Synthesis, characterization and biological properties of a novel copper complex

Article abstract of DOI:10.1016/j.ejmech.2003.08.002

The study of copper complex in relation to cancer is important in many ways. A novel copper complex has been synthesized with non toxic ligand, viz. potassium salt of N-(2-hydroxy acetophenone) glycinate (NHAG). The structure of the complex has been determined by spectroscopic means. Toxicity and antitumor property of the complex has been studied in vivo. Though the complex is toxic at higher doses, lower non toxic doses of the complex deplete glutathione (GSH). GSH depleting property of the complex may be utilized to sensitize drug resistant cells where resistance is due to elevated level of GSH.

Full text of DOI:10.1016/j.ejmech.2003.08.002

European Journal of Medicinal Chemistry 38 (2003) 893ꢀ898
/
www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis, characterization and biological properties of a novel
copper complex
Surajit Majumder 1, Gouri Sankar Panda 2, Soumitra Kumar Choudhuri 3,*
Department of Environmental Carcinogenesis and Toxicology (ECT), Chittaranjan National Cancer Institute (CNCI),
37 S.P. Mukherjee Road, Calcutta 700 026, India
Received 20 February 2003; received in revised form 6 August 2003; accepted 6 August 2003
Abstract
The study of copper complex in relation to cancer is important in many ways. A novel copper complex has been synthesized with
non toxic ligand, viz. potassium salt of N-(2-hydroxy acetophenone) glycinate (NHAG). The structure of the complex has been
determined by spectroscopic means. Toxicity and antitumor property of the complex has been studied in vivo. Though the complex
is toxic at higher doses, lower non toxic doses of the complex deplete glutathione (GSH). GSH depleting property of the complex
may be utilized to sensitize drug resistant cells where resistance is due to elevated level of GSH.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Coordination; Copper; Glutathione; N-(2-hydroxy acetophenone) glycinate
1. Introduction
Coordination behavior of sodium or potassium salt of
N-(2-hydroxy acetophenone) glycine (NHAG) towards
organotin moiety has recently been reported [11].
Antitumor property of the ligand [NHAG] and some
of its organotin complexes has been studied [12]. The
non-toxic nature, water solubility and strong coordina-
tion behavior of NHAG deserve attention [11,12].
The present work describes the synthesis of a novel
copper complex with potassium salt of NHAG; the
structure of the complex has been determined by
spectroscopic studies. Antitumor property, toxicity and
glutathione (GSH) depletion property in vivo of the
complex has also been reported.
Coordination of organic compound with metal i.e.,
chelation causes drastic change in the biological prop-
erty of the ligand and also the metal moiety [1]. It has
been reported that chelation is the cause and cure of
many diseases including cancer. The metal copper
[Cu(II)] is also involved in the causation and cure of
cancer [2ꢀ7]. Copper administration suppresses rat
/
hepatoma induced by chemical carcinogen [8]. Cop-
per(II) complexes cause tumor cells to redifferentiate
into normal cells [9]. Although physiological role of
copper is controversial, it is proved that copper is an
essential component of several endogenous antioxidant
enzymes [10].
2. Chemistry
2.1. Materials and methods
Abbreviations: NHAG, N-(2-hydroxy acetophenone) glycine;
CuNG, copper N-(2-hydroxy acetophenone) glycinate; BSO,
buthionine sulphoxamine; EA, ethacrynic acid; DTNB, 5,5?-dithio
bis (2-nitrobenzoic acid); DMSO, dimethyl sulphoxide; EAC, Ehrlich
ascites carcinoma.
2.1.1. Chemicals
N-(2-hydroxy) acetophenone, copper sulphate, gly-
cine, DMSO was purchased from Aldrich, NY. Etha-
crynic acid (EA), buthionine sulphoxamine (BSO),
verapamil, dithio nitrobenzene (DTNB) were purchased
from Sigma Chemical Compamy, St. Louis, MO. Other
chemicals used were of highest purity available.
* Correspondence and reprints.
E-mail address: soumitra01@vsnl.net (S. Kumar Choudhuri).
1
Junior Research Fellow.
