Co(II), Ni(II), Cu(II) and Zn(II) complexes of isatinyl-2-aminobenzoylhydrazone: Synthesis, characterization and anticancer activity

Article abstract of DOI:10.1002/aoc.3252

This article describes the synthesis, structural aspects and biological studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes of a new hydrazone derived from the condensation of isatin and 2-aminobenzoylhydrazide. The ligand is well characterized using ¹H NMR, ¹³C NMR, 2D HETCOR, mass and IR spectral studies. The chelating tendency of the ligand towards transition metal ions is established using analytical and spectral studies, which reveal the monobasic tridentate nature of the ligand. Octahedral geometry for Co(II), Cu(II) and Zn(II) and tetrahedral geometry for Ni(II) are tentatively proposed. All the synthesized compounds were screened for in vitro anticancer activity against Ehrlich ascites carcinoma and human cancer cell lines (adenocarcinoma HT29, kidney cancer cell line K293 and breast cancer cell line MDA231) using tryphan blue exclusion method and MTT assay.

Full text of DOI:10.1002/aoc.3252

Full Paper
Received: 8 October 2014
Revised: 25 October 2014
Accepted: 26 October 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/aoc.3252
Co(II), Ni(II), Cu(II) and Zn(II) complexes of
isatinyl-2-aminobenzoylhydrazone: synthesis,
characterization and anticancer activity
Rekha S. Hunoora, Basavaraj R. Patila, Dayananda S. Badigera,
Chandrashekhar V. M.b, I. S. Muchchandib and Kalagouda B. Gudasia*
This article describes the synthesis, structural aspects and biological studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes of a new
1
hydrazone derived from the condensation of isatin and 2-aminobenzoylhydrazide. The ligand is well characterized using H
NMR, 13C NMR, 2D HETCOR, mass and IR spectral studies. The chelating tendency of the ligand towards transition metal ions is
established using analytical and spectral studies, which reveal the monobasic tridentate nature of the ligand. Octahedral geom-
etry for Co(II), Cu(II) and Zn(II) and tetrahedral geometry for Ni(II) are tentatively proposed. All the synthesized compounds were
screened for in vitro anticancer activity against Ehrlich ascites carcinoma and human cancer cell lines (adenocarcinoma HT29, kid-
ney cancer cell line K293 and breast cancer cell line MDA231) using tryphan blue exclusion method and MTT assay. Copyright ©
2014 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: isatin; hydrazone; transition metal complex; 2-aminobenzoylhydrazone; anticancer activity
anticancer activity[17–21] and have proven to be promising candi-
dates. This prompted us to synthesize and characterize them
Introduction
Hydrazones derived from organic acid hydrazides are azomethines
characterized by the presence of the triatomic group >C¼N–N<
and form an interesting class of compounds which finds extensive
applications in biological, clinical, analytical and various other
fields.[1–4] The hydrazone functional group >C¼N–N< is very im-
portant for a compound to exhibit biological activity since this
group forms hydrogen bonds with lipid layers and thereby
increases the lipophilic nature of the compound. It has also been
reported that the introduction of a primary amino group on an
aromatic acid hydrazide at the ortho position makes it strongly
active against tuberculosis mycobacteria, and metal complexes of
2-aminobenzoylhydrazide have shown significant anti-tubercular
activity compared to the parent ligand.[5] A great deal of work has
been done on the metal complexes of 2-aminobenzoylhydrazone
due to their enhanced activity compared with the parent
ligand.[6–8]
and evaluate their preliminary cytotoxicity against Ehrlich ascites
carcinoma (EAC) and human cancer cell lines using tryphan blue
exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay.
Keeping in mind the wide spectrum of pharmacological activities
exhibited by indole-2,3-dione and 2-aminobenzoylhydrazide, we have
designed a new hydrazine, namely isatinyl-2-aminobenzoylhydrazone
(ISABH, L) with a free amino group at the ortho position of the hydra-
zide moiety, to enhance the biological activity. Considering the role of
transition metals in the enhancement of biological activity of the li-
gand, metal complexes of ISABH have been synthesized, thoroughly
characterized and screened for their anticancer activity.
