Synthesis, cytotoxicity, and DNA-binding property of berberine derivatives

Article abstract of DOI:10.1007/s00044-013-0796-9

9-Substituted berberine derivatives (4a-4f) with polyethylene glycol side chain and terminal group were synthesized and characterized by elemental (C, H, and N) and spectral analysis (NMR, HRMS and FTIR). These compounds were tested for their in vitro cytotoxic activity against four human tumor cell lines: granulocyte leukemia (HL-60), gastrocarcinoma (BGC-823), carcinoma (Bel-7402), and nasopharyngeal carcinoma (KB) by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The DNA-binding properties were investigated by UV-Vis absorption, fluorescence, CD spectroscopies, and thermal denaturation measurements. The results indicated that 4a-4f exerted cytotoxic effects with selectivity against tested cell lines. 4a exhibited higher cytotoxicity than cisplatin, berberine, and berberrubine against HL-60 and BGC-823 cell lines. The length of side chains and nature of terminal groups played an important role in the cytotoxicity. Berberine derivatives binded to CT-DNA in an intercalating mode. The binding affinities decreased with the increasing length of side chains. Compounds 4a-4c and 4e could change the DNA conformation from B to A-like form. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0796-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1899–1907
DOI 10.1007/s00044-013-0796-9
ORIGINAL RESEARCH
Synthesis, cytotoxicity, and DNA-binding property
of berberine derivatives
•
Cui Liu Siyuan Liu Yuechai Wang Shuxiang Wang
•
•
•
•
•
Jinchao Zhang Shenghui Li Xinying Qin Xiaoliu Li
•
•
•
Kerang Wang Quoqiang Zhou
Received: 19 April 2013 / Accepted: 12 September 2013 / Published online: 24 September 2013
Ó Springer Science+Business Media New York 2013
Abstract 9-Substituted berberine derivatives (4a–4f)
with polyethylene glycol side chain and terminal group
were synthesized and characterized by elemental (C, H,
and N) and spectral analysis (NMR, HRMS and FTIR).
These compounds were tested for their in vitro cytotoxic
activity against four human tumor cell lines: granulocyte
leukemia (HL-60), gastrocarcinoma (BGC-823), carcinoma
(Bel-7402), and nasopharyngeal carcinoma (KB) by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. The DNA-binding properties were
investigated by UV–Vis absorption, fluorescence, CD
spectroscopies, and thermal denaturation measurements.
The results indicated that 4a–4f exerted cytotoxic effects
with selectivity against tested cell lines. 4a exhibited
higher cytotoxicity than cisplatin, berberine, and berb-
errubine against HL-60 and BGC-823 cell lines. The length
of side chains and nature of terminal groups played an
important role in the cytotoxicity. Berberine derivatives
binded to CT-DNA in an intercalating mode. The binding
affinities decreased with the increasing length of side
chains. Compounds 4a–4c and 4e could change the DNA
conformation from B to A-like form.
Keywords 9-Substituted berberine derivatives ꢀ
Cytotoxicity ꢀ DNA binder
Introduction
Many diseases, including cancer, are often treated with drugs
that bind to DNA and/or interfere with its biological func-
tions. Unfortunately, toxic side effects always accompany
treatment using DNA-binding drugs owing to their poor
selectivity towards disease-affected cells. Modification of
existing drugs or natural compounds has been proven to be a
feasible way for developing more promising DNA-binding
candidate compounds (Paterson and Anderson, 2005).
Berberine (Fig. 1), originating from Chinese herb Hu-
anglian and many other plants, is an isoquinoline alkaloid
which has been used over many generations as an antibiotic
and disinfectant. Berberine displays a wide range of bio-
chemical and pharmacological actions such as antibiotic,
immunostimulant, antitumor, and antimotility properties,
and is relatively nontoxic to humans (Lau et al., 2001;
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0796-9) contains supplementary
material, which is available to authorized users.
C. Liu ꢀ S. Liu ꢀ Y. Wang ꢀ S. Wang ꢀ J. Zhang ꢀ S. Li ꢀ
X. Qin ꢀ X. Li ꢀ K. Wang ꢀ Q. Zhou
´
Jantova et al., 2003). In addition, berberine is proven to be
Chemical Biology Key Laboratory of Hebei Province, College of
Chemistry and Environmental Science, Hebei University,
Baoding 071002, China
a DNA-binding agent and has the ability to induce apop-
tosis in HL-60 cells (Kuo et al., 1995; Chen et al., 2005).
X-ray data certified that the berberine bond to DNA by
intercalation mode (Ferraroni et al., 2011). The computer-
aided modeling study on the berberine–DNA complex
suggested that berberine binds to DNA from its C5–C6–
N?–C8 side (Mazzini et al., 2003). Berberine derivatives
with substitutions at 9-position were prepared as chemical
modified compounds to enhance the pharmacological
S. Wang (&) ꢀ J. Zhang (&) ꢀ S. Li ꢀ X. Li
Key Laboratory of Medicinal Chemistry and Molecular
Diagnosis of Ministry of Education, Hebei University,
Baoding 071002, China
e-mail: wsx@hbu.edu.cn
J. Zhang
e-mail: jczhang6970@yahoo.com.cn
123

1900
Med Chem Res (2014) 23:1899–1907
Ia
Ib
5
O
H
4
1
=
R
O
O
O
O
6
O
O
Cl-
8
Cl-
7
NH2
N
N+
N+
N+
O
O
Ic
OCH3
OCH3
O
9
NH
n Ph
13
N
R
OCH3
IIa: n=0; IIb: n=1; IIc: n=2
OCH3
12
Id
N
N
10
11
Berberine
Fig. 1 Berberine and its 9-substituted derivatives
activities, as well as DNA-binding affinities. Compounds
Ia–Id (Fig. 1) showed strong binding to CT-DNA than
berberine (Pang et al., 2005). Introduction of different
spacers of alkyl ether chains and amino groups on the
9-position of berberine may enhance the DNA-binding
affinity via hydrogen bonding and/or electrostatic interac-
tion (Pang et al., 2005). Basu et al. synthesized three
9-substituted berberine derivatives (IIa–IIc, Fig. 1), which
displayed the DNA-binding affinity by more than six times
compared with berberine (Basu et al., 2012). All of the
above prompted us to design and synthesize new
9-substituted berberine derivatives with different length of
polyethylene glycol side chains and terminal groups (such
as piperidine, morpholine), and investigate the DNA-
binding affinity as well as their cytotoxicity.
