Design, synthesis and biological evaluation of novel benzimidazole amidines as potent multi-target inhibitors for the treatment of non-small cell lung cancer

Article abstract of DOI:10.1016/j.ejmech.2017.10.061

A series of novel amidino 2-substituted benzimidazoles linked to 1,4-disubstituted 1,2,3-triazoles were synthesized by implementation of microwave and ultrasound irradiation in click reaction and subsequent condensation of thus obtained 4-(1,2,3-triazol-1

Full text of DOI:10.1016/j.ejmech.2017.10.061

European Journal of Medicinal Chemistry xxx (2017) 1e19
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of novel benzimidazole
amidines as potent multi-target inhibitors for the treatment of
non-small cell lung cancer
c
,
**
ꢀ a
d
ꢀ b
ꢁ ꢀ c
ꢀ c
Andrea Bistrovic , Luka Krstulovic , Anja Harej , Petra Grbcic , Mirela Sedic
,
,
*
ꢁ
ꢀ
ꢀ c
ꢀ b
ꢀ
ꢀ a
Sanja Kostrun , Sandra Kraljevic Pavelic , Miroslav Bajic , Silvana Raic-Malic
a
ꢀ
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev Trg 20, HR-10000 Zagreb, Croatia
b Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, HR-10000 Zagreb, Croatia
c
ꢁ ꢀ
Department of Biotechnology, Center for High-Throughput Technologies, University of Rijeka, Ulica Radmile Matejcic 2, HR-51000 Rijeka, Croatia
Chemistry Department, Fidelta Ltd., Prilaz Baruna Filipovica 29, HR-10000 Zagreb, Croatia
d
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel amidino 2-substituted benzimidazoles linked to 1,4-disubstituted 1,2,3-triazoles were
synthesized by implementation of microwave and ultrasound irradiation in click reaction and subse-
quent condensation of thus obtained 4-(1,2,3-triazol-1-yl)benzaldehyde with o-phenylenediamines.
In vitro antiproliferative screening of compounds performed on human cancer cell lines revealed that p-
chlorophenyl-substituted 1,2,3-triazolyl N-isopropylamidine 10c and benzyl-substituted 1,2,3-triazolyl
imidazoline 11f benzimidazoles had selective and potent cytostatic activities in the low nM range
against non-small cell lung cancer cell line A549, which could be attributed to induction of apoptosis and
primary necrosis. Additional Western blot analyses showed different mechanisms of cytostatic activity
between compounds 10c and 11f that could be associated with the nature of aromatic substituent at 1-
(1,2,3-triazolyl) and amidino moiety at C-5 position of benzimidazole ring. Specifically, compound 11f
abrogated the activity of several protein kinases including TGM2, CDK9, SK1 and p38 MAPK, whereas
compound 10c did not have profound effect on the activities of CDK9 and TGM2, but instead showed
moderate downregulation of SK1 activity concomitant with a significant reduction in p38 MAPK. Further
in silico structural analysis demonstrated that compound 11f bound slightly better to the ATP binding site
of p38 MAPK compared to 10c, which correlated well with observed stronger decrement in the
expression level of phospho-p38 MAPK elicited by 11f in comparison with 10c.
Received 17 May 2017
Received in revised form
18 October 2017
Accepted 20 October 2017
Available online xxx
Keywords:
Benzimidazole
1,2,3-triazole
Kinase
p38 MAPK
Multitarget
Non-small cell lung cancer (A549)
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
chemotherapy produces only a small improvement in the survival
of patients with advanced NSCLC [6]. Thus, novel treatment stra-
Lung cancer is the leading cause of cancer-related mortality
worldwide [1,2]. Non-small cell lung cancer (NSCLC), which in-
cludes adenocarcinoma, squamous cell carcinoma, and large cell
carcinoma, accounts for approximately 85% of all lung cancers, with
only 17% predicted 5-year survival rate in all stages, and approxi-
mately 2% predicted 5-year survival rate in stage IV [3e5]. Although
the treatment of non-small cell lung cancer has evolved over the
past decade resulting in a delivery of different therapeutic options,
tegies for combating this disease are urgently needed.
Protein kinases play important roles in diverse molecular
mechanisms regulating cell division, growth, and death [7,8]. They
are involved in intracellular signaling processes by catalyzing the
transfer of the
g-phosphate of ATP to downstream protein sub-
strates [9]. In addition to their key roles in cell physiology, about
half of protein kinases are linked to pathological states including
cancer [10,11]. This fact makes kinases attractive targets for thera-
peutic intervention. The identification of small molecules targeting
protein kinases has become one of the milestones in the develop-
ment of anticancer drugs [12].
* Corresponding author.
** Corresponding author.
Over the past years, in the search for novel chemotherapeutic
agents,
a new generation of versatile benzimidazole-based
ꢀ
ꢀ
E-mail address: sraic@fkit.hr (S. Raic-Malic).
https://doi.org/10.1016/j.ejmech.2017.10.061
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
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Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
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A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
2
derivatives with potent antitumor activity has been developed.
Besides their function as DNA binding agents [13,14], benzimid-
azoles have been identified as potent inhibitors of protein kinases
[12]. Majority of the clinically approved kinase inhibitors include
bicyclic nitrogen hetrocycles, such as purine or purine isosters [15].
Besides, benzimidazole scaffold is recognized as a common moitey
that interacts with kinases by multiple binding modes [16e18]. The
substitution at 1, 2, 5 and/or 6 positions of the benzimidazole
moiety was crucial for their activities towards several kinases, such
as mitogen activated protein kinase (MAPK), aurora kinase, polo
like kinase and Tie kinase [12]. Furthermore, some benzimidazole-
based derivatives with anticancer and proapoptotic activity were
developed as selective inhibitors of protein kinase CK2 (casein ki-
nase II) [19e21]. Compounds containing fluoro-substituted benz-
imidazole core were in clinical trials as cyclin-dependent kinase 9
(CDK9) inhibitors for treatment of cancer [22]. Over the years,
protein kinase inhibitors have shown therapeutic limitations
mainly resulting from drug resistance due to mutations or the
activation of alternative pathways, poor selectivity, and off-target
effects [23]. For these reasons, in recent years, multitarget ap-
proaches directed towards inhibition of kinases and targets of
different families have received increasing attention [24e26]. Thus,
some benzimidazole derivatives have been also developed as
multi-target inhibitors [27e31]. Moreover, dovitinib (TKI258/
CHIR258) is a multi-kinase inhibitor in phase III clinical trials for the
treatment of several cancers [32].
On the other hand, 1,2,3-triazole core was also found as a
binding motif in potent inhibitors of different protein kinases
[33e40]. 1,2,3-Triazole heterocycles have been recognized as good
amide bioisosteres [41]. It has been found recently that 1,2,3-
triazolyl derivative inhibits the activity of transglutaminase 2
(TGM2) by inducing its conformational change to closed form [42].
Moreover, cinnamoyl triazole derivatives acting as reversible in-
hibitors showed to compete with acyl donor TGM2 substrates [43].
In our previous study of N-heterocyclic compounds as anti-
cancer agents, we found that combination of benzimidazole and
1,2,3-triazole moieties exerted potent and selective cytostatic effect
against hepatocellular carcinoma cells in nM range [44]. Among
diverse C-4 substituents at 1,2,3-triazole ring, the p-substituted
phenyl led to increased antiproliferative activity compared to
aliphatic branched or unbranched C-4 side chains. Moreover,
halophenyl-substituted 1,2,3-triazolyl in bioactive hybrids
contributed to strong cytostatic activity in hepatocellular carci-
noma cells associated with Wee-1 kinase inhibition [33]. In addi-
tion, amongst the aromatic diamidines, bis(amidinophenyl)-
derived heterocycles showed strong antiproliferative activity
against cervical carcinoma cells [45]. In view of the biological
importance of benzimidazoles and 1,2,3-triazoles, and as a part of
ongoing research focused on the development of new anticancer
agents, we aimed to design and synthesize multi-target hybrid
chemical entities by the fusion of both pharmacophoric ring sys-
tems in a single molecular framework that would result in pro-
nounced antiproliferative activities and reduced chemoresistance
by inhibiting multiple molecular targets (Fig. 1).
Fig. 1. Design and synthesis of amidine derivatives of benzimidazole-1,2,3-triazole
conjugates containing non-substituted and halogen-substituted aromatic moiety.
apoptosis and validation of potential protein targets for 10c and 11f
by Western blot were carried out. Since p38 mitogen-activated
protein kinase (MAPK) was identified as one of the possible pro-
tein targets for both compounds, structural analysis of binding in-
teractions within the ATP-binding pocket was performed by
molecular docking.
2. Results and discussion
2.1. Chemistry
The synthesis of novel 1,2,3-triazolyl linked 2-aryl benzimid-
azole derivatives (9ae9f, 10ae10g, 11ae11g, 12c and 13c) was
carried out as shown in Scheme 1. 4-O-Propargylated benzaldehyde
(2) was synthesized from the 4-hydroxy benzaldehyde with prop-
argyl bromide, which was then used as a dipolarophile in the
regioselective Cu(I) catalyzed cycloaddition with unsubstituted,
para-halogen- and para-trifluoromethyl-substituted phenyl azides
In this context, amidino 2-substituted benzimidazoles were
linked via phenoxymethylene, ethylene spacer or directly to the
1,2,3-triazole ring with a diverse p-substituted phenyl or benzyl
subunit at N-1, thus containing distributed highly hydrophilic and
hydrophobic part of the structure. Antiproliferative effects of
benzimidazoles with 1,4-disubstituted 1,2,3-triazole on selected
human tumor cell lines were evaluated, and the structure-activity
relationship (SAR) was discussed. The compounds 10c and 11f
with potent and selective antiproliferative activity on non-small
cell lung cancer (A549) were selected for more detailed investiga-
tion of their mechanism of action. Specifically, induction of
Scheme 1. Reagents and reaction conditions: (i): propargyl bromide, K2CO3, EtOH,
reflux, 24 h; (ii): azide, CuSO4, Cu(0), DMF, t-BuOH: H2O ¼ 1: 1, 80 ꢀC, 1.5 h, MW/US;
(iii): a) 1,2-phenylenediamine (4e8), NaHSO3, EtOH, reflux, 6 h; b) HCl/MeOH, rt, 4 h.
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Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
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A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
3
to afford 3ae3g. In the recent years, microwave and sonochemical
methods have proven to be powerful techniques for facilitating
various chemical reactions [46e48]. Therefore, greener methods
using ultrasound and microwave irradiation were applied in click
chemistry of 1,2,3-triazoles 3ae3g in the presence of Cu(I) catalyst,
which was generated in situ from Cu(II) sulfate and metallic copper.
It was indicated that acoustic cavitation effect of ultrasound-
assisted reaction was more beneficial for the heterogeneous
copper-catalyzed click reactions affording 1,2,3-triazole products
3ae3g in higher yields (62e99%) than those performed under
microwave irradiation (30e71%). Condensation of various o-phe-
nylenediamines (4e8) with 4-(1,2,3-triazol-1-yl)benzaldehyde
derivatives (3ae3g) using NaHSO3 [49], as an oxidative reagent,
afforded the target 5-amidino-substituted benzimidazoles 9ae9f,
10ae10g and 11ae11g, non-substituted benzimidazole 12c and 5-
chloro-substituted benzimidazole 13c. Amidino-substituted o-
phenylenediamines (4e6) were prepared by the Pinner method as
previously reported in the literature [50].
amidine 9c, N-isopropyl amidine 10c and imidazoline 11c exhibited
remarkable antiproliferative activities with IC50 of 0.05 and 0.06
in non-small cell lung cancer cells A549. Interestingly, benzyl-
mM
substituted 1,2,3-triazolyl analogue of imidazoline 11f exerted
both, strong and selective inhibitory effect (IC50 ¼ 0.07
mM) on
A549 cells. Besides, 1-(p-iodophenyl)-1,2,3-triazolyl N-iso-
propylamidine 10d showed also marked cytostatic effects on
A549 cells albeit with less selectivity. Taking into account the
submicromolar and low micromolar (<5 mM) potencies, relation-
ship between the nature of p-substituent at phenyl ring and their
activities revealed that cytostatic effects decreased in the following
order: I > CF3 > Cl > F. On the contrary to this, unsubstituted phenyl
(in 9a, 10a, 11a) and benzyl subunits (in 9f, 10f, 11f) led to reduced
or loss of the activity, with only exception of anti-lung cancer ac-
tivity of imidazoline 11f. Introduction of methylene linker between
p-chlorophenyl and 1,2,3-triazole ring in 10g and 11g generally
reduced their activity relative to their counterparts 10c and 11c,
respectively. However, this structural modification resulted in an
With the aim to assess the influence of the phenoxymethylene
linker between the benzimidazole and 1,2,3-triazole moieties on
the antiproliferative activities, the 1,2,3-triazole ring was intro-
duced directly (19a and 19b) or via an ethylene linker (20a) to the
benzimidazole ring, as displayed in Scheme 2.
1-(p-Chlorophenyl)-1,2,3-triazolyl aliphatic alcohols (15 and 16)
were prepared in excellent yields in ultrasound-assisted reaction of
4-chlorophenylazide and propargyl alcohol or pentynol using
Cu(OAc)2. 1-(p-Chlorophenyl)-1,2,3-triazolyl aldehydes (17 and 18),
as intermediates for the synthesis of 5-amidino benzimidazoles
(19a, 19b and 20a), were subsequently obtained by the Swern
oxidation [51].
extremely potent activity (IC50 ¼ 0.05
mM) of p-chlorobenzyl-
substituted 1,2,3-triazolyl analog of imidazoline 11g against colo-
rectal adenocarcinoma (SW620). This compound also showed
selectivity in its antiproliferative effect exhibiting >100-fold lower
activities on A549, CFPAC-1 and HeLa cells, relative to SW620 cells.
p-Iodophenyl subunit in 9d, 10d, 11d contributed to enhanced
antiproliferative effect on SW620 cells displaying activities in
submicromolar range (9d, IC50 ¼ 0.95
m
M; 10d, IC50 ¼ 0.69
mM; 11d,
IC50 ¼ 0.37 M). The type of cationic moiety at the benzimidazole
m
core had also impact on the antiproliferative activities. Thus,
considering the submicromolar activities among the amidino-
substituted benzimidazole series, imidazolines 11ae11f were the
most potent, whereas N-isopropylamidines 10ae10f showed the
lowest overall activity. Besides the abovementioned highly potent
effects of imidazolines 11c and 11f on A549 cells, imidazolino
fragment was mainly responsible for the inhibitory effect on cer-
vical carcinoma cells HeLa. Thus, imidazolines 11ae11g showed
antiproliferative activity against HeLa cells with IC50 in the range of
2.2. Biological profiling
2.2.1. In vitro antiproliferative activity
The results of antiproliferative evaluations of 9ae9f, 10ae10g
and 11ae11g, 12c, 13c, 19a, 19b and 20a performed on human tu-
mor cell lines including non-small cell lung cancer (A549), ductal
pancreatic adenocarcinoma (CFPAC-1), cervical carcinoma (HeLa)
and metastatic colorectal adenocarcinoma (SW620) as well as on
normal human lung fibroblasts (WI38) are presented in Table 1. 5-
Fluorouracil was used as the reference drug.
0.22e7.78 mM. In addition, p-iodophenyl and p-(trifluoromethyl)
phenyl analogs 11d and 11e of imidazolines exhibited sub-
micromolar activity on pancreatic carcinoma (CFPAC-1). Except for
10b, all amidines (9ae9f) and N-isopropylamidines (10a, 10ce10g)
either exhibited reduced potencies or were deprived of any activ-
ities. Replacement od phenoxymethylene with ethylene linker or
The p-chlorophenyl-substituted 1,2,3-triazolyl derivatives of
Scheme 2. Reagents and reaction conditions: (i): propargyl alcohol/4-pentyn-1-ol, Cu(OAc)2, MeOH, 80 ꢀC, US, 1.5 h; (ii): (COCl)2, DMSO, Et3N, CH2Cl2, -78 ꢀC to rt 45 min; (iii): a) 1,2-
phenylenediamine (5 and 6), NaHSO3, EtOH, reflux, 6 h; b) HCl/MeOH, rt, 4 h.
