Evaluation of the antioxidant, DNA interaction and tumor cell cytotoxicity activities of Copper(II) complexes with Paeonol Schiff-base

Article abstract of DOI:10.1016/j.inoche.2010.03.030

Herein, we report for the first time on the preparation of four Paeonol Schiff-base ligands: 1,2-Bis(2- hydroxy-4-methoxy-a-methylbenzylideneimino) ethane (H₂L¹), 2-hydroxy-4-methoxy-α- methylbenzylidene (benzoyl) hydrazone (HL²

Full text of DOI:10.1016/j.inoche.2010.03.030

Inorganic Chemistry Communications 13 (2010) 727–729
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Evaluation of the antioxidant, DNA interaction and tumor cell cytotoxicity activities
of Copper(II) complexes with Paeonol Schiff-base
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a
Dong-Dong Qin a, Zheng-Yin Yang a, , Fu-Hong Zhang , Bo Du , Ping Wang , Tian-Rong Li
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a
College of Chemistry and Chemical Engineering and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
Otolaryngology-Head & Neck Surgery, First Hospital of Lanzhou University, Lanzhou 730000, PR China
Otolaryngology-Head & Neck Surgery, First Hospital of Jilin University, Jilin 130000, PR China
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c
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report for the first time on the preparation of four Paeonol Schiff-base ligands: 1,2-Bis(2-
hydroxy-4-methoxy-α-methylbenzylideneimino) ethane (H2L1), 2-hydroxy-4-methoxy-α-methylbenzyli-
dene (benzoyl) hydrazone (HL2), 2-hydroxy-4-methoxyacetophenone-ethanolamine Schiff-base (HL3), and
2-hydroxy-4-methoxyacetophenone(2′-hydroxybenzoyl) hydrazone (H2L4), and their Cu(II) complexes, of
which the structure of C11N2O6H14Cu(CuL3.NO3) was confirmed by X-ray diffraction. Antioxidant activities of
four Cu(II) complexes were studied by the standard non-enzymatic NBT method and Fenton system, used to
produce superoxide anion radicals and hydroxyl radicals, respectively. The results show these Cu(II)
complexes have excellent antioxidant activities. DNA-binding and in vitro cytotoxicity experiments were
utilized to further investigate the biological activities of C11N2O6H14Cu(CuL3.NO3). These results support
Received 16 January 2010
Accepted 15 March 2010
Available online 22 March 2010
Keywords:
Antioxidant
DNA-binding
Cytotoxicity
Paeonol
Crystal structure
C11N2O6H14Cu as a potential candidate for biological use because it shows not only high antioxidant and
moderate DNA-binding activities, but also good tumor cell cytotoxicity activities in human cell lines
carcinomas Hep-2 (larynx), for the range of concentrations tested.
© 2010 Elsevier B.V. All rights reserved.
Introduction
Copper(II) complexes termed CuL1, Cu(L2)2, CuL3NO3 and Cu(HL4)2
were synthesized and their biological activities were evaluated.
Paeonol has been used in Asian countries for more than a thousand
years as a herbal medicine and now is being used as a tranquillizer and
an anti-hypertensive in Europe [1]. Recently, interactions of Paeonol
with human serum albumin (HSA) have been investigated [2].
However, metal complexes of Paeonol's Schiff-base ligand have not
been reported yet and their biological activities including antioxidant,
DNA-binding and cytotoxicity etc. have not been studied compre-
hensively. It is well known that cancer is currently the second leading
cause of death in industrialized countries, accounting for roughly a
quarter of all deaths [3]. Hence, the development of new anticancer
therapies is one of the fundamental goals in medicinal chemistry. DNA
is one of the main molecular targets in the design of anticancer
compounds [4]. Additionally, it has been demonstrated that free
radicals can damage proteins, lipids, and DNA of bio-tissues, leading to
increased rates of cancer [5]. Fortunately, antioxidants can prevent
this damage, due to their free radical scavenging activity [6]. Hence, it
is very important to develop compounds with both strong antioxidant
and DNA-binding properties for effective cancer therapy. Copper
complexes have shown remarkable performance in antioxidant [7],
DNA-binding [8] and anticancer studies [9]. In this effort, four new
Schiff-base ligands (H2L1, HL2, HL3, H2L4) as shown in Fig. 1 were
prepared by condensing Paeonol and different amine or hydrazide
directly, all of ligands show high purity after recrystallizing from a
suitable solvent. Due to the lack solubility of H2L and H2L4 in general
solvent, we had to prepare their complexes CuL1 and Cu(HL4)2 in
DMF, and then used water to precipitate the product. Cu(L2)2 was
prepared by refluxing HL2 and copper nitrate in methanol for several
hours till green precipitation appeared. We successfully got the
CuL3NO3 at room temperature in ethanol, using methanol could just
get green solution, not precipitation. We got the likely composition of
complexes: CuL1, Cu(L2)2 and Cu(HL4)2, through IR, MS and elemental
analyses, structure of CuL3NO3 was confirmed by X-Ray diffraction.
Green and stick like, single crystal of CuL3NO3 with the structure
depicted in Fig. 2, was obtained by slowly evaporating its methanol
solution. (See selected crystal data and experimental data in Table S1
and S2).
The scavenging abilities of the four Cu(II) complexes against
hydroxyl radicals were tested as a function of concentration, ranging
from 0.3 to 15 μM, shown in the Fig. 3. It can be seen that the
inhibitory effects of complexes on the hydroxyl radical are related
to concentration, and saturate at a concentration of 9 μM. Obviously,
the scavenging activities of four Cu(II) complexes follow the order
of CuL1(IC50 =2.42)NCu(L2)2(IC50 =4.28)NCuL3NO3(IC50 =9.00)N
Cu(HL4)2(IC50=42.00) (Detailed data is shown in Table S3) throughout
⁎
Corresponding author. Tel.: +86 931 8913515; fax: +86 931 8912582.
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
1387-7003/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2010.03.030

