Synthesis of piperazine based N-Mannich bases of berberine and their antioxidant and anticancer evaluations

Article abstract of DOI:10.1007/s13738-015-0762-1

Isoquinoline alkaloids possess versatile biological activities. Hence, in the current research an effort has been made to improve structurally important part of isoquinoline alkaloid berberine because it is recognized as the marking compound for the crude drugs. Synthesis of rationalized berberine has been performed via substituting various piperazine moieties bearing disubstituted electron withdrawing and electron donating groups to the berberine core via Mannich reaction. Intended scaffolds were inspected for their in vitro antioxidant potential using different bioassays, FRAP, DPPH and ABTS as well as anticancer efficacies against cervical cancer cell lines HeLa, CaSki adapting SRB assay. Also, an inspection of the cytotoxic nature of titled analogues has been carried out towards Madin-Darby canine kidney (MDCK) cell lines. Radical scavenging potential of the final derivatives 4a-i was found to be excellent with IC_50s, <20 and <12 μg/mL in DPPH and ABTS assay, respectively, whereas some dichlorophenyl piperazine analogues revealed important Fe³⁺ decreasing power with absorption at around 2 nm in FRAP assay. Moreover, compounds 4a-i appeared with significant inhibitors of the cervical cancer cell lines HeLa and CaSki with IC_50s ranging 4.346-6.321 and 3.408-6.081 μg/mL, with low level of cytotoxic values and higher therapeutic indices ranging 20.42-42.45 and 21.23-43.25, respectively. Therefore, from the bioassay results it can be mentioned that these analogues are effective double agents as the scavengers of reactive oxygen species and inhibitors of the cancerous cells. The correct structure of the final compounds was adequately confirmed on the basis of FT-IR, ¹H NMR and mass spectroscopy data as well as elemental analyses.

Full text of DOI:10.1007/s13738-015-0762-1

J IRAN CHEM SOC
DOI 10.1007/s13738-015-0762-1
ORIGINAL PAPER
Synthesis of piperazine based N‑Mannich bases of berberine
and their antioxidant and anticancer evaluations
1
1
1
1
Bhupendra Mistry · Young‑Soo Keum · Rafi Noorzai · Enkhtaivan Gansukh ·
Doo Hwan Kim
1
Received: 7 May 2015 / Accepted: 16 October 2015
©
Iranian Chemical Society 2015
Abstract Isoquinoline alkaloids possess versatile biologi-
cal activities. Hence, in the current research an effort has
been made to improve structurally important part of iso-
quinoline alkaloid berberine because it is recognized as the
marking compound for the crude drugs. Synthesis of ration-
alized berberine has been performed via substituting vari-
ous piperazine moieties bearing disubstituted electron with-
drawing and electron donating groups to the berberine core
via Mannich reaction. Intended scaffolds were inspected
for their in vitro antioxidant potential using different bioas-
says, FRAP, DPPH and ABTS as well as anticancer effica-
cies against cervical cancer cell lines HeLa, CaSki adapting
SRB assay. Also, an inspection of the cytotoxic nature of
titled analogues has been carried out towards Madin-Darby
canine kidney (MDCK) cell lines. Radical scavenging
potential of the final derivatives 4a–i was found to be excel-
lent with IC50s, <20 and <12 µg/mL in DPPH and ABTS
assay, respectively, whereas some dichlorophenyl pipera-
correct structure of the final compounds was adequately
confirmed on the basis of FT-IR, H NMR and mass spec-
troscopy data as well as elemental analyses.
1
Keywords Berberine · Mannich bases · Piperazine ·
Antioxidant · Cervical cancer
Introduction
Free radicals are atoms or compounds that hold an unpaired
electron and are extremely reactive, able of eliciting a fast
change response that destabilizes other elements and pro-
duces many more free radicals. Scientific evidence suggests
that antioxidants prevent chronic diseases including cancer
and cardiovascular disease [1–5]. Among the chronic dis-
eases, cancer is the most common epidemic all over the
world. Cancers figure among the major reasons of fatalities
and death rate globally, with approximately 14 million new
cases and 8.2 million cancer-related fatalities in 2012 and
the number of new cases is predicted to increase by about
70 % over the next two decades [6, 7]. Natural products,
like plant extract, either as pure compounds or as consistent
extracts, provide unlimited possibilities for new drug find-
ings due to the unrivaled chemical variety the can offer [8].
Various researches suggest that therapeutic plants contain
substances like proteins, unsaturated long chain fatty acids,
aldehydes, alkaloids, essential oils, phenols and water or
soluble ethanol compounds. These compounds are signifi-
cant in therapeutic application against human and animal
pathogens, including bacteria, fungi and viruses [9, 10].
In the plant, alkaloids are available in the free state, as salt
or as amine or alkaloid N-oxides [11]. They are identified
based on the amino acids that provide their nitrogen atom
zine analogues revealed important Fe3 decreasing power
with absorption at around 2 nm in FRAP assay. Moreover,
compounds 4a–i appeared with significant inhibitors of the
cervical cancer cell lines HeLa and CaSki with IC50s rang-
ing 4.346–6.321 and 3.408–6.081 µg/mL, with low level
of cytotoxic values and higher therapeutic indices ranging
+
20.42–42.45 and 21.23–43.25, respectively. Therefore, from
the bioassay results it can be mentioned that these analogues
are effective double agents as the scavengers of reactive
oxygen species and inhibitors of the cancerous cells. The
*
Bhupendra Mistry
bhupendra.mistry84@gmail.com
1
Organic Research Laboratory, Department of Bioresources
and Food Sciences, College of Life and Environmental
Sciences, Konkuk University, Seoul, South Korea
1
3

J IRAN CHEM SOC
and aspect of their skeleton and normally easily separable
from the other metabolites due to their basicity [12].
TLC was carried out using appropriate mobile phase sys-
tems and silica gel-G coated microscopic glass slides
(2 × 7.5 cm), and TLC spots were observed in UV light
Berberine is an isoquinoline alkaloid, with a bright yel-
low color, that is simply seen in most of the natural herb
components, which contain any significant amount. Ber-
berine is primary alkaloid from origins and stem-bark of
Berberis varieties. It is produced mainly from roots of B.
aristata (5 % in roots and 4.2 % in stem-bark), B. Petiola-
ris (0.43 %), B.vulgaris, B. aquifolium, B. thunbergii and
B. asiatica [13–15], C. teeta (rhizome 8–9 %) and Hydras-
tis Canadensis [16]. Berberine has been associated with
tremendous free-radical scavenging potential as an anti-
oxidant agent as reported by numerous research studies
−1
chamber. FT-IR bands were presented in cm as well as
1
H NMR spectral results were furnished in ppm downfield
from TMS with s, singlet; d, doublet; m, multiplet and br s,
broad singlet patterns. Elemental analyses (C, H, N) were
performed using a Heraeus Carlo Erba 1180 CHN analyzer.
General procedure for the synthesis of berberrubine (2)
Berberine hydrochloride (10 g, 0.01 mol) was added to a
50 mL round bottom flask. The reaction system was kept
under reduced pressure (20–30 mmHg) using an oil pump,
and heated to 190 °C followed by reacting for 40 min.
