Synthesis, in vitro and in silico Anti-Proliferative Studies of Novel Piperiene-Oxadiazole and Thiadiazole Analogs

Article abstract of DOI:10.1134/S1070363219110227

Piperine is a component of pepper which has earlier been reported as anticancer active compound. This work is emphasized on the design and synthesis of new hybrid piperine analogs by coupling piperine with the amine group of oxadiazoles and thiadiazoles.

Full text of DOI:10.1134/S1070363219110227

ISSN 1070-3632, Russian Journal of General Chemistry, 2019, Vol. 89, No. 11, pp. 2301–2307. © Pleiades Publishing, Ltd., 2019.
Synthesis, in vitro and in silico Anti-Proliferative Studies
of Novel Piperiene-Oxadiazole and Thiadiazole Analogs
K. R. Amperayania,* and U. D. Parimia
a Department of Chemistry, GVP Degree and PG College, Visakhapatnam, Andhra Pradesh, 530045 India
*e-mail: a.karteek.akrao@gmail.com
Received May 31, 2019; revised November 4, 2019; accepted November 5, 2019
Abstract—Piperine is a component of pepper which has earlier been reported as anticancer active compound.
This work is emphasized on the design and synthesis of new hybrid piperine analogs by coupling piperine with
the amine group of oxadiazoles and thiadiazoles. The new series of twelve piperine analogs was been tested for
in vitro anti-proliferative activity using sulforhodamine B (SRB) assay test against MCF-7, PC-3, and HeLa cell
lines. Among the twelve synthesised molecules piperine derivative with oxadiazole baring hydroxyl group (3) ex-
hibits the higher activity against MCF-7 cell line than the reference drug Adriamycin and also displays the highest
binding energy in the in silico studies. The other analogs are moderately active.
Keywords: piperine; oxadiazole; thiadiazoles; anti proliferative activity
DOI: 10.1134/S1070363219110227
INTRODUCTION
and thiadiazoles with piperic acid by the amide linkage
for testing anti proliferative effect on MCF-7, PC-3, and
HeLa cell lines. Actually, this is the first report on anti
proliferative activity of piperine derivatives of oxadia-
zoles and thiadiazoles.
Application of natural products as anticancer agents
is significantly developing due to their low side effects.
Combination of natural products with synthetic drugs
can produce hybrid molecules [1, 2] with low side ef-
fects and high biological activity. Such approach had
been employed by us in the earlier studies [3] and led
to positive results. So, we designed hybrid molecules of
piperine with oxadiazole and thiadiazole as promising
anticancer agents.
EXPERIMENTAL
All reagents were ofAR grade and used directly with-
out further purification. Piperine (97%) was purchased
from Sigma Aldrich chemicals. Melting points were
determined in open capillary tubes. FTIR spectra were
recorded on a Perkin Elmer FTIR spectrophotometer
using KBr pellets. 1H and 13C NMR spectra were mea-
sured on a Bruker Avance 400 and 100 spectrometers
respectively using TMS as an internal standard and CDCl3
(1H NMR) and CD3OD (13C NMR) as solvents. HRMS
were measured on a Bruker Micro TOF-Q II ESI spec-
trometer. Ultra sonication reactions were carried out on
a PCI Analytics sonicator, frequency 25 KHz and power
220 W at room temperature. Progress of the reactions
was monitored by TLC on Merck TLC silica gel plates
using hexane: ethylacetate (3 : 2) mixture as an eluent.
The spots were visualized under UV light.
Some earlier studies on piperine proved its substantial
anticancer effect [4] including prostate cancer [5], MCF-7
andA-549 [6], HeLa and breast cancer [7] and some more
[8, 9]. The activity of piperine resides on the amide bond
with the piperidine moiety [10]. Alterations in the amide
bond can affect the biological activity of the entire mol-
ecule. Piperine derivatives could possess activity superior
than the parent molecule [11, 12]. Piperine–amino acid
ester conjugates showed good cytotoxic activity against
human cancer cell lines [13].Accordingly, we have modi-
fied the amide functionality of piperine for the following
anti proliferative activity studies.
