Synthesis, DNA-binding, cytotoxicity, and cleavage studies of unsymmetrical oxovanadium complexes

Article abstract of DOI:10.1080/00958972.2011.645204

An unsymmetrical oxovanadium complex [VO(SAA)(phen)] (1) (SAA=salicylidene anthranilic acid, phen=phenanthroline) and its derivative [VO(MOSAA)(phen)] (2) (MOSAA=2-hydroxy-4-methoxysalicylidene anthranilic) have been synthesized and characterized by eleme

Full text of DOI:10.1080/00958972.2011.645204

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Synthesis, DNA-binding, cytotoxicity,
and cleavage studies of unsymmetrical
oxovanadium complexes
Haiwei Guo a , Jiazheng Lu a , Zhigang Ruan b , Yongli Zhang b ,
Yunjin Liu a , Linquan Zang a , Jing Jiang a c & Jinwang Huang c
a School of Pharmacy, Guangdong Pharmaceutical University,
Guangzhou 510006, People's Republic of China
b School of Basic Courses, Guangdong Pharmaceutical University,
Guangzhou 510006, People's Republic of China
c State Key Laboratory of Optoelectronic Material and
Technologies & School of Chemistry and Chemical Engineering,
Sun Yat-Sen University, Guangzhou 510275, People's Republic of
China
Version of record first published: 21 Dec 2011.
To cite this article: Haiwei Guo, Jiazheng Lu, Zhigang Ruan, Yongli Zhang, Yunjin Liu, Linquan
Zang, Jing Jiang & Jinwang Huang (2012): Synthesis, DNA-binding, cytotoxicity, and cleavage
studies of unsymmetrical oxovanadium complexes, Journal of Coordination Chemistry, 65:2,
191-204
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Journal of Coordination Chemistry
Vol. 65, No. 2, 20 January 2012, 191–204
Synthesis, DNA-binding, cytotoxicity, and cleavage studies of
unsymmetrical oxovanadium complexes
HAIWEI GUOy, JIAZHENG LU*y, ZHIGANG RUANz, YONGLI ZHANGz,
YUNJIN LIUy, LINQUAN ZANGy, JING JIANGyx and JINWANG HUANGx
ySchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
People’s Republic of China
zSchool of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006,
People’s Republic of China
xState Key Laboratory of Optoelectronic Material and Technologies & School of Chemistry
and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275,
People’s Republic of China
(Received 5 September 2011; in final form 11 November 2011)
An unsymmetrical oxovanadium complex [VO(SAA)(phen)] (1) (SAA ¼ salicylidene anthranilic
acid, phen ¼ phenanthroline) and its derivative [VO(MOSAA)(phen)] (2) (MOSAA ¼
2-hydroxy-4-methoxysalicylidene anthranilic) have been synthesized and characterized by
elemental analysis, UV-Vis, ES-MS, IR, and 1H NMR. The interaction of these two complexes
with calf thymus DNA (CT-DNA) was investigated by absorption titration, fluorescence
spectra, viscosity measurements, and thermal denaturation. Their photocleavage reactions with
pBR322 supercoiled plasmid DNA were investigated by gel electrophoresis. The cytotoxicity of
these two complexes against myeloma cell (Ag8.653) and gliomas cell (U251) have been
assessed by MTT assay. The results show that both 1 and 2 bind to CT-DNA in classical
intercalation, and the DNA-binding affinity of 1 is larger than that of 2. These complexes
enhance the oxidative cleavage of supercoiled pBR322 DNA and both complexes have
cytotoxic activities against Ag8.653 and U251 cell lines. Complex 1 has more potent inhibitory
effect against the two cell lines than 2.
Keywords: Oxovanadium complexes; DNA-binding; Cytotoxicity; Cleavage
1. Introduction
Transition metal complexes have been exploited for design of new drugs due to their
diverse biological activities. Most of these properties have been found to arise from
complexes binding DNA and cleaving DNA [1–3]. DNA-binding metal complexes,
especially those with small molecular weight, have been extensively studied as DNA
structural and conformational probes, DNA-dependent electron transfer and sequence-
specific cleaving agents, and potential anti-cancer drugs [4–6].
*Corresponding author. Email: lujia6812@163.com
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2011.645204

