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Synthesis X-ray powder structure analysis and biological properties of a mononuclear Cu(II) complex of N-2-hydroxyhippuric acid.

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Full Paper
Received: 15 June 2009
Revised: 12 September 2009
Accepted: 12 September 2009
Published online in Wiley Interscience: 21 October 2009
( DOI 10.1002/aoc.1565
Synthesis, X-ray powder structure analysis
and biological properties of a mononuclear
Cu(II) complex of N-2-hydroxyhippuric acid
Soumya Basua , Basab Chattopadhyayb , A. Gangulya , P. Chakrabortya ,
P. Roy Chowdhuryc , S. Samantac , M. Mukherjeeb, A. K. Mukherjeed
and S. K. Choudhuria∗
A mononuclear copper (II) complex of N-2-hydroxyhippuric acid (2HHA), [Cu(HA)(H2 O)2 ], has been synthesized and characterized
by spectroscopic and X-ray powder diffraction studies. Crystal structure of [Cu(HA)(H2 O)2 ] reveals a distorted square-pyramidal
geometry around the metal center. The crystal packing in the complex exhibits a three-dimensional framework formed by
intermolecular O–; H· · ·O and C–H· · ·O hydrogen bonds. Toxicity and antitumor properties of the complex have been studied
in vivo. The complex, capable of depleting glutathione (GSH) at nontoxic doses, may be utilized to sensitize drug-resistant cells
c 2009 John Wiley & Sons, Ltd.
where resistance is due to an elevated level of GSH. Copyright Supporting information may be found in the online version of this article.
Keywords: 2-hydroxy hippuric acid (2HHA); copper (II) 2-hydroxy hippurate dehydrate [Cu(HA)(H2 O)2 ](CuHA); BSO; Ehrlich ascites
carcinoma; powder diffraction structure analysis
Appl. Organometal. Chem. 2009 , 23, 527–534
Correspondence to: S. K. Choudhuri, Chittaranjan National Cancer Institute,
IVCCC, 37 S P Mukherjee Road, Calcutta 700 026, India.
a Department of In-vitro Carcinogenesis and Cellular Chemotherapy (IVCCC),
Chittaranjan National CancerInstitute(CNCI),37S.P.MukherjeeRoad,Calcutta700026, India
b Department of Solid State Physics, Indian Association for the Cultivation of
Science, Jadavpur, Kolkata-700032, India
c Chembiotek, Salt lake, Sector-V, Calcutta-700091, India
d Department of Physics, Jadavpur University, Jadavpur, Kolkata-700032, India
c 2009 John Wiley & Sons, Ltd.
Copyright 527
Copper is an essential trace element required for survival of all
organisms from bacterial cells to human being.[1] The ability of
copper ion to exist in oxidized Cu(II) or reduced Cu(I) states
facilitates its diverse redox chemistry including its role as a catalytic
cofactor for proteins.[2] The metallic copper is reported to induce
cancer and can also be used in cancer treatment.[3,4] Copper
administration suppresses rat hepatoma induced by chemical
carcinogens.[5] Several copper–Schiff base complexes have been
used widely in cancer therapy and these complexes cause
tumor cells to redifferentiate into normal cells.[6 – 8] Previously
we reported a copper complex of N-(2-hydroxy acetophenone)
glycine (NHAG) that can deplete glutathione (GSH) and glutathione
S-transferase (GST), inhibit multidrug resistance protein 1 (MRP1),
P-glycoprotein (P-gp) and overcome drug resistance.[9,10] Copper
N-(2-hydroxyacetophenone) glycinate (CuNG) can also increase
reactive oxygen species (ROS) generation, reduce MRP1 expression
in EAC/Dox cells, modulate superoxide dismutase (SOD), catalase
(CAT) and glutathione peroxidase (GPx) in different organs and
thereby reduce oxidative stress [11] . It has also been pointed out
by us that treatment of CuNG could resolve drug-resistant cancers
through induction of apoptogenic cytokines, such as IFN-γ and/or
tumor necrosis factor-α (TNF-α). IFN-γ and TNF-α released from
splenic mononuclear cells (SPMC) or patient peripheral blood
mononuclear cells (PBMC) can reduce the number of T-regulatory
marker bearing (T-reg) cells, while increasing infiltration of IFN-γ
producing T cells in the ascitic tumor size.[12]
2-Hydroxyhippuric acid (2HHA), Fig. 1, a glycine derivative
having similar chemical structure to NHAG, has been found in urine
of healthy human and is formed from tryptophan by bacterial
action in the colon. The isolation of 2HHA as a drug binding
inhibitor in uremia and its spectroscopic characterization and
coordination behavior have been reported in the literature.[13 – 16]
In continuation to our ongoing program of synthesis, characterization and biological activity of copper complexes of amino
acid derivatives,[9 – 12,17] the title compound has been synthesized.
