3-Aminomethyl pyridine chalcone derivatives: Design, synthesis, DNA binding and cytotoxic studies

Article abstract of DOI:10.1111/cbdd.13189

Herein we report design, synthesis, and anticancer activity of compounds 6a–h and 11a–j. Compounds 6a–f were designed based on 3-aminomethyl pyridine attached to different acetamide derivatives and in compounds 6g–h it was attached to coumarin moiety. Coumarin containing compounds 6g–h showed very poor anticancer activity against both A549 (Lungs cancer cell line), and MCF-7 (Breast cancer cell line) cell lines in MTT assay. Compounds 11a–j were designed as derivatives of 3-aminomethyl pyridine and 4-amino chalcones. A series of chalcone derivatives of 3-aminomethyl pyridine 11a–j have been synthesized and screened for their in vitro anticancer activity and DNA binding affinity. Most of the compounds showed very good antimitotic activity against A549 cell line as compared to fluorouracil. Compounds 11g and 11i were selected for DNA-binding studies as they showed excellent activity against cancer cell lines in MTT assay. CT-DNA binding affinity of compounds 11g and 11i have been investigated by UV based DNA titration and fluorescence emission study against DNA-EtBr complex. Interestingly, compound 11i has displayed excellent antiproliferative activity, with IC₅₀ 0.0067?±?0.0002?μm, against MCF-7 cell line. Compound 11i has been studied for its cytotoxicity using MTT, LDH, as well as EtBr/AO assay and was found to induce apoptosis in the cancerous cell line.

Full text of DOI:10.1111/cbdd.13189

DR SHUBHANGI S. SOMAN (Orcid ID : 0000-0002-1405-4064)
Article type : Research Article
3-Aminomethyl pyridine chalcone derivatives: Design, Synthesis, DNA binding and Cytotoxic
studies
Sunil Dutt Durgapal1, Rina Soni1, Shweta Umar2, Balakrishnan Suresh2, Shubhangi S Soman1*
1Department of Chemistry, 2Department of Zoology, Faculty of Science, The M. S. University
of Baroda, Vadodara - 390 002, India.
*Corresponding author’s e-mail: shubhangiss@rediffmail.com
Ph. No.: +91-265-2795552
Abstract
Herein we report design, synthesis and anticancer activity of compounds 6a-h and 11a-j. Compounds
6a-f were designed based on 3-aminomethyl pyridine attached to different acetamide derivatives
and in compounds 6g-h it was attached to coumarin moiety. Coumarin containing compounds 6g-h
showed very poor anticancer activity against both A549 (Lungs cancer cell line), and MCF-7 (Breast
cancer cell line) cell lines in MTT assay. Compounds 11a-j were designed as derivatives of 3-
aminomethyl pyridine and 4-amino chalcones. A series of chalcone derivatives of 3-aminomethyl
pyridine 11a-j have been synthesized and screened for their in vitro anticancer activity and DNA
binding affinity. Most of the compounds showed very good antimitotic activity against A549 cell line
as compared to fluorouracil. Compounds 11g and 11i were selected for DNA-binding studies as they
showed excellent activity against cancer cell lines in MTT assay. CT-DNA binding affinity of
compounds 11g and 11i have been investigated by UV based DNA titration and fluorescence
emission study against DNA-EtBr complex. Interestingly, compound 11i has displayed excellent
antiproliferative activity, with IC50 0.0067 ± 0.0002 µM, against MCF-7 cell line. Compound 11i has
been studied for its cytotoxicity using MTT, LDH, as well as EtBr/AO assay and was found to induce
apoptosis in the cancerous cell line.
Keywords: Aminomethyl pyridine, Anticancer activity, Cytotoxic studies, Chalcone derivatives, DNA
binding.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13189
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1. Introduction
Cancer is second leading cause of death worldwide. According to WHO report, the increase in
number of new cases is expected to rise by 70% over next two decades [1]. Cancer progresses via
multistep carcinogenesis, which involves various physiological processes of human body like
uncontrolled growth of cells due to deregulation of essential enzymes, cell signaling and apoptosis.