The vacuum pump was switched off after the tempera-
ture dropped to room temperature. The reaction product
was purified through silica gel column chromatography
(CHCl /CH OH:15:1 and 10:1, eluting until no compound
[
17–19]. Also, derivatization of berberine core structure has
resulted in the development of molecules with positively
enhanced biological effects [20–23]. Moreover, berberine
and its derivatives are associated with excellent cancerous
cell inhibitors effects [24–26]. In the design of new drugs,
the improvement of several components using the mixture
of various pharmacophores in one framework may cause
substances with exciting healing properties. Researchers
have recently reported the tremendous potential of pipera-
zine derivatives associated with different biological effica-
cies [27]. Moreover, substitution of piperazine derivatives
to the base nucleus bearing nitrogen ring has been found
to exert significant anticancer effects [28]. Also, piperazine
derivatization would lead to the development of scaffolds
furnishing enormous antioxidant efficacies [29]. Accord-
ing to the extensive variety of biological activities related
with berberine and the piperazine, the fusion of these two
moieties in the same compound is an exciting challenge
for the growth of new pharmacologically effective agents.
Derivatization has been conducted via developing N-Man-
nich bases as the biological results of this scaffold is well
known, particularly as anti-oxidant [30–32] and anticancer
providers [33, 34]. N-Mannich bases prepared with pipera-
zine linkage was recently tried and adapted in the existing
research for the efficient synthesis of berberine-piperazine
systems [35].
3
3
was observed in the eluent) to obtain a brownish red amor-
phous powder compound 2 (6.6 g, 85 %).
General procedure for the preparation of Mannich base
derivatives (4a–i)
Compound 2 (0.5 g, 0.01 mol) was dissolved in 25.0 ml
of the anhydrous ethanol, various piperazine derivatives
(0.015 mol) and aqueous formaldehyde solution (37 %,
0.05 mol) were added, stirred at room temperature or 80 °C
for 24 h. Concentrated under reduced pressure to give the
crude product, which was purified using flash silica gel col-
umn chromatography (CH Cl /MeOH = 20:1) to give final
2
2
compound 4a.
12‑(1‑(2,3‑Dichlorophenyl)
piperazine‑1‑ylmethyl)‑berberrubine (4a)
Light yellow solid, yield: 58 %. m.p. 241–243 °C. IR
−1
(KBr) cm : 3620 (–OH), 3027 (C–H, Ar), 1622–1547
1
(
C=C, Ar), 1261 (Ar–N), 768 (C–Cl). H NMR (400 MHz,
chloroform) δ 9.87 (s, 1H, –OH of berberine ring), 9.31
Experimental section
Materials and methods
(s, 1H, H-8), 7.78 (s, 1H, H-13), 7.51 (s, 1H, H-1), 7.15
(s, 1H, H-4), 6.93–6.75 (m, 3H, Ar–H, piperazine H-22,
H-23, H-24), 6.13 (s, 2H, –OCH O), 6.05 (s, 1H, H-11),
2
5
.95 (t, J = 5.4 Hz, 2H, H-6), 3.99 (s, 2H, CH , H-14
2
Highest quality chemicals and reagents were used in this
study without prior purification. Veego Open capillary
electronic apparatus VMP-D was utilized to obtain melt-
ing points of the synthesized compounds that were uncor-
rected. Bruker FT-IR spectrophotometer (KBr pellets) and
Mannich base), 3.93 (s, 3H, OCH ), 3.80 (br s, 4H, pip-
3
erazine, H-18, H-20), 3.49 (br s, 4H, piperazine, H-15,
1
3
H-17), 3.15 (t, J = 5.3 Hz, 2H, H-5). C NMR (DMSO,
400 MHz): δ 155.5, 152.2, 149.6, 147.4, 145.2, 142.3,
137.1, 134.3, 130.8, 128.1, 126.5, 124.7, 122.5, 121.4,
119.8, 118.6, 108.5, 105.7, 103.8, 101.2, 99.1, 80.8, 69.7,
Varian 400 MHz model spectrometer (CDCl and DMSO
3
+
as a solvent and TMS as internal standard) were used to
55.9, 52.1, 50.6, 26.8. HRMS m/z [M-Cl] calcd for
1
+
obtain FT-IR and H NMR spectra of the title compounds.
[C H Cl N O ] : 564.15; found: 564.20. Anal. calcd
3
0
28
2
3
4
1
3

J IRAN CHEM SOC
for C H Cl N O : C, 59.96; H, 4.70; N, 6.99. Found: C,
1H, –OH of berberine ring), 9.33 (s, 1H, H-8), 7.85 (s, 1H,
H-13), 7.56 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.98–6.78 (m,
3
0
28
3
3
4
5
9.84; H, 4.56; N, 6.87.
3
H, Ar–H, piperazine H-22, H-23, H-25), 6.09 (s, 2H, –
1
2‑(1‑(2,4‑Difluorophenyl)
OCH O), 6.01 (s, 1H, H-11), 5.93 (t, J = 5.4 Hz, 2H, H-6),
2
piperazine‑1‑ylmethyl)‑berberrubine (4b)
4.03 (s, 2H, CH , H-14 Mannich base), 3.95 (s, 3H, OCH ),
2
3
3
.86 (br s, 4H, piperazine, H-18, H-20), 3.48 (br s, 4H, pip-
Light yellow solid, yield: 61 %. m.p. 229–231 °C. IR
erazine, H-15, H-17), 3.24 (t, J = 5.4 Hz, 2H, H-5), 2.30 (s,
−
1
(
(
KBr) cm : 3632 (OH), 3041 (C–H, Ar), 1618–1558
C=C, Ar), 1252 (Ar–N). H NMR (400 MHz, chloro-
3H, Ar-CH of piperazine), 2.04 (s, 3H, Ar-CH of pipera-
3
3
1
13
zine). C NMR (DMSO, 400 MHz): δ 155.9, 152.4, 149.3,
147.6, 145.5, 142.5, 137.7, 134.2, 130.4, 128.6, 126.7,
124.8, 122.3, 121.6, 119.7, 118.7, 108.4, 105.0, 103.7,
101.3, 99.7, 80.4, 69.6, 55.8, 52.5, 50.1, 26.0, 22.4, 19.7.
form) δ 9.78 (s, 1H, –OH of berberine ring), 9.28 (s, 1H,
H-8), 7.71 (s, 1H, H-13), 7.47 (s, 1H, H-1), 7.13 (s, 1H,
H-4), 6.88–6.72 (m, 3H, Ar–H, piperazine H-22, H-23,
+ +
HRMS m/z [M-Cl] calcd for [C H N O ] : 524.25;
32 34 3 4
H-25), 6.17 (s, 2H, –OCH O), 6.08 (s, 1H, H-11), 5.91
2
(
t, J = 5.5 Hz, 2H, H-6), 3.98 (s, 2H, CH , H-14 Man-
found: 524.40. Anal. calcd for C H ClN O : C, 68.62; H,
6.12; N, 7.50. Found: C, 68.76; H, 6.26; N, 7.33.
