in triplicate. Significant vs untreated control cells: *p< 0.005.
Furthermore, since the apoptotic effect of IM is mediated by ROS formation in IM-sensitive cells
[23], here we measured the intracellular oxidants levels in K562R cells untreated and treated with
IM, 1, 2 imidazole compounds, and with compounds 6i and 6j. Results in Figure 7 clearly show that
IM was not effective, and treatment with compounds 1 and 2 slightly increased ROS levels. On the
contrary, treatment with 6i and 6j give the highest effect. This effect may be related to the HO-1
down-regulation observed for compounds 6i and 6j (Figure 6). Specifically, low levels of HO-1
protein and HO-1 activity decrease the protection of tumor cells against oxidant species. Then
compounds 6i or 6j treatment leads to an increase of ROS amount and of the percentage of
apoptotic cells (Figure 3 and 4). Therefore, the simultaneous inhibition/down-regulation of HO-1
and inhibition of TK may increase ROS and the elevated ROS levels may facilitate tumor killing.
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Figure 7. Intracellular oxidants in K562R cells untreated and treated with 10 µM IM, 1 and 2
imidazole compounds or 10 µM 6i and 6j. The fluorescence, corresponding to the oxidized radical
species 2',7'-dichlorofluorescein (DCF), was spectrofluorometrically monitored (excitation, λ = 488
nm; emission, λ = 525 nm). Values, expressed as fluorescence intensity (F.I.) of oxidized radical
species 2',7'-dichlorofluorescein (DCF)/mg protein, represent the means ± SD of four experiments
performed in triplicate. Significant vs untreated control cells: *p< 0.05.
2.3. Docking studies
In order to study the interaction of the new set of compounds 6a–n with HO-1 and with BCR-ABL
kinase, a molecular docking study was performed. We investigated the binding modes of 6a–n in
comparison
with
(2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((5-
trifluoromethylpyridin-2-yl)thio)methyl]-1,3-dioxolane (QC-80, Table 3, Figure S1, SI) in HO-1
and IM in BCR-ABL kinase. The X-ray crystal structures of the co-crystal HO-1/QC-80 (PDB code
3HOK) [30] and of the crystal structure of the BCR-ABL kinase domain in complex with IM (PDB
code 1IEP) [41] were used as the protein structures. Docking was performed using AutoDock [42].
To validate the docking model, we docked molecules QC-80 and IM to the original binding sites of
the respective proteins, to compare their experimental and calculated binding potencies.
Particularly, we used the same validated docking procedure as previously published by our research
group for the validation of the HO-1 model [43–45]. On the other hand, for the validation of the
BCR-ABL kinase model, we compared our results with the ones of Lin et al [46]. Results reported
in Tables 3 and 4 showed that our calculated binding potencies are in good agreement with the
experimental ones [30,47], and that the simulated complexes of QC-80 and IM show high
correspondence to the corresponding crystallographic structure with a RMSD value of 0.75 Å and
0.76 Å respectively. Once validated the model, two different set of molecules, representative of the
different potencies at the two different proteins, were chosen: 6a, 6f, 6g, 6j, 6l, and 6m were used in
the docking experiment for HO-1 whereas 6a, 6g, 6i, 6j, and 6l were docked into the binding site of
17

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BCR-ABL kinase. For molecules 6l and 6m, best binding energies for HO-1 were obtained after a
molecular dynamics simulation. The docked poses and the 2D-interaction inside HO-1 and BCR-
ABL are shown in Figures 8 and 9, and in Figures S3–S15 in the SI.
