Synthesis, characterization and antitumor studies of Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) complexes of N-salicyloyl-N′-o-hydroxythiobenzhydrazide

Article abstract of DOI:10.1016/S0968-0896(01)00373-X

A new ligand N-salicyloyl-N′-o-hydroxythiobenzhydrazide (H₂Sotbh) forms complexes [Mn(HSotbh)₂], [Fe(Sotbh-H)(H₂O)₂], [M(Scotbh)] [M = Co(II), Cu(II) and Zn(II)] and [Ni(Sotbh)(H₂O)₂], which were characterized by various physico-chemical techniques. Moessbauer spectrum of [Fe(Sotbh-H)(H₂O)₂] reveals the quantum admixture of 5/2 and 3/2 spin-states. Mn(II), Cu(II) and Ni(II) complexes showed reversal of tumor bearing mice. Tumor bearing mice administered with Mn(II), Cu(II) and Ni(II) complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implications of the H₂Sotbh and its metal complexes in tumor regression and the tumor growth associated immunosuppression. Copyright

Full text of DOI:10.1016/S0968-0896(01)00373-X

Bioorganic & Medicinal Chemistry 10 (2002) 887–895
Synthesis, Characterization and Antitumor Studies of Mn(II),
Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) Complexes of
N-Salicyloyl-N0-o-hydroxythiobenzhydrazide
Anuraag Shrivastav,a Nand K. Singha,* and Sukh Mahendra Singhb
aDepartment of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005, India
bSchool of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India
Received 18 June 2001; accepted 17September 2001
Abstract—A new ligand N-salicyloyl-N0-o-hydroxythiobenzhydrazide (H2Sotbh) forms complexes [Mn(HSotbh)2], [Fe(Sotbh-
H)(H2O)2], [M(Sotbh)] [M=Co(II), Cu(II) and Zn(II)] and [Ni(Sotbh)(H2O)2], which were characterized by various physico-chemical
techniques. Mossbauer spectrum of [Fe(Sotbh-H)(H2O)2] reveals the quantum admixture of 5/2 and 3/2 spin-states. Mn(II), Cu(II)
and Ni(II) complexes were observed to inhibit the growth of tumor in vitro, whereas, Fe(III), Co(II), Zn(II) complexes did not. In
vivo administration of Mn(II), Cu(II) and Ni(II) resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice
administered with Mn(II), Cu(II) and Ni(II) complexes showed reversal of tumor growth associated induction of apoptosis in
lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H2Sotbh and its metal complexes in
tumor regression and tumor growth associated immunosuppression. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
that Mn(II), Fe(III), Ni(II), Cu(II) and Zn(II) com-
plexes of N-aroyl-N0-thiohydrazide have antitumor
activity against different tumor cells.23À25 Many chemo-
therapeutic agents have also been reported to possess
immunomodulatory properties.26À28 However, the
immunomodulatory potential of the metal complexes
with antitumor activity has not been explored adequately.
Although, thiohydrazides are structurally quite similar
to thiosemicarbazides, scarcity of work on the anti-
neoplastic and immunomodulatory activity of transition
metal complexes of thiohydrazides prompted us to syn-
thesize, characterize and study the antitumor activity of
transition metal complexes of newly synthesized ligand
N-salicyloyl-N0-o-hydroxythiobenzhydrazide. Further,
we also investigated the immunomodulatory action of
these complexes in a tumor bearing host.
One of the major applications of the transition metal
complexes has been the testing of their antibacterial and
antititumor activities with an aim to find an effective
and safe therapeutic regimen for the treatment of cancer
and infections. The coordination chemistry of nitrogen–
sulfur donor ligands such as substituted thiosemicarba-
zide,1 thiosemicarbazones2À5 and dithiocarbazates6 is
well documented in literature. A few papers have
appeared on the synthetic and structural aspects of the
3d-metal complexes of thiohydrazides.7À11 In addition
to the antifungal12 and antibacterial13 properties, some
of the transition metal complexes of nitrogen–sulfur
donor ligands also exhibit antitumor activity against a
variety of tumor cells.14,15 Thiosemicarbazones such as
3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazonato)
copper(II) complexes (CuKTS) have been found to
exhibit antitumor activity16,17 due to their binding with
DNA.18 Thiosemicarbazones of 1-formylisoquinoline
and 2-formyl pyridine and their derivatives were also
demonstrated to be effective against animal tumor19,20
by inhibiting ribonucleoside diphosphate reductase
activity and synthesis of DNA.21,22 We have reported
Results and Discussion
The analytical data correspond to the 1:1 metal to
ligand stoichiometry, except for Mn(II) where it is 1:2,
and the complexes are colored. These complexes were
formed by loss of two or three protons from the ligand.
