Synthesis, anticancer and antibacterial activities of piperine analogs

Article abstract of DOI:10.1007/s00044-013-0541-4

A series of piperine analogs were synthesized by the condensation of piperic acid with different aminoacids and substituted aniline. The synthesized compounds (4a-4e) were evaluated for their anticancer activity against human cancer cell lines (MCF-7, Breast Cancer cell line, and Hela cervix cell line) and antibacterial activity against human pathogens (Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Shigella dysenteriae, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa). The efficacies of the synthesized compounds were superior to those of piperine in all tested human cancer cell lines. Among the tested conjugates, 4c showed significant anticancer activity against Hela cervix cell line with IC₅₀ of 0.736 μmol and 4a showed significant activity against breast cancer cell line. The antibacterial activity of the tested compounds was also found to be superior to that of piperine. The approach is novel as the abundantly available natural product piperine is utilized as precursor for the synthesis of new potential antimicrobial and anticancer agents.

Full text of DOI:10.1007/s00044-013-0541-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0541-4
ORIGINAL RESEARCH
Synthesis, anticancer and antibacterial activities of piperine
analogs
•
Parimi Umadevi Kolli Deepti Durvasula V. R. Venugopal
•
Received: 10 November 2012 / Accepted: 15 February 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of piperine analogs were synthesized
by the condensation of piperic acid with different amino-
acids and substituted aniline. The synthesized compounds
(4a–4e) were evaluated for their anticancer activity against
human cancer cell lines (MCF-7, Breast Cancer cell line,
and Hela cervix cell line) and antibacterial activity against
human pathogens (Escherichia coli, Klebsiella pneumo-
niae, Salmonella typhi, Shigella dysenteriae, Bacillus
subtilis, Staphylococcus aureus, Pseudomonas aerugin-
osa). The efficacies of the synthesized compounds were
superior to those of piperine in all tested human cancer cell
lines. Among the tested conjugates, 4c showed significant
anticancer activity against Hela cervix cell line with IC50 of
0.736 lmol and 4a showed significant activity against
breast cancer cell line. The antibacterial activity of the
tested compounds was also found to be superior to that of
piperine. The approach is novel as the abundantly available
natural product piperine is utilized as precursor for the
synthesis of new potential antimicrobial and anticancer
agents.
synthetic drugs made a comeback of herbal medicine to
improve the fulfillment of our health needs (Harun-
ur-rashid et al., 2002). Plants have a special place in the
treatment of cancer. It is estimated that plant-derived
compounds constitute more than 50 % of anticancer
agents, about 74 % of anticancer compounds being either
natural or natural product-derived products (Newman
et al., 2003). Due to an alarming increase in the rate of
infections with antibiotic resistant microorganism and due
to side effects (Yadav et al., 2011) of some synthetic
antibiotics, there is an increasing interest in medicinal
plants as a natural alternative to synthetic drugs (Deepa
et al., 2012).
Piperine, an alkaloid and hydrophobic amide, is a major
constituent of Piper nigrum Linn. (Black pepper, Pipera-
ceae family). Traditionally, pepper has been used for many
ailments. Piperine exhibited a wide spectrum of biological
and pharmacological activities (Mujumdar et al., 1990;
Birkmayer et al., 1985; Kong et al., 2004; Pradeep and
Kuttan, 2002; Sunila and Kuttan, 2004; Bezerra et al.,
2006; Selvendran et al., 2005) like anti-pyretic (Parmar
et al., 1997), antimetastatic (Pradeep and Kuttan, 2002),
antithyroid (Panda and Kar, 2003), antidepressant (Lee
et al., 2005), toxic effect against hepatocytes (Koul and
Kapil, 1993), antiapoptotic efficacy (Choi et al., 2007),
high immunomodulatory and antitumor activity (Sunila
and Kuttan, 2004).
