Synthesis and characterization of thiosemicarbazonato molybdenum(VI) complexes: In vitro DNA binding, cleavage, and antitumor activities

Article abstract of DOI:10.1016/j.poly.2014.02.048

Four dioxomolybdenum(VI) complexes were synthesized by the reaction of [MoO₂(acac)₂] with thiosemicarbazone ligands derived from 5-chloro-2-hydroxy-benzaldehyde (1), 2-hydroxy-5-methoxybenzaldehyde (2), 2-hydroxy-5-methylbenzaldehyde (3), or 5-tert-butyl-2-hydroxybenzaldehyde (4). In all complexes, the ligands were coordinated to molybdenum as tridentate ONS donors. X-ray crystallography results show that the distorted octahedral coordination of the molybdenum atom is completed by a methanol molecule (D), as in 1a, 3a, and 4a, or by H₂O, as in 4a. The molecular structures of 1, 2, and the complexes were determined by single-crystal X-ray crystallography. The binding properties of the ligand and complexes with calf thymus DNA (CT DNA) were investigated by UV titration, fluorescence titration, and viscosity measurements. Gel electrophoresis assay results show that the complexes can cleave the pBR 322 plasmid DNA. The cytotoxic activities of the complexes were investigated against human colorectal cell lines. All complexes showed high anticancer activities in the order, 4a > 1a 蠑 3a > 2a, according to their IC₅₀values (3.4, 3.5, 6.4, and 6.9 μM, respectively).

Full text of DOI:10.1016/j.poly.2014.02.048

Polyhedron 85 (2015) 93–103
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis and characterization of thiosemicarbazonato molybdenum(VI)
complexes: In vitro DNA binding, cleavage, and antitumor activities
a
a,
⇑
a
b
Mouayed A. Hussein , Teoh S. Guan , Rosenani A. Haque , Mohamed B. Khadeer Ahamed ,
b
Amin M.S. Abdul Majid
a
School of Chemical Science, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Four dioxomolybdenum(VI) complexes were synthesized by the reaction of [MoO (acac) ] with thiosem-
2
2
Received 29 October 2013
Accepted 5 February 2014
Available online 28 August 2014
icarbazone ligands derived from 5-chloro-2-hydroxy-benzaldehyde (1), 2-hydroxy-5-methoxybenzalde-
hyde (2), 2-hydroxy-5-methylbenzaldehyde (3), or 5-tert-butyl-2-hydroxybenzaldehyde (4). In all
complexes, the ligands were coordinated to molybdenum as tridentate ONS donors. X-ray crystallogra-
phy results show that the distorted octahedral coordination of the molybdenum atom is completed by
Keywords:
2
a methanol molecule (D), as in 1a, 3a, and 4a, or by H O, as in 4a. The molecular structures of 1, 2,
Molybdenum(VI) complexes
X-ray crystal structure
DNA binding
DNA cleavage
Antitumor activity
and the complexes were determined by single-crystal X-ray crystallography. The binding properties of
the ligand and complexes with calf thymus DNA (CT DNA) were investigated by UV titration, fluorescence
titration, and viscosity measurements. Gel electrophoresis assay results show that the complexes can
cleave the pBR 322 plasmid DNA. The cytotoxic activities of the complexes were investigated against
human colorectal cell lines. All complexes showed high anticancer activities in the order,
4
a > 1a ꢀ 3a > 2a, according to their IC50 values (3.4, 3.5, 6.4, and 6.9
lM, respectively).
Ó 2014 Elsevier Ltd. All rights reserved.
1
. Introduction
Molybdenum (Mo) is an essential trace element for almost all
[10–12]. These molybdoenzymes have oxo-groups that are
believed to be responsible for the oxotransferase activity of these
enzymes. These oxo-groups contain a Mo center, which has an oxi-
dation state of +6 or +4 during a reaction cycle, in their catalytic
site. This center is coordinated by two or more sulfur donor
ligands.
Thiosemicarbazones have attracted considerable pharmaceuti-
cal interest because of their antiviral, antifungal, and antitumor
activities [13]. The biological activities of thiosemicarbazones are
a function of their parent aldehyde or ketone moiety, which acts
as tridentate ONS (doubly deprotonated) donor ligands and pro-
vides a suitable complexation structure for many transition metals
[14,15].