2
32 34 3 4
nich base), 3.94 (s, 3H, OCH ), 3.83 (br s, 4H, pip-
3
erazine, H-18, H-20), 3.44 (br s, 4H, piperazine, H-15,
1
3
H-17), 3.18 (t, J = 5.5 Hz, 2H, H-5). C NMR (DMSO,
12‑(1‑(2,3‑Difluorophenyl)
piperazine‑1‑ylmethyl)‑berberrubine (4e)
4
1
1
5
00 MHz): δ 155.7, 152.0, 149.1, 147.8, 145.7, 142.7,
37.9, 134.6, 130.0, 128.8, 126.9, 124.1, 122.8, 121.7,
19.2, 118.4, 108.9, 105.6, 103.2, 101.4, 99.0, 80.1, 69.1,
5.0, 52.2, 50.8, 26.3. HRMS m/z [M-Cl] calcd for
C H F N O ] : 532.20; found: 532.60. Anal. calcd for
Light yellow solid, yield: 63 %. m.p. 247–249 °C. IR (KBr)
+
−1
cm : 3638 (OH), 3037 (C–H, Ar), 1620–1553 (C=C, Ar),
+
1
[
1254 (Ar–N). H NMR (400 MHz, chloroform) δ 9.76 (s,
3
0
28
2
3
4
C H ClF N O : C, 63.44; H, 4.97; N, 7.40. Found: C,
1H, –OH of berberine ring), 9.17 (s, 1H, H-8), 7.73 (s, 1H,
H-13), 7.61 (s, 1H, H-1), 7.18 (s, 1H, H-4), 6.80–6.68 (m,
3
0
28
2
3
4
6
3.59; H, 5.04; N, 7.56.
3
H, Ar–H, piperazine H-22, H-23, H-24), 6.11 (s, 2H, –
1
2‑(1‑(2,4‑Dimethoxyphenyl)
OCH O), 6.07 (s, 1H, H-11), 5.85 (t, J = 5.3 Hz, 2H, H-6),
2
piperazine‑1‑ylmethyl)‑berberrubine (4c)
3.97 (s, 2H, CH , H-14 Mannich base), 3.89 (s, 3H, OCH ),
2
3
3
.81 (br s, 4H, piperazine, H-18, H-20), 3.51 (br s, 4H, pip-
1
3
Light yellow solid, yield: 48 %. m.p. 232–234 °C. IR (KBr)
erazine, H-15, H-17), 3.16 (t, J = 5.3 Hz, 2H, H-5).
C
−1
cm : 3641(OH), 3034 (C–H, Ar), 1611–1560 (C=C, Ar),
NMR (DMSO, 400 MHz): δ 155.2, 152.9, 149.8, 147.9,
145.6, 142.2, 137.8, 134.5, 130.7, 128.5, 126.2, 124.3,
122.4, 121.1, 119.4, 118.2, 108.7, 105.4, 103.4, 101.6,
1
1
255 (Ar–N). H NMR (400 MHz, chloroform) δ 9.91 (s,
1
H, –OH of berberine ring), 9.21 (s, 1H, H-8), 7.82 (s, 1H,
+
H-13), 7.44 (s, 1H, H-1), 7.22 (s, 1H, H-4), 6.83–6.69 (m,
H, Ar–H, piperazine H H-22, H-23, H-25), 6.21 (s, 2H, –
99.2, 80.7, 69.9, 55.2, 52.6, 50.2, 26.1. HRMS m/z [M-Cl]
calcd for [C H F N O ] : 532.20; found: 532.30. Anal.
+
3
3
0
28
2
3
4
OCH O), 6.02 (s, 1H, H-11), 5.88 (t, J = 5.5 Hz, 2H, H-6),
calcd for C H ClF N O : C, 63.44; H, 4.97; N, 7.40.
Found: C, 63.35; H, 5.07; N, 7.62.
2
30 28 2 3 4
4
3
.05 (s, 2H, CH , H-14 Mannich base), 3.95 (s, 3H, OCH ),
2
3
.92 (s, 3H, Ar-OCH of piperazine), 3.86 (s, 3H, Ar-OCH
3
3
of piperazine), 3.78 (br s, 4H, piperazine, H-18, H-20), 3.45
br s, 4H, piperazine, H-15, H-17), 3.21 (t, J = 5.4 Hz, 2H,
H-5). C NMR (DMSO, 400 MHz): δ 155.1, 152.7, 149.0,
12‑(1‑(2,4‑Dichlorophenyl)
piperazine‑1‑ylmethyl)‑berberrubine (3f)
(
1
3
1
1
1
47.1, 145.8, 142.8, 137.4, 134.7, 130.3, 128.7, 126.8,
24.5, 122.1, 121.2, 119.5, 118.1, 108.0, 105.1, 103.5,
01.5, 99.6, 80.2, 69.0, 55.5, 54.8, 52.7, 51.1, 50.3, 26.6.
Light yellow solid, yield: 64 %. m.p. 217–219 °C. IR
−1
(KBr) cm : 3625 (OH), 3022 (C–H, Ar), 1613–1549
1
(C=C, Ar), 1257 (Ar–N), 781 (C–Cl). H NMR (400 MHz,
+
+
HRMS m/z [M-Cl] calcd for [C H N O ] : 556.24;
found: 556.40. Anal. calcd for C H ClN O : C, 64.91; H,
5
chloroform) δ 9.90 (s, 1H, –OH of berberine ring), 9.29
(s, 1H, H-8), 7.69 (s, 1H, H-13), 7.43 (s, 1H, H-1), 7.24
(s, 1H, H-4), 6.85–6.73 (m, 3H, Ar–H, piperazine H-22,
3
2
34
3
6
3
2
34
3
6
.79; N, 7.10. Found: C, 64.77; H, 5.91; N, 7.23.
H-23, H-25), 6.18 (s, 2H, –OCH O), 6.11 (s, 1H, H-11),
2
1
2‑(1‑(2,4‑Dimethylphenyl)
5.83 (t, J = 5.3 Hz, 2H, H-6), 4.01 (s, 2H, CH , H-14
2
piperazine‑1‑ylmethyl)‑berberrubine (4d)
Mannich base), 3.90 (s, 3H, OCH ), 3.79 (br s, 4H, pip-
3
erazine, H-18, H-20), 3.39 (br s, 4H, piperazine, H-15,
1
3
Light yellow solid, yield: 65 %. m.p. 259–261 °C. IR (KBr)
H-17), 3.22 (t, J = 5.5 Hz, 2H, H-5). C NMR (DMSO,
400 MHz): δ 155.4, 152.6, 149.5, 147.3, 145.3, 142.1,
137.3, 134.4, 130.6, 128.4, 126.3, 124.4, 122.7, 121.8,
−1
cm : 3628 (OH), 3021 (C–H, Ar), 1615–1555 (C=C, Ar),
1
1
263 (Ar–N). H NMR (400 MHz, chloroform) δ 9.84 (s,
1
3

J IRAN CHEM SOC
1
5
19.3, 118.5, 108.6, 105.3, 103.1, 101.0, 99.5, 80.3, 69.8,
12‑(1‑(3,5‑Dichlorophenyl)
piperazine‑1‑ylmethyl)‑berberrubin (4i)
+
5.1, 52.3, 50.7, 26.2. HRMS m/z [M-Cl] calcd for
+
[
C H Cl N O ] : 564.15; found: 564.60. Anal. calcd
3
0
28
2
3
4
for C H Cl N O : C, 59.96; H, 4.70; N, 6.99. Found: C,
Light yellow solid, yield: 67 %. m.p. 253–255 °C. IR
3
0
28
3
3
4
−1
6
0.05; H, 4.84; N, 7.08.
