Synthesis of nickel(II) complexes of isatin thiosemicarbazone derivatives: In vitro anti-cancer, DNA binding, and cleavage activities

Article abstract of DOI:10.1080/00958972.2014.959943

Six new nickel(II) complexes of thiosemicarbazone Schiff base with isatin moiety [Ni(L1)₂-Ni(L6)₂] were synthesized through reaction of Ni(II) with (Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide (L1H), (Z)-2-(5-methyl

Full text of DOI:10.1080/00958972.2014.959943

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Synthesis of nickel(II) complexes of
isatin thiosemicarbazone derivatives:
in vitro anti-cancer, DNA binding, and
cleavage activities
Amna Qasem Alia, Siang Guan Teoha, Naser Eltaher Eltayebb,
Mohamed B. Khadeer Ahamedc & Ams Abdul Majidc
a School of Chemical Sciences, Universiti Sains Malaysia, Minden,
Malaysia
b Department of Chemistry, Sciences & Arts College – Rabigh, King
Abdulaziz University, Rabigh, Saudi Arabia
c EMAN Research and Testing Laboratory, School of Pharmaceutical
Sciences, Universiti Sains Malaysia, Minden, Malaysia
Accepted author version posted online: 03 Sep 2014.Published
online: 01 Oct 2014.
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To cite this article: Amna Qasem Ali, Siang Guan Teoh, Naser Eltaher Eltayeb, Mohamed B. Khadeer
Ahamed & Ams Abdul Majid (2014): Synthesis of nickel(II) complexes of isatin thiosemicarbazone
derivatives: in vitro anti-cancer, DNA binding, and cleavage activities, Journal of Coordination
Chemistry, DOI: 10.1080/00958972.2014.959943
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Journal of Coordination Chemistry, 2014
http://dx.doi.org/10.1080/00958972.2014.959943
Synthesis of nickel(II) complexes of isatin thiosemicarbazone
derivatives: in vitro anti-cancer, DNA binding, and cleavage
activities
AMNA QASEM ALI†, SIANG GUAN TEOH*†, NASER ELTAHER ELTAYEB‡,
MOHAMED B. KHADEER AHAMED§ and AMS ABDUL MAJID§
†School of Chemical Sciences, Universiti Sains Malaysia, Minden, Malaysia
‡Department of Chemistry, Sciences & Arts College – Rabigh, King Abdulaziz University, Rabigh,
Saudi Arabia
§EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains
Malaysia, Minden, Malaysia
(Received 18 March 2014; accepted 25 July 2014)
Six new nickel(II) complexes of thiosemicarbazone Schiff base with isatin moiety [Ni(L1)2–Ni(L6)2]
were synthesized through reaction of Ni(II) with (Z)-2-(2-oxoindolin-3-ylidene)-N-phen-
ylhydrazinecarbothioamide (L1H), (Z)-2-(5-methyl-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbo-
thioamide (L2H), (Z)-2-(5-fluoro-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide (L3H),
(Z)-N-methyl-2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (L4H), (Z)-N-methyl-2-(5-
methyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (L5H), and (Z)-N-ethyl-2-(5-methyl-2-oxo-
indolin-3-ylidene)hydrazinecarbothioamide (L6H). The structures of the Ni complexes were character-
ized through elemental analysis, infrared, and mass spectral data. The structure of the NiL2 complex
was further characterized through single-crystal X-ray diffraction. The interaction of these complexes
with calf thymus (CT-DNA) exhibited high intrinsic binding constants (Kb = 1.4 ×
105–2.4 × 106 M−1), which reflected their intercalative activity toward CT-DNA. This result was also
confirmed by viscosity data. Electrophoresis studies revealed that these complexes could cleave the
DNA through the oxidative pathway. The in vitro anti-proliferative study establishes the anticancer
potency of these compounds against human colorectal carcinoma cell line.
Keywords: Nickel(II) complexes; Isatin moiety; Intercalative activity; Oxidative pathway;
Anti-proliferative activity
*Corresponding author. Email: sgteoh@usm.my
© 2014 Taylor & Francis

2
A.Q. Ali et al.
1. Introduction
Isatin thiosemicarbazones have numerous chemotherapeutic properties, such as anticancer,
antimicrobial, antituberculosis, antiulcer, antiviral, antiplasmodial, cytotoxic, and enzymatic
inhibition [1–12]. The biological activities of some thiosemicarbazone derivatives are
related to their ability to form chelates with transition metal ions through O, N, and S.
Metal ions are attracted to electron-rich molecules, such as proteins and DNA, leading to
cleavage of DNA [13], mutagenesis, and cancer [14]. Transition metal elements have func-
tions in medicinal chemistry, coordinating with different ligands to participate in biological
redox chemistry. The metal in the complex also increases the overall activity compared to
the free ligand. Nickel is an essential element for biological systems and is present in trace
quantities for bacteria, plants, animals, and humans and has an essential function in certain
enzymes such as urease. However, the biochemical activity of nickel has not been fully
explored, and nickel complexes have been rarely studied in medicinal biochemistry [15].
Nickel(II) Schiff base complexes containing sulfur donors have received attention due to
identification of a sulfur-rich coordination environment in biological nickel centres, such as
the active sites of certain ureases, methyl-S-coenzyme-M-methyl reductase, and hydroge-
nases [16]. The known biological activity of nickel includes antiepileptics, anticonvulsant
agents, vitamins, antibacterial, antifungal, antimicrobial, and anticancer/antiproliferative
activities. Several reports have described the reactivity of DNA with mononuclear nickel(II)
complexes [17]. The chemistry of nickel complexes with multidentate Schiff base ligands
has attracted attention because such complexes play an important role in bioinorganic chem-
istry and redox enzyme systems, and may provide the basis for models of the active sites of
biological systems or act as catalysts [18]. Our studies focused on the synthesis, physico-
chemical characterization, DNA binding, and nuclease activities of mononuclear Ni(II)
complexes with tridentate ligands. The in vitro cytotoxic activities of the compounds were
tested against human colorectal carcinoma cell line (HCT 116). The structures were proven
through IR and mass spectroscopy. The single-crystal X-ray structure of the nickel complex
C32H26NiN8O2S2, [Ni(L2)2] was determined to confirm its structure. Several techniques
were used to investigate the DNA-binding abilities of these complexes, including absorp-
tion, fluorescence spectrophotometry, and viscosity measurements. The nuclease activity
with supercoiled (SC) pBR322 DNA was also examined. The in vitro cytotoxic activities of
the compounds were tested against the human colorectal carcinoma cell line HCT 116.
2. Experimental
2.1. Instruments
The materials used in this study, such as isatin, 5-fluoroisatin, 5-methylisatin, 5-nitroisatin,
4-ethyl-3-thiosemicarbazide, 4-methyl-3-thiosemicarbazide, 4-phenyl-3-thiosemicarbazide,
and nickel(II) chloride (NiCl2·6H2O), were purchased from Aldrich Chemicals. Commer-
cial-grade solvents and reagents were used as supplied without purification. SC pBR322
DNA and loading dye were purchased from Fermentas. Calf thymus [CT-DNA, agarose
(molecular biology grade)] and ethidium bromide (EB) were from Sigma (St. Louis, MO,
USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was purchased
from Sigma-Aldrich, Germany. Elemental analysis was carried out using a Perkin-Elmer
2400 series-11 CHN/O analyzer (Waltham, MA, USA). The infrared, electronic, and