Material and Methods
Physical Measurements
Nitrogen- and oxygen-containing ligands have always fasci-
nated coordination chemists. Isatin (indole-2,3-dione; or 2,3-
dioxoindoline; or indoline-2,3-dione) is a most important deriva-
tive of oxindoles and its derivatives are known for a broad range
of biological activities, including antibacterial, antifungal, anticon-
vulsant, antiviral, antiproliferative and anticancer activities.[9–13]
Isatin is also present in biological systems whose metabolic path-
ways are still unknown.[14,15] A variety of metal complexes of isatin
hydrazones have been described in the literature. Many of them
did not reveal a biological relevance, but as soon as pharmacolog-
ical activities started to be demonstrated for these compounds,
their metal complexes gained biological importance and were
investigated for various activities.[16] Hydrazones of isatin and their
transition metal complexes have been extensively studied for their
Methyl anthranilate (SD Fine Chemicals, India), hydrazine hydrate
(SD Fine Chemicals, India) and isatin (Spectrochem, India) were
of AR grade and used without further purification. Solvents were
distilled before use. 2-Aminobenzoylhydrazide was prepared
*
Correspondence to: Kalagouda B. Gudasi, Department of Studies in Chemistry,
Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka, India. E-mail:
kbgudasi@gmail.com
a Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad
580 003, Karnataka, India
b HSK College of Pharmacy, Bagalkot, Karnataka, India
Appl. Organometal. Chem. (2014)
Copyright © 2014 John Wiley & Sons, Ltd.

R. S. Hunoor et al.
following earlier reports.[22] After wet ashing with HCl and HClO4,
cobalt, nickel and copper were determined gravimetrically; zinc
was determined volumetrically.[23] Chloride content was deter-
mined as silver chloride after decomposition with dilute HNO3.[23]
Elemental analysis (C, H, N) was carried out with a TruSpec
CHN/CHNS analyser. The mass spectrum of the ligand was ob-
tained with a Shimadzu GCMS-QP2010S. Magnetic susceptibility
measurements were made at room temperature with a Gouy bal-
ance using Hg[Co(SCN)4] as the calibrant and diamagnetic correc-
tions were made using Pascal’s constants. Electronic spectra were
recorded using a Varian CARY 50 Bio UV–visible spectrophotome-
ter with DMF as solvent. The IR spectra of ligand and complexes
were recorded as KBr pellets in the region 400–4000 cmꢀ1 with a
volume of DMSO was pegged below 0.1% of the total volume. The
control tube had 10 μl of solvent. The final volume was made up to
0.9 ml with MEM. To each tube, 100 μl of tryphan blue solution was
added. The live (without stain) and dead (with blue stain) cells were
counted using a haemocytometer and percentage inhibition of via-
ble cells was calculated using the formula
Number of viable cells
Inhibition of viable cells ð%Þ ¼
ꢁ100:
Total number of cells
1
Nicolet 170SX FT-IR spectrometer. H NMR and 13C NMR spectra
Cytotoxicity Assay using MTT Method (Effect on Cell
Proliferation)
of the ligand and its Zn(II) complex and 2D HETCOR of the ligand
were recorded in DMSO-d6 with a Bruker Avance 400MHz spec-
trometer using tetramethylsilane (TMS) as the internal standard.
Conductivity measurements of 10ꢀ3 M solutions of complexes in
DMF were made using an ELICO-CM82 conductivity bridge pro-
vided with a cell having a cell constant of 0.51. Simultaneous ther-
mogravimetric (TG) and differential thermal analysis (DTA) curves
were recorded with a PerkinElmer TGA7 analyser at a heating rate
of 10°Cminꢀ1 and maximum temperature of 1000°C in nitrogen
atmosphere. The electron paramagnetic resonance (EPR) spectra
of a polycrystalline Cu(II) complex were recorded at room temper-
ature and liquid nitrogen temperature with a Varian E-4 X-band
spectrometer using tetracyanoethylene as the calibrant.
Cells (adenocarcinoma, breast and kidney cancer cell lines) were
seeded at a density of 2 × 105 cells mlꢀ1 per well into a sterile 96-
well plate and allowed to adhere overnight. The solutions of com-
pounds of concentrations 10, 25, 50, 100 and 200 μg mlꢀ1 were pre-
pared in DMSO and 20 μl of the test compound solutions was
added to different wells. Cells were incubated with the test com-
pounds for a period of 24, 48, 72 or 96 h. After the required incuba-
tion period, 50 μl of MTT was added to each well and the plates
were incubated at 37°C in a humid atmosphere with 5% CO2 and
95% air for 4 h. The medium was then gently aspirated, and
150 μl of DMSO was added to dissolve the formazan crystals. The
amount of formazan product was measured spectrophotometri-
cally at 570 nm using an ELISA microplate reader (Biotech, USA).
