Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.103016

In our pursuit to develop novel non-carbohydrate small molecule Galectin-1 Inhibitors, we have designed a series of 1-benzyl-1H-benzimidazole derivatives and demonstrated their anticancer activity. The compound 6g, 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol

Full text of DOI:10.1016/j.bioorg.2019.103016

Accepted Manuscript
Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer
agents
Nerella Sridhar Goud, S. Mahammad. Ghouse, Jatoth Vishnu, D. Komal, Venu
Talla, Ravi Alvala, Jakkula Pranay, Janish Kumar, Insaf A. Qureshi, Mallika
Alvala
PII:
S0045-2068(18)30681-3
https://doi.org/10.1016/j.bioorg.2019.103016
103016
DOI:
Article Number:
Reference:
YBIOO 103016
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 July 2018
7 May 2019
26 May 2019
Please cite this article as: N. Sridhar Goud, S. Mahammad. Ghouse, J. Vishnu, D. Komal, V. Talla, R. Alvala, J.
Pranay, J. Kumar, I.A. Qureshi, M. Alvala, Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated
anticancer agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103016
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Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer
agents
a
a
b
a
Nerella Sridhar Goud , S. Mahammad. Ghouse , Jatoth Vishnu , D. Komal ,
Venu Talla b, Ravi Alvala c, Jakkula Pranay d, Janish Kumar d, Insaf A. Qureshi
d, Mallika Alvala a*
a Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500 037, India
b Department of Pharmacology and Toxicology, National Institute of
Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
c G. Pulla Reddy College of Pharmacy, Hyderabad 500 028, India.
d Department of Biotechnology & Bioinformatics, School of Life Sciences, University of
Hyderabad, Hyderabad-500046, India.
ABSTRACT
In our pursuit to develop novel non-carbohydrate small molecule Galectin-1
Inhibitors, we have designed a series of 1-benzyl-1H-benzimidazole derivatives
and demonstrated their anticancer activity. The compound 6g, 4-(1-benzyl-5-
chloro-1H-benzo[d]imidazol-2-yl)-N-(4-hydroxyphenyl) benzamide was found
to be most potent with an IC50 of 7.01 ± 0.20 µM and arresting MCF-7 cell
growth at G2/M phase and S phase. Induction of apoptosis was confirmed by
morphological changes like cell shrinkage, blebbing and cell wall deformation,
dose dependent increase in the mitochondrial membrane potential (∆Ψm) and
ROS levels. Further, dose dependent decrease in Gal-1 protein levels proves
Gal-1 mediated apoptosis by 6g. Molecular docking studies were performed to
understand the Gal-1 interaction with compound 6g. In addition, RP-HPLC
studies showed 85.44% of 6g binding to Gal-1. Binding affinity studies by
fluorescence spectroscopy and Surface Plasmon Resonance (SPR) showed that
6g binds to Gal-1 with binding constant (Ka) of 1.2x104 M-1 and equilibrium
constant KD value of 5.76 x10-4 M respectively.
Research Highlights

 Series of small molecules 1-benzyl-1H-benzimidazole analogues were
synthesized.
 Identified the synthetic molecule 6g which lowers the Gal-1 expression in
MCF-7 cells.
 Reporting 6g as anticancer agent with an IC50 of 7.01 ± 0.20 µM
mediated by Gal-1.
 Reporting the binding studies by SPR and fluorescence spectroscopy of
non-carbohydrate compound 6g to Gal-1.
Key words: 1-benzyl-1H-benzimidazoles, Galectin-1, cancer, Fluorescence
Spectroscopy, RP-HPLC, Surface Plasmon Resonance.
1. Introduction
Human Galectin-1 (Gal-1) is a family of carbohydrate-binding protein of S-type
(abundance in cysteine residues/ sulfhydryl residues) lectin having a high
affinity for β-galactosides on the cell surface and extracellular glycoproteins and
on glycolipids. Gal-1 is located both extracellularly and intracellularly and
found at the cell surface as secreted Gal-1 binds to lactosamines on
glycoconjugates expressed by the same cell or by neighbouring cells. Gal-1 is
expressed differentially by various normal and pathological tissues and appears
to be functionally multivalent, undertaking a wide range of biological activity
[1]. Reports from various studies show that Gal-1 contributes in tumour
metastasis, cell adhesion, increased invasiveness, angiogenesis and evasion of
the immune response. Gal-1 plays a key role in adhesion of prostate and ovarian
cancer cells to the Extra Cellular Matrix (ECM) and in turn mediates cancer
progression [2, 3], also mediate homotypic cell aggregation of human
melanoma cells in a carbohydrate-dependent manner and, it has been shown to
affect cell migration of cancer cells and influence their invasiveness [4].
Recombinant Gal-1 added extracellularly to melanoma cells induces a dose
dependent increase in cell-adhesion to laminin or fibronectin [5]. In fact,

exogenously added Gal-1 causes increased motility of glioblastoma cells in
vitro [6]. Gal-1 induces cell growth inhibition in human T-cells, inhibits T-cell
activation and promotes apoptosis of activated T cells [7]. Furthermore,
Matarrese et al. showed that Gal-1 sensitises resting T cells to CD95/Fas-
mediated cell death [8]. Intracellular Gal-1 plays a key role in the initiation of
transformed phenotype of tumours via interacting with signalling pathways such
as p21 [9], ganglioside GM1 receptor related pathways [10], RAS [11], RAF
and PI3/K [4]. It has been proven that Gal-1 is over expressed in pancreatic
ductal adenocarcinomas as compared to normal tissue and pancreatitis [12].
Tumour cells may impair T-cell effector functions through secretion of Gal-1
and this mechanism may contribute in tilting the balance towards an
immunosuppressive environment at the tumour site. The link between Gal-1
mediated immune regulation and its contribution to tumour-immune escape has
been well established through various knock down transfectant experiments.
Blanchard H et. al. summarized about monovalent- and multivalent-
carbohydrate-based inhibitors, peptides- and peptidomimetics as Gal-1
inhibitors and their potential therapeutic applications in their patent review [13].
Current study was initiated with an aim to develop non carbohydrate Gal-1
inhibitors as anticancer agents.
Benzimidazole (1H-benzo[d]imidazole) is a privileged scaffold, due to its
presence in a wide range of bioactive compounds by interacting with numerous
biomolecular targets and exhibit diverse activities like Antihypertensive [14],
Anti-inflammatory [15], Antimicrobial [16], Antiviral [17], Antiparasitic [18],
Antioxidants [19], Anticoagulants [20], CNS stimulants [21] and anticancer
activity [22]. The research in the development of bioactive molecules with
benzimidazole nucleus has accelerated during last 10 years hence it has
substantially been used as a lead compound to design and synthesize analogues
with improved activity. Bendamustine is a US FDA approved anti-cancer drug

