Synthesis of Oxovanadium(IV) Schiff base Complexes derived from C-substituted Diamines and Pyridoxal-5-Phosphate as Antitumor Agents

Article abstract of DOI:10.1111/j.1747-0285.2011.01265.x

Oxovanadium (IV) complexes of N,N'-bispyridoxyl-5, 5'-bis (phosphate) ethylenediimine (L1) and N,N'-bis(pyridoxyl)-5,5'-bis(phosphate)-1''-(p-nitrobenzyl)ethylenediimine (L2) were synthesized by condensation of optically active C-substituted diamines and

Full text of DOI:10.1111/j.1747-0285.2011.01265.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01265.x
Chem Biol Drug Des 2012; 79: 223–234
Research Letter
Synthesis of Oxovanadium(IV) Schiff base
Complexes derived from C-substituted Diamines
and Pyridoxal-5-Phosphate as Antitumor Agents
Puja Panwar Hazari1,*, Anand Kumar
chelating agent; U-87MG, U-87 malignant glioma; MTT, 3-(4,5-dimethyl-
thiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MPE, b-mercaptoethanol.
Pandey1,2, Shubhra Chaturvedi1, Anjani
Kumar Tiwari1, Sudhir Chandna3, Bilikere
Received 16 December 2010, revised 26 July 2011 and accepted for
publication 2 October 2011
Srinivasarao Dwarakanath3 and
Anil Kumar Mishra1,*
1Division of Cyclotron and Radiopharmaceutical Sciences, Institute
Current efforts are on to design transition metal complexes based
on vanadium that can act as chemical nucleases suitable for plas-
mid nicking or in a direct strand scission of the genomic DNA.
Potential Schiff base ligands derived from condensation reaction of
ethylenediamine with aldehyde group of pyridoxal-5-phosphate have
been prepared, and its oxovanadium complexes were investigated
for DNA cleaving activity. The preparation of early-transition metal
complexes with chelating ligands, such as derivatives of Schiff base
salen{bis(salicylidene)imino)}V=O complex, has been reported in lit-
erature (1,2). Antimicrobial ⁄ antitumor ⁄ cytotoxic activities of vana-
dium (IV), manganese (IV), iron (III), cobalt (II), and copper (II)
complexes have also been reported (3–6). These complexes are air
stable, good Lewis acids, and have been previously employed as
asymmetric catalysts. Vanadium is a transition element that exists
in several oxidation states, including +2, +3, +4, and +5. Only the
+3, +4, and +5 oxidation states are important in biological systems
(7–11). Several organometallic complexes of vanadium exhibit anti-
tumor properties both in vitro and in vivo, primarily via oxidative
damage (12,13). Vanadium compounds induce cell cycle arrest and
cytotoxic effects through DNA cleavage and plasma membrane lipo-
peroxidation (14,15). Vanadium may also exert inhibitory effects on
cancer cell metastases potentially through modulation of cellular
adhesive molecules, and reverse antineoplastic drug resistance.
Redox-active transition metal complexes in the presence of oxidants
have been extensively used for DNA cleavage reactions (16). In a
typical reaction, an oxidizing agent like O2, H2O2, or a peracid is
added in addition to the transition metal complex (17).
of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Road,
New Delhi 110054, India
2School of Biomedical Engineeering, Institute of Technology,
Banaras Hindu University, Varanasi 221005, Uttar pradesh, India
3Division of Radiation Biosciences, Institute of Nuclear Medicine
and Allied Sciences, Brig SK Mazumdar Road, New Delhi 110054,
India
*Corresponding authors: Anil Kumar Mishra, akmishra@
inmas.drdo.in, Puja Panwar Hazari, puja_panwar@yahoo.com
Oxovanadium (IV) complexes of N,N¢-bispyridoxyl-
5, 5¢-bis (phosphate) ethylenediimine (L1) and
N,N¢-bis(pyridoxyl)-5,5¢-bis(phosphate)-1¢¢-(p-nitrob-
enzyl)ethylenediimine (L2) were synthesized by con-
densation of optically active C-substituted diamines
and pyridoxal-5-phosphate. Oxovanadium (IV) com-
plexes derived from L1 and L2 were evaluated as
DNA cleavage agent (cleavage of supercoiled plas-
mid pBR322 DNA). Interestingly, both the oxovana-
dium (IV) complexes exhibited DNA nuclease
activity, and the extent of oxidation of DNA by
these vanadyl complexes was superior to VOSO4.
The significant reduction in primary tumor and
increased delay in tumor growth of 15 days was
seen in the tumor regression analysis with oxovana-
dium (IV) complex of L1. With the preliminary studies
performed with the pyridoxal-5-phosphate -based sa-
len derivatives including the cytotoxicity and tumor
regression, it is evident that the salen bifunctional
chelating agent has obtained therapeutic potential
if conjugated to a gene-specific targeting molecule
for the oxidation of guanine residue.
Previously, pyridoxal-based chelators have shown antitumor activity
when bound to metal ion; for instance, pyridoxal isonicotinoyl
hydrazone (PIH) compounds have a high affinity and specificity for
Fe(III), and some of its analogs have been shown to possess marked
Fe chelation efficacy in a wide variety of biological systems both in
vitro and in vivo. Antineoplastic activity of these ligands based on
PIH has been reported in literature (18). Attachment of the chelating
agents to the biologically active molecule is readily accomplished
because of the functionalization of the ligand N,N¢-bis(pyridoxyl)-
5,5¢-bis(phosphate)-1¢¢-(p-nitrobenzyl)ethylenediimine (L2) derived
from optically active amino acid. There is significant interest in the
Key words: bifunctional chelating agent, chemical nucleases, cyto-
toxicity, DNA modification, pyridoxal-5-phosphate, Schiff base, vanadium
complex
Abbreviations: [VO-(L1)], N,N¢-bispyridoxyl-5,5¢-bis (phosphate)
ethylenediimine vanadium complex; [VO-(L2)], N,N¢-bis(pyridoxyl)-5,5¢-
bis(phosphate)-1-(p-nitrobenzyl)ethylenediimine vanadium complex; P-5-P,
pyridoxal-5-phosphate; ROS, reactive oxygen species; BFC, bifunctional
223

Hazari et al.
synthesis and properties of Schiff bases bearing pendant arms, and
their transition metal complexes have an application as antitumor
agents.
Statistical methods
Data are reported as mean € standard deviation (SD ⁄ SE). One-way
ANOVA analysis was carried out to determine whether there is a sta-
tistically significant difference between combinations used to evalu-
ate the synergistic effect.
