Synthesis of berberine-piperazine conjugates as potential antioxidant and cytotoxic agents

Article abstract of DOI:10.1007/s00044-016-1662-3

Piperazine derivatives bearing different electron-withdrawing and electron-donating functional groups were linked to the well-known isoquinoline alkaloid derivative, berberine via efficient organic transformations. The entire target berberine-based analogues were examined for their in vitro antioxidant potency using 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid bioassays, and anticancer activities using sulforhodamine B assay against HeLa and CaSki cervical cancer cell lines in addition to the cytotoxicity using Madin-Darby canine kidney non-cancer cell lines and, ascorbic acid and berberine used as a control for antioxidant and anticancer activities, respectively. Bioassay results revealed that newer compounds were more active against CaSki and HeLa cell lines with therapeutic indices better than that of parent berberine and showed tolerable cytotoxicity to the normal cells. A final analogue 5a with 4-methylpiperazine substituent indicated most significant anticancer potency with a therapeutic index of 58.53 (HeLa) and 48.76 (CaSki), followed by those bearing meta-chloropiperazine rings with a therapeutic index of 41.83 (HeLa) and 47.35 (CaSki), respectively.In addition, newly synthesized analogues exerted a significant radical scavenging activity against 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid cation with IC₅₀ values of 8.917 μg/mL, and were good to moderate scavengers of 2,2-diphenyl-1-picrylhydrazyl radical with IC₅₀ values of 25.40 μg/mL. Synthesized compound was characterized using several techniques, fourier transform infrared spectroscopy, ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, mass spectroscopy and elemental (CHN) analyses.

Full text of DOI:10.1007/s00044-016-1662-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1662-3
ORIGINAL RESEARCH
Synthesis of berberine-piperazine conjugates as potential antioxidant and
cytotoxic agents
Bhupendra Mistry1 Young Soo Keum1 Muthuraman Pandurangan1
●
●
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Rahul V. Patel2 Doo Hwan Kim1
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Received: 31 July 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract Piperazine derivatives bearing different electron-
withdrawing and electron-donating functional groups were
linked to the well-known isoquinoline alkaloid derivative,
berberine via efficient organic transformations. The entire
target berberine-based analogues were examined for their
in vitro antioxidant potency using 2,2-diphenyl-1-picryl-
hydrazyl and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sul-
phonic acid bioassays, and anticancer activities using
sulforhodamine B assay against HeLa and CaSki cervical
cancer cell lines in addition to the cytotoxicity using Madin-
Darby canine kidney non-cancer cell lines and, ascorbic
acid and berberine used as a control for antioxidant and
anticancer activities, respectively. Bioassay results revealed
that newer compounds were more active against CaSki and
HeLa cell lines with therapeutic indices better than that of
parent berberine and showed tolerable cytotoxicity to the
normal cells. A final analogue 5a with 4-methylpiperazine
substituent indicated most significant anticancer potency
with a therapeutic index of 58.53 (HeLa) and 48.76
(CaSki), followed by those bearing meta-chloropiperazine
rings with a therapeutic index of 41.83 (HeLa) and
47.35 (CaSki), respectively.In addition, newly synthesized
analogues exerted a significant radical scavenging activity
against 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic
acid cation with IC50 values of 8.917 µg/mL, and were good
to moderate scavengers of 2,2-diphenyl-1-picrylhydrazyl
radical with IC50 values of 25.40 µg/mL. Synthesized
compound was characterized using several techniques,
1
fourier transform infrared spectroscopy, H nuclear mag-
netic resonance, 13C nuclear magnetic resonance, mass
spectroscopy and elemental (CHN) analyses.
●
●
●
Keywords Berberine Piperazine Antioxidant Cervical
cancer
Introduction
Oxygen species plays a vital role to perform essential bio-
logical functions in the human body as energy generation
for growth, metabolic activities, development and catabo-
lism of energy sources. Though they are beneficial for the
body, their disadvantages also appear in terms of toxic
agents for living cells. Oxygen may play a key role in
producing reactive oxygen species (ROS) found to occur
during important functions of the body like metabolism and
radiation through chemicals. ROS are responsible for gen-
erating disease conditions like cancers, inflammation and
cardiovascular harms (Kataki et al., 2012; Manikandan
et al., 2004) through their interaction with biomolecules.
Thus, a human being will always be in need of consuming
antioxidants regularly to get relieved out of these diseases
that emerge through ROS. Researchers have developed
various types of antioxidant molecules either purely syn-
thetic or prepared and inspired by natural product (NP)
fragments. They are found to have interaction with free
radicals, thereby destroying their side chain transformations
well in advance before the damage occurs in the vital
* Bhupendra Mistry
bhupendra.mistry84@gmail.com
* Doo Hwan Kim
kimdh@konkuk.ac.kr
1
Organic Research Laboratory, Department of Bioresources and
Food Sciences, College of Life and Environmental Sciences,
Konkuk University, Seoul, South Korea
2
Department of Food Science and Biotechnology, Dongguk
University, Biomedical Campus, 32 Dongguk-ro, Ilsandong-gu,
Goyang-si, Gyenggi-do, Republic of Korea

Med Chem Res
biomolecules (Ames et al., 1993) and known to have
potential effects as antioxidant drugs to counter diseases
caused by ROS functions like carcinogenesis, inflammation,
atherogenesis and aging in aerobic organisms (Tyagi et al.,
2005; Karegoudar et al., 2008). Cancer is the most threa-
tening disease that appears nowadays throughout the world.
Particularly, cervical cancer is among the world’s most
dangerous types of cancer for females, accounting for over
2,70,000 deaths yearly, 85 % of which happen in developing
countries (WHO, Internet). In the end, ROS are capable of
damaging biomolecules like DNA, proteins and lipids, thus
resulting in the development of cancer. It is, therefore, evi-
dent that the treatment of the above-mentioned pathophy-
siological conditions could benefit from the use of drugs that
exert antioxidant and anticancer activity.
Due to the aforementioned useful activities possessed by
berberine derivatives, we synthesized some novel piperazine-
based berberine analogues and evaluated them for their
pharmacological applications as anticancer agents (Patel
et al., 2012) and antioxidants (Andonovaa et al., 2014).