Each compound concentration had three replicates per assay, and
each experiment was carried out on three separate occasions. The
IC50 value, defined as the drug concentration causing a 50% reduc-
tion in cellular viability, was calculated for each test compound at
96h incubation. This value was used as a means for comparing
the cytotoxicity of the compounds for each of the three cell lines
used in this experiment.
Cell Culture
EAC-bearingBalb/C mice were obtained fromCentralAnimalHouse,
HSK College of Pharmacy, Bagalkot, Karnataka, India. The animal ex-
periments were performed according to the rules and regulations
of the Institutional Animal Ethical Committee (IAEC/Clearance/
2007/1-8), HSK College of Pharmacy, Bagalkot, Karnataka.
The cell lines were maintained in RPMI-1640 medium supple-
mented with 10% heat-inactivated foetal calf serum containing
5% of a mixture of gentamycin (3μg mlꢀ1), penicillin (100 units
mlꢀ1) and streptomycin (100μg mlꢀ1) in the presence of 5% CO2
in air at 37°C and routinely sub-cultured using a 0.25% trypsin–
0.02% ethylenediaminetetraacetic acid solution. Cells were
collected on 12–14 days of post-transplantation suspended in
phosphate buffer saline (pH= 7.4), centrifuged and washed with
cold phosphate buffer saline.
Results and Discussion
Chemistry
Synthesis of ISABH (L)
To a methanolic solution (20 ml) of 2-aminobenzoylhydrazide
(0.151g, 1 mmol), isatin (0.147g, 1 mmol) was added and stirred
for 3 h; a yellowish-orange solid separates (Scheme 1). The solid
was filtered, washed with ethanol and dried in air and recrystallized
from methanol. Yield 92%; m.p. 230–232°C.
Determination of Cytotoxicity of Compounds to EAC Cells
(In Vitro Studies) using Tryphan Blue Exclusion Method (Cell
Viability Test)
Synthesis of Co(II), Ni(II), Cu(II) and Zn(II) complexes of ISABH (C1–C4)
In order to transfer EAC cells from in vivo conditions to in vitro me-
dium, they were drawn with a sterile injector from the peritoneal
cavity of mouse and by diluting with HBSS; concentrations of 107
cells mlꢀ1 were thus obtained. Later these cells were transferred
to test tubes at a final concentration of 375 000 cells mlꢀ1. The test
tube contained Minimal Essential Medium (MEM; Gibco), 2.5% foe-
tal calf serum and 10 mM Hepes. After cells were brought to in the
in vitro method in this way, they were propagated at 37°C.
To 15ml of an ethanolic suspension of L (0.280g, 1 mmol), 5 ml of
an ethanolic solution of equimolar quantity of metal salt (1mmol)
was slowly added with constant stirring. The pH was adjusted to
6–7 by adding 2–3 drops of ammonia, followed by refluxing for
about an hour. The precipitates thus obtained were filtered, washed
repeatedly with ethanol and acetone and dried in air. Yield 60–65%.
The cells (1×106 cells in 0.1 ml of MEM) were incubated in clean
sterile tubes with the test compounds (0.01 ml) for 3 h at 37°C, keep-
ingthefinalvolumeat0.9ml.Thecompoundsweretestedatconcen-
trations of 5, 10, 25, 50, 100, 150, 300, 600 and 1200 μg mlꢀ1, and
these solutions were prepared in dimethylsulfoxide (DMSO). The
Scheme 1. Synthetic route for the preparation of ISABH.
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Copyright © 2014 John Wiley & Sons, Ltd. Appl. Organometal. Chem. (2014)

Complexes of isatinyl-2-aminobenzoylhydrazone
Physical measurements
The elemental analysis of L is consistent with the molecular formula
C15N4H12O2 and its mass spectrum shows m/z peak at 280 that cor-
responds to its molecular weight. The transition metal complexes of
ISABH are stable at room temperature and non-hygroscopic in na-
ture. Complexes are insoluble in common organic solvents but sol-
uble in coordinating solvents like DMF and DMSO. Formulation of
these complexes has been done by elemental analysis and molar
conductance. Analytical data for the complexes are given in Table 1.
Lower molar conductance values of the complexes measured in
DMF (10ꢀ3 M solutions) adequately confirm the non-electrolytic na-
ture of the complexes.