In an systemic effort to identify a potent anticancer agent, we syn-
thesized oxovanadium (IV) complexes of N,N¢-bispyridoxyl-5, 5¢-bis
(phosphate) ethylenediimine (L1) and N,N¢-bis(pyridoxyl)-5,5¢-
bis(phosphate)-1¢¢-(p-nitrobenzyl)ethylenediimine (L2) and examined
their cytotoxic activity against two different human cancer cell
lines [U-87 malignant glioma (U-87MG) and MDA-MB-468)]. The
Schiff base ligand and its oxovanadium (IV) complex were synthe-
sized based on the previously published chemistry. The studies,
including the cytotoxicity and tumor regression, performed with the
pyridoxal-5-phosphate (P-5-P)-based derivatives indicate that these
agents have obtained therapeutic potential against tumors of
human origin.
Synthesis of oxovanadium (IV) complex of N,N¢-
bispyridoxyl-5, 5¢-bis (phosphate)
ethylenediimine (1b)
Synthesis of N,N¢-bispyridoxyl-5, 5¢–bis (phosphate) ethylenediimine
[L1] (1a). A solution of pyridoxal-5-phosphate monohydrate (0.25 g;
0.94 mmol) in 10 mL methanol was stirred, and when a homoge-
neous solution was obtained, 30 lL of ethylenediamine (0.44 mmol)
was added. A bright yellow precipitate of the bis(imine) was formed
immediately (Scheme 1). The reaction mixture was allowed to stir
over night at 18–20 ꢀC, and the precipitate was filtered, washed
with methanol, and dried under vacuum. Yield 72% 1H NMR
(400 MHz, D2O pH 8) 8.97 (Ar, 1H, s), 7.68 (Ar, 1H, s), 5.09 (2H, m),
2.4 (4H, s). 13C NMR (100 MHz, D2O pH 8) 162.8, 153.1, 145.2,
144.7, 140.9, 131.4, 61.9, 29.6, 14.9. ESI-MS Found 517.2 [M-H]+;
Calcd m ⁄ e 518.35. Elemental Anal. Calcd for C18H24N4O10P2 C,
41.71; H, 4.67; N, 10.81. Found : C, 41.70; H, 4.68; N, 10.83.
Materials and Methods
Pyridoxal-5-phosphate monohydrate and vanadyl sulfate trihydrate
salt were obtained from Fluka. The corresponding diamine derivative
(2a) of optically active amino acid was synthesized in our
laboratory by the standard procedure (19). Salicylaldehyde, sodium
borohydride, stannous chloride dihydrate, and other solvents
(Sigma-Aldrich Co., St. Louis, MO, USA) were used as received. TLC
was run on plastic-backed silica gel plates (0.2-mm-thick silica gel
60 F-254; E. Merck, KGaA, Darmstadt, Germany) using a 10% w ⁄ v
aqueous ammonium acetate ⁄ CH3OH (1:1 v ⁄ v) solution as eluant.
Synthesis of oxovanadium (IV) complex of N,N¢-
bispyridoxyl-5, 5¢-bis (phosphate)
ethylenediimine [VO-(L1)] (1b)
To a hot methanol solution (20 mL) of vanadyl sulfate (0.347 g,
0.193 mmol) was added the ligand N,N¢-bispyridoxyl-5, 5¢-bis (phos-
phate) ethylenediimine (0.100 g, 0.193 mmol) and pyridine (0.35 mL,
0.34 g, 4.34 mmol), and the mixture was stirred for 1 h at 60 ꢀC. The
MDA-MB-468 kindly provided by Dr Normando, (CIMAB SA, Havana)
and human malignant glioma cells, U-87MG (NCCS, Pune, India), were
maintained at 37 ꢀC in a humidified CO2 incubator (5% CO2, 95% air)
in DMEM (Sigma-Aldrich Co.) supplemented with 10% fetal calf
serum (Biological industries, Kibbutz Beit Haemek, Israel), 50 U ⁄ mL
penicillin, 50 lg ⁄ mL streptomycin sulfate, and 2 lg ⁄ mL nystatin.
Cells were routinely subcultured twice a week using 0.05% Trypsin
(Sigma) in 0.02% EDTA.
H2O4P
O
H2N
NH2
H
N
OH
i
CH3
1
The H NMR spectra were determined by using Bruker Spectro spin
400 MHz at INMAS, Delhi. The 31P NMR spectra were determined
by using Bruker Spectro spin 400 MHz at INMAS, Delhi. The decou-
pled spectra were recorded with the decoupling frequency matching
that of proton. Mass spectrum was recorded on a JEOL SX
102 ⁄ DA-6000 Mass spectrometer using m-nitrobenzyl alcohol as the
matrix (Agilent 1100, Karlsrhue, Germany, and Thermofisher, Bombay
India). Animal protocols have been approved by Institutional Animal
ethics Committee. New Zealand Rabbits and albino BALB ⁄ c mice
were used for blood clearance, imaging, and biodistribution. Mice
and rabbits were housed under the conditions of controlled temper-
ature of 22 € 2 ꢀC and normal diet.
H2O4P
PO4H2
N
N
N
N
OH
HO
CH3
CH3
1a
ii
H2O4P
PO4H2
N
N
V
N
O
N
Cell cycle distribution was studied with the help of FACS Caliber
(Becton-Dickinson & Co., Mountain View, CA, USA) flow cytometer
using the CELL QUEST (version 3.0.1; Becton Dickinson and Co.) and
Mod fit L T (version 2.0 software House, Inc, Becton Dickinson and
Co.) software for acquisition and analysis, and a minimum of
10 000 cells per sample were analyzed.
O
O
CH3
1b
CH3
Scheme 1: Synthesis of oxovanadium (IV) complex of N,N¢-bis-
pyridoxyl-5, 5¢-bis (phosphate) ethylenediimine. Reagents (i) metha-
nol (ii) VOSO4 ⁄ pyridine.