Cancer is extremely complicated to combat [2]. The root cause was found out to be pattern of
lifestyle adopted such as excessive use of tobacco, physical inactivity and improper diet [3]. The
increase in cancer incidence proves that even today it is not curable with the available therapy and
medication. The major downsides of current therapy available to treat cancer include side effects,
lack of tumor specificity and multi drug resistance. Hence there is a need of potent anticancer agents
to overcome this hurdle. One of the important aspects in anticancer agent is designing of small
molecules targeting DNA. The interaction of small molecule with DNA depends not only on ability of
molecule to DNA recognition but also on type of interactions such as electrostatic, intercalation, and
groove binding [4-5].
α,β-Unsaturated ketones, commonly known as chalcones are important class of natural as well as
synthetic products which show variety of biological activities. During last few decades, chalcone
derivatives have been reported having potent anticancer activity with low side effects and better
solubility for therapeutic applications [6-7]. Simple structural modification in chalcone moiety with
heterocycles, polyarene compounds or organometal complexes may lead to new anticancer agents
with promising activity [8-9]. Chalcone based small molecules provide advantage over other due to
their low toxicity and mutagenicity profile. Yu et al showed that 4-(dimethylamino)-4-amino
chalcone can interact into base pair and created a new method to determine trace amount of DNA
[10].
Interestingly, pyridine derivatives have shown very good effect on anticancer activity when
substituted at 3rd position as in Entinostat 1 (MS-275), which is in phase II clinical trials for treatment
of various types of cancers [11]. Inci Gul et al have reported compound 2 as tumor selective
cytotoxin containing both chalcone and pyridine moieties in a molecule [12]. Recently, Wang et al
have reported pyridine containing compound 3 with very good antitumor activity against xenograft
A2780 ovarian cancer [13].
As a part of our ongoing research on anticancer agents [14-15], herein we report design, synthesis
and anticancer activity of compounds 6a-h and 11a-j. compounds 6a-f were designed based on 3-
aminomethyl pyridine attached to different acetamide derivatives and in compounds 6g-h it was
attached to coumarin moiety. Compounds 11a-j were designed as derivatives of 3-aminomethyl
pyridine and 4-amino chalcones.
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2. Experimental
2.1. Chemistry
Reagent grade chemicals and solvents were purchased from commercial supplier and used after
purification. TLC was performed on silica gel F254 plates (Merck). Acme’s silica gel (60-120 mesh)
was used for column chromatographic purification. All reactions were carried out in nitrogen
atmosphere. Melting points are uncorrected and were measured in open capillary tubes, using a
Rolex melting point apparatus. IR spectra were recorded as KBr pellets on Perkin Elmer RX 1
1
spectrometer. H NMR and 13C NMR spectral data were recorded on Advance Bruker 400
spectrometer (400 MHz) with CDCl3 or DMSO-d6 as solvent and TMS as internal standard. J values
are in Hz. Mass spectra were determined by ESI-MS, Mass spectra were determined by ESI–MS,
using a Shimadzu LCMS 2020 apparatus (Shimadzu Scientific Instruments, Inc., USA). Elemental
analyses were recorded on Thermo Finnigan Flash 11-12 series EA. All reactions were carried out
under nitrogen atmosphere. 7-amino-4-methyl coumarin, 3-amino coumarin and chalcones were
prepared as reported in literature [16-18].
2.1.1. Preparation of N-substituted bromoacetamide derivatives (5, 10)
To an ice-cold solution of substituted amines (10.0 mmol) in dichloromethane (DCM) (25 mL) was
added triethylamine (TEA) (15.0 mmol) and stirred for 5-10 min. To this bromoacetyl bromide (12.0
mmol) was added dropwise over a period of 10 min under cooling. The resulting solution was stirred
o
at 0-5 C for 30 min and at room temperature for 24 hrs. The reaction mixture was diluted with
water and extracted with DCM (2 x 30 mL). The organic layers were combined, washed with 0.5N HCl
solution (15 mL), dried over anhydrous Na2SO4, filtered and evaporated on a rotavapor to give
compounds 5 and 10. The substituted bromoacetamide 5 and 10 were directly used for next step
without any purification
2.1.2. General procedure for the preparation of compounds 6a-h and 11a-j
To a cold solution of substituted bromoacetamide 5/10 (1.0 eq) in DMF (20 mL) was added
of 3-aminomethyl pyridine 4 (0.5 g, 1.730 mmol) along with base triethylamine (1.5 eq) and
stirred for 30 min. The resulting mixture was stirred at room temperature for 14-16 h. The
completion of reaction was checked by TLC using DCM:MeOH (9:1). The reaction mixture
was poured into ice-cold water. The aqueous layer was extracted using ethyl acetate or
dichoromethane (3 x 25 mL). The organic layers were combined, dried over anhy. Na2SO4,
filtered and concentrated on a rotavapor to give crude product. The crude compound was
purified by column chromatography using DCM:MeOH (95:5).
