Anti-cancer potential of novel glycosylated 1,4-substituted triazolylchalcone derivatives

Article abstract of DOI:10.1016/j.bmcl.2019.08.019

A series of glycosylated 1,4-substituted triazolyl chalcone derivatives (8a-f and 14a-r) were synthesized in high yield using 1,3-cycloaddition (Click chemistry) of D-glucosyl azides with a variety of propargylated chalcone derivatives followed by de-O-ac

Full text of DOI:10.1016/j.bmcl.2019.08.019

Journal Pre-proofs
Anti-cancer potential of novel glycosylated 1,4-substituted triazolylchalcone
derivatives
Tapasi Manna, Kunal Pal, Kuladip Jana, Anup Kumar Misra
PII:
S0960-894X(19)30547-5
https://doi.org/10.1016/j.bmcl.2019.08.019
BMCL 26615
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 June 2019
9 August 2019
11 August 2019
Please cite this article as: Manna, T., Pal, K., Jana, K., Misra, A.K., Anti-cancer potential of novel glycosylated 1,4-
substituted triazolylchalcone derivatives, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
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0.1016/j.bmcl.2019.08.019
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Graphical Abstract
Anti-cancer potential of novel glycosylated
Leave this area blank for abstract info.
1
,4-substituted triazolylchalcone derivatives
Tapasi Manna, Kunal Pal, Kuladip Jana* and Anup Kumar Misra*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Anti-cancer potential of novel glycosylated 1,4-substituted triazolylchalcone
derivatives
1
1
*
*
Tapasi Manna, Kunal Pal, Kuladip Jana and Anup Kumar Misra
Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII M, Kolkata 700054, India; E-mail: Email: akmisra69@gmail.com; kuladip@jcbose.ac.in
1
Both authors contributed equally.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of glycosylated 1,4-substituted triazolyl chalcone derivatives (8a-f and 14a-r) were
synthesized in high yield using 1,3-cycloaddition (Click chemistry) of D-glucosyl azides with a
variety of propargylated chalcone derivatives followed by de-O-acetylation. The synthesized
compounds were evaluated for their cytotoxic potential against the human breast carcinoma cell
lines and non-cancerous cells. The MTT assay identified three promising cytotoxic compounds
(
14c, 14i and 14l) and further biochemical and microscopic studies were carried out with the
Keywords:
Triazole
chalcone
glycosyl
best compound 14i among the active compounds.
2
009 Elsevier Ltd. All rights reserved.
anti-cancer
apoptosis
Cancer is one of the most deadly diseases known to mankind,
which remains a global healthcare issue for a long time. It still
continues to be a major threat in the developing countries like
India with limited awareness of the disease. Among the various
interventions used therapeutically against cancer, chemotherapy
along with radiotherapy has been the most widely used and
potential strategies for treating this disease.1,2 Over the last few
decades, several bio-molecular interactions have been identified
through different biochemical techniques. Protein-protein
name a few. Conventionally, chalcones are prepared by Claisen-
Schmidt condensation of aryl aldehyde and acetophenone
1
3
derivatives in the presence of a base. Chalcones possess a wide
range of therapeutic potential, which include antitumor, antiviral,
antitubercular, antifungal, enzyme inhibitor, antiplatelet and
1
4, 15
many more.
In another aspect, glycosylated 1,2,3-triazole derivatives are
1
6,17
privileged class of compounds
showing wide variety of
pharmacological properties such as anticancer, anti-tubercular,
enzyme inhibitors, reverse transcriptase inhibitors, -lactum
antibiotics etc. Because of their remarkable synthetic utility
3
,4
5
6
interactions, Protein-DNA and RNA interactions have given
enormous idea about their decisive role in oncogenic
transformation, tumor progression and metastasis. 7 All together
the genomic, proteomic and structural information has provided
several new targets and opportunities for future drug discovery
against cancer. Chemotherapy is unable to provide complete
protection against cancer, regardless of remarkable advancement
in modern medical research. Most of the chemotherapeutic agents
have been found to be associated with poor prognosis, largely
owing to their cytotoxicity on normal cells.8,9 Additionally, high
toxicities and undesirable side-effects associated with some
cancer chemotherapy drugs increase the need for novel anti-
tumor drugs, with fewer side-effects and greater therapeutic
1
,2,3-triazole derivatives have been extensively studied by
1
8
medicinal chemists. Conventionally, they have been prepared
under copper mediated click chemistry condition.