Materials and methods
Highest quality chemicals and reagents were used in this
study without prior purification (Sigma). Melting points
were determined on Veego Open capillary electronic
apparatus (model: VMP-D, Veego Instrument Corporation,
Mumbai, India) to obtain melting points of the synthesized
compounds and were uncorrected. Bruker spectro-
photometer (KBr pellets) were used to obtain fourier
transform infrared spectroscopy (FT-IR) and were provided
in cm−1.1H nuclear magnetic resonance (NMR) and 13C
NMR spectra were recorded with Tetramethylsilane as the
internal standard on a Varian 400 spectrometer in CDCl3 or
dimethyl sulfoxide (DMSO). Chemical shifts (δ) were pre-
NPs have been the primary sources of chemical diversity
for pharmaceutical discoveries over the past century. NPs
and their analogues can interact with many specific targets
within the cell, thereby exhibiting curable effects against
varieties of diseases (Mishra and Tiwari, 2011). NP synth-
esis provides an essential validation of newly developed
reaction technologies leading to the development of new
chemotherapeutic agents. More than 2,50,000 entries of
natural substances were reported in the last few years
(Dictionary of Natural Products, the internet). Polyketides,
carbohydrates, flavonoids, terpenes-terpenoids, steroids,
alkaloids, amino acids, peptides are present in nature with a
long-lasting diversity (James, 2003). Moreover, NPs have
received immense attention in the research of drug dis-
covery. To develop their derivatives containing new classes
of active drug molecules that provide decreased drug level
of resistance has also been mostly accepted these days
(Breinbauer et al., 2002; Paul et al., 2008). Among the most
beneficial classes of plant-derived compounds, alkaloids are
the most diverse plant secondary metabolites. More speci-
fically, the quaternary ammonium salt from the proto-
berberine group of isoquinoline alkaloids compounds,
namely berberine has received enormous importance as a
drug molecule associated with plenty of pharmacological
actions as antimicrobial, antileukemic, antiulcerous, and
enzyme-inhibiting (Verpoorte, 1998; Bodiwala et al.,
2011), anti-inflammatory (Kuo et al., 2004), anti-diarrhea
(Yin et al., 2002), glucose-lowering (Leng et al., 2004),
cholesterol-lowering (Peng et al., 2007), neuroprotective
(Cui et al., 2009), antidepressant (Kulkarni and Dhir, 2007),
Alzheimers disease-ameliorating (Asai et al., 2007), anti-
hyperlipidemic, antibiotics (Zuo et al., 2014), pancreatic
lipase (Mohammad et al., 2013) and anticancer effects
(Orfila et al., 2000; Liu et al., 2014). It is found in plants
such as Berberis, Berberis aristata, Hydrastis canadensis,
Phellodendron amurense, Coptis chinensis, Tinospora
cordifolia, and to a smaller extent in Argemone mexicana
and Eschscholzia californica (Lo et al., 2013).
1
sented in ppm and coupling constants (J) in Hz. H NMR
spectral data were reported in the following order: chemical
shift, multiplicity (s: singlet, d: doublet, t: triplet, m multi-
plet, br s: broad singlet patterns). Mass spectrometry (MS)
were recorded on an Agilent liquid chromatography–mass
spectrometry 1100 instrument with Varian 500-MS selec-
tive detector. Flash column chromatography on silica gel
(200–300 mesh) was used for the routine purification of
reaction products. Thin-layer chromatography (TLC) was
performed out using appropriate mobile phase systems
silica gel-G coated microscopic glass slides (2 × 7.5 cm),
and TLC spots were seen in ultraviolet (UV) light chamber.
Elemental analyses (C, H, N) were conducted using a
Heraeus Carlo Erba 1180 CHN analyzer.
General procedure for the synthesis of berberrubine (2)
Berberine chloride (10 g, 0.01 mmol) was included in a
50 mL round bottom flask and the reaction system was
maintained at reduced pressure 20–30 mmHg applying an
oil pump and warmed to 190 °C followed by reacting for
40 min to afford dark-wine solid, which was recrystallized
with anhydrous ethanol twice than the product was purified
via silica gel column chromatography (CHCl3/CH3OH:15:1
and 10:1, eluting) to acquire a brownish red amorphous
powdered compound (8.5 g, 85 %).
Synthesis of bromopentyl berberrubine (3)
In a flask charged with dry acetonitrile, compound 2 (8 g,
0.029 mmol) and 1,5-dibromopentan (3.38 mL, 0.029
mmol) were warmed at reflux temperature for 6 h, and then

Med Chem Res
diethyl ether was added. The resulting solid was filtered and
then subject to anion-exchange into chloride form to give
compound 3. Yield: 61 %, m.p. 195–197 °C; IR (KBr)
cm−1: 3065 (C–H, Ar), 1615–1565 (C=C, Ar), 1115–1050
(C–O–C); 1H NMR(CDCl3, 400 MHz): δ 9.86 (s, 1H, H-8),
8.49 (s,1H, H-13), 7.86 (s, 1H, H-1), 7.54 (s, 1H, H-12),
7.54 (s, 1H, H-4), 6.84 (s, 1H, H-11), 6.03 (s, 2H,-OCH2O),
4.93 (t, 2H, J = 6.3, H-6), 4.35 (t, 2H, J = 6.5, H-15), 4.03
(s, 3H, -OCH3), 3.19 (t, 2H, J = 6.4, H-19), 2.58 (t, 2H, J =
7.4, H-5), 2.43 (br s, 4H, H-17, H-18), 2.21 (m, 2H, H-16);
high resolution mass spectrum (HRMS) m/z [M-Cl]+ calcd.
for [C24H25BrNO4]+: 471.36; found: 471.75; Anal. Calcd.
for C24H25BrClNO4: C, 56.88; H, 4.97; N, 2.76. Found: C,
56.75; H, 5.07; N, 2.54.
