Synthesis, structure, electrochemistry and spectral characterization of (d-glucopyranose)-4-phenylthiosemicarbazide metal complexes and their antitumor activity against Ehrlich Ascites Carcinoma in Swiss albino mice

Article abstract of DOI:10.1016/j.ejmech.2009.09.031

The novel glycosyl saccharide derivative, (d-glucopyranose)-4-phenylthiosemicarbazide (LH) and its complexes, with cobalt(II), nickel(II), copper(II) and zinc(II) were synthesized, characterised and tested for cytotoxic effects. The copper complex, [CuLCl

Full text of DOI:10.1016/j.ejmech.2009.09.031

European Journal of Medicinal Chemistry 45 (2010) 106–113
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structure, electrochemistry and spectral characterization
of ( -glucopyranose)-4-phenylthiosemicarbazide metal complexes and their
D
antitumor activity against Ehrlich Ascites Carcinoma in Swiss albino miceq
M.P. Sathisha a, Srinivasa Budagumpi a, Naveen V. Kulkarni a, Gurunath S. Kurdekar a,
,
b
V.K. Revankar a , K.S.R. Pai
*
a Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka, India
b Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576 104, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel glycosyl saccharide derivative, (D-glucopyranose)-4-phenylthiosemicarbazide (LH) and its
Received 24 February 2009
Received in revised form
9 September 2009
Accepted 17 September 2009
Available online 28 September 2009
complexes, with cobalt(II), nickel(II), copper(II) and zinc(II) were synthesized, characterised and tested
for cytotoxic effects. The copper complex, [CuLCl] inhibited Ehrlich Ascites Carcinoma (EAC) induced
cancer cell lines in Swiss albino mice at LC50 ¼ 1.94 ꢀ 10ꢁ8 (LC50 ¼ 2.76 ꢀ 10ꢁ8 for cisplatin) and so
distinctly better than free ligand and other complexes.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Transition metal complexes
Thiosemicarbazide
Antitumor activity
Cytotoxicity
1. Introduction
complexation of carbohydrates to metals is one of the main
objectives of carbohydrate coordination chemistry.
Biosynthesis of carbohydrates is a basic process of life and
quantitatively the most important one. Monosaccharides are
fundamental biomolecules in that they are the building blocks of
polysaccharides. They are the constitutional parts of complex lipids
(glycolipids) and proteins (glycoproteins). They are also building
blocks of nucleotides and, hence, of nucleic acids and of the
chemical ADP/ATP energy storage system. Carbohydrates are of
primary importance as energy sources for living organisms. Due to
the properties inherent to this class of molecules, carbohydrates
have been utilized to prepare bioactive materials [1], better-tar-
geted drugs [2], as well as for the functionalization of hydrophobic
materials [3]. Metal–carbohydrate interactions are also of signifi-
cant interest in bioinorganic chemistry [4–6]. Thus, carbohydrates
exert a wide range of functions in living organisms, and due to the
wide distribution of metals and their complex functions for all
forms of life, metal–carbohydrate interactions are a key for
understanding bioinorganic chemistry, and the study of
Present investigation focuses on the design and synthesis of
new saccharide derivative type of chelators by the introduction of
thiosemicarbazide as anchoring group, which can provide well-
defined binding environment as well as increase the stability of the
resultant metal complexes over the simple saccharide complexes
itself. In present study the anchoring groups introduced via
N-glycosylic linkage at the anomeric carbon at C-1 position of the
saccharide moiety. Thiosemicarbazide in the present investigation
are used hoping that the toxophoric functional group –C]S will be
away from the coordinating site so that these functional groups
could provide ‘point of attachment’, a system which mimics certain
classes of biological systems and in addition such groups could be
toxic to microbes when used as drugs. A potential benefit of
utilizing this approach is that the carbohydrate can remain
pendant, thereby being freely available to interact with carbohy-
drate transport and metabolic pathways in the body. From these
metal-binding properties of
D-glucose, it is hoped that such would
aid in the understanding of the structural chemistry of metal ions
interacting with saccharides, as an actual biological system, and
thereby in the interpretation of some particular biological
processes.
q
Dedicated in memory of Late Dr (Mrs) Geeta M. Kulkarni (1959–2009).
* Corresponding author. Tel.: þ91 836 2215286.
E-mail address: vkrevankar@rediffmail.com (V.K. Revankar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.031

M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
107
2. Results and discussion
the region 1120–1080 cmꢁ1 due to C]S absorption, which is
shifted to lower frequency side after complexation, indicating the
coordination of sulphur atom. The band observed between 2920
2.1. Chemistry
and 2930 cmꢁ1 are assigned to
nCH indicating the presence of
4-Phenylthiosemicarbazide [7] was prepared following the
literature procedure and D-glucose was purchased from Qualigens.