(KBr) cm : 3622 (OH), 3033 (C–H, Ar), 1616–1543
1
(
C=C, Ar), 1257 (Ar–N), 775 (C–Cl). H NMR (400 MHz,
1
2‑(1‑(2,3‑Dimethylphenyl)
chloroform) δ 9.94 (s, 1H, –OH of berberine ring), 9.35
piperazine‑1‑ylmethyl)‑berberrubine (4g)
(s, 1H, H-8), 7.83 (s, 1H, H-13), 7.42 (s, 1H, H-1), 7.08
(s, 1H, H-4), 6.78–6.65 (m, 3H, Ar–H, piperazine H-22,
Light yellow solid, yield: 46 %. m.p. 255–257 °C. IR
H-24, H-26), 6.16 (s, 2H, –OCH O), 6.07 (s, 1H, H-11),
2
−
1
(
(
KBr) cm : 3643 (OH), 3039 (C–H, Ar), 1608–1565
C=C, Ar), 1265 (Ar–N). H NMR (400 MHz, chlo-
5.92 (t, J = 5.5 Hz, 2H, H-6), 4.06 (s, 2H, CH , H-14
2
1
Mannich base), 3.96 (s, 3H, OCH ), 3.77 (br s, 4H, pip-
3
roform) δ 9.83 (s, 1H, –OH of berberine ring), 9.36
erazine, H-18, H-20), 3.40 (br s, 4H, piperazine, H-15,
1
3
(
7
s, 1H, H-8), 7.80 (s, 1H, H-13), 7.55 (s, 1H, H-1),
.11 (s, 1H, H-4), 6.79–6.67 (m, 3H, Ar–H, pipera-
zine H-22, H-23, H-24), 6.20 (s, 2H, –OCH O), 6.09
H-17), 3.20 (t, J = 5.4 Hz, 2H, H-5). C NMR (DMSO,
400 MHz): δ 155.7, 152.3, 149.4, 147.5, 145.0, 142.6,
137.2, 134.9, 130.5, 128.9, 126.6, 124.2, 122.0, 121.5,
119.9, 118.1, 108.1, 105.8, 103.0, 101.7, 99.6, 80.9, 69.5,
2
(
s, 1H, H-11), 5.96 (t, J = 5.4 Hz, 2H, H-6), 4.08 (s,
+
2
3
4
H, CH , H-14 Mannich base), 3.88 (s, 3H, OCH ),
55.6, 52.8, 50.0, 26.7. HRMS m/z [M-Cl] calcd for
2
3
+
.85 (br s, 4H, piperazine, H-18, H-20), 3.46 (br s,
[C H Cl N O ] : 564.15; found: 564.30. Anal. calcd
3
0
28
2
3
4
H, piperazine, H-15, H-17), 3.19 (t, J = 5.3 Hz, 2H,
for C H Cl N O : C, 59.96; H, 4.70; N, 6.99. Found: C,
30 28 3 3 4
H-5), 2.21 (s, 3H, Ar-CH of piperazine), 1.90 (s, 3H,
Ar-CH of piperazine). C NMR (DMSO, 400 MHz): δ
59.87; H, 4.83; N, 7.12.
3
1
3
3
1
1
1
5
55.6, 152.1, 149.2, 147.7, 145.4, 142.4, 137.6, 134.1,
30.9, 128.7, 126.4, 124.9, 122.6, 121.3, 119.6, 118.8,
08.3, 105.2, 103.6, 101.8, 99.4, 80.5, 69.3, 55.4, 52.4,
Evaluation of antioxidant activity
Antioxidant capacity by DPPH assay
+
0.4, 26.5, 19.6, 17.7. HRMS m/z [M-Cl] calcd for
+
[
C H N O ] : 524.25; found: 524.50. Anal. calcd for
Radical scavenging potency of the compounds examined
was evaluated in vitro by DPPH analysis. DPPH is one
of the few constants and from the commercial perspec-
tive available organic nitrogen radicals. DPPH radicals
are viewed as an associate method for the initial testing
of compounds able to scavenge activated oxygen species.
They are more constant and easier to deal with than oxy-
gen free radicals and DPPH analysis has been mostly used
to evaluate the antioxidant action of different phenolic
compounds. The DPPH radical displays absorbance at
517 nm that reduces by the bleaching of a violet-coloured
methanol solution upon decrease by an anti-oxidant or a
radical via hydrogen atom or electron transfer to the odd
electron in DPPH generating the light yellow non-radical
form (DPPH-H). This reduces proportionately with the
improvement of non-radical types of DPPH [36]. The radi-
cal scavenging activity of berberine derivatives and start-
ing materials towards the radical DPPH was measured as
described [37] with modifications to adapt the screen for
96-well plates. Berberine derivatives (20 µL) was included
in 96-well microplate, and 180 µL of DPPH was included
to the wells. Methanol was used as the blank sample. The
mixtures were remaining for 30 min at room temperature
and the absorbances then were measured at 517 nm. The
control contains all reagents except the scavenger. The
3
2
34
3
4
C H ClN O : C, 68.62; H, 6.12; N, 7.50. Found: C,
3
2
34
3
4
6
8.45; H, 6.04; N, 7.38.
1
2‑(1‑(3,4‑Dichlorophenyl)
piperazine‑1‑ylmethyl)‑berberrubine (4h)
Light yellow solid, yield: 53 %. m.p. 244–246 °C. IR
−
1
(
(
KBr) cm : 3631 (OH), 3025 (C–H, Ar), 1610–1548
1
C=C, Ar), 1249 (Ar–N), 753 (C–Cl). H NMR (400 MHz,
chloroform) δ 9.77 (s, 1H, –OH of berberine ring), 9.30
(
(
s, 1H, H-8), 7.75 (s, 1H, H-13), 7.46 (s, 1H, H-1), 7.16
s, 1H, H-4), 6.84-6.77 (m, 3H, Ar–H, piperazine H-22,
H-23, H-26), 6.23 (s, 2H, –OCH O), 6.10 (s, 1H, H-11),
2
5
.84 (t, J = 5.3 Hz, 2H, H-6), 4.04 (s, 2H, CH , H-14
2
Mannich base), 3.91 (s, 3H, OCH ), 3.82 (br s, 4H, pip-
3
erazine, H-18, H-20), 3.43 (br s, 4H, piperazine, H-15,
1
3
H-17), 3.25 (t, J = 5.5 Hz, 2H, H-5). C NMR (DMSO,
4
1
1
5
00 MHz): δ 155.3, 152.8, 149.7, 147.2, 145.1, 142.9,
37.5, 134.8, 130.2, 128.2, 126.1, 124.6, 122.9, 121.0,
19.1, 118.3, 108.8, 105.5, 103.9, 101.1, 99.8, 80.6, 69.2,
+
5.3, 52.9, 50.9, 26.4. HRMS m/z [M-Cl] calcd for
+
[
C H Cl N O ] : 564.15; found: 564.40. Anal. calcd
3
0
28
2
3
4
for C H Cl N O : C, 59.96; H, 4.70; N, 6.99. Found: C,
3
0
28
3
3
4
5
9.81; H, 4.59; N, 6.83.
