Synthesis and biological evaluation of berberine derivatives bearing 4-aryl-1-piperazine moieties

Article abstract of DOI:10.3184/174751915X14381686689721

Piperazine moieties with disubstituted N-aryl groups are linked to the isoquinoline alkaloid, berberine, through a pentyloxy side chain, replacing its 9-methoxyl group. The nine synthesised compounds are screened for antioxidant potency, in vitro anticancer activities against Hela and Caski cervical cancer cell lines and for cytotoxicity towards Malin Darby canine kidney cell lines. Several compounds demonstrate significant antioxidant potency and most of the compounds exhibit equipotent, or better, anticancer activity when compared to berberine.

Full text of DOI:10.3184/174751915X14381686689721

470 RESEARCH PAPER
VOL. 39 AUGUST, 470–474
JOURNAL OF CHEMICAL RESEARCH 2015
Synthesis and biological evaluation of berberine derivatives bearing
4-aryl-1-piperazine moieties
Bhupendra Mistry*, Young-Soo Keum and Doo Hwan Kim
Organic Research Laboratory, Department of Bioresources and Food Sciences, College of Life and Environmental Sciences, Konkuk University, Seoul,
South Korea
Piperazine moieties with disubstituted N-aryl groups are linked to the isoquinoline alkaloid, berberine, through a pentyloxy side chain,
replacing its 9-methoxyl group. The nine synthesised compounds are screened for antioxidant potency, in vitro anticancer activities
against Hela and Caski cervical cancer cell lines and for cytotoxicity towards Malin Darby canine kidney cell lines. Several compounds
demonstrate significant antioxidant potency and most of the compounds exhibit equipotent, or better, anticancer activity when compared
to berberine.
Keywords: berberine, piperazine, antioxidant, cervical cancer
The occurrence of diseases like diabetes, cirrhosis, cancer
and cardiovascular defects is associated with the presence of
free radicals,1,2 termed as reactive oxygen species (ROS). An
imbalance between oxidants and antioxidants in favour of the
oxidants, potentially leading to damage towards lipids, protein
and nucleic acids, is termed oxidative stress.3Antioxidants are
molecules, natural or synthetic, capable of interfering with free
radicals and avoiding their sequence responses before essential
vital components are damaged.4 Research suggests that
oxidative stress and ROS have a direct correlation with cancer
and hence discovering new molecules capable of targeting both
ROS scavenging, and cancer cell inhibition are of immense
interest.
Natural products have been the single most useful source of
leads for the development of drugs. Berberine, an isoquinoline
alkaloid isolated from the roots and control debris of the
Berberis varieties, is commonly used as a conventional
medicine for treating diarrhoea and gastrointestinal disorders.5
It possesses a variety of pharmacological properties such as
antimicrobial, antileukaemic, antiulcerous, anti-inflammatory,6
and anti-diarrhoea activities.7 It also acts as an an enzyme
inhibitor,8,9 and as a glucose-lowering,10 cholesterol-lowering,11
neuroprotective,12 antidepressant,13 and Alzheimers disease-
ameliorating agent.14 It has also been reported that berberine
analogues exhibit activity on LDLR,15 and cytotoxicity against
Hela, SVKO3 and Hep-2 cells,16 and anti-leishmaniasis activity.17
Research on the anticancer action of berberine, in particular,
has attratcted extensive interest and there have been significant
results.18-24 However, since berberine is poorly absorbed in the
digestive system it had a weak inhibitory impact on cancer cell
growth, decreasing its effectiveness as an anticancer drug.25,26
Hence, discovery of its derivatives which show pharmacological
potential as anticancer agents better than berberine itself is of
key importance in the ongoing anticancer drug development
program. Because piperazines have useful pharmacological
applications as anticancer agents27 and antioxidants,28 and
inspired by the wide range of useful activities possessed by
berberine derivatives we report here the synthesis of some novel
piperazine-based berberine analogues.