Structural study of these complexes is an important step towards
understanding their mechanism of action in biological systems
that may provide useful information for designing ‘tailor made’
molecules with enhanced antitumor activity.
Although single crystal X-ray diffraction is undoubtedly the
most widely used technique for elucidating the structure of
organic compounds and metal chelates, an intrinsic limitation
of this technique is the requirement to prepare single crystals
of appropriate size and quality, which are not always met for
all compounds of interest. In such circumstances, X-ray powder
diffraction can be used as an alternative route for structural
analysis. Recent developments in the direct space methodologies
as implemented in FOX,[18] DASH[19] and EAGER [20] have shown
S. Basu et al.
N 4.90; O 32.47; Cu 21.91%. UV–vis (λmax , nm): 248. IR (KBr)
cm−1 : 3401(O–H), 3343(N–H), 1609[(COO− )as ], 1314(OCO). 1 H
NMR (300 MHz, DMSO-d6): δ 3.32(2H s, CH2 ), aromatic protons at
7.5, 7.4, 7.8-7.5, 8.4.
X-ray Crystallography
Figure 1. Chemical diagram of 2-hydroxyhippuric acid (2HHA).
considerable promise for structure solution from X-ray powder
diffraction data and the crystal structures of several molecular
compounds and metal–organic complexes have been determined
following the direct space methodologies.[21,22]
The present work describes the synthesis, spectroscopic
characterization and crystal structure analysis using X-ray powder
diffraction data of title Cu (II) complex CuHA, [Cu(HA)(H2 O)2 ]
of a glycine derivative, along with its in vivo antitumor activity,
hematological toxicity and glutathione (GSH) depletion property
in cancer therapy.
2-Hydroxyhippuric acid was purchased from ACROS; copper
sulfate, dimethyl sulfoxide (DMSO) were purchased from Aldrich,
NY, USA. Reduced glutathione (GSH) was purchased from Sigma
Chemical Company, St Louis, MO, USA. Other chemicals used were
of highest purity available.
Physical Measurements
Elemental analysis (C, H, N) was performed using a PerkinElmer 2400 Series II elemental analyzer. The estimation of Cu
was carried out in the clear supernatant in a flame atomic
absorption spectrophotometer (AAS) (Varian Spectra 200 FS,
hollow cathode lamp, flame type: air acetylene; replicate 3;
wavelength 324.8 nm). The UV–vis spectra (800–200 nm) were
recorded on a Shimadzu UV 160 A instrument. IR spectra
(4500–500 cm−1 ) were obtained (as KBr pellets) with the PerkinElmer RX-1-FTIR spectrophotometer. 1 H NMR spectra were
recorded on a Bruker ACF 300 spectrometer with d6-DMSO as
the solvent with tetramethylsilane as the reference.