Keywords Piper nigrum Á Piperine Á Piperic acid Á
Amino acid conjugates Á Anticancer activity Á
Antibacterial activity
Introduction
Irrespective of the potentiality of piperine, not much
work has been carried out to study the efficacy of piperine
analogs as anticancer and antimicrobial agents. The newly
synthesized analogs of piperine increased the potentiality
of the natural compound piperine, and it emerged as a new
approach in the discovery of new cytotoxic and antimi-
crobial agents. The anticancer activity of piperine lies in
the amide linkage (Matsuda et al., 2009); we made an
Cancer is one of the most serious threats against human
health in the world. The toxic and adverse side effects of
P. Umadevi (&) Á K. Deepti Á D. V. R. Venugopal
Department of Chemistry, GIS, GITAM University,
Visakhapatnam 530045, AP, India
e-mail: umadevi_parimi@gitam.edu
123

Med Chem Res
effort to replace the piperidine nucleus with different
amino acids and substituted aniline. The present study
deals with the synthesis of conjugated piperine derivatives,
and they are characterized by IR, 1H NMR, 13C NMR, and
MS spectral data. The biological activity of the synthesized
compounds was evaluated in comparison with the parent
compound piperine.
mixture, and the organic layer is separated and washed
with water (2 9 25 ml), dried over anhydrous sodium
sulfate and concentrated to give crude product. The crude
was column chromatographed with hexane:ethylacetate
(4:2) to get the pure compounds (4a–4e).
Determination of invitro anticancer activity
Cell lines and cell culture
Experimental
The human cancer cell lines were maintained in Dul-
becco’s modified essential medium (DMEM) supple-
mented with 4.5 g/l glucose, 2 mM L-glutamine, and 5 %
fetal bovine serum (FBS) (growth medium) at 37 °C in
5 % CO2 incubator.
The solvents were purified by distillation and used freshly.
Column chromatography was carried out using silica gel
100–200 mesh, and precoated silica gel plates (Merck, 60
F254) were used for TLC. All the chemicals and reagents
were purchased from MERCK, India. All the chemicals are
of analytical grade.
MTT assay
Extraction and Isolation of Piperine
The MTT assay developed by Mosmann (1983) was modi-
fied and used to determine the inhibitory effects of test
compounds on cell growth in vitro. The trypsinized cells
from T-25 flask were seeded in each well of 96-well flat-
bottomed tissue culture plate at a density of 5 9 103 cells/
well in growth medium and cultured at 37 °C in 5 % CO2 to
adhere. After 48 h incubation, the supernatant was discarded
and the cells were pretreated with growth medium and were
subsequently mixed with different concentrations of test
compounds (32, 64, 128, 256, and 500 lg/ml) in triplicates to
achieve a final volume of 100 ll and then cultured for 48 h.
The compound was prepared as 1.0 mg/ml concentration
stock solutions in PBS. Culture medium and solvent are used
as controls. Each well then received 5 ll of fresh MTT
(0.5 mg/ml in PBS) followed by incubation for 2 h at 37 °C.
The supernatant growth medium was removed from the
wells and replaced with 100 ll of DMSO to solubilize
the colored formazan product. After 30 min incubation, the
absorbance (OD) of the culture plate was read at a wave-
length of 570 nm on an ELISA reader, Anthos 2020 spec-
trophotometer. The IC50 values of the test samples were
calculated and tabulated in Table 1.
100 g of ground black pepper powder is taken in a 250 ml
round bottomed flask, and 500 ml of 95 % ethanol is added
and refluxed for 2 h. The mixture is filtered and concen-
trated using a rotatory evaporator. The concentrated pepper
extract is added to 10 ml of 10 % ethanolic KOH solution.
The resulting solution is heated and water is added drop
wise. Yellow precipitate separates out, allowing the mix-
ture to stand overnight. The solid precipitate is filtered and
recrystallized with 10–20 ml of acetone to obtain 3 g of
piperine (1).
Synthesis of piperic acid (2) from piperine (1) (Scheme 1)
Piperine (5 g, 17.5 mmol) was refluxed with methanolic
KOH (2 N, 20 ml) for 6 h, the methanol was evaporated
under reduced pressure and the solution was cooled in ice
bath. The gummy potassium salt of piperic acid was sus-
pended in water and gradually acidified with dil. HCl, dark
yellow precipitate was collected and stirred for 3–4 h in ice
cold bath, filtered and washed with cold water. The com-
pound was recrystallized from methanol yielding a yellow
crystalline compound with 80 % yield.
Determination of antibacterial activity
Synthesis of piperic acid amides (4a–4e) (Scheme 1)
The antimicrobial activities of the synthesized compounds
were tested against Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Bacillus subtilis, Salmonella
typhi, Shigella dysenteriae, andStaphylococcus aureus.
2.0 ml of freshly distilled thionyl chloride is added to
piperic acid (2.18 g, 10 mmol) in dry dichloromethane
(DCM) and the contents are refluxed for 1 h. Excess of
thionyl chloride is removed on rotator evaporator under
reduced pressure to get acid chloride of piperine.
