Synthesis of newer piperidinyl chalcones and their anticancer activity in human cancer cell lines

Article abstract of DOI:10.1007/s11164-015-2238-4

Newer tetrahydropyridine chalcones were synthesized and tested for their antioxidant and anticancer activity. These molecules showed significant anticancer activity at IC₅₀ < 50 μM in human cancer cell lines such as A549 (lung adenocarcinoma),

Full text of DOI:10.1007/s11164-015-2238-4

Res Chem Intermed
DOI 10.1007/s11164-015-2238-4
Synthesis of newer piperidinyl chalcones and their
anticancer activity in human cancer cell lines
B. S. Jayashree1 Harshkumar H. Patel1
•
•
Neethu Susan Mathew2 Yogendra Nayak2
•
Received: 23 June 2015 / Accepted: 19 August 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Newer tetrahydropyridine chalcones were synthesized and tested for
their antioxidant and anticancer activity. These molecules showed significant anti-
cancer activity at IC50 \ 50 lM in human cancer cell lines such as A549 (lung
adenocarcinoma), HepG2 (hepatocellular liver carcinoma), HeLa (cervical cancer),
HCT-116 (colon carcinoma cells), MCF-7 (breast cancer cell line), and MDA MB
231 (breast cancer cells) cancer cells. The test compounds significantly inhibited
cancer cell migrations in scratch wound assay for angiogenesis. Further, the test
compounds bearing naphthalene substituents on the main ring showed in vitro
antioxidant activity at \100 lM by 2,20-azino-bis(3-ethylbenzothiazoline-6-sul-
fonic acid) (ABTS) radical cation, superoxide radical scavenging, O-phenonthro-
line, and lipid peroxidation assays. In conclusion, two test compounds bearing
naphthalene substitutions exhibited potent anticancer activity that could be attrib-
uted to their ability to induce apoptosis, inhibit cancer cell migrations, provide anti-
angiogenic properties, and also partly by reducing oxidative stress by scavenging
free radicals.
Keywords Piperidinyl chalcones Á Anticancer Á Human cancer cell lines Á
Bio-isosterism Á Antioxidant
&
Yogendra Nayak
yogendranayak@gmail.com; yogendra.nayak@manipal.edu
1
2
Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences,
Manipal University, Karnataka 576104, India
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal
University, Karnataka 576104, India
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B. S. Jayashree et al.
Introduction
Cancer is a multifactorial disease characterised by loss of the normal control
mechanisms that govern cell survival, proliferation, and differentiation. Globally,
around 5.1 % of disease burden and 12.5 % of deaths are attributed to cancer [1].
The toxicities and resistance of anticancer cells limit the available anticancer drugs
in therapeutics. Naturally occurring chalcones such as licochalcone A, butein,
cardamonin, xanthoangelol, isoliquiritigenin, and flavokawain-A/B are reported to
be potential leads as anticancer agents. Hence, synthetic chalcones could become a
useful scaffold for anticancer activity [2].
Chalcones have the chemical structure 1,3-diaryl-2-propen-1-ones (Fig. 1), and
the anticancer activity is mainly attributed to the interaction of the enone system
with the thiol residues, making them free from mutagenic and carcinogenic effects
that are associated with many alkylating agents [3]. To substantiate this, some of the
open ring chalcones, such as curcumin, and closed ring structures of chalcones
(cyclic chalcones), such as colchicine [4] and flavopiridol [5], have shown
anticancer activity. Further, a number of modifications are made on the basic a, b
unsaturated ketones by introducing Mannich base [6], amino [7], nitro, hydroxyl,
and methoxy groups and have been evaluated for their anticancer potential. Another
approach used in exploring the anticancer potential of chalcones is in replacing the
existing phenyl group in B-ring with a heterocyclic moiety of the chalcone.
Selection of the heterocyclic moiety is based on the molecular properties resembling
that of a phenyl group. For example, a phenyl ring can be conveniently replaced
with a thiophene moiety, as they are ring counterparts. In other words, they are
isosteric to each other. This strategy has been used to generate newer series of
chalcones, such as thiophene [8], pyrrole [9], pyrazoline [10], piperidyl, indolyl
[11], and naphthyl chalcones [12]. Most of these compounds were found to be
superior in anticancer activity compared to the corresponding benzylidenechal-
cones, presumably because of the introduction of a smaller heterocyclic group as
mentioned above, which might cause variations in the hydrophobicity of chalcones.
Liu and Go [13] have demonstrated the effect of the piperidinyl group in the A-ring
of chalones and suggested that the position of the nitrogen provides selectivity and
is also responsible for the cell cycle arrests.
Our present work was planned by keeping the position of the nitrogen atom
intact, along with the double bond in the tetrahydropyridine ring, which is further
reduced in piperidinyl chalcones [12], and introducing various heterocyclic moieties
in the B-ring of chalcone. Introduction of such groups may cause variations in the
Fig. 1 Chemical structure of
chalcone: 1,3-diaryl-2-propen-
1-ones
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Synthesis of newer piperidinyl chalcones and their…
hydrophobicity of the chalcones. This in turn might facilitate better penetration for
the test compounds into the cancer cells. It is of interest to elucidate the contribution
of the nature of different heterocyclic rings on the biological activity of such
piperidinyl chalcones. Thus, in our present study, the effect of isomeric and isosteric
substitutions on the B-ring of chalcone was studied for anticancer activity.