Infrared spectral studies
Infrared spectral data for the ligand and its complexes are pre-
sented in Table 2 along with assignments. Sharp bands of medium
intensity at 3443 and 3349 cmꢀ1 in the ligand spectrum are due to
asymmetric and symmetric stretching vibrations of –NH2 group.[24]
A sharp absorption band at 3197cmꢀ1 is attributed to ν(–NH)
stretching. The absorption bands characteristic of ν(C¼O) vibra-
tions of isatinic and 2-aminobenzoylhydrazide fragments appear
at 1716 cmꢀ1[25] and 1648 cmꢀ1 [26]
respectively, and the absorp-
,
tion band at 1629 cmꢀ1 is assigned to azomethine ν(C¼N)
stretching. The carbonyl stretching frequency of hydrazide is ob-
served at lower frequency compared with ν(C¼O) of aromatic acid
hydrazides without –NH2 group at the ortho position in which it
has appeared at 1673 cmꢀ1 [25]
This may be due to the presence
.
of intramolecular hydrogen bonding between carbonyl oxygen
and hydrogen of –NH2 group, which is clear from the crystal struc-
ture of 2-aminobenzoylhydrazide.[27] In the IR spectra of all the
complexes, the stretching frequency of lactam carbonyl of isatin
has decreased by 36–66 cmꢀ1, indicating the involvement of oxy-
gen of lactam carbonyl in the coordination. On complexation the
ν(C¼O) band of hydrazide disappears and a new band appears at
1614–1618 cmꢀ1, which is attributed to ligation of carbonyl oxygen
via enolization and deprotonation.[24] Moreover in the IR spectra of
the complexes, new absorption bands at 1186–1189 cmꢀ1 are ob-
served and are assigned to the vibrations of C–O–M bonds. These
data indicate that the ligand is in the enolic form in the complexes.
This is also supported by the appearance of a new absorption band
at 1278–1308 cmꢀ1 due to ν(C–O) stretching in all the complexes.
The azomethine stretching frequency in all the complexes has
shifted to lower frequencies, which indicates the involvement of
the nitrogen atom of azomethine group in the coordination with
metal ions.
NMR spectral studies
1
The H NMR spectral assignments were done by comparing with
isatin and 2-aminobenzoylhydrazide.[26] The numbering system
for the assignments of protons is given in Scheme 2. Chemical shift
assignments of 1H NMR and 13C NMR spectra of L and Zn(II) com-
plex are given in Table 3.
The 1H NMR spectrum of L shows singlets at 13.84 and 11.3 ppm
integrating for one proton and 6.74 ppm integrating for two pro-
tons assigned to N2H, N4H and N1H2, respectively, and all are
D2O exchangeable. N1H2 protons appear downfield to TMS, due
to the presence of intramolecular hydrogen bonding between
N1H2 proton and C7 carbonyl oxygen, whereas N2H is sandwiched
between C7 carbonyl and N3¼C8 azomethine nitrogen and also in-
tramolecular hydrogen bonding with C15 carbonyl oxygen. Hence,
N2H proton is observed downfield to TMS owing to its electron
Appl. Organometal. Chem. (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
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R. S. Hunoor et al.
ν(N–H)
Table 2. Diagnostic IR bands of ISABH and its metal complexes
Compound
code
ν(–NH2)
ν(C¼O)
Hydrazide
ν(C¼N)
ν(C¼N)
ν(C–O)
(new)
νsymm
νsym
Lactone
L8
3472m
3406m
3407m
3471s
3349m
3291m
3303m
3362s
1716s
1675s
1656s
1698m
1663s
1648s
n.o.
1629s
1580m
1518s
1577s
1581s
—
—
3197s
3180s
3157m
3254w
3213s
C29
C30
C31
C32
1614s
1615s
1615s
1618s
1283s
1278s
1308s
1293m
n.o.
n.o.
3469s
3329s
n.o.
m = medium; s = strong; w = weak; n.o. = not observed.
carbonyl oxygen on complexation. C2H and C5H show downfield
shifts of 0.53 and 0.11ppm due to coordination through carbonyl
oxygen. No considerable change is observed in the chemical shifts
of remaining aromatic protons. In the 13C NMR spectrum of Zn(II)
complex, signals corresponding to carbonyl carbon C7, lactam car-
bon C15 and azomethine carbon C8 have shifted downfield to
13.64, 3.05 and 4.94 ppm indicating coordination through carbonyl
oxygen, lactam oxygen and azomethine nitrogen.
