Journal of Inorganic Biochemistry 105 (2011) 426–434

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Journal of Inorganic Biochemistry

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o

Synthesis, characterization and preliminary cytotoxicity evaluation of five

Lanthanide(III)–Plumbagin complexes
Zhen-Feng Chen a,⁎, Ming-Xiong Tan a,c, Yan-Cheng Liu a, Yan Peng a, Hong-Hong Wang a,
Hua-Gang Liu b,⁎, Hong Liang a,c,⁎
a
Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China),
School of Chemistry & Chemical Engineering of Guangxi Normal University, Guilin 541004, PR China
b
School of Pharmacy, Guangxi Medical University, Nanning 530021, PR China
c
School of Chemistry, South Central University, Changsha 410083, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, H-PLN) was isolated from Plumbago zeylanica, the
Received 6 July 2010 anticancer traditional Chinese medicine (TCM). Five new lanthanide(III) complexes of deprotonated
Received in revised form 7 December 2010 plumbagin: [Y(PLN)3(H2O)2] (1), [La(PLN)3(H2O)2] (2), [Sm(PLN)3(H2O)2]⋅H2O (3), [Gd(PLN)3(H2O)2] (4),
Accepted 8 December 2010
and [Dy(PLN)3(H2O)2] (5) were synthesized by the reaction of plumbagin with the corresponding lanthanide
Available online 21 December 2010
salts, in amounts equal to ligand/metal molar ratio of 3:1. The PLN–lanthanide(III) complexes were
Keywords:
characterized by different physicochemical methods: elemental analyses, UV–visible, IR and 1H NMR and ESI-
Plumbagin MS (electrospray ionization mass spectrum) as well as TGA (thermogravimetric analysis). The plumbagin and
Lanthanide(III) complex its lanthanide(III) complexes 1–5, were tested for their in vitro cytotoxicity against BEL7404 (liver cancer) cell
Cytotoxicity lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The five PLN–
DNA binding lanthanide (III) complexes 1–5 effectively inhibited BEL7404 cell lines growth with IC50 values of 11.0 ±
3.5, 5.1 ± 1.3, 6.1 ± 1.1, 6.4 ± 1.3, and 9.8 ± 1.5 μM, respectively, and exhibited a significantly enhanced
cytotoxicity compared to plumbagin and the corresponding lanthanide salts, suggesting a synergistic effect
upon plumbagin coordination to the Ln(III) ion. The lanthanide complexes under investigation also exerted
dose- and time-dependent cytotoxic activity. [La(PLN)3(H2O)2] (2) and plumbagin interact with calf thymus
DNA (ct-DNA) mainly via intercalation mode, but for [La(PLN)3(H2O)2] (2), the electrostatic interaction
should not be excluded; the binding affinity of [La(PLN)3(H2O)2] (2) to DNA is stronger than that of free
plumbagin, which may correlate with the enhanced cytotoxicity of the PLN–lanthanide(III) complexes.
© 2010 Elsevier Inc. All rights reserved.

1. Introduction
Since the success of cisplatin and related platinum complexes as
anticancer agents, developing other active transition metal anticancer
complexes with better efficiency has attracted many bioinorganic
chemists' interest and become a central research theme in bioinor-
ganic chemistry [1–5]. Lanthanide complexes have attracted medic-
inal inorganic chemists' attention, because lanthanides manifest an
antitumor activity and may be developed into future anticancer drugs.
In the past twenty years, a number of lanthanide complexes have been
synthesized and their cytotoxicities evaluated. Some examples are
La(III) complexes with 1,10-phenanthroline-2,9-bis-α-amino acid
conjugates [6] or 1,10-phenanthroline [7], coumarines [8–10], 3,5-
pyrazoledicarboxylic acid [11], Sm(III) and Gd(III) complexes with
acenocoumarol [12], cerium(III) and neodymium(III) complexes
⁎ Corresponding authors.
E-mail addresses: chenzfubc@yahoo.com (Z.-F. Chen), hgliu@263.net (H.-G. Liu),
hliang@gxnu.edu.cn (H. Liang).
with 5-aminooritic acid [13] and many other lanthanide complexes
[14,15].
In order to develop new metal-based anti-cancer drugs, recently,
new strategies have been applied in the designs of antitumor
coordination compounds as drugs, such as synthesizing new ligands
or metal complexes with different reaction mechanisms. Among
them, new coordination compounds based on the traditional Chinese
medicines (TCMs) provide a novel approach to potential (pro-)drugs
[16–23], which have been recently reviewed by Chen and Liang [24].
It is well known that over long-term folk practice, a large number
of TCMs have been screened and used for treating and preventing
various chronic conditions, such as cancer, atherosclerosis, aging,
diabetes, and other degenerative diseases [25,26]. Plumbagin, 5-
hydroxy-2-methyl-1,4-naphthoquinone (H-PLN, Scheme 1) is a
potent toxic natural product extracted from Plumbago zeylanica
L. (Plumbaginaceae), which has been used in China and other Asian
countries for the treatment of rheumatoid arthritis, dysmenorrhea,
injury by bumping, and even cancer [27–29]. Plumbagin is structurally
derived from quinones which are a broadly distributed class of