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multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
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A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
4
Table 1
In vitro growth inhibitory effects of compounds 9ae9f, 10ae10g, 11ae11g, 12c, 13c, 19a, 19b and 20a on human tumor cell lines.
a
Compd
R1
X
R2
IC50
(m
M)
A549
CFPAC-1
29.87
HeLa
SW620
31.51
WI38
8.08
9a
-PhOCH2-
53.31
80.97
0.05
18.84
9b
9c
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
>100
18.54
21.48
2.09
7.39
0.80
6.45
0.32
13.71
16.60
37.85
7.57
0.70
5.03
0.62
0.52
0.89
0.99
9d
9e
9f
0.45
0.95
0.84
0.36
9.47
34.44
>100
30.91
62.14
10a
>100
10b
10c
10d
10e
10f
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
8.37
0.05
0.08
85.76
6.18
4.98
49.69
0.71
0.59
37.85
48.69
0.69
0.66
8.04
0.20
0.32
65.41
0.70
0.10
>100
5.13
17.53
0.51
>100
>100
>100
26.21
13.72
8.80
50.15
58.61
36.48
29.63
10g
11a
>100
7.78
11b
11c
11d
11e
11f
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
-PhOCH2-
>100
0.06
>100
0.59
0.07
>100
16.88
0.86
0.98
0.22
0.42
2.69
6.67
2.31
3.65
0.37
6.35
28.05
0.09
2.24
0.22
0.69
6.89
0.65
29.17
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Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
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A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
5
Table 1 (continued )
a
Compd
R1
X
R2
IC50
(
mM)
A549
9.34
CFPAC-1
6.82
HeLa
5.06
SW620
0.05
WI38
5.77
11g
-PhOCH2-
12c
13c
19a
-PhOCH2-
-PhOCH2-
e
H
7.96
2.91
>100
3.74
>100
3.54
0.85
99.26
4.28
1.11
1.52
6.85
Cl
27.11
3.86
15.95
32.45
19b
20a
e
0.83
0.69
1.67
2.72
-CH2CH2-
33.37
2.80
36.09
0.14
3.11
8.81
56.82
0.08
3.71
0.94
5-FU
e
e
e
a
50% inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%.
direct fusion of benzimidazole to 1,2,3-triazole had detrimental
effect on the anti-lung cancer potency. However, benzimidazole
directly connected to 1,2,3-triazole contributed to some antitu-
moral activities. For example, N-isopropylamidine 19a was 6- and
21-fold more active on CFPAC-1 and HeLa cells, respectively,
compared to its counterpart 10c bearing phenoxymethylene linker.
Similarly, imidazolino-substituted benzimidazole 19b displayed
20-fold increased activity on CFPAC-1 cells relative to its structural
analog 11c. Furthermore, the comparison of inhibitory effects be-
tween amidine 20a with ethylene linker and its analog 10c with
phenoxymethylene linker lends support to presumption that
ethylene linker has no significant contribution on activities against
all evaluated tumor cell lines. However, compounds showing
cytostatic activity against cancer cells were also cytotoxic to normal
human lung fibroblasts (WI38), although 9c, 10c, 11c, 11f and 11g
were less cytotoxic (with selectivity index, SI, around 102)
compared to other compounds that exhibited antiproliferative ef-
fects in submicromolar range, including 5-FU. Some insights into
structural requirements including unsubstituted, alkyl-substituted
or cyclic amidino moiety at 5-benzimidazole, linkers between
1,2,3-triazole and benzimidazole ring, or 1,2,3-triazole and phenyl
ring, as well as type of p-substituents at phenyl ring that influenced
the cytostatic activities, are presented in Fig. 2.
of their mechanism of action and to focus additional biological
studies, the Prediction of Activity Spectra for Substances (PASS) [52]
was combined with the available literature data for biological ac-
tivities of structurally related chemical entities and known bio-
logical pathways associated with the growth-inhibition effects on
A549 cells. Phosphodiesterase 5A (PDE5), cyclin-dependent kinase
9 (CDK9), transglutaminase 2 (TGM2), p38 mitogen-activated
protein kinase (p38 MAPK) and sphingosine kinase 1 (SK1) were
identified as potential targets of 10c and 11f (Table S1, Supple-
mentary information), and their expression levels were analyzed by
Western blot (Fig. 4).
PDE5 is cGMP-specific phosphodiesterase that specifically hy-
drolyzes cGMP to 50-GMP thereby lowering intracellular cGMP
levels. It has been demonstrated that PDE5 is overexpressed in
human non-small cell lung tumors compared to normal bronchial
epithelium, which suggests that PDE5 plays an important role in
lung tumorigenesis [53]. Furthermore, inhibition of PDE5 activity
was shown to induce apoptosis in human non-small cell lung
cancer cells A549 [53]. Our results showed significant reduction in
the expression level of PDE5 in A549 cells treated with compound
11f, whereas such an effect was not detected with compound 10c.
Thus, PDE5 does not seem to be a putative target of compound 10c.
Another enzyme that was recognized by PASS as potential target
of 11f was CDK9 which is known to associate with cyclin T1 forming
cyclin-dependent kinase pair. Interestingly, it was previously found
that CDK9 signaling pathway was related to aberrant transcription
profiles observed in lung adenocarcinoma [54]. CDK9 is known to
prevent degradation of p53 [55]. Moreover, an overexpression of
CDK9 could lead to accumulation of p53 in astroglioma cells
pointing to CDK9-dependent regulation of p53 levels. In line with
literature data, our results revealed that compound 11f dramati-
cally reduced the expression level of CDK9/cyclin T1 in A549 cells
which was associated with a decline in phospho-p53 (Ser15) levels.
On contrary, compound 10c did not induce marked changes in
CDK9/cyclin T1 expression level albeit a trend towards a decrease in
phospho-p53 level was observed, suggesting that CDK9/cyclin T1
was not involved in the regulation of p53 activity in the response to
treatment with compound 10c.
2.2.2. Apoptosis detection
In order to investigate whether antiproliferative effects of
compounds 10c and 11f with strong and highly selective activity in
non-small cell lung cancer cell line A549 could be associated with
induction of apoptosis, Annexin V assay was performed as previ-
ously described [33]. Compound 10c induced marked reduction in
viable cell population by 70.59% concomitant with a profound in-
crease in early and late apoptotic/primary necrotic cell populations
by 27.81% and 40%, respectively (Table 2, Fig. 3).
Similarly, compound 11f resulted in a significant decrease in
viable cell population by 49.77%, which was accompanied by a
marked increase in early and late apoptotic/primary necrotic cell
populations by 26.97% and 16.37%, respectively (Table 2, Fig. 3).
TGM2, suggested by in silico analyses as a putative target of 10c
and 11f, is a multi-functional enzyme catalysing the formation of
intermolecular isopeptide bonds between glutamine and lysine
2.2.3. Validation of predicted protein targets of compounds 10c and
11f by Western blot analysis
In order to further characterize compounds 10c and 11f in terms
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Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
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A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
6
Fig. 2. Structure-activity relationship (SAR) of a series of amidine, N-isopropylamidine and imidazoline benzimidazoles coupled with several 1,4-disubstituted 1,2,3-triazolyl
moieties.
pathway is sufficient to activate p53 [63]. Taking into account this
finding and the abovementioned literature data, we propose that
down-regulation of p53 activity in A549 induced by compound 11f
could be associated with abrogation of activities of several kinases
including TGM2, CDK9 and p38 MAPK. On the other hand, com-
pound 10c did not have profound effect on the activities of CDK9
and TGM2, but instead showed moderate downregulation of SK1
activity concomitant with significant reduction in p38 MAPK ac-
tivity. Since both compounds reduced levels of phospho-p53
although to a different extent, it seems plausible that compounds
10c and 11f trigger p53-independent apoptosis. Assuming p38
MAPK as a common biological target for both compounds 10c and
11f more detailed structural analysis of possible intramolecular
interactions have been investigated.
Table 2
Results of Anexin V assay for apoptosis detection of 10c and 11f in A549 cells.
A549 cells (%)a
Control
10c
11f
Secondary necrotic cells
Early apoptotic cells
Viable cells
0.00
0.52
99.48
0.00
2.78
6.43
28.33
28.89
40.00
27.49
49.71
16.37
Late apoptotic/primary necrotic cells
a
The percentages of viable cells (PIꢁ/Ann V-), early apoptotic cells (PIꢁ/Ann Vþ),
late apoptotic/primary necrotic cells (PIþ/Ann Vþ) and secondary necrotic cells
(PIþ) after 48 h treatment with compounds 10c and 11f at their 2 ꢂ IC50 values are
shown.
side-chains. Importantly, this enzyme has been shown to possess
intrinsic serine/threonine kinase activity and to phosphorylate p53
tumor suppressor protein thus regulating its activity [56]. Its
expression was previously associated with enhanced invasive and
migratory properties of non-small cell lung cancer cells in vitro
pointing to its tumour-promoting role [57]. Our results showed that
TGM2 and phospho-p53 levels were markedly down-regulated
upon treatment with 11f, whereas their expression levels were
not significantly altered by compound 10c.
SK1 catalyses the phosphorylation of sphingosine to form
sphingosine-1-phosphate (S1P), a lipid mediator that regulates cell
proliferation and survival. Previous findings demonstrated that
expression of SK1 was greatly increased in non-small cell lung
cancer tissues and cells, and to correlate with tumour progression
and poor survival of patients with NSCLC [58,59], which indicates
that SK1 may represent a promising pharmacologic target for the
treatment of NSCLC. Moreover, several studies provided evidence
that SK1 was required for the activation of p38 MAPK in both
normal [60] and cancer cells [61]. Similarly, we found that com-
pound 11f significantly reduced the expression level of phospho-
SK1 which was accompanied by a marked decline in phospho-
p38 MAPK level (Fig. 4). The same effect was observed with com-
pound 10c as well, although to a lesser extent. It has been
demonstrated that p38 MAPK phosphorylates p53 at serine 15,
which seems to be critical for the stabilization, up-regulation, and
functional activation of p53 during cellular stress [62]. Further
evidence showed that inhibition of p38 precludes stimulation of
the transcriptional activity of p53 and that activation of the p38
2.3. Structural analysis of possible interactions of compounds 10c
and 11f with p38 MAPK
p38 MAPK was selected for further in silico molecular binding
studies based on our Western blot results revealing marked
reduction in its activity induced by both compounds 10c and 11f,
together with the fact that their structurally related purinomimetic,
ralimetinib (LY2228820) [64] found to be a selective inhibitor of
p38 MAPK that has been evaluated in clinical trials in patients with
advanced cancer. Ralimetinib has shown acceptable safety and
pharmacokinetics profiles, which encourages further development
of novel p38 MAPK inhibitors in cancer treatment. Here, we ana-
lysed the ability of compounds 10c and 11f to bind to the ATP-
binding site of p38 MAP kinase. Since both compounds incorpo-
rate a number of potential pharmacophoric features, their possible
interactions with the p38 MAPK active site have been investigated
in several steps to elucidate the most probable binding modes. Both
structure based-approach in terms of docking to the active site and
ligand based approach including field-based alignment were used
as described in the methods section.
Due to a large number of available X-ray structures, ligands were
grouped into 24 clusters to obtain a representative but chemicaly
diverse set for further analysis. Aligned representative structures
are shown in Fig. S2, Supplementary data, indicating structural
diversity and different binding modes of known inhibitors that fill
the ATP-binding region but also hydrophobic regions I and II, as
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Fig. 3. Detection of apoptosis induced by compounds 10c and 11f in non-small cell lung cancer cell line A549 using Annexin V assay. Cells were visualized by fluorescence mi-
croscope at 40ꢂ magnification before and after treatment with the concentration of 2 ꢂ IC50 for 48 h. PI staining was used as a nuclear marker. Shown here are bright-field images
(left micrographs) and late apoptotic/primary necrotic cells (right micrographs).
well as DFG-out pocket in case of DFG-out inhibitors [65].
hinge amino-acids Met109 and Gly110 were applied. In order to
explore possible Gyl flip observed for selective p38 inhibitors, 1OVE
and 2YIW structures were also used for docking [66,67]. Binding
poses of 10c and 11f collected from filed-based alignments and
from different docking experiments were further refined using
Embrace protocol and binding energies were estimated using MM-
GBSA protocol as described in the methods section. It was shown
previously [68] that the absolute calculated values are not neces-
sarily in agreement with experimental binding affinities. However,
the ranking of the ligands based on the calculated binding are ex-
pected to correlate with ranking based on experimental binding
affinity, particularly in the case of congeneric series. In order to
investigate correlation of MM-GBSA calculated binding energies
Representative ligands were divided into two sets of DFG-in
(active form) and DFG-out (in-active form) binders and their
structures are shown in Fig. S3 and Fig. S4, Supplementary data.
Field-based alignment was performed using bioactive conforma-
tions from representative X-ray structures as a reference while
structures of 10c and 11f were generated from conformational
anaylsis. Structures of 10c and 11f aligned with the 3LFA ligand,
with medium activity of 100 nM, and 3D83 ligand, with strong
activity of 8 nM, are presented in Fig. 5. It can be observed that good
structural alignment and overlap of major pharmacophoric points
have been achieved for both 3LFA and 3D83 reference ligands.
Conformations of 10c and 11f aligned with representative ligands
were sampled for further structural refinement and estimation of
binding energies using MM-GBSA method as described below.
The ligand docking studies were carried out with extra precision
using the apo-p38 X-ray structure for DFG-in binding mode
(pdb:5UOJ) and p38 in complex with a biphenyl amide inhibitor
(pdb:3D83) for analysis of DFG-out binding mode. Both uncon-
strained protocol and protocol with constrained interactions with
with experimentally determined inhibition, DG values for the set of
ligands with available X-ray structures and inhibition activities on
p38 MAK kinase were calculated. Very good agreement was ob-
tained for a set of 10 inhibitors as shown in Fig. 6. These results
indicated that the applied computational strategy could provide
plausible ranking of different binding poses for 10c and 11f and at
least approximate comparison with the activities of known
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Fig. 4. Western blot analysis of predicted protein targets of compounds 10c and 11f in non-small cell lung cancer A549. Representative Western blots are shown detecting the
cellular levels of selected proteins before and after treatment of A549 cells with indicated compounds at their 2 ꢂ IC50 values for 48 h. Approximate molecular weights (kDa) are
indicated. Relative protein expressions determined by densitometric analysis of protein bands and normalized to the alpha-tubulin loading control. Two independent experiments
were performed with similar results. Data are presented as mean values SD. Statistically significant (p < 0.05) differences in the expression levels were marked by an asterisk.
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Fig. 5. a) Structures of 10c and 11f as well as 3LFA and 3D83 ligands used as a reference structures; b) Field-based alignment of 10c (green) and 11f (magenta, spherical fields) with
3LFA ligand (grey, fields as surfaces) and c) Field-based alignment of 10c (green) and 11f (magenta, spherical fields) with 3D83 ligand (grey, fields as surfaces). Negative potential
fields e blue, positive potential fields e red, hydrophobic fields e yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Fig. 6. Correlation between experimental pIC50 values and dG values calculated by MMGBSA method for a set of kinase inhibitors (green). Predicted pIC50 values for different poses
of compounds 10c and 11f based on the observed correlation (orange). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
inhibitors. Based on the correlation obtained for the known ligands,
inhibitory values are predicted for different binding poses of 10c
and 11f. Predicted values for different poses all fall in the activity
range below 100 nM (Fig. 6.), with DFG-out “3D83-like” binding
mode being most active for both compounds; 4 nM for 11f and
8 nM for 10c. Compound 11f is consistently predicted to bind
slightly better to p38 MAPK compared to 10c, which is in good
agreement with stronger reduction in the expression level of
phospho-p38 MAPK in A549 cells elicited by 11f (Fig. 4).
Best predicted binding pose of compound 11f in comparisson
with 3D83 ligand is shown in Fig. 7.
The triazole moiety forms an H-bond with the backbone of Met
109 in the hinge region, while the amidino-benzimidazole group
forms number of H-bonds with polar amino-acids in the linker
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Fig. 7. Best predicted binding pose for compound 11f obtained from docking, reoptimising 11f-p38 complex with Embrace method and calculating interaction energies using MM-
GBSA protocol. a) Overlay of compound 11f (gray) and 3D83 ligand (green); b) 2D diagram of 3D83-p38 interactions; c) 2D diagram of compound 11f-p38 interactions. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
region; Glu71, Hid148 and Asp168. The phenyl moieties linked to
the triazole and benzimidazole are both placed in the hydrophobic
other tested human cancer cell lines including pancreatic adeno-
carcinoma (CFPAC-1), cervical carcinoma (HeLa) and colorectal
adenocarcinoma (SW620) were reduced approximately by two
orders of magnitude. Further biological studies of selected candi-
dates 10c and 11f revealed that their strong growth inhibitory ac-
tivity against A549 cells could be associated with induction of
apoptosis and primary necrosis. Further analyses provided evi-
dence for different regulation of cellular signalling by 10c and 11f.
