Antiproliferative effects of metal complexes of new isatin hydrazones against HCT116, MCF7 and HELA tumour cell lines

Article abstract of DOI:10.3109/14756366.2011.588950

New hydrazone ligands (HL) derived from 5-substituted isatins and 1-(4-(2-methoxybenzyl)-6-arylpyridazin-3-yl)hydrazines and its complexes with Co(II) and Cu(II) were synthesized. The new hydrazones and their complexes were characterized by means of eleme

Full text of DOI:10.3109/14756366.2011.588950

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(3): 330–338
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.588950
RESEARCH ARTICLE
Antiproliferative effects of metal complexes of new isatin
hydrazones against HCT116, MCF7 and HELA tumour
cell lines
Nadia G. Kandile, Mansoura I. Mohamed, and Hind M. Ismaeel
Department of Chemistry, Faculty of Women, Ain Shams University, Heliopolis, Cairo, Egypt
Abstract
New hydrazone ligands (HL) derived from 5-substituted isatins and 1-(4-(2-methoxybenzyl)-6-arylpyridazin-3-yl)
hydrazines and its complexes with Co(II) and Cu(II) were synthesized. The new hydrazones and their complexes were
characterized by means of elemental, spectral analyses and magnetic studies. Primary cytotoxicity evaluation of HL
5a and the new complexes showed that these complexes could act as anticancer agents since they reduced the
growth of samples of human tumour cell lines (HCT116(Colon), MCF7(Breast) and HELA(Cervix)) to ≤18.5 μg/mL for the new
complexes.
Keywords: Hyrazone ligands, 5-substituted isatins, cytotoxicity, anticancer drugs
Introduction
and growth factors that are up-regulated in cancer cells.
ese provide new targets for anticancer drug design.
e field of metal-based anticancer drug design can
be divided into two different approaches: classical and
non-classical chemotherapeutics3. Hydrazones, which
may also be considered as Schiff bases or imines, are
an important class of compounds in medicinal and
pharmaceutical field; they show biological applications
including antibacterial, antifungal4–6; it can act as ligands
in interaction with various metal ions. Hydrazone ligands
(HL) possessing potential donor sites that have coordi-
nation capability are biologically active may be due
to the presence of an azomethine and uncoordinated
α-nitrogen of the hydrazone chromosphere; this lead to
more research on the synthesis of metal complexes of
new hydrazones with significant biological activity7–9. One
of the most important goals of pharmacological research
is the search for new molecular structures, which exhibit
effective antitumour activities. is has driven inorganic
and organometallic chemists to look for new metal com-
pounds with good activities, preferably against tumours
that are responsible for high cancer mortality. In view of
Metals, in particular, transition metals offer potential
advantages over the more common organic-based drugs,
including a wide range of coordination numbers and
geometries, accessible redox states, ‘tune-ability’ of the
thermodynamics and kinetics of ligand substitution and
a wide structural diversity. Medicinal inorganic chem-
istry is a thriving area of research, which was initially
fuelled by the discovery of the metallopharmaceutical
cisplatin about 40 years ago1,2. Regardless of the achieve-
ments of current platinum drugs, there are some major
drawbacks: they are efficient only for a limited range of
cancers; some tumours can have acquired or intrinsic
resistance; and they often cause severe side effects, such
as nausea, bone marrow suppression and kidney toxicity.
Although ~10 other platinum compounds are currently
in clinical trials, the cisplatin derivatives have not been
able to address sufficiently many of the disadvantages
associated with cisplatin. ere is a need, therefore, for
new approaches that are purposefully designed to cir-
cumvent these drawbacks. Recent progress in the field
of cell biology has resulted in the discovery of receptors
Address for Correspondence: Prof. Nadia G. Kandile, Department of Chemistry, Faculty of Women, Ain Shams University, Heliopolis 11757,
Cairo, Egypt. Tel: +20 24157804. Fax: +20 24157804. E-mail: nadiaghk@yahoo.com
(Received 04 April 2011; revised 10 May 2011; accepted 11 May 2011)
330

Antiproliferative effects of metal complexes of new hydrazones 331
these observations and as a part of our ongoing research
programme for the synthesis of biologically active
compounds10–13, the present article includes synthesis,
spectral characterization and preliminary evaluation of
their antiproliferative activities of new hydrazones and
its metal complexes.
3-[2-(4-(2-Methoxybenzyl)-6-p-tolylpyridazin-3-yl)hydrazon]
indolin-2-one (5b). Orange. Yield 75%, mp 260–262°C14.