The UV spectra and thermal denaturation experiment were
measured in 50 mM Tris–HCl buffer (pH 6.35) using a
UV-3600 UV–Vis spectrophotometer at room temperature.
Fluorometric measurements were run on a Hitachi F-7000
PC spectrofluorophotometer. The CD spectra were recor-
ded on a MOS-450/SFM300 spectropolarimeter.
Synthesis
General procedures for the preparation of compounds 4a–
4f
Partial demethylation of berberine (1) at 190 °C under
vacuum for 20 min, according to the reported procedure
´
(Ouchi et al., 1990; Matıas et al., 2007), afforded berb-
errubine (2) in 60 % yield. Treatment of 2 with oligoethyl-
eneglycol ditosylate in DMF (Iwasa et al., 1996) generated
3a, 3b, and 3c in 41, 68, and 46 % yields, respectively.
To a stirred solution of 3a–3c (0.2 mmol) in dry ace-
tonitrile (5 mL), secondary amine (piperidine or morpho-
line, 2.0 mmol) and anhydrous K2CO3 (1.0 mmol) were
added. The mixture was stirred at 80 °C for 4–6 h. Com-
pletion of the reaction was monitored by TLC. The mixture
was then cooled to room temperature and filtered. The
crude product was chromatographed on an Al2O3 column,
eluted with CH2Cl2/MeOH to afford the proposed com-
pounds 4a–4f as yellow powders.
Experimental
Materials and methods
All chemicals and reagents were of analytical grade. All
reactions were carried out under N2 using general grade
solvents. Purification and handling of all compounds were
carried out under air with a minimum exposure to light.
Berberine was obtained from Si Chuan Hong Yi Sheng Wu
Gong Cheng. LTD (purity: 97 %). CT-DNA was obtained
from Sigma-Aldrich Corporation (St. Louis, MO, USA).
Four different human carcinoma cell lines: HL-60 (granu-
locyte leukemia), BGC-823 (gastrocarcinoma), Bel-7402
(liver carcinoma), and KB (nasopharyngeal carcinoma)
were obtained from American Type Culture Collection.
Melting points were determined on a XT-4 microscopic
melting point spectrometer and were uncorrected. Ele-
mental analyses were determined on an Elementar Vario
EL III elemental analyzer. The IR spectra were recorded
using KBr pellets and a Perkin-Elmer Model-683 spectro-
photometer. NMR spectra were recorded on a Bruker
AVIII 600 NMR spectrometer using TMS as an internal
standard in DMSO-d6 or CD3OD. The HRMS were
recorded using a Bruker CVII high-resolution mass spec-
trometer. The optical density (OD) was measured on a
microplate spectrophotometer (Bio-Rad Model 680, USA).
9-O-2-(4-Morpholinyl)ethylberberine chloride (4a)
Light yellow solid, yield 36 %; m.p. 194–195 °C; IR (KBr,
cm-1): 3,035 (C–H, Ar), 1,607–1,575 (C=C, Ar),
1
1,115–1,015 (C–O); H-NMR (600 MHz, CD3OD) d 9.90
(s, 1H, H-8), 8.73 (s, 1H, H-13), 8.14 (d, 1H, J = 9.0 Hz,
H-11), 8.04 (d, 1H, J = 9.0 Hz, H-12), 7.69 (s, 1H, H-1),
6.99 (s, 1H, H-4), 6.13 (s, 2H, OCH2O), 4.97 (t, 2H,
J = 6.0 Hz, H-6), 4.56 (t, 2H, J = 6.0 Hz, CH2), 4.13 (s,
3H, OCH3), 3.69 (t, 4H, J = 4.8 Hz, 2CH2), 3.29 (t, 2H,
J = 6.0 Hz, H-5), 2.92 (t, 2H, J = 6.0 Hz, CH2), 2.63 (t,
4H, J = 4.8 Hz, 2CH2); 13C-NMR (150 MHz, CD3OD) d
150.81 (1C, C-10), 150.73 (1C, C-3), 148.57 (1C, C-2),
145.30 (1C, C-8), 143.26 (1C, C-9), 138.27 (1C, C-13a),
133.72 (1C, C-12a), 130.45 (1C, C-4a), 128.35 (1C, C-11),
123

Med Chem Res (2014) 23:1899–1907
1901
125.55 (1C, C-12), 122.39 (1C, C-8a), 120.49 (1C, C-13b),
120.12 (1C, C-13), 108.00 (1C, C-4), 105.17 (1C, C-1),
102.28 (1C, OCH2O), 70.01 (1C, OCH2–CH2–N), 66.37
(2C, CH2-O-CH2, morpholin), 58.02 (1C, OCH3), 56.21
(1C, C-6), 55.95 (1C, OCH2–CH2–N), 53.38 (2C, CH2–N–
CH2, morpholin), 26.84 (1C, C-5); HRMS (?)-ESI m/z [M-
Cl]? calcd for [C25H27N2O5]?: 435.19145; found:
435.19129; anal. calcd for C25H27ClN2O5: C, 63.76; H,
5.78; N, 5.95. Found: C, 63.71; H, 5.81; N, 5.92.
C-4a), 126.56 (1C, C-11), 123.32 (1C, C-12), 122.38 (1C,
C-8a), 120.45 (1C, C-13b), 120.15 (1C, C-13), 108.02 (1C,
C-4), 105.16 (1C, C-1), 102.28 (1C, OCH2O), 72.95 (1C, Ar–
O–CH2CH2O), 69.92 (1C, Ar–O–CH2CH2O), 67.73 (1C,
OCH2–CH2–N), 66.06 (2C, CH2–O–CH2, morpholin), 57.79
(1C, OCH3), 56.29 (1C, C-6), 56.01 (1C, OCH2–CH2–N),
53.61 (2C, CH2–N–CH2, morpholin), 26.85(1C, C-5); HRMS
(?)-ESI m/z [M-Cl]? calcd for [C27H31N2O6]?: 479.21766;
found:479.21742;anal. calcd for C27H31ClN2O6: C, 62.97;H,
6.07; N, 5.44. Found: C, 62.86; H, 6.14; N, 5.37.