Nickel(II) isatin thiosemicarbazone
3
fluorescence spectra were recorded on Perkin-Elmer 2000, Perkin-Elmer Lambda 25, and
Jasco FP-750 spectrophotometers, respectively. Viscosity measurements were carried out
using a Cannon Manning semi-micro viscometer (State College, PA, USA). ESI-mass spec-
tra were obtained through liquid chromatography/mass spectrometry using Trap VL (Agi-
lent Technologies) operated in either positive ion or negative ion detection mode.
2.2. Synthesis of ligands
General procedure. The thiosemicarbazone derivatives (L1H–L6H) were previously synthe-
sized and characterized in our laboratory [19–24]. Equimolar quantities of 5-unsubstituted
or 5-substituted isatin were refluxed with thiosemicarbazide derivatives in ethanol for 2 h
(scheme 1). The resulting precipitates were then filtered and washed with cold ethanol.
2.2.1. Synthesis of (Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide
(L1H). C15H12N4OS (L1H): Yellow crystals; m.p.: 237.2–238.6 °C; yield: 90%; Anal.
Calcd values: C (60.79%), H (4.08%), and N (18.91%); analytical results (experimental):
C (60.50%), H (4.19%), and N (18.23%); selected IR data (KBr pellet, υmax/cm−1): 3300 to
Scheme 1. Synthetic route and structures for the Schiff bases L1H–L6H.

4
A.Q. Ali et al.
1
3190 (NH), 1694 (C=O), 1621 (C=N), 1594 (C=C), 1138 (N–N), and 1208/790 (C=S); H
NMR (500 MHz, DMSO-d6) [δ (ppm)]: 12.79 (s, 1 H, thiosemicarbazide N-NH),11.26 (s, 1
H, indole N–H), 10.98 (s, 1 H, CS-NH), 7.78 (d, 1 H, indole C2-H, J = 7.5 Hz), 7.62 (d,
2 H, thiosemicarbazide C11–H, C15–H, J = 7.7 Hz), 7.44–7.41 (t, 2 H, thiosemicarbazide
C12-H, C14-H, J = 7.8), 7.39–7.36 (dt, 1 H, indole C3-H, J = 7.7, 1.0 Hz), 7.29–7.26 (t,
1 H, thiosemicarbazide C13-H, J = 7.4), 7.13–7.10 (t,1 H, indole C4-H, J = 7.5 Hz), 6.95
(d, 1 H, indole C5-H, J = 7.8); 13C NMR (126 MHz, DMSO-d6 ((δ (ppm)): 176.33, 162.68,
142.47, 138.43, 132.26, 131.41, 128.36, 126.07, 125.65, 124.65, 122.34, 121.37, 119.89,
111.07, and ESI-MS (m/z): 295(M−).
2.2.2. Synthesis of (Z)-2-(5-methyl-2-oxoindolin-3-ylidene)-N-phenylhydrazine-carbo-
thioamide (L2H). C16H14N4OS (L2H): Orange crystals; m.p.: 238.6–239.1 °C; yield:
80%; Anal. Calcd values: C (61.92%), H (4.55%), and N (18.05%); analytical results
(experimental): C (61.89%), H (4.65%), and N (18.01%); selected IR data (KBr pellet,
υ
max/cm−1): 3308–3169 (NH), 1691 (C=O), 1627 (C=N), 1593 (C=C), 1135 (N–N), and
1
1212/762 (C=S); H NMR (500 MHz, DMSO-d6) [δ (ppm)]: 12.77 (s, 1 H, thiosemicarba-
zide N-NH), 11.15 (s, 1 H, indole N–H),10.81 (s, 1 H, CS–NH), 7.61 (d, 3 H, indole C5-H,
thiosemicarbazide C11-H, C15-H, J = 7.7 Hz), 7.44–7.41 (t, 2 H, thiosemicarbazide C12-H,
C14-H, J = 7.8 Hz), 7.29–7.26 (t, 1 H, thiosemicarbazide C13-H, J = 7.4 Hz), 7.19 (d,1 H,
indole C3-H, J = 8 Hz), 6.84 (d, 1 H, indole C2-H, J = 7.9), 2.3 (s, 3 H, CH3); 13C NMR
(126 MHz, DMSO-d6 ((δ (ppm)): 176.30, 162.75, 140.25, 138.43, 132.37, 131.82, 131.36,
128.35, 126.06, 125.64, 121.74, 119.92, 110.82, 20.59, and ESI-MS (m/z):309 (M−).
2.2.3. Synthesis of (Z)-2-(5-fluoro-2-oxoindolin-3-ylidene)-N-phenylhydrazine-carbo-
thioamide (L3H). C15H11FN4OS (L3H): Orange crystals; m.p.: 244.2–244.8 °C; yield:
77%; Anal. Calcd values: C (57.31%), H (3.52%), and N (17.82%); analytical results
(experimental): C (57.42%), H (3.31%), and N (17.76%); selected IR data (KBr pellet,
υ
max/cm−1): 3313–3183 (NH), 1691 (C=O), 1594 (C=N), 1535 (C=C), 1140 (N–N), and
1
1202/797 (C=S); H NMR (500 MHz, DMSO-d6) [δ (ppm)]: 12.67 (s, 1 H, thiosemicarba-
zide N–NH), 11.26 (s, 1 H, indole N–H), 10.85 (s, 1 H, CS–NH), 7.65–7.63 (dd, 1 H,
indole C3-H, J = 8.1, 2.6 Hz), 7.61 (d, 2 H, thiosemicarbazide C11-H, C15-H, J = 7.9 Hz),
7.45–7.42 (t, 2 H, thiosemicarbazide C12-H, C14-H, J = 7.8 Hz), 7.30–7.27 (t, 1 H, thio-
semicarbazide C13-H, J = 7.4 Hz), 7.23–7.19 (dt, 1 H, indole C5-H, J = 7.7, 2.5 Hz), 6.95–
6.93 (dd, 1 H, indole C2-H, J = 8.6, 4.1 Hz); 13C NMR (126 MHz, DMSO-d6 ((δ (ppm)):
176.79, 163.26, 159.69, 132.12, 128.92, 126.70, 126.12, 121.88, 121.81, 118.21, 118.01,
112.88, 112.61, 108.88, 108.68, and ESI-MS (m/z): 313 (M−).
2.2.4. Synthesis of (Z)-N-methyl-2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinecarbothioa
mide (L4H). C10H9N5O3S (L4H): Orange crystals; m.p.: 306.6–307.1 °C; yield: 80%;
Anal. Calcd values: C (43.01%), H (3.25%), and N (25.08%); analytical results (experimen-
tal): C (43.12%), H (3.17%), and N (24.92%); Selected IR data (KBr pellet, υmax/ cm−1):
3437–3342 (NH), 1702 (C=O), 1623(C=N), 1551 (C=C), 1049 (N–N), and 1212/762
(C=S); 1H NMR (500 MHz, DMSO-d6) [δ (ppm)]: 12.32 (s, 1 H, thiosemicarbazide
N–NH), 11.80 (s, 1 H, indole N–H), 9.53–9.50 (q, 1 H, CS–NH, J = 9.0, 4.5 Hz), 8.50
(d, 1 H, indole C5-H, J = 2.3 Hz), 8.25–8.23 (dd, 1 H, indole C3-H, J = 8.6, 2.5 Hz), 7.10