3-[2-(4-(2-Methoxybenzyl)-6-(4-chlorophenyl)pyridazin-3-yl)
hydrazono]-indolin-2-one (5c) Orange. Yield 83%, mp
270–272°C. IR [cm−1]: 3406 NH(hydrazone), 3155 NH(oxindole)
,
1680 C=O(oxindole), 1616, 1546 C=N. GC-MS (m/z): 469.75
(M+). Calcd. for C26H20ClN5O2: C, 66.39; H, 4.25; N, 14.89;
Cl, 7.54; Found: C, 66.35; H, 4.25; N, 14.64; Cl, 7.22.
Experimental
Chemistry
Chemicals and solvents
3-[2-(4-(2-Methoxybenzyl)-6-phenylpyridazin-3-yl)
All the chemicals used were of reagent grade. 5-Sub-
stituted isatins were synthesized using Sandemyer
methodology starting from the corresponding amines.
1-(4-(2-Methoxybenzyl)-6-arylpyridazin-3-yl)hydra-
zines were synthesized according to earlier reports13.
Metal salts were of analytical reagent (E. Merk) as
chloride salts. e organic solvents used were either
spectroscopically pure or purified by the recommended
methods.
hydrazon]-5-methylindolin-2-one (5d) Red. Yield 51.9%,
mp 228°C. IR [cm−1]: 3420 NH(hydrazone), 3180 NH(oxindole)
,
1686 C=O(oxindole), 1625, 1545 C=N. 1H NMR [DMSO d6, 300
MHz] δ:11.14 (s, 1H, NH(oxindole)), 10.37 (s, 1H, NH(hydrazone)),
7.83–7.15 (m, 12H, 3Ar-H), 6.70 (s, 1H, CH), 4.34 (s, 2H,
CH2), 3.80 (s, 3H, OCH3 of CH3O-Ar), 2.56 (s, 3H, CH3 of
CH3-Ar). GC-MS (m/z): 450 (M+). Calcd. for C27H23N5O2:
C, 72.14; H, 5.16; N, 15.58; Found: C, 72.25; H, 5.15; N,
15.32.
Instrumental measurement
3-[2-(4-(2-Methoxybenzyl)-6-p-tolylpyridazin-3-yl)
Elemental analyses (C, H, N and Cl) of the synthesized
compounds were carried out by Microanalytical Center,
Cairo University. e metal percentage was estimated
using Inductively Coupled Argon Plasma (ICP) tech-
nique of the type 6500 Duo, ermo Scientific, England.
e 1000 mg/L multi-element and certified standard
solution (Merck, Germany) were used as stock solution
for instrument standardization. Microwave Digestion
Lab station closed system, Ethos Pro, Milestone, Italy
was used to digest the organic matter in aqua regia.
e infrared spectra were measured on Jasco instru-
ment (Model 6100, Japan) in the transmittance mode
at a resolution of 4 cm−1 using potassium bromide
Wafer technique. e mass spectra were measured on
mass spectrophotometer HP Model GC-MS-QPL000EX
(Shimadzu) at 70 eV. UV–vis spectra were measured
on UV-1600 spectrophotometer using dimethylfor-
mamide (DMF) as solvent. e 1H NMR were measured
on Varian Genini-300 MHz spectrophotometer using
dimethyl sulphoxide (DMSO) as solvent. e magnetic
moment measurement was performed using Faraday
method.
hydrazono]-5-methylindolin-2-one (5e) Red. Yield 76.7%,
mp 248°C. IR [cm−1]: 3398 NH(hydrazone), 3167 NH(oxindole)
,
1680 C=O(oxindole), 1618, 1584 C=N. GC-MS (m/z): 464
(M++1). Calcd. for C28H25N5O2: C, 72.55; H, 5.44; N, 15.11;
Found: C, 72.52; H, 5.45; N, 15.00.
3-[2-(4-(2-Methoxybenzyl)-6-(4-chlorophenyl)pyridazin-
3-yl)hydrazono]-5-methylindolin-2-one (5f). Red. Yield
70%, mp 234–235°C. IR [cm−1]: 3406 NH(hydrazone), 3146
NH(oxindole), 1682 C=O(oxindole), 1621, 1582 C=N. GC-MS
(m/z): 484 (M+), 486 (M++2). Calcd. for C27H22ClN5O2: C,
67.00; H, 4.58; N, 14.47; Cl, 7.33; Found: C, 67.15; H, 4.60;
N, 14.32; Cl 7.30.