For compounds 6a-f: To a cold solution of compounds 6a-f in methanol (15 mL) was added
o
2M HCl in methanol (1.2 eq). The resulting solution was stirred at 0-5 C for 30 min and
concentrated on a rotavapor to give solid. The solid was triturated in diethyl ether, filtered
and dried to give compounds 6a-f.
For compounds 6g-h and 11a-j: Compounds 6g-h and 11a-j were obtained as a solid after
column purification.
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2.2. Materials and methods for biological assays
2.2.1. Reagent and cell culture
MTT, LDH assay kit (Pierce LDH Cytotoxicity Assay), N,N-dimethylformamide (DMF), EtBr and
Acridine Orange, were purchased from Sigma-Aldrich, India. DMEM, Fetal Bovine Serum (FBS) and
trypsin were sourced from (Gibco USA). Human lung carcinoma cell line A549 and human breast
cancer cell line MCF-7 were purchased from NCCS, Pune, India. Cell line was maintained in
Dulbecco’s modified eagles medium (DMEM) supplemented with 2 mM l-glutamine and 10% fetal
bovine serum (GIBCO, USA). Penicillin and streptomycin (100 IU/100g) were adjusted to 1 mL/L. The
cells were maintained at 37 oC with 5% CO2 in a humidified CO2 incubator.
Stock solutions of derivatives were prepared in DMF and were diluted with PBS (Phosphate-buffered
saline) to achieve working concentration. However, care was taken to maintain the final
concentration of DMF not exceed more than 0.5% in any case.
2.2.2. MTT assay
The half minimal inhibitory concentration was evaluated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay as per standard protocol. Cells were plated in a 96-well plate (1
x 104 cells/well) and incubated overnight in 100 µl DMEM media supplemented with 10% FBS. Each
compound was added in 0.5, 1, 10, 25, 50, 75, 100 µM conc. and incubated further for 48h. 20 µl of
MTT solution (5 mg/mL in PBS) was added and further plate was incubated for 4h. Supernatant
solution was removed and the blue formazan was dissolved in 100µl of acidified isopropanol. The
absorbance was measured using microplate reader at 570 nm (Metertech Ʃ960)
Cell viability (%) = (average absorbance of treated groups/average absorbance of control group) ×
100%. IC50 values were calculated using GraphPad Prism. Each experiment was performed in
triplicates.
2.2.3. UV-Based Assay
PerkinElmer Lambda- 35 dual beam UV–Vis spectrophotometer was used for absorption
spectral studies. Solution of calf thymus DNA (CT DNA) was prepared in water. The UV
absorbance at 260 was found to 0.277 which is used to calculate the DNA concentrations (Ɛ =
6600 M-1) and was expressed in terms of base molarity. UV absorption titrations were carried
out by keeping the concentration of compounds 11g and 11i (dissolved in DMSO) fixed and
by adding a known concentration of CT DNA solution in both the cuvettes in increasing
amount until hypochromism saturation was observed. Absorbance values were recorded after
each successive addition of DNA solution and equilibration.
2.2.4. Ethidium Bromide Displacement Assay
DNA (100 µL, 8.4 x 10-4 M), EtBr (100 µL, 4.2 x 10-4 M), and Tris-HCl buffer pH 7.2 was
used to make a total volume of 3 mL EtBr displacement fluorescence assay was employed to
find DNA intercalation. Fluoroscence emission spectra (λmax= 600 nm, excitation
wavelength 546nm, slit width 10nm, 1cm path length) were obtained at 30oC on a JASCO
FP-6300 fluorescence spectrophotometer. The assays were performed by using different
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concentrations of compounds 11g and 11i in buffer solution (3 mL). I0/I are plotted along
with y axis against the concentration of compound, where in I and I0 are the fluorescence
intensities of the DNA-EtBr complex in the presence of and in the absence of compounds,
respectively.
2.2.5. Trypan blue
5 × 105 cells per well were seeded in 12 well plate and kept overnight for attachment. Next day cells
were treated with IC50 conc. of compound 11i, DMF and TritonX-100 and were incubated for 48 h.