192
H. Guo et al.
Vanadium complexes have antibacterial, antitumor, insulin-enhancing, and antipar-
asitic effects [7–10]. Vanadium takes part in various DNA maintenance reactions and
thereby prevents genomicin stability which otherwise leads to cancer [11–15]. Vanadium
complexes also suppress the growth and spread of existing tumors by inhibiting tumor
cell proliferation, inducing apoptosis, and limiting the invasion and metastatic potential
of neoplastic cells. Thus, DNA-binding and antitumor activities of oxovanadium
complexes and multifunctional bridging ligands have been investigated [8, 9]. Schiff
bases have been extensively studied because of their potential antibacterial, antifungal,
anti-malarial, and anticancer activities [10, 13, 16]. In this context, salicylidene
anthranilic acid (SAA) and its derivative are chosen as ligands for oxovanadium
complexes. The presence of a rigid aromatic system in the Schiff base structure gives
particular spectroscopic properties, which make it a potential probe for nucleic acids.
Electronic effects of Schiff-base complexes are important in this field in order to design
complexes with better DNA-binding characteristics [11, 17]. Increasing interest in
chemistry of Schiff base derivatives has prompted development of efficient synthetic
procedures for functionalization at the various ring positions of Schiff bases which
involves phen. Relatively simple Schiff-base ligands are building blocks for various
purposes, as well as new intercalating agents for polynucleotides [16–19].
Previously, we studied the interactions of some oxovanadium complexes,
[VO(satsc)(bipy)] (satsc ¼ salicylaldehyde thiosemicarbazone; bipy ¼ 2,20-bipyridine)
and its derivative [VO(3,5-dibrsatsc)(bipy)] (3,5-dibrsatsc ¼ 3,5-dibromosalicylaldehyde
thiosemicarbazone) with calf thymus DNA (CT-DNA), and found that these
oxovanadium complexes interact with CT-DNA through intercalation [18, 19]. In
continuation of our investigations of biological activities of oxovanadium complexes,
we synthesized and characterized two other oxovanadium complexes, [VO(SAA)(phen)]
(1) (phen ¼ 1,10-phenanthroline) and [VO(MOSAA)(phen)] (2) (MOSAA ¼ 2-hydroxy-
4-methoxysalicylidene anthranilic; phen ¼ 1,10-phenanthroline), scheme 1. The
DNA-binding behaviors of the two complexes were explored by absorption titration,
fluorescence spectra, viscosity measurements, and thermal denaturation. Their
photocleavage reactions with pBR322 supercoiled plasmid DNA were investigated by
gel electrophoresis. The cytotoxicity of these two complexes against the myeloma cell
(Ag8.653) and gliomas cell (U251) were assessed by MTT assay.
R1
R1
R1
N
N
OH
O
V
VO(acac)2,
OH
O
EtOH, 50ºC, 3h
CHO
N
+
MeOH, 4h, reflux
COOH
N
N
NH2
N
O
COOH
O
(1): R1 = H
(2): R1 = CH3O
Scheme 1. The synthetic routes for ligands and complexes.

Oxovanadium complexes
193
2. Experimental
2.1. Materials
VO(acac)2 (acac ¼ acetylacetonate) and 1,10-phenanthroline were purchased
from Shanghai Jingchun company. CT-DNA and pBR 322 DNA were obtained
from Sigma. Other materials were obtained from commercial sources and used
as received (analytical reagents). Tris-HCl buffer A (5 mmol Lꢀ1 Tris (hydroxymethy-
laminomethane)-HCl, 50 mmol Lꢀ1 NaCl, pH ¼ 7.2) was used for absorption titration,
luminescence titration, and viscosity experiments. Tris-HCl buffer B (50 mmol Lꢀ1
Tris-HCl and 18 mmol Lꢀ1 NaCl, pH ¼ 7.2) was used for DNA-cleavage
experiments. Buffer
0.25 mmol Lꢀ1
C
(1.5 mmol Lꢀ1 NaHPO4, 0.5 mmol Lꢀ1 NaH2PO4, and
Na2H2EDTA ¼ (H4EDTA ¼ N,N0-ethane-1,2-diylbis[N-(carboxy-
methyl)glycine]) (pH ¼ 7.0) was used for thermal denaturation. All buffers were prepared
using doubly distilled water. A solution of CT-DNA in the buffer gave a ratio of UV
absorbance at 260 and 280 nm of ca 1.8–1.9, indicating that the DNA was sufficiently free
of protein [18–20]. The DNA concentration per nucleotide was determined by absorption
spectroscopy using the molar absorption coefficient (6600 (mol L
260 nm [19, 21].
)
ꢀ1 ꢀ1 cmꢀ1) at
2.1.1. Synthesis of SAA. SAA was synthesized through modification of a previously
reported procedure [18, 19]. A stirring solution of salicylaldehyde (521 mL, 5 mmol) in
10 cm3 of absolute alcohol was added dropwise to anthranilic acid (0.686 g, 5 mmol)
which was dissolved in 10 cm3 of absolute alcohol; the mixture was continuously stirred
at 50ꢁC for 3 h giving a pink precipitate. Yield: 57%. Anal. Calcd for C14H11NO3 (%):
C, 69.70; H, 4.60; N, 5.81; Found (%): C, 69.61; H, 4.79; N, 5.74. ES-MS (CH3OH,
m/z): 242.0 ([M þ 1]þ). UV, ꢀmax, nm (", (mol L
)
ꢀ1 ꢀ1 cmꢀ1) in DMSO: 258 (34,300),
338 (30,150). IR (KBr)(ꢁmax/cmꢀ1): 3500 (br) (OH), 3069, 3039 (m, ArH), 1682 (s)
(COOH), 1618 (vs) (C¼N þ C¼C), 1570 (s) (C¼C), 1457 (m) (C¼C), 1389 (m), 1363 (s),
1242 (vs) (C–O), 811 (s), 756 (vs), 678 (s), 576 (m), 486 (s). 1H NMR (500 MHz,
DMSO-d6): 13.05 (br s, 1H, COOH), 10.27 (s, 1H, OH), 8.87 (s, 1H, –CH¼N–), 7.87
(s, 1H, ArH), 7.65 (d, 1H, ArH, J ¼ 8.2 Hz), 7.52 (t, 1H, ArH, J ¼ 8.7 Hz), 7.46 (s, 1H,
ArH), 7.22 (t, 1H, ArH), 6.97 (t, 1H, ArH, J ¼ 8.0 Hz), 6.74 (d, 1H, ArH, J ¼ 8.4 Hz),
6.50 (t, 1H, ArH, J ¼ 7.8 Hz).
2.1.2. Synthesis of MOSAA. This ligand was prepared using a similar procedure
described for SAA, but from 4-methoxysalicylaldehyde (0.761 g, 5 mmol). Yield: 65%.
Anal. Calcd for C15H13NO4 (%): C, 66.41; H, 4.83; N, 5.16; Found (%): C, 66.33;
H, 4.97; N, 5.10. ES-MS (CH3OH, m/z): 272.0 ([M þ 1]þ). UV, ꢀmax, nm
(", (mol L )
ꢀ1 ꢀ1 cmꢀ1) in DMSO: 257 (13,350), 340 (29,250). IR (KBr)(ꢁmax/cmꢀ1):
3470 (br) (OH), 3055 (m) (ArH), 2951 (m), 2847 (w) (CH3O–), 1697 (s) (COOH), 1615
(br, vs) (C¼N þ C¼C), 1564 (s) (C¼C), 1514 (C¼C), 1457 (m) (C¼C), 1392 (m) (–CH3),
1359 (s), 1267 (vs) (COOH), 1240 (s) (C–O), 1022 (s), 841 (s), 784 (m), 762 (s), 732 (m),
1
671 (s), 601 (m), 483 (s). H NMR (500 MHz, DMSO-d6): 13.31 (br s, 1H, COOH),
10.02 (s, 1H, OH), 8.76 (s, 1H, –CH¼N–), 7.88 (d, 1H, ArH, J ¼ 8.2 Hz), 7.63
(t, 1H, ArH, J ¼ 8.0 Hz), 7.54 (d, 1H, ArH, J ¼ 7.9 Hz), 7.46 (d, 1H, ArH, J ¼ 8.4 Hz),