X-ray powder diffraction data were recorded on a Bruker D8
Advance powder diffractometer using CuKα radiation (λ = 1.5418
Å). The diffraction pattern was scanned with a step size of 0.01◦ (2θ )
and counting time 40 s per step over an angular range 6.0–78.0◦
(2θ ) using the Bragg–Brentano geometry. The powder diffraction
pattern was indexed using the program TREOR[23] to a monoclinic
cell with a = 13.24, b = 9.07, c = 9.61Å, β = 108.3◦ [M
(20) = 49, F (20) = 101]. Statistical analysis of powder data using
the program EXPO2004[24] indicated P21 /c as the most probable
space group. To assess the choice of unit cell and space group, and
to estimate the shape and width of Bragg reflections along with
the instrumental shifts, full pattern decomposition was performed
with the program FOX[18] following the Le Bail algorithm[25] using a
pseudo-voigt[26] profile function, which converged to Rp = 0.0359
and Rwp = 0.0397, respectively. The structure was solved by global
optimization of structural model in direct space using the program
FOX[18] operating in the parallel tempering mode. Lattice and
profile parameters, zero-point and interpolated background calculated from a previous powder-pattern decomposition based on
the Le Bail algorithm, were introduced into the program FOX. The
molecular geometry used as input for structure solution with the
program FOX was optimized a priori by the energy minimization
procedure as incorporated in the MOPAC 5.0 program package.[27]
Rietveld refinement was carried out using the program GSAS[28]
with soft constraints on bond lengths and bond angles; planar
restraint were applied on the phenyl ring. The background was
described by the shifted Chebyshev function of first kind with
36 points regularly distributed over the entire 2θ range. A fixed
isotropic displacement parameter of 0.04 Å 2 for all nonhydrogen
atoms was maintained. Hydrogen atoms were placed in calculated
positions with Biso = 0.06 Å 2 , and C–H, N–H and O–H distances
restrained. Final Rietveld refinement converged to Rp = 0.0515
and Rwp = 0.0741 with excellent agreement between the
observed and the calculated patterns (Fig. 2). Relevant crystallographic data and refinement parameters are summarized in
Table 1. A molecular view of the compound [Cu(HA)(H2 O)2 ] with
the atom numbering scheme is shown in Fig. 3.
Synthesis of Copper Complex CuHA
CuSO4 .5H2 O (1.25g) was dissolved in 5 ml of deionised water and
2HHA (1.0 g) was dissolved in 1 M NaOH (15 ml) solution kept at
60 ◦ C. The alkaline solution of 2HHA (colorless) was slowly added
to blue-colored CuSO4 solution at room temperature (25 ◦ C) with
constant magnetic stirring. The mixture was further stirred for 1 h
at 45–50 ◦ C until the color of the mixture turned light green. The
mixture was cooled to room temperature and allowed to settle for
half an hour. The green precipitate was separated out by filtration.
The compound was dried in vacuuo and recrystallized from DMSO
to obtain CuHA as fine crystalline powder.
Yield 65%, m.p. 202–204 ◦ C. Anal. calcd for CuC9 H11 O6 N: C 36.93;
H 3.79; N 4.78; O 32.79; Cu 21.71%. Found: C 37.40; H 3.32;
Biological materials
All animals used in the present study were collected from our
animal colony. Ehrlich ascites carcinoma (EAC) was maintained as
an ascitic tumor in female Swiss albino mice weighing 18–20 g
(6–8 weeks old). The experimental protocols described herein
were approved by the Institutional Animal Ethics Committee
of Chittaranjan National Cancer Research Institute, Kolkata,
in accordance with the ethical guidelines laid down by the
Committee for the purpose of Control and Supervision of
Experiments on Animals (CPCSEA) by the Ministry of Social Justice
and Empowerment, Government of India. For isolation of bone
marrow and spleen, the animals were euthanized by overdosing
with sodium thiopentone (100 mg kg−1 body weight).
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2009, 23, 527–534
Mononuclear Cu(II) complex of N-2-hydroxyhippuric acid
Figure 2. Final Rietveld plot for [Cu(HA)(H2 O)2 ]. Brown crosses: observed pattern, black curve: calculated pattern, blue curve: difference curve.
Table 1. Crystal data and Rietveld refinement parameters for CuHA
Empirical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
Volume (V)
Density (calculated)
2θ range for data collection
Step size
No. of variable parameters
No. of background points refined
RF 2
CuC9 H11 NO6
293 K
P21 /c
a = 13.2547(24) Å
b = 9.0835(10) Å
c = 9.6207(16) Å
β = 108.27(2)◦
1100.0(4) Å 3
1.768 g cm−3
0.01◦ (2θ)
Study of in Vivo Toxicity
comparison to the control group was recorded. The experiment
was repeated three times and the results are presented in Fig. 4.