mm Hg pressure in 85% yield using a lietarture method.29 In the
final steps, dibromopentane in dry acetonitrile was employed
for the alkylation of the 9-hydroxy group to yield intermediate
compound 3 which was coupled with various piperazine
moieties by a nucleophilic substitution reaction in DMF for 6–8
h to produce 4a–i in reasonable yields.30
Spectroscopic techniques (IR, 1H NMR, and mass
spectrometry) and elemental analysis (CHN) were used to
characterise compounds 4a–i. The presence of C–H stretching
for aromatics was confirmed on the basis of peaks observed
around 3037–3061 cm-1in the FTIR spectra of 4a–i, and C=C
stretching by bands at 1622–1531 cm-1. The C–O–C band was
seen as a sharp peak at 1121–1077 cm-1 along with , when
appropriate, sharp chlorine bands at 757–781 cm-1. The presence
of the berberine ring was established on the basis of results of 1H
NMR spectra. For example, (4b) showed protons corresponding
to H-8, H-13, H-1, H-12, H-4, H-11 as singlet peaks at 9.75, 8.66,
7.92, 7.51, 7.47, 6.77 ppm, respectively, as well as a singlet at
6.08 ppm due to the –OCH2O of the berberine ring. The H-6 and
H-5 methylate protons of the berberine ring appeared as triplets
at 4.86 ppm and 2.55 ppm. A triplet for the alkyl chain protons
(H-15) was observed at 4.41 ppm. A triplet signal at 3.25 ppm,
a broad singlet at 2.41 ppm as well as multiplet signals at 2.24
ppm were due to the alkyl chain protons H-19, H-17 and H-18
and H-16, respectively. The presence of a methoxy functional
group was confirmed by its characteristic peak at 4.05 ppm
and the protons (H-23, H-25, H-20 and H-22) present on the
piperazine moiety resonated as a broad singlet at around 3.79
ppm as well as at 3.45 ppm. The protons of the aromatic rings
linked to the piperazine ring appeared as multiplets in the range
7.41–7.20 ppm. Mass spectrometric data (M+ ion values) for
4a–i were in accordance with their structures.
Evaluation of biological activities
Antioxidant activities
The radical scavenging activity of berberine compounds 4a–i
towards radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)
was measured with ascorbic acid as a standard compound.
DPPH radical scavenging is considered a good in vitro model
and is widely used to assess antioxidant efficacy conveniently.31
Attachment of disubstituted piperazine derivatives to the
berberine nucleus through a pentyl chain has an enhanced
antioxidant effect as displayed in both the DPPH and ABTS
bioassays. The activity level was found to vary according to
Results and discussion
Scheme 1 shows the synthesis of the titled berberine-piperazine
derivatives 4a–i. Berberrubine (2), a demethylated derivative
of berberine and a key intermediate was prepared by heating
o
berberine in a vacuum oven at 190 C temperature and 20–30
* Correspondent. E-mail: bhupendra.mistry84@gmail.com

JOURNAL OF CHEMICAL RESEARCH 2015 471
O
O
l-
O
O
N+
C
l-
o
N+
H N
C 3C
C
1
90 C
H
O
H
H
OC
r
r
B
3
20-30 mm Hg
B
H
OC
3
1,5-dibromopentane
2
OC
3
1
O
O
l-
O
N+
C
l-
N+
C
X
O
N-aryl-piperazine
DMF
H
O C
N
N
(
)
2 5
r
2 5B
)
H
O C
(
H
OC
3
4
H
OC
3
3
X
4a
4b
4c
4d
4e
4f
2,3-Cl2
2,4-F2
2,4-(MeO)2
2,4-(Me)2
2,3-F2
2,4-Cl2
4g
4h
4i
2,3-(Me)2
3,4-Cl2
3,5-Cl2
Scheme 1 Synthesis of piperazine linked berberine derivatives.