Fig. 2 Synthesis of newer chalcones analogues. Reagents: (i) Hydrochloric acid, Glacial acetic acid, rt
24 h, 95–100 °C, 3 h. (ii) Boron triflouride ethereate, acetic anhydride, dichloromethane, rt, 24–72 h. (iii)
10 % w/v sodium hydroxide, methanol, rt, 12–24 h
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B. S. Jayashree et al.
Materials and methods
Chemicals, reagents, and instruments
All the chemicals required for the synthesis and purification of the intermediates and
final compounds were purchased from Sigma-Aldrich and Merck. Melting points
were determined using a melting point apparatus from Shital Scientific Industries
and are uncorrected. The reactions were monitored by TLC plates, precoated with
Kieselgel 60 F254 (Merck), and spots were detected using short- and long-UV.
Column chromatography was performed on silica gel (120–200 mesh, 230–400
mesh). kmax, emax for the synthesized compounds were established by UV–visible
spectrophotometer UV-1650 PC (Shimadzu, Japan). The IR studies were deter-
mined using Shimadzu FTIR 8310. 1HNMR were recorded on Bruker Avance II and
III and a 400 MHz spectrometer, using TMS as internal standard; all values are
reported in parts per million. Mass spectral studies were done using GC–MS
(Shimadzu GCMS QP5050) and ESI MS [Applied Biosystem, QSTAR Elite
(Q-TOF)]. The test compounds are dissolved in dimethyl sulfoxide (DMSO) for
testing of biological activity.
Synthesis
General Procedure for the synthesis of 1-methyl-4-(2,4,6-trimethoxyphenyl)-1,2,3,6-
tetrahydropyridine (I; Fig. 2, reaction i) 1-Methyl-4-piperidone (0.005 mol) was
added to a solution of 1,3,5-trimethoxybenzene (0.005 mol) in glacial acetic acid
(2 mL) with constant stirring, and the temperature was maintained below 25 °C.
Further, concentrated HCl (0.75 mL) was added drop-wise. The reaction mixture
was then stirred for 24 h at 25 °C, and later at 95–100 °C for 3 h. It was then
allowed to cool to 40 °C, poured over crushed ice, and stirred for 10 min. The
precipitated 1,3,5-trimethoxy benzene was filtered off. Filtrate was basified at below
10 °C, with pH set at 11–12 using 50 % aqueous sodium hydroxide. The white solid
obtained was filtered, washed with water, and dried. Recrystallization was done with
acetone/water (1:2), which gave 1,2,3,6-tetrahydro-4-(2,4,6-trimethoxyphenyl)-1-
methylpyridine (I).
General Procedure for the synthesis of 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-
yl)-2-hydroxy-4,6-dimethoxyphenyl)ethanone (II; Fig. 2, reaction ii) Boron triflu-
oride diethyl ethereate (3.5 mL, 0.065 mol) was added drop-wise to a previously
cooled solution of compound I (0.8 g, 0.003 mol) in dichloromethane (15 mL)
under constant stirring. After stirring for 2–4 h in an ice bath, acetic anhydride
(5 mL, 0.049 mol) was added drop-wise to the solution, and the stirring was
continued for 24–72 h at room temperature. The concentrated reaction mixture was
diluted with cold water and rendered alkaline with sodium carbonate (reaction ii).
The precipitate obtained was then filtered and dried. It was further purified by
column chromatography using solvents such as chloroform and methanol in
gradient flow (up to 3.5 % methanol) to obtain the acetylated product.
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Synthesis of newer piperidinyl chalcones and their…
General Procedure for the synthesis of (3-(1,2,3,6-tetrahydro-1-methylpyridin-4-
yl)-2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one analogues (III; Fig. 2, reac-
tion iii) A solution of aldehyde (1.2 mmol) in methanol (5 mL) was added drop-
wise to a stirred solution of acetophenone-II (1 mmol) dissolved in 10 % (w/v)
NaOH in methanol (20 mL). Alternatively, the base was added drop-wise to a
solution of different aldehydes and acetophenones under stirring. The same was
stirred at room temperature (28 °C) for 12–24 h, and then the solvent was removed
under pressure. The resulting residue was then dissolved in 1 M HCl and extracted
with ether. The aqueous layer was rendered alkaline with saturated sodium
carbonate solution to give a precipitate that was filtered, washed with water, and
dried. Final purification was done by column chromatography, solvent as
chloroform:methanol; gradient flow (up to methanol 3.5 %).
Antioxidant activity
The ABTS radical scavenging assays were performed as per the procedure
explained in the literature [14]. The superoxide generated by alkaline DMSO was
assessed in the presence and absence of test compounds. The reduction of nitroblue
tetrazolium (NBT) by superoxide was measured spectrophotometrically at 560 nm.
Similarly, the chelating iron by O-phenanthroline and lipid peroxidation assay was
performed in rat brain homogenate as per the previously reported method [15].
Anticancer activity
Reagents and media
The media for cell culture (MEM and DMEM) were purchased from Sigma-Aldrich.
MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and
fetal bovine serum (FBS) was purchased from Himedia. Gentamycin sulphate was
procured from Kasturba Hospital, Manipal.