Effect of CuHA on survival of animals
Mean survival time after CuHA administration
Eighty healthy female Swiss albino mice, 6–8 weeks of age, were
divided into eight groups with one control group and seven
drug treated groups (10 mice in each group). Different doses
of CuHA (5, 10, 20, 30, 40, 50 and 60 mg kg−1 ) dissolved in
DMSO–DDW (1 : 1) solution were injected i.p. to seven groups
of healthy Swiss albino mice. The control group was injected i.p.
with the vehicle DMSO–DDW (1 : 1). Animals were observed over
a period of 72 h. The experiment was repeated three times, and
the average percentage of animals surviving against CuHA doses
along with the IC50 value (IC50 is the drug concentration that
inhibits 50% growth) is shown in Fig. 5.
c 2009 John Wiley & Sons, Ltd.
Sixty healthy female Swiss albino mice, 6–8 weeks of age, were
divided into six groups with one control and five drug-treated
groups (10 mice in each group). Various doses of CuHA (5, 10, 20, 30
and 40 mg kg−1 ) dissolved in dimethyl sulfoxide (DMSO)–double
distilled water (DDW) (1 : 1) were injected intraperitoneally (i.p.)
to five groups of Swiss albino mice. A DMSO–DDW autoclaved
solution (vehicle) was also injected i.p. to the control group of mice.
Life monitoring was restricted to daily body weight measurement,
recording time of death. Animals were observed over a period of
42 days. Mean survival time (MST) of the treated group of mice in
Appl. Organometal. Chem. 2009, 23, 527–534
Figure 3. A molecular view of [Cu(HA)(H2 O)2 ] with the atom submerging
S. Basu et al.
Figure 4. Effect of CuHA on Mean Survival Time (MST) of Swiss Albino Mice.
spleen was removed aseptically and a small amount of PBS was
injected into it; the spleen was rubbed against the fine wire mesh
of the tissue grinder. The cell suspension formed was spun at
1000–1500 rpm for 5–10 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 four times.
Effect of CuHA on bone marrow
CuHA (5 mg kg−1 ) in DMSO–DDW was injected into 10 female
Swiss mice. Ten untreated animals injected with vehicle were kept
as controls.
Figure 5. Effect of CuHA on survival of animals.
Effect of CuHA on blood
CuHA (1 mg) was dissolved in 1 ml DMSO–DDW and the solution
(0.1 ml) was injected i.p. to female Swiss albino mice (5 mg kg−1 )
(number of animals, n = 10). Ten animals injected with 100 µl
DMSO-DDW were kept as untreated controls. Blood was collected
from normal and also from treated mice (n = 10) after different
time intervals of CuHA injection. Blood was obtained via closed
cardiac puncture by means of a 22-gauge hypodermic needle and
a subxiphoid approach. Blood from each group (CuHA-treated and
untreated) was pooled into separate glass tubes and treated with
anticoagulant (heparin). Normal and differential blood count was
measured for the treated and normal mice. The experiment was
repeated four times.
Effect of CuHA on spleen
CuHA (5 and 10 mg kg−1 ) in DMSO-DDW was injected into female
Swiss albino mice divided into two groups, each group containing
10 animals. Ten untreated animals injected with vehicle were kept
as control.
Preparation of spleen cell suspension
Normal and CuHA-treated female Swiss mice were euthanized
and 70% alcohol was sprayed onto the abdominal region. The
Separation of bone marrow cells
Normal and CuHA treated mice were euthanized and the femur
bones were 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 ◦ C. Cells were counted under microscope for
treated and untreated animals. The experiment was repeated four
Study of Antitumor Property of CuHA in Vivo
Fifty-five female Swiss albino mice of 6–8 weeks age were divided
into six groups, one control group with five mice and five drugtreated groups with 10 mice in each group. All mice were kept
in plastic cages under standard conditions of light, temperature
and humidity. Food was given in the form of standard pellets
and water ad libitum. Mice were acclimatized for one week before
EAC cells (1 × 106 ) were injected i.p. into all mice on day
one. On day 2, different doses of CuHA dissolved in DMSO and
diluted in deionized and autoclaved water were injected i.p. to
mice of various groups. Only vehicle was administered to the
control group. Life monitoring was restricted to daily body weight
measurement and recording the time of death. Animals were
observed over a period of 60 days. Cell yield, ascites volume,
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2009, 23, 527–534
Mononuclear Cu(II) complex of N-2-hydroxyhippuric acid
Table 2. Hydrogen bond geometry (Å, deg) for CuHA
D–H· · ·A
O6–H6b· · ·O4
C8–H8a· · ·O2
C4–H4· · ·O5
C8–H8b· · ·O4
d (D–H)
d (H· · ·A)
d (D· · ·A)
packed cell volume, MST and change in the life span of the treated
mice in comparison to control (T/C value) were recorded. The
experiment was repeated four times.