Nickel(II) isatin thiosemicarbazone
5
(d, 1 H, indole C2-H, J = 8.6 Hz), 3.40 (d, 3H, thiosemicarbazide CH3, J = 4.6 Hz); 13C
NMR (126 MHz, DMSO-d6 ((δ (ppm)): 177.54, 162.86, 147.19, 142.70, 129.47, 126.72,
120.92, 115.97, 111.15, 31.33, and ESI-MS (m/z): 278 (M−).
2.2.5. Synthesis of (Z)-N-methyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-carbo-
thioamide (L5H). C11H12N4OS (L5H): Yellow crystals; m.p.: 278.7–279.2 °C; yield:
94%; Anal. Calcd values: C (53.21%), H (4.87%), and N (22.56%); analytical results
(experimental): C (53.45), H (4.21%), and N (22.51%); selected IR data (KBr pellet, υmax
/
cm−1): 3350–3248 (NH), 1693 (C=O), 1665(C=N), 1561 (C=C), 1042 (N–N), and 1205/
1
791 (C=S); H NMR (500 MHz, DMSO-d6) [δ (ppm)]: 12.56 (s, 1 H, thiosemicarbazide N-
NH), 11.09 (s, 1 H, indole N–H), 9.23–9.21 (q, 1 H, CS–NH, J = 9.0, 4.6 Hz), 7.50 (s, 1
H, indole C5-H), 7.16–7.14 (dd, 1 H, indole C3-H, J = 7.9, 0.9 Hz), 6.82 (d, 1 H, indole
C2-H, J = 7.9 Hz), 3.09 (d, 3 H, thiosemicarbazide CH3, J = 4.6 Hz), 2.30 (s, 3 H, indole
CH3); 13C NMR (126 MHz, DMSO-d6 ((δ (ppm)): 177.68, 162.65, 136.97, 131.64, 131.50,
131.31, 121.02, 119.98, 110.78, 31.27, 20.57, and ESI-MS (m/z): 247 (M−).
2.2.6. Synthesis of (Z)-N-ethyl-2-(5-methyl-2-oxoindolin-3-ylidenehydrazinecarbothioa-
mide (L6H). C12H14N4OS (L6H): Yellow crystals; m.p.: 260.0–261.8 °C; yield: 80%;
Anal. Calcd values: C (54.94%), H (5.38%), and N (21.36%); analytical results (experimen-
tal): C (54.83%), H (5.41%), and N (21.46%); selected IR data (KBr pellet, υmax/cm−1):
3343–3190 (NH), 1691 (C=O), 1669(C=N), 1543 (C=C), 1052 (N–N), and 1206/794
1
(C=S); H NMR (500 MHz, DMSO-d6) [δ (ppm)]: 12.53 (s, 1H, thiosemicarbazide N-NH),
11.09 (s, 1H, indole N–H), 9.28–9.25 (t, 1H, CS–NH, J = 5.8 Hz), 7.50 (s, 1 H, indole C5-
H), 7.17 (d, 1 H, indole C2-H, J = 7.9 Hz), 6.83 (d, 1 H, indole C3-H, J = 7.9 Hz), 3.68–
3.62 to (p, 2 H, thiosemicarbazide CH2, J = 6.8 Hz), 2.31 (s, 3H, indole CH3), 1.21–1.19 (t,
3H, thiosemicarbazide CH3, J = 7); 13C NMR (126 MHz, DMSO-d6 ((δ (ppm)): 176.70,
162.66, 139.99, 131.70, 131.52, 131.29, 121.14, 119.98, 110.78, 40.11, 20.56, 14.00, and
ESI-MS (m/z): 263 (M+).
2.3. Synthesis of complexes
The Ni(II) complexes were synthesized by refluxing the reaction mixture of hot ethanolic
solutions (30 mL each) of NiCl2 (0.01 M) and appropriate ligand (0.02 M) for 2 h. The
precipitates that formed during the reflux were filtered and washed with cold ethanol
(scheme 2).
2.3.1. Bis[2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamidato-κ3O,N2,S]-
nickel(II) dimethylformamide monosolvate [25]. Ni(L1)2. C30H22NiN8O2S2 [Ni(L1)2]:
Green crystal; m.p.: >300 °C; yield: 67%; Anal. Calcd values: C (55.49%), H (3.41%), N
(17.26%), and Ni (9.04%); analytical results (experimental): C (55.67%), H (3.14%), N
(16.93%), and Ni (9.20%); selected IR data (KBr pellet, υmax/cm−1): 3274–3134 (NH),
1668 (C=O), 1597 (C=N), 1048 (N–N), and 779 (C–S); UV–vis [DMSO, λmax, nm, ε
(dm3 M−1 cm−1)]: 309.5 (10.0 × 103), 409.0 (10.0 × 103), and 505.0 (7.2 × 103); and ESI-
MS (m/z): 649 (M + H)+.