The complexes are soluble in polar coordinating sol-
vent, DMSO, and melt above 300 ꢀC, except
[Cu(Sotbh)] which melts at 223 ꢀC. The composition of
*Corresponding author. E-mail: anuraagshrivastav@yahoo.com
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00373-X

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A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
Table 1. Analytical and Physical Properties of H2Sotbh and its Complexes
Compound
Colour
m.p.
(ꢀC)
Yield
(%)
Found (calcd) %
C
meff
B.M.
M
S
H
N
H2Sotbh
White
220
>300
>300
>300
210d
61
54
66
62
64
70
71
–
11.4
58.73.9
(58.3)
53.6
(53.4)
44.2
(44.6)
48.2
(48.7)
44.3
(44.1)
48.2
(48.1)
47.7
(47.8)
9.5
(4.1)
3.1
(3.4)
3.5
(3.4)
2.8
(2.9)
3.2
(3.6)
2.7
(2.9)
2.4
(2.8)
–
(11.1)
10.5
(10.1)
8.9
(8.5)
8.9
(9.2)
8.9
(8.4)
9.4
(9.7)
9.1
(8.9)
7.5
(7.4)
7.7
(8.1)
7.6
(7.4)
7.7
(8.0)
8.1
[Mn(Sotbh)2]
[Fe(Sotbh-H)(H2O)2]
[Co(Sotbh)]
Greenish Black
Black
8.9
(8.7)
14.4
(14.8)
16.8
(17.1)
15.1
(15.4)
17.9
5.9
4.7
Greenish Brown
Light Green
Blackish Brown
Light Yellow
2.43
2.83
1.9
[Ni(Sotbh)(H2O)2]
[Cu(Sotbh)]
223
(18.2)
19.1
(18.6)
(9.1)
8.8
(9.1)
[Zn(Sotbh)]
>300
Dia
(8.0)
d=decompose
the complexes along with color, mp magnetic
susceptibility and elemental analyses are given in Table
1. The proposed structure for the complexes is given in
Figure 1.
spin, octahedral Mn(II). A magnetic moment of 2.83
B.M. for [Ni(Sotbh)(H2O)2] suggests an octahedral
geometry around Ni(II). It displays bands at 16,780 and
3
3
3
25,510 cmÀ1 for the A2g ! T1g, T1g(P) transitions,
respectively, characteristic of an octahedral system. In
addition two spin forbidden bands at 15,575 and 24,040
Magnetic properties and electronic spectra
1
3
cmÀ1, one due to the transition to Eg (near T1g, for
common spin allowed transition systems with Dq/B
near unity), and the second, primarily due to the tran-
The magnetic moments of Mn(II) and Ni(II) complexes
suggest their octahedral geometry which is further sup-
ported by the electronic spectral data. [Mn(HSotbh)2]
exhibits a meff of 5.9 B.M. and shows two electronic
1
3
3
sition T2g [between the T1g and T1g(P) spin allowed
transitions]29 were also observed. The bands at 26,880
and 28,250 cmÀ1 are assigned to the charge transfer and
intraligand transitions, respectively.30 A magnetic moment
bands at 15,430 and 22,320 cmÀ1 assigned to the 6A1g
!
4
4T1g and 6A1g ! T2g transitions, respectively, for high-
of 4.7B.M. for [Fe(Sotbh-H)(H O)2] suggests an inter-
2
mediate spin state for iron(III) centre with a distorted
octahedral geometry and the presence of a band at
4
19,840 cmÀ1 assigned to the 6A1g ! T2g transition further
supports the above geometry around Fe(III). The mag-
netic moments of 2.43 and 1.9 B.M. for [Co(Sotbh)] and
[Cu(Sotbh)], respectively, suggest
a square-planar
geometry around Co(II) and Cu(II). [Cu(Sotbh)] displays
a broad band at 17,480 cmÀ1 assigned to the envelope of
2
2
2
2B1g ! A1g, B2g, Eg transitions further supporting a
square-planar geometry around Cu(II).31 Very limited
information is available on the electronic spectra of
square-planar complexes of Co(II). Nishida and Kida32
have reported that the electronic spectra of square-pla-
nar Co(II) complexes exhibit three bands at 4000, 7000
and 18000 cmÀ1 mainly due to the transitions A1g
2
!