While cytostatic effect of imidazoline benzimidazole 11f may be
ascribed to down-regulation of TGM2, CDK9/cyclin T1, SK1 and
particularly p38 MAPK activity, N-isopropylamidine benzimidazole
10c had a profound impact on the activities of only two kinases
including SK1 and p38 MAPK, as demonstrated by the reduction in
their activities in A549 cells treated with 10c. Since both com-
pounds inhibited p38 MAPK although with slightly different po-
tency, this enzyme was identified as the common target of 10c and
11f. Further in silico structural analysis confirmed that both com-
pounds could indeed efficiently inhibit p38 MAP kinase with
compound 11f consistently predicted to bind slightly better to p38
MAPK compared to 10c, which correlated well with observed
stronger decrement in the expression level of phospho-p38 MAPK
elicited by 11f in comparison with 10c.
environment, the first one forming nice
p-p stacking with the
Phe169. Other predicted binding poses for compounds 10c and 11f
are shown in Fig. S5, Supplementary data.
3. Conclusions
Target monocationic and non-cationic benzimidazoles 9ae9f,
10ae10g, 11ae11g, 12c, 13c, 19a, 19b and 20a were designed and
synthesized by oxidative coupling of o-phenylenediamines with
benzaldehydes 3ae3g bearing 1,4-disubstituted-1,2,3-triazole that
were provided by implementation of environmentally benign
synthetic protocols using nonconventional energy sources, micro-
wave and ultrasound irradiation. Compounds 3ae3g were obtained
in overall higher yields under ultrasound irradiation compared to
reactions performed under microwave irradiation. The present
work led to the development of novel p-chlorophenyl-substituted
1,2,3-triazolyl N-isopropylamidine 10c and benzyl-substituted
1,2,3-triazolyl imidazoline 11f benzimidazoles with selective and
potent cytostatic activities against non-small cell lung cancer
(A549) in the low nM range. Their growth-inhibitory effects on
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4. Experimentals
7.93e7.89 (4H, m, Ph), 7.61 (2H, t, J ¼ 7.9 Hz, Ph), 7.51 (1H, t,
J ¼ 7.5 Hz, Ph), 7.29 (2H, d, J ¼ 8.7 Hz, Ph), 5.38 (2H, s, OCH2).13
C
4.1. Materials and methods
NMR (75 MHz, DMSO) d 191.63, 163.01, 143.37, 136.62, 132.01,
130.12, 129.05, 123.32, 120.38, 115.40, 61.46.
All solvents were purified following recommended drying
agents and/or distilled over 3 Å molecular sieves. For monitoring
the progress of a reaction and for comparison purpose, thin layer
chromatography (TLC) was performed on pre-coated Merck silica
gel 60F-254 plates using appropriate solvent system and the spots
were detected under UV light (254 nm). For column chromatog-
raphy silica gel (Fluka, 0.063e0.2 mm) was employed, glass column
was slurry-packed under gravity. Melting points (uncorrected)
were determined with Kofler micro hot-stage (Reichert, Wien). 1H
and 13C NMR spectra were acquired on a Bruker 300 and 600 MHz
NMR spectrometer as well as 300 MHz Agilent Technologies DD2
NMR spectrometer. All data were recorded in DMSO-d6 at 298 K.
Chemical shifts were referenced to the residual solvent signal of
4.2.1.2. 4-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)benz-
aldehyde (3b). Compound 3b was prepared using the above
mentioned procedure from 2 (200 mg, 1.15 mmol) and 1-azido-4-
fluorobenzene (2.76 mL, 1.38 mmol). After purification by column
chromatography compound 3b was isolated as white powder
(Method A: 161.1 mg, 47%; Method B: 325.6 mg, 95%;
m.p. ¼ 140e142 ꢀC, m.p. lit. ¼ 138e140 ꢀC) [70]. 1H NMR (300 MHz,
DMSO)
7.47 (2H, t, J ¼ 8.8 Hz, Ph), 7.28 (2H, d, J ¼ 8.7 Hz, Ph), 5.38 (2H, s,
OCH2). 13C NMR (75 MHz, DMSO)
191.49, 163.44; 160.18 (d,
d
9.89 (1H, s, CHO), 8.96 (1H, s, H50), 8.00e7.85 (4H, m, Ph),
d
JCF ¼ 248.8 Hz), 162.92, 143.32, 133.15; 133.11 (d, JCF ¼ 2.9 Hz),
131.91, 130.03, 123.49, 122.76, 122.65, (d, JCF ¼ 8.8 Hz), 117.01, 116.70
(d, JCF ¼ 23.2 Hz), 115.32, 61.40.
DMSO at
d d
2.50 ppm for 1H and 39.50 ppm for 13C. Individual
resonances were assigned on the basis of their chemical shifts,
signal intensities, multiplicity of resonances and HꢁH coupling
constants. High performance liquid chromatography was per-
formed on an Agilent 1100 series system with UV detection
(photodiode array detector) using Zorbax C18 reverse-phase
4.2.1.3. 4-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)benz-
aldehyde (3c). Compound 3c was prepared using the above
mentioned procedure from 2 (200 mg, 1.15 mmol) and 1-azido-4-
chlorobenzene (2.76 mL, 1.38 mmol). After purification by column
chromatography compound 3c was isolated as white powder
(Method A: 122.3 mg, 34%; Method B: 224.2 mg, 62%;
m.p. ¼ 151e154 ꢀC, m.p. lit. ¼ 151e153 ꢀC) [70]. 1H NMR (600 MHz,
analytical column (2.1 ꢂ 30 mm, 3.5
mm). All compounds used for
biological evaluation showed >95% purity in this HPLC system.
4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems Inc., Foster
City, CA, USA) equipped with a 200 Hz, 355 nm Nd:YAG laser was
used for mass accuracy analysis of compounds. Acquisition was
performed in positive ion reflector mode. The ultrasound-assisted
reactions were carried out in a Bandelin Bath Cleaner (Sonorex
digital 10 P) with a nominal power of 1000 W and frequency of
35 kHz. The reactions were carried out in a round-bottomed flask of
25 mL capacity suspended at the centre of the cleaning bath, 5 cm
below the surface of the liquid. Microwave-assisted syntheses were
performed in a Milestone start S microwave oven using glass cu-
vettes at 80 ꢀC and 300 W under pressure of 1 bar.
DMSO)
d
9.89 (1H, s, CHO), 9.01 (1H, s, H50), 7.96 (2H, d, J ¼ 8.8 Hz,
Ph), 7.90 (2H, d, J ¼ 8.7 Hz, Ph), 7.69 (2H, d, J ¼ 8.8 Hz, Ph), 7.28 (2H,
d, J ¼ 8.7 Hz, Ph), 5.38 (2H, s, OCH2).13C NMR (151 MHz, DMSO)
d
191.44, 162.87, 143.45, 135.34, 133.18, 131.87, 130.02, 129.94,
123.28, 121.98, 115.30, 61.37.
4.2.1.4. 4-((1-(4-Iodophenyl)-1H-1,2,3-triazol-4-yl)methoxy)benzal-
dehyde (3d). Compound 3d was prepared using the above
mentioned procedure from 2 (200 mg, 1.15 mmol) and 1-azido-4-
iodobenzene (2.76 mL, 1.38 mmol After purification by column
chromatography compound 3d was obtained as white powder
(Method A: 270.6 mg, 56%; Method B: 381.7 mg, 82%;
4.2. Experimental procedures for the preparation of compounds
m.p. ¼ 259e262 ꢀC). 1H NMR (600 MHz, DMSO)
d 9.89 (1H, s, COH),
4-(Prop-2-ynyloxy)benzaldehyde (2) [69] and amidino
substituted o-phenylendiamines (4, 5, 6) [50] were prepared ac-
cording to known procedure, while compounds 4-(1,2,3-triazol-4-
yl)methoxy)benzaldehydes (3a, 3b, 3c, 3f) [70], 4-(1,2,3-triazol-4-
yl)methoxy)benzaldehyde (3g) [71], (1-(4-chlorophenyl)-1H-
1,2,3-triazol-4-yl)methanol (16) [72] and 1-(4-chlorophenyl)-1H-
1,2,3-triazole-4-carbaldehyde (18) [72] were synthesized according
to modified procedures given in the literature.
9.01 (1H, s, H50), 7.97 (2H, d, J ¼ 8.7 Hz, Ph), 7.90 (2H, d, J ¼ 8.7 Hz,
Ph), 7.74 (2H, d, J ¼ 8.7 Hz, Ph), 7.28 (2H, d, J ¼ 8.7 Hz, Ph), 5.38 (2H,
s, OCH2). 13C NMR (151 MHz, DMSO)
d 191.67,163.00,143.59,138.84,
136.32, 132.04, 130.13, 123.25, 122.34, 115.43, 94.63, 61.43.
4.2.1.5. 4-((1-(4-(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)
methoxy)benzaldehyde (3e). Compound 3e was prepared using the
above mentioned procedure from 2 (200 mg, 1.15 mmol) and 1-
azido-4-(trifluoromethyl)benzene (2.76 mL, 1.38 mmol). After pu-
rification by column chromatography compound 3e was isolated as
white powder (Method A: 120.4 mg, 30%; Method B: 396.3 mg,
4.2.1. General procedure for the synthesis of 4-(1,2,3-triazol-4-yl)
methoxy)benzaldehydes (3ae3g)
The reaction mixture of compound 2, Cu(0) (0.8 eq), 1 M CuSO4
(0.3 eq) and the corresponding azide (1.2 eq) was dissolved in 1 mL
DMF and a mixture of t-BuOH: H2O ¼ 1: 1 (3 mL). Method A: The
reaction mixture was stirred under microwave irradiation (300 W)
at 80 ꢀC during 1.5 h. Method B: The reaction mixture was placed in
an ultrasonic bath cleaner (1000 W, 35 kHz) at 80 ꢀC for 1.5 h. The
solvent was removed under reduced pressure and purified by col-
umn chromatography with CH2Cl2.
99%; m.p. ¼ 251e253 ꢀC). 1H NMR (600 MHz, DMSO)
d 9.90 (1H, s,
CHO), 9.14 (1H, s, H50), 8.19 (2H, d, J ¼ 8.5 Hz, Ph), 8.01 (2H, d,
J ¼ 8.6 Hz, Ph), 7.91 (2H, d, J ¼ 8.7 Hz, Ph), 7.29 (2H, d, J ¼ 8.7 Hz, Ph),
5.41 (2H, s, OCH2). 13C NMR (151 MHz, DMSO)
d 191.57, 162.93,
143.77, 131.97, 130.12, 129.32; 129.11; 128.89; 128.70 (q,
JCF ¼ 32.4 Hz), 127.40; 127.38; 127.36; 127.33 (q, JCF ¼ 3.5 Hz),
124.80; 123.00 (d, JCF ¼ 271.4 Hz), 123.53, 120.86, 115.38, 61.38.
4.2.1.6. 4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde
(3f). Compound 3f was prepared using the above mentioned pro-
cedure from 2 (200 mg, 1.15 mmol) and benzyl azide (2.76 mL,
1.38 mmol). After purification by column chromatography 3f was
obtained as white solid (Method A: 239.1 mg, 71%; Method B:
303.6 mg, 90%; m.p. ¼ 101e103 ꢀC, m.p. lit. ¼ 99e100 ꢀC) [70]. 1H
4.2.1.1. 4-((1-Phenyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde
(3a). Compound 3a was prepared using the above mentioned
procedure from 2 (200 mg, 1.15 mmol) and azidobenzene (2.76 mL,
1.38 mmol) to obtain 3a as white solid (Method A: 229.5 mg, 71%;
Method B: 310.1 mg, 97%; m.p. ¼ 99e101 ꢀC (m.p. lit. ¼ 101e103 ꢀC)
[70]. 1H NMR (600 MHz, DMSO)
d
9.89 (1H, s, COH), 8.99 (1H, s, H50),
NMR (300 MHz, DMSO) d
9.88 (1H, s, CHO), 8.33 (1H, s, H50), 7.87
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(2H, d, J ¼ 8.7 Hz, Ph), 7.44e7.27 (5H, m, Ph), 7.23 (2H, d, J ¼ 8.7 Hz,
4.2.2.3. 2-(4-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-1H-benzo[d]imidazole-5-carboximidamide hydrochloride
Ph), 5.62 (2H, s, CH2), 5.27 (2H, s, CH2). 13C NMR (75 MHz, DMSO)
d
191.32, 162.91, 142.32, 135.94, 131.75, 129.84, 128.76, 128.16,
(9c). Compound 9c was prepared using the above described
method from 3c (200 mg, 0.64 mmol) and o-phenylenediamine 4
(86.5 mg, 0.58 mmol) to give 9c as brown powder (142.1 mg, 41%,
127.96, 124.94, 115.19, 61.42, 52.84. MS (m/z) 294.1 [MþH]þ.
m.p. ¼ 212e213 ꢀC). 1H NMR (300 MHz, DMSO)
d 9.53 (2H, bs, NH),
4.2.1.7. 4-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benz-
aldehyde (3g). Compound 3g was prepared using the above
mentioned procedure from 2 (200 mg, 1.15 mmol) and p-chlor-
obenzyl azide (2.76 mL, 1.38 mmol). After purification by column
chromatography 3g was obtained as white powder (Method B:
302.2 mg, 80%; m.p. ¼ 106e108 ꢀC, m.p. lit. ¼ 108e111 ꢀC) [71]. 1H
9.22 (2H, bs, NH), 9.07 (1H, s, H50), 8.45 (2H, d, J ¼ 8.9 Hz, Ph), 8.23
(1H, s, H4), 7.98 (2H, d, J ¼ 8.9 Hz, Ph), 7.91 (1H, d, J ¼ 8.5 Hz, H7),
7.83 (1H, dd, J ¼ 8.6, 1.4 Hz, H6), 7.69 (2H, d, J ¼ 8.9 Hz, Ph), 7.40 (2H,
d, J ¼ 8.9 Hz, Ph), 5.42 (2H, s, CH2).13C NMR (75 MHz, DMSO)
d
165.60, 161.40, 152.38, 143.41, 135.31, 133.10, 130.02, 129.89,
d
9.88 (1H, s, CHO), 8.33 (1H, s, H50), 7.87
123.99, 123.46, 123.30, 121.89, 115.70, 115.01, 114.28, 61.35. Anal.
calcd. for C23H18ClN7O x 2 HCl x 2.6 H2O (Mr ¼ 536.66): C 49.01, H
4.51, N 17.39; found: C 49.33, H 4.27, N 17.48%. HRMS: calcd. for
NMR (600 MHz, DMSO)
(2H, d, J ¼ 8.7 Hz, Ph), 7.45 (2H, d, J ¼ 8.4 Hz, Ph), 7.35 (2H, d,
J ¼ 8.4 Hz, Ph), 7.23 (2H, d, J ¼ 8.7 Hz, Ph), 5.63 (2H, s, CH2), 5.27 (2H,
C
23H18ClN7O (M þ H)þ: 444.1340; found: 444.1346.
s, CH2). 13C NMR (75 MHz, DMSO)
d 191.33, 162.90, 142.38, 134.92,
132.90, 131.76,129.93,129.86, 128.77,124.98,115.19, 61.41, 52.04.MS
(m/z) 328.1 [MþH]þ.