Dioxomolybdenum(VI) complexes with tridentate ONS donor
ligands have attracted considerable attention as model systems
for molybdoenzyme active sites. In such systems, Mo exhibits a
normal octahedral coordination that is usually completed by addi-
tional nitrogen donor ligands [16]. Various classes of Mo com-
plexes have been prepared, tested, and developed for diverse
pharmaceutical purposes. The anticancer activities of these com-
plexes have received particular attention [17,18].
biological systems [1–3]. It is required as a cofactor for a number
of enzymes that catalyze important chemical transformations in
the global carbon, nitrogen, and sulfur cycles [4]. Mo functions as
a cofactor for three enzymes called molybdoenzymes. Sulfite oxi-
dase catalyzes the transformation of sulfite to sulfate, a reaction
that is necessary for the metabolism of sulfur-containing amino
acids such as cysteine [5,6]. Xanthine oxidase catalyzes the oxida-
tion of hypoxanthine to xanthine and can further catalyze the oxi-
dation of xanthine to uric acid [7,8]. This enzyme plays an
important role in the catabolism of purines in some species, includ-
ing humans. Aldehyde oxidase-catalyzed hydroxylation reactions
involve a number of different molecules with similar structures
[
9]. Xanthine oxidase catalyzes the breakdown of nucleotides (pre-
cursors to DNA and RNA) to form uric acid, which contributes to
the antioxidant capacity of the blood. Xanthine oxidase and alde-
hyde oxidase also play a role in the metabolism of drugs and toxins
⇑
Corresponding author. Address: School of Chemical Science, Universiti Sains
In this work, we describe the synthesis and characterization of a
series of cis-dioxomolybdenum(VI) complexes with thiosemicarba-
Malaysia, Penang 11800, Malaysia. Tel.: +60 4 6577888; fax: +60 4 6574854.
E-mail address: sgteoh@usm.my (T.S. Guan).
http://dx.doi.org/10.1016/j.poly.2014.02.048
0
277-5387/Ó 2014 Elsevier Ltd. All rights reserved.

9
4
M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
zone ligands (Scheme 1). The complexes and their respective
ligands were used to cleave/bind DNA using spectral and physical
methods. The complexes were also tested against human colorectal
were obtained in the 230 nm to 600 nm wavelength range using a
spectrophotometer (Perkin Elmer, lambda-35).
Fluorescence emission assay was performed using the above-
mentioned method. The fluorescence spectra were scanned in the
200–800 nm wavelength range using a spectrophotometer (Jasco
FP-750).
(
HCT 116) cell lines. The structures of all complexes were investi-
gated in detail using X-ray crystallography.
Viscosity measurements were performed using a Cannon Man-
ning Semi-micro viscometer immersed in a thermostatic water
bath at 37 °C. Flow times were manually measured with a digital
stopwatch. The viscosity values were calculated from the observed
flow times of the DNA-containing solutions (t), which were cor-
2
. Experimental
2.1. Materials and instrumentation
Melting points were measured using a Stuart Scientific SMP1
rected for that of the solvent mixture used (t
= t – t . Viscosity data were presented as (
plex]/[DNA], where and are the viscosity of the complex in
the presence of DNA and the viscosity of DNA alone, respectively
20].
0
) using the equation,
melting point apparatus. Infrared (IR) spectra were recorded on a
Perkin Elmer system 2000 spectrophotometer using the KBr disc
method. H and C NMR spectra were recorded on a Bruker spec-
trophotometer at 500 and 400 MHz, respectively, using tetrameth-
1
/3
g
0
g
/g
0
)
versus [com-
1
13
g
g
0
[
ylsilane as an internal standard and DMSO-d
6
as the solvent.
Elemental analysis was performed using a Perkin Elmer 2400 ser-
ies-11 CHN analyzer. Electronic and fluorescent spectra were
recorded on Perkin Elmer lambda-35 and Jasco FP-750 spectropho-
tometers, respectively. X-ray crystallographic data were recorded
on a Bruker SMART APEXII CCD area-detector diffractometer. All
chemicals, including thiosemicarbazide, aldehydes, and solvents,
were purchased from Sigma–Aldrich.
2
.3. DNA cleavage assay
The activities of the ligands and the complexes in cleaving
pBR322 plasmid DNA were determined by agarose gel electropho-
resis in a Tris/EDTA buffer solution. The samples were incubated at
3
7 °C for 2 h, treated with a loading dye, and then electrophoresed
on a 1% agarose gel for 1 h at 50 V. The gel was then stained using
ethidium bromide before being photographed under UV light.