2B2g, Eg and E02g, respectively. [Co(Sotbh)] exhibits a
2
2
band at 18,115 cmÀ1 assigned to the A1g ! E02g tran-
sition supporting square-planar geometry around
Co(II). Other bands at 23,585 and 25,640 cmÀ1 have
been attributed to the charge-transfer transitions.
2
2
IR spectra
The IR spectrum of H2Sotbh shows bands at 3452 and
3171 cmÀ1 assigned to n(OH) and n(NH), respectively.
The bands occurring at 1630, 1439, 1346 and 958 cmÀ1
are assigned to n(C¼O), thioamide I [b(NH)+n(CN)],
thioamide II [n(CN)+b(NH)] and n(C¼S), respectively.
A broad band at 3427cm À1 observed in the spectrum of
Figure 1. Proposed structure of the complexes of H2Sotbh.

A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
889
[Mn(HSotbh)2] may be assigned to n(OH) of the ligand.
Mossbauer spectra
A new band appears at 1608 cmÀ1, due to the n(C¼N)
of NCO in place of n(C¼O), suggesting that one
hydrazinic proton is lost via enolization and bonding of
the resulting enolic oxygen takes place with the metal
ion. The thioamide I and II bands at 1439 and 1346
cmÀ1 in the free ligand undergo a positive shift of 36
and 14 cmÀ1, respectively, in the complex, suggesting
the involvement of thione sulfur in bonding. Absence of
n(NH) and n(OH) bands in the spectrum of [Fe(Sotbh-
H)(H2O)2] suggests the involvement of these group as
bonding site, by loss of both the hydrazinic protons via
enolization and thioenolization and one phenolic pro-
ton. Presence of new bands at 1619 and 850 cmÀ1
assigned to the n(C¼N) of NCO and n(C–S) further
supports the above observation. [Co(Sotbh)] displays a
band at 3435 cmÀ1 assigned to n(OH), ruling out the
involvement of phenolic OH as bonding site. Absence of
The Mossbauer spectra (Fig. 2a and b) of [Fe(Sotbh-
H)(H2O)2] at room and liquid nitrogen temperatures
were studied. The Mossbauer spectra of the complex
show three lines at both the temperatures. The two sites
observed at room and liquid nitrogen temperature
reveals the spin-cross over phenomenon exhibited by
Fe(III) complex. The Mossbauer parameters at room
temperature are isomer shift, d=0.35 mm sÀ1 and
quadrupole splitting, ꢀ=0.48 mm sÀ1 for site 1 aÀn1d the
ꢀ=1.29 mm sÀ1. The MB parameters for site 1 are
characteristic of high-spin state S=5/2 and for site 2 are
characteristics of low spin state S=3/2. They are present
in 3:1 ratio. On lowering the temperature to 78 K, the
corresÀp1onding values for site 1 are found to be d=0.51
mm s ꢀ=0.51 mm sÀ1, whereas, for site 2, d=0.53
mm sÀ1 ꢀ=1.13 mm sÀ1 in 1:1 ratio. These values sug-
gest the quantum admixture of S=5/2 and S=3/2 spin
states. They also follow the usual trend of high-spin
Fe(III) ion going into more of low spin state on low-
ering the temperature as suggested by the data that both
iron species are present in equal quantity at 78 K.