3-[2-(4-(2-Methoxybenzyl)-6-phenylpyridazin-3-yl)
hydrazon]-5-chloroindolin-2-one (5g) Orange. Yield 60%,
mp 240°C. IR [cm−1]: 3370 NH(hydrazone), 3156 NH(oxindole)
,
1704 C=O(oxindole), 1618, 1589 C=N. GC-MS (m/z): 470 (M+),
472 (M++2). Calcd. for C26H20ClN5O2: C, 66.39; H, 4.25; N,
14.89; Cl, 7.54; Found: C, 66.69; H, 4.52; N, 14.90; Cl 7.55.
3-[2-(4-(2-Methoxybenzyl)-6-p-tolylpyridazin-3-yl)
hydrazono]-5-chloroindolin-2-one (5h) Violet. Yield 76%,
mp268–270°C. IR[cm−1]:3413NH(hydrazone), 3182NH(oxindole)
,
Synthesis of hydrazones (5a–5r)
1705 C=O(oxindole), 1624, 1566 C=N. 1H NMR [DMSO d6, 300
MHz] δ:11.12 (s, 1H, NH(oxindole)), 10.58 (s, 1H, NH(hydrazone)),
7.98–7.13 (m, 11H, 3Ar-H), 6.82 (s, 1H, CH), 4.33 (s, 2H,
CH2), 3.38 (s, 3H, OCH3 of CH3O-Ar), 2.51 (s, 3H, CH3 of
CH3-Ar). GC-MS (m/z): 484 (M+). Calcd. for C27H22ClN5O2:
C, 67.00; H, 4.58; N, 14.47; Cl, 7.33; Found: C, 66.90; H,
4.48; N, 14.40; Cl, 7.33.
A mixture of a 1-[4-(2-methoxybenzyl)-6-arylpyridazin-
3-yl]hydrazines (4a–4c) (0.01 mol) and 5-substituted
isatin (0.01 mol) was heated under reflux in absolute
ethanol (20 mL) in presence of catalytic amount of glacial
acetic acid for 3 h. During this period, the product was
separated from the reaction mixture as a precipitate was
collected and crystallized from ethanol to give the title
compounds.
3-[2-(4-(2-Methoxybenzyl)-6-(4-chlorophenyl)pyridazin-3-
yl)hydrazono]-5-chloroindolin-2-one (5i) Violet. Yield
73%, mp 264–265°C. IR [cm−1]: 3433 NH(hydrazone), 3145
NH(oxindole), 1689 C=O(oxindole), 1624, 1546 C=N. GC-MS
3-[2-(4-(2-Methoxybenzyl)-6-phenylpyridazin-3-yl)
hydrazono]indolin-2-one (5a). Orange. Yield 70%, mp
208–210°C14.
© 2012 Informa UK, Ltd.

332 N. G. Kandile et al.
(m/z): 504 (M+), 505 (M++1). Calcd. for C26H19Cl2N5O2: C,
61.91; H, 3.80; N, 13.89; Cl, 14.06; Found: C, 61.85; H, 3.68;
N, 13.72; Cl 14.14.
NH(oxindole), 1699 C=O(oxindole), 1624, 1585 C=N. GC-MS
(m/z): 454 (M+), 455 (M++1), 456 (M++2). Calcd. for
C26H20FN5O2: C, 68.86; H, 4.45; N, 15.45; Found: C, 68.99;
H, 4.55; N, 15.10.
3-[2-(4-(2-Methoxybenzyl)-6-phenylpyridazin-3-yl)
hydrazono]-5-methoxyindolin-2-one (5j) Red. Yield 90%,
3-[2-(4-(2-Methoxybenzyl)-6-p-tolylpyridazin-3-yl)
hydrazono]-5-fluoroindolin-2-one (5q) Orange. Yield
88.23%, mp 300°C. IR [cm−1]: 3398 NH(hydrazone), 3158
NH(oxindole),1697C=O(oxindole),1624,1542C=N.GC-MS(m/z):
467 (M+), 468 (M++1). Calcd. for C27H22FN5O2: C, 69.36; H,
4.74; N, 14.98; Found: C, 69.31; H, 4.72; N, 14.88.
mp210–211°C. IR[cm−1]:3422NH(hydrazone), 3152NH(oxindole)
,
1680 C=O(oxindole), 1625, 1587 C=N. 1H NMR [DMSO d6, 300
MHz] δ:10.97(s, 1H, NH(oxindole)), 10.30 (s, 1H, NH(hydrazone)
)
8.00–6.85 (m, 12H, 3Ar-H), 6.77 (s, 1H, CH), 4.11 (s, 2H,
CH2), 3.38–3.16 (s, 6H, 2OCH3 of 2CH3O-Ar). GC-MS
(m/z): 466 (M+). Calcd. for C27H23N5O3: C, 69.66; H, 4.98;
N, 15.05; Found: C, 69.67; H, 4.95; N, 14.92.