1
3

J IRAN CHEM SOC
DPPH radical scavenging activity of ascorbic acid was also
assayed for comparison; all tests were performed in tripli-
cate. The results of this bioassay, RSA % (the radical scav-
enging activity in percentage) was determined according to
Mensor et al. [38] as described in below equation.
via mixing acetate buffer (25 mL), TPTZ (2.5 mL), and
ferric chloride hexahydrate solution (2.5 mL). The tem-
perature of the solution was raised to 37 °C before use and
allowed to react with the FRAP solution (300 μL) in a vol-
ume ratio of 10:1:1, respectively. The 1 ml of methanol as
blank, test samples dissolved in methanol and ascorbic acid
as standard dissolved in water. The reaction was permit-
ted to run for 30 min. The colored product (ferrous tripy-
ridyl triazine complex) was monitored at a wavelength of
593 nm. The experiments were performed in triplicate, and
their mean was calculated for each compound.
Absorbance of blank − Absorbance of test
%
Scavenging =
× 100
Absorbance of blank
A plot of concentration of test compounds and %scav-
enging introduced IC s in the presence of an ascorbic acid
5
0
as standard.
Antioxidant capacity by ABTS assay
In vitro anticancer bioassay
The second analysis is by the capability of antiradical ele-
Cell cultures
·+
ments to satisfy the ABTS , a blue-green chromophore
with attribute absorption at 734 nm. The addition of anti-
oxidants to the performed radical cation reduces it to
ABTS, determining a decolorization. In this technique, an
antioxidant is included in a pre-formed ABTS radical solu-
Human cervical cancer cell line (HeLa) was maintained
in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-
BRL, Carlsbad, CA, USA) supplemented with 10 % heat-
inactivated fetal bovine serum (FBS) and 1 % of antibiotic–
antimycotic solution (100×). CaSki and MDCK cells were
cultured in RPMI-1640 medium (HyClone) supplemented
with 10 % FBS, and 1 % of antibiotic–antimycotic solution
(100×). Both of the cells were cultured at 37 °C in a 5 %
·+
tion, and after a set period, the staying ABTS is quanti-
fied spectrophotometrically at 734 nm, according to ABTS
method as previously described [39] with some modifica-
tions [37]. In brief, 20 L of sample was combined with
1
80 μL of ABTS radical solution followed by 10 min of
CO incubator.
2
incubation under dark conditions and the absorbance was
measured at 734 nm whereas ascorbic acid was used as a
reference drug. The UV absorption data represented the
radical scavenging rates which give the corresponding IC50s
for the test compounds. All measurements were created in
The effects of Mannich base berberine derivatives on
the different cell lines were determined by the SRB assay
3
as described previously [37, 42]. Briefly, 5 × 10 cells
were incubated in triplicate on a 96-well plate under nor-
mal culture conditions overnight. HeLa, CaSki and MDCK
were treated with different concentrations of Mannich base
berberine derivatives (0.1, 1, 10 and 100 μL). Untreated
cells served as control. After 48 h, cells were fixed with
ice-cold 70 % (w/v) acetone (100 μL/well) for 1 h at 4 C.
After incubation, the solvent was removed and plates were
dried in an oven at 60 °C. Cells were then stained with SRB
·+
triplicate and for each analysis a fresh ABTS stock solu-
tion was prepared.
·+
The scavenging capability of ABTS radical was calcu-
lated using the following equation:
Absorbance of blank − bsorbance of test
%
Scavenging =
× 100
Absorbance of blank
(0.4 % w/v in 1 % acetic acid, 100 μL/well) followed by
FRAP assay
SRB removal and washing thrice with 1 % of acetic acid
and dried again under hot air oven at 60 °C. Microscopic
observation was carried out to determine the morphology
of the cells. Bound dye was solubilized with 10 mM Tris
base (100 μL/well) and absorbance was read at 540 nm to
calculate the inhibition concentration of 50 % (IC ), cyto-
The FRAP assay of the compounds performed using modi-
fied method as described by Benzie and Strain [40] with
some modifications [41]. FRAP analysis conducted the
in vitro antioxidant action for all the newly synthesized
5
0
3
+
compounds by the decrease of a colorless Fe tripyridyl
toxic concentration of 50 % (CC ) and therapeutic index
50
2
+
triazine complex into a blue-colored Fe -tripyridyltriazine
complex. The antioxidant potentials of the compounds were
estimated as their power to reduce the TPTZ–Fe(III) com-
plex to TPTZ–Fe(II) complex, which is simple, fast, and
reproducible. The stock solutions contained 300 mM ace-
(TI).
Results and discussion
Chemistry
·
tate buffer (3.1 g C H NaO 3H O and 16 mL C H O ), pH
2
3
2
2
2
4
2
3
4
.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in
0 mM hydrochloric acid and 20 mM ferric chloride hexa-
The synthesis route of Mannich berberine derivatives was
conveniently undertaken as outlined in Scheme 1. To begin
hydrate solution. The fresh working solution was prepared
1
3

J IRAN CHEM SOC
O
O
O
O
Cl-
N+
Cl-
+ R-H
N+
i
OH
3
OCH3
OCH3
2
OCH3
1
O
O
Cl-
N+
ii
OH
OCH3
R
4
a-i
o
o
Reagents & conditions: i. 190 C, 20-30 mm Hg 40 min: ii. C H OH, HCHO, 80 C, 24 h
2
5
Cl
Cl
F
H CO
3
R
HN
HN
HN
N
HN
HN
HN
N
N
N
F
HN
N
OCH3
4
a
4b
4c
H C
F
F
Cl
N
3
N
CH3
HN
Cl
4
d
4e
4f
H C
CH3
Cl
Cl
Cl
3
N
Cl
HN
N
4
g
4h
4i
Scheme 1 Synthesis of piperazine linked berberine derivatives
with, berberrubine 2 was obtained in 85 % of yield through
selective demethylation of berberine hydrochloride 1 at
peak at around 3.92–4.15 ppm (s, 2H), for the CH group,
2
which confirms the formation of Mannich bases. The
1
1
90 °C temperature and 20–30 mmHg pressure [43] and
HNMR spectrum of berberine ring displayed singlet peaks
then the appropriate piperazine derivatives with formalde-
hyde were added, stirred at 80 °C or room temperature to
obtain the aminomethylation berberrubine derivatives 4a–i
at 9.31, 7.78, 7.51, 7.15, and 6.05 ppm for H-8, H-13, H-1,
H-4, and H-11 protons, respectively, as well as singlet peak
observe at 6.13 ppm presence of –OCH O– of berberine
2
[
44].
ring. H-5 and H-6 proton atoms of berberine ring exerted a
triplet peak at 3.15 and 5.95 ppm. The presence of methoxy
group was confirmed while observing its characteristic peak
as a singlet at around 3.92–4.17 ppm. The protons atom
(H-18, H-20, H-15 and H-17) present on the piperazine moi-
ety were resonated as a broad singlet at around 3.80 ppm as
well as 3.49 ppm. Moreover, aromatic rings linked to the
piperazine ring appeared to have a corresponding signal as
multiples in the range 6.93–6.75 ppm. Mass spectrometric
The adequate production of titled analogues (4a–i)
1
was confirmed adapting spectroscopic techniques (IR, H
NMR, and Mass spectrometry) and analyzing elemental
quantification (CHN).