The results of the docking calculation and the docking poses for HO-1 are reported in Table 3 and
in Figures 8 and S3–S9 (SI), respectively. The calculated binding potencies are in good agreement
with the experimental values in the HO-1 inhibition assay, as it is possible to see from a comparison
between the calculated Ki and the experimental IC50 (Figure S16 A). In all of the docked
compounds, the iron(II) of the heme in HO-1 is correctly coordinated by the nitrogen atom of the
imidazole ring of the studied compounds in the eastern pocket. By means of this coordination
binding, iron(II) 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. On the other hand, the position of the
oxylbutylimidazole (OR2) and the R1 group greatly influence the potency and the dual activity of
molecules. Looking at the poses of the docked compounds (Figure 8, left), we can see that when the
oxylbutylimidazole group is in 2 position, the molecules (6a, 6f, and 6g) form a series of
interactions with the internal hydrophobic pocket of the protein (Figure 8, right), particularly in the
western region [30], and the flexible linker capped with imidazole points to the heme group, making
possible the important interaction with the iron(II). On the other hand, when the oxylbutylimidazole
group is in 4 position (6l and 6m), the same interactions are not allowed due to steric hindrance in
the positioning of the molecules inside the internal pockets. However, the potent compound 6m
performs stabilizing interactions on the surface of the protein. In particular, the bromine atom is
easily located into a pocket formed by Phe37, Leu147 and Ile150 and a hydrogen bond is formed
with Gln38. In the case of the less potent compound 6l, its bigger iodine atom cannot be located
inside the same pocket and the molecule, flipping itself, does not get this set of interactions, that are
the probable cause of the potency of the bromine derivative. When the oxylbutylimidazole group is
in 3-position (6j), the “IM-like portion” of the molecule is pushed to the external portion of the
18

ACCEPTED MANUSCRIPT
western pocket, but the major part of the molecule is still inside the pocket. Regards the R1
substituent, small substituents are easily tolerated and can increase the activity when they are
localized in 4- or 5-position and the oxylbutylimidazole group is in 2-position. Particularly, these
substituents are located into an internal hydrophobic pocket; for example, in molecules 6f and 6g,
two of the most active compounds, the methyl and the bromine group are easily accommodated in a
pocket formed by Val50, Arg136, Phe166 and Ser14, and in the case of compound 6f, the bromine
atom can interact through a halogen bond interaction with Asn210.
Table 3. Docking results for the studied molecules in 3HOK.
Compound
IC50 (µM) ∆GB (kcal/mol) Calc. Ki (µM)
QC-80a
6a
2.1b
28
–7.55
–6.39
–7.89
–8.50
–6.70
–5.89
–7.58
2.9
20.59
1.63
0.58
12.2
47.9
2.76
0.95
0.95
15
74
0.9
6f
6g
6j
6l
6m
aFigure S1; bValue from reference [30]
Figure 8. Left: Docked poses inside HO-1 (3HOK) of: 6a (light pink), 6f (light blue), 6g (dark
orange), 6j (light orange), 6l (dark green), and 6m (light gray). Right: Binding regions of the
hydrophobic pocket inside the HO-1 with the QC-80 ligand (PDB ID: 3HOK).
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Regarding the activity towards BCR-ABL, the calculated binding energies and the poses of the
studied compounds are shown in Table 4 and in Figures 9 and S10–S15 (SI), respectively. All the
studied molecules are able to interact with the protein in a similar way to that of reference
compound IM. Results of calculated binding energies showed in Table 4 point out the high
correlation between the estimated free energies of binding and percentage of inhibitory efficiency
values of our compounds. Particularly, compounds 6i, 6j, and 6l resulted better than IM in reducing
TK activity (Figure 9), rather than compounds 6a and 6g that possess a lower activity than IM
(Figure S16 B). The N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl) moiety is
always located in the same pocket formed by Ile313, Tyr253, Leu248, Phe317, Met318, Leu370,
Val256 and Thr315. Conversely, the oxylbutylimidazole group is pointing to the external surface of
the protein in all docked compounds and can be oriented in different ways in relation to the position
of insertion on the benzamide ring. Judging by the docking results, and looking at the biological
results, we can conclude that the oxylbutylimidazole moiety can be positioned in 2, 3 and 4 position
without any influence in the positioning of the central core of the “IM-like portion” of the molecule,
that in all case can interact in the right way with the protein. Nevertheless, some important
differences can be obtained with the R1 substituents. For example, the methyl group in 5-position of
6g decreases the activity of the molecule for a steric clash with Leu298 and Ile293, whereas the
bromine in 5-position of 6i is well accommodated in a near pocket formed by Leu354, Ile293,
Val289 and Phe359 [48].
Table 4. Docking results for the studied molecules in 1IEP
Compound
∆GB (kcal/mol) Calc. Ki (µM)
–8.99
–7.15
–7.93
–10.87
–10.67
–10.21
0.25
5.70
1.53
0.01
0.01
0.03
IM
6a
6g
6i
6j
6l
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Figure 9. Docked poses inside BCR-ABL (1IEP) of: 6a (light orange), 6g (light blue), 6i (dark
green), 6j (dark orange), and 6l (light pink).