194
H. Guo et al.
7.34 (t, 1H, ArH, J ¼ 8.2 Hz), 6.55 (d, 1H, ArH, J ¼ 7.8 Hz), 6.48 (d, 1H, ArH,
J ¼ 8.0 Hz), 3.82 (s, 3H, CH3O–).
2.1.3. Synthesis of [VO(SAA)(phen)] (1). A mixture of SAA (0.121 g, 0.5 mmol) and
1,10-phenanthroline (0.090 g, 0.5 mmol) in absolute methanol (15 cm3) was heated at
80ꢁC under argon for 2 h. After dissolution, a 10 cm3 methanolic solution of VO(acac)2
(0.1325 g, 0.5 mmol) was added dropwise to this mixture. The mixture was refluxed for
another 4 h to give a reddish-brown precipitate. Then the solid powder was filtered from
the hot solution, washed with absolute methanol and diethyl ether, respectively, and
dried in vacuum. Yield: 80.4%. Anal. Calcd for C26H17N3O4V (%): C, 64.19; H,
3.52; N, 8.64; Found (%): C, 62.24; H, 3.77; N, 8.36. ꢀM (ꢀꢀ1 cm2 molꢀ1, in DMF):
19.9. ES-MS (CH3OH, m/z): 487.0 ([M þ 1]þ). UV, ꢀmax, nm (", (mol L
) )
ꢀ1 ꢀ1 cmꢀ1
in DMSO: 264.5 (34,150), 404 (5200). IR (KBr)(ꢁmax/cmꢀ1): 3062 (w) (ArH), 3014 (w)
(ArH), 1601 (vs) (C¼N þ C¼C), 1531 (s) (C¼C), 1433 (s) (C¼C), 1383 (m), 1349 (m),
1217 (w) (C–O), 1180 (w), 1146 (m), 964 (s) (VO), 873 (m), 846 (m), 771 (m), 721 (m),
611 (w), 559 (w), 538 (w), 465 (w). 1H NMR (500 MHz, DMSO-d6): 9.08
(s, 1H, –CH¼N–), 8.13–8.49 (m, 7H, ArH), 8.00 (s, 2H, ArH), 7.77 (s, 1H, ArH),
6.50–7.32 (br m, 6H, ArH). Magnetic moment: ꢂeff: 1.69 BM.
2.1.4. Synthesis of [VO(MOSAA)(phen)] (2). This complex was prepared using a
similar procedure described for 1. Yield: 84.7%. Anal. Calcd for C27H19N3O5V (%):
C, 62.80; H, 3.71; N, 8.14; Found (%): C, 62.51; H, 3.92; N, 8.09. ꢀM (ꢀꢀ1 cm2 molꢀ1):
21.0. ES-MS (CH3OH, m/z): 517.0 ([M þ 1]þ). UV, ꢀmax, nm (", (mol L
)
ꢀ1 ꢀ1 cmꢀ1) in
DMSO: 264 (36,400), 370 (4950). IR (KBr)(ꢁmax/cmꢀ1): 3063 (w) (ArH), 1597 (vs)
(C¼N þ C¼C), 1512 (s) (C¼C), 1421 (w) (C¼C), 1375 (m), 1326 (s), 1225 (s) (C–O),
1180 (s), 1114 (s), 1024 (w), 970 (s) (VO), 852 (m), 766 (m), 721 (m), 553 (w), 539 (w),
1
420 (w). H NMR (500 MHz, DMSO-d6): 9.11 (s, 1H, –CH¼N–), 8.36–8.49 (m, 8H,
ArH), 7.99 (s, 2H, ArH), 7.79 (s, 2H, ArH), 6.48–6.96 (br m, 3H, ArH), 3.71 (s, 3H,
CH3O). Magnetic moment: ꢂeff: 1.73 BM.
2.2. Physical measurements
Microanalyses (C, H, and N) were carried out with a Perkin-Elmer 240Q elemental
analyzer. Electrospray mass spectra (ES-MS) were recorded on a LCQ system
(Finnigan MAT, USA) using methanol as mobile phase. 1H NMR spectra were
recorded on a Varian-500 spectrometer with chemical shifts given relative to
tetramethylsilane (TMS). Infrared (IR) spectra were recorded on a Perkin-Elmer
Lambda 35 instrument using KBr pellets. UV-Vis spectra were recorded on a Shimadzu
UV-3101 PC spectrophotometer at room temperature. Emission spectra were recorded
on a Perkin-Elmer Lambda 55 spectrofluorophotometer. Molar conductivities were
measured using a DDS-307 digital direct reading conductivity meter in DMF solution
(1 mmol Lꢀ1) at room temperature. Magnetic susceptibility measurements were
recorded on an MPMSXL-7 (Quantum Design, USA) at room temperature.