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D.-D. Qin et al. / Inorganic Chemistry Communications 13 (2010) 727–729
Fig. 1. Structure of Paeonol and four Schiff-base ligands.
the range of concentrations tested. The relatively small standard
deviations (SD) obtained provide evidence of the reliability in our
experimental conditions using the Fenton system to generate hydroxyl
radicals.
method, as described in our previous work [10]. The inhibitory effects
on the superoxide radical are increased for greater concentrations of
Cu(II) complexes, ranging from 0.03 to 1.5 μM as shown in Fig. 4. It
is noteworthy that the scavenging effect of the four complexes is
comparable at the concentration of 1.5 μM, with a measured value
of approximately 90%. These results indicate that the four complexes
will show almost the same activities when the concentration of
complexes is as high as 1.5 μM, the differences can only be seen in low
concentrations (b1.5 μM). However, in our tested concentration,
activities of four complexes are also in the order of CuL1 NCu(L2)2 N
CuL3NO3 NCu(HL4)2, with the IC50 value of 0.079, 0.140, 0.194 and
0.238 μM (see Table S4).
The superoxide radicals (O2−•) were generated in vitro in a non-
enzymatic (NBT/Riboflavine assay) system and determined spectro-
photometrically by the nitro blue tetrazolium (NBT) photoreduction
Compared to inhibitory effects on the hydroxyl radical, the four
Cu(II) complexes exhibit greater activity (lower IC50 value) on the
Fig. 2. Molecule structure of CuL3NO3. Selected important bond lengths [Å] and bond
angles [°]: Cu(1)–O(4) 1.994(2); Cu(1)–O(2) 1.895(3); Cu(1)–N(1) 1.925(3); Cu(1)–O(3)
1.975(3); O(2)–Cu(1)–N(1) 93.73(13); O(2)–Cu(1)–O(3) 176.57(14); O(3)–Cu(1)–N(1)
84.80(14); O(2)–Cu(1)–O(4) 90.91(12); O(4)–Cu(1)–N(1) 175.23(13); O(3)–Cu(1)–O(4)
90.50(13).
Fig. 3. Scavenging effect of tested four Cu(II) complexes on hydroxyl radical. Values are
means SD (n=3). CuL1 (blue), Cu(L2)2 (red), CuL3NO3 (green) and Cu(HL4)2
(purple).