Cell lines
The human cancer cell lines A549 (lung adenocarcinoma epithelial cell line),
HepG2 (hepatocellular liver carcinoma cell line), HeLa (cervical cancer cell line),
HCT-116 (colon carcinoma cell line), MCF-7 (breast cancer cell line—ER-
positive), MDA MB 231 (breast carcinoma cell line—ER-negative), and one normal
cell line [Vero cells (money kidney epithelial cells/normal cells)] were procured
from National Centre for Cell Sciences, Pune, India. The cells were cultured in
MEM medium supplemented with 10 % FBS, 1 % L-glutamine, and 50 lg/mL
gentamicin sulphate in a humidified atmosphere of 5 % CO2 and 95 % air.
MTT Assay
This assay was carried out as explained in the literature [16] with minor
modifications. Exponentially growing cell lines were harvested from 25 cm2 tissue
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B. S. Jayashree et al.
culture flasks and a stock cell suspension (1 9 105 cell/mL) was prepared with
respective media. A 96-well flat bottom tissue culture plate was seeded with
1 9 104 cells in 0.1 mL of MEM and DMEM medium supplemented with 10 %
FBS and allowed to attach for 24 h. Test compounds were prepared just prior to the
experiment in 0.4 % DMSO and serially diluted with medium to get working stocks
of 100, 50, 25, 1.0 lmol/mL. After 24 h of incubation, cells were treated with 100
lL of test compounds, and the plates were again incubated for 48 h. The cells in the
control group received only the medium containing the 0.1 % DMSO (vehicle).
Each treatment was performed in triplicates. After the treatment, test compounds/s-
tandard drug containing-media was removed by inverting the plate onto a tissue
paper. To each well of the 96-well plate, 100 lL of MTT reagent (stock: 1.0 mg/mL
in PBS) was added and incubated for 4 h at 37 °C. After 4 h of incubation the plates
were inverted on tissue paper to remove the MTT reagent. To solubilize formazan
crystals in the wells, 100 lL of 100 % DMSO was added to each well. The optical
density (OD) was measured by an ELISA plate reader (Biotek FLX800) at 540 nm.
The percentage anticancer activity was calculated using the formula
ðControl OD À Blank ODÞ À ðTest OD À Blank ODÞ
%anticancer activity ¼
ðControl OD À Blank ODÞ
 100
The IC50 was calculated from the graph plotted by taking concentration against %
anticancer activity using Microsoft Excel.
Chromatin condensation studies (Hoechst 33342 staining)
Condensation of chromatin is a late event in apoptosis and can be detected by
Hoechst 33342 dye staining. Hoechst 33342 stain easily crosses the membrane and
binds nuclear adenine–thymine-rich regions of DNA and emits blue fluorescence at
UV range [17]. HepG2 cells were grown in culture media with a suitable
concentration of DMSO in 96-well plates and subjected to the treatment with test
compounds at their IC50 concentrations (lowest IC50 in MTT assay). After 24-h
incubation, the media containing the test samples was removed and 100 lL of 1 %
paraformaldehyde solution was added and incubated for 2 h at room temperature.
The excess paraformaldehyde solution was removed, and the plate was dried. Fifty
microliters of Hoechst 33342 stain was added, and plate was wrapped in aluminium
foil and incubated at 60 °C for 15 min. The plate was then washed with PBS/HBSS,
dried and observed under Nikon eclipse TS100 inverted microscope with excitation
filter 480/530 nm and emission filter 535/540 nm. The chromatin condensation and
apoptotic bodies were viewed and comparison was made with DMSO-treated
control and doxorubicin-treated positive control.
Scratch wound anti-migration assay
A scratch wound in a monolayer is made with the help of the micro-tip of predefined
size and images are captured at zero time point and then onwards depending upon
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Synthesis of newer piperidinyl chalcones and their…
the cell cycle time. Closures of the scratch are compared and the images are snapped
to quantify the migration rate of the cells. This method has been used to measure the
migration of individual cells in the leading edge of the scratch [18, 19]. HeLa cell
suspension containing 1 9 107 cells/mL was prepared in DMEM. The tissue culture
grade 6-well plate was seeded with 2 mL of the above cell suspension, and 1 mL of
fresh medium was added into each well. The cells were allowed to attach for 24 h.
The spent medium was removed by decantation and by using a micro-pipette and
1.5 mL of low-serum medium (medium containing 0.5–0.1 % FBS) was added to
each well and starved overnight. After 24 h, medium from each well was removed,
the cells were washed with 2 mL of sterile PBS and a wound was created with a
sterile 200 lL micro tip. The test compounds/standards were suitably diluted with
the medium to get IC50 concentration (lowest IC50 of MTT) and 1.5 mL of the
diluted test sample solution was added to each well. Images were taken in 910
magnification using an inverted microscope at 0, 6, 12, 24 h after incubation with
test samples. The wound area was roughly measured using a scale in a standard
magnification of the image and the mean wound width was expressed in centimeters
is compared with standard drug methotrexate.
Results
Chemistry
The synthesis of all the test compounds was achieved in three steps as previously
described [13, 20]. The first step involved an electrophilic substitution on 1,3,5-
trimethoxy benzene in the presence of hydrochloric acid by the carbonyl carbon of
N-methyl piperidone to get intermediate compound (I). The melting point was
121–123 °C; the spectral details were as follows.
1
IR: 2937 (C–H), 1585 (C=C), 1126 (C–O); HNMR (DMSO-d6) d 6.19 (2H, s),
5.32 (1H, s), 3.75 (3H, s), 3.68 (6H, s), 2.89 (2H, m), 2.5 (2H, m), 2.15 (3H, s),
2.147 (2H, m); MS: m/z [M]? 263.