1
9-21
Molecular hybridization of multiple pharmacophores for the
development of a single molecule with enhanced biological
activities has become an attractive area in medicinal chemistry.
Several reports have appeared in the literature on the synthesis of
hybrid class of molecules containing triazole and chalcone
moieties having significant improvement in the medicinal
2
2
properties, which include chalcone-triazole derivatives,
1
0
23
efficacy. Therefore, targeting the molecular mechanism that
contributes to the hallmarks of cancer through small molecule
from synthetic or natural source can be an useful tool for future
cancer therapeutics.
chalcone--lactum-triazole derivative, chalcone-pyrrolo-[2,1-
2
4
c][1,4]-benzodiazepine conjugate and their anticancer potential
against a variety of cancer cells. Taking cues from the earlier
reports, it was decided to design hybride class of molecules
incorporating chalcone, triazole and carbohydrate in them to
develop more potent anti-cancer agents. It is envisioned that
presence of carbohydrate moiety could improve the
bioavailability of the compound by improving its solubility in
1
,3-Diaryl-2-propenones namely, chalcones are important
1
1,12
class of molecules
to act as precursors of the medicinally
important heterocyclic compounds such as, pyrazolines,
quinoxalines, flavones, flavanones, isoxazolines, pyrimidines to

aqueous medium. Synthesis of a series of a glycosylated 1,4-
substituted triazolyl chalcone derivatives and their evaluation as
anti-proliferative agent against human breast carcinoma cell lines
is presented here. Several biochemical and microscopic
experiments were also been executed to demonstrate the potential
of the most promising compound as anti-cancer agent.
confirmed from the NMR spectral analysis. Finally, de-O-
acetylation of compounds 13a-r using sodium methoxide
furnished compounds 14a-r with free hydroxyl groups in the
sugar moieties in quantitative yield (Scheme 2).
Propargyl
OH bromide (2)
O
Ar
ArCHO (11a-r)
K2CO3, CH3CN
O
In order to prepare the chalcone derivatives containing a
propargyl functionality two starting materials were selected.
Vaniline (1) was treated with propargyl bromide (2) in the
presence of potassium carbonate at 70 C to give compound 3 in
70 oC, 15 h
81%
O
aq. NaOH
O
O
EtOH
12a: Ar = 2-Methoxyphenyl (77%)
2b: Ar = 3-Methoxyphenyl (80%)
12c: Ar = 4-Methoxyphenyl (75%)
2d: Ar = 3,5-Dimethoxyphenyl (80%)
1
9
10
1
OAc
O
12e: Ar = 3,4-Dimethoxyphenyl (72%)
AcO
1
1
2f: Ar = 3,4,5-Trimethoxyphenyl (70%)
2g: Ar = 4-Nitrophenyl (80%)
OAc
AcO
N3
8
2% yield. Compound 3 was treated with a series of
acetophenone derivatives (4a-f) in the presence of aq. sodium
N
O
6OAc
AcO
AcO
N
N
12h: Ar = 3-Nitrophenyl (82%)
OAc
12i: Ar = 4-Dimethylaminophenyl (77%)
12j: Ar = 4-Ethoxyphenyl (74%)
CuSO4 (1M),
Na-Ascorbate
DMSO, r t
2
5
hydroxide in ethanol to furnish a series of chalcone derivatives
5a-f) in satisfactory yield. It is noteworthy that the chalcone
O
12k: Ar = 4-Benzyloxyphenyl (75%)
12l: Ar = 3-Benzyloxy-4-methoxyphenyl (72%)
2m: Ar = 2-Chlorophenyl (82%)
12n: Ar = 3,4-Dichlorophenyl (80%)
(
Ar
1
derivatives (5a-f) were obtained exclusively as trans-olefins.