2.4. ADME and toxicity risk assessment
To evaluate if the designed compounds could display a good pharmacokinetic profile and no
adverse side effects (ADME-Toxicity), we conducted an in silico study for the two selected
compounds 6i and 6j. The results are reported in Tables 5 and 6. The in silico ADME results (Table
5) clearly show that compounds 6i and 6j should exhibit a good oral availability (human intestinal
absorption, HIA >95%) and a discrete Caco-2 cell permeability (35.36 nm s–1 for 6i and 48.10 nm
s–1 for 6j) but with a strong plasma protein binding (PPB >95%). Interestingly, both compounds 6i
and 6j are predicted to barely permeate the blood-brain barrier with an in vivo BBB penetration
(Cbrain/Cblood) of 0.05 and 0.11, respectively. Moreover, our new molecules resulted non-mutagen,
non-tumorigenic, non-irritant and without negative effects on the reproductive system (Table 6).
Finally, of the two compounds, only 6j has a positive value of drug-likeness, establishing that the
molecule predominantly contains common fragments that are present in commercial drugs; both
compounds have a positive value of drug-score, encompassing the contributions of partition
coefficient, solubility, molecular weight, drug-likeness and the four toxicity risks.
Table 5. Selected in silico ADME profiling for molecules 6i and 6j.a
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Absorption
Distribution
Compound HIA (%) In vitro Caco-2 cell
In
vitro In vivo BBB penetration
permeability (nm s–1)
PPB (%)
100.00
95.88
(Cbrain/Cblood
)
96.90
95.79
35.36
48.10
0.05
0.11
6i
6j
aThe properties related to ADME were predicted using PreADMET web-based application (http://preadmet.bmdrc.kr).
Table 6. In silico toxicity parameters for molecules 6i and 6j.a
Drug-
Likeness
–1.51
Compound Mutagenic Tumorigenic Reproductive Effects Irritant
Drug-Score
none
none
none
none
none
none
none
none
0.16
0.30
6i
6j
0.34
aThe properties related to toxicity were predicted using DataWarrior software [49].
3. Conclusion
In conclusion, in the present study, we discovered novel HO-1/TKI hybrid compounds 6a–n able to
inhibit both the enzymatic activity of HO-1 and BCR-ABL. The new compounds target
simultaneously the pathway responsible for the development of CML disease and one of the
pathways accountable for the onset of IM-resistance. To the best of our knowledge, it is the first
time that the structure of a HO-1 inhibitor was combined into a hybrid compound. Binding mode of
these hybrids with HO-1 is very similar to that observed for azole-based HO-1 inhibitors described
so far. Specifically, the position of the oxybutylimidazole moiety is critical for HO-1 interaction,
since 2-substituted compounds show better poses within the HO-1 protein, according to
experimental values of HO-1 inhibition. Also interactions to BCR-ABL protein are similar to those
observed for the reference compound IM. Many of these hybrids reduce cell viability of CML
resistant cells to a greater extend with respect to IM. The most interesting compounds 6i and 6j
enhanced cellular death by an apoptotic mechanism and by an increase of the ROS levels, and are
able to cross cellular membrane since they inhibit HO-1 also in intact cells. In addition, they are
able to reduce mRNA HO-1 expression, differently to Mps based HO-1 inhibitors. These results
confirm that HO-1 inhibition may help to overcome resistance in CML and suggest further
application of HO-1 inhibitors in other cancers in which HO-1 overexpression is involved [50].
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Further studies, supported by the good predicted values of drug-score observed for 6i and 6j, may
be designed to validate the potential clinical application of such hybrid compounds to overcome
IM-resistance in vivo.
4. Experimental section
4.1. Chemistry
4.1.1. General details
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 Mod. 1108 apparatus. The IR spectra were
recorded in KBr disks or Neat, on a Perkin Elmer 1600 series FT-IR spectrometer. 1H NMR spectra
were determined with Varian Inova Unity (200 MHz) spectrometer and 13C NMR spectra were
determined with Varian Inova Unity (500 MHz) spectrometer in DMSO-d6 solution. Chemical
shifts are in δ
values (ppm) using tetramethylsilane and DMSO as the internal standard for 1H NMR
and 13C NMR spectra, respectively; 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). ESI-
HRMS were determined with a Thermo Fischer Scientific LTQ Orbitrap XL. Reactions were
monitored by thin-layer chromatography (TLC) on Merck plates (Kieselgel 60 F254) and spots were
visualized under the UV light (λ = 254 and 366 nm) or in an iodine chamber. The purity of all final
compounds was 95% or higher. Preparative column chromatography was performed using Merck
silica-gel 60 (230–400 mesh) or Biotage® SNAP cartridge KP-Sil. Microwave 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.