Scheme 2. Numbering scheme for ISABH.
deficiency. Four doublets at 6.97, 7.60, 7.46 and 6.85 ppm each inte-
grating for one proton are assigned to C2H, C5H, C13H and C10H,
respectively. Four triplets at 7.28, 6.63, 7.11 and 7.38 ppm are attrib-
uted to C3H, C4H, C11H and C12H, respectively. The 13C NMR spec-
trum of ISABH shows 15 signals corresponding to the total number
of carbon atoms present in L. Thus the 1H NMR and 13C NMR anal-
yses indicate the formation of the ligand. Carbon signals due to car-
bonyl carbon C7, azomethine carbon C8 and lactonyl carbon C15
are observed at 165.72, 137.89 and 163.91 ppm, respectively. Re-
maining aromatic carbons are observed in the anticipated region.
Electronic spectral studies and magnetic properties
The electronic spectrum of the ligand shows two prominent ab-
sorptions at 401 and 708 nm. The band at 401 nm correspond to
the azomethine group and the broad band at 708nm corresponds
to the n → π* transition from amide oxygen of isatin to
azomethine group.[28,29] The spectra of all the complexes show
π → π* transitions in the range 372–390nm. In addition, they also
display bands at 455, 446, 455 and 462 nm in the case of Co(II),
Ni(II), Cu(II) and Zn(II) complexes, respectively, and are assigned
to ligand-to-metal charge transfer transitions. A broad band
around 870–894 nm appearing as an envelope in the spectrum
of the Cu(II) complex is assigned to 2Eg → 2T2g transition which re-
veals octahedral geometry.[28,29] The Ni(II) complex exhibits an
1
The H NMR spectrum of Zn(II) complex shows the disappear-
ance of N2H indicating the deprotonation of N2H proton via
enolization and supporting the coordination of the ligand in the
enol form. N1H has shifted upfield by 0.28 ppm indicating the
breakdown of intramolecular bonding between N1H2 proton and
Table 3. 13C NMR and 1H NMR chemical shifts (ppm) of ISABH and its Zn(II) complex
Position
13C NMR
1H NMR
ISABH
Zn(II) complex
ISABH
Zn(II) complex
C1
152.05
112.01
134.44
116.16
121.54
116.16
165.72
137.89
120.89
127.98
123.54
132.27
118.11
143.02
163.91
—
151.22
111.83
133.54
116.20
123.10
114.89
179.36
142.83
116.64
131.01
125.21
133.40
113.82
131.98
166.96
—
—
C2
6.97 (d, 1H) J2,3 = 7.6 Hz
7.08 (d, 1H) J2,3 = 7.6 Hz
C3
7.28 (dd, 1H) J2,3 = 7.6 Hz, J3,4 = 7.5 Hz
7.22 (dd, 1H) J2,3 = 7.6 Hz, J3,4 = 7.5 Hz
C4
6.63 (dd, 1H) J3,4 = 7.5 Hz, J4,5 = 7.5 Hz
6.57 (dd, 1H) J3,4 = 7.5 Hz, J4,5 = 7.5 Hz
C5
7.60 (d, 1H) J4,5 = 7.5 Hz
8.13 (d, 1H) J4,5 = 7.5 Hz
C6
—
—
C7
—
—
C8
—
—
—
C9
—
C10
C11
C12
C13
C14
C15
N1
N2
N4
7.46 (d, 1H) J10,11 = 7.6 Hz
8.05 (d, 1H) J10,11 = 7.6 Hz
7.11 (dd, 1H) J10,11 = 7.6 Hz, J11,12 = 7.7 Hz
7.22 (t, 1H) J10,11 = 7.6 Hz, J11,12 = 7.7 Hz
7.38 (dd, 1H) J11,12 = 7.7 Hz, J12,13 = 7.5 Hz
7.43 (t, 1H) J11,12 = 7.7 Hz, J12,13 = 7.5 Hz
6.85 (d, 1H) J12,13 = 7.5 Hz
6.77(d, 1H) J12,13 = 7.5 Hz
—
—
—
—
6.74 (s, 2H)
13.84 (s, 1H)
11.32 (s, 1H)
7.02 (s, 2H)
n.o.
—
—
11.22 (s, 1H)
s = singlet, d = doublet, dd = doublet of doublets, n.o. = not observed.
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Appl. Organometal. Chem. (2014)

Complexes of isatinyl-2-aminobenzoylhydrazone
absorption at 613–678 nm and this absorption is observed as a
split band, which is evidence of spin–orbit coupling in mixing a
Pharmacology
Tryphan blue exclusion method (cell viability test)
1
3
spin singlet Eg with T1g(F) thereby allowing the spin-forbidden
transition to gain intensity from the spin-allowed one. The low
intensities of the d–d bands compared with intra-ligand and
metal-to-ligand charge transfer absorptions in DMF solution
prevent an accurate assignment for the Co(II) complex.