(C–O–C), 760 (C–Cl). 1H NMR (CDCl3, 400 MHz): δ 9.58
(s, 1H, H-8), 8.62 (s, 1H, H-13), 8.03 (s, 1H, H-1), 7.51 (s,
1H, H-12), 7.48 (s, 1H, H-4), 7.38–7.15 (m, 4H, Ar–H,
piperazine H-27, H-28, H-29, H-30), 6.95 (s, 1H, H-11),
6.16 (s, 2H, –OCH2O), 4.82 (t, 2H, J = 6.3, H-6), 4.47 (t,
2H, J = 6.5, H-15), 4.14 (s, 3H, OCH3), 3.86 (br s, 4H,
piperazine, H-23, H-25), 3.56 (br s, 4H, piperazine, H-20,
H-22), 3.43 (t, 2H, J = 6.4, H-19), 2.73 (t, 2H, J = 7.6, H-5),
2.47 (br s, 4H, H-17, H-18), 2.18 (m, 2H, H-16); 13C NMR
(DMSO, 400 MHz): δ 153.9, 152.2, 149.7, 147.4, 143.5,
142.6, 139.3, 136.8, 133.1, 132.0, 131.9, 129.2, 123.7,
121.4, 120.5, 119.6, 116.3, 110.8, 107.1, 103.0, 73.9, 59.2,
56.7, 54.4, 52.5, 50.6, 28.3, 27.8, 25.1, 22.0. HRMS m/z
[M–Cl]+ calcd. for [C34H37ClN3O4]+: 587.13; found:
587.36. Anal. calcd. for C34H37Cl2N3O4: C, 65.59; H, 5.99;
N, 6.75. Found: C, 65.73; H, 6.11; N, 6.46.
General procedure for the preparation of derivatives
(5a–j)
9-O-3-(1-(3-Chlorophenyl)piperazine)pentylberberine (5c)
In a flask charged with dimethylformamide (DMF) (25 mL)
then add substituent piperazine (0.01 mmol), compound 3
(0.01 mmol) and anhydrous K2CO3 (0.012 mmol). The
reaction mixture was heated at 80 °C for 6–8 h, and TLC
monitored the reaction. The resulting solid was filtered at
room temperature and subjected to anion exchange into
chloride form. The crude product was chromatographed on
an Al2O3 column, eluted with CHCl3/CH3OH (9:1, v/v) to
give the proposed compound.
Light yellow solid, yield: 58 %. m.p. 228–230 °C. IR (KBr)
cm−1: 3038 (C–H, Ar), 1617–1547 (C=C, Ar), 1126–1067
(C–O–C), 747 (C–Cl). 1H NMR (CDCl3, 400 MHz): δ 9.78
(s, 1H, H-8), 8.68 (s, 1H, H-13), 7.88 (s, 1H, H-1), 7.62 (s,
1H, H-12), 7.51 (s, 1H, H-4), 7.30–7.04 (m, 4H, Ar–H,
piperazine H-27, H-28, H-29, H-31), 6.92 (s, 1H, H-11),
6.12 (s, 2H, –OCH2O), 4.89 (t, 2H, J = 6.2, H-6), 4.51 (t,
2H, J = 6.4, H-15), 4.12 (s, 3H, OCH3), 3.72 (br s, 4H,
piperazine, H-23, H-25), 3.51 (br s, 4H, piperazine, H-20,
H-22), 3.31 (t, 2H, J = 6.3, H-19), 2.71 (t, 2H, J = 7.8, H-5),
2.45 (br s, 4H, H-17, H-18), 2.11 (m, 2H, H-16); 13C NMR
(DMSO, 400 MHz): δ 153.6, 152.1, 149.8, 147.3, 143.0,
142.9, 139.2, 136.5, 133.4, 132.7, 131.6, 129.1, 123.8,
121.3, 120.0, 119.9, 116.2, 110.5, 107.4, 103.7, 73.6, 59.1,
56.8, 54.3, 52.0, 50.9, 28.2, 27.5, 25.4, 22.7. HRMS m/z
[M–Cl]+ calcd. for [C34H37ClN3O4]+: 587.13; found:
587.29. Anal. calcd. for C34H37Cl2N3O4: C, 65.59; H, 5.99;
N, 6.75. Found: C, 65.44; H, 6.07; N, 6.91.
9-O-3-(1-(4-Methylphenyl)piperazine)pentylberberine (5a)
Light yellow solid, yield: 55 %. m.p. 234–236 °C. IR (KBr)
cm−1: 3031 (C–H, Ar), 1610–1553 (C=C, Ar), 1117–1080
(C–O–C). 1H NMR (CDCl3, 400 MHz): δ 9.81 (s, 1H, H-8),
8.47 (s, 1H, H-13), 7.86 (s, 1H, H-1), 7.53 (s, 1H, H-12),
7.43 (s, 1H, H-4), 7.34–7.23 (m, 4H, Ar–H, piperazine H-
27, H-28, H-30, H-31), 6.75 (s, 1H, H-11), 6.02 (s, 2H,
–OCH2O), 4.98 (t, 2H, J = 6.4, H-6), 4.31 (t, 2H, J = 6.6,
H-15), 4.13 (s, 3H, OCH3), 3.81 (br s, 4H, piperazine, H-23,
H-25), 3.46 (br s, 4H, piperazine, H-20, H-22), 3.18 (t, 2H,
J = 6.4, H-19), 2.54 (t, 2H, J = 7.5, H-5), 2.43 (br s, 4H, H-
17, H-18), 2.15 (m, 2H, H-16), 1.90 (s, 3H, Ar–CH3); 13C
NMR (DMSO, 400 MHz): δ 153.2, 152.9, 149.4, 147.7,
143.6, 142.5, 139.8, 136.3, 133.0, 132.1, 131.2, 129.9,
123.4, 121.7, 120.6, 119.5, 116.8, 110.3, 107.0, 103.1,
73.2, 59.9, 56.4, 54.7, 52.6, 50.5, 28.8, 27.3, 25.0, 22.1,
21.3. HRMS m/z [M–Cl]+ calcd. for [C35H40N3O4]+:
566.71; found: 566.48. Anal. calcd. for C35H40ClN3O4: C,
69.81; H, 6.70; N, 6.98. Found: C, 69.75; H, 6.82; N, 6.72.