9-O-2-(1-Piperidinyl)ethylberberine chloride (4b)
9-O-5-(4-Piperidine)diethylene glycol berberine chloride
Light yellow solid, yield 25 %; m.p. 194–196 °C; IR (KBr,
cm-1): 3,039 (C–H, Ar), 1,607–1,575 (C=C, Ar),
1,120–1,085 (C–O–C); 1H-NMR (600 MHz, CD3OD) d
10.09 (s, 1H, H-8), 8.73 (s, 1H, H-13), 8.15 (d, 1H,
J = 9.0 Hz, H-11), 8.05 (d, 1H, J = 9.0 Hz, H-12), 7.69
(s, 1H, H-1), 6.99 (s, 1H, H-4), 6.13 (s, 2H, OCH2O), 4.99
(t, 2H, J = 6.0 Hz, H-6), 4.60 (t, 2H, J = 6.0 Hz, CH2),
4.14 (s, 3H, OCH3), 3.29 (t, 2H, J = 6.0 Hz, H-5), 3.13 (t,
2H, J = 6.0 Hz, CH2), 2.83 (m, 4H, 2CH2), 1.72 (m, 4H,
2CH2), 1.58 (m, 2H, 2CH2); 13C-NMR (150 MHz,
CD3OD) d 150.84 (1C, C-10), 150.58 (1C, C-3), 148.53
(1C, C-2), 145.24 (1C, C-8), 141.98 (1C, C-9), 138.47 (1C,
C-13a), 133.75 (1C, C-12a), 130.61 (1C, C-4a), 126.00
(1C, C-11), 124.24 (1C, C-12), 121.79 (1C, C-8a), 120.40
(1C, C-13b), 120.12 (1C, C-13), 108.02 (1C, C-4), 105.19
(1C, C-1), 102.28 (1C, OCH2O), 67.70 (1C, OCH2-CH2–
N), 56.63 (1C, OCH3), 56.32 (1C, OCH2-CH2–N), 55.77
(1C, C-6), 53.52 (2C, N(CH2CH2)2, piperidinyl), 26.77
(1C, C-5), 22.94 (2C, N(CH2CH2)2, piperidinyl), 21.58
(1C, CH2, piperidinyl); HRMS (?)-ESI m/z [M-Cl]? calcd
for [C26H29N2O4]?: 433.21218; found: 433.21196; anal.
calcd for C26H29ClN2O4: C, 66.59; H, 6.23; N, 5.97.
Found: C, 66.49; H, 6.18; N, 6.02.
(4d)
Light yellow solid, yield 29 %; m.p. 127–129 °C; IR (KBr,
cm-1): 3,035 (C–H, Ar), 1,610–1,575 (C=C, Ar),
1,117–1,088 (C–O–C); 1H-NMR (600 MHz, CD3OD) d
9.82 (s, 1H, H-8), 8.74 (s, 1H, H-13), 8.14 (d, 1H,
J = 9.0 Hz, H-11), 8.05 (d, 1H, J = 9.0 Hz, H-12), 7.68 (s,
1H, H-1), 6.98 (s, 1H, H-4), 6.12 (s, 2H, OCH2O), 5.02 (t,
2H, J = 6.0 Hz, H-6), 4.59 (t, 2H, J = 4.8 Hz, CH2), 4.12
(s, 3H, OCH3), 4.00 (t, 2H, J = 4.8 Hz, CH2), 3.93 (t, 2H,
J = 4.8 Hz, CH2), 3.29 (t, 2H, J = 6.0 Hz, H-5), 3.21–3.15
(m, 2H, 2CH2), 3.11 (s, 2H, CH2), 1.83–1.78 (m, 4H,
2CH2), 1.62 (s, 2H, CH2), 1.35–1.26 (m, 2H, CH2); 13C-
NMR (150 MHz, CD3OD) d 150.82 (1C, C-10), 150.66
(1C, C-3), 148.54 (1C, C-2), 145.03 (1C, C-8), 143.15 (1C,
C-9), 138.39 (1C, C-13a), 133.82 (1C, C-12a), 130.55 (1C,
C-4a), 126.43 (1C, C-11), 123.45 (1C, C-12), 122.24 (1C,
C-8a), 120.48 (1C, C-13b), 120.19 (1C, C-13), 108.00 (1C,
C-4), 105.19 (1C, C-1), 102.26 (1C, OCH2O), 72.95 (1C,
Ar–O–CH2CH2O), 70.06 (1C, Ar–O–CH2CH2O), 65.37
(1C, OCH2–CH2–N), 56.65 (1C, OCH3), 56.30 (1C, OCH2–
CH2–N), 56.01 (1C, C-6), 53.65 (2C, N(CH2CH2)2, pipe-
ridinyl), 26.86 (1C, C-5), 23.20 (2C, N(CH2CH2)2, pipe-
ridinyl), 21.81 (1C, CH2, piperidinyl); HRMS (?)-ESI m/z
[M-Cl]? calcd for [C28H33N2O5]?: 477.23840; found:
477.23792; anal. calcd for C28H33ClN2O5: C, 65.55; H,
6.48; N, 5.46. Found: C, 65.43; H, 6.53; N, 6.40.