1,3,4-Oxadiazoles [14–16] and some thiadiazole
[17–19] derivatives are promising molecules with diverse
biological activities including anticancer. Based on the
above, we have coupled the amine functional group of oxa
Piperic acid. Piperine (10 g) was dissolved in
300 mL of anhydrous ethanol containing 20% KOH and
the mixture was refluxed upon stirring for 10 h to give
the precipitate of potassium piperate, which was filtered
2301

2302
AMPERAYANI, PARIMI
Scheme 1.
N
COOH
R
N
H2SO4
reflux
X
NH2
X
sonication
HN
NH2
NH2
R
X = O or S; R = H, p-Cl, p-OH, p-NO , o-OH, o-NO .
2
2
Scheme 2.
N
N
O
NH2
+
O
O
X
OH
R
O
R
EDC·HCl
DIPEA
O
X
NH
O
N
N
PIP 1ꢀ12
X = O and S; R= H, p-Cl, p-NO , o-OH, o-NO .
2
2
off and washed with anhydrous ethanol. The precipitate
was dissolved in distilled water and precipitated upon
addition of HCl solution (0.1 M). The yellow precipitate
of piperic acid was filtrated off and washed with distilled
water (200 mL) to give powdery yellow piperic acid [20],
yield 86.6%, mp 214–216°C.
washed with water (2×10 mL), aqueous solution of 1%
H3PO4 (2×10 mL), an aqueous solution of 2.5% K2CO3
(2×10 mL), and concentrated to give the corresponding
crude product. This was purified by column chromatog-
raphy using hexane:ethylacetate as an eluent (Scheme 2).
(2E,4E)-5-(Benzo[d][1,3]dioxo-5-yl)-N-(5-phenyl-
1,3,4-oxadiazol-2-yl)penta-2,4-dienamide (1). Brown
crystals, yield 68%, mp 265°C. IR spectrum, ν, cm–1:
1710 (amide CONH), 3100–3500 (amide NH). 1H NMR
spectrum, δ, ppm: 5.19 d.d (J = 14.7, 0.8 Hz, 1H), 6.01
d (J = 2.3 Hz, 1H), 6.07 d (J = 2.3 Hz, 1H), 6.66 d.d.d
(J = 14.0, 7.0, 0.9 Hz, 1H), 6.70–6.82 m (2H), 6.82 d.d
(J = 8.5, 1.9 Hz, 1H), 7.04 d (J = 1.8 Hz, 1H), 7.37–7.47
m (2H), 7.60–7.50 m (2H), 7.97–7.89 m (2H), 8.21 s
(1H). 13C NMR spectrum, δ, ppm: 100.99. 106.96, 108.54,
120.68, 123.68, 126.01, 126.65, 126.74, 128.84, 130.40,
131.15, 138.30, 140.31, 147.05, 147.90, 152.42, 158.79,
168.74. HRMS: m/z: 362.1169 [H]+.
Substituted oxadiazoles and thiadiazoles. Amixture
of substituted benzoic acid (2 g) and 2 g of semicarbazide
or thiosemicarbazide was dissolved in 10 mL of H2SO4
and subjected to ultrasonication for 30–45 min at room
temperature (TLC). The product obtained was cooled
down and poured onto crushed ice. The residue thus ob-
tained was separated, washed with water, recrystallized
from ethanol and chromatographed with hexane : ethyl-
acetate to get the corresponding pure product (Scheme 1).
Coupling of piperine with oxadiazoles or thiadia-
zoles. The solution of 2.18 g of piperic acid in 20 mL of
anhydrous CH2Cl2 was mixed with 1.92 g of 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide HCl (EDC·HCl)
and 2.60 mL of N,N-diisopropylethylamine (DIPEA)
and stirred at 0–5°C. for 30 min. 1 mol of oxadiazole or
thiadiazole derivative was added to the above mixture
and stirred at room temperature for 2 h (TLC). After
completion of the process (TLC), the reaction mixture was
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(4-
chlorophenyl)-1,3,4-oxadiazol-2-yl]penta-2,4-dien-
amide (2). Brown crystals, yield 72%, mp 256°C. IR
spectrum, ν, cm–1: 1675 (amide CONH), 3100–3300
1
(amide NH). H NMR spectrum, δ, ppm: 5.19 d.d (J =
14.7, 0.8 Hz, 1H). 6.01 d (J = 2.3 Hz, 1H), 6.07 d (J =
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SYNTHESIS, IN VITRO AND IN SILICO ANTI-PROLIFERATIVE STUDIES
2303
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(2-
nitrophenyl)-1,3,4-oxadiazol-2-yl]penta-2,4-dien-
amide (6). Colourless crystals, yield 65%, mp 230°C.