9-O-3-(1-(4-Chlorophenyl)piperazine)pentylberberine (5d)
Light yellow solid, yield: 67 %. m.p. 251–253 °C. IR (KBr)
cm−1: 3043 (C–H, Ar), 1622–1566 (C=C, Ar), 1104–1075
(C–O–C), 783 (C–Cl). 1H NMR (CDCl3, 400 MHz): δ 9.75
(s, 1H, H-8), 8.67 (s, 1H, H-13), 8.06 (s, 1H, H-1), 7.67 (s,
1H, H-12), 7.45 (s, 1H, H-4), 7.39–7.06 (m, 4H, Ar–H,
piperazine H-27, H-28, H-30, H-31), 6.79 (s, 1H, H-11),
6.08 (s, 2H, –OCH2O), 4.86 (t, 2H, J = 6.1, H-6), 4.38 (t,
2H, J = 6.2, H-15), 4.02 (s, 3H, OCH3), 3.75 (br s, 4H,
piperazine, H-23, H-25), 3.49 (br s, 4H, piperazine, H-20,
H-22), 3.23 (t, 2H, J = 6.5, H-19), 2.58 (t, 2H, J = 7.5, H-5),
2.39 (br s, 4H, H-17, H-18), 2.21 (m, 2H, H-16); 13C NMR
(DMSO, 400 MHz): δ 153.3, 152.6, 149.5, 147.8, 143.7,
142.0, 139.9, 136.1, 133.2, 132.4, 131.3, 129.6, 123.5,
9-O-3-(1-(2-Chlorophenyl)piperazine)pentylberberine (5b)
Light yellow solid, yield: 64 %. m.p. 241–243 °C. IR (KBr)
cm−1: 3025 (C–H, Ar), 1604–1560 (C=C, Ar), 1104–1091

Med Chem Res
121.8, 120.7, 119.0, 116.9, 110.1, 107.2, 103.4, 73.3, 59.6,
56.5, 54.8, 52.7, 50.0, 28.9, 27.1, 25.2, 22.4. HRMS m/z
[M–Cl]+ calcd. for [C34H37ClN3O4]+: 587.13; found:
587.02. Anal. calcd. for C34H37Cl2N3O4: C, 65.59; H, 5.99;
N, 6.75. Found: C, 65.42; H, 5.89; N, 6.57.
(C–O–C).1H NMR (CDCl3, 400 MHz): δ 9.67 (s, 1H, H-8),
8.61 (s, 1H, H-13), 8.11 (s, 1H, H-1), 7.61 (s, 1H, H-12),
7.55 (s, 1H, H-4), 7.32–7.11 (m, 4H, Ar–H, piperazine H-
27, H-28, H-30, H-31), 6.81 (s, 1H, H-11), 6.05 (s, 2H,
–OCH2O), 4.85 (t, 2H, J = 6.1, H-6), 4.41 (t, 2H, J = 6.4,
H-15), 4.04 (s, 3H, OCH3), 3.68 (br s, 4H, piperazinee, H-
23, H-25), 3.39 (br s, 4H, piperazinee, H-20, H-22), 3.18 (t,
2H, J = 6.5, H-19), 2.44 (t, 2H, J = 7.6, H-5), 2.41 (br s, 4H,
H-17, H-18), 2.17 (m, 2H, H-16); 13C NMR (DMSO, 400
MHz): δ 153.1, 152.5, 149.0, 147.6, 143.4, 142.3, 139.7,
136.2, 133.8, 132.9, 131.1, 129.5, 123.0, 122.4, 121.6,
120.4, 119.3, 116.7, 110.2, 107.8, 103.9, 73.1, 59.5, 56.0,
54.6, 52.4, 50.3, 28.7, 27.2, 25.8, 22.9. HRMS m/z [M–Cl]+
calcd. for [C35H37F3N3O5]+: 636.68; found: 636.84. Anal.
calcd. for C35H37ClF3N3O5: C, 62.54; H, 5.55; N, 6.25.
Found: C, 62.68; H, 5.41; N, 6.41.
9-O-3-(1-(2-Fluorophenyl)piperazine)pentylberberine (5e)
Light yellow solid, yield: 53 %. m.p. 218–220 °C. IR (KBr)
cm−1: 3019 (C–H, Ar), 1617–1564 (C=C, Ar), 1116–1093
(C–O–C), 760 (C–Cl). 1H NMR (CDCl3, 400 MHz): δ 9.82
(s, 1H, H-8), 8.77 (s, 1H, H-13), 7.84 (s, 1H, H-1), 7.57 (s,
1H, H-12), 7.61 (s, 1H, H-4), 7.26–7.13 (m, 4H, Ar–H,
piperazine H-27, H-28, H-29, H-30), 6.76 (s, 1H, H-11),
6.15 (s, 2H, –OCH2O), 4.83 (t, 2H, J = 6.4, H-6), 4.53 (t,
2H, J = 6.5, H-15), 4.17 (s, 3H, OCH3), 3.79 (br s, 4H,
piperazine, H-23, H-25), 3.53 (br s, 4H, piperazine, H-20,
H-22), 3.16 (t, 2H, J = 6.6, H-19), 2.48 (t, 2H, J = 7.3, H-5),
2.33 (br s, 4H, H-17, H-18), 2.25 (m, 2H, H-16); 13C NMR
(DMSO, 400 MHz): δ 153.7, 152.4, 149.1, 147.5, 143.3,
142.2, 139.0, 136.9, 133.6, 132.8, 131.7, 129.4, 123.1,
121.5, 120.3, 119.3, 116.0, 110.9, 107.6, 103.8, 73.7, 59.4,
56.1, 54.5, 52.3, 50.2, 28.0, 27.9, 25.6, 22.8. HRMS m/z
[M–Cl]+ calcd. for [C34H37FN3O4]+: 570.67; found: 570.34.