4.1.2. General procedure for the synthesis of OH-1/TKI hybrid final compounds 6a–n.
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A solution of methyl imidazolylbutoxybenzoates 4a–n (1 mmol) and 5 (0.8 mmol) in 10 mL of
anhydrous THF, was added with tBuOK (6 mmol) under nitrogen atmosphere. The reaction mixture
was stirred at room temperature for 1/2 h, quenched by adding brine, and extracted by ethyl acetate
(3 × 70 mL). The combined extracts were washed with water, dried on anhydrous sodium sulfate,
and evaporated to obtain a residue which was purified by flash column chromatography on silica
gel using mixtures of ethyl acetate/methanol as eluent.
4.1.2.1.
2-[4-(1H-Imidazol-1-yl)butoxy]-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6a).
Pale yellow solid; mp 85.5–86.1 °C; yield 30%. IR (KBr) cm–1 3566, 3351, 2942, 1654, 1578, 1558,
1450, 1418, 1294. 1H NMR (DMSO-d6) δ 10.09 (s, 1H), 9.28 (d, J = 1.6 Hz, 1H), 9.04 (s, 1H), 8.69
(dd, J = 4.6, 1.4 Hz, 1H), 8.52–8.44 (m, 2H), 8.07 (d, J = 1.6 Hz, 1H), 7.64 (dd, J = 7.6, 1.6 Hz,
1H), 7.54–7.42 (m, 4H), 7.34 (dd, J = 8.0, 1.8 Hz, 1H), 7.21-7.02 (m, 4H), 6.81 (br s, 1H), 4.10 (t, J
13
= 6.2 Hz, 2H), 3.94 (t, J = 6.6 Hz, 2H), 2.22 (s, 3H), 1.88–1.64 (m, 4H). C NMR (DMSO-d6) δ
164.16, 161.57, 161.10, 159.39, 155.75, 151.32, 148.15, 137.91, 137.08, 137.01, 134.35, 132.17,
131.86, 130.18, 129.70, 128.29, 127.40, 125.15, 123.72, 120.52, 119.00, 116.14, 115.72, 112.88,
107.52, 67.69, 45.44, 27.18, 25.59, 17.63. Anal. Calcd. for C30H29N7O2: C, 69.35; H, 5.63; N,
+
18.87. Found: C, 69.04; H, 5.78; N, 18.99. HRMS (ESI) m/z (M+H)+ calcd. for C30H30N7O2 :
520.2461; found: 520.2459.
4.1.2.2.
3-[4-(1H-Imidazol-1-yl)butoxy]-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6b).
Yellow solid; mp 85.6–86.3 °C; yield 30%. IR (KBr) cm–1 3568, 3274, 2942, 1653, 1581, 1557,
1448, 1417, 1289.1H NMR (DMSO-d6) δ 10.18 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 9.01 (s, 1H), 8.68
(dd, J = 4.6, 1.4 Hz, 1H), 8.53–8.45 (m, 12H), 8.08 (dd, J = 2.0 Hz, 1H), 7.65 (s, 1H), 7.55–7.38
(m, 6H), 7.23–7.11 (m, 3H), 6.89 (s, 1H), 4.07–4.00 (m, 4H), 2.22 (s, 3H), 1.92-1.81 (m, 2H), 1.73-
24

ACCEPTED MANUSCRIPT
1.63 (m, 2H). 13C NMR (DMSO-d6) δ 164.99, 161.55, 161.1, 159.42, 158.41, 151.32, 148.1,
137.75, 137.20, 137.03, 136.40, 134.35, 132.16, 129.97, 129.47, 128.34, 127.61, 123.72, 119.80,
119.19, 117.51, 117.22, 116.75, 113.45, 107.48, 67.09, 45.58, 27.34, 25.70, 17.60. Anal. Calcd. for
C30H29N7O2: C, 69.35; H, 5.63; N, 18.87. Found: C, 69.19; H, 5.88; N, 19.12. HRMS (ESI) m/z
+
(M+H)+ calcd. for C30H30N7O2 : 520.2461; found: 520.2458.