4.2.2.4. 2-(4-((1-(4-Iodophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-1H-benzo[d]imidazole-5-carboximidamide hydrochloride
(9d). Compound 9d was prepared using the above described
method from 3d (100 mg, 0.25 mmol) and o-phenylenediamine 4
(37.06 mg, 0.25 mmol) to obtain red powder 9d (63.2 mg, 42%,
4.2.2. General procedure for the synthesis of 5-amidino-2-phenyl
benzimidazoles (9ae9f, 10ae10g and 11ae11g)
The reaction mixture of 4-triazolylbenzaldehyde derivatives
(3aeg), o-phenylenediamine (4, 5 or 6) and 40% NaHSO3 (aq) was
dissolved in 15 mL EtOH and stirred under reflux for 6e8 h. After
completition of the reaction NaHSO3 was filtered and the reaction
mixture was evaporated to dryness. Water was added (5 mL) and
the mixture was stirred over night and filtered. The crude residue
was dissolved in HCl saturated EtOH (8e10 mL) and stirred over
night. Addition of ether resulted in precipitation of products 9ae9f,
10ae10g and 11ae11g. Solid was collected by filtration, washed
with anhydrous ether, and dried under vacuum.
m.p. ¼ 229e231 ꢀC). 1H NMR (300 MHz, DMSO)
d 9.41 (2H, bs, NH),
9.05 (3H, bs, NH, H50), 8.32 (2H, d, J ¼ 8.5 Hz, Ph), 8.17 (1H, s, H4),
7.98 (1H, d, J ¼ 8.6 Hz, H7), 7.79e7.73 (3H, m, H6, Ph), 7.36 (2H, d,
J ¼ 8.8 Hz, Ph), 5.39 (2H, s, OCH2).13C NMR (75 MHz, DMSO)
d 165.70
(CNH), 160.87, 153.14, 143.52, 138.62, 136.16, 129.97, 129.48, 123.18,
123.07, 122.57, 122.06, 115.80, 115.57, 94.54, 61.27. Anal. calcd. for
C
23H18N7IO x 2 HCl x 3.75 H2O (Mr ¼ 675.82): C 40.88, H 4.10, N
14.51; found: 40.64, H 3.96, N 14.78%. HRMS: calcd. for C23H18N7IO
(M þ H)þ: 536.0696; found: 536.0679.
4.2.2.5. 2-(4-((1-(4-(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)
methoxy)phenyl)-1H-benzo[d]imidazole-5-carboximidamide hydro-
chloride (9e). Compound 9e was prepared using the above
described method from 3e (200 mg, 0.58 mmol) and o-phenyl-
enediamine 4 (86.5 mg, 0.58 mmol) and to obtain 3e as grey solid
(144.0 mg, 39%, m.p. ¼ 247e250 ꢀC). 1H NMR (300 MHz, DMSO)
4.2.2.1. 2-(4-((1-Phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-
benzo[d]imidazole-5-carboximidamide
hydrochloride
(9a).
Compound 9a was prepared using the above described method
from 3a (200 mg, 0.72 mmol) and o-phenylenediamine 4 (113.4 mg,
0.64 mmol) to obtain 9a as brown powder (100.3 mg, 28%,
m.p. ¼ 159e162 ꢀC). 1H NMR (300 MHz, DMSO)
d 9.42 (2H, bs, NH),
d
9.43 (2H, bs, NH), 9.18 (1H, s, H50) 9.07 (2H, bs, NH), 8.35 (2H, d,
9.13e8.99 (3H, m, NH, H50), 8.33 (2H, d, J ¼ 8.8 Hz, Ph), 8.18 (1H, s,
H4), 7.92 (2H, d, J ¼ 7.6 Hz, Ph), 7.86 (1H, d, J ¼ 8.5 Hz, H7), 7.76 (1H,
dd, J ¼ 8.6, 1.4 Hz, H6), 7.62 (2H, t, J ¼ 7.7 Hz, Ph), 7.56e7.45 (1H, m,
Ph), 7.37 (2H, d, J ¼ 8.9 Hz, Ph), 5.40 (2H, s, CH2).13C NMR (151 MHz,
J ¼ 8.9 Hz, Ph), 8.26e8.14 (3H, m, H4, Ph), 8.02 (2H, d, J ¼ 8.8 Hz, Ph),
7.88 (1H, J ¼ 8.7 Hz, H7), 7.78 (1H, dd, J ¼ 8.5,1.6 Hz, H6), 7.39 (2H, d,
J ¼ 8.9 Hz, Ph), 5.44 (2H, s, OCH2). 13C NMR (75 MHz, DMSO)
d
166.02, 160.91, 153.73, 144.04, 140.38, 139.48, 137.43, 130.04,
DMSO)
d 165.45, 162.11, 151.80, 143.22, 136.56, 136.20, 132.72,
129.78; 129.35; 128.92; 128.91 (q, JCF ¼ 32.4 Hz), 129.50, 127.59,
127.54; 127.50; 127.45 (q, JCF ¼ 3.7 Hz), 127.45, 125.81; 122.21 (d,
JCF ¼ 272 Hz), 123.64, 123.22, 122.59, 120.98, 120.16, 115.84, 114.73,
61.38. Anal. calcd. for C24H18F3N7O x 2 HCl x 2.6 H2O (Mr ¼ 597.21):
C 48.27, H 4.25, N 16.42; found: 48.52, H 4.32, N 16.18%. HRMS:
calcd. for C24H18F3N7O (M þ H)þ: 478.1603; found: 478.1611.
130.57, 130.03, 128.95, 124.96, 124.56, 123.34, 120.29, 116.18, 116.00,
114.71, 114.33, 61.52. Anal. calcd. for C23H19N7O x 2 HCl x 5 H2O
(Mr ¼ 572.45): C 48.26, H 5.46, N 17.13; found: C 48.53, H 5.76, N
17.28%. HRMS: calcd. for C23H19N7O (M þ H)þ: 410.1729; found:
410.1740.
4.2.2.2. 2-(4-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-1H-benzo[d]imidazole-5-carboximidamide
hydrochloride
4.2.2.6. 2-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-
(9b). Compound 9b was prepared using the above described
method from 3b (200 mg, 0.67 mmol) and o-phenylenediamine 4
(101.0 mg, 0.67 mmol) to obtain 9b as brown powder (87 mg, 27%,
benzo[d]imidazole-5-carboximidamide
hydrochloride
(9f).
Compound 9f was prepared using the above described method
from 3f (90 mg, 0.31 mmol) and o-phenylenediamine 4 (46.56 mg,
0.31 mmol) to obtain 9f as grey powder (140.6 mg, 90%,
m.p. ¼ 218e220 ꢀC). 1H NMR (300 MHz, DMSO)
d 9.37 (2H, bs, NH),
9.02e8.94 (3H, m, NH, H50), 8.28 (2H, d, J ¼ 8.7 Hz, Ph), 8.15 (1H, s,
H4), 7.90 (2H, d, J ¼ 8.7 Hz, Ph), 7.83 (1H, d, J ¼ 8.4 Hz, H7), 7.72 (1H,
d, J ¼ 8.0 Hz, H6), 7.35 (2H, d, J ¼ 8.9 Hz, Ph), 7.28 (2H, d, J ¼ 8.7 Hz,
m.p. ¼ 204e206 ꢀC). 1H NMR (300 MHz, DMSO)
d 9.48 (2H, s, NH),
9.12 (2H, s, NH), 8.40e8.32 (3H, m H50, Ph), 8.20 (1H, s, H4), 7.89
(1H, d, J ¼ 8.5 Hz, H7), 7.80 (1H, dd, 1H, J ¼ 8.6 Hz, H6), 7.47e7.27
(7H, m, Ph), 5.64 (2H, s, CH2), 5.30 (2H, s, CH2).13C NMR (75 MHz,
Ph), 5.38 (2H, s, CH2). 13C NMR (75 MHz, DMSO)
d 165.84, 163.34;
160.08 (d, JCF ¼ 245.8 Hz), 162.83, 153.50, 143.42, 143.23, 133.07,
131.81, 129.95, 129.22, 123.43, 122.66; 122.54 (d, JCF ¼ 8.8 Hz),
116.61; 116.92 (d, JCF ¼ 23.3 Hz),115.48,115.24, 61.35. Anal. calcd. for
DMSO) d 166.27, 161.19, 153.97, 142.94, 140.49, 137.57, 136.19, 129.61,
129.27, 128.74, 128.43, 125.37, 123.43, 122.83, 120.07, 116.01, 115.93,
114.95, 61.63, 53.39. Anal. calcd. for C24H21N7O x 2 HCl x 0.3 H2O
(Mr ¼ 501.80): C 47.44, H 4.74, N 19.54; found: 47.71, H 4.58, N
19.27%. HRMS: calcd. for C24H21N7O (M þ H)þ: 424.1886; found:
424.1881.
C
23H18FN7O x 2 HCl x 3 H2O (Mr ¼ 554.41): C 49.83, H 4.73, N 17.68;
found: C 49.82, H 4.78, N 17.54%. HRMS: calcd. for C23H18FN7O
(M þ H)þ: 428.1635; found: 428.1619.
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13
4.2.2.7. N-Isopropyl-2-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-1H-benzo[d]imidazole-5-carboximidamide hydrochloride
(10a). Compound 10a was prepared using the above described
method from 3a (200 mg, 0.72 mmol) and o-phenylenediamine 5
(114.2 mg, 0.72 mmol) to obtain 10a as brown powder (144.5 mg,
d 162.18,161.05,152.95,147.73,143.61,142.84,138.75,136.25,131.12,
129.59, 124.25, 123.67, 123.17, 122.21, 115.76, 114.38, 94.60, 61.35,
45.21, 21.32. Anal. calcd. for C26H24IN7O x 2 HCl x 1.3 H2O
(Mr ¼ 673.77): C 46.35, H 4.28, N 14.55; found: C 46.68, H 4.33, N
14.84%. HRMS: calcd. for C26H24IN7O (M þ H)þ: 578.1165; found:
578.1140.
34%, m.p. ¼ 194e197 ꢀC). 1H NMR (300 MHz, DMSO)
d 9.65 (1H, d,
J ¼ 7.8 Hz, NH), 9.50 (1H, s, NH), 9.10e9.01 (2H, m, NH, H50), 8.38
(2H, d, J ¼ 8.5 Hz, Ph), 8.08 (1H, s, H4), 7.99e7.82 (3H, m, H7, Ph),
7.56e7.48 (1H, m, H6), 7.39 (2H, d, J ¼ 8.7 Hz, Ph), 5.41 (2H, s, OCH2),
4.22e3.97 (1H, m, CH), 1.31 (6H, d, J ¼ 6.2 Hz, CH3CCH3).13C NMR
4.2.2.11. N-Isopropyl-2-(4-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole-5-
carboximidamide hydrochloride (10e). Compound 10e was prepared
using the above described method from 3e (200 mg, 0.58 mmol)
and o-phenylenediamine 5 (78.1 mg, 0.52 mmol) to obtain 8e as
brown powder (139.4 mg, 34%, m.p. ¼ 204e206 ꢀC). 1H NMR
(75 MHz, DMSO)
d 162.81, 160.88, 153.96, 143.85, 140.65, 138.07,
136.81, 130.46, 129.43, 123.87, 123.55, 123.27, 120.84, 120.65, 116.01,
114.86, 61.52, 45.54, 21.58. Anal. calcd. for C26H25N7O x 2 HCl x 3.75
H2O (Mr ¼ 592.01): C 52.75, H 5.87, N 16.56; found: 52.78, H 5.72, N
16.50%. HRMS: calcd. for C26H25N7O (M þ H)þ: 452.2199; found:
452.2202.
(300 MHz, DMSO)
d
9.69 (1H, d, J ¼ 7.7 Hz, NH), 9.53 (1H, s, NH),
9.20 (1H, s, H50), 9.11 (1H, s, NH), 8.43 (2H, d, J ¼ 8.6 Hz, Ph), 8.20
(2H, d, J ¼ 8.4 Hz, Ph), 8.09 (1H, s, H4), 8.01 (2H, d, J ¼ 8.5 Hz, Ph),
7.89 (1H, d, J ¼ 8.5 Hz, H7), 7.70 (1H, d, J ¼ 8.4 Hz, H6), 7.40 (2H, d,
J ¼ 8.7 Hz, Ph), 5.44 (2H, s, CH2), 4.17e4.03 (1H, m, CH), 1.31 (6H, d,
4.2.2.8. 2-(4-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-N-isopropyl-1H-benzo[d]imidazole-5-carboximidamide hy-
drochloride (10b). Compound 10b was prepared using the above
described method from 3b (200 mg, 0.67 mmol) and o-phenyl-
enediamine 5 (119.2 mg, 0.67 mmol) to obtain 10b as white powder
(243.7 mg, 60%, m.p. ¼ 214e216 ꢀC). 1H NMR (300 MHz, DMSO)
J ¼ 6.3 Hz, CH3CCH3).13C NMR (75 MHz, DMSO)
d 162.51, 160.80,
153.58, 144.05, 140.13, 139.48, 137.46, 132.13, 129.38, 129.07;
128.92; 128.49; 128.22 (q, JCF ¼ 33.0 Hz), 127.60; 127.55; 127.50;
127.45 (q, JCF ¼ 3.8 Hz), 125.81; 122.20 (d, JCF ¼ 272.4 Hz) 123.84,
123.63, 123.28, 120.97, 120.42, 115.83, 114.61, 61.36, 45.33, 21.45.
Anal. calcd. for C27H24F3N7O x 2 HCl x 3.75 H2O (Mr ¼ 696.47): C
49.47, H 5.07, N 14.96; found: C 49.56, H 4.99, N 14.88%. HRMS:
calcd. for C27H24F3N7O (M þ H)þ: 520.2073; found: 520.2059.
d
9.65 (1H, d, J ¼ 7.6 Hz, NH), 9.50 (1H, s, NH), 9.07 (1H, s, NH), 9.01
(1H, s, H50), 8.38 (2H, d, J ¼ 8.6 Hz, Ph), 8.08 (1H, s, H4), 8.02e7.93
(2H, m, Ph), 7.87 (1H, d, J ¼ 8.5 Hz, H7), 7.67 (1H, dd, J ¼ 8.5, 1.1 Hz,
H6), 7.48 (2H, t, J ¼ 8.8 Hz, Ph), 7.38 (2H, d, J ¼ 8.8 Hz, Ph), 5.40 (2H,
s, OCH2), 4.31e3.94 (1H, m, CH), 1.32 (6H, d, J ¼ 6.3 Hz, CH3CCH3).
4.2.2.12. N-Isopropyl-2-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)
methoxy)phenyl)-1H-benzo[d]imidazole-5-carboximidamide hydro-
chloride (10f). Compound 10f was prepared using the above
described method from 3f (200 mg, 0.68 mmol) and o-phenyl-
enediamine 5 (120.5 mg, 0.68 mmol) to obtain 10f as brown
powder (195.9 mg, 53%, m.p. ¼ 198e201 ꢀC). 1H NMR (600 MHz,
13C NMR (75 MHz, DMSO)
d
163.47; 160.21 (d, JCF ¼ 245.9 Hz),
162.21, 160.99, 153.03, 143.48, 133.18; 133.14 (d, JCF ¼ 2.9 Hz),
130.09, 129.55, 124.12, 123.53, 122.79; 122.67 (d, JCF ¼ 8.8 Hz),
119.45, 117.06; 116.75 (d, JCF ¼ 23.3 Hz), 115.74, 115.49, 114.41, 61.36,
45.21, 21.35. Anal. calcd. for C26H24FN7O x 2 HCl x 3.5 H2O
(Mr ¼ 605.40): C 51.57, H 5.49, N 16.19; found: C 51.57, H 5.24, N
16.48%. HRMS: calcd. for C26H24FN7O (M þ H)þ: 470.2105; found:
470.2082.
DMSO)
d
9.75 (1H, d, J ¼ 7.7 Hz, NH), 9.59 (1H, s, NH), 9.21 (1H, s,
NH), 8.51 (2H, d, J ¼ 8.5 Hz, Ph), 8.37 (1H, s, H50), 8.12 (1H, d,
J ¼ 0.9 Hz, H4), 7.90 (1H, d, J ¼ 8.5 Hz, H7), 7.74 (1H, dd, J ¼ 8.3,
1.1 Hz, H6), 7.40e7.32 (7H, m, Ph), 5.64 (2H, s, CH2), 5.31 (2H, s,
4.2.2.9. 2-(4-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-N-isopropyl-1H-benzo[d]imidazole-5-carboximidamide hy-
drochloride (10c). Compound 10c was prepared using the above
described method from 3c (200 mg, 0.64 mmol) and o-phenyl-
enediamine 5 (113.4 mg, 0.64 mmol) to obtain 10c as brown
powder (92.7 mg, 25%, m.p. ¼ 218e220 ꢀC). 1H NMR (300 MHz,
CH2), 4.17e4.10 (1H, m, CH), 1.31 (6H, d, J ¼ 6.4 Hz, CH3CCH3).13
C
NMR (75 MHz, DMSO)
d 165.63, 161.85, 152.32, 147.18, 145.76,
142.39, 142.36, 135.95, 129.81, 129.71, 128.77, 128.17, 127.98, 124.97,
124.58, 124.04, 115.62, 114.17, 61.41, 52.85, 45.12, 21.25. Anal. calcd.
for C27H27N7O x 2 HCl x 0.2 H2O (Mr ¼ 542.08): C 59.82, H 5.47, N
18.09; found: C 59.81, H 5.79, N 18.41%. HRMS: calcd. for C27H27N7O
(M þ H)þ: 466.2355; found: 466.2374.