9-O-5-(4-Morpholinyl) diethylene glycol berberine
chloride (4c)
Light yellow solid, yield 43 %; m.p. 153–154 °C; IR (KBr,
cm-1): 3,035 (C–H, Ar), 1,605–1,573 (C=C, Ar),
1,113–1,014 (C–O–C); 1H-NMR (600 MHz, CD3OD) d
9.83 (s, 1H, H-8), 8.75 (s, 1H, H-13), 8.14 (d, 1H,
J = 9.0 Hz, H-11), 8.04 (d, 1H, J = 9.0 Hz, H-12), 7.69
(s, 1H, H-1), 6.99 (s, 1H, H-4), 6.13 (s, 2H, OCH2O), 4.98
(t, 2H, J = 6.0 Hz, H-6), 4.58 (t, 2H, J = 4.2 Hz, CH2),
4.13 (s, 3H, OCH3), 3.91 (t, 2H, J = 4.2 Hz, CH2), 3.71 (t,
2H, J = 5.4 Hz, CH2), 3.66 (t, 4H, J = 4.8 Hz, 2CH2),
3.30 (t, 2H, J = 6.0 Hz, H-5), 2.60 (t, 2H, J = 5.4 Hz,
CH2), 2.52 (t, 4H, J = 4.8 Hz, 2CH2); 13C-NMR
(150 MHz, CD3OD) d 150.83 (1C, C-10), 150.74 (1C,
C-3), 148.57 (1C, C-2), 145.11 (1C, C-8), 143.27 (1C,
C-9), 138.30 (1C, C-13a), 133.78 (1C, C-12a), 130.46 (1C,
9-O-8-(4-Morpholinyl)triethylene glycol berberine
chloride (4e)
Light yellow solid, yield 16 %; m.p. 152–154 °C; IR (KBr,
cm-1):3,035(C–H, Ar),1,604–1,572(C=C, Ar),1,117–1,040
(C–O–C); 1H-NMR (600 MHz, CD3OD) d 9.87 (s, 1H, H-8),
8.75 (s, 1H, H-13), 8.15 (d, 1H, J = 9.0 Hz, H-11), 8.05 (d,
1H, J = 9.0 Hz, H-12), 7.69 (s, 1H, H-1), 7.00 (s, 1H, H-4),
6.13 (s, 2H, OCH2O), 4.99 (t, 2H, J = 6.6 Hz, H-6), 4.55 (t,
2H, J = 4.2 Hz, CH2), 4.13 (s, 3H, OCH3), 3.90 (t, 2H,
J = 4.8 Hz, CH2), 3.64 * 3.61 (m, 6H, 3CH2), 3.59 (t, 2H,
J = 6.0 Hz, CH2), 3.30 (t, 2H, J = 6.6 Hz, H-5); 13C-NMR
(150 MHz, CD3OD) d 150.96 (1C, C-10), 150.81 (1C, C-3),
123

1902
Med Chem Res (2014) 23:1899–1907
148.57 (1C, C-2), 145.43 (1C, C-8), 143.20 (1C, C-9), 138.19
(1C, C-13a), 133.67 (1C, C-12a), 130.47 (1C, C-4a), 126.35
(1C, C-11), 123.47(1C, C-12), 122.48(1C, C-8a), 120.47(1C,
C-13b), 120.10 (1C, C-13), 108.04 (1C, C-4), 105.17 (1C,
C-1), 102.30 (1C, OCH2O), 73.10 (1C, Ar–O–CH2CH2O),
69.90 (1C, Ar–O–CH2CH2O), 69.77 (1C, Ar–O–CH2CH2O–
CH2–CH2O), 69.71 (1C, Ar–O–CH2CH2O–CH2–CH2O),
67.51 (1C, OCH2–CH2–N), 66.01 (2C, CH2–O–CH2, mor-
pholin), 57.57 (1C, OCH3), 56.23 (1C, C-6), 55.97
(1C, OCH2–CH2–N), 53.56 (2C, CH2–N–CH2, morpholin),
26.85 (1C, 5-C); HRMS (?)-ESI m/z [M-Cl]? calcd for
[C29H35N2O7]?: 523.24388; found: 523.24352; anal. calcd
for C29H35ClN2O7: C, 62.30; H, 6.31; N, 5.01. Found: C,
62.18; H, 6.38; N, 4.97.
medium supplemented with 10 % fetal bovine serum, 100
units/mL of penicillin and 100 mg/mL of streptomycin.
Cells were maintained at 37 °C in a humidified atmosphere
of 5 % CO2 in air.
Solutions
The compounds were dissolved in DMSO at a concentra-
tion of 5 mM as stock solution, and diluted in culture
medium at concentrations of 0.1, 1.0, 10, and 100 mM as
working solution. To avoid DMSO toxicity, the concen-
tration of DMSO was less than 0.1 % (v/v) in all
experiments.
Cytotoxicity analysis
9-O-8-(4-Piperidine)triethylene glycol berberine chloride
(4f)
The cells harvested from exponential phase were seeded
equivalently into a 96-well plates, and then the compounds
were added to the wells to achieve final concentrations.
Control wells were prepared by addition of culture med-
ium. Wells containing culture medium without cells were
used as blanks. All experiments were performed in quin-
tuplicate. The MTT assay was performed as described by
Mosmann (1983). Upon completion of the incubation for
48 h, stock MTT dye solution (20 lL, 5 mg/mL) was
added to each well. After 4-h incubation, DMSO (100 lL)
was added to solubilize the MTT formazan. The optical
density (OD) of each well was measured on a microplate
spectrophotometer at wavelength of 570 nm. The IC50
value was determined from plot of percentage viability
against dose of compounds added.
Light yellow solid, yield 18 %; m.p. 151–152 °C; IR (KBr,
cm-1): 3,039 (C–H, Ar), 1,601–1,569 (C=C, Ar),
1,117–1,046 (C–O–C); 1H-NMR (600 MHz, CD3OD) d
9.82 (s, 1H, H-8), 8.75 (s, 1H, H-13), 8.15 (d, 1H,
J = 9.0 Hz, H-11), 8.06 (d, 1H, J = 9.0 Hz, H-12), 7.69
(s, 1H, H-1), 6.99 (s, 1H, H-4), 6.13 (s, 2H, OCH2O), 4.99
(t, 2H, J = 6.0 Hz, H-6), 4.56 (t, 2H, J = 4.8 Hz, CH2),
4.13 (s, 3H, OCH3), 3.98 (t, 2H, J = 4.2 Hz, CH2), 3.88 (t,
2H, J = 4.8 Hz, CH2), 3.80 (t, 2H, J = 5.4 Hz, CH2), 3.74
(t, 2H, J = 5.4 Hz, CH2), 3.62–3.58 (m, 2H, CH2), 3.31 (t,
2H, J = 6.0 Hz, H-5), 3.04–2.97 (m, 2H, CH2), 1.94–1.78
(m, 6H, 3CH2), 1.38 (t, 2H, J = 6.0 Hz, CH2); 13C-NMR
(150 MHz, CD3OD) d 150.87 (1C, C-10), 150.85 (1C,
C-3), 148.59 (1C, C-2), 145.00 (1C, C-8), 143.25 (1C,
C-9), 138.42 (1C, C-13a), 133.90 (1C, C-12a), 130.52 (1C,
C-4a), 126.64 (1C, C-11), 123.52 (1C, C-12), 122.39 (1C,
C-8a), 120.46 (1C, C-13b), 120.22 (1C, C-13), 108.03 (1C,
C-4), 105.20 (1C, C-1), 102.27 (1C, OCH2O), 73.12 (1C,
Ar–O–CH2CH2O), 70.03 (1C, Ar–O–CH2CH2O), 69.96
(1C, Ar–O–CH2CH2O–CH2-CH2O), 69.83 (1C, Ar–O–
CH2CH2O–CH2–CH2O), 64.33 (1C, OCH2-CH2–N), 56.39
(1C, OCH3), 56.12 (1C, OCH2–CH2-N), 56.10 (1C, C-6),
53.37 (2C, N(CH2CH2)2, piperidinyl), 26.89 (1C, 5-C),
22.60 (2C, N(CH2CH2)2, piperidinyl), 21.19 (1C, CH2,
piperidinyl); HRMS (?)-ESI m/z [M-Cl]? calcd for
[C30H37N2O6]?: 521.26461; found: 521.26414; anal. calcd
for C30H37ClN2O6: C, 64.68; H, 6.69; N, 5.03. Found: C,
64.53; H, 6.74; N, 5.05.