Oxovanadium complexes
2.3. DNA-binding and cleavage experiments
195
Absorption titration experiments were performed with fixed concentration of the
complexes (20 mmol Lꢀ1) while gradually increasing the concentration of CT-DNA.
V-DNA solutions were allowed to incubate for 5 min before the absorption spectra were
recorded. In order to compare the binding strength of the complexes, their intrinsic
binding constants (Kb) were determined by monitoring changes of absorbance in the
ligand transfer band with increasing concentrations of CT-DNA. Kb was then
calculated using the following equation [18, 22, 23]:
½DNAꢂ ½DNAꢂ
1
¼
þ
,
ð1Þ
"a ꢀ "f
"b ꢀ "f Kbð"b ꢀ "fÞ
where [DNA] is the concentration of DNA in the base pairs, "a is the extinction
coefficient observed for Aobsd/[V], "b is the extinction coefficient of the complex when
fully bound to DNA, "f is the extinction coefficient of the complex free in solution. The
non-linear least-squares analysis was done using Origin Lab, version 7.5.
Viscosity measurements were carried out with an Ubbelohde viscometer maintained
at a constant temperature (28 ꢃ 0.1)ꢁC in a thermostatic bath. A digital stopwatch was
used for flow time and each sample was measured five times to obtain the average flow
time. Data are presented as (ꢃ/ꢃ0)1/3 versus binding ratio, where ꢃ is the viscosity of
DNA in the presence of complexes while ꢃ0 is the viscosity of DNA alone [18, 24].
Thermal denaturation studies were carried out with a Shimadzu UV-3101 PC
spectrophotometer equipped with a Peltier temperature-controlling programmer
(ꢃ0.1ꢁC). The melting curves were obtained by measuring the absorbance at 260 nm
for solutions of CT-DNA (80 mmol Lꢀ1) in the absence and presence of oxovanadium
complex (20 mmol Lꢀ1) as a function of temperature. The temperature was scanned
from 50ꢁC to 90ꢁC at a speed of 5ꢁC minꢀ1
.
The cleavage of supercoiled pBR322 DNA by the complexes was studied by gel
electrophoresis; pBR322 DNA (0.1 mg) was treated with the oxovanadium complexes in
buffer B, and the solution was incubated at 37ꢁC in the incubator. The samples were
analyzed by electrophoresis for 1.5 h at 85 V on a 0.8% agarose gel in TBE
(89 mmol Lꢀ1 Tris-borate acid, 2 mmol Lꢀ1 EDTA, pH ¼ 8.3). The gel was stained
with 1 mg mLꢀ1 ethidium bromide and photographed on an Alpha Innotech IS-5500
fluorescence chemiluminescence and visible imaging system [2, 18, 19, 25].
2.4. Cell viability assay
The capacities of compounds to interfere with the growth of myeloma cells (Ag8.653)
and gliomas cells (U251) were determined with the aid of MTT dye assay. Compounds
were dissolved in DMSO and diluted with RPMI 1640 to the required concentrations
prior to use. The control was prepared by addition of culture medium (100 mL). Wells
containing culture medium without cells were used as blanks. Myeloma cells (Ag8.653)
and gliomas cells (U251) with a density 2 ꢄ 104 cells per well were seeded in 96-well
microtiter plates and then treated with varying doses of the vanadium complex and the
reference drug cisplatin in a highly humidified atmosphere of 95% air with 5% CO2 at
37ꢁC for 48 h. For each of the variants tested, four wells were used. Upon completion of
the incubation, stock MTT dye solution was added to each well. After 4 h incubation,