containing benzimidazole moiety (Figure-1). Telmisartan possessing an N-
benzylated benzimidazole moiety is reported to induce early apoptosis in the
prostate cancer cells [23]. Tsung-Chieh Shih et al. reported a novel Gal-1
inhibitor named LLS2 which was discovered through One-Bead-Two
Compound library and potentiated the anti-tumour effects of Paclitaxel in
vivo [24]. The same group reported another novel Gal-1 inhibitor named LLS30
which showed synergistic anti-tumor activity with docetaxel and also
effectively inhibit the metastasis and invasion of prostate cancer in vivo [25].
Cl
Cl
Cl
CH3
CH3
N
N
N
NH2
NH
N
N
HO
N
AcO
AcO
O
N
Cl
O
OH
N
N
O
N
Telmisartan
Bendamustine
O
NH2
LLS2
F
F
O
N
N
N
Heterocyclic/
Aromatic Rings
HN
Cl
N
Cl
R
Designed benzimidazoles (6i-6r)
Designed benzimidazoles (6a-6h)
Figure 1. Representative examples of available benzimidazole drugs, reported
derivatives and rationale for the designed target compounds 6a-6r.
With our interest to develop more potent cytotoxic molecules via Gal-1
inhibition, the present study aims in synthesis of non-carbohydrate small
molecules 1-benzyl-1H-benzimidazoles.
2. Results and Discussion
2.1. Chemistry
The synthetic strategy for the preparation of 1-benzyl-1H-benzimidazole
analogues 6a-6r is shown in Scheme-1. The N1-benzyl-4-chlorobenzene-1,2-
diamine (4) was condensed with various 4-formyl-N-phenylbenzamides (5a-

5h), various aromatic and heterocyclic aldehydes. Initially the N-benzyl-4-
chloro-2-nitroaniline (3) was synthesized from commercially available 4-
chloro-2-nitro aniline with benzyl bromide [26] and the nitro group of
compound (3) was further reduced to amino functional group by using stannous
chloride dihydrate offered N1-benzyl-4-chlorobenzene-1,2-diamine (4). On the
other hand, the 4-formyl-N-phenylbenzamides (5a-5h) were synthesized from
the commercially available 4-formyl benzoic acid and different aniline
derivatives [27]. Finally, refluxing 4-formyl-N-phenylbenzamides (5a-5h),
different aromatic and heterocyclic aldehydes and N1-benzyl-4-chloro
orthophenylene diamine in ethanol in the presence of sodium metabisulphite
[28] furnishing the target compounds 6a-6r with moderate to good yields.
Br
H
NH2
NO2
N
H2O, Reflux, 2h
85-88%
Cl
Cl
NO2
(1)
(2)
(3)
SnCl2.2H2O
EtOH, rt, 12h, 70-72%
H
N
Cl
NH2
(4)
O
OH
NH2
O
R
HATU, DIPEA, DMF, 3h
65-68%
N
H
OHC
R
CHO
(1a)
(5a-5h)
(2a)
Cl
O
N
N
H
N
HN
Intermediates 5a-5h
R
Cl
NH2
Na2S2O5, EtOH, reflux
7h, 80-82%
(6a-6h)
(4)

Cl
N
N
H
N
R
R CHO
Cl
NH2
Na2S2O5, EtOH, reflux
7h, 85-88%
(6i-6r)
(4)
6i R = indole-3-yl
6j R = pyrrole-2-yl
6k R = benzo[d] [1,3] dioxole-5-yl
6l R = furan-2-yl
6m R= pyridine-2-yl
6n R= thiophene-2-yl
6o R = 8-hydroxyquinolin-2-yl
6p R = 3-phenoxyphen-1-yl
6q R = 3-ethoxy-4-hydroxyphen-1-yl
6a R = H
6b R = 4-OCH3
6c R = 4-CH(CH3)2
6d R = 4-F
6e R = 3,4,5-tri-OCH3
6f R = 3,4,5-tri-CH3
6g R = 4-OH
6h R = 4-Cl
6r R = 3,5-dimethoxy-4-hydroxyphen-1-yl
Scheme 1. Synthesis of different 1-benzyl-1H-benzimidazole analogues 6a-6r.
All synthesized compounds (6a-6r) were characterized by analytical techniques
1
13
1
viz. HRMS, H and C-NMR Spectroscopy. The H-NMR spectrum of 6a
showed a sharp singlet of CH2 protons of benzyl group at δ 5.60 and a sharp
singlet of N-attached proton (amidic proton) at δ 10.45 and remaining all
13
protons appeared in the range of δ 6.90-8.45. In the C-NMR spectrum of 6a,
the N-attached methylene carbon of benzyl group appeared at δ 48.22 and the
carbonyl carbon appeared at δ 165.21. The remaining all carbons appeared in
the range of δ 113.29-154.19. Similar fashion was observed in the 1H-NMR and
13C-NMR spectra of all other compounds (6a-6r). The HRMS (ESI) of 6a-6r
showed corresponding [M+H] + peaks based on their molecular weights.
2.2. Biological Activity
2.2.1. In vitro cytotoxic activity

The synthesized 1-benzyl-1H-benzimidazole derivatives (6a-6r) were screened
for their in vitro cytotoxicity against a panel of human cancer cell lines viz. Lung
cancer (A-549), prostate cancer (DU-145), breast cancer (MCF-7 and MDA-
MB-231) and colorectal cancer (HCT-116 and HT-29), using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [29]. The
IC50 (µM) values of compounds 6a-6r and the standard, 5-fluorouracil are
displayed in Table-1.
Table 1. IC50 (µM) valuesa for the derivatives 6a-6r by MTT assay.
A-549 b
DU-145 c MCF-7 d
HCT-116 e
HT-29 f
>15
Compound
MDA-MB-
231 d
6a
6b
6c
6d
6e
12.14 ±
0.21
>15
>15
>15
>15
>15
>15
>15
10.03 ±
0.50
11.81 ±
0.75
10.09 ±
0.51
11.52 ±
0.45
>15
14.32 ±
0.37
>15
>15
>15
>15
>15
>15
>15
12.84 ±
0.32
>15
>15
>15
>15
>15
>15
14.12 ±
0.55
13.39 ±
0.21
>15
37.86 ±
0.42
>15
>15
6f
6g
>15
10.69 ±
0.14
9.30 ± 0.42
7.01 ± 0.20
13.89 ±
0.74
14.04 ±
0.62
12.91 ±
0.52
6h
6i
6j
6k
6l
6m
6n
6o
>15
>15
>15
>15
7.29 ± 0.71
>15
9.61 ± 0.24
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
>15
11.84 ±
0.82
6p
13.46 ±
0.17
>15
11.63 ±
0.24
>15
>15
>15
6q
6r
>15
>15
>15
>15
7.50 ± 0.83
7.14 ± 0.70
>15
>15
>15
>15
>15
>15
5-
1.60
1.92 ± 0.16 3.20 ± 0.50 0.60 ± 0.30 1.16 ± 0.06 3.90 ± 1.10
fluorouracil
±0.10