Compounds (5a-f) allowed to react with 2,3,4,6-tetra-O-acetyl--
D-glucopyranosyl azide (6) 21 using “Click chemistry” in the
presence of a combination of copper(II) sulphate and sodium L-
O
1
1
2o: Ar = 3,5-Dichlorophenyl (78%)
2p: Ar = Napthyl (71%)
1
3a: Ar = 2-Methoxyphenyl (76%)
13b: Ar = 3-Methoxyphenyl (74%)
3c: Ar = 4-Methoxyphenyl (75%)
3d: Ar = 3,5-Dimethoxyphenyl (76%)
12q: Ar = Thiophenyl (68%)
2r: Ar = Furanyl (70%)
1
1
1
2
1
13e: Ar = 3,4-Dimethoxyphenyl (72%)
0.1 M NaOCH3
OH
O
ascorbate to give glycosylated 1,4-substituted triazolylmethyl
chalcone derivatives (7a-f) in satisfactory yield. Exclusive
formation of 1,4-substituted 1,2,3-triazole derivatives (7a-f) was
observed under the reaction conditions, which was confirmed
from the NMR spectral analysis. Finally, de-O-acetylation of
compounds 7a-f using sodium methoxide furnished compounds
N
1
3f: Ar = 3,4,5-Trimethoxyphenyl (72%) CH3OH
HO
HO
N
N
13g: Ar = 4-Nitrophenyl (75%)
Quantitative
OH
O
13h: Ar = 3-Nitrophenyl (74%)
O
13i: Ar = 4-Dimethylaminophenyl (76%)
3j: Ar = 4-Ethoxyphenyl (77%)
13k
1
4a: Ar = 2-Methoxyphenyl
1
Ar
14b: Ar = 3-Methoxyphenyl
4c: Ar = 4-Methoxyphenyl
14d: Ar = 3,5-Dimethoxyphenyl
4e: Ar = 3,4-Dimethoxyphenyl
14f
:
Ar = 4-Benzyloxyphenyl (74%)
3l: Ar = 3-Benzyloxy-4-methoxyphenyl
70%)
3m: Ar = 2-Chlorophenyl (77%)
3n: Ar = 3,4-Dichlorophenyl (74%)
13o: Ar = 3,5-Dichlorophenyl (75%)
3p: Ar = Napthyl (70%)
13q: Ar = Thiophenyl (68%)
3r: Ar = Furanyl (70%)
1
1
(
1
1
1
:
Ar = 3,4,5-Trimethoxyphenyl
4g: Ar = 4-Nitrophenyl
4h: Ar = 3-Nitrophenyl
4i: Ar = 4-Dimethylaminophenyl
4j: Ar = 4-Ethoxyphenyl
4k: Ar = 4-Benzyloxyphenyl
14l: Ar = 3-Benzyloxy-4-methoxyphenyl
4m: Ar = 2-Chlorophenyl
1
8
a-f with free hydroxyl groups in the sugar moieties in
1
1
1
quantitative yield (Scheme 1).
1
1
1
Propargyl
1
bromide (2)
K2CO3,
O
14n: Ar = 3,4-Dichlorophenyl
14o: Ar = 3,5-Dichlorophenyl
14p: Ar = Napthyl
14q: Ar = Thiophenyl
14r: Ar = Furanyl
CHO
CHO
Ar
OCH3
O
CH3CN
4a-f
Ar
o
7
0 C, 15 h
OCH3
OCH3
O
aq. NaOH
EtOH
OH
O
8
2%
Scheme 2: Synthesis of glycosylated 1,4-substituted
triazolylmethyl chalcone derivatives (14a-14r). Isolated yield is
presented in parenthesis.