Minimum inhibitory concentration (MIC)
10 mmol of amine in 20 ml of DCM is added to
10 mmol of acid chloride in DCM (20 ml); the contents are
stirred for 1 h. 50 ml of water is added to the reaction
Minimum inhibitory concentration was determined by
serial dilution method (Stokes, 1975). Two fold serial
dilutions of test compounds were carried out in nutrient
123

Med Chem Res
Scheme 1 Synthesis of piperic acid
and piperine analogs from piperine
OH
N
ethanolic KOH, Reflux
Hydrolysis, Con. HCl
O
O
O
O
O
O
Piperic Acid (2)
Piperine (1)
SOCl2
Reflux
Cl
O
O
O
O
O
R-H
O
R
CH2Cl2
Stirring, 1h, RT
Piperic Acid chloride (3)
4a-4e
R
4a-
Histidine
4b- Phenylalanine
4c- Tryptophan
4d- p-ChloroAniline
4e- p- BromoAniline
2-[[(2E,4E)-5-(1,3-benzodioxol-5-yl)penta-2,4-dienoyl]amino]-3-(1H-imidazol-4-yl)
propanoic acid (4a):
O
O
N
O
O
O
N
N
2-[[(2E, 4E)-5-(1,3-benzodioxol-5-yl)penta-2,4-dienoyl]amino]-3-phenyl-propanoic acid
(4b):
O
O
N
O
O
O
2-[[(2E,4E)-5-(1,3-benzodioxol-5-yl)penta-2,4-dienoyl]amino]-3-(1H-indol-3-
yl)propanoic acid (4c):
O
O
N
O
O
O
N
broth. To each test tube, 105 CFU/ml of active bacterial
cultures was inoculated. Ampicillin was used as reference
drug. The culture tubes were incubated at 37 °C for 24 h.
After the incubation, the tubes were checked for bacterial
growth and MIC was determined and expressed in lg/ml.
The results are tabulated in Table 2.
123

Med Chem Res
Scheme 1 continued
(2E,4E)-5-(1,3-benzodioxol-5-yl)-N-(4-chlorophenyl)penta-2,4-dienamide (4d):
O
O
N
O
Cl
(2E,4E)-5-(1,3-benzodioxol-5-yl)-N-(4-bromophenyl)penta-2,4-dienamide (4e):
O
O
N
O
Br
Table 1 Determination of anticancer activity of piperine and its analogs given as IC50 in lmol
Cell line
Piperine
(1)
Piperine–His
(4a)
Piperine–Phe
(4b)
Piperine–Try
(4c)
Piperine–Cl–aniline
(4d)
Piperine–Br–aniline
(4e)
Hela cervix cell line
Breast cancer cell line
0.95
0.99
1.052
0.74
0.87
0.96
0.736
0.87
0.89
1.056
0.93
1.089
Table 2 Determination of minimum inhibitory concentration (MIC) of piperine and its analogs (lg/ml)
Bacteria
Piperine
(1)
Piperine–His
(4a)
Piperine–Phe
(4b)
Piperine–Try
(4c)
Piperine–
Cl–aniline (4d)
Piperine–
Br–aniline (4e)
Ampicillin
E. coli
300
200
200
600
500
300
100
200
300
300
200
300
300
300
300
300
200
150
400
400
400
400
200
200
300
400
300
200
100
300
400
200
500
200
200
200
100
300
300
500
300
500
5
8
Klebsiella pneumoniae
Salmonella typhi
15
16
10
11
50
Shigella dysentriae
Bacillus subtilis
Staphylococcus aureus
Pseudomonas aeruginosa
Spectral data of synthesized compounds (4a–4e)
10.96 (s, 1H), 8.42 (s, 1H), 8.14 (d, J = 2.0 Hz, 1H),
7.15–7.02 (m, 3H), 6.94 (d, J = 7.5 Hz, 1H), 6.85 (dt,
J = 15.0, 0.9 Hz, 1H), 6.71 (d, J = 15.1 Hz, 1H), 6.06 (s,
2H), 5.48 (d, J = 15.0 Hz, 1H), 4.83 (t, J = 5.4 Hz, 1H),
3.03 (dd, J = 12.4, 5.4 Hz, 1H), 2.81 (dd, J = 12.4, 5.4 Hz,
1H); 13C NMR (100 MHz, CDCl3): d 106.3, 131, 122.5,
108.5, 148.2, 147.7, 174.6, 165.1, 142.8, 138.2, 134.9, 132.4,
126.4, 122.8, 122.5, 117.9, 101.3, 51.9, 29.12; m/z 355.12.
