Novel benzimidazole-triazole hybrids as apoptosis inducing agents in lung cancer: Design, synthesis, 18F-radiolabeling & galectin-1 inhibition studies

Article abstract of DOI:10.1016/j.bioorg.2020.104125

In this study, we have synthesized a new series of benzimidazole-triazole hybrids as galectin-1 (gal-1) mediated apoptosis-inducing agents, and evaluated for their potential anticancer activity against a panel of human cancer cell lines viz. breast cancer

Full text of DOI:10.1016/j.bioorg.2020.104125

Journal Pre-proofs
Novel Benzimidazole-Triazole Hybrids as Apoptosis Inducing Agents in
Lung Cancer: Design, Synthesis, 18F-Radiolabeling & Galectin-1 Inhibition
Studies
Nerella Sridhar Goud, Venkatesh Pooladanda, K. Muni Chandra, P.S.
Lakshmi Soukya, Ravi Alvala, Pardeep Kumar, Chandana Nagraj, Rose Dawn
Bharath, Insaf A. Qureshi, Chandraiah Godugu, Mallika Alvala
PII:
S0045-2068(20)31422-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104125
YBIOO 104125
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Accepted Date:
2 June 2020
19 July 2020
Please cite this article as: N. Sridhar Goud, V. Pooladanda, K. Muni Chandra, P.S. Lakshmi Soukya, R. Alvala, P.
Kumar, C. Nagraj, R. Dawn Bharath, I.A. Qureshi, C. Godugu, M. Alvala, Novel Benzimidazole-Triazole
Hybrids as Apoptosis Inducing Agents in Lung Cancer: Design, Synthesis, 18F-Radiolabeling & Galectin-1
Inhibition Studies, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104125
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Novel Benzimidazole-Triazole Hybrids as Apoptosis Inducing Agents in Lung Cancer:
Design, Synthesis, 18F-Radiolabeling & Galectin-1 Inhibition Studies.
Nerella Sridhar Goud a, b, Venkatesh Pooladanda c, Muni Chandra K a, P.S. Lakshmi Soukya
d
b
b
b
a, Ravi Alvala , Pardeep Kumar , Chandana Nagraj , Rose Dawn Bharath , Insaf A.
Qureshi e, Chandraiah Godugu c, Mallika Alvala a*
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 03 7, India
b Department of Neuroimaging and Interventional Radiology (NI & IR), National Institute
of Mental Health and Neuro Sciences (NIMHANS), Bangalore 560 029, India.
c Department of Regulatory Toxicology, National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad 500 037, India
d G. Pulla reddy College of Pharmacy (GPRCP), Hyderabad 500 028, India
e Department of Biotechnology & Bioinformatics, School of Life Sciences, University of
Hyderabad (UOH), Hyderabad-500046, India.
*Corresponding Address:
Dr. Mallika Alvala, PhD
Department of Medicinal Chemistry
NIPER-Hyderabad
Email: mallikaalvala@yahoo.in
1

Abstract
In this study, we have synthesized a new series of benzimidazole-triazole hybrids as
galectin-1 (gal-1) mediated apoptosis-inducing agents, and evaluated for their potential
anticancer activity against a panel of human cancer cell lines viz. breast cancer (MCF-7 and
MDA-MB-231) lung cancer (A-549 and NCI-H460), and human keratinocyte cancer
(HaCaT), using MTT assay. The target compound 7c exhibited an excellent growth
inhibition against lung cancer (A-549 and NCI-H460) cells with an IC50 value of 0.63±0.21
µM, and 0.99±0.01 µM respectively. The target compound 7c also showed a significant
growth inhibition against breast cancer (MCF-7 and MDA-MB-23) with an IC50 value of
1.3±0.18 µM, and 0.94±0.02 µM respectively. In addition, the radiochemical synthesis has
been performed using fluorine-18 radionuclide in the GE Tracer-lab FX2N module to prove
the target compound 7c as a PET imaging agent. In the final stage, the 18F-7c target
compound was successfully purified with 60% ethanol in water. The radiochemical purity
was achieved > 95 % using HPLC, and the residual solvent DMF limit was around 78  3
ppm confirmed by GC analysis. Further, the apoptosis induction by 7c in lung cancer (A-
549) cells was confirmed as a result of the decrease in MMP levels, increased percentage of
apoptotic cells, and sub G1 phase arrest by JC-1 staining, DAPI staining, annexin V-
FITC/PI, and flow cytometric analysis. In addition, the target compound 7c significantly
reduced the gal-1 protein levels in a dose-dependent manner as confirmed by ELISA studies.
The protein binding studies like Surface Plasmon Resonance (SPR) and Fluorescence
Spectroscopy (FS) studies indicated that the target compound 7c is capable of binding to
gal-1 with an equilibrium constant (KD) value of 1.19E-06 M, and binding constant (Ka) of
9.5 x 103 M-1 respectively. The in-silico computational studies also revealed possible
interactions and pharmacokinetic properties (ADMET) of compound 7c with the binding
domain of gal-1. Therefore, the novel benzimidazole-triazole hybrids as apoptosis-inducing
agents in lung cancer would be potential cytotoxic and PET imaging agents via gal-1.
Key Words: benzimidazole-triazole, galectin-1, apoptosis, fluorine-18, positron emission
tomography.
1

Research Highlights
 The 7c is a potent cytotoxic agent against global major leading lung cancer burden.
 It has shown IC50 of 0.63±0.21 µM, and 0.99±0.01 µM in lung cancer cells (A-549
and NCI-H460) mediated by gal-1.
 The 7c is capable of binding with gal-1 based on the confirmation studies of SPR,
and FS.
 The 18F-7c radiotracer is considered as an ideal PET tracer due to its 110 min half-
life.
 The radiochemical purity & identity of 18F-7c was validated as per cGMP guidelines
for clinical evaluation.
1. Introduction
Cancer is the second major dreadful disease globally and, around 1 in 6 persons are being
diagnosed with cancer as per recent reports of the World Health Organization (WHO).
Globocan 2018 reported that the top three high frequent cancer types mainly lung, female
breast, and colorectal cancer, which are responsible for one-third of the cancer incidence
and mortality burden worldwide [1, 2]. The most commonly diagnosed cancer case is lung
cancer with the highest ratio of 11.6 % new cases and 18.4 % death cases followed by female
breast, and colorectal cancer globally. Lung cancer accelerated over the decades from 3 %
during 2008-09 to 11% 2019-20 [3]. Hence, it is necessary to develop highly effective and
selective chemotherapeutic agents for lung cancer treatment.
Galectin-1(gal-1), 14KDa protein belongs to galectin group of lectin family. It affects
cellular processes like extracellular & intracellular signaling pathways via protein-protein
interactions with other cytoplasmic, and nuclear proteins through specific binding to cell-
surface, and extracellular matrix glycans [4]. A wide variety of gal-1 biological phenomena
has been reported such as cell adhesion, proliferation, apoptosis, T cell receptor counter-
stimulation, immunomodulatory effects, cell cycle arrest, pre-B cell signaling, RNA splicing,
and promotion of H-Ras membrane anchorage etc. [5, 6]. Among the 15 mammalian
galectins, gal-1 is the first discovered protein in the family which is expressed ubiquitously
in the mammalian organism. It has been reported to overexpress/upregulated in many tumor
types including lung, astrocytoma, melanoma, prostate, thyroid, colon, bladder, and ovary
carcinomas [7, 8]. Gal-1 is regarded as a biomarker in various events like diagnosis, prognosis
and treatment condition of different tumor types [9]. Wei-an chang et al, reported the role of
1