Senior Research Fellow.
Senior Scientific Officer.
2
3
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.08.002

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S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893ꢀ898
/
2.2. Synthesis of the ligand
Blood was obtained via closed cardiac puncture by
means of a 22-guage hypodermic needle and a sub-
xiphoid approach. Blood from each group (CuNG
treated and untreated) was pooled into separate glass
tubes and treated with anticoagulant (heparin).
Normal and differential blood count was done for
treated and normal mice. The experiment was repeated
for four times.
The ligand, potassium N-(2-hydroxy acetophenone)
glycinate was prepared according to the reported
methods [11]. In brief, a cold aqueous solution of
KOH (1.03 g, in 12 mL) was mixed with cold aqueous
solution of glycine (1.38 g in 12 mL) and held at 15ꢀ
/
20 8C in an ice bath with continuous stirring. An
ethanolic solution of 2-(hydroxy) acetophenone (2.5 g
in 25 mL) was added dropwise. Deep yellow color was
developed and stirring was continued for 1 h followed
by 5 h at room temperature. The solvent was removed
by rotary evaporator. The yellow mass was washed with
3.2.2. Effect of CuNG on spleen
CuNG (5 mg kgꢁ1) in DMSO was injected into male
Swiss mice.
petꢀ
/
ether and precipitated with methanolꢀ/diethyl ether
3.3. Preparation of spleen cell suspension
mixture. The crude product was recrystallised from
methanol to yield potassium N-(2-hydroxyacetophe-
Normal and CuNG treated male Swiss mice were
anaesthetized and 70% alcohol was sprayed on abdom-
inal region. Spleen was removed aseptically and small
amount of PBS was injected to it; Spleen was rubbed
against the fine wire mesh of the tissue grinder. The cell
none) glycinate. Yield 75%, m.p. 258ꢀ
/
260 8C.
2.3. Synthesis of the copper complex
suspension formed is spinned at 1000ꢀ
/
1500 rpm for 5ꢀ
/
CuNG was synthesized from the ligand, potassium N-
(2-hydroxy acetophenone) glycinate by its reaction with
copper sulphate; in brief, 0.68 g CuSO4, 5H2O was
dissolved in 5 mL deionised water. 0.785 g potassium N-
(2-hydroxy acetophenone) glycinate was dissolved in 25
mL ethanol. The solution of N-(2-hydroxy acetophe-
none) glycinate (yellow color) was slowly added to
CuSO4 solution (blue color) at room temperature with
continuous stirring by a magnetic stirrer. The mixture
10 min. The supernatant was discarded and the cells
were washed by spinning in PBS twice at room
temperature.
Cell viability was tested by trypan blue and cells were
counted in a phase contrast microscope. The experiment
was repeated for four times.
3.3.1. Effect of CuNG on bone marrow
CuNG (5 mg kgꢁ1) in DMSO was injected to male
Swiss mice.
was further stirred in the magnetic stirrer for 1 h at 45ꢀ
/
50 8C. The color of the mixture changed to deep green.
The mixture was cooled to room temperature and the
green precipitate was separated by filtration. The
compound was dried and recrystallised from DMSO.
Yield 40%, m.p.2428C. Anal. Calc. C10H15O6NCu: C,
39.0; H, 4.87; N, 4.54; Cu, 20.45. Found: C, 40.57; H,
5.04; N, 4.65; Cu, 21.24%.
3.4. Separation of bone marrow cells
Normal and CuNG treated mice were anaesthetized
and the femur bone was cut with the help of a vertebrate
scissor. Bone marrow was flushed with 0.56% KCl
solution and centrifuged at 3000 rpm for 15 min at
37 8C.
Cells were counted under microscope for treated and
untreated animals. The experiment was repeated for
four times.
3. Pharmacology
3.1. Biological materials
3.5. Study of antitumor property of CuNG in vivo
All animals were collected from our animal colony.
EAC was maintained as an ascitic tumor in male
Fifty-five male Swiss albino mice 6 weeks of age were
divided into six groups; one control group with five mice
and five drug treated groups with 10 mice in each group.