DNA-binding study
UV–Vis
Absorption spectra were conducted using a quartz cell of
1-cm optical path length. The DNA and compound-DNA
samples were dissolved in Tris–HCl buffer (50 mM,
pH 6.35) and incubated at room temperature for 2 h. The
concentrations of compounds were fixed at 20 lM, and the
concentrations of DNA varied from 0 to 400 lM. The
absorbance of samples was detected on UV-3600 spectro-
photometer during the wavelength range of 300–510 nm.
Fluorescence
Determination of in vitro cytotoxic activity
Fluorescence spectra were conducted using a quartz cell
of 1-cm optical path length. The DNA and compound-
DNA samples were dissolved in Tris–HCl buffer
(50 mM, pH 6.35) and incubated at room temperature for
2 h. The concentrations of compounds were fixed at
20 lM, and the concentrations of DNA varied from 0 to
Cell culture
Four different human carcinoma cell lines: Bel-7402, KB,
HL-60, and BGC-823 were cultured in RPMI-1640
123

Med Chem Res (2014) 23:1899–1907
1903
400 lM. The fluorescence intensity of samples was detected
on F-7000 spectrophotometer during the wavelength range of
425–690 nm (kex = 355 nm).
ditosylates in DMF afforded intermediates 3a–3c, respec-
tively. Oligoethyleneglycol ditosylates were synthesized
according to the reported procedure (Garcia et al., 1999).
These desired compounds 4a–4f were prepared by substi-
tution reactions of 3a–3c with commercially available
secondary amines (Huang et al., 2010).
Circular dichroism
CD spectra were recorded using a quartz cell of 1-cm
optical path length and an instrument scanning speed of
100 nm/min with a response time of 1 s, using 0.5 points/s
from 220 to 510 nm and 1 nm bandwidth. The CD spectra
were obtained by taking the average of two scans made
from 220 to 510 nm. The DNA and compound-DNA
samples were dissolved in Tris–HCl buffer (50 mM,
pH 6.35) and incubated at room temperature for 2 h. The
compounds were dissolved by Tris–HCl buffer (50 mM,
pH 6.35) and titrated into the DNA samples at 0.5 mol
equiv up to 20 mol equiv.
The ESI-HRMS of 4a–4f exhibit the molecular ion
peaks ([M-Cl]?) which correspond to their molecular
weight. The elemental analysis data of 4a–4f are in good
1
agreement with the calculated values. The H-NMR spec-
troscopic data of these compounds showing the signals at
6.5–8.5 and 8.5–10 ppm suggest the presence of aromatic
and N-heteroaromatic protons, which are accorded with the
skeletal structure of berberine derivatives. In addition, the
signals at 3–6 ppm are attached to the protons of methylene
(-CH2-). 13C-NMR and IR also further confirmed the
structure of the target products.
Thermal melting
Cytotoxicity
Melting curves of DNA and berberine derivative-DNA
complexes were measured in Tris–HCl buffer (50 mM,
pH 6.35). The CT-DNA and compound-DNA complexes
solutions were added into a quartz cell of 1-cm optical path
length. The temperature was ramped from 35 to 45 °C at a
scan rate of 2 °C/s and 45–97 °C at a scan rate of 1 °C/s
monitoring the absorbance changes of DNA at 260 nm.
The cytotoxicity of compounds 4a–4f, berberine, berb-
errubine, and cisplatin against HL-60, BGC-823, Bel-7402,
and KB cell lines was investigated. As listed in Table 1,
compounds 4a–4f displayed cytotoxicity with lower values
of IC50 (\50 lM) against tested cell lines. Compound 4a
exhibited higher cytotoxicity than cisplatin against HL-60
and BGC-823 cell lines.
Accordingly, for 9-substituted berberine derivatives 4a–
4f, the structure–activity relationships are summarized as
follows: (1) The polyethylene glycol side chain and ter-
minal group on the 9-position of berberine enhance the
cytotoxicity against Bel-7402 cell line. For example,
compounds 4a, 4c and 4e showed higher cytotoxicity than
1 against Bel-7402 cell line; compounds 4a–4f exhibited
higher cytotoxicity than 2 against Bel-7402 cell line;
compound 4a exhibited higher cytotoxicity than 1 and 2
against HL-60, BGC-823, Bel-7402 and KB cell lines. (2)
The length of the side chains on the 9-position of berberine
has an important effect on the cytotoxicity. For example,
Results and discussion
Chemistry
The synthesis of 9-substituted berberine derivatives (4a–4f)
with different polyethylene glycol side chains and terminal
groups (piperidine or morpholine) involved three steps
(Scheme 1). Partial demethylation of 1 was carried out at
about 190 °C under vacuum for 20 min to generate 2.