IR spectrum, ν, cm–1: 1680 (amide CONH), 3200–3500
2.3 Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H), 6.82–
6.70 m (2H), 6.82 d.d (J = 8.5, 1.9 Hz, 1H), 7.04 d (J =
1.8 Hz, 1H), 7.45 d.d (J = 14.7, 7.1 Hz, 1H), 7.47–7.55 m
(2H), 8.03–8.11 m (2H), 8.21 s (1H). 13C NMR spectrum,
δ, ppm: 100.99. 106.96, 108.54, 120.68, 123.68, 126.01,
127.90, 128.38, 129.29, 130.40, 138.30, 138.60, 140.31,
147.05, 147.90, 152.42, 158.79, 168.74. HRMS: m/z:
396.1047 of [H]+.
1
(amide NH). H NMR spectrum, δ, ppm: 5.19 d.d (J =
14.7, 0.8 Hz, 1H), 6.01 d (J = 2.3 Hz, 1H), 6.07 d (J =
2.3 Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H),
6.70–6.82 m (2H), 6.82 d.d (J = 8.5, 1.9 Hz, 1H), 7.04
d (J = 1.8 Hz, 1H), 7.36–7.48 m (2H), 7.73 t.d (J = 7.6,
1.3 Hz, 1H), 7.79 d.d.d (J = 12.6, 7.9, 1.3 Hz, 2H), 8.14 s
(1H). 13C NMR spectrum, δ, ppm: 100.99, 106.96, 108.54,
120.68, 123.68, 125.71, 126.01, 126.59, 127.54, 128.77,
129.28, 130.40, 138.30, 140.31, 145.17, 146.16, 147.05,
147.90, 158.79, 168.74. HRMS: m/z: 407.0208 [H]+.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-[5-(4-
hydroxyphenyl)-1,3,4-oxadiazol-2-yl]penta-2,4-dien-
amide (3). Colourless crystals, yield 68%, mp 260°C.
IR spectrum, ν, cm–1: 1650 (CONH), 3100–3300 (amide
1
NH). H NMR spectrum, δ, ppm: 5.19 d.d (J = 14.7,
0.9 Hz, 1H), 6.10–6.02 m (2H), 6.66 d.d.d (J = 14.0,
7.0, 0.8 Hz, 1H), 6.70–6.79 m (1H), 6.79–6.86 m (2H),
7.07–6.94 m (3H), 7.34–7.27 m (1H), 7.35–7.51 m (2H),
8.16 s (1H), 9.10 s (1H). 13C NMR spectrum, δ, ppm:
100.99, 106.96, 108.54, 111.69, 116.86, 119.02, 120.68,
123.68, 126.01, 128.97, 130.40, 133.70, 138.30, 140.31,
143.82, 147.05, 147.90, 158.75, 158.79, 168.74. HRMS:
m/z: 378.2038 [H]+.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-(5-phenyl-
1,3,4-thiadiazol-2-yl)penta-2,4-dienamide. Grey crys-
tals, yield 60%, mp 230°C. IR spectrum, cm–1: 1690
1
(amide-CONH), 3100–3400 (amide NH). H NMR
spectrum, δ, ppm: 5.19 d.d (J = 14.7, 0.8 Hz, 1H), 6.01
d (J = 2.3 Hz, 1H), 6.07 d (J = 2.3 Hz, 1H), 6.66 d.d.d
(J = 14.0, 7.0, 0.9 Hz, 1H), 6.70–6.82 m (2H), 6.82 d.d
(J = 8.5, 1.9 Hz, 1H), 7.04 d (J = 1.8 Hz, 1H), 7.38–7.49
m (1H), 7.45–7.56 m (3H), 8.00–8.11 m (2H), 8.29 s
(1H). 13C NMR spectrum, δ, ppm: 100.99, 106.96, 108.54,
120.68, 123.68, 126.01, 127.34, 127.88, 130.27, 130.40,
133.75, 138.30, 140.31, 147.05, 147.90, 158.01, 163.17,
168.02. MS: (m/z): 378.2038 [H]+.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(4-
nitrophenyl)-1,3,4-oxadiazol-2-yl]penta-2,4-dien-
amide (4). Colorless crystals, yield 58%, mp 274°C. IR
spectrum: 1700 (amide CONH), 3100–3500 (amide NH).