6
A.Q. Ali et al.
Scheme 2. Synthetic route and structures for NiL1–NiL6.
2.3.2. Bis[2-(5-methyl-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamidato-
κ3O,N2,S]nickel(II). Ni(L2)2. C32H26NiN8O2S2 [Ni(L2)2]: Reddish brown powder; m.p.:
>300 °C; yield: 77%; Anal. Calcd values: C (56.74%), H (3.87%), N (16.54%), and Ni
(8.66%); analytical results (experimental): C (56.96%), H (4.01%), N (16.64%), and Ni
(8.90%); selected IR data (KBr pellet, υmax/cm−1): 3375–3276 (NH), 1669 (C=O), and
1595 (C=N), 1045 (N–N), and 750 (C–S); UV–vis [DMSO, λmax, nm, ε (dm3 M−1 cm−1)]:
306.0 (10.0 × 103), 408.5 (10.0 × 103), and 517.5 (10.0 × 103); and ESI-MS (m/z): 677
(M + H)+.
2.3.3. Bis[2-(5-fluoro-2-oxoindolin-3-ylidene)phenylhydrazinecarbothioamidato-κ3O,
N2,S]-nickel(II). Ni(L3)2. C30H20NiF2N8O2S2 [Ni(L3)2]: Reddish brown powder; m.p.:
>300 °C; yield: 80%; Anal. Calcd values: C (52.57%), H (2.94%), N (16.35%), and Ni
(8.56%); analytical results (experimental): C (52.81%), H (3.12%), N (16.37%), and Ni
(8.40%); selected IR data (KBr pellet, υmax/cm−1): 3407–3270 (NH), 1665 (C=O), 1595
(C=N), 1039 (N–N), and 749 (C–S); UV–vis [DMSO λmax, nm, ε (dm3 M−1 cm−1)]: 313.0
(10.0 × 103), 399.0 (10.0 × 103), and 477.5 (10.0 × 103); and ESI-MS (m/z): 685 (M + H)+.
2.3.4. Bis[N-methyl-2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinecarbothioamidato-κ3O,
N2,S]nickel(II). Ni(L4)2. C20H16NiN10O6S2 [Ni(L4)2]: Reddish brown powder;

Nickel(II) isatin thiosemicarbazone
7
m.p.: >300 °C; yield: 83%; Anal. Calcd values: C (39.04%), H (2.62%), N (22.77%), and
Ni (9.54%); analytical results (experimental): C (39.02%), H (2.48%), N (22.80%), and Ni
(9.20%); selected IR data (KBr pellet, υmax/cm−1): 3341 (NH), 1661 (C=O), 1521 (C=N),
1029 (N–N), and 765 (C–S); UV–vis [DMSO λmax, nm, ε (dm3 M−1 cm−1)]: 311.0
(10.0 × 103), 402.5 (10.0 × 103), and 493.5 (5.7 × 103); and ESI-MS (m/z): 614 (M−).
2.3.5. Bis[N-methyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamidato-
κ3O,N2,S]nickel(II). Ni(L5)2. C22H22NiN8O2S2 [Ni(L5)2]: Reddish brown powder; m.p.:
>300 °C; yield: 74%; Anal. Calcd values: C (47.76%), H (4.01%), N (20.25%), and Ni
(10.61%); analytical results (experimental): C (47.43%), H (4.12%), N (20.42%), and Ni
(10.42%); selected IR data (KBr pellet, υmax/cm−1): 3418–3295 (NH), 1651(C=O), 1513
(C=N), 1033 (N–N), and 745 (C–S); UV–vis [DMSO λmax, nm, ε (dm3 M−1 cm−1)]: 331.0
(10.0 × 103), 409.5 (10.0 × 103); and 471.5 (8.8 × 103); and ESI-MS (m/z): 552 (M + H)+.
2.3.6. Bis[N-ethyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamidato-κ3O,
N2,S]nickel(II). Ni(L6)2. C24H26NiN8O2S2 [Ni(L6)2]: Reddish-brown powder; m.p.:
>300 °C; yield: 79%; Anal. Calcd values: C (49.58%), H (4.51%), N (19.28%), and Ni
(10.10%); analytical results (experimental): C (49.86%), H (4.28%), N (19.14%), and Ni
(10.36%); selected IR data (KBr pellet, υmax/cm−1): 3265 (NH), 1671 (C=O), 1599 (C=N),
1042 (N–N), and 749 (C–S); UV–vis [DMSO λmax, nm, ε (dm3 M−1 cm−1)]: 348.5
(10.0 × 103), 403.5 (10.0 × 103), and 434.5 (4.65 × 103); and ESI-MS (m/z): 581 (M + H)+.
2.4. X-ray crystallography
Single crystals of Ni(L2)2 suitable for diffraction were grown by slow evaporation of 3 : 1
mixtures of acetone and DMF. Ni(L2)2 was crystallized with two molecules of DMF.
Selected crystal data and data collection parameters are presented in table 1. The data of the
Ni(L2)2 complex were collected on a Bruker SMART APEX II CCD diffractometer [26]
equipped with graphite monochromated Mo Kα radiation (λ = 0.71073) at 100(1) K. Multi-
scan absorption corrections were applied using the SADABS program [26]. The structure
was solved by direct methods using the SHELXS-97 program [27]. Refinements on F2 were
performed using SHELXL-97 by full-matrix least-squares with anisotropic thermal parame-
ters for all non-hydrogen atoms. N-bound hydrogens were located in a difference Fourier
map and were refined freely. The remaining hydrogens were positioned geometrically and
refined using a riding model. SHELXTL [27] was used in the preparation of figure 1.
2.5. DNA-binding studies
DNA-binding experiments, which include absorption, emission spectral studies, and viscos-
ity measurements, were performed in accordance with standard methods [28–30]. Concen-
trated CT-DNA stock solution was prepared in 5 mM Tris–HCl/50 mM NaCl in water at pH
7.2. The DNA concentration per nucleotide was determined by absorption spectroscopy
using the molar absorption coefficient (6600 M−1 cm−1) at 260 nm [31]. The purity of the
CT-DNA was verified by taking the ratio of the absorbance values at 260 and 280 nm in the
respective buffer, always >1.9. This finding indicates that the DNA was sufficiently free of
protein. The stock solutions were stored at 4 °C and used within 4 days. DNA-binding