The free saccharide was treated with 4-phenylthiosemicarbazide in
refluxing methanol in presence of a small amount of NH4Cl as
saccharide moieties in the product. The sharp absorption band in
the region 1640–1590 cmꢁ1 in the ligand and complexes is
assigned to NH deformation vibration. Absorption bands corre-
sponding to the anomeric properties of the N-glycosyl amines
were observed in the spectra. The strong bands observed in all the
complexes viz., 753–751 and 695–690 cmꢁ1, indicate the presence
shown in Scheme 1, to get
(D-glucopyranose)-4-phenyl-
thiosemicarbazide (LH). Cobalt(II), Nickel(II), Copper(II) and Zinc(II)
complexes of the ligand were prepared. The reaction sequences are
outlined in Scheme 2. The characterization of the ligand and
complexes cover both in the solution as well as in the solid state
using a variety of analytical and spectral techniques. The complexes
are mononuclear with M:L ratio being 1:1. All the complexes were
found to be soluble in ethanol, methanol, chloroform, acetone,
dimethylsulphoxide, dimethylformamide and were found to be
non-electrolytic in nature. The growth of single crystals of these
complexes for X-ray studies is very difficult owing to their amor-
phous nature and we were unsuccessful in our attempts to do so.
The composition and coordination geometry of these complexes
have been established on the following experimental observations.
The molar conductance values in dimethylsulphoxide fall in the
expected range (20–31 mho cm2molꢁ1) of non-electrolytes [8]
indicating that chloride ions are inside the coordination sphere. The
complexes were analyzed for metal, carbon, hydrogen, nitrogen,
sulfur and chloride. The analytical data, conductivity and magnetic
moment of the complexes are given along with their synthetic
procedures.
of both
a and b anomers [10], which is in consistent with the
observations made by NMR studies.
2.3. 1H NMR spectra
The 1H NMR spectra of the ligand (LH) and the zinc complex
were recorded in DMSO-d6 and the assignments were made by
relative comparison. The comparison clearly demonstrates the
formation of metal-saccharide complexes.
As the glycosylation occurs at C-1 centre by condensation of the
saccharide with the 4-phenylthiosemicarbazide, the spectrum of
the N-glycoside was devoid of C-1-OH resonance which otherwise
present in the corresponding saccharide spectrum around
6.49–6.78 ppm. Signals corresponding to the OH and CH groups of
the saccharide moiety were identified in the region 2.0–3.7 ppm from
the spectrum of (D-glucopyranose)-4-phenylthiosemicarbazide. The
down field shift in the glycosylic –NH protonwas observed in the zinc
complex (9.2–9.8 ppm) compared with the ligand (observed at
8.9 ppm). The observed downfield shift of the glycosylic N–H in the
metal ion complex indicates the coordinationof glycosylic nitrogen to
the metal centre. It is obvious from the comparison of the chemical
shift values of saccharide–OH group of the ligand with corresponding
zinc complex shows that 2–OH group could have disappeared as this
is involved in the formation of a five membered chelate with the
metal ion after deprotonation. However, in the complex it is very
difficult to trace out the number of –OH groups [10,11]. In the ligand
and zinc complex, the C–1–H resonance appears as doublet at
2.2. FTIR spectra
Formation of the N-glycoside was observed by comparing
the FTIR spectrum of the ligand, with the spectra of the saccharide,
D-glucopyranose and the corresponding 4-phenylthiosemicarbazide.
When 4-phenylthiosemicarbazide reacts with the saccharide, the
band corresponding to the primary amine was absent, and that of
the secondary amine was shifted to higher frequency in the ligand
due to the formation of the N-glycoside. The spectra of complexes
are indicative of the cleavage of extensive intermolecular
hydrogen bonds existing in the solid state of the parent ligand
[9,10], thus resulting in a broad band 3410 ꢂ 10 cmꢁ1 region with
a low frequency component as a shoulder at 3290 ꢂ 10 cmꢁ1 upon
complex formation. The sharp bands at 3335 and 3444 cmꢁ1 were
observed in the copper complex due to secondary amine and
hydroxy groups of the ligand moiety. Broad band in the remaining
complexes in the region 3413–3550 cmꢁ1 clearly demonstrate the
presence of aqua molecules. Coordinated mode of aqua molecules
in case of nickel complex and lattice held water molecules in case
of cobalt and zinc complexes were confirmed by their thermal
studies. Ligand and all the complexes show a prominent band in
4.99 ppm corresponding to the presence of both
a and b anomers.