Compound I, upon acylation using boron triflouride as a catalyst for demethylation
of the adjacent methoxy group gave corresponding acetophenone (II). The purified
acetophenone (II; mp 170–171 °C) was subjected to spectral characterization.
1
IR: 3441 (O–H), 2976 (C–H), 1626 (C=O), 1587 (C=C), 1130 (C–O); HMNR
(DMSO-d6) d 14 (1H, s), 6.28 (1H, s), 5.49 (1H, s), 3.99 (3H, s), 3.87 (3H, s), 3.68
(2H, m), 3.24-3.32 (2H, m), 2.77 (3H, s), 2.57 (3H, s), 2.42–2.5 (2H, m); MS: m/z
[M]?291.
Finally, the desired chalcones (III) were obtained by Claisen-Schmidt conden-
sation of the synthesized acetophenone (II) with various aldehydes (Fig. 2, reaction
iii).
All the synthesised test compounds showed a characteristic bathochromic shift
and exhibited kmax above 330 nm when compared to that of acetophenone at kmax
272 nm. This bathochromic shift indicated the formation of a-b unsaturated
carbonyl functional group, characteristic for chalcones [21]. The IR spectra of all
the compounds clearly showed a sharp C=O peak at around 1630 cm-1 and a broad
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B. S. Jayashree et al.
peak at above 3300 cm-1 indicating the presence of the hydroxyl group. The mass
spectra of all the test compounds showed the molecular ion peak as M ? 1, which
was also the base peak, indicating the stability of the synthesised chalcones. The
1HNMR spectra of all the test compounds showed the presence of the phenolic
hydroxyl proton at around d 14 along with the aromatic and aliphatic protons. The
chalcone characteristic Ha and Hb protons showed two sharp doublets at around d
7.67–8.62 and d 7.2–7.95, respectively. As the coupling constant for these two
adjacent protons is around 15.2–15.6 Hz, the protons are trans to each other and the
synthesised chalcones are assigned ‘E’ configuration [22] as shown in Fig. 3.
Similarly, the IR, 1HNMR and mass spectral details are given below along with the
physical parameters such as melting point, kmax and emax under the sub-headings of
each test compounds.
Physicochemical and spectral details of the newly synthesized chalcones
FS-3-S: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(thiophen-2-yl)prop-2-en-1-one Yield: 68 %; mp: 155–156 °C; kmax
:
347 nm; emax: 19635; IR: 3414 (O–H), 1631 (C=O), 1564 (C=C), 1126 (C–O);
1HNMR (DMSO-d6) d 13.8 (1H,s), 7.93 (1H, d, J = 15.6 Hz), 7.77 (1H, d,
J = 15.6 Hz), 7.65 (1H, br), 7.58–7.6 (1H, m), 7.17–7.2 (1H, m), 6.295 (1H, s),
5.44 (1H, s), 3.99 (3H, s), 3.87 (3H, s), 3.07 (2H, m), 2.78 (2H, m), 2.24 (3H, s),
1.91 (2H, m); MS: m/z [M ? 1]? 386.17.
FS-3-S1: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(thiophen-3-yl)prop-2-en-1-one Yield: 82 %; mp: 155–156 °C; kmax
:
349 nm; emax: 20,650; IR: 3437 (O–H), 1624 (C=O), 1568 (C=C), 1124 (C–O);
1HNMR (CDCl3) d 14.03 (1H, s), 7.82 (1H, d, J = 15.52 Hz), 7.72 (1H, d,
J = 15.52 Hz), 7.57 (1H, s), 7.36–7.39 (2H, m), 6.02 (1H, s), 5.6 (1H, br, s), 3.98
(3H, s), 3.87 (3H, s), 3.2 (2H, m), 2.75–2.78 (2H, m), 2.48 (3H, s), 2.43 (2H, m);
MS: m/z [M ? 1]? 386.17.
FS-3-SM3: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethox-
yphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one Yield: 50 %; mp: 121–122 °C;
k
max: 375 nm; emax: 22,155; IR: 3433 (O–H), 1614 (C=O), 1556 (C=C), 1120 (C–O);
1HNMR (CDCl3) d 14.06 (1H, s), 8 (1H, d, J = 16 Hz), 7.67 (1H, d, J = 15.52 Hz), 7.2
(1H, d, J = 2.8 Hz), 6.89 (1H, d, J = 4.8 Hz), 6 (1H, s), 5.59 (1H, s), 3.96 (3H, s), 3.85
(3H, s), 3.19 (2H, m), 2.7 (2H, m), 2.46 (3H, s), 2.41 (2H, m), 2.38 (3H, s); MS: m/z
[M ? 1]? 400.15.
Fig. 3 Configuration of chalcones; characteristic Ha and Hb protons shows two sharp doublets at around
d 7.67–8.62 and d 7.2–7.95, respectively, in NMR spectral studies
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Synthesis of newer piperidinyl chalcones and their…
FS-3-SM5: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethox-
yphenyl)-3-(5-methylthiophen-2-yl)prop-2-en-1-one Yield: 57 %; mp: 124–125 °C;
k
max: 380 nm; emax: 21450; IR: 3419 (O–H), 1614 (C=O).1556 (C=C), 1120 (C–O);
1HNMR (CDCl3) d 14.23 (1H, s), 7.88 (1H, d, J = 15.52 Hz), 7.6 (1H, d,
J = 15.16 Hz), 7.12–7.15 (1H, m), 6.7 (1H, d, J = 3.56 Hz), 6.02 (1H, s), 5.58 (1H,
br, s), 3.98 (3H, s), 3.88 (3H, s), 3.36 (2H, m), 2.9 (2H, m), 2.6 (3H, s), 2.5 (3H, s), 2.18
(2H, m); MS: m/z [M ? 1]?400.15.