196
H. Guo et al.
a solution containing DMF (50%) sodium dodecyl sulfate (20%) was added to
solubilize the MTT formazan. The cell viability was determined by measuring the
absorbance of each well at 490 nm using a Multiskan SSCENT microplate reader. IC50
values were determined by plotting the percentage viability versus concentration on a
logarithmic graph and reading off the concentration at which 50% of cells remain
viable relative to the control [17, 26, 27].
3. Results and discussion
3.1. Synthesis and characterization
The ligands SAA and 4-CH3O-SAA were prepared by the reaction of anthranilic acid
with salicylaldehyde and 4-methoxysalicyladehyde, respectively, in the appropriate
mole ratios using absolute ethanol as solvent [19, 27]. The complexes [VO(SAA)(phen)]
(1) and [VO(MOSAA)(phen)] (2) were prepared by refluxing of corresponding ligand,
VO(acac)2 and 1,10-phenanthroline in absolute methanol. The desired complexes were
purified by recrystallization. The structures of ligand and its corresponding complexes
were confirmed by elemental analysis, ES-MS, IR, and 1H NMR spectroscopies.
Synthetic routes of ligands and their complexes are shown in scheme 1.
As previously described for related mixed-ligand VIVO-complexes [15–17], IR spectra
show absorptions at 3500–3470 cmꢀ1 for (–OH), at 2951–3069 cmꢀ1 for aromatic (C–
H), at 1682–1697 cmꢀ1 for aromatic (COOH), at 1597–1618 cmꢀ1 for azomethine (–
CH¼N) and (C¼C), at 1457–1570 cmꢀ1 for azomethine group (C¼C), and at 1217–
1242 cmꢀ1 for (C–O) [15–19, 27]. In IR spectra of complexes, the absorption of the
azomethine (–CH¼N) was reduced 17–18 cmꢀ1 compared with the ligands, confirming
coordination of azomethine (–CH¼N) [16, 28, 29]. The strong (VO) band at 965 cmꢀ1
could be clearly identified for the complexes [16, 17].
Electronic spectra of complexes and ligands are shown in table 1. Complexes 1 and 2
show an intense band at 264.5 nm assignable to ꢄ–ꢄ* transition of aromatic rings of
phenanthroline [16, 30–32]. A medium band is observed near 400 nm, attributed to a
ligand-to-metal charge-transfer (LMCT) from a p-orbital on the ligand oxygen to the
empty d-orbital of vanadium [24, 29]. Bands in the region 748.5 and 772.0 nm of low
extinction coefEcient values have been assigned to metal d ! d transfer. Bands in the
UV-Vis region (320–350 nm) are assignable to intraligand transitions of the Schiff base
[30–33]. Five- and six-coordinate complexes of oxovanadium(IV) are usually square
pyramidal/trigonal bipyramidal and distorted octahedral, respectively [24, 34, 35].
From the above spectral data, the Schiff bases bond through the phenolate oxygen,
imine nitrogen, and carboxyl oxygen leaving the carboxyl as a pendant group. These
complexes have square-pyramidal geometry around the central V(IV) [17, 24, 31–35].
This is also supported by the magnetic moment of 1.69–1.73 BM observed for Eve-
coordinate VO(IV) complexes [24, 31]. Suggested structures of the complexes are given
in scheme 1.
1H NMR spectra of Schiff bases showed peaks of hydroxyl (OH), carboxyl (COOH),
1
imine (CH¼N) proton, and (CH3O–) [35, 36]. However, in the H NMR spectra of
complexes, the peaks of (COOH) and hydroxyl (OH) were not observed, affirming that
the ligand was coordinated to metal [18, 37]. In ES-MS spectra, Schiff bases showed