1H NMR spectrum, δ, ppm: 5.19 d.d (J = 14.7, 0.8 Hz,
1H), 6.01 d (J = 2.3 Hz, 1H), 6.07 d (J = 2.3 Hz, 1H), 6.66
d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H), 6.70–6.82 m (2H), 6.82
d.d (J = 8.5, 1.9 Hz, 1H), 7.04 d (J = 1.8 Hz, 1H), 7.45
d.d (J = 14.7, 7.1 Hz, 1H), 8.23 d.d (J = 7.4, 1.0 Hz, 3H),
8.37–8.45 m (2H). 13C NMR spectrum, δ, ppm: 100.99,
106.96, 108.54, 120.68, 123.68, 124.12, 126.01, 127.58,
130.40, 132.07, 138.30, 140.31, 147.05, 147.90, 149.36,
152.42, 158.79, 168.74. HRMS: m/z: 407.0208 [H]+.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(4-
chlorophenyl)-1,3,4-thiadiazol-2-yl]penta-2,4-dien-
amide (8). Colourless crystals, yield 64%, mp 256°C.
IR spectrum, ν, cm–1: 1670 (amide CONH), 3100–3500
1
(amide NH). H NMR spectrum, δ, ppm: 5.19 d.d
(J = 14.7, 0.8 Hz, 1H), 6.01 d (J = 2.3 Hz, 1H), 6.07 d
(J = 2.3 Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H),
6.70–6.79 m (1H), 6.79–6.86 m (2H), 7.04 d (J = 1.8 Hz,
1H), 7.40–7.52 m (3H), 7.87–7.95 m (2H), 8.22 s (1H).
MS: m/z: 412.1078 [H]+.
(2E,4E)-N-[5-(2-Hydroxyphenyl)-1,3,4-oxadiazol-
2-yl]-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienamide
(5). Green crystals, yield 65%, mp 260°C. IR spectrum,
cm–1: 1675 (amide CONH), 3100–3300 (amide NH). 1H
NMR spectrum, δ, ppm: 5.19 d.d (J = 14.7, 0.8 Hz, 1H).
6.01 d (J = 2.3 Hz, 1H), 6.07 d (J = 2.3 Hz, 1H), 6.66
d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H), 6.70–6.82 m (2H), 6.82
d.d (J = 8.5, 1.9 Hz, 1H), 6.94-7.07 m (3H), 7.27–7.34 m
(1H), 7.40 t.d (J = 7.6, 1.3 Hz, 1H), 7.46 d.d (J = 14.7,
7.1 Hz, 1H), 8.16 s (1H), 9.10 s (1H). 13C NMR spectrum,
δ, ppm: 100.99, 106.96, 108.54, 111.69, 116.86, 119.02,
120.68, 123.68, 126.01, 128.97, 130.40, 133.70, 138.30,
140.31, 143.82, 147.05, 147.90, 158.75, 158.79, 168.74.
HRMS: m/z: 378.0818 [H]+.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(4-
hydroxyphenyl)-1,3,4-thiadiazol-2-yl]penta-2,4-
dienamide (9). Colorless crystals, yield 58%, mp 280°C.
IR spectrum, ν, cm–1: 1690 (amide CONH), 3100–3500
1
(amide NH). H NMR spectrum: δ, ppm: 5.19 d.d (J =
14.7, 0.8 Hz, 1H), 5.42 s (1H), 6.01 d (J = 2.3 Hz, 1H),
6.07 d (J = 2.3 Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9
Hz, 1H), 6.70–6.82 m (2H), 6.82 d.d (J = 8.5, 1.9 Hz,
1H), 6.87–6.95 m (2H), 7.04 d (J = 1.8 Hz, 1H), 7.45 d.d
(J = 14.7, 7.1 Hz, 1H), 7.71–7.63 m (2H), 8.27 s (1H). 13C
NMR spectrum, δ, ppm: 100.99, 106.96, 108.54, 116.03,
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2304
AMPERAYANI, PARIMI
120.68, 123.68, 125.00, 126.01, 128.78, 130.40, 138.30,
140.31, 147.05, 147.90, 158.01, 160.07, 163.17, 168.02.