C–H Stretching and C=C of aromatics delivered their
−
1
characteristic absorption bands at around 3043–3017 cm
and 1622–1546 cm , respectively in FT-IR spectra of 4a–
i. The Ar–N bands were observed as a sharp peak nearby
−
1
−1
+
1
250–1270 cm in Mannich bases along with sharp chlo-
data were inaccurate as observed from the M ion values for
−1
rine signal at 795–742 cm while the broad band found
at 3610–3650 cm confirms the presence of –OH group.
In the HNMR spectra of Mannich bases 4a–i show singlet
the final compounds 4a–i. All of the novel compounds gave
C, H and N analyses within 0.4 % points from the theoreti-
cal values, i.e. in an acceptable range.
−
1
1
1
3

J IRAN CHEM SOC
Table 1 Screening results of DPPH, ABTS and FRAP radical scav-
enging activity of berberine derivatives (4a–i)
chlorine-based compounds with 3,4-dichlorophenyl pipera-
zine (4h) and 3,5-dichlorophenyl piperazine (4i) moieties
appeared with appreciable antioxidant effects in DPPH bio-
assay with IC50s20.75 ± 0.56 and 20.90 ± 0.37 µM, respec-
tively. Most importantly, these two derivatives established
the most powerful antioxidant action in ABTS bioassay
with 7.774 ± 0.53 and 8.189 ± 0.06 µM of IC50s, respec-
tively. In addition, a compound (4d) with electron releas-
ing dimethyl functionality showed 22.63 ± 1.63 µM of
Compounds
aIC μM ± SD
FRAP
IC in DPPH assay, whereas a compound (4b) with highly
50
50
electronegative fluorine atoms exerted 9.052 ± 0.07 µM of
DPPH
ABTS
Mean ± SD
IC in ABTS bioassay, hence these two compounds can be
5
0
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
19.80 ± 0.94
42.04 ± 2.56
27.04 ± 0.61
22.63 ± 1.63
31.49 ± 1.74
37.54 ± 2.34
34.54 ± 1.26
20.75 ± 0.56
20.90 ± 0.37
89.18 ± 1.60
61.03 ± 0.66
11.64 ± 0.40
9.052 ± 0.07
18.84 ± 0.05
11.52 ± 0.05
15.24 ± 0.32
12.24 ± 0.75
16.52 ± 0.08
7.774 ± 0.53
8.189 ± 0.06
230.1 ± 2.11
27.02 ± 0.05
0.822 ± 0.07
0.122 ± 0.01
0.566 ± 0.03
0.003 ± 0.02
0.201 ± 0.06
1.987 ± 0.15
0.075 ± 0.21
1.257 ± 0.31
2.078 ± 0.27
0.001 ± 0.66
2.140 ± 0.78
considered to have appreciable level of antioxidant power
in the respective assay. The rest of the compounds showed
better level of radical scavenging power when compared
to the control ascorbic acid. Nevertheless, all compounds
have better antioxidant efficacies than parent berberine as
assayed by DPPH and ABTS tests.
g
h
i
Titled compounds 4a–i were also evaluated for their
3
+
antioxidant effects in FRAP assay. Fe ion, biologically
2
+
inactive form of iron, can be converted to Fe ion via
reduction depending on pH conditions and can be further
oxidized through Fenton-type reaction producing hydroxyl
radical via superoxide anions production. An antioxidant
compound has reductive capabilities and is evaluated by
Berberine
Ascorbic acid
a
Antioxidant activities are shown as IC50 values in μg/mL. All
assays were carried out in triplicate, and the results expressed as an
average ± standard deviation
3
+
2+
Fe to Fe transformation upon releasing an electron in
the presence of test compounds. So, the concentration of
DPPH 2,2-diphenyl-1-picrylhydrazyl, ABTS 2,2′-azino-bis-3-eth-
ylbenzthiazoline-6-sulphonic acid, FRAP ferric reducing ability of
plasma
2
+
2+
Fe can be monitored by measuring the formation of Fe -
tripyridyltriazine complex with blue color. The higher the
absorbance of the compounds, greater the reducing power
and a compound 4i with 3,5-dichlorophenyl piperazine
entity reduced metal ion complexes to their lower oxidation
state or takes part in electron transfer reaction very effec-
tively with a absorption value of 2.078 nm as compared to
that of the control ascorbic acid with absorption value of
2.140 nm at a concentration 10 µg/mL. This compound has
significantly increased antioxidant potential in FRAP assay
when compared to berberine with the absorption value of
0.001 nm. A compound 4f with 2,4-dichlorophenyl pipera-
zine moiety attached to the berberine ring showed tremen-
dous ability of electron donor to scavenge free radicals in
FRAP assay with the absorption value of 1.987 nm nearer
to that of control ascorbic acid at a concentration 10 µg/
mL. The rest of the compounds showed lower absorbance
as compared to the standard but were of high efficacies
when compared to berberine.
Evaluation of biological activities
Antioxidant activities
Examination of antioxidant potential of final berberine–
piperazine adducts 4a–i was carried out adapting three
different antioxidant assays, DPPH, ABTS and FRAP
shown in Table 1. From the results of these bioassays,
it is worth mentioning that variation in functional groups
presents on piperazine entity leads to the difference in
antioxidant potential of the resultant molecules. Overall,
the results suggested that the final compounds demon-
strated significant antioxidant power as free-radical scav-
engers with IC50s ranging 19.80 ± 0.94–42.04 ± 2.56 and
7
.774 ± 0.53–18.84 ± 0.05 µM in DPPH and ABTS bioas-
say, respectively, when compared to that of berberine itself.