The corrected magnetic moments at room temperature for
diamagnetism are given in Table 1. The magnetic moment of
the Co(II) complex is 4.72 BM which is greater than the spin-only
value (3.46 BM) indicating the high-spin state of the complex and
an octahedral geometry.[28] The magnetic moment observed for
the Ni(II) complex of 2.84 BM reveals the high-spin state of the
complex. The magnetic moment value for the Cu(II) complex is
well within the range corresponding to a spin-only value for
one unpaired electron with an octahedral geometry and indicat-
ing no metal–metal interaction.[30]
The compounds were tested using the short-term in vitro cytotoxic-
ity towards EAC cells as a preliminary screening technique of
tryphan blue exclusion method (cell viability test) for their cytotoxic
potential.[32] This is one of the methods for assessing the cytotoxic-
ity of anticancer compounds. This test is based on the principle that
a living cell membrane has the ability to prevent the entry of a dye.
Hence, the cells remain unstained and can be easily distinguished
from dead cells that take up the dye. The percentage of viable cells
was determined. Results for the short-term in vitro cytotoxicity of
the compounds are shown in Table 4. These preliminary experi-
ments were carried out at nine different concentrations of the com-
pounds. The ligand and all the complexes are found to exhibit
higher cytotoxicity at higher concentration. The ligand is found to
be moderately active and to exhibit the highest percentage of inhi-
bition (56.55%) at 1200 μg mlꢀ1. All the complexes exhibit higher
cytotoxicity compared to the parent ligand. The Ni(II) and Zn(II)
complexes (C2 and C4) are found to exhibit moderate activity with
highest percentage of inhibition of 57.41 and 66.76%, respectively,
at the highest concentration. The Co(II) and Cu(II) complexes show
very good activity. The Cu(II) complex exhibits promising activity
causing 52.08% cell death at 10μg mlꢀ1 and percentage of inhibi-
tion of viable cells increases with increasing concentration. At
1200 μg mlꢀ1, the percentage inhibition is found to be 99.52%
which is the highest among all the tested compounds.
EPR spectral studies
The EPR spectra of a polycrystalline sample of Cu(II) complex at
room temperature (300 K) and liquid nitrogen temperature (77 K)
exhibit similar features with g1 = 2.04, g2 = 2.06 and g3 = 2.13 sug-
gesting a rhombic pattern. The observed g values are less than
2.3 indicating the covalent nature of the metal–ligand
bonding.[30,31]
Thermal studies
The IC50 values of all the compounds show that the Cu(II) com-
plex exhibits the highest activity, showing 50% inhibition of EAC
cells at 15.35 μg mlꢀ1 (Fig. 1). The next highest activity is observed
for Co(II) complex with IC50 of 32.87 μg mlꢀ1. The order of cytotox-
icity is found to be C3> C1 > C4> C2 > L.
The Ni(II) complex shows a weight loss of 4.02% (calc. 4.60%) be-
tween 50 and 100°C indicating the presence of one uncoordinated
water molecule. Weight loss of 9.40% (calc. 9.25%) around 300–
350°C corresponds to the loss of one coordinated chlorine atom.
The plateau obtained after heating the Ni(II) complex above
900°C corresponds to the formation of stable NiO, and the metal
content calculated from this residue (15.08%) tallies with the metal
analysis (15.27%). In the case of the Zn(II) complex, the residue
weight obtained after heating the complex above 900°C corre-
sponds to the formation of stable ZnO, and the metal content thus
calculated (11.03%) is in good agreement with the metal analysis
(10.85%). No weight loss is observed in the temperature range
30–200°C, indicating the absence of lattice-held and coordinated
solvent molecules. Thus thermal studies support the suggested
compositions for the complexes.
MTT assay (cell viability test)
The effects of ISABH and its Co(II), Ni(II), Cu(II) and Zn(II) complexes
on cell proliferation were tested against various human cancer cell
lines, namely human adenocarcinoma HT29, kidney cancer cell
line K293 and breast cancer cell line MDA231, at five concentra-
tions, determined following incubation of model cells using the
MTT assay.[33] This is a colorimetric determination to test the
in vitro growth inhibition effect of the test compounds in which
MTT is converted into formazan blue by living cells. The assay uses
a tetrazolium salt, MTT, to assess cellular metabolism and hence
viability. In metabolically active cells, MTT is reduced by the mito-
chondrial enzyme succinate dehydrogenase with the formation of
insoluble purple formazan crystals. These are then solubilized, and
Fast atom bombardment mass spectral studies
The fast atom bombardment mass spectra of complexes C2 and
C4 show peaks with m/z values of 391 and 621 corresponding to
the molecular weight of the respective complexes. These values
are in good agreement with the proposed compositions for the
complexes.