2.2. DNA binding assay
2
2
.4. Cytotoxicity assay
The binding of the complexes with calf thymus DNA (CT DNA)
was performed in a 6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7).
The stock DNA solution was prepared by dissolving a suitable
amount of DNA in the 6.3 mM Tris–HCl/50 mM NaCl buffer (pH
.4.1. Cell culture preparation
HCT 116 cells were grown under optimal incubator conditions.
Cells that have reached a confluency of 70% to 80% were chosen for
cell plating. The old medium was replaced by aspiration. The cells
were then washed with sterile phosphate-buffered saline (PBS)
7
) at room temperature and subsequently storing in a refrigerator
for two days. A solution of CT DNA in the buffer showed a ꢁ1.9:1
ratio of UV absorbances at 260 and 280 nm, suggesting that the
DNA was sufficiently free of protein. The DNA concentration was
estimated by the UV absorbance at 260 nm using the known molar
(
pH 7.4) 2 to 3 times. Afterward, the intact layer of the attached
cells was subjected to trypsinization. The cells were incubated in
% CO at 37 °C for 3–5 min. The flasks containing the cells were
5
2
À1
À1
absorption coefficient value of 6600 M cm [19]. UV–Vis spectra
gently tapped to promote cell segregation. The cells were then
observed under an inverted microscope to confirm that cell segre-
gation was complete. Trypsin activity was inhibited by adding 5 ml
of fresh complete media supplied with 10% fetal bovine serum
(
FBS). The cells were counted and then diluted to obtain a final
5
concentration of 2.5 Â 10 cells/ml. Then, the cells were inoculated
into wells (100 l/well). The plates containing the cells were then
incubated at 37 °C under an internal atmosphere of 5% CO
l
2
.
2.4.2. MTT assay
The cytotoxicity of complexes against HCT 116 cells was evalu-
5
ated using the MTT assay [21]. The HCT 116 cells (1.5 Â 10 cells/
ml, 100 l/well) were seeded in a 96-well microtiter plate, which
was then incubated overnight in a CO incubator to allow cell
attachment. Exactly 100 L of the test substance was added into
l
2
l
each well containing the cells. From the stock solution, the test
substance was diluted with media into the desired concentrations.
The plates were then incubated at 37 °C for 72 h under an internal
atmosphere of 5% CO
added into each well. The plates were again incubated for 4 h. After
this incubation period, 50 L of the MTT lysis solution (DMSO) was
added into the wells. The plates were further incubated for 5 min
in a CO incubator. Finally, the plates were read at 570 and
20 nm using a standard ELISA microplate reader. Data were
2
. Afterward, 20 ll of the MTT reagent was
l
2
6
recorded and analyzed to assess the effects of the test substance
on cell viability. The percentage of growth inhibition was then cal-
culated. The optical density (OD) was also obtained from the MTT
assay by dividing the hundredth multiple of the subtracted OD
value of the control and survived cells by the OD of the control
cells.
Scheme 1. General synthetic procedure of ligands and complexes.

M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
95
2
2
.5. Ligand synthesis
.5.1. Synthesis of N-ethyl-2-(5-chloro-2-hydroxybenzylidene)hydra-
2.6. Synthesis of complexes
2.6.1. Synthesis of N-ethyl-2-(5-chloro-2-hydroxybenzylidene)hydra-
zinecarbothioamide (1)
zinecarbothioamide dioxomolybdenum(VI) (1a)
The ligand 1 was synthesized as reported in literature [22]. M.p:
2 2
A solution of MoO (acac) (0.126 g, 0.387 mmol) in methanol
1
4
75–177 °C, (0.84 g, 78%). Anal. Calc. for C10
H
12ClN
3
OS: C, 46.55; H,
(25 ml) was added to a solution of N-ethyl-2-(5-chloro-2-hydrox-
ybenzylidene)hydrazinecarbothioamide (0.1 g, 0.387 mmol) in
methanol (25 ml). The resulting red solution was refluxed for 2 h
and then filtered. The filtrate was left to stand at room temperature
for three days, subsequently obtaining orange, plate-like crystals.
.65; N, 16.29. Found: C, 46.21; H, 4.77; N, 16.18%; IR(KBr) (
m
max
/
À1
cm ): 3301 (m, NH), 3146 (m, OH), 1601–1616 (m, C@N–N–C),
1
(
(
3
(
557 (s, CaroO), 1269 (m, C@S); 1H NMR (DMSO-d
CH ), 3.56 (CH ), 6.84–7.97 (H-aromatic), 8.27 (CS-NH), 8.58
CH@N), 11.30 (N–NH); C NMR (DMSO-d
0.54 (CH ), 117.72–137.66 (C-aromatic), 154.97 (C@N), 176.36
C@S).