0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.12.003
 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434
Scheme 1. Plumbagin (H-PLN).
naturally occurring substances with a variety of biological activities,
e.g. antibacterial, anticancer, antioxidant and anti-inflammatory.
Earlier studies have demonstrated that the metal complexes of
many quinones chemotherapeutic drugs such as daunorubicin and
adriamycin used today, display less cardiotoxicity than the parent
drug but effectively kill p388 leukemia [30]. These findings have
stimulated increasing interest in the study of the metal complexes of
plumbagin with an antitumor activity. Although the lanthanide
plumbaginates and their antibacterial activity have been reported
previously [31–35], but firstly, the cytotoxicity of the traditional
Chinese medicine (TCM) plumbagin in its copper chemistry has
been reported recently. It was observed that these copper(II)
plumbaginates exhibited a significantly enhanced cytotoxicity vs.
free plumbagin [20].
As a part of our continuing work on the synthesis, characterization
and application of metal complexes with plumbagin [20], herein, we
report the synthesis, characterization and in vitro cytotoxicity against
BEL7404 cell lines of five new lanthanide plumbaginates. The binding
properties of plumbagin and [La(PLN)3(H2O)2] (2) to DNA were
investigated by means of UV–visible (UV–vis), fluorescence, circular
dichroism (CD) spectroscopy, and agarose gel electrophoresis assay.
2. Experimental section
2.1. Materials
All the metallic salts were purchased from Alfa co. Ltd. The solvents
used were analytical grade. All the materials were used as received
without further purification unless noted specifically. Tris–HCl–NaCl
buffer solution (5 mM Tris, 50 mM NaCl), pH was digitally adjusted to
7.35 by titration with hydrochloric acid with Sartorius professional
meter, Tris was prepared using double distilled water. Calf thymus
DNA (ct-DNA) was purchased from Sino-American Biotech. co. Ltd,
Beijing of China. They were both used without further purification. A
Tris-buffer solution of ct-DNA gave ratios of UV absorbance at 260 and
280 nm of ca. 1.8 − 1.9:1, indicating that the DNA was sufficiently free
of protein. The DNA concentration per nucleotide in base pairs was
determined spectrophotometrically by employing a molar absorptiv-
ity (6600 M− 1 cm− 1) at 260 nm. Stock solution were stored at 4 °C
and used for no more than 4 days.
Traditional Chinese medicine material, roots of P. zeylanica were
collected in Guangxi province of China, in September, 2004 and
identified by Prof. S. Q. Tang (School of Life Science, Guangxi Normal
University). A voucher specimen was deposited at the School of
Chemistry & Chemical Engineering, Guangxi Normal University of
China.
2.2. Instrumentation
Infrared spectra were obtained on a Perkin-Elmer FT-IR Spectrom-
eter. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-500
NMR spectrometer using CD3Cl or DMSO-d6 as a solvent. Elemental
427
analyses (C, H) were carried out on a Perkin Elmer Series II CHNS/O
2400 elemental analyzer. ESI-MS (electrospray ionization mass
spectrum) were recorded on a Bruker HCT Electrospray Ionization
Mass Spectrometer. TGA (thermogravimetric analysis) were recorded
on Perkin-Elmer Pyris Diamond TG/DTA analyzer. UV–visible (UV–
vis) absorption spectra were performed on a Varian Cary100 UV–
Visible spectrophotometer. Fluorescence measurements were per-
formed on a Shimadzu RF-5301/PC spectrofluorophotometer. The
circular dichroic spectra of DNA were obtained by using a JASCO J-810
automatic recording spectropolarimeter operating at 25 °C. The
region between 220 and 320 nm was scanned for each sample.
Microscope images were recorded on Nikon TE2000.
2.3. Isolation and structure identification of plumbagin
Dried and powdered roots (15 kg) of P. zeylamca were successively
and exhaustively extracted with 95% ethanol at 50 °C. The ethanol
extracted solution was concentrated under reduced pressure to give a
dark brown residue. The residue was suspended in water, and then
partitioned successively with n-hexane and EtOAc, respectively, to
afford the residues of n-hexane (23 g) and EtOAc (252 g), respective-
ly. The EtOAc soluble-extract (252 g) was purified by a silica gel
column chromatography using n-hexane-EtOAc (from 9:1 to 0:1, v/v)
as a solvent system to give six fractions (F1–F6). Orange needle-like
crystals of plumbagin were obtained from fraction 1 (F1) by re-