Analogue 4a with two electron withdrawing chlorine atoms
on the piperazine entity attached to the berberine ring pre-
sented most active radical scavenging potential in DPPH
assay with 19.80 ± 0.94 µM of IC level, comparable to
Anticancer activities
Results of sulforhodamine B (SRB) bioassay adapted
to screen the cervical cancer cell line (HeLa and CaSki)
potential of title compounds 4a–i are furnished in Tables 2
and 3. Results of anticancer examination were worth stating
5
0
that of control drug ascorbic acid at 61.03 ± 0.66 µM and
far better than berberine at 89.18 ± 1.60 µM. Another two
1
3

J IRAN CHEM SOC
Table 2 Anticancer activity of synthesized compounds against HeLa
cancer cell and their toxicity
Table 3 Anticancer activity of synthesized compounds against CaSki
cancer cell and their toxicity
Compounds
aIC μM ± SD
CC50 μM ± SD
MDCK
TI
5
0
Compounds
aIC μM ± SD
CC50 μM ± SD
MDCK
TI
5
0
HeLa
CaSki
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
7.753 ± 0.06
11.12 ± 0.07
7.340 ± 0.04
8.257 ± 0.03
8.577 ± 0.05
7.896 ± 0.17
9.357 ± 0.32
7.526 ± 0.18
7.327 ± 0.08
13.42 ± 0.23
0.486 ± 0.04
312.6 ± 0.59
227.2 ± 1.07
248.9 ± 2.03
214.6 ± 0.46
238.7 ± 1.08
212.1 ± 0.75
279.0 ± 1.34
320.1 ± 0.08
310.6 ± 1.42
300.4 ± 0.47
250.2 ± 0.57
40.31
20.43
33.91
25.99
27.83
26.86
29.81
42.53
42.39
22.38
514.8
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
7.798 ± 0.25
10.70 ± 0.16
5.755 ± 0.17
7.856 ± 0.24
10.58 ± 0.56
8.932 ± 0.47
10.43 ± 1.05
8.090 ± 0.37
7.606 ± 0.08
15.17 ± 0.19
1.076 ± 0.04
312.6 ± 0.59
227.2 ± 1.07
248.9 ± 2.03
214.6 ± 0.46
238.7 ± 1.08
212.1 ± 0.75
279.0 ± 1.34
320.1 ± 0.08
310.6 ± 1.42
300.4 ± 0.47
250.2 ± 0.57
40.08
21.23
43.24
27.31
22.56
23.74
26.74
39.56
40.83
19.80
232.5
g
h
i
g
h
i
Berberine
Berberine
Doxorubicin
Doxorubicin
a
Anticancer activities are shown as IC50 values in μg/mL. All assays
a
were carried out in triplicate, and the results expressed as an average
Anticancer activities are shown as IC50 values in μg/mL. All assays
±
standard deviation
were carried out in triplicate, and the results expressed as an average
±
standard deviation
CC cytotoxicity concentration of 50 %, TI therapeutic index
5
0
CC cytotoxicity concentration of 50 %, TI therapeutic index
5
0
as overall all compounds exhibited a good level of cancer-
ous cell inhibitory potential with IC50s ranging 7.327–11.12
and 5.755–10.70 µM against HeLa and CaSki cell lines,
respectively, when compared to that of berberine itself with
IC50s of 13.42 µM (HeLa) and 15.17 µM (CaSki). Moreo-
ver, similar to the antioxidant efficacies, it can be said that
variation in the functionalities of groups attached to the
piperazine ring led to the change in the anticancer efficacies
of the resultant molecules. Secondly, final compounds 4a–i
exerted tolerable cytotoxic nature with cytotoxicity values
ranging 212.1–320.1 µM against MDCK cell lines thereby
exhibiting potent therapeutic index (TI) in the range 20.43–
cell inhibitory effects with 7.753 ± 0.06 µM of IC ,
50
3
12.6 ± 0.59 µM of cytotoxicity and 40.31 of the thera-
peutic index. All the remaining analogues except 4b dis-
played TI level (25.99–33.91 µM) greater than berberine
with 24.83. Finally, an analogue with electron donating
alkoxy functional groups, 4c, bearing 2,4-dimethoxyophe-
nyl piperazine entity furnished a significant level of CaSki
cell line inhibitory efficacy with 5.755 ± 0.17 µM of IC
50
and 248.9 ± 2.03 µM of CC thereby releasing high
50
therapeutic index of 43.24. Furthermore, compounds 4h
and 4i had an appreciable level of CaSki cell line inhibi-
tory potential with 8.090 ± 0.37 and 7.606 ± 0.08 µM of
4
2.53 for HeLa and 21.23–43.24 for CaSki cell lines. From
IC , respectively again. These compounds have shown
the TIs, it can be stated that the title compounds demon-
strated better anticancer efficacies against HeLa cell line
than CaSki cell lines of cervical cancer. Doxorubicin was
used as a control [45, 46].
50s
3
20.1 ± 0.08 and 310.6 ± 1.42 µM of cytotoxicity levels,
thereby introducing better TIs as 39.56 and 40.83, which
were considered better than parent compound berberine
with TI level of 19.80 against CaSki cell lines. A com-
pound (4a) was also found to fall in a considerable range
of anticancer effects against CaSki cell lines with 40.08
of TI. All remaining analogues showed TIs in the range
Two analogues bearing 3,4-dichlorophenyl pipera-
zine (4h) and 3,5-dichlorophenyl piperazine (4i) moiety
appeared with most potent anticancer action against HeLa
cell lines with 7.526 ± 0.18 and 7.327 ± 0.08 µM of IC
5
0s
2
1.23–27.31 that was good when compared to that of ber-
and 42.53 and 42.39 of TIs, respectively. They had lower
cytotoxic values of 320.1 ± 0.08 and 310.6 ± 1.42 µM,
respectively. Moreover, an analogue with 2,3-dichlorophe-
nyl piperazine entity showed a good level of HeLa cancer
berine. Concerning the activity concerning the functional
group attached to the piperazine ring, the order falls in the
way chloro > methoxy > fluoro > methyl.
1
3

J IRAN CHEM SOC
1
4. A.K. Nadkarni, Nadkarni’s Indian Materia Medica (PoPular
Prakashan, Pvt. Ltd, Bombay, 1976)
Conclusion
1
5. R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary of Indian
Medicinal Plants (Council of Scientific and Industrial Research,
New Delhi, 1996), p. 247
6. J. Gruenwald, T. Brendler, C. Jaenicke, PDR for herbal medi-
cines (Medical Economics Company, NJ, 2000), p. 313
7. W.J. Shan, L. Huang, Q. Zhou, F.C. Meng, X.S. Li, Eur J Med
Chem 46(12), 5885–5893 (2011)
To sum up, Mannich bases introducing piperazine moieties
enriched with different electron withdrawing and electron
releasing functional groups were furnished on the berber-
ine core to evaluate the antioxidant and anticancer poten-
tials. Efficient synthesis of final analogues was confirmed
using different spectroscopy methods and elemental analy-
sis as well as physical tests. Compounds 4h and 4i with
1
1
18. T. Su, S. Xie, H. Wei, J. Yan, L. Huang, X. Li, Bioorg Med Chem
2
1(18), 5830–5840 (2013)
1
2
9. A. Singh, S. Duggal, N. Kaur, J. Singh, J Nat Prod 3, 64–75 (2010)
0. L. Zhang, J. Li, F. Ma, S. Yao, N. Li, J. Wang, Y. Wang, X. Wang,
Q. Yao, Molecules 17, 11294–11302 (2012)
3
,4-dichloro and 3,5-dichloro functional groups performed
well in all the bioassay tested and can be considered as a
most potent antioxidant and anticancer agents for cervical
cancer cell lines. All compounds exercised low cytotoxic
values against normal cell lines. Final compounds exhib-
ited better pharmacological action tested in this study
than the parent berberine and this modification is likely to
enhance their ability to bind to the target of drug action
mainly through hydrophobic effect, conjugation effect and
hydrogen bond on 9-hydroxyl. The presence of dichloro
function was found beneficial for the antioxidant potencies
as well as against HeLa cell lines, whereas dialkoxy sub-
stitution was crucial to enhancing the anticancer potential
against CaSki cell lines. Thus, the rationalized molecular
designs presented in this study would be of considerable
interest for ongoing drug developing progress.