b
a50% Inhibitory concentration after 48 hr of drug treatment. Human lung
c
d
cancer. Human prostate cancer. Human breast cancer. e, f Human colorectal
cancer.
Among all, the compound 6g had shown significant growth inhibition with an
IC50 of 10.69 ± 0.14 µM, 13.89 ± 0.74 µM, 7.01 ± 0.20 µM, 14.04 ± 0.62 µM,
12.91 ± 0.52 µM in A-549, DU-145, MCF-7, MDA-MB-231 and HCT-116 cell
lines, respectively with 7.73% inhibition on Normal Rattus Norvegicus Kidney
(NRK) cells at 20 µM concentration. The compound 6g displayed no
cytotoxicity against NRK-52E (normal kidney cells) and L-132 (normal lung
cells) with an IC50 values of 126.45 µM and 220.56 µM respectively, which
clearly indicates that the compound is nontoxic. Further mechanistic studies of
6g were carried in MCF-7 cell line at 1.25, 2.5, 5 and 10 µM concentrations.
2.2.2. Acridine Orange/Ethidium Bromide (AO/EB) staining
Acridine Orange (AO)/Ethidium Bromide (EB) staining assay is performed to
differentiate between live, apoptotic and necrotic cells [30]. Acridine Orange
(AO) can permeate the intact cell membrane and stain the nuclei green, whereas
Ethidium Bromide (EB) can only stain the nucleus of cells that have lost
membrane integrity in red. It can be inferred from Figure 2 that the control cells
display normal morphology and appeared green in colour. Fluorescence
microscopic images of MCF-7 cells treated with compound 6g clearly showed
morphological changes such as membrane blebbing, cell shrinkage, chromatin
condensation and apoptotic body formation, suggesting that the compound 6g
induced apoptosis in MCF-7 cancer cells in dose dependent manner.
Figure 2
2.2.3. DAPI nucleic acid staining
DAPI (4',6-diamidino-2-phenylindole), a fluorescent dye capable of strong
binding to A–T rich sequences of DNA and helps in the visualization of

chromatin condensation or nuclear damage [31]. It distinguishes live cells from
apoptotic cells by staining the characteristic condensed nuclei of the latter bright
blue. Therefore, this staining technique was considered of interest to detect the
induction of apoptosis by the compound 6g in MCF-7 cells. The results from
Figure 3 demonstrated that the nuclear structure of untreated control cells was
intact whereas cells treated with 6g displayed condensed, fragmented nuclei.
Figure 3
2.2.4. Cell cycle analysis
Loss of DNA content is a typical characteristic feature of apoptosis. Treatment
with 6g that blocks cell division led to changes in the frequency of cells in every
phase relative to untreated cells. MCF-7 cells were treated with compound 6g,
at concentrations of 1.25, 2.5, 5 and 10 µM for 24 h, and stained with propidium
iodide and analysed by using BD FACSVerseTM flow analyser [32]. The
results from Figure 4 indicates that the untreated control cells exposed to
DMSO showed 20.53% cells in G2/M phase, whereas compound 6g treatment
resulted in increased G2/M population to 21.01% at 1.25µM, 22.18% at 2.5µM,
24.44% at 5µM, and 26.82% at 10µM concentration and S phase population
from 10.73% in control to 11.76% at 1.25µM, 12.98% at 2.5µM, 13.75% at
5µM, and 15.79% at 10µM concentration. These results clearly indicated that
treatment of MCF-7 cells with the compound 6g arrest cell growth at G2/M
phase and S phase.
Figure 4
2.2.5. Mitochondrial membrane potential
It is known mitochondria play a pivotal role in apoptosis in response to stress
and the loss of mitochondrial membrane potential (MMP) is a sensitive marker
of early mitochondrial damage during apoptosis which can be studied by JC1
staining assay [33]. MCF-7 cells upon treatment with compound 6g at

concentrations of 1.25, 2.5, 5 and 10 μM for 24 hours were stained with JC1 dye
and analysed with flow cytometer. Figure 5 indicated that increase in apoptotic
cells (depolarized cell population) from 14.56% in control to 48.95%, 37.15 %,
29.08 % and 28.36% in 10, 5, 2.5 and 1.25μM indicates decreased mitochondrial
membrane potential in dose dependent manner by compound 6g in MCF-7 cells.
Figure 5
2.2.6. AnnexinV/Propidium iodide dual staining assay
To quantify the percentage of apoptosis induced by compound 6g in MCF-7
cells, the Annexin V/Propidium iodide dual staining assay [34] was carried. The
Annexin V-Alexa Flour 488/PI dual staining assay facilitates the detection of
live cells (LC; AV-/ PI-), early apoptotic cells (EA; AV+/PI-), late apoptotic
cells (LA; AV+/PI+) and necrotic cells (NC; AV-/PI+). Figure 6 shows that
MCF-7 cells with compound 6g displayed rise in total percentage of apoptotic
(early and late apoptotic cells-Annexin v + cells) and dead cells from 10.77% in
control to 11.43% in 1.25μM, 12.98% in 2.5μM, 21.06% in 5μM, and 36.47% in
10μM respectively in a dose dependent manner.
Figure 6
2.2.7. Dichlorofluorescein (DCF) fluorescence
The effect of compound 6g on cellular Reactive Oxygen Species (ROS) levels in
MCF-7 cells was studied using cell permeant fluorogenic dye DCFDA (2',7' –
Dichlorofluorescin diacetate) [35]. A significant increase of ROS levels was
observed in cells treated with 6g at concentrations of 1.25, 2.5, 5 and 10µM. The
percentage of ROS generation by compound 6g in dose dependent manner was
confirmed by intensity of DCF as showed in Figure 7.
Figure 7

2.2.8. Effect of compound 6g on Gal-1 expression:
Human Gal-1, protein of lectin family showing affinity towards β-galactosides
has emerged as a critical regulator of tumor progression and metastasis, by
modulating diverse biological events including homotypic cell aggregation,
migration, apoptosis, angiogenesis and immune escape. Recent reports
demonstrated that knockdown of Gal-1 suppresses the growth and invasion of
osteosarcoma cells through inhibition of the MAPK/ERK pathway [36], Immune
escape due to the suppression of T-Cells [37], Increasing Heparin-binding
epidermal growth factor (HB-EGF) [38]. Compagno et. al., showed importance
of glycan and galectins in prostate cancer angiogenesis and metastasis [39].
Compound 6g was examined for its effect on Gal-1 levels. Briefly, MCF-7 cells
were treated with different concentration of compound 6g (10, 30, 100 and
300μM) and after 24hr of treatment, supernatant was collected. Equal amounts of
supernatant were subjected to quantitative enzyme immunoassay as per
manufacture’s protocol [DGAL10, R&D Systems, USA]. Reduced levels of Gal-
1 expression by compound 6g (Figure 8) in dose dependent manner was
observed. Table 2 represents the amount of Gal-1 protein expression in control
and 6g treated cells. The Gal-1 protein expression by compound 6g was
calculated against standard graph produced as per the supplier’s instructions.
Figure 8
Table 2: Amount of Gal-1 protein expression in control and 6g treated MCF-7
cells.
concentration
Absorbance at
450 nm
Gal-1 concentration(µg/mL)
Control
0.5323
3.337825
1.550186
1.521375
6g (10 µM)
6g (30 µM)
0.3400
0.3369