Swiss albino mice weighing 18ꢀ
/
20 g (6ꢀ8 weeks old).
/
EAC cells (1ꢂ
106) were injected i.p. to all mice on day
/
3.2. Study of in vivo toxicity
1. On day 2, various doses of the ligand (NHAG)
dissolved in deionised and autoclaved water were
injected i.p. to mice of various groups. No drug was
administered to the control group. Life monitoring was
restricted to daily body weight measurement, recording
time of death. Animals were observed for a period of 60
days. Cell yield, ascites volume, packed cell volume,
3.2.1. Effect of CuNG on blood
CuNG (1 mg) was dissolved in 1 mL DMSO. The
solution (0.1 mL) was injected to male Swiss albino mice
(i.e. 5mg kgꢁ1). Blood was collected from normal and
also from treated mice after different time interval of
CuNG injection intraperitoneally.

S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893ꢀ
/
898
895
mean survival time (MST) and change in life span of the
treated mice in comparison to control (T/C value) were
recorded. The experiment was repeated for four times.
bonding between H and nitrogen [11]. In the copper
complex the band shifted to 2700ꢀ
3407 cmꢁ1. The alkyl
groups CH2, CH3 show characteristic deforming bands
at 1466ꢀ 730
1395 cmꢁ1 and the rocking modes at ꢀ
/
/
/
3.5.1. Effect of CuNG on survival of animals
Various doses of CuNG dissolved in DMSO were
injected ip to male Swiss mice. Animals were observed
for a period of 72 h. The average value of the animals
living (percentage) of three independent experiments
with respect to the doses of CuNG was plotted (Fig. 2).
The amount of CuNG required to kill 50% of the
animals (IC50 value) in vivo was determined from the
plot.
cmꢁ1 in the ligand. The rocking mode of the ligand
shifts to 762 cmꢁ1 in the complex. The band at 1604
cmꢁ1 is assigned due to nCN in the complex (in the
ligand the band appears at 1619). The sharp band at
1395 cmꢁ1 in the ligand may be assigned due to nOCO
symmetric that has been shifted to 1364 cmꢁ1 in the
complex indicating coordination with the Cu-atom
through the COOꢁ group of the ligand. The band at
1269 cmꢁ1 in the ligand may be assigned due to
nPh(CO) which has been shifted to 1237 cmꢁ1 in the
complex indicating coordination of the phenolic oxygen
to the Cu-atom. The band at 1689 cmꢁ1 in the ligand
may be assigned due to a n(OCO) asymmetric vibration
and in complex this vibration appears as broad band at
1630 cmꢁ1 [12].
3.6. GSH-depleting properties of CuNG in vivo
Cells (1ꢂ
106) were injected i.p. into male Swiss mice
/
6 weeks of age. On the second day, 1 mg kgꢁ1 CuNG
was injected to mice. On the 12th day animals were
anesthetized, killed and EAC cells were collected. Cells
Thus, the IR spectra results provide strong evidence
for the complexation of the potentially multidentate
ligand.
(1ꢂ
106) were washed in PBS twice. Cells were homo-
genized and divided into two equal amounts for
/
measuring GSH and protein.
EA, BSO, verapamil were also injected in the similar
manner and cells were collected.
Normal cells were taken from EAC bearing mice 8
weeks of age not undergone any drug treatment.
Experiment for each drug treated and control group
were repeated for four times.
Proton NMR peak of the ligand in D2O appears at
7.38ꢀ
protons. CH2 protons appear at 4.09 (1H, m). CH3
protons appear at 2.26ꢀ2.29 (4H, m).
/
7.51 (s, 5H) and 6.59ꢀ
/
7.6 (s, 3H) for aromatic
/
Proton NMR peak of CuNG in DMSO-d6 appear at
5.59 (S) for aromatic protons. CH2 protons appear 3.40
(B). CH3 protons appear at 2.51ꢀ2.79 (m, 4H). In NMR
/
GSH was measured by the method of Sedlack and
Lindsay [13] and protein was measured by Lowry [14].
spectra we observe a shift of electron density from the
ligand to metal moiety. Aromatic protons shifts from
the range of 7.38ꢀ7.51 in the ligand to 5.99 in the
/
complex. Methelene protons shift from 4.09 in the
ligand to 3.40 in the complex. The shielding of the
aromatic and methylene protons indicate the formation
of the complex. However, the methyl protons in the
4. Results and discussions
UV spectrum for the complex and the ligand was
taken in methanol.