galectins mainly gal-1 in lung cancer and indicated that gal-1 is highly expressed in invasive
lung cancer cells and leads to promote lung cancer cell migration and invasion [10].
Therefore, the development of novel anticancer agents via targeting gal-1 protein would be a
fruitful strategy in lung cancer treatment.
The Positron Emission Tomography (PET) is regarded as a remarkable molecular imaging
modality to study the pathophysiology, and pharmacological conditions in the animals,
human subjects, and to evaluating drug development [11]. The PET modality plays a wide
role in medical diagnosis, mainly in oncology, psychiatry and neurology. The major
advantage of PET is to measure very low concentration of radiotracers at a specific region of
interest in a living subject, either animal or human or, throughout a whole body [12].
Therefore, it is essential to focus on radiochemical synthesis and its quality control studies to
achieve good PET imaging agents for early diagnosis. Nevertheless, the availability of 11C,
13N, 15O, the 18F isotope is widely used radionuclide in the radiolabelling of various bioactive
molecules for PET imaging due to its positron-emitting property and suitable half-life of 110
min [13]. In clinical practice, the 18F-FDG (Fluorodeoxyglucose-FDG) is being used
routinely in the diagnosis of different tumor types & neurological diseases using PET
modality [14]. The clinically available anticancer drugs like doxorubicin, paclitaxel,
erlotinib, and gefitinib are also being used as PET imaging agents in human subjects, which
are successfully labeled with 18F radionuclide or technetium-99m etc. These drugs are
capable to exhibit their potential side effects when used as cytotoxic agents due to high
concentration, but still being used as PET tracers without major side effects due to nanomole
concentration range for PET diagnosis [15-18]. Here, our goal is to prove the feasibility of
the radiochemical synthesis of the target molecule in addition to its chemical synthesis,
computational studies, and in-vitro biological evaluation. Further to develop this 18F-7c tracer
as a real PET imaging agent for clinical use, the preclinical, toxicity and tracer distribution
studies need to be assessed.
Benzimidazole is a fused heterocyclic ring system consisting of benzene, and imidazole
rings, it forms an integral part of vitamin B12 [19]. It is known for its crucial role in numerous
diseases like cancer, microbial infection, tuberculosis, Alzheimer's etc. through various
mechanisms like apoptosis, microtubulin inhibition, DNA intercalation, protein kinase
inhibition, aromatase inhibition, and HSP90 inhibition [20-21]. Some USFDA approved
drugs contain benzimidazole nucleus are already well established and successfully used in
the treatment of multiple diseases [22]. The substitution pattern of benzimidazole is essential
1

for selectivity, and it is reported that the N-1, C-2, C-5, and C-6 positions are widely explored
for anticancer potentials [23-24]. Tsung-Chieh Shih et al. reported a novel gal-1 inhibitor
LLS30 (I), it has exhibited synergistic anti-cancer activity with docetaxel, and also
potentially inhibited the invasion and metastasis of prostate cancer in vivo [25]. The same
group had also reported another novel gal-1 inhibitor LLS2 (II), it was reported to potentiate
the anticancer potentials of Paclitaxel in vivo [26]. Recently, our research group reported 1-
benzyl-1H-benzimidazole derivatives as gal-1 mediated anticancer agents, and the target
compound (III) had shown a significant growth inhibition against breast cancer (MCF-7)
cells with an IC50 value of 7.01 ± 0.20 µM. The binding affinity of target compound with
gal-1 was confirmed by Fluorescence Spectroscopy (FS), and Surface Plasmon Resonance
(SPR) with their respective inhibition (KD and Ki) values [27]. A new series of morpholines
linked coumarin-triazole hybrids have been reported as potential anticancer agents via gal-1
inhibition, and the target compound (IV) exhibited a significant growth inhibition against
bone cancer (MG-63) cells with an IC50 of 0.80 ± 0.22 µM. The FS, and SPR studies
confirmed the binding of target compound to gal-1 [28] (Fig. 1).
<insert Figure 1 here>
The role of gal-1 in various stages of tumor development, and the importance of PET
modality for early diagnosis have triggered our interest to develop highly selective target-
specific anticancer agents. Hence, the current study mainly focused on novel benzimidazole-
triazole hybrids as apoptosis-inducing agents in lung cancer, their design, synthesis, 18F-
radiolabeling & galectin-1 inhibition studies.
2. Results and discussion
2.1. Chemistry
The target benzimidazole-triazole hybrids (7a-t) were synthesized by utilizing Huisgen 1,3-
dipolar cycloaddition reaction of terminal alkyne of benzimidazole intermediate (6) with
various benzyl (or) phenyl azides in the presence of copper(I)-catalyst (Scheme 1) [29].
Initially, the N-benzyl-4-chloro-2-nitroaniline (3) was synthesized through a green approach
using 4-chloro-2-nitro aniline with benzyl bromide. Further, the compound (3) was reduced
with stannous chloride dehydrate offered N1-benzyl-4-chlorobenzene-1,2-diamine (4) [30].
In the next step, the 4-hydroxy benzaldehyde was reacted with N1-benzyl-4-chlorobenzene-
1,2-diamine (4) using ethanol in the presence of sodium metabisulphite furnished 4-(1-
benzyl-6-chloro-1H-benzo[d]imidazol-2-yl) phenol intermediate (5) [31], it had further
1

offered 1-benzyl-6-chloro-2-(4-(prop-2-yn-1-yloxy) phenyl)-1H-benzo[d]imidazole (6)
upon treatment with propargyl bromide in the presence of potassium bromide using acetone.
Finally, the 1,3-dipolar cycloaddition of benzimidazole intermediate (6) with various benzyl
(or) phenyl azides furnishing target compounds 7a-t with moderate to good yields.
<Insert scheme 1 here>
1
All synthesized compounds (7a-t) were characterized by spectral techniques viz. H-
NMR, 13C NMR and HRMS. The 1H NMR spectrum of 7a showed characteristic protons of
O- and N-attached methylene groups at δ 5.60 and 5.33 ppm respectively. All the remaining
protons appeared in the range of δ 7.00-8.98 ppm. 13C NMR spectrum showed the
characteristic O- and N-attached methylene carbons at δ 61.63 and δ 48.23 ppm respectively,
and all the remaining carbons appeared in the range of δ 112.95-159.86. A similar pattern
was observed for the rest of the compounds (7a-t).
2.2. Radiochemistry
The radiochemical synthesis is an essential task, and it shows a major impact on the
quality of PET images for the diagnosis, and the quality control studies like colour
appearance, pH, purity, residual solvents, stability and, sterility etc. must be passed as per
the limitations of the Food Drug Administration (FDA) Q3C(R6) and Current Good
Manufacturing Practice (cGMP) guidelines for clinical use. Dolle et al, have reported
18
different methods and reaction conditions of F labeled fluoropyridines through aromatic
and, heteroaromatic nucleophilic substitution mechanism, and reported the favourable
conditions like an activated Kriptofix K[18F] F-K222 complex and aprotic solvent like
Dimethyl Sulfoxide (DMSO) with the no-carrier-added, at high temperature around 120-150
°C for 2-3 minutes for 18F radiolabeling of pyridine by replacing chlorine atom at C-2
position, and offered high radiochemical yields [32].
18
Here, we adopted the above method of radiochemical synthesis for F radiolabeling of
the target molecule 7c with a chloro substitution at the C-6 position through the aromatic
nucleophilic substitution mechanism, and succeeded with high radiochemical purity and
18
good yields. Initially, the [18F] radionuclide was generated through O (p, n)18F nuclear
reaction in the cyclotron. The radiochemical synthesis was carried out in the GE tracer lab
FX2N synthesis module using 350  20 mCi of 18F radionuclide from the cyclotron. In the
hot cell, the reaction was started with trapping of 18F radionuclide in the Quaternary
1

Ammonium Anion exchange (QMA) cartridge and further, it was eluted by kryptofix (K222)
solution through QMA into the reactor vial (Fig. 2). The activated Kriptofix K[18F] F-K222
complex was dried at a high temperature of 90 °C for 10 minutes. Finally, the radiolabeling
18
of F-7c was carried out by reacting the precursor molecule (7c) in Dimethylformamide
(DMF) with dried Kriptofix K[18F] F-K222 complex (Scheme-2) [33].
<Insert Figure 2 here>
<Insert scheme 2 here>
After completion of synthesis, the purification was achieved by washing the reaction mixture
with purified warer followed by treatment with 20%, and 40% ethanol, and the maximum
elution was achieved with 60% ethanol solution. The radiochemical purity was confirmed
by the High-Pressure Liquid Chromatography (HPLC) analytical method, and the traces of
free 18F radionuclide always appears at low retention time, and the target compound 18F-7c
appeared at 9.830 min using radio detector (Fig. 3A), whereas the cold target compound
retention time was found to be 9.267 min by HPLC method using Uv detector (Fig. 3B).
Thus, almost equal retention time of 18F-7c with its cold target compound validated the 18F
radiolabeling with the precursor molecule (7c), and proven it was our radiolabeled molecule
of interest 18F-7c. The yield of the 18F-7c was around 6  1% after purification through HLB
cartridge, and the radiochemical purity was achieved > 95 % by HPLC method. The stability
studies were also performed post synthesis 1 hr, 2 hr, and found that there were no additional
metabolite peaks of the final product 18F-7c on the HPLC chromatogram indicated that the
18F-7c is metabolically stable. The Dimethylformamide (DMF) residual solvent levels were
found to be 78  3 ppm in the final formulation using Gas Chromatography (GC) analysis
(Fig. 4), the DMF levels were within the acceptable limits as per the guidelines of Food
Drug Administration (FDA) Q3C(R6) because the DMF is a class-II solvent, and the
recommended limit is 880 ppm. The ethanol residual solvent was found to be in higher ppm
concentration in GC analysis since it was an eluting solution. The European Medicines
Agency (EMA), and United States Food and Drug Administration (USFDA) had found a
greater ppm concentration of ethanol in the final formulation of many radiopharmaceuticals,
it might be considerable as which are synthesized, and radiolabeled in cGMP conditions and
the final radiopharmaceutical formulations are further diluted with saline or water to reduce
the radioactive concentration [34]. Therefore, our study successfully proved the
radiolabeling of synthesized 7c with 18F radionuclide, and the final radiolabeled 18F-7c
1