6g (100 µM)
6g (300 µM)
0.3005
0.2500
1.183086
0.713755
Figure 9
Binding of 6g to Gal-1 protein was studied by chromatographic analysis using
RP-HPLC [40, 41]. Initially the RP-HPLC method was developed for
compound 6g using chromatographic parameters as mentioned in the
experimental section, where 6g showed 85.44 ± 0.91% binding efficiency with
Gal-1 protein, which indicates there is an existence of the strong interaction of
compound 6g with Gal-1 protein and the chromatographic results were shown
in Figure 9. These results stipulate that the molecular target of 1-benzyl-1H-
benzimidazole derivatives might be Gal-1.
2.2.9. Fluorescence Spectroscopy study
The maximum emission spectra of Gal-1 was at 343 nm and there was decrease
in fluorescence intensity with increasing concentration of the compound
(Figure 10A). The plot of log[6g] versus log(Fo-F)/F had given a linear
relationship (Figure 10B) and the number of binding sites were calculated from
the slope, which were found to be 1 and infers interaction of protein and
Figure 10
compound in 1:1 ratio. The binding constant (Ka) was calculated from the
intercept value which was observed as 1.2x104 M-1. The intensity of the
fluorescence was quenched upon increase of ligand concentration and the
bimolar quenching constant (Kq) was calculated to be 2.3 x 1012 M-1 S-1 which
is larger than diffusion control limit [42] suggesting interaction of protein and
ligand as well as the mode of quenching to be static.
2.2.10. Surface Plasmon Resonance (SPR) studies

The Gal-1/6g interaction was analyzed through SPR using immobilized Gal-1
(ligand, 8070 RU) and compound 6g (analyte) at various concentrations.
Sensorgram was measured for each of concentration of compound 6g (Figure
11) and fitted using 1:1 interaction steady state affinity model. The equilibrium
constant KD value was found to be 5.76 x10-4 M using Biacore T200 Evaluation
software version 2.0. SPR analysis has shown interaction of Gal-1/6g, which is
in corroboration with florescence studies.
Figure 11
2.2.11. Molecular docking
The interaction of Gal-1 with the compound 6g was studied using molecular
docking calculations using the Glide docking module of Schrödinger suite. The
3D crystal co-ordinates of human Gal-1 were retrieved from protein data bank
(PDB ID: 1GZW). The ligand interaction diagram of 6g was shown in Figure
12. Hydrogen bonding interaction was found at a distance of 2.58Å between
phenolic OH acting as a donor (atom no. 20152) and C=O of ASP125 acting as
Figure 12
an acceptor (atom no. 950). Additional hydrogen bond was found at a distance
of 4.23Å between C=NH2+ of ARG48 that acts as donor (atom no. 360) and
benzimidazole nitrogen acting as an acceptor (atom no. 2028). Further, π-cation
interaction was found between nitrogen of ARG48 and aromatic group of 6g
and three π- π stacking between indole ring of TRP68 and benzimidazole as
well as aromatic ring system of 6g.
A set of ADMET related properties were calculated using qikprop program
(Qikprop, version 6.5, Schrödinger, LLC, New York, NY, 2014). The
physicochemical properties of 6g are marched with the prescribed ranges as
represented in Table 3. Additionally, the molecular weight (mol. Wt.),
hydrogen bond donors (donor HB), hydrogen bond acceptors (accpt HB),

partition coefficient (QPlogPo/w), exhibited acceptable values that followed
Lipinski rule of five.
Table 3: The ADMET properties of compound 6g.
S. No Properties or
Descriptors
Recommended
Values
Compound 6g
1
2
3
4
5
6
7
8
9
Molecular weight
Dipole moment
Total SASA
Molecular Volume
No. of rotatable bonds
Donor HB
Acceptor HB
QP Polarizability
QP logP o/w
130.0 – 725.0
1.0 – 12.5
300.0 – 1000.0
500.0 – 2000.0
0 – 15
453.927
2.796
764.191
1368.395
5
2
4.75
50.638
5.793
0.0 – 6.0
2.0 – 20.0
13.0 – 70.0
2.0 – 6.5
10
11
12
13
QP log BB
QP log HERG
Human Oral Absorption
Percent Human Oral
Absorption
-3.0 – 1.2
Concern below -5
1-3
-0.863
-7.708
1
>80% is high
100
14
15
Rule of Five violations
No. of metabolites
<25% is low
Maximum of 4
1
2
From above studies, it is clear that compound 6g is not toxic and following
Lipinski’s rule of 5.
3. Conclusion
Human Gal-1 plays an essential role in various biological processes including
metastasis, tumour progression and host-pathogen interaction. There are many
Gal-1 inhibitors reported in literature which are of monovalent- and multivalent-
carbohydrate-based inhibitors, peptides- and peptidomimetics. Current work
demonstrates synthesis of a series of 1-benzyl-1H-benzimidazole (6a-6r)
derivatives and anticancer potentials of compound 6g on MCF-7 cell lines with
an IC50 of 7.01 ± 0.20 µM. It also explains the underlying molecular events

occurring in the presence of compound 6g. Together, morphological changes
such as cell shrinkage, rounding of cell, membrane blebbing, cell cycle arrest at
G2/M and S phase, decrease in MMP and increased ROS levels demonstrates
the induction of apoptosis by compound 6g. A dose dependent decrease in Gal-
1 protein expression explains Gal-1 mediated apoptosis by compound 6g.
Further binding affinity studies using fluorescence, SPR and RP-HPLC
demonstrate the 6g binding with Gal-1. Molecular docking studies show the
binding orientation of 6g and its interaction with binding site amino acids. Our
data provide new light into design and development of non-carbohydrate, non
peptidomimetics small molecule Gal-1 inhibitors.
4. Materials and Methods
4.1. Chemistry
All the starting materials, reagents and solvents were purchased from
commercial suppliers. Analytical thin layer chromatography (TLC) was
performed on MERCK pre-coated silica gel 60-F254 aluminum plates.
Visualization of the spots on TLC plates was achieved either by exposure to
iodine vapour and UV light. All melting points were recorded on Stuart®
SMP30 melting point apparatus and are uncorrected. Column chromatography
was performed using silica gel (60-120 mesh) and was eluted with ethyl acetate-
hexane. NMR spectra were recorded on Bruker 500 (500 MHz for 1H-NMR and
13
125 MHz for C NMR) using CDCl3 and DMSO as solvents. Chemical shift
was reported in parts per million (ppm) with respect to internal standard Tetra
Methyl Silane (TMS). Coupling constants were quoted in Hertz (Hz). High
Resolution Mass Spectra (HRMS) were obtained on Agilent Q-TOF-Mass
Spectrometer 6540-UHD LC/HRMS operating at 70 eV using direct inlet.
4.1.1. Synthetic procedures and spectral data
4.1.2. 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 °C 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, 1H), 6.94 (d, J = 9.3 Hz, 1H), 4.65 (d, J = 6.2 Hz,
13
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.
4.1.3. 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
purified by column chromatography.
Brown solid, yield 82%; mp 172-174 °C; 1H 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 Hz, 1H), 6.27 (d, J
13
= 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.
4.1.4. Synthesis of 4-formyl-N-substitutedphenylbenzamides (5a-5h)