Table 1 Screening results for radical scavenging activity of berberine
derivatives 4a–i (Scheme 1)
the piperazine entity attached to the berberine ring was found
to be beneficial for very good radical scavenging activity both
in the DPPH assay (IC50 17.90 µg mL-1) and the ABTS assay
(IC50 of 6.303 µg mL-1). For the remaining analogues, variation
in the activity level was observed in both the bioassays. For
example an analogue 4a with 2,3-dichloro substituents on
the piperazine ring exhibited IC50=6.501 µg mL-1 and 4f with
2,4-dichloro sunstituents showed with IC50=18.63 µg mL-1
in the ABTS and DPPH bioassays, respectively. Overall,
the chlorine-based compounds emerged with significant
radical scavenging efficacies, and their activity order falls as
3,4-Cl2>3,5-Cl2>2,3Cl2>2,4-Cl2. All the remaining compounds
exhibited IC50 around 17–24 µg mL-1 in the DPPH assay as well
as around 7 µg mL-1 in the ABTS assay which are considered
to be good to moderate potencies when compared to the parent
scaffold berberine with IC50=34.29 µg mL-1 and 82.17 µg mL-1
and ascorbic acid with IC50=10.75 µg mL-1 and 5.528 µg mL-1 in
the DPPH and ABTS bioassays, respectively.
-1
Compound
IC50a/μg mL SD
DPPH
ABTS
4a
4b
4c
4d
4e
4f
4g
4h
4i
21.02 0.82
24.40 1.56
23.66 1.83
17.90 2.06
20.56 0.52
18.63 3.02
22.21 1.24
11.08 0.47
12.23 068
34.29 1.73
10.75 0.86
6.501 2.02
7.046 3.11
7.522 1.35
6.303 0.74
7.681 0.68
7.354 1.26
7.852 2.44
4.768 0.66
4.876 0.48
82.17 1.08
5.528 0.88
Berberine
Ascorbic acid
aAntioxidant activities are shown as IC50 values in μg mL . All assays were carried out in
triplicate and the results expressed as average standard deviation.
ABTS, 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid; DPPH, 2,2-diphenyl-1-
picrylhydrazyl.
-1
Anticancer activities
The anticancer potential of final berberine-based analogues
4a–i was assayed against cervical cancer cell lines Hela and
Caski, and the results of Sulforhodamine B colorimetric
assay are presented in Tables 2 and 3. Overall, the compounds
demonstrated low cytotoxicity towards the normal cell line,
Malin Darby canine kidney (MDCK). But they showed excellent
inhibitory effects against cervical cancer cell line Caski when
compared to their potencies against Hela cell lines. Compounds
4a–i demonstrated IC50=5.697–6.807 µg mL-1 against Caski cell
line as well as 5.923–7.849 µg mL-1 against the Hela cell line of
cervical cancer. In addition, cytotoxicity levels were observed
in the range 243.7–331.7 µg mL-1 and 4a–i exhibited impressive
the type of functional groups present on the piperazine moiety.
Overall, as can be seen from Table 1, most of the compounds
showed significant antioxidant activity with IC50s ranging from
11.08 to 24.40 µg mL-1 and 4.768 to 7.852 µg mL-1 in DPPH
and ABTS bioassays, respectively. The highest scavenger
activity observed in compounds 4h and 4i is probably due to the
presence of dichloro functionality on piperazine rings system
with IC50 levels of 11.08 µg mL-1 and 12.23 as well as 4.76 µg
mL-1 and 4.876 µg mL-1, respectively, in DPPH and ABTS
bioassay. The, presence of electron donating dimethyl groups on

472 JOURNAL OF CHEMICAL RESEARCH 2015
Table 2 Anticancer activity of synthesised compounds 4a–i against
HeLa and MDCK cancer cell lines and their toxicity
Table 3 Anticancer activity of synthesised compounds 4a–i against
Caski and MDCK cancer cell lines and their toxicity
-1
-1
-1
-1
Compound IC50a/μg mL SD CC50/μg mL SD
TI
Compound
IC50a/μg mL SD CC50/μg mL SD
TI
HeLa
MDCK
Caski
MDCK
4a
4b
4c
4d
4e
4f
4g
4h
4i
5.578 0.03
7.503 0.07
7.849 0.05
6.843 0.02
7.576 0.65
7.452 1.07
7.237 1.21
5.782 0.55
5.923 0.68
5.725 0.01
243.7 0.88
331.7 2.02
324.1 2.34
306.1 0.09
330.5 1.53
308.3 0.45
286.6 0.78
320.7 1.04
311.4 0.63
43.69
44.21
41.29
44.73
43.62
41.37
39.60
55.47
52.57
28.16
4a
4b
4c
4d
4e
4f
4g
4h
4i
5.697 0.37
6.133 1.05
6.075 1.58
6.405 1.32
6.807 2.06
6.412 2.41
6.078 0.92
5.634 0.53
5.846 0.48
5.983 0.72
243.7 0.88
331.7 2.02
324.1 2.34
306.1 0.09
330.5 1.53
308.3 0.45
286.6 0.78
320.7 1.04
311.4 0.63
42.78
54.08
53.35
47.79
48.55
48.08
47.15
56.92
53.27
26.94
Berberine
161.2 0.76
Berberine
161.2 0.76
aAnticancer activities are shown as IC50 values in μg mL . All assays were carried out in
triplicate and the results expressed as average standard deviation.