224
Chem Biol Drug Des 2012; 79: 223–234

Schiff base Oxovanadium(IV) Complexes as Antitumor Agents
suspension was then kept at room temperature, and the resulting
IR(KBr) m, ⁄ cm 3395, 2089, C=N stretch; 1639, 1510, 1392, 1208,
1079, 987, 844, 722,621, V-N stretch; 467. ESI-MS: Found 721
[M + 3H]+; Calcd for C25H27N5O12P2V: m ⁄ e 718.(Scheme 2). Elemental
Anal. Calcd. for C23H19N5O11P2V; C, 43.75; H, 3.96; N, 10.20; Found: C,
43.76; H, 3.94; N, 10.22.
green precipitate was collected by filtration and washed thoroughly
with water, methanol, and diethyl ether. Yield 84% calc., IR(KBr) m,
⁄ cm 3401, 2921, C=N stretch was observed at 1637 ⁄ cm, 1591,1508,
1424, 1387, 991,V-O (614 ⁄ cm), 495, V-N (466 ⁄ cm) ESI-MS: Found
585.3 [M + 2H]+, Calcd for C18H22N4O11P2V: m ⁄ e 583.28. 31P NMR:
(D2O) 4.349 (br). Elemental Anal. Calcd for C18H22N4O11P2V C, 37.07;
H, 3.80; N, 9.61 Found: C, 37.09; H, 3.83; N, 9.62.
EPR measurements
EPR spectra were recorded on an X-band Bruker ER200D-SCR spec-
trometer. Measurements were recorded in fluid solution at 298 K
(1 mM in H2O ⁄ DMSO 80 ⁄ 20) and in frozen solution at 77 K with
incident power sufficiently low (12 mW) to avoid saturation effects
and a modulation frequency of 100 kHz. Modulation amplitudes
from 1 to 10 G were used. The g values were determined by mea-
suring the magnetic field, H, and the microwave frequency. 2,2-
diphenyl-1-picrylhydrazyl is taken as standard.
Synthesis of N,N¢-bis(pyridoxyl)-5,5¢-
bis(phosphate)-1¢¢-(p-nitrobenzyl)
ethylenediimine [L2] (2b)
p-Nitrobenzylethylenediamine (2a) was synthesized as reported previ-
ously (19). A solution of pyridoxal-5-phosphate monohydrate (0.598 g;
2.2 mmol) in 10 mL methanol was stirred, and when a homogeneous
solution was obtained, 0.2 g of (2a) (1.02 mmol) was added. A bright
yellow precipitate of the bis(imine) was formed immediately. The
reaction mixture was allowed to stir over night at 18–20 ꢀC. The pre-
cipitate was filtered, washed with methanol, and dried under vacuum.
Yield 82% IR(KBr) m, ⁄ cm 3398, C=N stretch; 1645, 1473, 1321, 1157,
1029, 825 734 590 ESI-MS: Found 654.80 [M + H]+Calcd for
MTT and clonogenic assays
The cytotoxicity of oxovanadium (IV) complexes was tested in
human cancer cell lines using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphe-
nyl-2H-tetrazolium bromide (MTT) assay; 1 · 104 cells ⁄ well were
plated in 200 lL of the complete medium, and treatment was given
24 h after plating. Cells were exposed continuously with varying
concentrations of the complex (0.1–500 lM) in PBS in a volume of
50 lL, and MTT assays were performed at the end of fourth day.
At the end of treatment, cells were incubated with MTT at a final
concentration of 0.05 mg ⁄ mL (20 lL) for 2 h at 37 ꢀC, and the
medium was removed. The cells were lysed and the formazan crys-
tals dissolved using 150 lL of DMSO. Optical density was mea-
sured at 570 with 630 nm as reference filter.
1
C25H29N5O12P2: m ⁄ e 653.14 H NMR (400 MHz,D2O) 8.90(Py-Ar,2H,s),
8.51–8.49 (Ar, 2H,d; J = 12Hz), 7.81–7.79 (Ar,2H, J = 12Hz), 7.58(2H,
s), 3.4 (1H, t), 3.27(2H, s), 1.98(s). 13C NMR (100 MHz, D2O) 162.3,
145.6, 144.3, 134.2, 130.3, 122.2, 61.7, 56.7, 42.1, 28.9, 14.2. Elemen-
tal Anal. Calcd for C25H29N5O12P2 C, 45.95; H, 4.47; N, 10.72; Found:
C, 45.91; H, 4.44; N, 10.71.
Synthesis of oxovanadium (IV) complex of N,N¢-
bis(pyridoxyl)-5,5¢-bis(phosphate)-1¢¢-(p-
nitrobenzyl)ethylenediimine [VO-(L2)] (2c)
To a hot methanol solution (20 mL) of vanadyl sulfate (0.026 g,
0.16 mmol), 2b (0.1 g; 0.16 mmol) and pyridine (0.3 mL, 3.62 mmol)
were added and the mixture was stirred for 1 h at 60ꢀC. Yield 88%
Macrocolony assay
Monolayer cultures of MDA-MB-468 and U-87MG cell lines were
trypsinized, and 100–1000 cells were plated in 60-mm Petri dishes
NO2
PO4H2
NO2
OHC
N
HO
H
H2N
i
CH3
2a
NH2
PO4H2
H2O4P
H
N
N
NO2
N
N
ii
OH
HO
CH3
CH3
2b
H2O4P
PO4H2
H
N
N
V
N
O
N
O
O
2c
CH3
CH3
Scheme 2: Synthesis of oxovanadium complex of N,N¢-bis(pyridoxyl)-5,5¢-bis(phosphate)-1¢¢-(p-nitrobenzyl)ethylenediimine. Reagents (i)
methanol, pyridoxal-5-phosphate (ii)VOSO4 ⁄ pyridine.
Chem Biol Drug Des 2012; 79: 223–234
225

Hazari et al.
depending on the concentrations of the complex and incubated at
37 ꢀC in 5% CO2 humidified atmosphere. Growth media were chan-
ged every third day. Colonies were fixed in methanol and stained
with 1% crystal violet. Plating efficiency was calculated as P.E.
(%) = No. of colonies ⁄ No. of cells plated · 100. Surviving fraction
(SF) was calculated.
ing the P-5-P hydroxy groups (Schemes 1 and 2). Both ligands were
easily synthesized in high yield and were characterized by IR, ele-
mental analyses, mass, and NMR spectroscopy. Signal for (V-N)
stretch was observed for [VO-(L1)] and [VO-(L2)] in the region at 466
and 467 ⁄ cm, respectively, and m (V-O) stretching for [VO-(L1)]
occurred at 614 ⁄ cm as reported for other oxovanadium derivatives
(20). P-NMR chemical shifts are independent of formal oxidation
numbers. Rather, they depend on the actual electron density on
phosphorous, the bond angles, and pi-bonding. After complexation,
31P-NMR of the complex shows a downfield change in chemical
shift, which confirms the metal complexation. The elemental analy-
ses demonstrate the stoichiometric relation of atoms in the com-
pounds. The molecular ion peak (ESI-MS) at 585.3 and 721 for [VO-
(L1)] (Figure 1) and [VO-(L2)] (Figure 2) reflects that there is no sol-
vent bounded in the axial position of the ligand.