Correspondingly, two different signals were observed for the C-1-NH
(glycosyl) around 9.2–9.8 ppm indicating the presence of mixtures of
anomers [11].
2.4. Electronic spectra
The electronic spectra were recorded in DMSO solution and data
are given in the Table 1. The observation made by comparing the
infrared spectrum of the ligand with those of metal complexes it
was observed that the substantial energetic and charge distribu-
tional changes caused by complex formation. Hence it could be
presumed that such changes also appear in the spectra of
complexes in the ultra-violet and visible frequency range. The
OH
H
H2C
O
H
HO
H2N
HO
NH
OH
H
H
H
H
N
OH
H
H2C
H
N-Glycosidic
O
H
NH
S
NH
+
HO
Refluxing MeOH
NH4Cl
HO
H,OH
OH
S
NH
Scheme 1. Synthesis of (D-glucopyranose)-phenylthiosemicarbazide LH.

108
M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
HO
OH
H
H
CH2
H
H2C
H
H
H
O
O
OH
HO
OH
HO
H
H
HO
O
NH
S
N
H
CuCl2
HN
NH
Cu
Refluxing MeOH
Cl
S
HN
NH
Scheme 2. Synthesis of [CuLCl].
ligand exhibits absorption bands in UV-visible region around 274,
320 and 375 nm. The first band below 300 nm is assigned to
2.6. Circular dichroism studies (CD)
a ligand
p
/
p* transition. The band around 320 nm is assigned to
CDspectra ofligand and all the complexes were recorded in DMSO
in the region of 200–900 nm. Though the Cotton effect is not prom-
inent in ligand, they show a characteristic (þ) curve in the region
280–300 nm indicating the dextrorotatory nature having the ‘S’
configuration at the C-2 carbon [16]. The CD spectra of complexes
showed conspicuous Cotton effects in the d–d transition region and
strongly indicated the coordination of sugar moieties to the metal
center. Further the bimodal shape of peaks in complexes with (ꢁ)ve
peak (negative Cotton effect) in the region 600–610 nm and corre-
sponding (þ)ve peak (positive Cotton effect) in the region 700–750 nm
and the observation of negative CD couplet in the region 300 nm
running into vacuum UV (VUV) region indicates an ‘‘exiton
coupling’’ [17] probably arising due to a rapid equilibrium in which
both the anomeric form of the sugar moieties interact with the metal
centeralternatively. Almost sameCD patternwasobservedforall the
complexes. The spectral results indicate similarities in the configu-
ration around the metal centre in these complexes. The study
confirms that there seems to exist certain preferential orientations
for particular saccharides in their binding to metal ions Fig. 1.
n / p* transition associated with imine function of thio-
semicarbazide. Another band observed at 375 nm is assigned to
n / p* transition originating from the thioamide function [12] of
thiosemicarbazide. Comparison of the absorption spectrum of the
ligand with that of the metal complexes indicated the complex
formation. The band around 370 nm in ligand shifted to lower
wavelength by 20–30 nm in the metal complexes. In addition to
this, the metal complexes, except zinc complex, exhibit another
band in the range 415–474 nm is characteristic of d-d transitions.
However, the ratio of the intensities of all the bands slightly varies
in complexes [11]. Such intensity ratio analysis would be used in
establishing the binding of metal ions with the saccharide based
polymers and antibiotics.
2.5. Magnetic moment studies
The magnetic moment value for the cobalt(II) complex is found
to be 4.5 BM, which lie within the expected range for four-coordi-
nated tetrahedral cobalt(II) complexes. The observed room
temperature magnetic moment of Ni(II) complex is found to be
2.96 BM, which is consistent with high-spin octahedral geometry
[13]. The slightly higher than spin-only value (2.83 BM) of observed
moment may be due to mixing of upper states via spin orbit
coupling [14]. For the copper (II) complex, magnetic moment value
was found to be 1.93 BM, which lie appreciably above the spin-only
value of 1.73 BM for Cu(II) ion, indicating the presence of single
unpaired electron and suggesting the square planar structure for
the complex [15].
2.7. EPR study
The solid state EPR spectrum of the mononuclear copper(II)
complex was recorded in the X-band region at room temperature.
The EPR spectrum of the mononuclear copper(II) complex shows
two lines having g value at 2.11 and gt at 2.03. g value being less
jj
jj
than 2.3, witnesses more covalency in metal-ligand interaction
[18]. Further it is noteworthy that the relation g > gt > ge (2.0027)
jj
is typical of axially symmetric d9 CuII ion having one unpaired
electron in dx2ꢁy2 orbital [19].