UV bands for the ligand lmax (methanol): 271, 350,
ligand shifted to higher ppm; from ligand 2.26ꢀ
2.51ꢀ2.79 in the complex. Probably, methyl protons
were not pulled from ligand to metal moiety.
/2.29 to
/
411.
UV bands for the complex lmax (methanol): 271, 337,
380.
The change in the UV peak from 350 in the ligand to
Mass spectral data is presented in Fig. 1. The
formation of molecular ion peaks indicates that the
structure of the complex is A in Fig. 1.
337 in the complex indicates pꢀ
of the peak from 411 in the ligand to 380 in the complex
also indicates pꢀp* transition.
Important infrared (IR) bands for the ligand appear
at: 3410ꢀ3360, 1689, 1619, 1524, 1466, 1421, 1395, 1318,
1269, 1205, 1163, 969, 931, 752, 730.
/
p* transition. The shift
Electron paramagnetic resonance (EPR) spectrum of
the complex has been recorded in the polycrystalline
state at room temperature. The spectrum shows the
/
value of gÞ ꢃ
than 2.3 indicates a covalent metalꢀ
[15]. The value of g B2.3 in the present Cu-complex
gives a clear indication of covalent character of the
metalꢀligand bond and delocalisation of the unpaired
electron into the ligand. The trend of g ꢁgÞ ꢁ2.0023
describes the axial symmetry with the unpaired electron
/
2.06 and g ꢃ2.20. The value of g lower
/
jj
jj
/
/
ligand environment
/
jj
Important IR bands for the complex appear at: 3407ꢀ
2700, 2340, 1630, 1604, 1541, 1465, 1444, 1364,
1309,1237, 1160, 1088, 1071, 1035, 965, 941, 861, 785,
762, 722, 622, 595, 537, 526, 453.
/
/
/
/
jj
2
2
residing in the dx ꢁy orbital [16].
The IR spectrum of the ligand shows broad band at
3360ꢀ
nOH is generally observed between 3400 and 3500 cmꢀ1
The observed low value is due to intramolecular H-
/
3410 cmꢁ1 which may be due to nOH. The free
Magnetic susceptibility study of the present Cu-
complex gives a magnetic moment value (meff) of 1.98
BM at room temperature. meff
.
ꢃ1.98 BM indicates one
/

896
S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893ꢀ898
/
Fig. 1. Mass fragmentation of CuNG.
electron paramagnetism. This value is higher than the
spin-only value of 1.73 BM for one unpaired electron.
The higher value of the magnetic moment indicates that
Table 2
Effect of CuNG on spleen and bone marrow of male Swiss albino mice
Drug
Dose (mg
kgꢁ1
Spleen cell (ꢂ
/
Bone marrow cell (ꢂ
/
CuNG is monomeric in nature and there is no metalꢀ
/
)
103)
103)
metal interaction along the axial position in the complex
[17].
10D
10D
Study of hematological toxicity in vivo is presented in
Table 1.
ꢀ
/
ꢀ
50
/
4.79
4.659
4.459
4.659
4.619
3.959
3.179
/
0.82
1.82
0.41
0.45
0.31
1.15
0.03
1.279
1.29
1.39
1.019
0.979
0.79
0.659
/
0.06
0.82
0.82
0.13
0.08
0.08
0.04
Logan
Ligand 100
CuNG
/
/
The ligand is nontoxic up to 100 mg kgꢁ1. There is no
change in hematological parameters at a dose of 50 and
100 mg kgꢁ1 of the ligand. The effect was studied at
various time intervals like 1, 10 and 25 days of the
ligand. CuNG is toxic at a dose of 25 mg kgꢁ1. Lower
doses like 6 or 10 mg kgꢁ1 do not show any toxic effect.