To a solution of 4-formylbenzoic acid (1 eq) in dimethylformamide (3 mL)
was added HATU (1.5 eq) portion wise. The reaction mixture was then stirred
at cooled temperature 0 °C for 30 min, furthermore added substituted anilines
(1.1 eq) and DIPEA (3 eq) to the reaction mixture and stirred at room
temperature for 2 hours. On cooling at room temperature 20-30 mL of cold
water was added and stirred for 10 min. The solid separated out was filtered
with pump and dried. The products obtained were recrystallized from ethanol.
4.1.5. General procedure for the synthesis of final compounds (6a-6r).
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 of aromatic, heterocyclic aldehydes and 4-formyl-N-
substituted phenyl benzamides (5a-5h) (1 eq). The reaction mixture was stirred
under reflux 80 °C 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.
4.1.5.1. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-phenylbenzami
de (6a)
1
Off white solid, yield 81%; mp 257-259 °C; H NMR (500 MHz, DMSO) δ
10.40 (s, 1H), 8.11 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 8.3 Hz, 2H), 7.86 (d, J = 1.8
Hz, 1H), 7.80 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.39 – 7.25 (m, 6H),
13
7.12 (t, J = 7.4 Hz, 1H), 7.01 (d, J = 7.3 Hz, 2H), 5.69 (s, 2H). C NMR (125
MHz, DMSO) δ 165.21, 154.19, 143.53, 139.47, 136.93, 136.65, 135.29,
132.62, 129.59, 129.36, 129.12, 128.62, 128.13, 127.58, 126.56, 124.34,
123.81, 120.87, 119.18, 113.29, 48.22. HRMS (ESI): m/z calcd for
C27H20ClN3O, 437. 1295, found 438.1385 [M+H] +.

4.1.5.2. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(methoxyphenyl)
benzamide (6b)
1
Pale brown solid, yield 80%; mp 206-208 °C; H NMR (500 MHz, DMSO) δ
10.28 (s, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 8.1 Hz, 2H), 7.86 (s, 1H),
7.70 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 8.7 Hz, 1H), 7.38-7.26 (m, 4H), 7.01 (d, J
13
= 7.4 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 5.68 (s, 2H), 3.75 (s, 3H). C NMR
(125 MHz, DMSO) δ 164.74, 156.15, 154.26, 143.67, 136.96, 136.69, 135.33,
132.55, 132.52, 129.55, 129.35, 128.50, 128.12, 127.51, 126.56, 123.74,
122.46, 119.22, 114.25, 113.25, 55.66, 48.20. HRMS (ESI): m/z calcd for
C28H22ClN3O2, 467. 1401, found 468.1492 [M+H] +.
4.1.5.3. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-isopropyl phe
nyl) benzamide (6c).
1
Off white solid, yield 84%; mp 218-220 °C; H NMR (500 MHz, DMSO) δ
10.39 (s, 1H), 8.10 (d, J = 6.9 Hz, 2H), 7.95 – 7.88 (m, 2H), 7.86 (d, J = 1.6 Hz,
1H), 7.84 – 7.78 (m, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.35
– 7.21 (m, 6H), 7.01 (d, J = 6.2 Hz, 2H), 5.68 (s, 2H), 2.83 (d, J = 38.7 Hz, 1H),
13
1.20 (d, J = 6.8, 3H), 1.06 (d, J = 7.0, 3H). C NMR (125 MHz, DMSO) δ
165.13, 154.18, 143.57, 136.93, 136.45, 135.30, 132.70, 129.60, 129.35,
128.59, 128.13, 127.57, 126.83, 126.55, 123.80, 122.74, 120.98, 119.19,
115.80, 113.28, 48.21, 33.40, 24.42, 19.02. HRMS (ESI): m/z calcd for
C30H26ClN3O, 479. 1764, found 480.1859 [M+H] +.
4.1.5.4. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-fluorophenyl)
benzamide (6d)
1
white solid, yield 82%; mp 222-224 °C; H NMR (500 MHz, DMSO) δ 10.43
(s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 1.9 Hz,
1H), 7.83 – 7.79 (m, 2H), 7.60 (d, J = 8.7 Hz, 1H), 7.33 – 7.20 (m, 6H), 7.00 (d,
13
J = 7.2 Hz, 2H), 5.67 (s, 2H). C NMR (125 MHz, DMSO) δ 165.15, 154.31,

144.45, 143.95, 137.01, 135.41, 132.95, 129.57, 129.35, 128.57, 128.11,
127.42, 126.83, 126.53, 123.68, 122.74, 120.97, 119.33, 115.81, 113.20, 48.17.
HRMS (ESI): m/z calcd for C27H19ClFN3O, 455. 1201, found 456.1284 [M+H]
+.
4.1.5.5. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(3,4,5-trimethox
yphenyl) benzamide (6e)
1
Off white solid, yield 91%; mp 185-187 °C; H NMR (500 MHz, DMSO) δ
10.28 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 1.9
Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.34 – 7.24 (m, 6H), 7.00 (d, J = 7.2 Hz, 2H),
5.68 (s, 2H), 3.78 (s, 6H), 3.65 (s, 3H). 13C NMR (125 MHz, DMSO) δ 164.96,
154.27, 153.10, 143.95, 137.02, 136.48, 135.60, 135.43, 134.32, 132.91,
129.54, 129.35, 128.48, 128.11, 127.42, 126.54, 123.68, 119.34, 113.19, 98.55,
60.60, 56.22, 48.18. HRMS (ESI): m/z calcd for C30H26ClN3O4, 527. 1612,
found 528.1702 [M+H] +.
4.1.5.6. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(3,4,5-trimethyl
phenyl) benzamide (6f)
white solid, yield 81%; mp 268-270 °C; 1H NMR (500 MHz, DMSO) δ 9.83 (s,
1H), 8.13 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 1.9 Hz, 1H),
7.57 (d, J = 8.7 Hz, 1H), 7.33 – 7.26 (m, 4H), 7.01 (d, J = 7.3 Hz, 2H), 6.94 (s,
13
2H), 5.68 (s, 2H), 2.26 (s, 3H), 2.15 (s, 6H). C NMR (125 MHz, DMSO) δ
164.82, 154.38, 143.99, 137.04, 136.22, 136.10, 135.71, 135.39, 132.94,
132.80, 129.65, 129.36, 128.82, 128.42, 128.10, 127.40, 126.52, 123.64,
119.34, 113.21, 48.18, 21.01, 18.46. HRMS (ESI): m/z calcd for C30H26ClN3O,
479. 1764, found 480.1853 [M+H] +.
4.1.5.7. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-hydroxyphen
yl) benzamide (6g)