FS-3-F: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
:
phenyl)-3-(furan-2-yl)-prop-2-en-1-one Yield: 71 %; mp: 146–147 °C; kmax
366 nm; emax: 19965; IR: 3435 (O–H), 1624 (C=O), 1595 (C=C), 1126 (C–O);
1HNMR (CDCl3) d 14.07 (1H, s), 7.77 (1H, d, J = 15.2 Hz), 7.59, (1H, d,
J = 15.2 Hz), 7.5–7.51 (1H, m), 6.66 (1H, d, J = 3.6 Hz), 6.48–6.5 (1H, m), 6 (1H,
s), 5.58 (1H, s), 3.96 (3H, s), 3.85 (3H, s), 3.15 (2H, m), 2.71 (2H, m), 2.43 (3H, s),
2.39–2.4 (2H, m); MS: m/z [M ? 1]? 370.15.
FS-3-Br: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(5-bromofuran-2-yl)-prop-2-en-1-one Yield: 40 %; mp: 135–135 °C;
k
max: 372 nm; emax: 22600; IR: 3435 (O–H), 2935 (C–H), 1624 (C=O), 1124 (C–O);
1HNMR (CDCl3) d 14.01 (1H, s), 7.75 (1H, d, J = 4 Hz), 7.71 (1H, d, J = 4 Hz),
6.59 (1H, s), 6.43 (1H, m), 5.99 (1H, s), 5.58 (1H, m), 3.97 (3H, s), 3.85 (3H, s), 3.1
(2H, m), 2.7 (2H, m), 2.3 (3H, s), 2.1 (2H, m); MS: m/z [M ? 1]? 448.06.
FS-3-P: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
:
phenyl)-3-(pyridin-3-yl)prop-2-en-1-one Yield: 38 %; mp: 160–161 °C; kmax
339 nm; emax: 18652; IR:3435 (O–H), 1631 (C=O), 1566 (C=C), 1124 (C–O);
1HNMR (CDCl3) d 13.87 (1H, s), 8.87 (1H, s), 8.62 (1H, d, J = 15.7 Hz), 7.95 (1H,
d, J = 15.72 Hz), 7.34–7.37 (1H, m), 3.99 (3H, s), 3.88 (3H, s), 3.18 (2H, m),
2.72–2.75 (2H, m), 2.46 (3H, s), 2.19 (2H, m); MS: m/z [M ? 1]? 381.17.
FS-3-DCl: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethox-
yphenyl)-3-(2,4-dichlorophenyl)-prop-2-en-1-one Yield: 72 %; mp: 186–187 °C;
k
max: 373 nm; emax: 19665; IR: 3433 (O–H), 2933 (C–H), 1620 (C=O), 1122 (C–O);
1HNMR (DMSO-d6) d 7.5–7.96 (5H, m), 6.3 (1H, s), 5.44 (1H, s), 3.98 (3H, s), 3.87
(3H, s), 2.98 (2H, s), 2.57 (2H, m), 2.3 (3H, s), 2.21 (2H, s); molecular weight: 448;
MS: m/z [M ? 1]? 448.10.
FS-3-M: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(2-methoxyphenyl)prop-2-en-1-one Yield: 71 %; mp: 126–127 °C;
k
max: 366 nm; emax: 20560; IR: 3423 (O–H), 1620 (C=O), 1564 (C=C), 1116
1
(C–O); HNMR (CDCl3) d 14 (1H, s), 7.9 (2H, m), 7.7 (1H, m), 7.4 (1H, m), 7.1
(1H, m), 7 (1H, m), 6.2 (1H, s), 5.4 (1H, s), 3.8–3.9 (9H, s), 3.12 (2H, br), 2.7 (2H,
?
br), 2.39 (3H, s), 2.26 (2H, br); MS: m/z [M ? 1] 410.18.
FS-3-E: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(4-ethoxyphenyl)-prop-2-en-1-one Yield: 73 %; mp: 155–156 °C; kmax
:
368 nm; emax: 23210; IR: 3425 (O–H), 1627 (C=O), 1556 (C=C), 1122 (C–O);
1HNMR (DMSO-d6) d 7.67–7.77 (4H, m), 6.99–7.01 (2H, m), 6.35 (1H, s), 5.48
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B. S. Jayashree et al.
(1H, s), 4.08 (2H, q, J = 6.08 Hz), 4.02 (3H, s), 3.9 (3H, s), 3.21 (2H, br), 2.69 (2H,
br), 2.38 (3H, s), 2.26 (2H, br), 1.34 (3H, t, J = 12.8 Hz); MS: m/z [M ? 1]?
424.20.
FS-3-DA: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(4-(dimethylamino)phenyl)-prop-2-en-1-one Yield: 30 %; mp: 145–146 °C;
k
max: 335 nm; emax: 24255; IR: 3435 (O–H), 2935 (C–H), 1614 (C=O), 1126 (C–O);
1HNMR (CDCl3) d 14.03 (1H, s), 7.76 (1H, d, J = 15.4 Hz), 7.65 (1H, d,
J = 15.4 Hz), 7.45 (2H, d, J = 8.8 Hz), 6.63 (2H, d, J = 8.8 Hz), 5.93 (1H, s), 5.5
(1H, s), 3.89 (3H, s), 3.84 (3H, s), 3.09 (2H, m), 2.9 (6H, s), 2.66 (2H, m), 2.36 (3H,
s), 2.1 (2H, m); MS: m/z [M ? 1]? 423.22.