OH
H
OH
H
H2C
H2C
H
H
O
O
HO
HO
HO
HO
H
H
O
NH
O
OH2
M
H
NH
S
H
nH2O
NH
M
NH
Cl
Cl
NH
S
OH2
NH
M = Co(II), Cu(II) and Zn(II)
n = 2 for Co(II) and nil for Cu(II) and Zn(II)
M = Ni(II)

M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
109
Table 1
system in DMSO (0.1 M tetrabutylammoniumperchlorate as sup-
porting electrolyte). No peaks corresponding to the oxidation/
reduction have been observed in the working potential range, as
the ligand was shown to be electrochemically inactive. The redox
properties of all the complexes were explored by cyclic voltam-
metry. The copper and cobalt complexes are shown to be redox
active. The voltammogram (Fig. 4) data are summarized in Table 2.
The copper complex undergoes one step one-electron reduction
process (CuII / CuI) in forward scan and one step one-electron
oxidation (CuI / CuII) process in reverse scan. The electrochemical
process is found to be quasi-reversible, as evidenced by (i) the peak-
Electronic spectral data of the compounds.
Compound
lmax (nm)
274, 320, 375
273, 327, 373, 619
270, 300, 373, 474
260, 327, 340, 421
268, 373, 378
LH
[CoLCl]$2H2O
[NiLCl$2H2O]
[CuLCl]
[ZnLCl]
2.8. FAB mass and thermal studies
The analyses of FAB mass spectra, without any ambiguity,
conclude the monomeric nature for all the complexes, which
parallels the results of elemental analyses. The FAB mass spectrum
of copper(II) complex (as a representative) shows the molecular ion
peak at m/z 427. The spectrum also shows some prominent peaks
due to molecular cations, various fragments of the complexes are
due to isotopic atoms like copper and chloride ions (Fig. 2).
Room temperature stability for all the complexes was revealed
by their thermogravimetric analysis. All the complexes show
a similar pattern of decomposition at higher temperature. All the
complexes except cobalt and nickel complexes show an initial
weight loss of 7.2% in the temperature range 240–270 ꢃC, which
corresponds to loss of coordinated chloride ion. On increasing the
temperature further, weight loss was observed up to 510 ꢃC. The
observed weight loss is attributed to the loss of ligand moiety in all
the complexes. No weight loss was observed beyond 510 ꢃC,
possibly due to the formation of stable metal oxides. The obser-
vation at the same temperature range for nickel complex shows
a weight loss of 16.2%, which corresponds to combined loss of
coordinated chloride ion along with two molecules of coordinated
water. In case of cobalt complex, the initial weight loss of 8.01% in
the temperature range 100–110 ꢃC corresponding to the loss of
lattice held water molecules. These observations further supports
the composition of Ni(II) and Co(II) complexes, which is fixed as
a result of elemental and FAB mass studies. The decomposition
pattern at these temperature range rules out the possibility of
lattice held water molecules (Fig. 3).
to-peak separation DEp is greater than 59 mV, (ii) the current ratios
ipa/ipc is less than 1, and (iii) Epc shifts negatively with increasing
scan rate [20]. Cobalt complex exhibits a two one-electron quasi-
reversible oxidation processes (CoII / CoIII, CoIII / CoIV), when
scanned in anodic direction, two cathodic peaks had followed
respective cathodic peak in reverse scan (CoIV / CoIII, CoIII / CoII).
The structural reorganizations accompanying a redox change
should affect essentially the kinetics of the heterogeneous electron-
transfer process. According to the Marcus theory, inner-sphere
rearrangements, enhancing the activation barrier of the electron-
transfer, slow down the rate of the process, so causing a departure
from electrochemical reversibility [21]. In the present series of
complexes the largest rearrangement is likely experienced by the
planar copper complex.
3. Biochemistry
3.1. Antitumor activity of complexes against Ehrlich Ascites
Carcinoma in Swiss Albino mice
The compounds were tested using the short term in vitro cyto-
toxicity towards EAC (Ehrlich Ascites Carcinoma) cells as a prelim-
inary screening technique of tryphan blue exclusion method (Cell
Viability Test) for their cytotoxic potential [22]. Results of the short
term in vitro cytotoxicity of the compounds are shown in Table 3.
These preliminary experiments were carried out mainly with five
different concentrations of the compounds. All the compounds
were found to be cytotoxic and produced 50% cell death at
a concentration of 19.1
dard (Cisplatin) showed 96% cell death. At 50
the copper(II) complex showed more than 85% cell death while
ligand and other complexes showed w70% cell death at 50 g/ml
concentration. All the compounds were found to have considerable
cytotoxicity in the cell viability test.
m
g/ml. At 50
m
g/ml concentration the stan-
2.9. Electrochemical studies
m
g/ml concentration
The redox activity of ligand and all the complexes were studied
in the range þ0.2 to ꢁ0.8 V versus the ferrocene-ferrocenium redox
m
The tumor inoculated control animals gained a substantial
weight by day-0. They gained a maximum weight of 19% by day-15.