Selective cytotoxicity of synthesized compounds towards
the cancer cells
1
5a: Ar = Phenyl (76%)
b: Ar = 4-Nitrophenyl (82%)
c: Ar = 3,4,5-Trimethoxyphenyl (74%)
d: Ar = 3,4-Dimethoxyphenyl (75%)
e: Ar = 4-Cyanophenyl (78%)
f: Ar = 4-Bromophenyl (76%)
3
5
5
5
5
5
OAc
O
CuSO4 (1M),
AcO
AcO
N3 Na-Ascorbate
Primarily, 24 synthesized compounds (8a-f and 14a-r) along
with etoposide and paclitaxel were assessed for their cytotoxic
ability on MDA-MB-468 cells [human triple negative (ER-, PR-
and HER2-) breast cancer cells], MCF-7 [human (ER+, PR+ and
HER2-) breast cancer cells] and WI-38 cells (non-cancerous lung
fibroblast cell). For this experiment, cells were treated with
varying concentrations of the compounds (0-50 µM) and cell
OAc
6
DMSO, r t
0
.1 M NaOCH3
CH3OH
Quantitative
OH
O
OAc
N
N
HO
HO
AcO
O
N
N
N
N
AcO
OH
OAc
O
O
Ar
Ar
OCH3
a: Ar = Phenyl
b: Ar = 4-Nitrophenyl
c: Ar = 3,4,5-Trimethoxyphenyl
d: Ar = 3,4-Dimethoxyphenyl
e: Ar = 4-Cyanophenyl
f: Ar = 4-Bromophenyl
OCH3
O
O
8
7a: Ar = Phenyl (76%)
2
6
8
7b: Ar = 4-Nitrophenyl (72%)
7c: Ar = 3,4,5-Trimethoxyphenyl (75%)
7d: Ar = 3,4-Dimethoxyphenyl (76%)
7e: Ar = 4-Cyanophenyl (74%)
7f: Ar = 4-Bromophenyl (77%)
cytotoxicity was measured by MTT assay. The cytotoxicity of
the tested compounds in terms of LD50 were presented in Table 1.
From the MTT assay it was observed that three compounds, 14c,
8
8
8
8
1
4i and 14l showed significantly higher efficacy in MDA-MB-
Scheme 1: Synthesis of glycosylated 1,4-substituted
triazolylmethyl chalcone derivatives (8a-8f). Isolated yield is
presented in parenthesis.
4
68 cells with LD50 values of (39 ± 1.87; 28 ± 1.9 and 64 ± 3.8
µM) respectively. Similar efficacy of 14c, 14i and 14l was found
in MCF-7 cells with LD50 values of (53 ± 1.13; 31 ± 2.3 and 84 ±
3
.4 µM) respectively. Subsequently, when the selectivity index
In another experiment, 4-hydroxyacetophenone (9) was
treated with propargyl bromide (2) in the presence of potassium
carbonate at 70 C to give compound 10 in 81% yield.
Compound 10 was treated with a series of aromatic aldehyde
derivatives (11a-r) in the presence of aq. sodium hydroxide in
(SI) of the compounds were calculated by comparing the
cytotoxic LD50 value of the compound in normal cell (WI-38)
versus cancer cells (MDA-MB-468 and MCF-7), it was found
that compound 14i possessed higher SI (SI = 2.11 ± 0.08 and 1.9
±
0.07) as compared to compound 14c (1.84 ± 0.024 and 1.36 ±
2
5
ethanol to furnish a series of chalcone derivatives (12a-r) as
exclusively trans-olefin in satisfactory yield. The chalcone
derivatives (12a-r) were allowed to react with 2,3,4,6-tetra-O-
acetyl--D-glucopyranosyl azide (6) 21 using “Click chemistry”
in the presence of a combination of copper(II) sulphate and
sodium L-ascorbate21 to give glycosylated 1,4-substituted
triazolylmethyl chalcone derivatives (13a-r) in satisfactory yield.
Exclusive formation of 1,4-substituted 1,2,3-triazole derivatives
0
.011) and compound 14l (SI = 1.64 ± 0.023 and 1.25 ± 0.007).
Considering the MTT data, compound 14i was found as a potent
cytotoxic agent against cancer cells in comparison to non-
cancerous cells based on its highest selectivity index (SI) (Figure
1
).
(13a-r) was observed under the reaction conditions, which was

Table 1: Cytotoxic activities (LD50 in M) of compounds against
cancer cell lines (Data for active compounds are mentioned in
bold).
Compd.