Slight toxicity is shown at 12 mg kgꢁ1. The results
presented in Table 1 are based on four independent
experiments.
/
/
6
/
/
CuNG 10
CuNG 12
CuNG 25
/
/
/
/
/
/
The data are means9
at a dose of 10 mg kgꢁ1 is significantly non toxic to spleen and bone
marrow when compared with untreated control (p B0.001).
/
S.D. of four independent experiments. CuNG
/
The effect of the complex on spleen and bone marrow
is presented in Table 2. The ligand is nontoxic up to 100
mg kgꢁ1. There is no change in spleen or bone marrow
cells at a dose of 50 and 100 mg kgꢁ1 of the ligand.
CuNG is toxic at a dose of 25 mg kgꢁ1. Lower doses
like 6 or 10 mg kgꢁ1 do not show any toxic effect; 12 mg
kgꢁ1 shows slight toxicity.
Toxicity of CuNG in vivo is presented in Fig. 2.
CuNG at a dose of 35 mg kgꢁ1 kills 50% of the animals
Table 1
Effect of CuNG on hematological parameters of male albino Swiss mice
Drug
Dose (mg kgꢁ1
)
Haemoglobin (Hb) (gm/dl)
1D 10D
WBC (ꢂ
/
103)
RBC (ꢂ
/
106)
Lymphocyte (%)
10D
Neutrophil (%)
10D
25D
10D
10D
ꢀ
/
ꢀ
/
11.779
11.5790.49 11.629
11.8390.29 11.5990.14 11.919
11.990.96 11.3890.28 11.9390.31 2.899
11.7190.32 11.2890.13 11.690.51 2.799
11.990.96 10.9290.31 10.7390.47 2.369
8.390.22 8.4390.35 8.4190.31 1.259
/
0.31 11.899
0.31 11.819
0.41 2.849
/
0.35 11.919
/
0.41
2.99
/
0.08
5.69
5.879
5.779
5.69
5.59
3.99
2.69
/
0.21
59.29
52.039
51.679
59.09
58.69
57.29
51.539
/
0.83
36.079
42.079
40.079
41.079
40.109
36.879
28.029
/
0.33
1.41
3.33
0.8
Ligand 50
Ligand 100
/
/
/0.37 2.939
/
0.12
0.21
0.17
0.13
0.25
0.04
/
0.25
0.13
0.08
0.11
0.21
0.08
/053
/
/
/
/
/
/
/
1.83
2.83
2.1
/
CuNG
CuNG
CuNG
CuNG
6
10
12
25
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0.72
1.66
1.67
/
/
/
/
/
/2.83
/
/
/
/
/
/
/1.01
/
The data are means9
/
S.D. of four independent experiments. CuNG at a dose of 10 mg kgꢁ1 has no toxic effect on hematological parameters (p B
/
0.001) when compared with untreated control.

S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893ꢀ
/
898
897
GSH. The study of the effect of CuNG on various
sensitive and drug resistant cells are in progress.
6. Experimental protocols
6.1. Chemistry
UVꢀ
/
vis spectra was recorded in Shimadzu UV 160 A
200
and in Varian Cary 100 Scan in the range of 800ꢀ
/
Fig. 2. In vivo toxicity of CuNG. The plot is drawn based upon the
average value of three independent experiments of animals living
(percentage) with respect to dose of CuNG. IC50 value of CuNG is 35
mm.
IR spectra were recorded in Perkinꢀ
/
Elmer RX 1 FT
mg kgꢁ1
.
spectrophotometer in KBR discs in the range 4500ꢀ
/
500
cmꢁ1
.
Proton NMR spectra was recorded in DMSO-d6 on a
Bruker ACF 300 spectrometer at 300.13 MHz reference
to Me4Si (0.0 ppm).
Mass spectrum was recorded in an AEI MS-30 mass
spectrometer.
(IC50 value). The graph is based on the average value of
three independent experiments.
The antitumor property of CuNG is presented in
Table 3. Neither the ligand nor the complex showed any
antitumor property in vivo as reflected in T/C value. The
ligand is non toxic at 120 mg kgꢁ1. CuNG is quite toxic
C, H, N was measured by Perkinꢀ/Elmer 2400 Series
at a dose of 35 mg kgꢁ1
.