the absorbance measured spectrophotometrically at 570nm.[34]
A
concentrated stock solution of each test compound was prepared
in DMSO and stored at ꢀ20°C until required for use. Prior to the
Table 4. Short-term in vitro cytotoxicity of compounds towards EAC cells
Compound
Percentage cell death at different concentrations after 3 h
5 μg mlꢀ1 10 μg mlꢀ1 25 μg mlꢀ1 50 μg mlꢀ1 100 μg mlꢀ1 150 μg mlꢀ1 300 μg mlꢀ1 600 μg mlꢀ1 1200 μg mlꢀ1
IC50
(μg mlꢀ1
)
L
23.65
27.87
23.17
40.71
27.23
32.21
29.15
23.86
52.08
28.05
36.63
48.81
24.44
52.30
43.48
36.78
61.57
25.64
53.25
45.64
42.68
96.42
30.87
94.60
51.36
41.78
97.12
46.78
97.48
57.46
50.81
95.49
53.68
97.69
60.67
49.73
98.31
54.33
98.85
67.78
56.55
99.404
57.41
99.52
66.76
263.66
32.87
246.96
15.35
82.44
C1
C2
C3
C4
Appl. Organometal. Chem. (2014)
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R. S. Hunoor et al.
Table 6. IC50 values of compounds against human cancer cell lines
IC50 values of compounds against EAC cells
300
250
200
150
100
50
Compound
IC50 (μg mlꢀ1
)
263.66
246.96
HT29
K293 (kidney
MDA231 (breast
cancer cell line)
(adenocarcinoma) cancer cell line)
L
41.42
56.03
261.42
307.25
160.99
192.53
570.95
106.34
324.18
145.93
158.06
150.34
C1
C2
C3
C4
104.18
60.87
82.44
C4
148.38
32.87
C1
15.35
C3
0
L
C2
cancer cell lines and its cytotoxicity against human adenocarci-
noma HT29 is significant with 76.49% inhibition. Its activity against
kidney cancer K293 is not notable but considerable inhibitory activ-
ity is observed against breast cancer MDA231 (67.85%). The IC50 of
the ligand (Table 6) against HT29, K293 and MDA231 clearly illus-
trates that the ligand is potent against HT29 with IC50 of
41.42μg mlꢀ1. The Co(II) complex exhibits promising cytotoxicity
against HT29 with inhibiting proliferation rate of 91.69% which is
also obvious from its IC50 of 56.03 μg mlꢀ1. Its cytotoxicity against
K293 and MDA231 is found to be poor. All the other complexes
are found to be least potent against K293 and MDA231 while they
exhibit promising activity against HT29. The Cu(II) complex exhibits
Compounds
Figure 1. In vitro cytotoxicity against EAC cells.
assay, the stock solution was diluted to the required dilutions
using blank RPMI-1640 media. The maximum percentage of
DMSO present in the wells was 0.5% (v/v) and this was incorpo-
rated as a negative control in all the experiments. Compounds
were tested against a panel of human tumour cell lines of differ-
ent origins (breast, kidney) at five concentrations: 10, 25, 50, 100
and 200 μg mlꢀ1 (Table 5).
noteworthy cytotoxicity against HT29 with IC50 of 60.87μg mlꢀ1
.
All the complexes exhibit concentration-dependent cytotoxicity.
The activity of the Zn(II) complex is lower against all the strains.