Cisplatin administration (on 10th post inoculation day) signifi-
cantly (p < 0.05) reduced weight gain as compared to control on
day-15. The compounds significantly (p < 0.05) reduced the weight
gain on day-15 as compared to control (Table 4).
The effect of compounds on survival of tumor bearing mice is
shown in Table 5. Cisplatin significantly prolonged the mean
survival time (p < 0.05) with respect to its control. It shows
a significant increase in the percentage life span of animals
(ILS > 50). On the other hand, all the compounds significantly
prolonged the mean survival time. The influence of all the
compounds on %ILS was more than 25%. By convention, a 25%
increase in life span is considered as possible anticancer activity of
a test compound [23].
4. Conclusion
The present study demonstrates the binding nature of the newly
synthesized unprotected N-glycosyl saccharide derived from
Fig. 1. CD spectra of Ligand and [CuLCl].

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M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
Fig. 2. FAB mass spectrum of [CuLCl].
4-phenylthiosemicarbazide, with the Co(II), Ni(II), Cu(II) and Zn(II)
chlorides. Elemental analysis and molar conductance studies reveal
that, the complexes are mononuclear and are non-electrolytes. All
the trends observed with the 1H NMR chemical shifts and CD
measurements together indicate that the ligand is found to be
thereby in the interpretation of some particular biological
processes.
The compounds showed considerable cytotoxic activity in the
tryphan blue exclusion method. In the in vivo cancer model, the
compounds significantly (p < 0.05) reversed the tumor induced
changes in the parameters monitored, viz., percentage increase in
body weight, percentage increase in life span, tumor viable count
and haematological parameters (total and DLC of WBC, total RBC
and Hb). These effects were almost comparable to cisplatin. The
compounds however were found to have good effect in prolonging
the life span as compared to standard. These findings imply that the
compounds might be having some anticancer principles. Based on
the data of the present study, it is very difficult to suggest the
possible mechanism for the anticancer effects. The compounds
tested in present study have shown promising cytotoxic activity
when screened using the in vitro method and at the same time were
shown to have good activity when tested using the Ehrlich Ascites
Carcinoma model. Though it is very difficult to conclude anything at
this stage, it can be assumed that after testing against various other
a mixture of both
a and b anomers. The specific rotation of the
ligand is positive (dextrorotatory), which is consistent with their
corresponding anomeric assignments. The study of complexation
of ligand with different metal ions show binding of the glycosylic-
NH, C-2-OH group from one of the saccharide residue and –C]S
group of the thiosemicarbazide unit as revealed by the FTIR and
NMR studies. Therefore the ligand acts as monobasic, tridentate
‘SNO’ donor. All the above observations together allow structural
motif to be proposed for the binding of metal ions to the ligand.
Although M(II)-saccharide complexes exist at physiological pH, the
complexation is favored under alkaline conditions. From these
metal-binding properties of D-glucose, it is hoped that such would
aid in the understanding of the structural chemistry of metal ions
interacting with saccharides, as an actual biological system, and
Fig. 3. Thermograms of complexes.

M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
111
Table 3
Short term in vitro cytotoxicity of compounds towards EAC cells.
Compounds Percentage cell death at different
concentrations after 3 h
LC50 in M ꢀ 10ꢁ8
1
m
g/ml 5
mg/ml 10
m
g/ml 25
m
g/ml 50 mg/ml
Cisplatin
LH
[CoLCl]$2H2O 12
[NiLCl$2H2O] 14
[CuLCl]
[ZnLCl]
18
15
31
47
44
39
44
58
46
85
67
52
63
69
63
96
75
74
72
84
76
2.76
3.14
3.69
2.89
1.94
2.56
32
28
31
39
34
18
19
measurements of the complexes were recorded in nitrogen atmo-
sphere on Universal V2.4F TA Instrument keeping final temperature
at 800ꢃ C and heating rate was 10ꢃ C/min. The FAB mass spectra
were drawn from JEOL SX 102/DA-6000 mass spectrometer using
Argon/Xenon (6 kV, 10mA) as the FAB gas.
Fig. 4. Cyclic voltammogram of [CuLCl].
5.1. Chemistry
cancer models and at different doses these compounds may prove
to be safer drugs for tomorrow.