II CHN analyzer.
Copper was measured by atomic absorption spectro-
photometer Varian Spectra 200 FS, hollow cathode
lamp, flame type: air acetylene; replicate 3; wavelength
324.8 nm.
Depletion of GSH by CuNG is shown in Table 4.
CuNG is stronger GSH depleting agent than the
recommended drug EA [18] at a dose of 10 mg kgꢁ1
Most of the GSH depleting agents reported in literature
are effective in vitro and are toxic at required doses
.
EPR spectrum (Fig. 3) was recorded in Varian E 109C
at room temperature at field set: 3200G; scan range 4ꢂ
1 K; receiver gain 4.0ꢂ
103; scan time 4 min; modula-
tion 1.25ꢂ10; time constant 0.250; frequency 9.1 GHz,
/
when applied in vivo [18ꢀ
higher doses, low non-toxic doses like 2ꢀ
be effective to deplete GSH.
/
20]. Though CuNG is toxic at
5 mg kgꢁ1 may
/
/
/
power 30 dB; temp. 295 K; state: microcrystalline.
Magnetic moment was measured in EG & G vibrating
sample magnetometer (model 155).
5. Conclusions
CuNG is toxic at higher doses and depletes GSH even
in nontoxic doses. It has been reported that a number of
drug resistant cells have higher level of GSH compared
to drug sensitive cells and modulation of cellular GSH
homeostasis sensitizes drug resistant cancer cells to a
wide range of chemotherapeutic drugs [21]. The non
toxic dose of CuNG may be utilized to deplete GSH in a
number of drug resistant cell lines and thereby overcome
drug resistance where resistant is due to higher level of
6.2. Biological evaluation
GSH was measured following the method of Sedlack
and Lindsay [13]. Briefly, to 2ꢂ
105 cells in 0.2 mL PBS,
4.8 mL EDTA (0.2 M) was added and kept on ice bath
for 10 min. Then 4 mL deionised water and 1 mL of 5%
trichloroacetic acid (TCA) were added. The mixture was
/
again kept on ice for 10ꢀ
/
15 min and then centrifuged at
3000 rpm for 15 min. Two milliliters of supernatant was
Table 3
Effect of CuNG on EAC bearing male Swiss mice (10 days after CuNG injection)
Parameters
Control
Ligand
50
CuNG (mg kgꢁ1
)
100
6
12
35
Cells in whole ascites fluid (106)
1709
/
10.2
1739
7.139
5.490.16
3691.63
107.46
/
1.7
1719
7.779
5.079
349
107.46
/
2.49
174.49
7.2790.13
4.9390.19
3091.63
89.55
/
1.63
1549
5.490.52
490.16
2391.26
69.64
/
3.27
1389
5.379
3.490.16
1790.82
50.75
/
1.67
Total volume (mL) (tumor cellsꢄ
Total packed cell volume (mL)
Mean survival time (MST) (days)
T/C (%)
/ascites fluid)
7.59
/
1.48
/0.2
/0.13
/
/
/
0.17
5.39
/
0.43
/
/0.13
/
/
/
33.59
100
/2.73
/
/
1.63
/
/
/
The data are means9
with untreated control (p B
/
S.D. of four independent experiments. CuNG at non toxic dose (10 mg kgꢁ1) has no antitumor property when compared
0.005).
/

898
S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893ꢀ898
/
Table 4
Depletion of glutathione (GSH) by CuNG
Drugs
Dose (mg kgꢁ1
)
GSH (ng mgꢁ1 of protein)
% of GSH depletion
No drug (control)
Ethacrynic acid (EA)
Buthionnine sulphoxamine (BSO)
Verapamil
ꢀ
/
2259
/
31
0
73
20
20
15
2
599
/9
339
/5
85
2219
151.339
102.679
57.679
/
21
1.78
39.5
58.93
76.93
CuNG
CuNG
CuNG
/
5.31
6.38
5.12
5
/
10
/
The values are means9
treated cases when compared with untreated control (p B
/
S.D. of four independent experiments. The values of GSH are significantly lowered in EA, BSO and CuNG (10 mg kgꢁ1
0.001).