DNA cleaving experiments
Experiments with plasmid DNA pBR322 on
agarose gels
Each reaction mixture contained 2 lL of supercoiled pLAZ DNA
pBR322 (1 lg), 2 lL of Tris–HCl ⁄ NaCl 10 mM buffer at pH 7.0, 6 lL
of water, and 10 lL of the [VO-(L1)] and [VO-(L2)] compound at the
desired concentration (0.1 and 0.25 mM) that were prepared in 100%
PBS immediately before use. After 2-h incubation at 37 ꢀC or over-
night at room temperature, 5 lL of loading buffer (0.25% bromophe-
nol blue, 0.25% xylene cyanol, and 30% glycerol in water) was added
to each tube and the solution was loaded onto 1% agarose gel. The
electrophoresis was carried out for about 2 h at 120 V in TAE buffer
(89 mM Tris–acetate, 1 mM EDTA, pH 8.3). Gels were stained with
ethidium bromide (11 g ⁄ mL) and destained with water prior to being
photography of following UVA excitation of 360 nM.
Figure 3 shows the EPR spectrum of the compound [VO-(L1)] and
[VO-(L2)]. The spectra were recorded at 293 K and found to consist
of eight well-resolved hyperfine splitting lines, which were not
equivalent in intensity. The G and A values obtained in the spectra
give an idea of the environment around V(IV): g is 1.972 and A is
Cell cycle analysis
[M + 2H]+
Exponentially growing cells were incubated with 0.1 mM of oxovana-
dium compounds for 24 h at 37 ꢀC. Cells were harvested by trypsin
release and resuspended in DNA staining solution (10 lg ⁄ mL RNAse,
0.1% Triton X-100, 0.1 mM EDTA, 0.1% sodium citrate, 50 lg ⁄ mL PI,
and 1 mM Tris–HCl) 1–2 h prior to flow cytometric analysis. The fluo-
rescence of 10 000 cells was measured using flow cytometer (Fac-
scalibur; Becton Dickinson) with excitation of 488 nm. The percentage
of cells in G1, S, and G2-M was determined by analyzing the DNA his-
tograms with modfit software provided by the manufacturer.
Figure 1: Mass spectrum of oxovanadium (IV) complex of N,N¢-
bispyridoxyl-5, 5¢-bis (phosphate) ethylenediimine [VO-(L1)].
Tumor regression analysis
Seven-week-old female BALB ⁄ c mice (n = 10, weight = 28–30 g)
received a subcutaneous injection of 105 Ehrlich ascites tumor (EAT)
cells on the right hind leg. Complex was administered via i.v. injec-
tions. The study began treatment after tumor volume has reached
0.5–0.7 cm3. [VO-(L1)] treatment was given in volume of 100 lL at
400 mg ⁄ kg of dose every 48 h beginning on day one and continuing
for 10 doses. The mice were weighed once a week throughout the
trial. The day of tumor detection was recorded, and the dimensions
of the tumors were measured every 48 h. As life span was one of
the parameters considered in the study, the mice were brought to
survival approximately 2–3 days before their natural death. The
tumor development study lasted 40 days.
Results
Spectroscopic characterization
The ligand N,N¢-bispyridoxyl-5, 5¢-bis (phosphate) ethylenediimine
and C-substituted diimine form stable water-soluble chelates involv-
Figure 2: Mass spectrum of oxovanadium (IV) complex of N,N¢-
bis(pyridoxyl)-5,5¢-bis(phosphate)-1-(p-nitrobenzyl)ethylenediimine [VO(L2)].
226
Chem Biol Drug Des 2012; 79: 223–234

Schiff base Oxovanadium(IV) Complexes as Antitumor Agents
(25 ꢀC) yielded the stability constants 16.4 and 17.2, respectively,
when metal and ligand were taken in 1:1 ratio (1 mM each).
Plasmid DNA on agarose gel
We have investigated vanadium complexes capable of reacting with
O2 in the presence of a reductant to generate V(III) species leading
to oxidative damage at guanine residue of DNA (G). With redox-
active ligands, the role of V(III) is to trigger formation of ligand rad-
icals capable of generating covalent adducts (17).
DNA cleavage was analyzed by monitoring the conversion of super-
coiled plasmid DNA pBR322 (Sc) to nicked circular DNA (Nck) and
linear DNA (Lin) (Figure 4). Under aerobic conditions, the [VO-L1]
was able to trigger oxidative cleavage of DNA by 38% in the
absence of any activating agent (Table 2).
Figure 3: EPR spectrum of VO-L1 (red) and VO-L2 (blue)
recorded at 293 K in H2O ⁄ DMSO (80 ⁄ 20).
Table 1: Potentiometric species distribution pattern of VOSO4,
[VO-(L1)] system in the presence and absence of oxidant and reductant
Vanadium complexes caused molecular oxygen-dependent DNA
strand break in cooperation with H2O2-generated OH radicals. Incu-
bation of the plasmid with the vanadium complexes in the presence
of H2O2 at 37 ꢀC for 2 h or at rt for 24 h caused conversion of the
Sc form to the Nck form. The cleavage reaction could be activated
by the addition of either a reducing agent or an oxidant with the
complex significantly promoting DNA cleavage in the presence of
b-mercaptoethanol (MPE) (Figure 4B).