crystallizing in n-hexane-EtOAc (5:1), with a purity of over 98% as
determined by HLPC (by peak area normalization). The total yield of
plumbagin obtained after extraction and purification by column
chromatography was 13.3 g (0.09% basing on the dried and powdered
roots (15 Kg) of P. zeylamca). IR (v, cm− 1, KBr, s = strong, m =
medium, w = weak): 3445 m, 1663 s, 1645 s, 1567 m, 1456 m,
1365 m, 1229 m, and 752w. 1H NMR (CDCl3, 500 M Hz) (δ ppm):
δ2.22 (3 H, s(singlet), -CH3), δ 6.83 (1H, s, H-3), δ 7.28 (1H, d
(doublet), J = 8.5 Hz, H-6), δ 7.60 (1H, d, J = 6.2 Hz, H-8), δ 7.66 (1H, d,
J = 7.5 Hz, H-7), δ11.99(1H, s, -OH), 13 C NMR (CDCl3, 125 M Hz)
(δ, ppm): δ 16.5(-CH3), δ 115.2(C-10), δ119.3(C-8), δ124.2(C-6),
δ 132.0 (C-9), δ135.5(C-7), δ 136.1(C-3), δ149.6(C-2), δ161.2(C-5),
δ184.8(C-1), δ190.3(C-4).
2.4. Synthesis of [Y(PLN)3(H2O)2] (1)
An ethanolic solution (15 mL) of plumbagin (0.564 g, 3 mmol) was
added to an aqueous solution (15 mL) of YCl3·6H2O (0.303 g,
1 mmol), and was adjusted pH to 6.0 with dilute ammonia solution.
The reaction mixture was refluxed and stirred with an electromag-
netic stirrer for 2 h. At the moment of mixing of the solutions, red
brown precipitate was obtained. After cooling to room temperature,
the precipitate was filtered, washed three times with water and
ethanol, and dried in a desiccators containing P2O5 to constant weight.
Finally the red brown solid of [Y(PLN)3(H2O)2] (1) was obtained.
Yield: 0.38 g (56% yield basing on plumbagin); Anal. Calc. for
C33H25O11Y: C, 57.74; H, 3.67. Found: C, 57.65; H, 3.53%. IR (v, cm− 1,
KBr): 3433 m, 1642 m, 1604 s, 1424 m, 1254 m, 622w. ESI-MS (in
DMSO-H2O-CH3OH): m/z 668.9 (Calc. 669.4) [Y(PLN)3 + H2O + H]+,
m/z 463.1 (Calc. 463.2) [Y(PLN)2]+. UV–Vis (DMSO): λmax = 270,
λmax = 424 nm.
2.5. Synthesis of [La(PLN)3(H2O)2] (2)
A purple solid of [La(PLN)3(H2O)2] (2) was synthesized by the
same method as that employed for [Y(PLN)3(H2O)2] (1) using
LaCl3·6H2O to replace YCl3·6H2O (plumbagin 3 mmol, LaCl3·6H2O
1 mmol). Yield: 0.40 g (55% yield basing on plumbagin); Anal. Calc. for
C33H25LaO11: C, 53.82; H, 3.42. Found: C, 54.71; H, 3.31%. IR (v, cm− 1,
KBr): 3433 m, 1642 m, 1610 s, 1424 m, 1251 m, 614w. 1 H NMR(DMSO-
d6, 500 M Hz) (δ ppm): δ2.01 (3 H, s, -CH3), δ 6.63 (1 H, s, H-3), δ 6.87
428 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434
(1 H, m(multiplet), H-6), δ 7.13 (1 H, m, H-8), δ 7.43 (1 H, m, H-7). ESI-
MS (in DMSO-H2O-CH3OH): m/z 737.0 (Calc. 737.5), [La(PLN)3 + H2O +
Na]+, m/z 669.8 (Calc. 669.5) [La(PLN)2 + 2DMSO]+. UV–Vis (DMSO):
λmax = 265, λmax = 420 nm.
2.6. Synthesis of [Sm(PLN)3(H2O)2]⋅H2O (3)
A yellow brown solid of [Sm(PLN)3(H2O)2]⋅H2O (3) was synthe-
sized by the same method as that employed for [Y(PLN)3(H2O)2] (1)
using SmCl3·6H2O to replace YCl3·6H2O (plumbagin 3 mmol,
SmCl3·6H2O 1 mmol). Yield: 0.41 g (54% yield basing on plumbagin);
Anal. Calc. for C33H27O12Sm: C, 51.75; H, 3.55. Found: C, 52.62; H,
3.45%. IR (v, cm− 1, KBr): 3399 m, 1640 m, 1603 s, 1423 m, 1253 m,
617w. ESI-MS (in DMSO–H2O–CH3OH): m/z 752.8 (Calc. 752.9), [Sm
(PLN)3 + H2O + Na]+, m/z 656.9 (Calc. 657.2) [Sm(PLN)2 + DMSO +
CH3ONa]+. UV–Vis (DMSO): λmax = 267, λmax = 416 nm.
2.7. Synthesis of [Gd(PLN)3(H2O)2] (4)
A mauve solid of [Gd(PLN)3(H2O)2] (4) was synthesized by the
same method as that employed for [Y(PLN)3(H2O)2] (1) using
GdCl3·6H2O to replace YCl3·6H2O (plumbagin 3 mmol, GdCl3·6H2O
1 mmol). Yield: 0.43 g (57% yield basing on plumbagin). Anal. Calc. for
C33H25GdO11: C, 52.51; H, 3.34; Found: C, 53.43; H, 3.21%. IR (v, cm− 1,
KBr): 3442 m, 1642 m, 1611 s, 1426 m, 1253 m, 616w. ESI-MS (in
DMSO–H2O–CH3OH): m/z 769.6 (Calc.769.8), [Gd(PLN)3 + CH3OH +
H2O + H]+, m/z 581.4 (Calc. 581.7), [Gd(PLN)2 + CH3OH+ H2O]+. UV–
Vis (DMSO): λmax = 268, λmax = 415 nm.
2.8. Synthesis of [Dy(PLN)3(H2O)]2 (5)
A yellow brown solid of [Dy(PLN)3(H2O)2] (5) was synthesized by
the same method as that employed for [Y(PLN)3(H2O)2] (1) using
DyCl3·6H2O to replace YCl3·6H2O (plumbagin 3 mmol, DyCl3·6H2O
1 mmol). Yield: 0.38 g (50% yield basing on plumbagin). Anal. Calc. for
C33H25DyO11: C, 52.15; H, 3.32; Found: C, 52.33; H, 3.28%. IR (v, cm− 1,
KBr): 3435 m, 1640 m, 1598 s, 1423 m, 1254 m, 620w. ESI-MS (in
DMSO–H2O–CH3OH): m/z 783.4 (Calc. 783.0), [Dy(PLN)3 + 2H2O +
Na]+, m/z 693.0 (Calc. 693.1), [Dy(PLN)2 + 2DMSO] +. UV–Vis
(DMSO): λmax = 268, λmax = 423 nm.
2.9. In vitro cytotoxicity