5.1.1. Synthesis of (
carbazide LH
D-glucopyranose)-4-phenylthiosemi-
D-Glucose (0.01 mol) is treated with 4-phenylthiosemicarbazide
5. Experimental protocols
(0.01 mol) in presence of a small amount of NH4Cl (0.004 mol) in
refluxing methanol. Reaction was monitored through TLC at a time
interval of 20 min. The reaction proceeds almost to completion
after about 1.5 h. Thus separated product was filtered off washed
with methanol and diethyl ether and dried under vacuum. Yield
w65%, Mp 153–156 ꢃC. Elemental analysis: Calculated (found) for
C13H19N3O5S: C, 47.41 (47.39); H, 5.77 (5.72); N, 12.76 (12.73); S,
All chemicals used were of reagent grade. Solvents were distilled
prior to use in the synthetic part. The metal content of the
complexes was estimated by using standard methods. All the
compounds were analyzed for carbon, hydrogen, nitrogen and
sulfur by Thermo quest elemental analyzer at STIC Cochin Univer-
sity of Science and Technology, Cochin. Magnetic susceptibility of
complexes was measured at room temperature on a Faraday
balance using Hg[Co(SCN)4] as a calibrant. Electronic spectra were
recorded using Varian Cary 50 Bio UV-visible spectrophotometer in
DMSO. The IR spectra of ligand and its complexes were recorded as
KBr pellets in the region 4000–400 cmꢁ1 on Nicolet 170 S ꢀ FT-IR
spectrometer. Proton magnetic resonance spectra were recorded on
a Bruker 300 M Hz spectrometer in DMSO-d6 using TMS as an
internal standard. The EPR spectrum of copper(II) complex was
recorded at both room on Varian E-4 X-band spectrometer using
TCNE as g-marker. Conductivity measurements were measured at
10ꢁ3 M solutions of complexes in DMSO using ELICO-CM82
conductivity bridge provided with a cell having cell constant 0.51.
CD measurements of saccharide ligand and their complexes were
made on a JASCO-J-715 spectropolarimeter. The cyclic voltam-
metric experiments were carried out with a three electrode appa-
ratus using a CHI1110A electrochemical analyzer (USA). Cyclic
voltammetric data were recorded using a glassy carbon working
electrode (0.082 cm2), a platinum counter electrode, and an Ag/
Agþ reference electrode. Glassy carbon electrode surfaces were
polished with 0.05 mm alumina, rinsed in water, and air-dried
immediately before use. The electrochemical experiments were
carried out and the positions of the waves were compared to the
potential of the ferrocene/ferrocenium couple. The DMSO solution
(containing 0.1 M tetrabutylammoniumperchlorate, as supporting
electrolyte, 10ꢁ3 molar concentration of the ligand and each of
the complexes) was placed in a single-compartment electro-
chemical cell and degassed by bubbling with N2(g) saturated with
DMSO. A N2 atmosphere was continuously maintained above
the solution while the experiments were in progress. TG and DTA
9.72 (9.69). FT-IR (KBr) cmꢁ1: 3448 (br s)
(CH), 1641 (vs) (N–H), 1045 (s) d
(C]S). 1H NMR (DMSO)
n
(OH), 2978–2897 (m)
: 10.50
n
d
n
(s, 1H, hydrazide NH), 8.90–9.00 (2s, 1H, glycosylic N–H), 7.12–7.60
(m, 5H, Ar), 4.99 (d, 1H, C–1–H), 4.60 (s, 1H, Ar–C–NH), 3.07–3.67
(m, –OH and –H of saccharide moiety).
5.2. Synthesis of complexes
In a representative preparation, the complex was prepared by
the addition of methanolic solution of metal(II) chloride
(0.003 mol) with constant stirring to the ligand (0.003 mol) in the
same solvent at room temperature. Ammonia gas is passed through
the reaction mixture. After a sufficient time of stirring the reaction
mixture was concentrated. All the complexes were filtered and
washed with methanol and dried in vacuum over P2O5.
5.2.1. [CoLCl]$2H2O
Yield w70%, Mp > 280 ꢃC. Elemental analysis: Calculated
(found) for [Co(C13H18N3O5S) Cl].2H2O: C, 35.45 (35.41); H, 4.54
(4.50); N, 9.54 (9.49); S, 7.27 (7.24); Co, 13.39 (13.36); Cl, 8.05 (8.01).
FT-IR (KBr) cmꢁ1: 3268 (br s)
H), 1030 (s)
n
(OH), 2920 (m)
n(CH), 1598 (vs) d(N–
n
(C]S). Molar conductance: 31 mho cm2molꢁ1
.
Magnetic moment: 4.5 BM.
5.2.2. [NiLCl$2H2O]
Yield w70%, Mp > 280 ꢃC. Elemental analysis: Calculated
(found) for [Ni(C13H18N3O5S) Cl.2H2O]: C, 34.06(33.99); H, 4.80
(4.78); N, 9.17 (9.13); S, 6.98 (6.95); Ni, 12.81 (12.78); Cl, 7.74 (7.71).