FS-3-Dm: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one Yield: 62 %; mp: 168–169 °C;
k
max 374 nm; emax: 22140; IR: 3435 (O–H), 1625 (C=O), 1566 (C=C), 1126 (C–O);
1HNMR (DMSO-d6) d7.78 (1H, d, J = 15.6 Hz), 7.7 (1H, d, J = 15.32 Hz),
7.27–7.32 (2H, m), 7.02 (1H, m), 6.28 (1H, s), 5.43 (1H, s), 3.99 (3H, s), 3.86 (3H, s),
3.83 (3H, s), 3.79 (3H, s), 2.98 (2H, m), 2.56 (2H, m), 2.29 (3H, s), 2.08 (2H, m); MS:
m/z [M ? 1]? 440.19.
FS-3-N:1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxyphe-
nyl)-3-(naphthalen-1-yl)prop-2-en-1-one Yield: 78 %; mp: 160–161 °C; kmax
:
Table 1 Antioxidant activity of the synthesised test compounds
Radical scavenging activity (IC50) in lM SEM
Compound code ABTS radical Superoxide radical O-Phenanthroline Lipid peroxidation
1
2
3
4
5
6
7
8
9
FS-3-S
81.21 3.25
92.13 2.15
134.2 3.62
130.5 4.55
80.19 0.29
83.20 1.24
79.47 1.61
82.55 1.35
68.45 1.45
71.99 1.06
69.45 2.12
66.35 1.55
73.24 2.06
72.50 1.56
37.00 1.76
–
73.42 1.39
98.32 2.03
142.3 5.23
139.6 3.34
107.9 1.86
110.3 1.05
79.59 1.04
80.50 2.02
64.22 2.23
79.98 1.31
75.29 1.43
65.02 1.43
77.28 2.01
75.44 1.45
27.25 1.56
–
33.42 1.27
48.23 2.30
78.43 4.21
80.03 3.50
22.50 1.16
25.35 1.25
53.61 1.56
55.65 1.24
28.33 1.46
30.31 1.13
28.23 2.03
27.50 2.04
32.03 2.23
29.45 1.54
50.31 1.63
–
46.43 0.41
54.53 1.35
82.30 2.31
85.20 2.42
47.09 1.85
49.10 2.31
46.44 2.44
50.05 3.00
14.09 1.65
15.47 1.80
14.27 2.08
13.90 1.51
14.91 2.05
17.55 2.90
–
FS-3-S1
FS-3-SM3
FS-3-SM5
FS-3-F
FS-3-Br
FS-3-P
FS-3-DCl
FS-3-M
10 FS-3-E
11 FS-3-DA
12 FS-3-Dm
13 FS-3-N
14 FS-3-N1
Ascorbic acid
Quercetin
56.25 2.70
123

Synthesis of newer piperidinyl chalcones and their…
374 nm; emax: 19660; IR: 3435 (O–H), 1618 (C=O), 1566 (C=C), 1120 (C–O);
1HNMR (CDCl3) d 13.93 (1H, s), 8.54 (1H, d, J = 15.6 Hz), 8.25 (1H, d,
J = 15.6 Hz), 7.4–7.9 (7H, m), 5.95 (1H, s), 5.54 (1H, br, s), 3.9 (3H, s), 3.8 (3H, s),
3.1 (2H, m), 2.7 (2H, m), 2.37 (3H, s), 2.36 (2H, m); MS: m/z [M ? 1]? 430.19.
FS-3-N1: 1-(3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2-hydroxy-4,6-dimethoxy-
phenyl)-3-(naphthalen-2-yl)prop-2-en-1-one Yield: 73 %; mp: 172–173 °C; kmax
:
356 nm; emax: 19885; IR: 3435 (O–H), 1624 (C=O), 1556 (C=C), 1118 (C–O);
1HNMR (CDCl3) d 14 (1H, s), 7.99 (1H, br), 7.95–7.96 (2H, m), 7.82–7.89 (3H, m),
7.74–7.76 (1H, m), 7.49–7.53 (2H, m), 6.03 (1H, s), 5.61 (1H, s), 4 (3H, s), 3.87
(3H, s), 3.17 (2H, m), 2.75 (2H, m), 2.4 (2H, m), 1.8 (3H, s); MS: m/z [M ? 1]?
430.19.