would be a potential PET imaging against upregulated gal-1 protein in lung cancer through
validating further confirmation studies like preclinical, toxicity and tracer distribution
studies.
<Insert Figure 3A & 3B here>
<Insert Figure 4 here>
2.3. Biological evaluation
2.3.1. In vitro cytotoxic activity
The synthesized benzimidazole-triazole derivatives (7a-t) were screened for their in vitro
cytotoxicity against a panel of human cancer cell lines viz. breast cancer (MCF-7 and MDA-
MB-231) lung cancer (A-549 and NCI-H460), and Human keratinocyte cancer (HaCaT),
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [35]. The
IC50 (µM) values of synthesized compounds (7a-t) and the standard, 5-fluorouracil are
displayed in Table-1. Among all, the compound 7c had shown significant growth inhibition
with an IC50 of 1.3±0.18µM, 0.99±0.01 µM, 0.94±0.02 µM, 0.63±0.21µM, 2.99±0.09 µM
against MCF-7, NCI-H460, MDA-MB-231, A-549, and HaCaT cell lines respectively with
8.43% inhibition on normal Rattus Norvegicus Kidney (NRK) cells. Further mechanistic
studies of 7c were carried in A-549 cell line at different concentrations based on its potency
towards lung cancer cell lines.
The Structural activity relationship (SAR) revealed that all synthesized compounds had
shown excellent to good activity in all selected cancer cell lines. The compounds namely
7b, 7c, 7f, 7g, 7i and 7n exhibited prominent growth inhibition against breast cancer (MCF-7
and MDA-MB-231) lung cancer (A-549 and NCI-H460), and Human keratinocyte cancer
(HaCaT) in the range of 0.63-8.58 µM. The target compound 7c with 3-hydroxy phenyl
substitution exhibited an excellent growth inhibition against lung cancer (A-549 and NCI-
H460) cells with an IC50 value of 0.63±0.21 µM, and 0.99±0.01 µM respectively. It also
showed a significant growth inhibition against breast cancer (MCF-7 and MDA-MB-23)
with an IC50 value of 1.3±0.18 µM, and 0.94±0.02 µM respectively. The compound 7g with
3-fluoro substitution exhibited good cytotoxicity against lung cancer (A-549) cells with an
IC50 value of 1.11±0.12 µM, whereas, another potent compound 7n with 4-nitro substitution
had shown good cytotoxicity against lung cancer (NCI-H460) and Human keratinocyte
cancer (HaCaT) cells with an IC50 value of 1.98±0.08 µM and 1.35±0.09 µM respectively.
The compound 7f with 4-fluoro substitution had shown good cytotoxicity against breast
1

cancer (MCF-7) cells with an IC50 value of 1.21±0.17 µM. The compound 7i with 4-nitro-
2-chloro substitution exhibited good cytotoxicity against breast cancer (MDA-MB-23) cells
with an IC50 value of 1.65±0.05 µM. The rest compounds have shown less cytotoxicity
against selected cell lines. Over all, compound 7c was proved to be the best cytotoxic agent
among all.
<Insert Table 1 here>
2.3.2. Observations of morphological changes using phase contrast microscopy
The distinic features of programmed cell death or apoptosis are recognized by the changes
in the molecular, biochemical and morphology of a cell [36]. The cells (A-549) treated with
compound 7c at different concentrations of 0.5, 1 and 2.5µM showed promising
morphological changes such as formation of membrane blebs, and cell shrinkage with cells
rounding as evidenced by phase contrast microscopy studies and corresponding to the
concentration (Fig. 5). However, there are no distinctive features of morphological changes
found in the controlled cells.
<Insert Figure 5 here>
2.3.3. DAPI nucleic acid staining
This study distinguishes normal cells from apoptotic cells by staining the condensed nuclei
of apoptotic cells using DAPI (4',6-diamidino-2-phenylindole), a blue fluorescent dye assists
in the visualization of nuclear damage or chromatin condensation of apoptotic cells, which
are of high interest to detect the apoptosis induction by compound 7c in lung cancer (A-549)
cells [37]. The results from Fig. 6 clearly suggested that the treated cells with compound 7c
at different concentrations of 0.5, 1 and 2.5µM showed condensed, fragmented or horse-
shoe shaped nuclei in the cells, whereas the nuclear structure of controlled cells was intact.
<Insert Figure 6 here>
2.3.4. Cell cycle analysis
Damage of DNA content is an essential feature of apoptosis. The changes in the frequency
of treated cells with compound 7c in every phase have been observed corresponding to
control cells. A-549 cells were treated with compound 7c, at different concentrations of 0.25,
1 and 2.5 µM for 24 h, followed by stained with Propidium Iodide (PI), and cell death was
quantified by using BD FACSVerseTM flow analyser [38]. The PI staining of treated cells
with 7c at different concentrations exhibited an increase in apoptotic cells in a dose
1

dependent manner (Fig. 7). Control cells exposed to DMSO showed 1.97 % cells in sub G1
phase, whereas treated cells with compound 7c showed a spike in the sub G1 population to
6.20% at 0.5 µM, 30.93% at 1 µM and 36.15% at 2.5 µM concentration in 24 h. These results
clearly demonstrate that 7c induce apoptosis by sub G1 phase arrest of cell cycle.
<Insert Figure 7 here>
2.3.5. Mitochondrial Membrane Potential (MMP)
The hallmark of apoptosis is the loss of mitochondrial membrane potential. Mitochondria
plays a major role in apoptosis corresponding to stress, and the loss of MMP is a signaling
marker of early mitochondrial damage in the process of apoptosis. The study was performed
using JC1 (a carbocyanine cationic dye) staining to detect the mitochondrial depolarization
occurs during early stages of apoptosis in treated cells with compound 7c at different
concentrations of 0.5, 1 and 2.5 µM. It shows potential based aggregation in mitochondria,
as indicated by a fluorescence emission changes. Enhanced ROS levels induce the loss of
MMP which further activates the apoptotic signalling and leads to cell death. The polarised
cells mitochondria exhibits red colour (P1-phase) due to J-aggregates formation with it,
whereas the depolarised cells mitochondria exhibits green colour (P2-phase) due to J-
monomers formation [39]. The results from Fig. 8 showed an increase in the apoptotic cell
population of P2-phase (depolarized cell population) from 1.04% in control to 23.85% at 0.5
µM, 52.80% at 1 µM and 88.91% at 2.5 µM. therefore, A-549 cells treated with compound
7c for 24 hrs exhibited decreased MMP in dose dependent manner.
<Insert Figure 8 here>
2.3.6. AnnexinV/Propidium iodide dual staining assay
The percentage of apoptotic cells induced by compound 7c can be easily calculated using
Annexin V/Propidium iodide dual staining assay [40]. The Annexin V-Alexa Flour 488/PI
dual staining assay provide detection of live cells (LL; AV-/PI-), early apoptotic cells (LR;
AV+/PI-), late apoptotic cells (UR; AV+/PI+) and necrotic cells (UL; AV-/PI+). The results
from Fig. 9 showed that the compound 7c induced the percentage of late apoptotic cells from
1.11% in control to 14.80% at 0.5 µM, 31.41% at 1 µM and 59.36% at 2.5 µM respectively.
Therefore, it is indicated that the compound 7c induces apoptosis in A-549 cells in a dose
dependent manner.
1