MS: m/z: 394.0970 [H]++.
plates in 100 μL at plating densities, depending on the
doubling time of individual cell line. After cell inocula-
tion, the microtiter plates were incubated at 37°C, 5%
CO2, 95% air and 100% relative humidity for 24 h prior
to addition of experimental drugs [21, 22]. Experimental
drugs were initially solubilized in DMSO at 100 mg/mL
and diluted to 1mg/mLusing water and stored frozen prior
to use. At the time of drug addition, an aliquot of frozen
concentrate (1 mg/mL) was diluted to 100, 200, 400, and
800 μg/mLwith complete medium containing test article.
Aliquots of 10 μL of these different drug dilutions were
added to the appropriate microtiter wells already contain-
ing 90 μL of medium, resulting in the required final drug
concentrations 10, 20, 40, and 80 μg/mL.After addition of
the compound, plates were incubated at standard condi-
tions for 48 h and assay was terminated by the addition of
cold TCA. Cells were fixed in situ by the gentle addition
of 50 μLof cold 30% (w/v) TCA(final concentration, 10%
TCA) and incubated for 60 min at 4°C. The supernatant
was discarded; the plates were washed five times with tap
water and air dried. Sulforhodamine B (SRB) solution
(50 μL) at 0.4% (w/v) in 1% acetic acid was added to
each of the wells, and plates were incubated for 20 min
at room temperature. After staining, unbound dye was
recovered and the residual dye was removed by washing
five times with 1% acetic acid. The plates were air dried.
Bound stain was subsequently eluted with 10 mM trizma
base, and the absorbance was read on a plate reader at a
wavelength of 540 nm with 690 nm reference wavelength.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(4-
nitrophenyl)-1,3,4-thiadiazol-2-yl]penta-2,4-dien-
amide (10). Brown crystals, yield 62%, mp 272°C. IR
spectrum, ν, cm–1: 1680 (amide CONH), 3100–3300
1
(amide NH). H NMR spectrum, δ, ppm: 5.19 d.d (J =
14.7, 0.8 Hz, 1H). 6.01 d (J = 2.3 Hz, 1H), 6.07 d (J =
2.3 Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H), 6.70–
6.82 m (2H), 6.82 d.d (J = 8.5, 1.9 Hz, 1H), 7.04 d (J =
1.8 Hz, 1H), 7.45 d.d (J = 14.6, 7.0 Hz, 1H), 8.16–8.24
m (2H), 8.29–8.40 m (3H). 13C NMR spectrum, δ, ppm:
100.99, 106.96, 108.54, 120.68, 123.68, 123.86, 126.01,
127.21, 130.40, 138.30, 139.45, 140.31, 147.05, 147.90,
149.37, 158.01, 163.17, 168.02. MS: m/z: 423.1164 [H]+.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(2-
hydroxyphenyl)-1,3,4-thiadiazol-2-yl]penta-2,4-
dienamide (11). Grey crystals, yield 64%, mp 286°C.
IR spectrum, ν, cm–1: 1690 (amide-CONH), 3100–3400
1
(amide NH). H NMR spectrum, δ, ppm: 5.19 d.d (J =
14.7, 0.8 Hz, 1H), 6.01 d (J = 2.3 Hz, 1H), 6.07 d (J = 2.3
Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H), 6.70–6.82
m (2H), 6.82 d.d (J = 8.5, 1.9 Hz, 1H), 6.88–7.03 m (2H),
7.04 d (J = 1.8 Hz, 1H), 7.30 d.d (J = 7.8, 1.2 Hz, 1H),
7.40 t.d (J = 7.7, 1.3 Hz, 1H), 7.46 d.d (J = 14.7, 7.1 Hz,
1H), 7.78 s (1H), 8.23 s (1H). 13C NMR spectrum, δ,
ppm: 100.99, 108.54, 116.66, 119.44, 120.68, 123.16,
123.68, 126.01, 127.61, 129.20, 130.40, 138.30, 140.31,
147.05, 147.90, 156.27, 157.41, 158.01, 168.02. MS:
m/z: 394.1161 [H]+.