DMSO)
d
9.65 (1H, d, J ¼ 7.8 Hz, NH), 9.50 (1H, s, NH), 9.07 (2H, s, NH
and H50), 8.38 (2H, d, J ¼ 8.6 Hz, Ph), 8.08 (1H, s, H4), 7.98 (2H, d,
J ¼ 8.8 Hz, Ph), 7.87 (1H, d, J ¼ 8.4 Hz, H7), 7.73e7.64 (3H, m, H6, Ph),
7.37 (2H, d, J ¼ 8.8 Hz, Ph), 5.40 (2H, s, OCH2), 4.16e3.95 (1H, m, CH),
4.2.2.13. N-Isopropyl-2-(4-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-1H-benzo[d]imidazole-5-carboximidamide hy-
drochloride (10g). Compound 10g was prepared using the above
described method from 3g (200 mg, 0.61 mmol) and o-phenyl-
enediamine 5 (117.32 mg, 0.61 mmol) to obtain 10g as white
powder (184.6 mg, 53%, m.p. ¼ 183e186 ꢀC). 1H NMR (300 MHz,
1.31 (6H, d, J ¼ 6.3 Hz, CH3CCH3).13C NMR (75 MHz, DMSO)
d 161.95,
161.10, 152.53, 143.46, 135.31, 133.10, 129.90, 129.72, 126.48, 124.35,
123.82, 123.29, 121.89, 116.35, 61.30, 45.10, 21.26. Anal. calcd. for
C
26H24ClN7O x 2 HCl x 0.75 H2O (Mr ¼ 572.41): C 54.56, H 4.84, N
17.13; found:
C
C
54.25,
H
4.92,
N
17.16%. HRMS: calcd. for
DMSO)
d
9.84 (1H, d, J ¼ 7.8 Hz, NH), 9.67 (1H, s, NH), 9.30 (1H, s,
26H24ClN7O (M þ H)þ: 486.1809; found: 486.1818.
NH), 8.60 (2H, d, J ¼ 8.8 Hz, Ph), 8.40 (1H, s, H50), 8.15 (1H, s, H4),
7.96 (1H, d, J ¼ 8.5 Hz, H7), 7.80 (1H, dd, J ¼ 8.5,1.0 Hz, H6), 7.45 (2H,
d, J ¼ 8.4 Hz, Ph), 7.41e7.30 (4H, m, Ph), 5.65 (2H, s, CH2), 5.33 (2H, s,
4.2.2.10. 2-(4-((1-(4-Iodophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-N-isopropyl-1H-benzo[d]imidazole-5-carboximidamide hy-
drochloride (10d). Compound 10d was prepared using the above
described method from 3d (200 mg, 0.49 mmol) and o-phenyl-
enediamine 5 (86.8 mg, 0.49 mmol) to obtain 10d as brown powder
(93.2 mg, 28%, m.p. ¼ 219e221 ꢀC). 1H NMR (300 MHz, DMSO)
CH2), 4.22e4.07 (1H, m, CH), 1.31 (6H, d, J ¼ 6.3 Hz, CH3CCH3).13
C
NMR (75 MHz, DMSO)
d 162.18, 161.50, 151.06, 142.28, 135.19,
134.94, 132.90, 131.94, 130.68, 129.98, 128.78, 125.88, 125.36, 125.13,
115.82, 115.64, 114.52, 113.93, 61.52, 52.05, 45.26, 21.25. Anal. calcd.
for C27H26ClN7O x 2 HCl x 0.2 H2O (Mr ¼ 576.53): C 56.25, H 4.97, N
d
9.70 (1H, d, J ¼ 7.6 Hz, NH), 9.55 (1H, s, NH), 9.16 (1H, s, NH), 9.07
17.00; found: C 56.21, H 4.16, N 16.88%. HRMS: calcd. for
(1H, s, H50), 8.46 (2H, d, J ¼ 8.6 Hz, Ph), 8.09 (1H, s, H4), 7.97 (2H, d,
J ¼ 8.7 Hz, Ph), 7.88 (1H, d, J ¼ 8.5 Hz, H7), 7.79e7.67 (3H, m, H6, Ph),
7.38 (2H, d, J ¼ 8.8 Hz, Ph), 5.40 (2H, s, CH2), 4.18e4.06 (1H, m, CH),
1.31 (6H, d, J ¼ 6.3 Hz, CH3CCH3).13C NMR (151 MHz, DMSO)
C
27H26ClN7O (M þ H)þ: 500.1966; found: 500.1956.
4.2.2.14. 5-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-((1-phenyl-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole
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14
hydrochloride (11a). Compound 11a was prepared using the above
described method from 3a (200 mg, 0.72 mmol) and o-phenyl-
enediamine 6 (127.6 mg, 0.64 mmol) to obtain 11a as white powder
(199.6 mg, 50%, m.p. ¼ 171e173 ꢀC). 1H NMR (600 MHz, DMSO)
4.2.2.18. 5-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-((1-(4-(tri-
fluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-
benzo[d]imidazole hydrochloride (11e). Compound 11e was pre-
pared using the above described method from 3e (200 mg,
0.58 mmol) and 6 (92.2 mg, 0.52 mmol) to obtain 11e as white
powder (77.4 mg, 22%, m.p. ¼ 169e171 ꢀC). 1H NMR (300 MHz,
d
10.69 (2H, s, NH), 9.02 (1H, s, H50), 8.38 (1H, s, H4), 8.33 (2H, d,
J ¼ 8.5 Hz, Ph), 7.94e7.86 (4H, m, H6, H7 and Ph), 7.62 (2H, t,
J ¼ 7.8 Hz, Ph), 7.52 (1H, t, J ¼ 7.4 Hz, Ph), 7.36 (2H, d, J ¼ 8.7 Hz, Ph),
5.40 (2H, s, OCH2), 4.03 (4H, s, CH2CH2). 13C NMR (151 MHz, DMSO)
DMSO)
d
10.50 (2H, s, NH), 9.17 (1H, s, H50), 8.32e8.18 (5H, m, H4,
Ph), 8.02 (2H, d, J ¼ 8.7 Hz, Ph), 7.83 (2H, s, H7 and H6), 7.34 (2H, d,
d
165.12, 160.80, 153.69, 143.34, 138.43, 137.80, 136.50, 129.89,
J ¼ 8.9 Hz, Ph), 5.41 (2H, s, OCH2), 4.05 (4H, s, CH2CH2). 13C NMR
129.45, 128.79, 123.30, 123.11, 121.39, 120.17, 116.35, 115.51, 115.46,
114.92, 61.30, 44.28. Anal. calcd. for C25H21N7O x 2 HCl x 2.5 H2O
(Mr ¼ 553.45): C 54.25, H 5.10, N 17.72; found: 54.71, H 4.95, N
17.62%. HRMS: calcd. for C25H21N7O (M þ H)þ: 436.1886; found:
436.1880.
(151 MHz, DMSO) d 165.12, 160.71, 153.74, 143.77, 139.27, 138.56,
135.63, 131.77, 129.42, 129.10; 128.88; 128.67; 128.45 (q,
JCF ¼ 32.4 Hz),127.24; 127.22; 127.19; 127.17 (q, JCF ¼ 3.5 Hz),124.66;
122.86 (d, JCF ¼ 272.4 Hz), 123.38, 123.26, 121.04, 120.65, 116.28,
115.50, 115.22, 61.22, 44.28. Anal. calcd. for C26H20F3N7O x 2 HCl x 2
H2O (Mr ¼ 612.44): C 50.99, H 4.28, N 16.01; found: 51.13, H 4.21, N
15.87%. HRMS: calcd. for C26H20F3N7O (M þ H)þ: 504.1760; found:
504.1741.
4.2.2.15. 5-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-((1-(4-
fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]
imidazole hydrochloride (11b). Compound 11b was prepared using
the above described method from 3b (200 mg, 0.67 mmol) and 6
(106.87 mg, 0.60 mmol) to obtain 11b as brown powder (103.3 mg,
4.2.2.19. 2-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-
(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole hydrochloride
(11f). Compound 11f was prepared using the above described
method from 3f (125 mg, 0.43 mmol) and 6 (70.9 mg, 0.40 mmol) to
obtain 11f as brown powder (130.2 mg, 51%, m.p. ¼198e201 ꢀC).1H
27%, m.p. ¼ 215e218 ꢀC). 1H NMR (600 MHz, DMSO)
d 10.74 (2H, s,
NH), 9.00 (1H, s, H50), 8.40 (1H, s, H4), 8.36 (2H, d, J ¼ 8.6 Hz, Ph),
8.00e7.92 (3H, m, H7, Ph), 7.89 (1H, d, J ¼ 8.5 Hz, H6), 7.47 (2H, t,
J ¼ 8.7 Hz, Ph), 7.36 (2H, d, J ¼ 8.7 Hz, Ph), 5.39 (2H, s, CH2), 4.03 (4H,
NMR (600 MHz, DMSO)
d
10.92 (2H, s, NH), 8.49 (1H, s, H50), 8.44
(2H, d, J ¼ 8.5 Hz, Ph), 8.38 (1H, s, H4), 8.04 (1H, d, J ¼ 8.3 Hz, H7),
7.96 (1H, d, J ¼ 8.5 Hz, H6), 7.41e7.32 (7H, m, Ph), 5.64 (2H, s, CH2),
5.31 (2H, s, CH2), 4.04 (4H, s, CH2CH2). 13C NMR (75 MHz, DMSO)
s, CH2CH2).13C NMR (75 MHz, DMSO)
d 165.41, 163.41, 160.15, d,
JCF ¼ 245.9 Hz), 160.54, 154.25, 143.51, 143.29, 134.22, 133.15; 133.11
(d, JCF ¼ 2.9 Hz), 129.59, 129.17, 123.42, 122.84, 122.72; 122.60 (d,
JCF ¼ 8.8 Hz), 120.76, 116.99; 116.68 (d, JCF ¼ 23.3 Hz), 116.18, 115.87,
115.53, 61.28, 44.35. Anal. calcd. for C25H20FN7O x 2 HCl x 1.7 H2O
(Mr ¼ 563.66): C 59.91, H 4.60, N 17.60; found: C 59.73, H 4.66, N
17.93%. HRMS: calcd. for C25H20FN7O (M þ H)þ: 454.1792; found:
454.1805.
d
165.03, 161.48, 153.21, 142.49, 139.07, 136.02, 135.62, 129.93,
128.89, 128.31, 128.09, 125.08, 124.13, 118.25, 117.30, 115.73, 114.81,
61.50, 52.98, 44.46. Anal. calcd. for C26H23N7O x 2 HCl x 2.4 H2O
(Mr ¼ 594.93): C 55.21, H 5.31, N 17.33; found: C 52.13, H 4.99, N
17.58%. HRMS: calcd. for C26H23N7O (M þ H)þ: 405.2042; found:
405.2029.
4.2.2.20. 2-(4-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-5-(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole
hydrochloride (11g). Compound 11g was prepared using the above
described method from 3g (200 mg, 0.61 mmol) and 6 (139.52 mg,
0.61 mmol) to obtain 11g as white powder (321.3 mg, 92%,
4.2.2.16. 2-(4-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-5-(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole
hydrochloride (11c). Compound 11c was prepared using the above
described method from 3c (200 mg, 0.64 mmol) and 6 (112.98 mg,
0.64 mmol) to obtain 11c as brown powder (227.3 mg, 63%,
m.p. > 250 ꢀC). 1H NMR (600 MHz, DMSO)
d 10.89 (2H, s, NH), 8.47
m.p. ¼ 207e209 ꢀC). 1H NMR (300 MHz, DMSO)
d 10.77 (s, 1H), 9.06
(1H, s, H50), 8.42 (2H, d, J ¼ 8.2 Hz, Ph), 8.38 (1H, s, H4), 8.02 (d,
J ¼ 8.2 Hz, 1H), 7.92 (d, J ¼ 8.3 Hz, 1H), 7.45 (d, J ¼ 8.0 Hz, 1H), 7.36
(d, J ¼ 8.0 Hz, 1H), 7.33 (d, J ¼ 8.4 Hz, 1H), 5.64 (s, 2H), 5.30 (s, 1H),
(s, 1H), 8.42 (s, 1H), 8.37 (d, J ¼ 8.7 Hz, 1H), 8.03e7.92 (m, J ¼ 10.4,
7.3 Hz, 3H), 7.69 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 8.7 Hz, 1H), 5.40 (s,
1H), 4.04 (s, 2H).13C NMR (75 MHz, DMSO)
d 165.47, 160.44, 154.37,
4.03 (s, 4H).13C NMR (75 MHz, DMSO)
d 187.52, 165.05, 161.09,
143.68, 135.37, 133.78, 133.37, 133.19, 131.90, 129.98, 129.10, 123.27,
122.72, 121.97, 121.04, 115.74, 115.52, 115.31, 61.25, 44.35. Anal.
calcd. for C25H20ClN7O x 2 HCl x 1.3 H2O (Mr ¼ 566.27): C 53.03, H
4.38, N 17.31; found: C 52.82, H 4.33, N 17.33%. HRMS: calcd. for
153.47,142.52,135.50,134.96,134.46,132.92,129.98,129.63,128.80,
126.27, 125.03, 123.65, 116.73, 115.56, 114.77, 110.63, 61.39, 52.07,
44.34. Anal. calcd. for C26H22ClN7O x 2 HCl x 0.7 H2O (Mr ¼ 569.49):
C 54.84, H 4.49, N 17.22; found: C 55.09, H 4.20, N 17.28%. HRMS:
calcd. for C26H22ClN7O (M þ H)þ: 484.1653; found: 484.1638.
C
25H20ClN7O (M þ H)þ: 470.1496; found: 470.1492.
4.2.2.17. 5-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-((1-(4-iodophenyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole hydro-
chloride (11d). Compound 11d was prepared using the above
described method from 3d (200 mg, 0.49 mmol) and 6 (87.5 mg,
0.49 mmol to obtain 11d as brown powder (95.0 mg, 28%,
4.2.3. General procedure for the synthesis of compounds 12c and
13c
The reaction mixture of 4-triazolylbenzaldehyde derivative (3c),
o-phenylenediamine (7 or 8) and 40% NaHSO3 was dissolved in
15 mL EtOH and stirred under reflux for 6e8 h. After completition
of the reaction NaHSO3 was filtered and the reaction mixture was
evaporated to dryness. Water was added (5 mL) and the mixture
was stirred over night and filtered. The crude residue was dissolved
in acetone addition of water resulted in precipitation of products
12c and 13c.
m.p. ¼ 152e154 ꢀC). 1H NMR (600 MHz, DMSO)
d 10.67 (2H, s, NH),
9.04 (1H, s, H50), 8.37 (1H, s, H4), 8.31 (2H, d, J ¼ 8.6 Hz, Ph), 7.97
(2H, d, J ¼ 8.6 Hz, Ph), 7.90 (1H, d, J ¼ 8.1 Hz, H7), 7.85 (1H, d,
J ¼ 8.4 Hz, H6), 7.75 (2H, d, J ¼ 8.6 Hz, Ph), 7.34 (2H, d, J ¼ 8.7 Hz, Ph),
5.38 (2H, s, CH2), 4.03 (4H, s, CH2CH2). 13C NMR (75 MHz, DMSO)
d
165.17, 160.58, 153.89, 143.56, 138.60, 136.15, 129.29, 128.42,
126.56, 123.04, 122.04, 120.87, 116.04, 115.45, 94.52, 61.23, 44.27.
Anal. calcd. for C25H20IN7O x 2 HCl x 1.9 H2O (Mr ¼ 688.53): C 44.91,
H 3.89, N 14.66; found: C 45.18, H 3.93, N 14.82%. HRMS: calcd. for
4.2.3.1. 2-(4-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-1H-benzo[d]imidazole (12c). Compound 12c was prepared
using the above described method from 3c (200 mg, 0.64 mmol)
and 7 (69.21 mg, 0.64 mmol) to obtain 12c as white crystals
C
25H20IN7O (M þ H)þ: 562.0852; found: 562.0850.