IR spectra were recorded on Nicolet-740 spectrometer with
KBr Pellets. The 1H and 13C NMR spectra were recorded on a
1
Bruker FT-400 MHz spectrometer at 400 MHz for H and
100 MHz for 13C, respectively, using TMS as internal stan-
dard. Mass spectra were measured on LC–MS instrument.
2-[[(2E,4E)-5-(1,3-benzodioxol-5-yl)penta-2,4-
dienoyl]amino]-3-(1H-imidazol-4-yl)propanoic acid (4a)
2-[[(2E, 4E)-5-(1,3-benzodioxol-5-yl)penta-2,4-
dienoyl]amino]-3-phenyl-propanoic acid (4b)
It is a colorless solid, molecular formula: C18H17N3O5; m.p.
140–142 °C; IR (KBr) tmax: 3550, 1640, 1600, 1515, 1500,
1453, 1374; H NMR (400 MHz, CDCl3): d 13.16 (s, 1H),
It is a pale white colored solid, molecular formula:
1
C21H19NO5; m.p. 165–167 °C; IR (KBr) tmax: 3600, 2737,
123

Med Chem Res
1670, 1628, 1623, 1600, 1557, 1515, 1500, 1306, 1225,
1074, 1025, 913, 849, 745, 698; 1H NMR (400 MHz,
CDCl3): d 10.97 (s, 1H), 8.42 (s, 1H), 7.20 (dd, J = 3.6,
1.9 Hz, 5H), 7.15–6.90 (m, 4H), 6.86–6.77 (m, 1H), 6.47
(d, J = 15.2 Hz, 1H), 6.06 (s, 2H), 4.94 (d, J = 15.2 Hz,
1H), 4.73 (t, J = 6.0 Hz, 1H), 3.30 (dd, J = 12.4, 6.1 Hz,
1H), 3.05 (dd, J = 12.4, 6.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d 106.3, 131, 122.5, 108.5, 148.2, 147.7, 101.3,
138.2, 126.4, 142.8, 122.8, 165.1, 135.1, 127.0, 128.8, 129.4,
55.5, 175.0, 37.8; m/z 365.13.
148.2, 147.7, 164.9, 142.3, 138.2, 136.6, 132.4, 126.4,
122.7, 122.3, 119.4, 101.3; m/z 371.02.
Results and discussion
Combining different active molecules present in nature,
results in enhancement of their bioavailability. These mol-
ecules contain the unique properties like chirality, hydro-
philicity/hydrophobicity, and optical properties (Frank-Frut
and Krishnan, 2003; Thiantanwat et al., 2000). Piperine (1),
obtained from the extraction of dried seeds of Piper nigrum,
was converted into acid by hydrolysis using ethanolic KOH
and Con. HCl. The acid is converted into acid chloride using
thionyl chloride which provides an electron-deficient acid
carbonyl center followed by condensation with amine in
dichloromethane. The piperidine amide of natural piperine
was replaced by different aminoacids and substituted aniline
to obtain compounds 4a–4e. All the tested compounds
(4a–4e) were characterized by 1H, 13C NMR, IR, and LCMS
analysis. The IR peaks at 1,630–1,690 cm-1 for all the
synthesized compounds arise due to the t C=O and those at
3,500–3,700 cm-1 are due to the t N–H.
The cytotoxic activity was carried out by MTT assay
and the results were summarized in Table 1. The synthe-
sized compounds (4a–4e) were screened for their cytotoxic
activity against Hela and Breast cancer cell lines. The IC50
value for piperine is 0.95 and 0.99 lmol against hela and
breast cancer cell lines, respectively. Compound 4c, a
tryptophan analog of piperine which possesses heterocyclic
aromatic ring, showed highest growth inhibition against
hela cell lines (IC50—0.736 lmol) and breast cancer cell
lines (IC50—0.87 lmol). Compound 4a, a histidine analog
of piperine containing imidazole ring structure, showed
highest cytotoxic activity against breast cancer cell line
(IC50—0.74 lmol). From the data, we summarize that the
activity of all the synthesized compounds was found to be
better than that of piperine. The antibacterial assay was
carried out by serial dilution method, and the MIC values
were recorded and summarized in Table 2. Among all the
tested compounds, compound 4e containing p-bromoaniline
nucleus was found to be the best against K. pneumoniae.