CC50, cytotoxicity concentration of 50%; MDCK, Malin Darby canine kidney; TI,
therapeutic index.
aAnticancer activities are shown as IC50 values in μg mL . All assays were carried out in
triplicate and the results expressed as average standard deviation.
CC50, cytotoxicity concentration of 50%; MDCK, Malin Darby canine kidney; TI,
therapeutic index.
-1
-1
levels of therapeutic index ranging from 42.78 to 56.92 for
Caski cell lines as well as 39.60 to 55.47 for Hela cell lines.
Compound 4h with a 3,4-dichlorophenyl piperazine entity was
found to be the most active analogue among all those tested
with IC50=5.782 0.55 µg mL-1 and CC50=320.7 1.04 µg mL-1
and the highest TI of 55.47 against Hela cell lines. The potency
of this compound was double that of the parent berberine which
showed a TI of 28.16. 4h aslo has the most active effects against
Caski cell lines with IC50=5.634 0.53 µg mL-1 and CC50=
320.7 1.04 µg mL-1 and a TI of 56.92, again more than double
that of berberine with TI=26.94. The dichloro functionality
was again found to be beneficial by contributing significant
anticancer potential, as compound 4i with a 3,5-dichlorophenyl
piperazine entity appearing with IC50=5.923 0.68 µg mL-1
and 5.846 0.48 µg mL-1 against Hela and Caski cell lines,
respectively, as well as CC50=311.4 0.63 and TI=52.57 and
53.87, respectively. Compound (4b) with strong electronegative
2,4-difluoro substituents demonstrated IC50=6.133 1.05 µg
mL-1 and CC50=331.7 2.02 µg mL-1 and TI= 54.08 against the
Caski cell line. Compound 4c with electron-donating alkoxy
functionality exerted TI=53.35, the same as 4i against Caski
cell lines. Hence, the results clearly indicated that the berberine-
piperazine adducts were more active against Caski cell lines
than Hela. Overall, concerning the functionality, the anticancer
activity order falls as Cl>F>OCH3>CH3. All the compounds
exhibited similar double anticancer potentials against both the
cervical cancer cell lines than did the parent berberine.
when compared to that of berberine against Hela and Caski,
respectively. Overall, all compounds gave enhanced antioxidant
and anticancer effects compared to the parent berberine, and
equipotent potencies when compared to the control drugs..
Experimental
Highest quality chemicals and reagents (Sigma) were used in this study
without prior purification. Veego Open capillary electronic apparatus
VMP-D was utilized to obtain melting points which were uncorrected.
A Shimadzu 8400-S FTIR spectrophotometer (KBr pellets) were used
to obtain IR spectra. 1H NMR spectra were obtained on a Varian 400
spectrometer in CDCl3 using TMS as an internal reference. Chemical
shifts (δ) are given in ppm and coupling constants (J) in Hz. TLC
was carried out using appropriate mobile phase systems and silica
gel-G coated microscopic glass slides (2x7.5 cm), and TLC spots were
observed in a UV light chamber. Elemental analyses (C, H, N) were
performed using a Heraeus Carlo Erba 1180 CHN analyser.
Berberrubine (2): Berberine hydrochloride (10 g, 0.01 mol) was
added to a 50 mL round bottomed flask. The mixture was kept under
reduced pressure (20–30 mmHg) and heated to 190° C for 40 min.