3-[2-(4-(2-Methoxybenzyl)-6-(4-chlorophenyl)pyridazin-3-
yl)hydrazon]-5-fluoroindolin-2-one (5r) Orange. Yield
55.24%, mp 296–298°C. IR [cm−1]: 3334 NH(hydrazone), 3156
NH(oxindole), 1698 C=O(oxindole), 1621, 1579 C=N. 1H NMR
[DMSO d6, 300 MHz] δ: 11.12 (s, 1H, NH(oxindole)), 10.45 (s,
1H, NH(hydrazone)), 8.06–7.02 (m, 11H, 3Ar-H), 6.84 (s, 1H,
CH), 4.19 (s, 2H, CH2), 3.82 (s, 3H, OCH3 of CH3O-Ar).
GC-MS (m/z): 488 (M+), 489 (M++1), 490 (M++2). Calcd.
for C26H19ClFN5O2: C, 64.00; H, 3.93; N, 14.36; Cl, 7.27;
Found: C, 64.11; H, 4.18; N, 14.02; Cl, 7.33.
3-[2-(4-(2-Methoxybenzyl)-6-p-tolylpyridazin-3-yl)
hydrazono]-5-methoxyindolin-2-one (5k) Brick red. Yield
94%, mp 220–222°C. IR [cm−1]: 3362 NH(hydrazone), 3165
NH(oxindole), 1683 C=O(oxindole), 1627, 1586 C=N. GC-MS
(m/z): 480 (M+). Calcd. for C28H25N5O3: C, 70.13; H, 5.26;
N, 14.61; Found: C, 69.58; H, 5.21; N, 14.57.
3-[2-(4-(2-Methoxybenzyl)-6-(4-chlorophenyl)pyridazin-
3-yl)hydrazon]-5-methoxyindolin-2-one (5l) Brick red.
Yield 94%, mp 250°C. IR [cm−1]: 3415 NH(hydrazone), 3153
NH(oxindole), 1683 C=O(oxindole), 1625, 1590 C=N. GC-MS
(m/z): 500 (M+). Calcd. for C27H22ClN5O3: C, 64.86; H, 4.44;
N, 14.01; Cl, 7.09; Found: C, 64.57; H, 4.33; N, 13.80; Cl,
7.09.
Synthesis of Co(II) and Cu(II) complexes (6a–6k)
Warm ethanolic solutions of the ligand (20 mmol) and
the metal chloride (10 mmol) were mixed. e reaction
mixture was refluxed and stirred for 3 h. e solid prod-
uct obtained after evaporation under reduced pressure
was filtered and crystallized from ethanol to give the
complexes (6a–6k).
3-[2-(4-(2-Methoxybenzyl)-6-phenylpyridazin-3-yl)
hydrazono]-5-nitroindolin-2-one (5m) Orange. Yield
74.44%, mp 280–282°C. IR [cm−1]: 3403 NH(hydrazone), 3162
NH(oxindole), 1699 C=O(oxindole), 1624, 1585 C=N. GC-MS
(m/z): 480 (M+). Calcd. for C26H20N6O4: C, 64.99; H, 4.20;
N, 17.50; Found: C, 64.80; H, 4.50; N, 17.25.
Copper(II) complex (6a) Brick red. Yield 60%, mp
280–281°C. IR [cm−1]: 3327 NH(hydrazone), 3178 NH(oxindole)
,
1645 C=O(oxindole), 1575, 1540 C=N, 604, 509 (M-N), 426
(M-O). λmax [nm]: 483, 394, 265. μeff: 1.87 B.M. Calcd.
for C56H50CuN10O6: C, 65.77; H, 4.93; N, 13.70; Cu, 6.21;
Found: C, 66.40; H, 4.47; N, 13.44; Cu, 6.57.