BEL7404 cell lines (liver cancer) were obtained from Shanghai Cell
Bank in Chinese Academy of Sciences. Cell lines were grown in DMEN
(Gibco, Scotland, UK) at 37 °C in a humidified atmosphere of 5% CO2/
95% air. In order to investigate the potential of these synthetic
complexes, cisplatin, a commonly used anti-cancer drug was selected
as a reference metallodrug.
Assays of cytotoxicity were conducted in 96-well, flat bottomed
microtitre plates. The supplemented culture medium with cell lines was
added to the wells. Complexes 1–5, plumbagin, cisplatin, and
corresponding lanthanide salts were dissolved in the culture medium
with 1% DMSO to give various concentrations (1.25, 2.5, 5, 10, 20 μg/mL,
respectively). The resultant solutions were subsequently added to a set
of wells. Control wells contained supplemented media with 1% DMSO.
The microtitre plates were incubated at 37 °C in a humidified
atmosphere of 5% CO2/95% air for a further 3 day. Cytotoxic screening
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was carried out. At the end of each incubation period, the
MTT solution (10 μL, 5 mg/mL) was added into each well and the
cultures were incubated further for 48 h (for the time-dependent
cytotoxic effects studies, the treatment time is 24, 48, and 72 h, re-
spectively) at 37 °C in a humidified atmosphere of 5% CO2/95% air. After
removal of the supernatant, DMSO (150 μL) was added to dissolve the
formazan crystals. The absorbance was read by enzyme labelling
instrument with 570/630 nm double wavelength measurement. The
cytotoxicity was evaluated based on the percentage cell survival in a
dose-dependent manner relative to the negative control. The final IC50
values were calculated by the Bliss method (n = 5). All the tests were
repeated by at least three independent experiments.
2.10. UV–visible (UV–vis) absorption titration
Absorption titrations were performed by using a fixed compound's
concentration (6.7 × 10− 5 M [La(PLN)3(H2O)2] (2), 6.0 × 10− 4 M
plumbagin) and varying the concentration of DNA (50 μL DNA per
scan for [La(PLN)3(H2O)2] (2), 10 μL DNA per scan for plumbagin).
While measuring the absorption spectra, the solutions were allowed
to incubate for 10 min before the absorption spectra were recorded
and an equal amount of ct-DNA was added to both the compound
solution and the reference solution to eliminate the absorbance of ct-
DNA itself.
2.11. Fluorescence emission titration
Fluorescence emission spectra of the DNA–EB system were
determined with DNA pre-treated with ethidium bromide (EB) at a
ratio of [DNA]/[EB] = 6:1 for 30 min. To the DNA-EB solution in-
creasing amounts of compounds (7.0 × 10− 4 M for [La(PLN)3(H2O)2]
(2), 6.0 × 10− 4 M for plumbagin, 50 μL per scan, respectively) were
added and their effects on the emission intensity were measured.
Samples were excited at 350 nm and the emissions were observed
between 500 and 700 nm.
2.12. Circular dichroism spectra
The CD absorption spectra of ct-DNA were recorded in the absence
and presence of [La(PLN)3(H2O)2] (2) and plumbagin with [com-
pound]/[ct-DNA] = 0.25 in Tris–HCl buffer, pH = 7.35. The sample
solution was mixed and was allowed at room temperature. Each
sample solution was scanned in the range 200–400 nm UV–vis region
with a screening rate of 100 nm/min at room temperature.
2.13. Agarose gel electrophoresis assay
For agarose gel electrophoresis assay experiments, supercoiled
plasmid PUC19 DNA was treated with the test compounds in 5 mM
Tris–HCl, 50 mM NaCl buffer, pH 7.2 and the solution was incubated
for 2 h in the dark at 37 °C. The samples were electrophoresed for 1 h
at 110 V on a 0.8% agarose gel in Tris–HCl/NaCl buffer. The gel was
stained with 0.5 μg/mL ethidium bromide and then photographed
under UV light and visualized by a Bio-Rad Gel Imaging System.
2.14. Statistics
The data processing included the Student's t-test with P ≤ 0.05
taken as significance level, using SPSS 13.0.
3. Results and discussion
3.1. Isolation and structure identification of plumbagin (H-PLN)
Plumbagin is a naphthoquinone, which was isolated from the
ethanol extract of roots of P. zeylanica, and was identified by 1H NMR
and 13C NMR.
3.2. Characterization of complexes 1–5
Complexes 1–5 were synthesized by mixing ethanol and water
solutions of plumbagin and the corresponding Ln(III) salts, in amounts
equal to a ligand/metal molar ratio of 3:1. The PLN–lanthanide(III)
 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434 429