GSH-depleting Properties of CuHA in Vivo
Cells (1 × 106 ) were injected i.p. into female Swiss mice of
6–8 weeks age. On the second day, 1 mg kg−1 CuHA was injected
to mice. On the twelfth day animals were anesthetized, killed and
EAC cells were collected. Cells (1 × 106 ) washed in PBS twice were
homogenized and divided into two equal amounts for measuring
GSH and protein. Ethacrynic acid (EA), buthionine sulfoxamine
(BSO) and verapamil were also injected in similar manner and
cells were collected. Normal cells were taken from EAC-bearing
mice that had not undergone any drug treatment. Experiments for
each drug-treated and control group were repeated four times.
GSH and protein were measured by the methods of Sedlack and
Lindsay[29] and Lowry et al.,[30] respectively.
Results and Discussion
Spectroscopic Properties
Appl. Organometal. Chem. 2009, 23, 527–534
x, −y + 1/2, z − 1/2
x, −y + 1/2, z + 1/2
1 − x, −y, −z
−x, −y, −z
copper complex, the methylene protons are not adjacent to the
copper ion and are detected at 3.32 ppm. This is consistent with
the earlier reports,[17,38] where methylene proton signals appear
at around 3.2–3.4 ppm. The aromatic protons in the ligand and
Cu-complex appear at 7.0, 7.8–7.5, 7.8 and 7.9 ppm[13] and 7.5,
7.4, 7.8–7.5 and 8.4 ppm, respectively. The signal due to water
molecules appears at 4.0 ppm in the complex.
Crystal Structure of CuHA
The coordination geometry around the Cu(II) atom bonded to
three donor atoms (N1, O1 and O2) of hydroxy hippuric acid ligand
and two oxygen (water) atoms (O5 and O6) can be best described
as distorted square pyramidal with the basal plane defined by N1,
O1, O2 and O5 atoms (r.m.s deviation 0.129 Å); the other aqua O
atom (O6) occupies the apical site. The Cu atom is displaced by
0.171(6) Å towards O6 from the basal plane. Similar distortion in
metal coordination geometry, a consequence of steric hindrance
induced by the tridentate HHA ligand, has been observed in
other Cu(II)–Schiff base complexes, which are reported to act as
antiradical, antimicrobial agents, and as inhibitors of enzymatic
systems.[39 – 42] The equatorial Cu–N [2.069(6) Å] and Cu–O
[1.925(8)–1.972(1) Å] bond distances in the basal plane of the
pyramid are comparable to the corresponding values reported
for related structures in the Cambridge Structural Database
(version 5.30, November CSD 2008 release). The lengthening of
the Cu–O (axial) bond length [2.058(9) Å] in comparison to the
Cu–O (equatorial) distances is also consistent with similar Cu(II)
complexes exhibiting square-pyramidal geometry.[43 – 46]
The crystal packing is stabilized by a combination of intermolecular O–H· · ·O and C–H· · ·O hydrogen bonds (Table 2). A
pair of C8–H8B· · ·O4 hydrogen bonds between molecules related
by inversion and translation generates a centrosymmetric R2 2 (10)
dimeric ring (M) centered at (1/2, 0, 0). Another type of centrosymmetric R2 2 (14) dimeric ring(N) is formed by a pair of intermolecular
C4–H4· · ·O5 hydrogen bonds. The R2 2 (10) and R2 2 (14) rings are alternately linked into infinite one-dimensional MNMN· · ·polymeric
chain propagating along the [100] direction (Fig. 6, inset II). A similar polymeric chain running along the [001] direction is generated
by intermolecular O6–H(O6)· · ·O4 and C8–H(C8)· · ·O2 hydrogen
bonds (Fig. 6, inset III). The combination of [100] and [001] chains
results in a three-dimensional framework structure (Fig. 6, inset I).