Anal. calcd. for C34H37ClFN3O4: C, 67.37; H, 6.15; N,
6.93. Found: C, 67.51; H, 6.32; N, 6.78.
9-O-3-(4-Trifluoromethylphenyl)piperazine)pentylberberine
(5h)
Light yellow solid, yield: 58 %. m.p. 221–223 °C. IR (KBr)
cm−1: 3017 (C–H, Ar), 1605–1563 (C=C, Ar), 1108–1086
(C–O–C). 1H NMR (CDCl3, 400 MHz): δ 9.63 (s, 1H, H-8),
8.52 (s, 1H, H-13), 8.07 (s, 1H, H-1), 7.59 (s, 1H, H-12),
7.49 (s, 1H, H-4), 7.29–7.17 (m, 4H, Ar–H, piperazine H-
27, H-28, H-30, H-31), 6.91 (s, 1H, H-11), 6.02 (s, 2H,
–OCH2O), 4.81 (t, 2H, J = 6.2, H-6), 4.46 (t, 2H, J = 6.3,
H-15), 4.16 (s, 3H, OCH3), 3.78 (br s, 4H, piperazine, H-23,
H-25), 3.55 (br s, 4H, piperazine, H-20, H-22), 3.32 (t, 2H,
J = 6.3, H-19), 2.49 (t, 2H, J = 7.3, H-5), 2.37 (br s, 4H, H-
17, H-18), 2.06 (m, 2H, H-16); 13C NMR (DMSO, 400
MHz): δ 153.5, 152.8, 149.6, 147.0, 143.2, 142.4, 139.1,
136.7, 133.9, 132.3, 131.5, 129.8, 125.2, 123.6, 121.0,
120.2, 119.4, 116.1, 110.7, 107.9, 103.3, 73.5, 59.8, 56.6,
54.0, 52.2, 50.4, 28.1, 27.7, 25.9, 22.3. HRMS m/z
[M–Cl]+ calcd. for [C35H37F3N3O4]+: 620.68; found:
620.51. Anal. calcd. for C35H37ClF3N3O4: C, 64.07; H,
5.68; N, 6.40. Found: C, 63.98; H, 5.53; N, 6.63.
9-O-3-(1-(4-Fluorophenyl)piperazine)pentylberberine (5f)
Light yellow solid, yield: 55 %. m.p. 234–236 °C. IR (KBr)
cm−1: 3033 (C–H, Ar), 1611–1553 (C=C, Ar), 1128–1072
(C–O–C), 773 (C–Cl). 1H NMR (CDCl3, 400 MHz): δ 9.73
(s, 1H, H-8), 8.53 (s, 1H, H-13), 8.04 (s, 1H, H-1), 7.69 (s,
1H, H-12), 7.47 (s, 1H, H-4), 7.33–7.12 (m, 4H, Ar–H,
piperazine H-27, H-28, H-30, H-31), 6.84 (s, 1H, H-11),
6.18 (s, 2H, –OCH2O), 4.95 (t, 2H, J = 6.3, H-6), 4.35 (t,
2H, J = 6.6, H-15), 4.23 (s, 3H, OCH3),3.73 (br s, 4H,
piperazine, H-23, H-25), 3.42 (br s, 4H, piperazine, H-20,
H-22), 3.12 (t, 2H, J = 6.4, H-19), 2.53 (t, 2H, J = 7.5, H-5),
2.48 (br s, 4H, H-17, H-18), 2.03 (m, 2H, H-16). 13C NMR
(DMSO, 400 MHz): δ 153.8, 152.0, 149.3, 147.9, 143.1,
142.7, 139.4, 136.6, 133.5, 132.2, 131.8, 129.0, 123.3,
121.9, 120.1, 119.7, 116.4, 110.6, 107.5, 103.2, 73.8, 59.0,
56.3, 54.9, 52.1, 50.7, 28.4, 27.6, 25.5, 22.2. HRMS m/z
[M–Cl]+ calcd. for [C34H37FN3O4]+: 570.67; found: 570.85.
Anal. calcd. for C34H37ClFN3O4: C, 67.37; H, 6.15; N,
6.93. Found: C, 67.21; H, 6.03; N, 6.75.
9-O-3-(1-(2-Nitrophenyl)piperazine)pentylberberine (5i)
Light yellow solid, yield: 61 %. m.p. 251–253 °C. IR (KBr)
cm−1: 3024 (C–H, Ar), 1618–1573 (C=C, Ar), 1533 (N–O
stretch), 1347 (N–O stretch), 1117–1080 (C–O–C). 1H
NMR (CDCl3, 400 MHz): δ 9.78 (s, 1H, H-8), 8.71 (s, 1H,
H-13), 7.90 (s, 1H, H-1), 7.65 (s, 1H, H-12), 7.57 (s, 1H, H-
4), 7.37–7.01 (m, 4H, Ar–H, piperazine H-27, H-28, H-29,
H-30), 6.88 (s, 1H, H-11), 6.14 (s, 2H, –OCH2O), 4.87 (t,
2H, J = 6.3, H-6), 4.37 (t, 2H, J = 6.5, H-15), 4.07 (s, 3H,
OCH3), 3.96 (br s, 4H, piperazine, H-23, H-25), 3.59 (br s,
4H, piperazinee, H-20, H-22), 3.17 (t, 2H, J = 6.2, H-19),
9-O-3-(4-Trifluoromethoxyphenyl)piperazine)
pentylberberine (5g)
Light yellow solid, yield: 51 %. m.p. 244–246 °C. IR (KBr)
cm−1: 3037 (C–H, Ar), 1613–1546 (C=C, Ar), 1121–1076

Med Chem Res
2.61 (t, 2H, J = 7.5, H-5), 2.44 (br s, 4H, H-17, H-18), 2.19
(m, 2H, H-16); 13C NMR (DMSO, 400 MHz): δ 153.0,
152.3, 149.2, 147.1, 143.9, 142.8, 139.5, 136.4, 133.7,
132.6, 131.0, 129.3, 123.2, 121.1, 120.9, 119.8, 116.5,
110.4, 107.7, 103.6, 73.0, 59.3, 56.2, 54.1, 52.9, 50.8, 28.5,
27.4, 25.7, 22.6. HRMS m/z [M–Cl]+ calcd. for
[C34H37N4O6]+: 597.68; found: 597.45. Anal. calcd. for
C34H37ClN4O6: C, 64.50; H, 5.89; N, 8.85. Found: C,
64.33; H, 5.76; N, 8.67.
was operated according to the methodology described by
Brand-Williams et al. (1995) with some modifications
(Mistry et al., 2016). A stable free radical, DPPH, was
allowed to react with test compounds in methanol as 20 µg/
mL (100, 10, 1 and 0.1) quantities of title compounds were
mixed up with 180 µg/mL of DPPH in methanol. Titled
compounds donated hydrogen during the mixing thereby
introduced the reduction of DPPH and hence a change in
the color was observed from deep violet to light yellow
at 517 nm after 25 min of reaction in a UV–visible spec-
trophotometer (Perkin Elmer). The blank reading was also
performed using the mixture of methanol (20 µg/mL) and
sample (180 µg/mL of DPPH). Ascorbic acid served
as a control drug in this assay, and its solution was prepared
by mixing methanol (20 µg/mL) and DPPH radical solution
(180 µg/mL). The results of this bioassay, RSA % (the
radical scavenging activity in percentage) was determined
according to Mensor et al. (2001) as described in the
below equation.