complexes were characterized by different physicochemical methods: Table 1

elemental analyses, FT–IR and UV–visible spectroscopy, 1H NMR and Significant IR peaks (cm− 1).

ESI-MS and TGA measurements. It should be pointed out that the Compounds v(O–H) v(C = O) v(C–O)
syntheses of the complexes took place in aqueous solution (ethanol:
Chelated Free
water = 1:1, v:v), but the complexes were insoluble in water, slightly
Plumbagin 3445 1645 1663 1229
soluble in methanol, acetone, and well soluble in DMSO. Therefore,
1 [Y(PLN)3(H2O)2] (1) 3433 1604 1642 1254
the experiments involving solution properties studies need dissolu- 2 [La(PLN)3(H2O)2] (2) 3433 1610 1642 1251
tion in DMSO. 3 [Sm(PLN)3(H2O)2]⋅H2O (3) 3399 1603 1640 1253
4 [Gd(PLN)3(H2O)2] (4) 3442 1611 1642 1253
3.3. Electronic spectra 5 [Dy(PLN)3(H2O)2] (5) 3435 1598 1640 1254

As shown in Fig. 1, the electronic spectra of plumbagin in the

region 200–600 nm present two characteristic bands, an intensive
band at 265 nm and a less intensive band at 418 nm, which are
conveniently described in terms of quinonoid electronic transitions
(QET) and n → π transition [36,37]. The band observed at 265 nm for
plumbagin, due to QET, is red-shifted in the spectra of the PLN–
lanthanide chelates. The amounts of these shifts however are small,
i.e., 2–5 nm. The intensity has a considerable decrease in comparison
to free plumbagin. The band due to n → π transition, observed in the
region 400–500 nm, is very broad and weak but shows definite trends
similar to those for QET. For lanthanide plumbaginates, it shows small
amount of red shift or blue shift, i. e., 3–6 nm. The intensity has a
considerable decrease in comparison to free plumbagin. The occur-
rence of red shifts or blue shifts for PLN–lanthanide chelates indicates
the decrease or increase in the corresponding energy gaps in the
electronic energy levels of the plumbagin as a result of chelation [37].
3.4. IR spectra
The significant IR peaks of the free plumbagin and their lanthanide
chelates are shown in Table 1. These peaks are examined particularly
to assess the effect of chelation on their peak position and peak
intensity leading to the following important conclusions.
(i) The lanthanide plumbaginates are strongly hydrated as
indicated by the presence of characteristic bands due to O–H
stretching frequency in the region 3600–3300 cm−1.
(ii) The chelated as well as unchelated C=O stretching frequencies
[6,7] are shifted to the lower frequency region for lanthanide
plumbaginates. The large and red shift of the chelated C=O
frequency by an amount of ca. 40 cm− 1 units indicates that the
coordination bonding to lanthanide is via carbonyl oxygen [38].
(iii) A strong v(C–O) phenolic vibration at approx. 1229 cm− 1 for
free plumbagin is shifted to higher frequency region for lan-
thanide plumbaginates (1251–1254 cm− 1) (Δ22–25 cm− 1),
confirming the participation of the C(5) oxygen atom in the
coordination [37,39].
3.5. 1H NMR spectra
Plumbagin and its [La(PLN)3(H2O)2] (2) were examined by 1H
NMR spectroscopy; the chemical shift differences between [La(PLN)3
(H2O)2] (2) and plumbagin were shown in Table 2. Due to an electron
transfer from the hydroxyl and the carbonyl oxygen atoms to La(III),
[La(PLN)3(H2O)2] (2) exhibited upfield shifts and the peaks were
broadened (Fig. S1, ESI*), indicating the complexation of plumbagin.
This observation is consistent with previously reported on a series of
lanthanide complexes with coumarin derivatives (L), such as La(L)
(OH)⋅H 2 O (L = 4-hydroxycoumarin) [10,40], Ce(L)(OH)⋅H 2 O
(L = bis-coumarin) [41], La(L)3⋅ H2O (L = acenocoumarol) [42]. In
addition, compared with the 1H NMR of plumbagin, for La(III) com-
plex, the single peak ascribing hydroxyl proton (5-OH) of the free
ligand disappeared [14], which indicated that the hydroxyl group of
the ligand coordinated to the La(III) ions. On the basis of the results
obtained, it suggests that the plumbagin chelated to the La(III) ion via
both 4-C–O and 5-OH, resulting in PLN–La(III) complex.
3.6. Thermogravimetric analysis (TGA)
TGA traces of complexes 1–5 exhibit similar weight losses upon
heating. The results indicate that they at first lost the co-crystallized
water molecules by 110 °C, such as the first weight loss 2.6%
(calculated 2.4%) for [Sm(PLN)3(H2O)2]⋅H2O (3); then coordinated
aqua ligands loss were completed by about 160 °C, such as the second
weight loss 5.5% (calculated 5.2%) for [Y(PLN)3(H2O)2] (1), 5.2%
(calculated 4.9%) for [La(PLN)3(H2O)2] (2), 5.1% (calculated 4.7%) for
[Sm(PLN)3(H2O)2]⋅H2O (3), 5.2% (calculated 4.7%) for [Gd(PLN)3
(H2O)2] (4), and 5.1% (calculated 4.7%) for [Dy(PLN)3(H2O)2] (5);
Further heating to 260 °C led to a sharp decomposition of these
complexes. The TGA results of complexes 1–5 are in agreement with
the structural characterizations mentioned previously.
On the basis of the experimental results, our previously published
PLN-copper(II) crystal structure analysis, where the plumbagin
chelated to the Cu(II) ion via both 4-C–O and 5-(OH) [20], we are
able to suggest the most probable structure of the new complexes
(Scheme 2). The PLN–lanthanide complexes 1–5 may possess a
general formula [Ln(PLN)3(H2O)2]⋅nH2O, where Ln are lanthanide(III)
cations (n = 0 for Y(III) (1), La(III) (2),Gd(III) (4), Dy(III) (5); n = 1 for

Table 2
1
H NMR chemical shift differences between plumbagin and its complex 2.