MDA-
MB 468
MCF-
7
WI38
S.I.(WI S.I.(WI38/
38/
MCF-7)
MDA-
MB
4
68)
8
a
89 ± 3.2 123 ±
.59
68 ± 2.3 76 ±
.6
73 ± 3.2 87 ±
.49
66 ±
.17
82 ± 3.1 78±
.65
98
1.10 ±
0.02
1.02 ±
0.09
0.92 ±
0.016
1.43 ±
0.004
1.07 ±
0.02
1.37 ±
0.008
1.3 ±
0.025
0.94 ±
0.01
1.84 ±
0.024
1.56 ±
0.04
1.08 ±
0.004
1.06 ±
0.04
1.49 ±
0.06
0.80 ±
0.01
0.99 ±
0.014
0.77
±0.0002
1.45 ±
0.01
1.13 ±
0.003
0.90 ±
0.006
1.06
±0.008
1.16 ±
0.003
1.36 ±
0.011
0.72 ±
0.009
0.83 ±
0.005
0.64
±0.018
1.19 ±
0.003
1.17 ±
0.04
Figure 1: (a) Cytotoxicity assay of compound 14i on cancer
3
±1.89
75 ±
2.9
67 ±
1.89
96 ±
3.63
88±
1.78
89
±2.9
103 ±
2.6
73 ±
1.8
72 ±
2.23
52 ±
1.8
95 ±
2.25
54 ±
2.39
64 ±
2.34
69 ±
1.6
(MDA-MB-468 and MCF-7) and non-cancerous cells (WI-38);
8
b
(b) LD50 values of compound 14i in cancer (MDA-MB-468 and
1
MCF-7) and non-cancerous cells (WI-38).
8
c
2
Induction of apoptosis with the treatment of compound 14i
in cancer cells
8
d
67±2.5
2
8
e
In order to study the mechanism of action of the cytotoxicity of
the most promising compound 14i against the cancer cells the
induction of apoptosis was measured using Annexin V-FITC/PI
1
8
f
65 ±
.7
99 ±
2.4
1
27
staining. After the treatment of MDA-MB-468, MCF-7 and
1
4a
4b
4c
4d
4e
4f
4g
4h
79 ± 3.8 97 ±
.9
63 ±
WI38 cells with different concentrations of compound 14i (0, 20,
1
4
0 and 60 μM) for 24 h and staining with Annexin V-FITC/PI,
1
77 ±1.6
the flow cytometry analysis showed significant induction of
apoptosis in a concentration dependent manner in the cancer cells
in comparison with the non-cancer cells (WI38). Quite evidently,
compound 14i was able to induce nearly 60% apoptosis in MDA-
MB-468 cells and 63% in MCF-7 cells at 60 μM dose (Figure 2).
From the Annexin V-FITC/PI staining on the cancer cells (MDA-
MB-468 and MCF-7) and non-cancer cell lines (WI38), it was
observed that there was no substantial apoptosis in the case of the
non-cancer cells treated with compound 14i, while in case of the
cancer cells the percentage of apoptosis was quite high.
Moreover, compound 14i was found comparatively non-toxic to
the non-cancerous cells in comparison to cancerous cells. Hence,
this experiment confirmed that the compound 14i kills the
cancerous cells via apoptosis sparing any adverse effects on the
non-cancerous cells. Therefore, it was decided to identify the
mechanism of the induction of apoptosis in cancer cells by
compound 14i employing different types of biochemical and
microscopic analysis.
1
.3
1
39 ±
53 ±
1.13
72 ±
2.65
1
.87
33 ±
.3
1
2
1
88 ± 2.4 115 ±
.43
1
1
51 ±
.4
43 ±
.4
64 ±
.1
84 ±
1.68
54 ±
1.9
59 ±
2.3
2
1
3
1
1.07 ±
0.018
2.11 ±
0.08
3
1
4i
28 ± 1.9 31 ±
.3
68 ±
.7
59 ±
1.8
78 ±
2.7
68 ±
4.5
105 ±
3.6
1.9 ±
0.07
1.15 ±
0.04
2
1
4j
74± 1.5
57 ±
1.05 ±
0.019
1.19 ±
0.016
1.64 ±
0.023
1.06 ±
0.009
0.93 ±
0.0008
1.03 ±
0.01
4
79
1
4k
4l
4m
4n
0.86 ±
0.025
1.25 ±
0.007
0.94 ±
0.02
0.85 ±
0.012
0.86 ±
0.023
1.00
±0.01
0.96 ±
0.008
1.05 ±
0.01
3
.13
±2.9
1
64 ± 3.8 84 ±
.4
64 ± 2.9 72
2.5
3
1
68
±3.5
±
1
59± 3.5
65±2.4 55±
2
3.12
64
±3.9
69 ±
1.7
88 ±
3.7
87
1
4o
4p
4q
62 ±
74 ±
2.9
3
.1
1
65 ± 3.7 69
1.06 ±
0.03
±
2.4
1
76 ± 2.8 92 ±
1.16 ±
0.004
0.98 ±
0.008
1.06 ±
0.19
2
.7
89 ± 3.5 83
1.7
Etoposide 34 ± 2.7 38
1
4r
±
±2.9
36
±1.2
0.95 ±
0.025
±
2.2
Paclitaxel 27 ± 1.8 29
35
±1.9
1.29 ±
0.21
1.20 ±
0.042
±
2.6
Figure 2: Annexin V-FITC/PI analysis after 24 h of different
concentrations of compound 14i treatment in cancer cells and
normal cells. (a) Dot plot of Annexin V-FITC/PI for evaluation
of apoptosis in MDA-MB 468, MCF-7 and WI-38 cells treated
with the compound 14i; (b) Quantification of apoptotic cells for
evaluation of apoptosis in these cells treated with the compound
1
4i at doses 20µM, 40 µM, 60 µM.