9-O-3-(1-(4-Nitrophenyl)piperazine)pentylberberine (5j)
Light yellow solid, yield: 63 %. m.p. 244–246 °C. IR (KBr)
cm−1: 3041 (C–H, Ar), 1603–1564 (C=C, Ar), 1542 (N–O
stretch), 1331 (N–O stretch), 1108–1091 (C–O–C). 1H
NMR (CDCl3, 400 MHz): δ 9.76 (s, 1H, H-8), 8.63 (s, 1H,
H-13), 8.08 (s, 1H, H-1), 7.58 (s, 1H, H-12), 7.63 (s, 1H, H-
4), 7.39–7.09 (m, 4H, Ar–H, piperazine H-27, H-28, H-30,
H-31), 6.90 (s, 1H, H-11), 6.17 (s, 2H, –OCH2O), 4.90 (t,
2H, J = 6.4, H-6), 4.56 (t, 2H, J = 6.4, H-15), 4.03 (s, 3H,
OCH3), 3.91 (br s, 4H, piperazine, H-23, H-25), 3.54 (br s,
4H, piperazine, H-20, H-22), 3.28 (t, 2H, J = 6.5, H-19),
2.71 (t, 2H, J = 7.5, H-5), 2.52 (br s, 4H, H-17, H-18), 2.23
(m, 2H, H-16); 13C NMR (DMSO, 400 MHz): δ 153.4,
152.7, 149.9, 147.2, 143.8, 142.1, 139.6, 136.0, 133.3,
132.5, 131.4, 129.7, 123.9, 121.2, 120.8, 119.1, 116.6,
110.0, 107.3, 103.5, 73.4, 59.7, 56.9, 54.2, 52.8, 50.1, 28.6,
27.0, 25.3, 22.5. HRMS m/z [M–Cl]+ calcd. for
[C34H37N4O6]+: 597.68; found: 597.83. Anal. calcd. for
C34H37ClN4O6: C, 64.50; H, 5.89; N, 8.85. Found: C,
64.32; H, 5.98; N, 8.94.
% Scavenging
Absorbance of blank ꢀ Absorbance of test
¼
´ 100
Absorbance of blank
A plot of concentration of test compounds and %
scavenging introduced IC50s in the presence of an ascorbic
acid as standard.
2,2′-Azino-bis-3-ethylbenzthiazoline-6-sulphonic acid
(ABTS) radical scavenging assay
The ABTS•+ radical cation scavenging efficacies of the test
compounds was determined according to the method
described earlier (Re et al., 1999) with some modifications
(Mistry et al., 2016). Mixing of an equal amount of
7 mM ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid)) stock solution with 2.45 mM potassium
persulfate stock solution produces the ABTS•+ cation. The
mixture was kept in a dark place at 0 °C temperature for 12
h and ABTS solution was diluted with MeOH so that it
gives UV absorption value of 0.700 ( 0.200) at the 734 nm.
The 1000 µL stock solutions of titled compounds 5a–j was
established upon dissolving in methanol and further dilu-
tions furnishes 100, 10, 1 and 0.1 µg/mL quantities of test
samples. In all 180 µg/mL solutions of compounds to be
evaluated and 20 µg/mL of the ABTS solution were mixed
in 96-well plates in a dark place and then incubated for 10
min to measure UV absorption at 734 nm. A mixture of 180
µg/mL ABTS and 20 µg/mL methanol was used as a control
determination, while ascorbic acid was used as a reference
drug. The UV absorption data represented the radical
scavenging rates that give the corresponding IC50s for the
test compounds.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging assay
Reduction of a stable free radical, 2,2-diphenyl-1-picryl-
hydrazyl is the base of the DPPH antioxidant bioassay. It
has an odd electron that shows a maximum absorption band
of 517 nm (deep violet color) in ethanol. The DPPH
bioassay is the widely used and acceptable method for
evaluating the free radical scavenging action of the tested
compounds. Such substances donate a hydrogen atom when
mixed with the DPPH, thereby introducing its reduced
congener, diphenylpicrylhydrazine (non-radical) with the
loss of violet color.
In the present study, DPPH bioassay was adopted to
screen the berberine-based compounds for their in vitro
antioxidant profiles. The results of this bioassay investiga-
tion were introduced in the form of the percentage of radical
scavenging antioxidant activity (RSA %) of each substance.
The investigation of the DPPH radical scavenging activity

Med Chem Res
The scavenging capability of ABTS•+ radical was cal-
culated using the following equation:
incubated overnight (Adaramoye et al., 2011; Mistry et al.,
2016). Spectrophotometric data were recorded at 510 nm to
calculate the inhibitory concentration of 50 % (IC50),
cytotoxic concentration of 50 % (CC50) and therapeutic
index (TI).
% Scavenging
Absorbance of blank ꢀ Absorbance of test
¼
´ 100
Absorbance of blank
Results and discussion
Chemistry
In vitro anticancer bioassay
Cell cultures
Scheme 1 represents the synthetic pathway followed to
generate berberine-piperazine derivatives (5a–j). At first,
demethylation of berberine hydrochloride itself under
vacuum oven at 190 oC temperature and 20–30 mmHg
pressure introduced berberrubine (2) with 85 % of yield
(Iwasa et al., 1996). Alkylation was followed in the further
step in the presence of dibromoalkanes in dry acetonitrile
(Mistry et al., 2015; Mistry et al., 2016a; Mistry et al.,
2016b) yielding final intermediate 3. In the last step, dif-
ferent piperazine moieties were coupled with intermediate 3
in DMF for 6–8 h to construct 5a–j with (51–67 %) rea-
sonable yields.
The test compounds 5a–j were checked for their in vitro
anticancer action against cervical cancer cell line HeLa,
CaSki and non-cancer Madin-Darby canine kidney
(MDCK) cells which were purchased from American Type
Culture Collection. All the cell lines were well maintained
in a humidified cell culture incubator in the presence of 5 %
of CO2 at 37 °C temperature for cell growth. Dulbecco’s
Modified Eagle’s Medium (DMEM) and RPMI-1640
medium supplemented with 10 % of fetal bovine serum
(FBS) and 1 % of Antibiotic-Antimycotic Solution
(100×) were used for HeLa, CaSki and MDCK cell
growth, respectively. DMEM, RPMI-1640, trypsin–EDTA,
Antibiotic-Antimycotic Solution 100× and FBS were pur-
chased from Welgene (150-Seongseo Industrial complex
Bukro, Dalseogu, Daegu, 704–948 Republic of Korea).