FT-IR (KBr) cmꢁ1: 3323 (br s)
(N–H), 1036 (s)
n
(OH), 2932 (m)
n(CH), 1592 (vs)
d
n
(C]S). Molar conductance: 29 mho cm2molꢁ1
.
Table 2
Magnetic moment: 2.96 BM.
Cyclic voltammogram data for the complexes.
Complex
E1/2/mV (DEp/mV)
5.2.3. [CuLCl]
[CuLCl]
[CoLCl]$2H2O
399(143)
159(222), 149(199)
Yield w75%, Mp > 280 ꢃC. Elemental analysis: Calculated
(found) for [Cu(C13H18N3O5S)Cl]: C, 36.53(36.59); H, 4.21 (4.19); N,

112
M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
Table 4
Effect of drugs on body weight changes in tumor induced mice.
Group
Dose (mg/Kg) i.p.
% increase in weight as compared to day-0 (mean ꢂ SE)
Day-3
Day-6
Day-9
Day-12
Day-15
Control
Cisplatin
LH
[CoLCl]$2H2O
[NiLCl$2H2O]
[CuLCl]
–
3.96 ꢂ 1.77
3.81 ꢂ 1.55
4.45 ꢂ 1.62
3.81 ꢂ 1.21
4.21 ꢂ 1.34
4.62 ꢂ 1.48
4.34 ꢂ 1.45
6.84 ꢂ 1.69
13.04 ꢂ 1.22
13.29 ꢂ 1.27
11.34 ꢂ 1.33
12.34 ꢂ 2.56
17.54 ꢂ 2.23
16.43 ꢂ 1.58
16.43 ꢂ 1.45
18.27 ꢂ 1.22
19.08 ꢂ 3.41
19.46 ꢂ 3.61
18.49 ꢂ 3.51
21.32 ꢂ 1.72
18.32 ꢂ 1.53
18.15 ꢂ 1.59
8.72 ꢂ 2.22a
16.54 ꢂ 2.35
18.71 ꢂ 4.33
13.56 ꢂ 2.39
16.42 ꢂ 3.03
16.54 ꢂ 1.65
21.25 ꢂ 2.34
1.53 ꢂ 3.24a
7.43 ꢂ 2.45a
14.61 ꢂ 1.82
7.24 ꢂ 1.65a
7.55 ꢂ 3.23a
8.45 ꢂ 2.39a
3.5
50
50
50
50
50
[ZnLCl]
a
p < 0.05 Vs Control.
9.83 (9.79); S, 7.49 (7.45); Cu, 14.88 (14.83); Cl, 8.30 (8.27). FT-IR
(KBr) cmꢁ1: 3445 (s)
(OH), 3335 (s) (NH), 2978–2897 (m) (CH),
(C]S). Molar conductance:
5th, 7th, 10th, 12th, and 14th day of tumor inoculation in the
volume of 0.1 ml/10 g mice. All the compounds were tested at the
dose of 50 mg/kg body weight. The dose of cisplatin selected was
3.5 mg/kg. This was calculated by using body mass Index and past
experience with the drug [24].
n
n
n
1590 (vs)
d
(N–H), 1035 (s)
n
20 mho cm2molꢁ1. Magnetic moment: 1.93 BM.
5.2.4. [ZnLCl]
Yield w75%, Mp > 280 ꢃC. Elemental analysis: Calculated
(found) for [Zn(C13H18N3O5S) Cl].2H2O: C, 34.89 (34.82); H, 4.47
(4.45); N, 9.39 (9.33); S, 7.15 (7.12); Ni, 12.81 (12.78); Cl, 7.74 (7.71).
5.3.4. Determination of cytotoxicity of compounds against EAC cells
(In vitro studies, Tryphan Blue Exclusion Method (Cell Viability
Test))
FT-IR (KBr) cmꢁ1: 3368 (br s)
n
(OH), 2929 (m)
n(CH),1600 (vs) d(N–H),
In vitro short-term cytotoxic activity of drug was determined
using EAC cells. The EAC cells that were collected from the animal
peritoneum by aspiration were washed repeatedly with PBS to free
it from blood. After checking the viability of the cells in a haemo-
cytometer, a cell (1 ꢀ106) in 0.1 ml PBS, 0.01 ml of various
1033 (s)
n
(C]S). 1H NMR (DMSO)
d: 10.50 (s, 1H, hydrazide NH),
8.90–9.00 (2s,1H, glycosylic N–H), 7.12–7.60 (m, 5H, Ar), 4.99 (d,1H,
C–1–H), 4.60 (s, 1H, Ar–C–NH), 3.77–3.07 (m, –OH and –H of
saccharide moiety). Molar conductance: 24 mho cm2molꢁ1
.