pH
VO2+
V(III)
V(IV)
V(V)
VOSO4
92
b-mercaptoethanol
0
5
–
NaIO4
2
3
5
5
1
2
3
4
5
6
7
8
7.6
11
17.7
23.5
40
33
30
29
25
21
11
7
72.3
70.4
30.6
18.1
10.2
5.1
3.2
3.1
2.9
2.7
5.9
7.6
6.5
0.6
0.5
0.5
0.4
8.5
7
6
0.8
0.8
1
1
6
11
15
15.5
13
Modification of DNA by salen complexed with vanadyl ion was also
studied in the presence of an oxygen donor compound, sodium
periodate. In this case, plasmid DNA modification was much more
effective than the reducing agent as complete conversion of Sc
form to Nck 52% and Lin 47% forms was observed at 0.25 mM
concentration. Indeed, in all the cases, the Sc form of the plasmid
DNA was practically converted into the Nck and Lin forms
(Figure 4). When the DNA was incubated for 2 h at 37 ꢀC in the
presence of oxidizing agent NaIO4, it caused substantial DNA cleav-
age, and with complete conversion of the Sc form to the Nck and
Lin DNA molecules (excess single-strand breaks enhance the proba-
bility of double-strand scissions). The DNA cleavage efficiency
increased with the concentration of the redox agent (0.15 or
0.2 mM) as well as the complex (0.1, 0.25, 0.5 mM). It is also worth
noting that all combinations and the complex alone exhibited nucle-
ase activity. Similar results were obtained with the bifunctional ox-
ovanadium complex as well. In this case, however, plasmid DNA
cleavage was effective in the presence of both reducing and oxidiz-
ing agent. The Sc form of plasmid DNA was completely converted
into the Nck (79%) and Lin (20%) forms. Presence of optically active
p-NO2-phenylalanine imparted cleavage with reducing agent by suc-
cessful conversion into Nck and Lin forms at 0.1 mM, which was
seen at 0.25 mM for VO-L1. This could be attributed to the pres-
ence of chiral center in VO-L2. Interestingly, 0.1 mM of the bifunc-
tional oxovanadium complex with NaIO4 in the presence of H2O2
completely digested DNA (Figure 4D).
9
10
11
12
13
14
1.5
1.4
1.3
0
0
0
6
2
ꢀ85 · 10)4 ⁄ cm, A ⁄ GI = 84, GI = 1.972, AI ⁄ G = 94 for [VO-(L1)],
and for [VO-(L2)], g is 1.989 and A is 89 · 10)4 ⁄ cm, which indi-
cated distorted octahedral symmetry. EPR data for [VO-(L1)] and
[VO-(L2)] revealed that g values are close to 2 which are for the
unpaired electron in a non-degenerate orbital. Any orbital angular
momentum is subsequently quenched resulting in a 'spin-only' sys-
tem. In addition, there should be little mixing in of an orbital
component from excited states through spin–orbit coupling, leading
to typical g values close to, but slightly less than, 2. Similar signals
have been reported by Verquin et al. (20) for vanadium (IV).
Potentiometric speciation in aqueous solution
Potentiometric titration was carried out in aqueous solution to check
the behavior in solution. The metal ligand system was described by
the formation of [VO-(L1)] 1:1 species. The speciation in solution at
different pH was obtained for VOSO4, [VO-(L1)] in the presence of
mercaptoethanol and NaIO4. At physiological pH, the species distri-
bution pattern of vanadium complexes in solution shows that only
one species V(IV) exist as a major species, while at lower and higher
pH, two species V(III) and V(V) were predominant, respectively, as
seen in pH ⁄ species distribution Table 1. The evaluation of the titra-
tion of the [VO-(L1)] and [VO-(L2)] systems at 0.1 M ionic structure
Cytotoxicity studies of oxovanadium
compounds against human tumor cell lines
[VO-(L1)] and [VO-(L2)] were tested for the ability to induce cytotoxic-
ity in the cancer cell lines using both an assay of mitochondrial activ-
Chem Biol Drug Des 2012; 79: 223–234
227

Hazari et al.
Table 2: Cleavage of supercoiled plasmid DNA (1 g) pBR322 (Sc)
by [VO-(L1)] (1b) and [VO-(L2)] (2c) complexes incubated for 2 h at
37ꢀC in the presence of H2O2, b-Mercaptoethanol (MPE) as reduc-
ing agent and NaIO4 as an oxidant
Drug
Reaction condition
MPE
DNA (form)
S. no. [VO-(L1)] [VO-(L2)] H2O2
NaIPO4 Sc Nck Lin
1
2
3
4
5
6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
82 18
79 21
72 28
–
–
–
–
–
0.01 lM
–
–
0.01 lM 200 lg
0.01 lM
200 lg
–
200 lg 70 30
63 37
200 lg 55 45
–
–
–
7
8
9
0.1 mM
0.1 mM
0.25 mM
0.25 mM
0.1 mM
0.25 mM
0.1 mM
0.25 mM
0.1 mM
0.25 mM
0.1 mM
0.25 mM
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
62 38
17 83
39 61
17 83
35 65
37 63
27 73
–
–
–
–
–
–
–
37
–
47
51
50
21
31
26
25
4
0.01 lM
–
0.01 lM
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
200 lg
200 lg
–
–
0.01 lM
0.01 lM 200 lg
–
–
0.01 lM
0.01 lM
–
–
–
63
–
–
–
–
–
200 lg 30 70
200 lg
200 lg
200 lg
–
–
–
–
–
–
–
–
–
52
49
50
79
60
74
75
3
0.1 mM
0.1 mM
–
–
–
–
200 lg
–
–
k
0.1 mM 0.01 lM 200 lg
0.1 mM
0.1 mM 0.01 lM
–
–
–
200 lg
200 lg
Nck and Lin refer to the nicked and linear DNA forms respectively. In all
cases SD was found to be <5%.
100
[VOL1]
[VOL2]
[VO-Salen(P-5-P)]
[VO-p-NO2-Salen(P-5-P)]
Nck
in
80
60
40
20
0
Sc
Figure 4: Cleavage of supercoiled plasmid DNA pBR322 (Sc) by
[VO-(L1)] (0.1 and 0.25 mM): 3(A) in the presence of H2O2; 3(B) b-
mercapotoethanol (MPE) as reducing agent; 3(C) NaIO4 as oxidant;
3(D) nuclease activity of bifunctional [VO-(L2)] complex. The lanes
marked DNA, H2O2, VOSO4, MPE, NaIO4, MPE + H2O2, and
NaIO4 + H2O2 refer to the plasmid DNA incubated without drug as
controls. The DNA was incubated for 2 h at 37 ꢀC.
0
100
200
300
400
500
Concentration in mM
Figure 5: Cytotoxic effects of [VO-(L1)] and [VO-(L2)] in human
glioma cell line (U-87 MG) studied by metabolic viability using 3-
(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay.
The data points represent the mean (€SD) values.
ity (in a MTT assay) and a clonogenic survival. Both the vanadium
complexes showed
a concentration-dependent cytotoxicity in
U-87MG cells (Figure 5). A known number of cells were treated with
increasing concentration (0.1–500 lM) of [VO-(L1)] and [VO-(L2)] for
48 h in DMEM and the metabolic viability assayed. A concentration-
dependent decrease in the fraction of the metabolically viable cells
was observed with 40% decrease in viability at 0.5 mM (Figure 5).