DMF treated cells were taken as vehicle control and TritonX-100 as positive control. Following
incubation, the supernatant pool was collected and adherent cells were trypsinized and collected.
Cell viability was performed by the dye exclusion test with 0.5% trypan blue using a hemocytometer.
Each experiment was performed in triplicates.
2.2.6. LDH assay
Lactate dehydrogenase enzyme remains in cytoplasm, however during necrosis due to plasma
membrane damage it leaches out. Cells were plated on 96 well plate (1 x 104 cells/well) for 24 h in
DMEM media without phenol red, then derivatives were added in the 0.5, 1, 10, 25, 50, 75, 100 µM
concentration range. Subsequently, they were incubated for 48 h. Assay was performed according to
the manufacture’s instruction (Pierce LDH Cytotoxicity Assay, Thermo Scientific, USA). Absorbance
was measured at 490 nm in a microplate reader and percentage cytotoxicity was calculated.
2.2.7. Ethidium Bromide/Acridine Orange staining assay
Morphological changes due to apoptosis and necrosis were visualized using EtBr/AO staining
technique [19]. Cells were treated with IC50 concentration of compound 11i. For positive control cell
were treated with Triton-X 100 and incubated for 48h. Cells were washed with PBS and stained with
1 µl of 1:1 ratio of EtBr and AO (100 µg/ml). Cell to stain ratio was maintained as 1:25 µl. 10 µl of cell
suspension was placed on microscopic slide and images were taken using Leica DM 2500
fluorescence microscope fitted with Leica EZ camera.
3. Results and Discussion
3.1.
Chemistry
Compounds 6a-h were synthesized by reaction of bromoacetamide derivative 5 with 3-aminomethyl
pyridine 4 (Scheme 1). Amino derivatives were reacted with bromoacetyl bromide to give
bromoacetamide derivative 5, which on reaction with 3-aminomethyl pyridine 4 using triethylamine
in anhydrous N,N-dimethylformamide gave derivatives 6a-h as shown in scheme 1.
Compounds 6a-f were isolated in the form of hydrochloride salts. The structures of compounds 6a-f
1
were confirmed by different analytical techniques such as H-NMR, 13C-NMR, IR, ESI-MS. In general,
the IR spectra of compounds 6a-f exhibited three strong bands in the range of 3421-3383 cm-1, 3255-
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3213 cm-1 and 3200-3176 cm-1 for secondary amine salt and N-H stretching respectively. Carbonyl
1
stretching of amide was observed in the range of 1691-1670 cm-1. In the H NMR spectra of 6a-f,
peak for methylene next to amide group was observed in range of δ ~3.94-4.02 while methylene next
to pyridine was observed in range of δ ~4.26-4.46, aromatic protons were observed in range of 6.92-
9.67 depending on effect of different substituents on aromatic ring. For compounds 6a-f, -NH
protons of secondary amine salts were observed as broad singlet in range of 9.82-10.09 ppm, while
13
amide -NH proton was observed in range of 10.75-11.30 ppm. In the C NMR spectra of 6a-f, peak
for methylene carbons observed around 46.96-47.95 and 48.10-48.37 ppm, aromatic carbons in
range of 106-161 ppm, amide carbonyl carbon in range of 164-165 ppm.
All compounds 6a-f were analyzed by ESI-MS analyses to give [M+H]+/[M+Na]+ peak corresponding
to their molecular weight. As modification in compounds 6a-f, 3-aminomethyl pyridine was attached
to comuarin moiety at 7th and 3rd position in compounds 6g and 6h respectively. Both compounds 6g
1
and 6h were also fully characterized by H-NMR, 13C-NMR, IR and ESI-MS analysis and all data
complies for formation of desired product.
As a structural modification of compounds 6g-h, 3-aminomethyl pyridine was linked to 4-amino
chalcone derivatives to study the effect of chalcone on activity profile. Compounds 11a-j were
synthesized by reaction of bromoacetamide derivative 10 with 3-aminomethyl pyridine 4 (Scheme
2). 4-Aminoacetophenone 7 on reaction with aldehyde 8 under basic conditions gave 4-amino
chalcone derivatives 9 as shown in scheme 2. 4-Amino chalcone 9 on reaction with bromoacetyl
bromide gave 10, which on reaction with 3-aminomethyl pyridine gave various derivatives 11a-j
(Scheme 2).