<Insert Figure 9 here>
2.3.7. Gal-1 ELISA enzymatic study with compound 7c
The Gal-1 levels in the healthy subjects are normal but it is upregulated in the tumour
environment, and leads to cause tumour proliferation and metastasis. Therefore, Gal-1 is
considered as a biomarker to target specific tumors like lung, breast, and prostate [41]. gal-
1 quantitative enzyme immunoassay was performed to see the effectiveness of compound
7c on gal-1 levels in the process of apoptosis. The treated A-549 cells with compound 7c
were incubated in 60mm cell culture dishes for 48 hrs, and the supernatant was collected.
The expression of gal-1 can be quantified from the cellular external environment as gal-1 is
an extracellular secreted protein. The collected equal quantities of supernatant were
subjected to quantitative enzyme immunoassay as per manufacture’s protocol [DGAL10,
R&D Systems, USA]. The supernatant solutions were incubated with human gal-1coated
plates, and washed to remove unbound protein. Furthermore, an enzyme attached antibody
was added into the reaction, specific to human gal-1 and incubated with substrate solution
about 30 minutes before terminating the reaction with 50µl of the stop solution. In the last
stage, the quantification of protein expression was detected using UV-spectrophotometer.
The results were stipulated in the graph (Table 2) and found to be a greater reduction in the
gal-1 protein levels in dose dependent manner (Fig. 10) at the concentrations of 10, 30, 100
and 300 µM. therefore, it is confirmed that the compound 7c significantly reduces the
expression of gal-1.
<Insert Figure 10 here>
<Insert Table 2 here>
2.3.8. Fluorescence Spectroscopy (FS) study
The FS is a simple, sensitive and widely used technique to evaluate the interactions, and
dynamics of proteins with ligands based on its fluorescence properties in solution. It
provides binding affinity based on the protein–ligand interactions qualitatively and
quantitatively in equilibrium conditions [42]. Fluorescence spectra of gal-1 in the increasing
concentrations of ligand from 0 to 65 μM at pH 7.5 was analysed, and the emission spectrum
of gal-1 was achieved at 343 nm. It is noticed that there is a decrease in the fluorescence
intensity of 7c with increasing its concentration (Fig. 11A). The graph of log[7c] versus log
(F o-F)/F showed a linear relationship (Fig. 11B), and the total number of binding sites were
found to be 1 based on the calculations from the slope, and concludes interaction of gal-1
1

protein and compound in 1:1 ratio. The binding constant (Ka) was calculated from the
intercept value is 9.5 x 103 M-1. The intensity of the fluorescence was quenched upon
increase of ligand concentration and the bimolar quenching constant (Kq) was calculated to
be 1.4 x 1012M-1 S-1 which is larger than diffusion control limit suggesting interaction of gal-
1protein with ligand as well as the mode of quenching to be static.
<Insert Figure 11A & 11B here>
2.3.9. Surface Plasmon Resonance (SPR) studies
It is a spectroscopic technique which is used to detect protein interactions mainly binding of
ligands in real-time without the use of labels by fixing or immobilizing the ligand on a thin
metal film, and measuring the change in refractive index based on binding of the analyte.
SPR instruments are primarily used to measure the binding kinetics and affinity of
molecular interactions [43]. The gal-1 interaction with compound 7c was analysed through
SPR using immobilized gal-1 (ligand, 8070 RU) and compound 7c (analyte) at various
concentrations. Sensorgram was measured for each of concentration of compound 7c (Fig.
12) and fitted using 1:1 interaction steady state affinity model. The equilibrium constant KD
value was found to be 1.19E-06 M using Biacore T200 Evaluation software version 2.0.
therefore, the SPR analysis showed interaction of gal-1 with compound 7c, which is in
validation with florescence studies.
<Insert Figure 12 here>
2.3.10. Molecular docking studies
To better understand the affinity of the compound 7c with the active site of the gal-1, we
have performed the molecular docking calculations using the Glide docking module of
Schrodinger suite [44]. The 3D crystal co-ordinates of human gal-1 were obtained from the
protein data bank (PDB ID: 4Y24). The 2D and 3D ligand interaction diagrams of compound
7c are shown in Fig. 13A, 13B & 13C. Hydrogen bonding interaction was found at a distance
of 1.75Å between C=O of Glu71 acting as acceptor (atom no. 489) and the phenolic OH
acting as donor (atom no. 2066). A π-cation interaction was found between NH of Lys63
(atom no. 464) and the phenyl ring of the N-benzyl substitution. Further, π- π stacking
between the indole ring of Trp68 and phenyl ring connecting the benzimidazole with the
triazole moiety and between imidazole moiety His44 and the benzimidazole moiety stabilize
the docking pose of the 7c molecule in the binding site.
1

A set of pharmacokinetic Absorption, Distribution, Metabolism, Elimination (ADMET)
related properties of the molecule 7c were calculated using qikprop [45]. The
physicochemical properties of 7c matched with the prescribed ranges as represented in
Table 3. Additionally, the molecular weight (mol. Wt.), hydrogen bond donors (donor HB),
hydrogen bond acceptors (accept HB), partition coefficient (QPlogPo/w), exhibited
acceptable values that followed Lipinski rule of five.
<Insert Figure 13A, 13B & 13C here>
<Insert Table 3 here>
3. Conclusion
In conclusion, a series of benzimidazole-triazole hybrids have been synthesized and
evaluated for their in vitro cytotoxic potential against various selected human cancer cell
lines. The initial screening analysis showed that some of the derivatives were active against
all selected cancer cell lines with IC50 values below 10 µM. The in vitro screening results
revealed the most active compound 7c with broad range of activity against all the tested cell
lines, and exhibited an excellent growth inhibition with an IC50 of 0.63±0.21 µM against A-
549 cell lines by inducing apoptosis via arresting cell cycle at Sub G1 phase, decreasing
MMP, increasing apoptotic cells by Annexin V. The morphological changes like nuclear
condensation, cell shrinkage, were observed through microscope DAPI staining, and also
compound 7c found to be non-toxic against normal NRK cells. The compound 7c
significantly reduced gal-1 protein expression in a dose dependent manner using enzymatic
ELISA study. Further, the gal-1 interaction with compound 7c was confirmed by surface
plasmon resonance and fluorescence spectroscopy studies. Molecular docking studies also
supported the possible mode of binding to the gal-1. we have succeeded in radiolabeling of
7c with 18F radionuclide to prove as a PET tracer in addition to its potential cytotoxicity in
low concentration. Overall, the current study indicates that these new series of
benzimidazole-triazole hybrids have the potential to be advanced as gal-1 targeting cytotoxic
agents as well as PET tracers for the diagnosis and, treatment of cancer.
4. Experimental protocols
4.1. Chemistry
All chemicals, reagents, and solvents were obtained from commercial suppliers, and
used without further purification. Analytical thin layer chromatography (TLC) was
performed on MERCK precoated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization
1

1
of the spots on TLC plates was achieved by UV light. H-NMR &13C-NMR spectra were
recorded on Bruker 500 MHz using DMSO or CDCl3 solvent with tetramethyl silane (TMS)
as the internal standard. Chemical shifts for 1H & 13C are reported in parts per million (ppm)
towards higher values from TMS position. Spin-spin splitting pattern was mentioned as s
(singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). The coupling constant (J)
values are mentioned in hertz (Hz). HRMS were calculated with Agilent QTOF mass
spectrometer 6540 series instrument. Column chromatography was performed wherever
required using silica gel 60-120 or 100-200 size. The evaporation of solvents was carried
out under reduced pressure on Heidolph rotary evaporator below 50 0C.
4.1.1. Synthesis of N-benzyl-4-chloro-2-nitroaniline (3)
The 4-chloro-2-nitroaniline (1 eq) was added directly into water (10 mL) followed by added
benzyl bromide (1.5 eq) and the reaction mixture was then stirred under reflux at 100 0C for
3-4 hours. After conformation by TLC the reaction mixture was removed and extracted with
ethyl acetate (3x20 ml) and dried over Na2SO4. The combined organic layer was
concentrated in vacuo to give a red colour solid purified by column chromatography. Red
solid, yield 85%; mp 186-188 °C; 1H NMR (500 MHz, DMSO) δ 8.75 (t, J = 6.0 Hz, 1H),
8.07 (d, J = 2.6 Hz, 1H), 7.50 (dd, J = 9.1, 2.4 Hz, 1H), 7.37 – 7.33 (m, 4H), 7.28 – 7.24 (m,
13
1H), 6.94 (d, J = 9.3 Hz, 1H), 4.65 (d, J = 6.2 Hz, 2H). C NMR (125 MHz, DMSO) δ
144.24, 138.64, 136.57, 131.68, 129.08, 127.59, 127.33, 125.44, 119.03, 117.49, 46.20.
4.1.2. Synthesis of N1-benzyl-4-chlorobenzene-1,2-diamine (4)
Stannous chloride (5 eq) was added to a solution of N-benzyl-4-chloro-2-nitroaniline (3) in
ethanol (5 ml) and the reaction mixture was stirred under reflux at 80 °C for 5-6 hours. The
reaction mixture was quenched with diluted NaHCO3 and formed sticky emulsion type
solution. It was filtered by suction filtration with celite bed on Buckner funnel to remove
precipitate. Finally, the filtrate was extracted with ethyl acetate (3x20 ml) and dried over
Na2SO4. The combined organic layer was concentrated in vacuo to give a purple colour solid
0
1
purified by column chromatography. Brown solid, yield 82%; mp 172-174 C; H NMR
(500 MHz, DMSO) δ 7.41 – 7.10 (m, 5H), 6.57 (d, J = 2.4 Hz, 1H), 6.38 (dd, J = 8.4, 2.4
13
Hz, 1H), 6.27 (d, J = 8.4 Hz, 1H), 4.92 (s, 2H), 4.28 (d, J = 5.8 Hz, 2H). C NMR (125
MHz, DMSO) δ 140.45, 137.51, 134.74, 128.73, 127.64, 127.12, 120.87, 116.56, 113.46,
111.41, 47.33.
4.1.3. Synthesis of 4-(1-benzyl-6-chloro-1H-benzo[d]imidazol-2-yl) phenol (5)
1