Twofold decrease was observed for the [VO-(L2)] as compared to the
[VO-(L1)] complex.
as to suppress its oxidizing effect (14). The cytotoxic effects of acti-
vating agents, reductant (2-mercaptoethanol), and oxidant (sodium
periodate) in combination with the complex were studied in the
absence and presence of H2O2.
U-87 malignant glioma cells were treated with minimal concentra-
Treatment of cells with hydrogen peroxide (0.01 lM) and activating
tion, 0.01 lM of H2O2 (ED50 of H2O2 reported to be 50–100 lM), so
agents in the absence of complex did not induce marked cytotoxic
228
Chem Biol Drug Des 2012; 79: 223–234

Schiff base Oxovanadium(IV) Complexes as Antitumor Agents
[VO-L1]
[VO-L1]
A
B
[VO-L2]
[VO-L2]
C
D
Figure 6: Cytotoxic effects of oxovanadium complexes in the presence of H2O2 in U-87 malignant glioma human glioma cell line deter-
mined by clonogenic survival assay (A) b-mercaptoethanol (MPE) + [VO-(L1)] (B) NaIO4 + [VO-(L1)] (C) MPE + [VO-(L2)] (d)NaIO4 + [VO-(L2)] in
HBSS. The data points represent the mean (€SE) values.
A
2 h
48 h
2 h
48 h
B
Figure 7: Cytotoxic activity of [VO-(L1)] against MDA-MB-468 human tumor cell line. Cells were incubated with increasing concentrations
(0.1–0.25 mM) for 2 and 48 h (A) Effect of reducing agent [b-mercaptoethanol (MPE)]; (B) effect of oxidizing agent (NaIO4) in the absence and
presence of hydrogen peroxide, and the cell survival was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
assay. The data points represent the mean (€SE) values.
Chem Biol Drug Des 2012; 79: 223–234
229

Hazari et al.
G0/G1
MDA-MB-468) to investigate the toxicity of [VO-salen(P-5-P)]. Com-
plex alone induced a molecular level of cytotoxicity with SF values
of 0.72 € 0.001 for a 2-h exposure and 0.68 € 0.009 for 48 h at
0.1 mM (Figure 7) implying time-independent toxicity. With the
increase in exposure time to 48 h in DMEM, a further increase in
the cytotoxicity (150%) was observed. NaIO4 was found to be more
effective than the reducing agent with the complex. Presence of
H2O2 further increased the toxicity by twofold as compared to the
treatment with activating agent in the presence of H2O2 without
the complex.
A
B
G2/M
S
Relative DNA content
Relative DNA content
C
D
Cell cycle analysis
As oxovanadium compounds cause DNA breaks (Figure 4), which
elicit cell cycle checkpoints response in cells, we investigated the
cell cycle perturbation by flow cytometric measurements of DNA
content of U-87 cells. Cells treated with 0.1 mM of the compound
for 2 h at 37 ꢀC were allowed to grow, and after removing the
compounds, cells were trypsinized, fixed, stained with propidium
iodide, and analyzed by flow cytometry for DNA content. The per-
centages of cells in each cell cycle were determined using the
CELLQUEST Software (shown as inset in histograms). A transient
delay in the progression of cells through the cell cycle (Both G1-S
and G2-M transition) was induced by the complex with a near
complete recovery by 48 h after removal of the complex (Fig-
ure 8).
Relative DNA content
Relative DNA content
Figure 8: Effect of [VO-(L1)] on the proliferation of U-87MG cell
cycle distribution (A) control, (B) 2 h (C) 24 h, (D) 48 h post-treat-
ment.
effect with SF values more than 0.90 (Figure 6). However, when
cells were treated with the complex in the presence of H2O2 for
2 h, the SF was significantly reduced (0.40 € 0.06 for 0.1 mM
and 0.25 € 0.02 for 0.25 mM). The cytotoxic effect of the complex
was found to be concentration dependent with SF values
of 0.48 € 0.007 and 0.39 € 0.02 for 0.1 and 0.25 mM, respectively
(Figure 6).
Tumor regression analysis
To study in vivo effects of the complex, we investigated the growth
of subcutaneous implanted EAT in mice following i.v. administration
of the complex at a dose of 400 mg ⁄ kg with the time required for
reaching five times the initial volume for nearly 10 days. The initial
growth of tumors in untreated controls was exponential in nature
(Figure 9). Administration of vanadium complex significantly delayed
the growth, and the time for reaching five times the initial volume
was 25 days. [VO-L1]-treated group caused a significant delay in
tumor growth to be an offset from that of the untreated control
which is evidenced in the peak tumor volumes of the control group
occurring on day 22, whereas the same climax was reached at day
40 by the treated mice.
It is worthwhile to note that the formation of macrocolonies follow-
ing exposure of 0.1 mM of vanadium complex was significantly
delayed (20 against 7 days) as compared to the untreated cells,
suggesting a long transient delay in the cell proliferation under
these conditions.
In the presence of H2O2, synergistic effect was observed with
reducing agent, and the SF was observed to be 0.09 € 0.004 at
0.25 mM concentration of the complex (Figure 6). One-way ANOVA
analysis was carried out to determine the significance of synergistic
effect. It was found that complex with MPE in the presence of
H2O2 was compared with control at 48 h, and the means were sig-
nificantly different with F = 40.63 and p = 0.003 at 0.05 signifi-
cance level.
Discussion
We have selected pyridoxal-5-phosphate-based salen ligand
described in the literature [Rocklage et al. (21)] as this Schiff base
ligand forms highly stable coordination complexes with transition
metal ions and subsequently stabilizes the aldimine Schiff base. We
have synthesized oxovanadium (IV) complexes of N,N¢-bis(pyridoxyl)-
5,5¢-bis(phosphate)ethylenediimine and N,N¢-bis(pyridoxyl)-5,5¢–bis
(phosphate)-1¢¢-(p-nitrobenzyl)ethylenediimine derivative and exam-
ined their cytotoxic activity against two different human cancer cell
lines. The formation of the complexes was confirmed by IR studies.
Oxidizing agent, NaIO4, was found to be more potent even at a
lower concentration of the complex. Nearly 13-fold decrease in the
cell survival was seen at 0.25 mM. The cytotoxic activity of the ox-
ovanadium (IV) complexes was strongly dependent on the presence
of H2O2 (Figure 6). Addition of the oxidizing and reducing agents
significantly improves the cytotoxic activity of the oxovanadium (IV)
complexes.