Then, treatment of 2 with various oligoethyleneglycol
5
4
1
3
2
6
O
O
O
O
Cl-
8
O
Cl-
N+
N+
Cl-
+ Cl-
N
O
N+
O
R
O
i
ii
Ts
9
O
iii
O
13
O
OH
m
OCH3
OCH3
O
n
12
OCH
10
OCH3
3
11
OCH3
R=
N
O
N
n=1, 2,
3
1
2
3a, 3b, 3c
m=0,
4a,
4c,
4e,
4b
4d
4f
1,
2,
Scheme 1 Synthesis of berberine derivatives. (i) 190 °C, 20–30 mmHg, 20 min; (ii) CH3CN, K2CO3, TsO(CH2CH2O)nTs (n = 1, 2, 3), 60 °C,
3 h; (iii)morpholine (or piperidine), CH3CN, K2CO3, 80 °C, 2–3 h
123

1904
Med Chem Res (2014) 23:1899–1907
Table 1 Cytotoxicity for
compounds against tumor cell
lines (HL-60, BGC-823, Bel-
7402, and KB)
Compound
IC50 (lM)
HL-60
BGC-823
Bel-7402
KB
6.03 0.24
1
5.83 0.26
11.36 0.42
2.71 0.23
6.51 0.47
16.39 0.68
46.80 1.02
14.82 0.53
45.76 1.32
2.9 0.3
7.04 0.29
7.66 0.34
2.82 0.31
10.17 0.62
11.89 0.56
22.18 1.17
10.84 0.64
20.59 1.13
6.5 0.8
16.28 0.68
34.31 0.53
11.58 0.65
27.53 0.86
9.44 0.38
17.86 0.76
9.66 0.58
33.43 0.89
2
6.92 0.36
5.46 0.48
17.12 0.63
9.93 0.43
19.68 0.85
10.03 0.72
33.94 0.76
2.6 0.3
4a
4b
4c
4d
4e
4f
Cisplatin
8.12
1
the cytotoxicity against HL-60, BGC-823 and KB cell lines
decreases in the sequences: 4a[4c[4d; the cytotoxicity
against HL-60 and BGC-823 cell lines decreases in the
sequences: 4b C 4e [ 4f. This suggests that compounds
with longer side chain may exhibit lower cytotoxicity. (3)
The nature of terminal groups in the side chains affect on
the cytotoxicity. For example, compound 4a linked with
morpholine displayed higher cytotoxicity than compound
4b linked with piperidine against HL-60, BGC-823, Bel-
7402, and KB cells lines. In summary, 9-substituted ber-
berine derivatives with different lengths of polyethylene
glycol side chains and terminal groups display selective
cytotoxicity against tested cells lines, both the length of the
polyethylene glycol side chains and the nature of the ter-
minal groups have important effects on the cytotoxicity.
molecules with DNA is important in the development of
DNA molecular probes and design of new and more effi-
cient drugs targeted to DNA (Erkkila et al., 1999; Ma-
heswari and Palaniandavar, 2004). The CT-DNA-binding
affinity of 9-substituted berberine derivatives 4a–4f was
investigated using UV–Vis, fluorescence, CD spectrosco-
pies, and thermal denaturation experiment.
UV–Vis absorption titration analysis
As shown in Fig. 2 and Fig. S1, the absorption intensity of
these compounds decreases with the increasing concentra-
tion of CT-DNA. The obvious hypochromicities (17–25 %)
and bathochromic shifts (5–8.5 nm) suggested that these
berberine derivatives can bind to CT-DNA in intercalative
mode. The values of red shifts and hypochromicities for
these compounds are higher than that of berberine (with the
value of hypochromicity is 5 %, and bathochromic shift is
1 nm), this indicates that the introduction of side chains and
terminal groups at 9-position of berberine enhances the
binding ability of berberine to CT-DNA. The binding
properties are similar to those previously observed inter-
calative ligand-DNA complexation (Friedman et al., 1991)
and are indication of intermolecular interaction involving
the p stacking with the base pairs of CT-DNA. Binding
DNA-binding study
DNA is an important cellular receptor; many compounds
exert their antitumor effects by binding to DNA thereby
changing the replication of DNA and inhibiting the growth
of the tumor cells, which is the basis of designing new and
efficient antitumor drugs (Barton et al., 1986; Pyle et al.,
1990; Friedman et al., 1991; Kozurkova et al., 2007).
Therefore, a clear understanding of the interaction of small
Fig. 2 Absorption spectra of
4a and 4f with increasing
concentration of CT-DNA
(0–400 lM) at the
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.45
0.40
0.39
0.38
0.37
0.36
0.35
0.34
0.33
4a
4b
0.40
0.39
0.38
0.37
0.36
0.35
0.34
0.33
0.32
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
concentration of 2.0 9 10-5
in 50 mM Tris–HCl buffer
(pH 6.35) at r.t. Arrows show
the absorbance changes upon
increasing DNA concentration.
Inset Plots of [DNA]/(ea–ef)
versus [DNA] for the
M
1
10-4
2
10-4
3
10-4
4
10-4
0
-4 3 10-4
4 10-4
1
10-4
Concentration of CT-DNA
2
10
Concentration of CT-DNA
0
compounds with CT-DNA
350
400
450
500
350
400
450
500
Wavelength/nm
Wavelength/nm
123

Med Chem Res (2014) 23:1899–1907
1905
to 6 nm (from 531 to 525 nm), which suggests that the
berberine backbones of these compounds intercalate into
the base pairs of DNA (Barton et al., 1986). The observed
fluorescence intensities were quantified by plotting F/F0
(F and F0 are the fluorescence intensity in the presence
and absence of CT-DNA) as a function of CT-DNA con-
centrations, and the value of F/F0 is obtained according to
Stern–Volmer analysis (Erkkila et al., 1999). As shown in
Table 3, the Stern–Volmer plots denote that these com-
pounds are sensitive to the concentration of CT-DNA.
Table 2 Binding constants (K) and photo physical properties of 4a–
4f with CT-DNA
Compound
K (M-1
)
Bathochromic
shifta (nm)
Hypochromicitya
(%)
4a
4b
4c
4d
4e
4f
a
3.55 9 108
1.27 9 108
6.32 9 106
1.49 9 106
3.45 9 105
1.86 9 105
8.0 (345–353)
17
21
21
19
25
21
8.5 (345–353.5)
7.0 (345.5–352.5)
5.5 (345.5–351)
8.5 (345–353.5)
5.0 (346–351)
Obtained at the absorption maximum
Circular dichroism studies
constants (K) of these compounds binding to CT-DNA are
calculated by the nonlinear least-squares curve-fitting
method (Kozurkova et al., 2007). As shown in Table 2, the
K values of these compounds binding to CT-DNA decrease
with the increasing length of the side chains. For example,
the K values decrease in the sequence: 4a[4c[4d and 4b
[4e[4f.