N1-benzyl-4-chlorobenzene-1, 2-diamine (4) (1 eq) was added to water and ethanol mixed
solvent (1:1) and to this added sodium metabisulphite (15 eq) and finally added 4-hydroxy
benzaldehyde (1 eq). The reaction mixture was stirred under reflux 80 0C for overnight. On
cooling at room temperature 20-30 mL of cold water was added and stirred for 10 min. The
solid separated out was filtered at pump and dried. The residues were purified by column
chromatography on silica gel. White solid, yield 85%; mp 242-244 0C; 1H NMR (500 MHz,
DMSO) δ 10.03 (s, 1H), 7.74 (d, J = 1.9 Hz, 1H), 7.59 – 7.55 (m, 2H), 7.45 (d, J = 8.6 Hz,
1H), 7.30 (t, J = 7.3 Hz, 2H), 7.27 – 7.21 (m, 2H), 6.99 (d, J = 7.2 Hz, 2H), 6.89 (d, J = 8.2
13
Hz, 2H), 5.57 (s, 2H). C NMR (125 MHz, DMSO) δ 159.61, 155.66, 144.08, 137.18,
135.25, 131.08, 129.28, 127.99, 126.97, 126.55, 122.77, 120.67, 118.80, 116.10, 112.73,
48.12.
4.1.4.
Synthesis
of
1-benzyl-6-chloro-2-(4-(prop-2-yn-1-yloxy)
phenyl)-1H
benzo[d]imidazole (6)
To the solution of compound 5 (1 mmol) in freshly distilled acetone (10 mL), added
anhydrous K2CO3 (2.5 mmol) as a base and propargyl bromide (1 mmol) was added drop
wise and stirred under reflux for 2-3 hr. The solvent was evaporated completely and the
obtained residue was extracted with ethyl acetate (3x20 mL) and dried over Na2SO4. The
combined organic layer was concentrated in vacuo and the residues were purified by column
chromatography on silica gel. White solid, yield 81%; mp 256-258 0C; 1H NMR (500 MHz,
DMSO) δ 7.77 (d, J = 2.0 Hz, 1H), 7.72 – 7.69 (m, 2H), 7.48 (d, J = 8.6 Hz, 1H), 7.32 –
7.28 (m, 2H), 7.27 – 7.23 (m, 2H), 7.15 – 7.12 (J = 8.0 Hz, 2H), 7.00 (d, J = 7.2 Hz, 2H),
5.60 (s, 2H), 4.89 (d, J = 2.4 Hz, 2H), 3.62 (t, J = 2.3 Hz, 1H). 13C NMR (125 MHz, DMSO)
δ 159.04, 155.07, 144.05, 137.10, 135.28, 130.97, 129.32, 128.02, 127.12, 126.51, 123.03,
118.99, 115.64, 112.83, 79.37, 79.00, 56.11, 48.15.
4.1.5. General procedure for the synthesis of final compounds 7a-t.
A 50 mL round bottom flask was charged with 1-benzyl-6-chloro-2-(4-(prop-2-yn-1-yloxy)
phenyl)-1H-benzo[d]imidazole (1 mmol), various phenyl and, benzyl azides (0.9 mmol),
sodium ascorbate (0.3 mmol), 2 mL of ethanol and 2 mL of distilled water. Then,
CuSO4.5H2O (0.15 mmol) was added. The reaction mixture was stirred under rt for
overnight. After completion of the reaction by TLC analysis, the mixture was filtered and
extracted with ethyl acetate (3x20 ml) and dried over Na2SO4. The combined organic layer
1

was concentrated in vacuo and the residues were purified by column chromatography on
silica gel, offered the final target compounds 7a-t with moderate to good yields.
4.1.5.1. 1-benzyl-5-chloro-2-(4-((1-phenyl-1H-1,2,3-triazol-4-yl) methoxy) phenyl)-
1H-benzo[d]imidazole (7a)
White solid, yield 83%; mp 287-289 0C; 1H NMR (500 MHz, DMSO) δ 8.97 (s, 1H), 7.91
(d, J = 6.1 Hz, 2H), 7.78 – 7.71 (m, 3H), 7.62 (s, 2H), 7.51 (d, J = 6.9 Hz, 2H), 7.29 (d, J =
6.1 Hz, 2H), 7.22 – 7. 16 (m, 4H), 7.00 (s, 2H), 5.60 (s, 2H), 5.33 (s, 2H). 13C NMR (125
MHz, DMSO) δ 159.86, 144.03, 136.97, 131.02, 130.93, 130.40, 129.31, 128.07, 127.14,
126.52, 123.50, 123.12, 122.74, 120.70, 119.19, 119.00, 118.96, 115.66, 115.62, 112.95,
61.63, 48.23. HRMS (ESI): m/z calcd for C29H22ClN5O, 491. 1513, found 492.1597 [M+H]
+.
4.1.5.2. 1-benzyl-5-chloro-2-(4-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7b)
White solid, yield 85%; mp 310-312 0C; 1H NMR (500 MHz, DMSO) δ 8.61 (s, 1H), 7.74
(d, J = 15.2 Hz, 3H), 7.63 (d, J = 7.2 Hz, 1H), 7.54 (d, J = 6.4 Hz, 2H), 7.35 – 7.19 (m, 7H),
7.15 (t, J = 7.3 Hz, 1H), 7.00 (d, J = 6.4 Hz, 2H), 5.60 (s, 2H), 5.30 (s, 2H), 3.85 (s, 3H).
13C NMR (125 MHz, DMSO) δ 160.04, 152.14, 142.65, 136.97, 136.90, 131.32, 131.06,
129.32, 128.09, 127.29, 127.28, 127.25, 126.56, 126.25, 126.09, 123.31, 121.39, 118.99,
118.91, 115.61, 113.54, 113.08, 61.58, 56.61, 48.29. HRMS (ESI): m/z calcd for
C30H24ClN5O2, 521. 1619, found 522.1700 [M+H] +.
4.1.5.3. 3-(4-((4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl) phenoxy) methyl)-1H-
1,2,3-triazol-1-yl) phenol (7c)
0
1
Light yellow solid, yield 81%; mp 274-276 C; H NMR (500 MHz, DMSO) δ 10.06 (s,
1H), 8.92 (s, 1H), 7.75 – 7.70 (m, 3H), 7.54 (s, 1H), 7.35 – 7.30 (m, 6H), 7.26 – 7.24 (m,
3H), 6.95 (d, J = 48.0 Hz, 3H), 5.60 (s, 2H), 5.30 (s, 2H). 13C NMR (125 MHz, DMSO) δ
159.93, 158.97, 143.87, 138.01, 136.94, 131.24, 130.99, 129.30, 128.06, 127.19, 126.56,
123.46, 123.25, 122.63, 119.01, 116.25, 115.58, 113.08, 111.04, 107.64, 61.66, 48.34.
HRMS (ESI): m/z calcd for C29H22ClN5O2, 507. 1462, found 508.1551 [M+H] +.
4.1.5.4. 1-benzyl-5-chloro-2-(4-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl) methoxy) phenyl)-
1H-benzo[d]imidazole (7d)
1