corresponding values for site 2 are d=0.47mm s
and
bands due to n(NH), n(C¼O) and n(C¼S) and appear-
À1
ance of a new band at 1597cm
due to the n(C¼N) of
NCO suggests that both NH protons are lost via enoli-
zation and thioenolization. The bonding of the resulting
enolic oxygen and thiolato sulfur takes place with the
metal ion. The thioamide I and II bands at 1491 and
1370 cmÀ1 undergo a positive shift of 52 and 25 cmÀ1
,
respectively, in the complex suggesting the involvement
of these groups as bonding sites. [Ni(Sotbh)(H2O)2]
shows the presence of a band at 3404 cmÀ1, assigned to
n(OH) and absence of n(NH), n(C¼O) and n(C¼S) and
appearance of new bands at 1606 and 860 cmÀ1 suggest
that both the hydrazinic protons are lost via enolization
and thioenolization and the bonding of the resulting
enolic oxygen and the thiolato sulfur takes place with
the metal ion. Thioamide I and II bands at 1489 and
Antitumor studies
To investigate the antitumor activity of H2Sotbh and its
metal complexes their effect on the growth of Dalton’s
lymphoma (DL) cells in vitro was measured by MTT
assay. It was observed that treatment of tumor cells
with [Mn(HSotbh)2], [Cu(Sotbh)] and [Ni(Sotbh)
1412 cmÀ1 undergo a positive shift of 50 and 66 cmÀ1
,
respectively, thereby supporting the involvement of
these groups as bonding sites.
The spectra of [Cu(Sotbh)] and [Zn(Sotbh)] display
bands at 3450 and 3427cm À1, respectively, for n(OH).
There is significant change in the bands due to
thioamide I, II and n(C¼S) as compared to the free
ligand in the spectra of [Cu(Sotbh)] and [Zn(Sotbh)].
The presence of new bands at 1597cm À1 for n(C¼N) of
NCO in place of n(C¼O) and at 840 cmÀ1 for n(C–S),
suggest bonding through enolic oxygen and thiolato
sulfur to the metal ion.
NMR spectra
1
The H NMR spectrum of H2Sotbh exhibits signals at
d13.0 and 12.2 ppm for the OH protons and at d 5.6–4.0
(br) ppm for the NH protons, which disappear on D2O
exchange. The protons due to the two aromatic rings
appear as multiplets between d 8.66 and 7.0 ppm. The
diamagnetic [Zn(Sotbh)] shows a broad signal at d 12.6–
11.86 ppm for both OH protons and a multiplet
between d 8.4 and 6.6 ppm for the aromatic ring pro-
tons. Absence of NH signals further support bonding
through the enolic oxygen and thiolato sulfur via enoli-
zation and thioenolization. The low solubility and
paramagnetic nature of other metal complexes pre-
cluded the recording of their NMR spectra.
Figure 2. (a) Mossbauer spectrum of [Fe(Sotbh-H)(H2O2] at room
temperature; (b) Mossbauer spectrum of [Fe(Sotbh-H)(H2O)2 at 78 K.

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A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
(H2O)2] caused maximum growth inhibition followed by
[Fe(Sotbh-H)(H2O)2] and the ligand (Table 2). The rea-
son for the observed inhibition of tumor cell growth by
the metal complexes is unclear however, several possi-
bilities could be considered. The cytostatic activity of
the metal complexes could be a direct result of the
interaction of the metal complexes with DNA, thus
interfering with the process of DNA replication. Indeed,
DNA binding of metal complexes has been docu-
mented.33 Furthermore, inhibition/activation of various
enzymes directly/indirectly involved in DNA replication
is not ruled out. Interaction of metal complexes with
protein components of viable cells has been repor-
ted.34,35 Binding of metal complexes with protein may
cause alterations in the structural and functional organ-
ization of proteins. However, more studies will be
needed to confirm the existence of one or more of such
possibilities in our system. These results may also indi-
cate a possible decline of the overall metabolic activity
of the tumor cells with a concomitant inhibition of the
activity of various enzymes involved in respiration.
limited to the tumor phenotype and not on all pro-
liferating cells. The killing of tumor cells specifically
could be attributed to the susceptibility of tumor cell
DNA to a damage by metal complexes as compared to
the normal cells. Indeed, in case of cisplatin, it has been
shown that the tumor cells are killed specifically because
they are unable to overcome the genetic load of muta-
tions caused by the drug because of their defective DNA
repair mechanism.39
In the next part of the investigation we checked the life
prolonging effect in DL bearing mice administered with
phosphate buffer saline (PBS) alone or containing
ligand or the metal complexes as indicated in the
Experimental. As shown in Table 3, minimal % T/C
was observed in mice administered with ligand alone as
compared to that of mice administered with metal
complexes. Maximum% T/C was found for [Cu(Sotbh)]
followed by that of [Mn(HSotbh)2] and [Ni(Sotbh)
(H2O)2]. Our observations show that H2Sotbh, [Fe(S-
otbh-H)(H2O)2] and [Zn(Sotbh)] did not have antitumor
activity whereas, significantly higher life prolonging
ability in tumor bearing mice were observed for
[Mn(HSotbh)2], [Cu(Sotbh)] and [Ni(Sotbh)(H2O)2].