In the 96-well plates, both the cancer cell lines HeLa,
CaSki and a non-cancer cell line MDCK were seeded, and
plates were concentrated as 2 × 104 cells per well plate.
Cancerous cells were allowed to grow for 1 day initially and
after that the 96-well plates were washed twice with phos-
phate buffer saline (PBS). DMEM and RPMI-1640 medium
containing trypsin–EDTA were used to dilute HeLa, CaSki
and MDCK cells upto 5 × 103 level which was used for the
infection followed by pacing of 10 µg/mL of compound
quantities and 90 µg/mL of cell solution onto the 96-well
plates in which HeLa, CaSki and MDCK cells were grown
the previous day. In all 0.1, 1, 10 and 100 µg/mL con-
centrations of the test compounds were used in 96-well
plates for the analysis with three replicates of observations.
Infected plates were incubated in a CO2 incubator for 48 h.
After incubation, the medium was taken out and cleaned
twice with PBS buffer. After that, 70 % of acetone was
added to fix the cells and were incubated for 1 h at 4 °C.
After incubation, the solvent was removed, and plates were
dried in an oven at 60 °C. The dried plates were overnight
incubated 100 µg/mL of sulforhodamine B (SRB; 0.4 mg/L)
followed by SRB removal and washing thrice with 1 % of
acetic acid and dried again under hot air oven at 60 °C.
Microscopic observation was performed to identify the
morphology of the cells and to follow this observation, the
SRB strain was dissolved with 10 mM of Tris base and
The correct formation of the structure of compounds
5a–j was confirmed utilizing spectroscopic techniques such
as FT-IR, 1H NMR, mass spectrometry and elemental
analysis (CHN). Absorption bands at 3043–3017 cm−1 for
the FT-IR of 5a–j confirmed the presence of C-H stretching
for aromatics, whereas C=C band of aromatic ring observed
in the range 1622–1546 cm−1. The C-O-C band indicated a
sharp peak nearby 1128–1067 cm−1 along with sharp
1
chlorine signal at 790–740 cm−1. In the H NMR spectra
(5a), proton atoms of the berberine ring resonated at 9.81,
8.47, 7.86, 7.53, 7.43, 6.75 ppm for H-8, H-13, H-1, H-12,
H-4, H-11, respectively, in the form of singlet along with
singlet peak observe at 6.02 ppm due to the presence of
-OCH2O of berberine. Also, a triplet was indicated at 4.98
and 2.54 ppm due to H-6 and H-5 proton atoms of the
berberine ring. An alkyl chain proton (H-15) falls at around
4.31 ppm in the form of the triplet. Furthermore, a triplet
signal at 3.18 ppm, a broad singlet at 2.43 ppm, as well as
multiplet signals at 2.15 ppm, were due to the alkyl chain
protons H-19, H-17 and H-18, as well as H-16, respectively.
The proton atoms belonging to the methoxy functional
group were found to resonate at 4.13 ppm, whereas methyl
protons of the piperazine ring were observed to have their
presence around 1.90 ppm. The protons (H-23, H-25, H-20
and H-22) of piperazine ring were resonated as a broad
singlet at around 3.81 and 3.46 ppm. Moreover, aromatic
rings linked to the piperazine ring appeared to have corre-
sponding signals as multiples in the range 7.34–7.23 ppm.
The 13C NMR data observed for compound 5a further
confirmed the correct formation of the synthesized structure

Med Chem Res
Scheme 1 Synthesis of piperazine-linked berberine derivatives
of the compounds. The high-resolution mass spectral data of
compounds 5a–j exhibited molecular ion peaks at their
respective molecular weights, which confirmed their for-
mation. All of the novel compounds gave C, H and N
analyses within 0.4 percent points from the theoretical
values, i.e., in an acceptable range.
scaffolding varies considerably based upon on the back-
bone structures and functional groups linked with the ber-
berine through a pentyl chain. Overall derivatives of
berberine had used enhanced efficacy in both the anti-
oxidant bioassays in evaluation to parent analog berberine.
Different electron-withdrawing functional groups such as
chloro, fluoro and nitro, as well as electron-donating func-
tional group such as methyl were present on the piperazine
moieties. Title analogues 5a–j exhibited 17.25–25.40 µg/mL
of IC50 levels in DPPH free radical scavenging assay in
which the highest scavenger activity seen in compound 5c is
probably due to the existence of meta-chloro piperazine
entity with 17.25 µg/mL of IC50. In addition, for all of the
remaining analogues IC50 level varied from 21 to 22 µg/mL,
which can be regarded to be antioxidant action when
Evaluation of biological activities
Antioxidant activities
Final analogues 5a–j was examined for their in vitro anti-
oxidant potencies using DPPH and ABTS bioassay and
results are summarized in Table 1. It was noticed that the
anti-oxidant activity of the piperazine based berberine a

Med Chem Res
Table 1 Screening results of DPPH and ABTS radical scavenging
activity of berberine derivatives (5a–j)
Table 2 Anticancer activity of synthesized compounds against HeLa
cancer cell and their toxicity
Compounds
aIC50 μg/mL SD
Compounds
aIC50 μg/mL SD
CC50 μg/mL SD
TI
HeLa
MDCK
DPPH
ABTS
5a
5.595 0.02
7.532 0.06
7.534 0.06
5.526 0.03
7.632 0.09
5.599 0.02
7.542 0.11
7.180 0.07
7.657 0.13
7.691 0.07
5.611 0.01
327.5 0.05
315.1 1.14
318.5 3.35
236.8 3.75
304.9 0.87
232.9 2.72
216.9 2.40
206.7 2.55
261.4 0.49
197.9 2.10
153.8 1.16
58.53
41.83
42.28
42.85
39.95
41.60
28.76
28.79
34.14
25.73
27.41
5a
25.40 1.03
25.28 3.12
17.25 0.79
21.51 2.15
21.87 4.17
21.82 1.21
22.40 0.68
24.19 2.07
22.37 3.07
22.72 1.06
34.29 2.75
11.52 1.23
7.512 1.95
7.267 1.34
7.470 0.76
4.778 0.86
6.010 1.88
6.543 2.24
6.737 1.65
7.184 1.18
6.803 2.04
8.917 0.78
82.17 4.02
5.528 2.04
5b
5b
5c
5c
5d
5d
5e
5e
5f
5f
5g
5g
5h
5h
5i
5i
5j
5j
Berberine
Berberine
Ascorbic acid
CC50 cytotoxicity concentration of 50 %, TI therapeutic index