1
Pale yellow solid, yield 83%; mp 223-225 °C; H NMR (500 MHz, DMSO) δ
10.27 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.74 – 7.66 (m,
3H), 7.49 (d, J = 8.7 Hz, 1H), 7.26-7.12 (m, 5H), 7.00 (d, J = 6.4 Hz, 1H), 6.89
13
(d, J = 7.3 Hz, 2H), 5.56 (s, 2H). C NMR (125 MHz, DMSO) δ 165.07,
153.95, 147.23, 143.94, 141.06, 136.93, 135.45, 134.85, 131.00, 130.47,
130.04, 129.34, 128.13, 127.49, 126.61, 123.83, 122.34, 119.42, 114.53,
113.31, 48.20. HRMS (ESI): m/z calcd for C27H20ClN3O2, 453. 1244, found
454.1330 [M+H] +.
4.1.5.8. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-chlorophenyl)
benzamide (6h)
1
Off white solid, yield 84%; mp 216-218 °C; H NMR (500 MHz, DMSO) δ
10.32 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.73 – 7.67 (m,
3H), 7.48 (d, J = 8.7 Hz, 1H), 7.22 – 7.08 (m, 6H), 6.88 (d, J = 7.2 Hz, 2H), 5.56
(s, 2H). 13C NMR (125 MHz, DMSO) δ 164.92, 153.90, 143.24, 139.17, 136.63,
136.36, 135.00, 132.32, 129.29, 129.06, 128.83, 128.32, 127.84, 127.28,
126.27, 124.04, 123.51, 120.58, 118.88, 113.00, 47.92. HRMS (ESI): m/z calcd
for C27H19Cl2N3O, 471. 0905, found 472.0977 [M+H] +.
4.1.5.9. 1-benzyl-5-chloro-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (6i)
1
white solid, yield 85%; mp 247-249 °C; H NMR (500 MHz, CDCl3) δ 10.52
(s, 1H), 8.05 (s, 1H), 7.87 (d, J = 7.1 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.40-
7.34 (m, 3H), 7.30 (d, J = 5.9 Hz, 1H), 7.16 (dd, J = 8.5, 2.6 Hz, 2H), 7.05 (dd, J
= 16.4, 8.9 Hz, 2H), 6.98 (d, J = 7.1 Hz, 2H), 5.44 (s, 2H). 13C NMR (125 MHz,
CDCl3) δ 151.16, 136.14, 135.99, 135.78, 133.92, 129.25, 128.54, 128.03,
126.23, 125.87, 123.35, 123.28, 123.20, 121.38, 120.60, 118.44, 111.79,
111.05, 48.37. HRMS (ESI): m/z calcd for C22H16ClN3, 357. 1033, found
358.1114 [M+H] +.

4.1.5.10. 1-benzyl-5-chloro-2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazole (6j)
1
Off white solid, yield 83%; mp 192-194 °C; H NMR (500 MHz, CDCl3) δ
11.22 (s, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.40-7.33 (m, 3H), 7.21 – 7.13 (m, 4H),
7.09 (d, J = 0.9 Hz, 1H), 6.56 (s, 1H), 6.29 (dd, J = 6.1, 2.5 Hz, 1H), 5.62 (s,
13
2H). C NMR (125 MHz, CDCl3) δ 148.36, 142.35, 135.28, 134.41, 129.28,
128.74, 128.06, 125.88, 123.17, 122.70, 119.93, 117.98, 111.44, 110.59,
110.56, 48.43. HRMS (ESI): m/z calcd for C18H14ClN3, 307. 0876, found
308.0956 [M+H] +.
4.1.5.11. 2-(benzo[d] [1,3] dioxol-5-yl)-1-benzyl-5-chloro-1H-benzo[d]imid
azole (6k)
white solid, yield 85%; mp 168-170 °C; 1H NMR (500 MHz, CDCl3) δ 7.89 (s,
1H), 7.38 – 7.32 (m, 3H), 7.25 – 7.19 (m, 3H), 7.12 (d, J = 8.6 Hz, 1H), 7.07 (d,
J = 6.6 Hz, 2H), 6.91 – 6.88 (m, 1H), 6.04 (s, 2H), 5.48 (s, 2H). 13C NMR (125
MHz, CDCl3) δ 154.51, 149.77, 148.24, 135.51, 135.43, 134.18, 129.28,
128.91, 128.16, 125.86, 123.96, 123.87, 122.10, 119.14, 111.48, 109.57,
108.82, 101.76, 48.72. HRMS (ESI): m/z calcd for C21H15ClN2O2, 362. 0822,
found 363.0908 [M+H] +.
4.1.5.12. 1-benzyl-5-chloro-2-(furan-2-yl)-1H-benzo[d]imidazole (6l)
1
Pale yellow solid, yield 82%; mp 164-166 °C; H NMR (500 MHz, CDCl3) δ
7.84 (s, 1H), 7.61 (d, J = 1.1 Hz, 1H), 7.35-7.24 (m, 4H), 7.25 – 7.19 (m, 2H),
7.12 (d, J = 6.7 Hz, 2H), 6.60 (dd, J = 3.5, 1.7 Hz, 1H), 5.72 (s, 2H). 13C NMR
(125 MHz, CDCl3) δ 145.02, 144.77, 144.15, 142.83, 135.66, 134.04, 129.08,
128.07, 126.23, 124.05, 119.22, 114.41, 112.32, 111.00, 48.70. HRMS (ESI):
m/z calcd for C18H13ClN2O, 308. 0716, found 309.0799 [M+H] +.

4.1.5.13. 1-benzyl-5-chloro-2-(pyridin-2-yl)-1H-benzo[d]imidazole (6m)
Off white solid, yield 84%; mp 140-142 °C; 1H NMR (500 MHz, CDCl3) δ 8.66
(d, J = 4.3 Hz, 1H), 8.57 (d, J = 7.5 Hz, 1H), 7.95 – 7.88 (m, 2H), 7.39 (dd, J =
6.9, 4.9 Hz, 1H), 7.30 – 7.26 (m, 3H), 7.26 – 7.22 (m, 2H), 7.16 – 7.13 (m, 2H),
13
6.22 (s, 2H). C NMR (125 MHz, CDCl3) δ 148.90, 137.45, 137.28, 136.58,
134.80, 129.23, 129.20, 128.78, 127.74, 126.76, 125.36, 124.77, 124.66,
119.31, 111.87, 49.31. HRMS (ESI): m/z calcd for C19H14ClN3, 319. 0876,
found 320.0957 [M+H] +.
4.1.5.14. 1-benzyl-5-chloro-2-(thiophen-2-yl)-1H-benzo[d]imidazole (6n)
white solid, yield 83%; mp 178-180 °C; 1H NMR (500 MHz, CDCl3) δ 7.87 (s,
1H), 7.54 (d, J = 5.0 Hz, 1H), 7.47 (s, 1H), 7.39 – 7.30 (m, 3H), 7.23 (d, J = 8.6
Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 12.5, 6.1 Hz, 3H), 5.61 (s, 2H).
13C NMR (125 MHz, CDCl3) δ 149.00, 135.37, 134.66, 129.63, 129.41, 129.30,
129.13, 128.90, 128.25, 128.15, 125.89, 125.79, 123.94, 119.35, 110.92, 48.47.
HRMS (ESI): m/z calcd for C18H13ClN2S, 324. 0488, found 325.0568 [M+H] +.
4.1.5.15. 2-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl) quinolin-8-ol (6o)
1
Yellow solid, yield 85%; mp 292-294 °C; H NMR (500 MHz, CDCl3) δ 8.45
(d, J = 8.6 Hz, 1H), 8.25 (d, J = 8.7 Hz, 1H), 7.87 (s, 1H), 7.44 – 7.24 (m, 6H),
7.19 (s, 1H), 7.15 (d, J = 7.3 Hz, 2H), 7.02 (d, J = 7.6 Hz, 1H), 6.91 (s, 1H),
13
5.95 (s, 2H). C NMR (125 MHz, CDCl3) δ 152.72, 137.70, 137.55, 137.34,
135.48, 130.81, 130.10, 129.59, 127.79, 127.43, 124.91, 124.84, 124.76,
122.94, 122.16, 120.00, 113.35, 112.61, 49.93. HRMS (ESI): m/z calcd for
C23H16ClN3O, 385. 0982, found 386.1060 [M+H] +.
4.1.5.16. 1-benzyl-5-chloro-2-(3-phenoxyphenyl)-1H-benzo[d]imidazole (6p)