3-[2-(4-(2-Methoxybenzyl)-6-p-tolylpyridazin-3-yl)
hydrazono]-5-nitroindolin-2-one (5n) Red. Yield 75.67%,
mp308–310°C. IR[cm−1]:3417NH(hydrazone), 3104NH(oxindole)
,
Cobalt(II) complex (6b) Violet. Yield 50%, mp 282–
1677 C=O(oxindole), 1614, 1591 C=N. 1H NMR [DMSO d6, 300
MHz] δ: 11.17 (s, 1H, NH(oxindole)), 9.19 (s, 1H, NH(hydrazone)),
7.96–7.02 (m, 11H, 3Ar-H), 6.92 (s, 1H, CH), 4.34 (s, 2H,
CH2), 3.84 (s, 3H, OCH3 of CH3O-Ar), 2.49 (s, 3H, CH3 of
CH3-Ar). GC-MS (m/z): 495 (M+). Calcd. for C27H22N6O4:
C, 65.57; H, 4.48; N, 17.00; Found: C, 65.64; H, 4.40; N,
16.77.
284°C. IR [cm−1]: 3401 NH(hydrazone), 3183 NH(oxindole)
,
1615 C=O(oxindole), 1567, 1524 C=N, 591, 545 (M-N), 462
(M-O). λmax [nm]: 485.5, 392, 265. μeff: 5.18 B.M. Calcd.
for C56H50CoN10O4: C, 68.21; H, 5.11; N, 14.21; Co, 5.98;
Found: C, 68.09; H, 5.34; N, 13.55; Co, 5.40.
Copper(II) complex (6c) Golden brown. Yield 68%,
mp 273°C. IR [cm−1]: 3316 NH(hydrazone), 3165 NH(oxindole)
,
3-[2-(4-(2-Methoxybenzyl)-6-(4-chlorophenyl)pyridazin-3
-yl)hydrazono]-5-nitroindolin-2-one (5o) Orange. Yield
58.92%, mp 304°C. IR [cm−1]: 3410 NH(hydrazone), 3168
NH(oxindole), 1716 C=O(oxindole), 1617, 1593 C=N. GC-MS
(m/z): 516 (M++2). Calcd. for C26H19ClN6O4: C, 60.64; H,
3.72; N, 16.32; Cl, 6.88; Found: C, 60.45; H, 3.92; N, 16.05;
Cl, 7.03.
1645 C=O(oxindole), 1568, 1523 C=N, 577, 504 (M-N), 467
(M-O). λmax [nm]: 477.5, 381, 264. μeff: 1.88 B.M. Calcd.
for C52H42CuN10O4: C, 66.83; H, 4.53; N, 14.99; Cu, 6.80;
Found: C, 66.78; H, 4.53; N, 14.66; Cu, 6.45.
Cobalt(II) complex (6d) Violet. Yield 56.9%, mp 164°C. IR
[cm−1]: 3324 NH(hydrazone), 3096 NH(oxindole), 1644 C=O(oxindole)
,
1565, 1524 C=N, 593, 549 (M-N), 425 (M-O). λmax [nm]:
478.5, 389, 266.5. μeff: 5.44 B.M. Calcd. for C52H42CoN10O4:
C, 67.16; H, 4.55; N, 15.07; Co, 6.34; Found, 66.89; H, 4.29;
N, 14.76; Co, 6.33.
3-[2-(4-(2-Methoxybenzyl)-6-phenylpyridazin-3-yl)
hydrazono]-5-fluoroindolin-2-one (5p) Orange. Yield
52%, mp 250°C. IR [cm−1]: 3399 NH(hydrazone), 3162
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative effects of metal complexes of new hydrazones 333
1. Cells were plated in 96-multiwell plate (105 cells/
well) for 24 h before treatment with the compound to
Copper(II) complex (6e) Golden brown. Yield 52.22%,
mp 310°C. IR [cm−1]: 3318 NH(hydrazone), 3155 NH(oxindole)
1676 C=O(oxindole), 1569, 1533 C=N, 586, 533 (M-N), 447
(M-O). λmax [nm]: 482.5, 264. μeff: 2.22 B.M. Calcd. for
C52H42Cl2CuN12O10: C, 55.30; H, 3.75; N, 14.89; Cu, 5.63; Cl,
6.28; Found, 55.31; H, 3.91; N, 15.22; Cu, 6.13; Cl, 7.12.
,
allow attachment of cell to the wall of the plate.
2. Different concentrations of the compound under
test (0, 1, 2.5, 5 and 10 µg/mL) were added to the cell
monolayer triplicate walls that were prepared for
each individual dose.
3. Monolayer cells were incubated with the compound
for 48 h at 37°C and in atmosphere of 5% CO2.
4. After 48 h, cells were fixed, washed and stained with
sulpho-rhodamine-B stain.
5. Excessstainwaswashedwithaceticacidandattached
stain was recovered with Tris–EDTA buffer.