4.1.2.3.
4-[4-(1H-Imidazol-1-yl)butoxy]-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6c).
Pale yellow solid; mp 150.5–151.4 °C; yield 23%. IR (KBr) cm–1 3499, 3262, 2924, 1658, 1605,
1580, 1504, 1449, 1247. 1H NMR (DMSO-d6) δ 10.06 (s, 1H), 9.28 (d, J = 1.6 Hz, 1H), 9.00 (s,
1H), 8.69 (dd, J = 4.6, 1.4 Hz, 1H), 8.52-8.45 (m, 2H), 8.07 (d, J = 1.8 Hz, 1H), 7.97–7.93 (m, 2H),
7.65 (br s, 1H), 7.55–7.42 (m, 3H), 7.21–7.17 (m, 2H), 7.06–7.01 (m, 2H), 6.90 (br s, 1H), 4.07–
4.00 (m, 4H), 2.22 (s, 3H), 1.91–1.87 (m, 2H), 1.84–1.63 (m, 2H). 13C NMR (DMSO-d6) δ 164.65,
161.56, 161.16, 161.08, 159.42, 151.33, 148.15, 137.71, 137.30, 137.20, 134.37, 132.18, 129.93,
129.48, 128.36, 127.36, 126.98, 123.73, 119.19, 117.20, 116.71, 113.98, 107.44, 67.15, 45.57,
27.27, 25.63, 17.59. Anal. Calcd. for C30H29N7O2: C, 69.35; H, 5.63; N, 18.87. Found: C, 69.09; H,
5.85; N, 19.15.
+
HRMS (ESI) m/z (M+H)+ calcd. for C30H30N7O2 : 520.2461; found: 520.2457.
4.1.2.4.
2-[4-(1H-Imidazol-1-yl)butoxy]-4-iodo-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6d).
Pale yellow solid; mp 168.0–169.4 °C; yield 40%. IR (KBr) cm–1 3587, 3343, 2944, 1648, 1600,
1580, 1533, 1443, 1414. 1H NMR (DMSO-d6) δ 10.06 (s, 1H), 9.27 (d, J = 1.6 Hz, 1H), 9.04 (s,
1H), 8.68 (dd, J = 4.8, 1.6 Hz, 1H), 8.51–8.44 (m, 2H), 8.04 (d, J = 1.8 Hz, 1H), 7.54–7.29 (m, 7H),
7.20–7.16 (m, 1H), 7.02 (s, 1H), 6.81 (s, 1H), 4.11 (t, J = 5.8 Hz, 2H), 3.94 (t, J = 6.8 Hz, 2H), 2.22
(s, 3H), 1.85-1.64 (m, 4H). 13C NMR (DMSO-d6) δ 163.57, 161.56, 161.07, 159.38, 156.12, 151.32,
25

ACCEPTED MANUSCRIPT
148.13, 137.92, 137.08, 136.83, 135.34, 132.15, 131.14, 130.20, 129.48, 128.28, 127.50, 124.98,
123.72, 121.58, 119.00, 116.11, 115.69, 107.54, 98.10, 68.09, 45.32 27.05, 25.43, 17.63. Anal.
Calcd. for C30H28IN7O2: C, 55.82; H, 4.37; N, 15.19. Found: C, 56.13; H, 4.15; N, 15.30. HRMS
+
(ESI) m/z (M+H)+ calcd. for C30H29IN7O2 : 646.1427; found: 646.1425.
4.1.2.5.
2-[4-(1H-Imidazol-1-yl)butoxy]-5-iodo-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6e).