1
Off white solid, yield 82%; mp 176-178 °C; H NMR (500 MHz, DMSO) δ
7.80 (d, J = 1.3 Hz, 1H), 7.58 – 7.52 (m, 2H), 7.49 (d, J = 7.6 Hz, 1H), 7.40 (t, J
= 7.8 Hz, 2H), 7.34 – 7.27 (m, 4H), 7.23 – 7.14 (m, 3H), 7.04 (d, J = 7.9 Hz,
13
2H), 6.95 (d, J = 6.7 Hz, 2H), 5.60 (s, 2H). C NMR (125 MHz, DMSO) δ
157.63, 156.51, 154.32, 143.67, 136.90, 135.26, 131.74, 131.14, 130.64,
129.56, 129.29, 128.03, 127.35, 126.49, 124.48, 124.29, 123.54, 120.67,
119.53, 119.20, 119.08, 113.09, 48.16. HRMS (ESI): m/z calcd for
C26H19ClN2O, 410. 1186, found 411.1270 [M+H] +.
4.1.5.17.
(6q)
4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-2-ethoxyphenol
white solid, yield 91%; mp 147-149 °C; 1H NMR (500 MHz, DMSO) δ 9.64 (s,
1H), 7.78 (d, J = 1.9 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.35 – 7.26 (m, 4H), 7.20
– 7.17 (m, 2H), 7.04 (d, J = 7.3 Hz, 2H), 6.93 (d, J = 8.7 Hz, 1H), 5.62 (s, 2H),
3.97 (q, J = 8.2 Hz, 2H), 1.26 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, DMSO)
δ 155.39, 149.41, 147.16, 137.24, 135.24, 129.60, 129.33, 127.99, 127.26,
126.45, 123.09, 122.68, 120.31, 118.54, 116.25, 114.46, 112.82, 64.21, 48.24,
15.03. HRMS (ESI): m/z calcd for C22H19ClN2O2, 378. 1135, found 379.1225
[M+H] +.
4.1.5.18. 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-2,6-dimethoxyphe
nol (6r)
1
Off white solid, yield 91%; mp 208-210 °C; H NMR (500 MHz, DMSO) δ
9.01 (s, 1H), 7.79 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.37 – 7.23 (m, 4H), 7.07 (d,
13
J = 7.2 Hz, 2H), 6.92 (s, 2H), 5.63 (s, 2H), 3.65 (s, 6H). C NMR (126 MHz,
DMSO) δ 155.53, 148.40, 143.68, 138.07, 137.52, 135.58, 129.35, 127.94,
127.14, 126.37, 123.05, 119.48, 118.75, 112.65, 107.00, 56.29, 48.29. HRMS
(ESI): m/z calcd for C22H19ClN2O2, 394. 1084, found 395.1171 [M+H] +.
4.2. Biology

4.2.1. Cell culture
Lung cancer (A-549), prostate cancer (DU-145), Breast cancer (MCF-7), Breast
cancer (MDA-MB-231), Colorectal cancer (HCT-116), Colorectal cancer (HT-
29) cell lines were procured from National Centre for Cell Science, Pune, India.
All the cell lines were grown in appropriate DMEM, MEM and RPMI-1640
medium (Sigma-Aldrich, USA). Cells were supplemented with 10% fetal
bovine serum stabilized with 1% antibiotic-antimycotic solution (Sigma-
Aldrich, USA) in a CO2 incubator at 37 oC in incubator. When the cells reached
up to 80-90% of confluency, they were sub-cultured using 0.25% trypsin/1 mM
EDTA solution for further passage.
4.2.2. MTT assay
This MTT assay is a colorimetric assay that measures the reduction of 3-(4,5-
dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by
mitochondrial succinate dehydrogenase. The MTT enters the cells and passes
into the mitochondria where it gets reduced and forms insoluble dark purple
formazan crystals. The crystals are then solubilized with an organic solvent
DMSO and the soluble formazan product is measured by reading absorbance at
570 nm with spectrophotometer (Spectra Max, M4 Molecular devices, USA).
Since reduction of MTT can only occur in metabolically active cells, the level
of activity is the measure of the viability of the cells. Briefly, cells were seeded
in 96-well plates at a density of 103 cells per well in 100 µl of complete medium
and allowed to grow overnight for attachment. Then the cells were treated with
various concentrations of the compounds for a period of 48 hrs. After the
o
treatment, 100 µl of MTT (0.5 mg/ml) was added and incubated at 37 C for 4
h. Then MTT reagent was aspirated and the formazan crystals formed were
o
dissolved by the addition of 200 µL of DMSO for 20 mins at 37 C. The
quantity of formazan product was measured by using a spectrophotometric
microtiter plate reader at 570 nm wavelength. Initially, cytotoxicity effects of

the synthetic derivatives were screened by MTT assay at 50 µM concentration.
Among these, the compound which shown IC50 value <20 µM was used for the
dose dependent studies at various concentrations ranging from 1.56 µM to 100
µM in serial dilutions and the percentage of cytotoxicity was calculated.
4.2.3. Acridine orange/ethidium bromide (AO/EB) staining
MCF-7 cells were treated with 1.25, 2.5, 5 and 10 µM of compound 6g. Plates
o
were incubated in an atmosphere of 5% CO2 at 37 C for 48 h, followed by
addition of fluorescent dyes containing AO/EB into each well in equal volumes
and within 10 min cells were visualized under fluorescence microscope (Nikon,
Inc. Japan) with excitation (488 nm) and emission (550 nm) at 200X
magnification.
4.2.4. DAPI staining
MCF-7 cells were cultured in 12-well plates and treated with 1.25, 2.5, 5 and 10
µM of compound 6g. 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 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.
4.2.5. Cell cycle analysis
MCF-7 (1 X 106 cells/well) in 6 well plate was treated with 1.25, 2.5, 5 and 10
µM of compound 6g for 24 h. Cells were collected by trypsinisation, washed
with PBS and fixed with 70% ethanol for 30 min at 4oC. After fixing, cells were
washed with PBS and stained with 400 µL of propidium iodide staining buffer
for 30 min in dark at room temperature. The samples were then analysed for
propidium iodide fluorescence from 10,000 events by flow cytometry using BD
Accuri C6 flow cytometer.