The vacuum pump was turned off after the temperature decreased to
room temperature. The reaction product was purified via silica gel
column chromatography (CHCl3/CH3OH:15:1 and 10:1, eluting until
no compound was observed in the eluent) to yield 2 asa brownish red
amorphous powder (6.6 g, 85%).29
Bromopentylberberrubine (3): A solution of 2 (5 g, 0.01 mol)l and
1,5-dibromopentane (0.01 mol) in dry acetonitrile was heated at
reflux for 6 h and then diethyl ether was added. The resulting solid
was filtered off and then subjected to anion-exchange to convert into
its chloride form 3. Yield 61%; m.p. 195–197°C (DMF); IR (KBr)
cm-1: 3065 (C–H, Ar), 1615–1565 (C=C, Ar), 1115–1050 (C–O–C);
1H NMR: 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); EMI–MS (m/z): 507.73
(M+); Anal. calcd for C24H25BrClNO4: C, 56.88; H, 4.97; N, 2.76;
found: C, 56.75; H, 5.07; N, 2.54%.
Conclusion
In summary, most of the synthesised compounds were potential
leads for antioxidant activity. On the basis of observed results,
it may be concluded that disubstitution groups favours activity.
The chloro and methyl disubstitution increases the DPPH as well
as ABTS free radical scavenging activity as compared to the
potency of the parent berberine. All analogues bearing dichloro
substituents showed a significant level of radical scavenging
efficacy, particularly the 3,4-dichloroa and 3,5-dichloro
compounds. All compounds exhibited antioxidant activities
(IC50=4.768–7.852 µg mL-1) equipotent to the control ascorbic
acid at IC50=5.528 µg mL-1, and hence can be considered further
for development as antioxidant drugs. In addition, the dichloro
compounds showed excellent anticancer effects against cervical
cancer cell lines Hela and Caski, proving the importance of
dichloro substitution on the piperazine rings system with around
IC50=5–6 µg mL-1 and TI level of >50 against both cell lines
Synthesis of derivatives (4a–i); general procedure
The substituted piperazine (0.01 mol) was added to a magnetically
stirred solution of compound 3 and anhydrous K2CO3 in dry DMF
(25 mL). The reaction mixture was heated at 80°C for 6-8 h and the
reaction was monitored by TLC. The resulting solid was filtered off at
room temperature and subjected to anion exchange to convert into its
chloride form. The crude product was chromatographed on an Al2O3
column, eluted with CHCl3/CH3OH (9:1, v/v) to give the proposed
compound.

JOURNAL OF CHEMICAL RESEARCH 2015 473
9-O-3-(1-(2 3-Dichlorophenyl)piperazine)pentylberberine (4a):
Light yellow solid; yield 61%; m.p. 217–219°C (DMF); IR (KBr) cm-1:
3052 (C–H, Ar), 1608–1559 (C=C, Ar), 1103–1080 (C–O–C), 781 (C–
Cl); 1H NMR: δ 9.82 (s, 1H, H-8), 8.41 (s,1H, H-13), 7.81 (s, 1H, H-1),
7.59 (s, 1H, H-12), 7.51 (s, 1H, H-4), 7.39–7.17 (m, 3H, ArH, piperazine
H-27, H-28, H-29), 6.81 (s, 1H, H-11), 6.11 (s, 2H, –OCH2O), 4.91 (t, 2H,
J = 6.3, H-6), 4.38 (t, 2H, J = 6.5, H-15), 4.11 (s, 3H, –OCH3), 3.89 (br
s, 4H, piperazine, H-23, H-25), 3.51 (br s, 4H, piperazine, H-20, H-22),
3.21 (t, 2H, J = 6.4, H-19), 2.61 (t, 2H, J = 7.4, H-5), 2.49 (br s, 4H, H-17,
H-18), 2.16 (m, 2H, H-16); EMI–MS (m/z): 656.37 (M+). Anal. calcd for
C34H36Cl3N3O4: C, 62.15; H, 5.52; N, 6.40; found: C, 62.27; H, 5.43; N,
6.53%.