8
A.Q. Ali et al.
Table 1. Crystallographic data for Ni(L2)2.
Formula
Fw
C32 H26 N8 Ni O2 S2, C4NO2, C3NO
837.53
Crystal system
Space group
a (Å)
Monoclinic
C2/c
22.161(3)
b (Å)
19.306(3)
c (Å)
10.6365(14)
α (°)
90
β (°)
115.222(2)
γ (°)
90
V (nm3)
Z
4116.9(10)
4
1.351
1720
DCalcd (Mg m−3
F (0 0 0)
)
Crystal dimensions (mm)
θ range (°)
0.102 × 0.269 × 0.681
1.5, 30.0
hkl ranges
−31 < h < 31
−27 < k < 27
−14 < l < 12
Data/parameters
5980/270
1.04
R1 = 0.0678
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
wR2 = 0.2104
Highest peak/deepest hole
Δρmax = 1.01e Å−3/Δρmin = −0.96e Å−3
experiments were performed at room temperature. All experiments were carried out by
keeping the concentration of nickel complexes constant (50 μM) while varying the DNA
concentration (0–200 μM). An equal amount of DNA was added to the nickel complex cuv-
ette and the reference cuvette to eliminate the absorbance of CT-DNA [32, 33]. Tris buffer
was subtracted through baseline correction. The UV–vis and the emission spectra were
recorded after equilibration for 10 min after each addition. Viscosity experiments were con-
ducted using a Cannon Manning semi-micro viscometer (State College, PA, USA) and
thermostated in a water bath maintained at 37.0 0.1 °C. The flow rates of the Tris–HCl
buffer (pH 7.2), CT-DNA (200 μM), and DNA in the presence of Ni(II) complex at various
concentrations (0 × 10−4–2.5 × 10−4 M) were measured three times each with a digital
stopwatch, and the average flow time was calculated. The resulting data are presented as
(η/ηo)1/3 versus[complex]/[DNA], where η is the viscosity of the DNA in the presence of Ni
(II) complex and ηo is the viscosity of the DNA. The viscosity values of DNA-containing
solutions were calculated from the observed flow time (t) corrected from that of the buffer
alone (Tris–HCl/NaCl, 5 : 50 mM) (to), η = (t – to).
2.6. DNA cleavage studies
Cleavage experiments of SC pBR322 DNA (0.5 μg μL−1) were performed at pH 7.2 in
Tris–HCl/NaCl (5 : 50 mM) buffer. Oxidative DNA cleavage was monitored by treating
pBR322 DNA with varying concentrations of Ni(II) complexes (0.1–1 mM) and H2O2, fol-
lowed by dilution with Tris–HCl/NaCl (5 : 50 mM) buffer to a total volume of 20 μL
(Lanes 4–12). To investigate the mechanism of DNA cleavage promoted by these com-
plexes, the experiment was carried out by adding scavenger for reactive oxygen species
(DMSO) to complex DNA mixture (Lane 3). The samples were incubated for 2 h at 37 °C.
A loading dye was added, and electrophoresis was carried out at 50 V for 1 h in Tris–HCl

Nickel(II) isatin thiosemicarbazone
9
Figure 1. The structure of Ni(L2)2 showing 50% probability displacement ellipsoids and atom numbering.
buffer using 1% agarose gel. The resulting bands were stained with EB before being photo-
graphed under UV light.
2.7. In vitro anti-proliferative activity
2.7.1. Preparation of cell culture. HCT 116 cells were allowed to grow under optimal
incubator conditions. Cells that reached a confluence of 70–80% were chosen for cell plat-
ing. The old medium was aspirated out of the plate, and then, the cells were washed two to
three times using sterile phosphate buffered saline (PBS; pH 7.4). PBS was discarded after
washing. Trypsin was added and evenly distributed onto the cell surface, and the cells were
incubated at 37 °C in 5% CO2 for 1 min. Afterwards the flasks containing the cells were
gently tapped to aid cell segregation and observed under an inverted microscope. If cell seg-
regation was not satisfactory, the cells were incubated for another minute. Trypsin activity

10
A.Q. Ali et al.
was inhibited by adding 5 mL of fresh complete medium (10% FBS). Cells were counted
and diluted to obtain a final concentration of 2.5 × 105 cells/mL and then seeded into wells
(100 μL cells/well). Finally, the cell-containing plates were incubated at 37 °C with an inter-
nal atmosphere of 5% CO2.
2.7.2. MTT assay. Cancer cells (100 μL cells/well, 1.5 × 105 cells/mL) were seeded on a
96-well microtiter plate incubated with CO2 overnight to allow cell attachment. The com-
pounds were diluted with media into the required concentrations from the stock. Various
concentrations (6.25–200 μM) of 100 μL test substances were separately added to each well
containing the cells. The plates were then incubated at 37 °C with an internal atmosphere of
5% CO2 for 48 h. After the treatment period, the plates were treated with 20 μL of MTT
reagent and incubated again for 4 h. After the incubation period, 50 μL of MTT lysis solu-
tion (DMSO) was added to the wells. The plates were further incubated for 5 min at room
temperature. Finally, the absorbances at 570 and 620 nm were measured using a standard
Infinite 200 PRO multimode microplate reader (Tecan, USA). Data were recorded and ana-
lyzed to estimate the effects of the test compounds on cell viability and growth inhibition.
The percentage inhibition of cell proliferation was calculated from the optical density
obtained from the MTT assay. 5-Fluorouracil (5-FU) was used as the standard reference
drug [34]. Statistical difference between the treatments and the control was evaluated by
one-way ANOVA, followed by Tukey’s multiple comparison test. Differences were consid-
ered significant at p < 0.05 and p < 0.01.
3. Results and discussion
The nickel(II) complexes Ni(L1)2–Ni(L6)2 were obtained in good yield from the reaction of
Ni(II) chloride with appropriate Schiff base (L1H–L6H) in 1 : 2 M ratio in ethanol with
reflux for 2 h (via scheme 2). These complexes were slightly soluble in common organic
solvents, but highly soluble in DMF and DMSO.
3.1. Spectral characterization
3.1.1. IR studies. Characteristic IR bands for the free ligands are different from those of
the related complexes, providing significant indications regarding the bonding sites of the
thiosemicarbazone ligands. Selected vibrational bands of the ligands and nickel complexes
are listed in table 2. Compared with the spectra of the Schiff bases, all the Ni(II) complexes
exhibit a band at 1651–1671 cm−1 assigned to ν(C=O). The shift of the band to lower wave-
numbers indicates that the carbonyl oxygen is coordinated to the metal ion. The ν(C=N)
band at 1513–1599 cm−1 in the metal complexes is shifted to lower wavenumbers, indicat-
ing that nitrogen of the azomethine group is coordinated. IR spectra of the free ligands have
ν(N–N) at 1042–1149. This band shifted to lower wavenumbers (1029–1048) in spectra of
the nickel complexes, indicating involvement of this group in complexation [35]. These
compounds also display out-of-plane C–H bending and ring bending absorptions at
650–900 cm−1 [36]. The band corresponding to the C=S stretch appears at 762–797 cm−1
in the ligands spectra, lowered by ≈20 cm−1 in spectra of the complexes, indicating