Compound 4b containing phenyl alanine skeleton was active
against S. dysenteriae compared with all other tested com-
pounds. The MIC values of most of all the tested compounds
were as good as or even better than piperine.
2-[[(2E,4E)-5-(1,3-benzodioxol-5-yl)penta-2,4-
dienoyl]amino]-3-(1H-indol-3-yl)propanoic acid (4c)
It is a colorless solid, molecular formula: C23H20N2O5;
m. p. 135–137 °C; IR (KBr) tmax: 1660, 1675, 700, 500,
1300–700 (indole ring), 1230, 1160, 1115, 1045, 770,
1
1375, 3410, 3650, 1680, 1628, 1600, 1515, and 1500; H
NMR (400 MHz, CDCl3): d 11.02 (s, 1H), 8.42 (s, 1H),
7.72 (s, 1H), 7.58 (dd, J = 7.5, 1.7 Hz, 1H), 7.36–7.29 (m,
1H), 7.20 (s, 1H), 7.15–6.86 (m, 6H), 6.72 (dt, J = 15.1,
1.1 Hz, 1H), 6.53 (d, J = 15.2 Hz, 1H), 6.06 (s, 2H), 5.31
(d, J = 15.2 Hz, 1H), 4.85 (t, J = 5.1 Hz, 1H), 3.38 (dd,
J = 12.4, 5.0 Hz, 1H), 3.12 (dd, J = 12.3, 5.1 Hz, 1H);
13C NMR (100 MHz, CDCl3): d 106.3, 112.0, 121.3, 119.6,
118.1, 124.7, 136.3, 127.9, 110.9, 28.2, 174.6, 52.5, 165.1,
122.8, 142.8, 126.4, 138.2, 101.3, 147.7, 148.2, 108.5,
122.5, 131.6; m/z 404.14.
(2E,4E)-5-(1,3-benzodioxol-5-yl)-N-(4-
chlorophenyl)penta-2,4-dienamide (4d)
It is a colorless solid, molecular formula: C18H14ClNO3;
m. p. 139–141 °C; IR (KBr) tmax: 3600, 1618, 1495, 1281,
1174, 1082, 1005, 821, 630, 500, 1670, 1628, 1685, 1600,
1
1515, 1500; H NMR (400 MHz, CDCl3): d 9.52 (s, 1H),
7.89–7.81 (m, 2H), 7.44–7.36 (m, 2H), 7.15–7.02 (m, 3H),
6.97–6.82 (m, 2H), 6.68 (d, J = 15.0 Hz, 1H), 6.06 (s, 2H),
5.27 (d, J = 15.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): d
106.3, 131.0, 122.52, 108.5, 148.2, 147.7, 101.3, 138.2, 126.4,
142.3, 122.3, 164.9, 137.0, 128.4, 127.8, 122.0; m/z 327.07.
(2E,4E)-5-(1,3-benzodioxol-5-yl)-N-(4-
bromophenyl)penta-2,4-dienamide (4e)
It is a pale white color solid, molecular formula:
C18H14BrNO3; m.p. 145–148 °C; IR (KBr) tmax: 3565,
1618, 1495, 1291, 1164, 1082, 1005, 821, 630, 500, 1650,
1628, 1670, 1600, 1515, 1500; 1H NMR (400 MHz,
CDCl3): d 9.52 (s, 1H), 7.54–7.46 (m, 2H), 7.42–7.34 (m,
2H), 7.15–7.02 (m, 2H), 6.94 (d, J = 7.5 Hz, 1H),
6.85–6.70 (m, 3H), 6.06 (s, 2H), 5.40 (d, J = 14.9 Hz, 1H);
13C NMR (100 MHz, CDCl3): d 106.3, 131.0, 122.5, 108.5,
Conclusion
We conclude that the synthetic analogs of piperine were
superior to piperine with respect to both cytotoxic and
antibacterial efficacy.
123

Med Chem Res
Acknowledgments The authors acknowledge TRIMS labs Pvt.
Ltd., Visakhapatnam for providing them with proper facilities to carry
out anticancer studies.
Matsuda H, Ninomiya K, Morikawa T, Yasuda D, Masayuki I,
Yoshikawa M (2009) Hepatoprotective amide constituents from
the fruit of Piper chaba, structural requirements, mode of action
and new amides. Bioorg Med Chem 17:7313–7323
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55
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