Oxovanadium complexes
197
Table 1. Electronic spectral data of the complexes and ligands.
ꢄ ! ꢄ*
n ! ꢄ*
LMCT
ꢀmax (nm)
d ! d
Compound
ꢀmax (nm)
ꢀmax (nm)
ꢀmax (nm)
MOSAA
SAA
VO(SAA)(p)
VO(MOSAA)(p)
258.0
264.5
265.5
264.5
342.5
347.5
386.5
324.0
398.5
391.5
748.5
772.0
(b)
(a) 1.2
1.0
1.2
3.2
3.0
2.8
2.6
2.4
2.2
2.0
2.0
1.0
0.8
0.6
1.6
1.2
0.8
0.8
0.6
0
4
8
12
16
–1
20
0
4
8
12
16
–1
20
5
5
[DNA]x10 mol
[DNA]x10 mol
0.4
0.4
0.2
0.0
0.2
0.0
250 300 350 400 450 500 550
Wavelength (nm)
250 300 350 400 450 500 550
Wavelength (nm)
Figure 1. Absorption spectral trace of 1 (a) and 2 (b) on addition of CT-DNA in Tris-HCl buffer A.
[V] ¼ 20 mmol Lꢀ1. Arrows show the decreasing absorbance with the increasing amounts of CT-DNA. Inset:
plots of [DNA]/("a – "f) vs. [DNA] for the titration of [V] with CT-DNA.
peaks at m/z 242.0 ([M þ 1]þ) (SAA) and 272.0 ([M þ 1]þ) (MOSAA), respectively. The
complexes showed peaks at m/z 487.0 ([M þ 1]þ) (1) and 517.0 ([M þ 1]þ) (2),
1
respectively. Elemental analysis, UV-Vis, ES-MS, IR, and H NMR results of all the
compounds are in agreement with the expected structures. All of the compounds are
neutral and non-conducting in DMF.
3.2. DNA-binding studies
3.2.1. Electronic absorption titration. Electronic absorption spectroscopy is one of the
most common techniques to monitor the interaction of complexes with DNA.
Complexes bound to the base pairs of DNA usually result in hypochromism and
bathochromism, which is correlated with the stacking interaction between planar
aromatic chromophore of complexes and the base pairs of DNA [38, 39]. The
extent of the hypochromism is consistent with strength of intercalative binding
interaction [9, 40–42].
The electronic absorption spectra of 1 and 2 in the absence and presence of CT-DNA
are shown in figure 1. Upon increasing CT-DNA concentration, the hypochromism and
bathochromic shift for 1 at 264.5 nm are 29.5% and 3 nm, respectively. However, under
the same conditions, with increasing CT-DNA concentration, 2 exhibits hypochromism

198
H. Guo et al.
of about 20.9%, and with a red shift of 2.5 nm at 273 nm. According to previous
reported results [39, 41], we believe that 1 and 2 are most likely to bind with DNA
through a stacking interaction between the aromatic chromophore of the ligands and
the base pairs of DNA.
In order to compare quantitatively the binding strength of these two complexes with
DNA, the intrinsic binding constant Kb was calculated by monitoring changes of
absorbance in the ligand transfer bands, with increasing amounts of CT-DNA. The
intrinsic binding constant Kb obtained for 1 and 2 were 4.50 ꢄ 104 (mol Lꢀ1 ꢀ1
)
and
2.95 ꢄ 104 (mol L
)
ꢀ1 ꢀ1, respectively. Such values of intrinsic binding constants indicate
that the interaction of 1 and 2 with DNA are weaker than reported complexes such as
[VO(dbdppo)(bpy)](SO4), [VO(dbdppo)(phen)](SO4), and other systems [17, 8, 33],
suggesting that the interaction of 1 and 2 with DNA are medium strength intercalation.
The difference may be due to steric hindrance of the phenolate group. To fully
understand the mechanism involved in the interaction of these two complexes with
DNA, further investigation will be needed. The Kb value of 1 is larger than that of 2,
due to the different substituents in the ligands. The electron-pushing substituent
(CH3O– in SAA for 2) on the intercalative ligand may decrease the DNA-binding
affinity of the original complex [11, 18]. The results indicate that the DNA-binding
affinities of oxovanadium complexes correlate with electronic effects of their
intercalative ligands.
3.2.2. Fluorescence spectroscopic studies. The emission spectra of the complexes in the
absence and presence of CT-DNA in Tris buffer A at room temperature are shown in
figure 2. The emission intensity of 1 and 2 increase with increasing concentrations of
CT-DNA, growing about 0.78 and 0.73 times for 1 and 2 at 602 nm, respectively. These
values were larger than those of complexes alone and the [DNA]/[V] ratios are 8.75 : 1
and 6.81 : 1 for 1 and 2 at 602 nm after saturation of their emission intensities. This
implies that these two complexes intercalate into the base pairs of the DNA helix. The
enhancement may be due to the hydrophobic environment inside the DNA helix
reducing the accessibility of water to the complex and complex mobility is restricted at
(a) 150
125
100
75
(b) 250
200
150
100
50
50
25
0
0
580
590
600
610
620
630
580
590
600
610
620
630
Wavelength (nm)
Wavelength (nm)
Figure 2. Emission spectra of 1 (a) and 2 (b) in Tris-HCl buffer A in the absence and presence of CT-DNA.
[V] ¼ 20 mmol Lꢀ1. Arrows show the increasing intensity with increasing concentrations of DNA.