Yellow-orange solid; mp 159.1–162.4 °C; yield 23%. IR (KBr) cm–1 3413, 3356, 2925, 1647, 1604,
1578, 1531, 1452, 1417. 1H NMR (DMSO-d6) δ 10.11 (s, 1H), 9.27 (d, J = 1.8 Hz, 1H), 9.03 (s,
1H), 8.68 (dd, J = 4.8, 1.6 Hz, 1H), 8.51–8.42 (m, 2H), 8.03 (d, J = 1.8 Hz, 1H), 7.83 (d, J = 2.2 Hz,
1H), 7.76 (dd, J = 8.6, 2.2 Hz, 1H), 7.54–7.42 (m, 3H), 7.32 (dd, J = 8.0, 1.8 Hz, 1H), 7.18 (d, J =
8.4 Hz, 1H), 7.01–6.96 (m, 2H), 6.80 (br s, 1H), 4.06 (t, J = 6.0 Hz, 2H), 3.92 (t, J = 6.8 Hz, 2H),
2.21 (s, 3H), 1.84–1.60 (m, 4H). ). 13C NMR (DMSO-d6) δ 162.79, 161.57, 161.08, 159.41, 155.58,
151.34, 148.17, 139.89, 137.92, 137.35, 137.07, 136.78, 134.35, 132.16, 130.20, 128.27, 127.95,
127.61, 123.73, 119.01, 116.22, 115.73, 107.56, 82.94, 67.95, 45.43, 40.09, 27.10, 25.44, 17.64.
Anal. Calcd. for C30H28IN7O2: C, 55.82; H, 4.37; N, 15.19. Found: C, 55.51; H, 4.66; N, 15.37.
HRMS (ESI) m/z (M+H)+ calcd. for C30H29IN7O2 : 646.1427; found: 646.1426.
+
4.1.2.6.
4-Bromo-2-[4-(1H-imidazol-1-yl)butoxy]-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6f).
Yellow solid; mp 177.4–179.5 °C; yield 33%. IR (KBr) cm-1 3425, 3348, 2953, 1662, 1604, 1582,
1526, 1452, 1413, 1233. 1H NMR (DMSO-d6) δ 10.06 (s, 1H), 9.27 (d, J = 2.2 Hz, 1H), 9.03 (s,
1H), 8.68 (dd, J = 4.8, 1.4 Hz, 1H), 8.51–8.41 (m, 2H), 8.04 (d, J = 1.6 Hz, 1H), 7.57–7.16 (m, 8H),
7.02 (s, 1H), 6.81 (s, 1H), 4.12 (t, J = 6.2 Hz, 2H), 3.93 (t, J = 7.0 Hz, 2H), 2.21 (s, 3H), 1.85–1.62
13
(m, 4H). C NMR (DMSO-d6)
δ 163.41, 161.58, 161.09, 159.40, 156.50, 151.34, 148.15, 137.94,
136.83, 134.36, 132.17, 131.20, 130.22, 128.30, 127.55, 124.66, 124.54, 123.74, 123.45, 119.03,
26

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116.14, 115.98, 115.73, 107.56, 68.20, 45.34, 27.07, 25.39, 17.64. Anal. Calcd. for C30H28BrN7O2:
C, 60.21; H, 4.72; N, 16.38. Found: C, 60.48; H, 4.49; N, 16.61. HRMS (ESI) m/z (M+H)+ calcd.
+
for C30H29BrN7O2 : 598.1566; found: 598.1561.
4.1.2.7.
2-[4-(1H-Imidazol-1-yl)butoxy]-5-methyl-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6g).
Yellow-orange solid; mp 65.5–67.9 °C; yield 28%. IR (KBr) cm–1 3445, 3349, 2944, 1662, 1578,
1557, 1448, 1418, 1236, 802. 1H NMR (DMSO-d6) δ 10.06 (s, 1H), 9.27 (br s, 1H), 9.04 (s, 1H),
8.69–8.67 (m, 1H), 8.51–8.04 (m, 2H), 8.04 (br s, 1H), 7.56–7.16 (m, 7H), 7.05–7.01 (m, 2H), 6.83
(br s, 1H), 4.09–3.91 (m, 4H), 2.28 (s, 3H), 2.21 (s, 3H), 1.87–1.66 (m, 4H). 13C NMR (DMSO-d6)
δ 164.15, 161.59, 161.11, 159.41, 153.74, 151.34, 148.15, 137.91, 137.07, 137.00, 134.36, 132.23,
132.19, 130.20, 130.03, 129.42, 128.19, 127.43, 124.66, 123.75, 119.07, 116.19, 115.77, 113.03,
107.54, 67.88, 45.50, 27.19, 25.64, 19.92, 17.64. Anal. Calcd. for C31H31N7O2: C, 69.77; H, 5.86;
+
N, 18.37. Found: C, 69.45; H, 5.99; N, 18.67. HRMS (ESI) m/z (M+H)+ calcd. for C31H32N7O2 :
534.2617; found: 534.2615.