4.2.6. Assay of mitochondrial membrane potential (Δψm):
The mitochondrial- specific cationic dye JC-1 (Invitrogen, USA), which
undergoes potential-dependent accumulation mitochondria, was used to detect
mitochondrial membrane potential. Briefly, cells were plated at a seeding
density of 2×105 cells/well in a 12-well plate. After 48 h of treatment with 6g
(1.25, 2.5, 5 and 10 µM), cells were incubated with 2 μM JC-1 for 30 min at
room temperature in the dark and the quantitative analysis of Δψm was done by
flow cytometry.
4.2.7. AnnexinV/Propidium iodide dual staining assay
The Annexin V/Propidium iodide dual staining assay was performed by using
MCF-7 cells, to quantify the percentage of apoptotic cells. Cells were plated in
12-well culture plates and allowed to grow for 24 h. After treatment with 1.25,
2.5, 5 and 10 µM of compound 6g for 48 h, cells were collected by
trypsinisation. The collected cells were washed twice with ice-cold PBS, then
incubated with 200 µL 1X binding buffer containing 5 µL Annexin V-FITC,
and then in 300 µL 1X binding buffer containing 5 µL Propidium iodide (PI) in
the dark for 5 min at room temperature and incubate for 15 min. After 15 min of
incubation, cells were analysed for percentage of apoptosis using BD-C6 accuri
flow-cytometer.
4.2.8. Detection of intracellular ROS
Intracellular ROS generation was measured by DCF-DA method. MCF-7 cells
were seeded into a 12-well plate and allowed to grow for 24 h. After treatment
with 1.25, 2.5, 5 and 10 µM of 6g for 48 h. then cells were incubated with 10
µM DCF-DA at 37οC for 15 min. The intracellular reactive oxygen species
(ROS) mediated oxidation of DCFDA to the fluorescent compound 2,7-
dichlorofluorescein (DCF) monitored by excitation (498 nm) and emission (530
nm) using Multimode Plate Reader (Spectra Max M4, Molecular devices,

USA). The images were taken by fluorescence microscope at 200X
magnification.
4.2.9. Statistical analysis
All the values were expressed as mean ± SEM. The intergroup variation
between different groups was measured by one-way ANOVA using the Graph
Pad Prism, version 6.01. Here, the results were considered statistically
significant when *p <0.05.
4.2.10. Quantikine ELISA Human Gal-1 Immunoassay
This assay employs the quantitative sandwich enzyme immunoassay technique.
A polyclonal antibody specific for human Gal-1 has precoated on a microplate.
Standards and samples are pipetted into the wells and Gal-1 present is bound by
the immobilized antibody. After washing away any unbound substances, an
enzyme-linked polyclonal antibody specific for human Gal-1 is added to the
wells. Following a wash to remove any unbound antibody-enzyme reagent, a
substrate solution is added to the wells and colour develops in proportion to the
amount of Gal-1 bound in the initial step. The colour development is stopped
and the intensity of the colour is measured at 450 nm in a multi detection plate
reader (Spectramax M4, molecular devices, USA). MCF-7 cells were seeded in
petri dish and allowed to attached for 24hr after that cells were treated with
different concentration of compound 6g (10, 30, 100 and 300μM) and after 24hr
of treatment cells supernatant was collected and cell lysis collected by adding
cell lysis buffer (RIPA) and cells are sonicated. The galectin quantikine ELISA
was done by using R & D human galectins quantikine ELISA kit.
4.2.10.1 Determination of binding efficiency of compound 6g with RP-
HPLC
The assay for 6g was carried out by high-performance liquid chromatography
(HPLC). The analysis was carried out by automatic RP-HPLC (Waters Corp,

USA) on X-bridge C18 column (250 mm X 4.6 mm, 5 μm) with a mobile phase
consisted of ammonium acetate (pH 6.3) and Acetonitrile (v/v). The mobile
phase was filtered through 0.22 μm membrane filter, sonicated and used. The
analysis was performed in gradient mode at a flow rate of 1 ml/min with 210
nm as detection wavelength and the injection volume of 20 μl. The HPLC
sample was prepared by using amicon Ultra-4 centrifugal filters containing an
Ultracel 10k regenerated cellulose membrane (10,000 MW cut-off), were
purchased from Millipore, USA. Before addition of compound 6g, each of these
filtration devices was filled with 1 ml of PBS. The membrane was washed by
spinning the filtration device at 10000 x g for 1 min. Compound 6g was
incubated with Gal-1 for 30 min at 37 °C to allow the binding interactions.
Immediately, after PBS washing, 0.5 µg/ml concentration of compound 6g
(control) or compound 6g with Gal-1 protein (0.5 µg / ml concentration) sample
was introduced into centrifugal filters. These devices were spun in the
centrifugal filters at 7500 x g and 25 °C for 30 min. The filtrates were collected
and their contents were analysed by RP-HPLC as per the chromatographic
conditions. In each case, ultrafiltration units were pre-treated with compound 6g
and washed with PBS several times to avoid non-specific binding and to ensure
complete removal of compound 6g from units before subjecting to actual test
samples. The binding efficiency is calculated by the following formula.
% Gal-1 binding efficiency = (Bound drug concentration/Free drug
concentration) x 100
4.2.10.2 Chromatographic conditions
Chromatographic analysis was performed on Alliance HPLC system e 2695
separation module equipped (Waters Corp., Milford, M.A, USA) with an in-line
degasser, quaternary pump, an autosampler, a column compartment with
temperature control facility and a photo diode array detector (model 2998). The

output signal was monitored and processed using Waters Empower 3 software
(Waters Corp.).
Mobile phase: Ammonium acetate (pH 6.3) (solvent A): Acetonitrile (solvent
B), Diluent: Acetonitrile, Column: X-bridge C18 column (250 mm X 4.6 mm, 5
μm), Lambda max (λmax): 299 nm, Flow rate: 1 ml/min, Injection volume: 20 µl
Optimized method
Time (min)
0.00
% A
90
% B
10
2.00
90
10
6.00
10
90
11.00
12.00
13.00
10
90
90
90
10
10
4.2.10.3. Fluorescence measurements
In order to perform fluorescence binding studies of Gal-1 with compound 6g,
full length ORF of Gal-1 was cloned into pET28a expression vector followed by
purification using Ni-NTA affinity chromatography as reported by Hsieh and
co-authors, 2015. Intrinsic fluorescence measurements were carried out on a
Jasco spectrofluorimeter equipped with peltier at 25 °C. Compound 6g was
dissolved in DMSO to prepare a stock of 10 mM and was used in the range of
0-65 µM. Purified Gal-1with concentration of 13 μM in 10 mM phosphate
buffer (pH 7.5) was excited at 280 nm and the emission was recorded from 300-
400 nm using a cuvette of 10 mm path length. Slit width 5 nm was used for
excitation and emission, while scan speed was maintained at 100 nm/min.
Buffer correction was made for each spectra. Binding constant (Ka) and
number of binding sites (n) were determined using modified Stern-Volmer
equation i.e. log(Fo-F)/F = logKa + nlog[Q] where Fo and F is the intensity of