Oxovanadium complexes
199
the binding site [17, 42]. The enhancement of emission intensities of both complexes
indicates that the complexes were protected by the hydrophobic environment of DNA
from water, leading to decrease of the vibrational modes of relaxation [42]. It also
shows that 1 interacts with DNA more strongly than 2, consistent with the above
absorption spectra results.
3.2.3. Viscosity measurements. In the absence of crystallographic structural data or
NMR spectra [24, 25], viscosity measurements that are sensitive to length change of
DNA are regarded as the most critical tests of binding mode in solution. Intercalation
of complexes into the base pairs of DNA leads to lengthening of the DNA helix as base
pairs are separated to accommodate the binding ligand, resulting in an increase in
viscosity of the DNA solution [43]. In contrast, partial and/or non-classical interca-
lation of ligand may bend or twist the DNA helix leading to a decrease in its effective
length and, concomitantly, its viscosity [44, 45]. To further clarify the nature of the
interaction between both complexes and DNA, viscosity measurements were carried
out. The effects of 1 and 2 on the viscosity of CT-DNA are given in figure 3. Upon
increasing concentrations of 1 and 2, the relative viscosity of CT-DNA increases
gradually. These results suggest that these two complexes bind to CT-DNA through
intercalation modes [46, 47].
3.2.4. Thermal denaturation studies. DNA melting experiments are usually used to
study extent of interaction. Figure 4 shows the changes of melting temperature of CT-
DNA in the absence and presence of 1 and 2. The melting temperature Tm was
determined from the hyperchromic effect on the absorption of DNA base pairs
(ꢀ ¼ 260 nm) resulting from the double-stranded DNA gradually dissociating to single
strands with increasing temperature. The Tm of CT-DNA in the absence of the
1.04
Complex 1
Complex 2
1.03
1.02
1.01
1.00
0.00
0.02
0.04
0.06
0.08
0.10
[V]/[DNA]
Figure 3. Effects of increasing amounts of 1 (g) and 2 (ꢅ) on the relative viscosity of CT-DNA at
28(ꢃ0.1)ꢁC. [DNA] ¼ 0.40 mmol Lꢀ1
.

200
H. Guo et al.
1.0
0.8
0.6
0.4
0.2
0.0
DNA
Complex 1
Complex 2
40
50
60
70
80
90
Temperature (°C)
Figure 4. Thermal denaturation of CT-DNA in the absence (g) and presence of 1 (ꢅ) and 2 (m).
[V] ¼ 20 mmol Lꢀ1, [DNA] ¼ 80 mmol Lꢀ1
.
complexes was 60.7ꢁC. The observed Tm in the presence of 1 and 2 were 64.8ꢁC and
63.7ꢁC, respectively. The moderate increase in Tm (DTm are 4.1ꢁC and 3.0ꢁC for the two
complexes) were comparable to those observed for classical intercalators and lend
strong support for their binding with DNA in intercalative modes [19, 48–50].
The results also reveal that 1 has a larger DNA-binding affinity than 2.
3.3. DNA cleavage
Cleavage reactions of 1 and 2 toward plasmid DNA were studied by agarose gel
electrophoresis. When circular plasmid DNA is subjected to electrophoresis, relatively
fast migration is observed for the intact supercoiled form (Form I). If scission occurs on
one strand (nicking), the supercoil form will relax to generate a slower moving open
circular form (Form II). If both strands are cleaved, a linear form (Form III) that
migrates between Forms I and II will be generated [51, 52].
In figure 5, the gel electrophoresis pattern of pBR322 DNA is shown after incubation
with 1 and 2 at 37ꢁC for 1 h in the dark. No DNA cleavage was observed for negative
control (lane 1) and/or (lane 2). In the presence of 1 and 2 with 30 mmol Lꢀ1 H2O2
(lanes 3–5 and 8–10, respectively), plasmid DNA was nicked as is evident from the
formation of Form II. These results indicate that the two oxovanadium complexes can
degrade pBR322 DNA through oxidative cleavage in the presence of H2O2.
To study the cleavage mechanism of pBR322 DNA induced by 1 and 2, the DNA
cleavage experiment has been carried out in the presence of L-histidine (lanes 6 and 11)
and DMSO (lanes 7 and 12). In the presence of L-histidine, a singlet oxygen quencher,
the inhibition of DNA cleavage was not observed under the same conditions, whereas,
complete inhibition of DNA cleavage occurred in the presence of hydroxyl radical
scavenger DMSO (lanes 7, 12), suggesting that . OH radical is likely the reactive species