4.1.2.8. 2-[4-(1H-Imidazol-1-yl)butoxy]-4-methoxy-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6h).
Yellow-orange solid; mp 164.7–165.5 °C; yield 41%. IR (KBr) cm–1 3436, 3355, 2944, 1662, 1602,
1
1581, 1525, 1455, 1412, 1249. H NMR (DMSO-d6) δ 9.91 (s, 1H), 9.28 (d, J = 1.4 Hz, 1H), 9.04
(s, 1H), 8.69 (dd, J = 4.8, 1.4 Hz, 1H), 8.52–8.46 (m, 2H), 8.05 (br s, 1H), 7.75 (d, J = 9.2 Hz, 1H),
7.56–7.43 (m, 3H), 7.33–7,28 (m, 1H), 7.19 (d, J = 8.2 Hz, 1H),7.05 (br s, 1H), 6.80 (br s, 1H),
6.68–6.65 (m, 2H), 4.14 (t, J = 5.6 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 3.82 (s, 3H), 2.22 (s, 3H),
1.85–1.73 (m, 4H). 13C NMR (DMSO-d6) δ 163.24, 162.78, 161.58, 161.10, 159.41, 157.51,
151.34, 148.14, 137.96, 136.97, 134.37, 132.18, 131.92, 130.25, 128.33, 127.22, 123.74, 119.05,
116.29, 116.04, 115.65, 107.55, 105.90, 99.33, 68.02, 55.54, 45.42, 27.18, 25.55, 17.62. Anal.
27

ACCEPTED MANUSCRIPT
Calcd. for C31H31N7O3: C, 67.74; H, 5.69; N, 17.84. Found: C, 67.44; H, 5.88; N, 18.03. HRMS
+
(ESI) m/z (M+H)+ calcd. for C31H32N7O3 : 550.2567; found: 550.2564.
4.1.2.9.
5-Bromo-3-[4-(1H-imidazol-1-yl)butoxy]-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6i).
Yellow solid; mp 142.0–157.8 °C; yield 45%. IR (KBr) cm–1 3446, 3301, 2932, 1648, 1583, 1527,
1438, 1400, 1284, 799. 1H NMR (DMSO-d6) δ 10.26 (s, 1H), 9.28 (d, J = 1.6 Hz, 1H), 9.00 (s, 1H),
8.68 (dd, J = 4.8, 1.6 Hz, 1H), 8.53–8.44 (m, 2H), 8.06 (d, J = 2.0 Hz, 1H), 7.70–7.64 (m, 2H),
7.55–7.43 (m, 4H), 7.37–7,35 (m, 1H), 7.23–7.19 (m, 2H), 6.80 (br s, 1H), 4.11–3.97 (m, 4H), 2.22
13
(s, 3H), 1.90–1.79 (m, 2H), 1.72–1.62 (m, 2H). C NMR (DMSO-d6) δ 163.44, 161.57, 161.11,
159.44, 159.36, 151.34, 148.17, 137.85, 137.80, 137.21, 136.74, 134.37, 132.15, 130.04, 128.34,
127.87, 123.73, 122.38, 122.10, 119.98, 119.20, 117.23, 116.77, 113.34, 107.54, 67.68, 45.50,
27.21, 25.52, 17.62. Anal. Calcd. for C30H28BrN7O2: C, 60.21; H, 4.72; N, 16.38. Found: C, 60.53;
+
H, 4.47; N, 16.59. HRMS (ESI) m/z (M+H)+ calcd. for C30H29BrN7O2 : 598.1566; found: 598.1563.
4.1.2.10. 3-[4-(1H-Imidazol-1-yl)butoxy]-4-methoxy-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6j).