Biological Properties of CuHA
The mean survival time of healthy Swiss Albino mice after
administration of CuHA (5 mg kg−1 ) was found to be 35 days. The
survivability gradually decreased with administration of higher
doses of CuHA [Fig. 5]. The IC50 value was found to be 30 mg
kg−1 after administration of CuHA. The results in Table 3 indicate
that CuHA up to a dose of 10 mg kg−1 has no toxic effect on
c 2009 John Wiley & Sons, Ltd.
The UV absorption band for complex (DMSO) was obtained at
248 nm, whereas for the ligand 2HHA the corresponding peaks
were obtained at 237 and 301 nm, respectively. The shift in the UV
peak from 301 nm in the ligand to 248 nm in the metal complex is
probably due to π –π ∗ transition.[13,17]
The peak at 3370 cm−1 in the IR spectrum of 2HHA can be
assigned to ν(O–H) stretching vibration, which is shifted to
3401 cm−1 in CuHA complex. The sharp peak at 3331 cm−1 in
the ligand due to N–H stretching is shifted in the complex to
3343 cm−1 .[31] In the complex, the disappearance of peak at
1753 cm−1 observed in 2HHA indicates that the C O group of
2HHA is coordinated with Cu2+ ion. The carbonyl stretching
frequency of the amide group[30,32] remains unchanged on
complexation and appears at 1608 cm−1 in both ligand and
complex. The sharp band at 1354 cm−1 assignable to ν (OCO)
symmetric stretching of 2HHA ligand has been shifted to
1314 cm−1 in the CuHA complex, indicating coordination of Cuatom through the COO− group.[28]
The 1 H NMR spectrum of 2HHA ligand has been reported by
Lichtenwalner et al.[13] The glycine methylene protons appears as a
doublet at 3.32 ppm.[33,34] In the title complex, the –CH2 –protons
also appear at 3.32 ppm as a doublet. The magnetic moment
associated with the unpaired electron on copper ion exerts a
‘non-negligible effect’ on the NMR parameters of nearby nuclei
through hyperfine coupling. The slow electronic relaxation of
bivalent Cu ions usually results in large line widths and poor
resolution of spectra and consequently the protons in close
proximity of copper ions experience a strong paramagnetic effect
in comparison to the protons in periphery.[35 – 37] In the present
[Symmetry transformation]
S. Basu et al.
Figure 6. I- Molecular Packing in [Cu(HA)(H2 O)2 ] viewed along c-axis; II- Infinite one-dimensional polymeric chain formed by C—H· · ·O hydrogen bonds
propagating along [100] direction; III- Infinite one-dimensional polymeric chain formed by C—H· · ·O and O—H· · ·O hydrogen bonds propagating along
[001] direction.
Table 3. Effect of CuHA on hematological parameters of female albino Swiss mice after 24 hr treatment
Dose (mg kg−1 )
(Hb)(g dl−1 )
WBC (× 103 µl−1 )
RBC (× 103 µl−1 )
Platelets (× 103 µl−1 )
Lymphocyte (%)
13.9 ± 1.5
13.9 ± 2.2
13.7 ± 3.1
5.2 ± 0.7
10.7 ± 3.2
10.2 ± 2.8
9.3 ± 2.5
9.0 ± 1.7
8.9 ± 1.6
452.1 ± 11.3
1020.0 ± 32.0
1059.0 ± 43.0
90.1 ± 6.1
85.6±. 6.1
81.6 ± 4.5
Data are means ± SD of four independent experiments.
Table 4. Effect of CuHA on spleen and bone marrow of female Swiss
albino mice
(mg kg−1 )
Spleen cell
(×106 per ml)
Bone marrow
(×106 per ml)
104.0 ± 22.1
105.00 ± 17.20
101.00 ± 19.10
72.50 ± 6.10
46.00 ± 4.10
3.7 ± 0.3
3.60 ± 0.30
3.50 ± 0.20
4.50 ± 0.30
1.60 ± 0.01
The data are means ± SD of four independent experiments.
hematological parameters (p > 0.001) when compared with the
untreated control.