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A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
15
(205.8 mg, 80%, m.p. > 250 ꢀC). 1H NMR (300 MHz, DMSO)
d
12.76
to room temperature. The reaction mixture was diluted with EtOAc,
washed with water and brine. The combined extracts were dried
over MgSO4, filtered, concentrated and purified by column chro-
matography with CH2Cl2:CH3OH ¼ 50:1 [51].
(1H, s, NH), 9.03 (1H, s, H50), 8.14 (2H, d, J ¼ 8.8 Hz, Ph), 7.97 (2H, d,
J ¼ 8.8 Hz, Ph), 7.69 (2H, d, J ¼ 8.8 Hz, Ph), 7.60e7.52 (2H, m, H5, H6),
7.26 (2H, d, J ¼ 8.8 Hz, Ph), 7.21e7.14 (2H, m, H4, H7), 5.34 (2H, s,
CH2).13C NMR (75 MHz, DMSO)
d 159.27, 151.20, 143.80, 135.34,
133.06, 129.88, 128.02, 123.14, 123.10, 121.87, 121.79, 115.15, 61.12.
Anal. calcd. for C22H16ClN5O x 2.6 H2O (Mr ¼ 448.69): C 58.89, H
4.76, N 15.61; found: C 58.62, H 4.55, N 15.67%. HRMS: calcd. for
4.2.5.1. 1-(4-Chlorophenyl)-1H-1,2,3-triazole-4-carbaldehyde (17).
Compound 17 was prepared using the above mentioned procedure
from 15 (165 mg, 0.79 mmol). After purification by column chro-
matography 17 was obtained as white powder (101.5 mg, 62%;
C
22H16ClN5O (M þ H)þ: 402.1122; found: 402.1134.
m.p. ¼165e169 ꢀC). 1H NMR (300 MHz, DMSO)
d 10.12 (1H, s, CHO),
9.60 (1H, s, H50), 8.03 (2H, d, J ¼ 8.9 Hz, Ph), 7.73 (2H, d, J ¼ 8.9 Hz,
4.2.3.2. 5-Chloro-2-(4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methoxy)phenyl)-1H-benzo[d]imidazole (13c). Compound 13c was
prepared using the above described method from 3c (200 mg,
0.64 mmol) and 7 (90.90 mg, 0.64 mmol) to obtain 13c as light
brown powder (185 mg, 66%, m.p. > 250 ꢀC) 1H NMR (600 MHz,
Ph).13C NMR (75 MHz, DMSO)
d 184.97, 147.59, 134.79, 133.89,
129.94, 126.30, 122.47. MS (m/z) 208.0 [MþH]þ.
4.2.5.2. 3-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)propanal (18).
Compound 18 was prepared using the above mentioned procedure
from 16 (150 mg, 0.63 mmol). After purification by column chro-
matography 17 was obtained as white powder (96.1 mg, 65%;
DMSO)
d
12.96 (1H, bs, NH), 9.01 (1H, s, H50), 8.13 (2H, d, J ¼ 8.6 Hz,
Ph), 7.97 (2H, d, J ¼ 8.7 Hz, Ph), 7.68 (2H, d, J ¼ 8.7 Hz, Ph), 7.60 (1H,
s, H4), 7.56 (1H, d, J ¼ 8.2 Hz, H7), 7.27 (2H, d, J ¼ 8.6 Hz, Ph), 7.20
(1H, dd, J ¼ 8.6, 1.3 Hz, H6), 5.35 (2H, s, CH2).13C NMR (151 MHz,
m.p. ¼ 57e62 ꢀC). 1H NMR (600 MHz, DMSO)
d 9.77 (1H, s, CHO),
DMSO)
d 159.54, 152.60, 143.73, 135.31, 133.05, 129.84, 128.21,
8.60 (1H, s, H50), 7.90 (2H, d, J ¼ 8.8 Hz, Ph), 7.66 (2H, d, J ¼ 8.9 Hz,
126.10, 123.07, 122.54, 122.01, 121.84, 115.21, 61.14. Anal. calcd. for
Ph), 2.98 (1H, t, J ¼ 7.1 Hz, CH2), 2.89 (2H, t, J ¼ 7.2 Hz, CH2).13C NMR
C
22H15Cl2N5O x 0.6 H2O (Mr ¼ 476.21): C 59.10, H 3.65, N 15.66;
(75 MHz, DMSzzO)
d 202.36, 147.03, 135.50, 132.63, 129.83, 121.45,
found: C 58.95, H 3.55, N 15.63%. HRMS: calcd. for C22H15Cl2N5O
120.43, 41.81, 17.85. MS (m/z) 236.0 [MþH]þ.
(M þ H)þ: 436.0732; found: 436.0743.
4.2.6. General procedure for the synthesis of compounds 19a, 19b
and 20a
4.2.4. General procedure for the synthesis of 1-(4-chlorophenyl)-
1,2,3-triazolyl alcohols 15 and 16
The reaction mixture 1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)
aldehyde derivatives (17 or 18), o-phenylenediamine (5 or 6) and
40% NaHSO3 was dissolved in 15 mL EtOH and stirred under reflux
for 6e8 h. After completition of the reaction NaHSO3 was filtered
and the reaction mixture was evaporated to dryness. Water was
added (5 mL) and the mixture was stirred over night and filtered.
The crude residue was dissolved in HCl saturated EtOH (8e10 mL)
and stirred over night. Addition of ether resulted in precipitation of
products 19a, 19b and 20a Solid was collected by filtration, washed
with anhydrous ether, and dried under vacuum.
1-Azido-4-chlorobenzene (1 eq), the corresponding terminal
alkyne (propargyl alcohol or 4-pentyn-1-ol, 1 eq) and Cu(OAc)2
(0.05 eq) were dissolved in methanol. The reaction mixture was
placed in an ultrasonic bath cleaner (1000 W, 35 kHz) at 55 ꢀC for
1.5 h. The solvent was removed under reduced pressure and puri-
fied by column chromatography with CH2Cl2: MeOH ¼ 50: 1.
4.2.4.1. (1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methanol
(15).
Compound 15 was prepared using the above mentioned procedure
from propargyl alcohol (0.07 mL, 1.2 mmol) and 1-azido-4-
chlorobenzene (2.76 mL, 1.2 mmol). After purification by column
chromatography 15 was obtained as white powder (184.4 mg, 88%;
4.2.6.1. 2-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)-N-isopropyl-
1H-benzo[d]imidazole-5-carboximidamide hydrochloride (19a).
Compound 19a was prepared using the above described method
from 17 (200 mg, 0.96 mmol) and 5 (204.87 mg, 0.96 mmol) to
obtain 19a as brown powder (280.7 mg, 77%, m.p. > 250 ꢀC). 1H
m.p. ¼ 145e146 ꢀC). 1H NMR (300 MHz, DMSO)
d
8.71 (1H, s, H50),
7.95 (2H, d, J ¼ 8.9 Hz, Ph), 7.66 (2H, d, J ¼ 8.9 Hz, Ph), 5.34 (1H, t,
J ¼ 5.6 Hz, OH), 4.61 (2H, d, J ¼ 5.5 Hz, CH2OH).13C NMR (75 MHz,
DMSO) d 149.30,135.53, 132.71, 129.83, 121.59, 121.06, 54.91. MS (m/
z) 210.0 [MþH]þ.
NMR (300 MHz, DMSO)
d
9.67 (1H, s, NH), 9.56 (1H, d, J ¼ 8.2 Hz,
NH), 9.41 (1H, s, NH), 9.02 (1H, s, H50), 8.15e7.97 (3H, m, H4, Ph),
7.84e7.69 (3H, m, Ph, H7), 7.59 (1H, d, J ¼ 8.8 Hz, H6), 4.24e3.90
(1H, m, CH), 1.31 (6H, d, J ¼ 5.8 Hz, CH3CCH3).13C NMR (75 MHz,
4.2.4.2. 3-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)propan-1-ol
(16). Compound 16 was prepared using the above mentioned
procedure from 4-pentyn-1-ol (0.11 mL, 1.2 mmol) and 1-azido-4-
chlorobenzene (2.76 mL, 1.38 mmol). After purification by column
chromatography 16 was obtained as white powder (235 mg, 99%;
DMSO)
d 162.39, 146.62, 139.75, 135.07, 133.58, 129.95, 123.37,
123.10, 122.50, 122.25, 45.00, 21.29. Anal. calcd. for C19H18ClN7 x 2
HCl x 5.2 H2O (Mr ¼ 546.45): C 41.76, H 5.61, N 17.94; found: C
42.00, H 5.24, N 17.88%. HRMS: calcd. for C19H18ClN7 (M þ H)þ:
380.1390; found: 380.1375.
m.p. ¼ 96e99 ꢀC). 1H NMR (300 MHz, DMSO)
d
8.60 (1H, s, H50),
7.92 (2H, d, J ¼ 8.9 Hz, Ph), 7.66 (2H, d, J ¼ 8.9 Hz, Ph), 4.53 (1H, t,
J ¼ 5.1 Hz, OH), 3.48 (2H, dd, J ¼ 11.6, 6.2 Hz, CH2CH2CH2OH), 2.74
(2H, t,
J
¼
7.7 Hz, CH2CH2CH2OH), 1.93e1.71 (1H, m,
4.2.6.2. 2-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)-5-(4,5-
CH2CH2CH2OH).13C NMR (75 MHz, DMSO)
d
148.24, 135.59, 132.51,
dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole
hydrochloride
129.80, 121.41, 120.18, 59.96, 32.02, 21.63. MS (m/z) 238.1 [MþH]þ.
(19b). Compound 19b was prepared using the above described
method from 17 (150 mg, 0.72 mmol) and 6 (165.24 mg, 0.72 mmol)
to obtain 19b as brown powder (135 mg, 52%, m.p. > 250 ꢀC). 1H
4.2.5. General procedure for the synthesis of 1-(4-chlorophenyl)-
1,2,3-triazolyl aldehydes 17 and 18
NMR (300 MHz, DMSO)
(1H, bs, H4), 8.10 (2H, d, J ¼ 7.4 Hz, Ph), 7.93e7.68 (4H, m, H6, H7,
Ph), 4.03 (4H, s,CH2CH2).13C NMR (75 MHz, DMSO)
165.39, 139.70,
d
10.62 (2H, s, NH), 9.70 (1H, s, H50), 8.39
DMSO (8 mmol) was added dropwise to a solution of oxalyl
chloride (4 mmol) in CH2Cl2 (4 mL) at ꢁ78 ꢀC. After 2 min, a so-
lution of the corresponding 1,2,3-triazolyl alcohol 15 or 16 (1 mmol)
in 0.11 mL CH2Cl2 was added slowly over 5 min. After stirring for an
additional 15 min Et3N (8 mmol) was added to the reaction mixture
and stirred at ꢁ78 ꢀC for 5 min and then the mixture was warmed
d
135.06, 133.60, 129.97, 129.82, 123.52, 122.68, 122.20, 121.77, 115.69,
44.26. Anal. calcd. for C18H14ClN7 x 2 HCl x 7 H2O (Mr ¼ 562.84): C
38.41, H 5.37, N 17.40; found: C 38.37, H 5.25, N 17.64%. HRMS: calcd.
for C18H14ClN7 (M þ H)þ: 364.1077; found: 364.1083.
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Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
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16
4.2.6.3. 2-(2-(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)ethyl)-N-iso-
propyl-1H-benzo[d]imidazole-5-carboximidamide hydrochloride
compounds at their 2 ꢂ IC50 concentrations for 48 h. Cells were
lysed in RIPA buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% NP-40 and 1% sodium
deoxycholate supplemented with protease inhibitor cocktail
(20a). Compound 20a was prepared using the above described
method from 18 (180 mg, 0.76 mmol) and 5 (162.44 mg, 0.76 mmol)
to obtain 20a as white powder (150.3 mg, 48%, m.p. ¼ 199e201 ꢀC).
(Roche). Total proteins (50 mg) were resolved on 12% Tris-glycine
1H NMR (600 MHz, DMSO)
d
9.63 (1H, d, J ¼ 8.5 Hz, NH), 9.48 (1H, s,
polyacrylamide gels and transferred to PVDF membranes. Subse-
quently, membranes were blocked for 1 h at room temperature
with 4% BSA in TBST [50 mmol/L Tris base, 150 mmol/L NaCl, 0.1%
Tween 20 (pH 7.5)] and probed overnight at 4 ꢀC with primary
antibody against either PDE5 (Cell Signaling Technology), CDK9/
cyclin T1 (Cell Signaling Technology), TGM2 ((Cell Signaling Tech-
nology), p-p53 (Ser15) (Abcam), p-SK1 (ECM Biosciences) and p-38
MAPK (Thr180/Tyr182). Membranes were washed with TBST and
incubated with either goat anti-mouse (Santa Cruz Biotechnology)
or goat anti-rabbit (Santa Cruz Biotechnology) horseradish perox-
idaseeconjugated secondary antibody at room temperature for 1 h.
Individual proteins were visualized by the BM Chemiluminescence
Western Blotting Substrate (POD) (Roche) using ImageQuant LAS
500 (GE Healthcare). Densitometry quantitation was determined
using the Quantity One 1-D Analysis Software (Bio-Rad, USA).
NH), 9.08 (1H, s, NH), 8.74 (1H, s, H50), 8.09 (1H, s, H4), 7.90 (2H, d,
J ¼ 8.9 Hz, Ph), 7.87 (1H, d, J ¼ 8.4 Hz, H7), 7.71 (1H, d, J ¼ 8.5 Hz,
H6), 7.66 (2H, d, J ¼ 8.8 Hz, Ph), 4.13e4.05 (1H, m, CH), 3.55 (2H, t,
J ¼ 7.2 Hz,CH2), 3.42 (2H, t, J ¼ 7.3 Hz, CH2), 1.31 (6H, d, J ¼ 6.4 Hz,
CH3CCH3).13C NMR (75 MHz, DMSO)
d 161.67, 156.58, 145.87, 141.03,
138.06, 135.41, 132.79, 129.87, 126.47, 124.30, 121.54, 120.91, 114.14,
45.15, 26.56, 22.66, 21.22. Anal. calcd. for C21H22ClN7 x 2 HCl x 1.5
H2O (Mr ¼ 507.85): C 49.67, H 5.36, N 19.31; found: C 49.73, H 5.32,
N 18.98%. HRMS: calcd. for C21H22ClN7 (M þ H)þ: 408.1703 found:
408.1687.
4.3. Cell culturing
Human cell lines A549 (lung carcinoma), HeLa (cervical carci-
noma), SW620 (colorectal adenocarcinoma, metastatic) and CFPAC-
1 (pancreatic cancer, derived from metastatic: liver) as well as WI38
(normal human lung fibroblasts) were cultured as monolayers and
maintained in Dulbecco's modified Eagle medium (DMEM) sup-
4.6. Apoptosis detection
Detection and quantification of apoptosis and differentiation
from necrosis at single cell level was carried out by Annexin-V-FITC
Staining kit (Santa Cruz Biotech) according to the manufacturer's
instructions. Briefly, cells were seeded into Lab-tek II Chamber Slide
with 8 wells and treated with test compounds at their 2 ꢂ IC50
concentrations for 48 h. The cells were washed with Incubation
buffer, and Annexin-V-FITC labelling solution was added. After in-
cubation at room temperature for 15 min, chambers and silicon
borders of the chamber slides were removed, the cells were fixed
with 20% glycerol and analyzed by fluorescence microscopy.
plemented with 10% fetal bovine serum (FBS), 2 mM
L-glutamine,
100 U/ml penicillin and 100
mg/mL streptomycin in a humidified
atmosphere with 5% CO2 at 37 ꢀC.
4.4. Proliferation assays
The panel cell lines were inoculated onto a series of standard 96-
well microtiter plates on day 0, at 5000 cells per well according to
the doubling times of specific cell line. Test agents were then added
in five, 10-fold dilutions (0.01e100 mM) and incubated for further
72 h. Working dilutions were freshly prepared on the day of testing
in the growth medium. The solvent (DMSO) was also tested for
eventual inhibitory activity by adjusting its concentration to be the
same as in the working concentrations (DMSO concentration never
exceeded 0.1%). After 72 h of incubation, the cell growth rate was
evaluated by performing the MTT assay: experimentally deter-
mined absorbance values were transformed into a cell percentage
growth (PG) using the formulas proposed by NIH and described
previously [73]. This method directly relies on control cells
behaving normally at the day of assay because it compares the
growth of treated cells with the growth of untreated cells in control
wells on the same plate - the results are therefore a percentile
difference from the calculated expected value. The IC50 and LC50
values for each compound were calculated from dose-response
curves using linear regression analysis by fitting the mean test
concentrations that give PG values above and below the reference
value. If, however, all of the tested concentrations produce PGs
exceeding the respective reference level of effect (e.g. PG value of
50) for a given cell line, the highest tested concentration is assigned
as the default value (in the screening data report that default value
is preceded by a “>” sign). Each test point was performed in
quadruplicate in three individual experiments. The results were
statistically analyzed (ANOVA, Tukey post-hoc test at p < 0.05).