21. Y. Wang, H. Fu, Y. Li, J. Jiang, D. Song, Acta Pharm Sin B 2(6),
81–587 (2012)
5
2
2
2
2
2. X. Deng, X. Zhao, J. Han, J. Wang, W. Huang, H. Qian, L. Ge,
Lett Drug Des Discov 9, 489–493 (2015)
3. X. Jin, T.H. Yan, L. Yan, Q. Li, R.L. Wang, Z.L. Hu, Y.Y. Jiang,
Q.Y. Sun, Y.B. Cao, Drug Design. Dev Ther 8, 1047–1059 (2014)
4. Y. Sun, K. Xun, Y. Wang, X. Chen, Anticancer Drugs 20(9),
7
57–769 (2009)
5. C.V. Diogo, N.G. Machado, I.A. Barbosa, T.L. Serafim, A. Bur-
geiro, P.J. Oliveira, Curr Drug Targets 12(6), 850–859 (2011)
26. L.M.G. Ortiz, P. Lombardi, M. Tillhon, A.I. Scovassi, Molecules
9, 12349–12367 (2014)
7. R.V. Patel, S.W. Park, Mini Rev Med Chem 13(11), 1579–1601
2013)
1
2
2
2
3
3
(
8. R.V. Patel, P. Kumari, D.P. Rajani, K.H. Chikhalia, Eur J Med
Chem 46(9), 4354–4365 (2011)
9. L. Mallesha, K.P. Harish, K.N. Mohana, N.D. Rekha, Can Chem
Trans 2(4), 518–525 (2014)
0. M. Malhotra, R. Sharma, M. Sanduja, R. Kumar, J. Jain, A.
Acknowledgments This article was supported by the KU Research
Professor Program of Konkuk University, Seoul, South Korea.
Deep, Acta Pol Pharm 69(2), 355–361 (2012)
1. L. Ma, Y. Xiao, C. Li, Z.L. Xie, D.D. Li, Y.T. Wang, H.T. Ma,
H.L. Zhu, M.H. Wang, Y.H. Ye, Bioorg Med Chem 21(21),
6
763–6770 (2013)
3
3
3
3
3
3
3
3
2. D.H. Parka, J. Venkatesan, S.K. Kim, V. Ramkumar, P. Parthi-
bana, Bioorg Med Chem Lett 22(20), 6362–6367 (2012)
3. S. Bala, N. Sharma, A. Kajal, S. Kamboj, V. Saini, Int. J. Med.
Chem. (2014) (Article ID 191072)
4. K. Venkateshwarlu, G. Chakradar Rao, V.M. Reddy, Y. Nar-
asimha Reddy, J Iran Chem Soc 11(6), 1619–1627 (2014)
5. R.V. Patel, J.K. Patel, P. Kumari, K.H. Chikhalia, Lett Org Chem
References
1
.
H. Haraguchi, H. Ishikawa, Y. Sanchez, T. Ogura, Y. Kubo, I.
Kubo, Bioorg Med Chem 5, 865–871 (1997)
2
3
.
.
Q. Cai, R.O. Rahn, R. Zhang, Cancer Lett 119, 99–107 (1997)
S. Cuzzocrea, D.P. Riley, A.P. Caputi, D. Salvemini, Pharmacol
Rev 53, 135–159 (2001)
1
2(10), 478–486 (2015)
4.
5.
6.
H. Ahsan, A. Ali, R. Ali, Clin Exp Immunol 131(3), 398–404 (2003)
W. Nickels, Eur J Biochem 270, 2109–2119 (2003)
WHO, World Cancer Report 2014. http://www.who.int/
mediacentre/factsheets/fs297/en/
6. K. Iwasa, M. Kamigauchi, M. Ueki, M. Taniguchi, Eur J Med
Chem 31, 469–478 (1996)
7. B. Mistry, Y.S. Keum, D.H. Kim, Res Chem Intermed (2015).
doi:10.1007/s11164-015-2208-x
7
.
J. Ferlay, H. R. Shin, F. Bray, D. Forman, D. M. Parkin, GLO-
BOCAN 2008, Cancer Incidence and Mortality Worldwide:
IARC CancerBase No. 10. Lyon, France: International Agency
for Research on Cancer, 2010. http://globocan.iarc.fr
P. Cos, A.J. Vlietinck, D.V. Berghe, L. Maes, J Ethnopharmacol
8. L.L. Mensor, F.S. Menezes, G.G. Leitao, A.S. Reis, T.C. Dos
Santos, C.S. Coube, Phytother Res 15, 127–130 (2001)
9. R. Re, N. Pellergini, A. Proteggenete, A. Pannala, M. Yang, C.
Rice Evans, Free Rad Biol Med 26, 1231–1237 (1999)
0. I.F.F. Benzie, J.J. Strain, Methods Enzymol 299, 15–27 (1999)
1. B. Mistry, Y.S. Keum, D.H. Kim, Saudi J Biol Sci (2015).
doi:10.1016/j.sjbs.2015.09.005
2. O.A. Adaramoye, J. Sarkar, N. Singh, S. Meena, B. Changkija, P.P.
Yadav, S. Kanojiya, S. Sinha, Phytother Res 25, 1558–1563 (2011)
3. O. Bekircan, H. Bektas, Molecules 13, 2126–2135 (2008)
4. P. Molyneux, Songklanakarin J Sci Technol 26(2), 211–219 (2004)
5. V. Kuete, H.K. Wabo, K.O. Eyong, M.T. Feussi, B. Wiench,
B. Krusche, P. Tane, G.N. Folefoc, T. Efferth, PLoS One 6(8),
e21762 (2011)
8
9
.
.
4
4
1
06, 290–302 (2006)
M. Pavrez, H.K. Mahboob, I. Zahuul, M.H. Shek, J Life Earth
Sci 1, 25–29 (2005)
4
1
1
1
0. M. Khan, M. Kibm, B. Oinoloso, Phannaceut Biol 41, 277–280
2003)
1. R.H.F. Manske, The Alkaloids. Chemistry and Physiology, vol.
VIII (Academic Press, New York, 1965), p. 673
2. T. Aniszewski, Alkaloids-Secrets of Life Alkaloids Chemistry,
Biological Significance Application and Ecological Role, 1st
edn. (Elsevier, Amsterdam, 2007)
(
4
4
4
4
6. R. Dharmatti, C. Phadke, A. Mewada, M. Thakur, S. Pandey, M.
Sharon, Mater Sci Eng C 44, 92–98 (2014)
1
3. G. Watt, Dictionary of Economic Products of India, Reprinted
edition periodical expert, Delhi, vol. VI (Pt. IV), 1972, p. 83
1
3