Nickel(II) isatin thiosemicarbazone
11
Table 2. Selected vibrational bands of the ligand and its metal complexes.
Compound
νC=O
νC=N
νN-N
νC=S
L1H
L2H
L3H
L4H
L5H
L6H
Ni(L1)2
Ni(L2)2
Ni(L3)2
Ni(L4)2
Ni(L5)2
Ni(L6)2
1694
1691
1691
1702
1693
1691
1668
1669
1665
1661
1651
1671
1621
1627
1625
1623
1629
1629
1597
1595
1595
1521
1513
1599
1138
1135
1140
1049
1042
1052
1048
1045
1039
1029
1033
1042
790
762
797
785
791
794
779
750
749
765
745
749
involvement of the thioketo sulfur in coordination. The band that appears at 745–779 cm−1
is thus assigned to ν(C–S) in IR spectra of the metal complexes [37]. IR spectral results pro-
vide strong evidence for tridentate complexation of the Schiff base with nickel.
3.1.2. UV–vis studies. UV–vis absorption spectra of the Ni(II) complexes were measured
from 200 to 800 nm using DMSO. Three absorptions with varying intensities can be
observed. The bands observed from 306.0 to 348.5 nm are probably due to the intra-ligand
π → π* transitions of the thiosemicarbazone ligands. The bands from 399.0 to 409.5 nm
can be attributed to ligand-to-metal charge transfer transitions from sulfur to nickel. Occur-
rence of S → M (LMCT) bands is quite common in electronic spectra of metal complexes
of thiosemicarbazones. The bands at the lower frequency (434.5–517.5 nm) of the spectra
correspond to d–d transitions [38, 39].
3.1.3. MS studies. The ESI-mass spectra of Ni(L1)2, Ni(L2)2, Ni(L3)2, Ni(L5)2, and Ni
(L6)2, which were recorded in positive mode, exhibit peaks at m/z 649, 677, 685, 552, and
581, respectively, indicating the presence of [M + H]+ complexes. By contrast, the ESI-
mass spectrum of Ni(L4)2, which was recorded in negative mode, exhibit a peak at m/z
614, indicating the presence of [M−] complex. These assignments are based on 58Ni. Fur-
ther confirmation for the molecular structure of the investigated complexes comes from the
appearance of other peaks containing 60Ni besides the peaks due to successive degradation
of the target compound to various fragments. Another confirmation came from the appear-
ance of sodium adduct [M + Na + H] as in the case of Ni(L4)2 (the peak at m/z 638). The
data of the elemental analysis and MS for the prepared complexes confirm the stoichiometry
of metal chelates as [ML2] type.
3.2. Crystal structure description
A crystal structure of Ni(L1)2 has been published in our previous study [25]. The current
study describes the crystal structure of Ni(L2)2 and compares it with Ni(L1)2. The asym-
metric unit of the Ni(L2)2 molecule (figure 1) contains one Ni(II) complex and two solvent
molecules that may be DMF. The solvent molecules are disordered and not solved com-
pletely, but the crystal quality is good, with R value of 0.0678. This study will not discuss

12
A.Q. Ali et al.
these solvent molecules and concentrates on the Ni(II) complex. Coordination spheres
around Ni(II) in both Ni(L1)2 and Ni(L2)2 are slightly distorted octahedral, with the coordi-
nation around Ni(II) formed by the S2N2O2 donors of the ligands. The Ni–O distance is
2.225 Å, with Ni–N distance of 2.003 Å and Ni–S distance of 2.370 Å, which fall in the
range observed for Ni(L1)2. For Ni(L1)2, the Ni–O distances are 2.189–2.244 Å, with Ni–N
distances of 2.034–2.037 Å and Ni–S distances of 2.356–2.387 Å.
The bond angle O1–Ni–O2 is 86.1°, S1–Ni–S2 is 94.9°, N1–Ni–N2 is 163°, S1–Ni–O1
is 160.7°, S2–Ni–O2 is 158.7°, and O1–Ni–N6 is 90°. These bond angles are comparable
with that of Ni(L2)2. Other bond lengths and angles observed in the structure are normal
[40].
3.3. DNA-binding studies
3.3.1. Absorption spectral studies. One of the most useful techniques for DNA-binding
studies of molecules is electronic absorption spectroscopy. DNA usually results in hypo-
chromism and red shift (bathochromism) because of intercalation involving a strong stack-
ing interaction between an aromatic chromophore and the DNA base pairs [41].
Hypochromism arises from the contraction of CT-DNA (calf thymus) in the helix axis and
its conformational changes [42]. Meanwhile, hyperchromism results from the secondary
damage of the DNA double helix structure [43, 44], in which the extent of hyperchromism
is indicative of partial or non-intercalative binding modes [45]. The absorption spectra
of 50 μM Ni(L2)2 and Ni(L3)2 complexes in the absence and presence of CT-DNA
(0–200 μM) are given in figures 2 and 3. The absorption spectra of Ni(L1)2, Ni(L4)2,
Ni(L5)2, and Ni(L6)2 are shown in Supplementary material (figures S1–S4, see online
supplemental material at http://dx.doi.org/10.1080/00958972.2014.959943). Absorption
spectra of Ni(L1)2–Ni(L6)2 display clear hypochromism with slight red shift of 2–7 nm.
After intercalation of the compounds into the DNA base pairs, the π* orbitals of the interca-
lated compounds are able to couple with the π orbitals of the base pairs, thereby decreasing
the π→π* transition energies [46]. These interactions result in the observed hypochromism
[47]. The hypochromism of the complexes with aromatic thiosemicarbazone is in the order
Ni(L3)2 > Ni(L2)2 > Ni(L1)2, whereas the hypochromism of the compounds with aliphatic
thiosemicarbazone is in the order Ni(L5)2 > Ni(L6)2 > Ni(L4)2, which may be due to a
strong stacking interaction between the aromatic chromophore of the ligand and the DNA
base pairs [48]. Remarkably, the Ni(L2)2 complex exhibits both hypochromism and hyper-
chromism at ~257 and 360 nm, respectively. This behavior has been reported for chiral
Schiff base complexes [49, 50]. The entity of hyperchromism behavior [42, 43], which
reflects the secondary damage in the complex (Ni(L2)2), can be attributed to the enhanced
hydrogen bonding between this complex and the DNA caused by the presence of an
aliphatic group in the isatin moiety.
The intrinsic binding constants of all the complexes with DNA were obtained by observ-
ing the changes in the absorbance of the complexes with increase in DNA concentration
using the following equation [51, 52]:
½DNAꢀ=ðea ꢁ ef Þ ¼ ½DNAꢀ=ðeb ꢁ ef Þ þ 1=Kbðeb ꢁ ef Þ
where εa, εf, and εb are the extinction coefficients observed for the absorption band at a
given DNA concentration, free and bound complexes, respectively, and [DNA] is the con-
centration of DNA in the base pairs. The binding constant Kb was determined from the ratio