6. Colour intensity was measured in an ELISA reader.
7. e relation between the surviving fraction and drug
concentration is plotted to give the survival curve of
each tumour cell line after the specified compound.
Cobalt(II) complex (6f) Dark brown. Yield 30.20%, mp
260°C. IR [cm−1]: 3316 NH(hydrazone), 3185 NH(oxindole)
,
1647 C=O(oxindole), 1566, 1527 C=N, 592, 507 (M-N), 456
(M-O). λmax [nm]: 480, 414, 267. μeff: 3.82 B.M. Calcd. for
C52H38Cl2CoF2N10O4: C, 60.35; H, 3.70; N, 13.54; Co, 5.70;
Cl, 6.85; Found: C, 60.29; H, 3.80; N, 13.26; Co, 5.51; Cl,
6.89
Cobalt(II) complex (6g) Violet. Yield 34.23%, mp
258–260°C. IR [cm−1]: 3421 NH(hydrazone), 3100 NH(oxindole)
,
1635 C=O(oxindole), 1569, 1492 C=N, 601, 551 (M-N), 443
(M-O). λmax [nm]: 485.5, 417, 265. μeff: 4.82 B.M. Calcd.
for C52H40Cl4CoN10O5: C, 58.50; H, 3.59; N, 13.12; Co, 5.52;
Cl, 13.28; Found: C, 58.30; H, 3.60; N, 12.96; Co, 6.16; Cl,
13.59.
e results of the in vitro cytotoxicity activity on human
tumour cell lines HCT116 (colon cell), MCF7 (breast cell)
and HELA (cervix cell) were determined according to the
dose values of the drug exposure required to reduce sur-
vival in the cell lines to 50%.
Copper(II) complex (6h) Brown. Yield 47.05%, mp
>350°C. IR [cm−1]: 3336 NH(hydrazone), 3070 NH(oxindole)
,
Results and discussion
1643 C=O(oxindole), 1569, 1500 C=N, 597, 509 (M-N), 455
(M-O). λmax [nm]: 483, 381, 265. μeff: 2.48 B.M. Calcd. for
C54H44CuF2N10O4: N, 14.03; Cu, 6.36; Found: N, 13.86; Cu,
6.42.
Chemistry
e sequence of reactions followed in the synthesis of
the target compounds is illustrated in Scheme 1. is
article describes a facile synthesis of the new hydrazones
(5a–5r) and its Co(II) and Cu(II) complexes (6a–6k). e
hydrazine derivatives (4a–4c) required for the synthesis
of the new hydrazone derivatives were obtained through
our reported methods10–14. 6-Aryl-4-(2-methoxybenzyl)
pyridazin-3-(2H)-ones (2a–2c) were obtained via reac-
tion of 4-methoxybenzaldehyde with 6-aryl-4,5-dihydro-
pyridazin-3(2H)-one (1a–1c) in alcoholic 5% KOH. en
reaction of (2a–2c) with phosphorus oxychloride yielded
the corresponding 3-chloropyridazin derivatives (3a–
3c), which on reaction with hydrazine hydrate afforded
the corresponding hydrazines 1-[4-(2-methoxybenzyl)-
6-arylpyridazin-3-yl]hydrazines (4a–4c). In the present
study, hydrazine derivatives or its tautomeric structures
(4a–4c) (Figure 1) were used as key starting materials for
the synthesis of new hydrazones derivatives (5a–5r).
Condensation of hydrazine derivatives (4a–4c) with
isatin and 5-substituted isatins afforded the correspond-
ing hydrazones (5a–5r).e structure of these hydrazones
were determined on the basis of elemental analysis and
Cobalt(II) complex (6i) Brick red. Yield 50.38%, mp
140°C. IR [cm−1]: 3314 NH(hydrazone), 3166 NH(oxindole)
,
1647 C=O(oxindole), 1599, 1491 C=N, 577, 504 (M-N), 423
(M-O). λmax [nm]: 485, 388, 267. μeff: 4.64 B.M. Calcd. for
C54H44CoF2N10O4: C, 65.25; H, 4.46; N, 14.09; Co, 5.82;
Found: C, 64.95; H, 4.62; N, 14.12; Co, 5.78.
Copper(II)complex (6j) Brown. Yield 34%, mp 254°C IR
[cm−1]: 3321 NH(hydrazone), 3056 NH(oxindole), 1647 C=O(oxindole)
,
1569, 1531 C=N, 594, 513 (M-N), 470 (M-O). λmax [nm]:
488, 381, 265. μeff: 2.28 B.M. Calcd. for C56H50CuN10O4: N,
14.14; Cu, 6.41; Found: N, 13.84; Cu, 6.25.