White solid, yield 82%; mp 293-295 0C; 1H NMR (500 MHz, DMSO) δ 8.92 (s, 1H), 7.75
(d, J = 34.9 Hz, 5H), 7.55 – 7.15 (m, 9H), 7.00 (s, 2H), 5.60 (s, 2H), 5.31 (s, 2H), 2.38 (s,
3H). 13C NMR (125 MHz, DMSO) δ 159.87, 144.09, 143.91, 138.91, 137.09, 134.81,
131.04, 130.71, 129.31, 128.03, 127.09, 126.54, 123.37, 123.04, 122.80, 120.56, 119.01,
115.59, 112.89, 61.71, 48.20, 21.03. HRMS (ESI): m/z calcd for C30H24ClN5O, 505. 1669,
found 506.1755 [M+H] +.
4.1.5.5. 1-benzyl-5-chloro-2-(4-((1-(4-(trifluoromethyl) phenyl)-1H-1,2,3-triazol-4-yl)
methoxy) phenyl)-1H-benzo[d]imidazole (7e)
Brown solid, yield 79%; mp 273-275 0C; 1H NMR (500 MHz, DMSO) δ 9.14 (s, 1H), 8.19
(d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 27.5 Hz, 3H), 7.51 (d, J = 8.0 Hz,
1H), 7.30 (t, J = 7.3 Hz, 2H), 7.25 (d, J = 7.5 Hz, 4H), 7.00 (d, J = 7.3 Hz, 2H), 5.61 (s, 2H),
5.35 (s, 2H). 13C NMR (125 MHz, DMSO) δ 159.82, 144.46, 139.82, 137.06, 131.01,
129.43, 129.31, 129.17, 128.02, 127.70, 127.67, 127.06, 126.54, 125.36, 123.77, 123.20,
123.07, 121.15, 119.09, 115.61, 112.98, 61.64, 48.29. HRMS (ESI): m/z calcd for
C30H21ClF3N5O, 559. 1387, found 560.1481 [M+H] +.
4.1.5.6. 1-benzyl-5-chloro-2-(4-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7f)
Light brown solid, yield 80%; mp 251-253 0C; 1H NMR (500 MHz, DMSO) δ 8.96 (s, 1H),
7.96 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 24.1 Hz, 3H), 7.52 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 8.6
Hz, 2H), 7.30 – 7.26 (m, 3H), 7.25 (d, J = 9.6 Hz, 3H), 7.00 (d, J = 7.2 Hz, 2H), 5.60 (s,
2H), 5.32 (s, 2H). 13C NMR (126 MHz, DMSO) δ 163.18, 161.23, 159.94, 144.04, 136.95,
133.61, 131.06, 129.31, 128.06, 127.26, 126.56, 123.75, 123.26, 123.11, 123.04, 122.59,
118.92, 117.30, 117.11, 115.59, 113.04, 61.69, 48.29. HRMS (ESI): m/z calcd for
C29H21ClFN5O, 509. 1419, found 510.1496 [M+H] +.
4.1.5.7. 1-benzyl-5-chloro-2-(4-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7g)
Light brown solid, yield 81%; mp 247-249 0C; 1H NMR (500 MHz, DMSO) δ 9.04 (s, 1H),
7.87 (d, J = 10.1 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 20.3 Hz, 2H), 7.70 – 7.63
(m, 1H), 7.53 (s, 1H), 7.38 (t, J = 7.2 Hz, 1H), 7.33 – 7.20 (m, 6H), 6.99 (d, J = 6.9 Hz, 2H),
5.61 (s, 2H), 5.33 (s, 2H). 13C NMR (125 MHz, DMSO) δ 163.86, 161.91, 159.87, 144.23,
138.26, 136.93, 132.39, 132.32, 130.99, 129.31, 128.07, 127.15, 126.55, 123.69, 123.23,
1

119.07, 116.62, 116.10, 115.93, 115.62, 113.11, 108.29, 108.08, 61.63, 48.38. HRMS (ESI):
m/z calcd for C29H21ClFN5O, 509. 1419, found 510.1504 [M+H] +.
4.1.5.8. 1-benzyl-2-(4-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl) methoxy) phenyl)-5-
chloro-1H-benzo[d]imidazole (7h)
0
1
Light red solid, yield 84%; mp 269-271 C; H NMR (500 MHz, DMSO) δ 9.01 (s, 1H),
7.90 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 27.7 Hz, 3H), 7.49 (d, J = 8.1
Hz, 1H), 7.29 (d, J = 7.2 Hz, 2H), 7.24 (t, J = 8.2 Hz, 4H), 7.00 (d, J = 7.0 Hz, 2H), 5.60 (s,
2H), 5.32 (s, 2H). 13C NMR (125 MHz, DMSO) δ 159.83, 144.22, 137.08, 136.24, 133.27,
131.05, 129.31, 128.03, 127.10, 126.53, 123.53, 123.04, 122.83, 122.60, 121.93, 119.01,
115.59, 112.89, 61.66, 48.20. HRMS (ESI): m/z calcd for C29H21BrClN5O, 569. 8750, found
572.0687 [M+2] +.
4.1.5.9.
1-benzyl-5-chloro-2-(4-((1-(2-chloro-4-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methoxy) phenyl)-1H-benzo[d]imidazole (7i)
White solid, yield 81%; mp 315-217 0C; 1H NMR (500 MHz, DMSO) δ 8.88 (s, 1H), 8.64
(d, J = 2.0 Hz, 1H), 8.41 (dd, J = 8.7, 2.0 Hz, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.82 – 7.66 (m,
3H), 7.50 (d, J = 8.3 Hz, 1H), 7.34 – 7.21 (m, 6H), 7.00 (d, J = 7.3 Hz, 2H), 5.61 (s, 2H),
5.37 (s, 2H). 13C NMR (125 MHz, DMSO) δ 159.84, 148.80, 143.43, 139.56, 137.05,
131.03, 129.77, 129.73, 129.32, 128.05, 127.45, 127.12, 126.54, 126.35, 124.05, 123.09,
122.85, 119.02, 115.62, 112.95, 61.43, 48.23. HRMS (ESI): m/z calcd for C29H20Cl2N6O3,
570. 0974, found 571.1061 [M+H] +.
4.1.5.10.
1-benzyl-5-chloro-2-(4-((1-(4-methyl-3-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methoxy) phenyl)-1H-benzo[d]imidazole (7j)
0
1
Off White solid, yield 82%; mp 295-297 C; H NMR (500 MHz, DMSO) δ 9.12 (s, 1H),
8.54 (s, 1H), 8.21 (d, J = 7.2 Hz, 1H), 7.84 – 7.64 (m, 4H), 7.53 (d, J = 7.4 Hz, 1H), 7.33 –
13
7.15 (m, 6H), 7.00 (d, J = 6.9 Hz, 2H), 5.60 (s, 2H), 5.34 (s, 2H), 2.59 (s, 3H). C NMR
(125 MHz, DMSO) δ 159.93, 149.74, 144.33, 136.88, 135.49, 134.80, 134.58, 133.46,
131.05, 129.31, 128.08, 127.32, 126.57, 124.78, 123.76, 123.37, 122.54, 118.92, 116.28,
115.57, 113.15, 61.63, 48.35, 19.57. HRMS (ESI): m/z calcd for C30H23ClN6O3, 550. 1520,
found 551.1604 [M+H] +.
1

4.1.5.11. 1-benzyl-5-chloro-2-(4-((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7k)
White solid, yield 85%; mp 285-287 0C; 1H NMR (500 MHz, DMSO) δ 9.22 (s, 1H), 8.75
(t, J = 2.1 Hz, 1H), 8.43 (dd, J = 8.1, 1.4 Hz, 1H), 8.35 (dd, J = 8.2, 1.6 Hz, 1H), 7.92 (t, J
= 8.2 Hz, 1H), 7.79 – 7.69 (m, 3H), 7.49 (d, J = 8.5 Hz, 1H), 7.31 – 7.24 (m, 6H), 7.01 (d, J
= 7.3 Hz, 2H), 5.59 (s, 2H), 5.36 (s, 2H). 13C NMR (125 MHz, DMSO) δ 159.81, 159.06,
149.02, 144.48, 137.60, 137.06, 132.02, 131.06, 130.95, 129.32, 128.03, 127.12, 126.68,
126.53, 123.98, 123.69, 123.09, 122.80, 118.98, 115.63, 115.60, 115.39, 112.92, 61.62,
48.20. HRMS (ESI): m/z calcd for C29H21ClN6O3, 536. 1364, found 537.1443 [M+H] +.
4.1.5.12. 1-benzyl-2-(4-((1-benzyl-1H-1,2,3-triazol-4-yl) methoxy) phenyl)-5-chloro-
1H-benzo[d]imidazole (7l)
0
1
Off white solid, yield 86%; mp 252-254 C; H NMR (500 MHz, DMSO) δ 8.31 (s, 1H),
7.77 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 6.8 Hz, 2H),
7.36 – 7.27 (m, 5H), 7.26 (d, J = 5.9 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 7.00 (d, J = 7.1 Hz,
2H), 5.62 (s, 2H), 5.59 (s, 2H), 5.22 (s, 2H). 13C NMR (125 MHz, DMSO) δ 159.90, 159.05,
144.04, 143.16, 137.08, 136.44, 131.00, 129.32, 129.24, 128.64, 128.44, 128.04, 127.10,
126.52, 125.31, 123.05, 122.62, 118.98, 115.64, 115.53, 112.90, 61.72, 53.36, 48.17. HRMS
(ESI): m/z calcd for C30H24ClN5O, 505. 1669, found 506.1752 [M+H] +.
4.1.5.13. 1-benzyl-5-chloro-2-(4-((1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7m)
White solid, yield 85%; mp 291-293 0C; 1H NMR (500 MHz, DMSO) δ 8.31 (s, 1H), 8.15
(d, J = 8.0 Hz, 1H), 7.81 – 7.68 (m, 4H), 7.64 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.4 Hz, 1H),
7.30 (t, J = 7.1 Hz, 2H), 7.26 (d, J = 6.6 Hz, 2H), 7.20 (d, J = 7.0 Hz, 2H), 7.09 (d, J = 7.5
Hz, 1H), 7.00 (d, J = 7.1 Hz, 2H), 5.99 (s, 2H), 5.59 (s, 2H), 5.26 (s, 2H). 13C NMR (125
MHz, DMSO) δ 159.88, 159.07, 148.08, 143.18, 137.02, 134.82, 131.17, 130.98, 130.65,
130.17, 129.32, 128.05, 127.13, 126.53, 126.11, 125.55, 123.11, 122.61, 119.00, 115.64,
115.59, 112.98, 61.68, 56.09, 50.48. HRMS (ESI): m/z calcd for C30H23ClN6O3, 550. 1520,
found 551.1600 [M+H] +.
4.1.5.14. 1-benzyl-5-chloro-2-(4-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7n)
1