Hydrogen site δ (plumbagin) δ(complex 2) Δδa

3 6.83 6.63 − 0.20

6 7.28 6.87 − 0.41
7 7.66 7.43 − 0.23
8 7.60 7.13 − 0.47
11 2.22 2.01 − 0.21
5-OH 11.99 − −

Fig. 1. UV–vis spectra of plumbagin and complexes 1–5 (in DMSO). a: Δδ = δ(complex 2) − δ (plumbagin).
430 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434
Scheme 2. The possible structure of complexes 1–5 (n = 0, Ln = Y(III) (1), La(III) (2),
Gd(III) (4), Dy(III) (5); n = 1, Ln = Sm(III) (3)).
Sm(III) (3)). It should be pointed out that the data presented may
indicate a structure, definitive structural proof, however, comes from
X-ray crystallography. Although we tried many methods, but we still
are not able to obtain good quality crystals suitable to X-ray diffraction
analysis. It is well known that a coordination structure in solid
(crystal) is often different from its dissolved form. The ESI-MS
analyses of complexes 1–5 revealed that two major species peaks
were observed, which existed in two cationic species with metal/
ligand molar ratios of 1:3 and 1:2, respectively, in solution. Therefore,
it should be taken into account that the spectra and pharmacology
were measured in DMSO and changes could occur with the complexes
because of the high coordination number of Ln(III) [42].
3.7. In vitro cytotoxicity
In vitro cytotoxicity of plumbagin and its five new lanthanide (III)
complexes was evaluated by MTT assay on BEL7404 cell lines. Under
the same experimental conditions, cisplatin was also evaluated as
reference metallodrug. The IC50 values towards BEL7404 were
reported in Fig.2.
As shown in Fig. 2, the PLN–lanthanide complexes 1–5 inhibited
the growth BEL7404 cell lines with IC50 values of 11.0 ± 3.5, 5.1 ± 1.3,
6.1 ± 1.1, 6.4 ± 1.3, and 9.8 ± 1.5 μM, respectively. Complexes [La
(PLN)3(H2O)2] (2), [Sm(PLN)3(H2O)2]⋅H2O (3), and [Gd(PLN)3
(H2O)2] (4) show better toxicity than that of [Y(PLN)3(H2O)2] (1)
and [Dy(PLN)3(H2O)2] (5), which are about 2 times higher than that
Fig. 2. IC50 values for complexes 1–5 against BEL7404 (μM). IC50 values are presented as
the mean ± SD (standard error of the mean) from five separated experiments. Cisplatin
was used as a reference metallodrug and DMSO was used as a solvent control.
of [Y(PLN)3(H2O)2] (1) and [Dy(PLN)3(H2O)2] (5). Meanwhile the
cytotoxicity of complexes 1–5 is about 3–7 times higher than that of
cisplatin (IC50 36.4 ± 7.6 μM) and 13-cis-retinoyl ferrocene deriva-
tives (IC50 22.3–42.6 μM) [43], and is slightly better than that of [Cu
(PLN)(bpy)(H2O)]2(NO3)2⋅4H2O (IC50 12.9 ± 3.6 μM) [20]. It can be
seen the synthetic PLN–lanthanide complexes have a better potential
than cisplatin, if we only judge from the in vitro cytotoxicity. As shown
in Table 3, complexes 1–5 exhibit a better cytotoxic activity than that
of plumbagin (at concentration of 10 μg/mL, the inhibitory rate of
plumbagin to BEL7404 only is 2.3%) and corresponding lanthanide
salts (at concentration of 10 μg/mL, the inhibitory rates of five
lanthanide salts towards BEL7404 are in range 10.7–36.0%). Also,
these lanthanide plumbaginates to BEL7404 are more cytotoxic than
lanthanide complexes with amino acid Schiff base ligand [44]. Similar
to copper(II) plumbaginates [20], these new PLN–lanthanide com-
plexes exhibit a significantly enhanced cytotoxicity vs. free plumbagin
towards BEL7404, suggesting that these PLN–lanthanides display a
synergistic effect upon the combination of lanthanide with plumba-
gin. The inhibition growth ability of these new PLN–lanthanides
against BEL7404 is confirmed further by the microscope images. As
shown in Fig. 3, in comparison with the untreated (image (a)) and
positive control (image (c)) and (image (d)), it can be seen that the
PLN–lanthanide(III) complex effectively inhibits the growth of
BEL7404.
To determine the dose- and time-dependent relationship, BEL7404
cell lines were exposed to increasing doses of [Y(PLN)3(H2O)2] (1)
and [La(PLN)3(H2O)2] (2) for increasing periods of time (24, 48 and
72 h), and cell viability was measured by the MTT assay. Cytotoxic
effects were achieved in concentrations of 1.25–20 μg/mL. As shown
in Figs. 4 and 5, BEL7404 cell lines exhibit increased sensitivity to the
complexes in a dose- and time-dependent manner. These findings are
comparable to copper(II) plumbaginate complexes to MCF-7 and 786-
O [20]. Towards BEL7404, [La(PLN)3(H2O)2] (2) exhibits a higher
cytotoxicity than [Y(PLN)3(H2O)2] (1). At the concentration of 20 μg/
mL, [Y(PLN)3(H2O)2] (1) and [La(PLN)3(H2O)2] (2) acquired the cell
growth inhibition rates of about 60–70% during the 72 h incubation
period.
3.8. DNA binding studies
Since DNA is the primary pharmacological target of many metal-
based antitumor agents [45,46], DNA–metal complex interaction has
paramount importance in understanding the mechanism of tumor
inhibition for the treatment of cancer. Because the five new PLN–
lanthanide complexes have similar chemical structure and analogous
cytotoxicity, [La(PLN)3(H2O)2] (2) was selected as representative to
investigate the interaction with DNA. The interactions of [La(PLN)3
(H2O)2] (2) and plumbagin with DNA were investigated by absorption
titration, fluorescence spectroscopy, circular dichroism, and agarose
gel electrophoresis assay.
Table 3
Inhibitory rates of plumbagin, its lanthanide complexes and lanthanide salts against
BEL7404 (the tested compounds with concentration of 10 μg/mL, treatment of 48 h
with the tested compounds).
Compounds Inhibitory Lanthanide Inhibitory
rate (%) salts rate (%)
Plumbagin 2.3 ± 0.2 − −
[Y(PLN)3(H2O)2] (1) 49.1 ± 2.8 Y(NO3)3⋅ 6H2O 36.0 ± 3.9
[La(PLN)3(H2O)2] (2) 52.2 ± 2.1 LaCl3⋅6H2O 23.8 ± 3.3
[Sm(PLN)3(H2O)2]⋅H2O (3) 52.5 ± 2.9 SmCl3⋅ 6H2O 29.5 ± 4.0
[Gd(PLN)3(H2O)2] (4) 55.6 ± 6.0 GdCl3⋅ 6H2O 16.2 ± 1.6
[Dy(PLN)3(H2O)2] (5) 52.0 ± 2.6 DyCl3⋅6H2O 10.7 ± 4.6
Results represent mean ± SD of at least three independent experiments. SD represents
the standard deviation.
 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434
Fig. 3. Microscope images of BEL7404 cells treated with [Dy(PLN)3(H2O)2] (5), (b) cisplatin (c) and adriamycin (d) (a: negative control; 200× magnification).
3.8.1. Absorption titration
It is well documented that intercalative π–π stacking of the
aromatic rings of the metal complexes with the DNA bases affects the
transition dipoles of the molecules and usually leads to a decrease in
its absorbance [47]. In order to determine the DNA binding constants
of [La(PLN)3(H2O)2] (2) and plumbagin in Tris–NaCl buffer, the
absorption titrations were carried out. As indicating in Fig. 6, [La
(PLN) 3 (H 2 O) 2 ] (2) has similar absorption peaks to those of
plumbagin with an intense absorption band at around 269 nm and
a weak absorption band at 415 nm. With increasing concentration of
ct-DNA, the strong absorbance bands of [La(PLN)3(H2O)2] (2) in the
UV region exhibit a hypochromism of 17.9% and a bathochromism of
3 nm, meanwhile that of plumbagin also appears a hypochromism of
24.3% and a bathochromism of 9 nm. These results suggest the main
action mode of plumbagin and its PLN–lanthanide complex binding
to DNA is the intercalation [48,49].
Fig. 4. Dose-dependent cytotoxic effects against BEL7404 cell lines of complexes: 1, 2
treated with various doses for 48 h. Results represent mean ± SD of at least five
independent experiments; the error bars represent the standard deviation.
431
The intrinsic binding constant, Kb, of the complex to DNA can be
calculated by the classic equation [50,51]: [DNA]/(εa − εf) = [DNA]/
(εb − εf) + 1/[Kb (εb − εf)] (1), where [DNA] is the concentration of
DNA, εa corresponds to the apparent extinction coefficient for the
compound in the presence of DNA, εf and εb represent the extinction
coefficients for the free compound and its fully DNA-bound
combination, respectively. In the plot of [DNA]/(εf − εa) versus
[DNA], Kb can be obtained by the ratio of the slope to intercept.
From the absorption spectra of [La(PLN)3(H2O)2] (2) at 266 nm
and plumbagin at 269 nm in the presence of increasing amounts of ct-
DNA, the plots of [DNA]/(εf − εa) versus [DNA] (Fig. 6 inset) are found
to be linear fitting, and the Kb value can be given by the ratio of the
slope to intercept. The binding constants Kb obtained for [La(PLN)3
(H2O)2] (2) and plumbagin are 1.74 × 104 (with standard deviation
Fig. 5. Time-dependent cytotoxic effects against BEL7404 cell lines treated with 10 μg/mL
of complexes 1 and 2 for 24, 48, 72 h, respectively. Results represent mean± SD of at least
five independent experiments; the error bars represent the standard deviation.
432 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434