)
/
References
[1] R.J.R. Sorenson (Ed.), Biology of Copper Complexes, Human
Press, Clifton, NJ, 1987.
[2] G.F. Hu, J. Cell Biochem. 69 (1986) 326ꢀ
[3] E. Parke, P. Bhattacharjee, R.M. Palmer, et al., Am. J. Clin.
Pathol. 137 (1988) 1121ꢀ1142.
[4] K.S. Raju, G. Alesandrii, M. Zinche, et al., J. Natl. Cancer Inst.
69 (1982) 1183ꢀ1188.
[5] M. Ziche, J. Jones, P.M. Gullino, J. Natl. Cancer Inst. 69 (1982)
475ꢀ482.
[6] D. Yoshida, Y. Ikeda, S. Nakazawa, J. Neuro Oncol. 17 (1993)
91ꢀ107.
[7] K.A. Engleka, T.J. Maciog, Biol. Chem. 267 (1994) 11307ꢀ
[8] Y. Kawamoto, S. Makiura, S. Sugihara, et al., Cancer Res. 33
(1973) 1129ꢀ1135.
[9] R.J.R. Sorenson (Ed.). Prog. Med. Chem. 26 (1989) 506ꢀ
[10] R.A. Di Silvestro, J.K. Greenson, Z. Liao, Proc. Soc. Exp. Biol.
Med. 201 (1992) 94ꢀ99.
[11] D. Dakternieks, T.S. Basu Baul, S. Dutta, E.R.T. Tiekink,
Organometallics 17 (1998) 3058ꢀ3062.
[12] T.S. Basu baul, S. Dutta, E. Rivarola, M. Scopelliti, S.K.
Choudhuri, Appl. Organometal. Chem. 15 (2001) 947ꢀ953.
[13] J. Sedlack, R.N. Lindsay, Anal. Biochem. 25 (1968) 192ꢀ205.
[14] O.H. Lowry, N.J. Rosenberg, A.L. Farr, R.J. Randal, J. Biol.
Chem. 196 (1951) 265ꢀ271.
/
335.
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/
/
/
/
11315.
/
/
7.
/
Fig. 3. EPR spectrum of CuNG.
/
taken and 4 mL of 0.4 M Tris buffer (pH 8.9) was
added. 5,5?-dithio-bis-(2-Nitrobenzoic acid) (DTNB)
solution (0.1 mL) was added and vortexed thoroughly.
/
/
Optical density (O.D.) was read (within 2ꢀ3 min after
/
/
[15] Procter I.M., Hathaway, B.J., Nichols P., J. Chem. Soc. A (1968)
addition of 0.1 mL 0.01 M DTNB) at 412 nm against a
reagent blank. Appropriate standards were taken and
protein was measured according to Lowry et al. [14].
The experiment was repeated for four times.
1678
[16] J.D. Frederick, B.C. Gilbert, R.O.C. Norman, M.C.R. Symons, J.
Chem. Soc. Parkin Trans. II (1983) 1763.
[17] S. Chandra, Sangeetika, Indian J. Chem. 41A (2002) 1629ꢀ
[18] J.H.T.M. Ploemen, B. Van Ommen, P.J. Van Bladeren, Biochem.
Pharmacol. 40 (1990) 1631ꢀ1635.
[19] J.A. Prezioso, G.B. Fitzgerald, M.M. Wick, J. Invest. Darmatol.
99 (1992) 289ꢀ293.
[20] F.P. La Creta, J.M. Brennman, T.C. Hamilton, R.F. Ozols, P.J.
O’Dwyer, Drug Metab Disp. 22 (1994) 835ꢀ842.
[21] L.I. McLellian, C.R. Wolf, Drug Resist. Update 2 (1999) 153ꢀ
164.
/33.
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Acknowledgements
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This investigation received financial support from
Indian Council of Medical Research (ICMR), New
Delhi, no. 5/13/17/2001-NCD-III; ICMR provides se-
nior research fellowship to one of the authors (G.S.P.).
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