Increase in the value of% T/C, an indicator of the effect
of drug administration on the survival of tumor bearing
mice, suggests that such effect could either result from
the direct cytotoxic/cytostatic action of the complexes
on tumor cells or due to the activation of certain host
derived mechanism resulting in a decrease of tumor
load.
Although the metal complexes showed cytostatic effects
on the tumor cells in vitro, these results do not necessa-
rily indicate if these cells are actually killed by the direct
action of the metal complexes. To check this in the next
part of the investigation we studied the effect of the
metal complexes on the tumor cell killing with respect to
the mode of cell death. Cisplatin has been reported to
induce apoptotic cell death in the tumor cells.36,37 It was
observed that the tumor cells were killed by the induc-
tion of apoptosis (Fig. 3a). [Mn(HSotbh)2] and
[Cu(Sotbh)] were found to be most effective in the
induction of tumor cell apoptosis. The mechanism of
the induction of apoptosis remains poorly understood
and is thought to be dependent on multiple mechanism(s)
ultimately culminating in the activation of DNA cleaving
endonucleases.36,38 As shown in Figure 3b, [Mn(HSotbh)2]
and [Cu(Sotbh)] cause an increase in% specific DNA
fragmentation, it is probable that these metal complexes
may act via the activation of endonucleases to kill the
tumor cells by inducing apoptotic cell death.
Progression of growth of various tumors including DL
is invariably associated with the onset of immunosup-
pression in tumor bearing host,40À43 one of the reasons
being induction of apoptosis and inhibition of pro-
liferation of hematopoietic precursor cells.44,45 Since in
vivo administration of these metal complexes prolonged
survival of tumor bearing animals and these metal
complexes did not show cytotoxicity against normal
cells in vitro, we were interested to investigate if the
administration of metal complexes could reverse tumor
growth associated induction of apoptosis in various
hematopoietic cells. For this DL bearing mice were
administered with metal complexes, and the% of apop-
totic thymocyte, spleenocyte and bone marrow cells
were enumerated. As shown in Figure 4a, administra-
tion of metal complexes in tumor bearing mice resulted
in the inhibition of tumor associated apoptosis of thy-
mocyte, splenocyte and bone marrow cells. Similar
results were obtained for % DNA fragmentation as well
(Fig. 4b). The reversal of tumor growth associated
induction of apoptosis of hematopoietic cells by metal
complexes is predicted to be due to two reasons:
(1) reduction of tumor load resulting due to the cyto-
toxic effect of metal complexes on tumor cells, leading
to a decrease in the tumor associated concentration of
apoptotic factors; (2) direct protective effect of metal
complexes on the hematopoietic cells. Although not
very clear, the probability of the latter could be due to
the fact that metal complexes can bind to DNA and
several proteins in cells, which could result in the pro-
tective effect.
Furthermore, the metal complexes at the concentration
checked did not inhibit the growth of normal spleeno-
cyte and bone marrow cells (data not shown), which
comprise a major proportion of proliferating cells indi-
cate that the cytostatic effect of the metal complexes was
Table 2. Effect of H2Sotbh and its metal complexes on tumor cell
growth in vitro (ID50 values in mg/mL)
Compounds
ID50
H2Sotbh
24.7
0.92
1.35
15.6
0.91
0.71
[Mn(Sotbh)2]
[Ni(Sotbh)(H2O)2]
[Fe(Sotbh-H)(H2O)2]
[Cu(Sotbh)]
Cisplatin
ID50=average drug concentration (mg/mL) for 50% inhibition of
tumor cell growth. Values are meanꢁSD of three experiments. r<
0.05 with respect to values of ID50 of H2Sotbh alone.