CD is a useful technique to investigate the conformational
changes in DNA morphology during small molecule-DNA
interactions. The CD spectra of compounds-DNA were
recorded and shown in Fig. 4. The spectra demonstrated
that the CT-DNA has the characterization of double helix
B-DNA conformation, with a positive band at 277 nm and
a negative band at 247 nm, which are generated by the
base stacking and right-handed helicity structure, respec-
tively. Increases in the intensity of the positive band and
decrease in the intensity of the negative band for CT-DNA
in the presence of compounds 4a, 4b, 4c, and 4e were
observed, which indicated that the compounds could
intercalate into the CT-DNA molecular, resulting in the
conformation of B-DNA change into A-like form (Ma-
heswari and Palaniandavar, 2004). On the other hand, both
positive and negative bands increase in intensity when the
compounds 4d and 4f are added to the CT-DNA solution,
which is typical intercalation involving p-stacking and
stabilization of the right-handed B form of CT-DNA (Xie
et al., 2011).
Fluorescence studies
To further ascertain the interaction of 4a–4f with CT-DNA,
the fluorescence spectra were detected in the range from
425 to 690 nm with an exciting wavelength at 355 nm at
room temperature. As shown in Fig. 3 and Fig. S2, ber-
berine derivatives have weak fluorescence properties at
around 525 nm. The fluorescence intensity of compounds
4a–4f increased largely with the increasing concentration of
CT-DNA, which indicated that these compounds strongly
interacted with CT-DNA. Furthermore, the maximum
emission wavelength undergoes an obvious blue shift of up
Fig. 3 Fluorescence spectra of
compounds 4a and 4f with
increasing concentration of CT-
DNA (0–400 lM) at the
3500
3000
2500
2000
1500
1000
500
5000
4500
4000
3500
45
30
25
20
15
10
5
4a
4b
40
35
30
25
20
15
concentration of 2.0 9 10-5
in 50 mM Tris–HCl buffer
(pH 6.35) at at r.t.,
M
10
5
3000
0
10-4
2 10-4
3 10-4
4 10-4
0
-44 10-4
0
1
1
10-4
Concentration of CT-DNA
2
10-4
3
10
Concentration of CT-DNA
2500
2000
1500
1000
500
kex = 355 nm. Arrow shows the
emission intensity increases
upon increasing DNA
concentration
0
0
450
500
550
600
650
450
500
550
600
650
Wavelength/nm
Wavelength/nm
Table 3 F/F0 of 4a–4f with CT-DNA
Compound
F/F0 (M-1
4a
4b
4c
4d
8.01 9 104
4e
1.79 9 105
4f
)
1.27 9 105
7.09 9 104
1.82 9 105
1.01 9 105
123

1906
Med Chem Res (2014) 23:1899–1907
Fig. 4 CD spectra of CT-DNA
and compound 4a–4f with the
fixed concentration of CT-DNA
(60 lM) at the concentration of
2.0 9 10-5 M in 50 mM Tris–
HCl buffer (pH 6.35) at r.t
12
10
8
6
4
12
10
8
6
4
CT-DNA
CT-DNA
4d+CT-DNA
4f+CT-DNA
4a+CT-DNA
4b+CT-DNA
4c+CT-DNA
4e+CT-DNA
2
0
2
0
-2
-4
-6
-8
-10
-2
-4
-6
-8
-10
250
300
350
400
450
500
250
300
350
400
450
500
Wavelength/nm
Wavelength/nm
Thermal denaturation studies
point of CT-DNA (Tm) increase obviously (Chaveerach
et al., 2010). As shown in Fig. 5, the Tm of CT-DNA is
75.8 °C in the absence of any compounds. The Tm of CT-
DNA increases in the presence of berberine derivatives 4a–
4f, with the values of increased melting point (4Tm) are 1.4,
1.7, 1.5, 2.2, 1.2, and 1.3 °C, respectively (as shown in
Table 4). These spectral properties suggested that these
9-substituted berberine derivatives could intercalate into the
base pairs of CT-DNA, and increase the stability of CT-DNA
(Waning, 1965; Neyhart et al., 1993).
To further verify the mode of interaction of berberine
derivatives with CT-DNA, thermal denaturation experiment
was conducted. As well known, double helix DNA is very
stable due to the presence of hydrogen bonds and base
stacking interactions. The two strands of DNA will disinte-
grate into single when the hydrogen bonds between the base
pairs are broken by heating. Small molecules, which can
intercalate into the base pairs, will make the value of melting
1.0
CT-DNA
CT-DNA+4a
Conclusions
CT-DNA+4c
0.8
CT-DNA+4d
CT-DNA+4b
CT-DNA+4e
We have described the synthesis of a new series of
9-substituted berberine derivatives with a varying length of
polyethylene glycol side chain and piperidine/morpholine as
a terminal group. The cytotoxicity assay suggested that
compound 4a exhibited higher cytotoxicity than cisplatin
against HL-60 and BGC-823 cell lines. Both the length of
polyethylene glycol side chain and the nature of terminal
group have important effects on the cytotoxicity. The cyto-
toxicity of these compounds decrease with the increasing
length of the side chains. Berberine derivative 4a linked with
morpholine exhibited higher cytotoxicity than 4b linked
with piperidine against HL-60, BGC-823, Bel-7402, and KB
cells lines. These semi-synthesized berberine derivatives
could intercalate into the base pairs of CT-DNA. The DNA-
binding affinities of these compounds decrease with the
increasing length of the side chains. Compounds 4a–4c and
4e could change the DNA conformation from the B to the
A-like form, and compounds 4d and 4f could stabilize the
right-handed B form of CT-DNA. These studies may provide
some guidance for the development of natural compounds as
anticancer agents with potential clinical value.
0.6
CT-DNA+4f
0.4
0.2
0.0
40
50
60
70
80
90
Temperature(°C)
Fig. 5 The melting curves of CT-DNA (10 lM) in the absence and
presence of compounds 4a–4f (100 lM) in 50 mM Tris–HCl buffer
(pH 6.35) at at r.t
Table 4 Tm and DTm for CT-DNA in the absence and presence of
compounds 4a–4f
Compound
Tm (°C)
DTm (°C)
CT-DNA
75.8
77.2
78
–
4a ? CT-DNA
4b ? CT-DNA
4c ? CT-DNA
4d ? CT-DNA
4e ? CT-DNA
4f ? CT-DNA
1.4
2.2
1.7
1.5
1.2
1.3
77.5
77.3
77
Acknowledgments This work was supported by the National Basic
Research 973 Program (No. 2010CB534913), the Natural Science
Foundation of Hebei Province (No. B2011201135), the Nature Sci-
ence Foundation for Distinguished Young Scholars of Hebei Province
(No. B2011201164), the Key Basic Research Special Foundation of
77.1
123

Med Chem Res (2014) 23:1899–1907
1907
´
´
´
´
´
´
Science Technology Ministry of Hebei Province (Grant No.