3.16 (t, 2H, J = 6.5, H-19), 2.48 (t, 2H, J = 7.4, H-5), 2.45 (br s, 4H, H-17,
H-18), 2.23 (m, 2H, H-16), 2.11 (s, 3H, Ar-CH3 of piperazine), 1.90 (s,
3H, Ar-CH3 of piperazine); EMI–MS (m/z): 616.52 (M+). Anal. calcd for
C36H42ClN3O4: C, 70.17; H, 6.87; N, 6.82; found: C, 70.05; H, 6.73; N,
6.95%.
9-O-3-(1-(3,4-Dichlorophenyl)piperazine)pentylberberine
(4h):
Light yellow solid; yield 62%; m.p. 259–261°C (DMF); IR (KBr) cm-1:
3037 (C–H, Ar), 1615–1575 (C=C, Ar), 1106–1083 (C–O–C) 757 (C–
Cl); 1H NMR: δ 9.83 (s, 1H, H-8), 8.44 (s,1H, H-13), 7.85 (s, 1H, H-1),
7.66 (s, 1H, H-12), 7.53 (s, 1H, H-4), 7.34–7.13 (m, 3H, ArH, piperazine
H-27, H-28, H-31), 6.83 (s, 1H, H-11), 6.07 (s, 2H, –OCH2O), 4.79 (t, 2H,
J = 6.5, H-6), 4.33 (t, 2H, J = 6.6, H-15), 4.12 (s, 3H, OCH3), 3.77 (br
s, 4H, piperazine, H-23, H-25), 3.43 (br s, 4H, piperazine, H-20, H-22),
3.15 (t, 2H, J = 6.3, H-19), 2.51 (t, 2H, J = 7.3, H-5), 2.35 (br s, 4H, H-17,
H-18), 2.15 (m, 2H, H-16); EMI–MS (m/z): 656.37 (M+). Anal. calcd for
C34H36Cl3N3O4: C, 62.15; H, 5.52; N, 6.40; found: C, 62.04; H, 5.42; N,
6.26%.
9-O-3-(1-(2,4-Difluorophenyl)piperazine)pentylberberine
(4b):
Light yellow solid; yield 49%; m.p. 219–221°C (DMF); IR (KBr) cm-1:
3048 (C–H, Ar), 1611–1547 (C=C, Ar), 1112–1085 (C–O–C); 1H NMR:
δ 9.75 (s, 1H, H-8), 8.66 (s,1H, H-13), 7.92 (s, 1H, H-1), 7.51 (s, 1H, H-12),
7.47 (s, 1H, H-4), 7.41–7.20 (m, 3H, ArH, piperazine H-27, H-28, H-30),
6.77 (s, 1H, H-11), 6.08 (s, 2H, –OCH2O), 4.86 (t, 2H, J = 6.5, H-6), 4.41
(t, 2H, J = 6.6, H-15), 4.05 (s, 3H, OCH3), 3.79 (br s, 4H, piperazine,
H-23, H-25), 3.45 (br s, 4H, piperazine, H-20, H-22), 3.25 (t, 2H, J = 6.5,
H-19), 2.55 (t, 2H, J = 7.5, H-5), 2.41 (br s, 4H, H-17, H-18), 2.24(m, 2H,
H-16); EMI–MS (m/z): 624.52 (M+). Anal. calcd for C34H36ClF2N3O4: C,
65.43; H, 5.81; N, 6.73; found: C, 65.59; H, 5.93; N, 6.58%.
9-O-3-(1-(3 5-Dichlorophenyl)piperazine)pentylberberine (4i):
Light yellow solid; yield 56; m.p. 245–247°C (DMF); IR (KBr) cm-1:
3045 (C–H, Ar), 1601–1555 (C=C, Ar), 1111–1089 (C–O–C) 782 (C–
Cl); 1H NMR: δ 9.69 (s, 1H, H-8), 8.53 (s,1H, H-13), 7.90 (s, 1H, H-1),
7.55 (s, 1H, H-12), 7.46 (s, 1H, H-4), 7.35-7.19 (m, 3H, ArH, piperazine
H-27, H-29, H-31), 6.78 (s, 1H, H-11), 6.09 (s, 2H, –OCH2O), 4.83 (t, 2H,
J = 6.4, H-6), 4.31 (t, 2H, J = 6.4, H-15), 4.19 (s, 3H, OCH3), 3.90 (br
s, 4H, piperazine, H-23, H-25), 3.47 (br s, 4H, piperazine, H-20, H-22),
3.24 (t, 2H, J = 6.3, H-19), 2.59 (t, 2H, J = 7.5, H-5), 2.33 (br s, 4H, H-17,
H-18), 2.21 (m, 2H, H-16); EMI–MS (m/z): 656.07 (M+). Anal. calcd for
C34H36Cl3N3O4: C, 62.15; H, 5.52; N, 6.40; found: C, 62.07; H, 5.66; N,
6.56%.