Cobalt(II) complex (6k) Violet. Yield 48.5%, mp 280–
282°C. IR [cm−1]: 3300 NH(hydrazone), 3177 NH(oxindole), 1627
C=O(oxindole), 1573, 1496 C=N, 613, 520 (M-N), 432 (M-O).
λmax [nm]: 485, 392, 266. μeff: 3.87 B.M. Calcd. for C54H46Co
N10O4: C, 67.71; H, 4.84; N, 14.62; Co, 6.15; Found: C, 67.67;
H, 4.54; N, 14.46; Co, 6.84.
1
spectroscopic methods such as IR, H NMR and mass
Biological activity
fragmentation. Support for the structure was evidenced
by the presence of prominent bands in the IR spectra
for NH (3433–3334 cm−1 and 3182–3104 cm−1), carbo-
nyl (1716–1677 cm−1) and C=N (1627–1542 cm−1). e
e primary evaluation of in vitro cytotoxicity against
human tumour cell of the complexes under investigation
has been tested in National Cancer Institute (NCI), Cairo
University using the method of Skehan and Storeng15. e
study also involved the cytotoxicity evaluation of vinblas-
tine sulphate16 as antitumour agent reference as follows:
1
structures were further established by H NMR spectral
data. e signal showed at δ: 12.35–10.97 for NH(oxindole)
,
10.58–9.19 for NH(hydrazone), 8.06–6.86 for Ar-H, 6.92–6.70
© 2012 Informa UK, Ltd.

334 N. G. Kandile et al.
Scheme 1. Experimental protocol for the synthesis of hydrazones (5a–5r).
Figure 1. Tautomeric forms of new Hydrazones (5a–5r).
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative effects of metal complexes of new hydrazones 335
for CH(hetero), 4.34–4.11 for CH2, 3.86–3.16 for OCH3 of
CH3O-Ar and 2.83–2.49 for CH3 of CH3-Ar.
Electronic spectra and magnetic moment studies
e Co(II) complexes exhibit two bands at 485–478 and
392–388 nm. e bands can be assigned to 4T1g(F)–4A2g and
4T1g(F)–4T1g(P), which are in accordance with Co(II) high-
spin octahedral geometry. e magnetic susceptibility
measurement for the solid Co(II) complexes (3.82–5.44
B.M.) are also indicative of three unpaired electrons per
Co(II) ion suggesting consistency with their octahedral
environment.
e hydrazones (5a–5r) reacted with the metal salt
of Co(II) and Cu(II) to give the corresponding Co(II),
Cu(II) complexes (6a–6k). e complexes were designed
in the aim of exploring their anticancer activity. e
structure of metal complexes (6a–6k) was confirmed by
elemental analysis and spectral studies. e elemental
analyses showed 1:2 (metal:ligand) stoichiometry for
all complexes (Figure 2). e analytical data of ligands
and complexes are given in the “Experimental” section,
which showed that they are agreed well with the general
formula [M(HL)2 yH2O] (where M = Co(II), Cu(II) and
y= 0–1).
e Cu(II) complexes exhibit two bands at 488–477
2
2
and 394–381 nm may be attributed to B1g–2A1g and B1g–
2Eg transitions, respectively. Also, the magnetic suscepti-
bility measurements (1.87–2.48 B.M.) for the copper (II)
complex are indicative of octahedral geometry17.
IR spectral studies and mode of coordination
In vitro cytotoxicity evaluation “antiproliferative
studies”
e important infrared frequencies of hydrazones and
metal complexes are reported in the “Experimental”
section. In the IR spectra of the complexes (6a–6k), the
band located at 1716–1680 cm−1 in all ligands attributed
to carbonyl group in oxindole moiety moved to the lower
frequency side by values up to 84 cm−1, also the band due
to (C=N) of azomethine moiety at 1627–1618 cm−1 low-
ered by value up to 51 cm−1 because of coordination of
these sites to the metal. New bands in the 613–577 cm−1,
551–504 cm−1 and 470–425 cm−1 regions are assigned to
stretching frequencies of (M-N), (M-N) and (M-O) bonds,
respectively. us the IR spectral results provide strong
evidence for the complexation of hydrazones with metal
ions in tridentate mode.