A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
891
Although more investigations will be required to con-
Experimental
Preparation of H2Sotbh
firm the mechanism of action of metal complexes on
tumor and normal cells, the study suggests that
[Mn(HSotbh)2] and [Cu(Sotbh)] can cause prolongation
of survival in tumor bearing animals by:
Sodium salt of o-hydroxydithiobenzoate. A solution of
o-hydroxybenzaldehyde (21 mL ꢂ 25 g) in 60 mL of
ethanol was heated to 65 ꢀC and 135 mL of the filtered
ammonium polysulfide was added in 10 mL portions
during 10 min keeping temperature at 65 ꢀC. The reac-
tion mixture was boiled to reflux for 1 h, immediately
cooled in ice, 100 mL of ether was added and the solu-
tion was acidified with concd HCl. The dithioacid which
separated as red oil, was filtered through suction to
remove the precipitated sulfur. The filtrate was trans-
ferred into a separating funnel, 100 mL of ether was also
added and the ethereal layer containing the dithioacid
1. directly killing tumor cells;
2. reversing tumor associated immunosuppression.
The finding of this investigation may have long lasting
clinical implication with the novel proposition that
metal complexes of N-salicyloyl-N0-o-hydroxythio-
benzhydrazide may have dual mechanism of action in
tumor regression.
Figure 3. (a) Effect of H2Sotbh and its metal complexes on the induction of apoptosis in tumor cells. DL cells were incubated in medium alone or
containing H2Sotbh or its metal complexes (10 mg/mL) for 24 h and the number of cell showing apoptotic morphology was enumerated. Values are mean
of three experiments. (b) Effect of H2Sotbh and its metal complexes on % DNA fragmentation of tumor cells. DL cells were incubated in medium alone
or containing H2Sotbh or its metal complexes (10 mg/mL) for 24 h and the % DNA fragmentation was evaluated. Values are mean of three experiments.

892
A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
was separated and washed with distilled water. The red
colored sodium salt of the dithioacid was extracted by
shaking the ethereal solution of the dithioacid twice
with aqueous NaOH solution (8 g in 100 mL).
Carboxymethyl-o-hydroxydithiobenzoate. To the sodium
salt of o-hydroxydithiobenzoate was added a solution of
chloroacetic acid (20 g) neutralized with sodium car-
bonate (pH of solution 7). After standing the reaction
Figure 4. (a) Effect of in vivo administration of H2Sotbh or its metal complexes on the induction of apoptosis in thymocyte, splenocyte and bone
marrow cell of normal or tumor bearing mice treated with ligand or its metal complexes. Thymocytes, splenocytes and bone marrow cells of normal
or tumor bearing mice or tumor bearing mice treated with ligand or its metal complexes were enumerated for the number of cell showing apoptotic
morphology. Values are mean of three experiments. (b) Effect of in vivo administration of H2Sotbh or its metal complexes on % DNA fragmenta-
tion of thymocyte, splenocyte and bone marrow cell of normal or tumor bearing mice treated with ligand or its metal complexes. Thymocytes,
splenocytes and bone marrow cells of normal or tumor bearing mice or tumor bearing mice treated with ligand or its metal complexes and checked
for % DNA fragmentation. Values are mean of three experiments.

A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
893
mixture overnight at room temperature, the dark solu-
tion was acidified with concd HCl and the ester which
separated on cooling was filtered off, washed with cold
water and recrystallized from hot ethanol in the presence
of animal charcoal, mp 120 ꢀC.
The Mossbauer spectra were collected using a Cryo-
physics MS-1 microprocessor controlled spectrometer
operating in the constant acceleration mode.
Antitumor screening
N-Salicyloyl-N0-o-hydroxythiobenzhydrazide (H2Sotbh)
was prepared by mixing an equivalent quantity of car-
boxymethyl-o-hydroxydithiobenzoate and salicylic acid
hydrazide, each dissolved separately in one equivalent
of 1 N NaOH solution and adding acetic acid dropwise
to the above ice cooled solution, after standing the
solution for ꢂ2 h. The product thus obtained was suc-
tion filtered, washed with water and recrystallized from
ethanol which yielded white compound, mp 220 ꢀC.
Mice. Inbred populations of BALB/c mice of either sex
of 8–12 weeks were used for the study. The mice were
fed food and water ad libitum under pathogen-free
conditions and were treated with utmost human care.