The effect of the CuHA on spleen and bone marrow is presented
in Table 4. CuHA at a dose of 5 mg kg−1 is nontoxic to spleen
and bone marrow when compared with the untreated control
(p < 0.001). There is no change in the spleen or bone marrow cells
after one or 10 days’ duration of administration of CuHA at a dose
of 5 mg kg−1 . However, the compound at a dose of 10 mg kg−1
shows toxicity for spleen and bone marrow for one and 10 days’
duration of administration. There is a significant decrease in cell
numbers in spleen in one day (30.29%) and in 10 days (55.77%).
In the case of bone marrow, initially there is an increase in cell
numbers (21.62%), but for longer duration the bone marrow cells
decrease to 56.76%, indicating a toxic effect of CuHA on spleen
and bone marrow at a dose of 10 mg kg−1 .
The antitumor activity of CuHA and its effect on GSH
level are shown in Tables 5 and 6, respectively. CuHA at a
nontoxic dose of 5 mg kg−1 cannot increase the life span
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2009, 23, 527–534
Mononuclear Cu(II) complex of N-2-hydroxyhippuric acid
Table 5. The antitumor property of CuHA
CuHA (mg kg−1 )
Cells in whole ascites fluid × 106
Total volume (tumor cells + ascites fluid), ml
Total packed cell volume, ml)
Mean survival time (MST), days
(2 mg kg−1 )
168.0 ± 9.6
7.7 ± 1.4
4.9 ± 0.6
161.0 ± 8.2
7.1 ± 1.4
4.6 ± 1.1
149.0 ± 6.8
6.1 ± 1.1
4.1 ± 0.8
137.0 ± 1.8
5.3 ± 0.9
3.7 ± 0.7
72 ± 7.7
2.5 ± 1.3
1.5 ± 0.7
The data are means ± SD of four independent experiments.
Table 6. Depletion of Gluathione (GSH) by CuHA
Control (no drug treatment)
Ethacrynic acid (EA)
Buthionine sulfoxamine (BSO)
(mg kg−1 )
GSH (ng µg−1 of protein)
Percentage of
GSH depletion
240.0 ± 27.0
65.0 ± 7.0
39.0 ± 6.2
162.0 ± 5.7
72.0 ± 2.5
55.0 ± 27.0
The data are means ± SD of four independent experiments.
of EAC cell-bearing mice when compared with untreated
control (p < 0.001). The doses of 10 and 15 mg kg−1 are
toxic and cause a decrease in the life span of cancer-bearing
The GSH values are significantly lowered in EA, BSO and
CuHA treated cases when compared with untreated control (p < 0.001). The overall nontoxic nature of CuHA in
hematological, spleen and bone marrow parameters at a
dose of 5 mg kg−1 body weight in vivo and its GSH depleting property (Fig. 7) warrants further study with this complex, which may have potentiality as a resistance modifying agent in combination with other anticancer drugs where
resistance occurs due to elevated level of GSH in the
Figure 7. GSH depletion property of CuHA at different doses.
CuHA is toxic at higher doses and depletes GSH even in
nontoxic doses. It has been reported that a number of drug
resistant cells has higher level of GSH compared with drugsensitive cells and modulation of cellular GSH homeostasis
sensitizes drug resistant cancer cells to a wide range of
chemotherapeutic drugs.[26] The nontoxic dose of CuHA may
be utilized to deplete GSH in a number of drug-resistant cell lines,
thereby overcoming drug resistance caused due to elevated GSH
Appl. Organometal. Chem. 2009, 23, 527–534
Supporting information
Supporting information may be found in the online version of
this article. Crystallographic data for the structure reported in this
article have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number CCDC 733 843.
Copies of the data can be obtained free of charge on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223336033; e-mail: (
c 2009 John Wiley & Sons, Ltd.
This investigation received financial support from Indian Council
of Medical Research (ICMR), New Delhi, no. 5/13/18/2007-NCDIII. Financial support from the University Grants Commission,
New Delhi, and the Department of Science and Technology,
Government of India, through DRS (SAP-I) and FIST programs
to the Department of Physics, Jadavpur University, for purchasing the X-ray powder diffractometer is gratefully acknowledged.
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acid, powder, structure, complex, synthesis, properties, hydroxyhippuric, biological, analysis, mononuclear, ray
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