Percent growth was calculated on a plate-by-plate
basis for test wells relative to control wells. Percent
growth was expressed as the ratio of average absorbance
of the test well to the average absorbance of the control
wells×100. Using the six absorbance measurements [time
zero (Tz), control growth (C), and test growth in the
presence of drug at the four concentration levels (Ti)];
the percentage growth was calculated at each of the drug
concentration levels. Percentage growth inhibition was
calculated as: [Ti/C]×100%.
(2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N-[5-(2-
nitrophenyl)-1,3,4-thiadiazol-2-yl]penta-2,4-dien-
amide (12). Colorless crystals, yield 64%, mp 284°C.
IR spectrum, ν, cm–1: 1665 (amide CONH), 3100–3400
1
(amide NH) H NMR spectrum, δ, ppm: 5.19 d.d (J =
14.7, 0.8 Hz, 1H), 6.01 d (J = 2.3 Hz, 1H), 6.07 d (J =
2.3 Hz, 1H), 6.66 d.d.d (J = 14.0, 7.0, 0.9 Hz, 1H),
6.70–6.82 m (2H), 6.82 d.d (J = 8.5, 1.9 Hz, 1H), 7.04
d (J = 1.8 Hz, 1H), 7.27 d.d.d (J = 7.9, 7.3, 1.3 Hz, 1H),
7.35–7.48 m (3H), 7.74 d.d (J = 7.9, 1.4 Hz, 1H), 8.26 s
(1H). 13C NMR spectrum, δ, ppm: 100.99, 106.96, 108.54,
120.68, 123.68, 126.01, 126.03, 127.59, 130.40, 130.82,
131.86, 132.39, 133.84, 138.30, 140.31, 147.05, 147.90,
158.01, 163.42, 168.02. MS: m/z: [H]+.
In silico docking. The possible docking modes be-
tween the piperine analog and cancer cell lines 3EU7
[23] for MCF-7 (Human breast cancer protein), 1Z8I [24]
for PC-3 cell line (Human prostate cancer) and 1XQH
[25] for HeLa cell line (Cervical cancer) was studied
using Autodock 2.0 software. The lead molecules were
designed using ChemDraw software and used to analyze
the binding affinity with the cancer proteins. Crystal
structure of all the cancer cell lines was downloaded from
SRB assay. The cell lines were grown in RPMI 1640
medium containing 10% fetal bovine serum and 2 mM
L-glutamine. Cells were inoculated into 96 well microtiter
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SYNTHESIS, IN VITRO AND IN SILICO ANTI-PROLIFERATIVE STUDIES
2305
Table 1. In vitro anti-proliferative activity of piperine deriva-
tives
Protein Data Bank website (http://www.rcsb.org) as PDB
format and converted to PDBQT format. We have taken
Lamarckian genetic algorithm (LGA) for ligand confor-
mations.An extended PDB format, termed as PDBQT file
was used for coordinate ligand and macromolecule which
includes atomic partial charges, polar bonds and hydrogen
bonds. An AutoDock tool was used for creating PDBQT
files from traditional PDB files.AutoDock was run several
times to get various docked conformations, and used to
analyze the predicted docking energy. AutoDock tools
provide various parameters to analyze the results of dock-
ing simulations such as binding energy, ligand efficiency,
inhibition constant and intramolecular energy. For each
ligand, ten best conformations were generated and scored
using AutoDock 4.2 scoring functions.
GI50a, μg/mL
Compound
MCF-7
28.2
>80
PC-3
50.9
64.3
48.0
>80
>80
78.1
>80
58.8
38.5
>80
41.8
>80
12.8
<10
HeLa
50.1
66.8
48.0
>80
>80
62.3
>80
60.1
33.4
>80
43.4
>80
14.5
<10
1
2
3
2.0
4
>80
5
18.6
27.4
23.2
36.7
12.9
>80
6
7
8
RESULTS AND DISCUSSION
9
10
All the novel molecules were characterized by FT-IR,
1H and 13C NMR, and Mass spectral data. Formation
of new compounds was confirmed by the amide bond
1H NMR characteristic singlet in the range of 9.15–
9.75 ppm, IR broad amide N–H stretching frequency band
at 3300–3500 cm–1 and amide C=O stretching frequency
band in the range of 1630–1680 cm–1.