D.-D. Qin et al. / Inorganic Chemistry Communications 13 (2010) 727–729
729
Fig. 4. Scavenging effect of tested four Cu(II) complexes on superoxide radicals. Values
are means
(purple).
SD (n=3). CuL1 (blue), Cu(L2)2 (red), CuL3NO3 (green) and Cu(HL4)2
Fig. 5. Cytotoxic activities of tested compounds against larynx tumor cell line. Ligand
(HL3), complex (CuL3NO3).
ligand is much less, showing roughly 20%. It is noteworthy that copper
nitrate also shows high activities, nearly as high as CuL3NO3, which can
deduce a conclusion that copper takes a key role in the entire process
through stimulating the directional migration of endothelial cells in
angiogenesis as the tumors are angiogenesis dependent [20]. This offers
anexplanation why CuL3NO3 shows superior activity than its free ligand.
Today many researchers focus their attention on the biological
activity of Cu(II) complexes. In this paper, four new Schiff-base ligands
and their Cu(II) complexes are synthesized and X-ray diffraction show
CuL3NO3 has square planar geometry. The results reveal that the Cu(II)
complex has a moderate DNA-binding ability and tumor cell cytotoxi-
city, but excellent antioxidant activity, especially for superoxide radical.
superoxide radical, which may be due to the higher activity of
the hydroxyl radical than the superoxide radical. It must be noted
that even at the lowest concentrations tested (1.5×10−7 mol/L),
CuL1, Cu(L2)2 and CuL3NO3 can also show scavenging ratio of more
than 50% (Cu(HL4)2 is 46.9%). These results are several orders of
magnitude higher than that of previous reported natural products [11],
and also higher than that of some copper [12] and rare earth [13] Schiff-
base complexes reported by Yang et al. It is not uncommon for copper
complexes to show excellent antioxidant activity, ever since the
discovery of Cu-SOD. Considering the mononuclear structure of the
four complexes, differences in the ligand structure are likely to induce
variations in antioxidant activities. The highest performance of CuL1 is
due to two coordinated phenolic group compared to Cu(L2)2 and
CuL3NO3 which have only one. Because the phenolic group in the 3′
position of Cu(HL4)2 does not coordinate with copper, approximately
87 kcal/mol [14] of extra energy is required in the first step [6] of the
radical scavenging process to make the proton dissociate from phenol,
according to theoretical value. Consequently, Cu(HL4)2 exhibits low
performance in our radical scavenging experiment.
Acknowledgements
This work is supported by the National Natural Science Foundation
of China (20975046).
Appendix A. Supplementary data
CuL3NO3 was chosen to explore the DNA-binding properties, which
are greatly affected by the structure of the complex and its solubility in
methanol. The nearly square planar geometry of CuL3NO3 allows itself to
easily intercalate into the base pairs of DNA. The electronic spectra of
CuL3NO3 intheabsence and presence of DNA show the absorption bands
at 223, 287 and 361 nm appear with different hypochromism of 26.6%,
18.0% and 9.4% (Fig. S1) caused by π* orbital of the complex coupling
with π orbit of the base pairs of DNA when intercalation interaction
happens. A 1.4 fold enhancement in fluorescence is observed when 50%
more DNA was added to original complex buffer solution, because the
complex can be protected more effectively by the non-polar environ-
ment of DNA. DNA-binding constant of 1.2×106 M−1 was obtained by
Scatchard equation (Fig. S2), the complex can also slightly quench the
emission of EB-DNA system with competitive constant of 5.2×103 M−1
determined by Stern–Volmer equation (Fig. S3). Further experiments
show the viscosity of DNA continuously increase with increasing
amounts of CuL3NO3 (Fig. S4). All of the results mentioned above
conclude a groove DNA-binding mode [15–19] and moderate binding
ability of CuL3NO3.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.inoche.2010.03.030.
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