concentrations of test compounds (1–50 mg/ml) were made (the
test compounds were dissolved in dimethylsulphoxide (DMSO), the
final concentration of DMSO not exceeding 0.1% of the total volume)
and phosphate buffered saline (0.1 mole/l, pH 7) in a total volume of
0.9 ml were incubated in clean sterile tubes for 3 h at 37 ꢃC. The
5.3. Antitumor activity
5.3.1. Cell lines
Cancer cell lines viz. Ehrlich’s Ascitic Carcinoma (EAC), to induce
cancer in animal model (mice) were obtained from Amala Cancer
Research Center, Amala Nagar, Thrissur, Kerala, India. The cells were
maintained as ascites tumor in Swiss albino mice by intraperitoneal
inoculation of 1 ꢀ106 viable cells.
control tube had 10
ml of solvent. The final volume was made up to
0.9 ml with PBS. To each 100
ml of Tryphan blue solution was added.
The live (without stain) and dead (with blue stain) cells were
counted using haemocytometer and percent cell death was calcu-
lated using the formula:
5.3.2. Animals
%Cytotoxicity [ 1003ðTdead L CdeadÞ=Ttot
Female Swiss albino mice of 6–8 weeks old (25 ꢂ 5 g body
weight) were selected. The animals were acclimatized to the
experimental room having temperature 23 ꢂ 2 ꢃC, controlled
humidity conditions, and 12:12 h light and dark cycle. The Mice
were housed in sterile polypropylene cages containing sterile
paddy husk as bedding material with maximum of 4 animals in
each cage. The mice were fed on autoclaved standard mice food
pellets (Hindustan Lever) and water ad libitum. The animal exper-
iments were performed according to the rules and regulations of
the Institutional Animal Ethics Committee (IAEC).
where, Tdead is the No. of dead cells in the treated group, Cdead is
that in the control group and Ttot is the total number of dead and
live cells in the test compound treated group. Cisplatin was used as
the standard [25].
5.3.5. Induction of Ehrlich Ascites Carcinoma [22]
Antitumor activities of compounds were determined using
Ehrlich Ascites Carcinoma (EAC) tumor model in mice. Female
Swiss albino mice were divided into groups of 6 animals each.
(A. Tumor bearing mice, B. Tumor bearing mice treated with one
dose of Cisplatin, C. Tumor bearing mice groups treated with
compounds). The ascites carcinoma-bearing mice (donor) were
used for the study, 15 days after tumor transplantation. The ascitic
fluid was drawn using an 18-gauge needle into sterile syringe.
A small amount was tested for microbial contamination. Tumor
viability was determined by Tryphan blue exclusion test and cells
were counted using haemocytometer. The ascitic fluid was suitably
diluted in normal saline to get a concentration of 106 cells/ml. of
tumor cell suspension. This was injected intraperitonealy to obtain
ascitic tumor. The mice were weighed on the day of tumor inocu-
lation and then once in three days thereafter. Treatment was star-
ted on the 3rd, 5th, 7th, 10th, 12th, and 14th day of tumor
inoculation. Cisplatin (one dose) was injected on 1st day intra-
peritonealy. The animals in each of the groups were kept to check
the mean survival time (MST) of the tumor bearing hosts.
5.3.3. Preparation of test solution of compounds
The solutions of the compounds were prepared by suspending
them in 2% acacia and administrated intraperitoneally on 3rd,
Table 5
Effect of drugs on the survival time in tumor induced mice.
Group
Dose (mg/kg) LC50 in M ꢀ 10ꢁ8 Mean survival time (days)
(Mean ꢂ SEM) %T/C
%ILS
Control
Cisplatin
LH
–
3.5
50
–
19.00 ꢂ 0.37
–
–
2.76
3.14
3.69
2.89
1.94
2.56
38.29 ꢂ 1.17a 201.53 101.53
27.83 ꢂ 0.48a 146.47 46.47
22.50 ꢂ 0.43a 118.42 18.42
25.83 ꢂ 0.43a 135.95 35.95
28.83 ꢂ 0.43a 151.74 51.74
27.67 ꢂ 0.42a 145.63 45.63
[CoLCl]$2H2O 50
[NiLCl$2H2O] 50
[CuLCl]
[ZnLCl]
50
50
a
p < 0.05 Vs Control Groups.

M.P. Sathisha et al. / European Journal of Medicinal Chemistry 45 (2010) 106–113
113
Antitumor effects of compounds were assessed by observation of
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