All the newly synthesized copper and cobalt complexes
(6a–6k) and HL (5a) are chosen as prototypes were
screened against a panel of three human cancer cell lines
HCT116 (colon cell), MCF7 (breast cell) and HELA (cervix
cell). Primary anticancer assay was performed in accor-
dance with the protocol of the National Cancer Institute,
Cairo University, Egypt by the method described in the
“Experimental” section. e cytotoxicity of the complexes
comparable with that obtained with widely used antican-
cer drug vinblastine sulphate16. e study also involved the
examination of the cytotoxicity of the ligand (5a) in order
to study the impact of metal complexation. e cobalt
complexes (6b and 6d) and copper complex (6c) are the
Figure 2. e proposed structure of complexes (6a–6k).
© 2012 Informa UK, Ltd.

336 N. G. Kandile et al.
most active on the colon tumour cell line (HCT116). In
addition as with HCT116 cell line copper complex (6c)
and cobalt complex (6d) showed the highest activity
towards the breast tumour cell line (MCF7) together with
the cobalt complex (6i). Metal complexes (6e–6h) are the
most active on cervix tumour cell line (HELA). All data of
the antiproliferative activity of metal complexes 6a–6k
and HL 5a virus vinblastin sulphate (in means of IC50 val-
ues) were recorded in (Figures 3–5) and Table 1.
characterized and screened for anticancer activity against
a panel of three human cancer cell lines. e new HL were
coordinated to the metal ion resulting octahedral mono-
nuclear complexes; they coordinated essentially through
the carbonyl oxygen of the oxoindole moiety, the nitrogen
atom of the azomethine group and the nitrogen of the
pyridazine fragments. e hydrazone acts as a monoba-
sic tridentate ligand. e two series of metal complexes
possessed considerable cytotoxicity activity using colon,
breast and cervix human cancer cell lines. e study
involved the cytotoxicity of the reference vinblastine
sulphate according to this arrangement the cobalt com-
plexes (6b and 6d) were more potent than the remaining
complexes on the colon human cancer cell line, for the
Conclusion
In the present study, a series of new hydrazones and
its metal complexes were designed, synthesized,
Figure 3. Cell viability dose–response curve of tested compounds against HCT116 cells.
Figure 4. Cell viability dose–response curve of tested compounds against MCF7 cells.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative effects of metal complexes of new hydrazones 337
Figure 5. Cell viability dose–response curve of tested compounds against HELA cells.
thus increases the lipophilic nature of the central metal
atom, which in turn is increasing the activity of the com-
plexes. Besides many other factors such as solubility,
dipole moment, conductivity influenced by metal ion
may be reasons for remarkable activities of these com-
plexes. Also, it was observed that some moieties such as
azomethine linkage or heteroaromatic introduced into
such compounds exhibit extensive biological activities
that may be responsible for the increase in hydrophobic
character and liposolubility of the molecules in crossing
the cell membrane human tumour cell and enhance bio-
logical utilization ratio and activity of complexes.
Table 1. e antiproliferative activity of metal complexes 6a–6k
and ligand 5a virus vinblastin sulphate (in means of IC50 values).
Compound
HCT116 (Colon) MCF7 (Breast) HELA (Cervix)
5a
6a
6b
6c
6d
6e
6f
6g
6h
6i
Negative
77.8 μg/mL
5.23
14.5 μg/mL
3.56
5.03
IC50 =0.54IC10 =9.6
9.53
9.33
0.94
0.69
3.95
7.25
4.52
4.02
8.77
13
0.54
6.88
0.74
8.82
4.62
1.03
4.85
1.14
4.68
1.71
Negative
0.896
1.66
0.656
12.9
6j
8.77
Declaration of interest
6k
14.4
9.8
18.5
14.6
e authors report no conflict of interest. e authors
alone are responsible for the content and writing of the
article.
Vinblastine
sulphate
11.6
10.9
breast human cancer cell line the two complexes (6b and
6c) with unsubstituted aromatic rings in their structure
showed the most potent cytotoxicity effect and finally the
two copper complexes (6e and 6h) were the most potent
complexes against the cervix human cancer cell line. In
conclusion, the cytotoxicity of the metal complexes on
the human cancer cells exhibited improved anticancer
activity as compared with the hydrazino ligand (5a) on
the selected human cancer cell lines. Compound (5a)
showed no cytotoxicity against colon cell line, very weak
cytotoxicity against breast and less cytotoxicity against
cervix cell line comparable with the metal complexes
(6a–6k).We can conclude that coordination reduces
the polarity of the metal ion mainly because of partial
sharing of its positive charge with donor group within
the whole chelate ring system. e process of chelation
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