Treatment of compound 14i promotes ROS generation,
mitochondrial permeability transition (MPT) in cancer cells
fluorescence upon binding to nucleic acids. In the present study,
the enhanced fluorescence was observed in case of cells treated
with different concentrations of compound 14i (40 µM and 60
Since the generation of reactive oxygen species (ROS) is a
critical phenomenon linked with several anti-proliferative
2 2
µM) as well as H O (positive control) (Figure 5). Therefore, it
can be concluded that compound 14i mediated cancer cell
apoptosis was observed due to the increased generation of
mitochondrial ROS. Altogether these results imply that 14i
mediated generation of mitochondrial ROS may have decisive
role in the mitochondrial permeability transition.
2
8
processes, the ROS production in response to the treatment
with compound 14i was measured using the DCFDA method.29
Briefly, cells were treated with varying doses (0, 20, 40 and 60
µM) of compound 14i for 12 h followed by microscopic analysis
after staining with DCFDA (100 µM) for 30 min. The
photomicrographs demonstrated concentration dependent
increase in the intensity of the green color in response to
compound 14i suggesting significant induction of ROS
generation by compound 14i in MDA-MB-468 and MCF-7 cells
2 2
as shown in Figure 3. Hydrogen peroxide (H O ) has been used
as a positive control to confirm the generation of ROS induced
by compound 14i (Figure 3). Moreover, it has been confirmed
that compound 14i induced oxidative damage of the cancer cells
through the production of ROS. Compound 14i induce pro-
oxidant activity leading to ROS generation as well as cancer cell
apoptosis, which was confirmed from the above experiment.
Figure 4: Mitochondrial permeability transition (MPT)
determination by JC-1 dye in (a) MDA-MB-468 cells and (b)
Quantitative analysis of red to green transition in MDA-MB 468
cells; (c) MPT determination by JC-1 dye in MCF-7 cells and (d)
Quantitative analysis of red to green transition in MCF-7 cells.
Figure 3: Assessment of ROS generation following treatment
with compound 14i and H O in cancer cells. (a) Fluorescence
2 2
microscopic image of treated cells ; (b) Quantitative analyses of
Fluorescence microscopic images; (c) Spectrophotometric
fluorescence intensity measurement which indicates the
enhancement of ROS in cells treated with the compound 14i at
doses 40 µM, 60 µM.
Since ROS plays a critical role in changing the mitochondrial
permeability transition (MPT)28 therefore,
the effect of
compound 14i on mitochondrial damage was investigated by JC-
staining, which showed a drastic alteration of the redox status
of cellular mitochondria in response to compound 14i in a
concentration dependent manner (Figure 4). The shift of
fluorescence from red to green or a decrease in the red/green ratio
indicated the increase in the mitochondrial permeability in
response to compound 14i. Altogether this result implies that
compound 14i mediated generations of ROS may have decisive
role in the mitochondrial permeability transition. Hydrogen
1
Figure 5: Measurement of mitochondrial ROS generation using
MitoSOX™ Red in response to the treatment with compound 14i
(a) Fluorescence microscopic image of treated cells and (b)
Spectrophotometric fluorescence intensity measurement which
indicates the enhancement of mitochondrial ROS in cells treated
with the compound 14i at doses 40 µM, 60 µM.