Tumor systems
DL (a spontaneous murine T cell lymphoma) were
maintained in culture in vitro as well as in ascites by
serial transplantation in BALB/c mice by an intraperi-
toneal injection of 5 ꢃ 105 cells/mouse.
Metal complexes of H2Sotbh. [Mn(HSotbh)2], [Fe(S-
otbh-H)(H2O)2], [M(Sotbh)] [M=Co(II), Cu(II) and
Zn(II)], [Ni(Sotbh)(H2O)2] were prepared by adding a
DMF solution (10 mL) of H2Sotbh (1.2 g, 5.2 mmol)
dropwise to the ethanolic solution (20 mL) of the
respective metal(II) acetate or to an aqueous solution of
Thymocyte preparation and culture
Thymuses obtained from normal and tumor bearing
mice with or without administration of complexes were
weighed on a chilled watch glass, diced on ice and pas-
sed through a stainless steel screen using a syringe
plunger. These cells, after washing with phosphate buf-
fered saline (PBS) by centrifugation at 200g for 10 min
at 4 ꢀC, were used directly for thymocyte counts. Cell
viability in the thymocyte preparation was determined
by mixing 10 mL sample with an equal volume of 0.4%
trypan blue-PBS solution49 and counting the cells on a
hemocytometer under light microscope. Cells that did
not excude trypan blue were considered nonviable. For
culturing thymocytes in vitro, thymocytes were main-
tained in complete RPMI 1640 medium at 37 ꢀC in
humidified atmosphere of 5% CO2 in air.
.
FeSO4 7H20 in an 1:1 molar ratio. Colored pre-
cipitate of the complexes started separating out after
5 min of stirring at room temperature but the reac-
tion mixture was further stirred at room temperature
for 2 h to ensure the completion of the reaction. The
insoluble complexes thus obtained were filtered off,
washed with ethanol and dried in vacuo. The products
obtained were characterized by various physico-chemi-
cal methods.
Physical measurements
The complexes were analysed for their metal and sulfur
contents as described elsewhere.46 Carbon, hydrogen
and nitrogen contents were estimated on a Perkin-
Elmer 240C microanalyzer. The electronic spectra were
recorded on a Cary-2390 UV–vis. spectrophotmeter in
DMSO solutions and as Nujol mulls.47 Room tempera-
ture magnetic susceptibility measurements were made
on a Cahn Faraday electrobalance using cobalt mercury
tetrathiocyanate as a calibrant and the experimental
magnetic susceptibilities were corrected for diamagnet-
ism by using the procedure of Figgis and Lewis.48 The
1H NMR spectra were obtained in DMSO-d6 on JEOL
FX-90Q spectrometer using TMS as internal reference.
Splenocyte preparation and culture
Spleens obtained from normal and tumor bearing mice
with or without administration of complexes were
weighed on a chilled watch glass, diced on ice and pas-
sed through a stainless steel screen using a syringe
plunger. These cells, after washing with phosphate buf-
fered saline (PBS) by centrifugation at 200g for 10 min
at 4 ꢀC, RBC were depleted by treatment with 0.84%
ammonium chloride for 10 min at room temperature.
Cells were again washed in PBS and then cultured in a
humidified atmosphere at 5% CO2, to remove adherent
cells. Non-adherent cells were collected and used for
assay.
Table 3. Effect of in vivo administration of H2Sotbh and its metal
complexes on the survival of tumor bearing mice
Compounds
Post inoculation life span (% T/C)
Bone marrow cell preparation and culture
H2Sotbh
148
248
209
168
267
250
[Mn(Sotbh)2]
[Ni(Sotbh)(H2O)2]
[Fe(Sotbh-H)(H2O)2]
[Cu(Sotbh)]
Bone marrow cells (BMC) were obtained from the
femurs of normal and tumor bearing mice with or
without administration of complexes, as described else-
where.50 Briefly, the mice were killed by cervical dis-
location and the BMC were obtained from the femoral
shafts by flushing it with serum-free medium. BMC
were then agitated gently to prepare a single cell sus-
pension and then washed thrice with serum-free medium
by centrifugation at 200g at 4 ꢀC. BMC were then incu-
bated in plastic tissue culture flask for 2 h at 37 ꢀC to
Cisplatin
Treatment responses (six mice per treatment group) presented as %
T/C, was calculated according to the equation: mean life span of
treated mice/mean life of control mice by 100. A % T/C ꢄ 125 is con-
sidered biologically significant. C=20ꢁ2 days, experiment terminated
after 50 days. Values are meanꢁSD of three experiments. r< 0.05
with respect to values of H2Sotbh alone.