Finally, the effects of the tested substances were evaluated by
plotting the mean percentage growth for each cell type in com-
parison to control on dose response graphs.
4.7. In silico analysis
Predictions of plausible biological targets and pharmacological
activities were made by web-service PASS (http://www.
pharmaexpert.ru/passonline/index.php) which is based on the
identification of substructure features typical for active molecules
[52,74].
4.8. Computational methods
Available crystal structures of apo and co-crystalised p38-a ki-
nase with various inhibitors were downloaded from protein data
bank [75,76]. Due to the large number of available crystal structures
(384 structures), downloaded ligands were clustered by Canvas
similarity and clustering protocol [77,78] in order to obtain repre-
sentative but chemicaly diverse set for further anaylsis. Represen-
tative ligands from the following DFG-in X-ray structure were used:
3LFA, 3MW1, 4EH3, 4FA2, 4KIN, 4ZTH, 4R3C, 3ZSH, 3C5U, 3FMK,
3HP5, 3BX5, 3ZYA, 1ZZ2. Representative ligands from the following
DFG-out X-ray structures: 3HV6, 3HEC, 3LFE, 3IW8, 3P79, 3D83,
3K3J, 3P5K, 3P7A, 3P7b, 3P7C. Gly flip structures: 1OUK, 1OUY AND
1OVE for DFG-in conformation and 2YIW AND 4KIN for DFG-out
conformation. IC50 inhibition values were collected from Bind-
ingDB [79]. Field based alignment of bio-active conformations of
representative ligands was performed using Torch software from
Cresset [80]. Conformational search was performed for compounds
10c and 11f while p38 inhibitors from Protein data bank were fixed
in the bio-active conformation. Protein structure was used to define
excluded volume and further restrict possible alignment options.
The ligand docking studies were carried out using Glide docking
protocol [81e83] within Schroedinger suite of software [84] with
4.5. Western blot analysis
Cells were cultured in 6-well plates at seeding density of
200000 cells/well and subjected to treatment with selected
ꢀ
Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
10.1016/j.ejmech.2017.10.061

ꢀ
A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
17
ꢀ
[19] K. Kubinski, M. Masłyk, A. Orzeszko, Benzimidazole inhibitors of protein ki-
nase CK2 potently inhibit the activity of atypical protein kinase Rio1, Mol. Cell.
Biochem. 426 (2017) 195e203, https://doi.org/10.1007/s11010-016-2892-x.
extra precision (XP). Docking was performed using both con-
strained interactions with hinge amino-acids Met109 and Gly110 as
well as unconstrained docking. Binding poses were refined and
binding energy was estimated using MM-GBSA [85e87] protocol
and OPLS3 force-field with flexible residues distance being 5 Å.
ꢀ
[20] E. Łukowska-Chojnacka, P. Winska, M. Wielechowska, M. Poprzeczko,
M. Bretner, Synthesis of novel polybrominated benzimidazole deriva-
tivesepotential CK2 inhibitors with anticancer and proapoptotic activity,
Bioorg. Med. Chem. 24 (2016) 735e741, https://doi.org/10.1016/
j.bmc.2015.12.041.
[21] M.A. Pagano, M. Andrzejewska, M. Ruzzene, S. Sarno, L. Cesaro, J. Bain,
M. Elliott, F. Meggio, Z. Kazimierczuk, L.A. Pinna, Optimization of protein ki-
nase CK2 inhibitors derived from 4,5,6,7-tetrabromobenzimidazole, J. Med.
Chem. 47 (2004) 6239e6247, https://doi.org/10.1021/jm049854a.
[22] Y.A. Sonawane, M.A. Taylor, J.V. Napoleon, S. Rana, J.I. Contreras, A. Natarajan,
Cyclin dependent Kinase 9 inhibitors for cancer therapy: miniperspective,
Acknowledgements
We greatly appreciate the financial support of the Croatian
Science Foundation (grant number IPe2013e11e5596), University
of Rijeka research grants 13.11.1.1.11. and 13.11.2.1.12., and the access
to equipment owned by the University of Rijeka within the project
RISK “Development of University of Rijeka campus laboratory
research infrastructure”, financed by the European Regional
Development Fund.
J.
Med.
Chem.
59
(2016)
8667e8684,
https://doi.org/10.1021/
acs.jmedchem.6b00150.
[23] L. Carlino, G. Rastelli, Dual Kinase-bromodomain inhibitors in anticancer drug
discovery: a structural and pharmacological perspective: miniperspective,
J.
Med.
Chem.
59
(2016)
9305e9320,
https://doi.org/10.1021/
acs.jmedchem.6b00438.
[24] Z.A. Knight, H. Lin, K.M. Shokat, Targeting the cancer kinome through poly-
pharmacology, Nat. Rev. Cancer 10 (2010) 130e137, https://doi.org/10.1038/
nrc2787.
Appendix A. Supplementary data
[25] N.P. Shah, J.M. Nicoll, B. Nagar, M.E. Gorre, R.L. Paquette, J. Kuriyan, C.L.
Sawyers, Multiple BCR-ABL kinase domain mutations confer polyclonal
resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase
and blast crisis chronic myeloid leukemia. Cancer Cell 2 (202) 117e125. doi:
10.1016/S1535-6108(02)00096-X.
[26] J.A. Engelman, K. Zejnullahu, T. Mitsudomi, Y. Song, C. Hyland, J.O. Park,
N. Lindeman, C.M. Gale, X. Zhao, J. Christensen, T. Kosaka, MET amplification
leads to gefitinib resistance in lung cancer by activating ERBB3 signaling,
Science 316 (2007) 1039e1043, https://doi.org/10.1126/science.1141478.
[27] M. Hasegawa, N. Nishigaki, Y. Washio, K. Kano, P.A. Harris, H. Sato, I. Mori,
R.I. West, M. Shibahara, H. Toyoda, L. Wang, Discovery of novel benzimid-
azoles as potent inhibitors of TIE-2 and VEGFR-2 tyrosine kinase receptors,
J. Med. Chem. 50 (2007) 4453e4470, https://doi.org/10.1021/jm0611051.
[28] J. Akhtar, A.A. Khan, Z. Ali, R. Haider, M.S. Yar, Structure-activity relationship
(SAR) study and design strategies of nitrogen-containing heterocyclic moi-
eties for their anticancer activities, Eur. J. Med. Chem. 125 (2017) 143e189,
https://doi.org/10.1016/j.ejmech.2016.09.023.
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2017.10.061.
References
[1] M. Reck, D.F. Heigener, T. Mok, J.C. Soria, K.F. Rabe, Management of non-small-
cell lung cancer: recent developments, Lancet 382 (2013) 709e719, https://
doi.org/10.1016/S0140-6736(13)61502-0.
[2] F.R. Khuri, Lung cancer and other pulmonary neoplasms, in: G. Lee,
I.S. Andrew (Eds.), Goldman's Cecil Medicine, 25th ed, Saunders, New York,
2016, pp. 1303e1313.
ꢂ
[3] C.C. Tieche, R.W. Peng, P. Dorn, L. Froment, R.A. Schmid, T.M. Marti, Prolonged
pemetrexed pretreatment augments persistence of cisplatin-induced DNA
damage and eliminates resistant lung cancer stem-like cells associated with
EMT, BMC Cancer 16 (2016) 125, https://doi.org/10.1186/s12885-016-2117-4.
[4] D.S. Ettinger, D.E. Wood, W. Akerley, L.A. Bazhenova, H. Borghaei,
D.R. Camidge, R.T. Cheney, L.R. Chirieac, T.A. D'Amico, T.J. Dilling,
M.C. Dobelbower, NCCN guidelines insights: nonesmall cell lung cancer,
version 4.2016, J. Natl. Compr. Canc. Netw. 14 (2016) 255e264, https://
doi.org/10.6004/jnccn.2016.0031.
[29] R. Determann, J. Dreher, K. Baumann, L. Preu, P.G. Jones, F. Totzke,
€
C. Schachtele, M.H. Kubbutat, C. Kunick, 2-Anilino-4-(benzimidazol-2-yl)
pyrimidineseA multikinase inhibitor scaffold with antiproliferative activity
toward cancer cell lines, Eur. J. Med. Chem. 53 (2012) 254e263, https://
doi.org/10.1016/j.ejmech.2012.04.007.
[30] B. Chu, F. Liu, L. Li, C. Ding, K. Chen, Q. Sun, Z. Shen, Y. Tan, C. Tan, Y. Jiang,
A benzimidazole derivative exhibiting antitumor activity blocks EGFR and
HER2 activity and upregulates DR5 in breast cancer cells, Cell Death Dis. 6
(2015) e1686, https://doi.org/10.1038/cddis.2015.25.
[31] Y. Li, C. Tan, C. Gao, C. Zhang, X. Luan, X. Chen, H. Liu, Y. Chen, Y. Jiang, Dis-
covery of benzimidazole derivatives as novel multi-target EGFR, VEGFR-2 and
PDGFR kinase inhibitors, Bioorg. Med. Chem. 19 (2011) 4529e4535, https://
doi.org/10.1016/j.bmc.2011.06.022.
[32] B.B. Hasinoff, X. Wu, J.L. Nitiss, R. Kanagasabai, J.C. Yalowich, The anticancer
multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisom-
erase II, Biochem. Pharmacol 84 (2012) 1617e1626, https://doi.org/10.1016/
j.bcp.2012.09.023.
[5] Y. Xiong, B.Y. Huang, J.Y. Yin, Pharmacogenomics of platinum-based chemo-
therapy in non-small cell lung cancer: focusing on DNA repair systems, Med.
Oncol. 34 (2017) 48e64, https://doi.org/10.1007/s12032-017-0905-6.
[6] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M.J. Thun, Cancer statistics, 2009, CA
Cancer J. Clin. 59 (2009) 225e249, https://doi.org/10.3322/caac.20006.
[7] E. Hafen, Kinases and phosphatases-a marriage is consummated, Science 280
(1998) 1212e1213, https://doi.org/10.1126/science.280.5367.1212.
[8] P. Cohen, The origins of protein phosphorylation, Nat. Cell Biol. 4 (2002)
e127ee130, https://doi.org/10.1038/ncb0502-e127.
[9] J.A. Ubersax, J.E. Ferrell, Mechanisms of specificity in protein phosphorylation,
Nat. Rev. Mol. Cell Biol. 8 (2007) 530e541, https://doi.org/10.1038/nrm2203.
[10] P. Blume-Jensen, T. Hunter, Oncogenic kinase signalling, Nature 411 (2001)
355e365, https://doi.org/10.1038/35077225.
[11] J. Brognard, T. Hunter, Protein kinase signaling networks in cancer, Curr. Opin.
Genet. Dev. 21 (2011) 4e11, https://doi.org/10.1016/j.gde.2010.10.012.
[12] P. Singla, V. Luxami, K. Paul, Benzimidazole-biologically attractive scaffold for
protein kinase inhibitors, RSC Adv. 4 (2014) 12422e12440.
[13] M. Munde, M. Lee, S. Neidle, R. Arafa, D.W. Boykin, Y. Liu, C. Bailly,
W.D. Wilson, Induced fit conformational changes of a “reversed amidine”
heterocycle: optimized interactions in a DNA minor groove complex, J. Am.
Chem. Soc. 129 (2007) 5688e5698, https://doi.org/10.1021/ja069003n.
[14] W.D. Wilson, B. Nguyen, F.A. Tanious, A. Mathis, J.E. Hall, C.E. Stephens,
D.W. Boykin, Dications that target the DNA minor groove: compound design
and preparation, DNA interactions, cellular distribution and biological activity,
ꢀ
ꢀ
ꢁ ꢀ
ꢀ
ꢀ
[33] T. Gregoric, M. Sedic, P. Grbcic, A.T. Paravic, S.K. Pavelic, M. Cetina, R. Vianello,
ꢀ
ꢀ
S. Raic-Malic, Novel pyrimidine-2,4-dionee1,2,3-triazole and furo [2,3-d]
pyrimidine-2-onee1,2,3-triazole hybrids as potential anti-cancer agents:
synthesis, computational and X-ray analysis and biological evaluation, Eur. J.
Med.
Chem.
125
(2017)
1247e1267,
https://doi.org/10.1016/
j.ejmech.2016.11.028.
[34] M. Liu, Y. Hou, W. Yin, S. Zhou, P. Qian, Z. Guo, L. Xu, Y. Zhao, Discovery of a
novel 6,7-disubstituted-4-(2-fluorophenoxy) quinolines bearing 1,2,3-
triazole-4-carboxamide moiety as potent c-Met kinase inhibitors, Eur. J.
Med.
Chem.
119
(2016)
96e108,
https://doi.org/10.1016/
j.ejmech.2016.04.035.
ꢁ
ꢀ
ꢁ
ꢀ
[35] M. Vojtickova, J. Dobias, G. Hanquet, G. Addova, R. Cetin-Atalay, D.C. Yildirim,
ꢀꢁ
A. Bohac, Ynamide Click chemistry in development of triazole VEGFR2 TK
Curr. Med. Chem. Anticancer Agents
10.2174/1568011054222319.
5 (2005) 389e408, https://doi.org/
modulators, Eur. J. Med. Chem. 103 (2015) 105e122, https://doi.org/10.1016/
j.ejmech.2015.08.012.
[15] P. Wu, T.E. Nielsen, M.H. Clausen, FDA-approved small-molecule kinase in-
hibitors, Trends Pharmacol. Sci. 36 (2015) 422e439, https://doi.org/10.1016/
j.tips.2015.04.005.
[16] L. Garuti, M. Roberti, G. Bottegoni, Benzimidazole derivatives as kinase in-
hibitors, Curr. Med. Chem. 21 (2014) 2284e2298, https://doi.org/10.2174/
0929867321666140217105714.
[17] W. Akhtar, M.F. Khan, G. Verma, G.M. Shaquiquzzaman, M.A. Rizvi, S.H. Mehdi,
M. Akhter, M.M. Alam, Therapeutic evolution of benzimidazole derivatives in
the last quinquennial period, Eur. J. Med. Chem. 126 (2017) 705e753.
[18] M.J. Akhtar, A.A. Siddiqui, A.A. Khan, Z. Ali, R.P. Dewangan, S. Pasha, M.S. Yar,
Design, synthesis, docking and QSAR study of substituted benzimidazole
linked oxadiazole as cytotoxic agents, EGFR and erbB2 receptor inhibitors,
Eur. J. Med. Chem. 126 (2017) 853e869, https://doi.org/10.1016/
j.ejmech.2016.12.014.
[36] Y. Kommagalla, S. Cornea, R. Riehle, V. Torchilin, A. Degterev, C.V. Ramana,
Optimization of the anti-cancer activity of the phosphatidylinositol-3 kinase
pathway inhibitor PITENIN-1: switching thiourea with 1,2,3-triazole, Med-
ChemComm 5 (2014) 1359e1363, https://doi.org/10.1039/C4MD00109E.
[37] A. Lebeau, C. Abrioux, D. Benimelis, Z. Benfodda, P. Meffre, Synthesis of 1,4-
disubstituted 1,2,3-triazole derivatives using click chemistry and their Src
Kinase activities, Med. Chem. 13 (2014) 40e48, https://doi.org/10.2174/
1573406412666160404125718.
[38] A. Kumar, I. Ahmad, B.S. Chhikara, R. Tiwari, D. Mandal, K. Parang, Synthesis of
3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates and evaluation of their
Src kinase inhibitory and anticancer activities, Bioorg. Med. Chem. Lett. 21
(2011) 1342e1346, https://doi.org/10.1016/j.bmcl.2011.01.047.