As a part of our efforts to develop bifunctional chelating agents
(BFC) (19), synthesis of new class of bifunctional Schiff base
ligands and complexation with vanadium was carried out. To
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium
assay was also carried out in another cell line (breast carcinoma,
bromide
230
Chem Biol Drug Des 2012; 79: 223–234

Schiff base Oxovanadium(IV) Complexes as Antitumor Agents
observed. The IR spectra demonstrate that final compound shows
sharp OH stretching band near 3400 ⁄ cm, while the reactant P-5-P
has broad OH stretching in the range of 3100–3400 ⁄ cm at lower
transmittances for phenolic compounds that disappear after com-
plexation. EPR allows investigation of paramagnetic species of vari-
ous compounds having one or more unpaired electron. Depending
on the synthetic procedure, the nature and the coordination symme-
tries of different V(IV) species could be detected by EPR. V(IV) clus-
10
Control
Treated
ters give rise to a broad signal owing to significant dipolar
interactions, whereas isolated V(IV) species exhibits hyperfine struc-
ture (HFS) derived from the interaction of free electrons (electronic
configuration 3d1) with the magnetic nuclear moment of 51V
(I = 7 ⁄ 2, 99.76% of natural abundance). In the study, the EPR signal
splits into eightfold lines, indicating the formation of single V(IV)
species (Figure 3). For both complexes at 298 K spectrum showed
isotropic nature.
1
0
5
10
15
20
25
30
35
40
45
50
Days (post treatment)
Figure 9: Effect of [VO-(L1)] on Ehrlich ascites tumor in BALB ⁄ c
mice. Mean values (€SD) from 10 animals are presented.
introduce bifunctionality in the Schiff base chelating system, opti-
cally active p-NO2 phenylalanine was used as a precursor to ren-
der a linking group in the system. Specificity can be generated
by direct reduction of nitro group in the presence of iron powder
and acetic acid by selective reduction as reported elsewhere (22)
followed by conjugation of biomolecule and vanadium complexa-
tion.
As a consequence of low radius ⁄ charge ratio, vanadium(V) centers
are usually strong Lewis acids, which make them suitable for the
activation of peroxidic reagents (23). In the presence of excess
peroxide, they are readily converted to the oxoperoxovanadium (V)
complexes. In the presence of strong oxidizing agent, dominant
structural unit in the coordination chemistry of vanadium in the
pentavalent state is the dioxo VO2+ moiety, and upon addition of
H2O2, the compounds are converted into the corresponding oxoper-
oxovanadium complexes that are known for their oxidizing proper-
ties (24). Investigations on the reactivity of dioxovanadium (V)
complexes with H2O2 by Hamstra et al. (23) suggest that peroxide
binds either to a protonated form of the vanadium complex or to
the complex itself. As a weak oxidant, hydrogen peroxide can
The synthesized compounds are characterized on the basis of vari-
ous spectroscopic techniques. IR studies of vanadium complexes
confirm the formation of aliphatic –C=N– (1637 and 1639 ⁄ cm for
L1 and L2, respectively) with P-5-P derivatives, as well as absence
of broad –NH2 stretching in the range of 3200–3600 ⁄ cm was
OH
N
N
O
O
HS
H
N
N
IV
III
S
V
OH
V
HO
S
O
O
O
O
OH
2
S
N
N
O
N
O
O
-O
O
O
V
O-
O
H2O
N
IV
OH
N
I
O
I
IV
N
V
V
O
V
O
-
O
O
IO3
O
O
O
O
H2O2
H2O2
N
N
O
V
H
N
N
O
H
O
O
N
O
IV
O
N
O
N
IV
N
V
O
V
O
O
V
V
V
O
H+
OH
H
OH
O
O
O
O
O
OH
R
N
H2O3P
N
PO3H2
N
N
N
OH
R = H (L1)
R = p-nitrobenzyl (L2)
N
OH
HO
CH3
CH3
OH
Scheme 3: Schematic representation of different oxidation states of vanadium in the presence and absence of redox agents.
Chem Biol Drug Des 2012; 79: 223–234
231

Hazari et al.
lead to cellular depletion of ATP, GSH, and NADPH, as well as
inducing rises in free cytosolic Ca2+ and activation of poly-ADP
ribose polymerase, events leading to apoptosis (25). As a powerful
oxidant, hydrogen peroxide decomposes in vivo into the extremely
reactive hydroxyl radical upon reduction by metals in the cytosol
or bound to lipids, proteins, and DNA. If generated at very close
proximity, hydroxyl radical rapidly oxidizes these essential cellular
constituents, accounting for much of the damage. In general, any
drug or biological process that generates superoxide can produce
hydrogen peroxide by dismutation. Hydrogen peroxide rapidly
decomposes into hydroxyl radical and hydroxide anion through
metal ion-catalyzed radical reactions, known as the Haber–Weiss
or Fenton reaction. Proposed mechanism of different oxidation
states of vanadium in the presence and absence of redox agents
is given above (Scheme 3).
The present study shows that oxidant and peracid are most effec-
tive in combination with the complex. Similar to clonogenic assay,
the data on MTT assay show potency of oxovanadium compound on
MDA-MB-468 cell line. Quantitative cytotoxicity was evident for the
combination used and with the complex alone, suggesting that the
redox-active compounds, [VO-(L1)] and [VO-(L2)], were able to gener-
ate oxidative stress, thereby producing cytotoxicity.
The in vitro results of U-87MG cells exposed to combination treat-
ment with the complex and H2O2 in the presence of reducing ⁄ oxi-
dizing agent showed an increased toxic effect (Figure 6). It
therefore appears that the significant reduction in primary tumor
incidence and increased delay in tumor growth were seen in the
initial study. Because the majority of cancer treatments involve
reducing tumor volume and prolonging life, the ability of treatment
regimens to increase life spans is important.
The sugar, the phosphate, and the base moieties constitute three
chemical targets for metal-mediated reactions leading to scission
of a DNA or RNA strand. Certain metal complexes, particularly
those of lanthanides, can act as Lewis acids to facilitate phospho-
diester hydrolysis. There are now a large number of redox-active
transition metal complexes, particularly those capable of forming
free or metal-bound electron-deficient oxygen species (metal-oxo,
HOÆ, O2–Æ, etc.) that trigger DNA and RNA strand scission follow-
ing hydrogen atom abstraction from the ribose moiety. The hetero-
cyclic bases like guanine in particular present a wealth of sites
for metal binding and oxidation or alkylation. In principle, any
metal complex that is sufficiently oxidizing to attack hydrogen
atoms of the ribose moiety is also capable of abstracting an elec-
tron from G. Base oxidation does not directly yield strand scission,
but rather creates a site that is sensitive to cleavage using either
chemical reagents (such as HO)) or enzymes, principally those
involved in DNA repair.