1
The structures of compounds 11a-j were confirmed by different analytical techniques such as H-
NMR, 13C-NMR, IR, ESI-MS. In general, the IR spectra of compounds 11a-j exhibited two strong bands
in the range of 3333-3236 cm-1 and 3290-3169 cm-1 for secondary amine and amide N-H stretching
vibrations respectively. Carbonyl group of chalcone exhibited stretching frequency in range of 1693-
1
1685 cm-1 and that of amide carbonyl in range of 1658-1649 cm-1. In the H NMR spectra of 11a-j,
peak for methylene next to amide group was observed in the range of δ ~3.34-4.13 while methylene
next to pyridine was observed in range of δ ~3.78-4.50, aromatic protons and chalcone protons
observed in range of 6.93-9.11 depending on effect of different substituents on aldehyde. For
compounds 11a-j -NH protons are observed between δ 9.42-11.49 ppm.
In the 13C NMR spectra of 11a-j, two peak for methylene carbons observed around 46.37-51.51 and
48.03-52.50 ppm, aromatic carbons between δ 113-161 ppm, amide carbonyl carbon around δ 164-
171 ppm, chalcone carbonyl carbon between δ 187-189 ppm. All compounds 11a-j were analyzed by
ESI-MS analysis to give [M+H]+/[M+Na]+ peak corresponding to their molecular weight. All these new
chemical entities were subjected to in-vitro studies for anticancer activity by MTT assay method.
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3.2 Biological activity
3.2.1 MTT assay:
Since the 70-80% of total cancer diagnosed all over world are breast cancer and lung cancer, we
have selected representative MCF7 and A549 cell lines for our study. 3-Aminomethyl pyridine
derivatives 6a-h were screened for their anticancer activity against A549 (Lungs cancer cell line), and
MCF-7 (Breast cancer cell line) cell lines using MTT assay and compared the results with that of
standard drug Fluorouracil (Table 1) [20].
Compound 6a with 4-methyl group found to be inactive against both the tested cell lines.
Replacement of 4-methyl to 4-fluoro in compound 6b, resulted in very good activity against A549 cell
lines with IC50 1.186 ± 0.024 µM, but remained inactive against MCF-7 cell line. Similar trend of
activity was observed for compound 6c with 4-chloro group with IC50 0.213 ± 0.0031 µM against
A549 cell lines. Interestingly, change in position of halogen from 4th to 3rd position in compounds 6d-
e, resulted in loss of anticancer activity. Compound 6f with 4-COCH3 group showed moderate activity
against tested cell lines. However, coumarin containing compounds 6g-h showed very poor
anticancer activity against both cell lines.
3-Aminomethyl pyridine linked to 4-amino chalcones showed very good activity profile for
anticancer activity in MTT assay against tested cell lines. Compounds 11a-b and 11g-j showed
moderately good activity against MCF-7 cell line as compared to A549 cell line. Compound 11a
showed very good activity against A549 cell line with IC50 6.18 ± 0.11 μM. On placing methyl group at
4th position on chalcone moiety in compound 11b resulted in loss of activity against tested cell lines.
Compound 11c with methoxy group on 4th position showed very good activity against A549 cell line
with IC50 0.269 ± 0.0089 μM, however remained inactive against MCF-7 cell line. On replacement of
methoxy group of compound 11c by nitro group for compound 11f, showed loss of activity against
A549 cell line, while remained inactive against MCF-7 cell line. Compound 11e having chloro at 4th
position, showed very good activity with IC50 5.14 ± 1.07 μM against A549 cell line. Further position
change of methoxy group from 4th position in compound 11c to 3rd position in compound 11g,
resulted in decrease in activity against A549 cell line, however this variation showed excellent
activity against MCF-7 cell line with IC50 0.174 ± 0.0076 μM.
On replacement of chloro from 4th to 2nd position in compound 11h showed loss of activity against
both tested cell lines. Interestingly, compound 11i with chloro on 3rd position showed very good
activity against A549 cell line and excellent activity against MCF-7 cell line. Further, compound 11j
containing 2,4-dichloro showed moderate activity against tested cell lines.