Treatment of compound 14i induces DNA fragmentation
and Cell cycle arrest in cancer cells.
peroxide (H
2 2
O ) has also been used as a positive control (Figure
4
). Thus from the above results we can conclude that the
Several reports suggested 28 that ROS generation and change
in MPT are linked with cell cycle arrest and DNA damage
induced apoptosis, which is an imperative process that controls
cellular growth. Therefore, the effects of compound 14i on cell
cycle analysis and DNA damage were assessed. Treatment of
MDA-MB-468 and MCF-7 cells with different doses of
compound 14i (20 µM, 40 µM and 60 µM), significantly
increased the percentage (%) of sub G1 cells populations up to
exposure to the compound 14i is responsible for inducing
intracellular ROS generation which subsequently results in the
mitochondrial membrane potential disruption.
Furthermore, the mitochondrial ROS production in response
to compound 14i was measured by the treatment with
MitoSOX™ Red reagent,28 which permeates live cells selectively
targeting mitochondria. MitoSOX™ Red can be rapidly oxidized
by superoxide anions and the oxidized product emits high red
6
2% for MDA-MB-468 cells and 67% for MCF-7 cells (in 60

µM dose) indicating cell cycle arrest (Figure 6). DAPI staining of
cancer cells after treatment with compound 14i showed
polynuclear fragmentation and apoptosis in comparison to the
untreated cells (Figure 7). The quantitative analysis confirmed
around 72% and 78% apoptotic cells in MDA-MB-468 and
MCF-7 cells respectively following the treatment with 60 µM
compound 14i, which were highly significant compared to
corresponding control cells as mentioned in Figure 7. Hydrogen
results suggested that significant change in Bax/Bcl-2 ratio and
activation of caspase-3 play a critical role in compound 14i
mediated cancer cell apoptosis (Figure 8).
In summary, a series of glycosylated 1,4-substituted triazolyl
chalcone derivatives (8a-f and 14a-r) were synthesized using a
generalized reaction condition in satisfactory yield. The
synthesized compounds (8a-f and 14a-r) were evaluated against
human breast cancer cells (MDA-MB-468 and MCF-7 cells) and
non-cancer cells (WI38). Three compounds (14c, 14i and 14l)
were found promising in terms of the cytotoxic activities against
cancer cells and less cytotoxic against non-cancer cells (WI38).
Among three active compounds, compound 14i has been
considered as the best based on the selectivity index. Further, a
number of microscopic experiments and fluorescence activated
cell sorter (FACS) studies confirmed that compound 14i exhibits
anti-cancer activities against several cancer cells following
apoptotic pathway.
2 2
peroxide (H O ) has also been used as a positive control. Hence,
we can conclude that compound 14i induces cell cycle arrest,
DNA fragmentation and apoptosis in cancer cells without
affecting the non-cancerous cells.
Figure 6: (a) Cell cycle analysis following the treatment of
compound 14i in cancer cells (MDA-MB 468 and MCF-7);
(b) bar diagram of the quantification of the sub G1 cells in the
cell cycle which is the indication of cellular apoptosis.
Figure 8: (a) Western blot of apoptotic protein cleaved
Caspase-3, Bax and Bcl2 which suggests the upregulation of
apoptic pathway upon treatment of the MDA-MB 468 cells
(b) Bar diagram of the quantitative analysis of the band
intensity.
Acknowledgements
T.M. and K.P. thank CSIR, India for providing Senior
Research Fellowships. This work was supported by SERB, New
Delhi, India (AKM) [Project No. EMR/2015/000282].
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Anti-cancer potential of novel glycosylated 1,4-substituted triazolylchalcone
derivatives
1
1
Tapasi Manna, Kunal Pal, Kuladip Jana* and Anup Kumar Misra*
Research highlights:





Glycosylated 1,4-substituted triazolylchalcone derivatives were prepared.
“Click chemistry” condition has been used to prepare compounds in high yield.
Synthetic compounds were evaluated for their potential as anticancer agents.
Three compounds showed promising cytotoxicity against human breast cancer.
Biochemical and microscopic experiments were carried out using the best compound.