0
1
Off White solid, yield 84%; mp 287-289 C; H NMR (500 MHz, DMSO) δ 8.39 (s, 1H),
8.24 (d, J = 8.4 Hz, 2H), 7.70 – 7.62 (m, 3H), 7.55 (d, J = 8.5 Hz, 3H), 7.30 – 7.24 (m, 4H),
7.18 (s, 2H), 6.99 (d, J = 7.3 Hz, 2H), 5.82 (s, 2H), 5.59 (s, 2H), 5.23 (s, 2H). 13C NMR (125
MHz, DMSO) δ 160.08, 147.76, 143.78, 136.72, 130.97, 129.54, 129.31, 128.13, 127.42,
126.59, 125.85, 124.39, 123.59, 122.27, 118.97, 118.90, 115.56, 113.37, 67.40, 61.73,
52.48. HRMS (ESI): m/z calcd for C30H23ClN6O3, 550. 1520, found 551.1591 [M+H] +.
4.1.5.15. 1-benzyl-5-chloro-2-(4-((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl) methoxy)
phenyl)-1H-benzo[d]imidazole (7o)
Light brown solid, yield 82%; mp 268-270 0C; 1H NMR (500 MHz, DMSO) δ 8.32 (d, J =
26.8 Hz, 1H), 7.77 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.6 Hz, 1H), 7.45 – 7.38
(m, 1H), 7.30 (t, J = 7.2 Hz, 2H), 7.24 (dd, J = 15.9, 7.7 Hz, 3H), 7.20 – 7.13 (m, 4H), 7.00
13
(d, J = 7.2 Hz, 2H), 5.67 (d, J = 16.5 Hz, 2H), 5.59 (s, 2H), 5.23 (d, J = 4.6 Hz, 2H). C
NMR (125 MHz, DMSO) δ 159.88, 144.05, 143.24, 143.07, 139.12, 139.06, 137.10, 131.37,
131.01, 129.32, 128.03, 127.09, 126.52, 125.45, 124.52, 123.01, 122.66, 118.97, 115.53,
115.24, 112.87, 61.71, 61.66, 52.67, 48.15. HRMS (ESI): m/z calcd for C30H22ClFN5O, 523.
1575, found 524.1660 [M+H] +.
4.1.5.16.
1-benzyl-5-chloro-2-(4-((1-(3,5-difluorobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy) phenyl)-1H-benzo[d]imidazole (7p)
Brown solid, yield 80%; mp 264-266 0C; 1H NMR (500 MHz, DMSO) δ 8.37 (s, 1H), 7.77
(s, 1H), 7.69 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 8.5 Hz, 1H), 7.30 (t, J = 7.2 Hz, 2H), 7.28 –
7.22 (m, 3H), 7.19 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 6.3 Hz, 2H), 7.00 (d, J = 7.2 Hz, 2H),
13
5.67 (s, 2H), 5.59 (s, 2H), 5.24 (s, 2H). C NMR (125 MHz, DMSO) δ 159.87, 144.07,
143.32, 140.60, 137.10, 131.01, 129.31, 128.03, 127.08, 126.53, 125.57, 123.01, 122.71,
118.97, 115.55, 112.86, 111.89, 111.84, 111.73, 111.68, 104.42, 104.21, 104.01, 61.72,
52.25, 48.15. HRMS (ESI): m/z calcd for C30H22ClF2N5O, 541. 1481, found 542.1558
[M+H] +.
4.1.5.17. 1-benzyl-2-(4-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl) methoxy) phenyl)-5-
chloro-1H-benzo[d]imidazole (7q)
0
1
Light red solid, yield 81%; mp 281-283 C; H NMR (500 MHz, DMSO) δ 8.32 (s, 1H),
7.77 (s, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 8.4 Hz, 1H),
7.32 – 7.23 (m, 6H), 7.18 (d, J = 7.8 Hz, 2H), 7.00 (d, J = 7.0 Hz, 2H), 5.61 (s, 2H), 5.59 (s,
1

2H), 5.22 (s, 2H). 13C NMR (125 MHz, DMSO) δ 159.87, 144.08, 143.22, 137.10, 135.84,
132.18, 131.00, 130.69, 129.32, 128.03, 127.08, 126.52, 125.37, 123.01, 122.68, 121.94,
118.98, 115.55, 112.87, 61.71, 52.60, 48.16. HRMS (ESI): m/z calcd for C30H23BrClN5O,
584. 9020, found 586.0839 [M+2] +.
4.1.5.18. 1-benzyl-2-(4-((1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl) methoxy) phenyl)-5-
chloro-1H-benzo[d]imidazole (7r)
0
1
Light red solid, yield 83%; mp 290-292 C; H NMR (500 MHz, DMSO) δ 8.35 (s, 1H),
7.75 (s, 1H), 7.69 (d, J = 7.9 Hz, 2H), 7.55 (d, J = 11.3 Hz, 2H), 7.48 (d, J = 8.5 Hz, 1H),
7.39 – 7.30 (m, 4H), 7.27 – 7.22 (m, 2H), 7.19 (d, J = 8.1 Hz, 2H), 7.00 (d, J = 7.3 Hz, 2H),
13
5.64 (s, 2H), 5.59 (s, 2H), 5.23 (s, 2H). C NMR (125 MHz, DMSO) δ 159.89, 144.01,
143.25, 139.05, 137.08, 131.54, 131.45, 131.23, 131.00, 129.32, 128.04, 127.57, 127.11,
126.53, 125.47, 123.04, 122.63, 122.33, 118.97, 115.64, 115.53, 112.89, 61.71, 52.51,
48.18. HRMS (ESI): m/z calcd for C30H23BrClN5O, 584. 9020, found 586.0840 [M+2] +.
4.1.5.19.
1-benzyl-5-chloro-2-(4-((1-(2,3-dimethylphenyl)-1H-1,2,3-triazol-4-yl)
methoxy) phenyl)-1H-benzo[d]imidazole (7s)
White solid, yield 85%; mp 298-300 0C; 1H NMR (500 MHz, DMSO) δ 8.57 (s, 1H), 7.77
(s, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.49 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.30 (d,
J = 7.2 Hz, 3H), 7.30 – 7.21 (m, 5H), 7.01 (d, J = 7.0 Hz, 2H), 5.60 (s, 2H), 5.33 (s, 2H),
2.35 (s, 3H), 1.94 (s, 3H). 13C NMR (125 MHz, DMSO) δ 159.89, 144.04, 142.91, 138.95,
137.09, 136.77, 132.68, 131.67, 131.04, 129.32, 128.04, 127.18, 127.10, 126.69, 126.54,
124.46, 123.04, 122.73, 118.97, 115.64, 112.89, 61.66, 48.16, 20.34, 14.33. HRMS (ESI):
m/z calcd for C31H26ClN5O, 519. 1826, found 520.1911 [M+H] +.
4.1.5.20.
1-benzyl-5-chloro-2-(4-((1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-1H-benzo[d]imidazole (7t)
0
1
Off white solid, yield 80%; mp 311-313 C; H NMR (500 MHz, DMSO) δ 8.82 (s, 1H),
8.18 (dd, J = 36.0, 8.1 Hz, 2H), 7.82 – 7.59 (m, 7H), 7.49 (dd, J = 16.4, 8.4 Hz, 2H), 7.34 –
7.20 (m, 6H), 7.01 (d, J = 7.2 Hz, 2H), 5.61 (s, 2H), 5.40 (s, 2H). 13C NMR (125 MHz,
DMSO) δ 159.98, 159.09, 143.80, 143.14, 137.03, 134.14, 133.64, 131.08, 130.97, 130.84,
129.32, 128.85, 128.51, 128.41, 128.06, 127.98, 127.62, 127.20, 126.55, 125.91, 124.47,
123.16, 122.61, 122.33, 118.93, 115.64, 112.96, 61.70, 48.21. HRMS (ESI): m/z calcd for
C33H24ClN5O, 541. 1669, found 542.1776 [M+H] +.
1