Out of all screened compounds 6a-h (Table 1) and 11a-j (Table 2), compounds 11g and 11i showed
excellent activity with IC50 0.174 ± 0.0076 µM and 0.0067 ± 0.0002 µM respectively, as compared to
standard drug Fluorouracil against MCF-7 cell line in MTT assay. Hence Compounds 11g and 11i were
selected to study their DNA binding studies.
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3.2.2 DNA binding studies
Compounds 11g and 11i were selected for DNA-binding studies as they showed excellent activity
against cancer cell lines in MTT assay. To explore mode of interaction of compounds 11g and 11i
with DNA in cell, DNA binding UV based DNA titration and fluorescence emission study against DNA-
EtBr complex were carried out [21]. UV based DNA binding studies gave information for possible
interaction taking place between DNA and the lead compounds, while fluorescence based assay, i.e.
EtBr displacement assay gave mode of interaction of our lead compounds is through intercalation.
The fixed concentration of compounds 11g and 11i was titrated against the known concentration of
CT-DNA solution with tris-HCl buffer (pH 7.2) (Figure 3). The strength of binding to CT-DNA was
determined through the calculation of intrinsic binding constant Kb which is obtained by monitoring
the changes in the absorbance of the compounds with increasing concentration of CT-DNA. Plot of
[DNA]/ (ƐA-Ɛf) versus [DNA] (equation 1) is used to find out Kb. Compound 11g and 11i showed the
hypochromic shift with the intrinsic binding (kb) values 2.12 x 104 M-1 and 1.06 x 104 M-1 respectively
in UV based DNA titrations indicated intercalative mode of binding (λmax = 325 nm) (Figure 3 & Table
3).
[DNA]/ (ƐA-Ɛf) = [DNA]/ (Ɛb-Ɛf) + 1/Kb(Ɛb-Ɛf)
(eq 1)
Fluorescence emmission based Ethidium bromide (EtBr) displacement assay was also performed
with compound 11g and11i. The emission spectra of DNA-EtBr (λex = 546 nm) in the absence and
presence of increasing amount of compound 11g and 11i were recorded at emission λmax 608 nm.
(Figure 4). The data were plotted according to the Stern-Volmer equation (eqation 2). Quenching of
fluoroscence intensity was observed for compound 11g and 11i with Ksv values 5.21 x 103 M-1 and
5.85 x 103 M-1 respectively (Table 3), which supports the DNA intercalating property of these
compounds (Figure 4).
I0/I = 1 + KSV[Q]
(eq 2)
3.2.3 Cytotoxic studies:
Compound 11i induced cytotoxic studies in A549, MCF-7 cancer cell lines and NIH/3T3 non cancer
cell line were performed. The cytotoxicity of compound 11i was estimated using MTT assay and IC50
conc. of compound 11i was measured in both the cancer cell line as well as in non-cancer cell line.
Mechanism of cytotoxicity was evaluated by Trypan blue and LDH assay in cancer cell lines. LDH
assay is based on the release of cytosolic enzyme into culture medium due to the damage of cell
membrane which is a hallmark of necrosis, while MTT assay is based on the activity of mitochondrial
dehydrogenase enzyme activity and represent the metabolic rate or percentage viability of cell [22-
24].
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In MTT assay, the percentage viability of cell line was decreased with increased concentration of
compound 11i. IC50 concentration of compound 11i was estimated using Graphpad Prism 5 software
viz. 0.245 ± 0.011, and 0.0067 ± 0.0002 µM in A549 and MCF-7 cell line respectively (Figures 5(a) and
5(b)). Compound 11i showed IC50 79.31 ± 0.08 µM (Figure 5(c)) for non-cancer mouse fibroblast cell
line (NIH/3T3), which was significantly high in compare to IC50 values against cancer cell lines A549
and MCF-7. These results prove beyond doubt that when cancer cell will be treated at such lower
concentration of compound 11i, will not be cytotoxic to nearby normal cells, hence, compound 11i
was very specific toward cancer cells. Therefore, it was taken further for the study of mechanism in
cancer cell lines against A549 and MCF-7 cell line.
There are several mechanisms by which an anticancer compound exerts its effect on cancer cell in
vitro. Apoptosis and necrosis are the most preferred mechanisms. To understand the mode of
cytotoxicity of compound 11i for cancer cell lines, LDH Assay was performed in both cancer cell lines.