11
39.8
>80
12
Piperine
Adriamycin
11.75
<10
a
(GI50) is a concentration of drug causing 50% inhibition of cell
growth.
In vitro anti-proliferative activity. Anti-proliferative
activity of newly synthesised piperine analogs 1–12 was
screenedagainstthreecelllinesMCF-7,PC-3,andHeLaus-
ing sulforhodamine B (SRB) colorimetric assay (Table 1).
The growth inhibitory concentration (GI50) values
were compared with the standard drugs Adriamycine.
All tested molecules demonstrated high to moderate
anti-proliferative activity. Among the entire series of
molecules piperine oxadiazole analog 3 exhibited very
high activity against MCF-7. The compounds 5 and 9
were also characterized by a noteworthy activity against
MCF-7 cancer cell lines. Remaining compounds showed
moderate activity against MCF-7, PC-3, and HeLa cell
lines. Some compounds 4, 10, and 12 were found to be
inactive against all cell lines. The anti proliferative activ-
ity of piperine analog 3 might be due to the presence of
the electron donating hydroxyl group on the benzene ring
which was characterized by strong hydrogen bonding with
protein molecule. Inactivity of some compounds could
be due to the presence of electron withdrawing groups
that could not form hydrogen bonds with the protein.
The accumulated data indicated that inhibition of cells
growth was concentration dependent. Docking studies
also singled out the derivative 3 as the most active with
the highest binding energy –15.23 kcal/mol.
In silico molecular docking. The synthesized piperine
analogs 1–12 were used as the objects of molecular dock-
ing studies. The targeted molecules were docked against
the cancer cell lines 3EU7 for MCF-7 (Human Breast
cancer protein), 1Z8I for PC-3 cell line (Human prostate
cancer) and 1XQH for HeLa cell line (Cervical cancer)
using Autodock 4.2 software (Table 2).
According to the in silico studies it was determined
that compound 3 could adopt a “U-shape” conformation
in the pocket of the 3EU7 protein molecule, and the
4-hydroxyphenyl group was surrounded by the residues
of Val-200, Try-300, Ala-278, and Phe-300, forming a
structure stabilized by H-bonding. The estimated binding
energy of the target molecule also showed strong binding
towards the protein moiety probably due to the presence
of the rigid molecule with amide linkage and hydroxyl
groups on heterocyclic moiety.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 89 No. 11 2019

2306
AMPERAYANI, PARIMI
Table 2. Binding energies between the representative piperine derivatives 1–4, 7, 12, and MCF-7, PC3, and HeLa
Binding energy, kcal/mol
Compound no.
Structure of compounds
MCF-7
PC-3
–9.56
HeLa
1
–13.54
–13.87
–15.23
–12.92
–12.92
–13.22
O
O
O
O
NH
NH
NH
NH
NH
NH
N N
2
3
–9.83
O
O
O
O
O
Cl
O
O
O
N N
–10.83
OH
NO
O
O
O
N N
4
7
–13.63
–14.06
–13.88
–9.13
–9.08
–5.83
–13.14
–13.25
–13.68
2
O
O
O
N N
O
O
S
N N
Cl
12
O
O
S
N N
CONCLUSIONS
ACKNOWLEDGMENTS
We also thank for in vitro SRB assay for the anti-
cancer activity, at Anti-Cancer Drug screening facil-
ity (ACDSF), ACTREC, Tata Memorial Centre, Navi
Mumbai on hiring basis.
A novel series of piperine analogs with oxadiazoles
and thiadiazoles is synthesized by coupling the natural
product piperine with amine substituted oxadiazoles
and thiadiazoles. The products have been tested for their
anti proliferative activity against three cell lines MCF-7,
PC-3, and HeLa. Among the entire synthesized analogs
piperine derivative 3 with oxadiazole baring the hydroxyl
group has been determined to be a potent anticancer
agent against MCF-7 cell line with binding energy of
–15.23 kcal/mol. All the remaining compounds are mod-
erately active against the tested cell lines. The results are
in accord with the in silico studies.
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