9-O-3-(1-(2,4-Dimethoxyphenyl)piperazine)pentylberberine
(4c):
Light yellow solid; yield 47%; m.p. 239–241°C (DMF); IR (KBr)
cm-1: 3061 (C–H, Ar), 1606–1531 (C=C, Ar), 1121–1088 (C–O–C);
1H NMR: δ 9.67 (s, 1H, H-8), 8.54 (s,1H, H-13), 7.80 (s, 1H, H-1), 7.61
(s, 1H, H-12), 7.43 (s, 1H, H-4), 7.37–7.15 (m, 3H, ArH, piperazine H-27,
H-28, H-30), 6.69 (s, 1H, H-11), 6.15 (s, 2H, –OCH2O), 4.95 (t, 2H, J =
6.4, H-6), 4.35 (t, 2H, J = 6.5, H-15), 4.21 (s, 3H, OCH3), 4.03 (s, 3H,
Ar–OCH3 of piperazine), 3.95 (s, 3H, Ar–OCH3 of piperazine), 3.83 (br
s, 4H, piperazine, H-23, H-25), 3.41 (br s, 4H, piperazine, H-20, H-22),
3.18 (t, 2H, J = 6.3, H-19), 2.49 (t, 2H, J = 7.3, H-5), 2.47 (br s, 4H, H-17,
H-18), 2.17 (m, 2H, H-16); EMI–MS (m/z): 648.06 (M+). Anal. calcd for
C36H42ClN3O6: C, 66.71; H, 6.53; N, 6.48; found: C, 66.63; H, 6.68; N,
6.61%.
DPPH free radical scavenging assay
Free radical scavenging is one of the best-known mechanisms by which
antioxidants inhibit lipid oxidation. DPPH and ABTS radical scavenging
activity evaluations are standard assays in antioxidant activity
studies. The antioxidant activity of the berberine derivatives 4a–i was
determined by these methods using ascorbic acid as standard.
9-O-3-(1-(2,4-Dimethylphenyl)piperazine)pentylberberine
(4d):
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of
the berberine derivatives 4a–i were evaluated by adjusting the process
of Kim et al.32 Berberine derivatives (20 µL) were included in a 96-well
microplate and 180 µL of DPPH was added to the wells. Methanol (20
µL) provided a blank and after 30 min incubation the optical density
was determined at l=517 nm. The control contains all reagents except
the scavenger. The DPPH radical scavenging activity of ascorbic acid
was also assayed for comparison; all tests were performed in triplicate.
The results of this bioassay, RSA % (the radical scavenging activity in
percentage) was determined as described in the following equation.
Light yellow solid; yield 55%; m.p. 227–229°C (DMF); IR (KBr)
cm-1: 3053 (C–H, Ar), 1617–1567 (C=C, Ar), 1107–1079 (C–O–C);
1H NMR: δ 9.86 (s, 1H, H-8), 8.61 (s,1H, H-13), 7.88 (s, 1H, H-1), 7.68
(s, 1H, H-12), 7.49 (s, 1H, H-4), 7.40–7.23 (m, 3H, ArH, piperazine H
H-27, H-28, H-30), 6.75 (s, 1H, H-11), 6.05 (s, 2H, –OCH2O), 4.88 (t, 2H,
J = 6.4, H-6), 4.39 (t, 2H, J = 6.4, H-15), 4.13 (s, 3H, OCH3), 3.78 (br
s, 4H, piperazine, H-23, H-25), 3.53 (br s, 4H, piperazine, H-20, H-22),
3.22 (t, 2H, J = 6.5, H-19), 2.62 (t, 2H, J = 7.5, H-5), 2.43 (br s, 4H, H-17,
H-18), 2.20 (m, 2H, H-16), 2.07 (s, 3H, Ar-CH3 of piperazine), 1.95 (s,
3H, Ar-CH3 of piperazine); EMI–MS (m/z): 616.44 (M+). Anal. calcd
for C36H42ClN3O4: C, 70.17; H, 6.87; Cl, 5.73; N, 6.82; found: C, 70.06; H,
6.74; N, 6.69%.