At lower concentration of compound 11i, the LDH release was very low which revealed that at lower
concentration the prevalent mechanism of cytotoxicity was apoptosis but with increased
concentration of compound 11i LDH release increases significantly therefore, at higher
concentration treated cells changed its fate towards the necrosis from apoptosis. As IC50 value of
compound 11i is very low for both cell lines therefore, it can be deduced that preferred mechanism
of cytotoxicity for compound 11i can be apoptosis (Figure 6 (a)-(b)).
To confirm, that loss of percentage viability was either due to cell death or due to cell proliferation
inhibition effect of compound 11i, Trypan blue assay was performed in both A549 and MCF-7 cell
lines. The percentage cell death at IC50 concentration of compound 11i was approx. 36% ± 3% in
A549 cell line and 45% ± 2.68% in MCF-7 cell line (Figure 7), from this it can be construed that
compound 11i is cytotoxic towards the MCF-7 whereas it might be cytotoxic or cytostatic toward
A549 cell line, further experimental confirmation is required to explain the effect of compound 11i
on cancer cell lines.
In order to reaffirm the findings of LDH assay, the EtBr/AO staining assay was performed with
compound 11i. Acridine orange is a vital dye that stains both live and dead cells however, ethidium
bromide stains only the cells that have lost their membrane integrity. EtBr/AO dye stains, necrotic
cells red, live cells green, early apoptotic cell’s nuclei green but with visible condensation whereas,
late apoptotic cell’s nuclei Orange with condensation and fragmentation.
Cells were treated with IC50 concentration of compound 11i and it was found that most of the cells of
A549 cell line (Figure 8(a)-(c)) were under late apoptosis, there were no cells under necrosis whereas,
in MCF-7 cell line (Figure 8(d)-(f)) most of the cells were under early apoptosis with few under
necrosis. Number of the cells under apoptosis and necrosis were negligible in control cell lines.
Which confirmed the LDH assay finding that compound 11i exhibits cytotoxic activity in both cell
lines via apoptosis. To confirm the possible interaction of compound with DNA, compound 11i was
studied for DNA binding activity.
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4. Conclusion
In conclusion, a series of chalcone of 3-aminomethyl pyridine derivatives were synthesized and
evaluated for their anticancer activity against A549 and MCF-7 cell lines. Based on MTT assay, one of
the compounds 11i has shown very good selectivity for MCF-7 cancer cell line as compared to
NIH/3T3 normal cell line (non-cancer mouse fibroblast cell line). DNA binding studies of compound
11i indicated intercalation mode of binding with CT-DNA. The cytotoxic studies of compound 11i
have shown the apoptosis in MCF-7 cell line using LDH assay and the EtBr/AO assay. From the Kb
binding values it can be concluded that compound 11g has two-fold higher affinity for CT-DNA
binding than compound 11i. Further study for sequence selectivity is undergoing for the lead
compounds.
Acknowledgements:
One of the authors (RS) is thankful to Department of Science & Technology, Government of India for
financial support vide reference no. SR/WOS-A/CS-1028/2014 under Women Scientist Scheme to
carry out this work. One of the authors (SU) is thankful to UGC, Government of India for UGC-JRF
vide reference no. 22/06/2014 (i) EU-V. Authors are thankful to The Head, Department of Chemistry
and Department of Zoology, Faculty of Science, The M. S. University of Baroda for providing
laboratory facilities, Zydus Research Centre, Ahmedabad, for the ESI-MS analyses.
Conflict of Interest
The authors declare no conflict of interest.
Supporting Information
Characterization data and spectral data for compounds 6a-h and 11a-j are provided in the
supporting Information.
Figure 1: Compounds with anticancer activity.
Figure 2: Designing of hybrid chalcone 11a-j derived from 3-aminomethyl pyridine and 4-amino chalcones.
Scheme 1: Synthesis of compound 6a-h. Reagents and Conditions: (i) TEA, DMF, 0-5 oC, 30 min, r.t 14-16 h.
Scheme 2: Synthesis of compound 11a-j. Reagents and Conditions: (i) 40% aq NaOH, ethanol, r.t 5-10 h; (ii)
bromoacetyl bromide, TEA, DCM, r.t, 16-18 h; (iii) TEA, DMF, 0-5 oC, 30 min, r.t 14-16 h.