11966412D, 12966418D), the Science and Technology Support Pro-
ject of Hebei Province (No. 11276431), Hebei Provincial Natural
Science Foundation of China-Shijiazhuang Pharmaceutical Group
(CSPC) Foundation (B2011201174), the Key Research Project
Foundation of Department of Education of Hebei Province (Grant No.
ZH2012041), the Key Research Project Foundation of Department of
Education of Hebei Province (Grant No. ZH2012041).
Jantova S, Cipak L, Cernakova M, Kost’alova D (2003) Effect of
berberine on proliferation, cell cycle and apoptosis in HeLa and
L1210 cells. J Pharm Pharmacol 55(8):1143–1149
Kozurkova M, Sabolova D, Paulikova H, Janovec L, Kristian P,
ˇ
Bajdichova M, Busa J, Podhradsky D, Imrich J (2007) DNA-
binding properties and evaluation of cytotoxic activity of 9,10-
bis-N-substituted (aminomethyl)anthracenes. Int J Biol Macro-
mol 41(4):415–422
Kuo CL, Chou CC, Yung BY-M (1995) Berberine complexes with
DNA in the berberine-induced apoptosis in human leukemic HL-
60 cells. Cancer Lett 93(2):193–200
Lau CW, Yao XQ, Chen ZY, Ko WH, Huang Y (2001) Cardiovas-
cular actions of berberine. Cardiovasc Drug Rev 19(3):234–244
Maheswari PU, Palaniandavar M (2004) DNA-binding and cleavage
activity of [Ru(NH3)4(diimine)]Cl2 complexes. Inorg Chim Acta
357(4):901–912
Mazzini S, Bellucci MC, Mondelli R (2003) Mode of binding of the
cytotoxic alkaloid berberine with the double helix oligonucleo-
tide d(AAGAATTCTT)2. Bioorg Med Chem 11(4):505–514
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65(1–2):55–63
References
Barton JK, Goldberg JM, Kumar CV, Turro NJ (1986) Binding modes
and base specificity of tris(phenanthroline)ruthenium(II) enanti-
omers with nucleic acids: tuning the stereoselectivity. J Am
Chem Soc 108(8):2081–2088
Basu A, Jaisankar P, Kumar GS (2012) Synthesis of novel 9-O-N-
aryl/aryl–alkyl amino carbonyl methyl substituted berberine
analogs and evaluation of DNA-binding aspects. Bioorg Med
Chem 20(8):2498–2505
Chaveerach U, Meenongwa A, Trongpanich Y, Soikum C, Chaveerach
P (2010) DNA-binding and cleavage behaviors of copper(II)
complexes with amidino-O-methylurea and N-methylphenyl-
amidino-O-methylurea, and their antibacterial activities. Polyhe-
dron 29(2):731–738
Chen WH, Pang JY, Qin Y, Peng Q, Cai Z, Jiang ZH (2005) Synthesis
of linked berberine dimers and their remarkably enhanced DNA-
binding affinities. Bioorg Med Chem Lett 15(10):2689–2692
Erkkila KE, Odom DT, Barton JK (1999) Recognition and reaction of
metallointercalators with DNA. Chem Rev 99(9):2777–2795
Ferraroni M, Bazzicalupi C, Bilia AR, Gratteri P (2011) X-Ray
diffraction analyses of the natural isoquinoline alkaloids Ber-
berine and Sanguinarine complexed with double helix DNA
d(CGTACG). Chem Commun 47(17):4917–4919
Friedman AE, Kumar CV, Turro NJ, Barton JK (1991) Luminescence
of ruthenium(II) polypyridyls: evidence for intercalative binding
to Z-DNA. Nucleic Acids Res 19(10):2595–2602
Garcia C, Pointud Y, Jeminet G, Dugat D (1999) Novel cryptand
analogs incorporating a chiral spiran moiety. Tetrahedron Lett
40(27):4993–4996
Neyhart GA, Grover N, Smith SR, Kalsbeck WA, Fairley TA, Cory
M, Thorp HH (1993) Binding and kinetics studies of oxidation of
DNA by oxoruthenium(IV). J Am Chem Soc 115(11):4423–
4428
Ouchi M, Inoue Y, Liu Y, Nagamune S, Nakamura S, Wada K,
Hakushi
K (1990) Convenient and efficient tosylation of
oligoethylene glycols and the related alcohols in tetrahydrofu-
ran-water in the presence of sodium hydroxide. Bull Chem Soc
Jpn 63:1260–1262
Pang JY, Qin Y, Chen WH, Luo GA, Jiang ZH (2005) Synthesis and
DNA-binding affinities of monomodified berberines. Bioorg
Med Chem 13(20):5835–5840
Paterson I, Anderson EA (2005) Chemistry. The renaissance of
natural products as drug candidates. Science 310:451–453
Pyle AM, Morri T, Barton JK (1990) Probing microstructures in
double-helical DNA with chiral metal complexes: recognition of
changes in base-pair propeller twisting in solution. J Am Chem
Soc 112(25):9432–9434
Waning MJ (1965) Complex formation between ethidium bromide
and nucleic acids. J Mol Biol 13:269–282
Huang L, Luo Z, He F, Shi A, Qin F, Li X (2010) Berberine
derivatives, with substituted amino groups linked at the
9-position, as inhibitors of acetylcholinesterase/butyrylcholines-
terase. Bioorg Med Chem Lett 20(22):6649–6652
Xie L, Cui J, Qian X, Xu Y, Liu J, Xu R (2011) 5-Non-amino
aromatic substituted naphthalimides as potential antitumor
agents: synthesis via Suzuki reaction, antiproliferative activity,
and DNA-binding behavior. Bioorg Med Chem 19:961–967
Iwasa K, Kamigauchi M, Ueki M, Taniguchi M (1996) Antibacterial
activity and structure-activity relationships of berberine analogs.
Eur J Med Chem 31(6):469–478
123