Antioxidant activity
The antioxidant activity of the synthesised test compounds are expressed in terms of
IC50 values as shown in Table 1. The test compounds have considerable free radical
scavenging activity when they were tested against ABTS and superoxide radical
Table 2 Anticancer activity of the synthesised test compounds
No
Compound code
IC50 (lM)
A549
HepG2
HeLa
HCT-116
MCF-7
MDA MB 231
1
FS-3-S
26.05
23.07
33.50
34.48
43.84
46.01
24.12
22.07
23.80
28.34
–
17.50
10.20
6.75
8.00
15.00
11.50
6.60
7.00
6.75
6.75
28.0
7.00
7.25
6.50
–
56.35
48.23
44.32
45.31
41.83
49.16
67.29
41.11
42.12
42.12
54.31
41.23
35.23
33.21
–
45.33
39.21
37.23
35.34
32.23
40.34
56.45
34.54
35.56
33.63
45.72
32.38
23.34
21.80
–
48.65
45.35
42.57
40.38
38.47
44.43
59.18
37.73
38.63
36.71
47.16
35.21
25.36
23.45
–
49.21
45.63
43.26
42.91
40.28
46.19
60.48
41.65
40.46
36.54
48.43
37.32
27.54
25.80
–
2
FS-3-S1
3
FS-3-SM3
FS-3-SM5
FS-3-F
4
5
6
FS-3-Br
FS-3-P
7
8
FS-3-DCl
FS-3-M
9
10
FS-3-E
11
FS-3-DA
FS-3-Dm
FS-3-N
12
27.07
4.69
4.28
0.7
13
14
FS-3-N1
Methotrexate
Cisplatin
Doxorubicin
Standards
–
10.00
–
–
–
–
–
–
3.9
0.07
0.12
3.2
IC50 values are calculated at 95% confidence interval (CI) and coefficient of variation \10
A549 human lung adenocarcinoma epithelial cell line, HepG2 human hepatocellular carcinoma cell line,
HeLa human cervical cancer cell line, HCT-116 human colon carcinoma cell line, MCF-7 human breast
cancer cell line (ER-positive), MDA MB 231 human breast carcinoma cell line (ER-negative)
123

B. S. Jayashree et al.
scavenging assay. Similarly, the test compounds showed good iron chelating
abilities in O-phenanthroline assay. The test compounds also showed considerable
inhibition of ferric induced lipid peroxidation in vitro. Further, all the test
compounds showed an overall free radical scavenging activity at IC50 \ 100 lM in
all the tests performed as shown in Table 1.
Toxicity on Vero cells
When the test compounds were tested on Vero cells, they showed IC50 above
800 lM suggesting the selective toxicity of the synthesized compounds for cancer
cells.
Anticancer activity
The results of anticancer activity of the test compounds performed by MTT assay
are presented in Table 2. Test compounds FS-3-S and FS-3-S1, bearing a thienyl
substitution, FS-3-SM3 and FS-3-SM5 possessing methyl thienyl group and
compounds such as FS-3-N and FS-3-N1 having naphthalene rings were isomeric
to each other. However, there was no significant difference in the anticancer activity
in two isomers of a particular substitution as shown in Table 2. Further, the test
compounds FS-3-N and FS-3-N1 exhibited potent anticancer activity comparable to
that of other molecules in all the human cancer cell lines.
The bio-isosteric replacement of the thienyl ring with that of furanyl moiety was
another objective of the present study, where the test compounds FS-3-S and FS-3-
S1 with thienyl ring were conveniently replaced by sulphur with oxygen in the
B-ring. Here, bio-isosteric modification was used as a tool in lead modification,
where one sulphur atom becomes equivalent to one oxygen atom (non-classical bio-
isosteres). With this rationale, the furanyl substituted compounds were designed,
speculating for their anticancer activity. From our studies it was found that the
furanyl substituted test compounds such as FS-3-F and FS-3-Br, resulted in a
slightly decreased activity against cell lines studied. Further, the six-membered
substitutions, namely, 3-pyridyl in test compound FS-3-P and other non-hetero
aromatic compounds such as FS-3-DCl, FS-3-M, FS-3-E, and FS-3-Dm, showed
potency equivalent to that of those bearing a thiophene nucleus, or even more, as
seen against the cancer cell lines studied. Test compounds FS-3-N and FS-3-N1
Fig. 4 Hoechst 33342 staining to study chromatin condensation in HepG2 cells, a control DMSO-
treated; b positive control doxorubicin-treated; c compound SF-3-N-treated; d SF-3-N1 -treated
123

Synthesis of newer piperidinyl chalcones and their…
bearing a naphthalene moiety were the most active compounds among the entire
group as shown in Table 2.
Hoechst staining technique was used for observing apoptosis in cancer cell lines.
The stained apoptotic cells would show a bright fluorescence. The Fig. 4 represents
the apoptosis induced by compound FS-3 N and FS-3-N1. The chromatin
condensations in apoptotic cells would show high fluorescence. This test inferred
the strong evidence for apoptosis induction by our test compounds.
Scratch wound antimigration assay was also used to assess the inhibition of
invasion and angiogenesis by tumour cells. The test compounds exhibited
antimigration activity at 24 h incubations. All the test compounds were equally
potent in inhibiting cell migrations compared to the control and the standard
methotrexate (Table 3). The Fig. 5 represents the photograph taken at different time
interval after control, standard and test compounds SF-3-N and SF-3-N1
inoculation.
Discussion
Chalcones have been reported for their anticancer activity by multimodal
mechanisms, such as inhibition of tubulin assembly, inhibition of angiogenesis,
induction of apoptosis, anti-estrogenic activity, and reversal of multidrug resistance,
Table 3 Scratch wound antimigration assay
Sl no.
Treatmenta
Wound width in mm (mean SEM)
0 h
6 h
12 h
24 h
1.