Fig. 6. UV–Vis spectra of [La(PLN)3(H2O)2] (2) (6.7 × 10− 5 M, 50 μL DNA per scan,
pH = 7.35) in the absence (----) and presence (—) of increasing amounts of DNA
(2.1 × 10− 3 M). Kb represents mean ± SD; SD represents the standard deviation.

(SD) of 0.524) and 5.07 × 103 M− 1 (with SD of 0.091, Fig. S2, ESI*),
respectively. The Kb value for [La(PLN)3(H2O)2] (2) is larger than that
for plumbagin, indicating that [La(PLN)3(H2O)2] (2) has a stronger
affinity to DNA than plumbagin. This is in agreement with the fact that
PLN–lanthanide complexes exhibit higher cytotoxcity than plumba-
gin. As mentioned previously, plumbagin and PLN–lanthanide com-
plexes mainly interact with DNA via intercalation fashion, but the
larger Kb value for [La(PLN)3(H2O)2] (2) may contributed from
intercalation and electrostatic interaction [52–54], while plumbagin
has no electrostatic interaction. Such an explanation is supported by
SDS experiments. In the control experiment with SDS instead of ct-
DNA, with the addition of SDS, the hypochromism for [Y(PLN)3
(H2O)2] (1), [La(PLN)3(H2O)2] (2) and [Gd(PLN)3(H2O)2] (4) was
observed obviously (Fig. S3, ESI*), but no significant differences were
observed in the absorption spectra of plumbagin in the absence and
presence of SDS. These observations indicate that the electrostatic
binding of the cation species in the solution of PLN–lanthanides to the
polyanionic DNA phosphate back-bone may exist [55], which perhaps
results from the acquired positive charge on the species of the PLN–
complexes in the solution (see ESI-MS results); but for plumbagin,
such an interaction may not exist.
3.8.2. Fluorescence emission titration
Binding of [La(PLN)3(H2O)2] (2) and plumbagin to ct-DNA was
further investigated by EthBr-competitive binding experiments. The
emission spectra of EthBr bound to ct-DNA in the absence and
presence of plumbagin, [La(PLN)3(H2O)2] (2) are shown in Fig. 7. The
Fig. 8. The Stern–Volmer quenching plots of EB bound to DNA by [La(PLN)3(H2O)2] (2)
and plumbagin, which give the quenching constants Ksq.
addition of [La(PLN)3(H2O)2] (2) and free plumbagin to the DNA-
bound EthBr solutions caused an obvious decrease in its emission
intensity, indicating that [La(PLN)3(H2O)2] (2) competed with EthBr
for binding to DNA. The relative binding intensity of [La(PLN)3(H2O)2]
(2) and plumbagin to ct-DNA was determined by the classical Stern–
Volmer equation [50,51]: I0/I = 1 + Ksqr, where I0 and I represent the
fluorescence intensities in the absence and presence of the compound,
respectively, r is the concentration ratio of the compound to DNA. Ksq
is the linear Stern–Volmer quenching constant.
The Ksq values obtained from the plot of I0/I & [compound]/[DNA]
(Fig. 8) for [La(PLN)3(H2O)2] (2) and plumbagin are 3.16 and 0.16,
respectively, which demonstrate that [La(PLN)3(H2O)2] (2) has a
stronger affinity to DNA than plumbagin. Such a trend is consistent
with the previous UV–vis absorption spectral results.
From the plot of these intensities against the compound con-
centrations, the values of the apparent DNA binding constant (Kapp)
were calculated using the equation KEB·[EB] = Kapp·[complex] [55], in