Oxovanadium complexes
201
Figure 5. Cleavage of pBR322 DNA by 1 and 2 (15–60 mmol Lꢀ1) in the absence and presence of H2O2
(30 mmol Lꢀ1) in buffer B (pH 7.2). Lane 1, DNA control; lane 2, DNA þ H2O2 (30 mmol Lꢀ1); lane 3,
DNA þ 1 (15 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1); lane 4, DNA þ 1 (30 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1); lane 5,
DNA þ 1 (60 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1); lane 6, DNA þ 1 (30 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1) þ
L-histidine (0.02 mol Lꢀ1); lane 7, DNA þ 1 (30 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1) þ DMSO (2 mL); lane 8,
DNA þ 2 (15 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1); lane 9, DNA þ 2 (30 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1);
lane 10, DNA þ 2 (60 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1); lane 11, DNA þ 2 (30 mmol Lꢀ1) þ H2O2
(30 mmol Lꢀ1) þ L-histidine (0.02 mol Lꢀ1); lane 12, DNA þ 2 (30 mmol Lꢀ1) þ H2O2 (30 mmol Lꢀ1) þ
DMSO (2 mL).
Table 2. The IC50 values for SAA, MOSAA, 1, and 2 against
Ag8.653 and U251 cell lines.
IC50 (mmol Lꢀ1
)
Compounds
Ag8.653
U251
Cisplatin
SAA
MOSAA
1
2
0.002 ꢃ 0.001
2.5 ꢃ 0.1
0.005 ꢃ 0.001
12 ꢃ 1
19 ꢃ 4
32 ꢃ 3
0.018 ꢃ 0.006
0.26 ꢃ 0.07
0.038 ꢃ 0.007
0.29 ꢃ 0.06
.
for cleavage. This may be explained by generation of OH radicals obtained from the
oxidation of VO2þ in the presence of H2O2 through the reaction as follows:
(VO2þ þ H2O2 ! VO2 þ OH þ H ) [2, 18, 52]. According to previously reported
results [2], we believe that DNA cleavage may be closely related to oxidation of vanadyl
ions of oxovanadium complexes. Further studies are currently underway to clarify the
cleavage mechanism.
þ
.
þ
3.4. Cytotoxic assay in vitro
The effects of 1 and 2 were examined on myeloma cells (Ag8.653) and gliomas cells
(U251) cell lines using the MTT assay [14, 17], a colorimetric determination of cell
viability during in vitro treatment with a drug. The IC50 values were derived after the
selected tumor cells incubated for 48 h in the presence of 1 and 2 and cisplatin in various
concentrations. The IC50 values of the vanadium complexes and cisplatin for growth
inhibition of Ag8.653 and U251 cells are given in table 2. The histograms of cell
viability assay for different compounds against selected tumor cells are shown in
figure 6. As shown in table 1 and figure 6, the antitumor activities of 1 and 2 are
concentration-dependent and 1 (IC50 ¼ 0.018 ꢃ 0.006, 0.038 ꢃ 0.007) possessed more
inhibitory effect against the two cell lines. The antiproliferative activity with increasing
concentrations of 1 is shown in figure 7. Comparing the cytotoxicity of the ligands and

202
H. Guo et al.
Figure 6. Cell viability of 1 and 2 on Ag8.653 and U251 cell proliferation in vitro. Each data point is the
mean ꢃ standard error obtained from three independent experiments.
Figure 7. Micrograph of the myeloma tumor (Ag8.653) cell line after treatment for 48 h in the absence I
(the control) and presence of different concentrations of 1; II: [V] ¼ 0.002 mmol Lꢀ1; III: [V] ¼ 0.02 mmol Lꢀ1
;
IV: [V] ¼ 0.2 mmol Lꢀ1
.
corresponding complexes, it can be concluded that antitumor activities against selected
tumor cell lines have been remarkably enhanced when ligands coordinate with
vanadium [16, 17, 53]. Complexes 1 and 2 exhibit in vitro cytotoxicity against the
selected cells lines, but lower than that of cisplatin which was determined in this work
through the same technique.

Oxovanadium complexes
203
4. Conclusion
Two unsymmetrical oxovanadium complexes, [VO(SAA)(phen)] (1) and
[VO(MOSAA)(phen)] (2), have been synthesized and characterized by elemental
analysis, UV-Vis, ES-MS, IR, and 1H NMR spectroscopy. The DNA-bindings of these
complexes were investigated by spectroscopic methods, viscosity measurements, and
thermal denaturation. The results show that 1 and 2 bind to CT-DNA in intercalation
mode. The DNA-binding affinity of 1 is larger than that of 2. These oxovanadium
complexes are capable of cleaving supercoiled plasmid DNA in the presence of H2O2.
Both 1 and 2 have cytotoxic activities against Ag8.653 and U251 cell lines, with 1
possessing more inhibitory effect. Further investigation is required to study the possible
cytotoxicity mechanisms of these two complexes.
Acknowledgments
We gratefully acknowledge financial support for this work by Guangdong
Pharmaceutical University (No. 43540119), the National Key Research Project of
China (No. 2011zx09102-001-31), and the National Natural Science Foundation of
P.R. China (Nos 81102753 and 31070858).
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