Control
MTX
0.83 0.037
0.75 0.04
0.60 0.01
0.64 0.02
0.65 0.03
0.67 0.05
0.80 0.04
0.76 0.06
0.64 0.04
0.76 0.03
0.77 0.02
0.73 0.01
0.76 0.03
0.79 0.04
0.73 0.07
0.72 0.05
0.46 0.057
0.72 0.03
0.50 0.03
0.54 0.05
0.54 0.06
0.56 0.03
0.70 0.041
0.72 0.06
0.58 0.01
0.68 0.03
0.74 0.04
0.68 0.03
0.64 0.04
0.65 0.05
0.65 0.02
0.68 0.03
0.16 0.02
0.68 0.01
0.44 0.01
0.46 0.03
0.45 0.05
0.46 0.06
0.65 0.02
0.66 0.07
0.53 0.01
0.54 0.02
0.55 0.05
0.53 0.01
0.58 0.03
0.59 0.04
0.63 0.03
0.65 0.02
0.06 0.057
0.66 0.042
0.41 0.045
0.43 0.05
0.42 0.03
0.43 0.02
0.63 0.015
0.44 0.02
0.45 0.042
0.43 0.034
0.42 0.06
0.44 0.042
0.48 0.021
0.52 0.031
0.60 0.032
0.63 0.022
2.
3.
FS-3-S
4.
FS-3-S1
FS-3-SM3
FS-3-SM5
FS-3-F
5.
6.
7.
8.
FS-3-Br
FS-3-P
9.
10.
11.
12.
13.
14.
15.
16.
FS-3-DCl
FS-3-M
FS-3-E
FS-3-DA
FS-3-Dm
FS-3-N
FS-3-N1
Inhibition of closure of scratch wound in HeLa cell line; MTX methotrexate
a
At concentration equal to the IC50 determined by MTT in HeLa cell lines
123

B. S. Jayashree et al.
Fig. 5 Scratch wound anti-migration assay; Photomicrograms of scratch wounds on HeLa confluent cell
layer and their closure by exposure without treatment and with test compounds for 8, 12, and 24 h;
a control treated with solvent DMSO; b methotrexate treated; c, d compound FS-3-N and FS-3-N1 treated
or a combination of these mechanisms [2]. A recent report on newer 3-phenylquino-
linylchalcone derivatives, which showed anticancer activity by polymerization of
tubulins, induced G2/M cell cycle arrest via modulation of the cyclin B1, cdk1, and
CDC25 [17]. The pyrazole chalcones and heterocyclic diamides have been reported
for anticancer activity in MCF-7 and HeLa cell lines [23]. The newly synthesized
test compounds have shown anticancer activity in human cancer cell lines by
induction of apoptosis. Hoechst stain enters the plasma membrane and binds to
apoptotic DNA, making the cell nucleus highly fluorescent, and confirms the
mechanism of action of test compounds as apoptotic agent [19]. However, further
studies have to be carried out to find alternative pathways for understanding the
apoptotic mechanism for anticancer activity in these test compounds.
Cell migration is an indicator of proliferation of tumors or tumor invasion and
metastasis [24]. The scratch wound healing assay is proven to be a good model to
screen anti-invasive or antimetastatic molecules. The scratch wound assay is also an
indicator for angiogenesis during tumor growth [18]. The inhibition of angiogenesis
has multiple therapeutic implications other than cancer [25]. Many of the synthetic
chalcones are reported for inhibition of angiogenesis and prevention of tumour
growth [26]. Thus, in the present study, considerable inhibition of scratch wound
123

Synthesis of newer piperidinyl chalcones and their…
closure by test compounds indicates their applications as antimetastatic or
antiangiogenic potentials.
Plant-derived and dietary phenolic antioxidants were proven to have anticancer
properties with different mechanisms along with their antioxidant activity [27].
However, the relevance of antioxidant molecule for anticancer activity has recently
been established. Along with intracellular free radical scavenging activity, the
majority of antioxidant molecules activates transcription factor Nrf2 (nuclear factor-
E2-related factor 2), which acts through antioxidant reactive element (NRE)
signaling pathways in cancer cells and increases antioxidant enzymes by gene
expression, whereby they contribute for anticancer activity [28]. In line with this,
there are reports of novel synthetic chalcones for anticancer activity by activation of
Nrf2. These chalcones have increased expression of Nrf2 dependent antioxidant
genes in human lung epithelial cells and in mouse model of cancer [29]. It has been
observed from our studies that these test compounds possesses antioxidants and this
could attributed to their anticancer activity hence it warrants further mechanistic
study.
Conclusions
The piperidinyl chalcones having naphthalene substitutions on the B-ring in
chalcone showed antioxidant activity in vitro and anticancer activity against human
cancer cell lines. The bio-isosteric replacement of the thienyl substituent with
furanyl moiety resulted in decreased anticancer activity. However, non-hetero
aromatic substitutions were equally potent as compared to their thienyl ring
counterpart. Furthermore, there was no significant difference in the activity of the
isomeric compounds bearing a thienyl group, methyl thienyl group and naphthalene
moiety. The tetrahydropyridine substituted chalcones, with the retention of double
bond in the piperidinyl ring were the newer bioisosterically modified derivatives of
piperidinyl chalcones, and could be considered as useful leads for further anticancer
investigations.
Acknowledgments Authors duly acknowledge the Department of Pharmacology, Manipal College of
Pharmaceutical Sciences, and Manipal University for providing the facility to carry out the research.
Authors also acknowledges the Indian Institute of Science, Bangalore, Sophisticated Analytical
Instrument Facility, Kochi University, Kerala and Panjab University, Chandigarh for providing the NMR
data of newly synthesized compounds. Authors would also like to thank AICTE—New Delhi for RPS and
MODROBS Grants for instrumentation.
Compliance with ethical standards
Conflict of Interest Authors declare that there is no conflict of interest.
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