894
A. Shrivastav et al. / Bioorg. Med. Chem. 10 (2002) 887–895
remove the adherent macrophage. The non adherent
BMC were then used for proliferation.
centrifugation at 13,000g for 10 min and then DNA in
each pellet was hydrolyzed with 80 mL of 5% tri-
chloroacetic acid by heating on water bath at 90 ꢀC for
15 min and 160 mL of freshly prepared diphenylamine
(150 mg diphenylamine in 10 mL glacial acetic acid, 150
mL concd H2SO4 and 50 mL of acetaldehyde solution)
was added and the tubes were allowed to stand over-
night at room temperature to develop color. 100 mL of
this colored solution was transferred to a 96-well
flat bottom ELISA plate (NUNC, Denmark) and
absorbance at 600 nm noted on Biorad plate reader
(Australia). Percent fragmented DNA was calculated
using formula:
Proliferation assay
Different cells obtained from normal or tumor bearing
mice treated with or without complexes were incubated
at a concentration of 1.5 ꢃ 106 cells per well in a 96-well
plastic tissue culture plate with medium containing sub-
mitogenic doses of concanavalian-A (1 mg/mL). Cul-
tures were then incubated at 37 ꢀC in CO2 incubator for
48 h and assayed for proliferation and growth inhibition
using MTT assay.
T
% fragmented DNA ¼
100
In vitro growth inhibitory assay
T þ B
The MTT assay was used to measure the cytotoxic effect
of the ligand and the complexes. The procedures
employed the pale yellow tetrazolium salt [3-(4,5-dime-
thylthiazol)-2-yl-2,5-diphenyl-2H-tetrazolium bromide]
(MTT), which was cleaved by active mitochondria to
form a dark blue formazon product that can be com-
pletely solubilized in acidic isopropanol.51 The assay
provides a simple way to detect living and growing cells
without use of radioactivity. Briefly, 5 ꢃ 104 tumor cells
were plated in triplicate in 96-well flat bottom tissue
culture plates, and treated with different concentrations
of drugs for the time indicated. MTT (0.005 g cmÀ3 in
PBS) was added to the cell culture and incubated for 4 h
in a 37 ꢀC, 5% CO2 humidified incubator. The for-
mazon crystals formed during the reaction were dis-
solved in 100 mL of 0.04 N HCl in isopropanol and
absorbance was read at 570 nm. The average drug con-
centration (mg/mL) for 50% inhibition (ID50) of tumor
cell-growth was determined by plotting the log of drug
concentration versus the growth rate (% control).
where T=absorbance of fragmented DNA, T+B=ab-
sorbance of total DNA.
In vivo studies
In order to aassess the antitumor activity of the com-
pounds, 6–8 groups of BALB/c mice were inoculated
intraperitoneally with DL (106) cells followed by treat-
ment with the metal complexes (10 mg/kg body weight)
in a single ip injection on days 1, 5.9 and 12 after tumor
transplantation. This treatment protocol was selected
for administration, as various previous studies54 have
shown that metal complexes of sulfur donor ligands
have shown optimal antitumor activity at a dose of 10
mg/kg. The antitumor efficacy of each agent is expressed
as % T/C and is given by,
Mean life span of treated mice
% T ꢀ C ¼
ꢁ 100
100 Mean life span of control mice
Morphological evaluation of apoptotic cells
References and Notes
Cells were air dried, fixed in methanol, stained with
Wright staining solution, mounted in glycerine and
analyzed under light microscope at 450ꢃ magnification.
Apoptotic cells were identified on the basis of morpho-
logical features that included contracted cell bodies,
condensed, uniformly circumscribed and densely stained
chromatin, or membrane-bound apoptotic bodies con-
taining one or more nuclear fragments.52 The percen-
tage of apoptotic cells was determined by counting more
than 300 cells in at least three separate visions.
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