Fig. 7. Emission spectra of DNA–EB of plumbagin (6.0 × 10− 4 M) and [La(PLN)3(H2O)2] (2) (7.0 × 10− 4 M) in the absence (----) and presence (—) of increasing amounts (3 mL
solution, [DNA]/[EB] ratio of 6:1, pH = 7.35, excited at 350 nm), 50 μL per scan.
 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434
which the complex concentration was the value at a 50% reduction of
the fluorescence intensity of EB and KEB = 1.0 × 107 M− 1([EB] =
1.3 μM). The Kapp values of [La(PLN)3(H2O)2] (2) and plumbagin are
2.97 × 105 and 2.07 × 104 M− 1, respectively. These results further
confirm that the DNA binding of [La(PLN)3(H2O)2] (2) and plumbagin
may be completed by intercalation mode [56,57].
3.8.3. Circular dichroism spectra
The CD spectrum of plumbagin (Fig. 9) exhibits a slight alteration
on both negative and positive absorptions, only a weak decrease in the
positive band and a weak increase in the negative band are observed,
indicating a relatively weak intercalation of plumbagin to ct-DNA.
However, with the addition of [La(PLN)3(H2O)2] (2), the intensity of
the positive band decreases and occurs a small red shift, which
indicates that [La(PLN)3(H2O)2] (2) can intercalate the neighboring
base pairs of ct-DNA to reduce the energy of the π–π electronic
transition, and lead to a decrease in the positive absorption of DNA.
Meanwhile, significant decreases in the negative absorption bands of
ct-DNA also are observed, suggesting that a decline of the right-hand
helicity character of ct-DNA, by which the intercalation mode of
[La(PLN)3(H2O)2] (2) and is further confirmed [58–60].
3.8.4. Agarose gel electrophoresis assay
The binding modes to DNA were further examined by an agarose
gel electrophoresis assay [61,62]. pUC19 plasmid DNA exists in
supercoiled (SC) and relaxed (RLX) conformation. Incubation with
plumbagin and [La(PLN)3(H2O)2] (2) for 1 h in the absence of any
external reagent or light, resulted in the supercoiled form (SC) of
pUC19 plasmid DNA being lengthened without obvious unwinding
(Fig. 10). For [La(PLN)3(H2O)2] (2), the mobility rate of supercoiled
form (SC) DNA was slightly decreased, whereas for plumbagin, that
was significantly decreased (Fig. 10), which could be because the
planar plumbagin more easily intercalated into the neighboring base
pairs of DNA than the nonplanar [La(PLN)3(H2O)2] (2). These results
are comparable to those found when the DNA solution was treated
with the classical intercalator ethidium bromide and also observed in
plumbagin copper(II) complexes [20], which is probably related to the
weak interaction of these compounds with DNA [63,64].
In summary, the UV–vis spectral titration analyses revealed that
the binding of plumbagin and [La(PLN)3(H2O)2] (2) to ct-DNA was
mainly attributed to planar plumbagin moiety intercalation into
neighboring base pairs of ct-DNA; but the SDS experiments
demonstrated that [La(PLN)3(H2O)2] (2) could also exist electrostatic
interaction with DNA; furthermore, the EB-competitive binding
studies indicated that [La(PLN)3(H2O)2] (2) exhibited a higher
competitive binding ability with EB in ct-DNA system than plumba-
Fig. 9. CD spectra of ct-DNA (3 mL solution, 2.0 × 10− 4 M) in the absence (a) and
presence of [La(PLN)3(H2O)2] (2) (b), plumbagin (c), with [compound]/[ct-DNA] =
0.25 in Tris–HCl buffer, pH = 7.35.
433
Fig. 10. Gel mobility shift of plasmid pUC19 DNA treated with increasing concentrations
of complex 2 and plumbagin after incubation for 2 h at 37 °C in Tris–HCl/NaCl buffer.
Lane 0: Control DNA, lanes 1–6: DNA + 30, 60, 90, 120, 150, 200 μM compounds
respectively. SC: supercoiled conformation, RLX: relaxed conformation.
gin; and [La(PLN)3(H2O)2] (2) induced significant CD spectral
perturbation of ct-DNA, but plumbagin just slightly. The agarose gel
electrophoresis assays suggested that [La(PLN)3(H2O)2] (2) and
plumbagin resulted in the supercoiled form (SC) of pUC19 plasmid
DNA being lengthened without obvious unwinding. For [La(PLN)3
(H2O)2] (2), the mobility rate of supercoiled form (SC) DNA was
slightly decreased, whereas for plumbagin, that was significantly
decreased due to their different planarity. Generally, PLN-lanthanide
complexes binding to DNA are more powerful than plumbagin. These
findings agree well with the fact that the cytotoxicity of PLN–
lanthanide complexes towards BEL7404 cell lines tested are more
sensitive than that of plumbagin.
4. Conclusion remarks
Five new PLN–lanthanide (III) complexes of Y(III) (1), La(III) (2),
Sm(III) (3), Gd(III) (4), and Dy(III) (5) were synthesized by the
reaction of plumbagin with the corresponding lanthanide salts, in
amounts equal to ligand/metal molar ratio of 3:1. They were
characterized by elemental analyses, UV–vis, IR, 1 H NMR and ESI-
MS as well as TGA. These new PLN-lanthanide complexes possess a
general formula [Ln(PLN)3(H2O)2]⋅nH2O (n = 0 for complexes 1, 2, 4,
5; n = 1 for complex 3), where three PLN anions chelated the
lanthanide via both 4-C=O and 5-OH of PLN, and two aqua ligands
participate to coordination, resulting in eight-coordinated complexes.
The results of in vitro cytotoxicity assays against BEL7404 showed that
the PLN–lanthanide complexes 1–5 exhibited a significantly enhanced
cytotoxicity, compared to plumbagin, cisplatin and the corresponding
lanthanide salts, suggesting a synergistic effect upon plumbagin
coordination to the Ln(III) ion. BEL7404 cell lines exhibit an increased
sensitivity to the complexes in a dose- and time-dependent manner.
Plumbagin and [La(PLN)3(H2O)2] (2) interacted with DNA mainly by
intercalation fashion, but for the PLN–lanthanide complex, electro-
static interaction could not be excluded due to cationic species
existence in solution. [La(PLN)3(H2O)2] (2) presents a higher affinity
to DNA than plumbagin, which may correlate to the enhanced
cytotoxicity of the PLN–lanthanide complex.
Abbreviations
CD circular dichroism
ct-DNA calf thymus DNA
EB ethidium bromide
ESI-MS electrospray ionization mass spectrum
BEL7404 human liver cancer cells
MTT 3-[4,5-dimentylthiazole-2-yl]-2,5-diphenpyltetra-zolium
bromide
PBS phosphate-buffered saline
PLN plumbagin anion
434 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 426–434

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