4.2. Radiochemistry
4.2.1 General Procedure:
All the chemicals, and solvents were procured from Loba chemie, India. The analytical
techniques were used for quality control studies like Gas chromatography (Scion 436 GC,
Netherlands) with flame ionization detector (FID), High performance liquid
chromatography (HPLC) system (Dionex, ICS- 5000+, +, California. USA), Dose calibrator
(Capintec CRC-25PET, New Jersey. USA), Radio Thin layer chromatography (Radio TLC)
scanner (EZ-SCAN, California, USA) with multimode radiation detector (OMNI-RAD,
California. USA), pH paper (Fisher Scientific, New Hampshire. USA). The 18F radionuclide
was produced with 16.5MeV in-house Cyclotron (PETtrace 860, GE Healthcare, USA) by
the proton bombardment on the enriched 18O water using 18O (p, n) 18F reaction. The proton
bombardment was carried out with the beam current of range of 30 µA -65 µA for 10-15
min, which depend on the requirement of 18F radioisotope. The radiolabeling was performed
with the produced 18F in the semiautomatic synthesizer module i.e., Tracerlab FX2N, GE
Healthcare, Chicago. USA using the Helium (UHP-5.5) as a carrier gas.
4.2.2 Radio synthesis of target compound
A stock solution for 18F radioisotope eluent was made by adding 12 mg of potassium
carbonate and 60 mg of Kryptofix (K222) into a small beaker containing 12 ml of
acetonitrile and 525 L of the deionized water. The 18F was eluted from conditioned QMA
(4 ml ethanol followed by 10 ml water) cartridge through a mixture of QMA eluent stock
solution (1.5 mL), and acetonitrile (0.5 mL) into the reactor vessel. Initially, the [K 2.2.2]
K+[18F]- complex was dried and, the reaction with the precursor (5±0.5 mg,) in 1.5 ml of
DMF was carried out at 160C for 30 min. After completion of the reaction, the reaction
mixture was diluted with acidic water (5 mL, pH=3.0) and passed through tC18 light
cartridge for primary purification. The cartridge was washed with 5 mL of water, and 4 mL
of 8% ethanol. The final purification was performed using 3 mL of 20-100% ethanol.
4.2.3. Quality control parameters
The radiochemical purity, identity & residual solvent limits of the final 18F-7c preparation
was determined by High-Performance Liquid Chromatography (HPLC), Thin-Layer
Chromatography (TLC), and Gas Chromatography (GC). In TLC study, 5 µL of sample was
spotted on TLC plate above the mobile phase level and run the TLC using acetonitrile/saline
1

(1:1). In HPLC study, gradient method was used for better resolution of peaks on the spectra.
The gradient method was started with 5% acetonitrile (0-2.5 min), 5% to 100% acetonitrile
(2.5-10 min), then100% acetonitrile (10-12.5 min) and again at 5% acetonitrile (21.5-15min)
with a flow rate of 2 mL/min. The C18 column (250 mm X 4.6 mm, 5 μm) was used, and
UV detection wavelength (λmax) was 280 nm. In GC study, 1 L sample was injected into
the injector, the column (BR-200ms, 0.32 mm ID) was operated at 40°C for initial 3 min
followed by 50°C/min up to 8 min, and the temperature was set at 240°C and, the makeup
gas consists of Helium (28 mL/min), hydrogen gas (30 mL/min), and zero air (300 mL/min)
at the flow rate of 2 mL/min.
4.3. Biology
4.3.1. Cell Culture
Cells were procured from National Centre for Cell Science (NCCS) Pune, India and stocks
were maintained in the sterile laboratory conditions. breast (MCF-7 and MDA-MB-231)
lung (A-549 and NCI-H460), and Human keratinocyte (HaCaT) cancer cells were grown in
tissue culture flasks in DMEM (Dulbecco modified Eagle medium, Sigma) or MEM
(Minimum Essential Medium, Sig1ma) supplemented with 10% fetal bovine serum with 1X
stabilized antibiotic-antimycotic solution (Sigma) in a CO2 incubator at 37 0C with 5% CO2
and 90% relative humidity.
4.3.2. MTT assay
For all the synthesized compounds 7a-t. The anticancer activity was determined using MTT
assay. 1x104 cells per well were seeded, supplemented with 10% FBS in each well of 96-
well microculture plates and incubated for 48 h, at 37 °C in a CO2 incubator. Compounds,
diluted to the required concentrations in culture medium, were added to the wells with
respective vehicle control. After 48 h of incubation, 10 µL MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to all the plates and incubated
for 4 h. Then, the supernatant from each well was carefully decanted, formazon crystals
were dissolved in 100 µL of DMSO and absorbance was recorded at 570 nm wavelength.
4.3.3. DAPI staining
A-549 cells were cultured in 12-well plates and treated with 0.5, 1 and 2.5 µM of compound
7c. The untreated and treated cells were washed two times with PBS, followed by fixation
with 4% paraformaldehyde and staining with 10 µg/mL of DAPI. The cells were observed
1

for apoptotic characteristics under fluorescence microscope (Nikon, Inc. Japan) with
excitation at 359 nm and emission at 461 nm using DAPI filter at 200X magnification.
4.3.4. Cell cycle analysis
A-549 cells (1 x 106 cells/well) in 6 well plate were treated with different 0.25, 1 and 2.5
µM concentrations of the compound 7c for 24 h and cells were collected by trypsinisation,
washed with 150 mM PBS and were fixed with 70% ethanol for 30 min at 4 oC. after fixing,
cells were washed with PBS and stained with 400 µL of propidium iodide staining buffer
[PI (200 µg), Triton X (100 µL), DNAse-free RNAse A (2 mg) in 10 mL PBS] for 15 min
at room temp in dark. The samples were then analyzed for propidium iodide fluorescence
from 15,000 events by flow cytometry using BD Accuri C6 flow-cytometer.
4.3.5. Measurement of mitochondrial membrane potential (DΨm)
A-549 cells were cultured in 6 well plates at 5 x 105 cells/mL density and allowed to adhere
overnight. The cells were treated with 0.5, 1 and 2.5 µM concentrations of compound 7c for
24h. After 24h of compound treatment, the adherent cells were collected by trpsinisation,
washed with PBS and resuspended in solution of PBS containing rhodamine-123 (10
µg/mL) and further incubated at room temperature for 30 minutes. Cells were washed with
PBS to remove excess dye and resuspended in PBS. The samples were analyzed for
rhodamine-123 fluorescence using spectrofluorometer with an excitation and emission
wavelengths of 480 and 530 nm.
4.3.6. Annexin V-FITC/Propidium iodide dual staining assay
The Annexin V-FITC/Propidium iodide dual staining assay was performed using A-549
cells. To quantify the percentage of apoptotic cells, A-549 cells (1 x 106 mL per well) were
plated in six-well culture plates and allowed to grow for 24 h. after treatment with increasing
concentrations of compound 7c (0.5, 1 and 2.5 µM) for 24 h, cells were collected by
trypsinisation. The collected cells were washed twice with ice-cold PBS, then incubated with
200 µL1 x binding buffer containing 5 µL Annexin V-FITC, and then in 300 µL1 x binding
buffer containing 5 µL Propidium iodide (PI) for 5 min at room temp in the dark. After 15
min of incubation, cells were analyzed for apoptosis using BD-c6 accuri flow-cytometer.
4.3.7. Quantikine ELISA Human Galectin-1 Immunoassay
This assay employs the quantitative sandwich enzyme immunoassay technique. A
polyclonal antibody specific for human Galectin-1 has precoated on a microplate. Standards
1

and samples are pipetted into the wells and galectin-1 present is bound by the immobilized
antibody. After washing away any unbound substances, an enzyme-linked polyclonal
antibody specific for human Galectin-1 is added to the wells. The galectin levels were
measured by using R & D human galectins quantikine ELISA kit (R & D Systems, USA).
4.3.8. Molecular modelling studies
The gal-1 was downloaded from PDB (PDB ID: 4Y24) for protein preparation wizard, and
ready for docking i.e. removing water molecules, addition of missing side chains and, energy
minimization by OPLS-2005 force field. The final compound 7c was sketched using
chemdraw, and converted to 3D using Ligprep. The Glide XP docking algorithm was
utilized using a grid box volume of 10x 10x 10 Å at the centre of TD-139 as standard.
Acknowledgements
Department of Biotechnology (DBT), Govt. of India, New Delhi, for the young investigators
in cancer biology start up grants (6242-19/RGCB/PMD/DBT /MLKA/2015).
Conflicts of Interest
Authors declared no conflicts
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