Oxidative stress created by the reactive oxygen and other species
like hydroxyl radicals damages many cellular targets besides caus-
ing DNA damage that contributes to the cell death. Damage to
mitochondria not only compromises the metabolic states through
fall in ATP production but also makes the cells susceptible to
intrinsic pathway of apoptosis induced by a variety of signals
including oxidative damage to DNA. Our in vitro results in both
the cell lines support the proposition that the cytotoxicity of ox-
ovanadium compounds arises on account of their oxidative stress
generation ability (Figures 5–7), as both the loss of mitochondrial
viability and clonogenic survival were augmented by the presence
of oxidizing agents like NaIO4 and H2O2. The in vivo implication
of these in vitro observations is clearly brought out in the studies
on tumor growth where a significant growth delay was noted (Fig-
ure 9), although a partial response in the form of tumor growth
delay is reported here.
Peroxovanadates (V) are known to generate superoxide and vana-
dium(IV) for DNA cleavage in the presence of additive amount of
H2O2 (23,24) and inhibit protein-tyrosine phosphatases, the func-
tion of which is essential for mitosis progression, thereby perturb-
ing the cell cycle progression (26) as observed here in this study
also (Figure 8). The vanadium (IV)-containing compound showed
substantial in vitro anticancer activity. This activity appears to be
mediated by the reactive oxygen intermediates inducing activity of
oxovanadium compounds. Although the molecular basis for the in
vitro anticancer properties of oxovanadium (IV) compound is not
completely understood at the present time, it has been shown
that this type of compounds can interact with DNA and cause
DNA cleavage (27). Hydroxyl radicals are generated in a pH-
dependent manner in the [VO-(L)]2+.H2O2 system, and the optimal
pH of the active species for DNA cleavage was found to be in
the region between 8.5 and 9.5. The synthesized 1:1 vana-
dyl ⁄ Schiff base complex was found to cleave supercoiled plasmid
pBR322 DNA more effectively in the presence of hydrogen perox-
ide (Figure 5). This DNA damaging potential appears to be partly
responsible for the oxovanadium complex–induced cytotoxicity
observed in human tumor cell lines which was concentration
dependent (Figure 6). This notion is further supported by the
observation that the presence of H2O2 substantially improved the
cytotoxic activity.
It is likely that under certain conditions (viz., higher dose or in com-
bination with other agents), these complexes may in fact elicit com-
plete response in the form of total tumor regression and cure
(tumor-free survival). These initial observations are indeed encourag-
ing to contemplate newer studies in future.
Conclusion
Our results (both in vitro and in vivo) provide experimental evidence
that oxovanadium (IV) complex has the potential to be anticancer
complex against human cancer cells. Redox-active vanadium (III)
complexes having planar N-donor heterocyclic ligands are found to
be nuclease active (28,29). Apart from antitumor activity, we have
developed cell homing peptides conjugated to the developed system
and evaluated that the complexes activate glycogen synthesis in rat
adipocytes. Thus, the development of these vanadium compounds
holds promise for explosion of novel antitumor ⁄ antidiabetic agents
by enhancing the positive effects of vanadium as an oral therapeu-
tic adjunct in diabetic control, while minimizing potential toxicity. It
is our further aim to optimize VO-L1 and VO-L2 and conjugate anti-
sense oligonucleotide, which may provide the basis for the design
of potentially effective target-specific complex for the treatment of
cancer.
232
Chem Biol Drug Des 2012; 79: 223–234

Schiff base Oxovanadium(IV) Complexes as Antitumor Agents
15. Sakurai H., Tamura H., Okatami K. (1995) Mechanism for a new
antitumor vanadium complex: hydroxyl radical-dependent DNA
Acknowledgments
cleavage by 1,10-phenanthroline vanadyl complex in the pres-
ence of hydrogen peroxide. Biochem Biophys Res Com-
mun;206:133–137.
We thank Dr R. P. Tripathi, Director INMAS, for providing necessary
facilities. The work was supported by Defence Research and Develop-
ment Organization, Ministry of Defence, under R&D project INM–311 and
Indian Council for Medical Research under project 45 ⁄3⁄2006 ⁄PHA ⁄BMS.
16. Keller R.J., Sharma R.P., Grover T.A., Piette L.H. (1988) Vanadium
and lipid peroxidation: evidence for involvement of vanadyl and
hydroxyl radical. Archiv Biochem Biophys;265:524–533.
17. Ozawa T., Hanaki A. (1989) ESR evidence for the formation of
hydroxyl radicals during the reaction of vanadyl ions with hydro-
gen peroxide. Chem Pharm Bull;37:1407–1409.
Declaration of Interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
18. Richardson D.R., Milnes K. (1997) The potential of iron chelators
of the pyridoxal isonicotinoyl hydrazone class as effective
antiproliferative agents II: the mechanism of action of ligands
derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-
naphthylaldehyde benzoyl hydrazone. Blood;89:3025–3038.
19. Mishra A.K., Panwar P., Chopra M., Sharma R.K., Chatal J.F.
(2003) Synthesis of novel bifunctional Schiff-base ligands derived
from condensation of 1-(p-nitrobenzyl)ethylenediamine and 2-(p-
nitrobenzyl)-3-monooxo-1,4,7-triazaheptane with salicylaldehyde.
New J Chem;27:1054–1058.
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Figure S3. Mass spectrum of p-nitrobenzylethylenediamine (2a).
Supporting Information
Figure S4. Mass spectrum of N,N¢-bis(pyridoxyl)-5,5¢-bis(phos-
phate)-1¢¢-(p-nitrobenzyl)ethylenediimine (L2).
Additional Supporting Information may be found in the online ver-
sion of this article:
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
Figure S1. Mass spectrum of N,N¢-bispyridoxyl-5, 5¢–bis (phos-
phate) ethylenediimine (L1).
Figure S2. 31P-NMR of Oxovanadium (IV) complex of N,N¢-bispyri-
doxyl-5, 5¢–bis (phosphate) ethylenediimine [VO-L1].
234
Chem Biol Drug Des 2012; 79: 223–234