a
Anticancer 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
ABTS 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid, DPPH
2,2-diphenyl-1-picrylhydrazyl
a Antioxidant activities are shown as IC50 values in μg/mL. All assays
were carried out in triplicate, and the results expressed as anaverage
standard deviation
Table 3 Anticancer activity of synthesized compounds against CaSki
cancer cell and their toxicity
compared to the potency of the parent compound berberine
at 34.29 µg/mL and control drug ascorbic acid at 11.52 µg/
mL. The least active scaffold was holding the methyl group
at 25.40 µg/mL of IC50. Overall, it can be stated that the
presence of chlorine atom was the most beneficial part of
the enhanced potency of the title compounds in DPPH
assay. Secondly, the said potency of compounds 5a–j was
further verified via examining in ABTS bioassay, in which
newer scaffolds demonstrated a significant level of anti-
oxidant efficacies with IC50s ranged from 4.778 to 8.917 µg/
mL. The most active free radical scavenger was compound
5d with 4.778 µg/mL of the IC50 level, hence again con-
firming the key role of chlorine atom(s). Remaining com-
pounds exhibited IC50s (6.010–7.512 µg/mL) near to that of
control drugs ascorbic acid at 5.528 µg/mL, and excellent
potencies when compared to the parent compound berberine
with 82.17 µg/mL of IC50.
Compounds
aIC50 μg/mL SD
CaSki
CC50 μg/mL SD
MDCK
TI
5a
6.716 0.05
6.654 0.09
6.878 0.24
5.742 0.07
6.439 0.08
5.989 0.09
6.602 0.15
6.515 0.05
6.769 0.13
6.739 0.25
5.951 0.06
327.5 0.05
315.1 1.14
318.5 3.35
236.8 3.75
304.9 0.87
232.9 2.72
216.9 2.40
206.7 2.55
261.4 0.49
197.9 2.10
153.8 1.16
48.76
47.35
46.31
41.24
47.35
38.89
32.85
31.73
38.62
29.37
25.84
5b
5c
5d
5e
5f
5g
5h
5i
5j
Anticancer activities
Berberine
CC50 cytotoxicity concentration of 50 %, TI therapeutic index
In the present work, compounds 5a–j were checked for their
in vitro anticancer efficacies using a SRB bioassay and the
results are summarized in Table 2 and Table 3. The results
a
Anticancer activities are shown as IC50 values in μg/mL. All assays
were carried out in triplicate, and the results expressed as anaverage
standard deviation

Med Chem Res
of anticancer screening suggested that such analogues are
potent inhibitors of the growth of cervical cancer cell
lines HeLa and CaSki with IC50 levels of around
5.595–7.691 µg/mL and 5.742–6.878 µg/mL, as well as CC50
levels of around 197.9–327.5 µg/mL, respectively. Overall,
tested compounds demonstrated the low cytotoxic nature
towards the non-cancer MDCK cell line and as a result
presented remarkable TIs of around 25.73–58.53 and
29.37–48.76, better than that of a parent compound berberine
with 27.41 and 25.84 of TI, respectively, against HeLa and
CaSki. An analogue 5a with piperazine entity bearing
electron-releasing methyl functional group displayed 5.595
0.02 µg/mL of IC50 and 327.5 0.05 µg/mL of CC50 level,
presenting the highest anticancer potential against HeLa cell
line with 58.53 of TI. Moreover, the same analogue had the
highest efficacy against CaSki cell line with 6.716 0.05 µg/
mL of IC50 and 327.5 0.05 µg/mL of CC50 and 48.76 of TI.
The inhibitory potential of cervical cancer cell lines was
followed by a group of final analogues bearing piperazine
moiety with electron-withdrawing chlorine functionality as
compound 5b, 5c and 5d exhibited TIs of 41.83, 42.28 and
42.85, as well as 47.35, 46.31 and 41.24 against HeLa and
CaSki cell lines, respectively. Overall, in regard with the TI,
title compounds were having higher inhibitory potential
against CaSki cell lines when compared to HeLa. Compound
5f with para-fluorophenylpiperazine moiety and 5e with
ortho-fluorophenylpiperazine moiety emerged with equipo-
tent anticancer effects as their chloro precursors with TI of
41.60 and 47.35 against HeLa and CaSki cell lines, respec-
tively. Compounds with para-trifluoromethoxy (5g), para-
trifluoromethyl (5h) and ortho and para-nitro (5i and 5j)
functional groups have the least activities observed in the
bioassay with TI levels 25–38 against both the cell lines, but
still it was equal or higher than that of berberine itself.
Concerning the activity regarding the functional group
attached to the piperazine ring, the order falls in the way
alkyl > chloro > fluoro > nitro.
improved antioxidant and anticancer effects than berberine
and good to moderate potencies when compared to the
control drugs. Compound 5a with methyl functionality and
5c with chloro functionality exhibited significant
anticancer and antioxidant potential, respectively, and can be
considered further in the ongoing drug discovery process.
The molecular designs and rationalization presented with this
can be a tool to process for further modification in the
molecular systems to obtain an advanced level of therapeutic
potencies.
Acknowledgments This article was supported by the KU Research
Professor Program of Konkuk University, Seoul, South Korea.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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Conclusion
In summary, isoquinoline alkaloid natural analogue berberine
has been utilized to furnish a new class of substituted
piperazine-based scaffolds. Efficient synthesis of final ana-
logues was verified using different spectroscopic methods,
elemental analyses and physical measurements. Final com-
pounds were tested for their antioxidant and anticancer
effects (HeLa and CaSki cervical cancer cell lines). More
specifically, meta-chloropiperazine was valuable for the
radical scavenging effects, whereas para-methyl piperazine
had applied better anticancer effects. All compounds exhib-
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