9-O-3-(1-(2 3-Difluorophenyl)piperazine)pentylberberine (4e): Light
yellow solid; yield 43; m.p. 211–213°C (DMF); IR (KBr) cm-1: 3044 (C–
H, Ar), 1622–1566 (C=C, Ar), 1102–1077 (C–O–C); 1H NMR: δ 9.66 (s,
1H, H-8), 8.71 (s,1H, H-13), 7.95 (s, 1H, H-1), 7.57 (s, 1H, H-12), 7.41 (s,
1H, H-4), 7.32–7.11 (m, 3H, ArH, piperazine H-27, H-28, H-29), 6.73 (s,
1H, H-11), 6.13 (s, 2H, –OCH2O), 4.78 (t, 2H, J = 6.3, H-6), 4.29 (t, 2H, J
= 6.4, H-15), 4.07 (s, 3H, OCH3), 3.85 (br s, 4H, piperazine, H-23, H-25),
3.46 (br s, 4H, piperazine, H- H-20, H-22), 3.11 (t, 2H, J = 6.3, H-19),
2.58 (t, 2H, J = 7.4, H-5), 2.39 (br s, 4H, H-17, H-18), 2.27 (m, 2H, H-16);
EMI–MS (m/z): 624.09 (M+). Anal. calcd for C34H36ClF2N3O4: C, 65.43;
H, 5.81; N, 6.73; found: C, 65.58; H, 5.68; N, 6.86%.
Absorbance of blank − Absorbance of test
%Scavenging =
×100
Absorbance of blank
A plot of concentration of test compounds and % scavenging
introduced IC50 in the presence of ascorbic acid as standard.
ABTS radical cation decolourization assay protocol: 2,2′-Azinobis(3-
ethylbenzothiazoline-6-sulfonic acid) Radical Scavenging Assay.
ABTS analysis was conducted by following the method of Lee et al.33
In brief, 20 휇L of sample was combined with 180 휇L of ABTS radical
solution followed by 10 min of incubation under dark conditions and
the absorbance was measured at l=734 nm. 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.
9-O-3-(1-(2,3-Dimethylphenyl)piperazine)pentylberberine
(4g):
Light yellow solid; yield 57; m.p. 223–225°C (DMF); IR (KBr)
cm-1: 3041 (C–H, Ar), 1603–1558 (C=C, Ar), 1115–1076 (C–O–C);
1H NMR: δ 9.77 (s, 1H, H-8), 8.51 (s,1H, H-13), 7.93 (s, 1H, H-1), 7.58
(s, 1H, H-12), 7.44 (s, 1H, H-4), 7.33–7.08 (m, 3H, ArH, piperazine H-27,
H-28, H-29), 6.70 (s, 1H, H-11), 6.17 (s, 2H, –OCH2O), 4.77 (t, 2H, J
= 6.3, H-6), 4.43 (t, 2H, J = 6.5, H-15), 4.09 (s, 3H, OCH3), 3.88 (br s,
4H, piperazine, H-23, H-25), 3.49 (br s, 4H, piperazine, H- H-20, H-22),
The scavenging capability of ABTS•+ radical was calculated using the
following equation:
Absorbance of blank − Absorbance of test
%Scavenging =
×100
Absorbance of blank

474 JOURNAL OF CHEMICAL RESEARCH 2015
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In vitro anticancer bioassay
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This article was supported by the KU Research Professor
Program of Konkuk University, Seoul, South Korea.
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Received 28 June 2015; accepted 14 July 2015
Paper 1503449 doi: 10.3184/174751915X14381686689721
Published online: 7 August 2015
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