Pale yellow solid; mp 168.0–169.7 °C; yield 38%. IR (KBr) cm–1 3566, 3359, 3100, 2962, 1647,
1585, 1557, 1515, 1418, 1269. 1H NMR (DMSO-d6) δ 10.04 (s, 1H), 9.28 (d, J = 2.0 Hz, 1H), 8.99
(s, 1H), 8.68 (dd, J = 4.8, 1.6 Hz, 1H), 8.52–8.46 (m, 2H), 8.05 (d, J = 1.8 Hz, 1H), 7.66–7.42 (m,
6H), 7.22–7.18 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.89 (br s, 1H), 4.08–4.01 (m, 4H), 3.84 (s, 3H),
2.22 (s, 3H), 1.90–1.80 (m, 2H), 1.70–1.63 (m, 2H). 13C NMR (DMSO-d6) δ 164.67, 161.56,
161.16, 159.42, 151.33, 150.75, 148.38, 148.16, 137.73, 137.22, 134.36, 132.17, 129.94, 128.35,
127.39, 127.11, 123.72, 120.92, 119.20, 117.31, 116.83, 111.91, 111.21, 107.46, 67.85, 55.65,
45.56, 27.43, 25.55, 17.59. Anal. Calcd. for C31H31N7O3: C, 67.74; H, 5.69; N, 17.84. Found: C,
28

ACCEPTED MANUSCRIPT
+
67.49; H, 5.88; N, 18.13. HRMS (ESI) m/z (M+H)+ calcd. for C31H32N7O3 : 550.2567; found:
550.2562.
4.1.2.11.
3-[4-(1H-Imidazol-1-yl)butoxy]-4-nitro-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6k).
Yellow solid; mp 103.2–105.4 °C; yield 38%. IR (KBr) cm–1 3420, 3406, 2936, 1664, 1582, 1526,
1449, 1416, 1235. 1H NMR (DMSO-d6) δ 10.42 (s, 1H), 9.29 (d, J = 2.0 Hz, 1H), 9.01 (s, 1H), 8.68
(dd, J = 4.6, 1.6 Hz, 1H), 8.53–8.45 (m, 2H), 8.08 (d, J = 1.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H),
7.81–7.78 (m, 1H), 7.67–7.63 (m, 2H), 7.56–7.43 (m, 3H), 7.26–7.17 (m, 2H), 6.88 (s, 1H,, 4.28 (t,
J = 6.0 Hz, 2H), 4.04 (t, J = 7.0 Hz, 2H), 2.22 (s, 3H), 1.91–1.80 (m, 2H, CH2), 1.75–1.65 (m, 2H,
CH2). 13C NMR (DMSO-d6) δ 163.61, 161.60, 161.10, 159.46, 151.37, 150.87, 148.19, 140.97,
140.10, 137.90, 137.19, 136.57, 134.40, 132.14, 130.15, 128.37, 128.04, 124.89, 123.77, 119.66,
119.16, 117.24, 116.83, 114.25, 107.62, 68.94, 45.44, 27.11, 25.35, 17.64. Anal. Calcd. for
C30H28N8O4: C, 63.82; H, 5.00; N, 19.85. Found: C, 63.55; H, 5.28; N, 20.12. HRMS (ESI) m/z
+
(M+H)+ calcd. for C30H29N8O4 : 565.2312; found: 565.2310.
4.1.2.12.
4-[4-(1H-Imidazol-1-yl)butoxy]-3-iodo-N-[4-methyl-3-[[4-(pyridin-3-yl)pyrimidin-2-
yl]amino]phenyl]benzamide (6l).
Pale yellow solid; mp 108.7–110.8 °C; yield 20%. IR (KBr) cm–1 3435, 3350, 2923, 1658, 1581,
1
1529, 1449, 1414, 1256, 801. H NMR (DMSO-d6) δ 10.14 (s, 1H), 9.28 (d, J = 1.4 Hz, 1H,, 9.00
(s, 1H), 8.69 (dd, J = 4.8, 1.4 Hz, 1H), 8.53–8.40 (m, 3H), 8.06–7.97 (m, 2H), 7.66 (s, 1H), 7.55–
7.42 (m, 3H), 7.22–7.08 (m, 3H), 6.90 (s, 1H), 4.16–3.99 (m, 4H), 2.22 (s, 3H), 2.06–1.87 (m, 2H),
1.71–1.65 (m, 2H). 13C NMR (DMSO-d6)
δ 163.27, 161.57, 161.15, 159.44, 159.41, 151.34,
148.15, 138.12, 137.75, 137.19, 137.06, 134.37, 132.17, 129.98, 129.77, 128.71, 128.39, 127.56,
123.74, 119.16, 117.22, 116.73, 111.71, 107.49, 86.25, 68.40, 45.57, 27.30, 25.47, 17.60. Anal.
29