Nickel(II) isatin thiosemicarbazone
13
Figure 2. Absorption spectra of Ni(L2)2 in the absence and presence of increasing amounts of DNA in Tris–HCl
buffer (pH 7.2). [Ni(L2)2] = 50 μM, [DNA] = 0–163.58 × 10−6 M. The arrow shows the effect of increasing of
CT-DNA concentration on the absorption intensity.
Figure 2a. Comparative plot of [DNA]/(εa − εf) vs. [DNA] for the absorption titration of CT-DNA with Ni(L2)2 in
Tris–HCl buffer (pH 7.2); Kp = 3.29 × 106 M−1 (R = 0.9066, n = 6 points).
of the slope to intercept by the [DNA]/(εa − εf) versus [DNA] plot (figures 2a and 3a). The
intrinsic binding constants (Kb) for Ni(L1)2–Ni(L6)2 were 7.39 × 105, 5.83 × 105,
1.07 × 106, 5.83 × 105, 1.41 × 105, and 2.44 × 106 M−1, respectively. Comparing the Kb
values of these complexes with those of the DNA-intercalative Ni(II) complexes, such as
the nickel complex of 6-hydroxy chromone-3-carbaldehyde thiosemicarbazone [53], [Ni
(sf)2(bipyam)]·7H2O, [Ni(oxo)2(bipyam)], [Ni(flmq)2(bipyam)], and [Ni(erx)2(bipyam)]
[54], these complexes can be assumed to bind to DNA by intercalation, but the binding
mode needs to be proven through more experiments.
3.3.2. Emission spectral studies. To investigate further, the interaction mode among the
Ni(II) complexes, [Ni(L1)2–Ni(L6)2], and CT-DNA fluorescence titration experiments were
performed. These complexes emit luminescence in Tris–HCl/NaCl buffer at room tempera-
ture with maximum wavelengths of approximately 379 nm (Supplementary material, figures
S5–S8). The emission spectra of Ni(L1)2 and Ni(L2)2 (50 μM) in the absence and presence
of CT-DNA (0–200 μM) are shown in figures 4 and 5. The emission intensity of

14
A.Q. Ali et al.
Figure 3. Absorption spectra of Ni(L3)2 in the absence and presence of increasing amounts of DNA in Tris–HCl
buffer (pH 7.2). [Ni(L3)2] = 50 μM, [DNA] = 0–163.58 × 10−6 M. The arrow shows the effect of increasing of
CT-DNA concentration on the absorption intensity.
Figure 3a. Comparative plot of [DNA]/(εa − εf) vs. [DNA] for the absorption titration of CT-DNA with Ni(L3)2 in
Tris–HCl buffer (pH 7.2); Kp = 3.29 × 106 M−1 (R = 0.92621, n = 5 points).
Ni(L1)2–Ni(L6)2 increases with increasing CT-DNA concentrations. These observations
imply that these complexes can strongly interact with CT-DNA and propose intercalative
modes with the base pairs of the DNA helix [55].
3.3.3. Viscosity measurements. Hydrodynamic measurements (i.e. viscosity and sedimen-
tation) that are sensitive to length changes are regarded as the most critical and the least
ambiguous tests of binding in a solution in the absence of crystallographic data [56, 57]. A
classical intercalator molecule such as EB can cause a significant increase in the viscosity
of the DNA solution because of an increase in the separation of base pairs at the interaction
site and an increase in overall double helix length. However, molecules that bind exclu-
sively in the DNA grooves, groove face, or electrostatic interactions typically cause a bend
in the DNA helix, thereby reducing its effective length and decreasing its viscosity [58–60].
The viscosity measurements of Ni complexes (Ni(L1)2–Ni(L6)2) and EB are shown in

Nickel(II) isatin thiosemicarbazone
15
Figure 4. Emission spectra of Ni(L1)2 in the absence and presence of increasing amounts of DNA in Tris–HCl
buffer (pH 7.2). [Ni(L1)2] = 50 μM, [DNA] = 0–163.58 × 10−6 M. The arrow shows the emission intensity
increases upon increasing CT-DNA concentration.
Figure 5. Emission spectra of Ni(L2)2 in the absence and presence of increasing amounts of DNA in Tris–HCl
buffer (pH 7.2). [Ni(L2)2] = 50 μM, [DNA] = 0–163.58 × 10−6 M. The arrow shows the emission intensity
increases upon increasing CT-DNA concentration.
figures 6 and 7. The plot of relative specific viscosity versus [complex]/[DNA] ratio signifi-
cantly increases upon the addition of ligand. These observations suggest the intercalative
binding of these complexes to the double helix [61].
3.4. DNA cleavage studies
The cleavage of SC pBR322 DNA (0.5 μg μL−1) was investigated through agarose gel elec-
trophoresis using different concentrations of Ni(II) complexes (see Supplementary material,
figures S9–S12) in 1% DMSO/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2, with and
without H2O2, and with 2 h incubation. The activities of the complexes were estimated by
the conversion of DNA from Form I to Forms II and III. The fastest migration is detected
in the SC form (Form I). If only one strand is cleaved, the supercoils relax to convert into a
slower-moving form (Form II). If both strands are cleaved, a linear form (Form III) is
produced, which migrates between Forms I and II [62].

16
A.Q. Ali et al.
Figure 6. Effect of increasing amounts of EB ( ), Ni(L1)2
viscosity of CT-DNA at 37.0 ( 0.1) °C.
( ), Ni(L2)2 ( ), and Ni(L3)2 ( ) on the relative
Figure 7. Effect of increasing amounts of EB ( ), Ni(L4)2
viscosity of CT-DNA at 37.0 ( 0.1) °C.
( ), Ni(L5)2 ( ), and Ni(L6)2 ( ) on the relative
3.5. Oxidative cleavage
The control experiment did not show any apparent cleavage of DNA (Lane 2). In the pres-
ence of the complexes [Ni(L1)2–Ni(L6)2] at different concentrations of H2O2 (Lanes 4–12),
the plasmid DNA is converted from Form I to Form II at 0.1 mM concentration [Lane 4 for
Ni(L1)2, Ni(L4)2, and Ni(L5)2] and at 0.2 mM concentration [Lane 5 for (Ni(L3)2), figure
8]. Meanwhile, the plasmid DNA is converted from Form I to Forms II and III at 0.1 and
0.4 mM concentrations [Lanes 4 and 7 for Ni(L6)2 and Ni(L2)2, respectively]. The DNA is
totally degraded at 0.2, 0.3, 0.4, and 0.4 mM concentrations [Lanes 5, 6, 7, and 8 for Ni
(L6)2, Ni(L4)2, Ni(L5)2, and Ni(L1)2, respectively]. These results indicate that the Ni(II)
complexes can degrade pBR322 DNA through oxidative cleavage in the presence of H2O2
via the formation of hydroxyl radical species [63, 64]. This phenomenon can be explained
by considering Fenton-type or Haber–Weiss-type reactions [65], in which a transition metal
ion reduces H2O2 to yield hydroxyl radical that can damage the DNA and lead to strand
breakage.