[39] D. Kumar, V.B. Reddy, A. Kumar, D. Mandal, R. Tiwari, K. Parang, Click
ꢀ
Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
10.1016/j.ejmech.2017.10.061

ꢀ
A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
18
chemistry inspired one-pot synthesis of 1,4-disubstituted 1,2,3-triazoles and
their Src kinase inhibitory activity, Bioorg. Med. Chem. Lett. 21 (2011)
serine 15 in response to UV radiation, J. Biol. Chem. 275 (2000) 20444e20449,
https://doi.org/10.1074/jbc.M001020200.
449e452, https://doi.org/10.1016/j.bmcl.2010.10.121.
[40] Z.Y. Cheng, W.J. Li, F. He, J.M. Zhou, X.F. Zhu, Synthesis and biological evalu-
ation of 4-aryl-5-cyano-2H-1,2,3-triazoles as inhibitor of HER2 tyrosine ki-
[63] R. Sanchez-Prieto, J.M. Rojas, Y. Taya, J.S. Gutkind, A role for the p38 mitogen-
activated protein kinase pathway in the transcriptional activation of p53 on
genotoxic stress by chemotherapeutic agents, Cancer Res. 60 (2000)
2464e2472.
nase,
Bioorg.
Med.
Chem.
15
(2007),
https://doi.org/10.1016/
j.bmc.2006.09.041, 533e1538.
[64] A. Patnaik, P. Haluska, A.W. Tolcher, C. Erlichman, K.P. Papadopoulos,
J.L. Lensing, M. Beeram, J.R. Molina, D. Rasco, R.R. Arcos, C.S. Kelly, A first-in-
human phase i study of the oral p38 MAPK inhibitor, ralimetinib (LY2228820
Dimesylate), in patients with advanced cancer, Clin. Cancer Res. 22 (2016)
1095e1102, https://doi.org/10.1158/1078-0432.CCR-15-1718.
[65] R.S.K. Vijayan, P. He, V. Modi, K.C. Duong-Ly, H. Ma, J.R. Peterson,
R.L. Dunbrack Jr., R.M. Levy, Conformational analysis of the DFG-out kinase
motif and biochemical profiling of structurally validated type II inhibitors,
J. Med. Chem. 58 (2015) 466e479, https://doi.org/10.1021/jm501603h.
[66] C.E. Fitzgerald, S.B. Patel, J.W. Becker, P.M. Cameron, D. Zaller, V.B. Pikounis,
[41] H.C. Kolb, K.B. Sharpless, The growing impact of click chemistry on drug
discovery, Drug Discov. Today 8 (2003) 1128e1137, https://doi.org/10.1016/
S1359-6446(03)02933-7.
[42] M. Song, H. Hwang, C.Y. Im, S.Y. Kim, Recent progress in the development of
transglutaminase 2 (TGase2) inhibitors, J. Med. Chem. 60 (2017) 554e557,
https://doi.org/10.1021/acs.jmedchem.6b01036.
[43] C. Pardin, J.N. Pelletier, W.D. Lubell, J.W. Keillor, Cinnamoyl inhibitors of tissue
transglutaminase, J. Org. Chem. 73 (2008) 5766e5775, https://doi.org/
10.1021/jo8004843.
ꢀ
ꢀ
ꢀ
ꢀ
ꢁ
ꢀ
[44] T.G. Kraljevic, A. Harej, M. Sedic, S.K. Pavelic, V. Stepanic, D. Drenjancevic,
S.J. O'Keefe, G. Scapin, Structural basis for p38a MAP kinase quinazolinone and
pyridol-pyrimidine inhibitor specificity, Nat. Struct. Mol. Biol. 10 (2003)
764e769, https://doi.org/10.1038/nsb949.
ꢀ
ꢀ
J. Talapko, S. Raic-Malic, Synthesis, in vitro anticancer and antibacterial ac-
tivities and in silico studies of new 4-substituted 1, 2, 3-triazoleecoumarin
hybrids, Eur. J. Med. Chem. 124 (2016) 794e808, https://doi.org/10.1016/
j.ejmech.2016.08.062.
[67] D.S. Millan, M.E. Bunnage, J.L. Burrows, K.J. Butcher, P.G. Dodd, T.J. Evans,
D.A. Fairman, S.J. Hughes, I.C. Kilty, A. Lemaitre, R.A. Lewthwaite, Design and
synthesis of inhaled p38 inhibitors for the treatment of chronic obstructive
pulmonary disease, J. Med. Chem. 54 (2011) 7797e7814, https://doi.org/
10.1021/jm200677b.
[68] P.A. Greenidge, C. Kramer, J.C. Mozziconacci, R.M. Wolf, MM/GBSA binding
energy prediction on the PDBbind data set: successes, failures, and directions
for further improvement, J. Chem. Inf. Model 53 (2012) 201e209, https://
doi.org/10.1021/ci300425v.
ꢀ
ꢁ
ꢀ
ꢁ
ꢁ
ꢀ
[45] I. Stolic, K. Miskovic, I. Piantanida, M.B. Loncar, Lj Glavas-Obrovac, M. Bajic,
Synthesis, DNA/RNA affinity and antitumour activity of new aromatic dia-
midines linked by 3,4-ethylenedioxythiophene, Eur. J. Med. Chem. 46 (2011)
743e755, https://doi.org/10.1016/j.ejmech.2010.12.010.
[46] R.N. Baig, R.S. Varma, Alternative energy input: mechanochemical, microwave
and ultrasound-assisted organic synthesis, Chem. Soc. Rev. 41 (2012)
1559e1584, https://doi.org/10.1039/C1CS15204A.
[47] C.O. Kappe, E. Van der Eycken, Click chemistry under non-classical reaction
conditions, Chem. Soc. Rev. 39 (2010) 1280e1290, https://doi.org/10.1039/
B901973C.
[69] N.T. Chandrika, S.K. Shrestha, H.X. Ngo, S. Garneau-Tsodikova, Synthesis and
investigation of novel benzimidazole derivatives as antifungal agents, Bioorg.
Med.
Chem.
24
(2016)
3680e3686,
https://doi.org/10.1016/
[48] G. Cravotto, E.C. Gaudino, P. Cintas, On the mechanochemical activation by
ultrasound, Chem. Soc. Rev. 42 (2013) 7521e7534, https://doi.org/10.1039/
C2CS35456J.
j.bmc.2016.06.010.
[70] R. Kumar, J. Arora, A.K. Prasad, N. Islam, A.K. Verma, Synthesis and antimi-
crobial activity of pyrimidine chalcones, Med. Chem. Res. 22 (2013)
5624e5631, https://doi.org/10.1007/s00044-013-0555-y.
[71] P.V. Chavan, K.S. Pandit, U.V. Desai, M.A. Kulkarni, P.P. Wadgaonkar, Cellulose
supported cuprous iodide nanoparticles (Cell-CuI NPs): a new heterogeneous
and recyclable catalyst for the one pot synthesis of 1,4-disubstitutede1,2,3-
triazoles in water, RSC Adv. 4 (2014) 42137e42146, https://doi.org/10.1039/
C4RA05080K.
[49] K.A. Ohemeng, V.N. Nguyen, Ortho Pharmaceutical Corporation, 2-substituted
Phenyl-benzimidazole Antibacterial Agents. U.S. Patent Application 08/
924,558, 1999. WO1999011627A1.
ꢁ
ꢀ
[50] M. Hranjec, K. Starcevic, B. Zamola, S. Mutak, M. Derek, G. Karminski-Zamola,
New amidino-benzimidazolyl derivatives of tylosin and desmycosin,
J. Antibiot. 55 (2002) 308e314, https://doi.org/10.7164/antibiotics.55.308.
[51] N.R. Glover, G.C. MacDonald, J. Entwistle, J. Cizeau, D.G. Bosc, F.C. Chahal,
Viventia Bio Inc, Tumor Specific Antibody. U.S. Patent Application 14/604,047,
2015. WO2015/092713.
[72] N. Boechat, V.F. Ferreira, S.B. Ferreira, M.D.L.G. Ferreira, F.D.C. da Silva,
M.M. Bastos, M.D.S. Costa, M.C.S. Loureņco, A.C. Pinto, A.U. Krettli, A.C. Aguiar,
Novel 1, 2, 3-triazole derivatives for use against Mycobacterium tuberculosis
H37Rv (ATCC 27294) strain, J. Med. Chem. 54 (2011) 5988e5999, https://
doi.org/10.1021/jm2003624.
[52] D.A. Filimonov, V.V. Poroikov, in: A. Varnek, A. Tropsha (Eds.), Chemo-
informatics Approaches to Virtual Screening, RSC Publishing, Cambridge, UK,
2008, pp. 182e216.
[53] C.M. Whitehead, K.A. Earle, J. Fetter, S. Xu, T. Hartman, D.C. Chan, T.L. Zhao,
G. Piazza, A.J. Klein-Szanto, R. Pamukcu, H. Alila, Exisulind-induced apoptosis
in a non-small cell lung cancer orthotopic lung tumor model augments
docetaxel treatment and contributes to increased survival, Mol. Cancer Ther. 2
(2003) 479e488.
ꢀ
ꢀ
ꢁ
ꢀ
ꢀ
[73] T. Gazivoda, S. Raic-Malic, V. Kristafor, D. Makuc, J. Plavec, S. Kraljevic-Pavelic,
ꢀ
K. Pavelic, L. Naesens, G. Andrei, R. Snoeck, J. Balzarini, M. Mintas, Synthesis,
cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 py-
rimidine nucleoside analogues, Bioorg. Med. Chem. 16 (2008) 5624e5634,
https://doi.org/10.1016/j.bmc.2008.03.074.
[74] K.R. Patil, P. Mohapatra, H.M. Patel, S.N. Goyal, S. Ojha, C.N. Kundu, C.R. Patil,
[54] T. Yin, M.J. Lallena, E.L. Kreklau, K.R. Fales, S. Carballares, R. Torrres,
G.N. Wishart, R.T. Ajamie, D.M. Cronier, P.W. Iversen, T.I. Meier, A novel CDK9
inhibitor shows potent antitumor efficacy in preclinical hematologic tumor
models, Mol. Cancer Ther. 13 (2014) 1442e1456, https://doi.org/10.1158/
1535-7163.MCT-13-0849.
[55] A. Bagashev, S. Fan, R. Mukerjee, P. Paolo Claudio, T. Chabrashvili, R.P. Leng,
S. Benchimol, B.E. Sawaya, Cdk9 phosphorylates Pirh2 protein and prevents
degradation of p53 protein, Cell Cycle 12 (2013) 1569e1577, https://doi.org/
10.4161/cc.24733.
[56] S. Mishra, L.J. Murphy, The p53 oncoprotein is a substrate for tissue trans-
glutaminase kinase activity, Biochem. Bioph. Res. Co. 339 (2006) 726e730,
https://doi.org/10.1016/j.bbrc.2005.11.071.
[57] C.M. Choi, S.J. Jang, S.Y. Park, Y.B. Choi, J.H. Jeong, D.S. Kim, H.K. Kim, K.S. Park,
B.H. Nam, H.R. Kim, S.Y. Kim, Transglutaminase 2 as an independent prog-
nostic marker for survival of patients with non-adenocarcinoma subtype of
non-small cell lung cancer, Mol. Cancer 10 (2011) 119, https://doi.org/
10.1186/1476-4598-10-119.
Pentacyclic triterpenoids inhibit IKKb mediated activation of NF-kB pathway:
in silico and in vitro evidences, PLoS One 10 (2015), e0125709, https://doi.org/
10.1371/journal.pone.0125709.
[75] RSC PDB, http://www.rcsb.org/pdb/, 2017 (accessed 31.03.17).
[76] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, The protein data bank, Nucleic Acids Res. 28
(2000) 235e242, https://doi.org/10.1093/nar/28.1.235.
[77] J. Duan, S.L. Dixon, J.F. Lowrie, W. Sherman, Analysis and comparison of 2D
fingerprints: insights into database screening performance using eight
fingerprint methods, J. Mol. Graph. Model 29 (2010) 157e170, https://doi.org/
10.1016/j.jmgm.2010.05.008.
[78] M. Sastry, J.F. Lowrie, S.L. Dixon, W. Sherman, Large-scale systematic analysis
of 2D fingerprint methods and parameters to improve virtual screening en-
richments, J. Chem. Inf. Model 50 (2010) 771e784, https://doi.org/10.1021/
ci100062n.
[79] X. Chen, Y. Lin, M.K. Gilson, The binding database: overview and user's guide,
Biopolymers 61 (2001) 127e141.
[58] L. Song, H. Xiong, J. Li, W. Liao, L. Wang, J. Wu, M. Li, Sphingosine Kinase-1
[80] Torch, Version 10.4.1., Cresset, Litlington, Cambridgeshire, UK, http://www.
cresset-group.com/torch/.
enhances resistance to apoptosis through activation of PI3K/Akt/NF-kb
pathway in human nonesmall cell lung cancer, Clin. Cancer Res. 17 (2011)
1839e1849, https://doi.org/10.1158/1078-0432.CCR-10-0720.
[81] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood,
T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and
scoring incorporating a model of hydrophobic enclosure for proteinꢁligand
complexes, J. Med. Chem. 49 (2006) 6177e6196, https://doi.org/10.1021/
jm051256o.
[82] T.A. Halgren, R.B. Murphy, R.A. Friesner, H.S. Beard, L.L. Frye, W.T. Pollard,
J.L. Banks, Glide: a new approach for rapid, accurate docking and scoring. 2.
Enrichment factors in database screening, J. Med. Chem. 47 (2004)
1750e1759, https://doi.org/10.1021/jm030644s.
[59] L. Zhu, Z. Wang, Y. Lin, Z. Chen, H. Liu, Y. Chen, N. Wang, X. Song, Sphingosine
kinase 1 enhances the invasion and migration of non-small cell lung cancer
cells via the AKT pathway, Oncol. Rep. 33 (2015) 1257e1263.
ꢀ
[60] V. Schenten, C. Melchior, N. Steinckwich, E.J. Tschirhart, S. Brechard, Sphin-
gosine kinases regulate NOX2 activity via p38 MAPK-dependent translocation
of S100A8/A9, J. Leukoc. Biol. 89 (2011) 587e596, https://doi.org/10.1189/
jlb.0510304.
[61] M.M. Adada, K.A. Orr-Gandy, A.J. Snider, D. Canals, Y.A. Hannun, L.M. Obeid,
C.J. Clarke, Sphingosine kinase 1 regulates tumor necrosis factor-mediated
RANTES induction through p38 mitogen-activated protein kinase but inde-
[83] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz,
M.P. Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, Glide: a new
approach for rapid, accurate docking and scoring. 1. Method and assessment
of docking accuracy, J. Med. Chem. 47 (2004) 1739e1749, https://doi.org/
10.1021/jm0306430.
pendently of nuclear factor
kB activation, J. Biol. Chem. 288 (2013)
27667e27679, https://doi.org/10.1074/jbc.M113.489443.
€ €
[84] Schrodinger Release 2017-1, MacroModel, Schrodinger, LLC, New York, NY,
[62] Q.B. She, N. Chen, Z. Dong, ERKs and p38 kinase phosphorylate p53 protein at
ꢀ
Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
10.1016/j.ejmech.2017.10.061

ꢀ
A. Bistrovic et al. / European Journal of Medicinal Chemistry xxx (2017) 1e19
19
2017.
organic liquids, J. Am. Chem. Soc. 118 (1996) 11225e11236, https://doi.org/
10.1021/ja9621760.
[87] D. Shivakumar, J. Williams, Y. Wu, W. Damm, J. Shelley, W. Sherman, Pre-
diction of absolute solvation free energies using molecular dynamics free
energy perturbation and the OPLS force field, J. Chem. Theory Comput. 6
(2010) 1509e1519, https://doi.org/10.1021/ct900587b.
[85] J. Li, R. Abel, K. Zhu, Y. Cao, S. Zhao, R.A. Friesner, The VSGB 2.0 model: a next
generation energy model for high resolution protein structure modeling,
Proteins 79 (2011) 2794e2812, https://doi.org/10.1002/prot.23106.
[86] W.L. Jorgensen, D.S. Maxwell, J. Tirado-Rives, Development and testing of the
OPLS all-atom force field on conformational energetics and properties of
ꢀ
Please cite this article in press as: A. Bistrovic, et al., Design, synthesis and biological evaluation of novel benzimidazole amidines as potent
multi-target inhibitors for the treatment of non-small cell lung cancer, European Journal of Medicinal Chemistry (2017), https://doi.org/
10.1016/j.ejmech.2017.10.061