The ligand is found to exhibit very good activity against all the
Table 5. Anticancer activity of test compounds from MTT assay
Compound
Concentration
Absorbance (percentage inhibition)
(μg mlꢀ1
)
HT29
K293
MDA231
(human adenocarcinoma)
(human kidney cancer cell line)
(human breast cancer cell line)
L
200
100
50
0.732 (00.00)
0.132 (76.49)
0.221 (60.68)
0.312 (44.42)
0.368 (34.40)
0.047 (91.69)
0.167 (70.27)
0.256 (54.42)
0.324 (42.34)
0.422 (24.96)
0.115 (79.50)
0.256 (54.43)
0.368 (34.51)
0.455 (19.21)
0.467 (16.90)
0.062 (88.95)
0.152 (72.22)
0.149 (73.52)
0.436 (22.41)
0.446 (20.61)
0.231 (58.86)
0.322 (42.64)
0.368 (34.51)
0.456 (18.85)
0.524 (06.76)
0.562
0.729 (40.71)
0.921 (25.08)
1.125 (08.49)
1.021 (16.96)
1.112 (09.57)
0.824 (32.99)
0.982 (20.15)
1.024 (16.73)
1.135 (07.75)
1.201 (02.33)
0.526 (57.11)
0.719 (41.51)
0.985 (19.93)
1.027 (16.53)
1.125 (08.56)
0.648 (47.29)
0.802 (34.75)
0.925 (24.75)
1.162 (05.48)
1.212 (01.43)
1.022 (16.97)
1.142 (07.10)
1.192 (03.04)
1.225 (0.439)
1.212 (01.43)
1.2299
0.152 (67.85)
0.198 (58.13)
0.261 (44.84)
0.351 (25.78)
0.398 (15.89)
0.310 (34.42)
0.350 (25.95)
0.382 (19.25)
0.412 (12.89)
0.414 (13.03)
0.198 (58.07)
0.268 (43.27)
0.302 (36.15)
0.324 (31.50)
0.350 (26.01)
0.202 (57.41)
0.285 (39.87)
0.332 (29.81)
0.362 (23.45)
0.389 (17.70)
0.212 (55.11)
0.254 (46.30)
0.291 (38.41)
0.352 (25.52)
0.421 (11.01)
0.473
25
10
C1
200
100
50
25
10
C2
200
100
50
25
10
C3
200
100
50
25
10
C4
200
100
50
25
10
Growth control
—
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Appl. Organometal. Chem. (2014)

Complexes of isatinyl-2-aminobenzoylhydrazone
IC50 values of compounds against human cancer cell lines
600
HT29
500
K293
MDA231
400
300
200
100
0
Figure 3. Proposed structure of the complexes.
presented in Fig. 3. The preliminary in vitro cytotoxicity activity of
the compounds is impressive and further studies are necessary to
elucidate their exact mechanism of action.
L
C1
C2
C3
C4
Compounds
Acknowledgements
Figure 2. Cytotoxicity of L and its transition metal complexes against
human cancer cell lines.
The authors thank USIC, Karnatak University, Dharwad for providing
spectral facilities. Thanks are due to IISc Bangalore, STIC Cochin and
IIT Mumbai for NMR, thermal and ESR analyses, respectively. The au-
thors also thank the Department of Physics, Karnatak University,
Dharwad for extending facilities to carry out magnetic susceptibility
measurements. One of the authors (R.S.H.) thanks Karnatak Univer-
sity, Dharwad for providing a Research Fellowship.
Among all the complexes, C1 and C3 exhibit impressive potency
with IC50 of 56.03 and 60.87 μg mlꢀ1 against HT29. For remaining
strains C2 is found to exhibit better activity compared to the other
complexes. The ligand is found be a good potential inhibitor of cell
proliferation of HT29, K293 and MDA231 cell lines compared to its
complexes. The in vitro cytotoxicity of the compounds against hu-
man cancer cell lines is displayed in Fig. 2 showing their IC50 values.
Anticancer drugs exert their action by various mechanisms. The
isatin hydrazones and their metal complexes, in particular copper
complexes, were reported to exert their action through anti-
proliferative and pro-apoptotic action. The protein kinases are crit-
ical components of signalling pathways in control of cell prolifera-
tion of many human cancers. The isatin derivatives selectively
inhibit this class of proteins and effects on cell kinase activity, cell
proliferation, cell cycle progression and apoptosis. These have been
reported as potent inhibitors of vascular endothelial growth factor
(VEGF) that stimulates angiogenesis. The oxindole derivatives have
been reported to act as angiogenesis inhibitors. Angiogenesis in-
hibitors stop the growth of blood vessels from surrounding tumour
tissues and VEGF exerts its action by binding to cell surface
receptors.
Compounds in the present investigation being derivatives of
isatin (L, C1–C4) exhibit their cytotoxicity activity against EAC and
human cancer cell lines possibly through the abovementioned
mechanisms, i.e. through pro-apoptosis, or through inhibition of
protein kinase or through inhibition of cyclin-dependent kinases.
Another possible mechanism could be through angiogenesis inhib-
itory activity. Thus the preliminary cytotoxicity results of ISABH and
its transition metal complexes show that these can be a potent class
of anticancer agents. Though the mode of action is unclear, it is en-
visaged that further molecular-level studies could elaborate on
their exact mode of action.
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version of this article at the publisher’s web site.
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