Nickel(II) isatin thiosemicarbazone
17
Figure 8. Cleavage of SC pBR322 (0.5 μg μL−1) Ni(L3)2 at different concentrations in Tris–HCl buffer pH (7.2)
for 2 h at 37 °C. Lane 1: DNA ladder; Lane 2: DNA + H2O2; Lane 3: DNA + Ni(L3)2 (1 mM) + DMSO; Lanes 4–
12: DNA with increasing the concentrations of Ni(L3)2 (0.1–1 mM) + H2O2 + 1% DMSO.
NiðIIÞ þ ꢂOꢁ2 ! NiðIÞ þ O2
NiðIÞ þ H2O2 ! NiðIIÞ þ OHꢁ þ ꢂOH
ꢂOꢁ2 þ H2O2 ! O2 þ OHꢁ þ ꢂOH
(1)
(2)
(3)
To investigate the mechanism of DNA cleavage promoted by these complexes, the reac-
tions were allowed to proceed in the presence of DMSO as hydroxyl radical scavengers
(Lane 3). The addition of hydroxyl radical scavenger completely inhibits DNA cleavage
activity (Ni(L5)2, figure 9), which is induced by these complexes. This observation suggests
the involvement of the hydroxyl radical in the cleavage [66], confirming the oxidative path-
ways of these complexes toward DNA [67]. The results revealed that the Ni(L6)2 complex
has more cleavage activity than the other complexes.
3.6. In vitro anti-proliferative activity
In vitro anti-proliferative study of the Ni(II) complexes of thiosemicarbazone analogs was
previously conducted on Ehrlich ascitic carcinoma cells (IC50 = 191 μM) [66] against three
human cancer cell lines (THP-1; IC50 = 89 μM, Raji; IC50 = 97 μM and Hela cells;
IC50 = 181 μM) [52]. In the present research, the activities of the new Ni(II) complexes were
studied against the human colorectal carcinoma cell line and are reported for the first time.
The in vitro anti-proliferative activity of the Ni(II) complexes of isatin thiosemicarbazone
derivatives was evaluated against a human colorectal cancer (HCT116) cell. The effect of
Figure 9. Cleavage of SC pBR322 (0.5 μg μL−1) Ni(L5)2 at different concentrations in Tris–HCl buffer pH (7.2)
for 2 h at 37 °C. Lane 1: DNA ladder; Lane 2: DNA + H2O2; Lane 3: DNA + Ni(L5)2 (1 mM) + DMSO; Lanes
4–12: DNA with increasing the concentrations of Ni(L5)2 (0.1–1 mM) + H2O2 + 1% DMSO.

18
A.Q. Ali et al.
different concentrations of Ni complexes [Ni(L1)2–Ni(L6)2] on human colorectal cancer
cells (HCT 116) after 72 h of treatment is given in figure 10. The anticancer efficiencies of
all tested compounds are presented in table 3. The cells treated with the standard drug 5-FU
(IC50 = 7.3 μM) show strong inhibitory effect on cell proliferation. The cells exhibit several
toxic signs, as all the cells are converted into round-shaped morphology after losing the nor-
mal pseudopodial cellular projections. These compounds show comparable cytotoxicities
against HCT116 cell lines to those of 5-FU. Figure 11 shows the image of the cancer cells
treated with these compounds for 48 h. The Ni(L6)2 compound has the strongest cytotoxic
effect (IC50 = 15 μM). The image shows clear signs of cytotoxicity of the compound on the
cells, as most of the cells are affected because of the treatment. The effect can be compared
with that of the standard reference 5-FU. The treatment of HCT 116 cells with the Ni(L3)2
compound has significant cytotoxicity (IC50 = 55 μM). The population doubling time is
reduced significantly compared with the negative control. The morphology of the cells
reveals that the cells become weak because of the treatment. The HCT 116 cells treated
with Ni(L1)2 display moderate inhibition in cell proliferation, with IC50 = 79 μM. The
image reveals that the number of cells considerably decreases compared with the negative
control. The Ni(L4)2 compound exhibits moderate inhibitory effect on the proliferation of
Figure 10. Effect of different concentrations of nickel(II) complexes of isatin thiosemicarbazone derivatives [Ni
(L1)2–Ni(L6)2] on human colorectal cancer cells (HCT 116) after 72 h treatment.
Table 3. IC50 values of selected
compounds.
Sample code
IC50 value/μM
Ni(L1)2
Ni(L2)2
Ni(L3)2
Ni(L4)2
Ni(L5)2
Ni(L6)2
5-FU
79
104
55
85
134
15
7.3

Nickel(II) isatin thiosemicarbazone
19
Figure 11. Picture of cancer cells treated with nickel(II) complexes of isatin thiosemicarbazone derivatives [Ni
(L1)2–Ni(L6)2] for 48 h.
the cells (IC50 = 85 μM). Finally, compounds Ni(L2)2 and Ni(L5)2 show negligible cytotox-
icity as the estimated IC50 is high (104 and 134 μM, respectively). The cellular morphology
was unaltered compared with the vehicle-treated cells.

20
A.Q. Ali et al.
4. Conclusion
Six octahedral Ni(II) complexes, Ni(L1)2–Ni(L6)2, with tridentate ligands were synthesized
and characterized using elemental analysis and various spectroscopic techniques. The crys-
tal structure of Ni(L2)2 showed that it was a six-coordinate complex with slightly distorted
octahedral geometry. The results of binding studies showed that these complexes interact
with CT-DNA through an intercalative mode, which was investigated by absorption (the
intrinsic binding constant (Kb) values were 1.4 × 105–2.4 × 106 M−1), fluorescence, and vis-
cosity measurement. Finally, the results of the in vitro anti-proliferative activity against
HCT 116 cells showed dose-dependent cytotoxicity of the synthesized complexes, with low
IC50 values ranging from 15 to 134 μM. The strongest IC50 value for Ni(L6)2 compared
with Ni(L1)2, Ni(L2)2, and Ni(L3)2, which have extended phenyl rings that may lead to
additional activity, is unexpected [68]. The relationships between structure and anticancer
potency are not yet well established. Elucidation of this behavior requires more experi-
ments, which will be undertaken in the near future. The Ni(II) complexes of thiosemicarba-
zone Schiff bases with isatin can be promising anti-neoplastic agents. The toxic potentials
of the most active complexes and their anti-tumor efficacies are under investigation in ani-
mal models, and will be reported in due course. The structure–activity relationship studies
were recommended for future work.
Acknowledgements
The authors thank the Malaysian Government and Universiti Sains Malaysia for the RU
research grant (1001/PKIMIA/815067).
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