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Table 1: Anticancer activity against A549 (Lungs cancer cell line), MCF-7 (Breast cancer cell line) for compounds
6a-h.
aIC50 values were determined based on MTT assay using GraphPad Prism
software. NA = Not active
Table 2: Anticancer activity against A549 (Lungs cancer cell line), MCF-7 (Breast cancer cell line) compounds
11a-j.
aIC50 values were were determined based on MTT assay using GraphPad Prism
software. NA = Not active
Figure 3: Titration plot of compounds 11g and 11i with DNA. Plot of Absorbance versus Wavelength (nm) (a)
for compound 11g and (b) compound 11i. Plot of [DNA]/(ƐA-Ɛf) versus [DNA] (c) for compound 11g and (d).for
compound 11i
Figure 4: Plot of Fluorescence emission intensity I versus Wavelength (nm) for DNA-EtBr complex at different
concentrations (a) for compound 11g and (b) for compound 11i. Stem-Volmer quenching plot of DNA-EtBr (c)
for compound 11g and (i) compound 11i.
Table 3: Kb and KSV values for compound 11g and 11i .
Figure 5: The cytotoxicity of compound 11i against A549, MCF-7 and NIH/3T3 cell line. In MTT assay (A-C) cells
were treated with 0.5, 1, 10, 25, 50, 75, 100 µM concentrations of compound 11i graph was plotted against %
viability v/s dose. Data were represented as mean ± SD from three independent experiments. ***P<0.001
Figure 6: In LDH assay (a-b): Representation of cytosolic enzyme LDH (a) activity of LDH in A549 cell line (b)
activity of LDH in MCF-7 cell line. Cells were treated with 0.5, 1, 10, 25, 50, 75, 100 µM concentrations of
compound 11i. Graph was plotted against LDH release versus dose. (***P ≤ .001, **p < .01 significance one-
way ANOVA (Tukey–Kramer). ANOVA, analysis of variance; LDH, lactic dehydrogenase.
Figure 7: Trypan blue assay- The percentages cell death of A549 and MCF-7 cell line treated with DMF, with
IC50 concentration of compound 11i and with positive control was plotted. Data was represented as mean ± SD
from three independent experiments.
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Figure 8: EtBr/AO assay: EtBr/AO assay was performed with A549 cell line (a,b,c) and MCF-7 (d,e,f). (a,b,c)
images represent control, IC50 conc. compound 11i treated and positive control in A549 cell line; (d,e,f) images
represent control, IC50 conc. compound 11i treated and positive control in MCF-7 cell line.
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Table 1: Anticancer activity against A549 (Lungs cancer cell line), MCF-7 (Breast cancer cell line) for compounds
6a-h.
IC50 in µMa
Compound no
Ar
A549
MCF-7
6a
6b
6c
NA
NA
1.186 ± 0.024
0.213 ± 0.0031
NA
950 ± 11.87
6d
NA
NA
6e
6f
NA
NA
32.63 ± 2.1
177.10 ± 18.2
90.78 ± 6.24
377.8 ± 29.32
6g
6h
69.1 ± 8.2
298.9 ± 21.2
45.04 ±1.02
fluorouracil
11.13 ±0.083
aIC50 values were determined based on MTT assay using GraphPad Prism
software. NA = Not active
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Table 2: Anticancer activity against A549 (Lungs cancer cell line), MCF-7 (Breast cancer cell line) compounds
11a-j.
IC50 in µMa
Compound no Ar
A549
MCF-7
11a
11b
11c
11d
11e
6.18 ± 0.11
53.27 ± 3.56
132.00 ± 9.86
0.269 ± 0.0089
16.04 ± 3.43
5.14 ± 1.07
90.61 ± 5.96
n/a
n/a
n/a
11f
28.19 ± 1.19
32.42 ± 2.08
n/a
11g
0.174 ± 0.0076
11h
11i
46.89 ± 6.08
71.72 ± 5.32
0.245 ± 0.011
0.0067 ± 0.0002
11j
62.26 ± 5.9
92.21 ± 7.21
45.04 ±1.02
Fluorouracil
11.13 ±0.083
aIC50 values were were determined based on MTT assay using GraphPad Prism software. NA = Not active
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Table 3: Kb and KSV values for compound 11g and 11i.
Compound
Emission
Fluorescence assay
KSV (M-1)
λmax
nm
UV based assay Kb
(M-1)
No
λmax nm
610
11g
11i
325
325
2.12 x 104
1.06 x 104
5.21 x 103
608
5.85 x 103
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