6
, ppm): 1.11
3
2
13
6
, ppm): 14.46 (CH
3
),
3 4
M.p: 242–244 °C, (0.11 g, 73%). Anal. Calc. for C11H14ClMoN O S:
2
C, 31.75; H, 3.36; N, 10.10; Mo, 23.08. Found: C, 31.76; H, 3.37;
À1
N, 10.44; Mo, 22.98%; IR(KBr) (
m
max/cm ): 3303 (s, NH), 1587 (s,
1
C@N), 1553 (s, CaroO), 1266 (m, C–S), 938,901 (s, Mo@O); H NMR
(DMSO-d , ppm): 1.11 (CH ), 3.25 (alcoholic-CH ), 3.56 (CH ),
6.85, 7.39, 8.03 (H-aromatic), 7.65 (alcoholic-OH), 8.28 (C–NH),
2.5.2. Synthesis of N-methyl-2-(5-methoxy-2-hydroxybenzylidene)-
6
3
3
2
hydrazinecarbothioamide (2)
solution of 5-methoxy-2-hydroxybenzaldehyde (0.72 g,
.75 mmol) in ethanol (20 ml) was added to a solution of 4-
methyl-3-thiosemicarbazide (0.5 g, 4.75 mmol) in ethanol
20 ml). The resulting colorless solution was refluxed with stirring
1
3
A
8.55 (CH@N); C NMR (DMSO-d
6
,ppm): 14.54 (CH
3 2
), 30.65 (CH ),
4
48.15 (alcoholic-CH
157.29 (C–S).
3
), 117.68–137.18 (C-aromatic), 149.94 (C@N),
(
for 2 h. A white, fluffy product was formed when the solution
cooled to room temperature. The solution was then filtered,
washed with ethanol, and then air-dried. Colorless, plate-like
crystals were obtained by slow evaporation of a DMF solution at
room temperature. M.p: 228–230 °C, (0.85 g, 75%). Anal. Calc. for
2.6.2. Synthesis of N-methyl-2-(5-methoxy-2-hydroxybenzylidene)-
hydrazinecarbothioamide dioxomolybdenum(VI) (2a)
2 2
A solution of MoO (acac) (0.136 g, 0.417 mmol) in ethanol
(25 ml) was added to a solution of N-methyl-2-(5-methoxy-2-
hydroxybenzylidene)hydrazinecarbothioamide (0.1 g, 0.417 mmol)
in ethanol (25 ml). The resulting red solution was refluxed for 2 h
and then filtered. The filtrate was left to stand at room temperature
for one week, subsequently obtaining brown, needle-like crystals.
C
5
1
10
H
13
N
3
O
2
S: C, 50.14; H, 5.43; N, 17.55. Found: C, 50.17; H,
À1
.48; N, 17.44%; IR(KBr) (
m
max/cm ): 3379 (s, NH), 3250 (s, OH),
1
610–1622 (m, C@N–N–C), 1559 (s, CaroO), 1271 (s, C@S);
ppm): 3.02 (CH ), 3.37 (O–CH ), 6.78–7.49
H-aromatic), 8.34 (CS–NH), 8.43 (CH@N), 9.46 (OH), 11.41
H
NMR (DMSO-d
6
,
3
3
3 5
M.p: 225–227 °C, (0.12 g, 81%). Anal. Calc. for C10H13MoN O S: C,
31.31; H, 3.39; N, 10.95; Mo, 25.03. Found: C, 31.54; H, 3.0; N,
(
(
1
3
À1
N–NH); C NMR (DMSO-d
6
, ppm): 30.83 (CH
3
), 55.61 (O–CH
3
),
10.94; Mo, 24.96%; IR(KBr) (
m
max/cm ): 3343 (s, NH), 1592 (m,
1
1
10.27–152.35 (C-aromatic), 152.35 (C@N), 177.56 (C@S).
C@N), 1564 (s, CaroO), 1269 (s, C–S), 933,898 (s, Mo@O); H NMR
DMSO-d , ppm): 3.02 (CH ), 3.81 (O–CH ), 6.78, 7.05, 7.19
(H-aromatic), 7.46 (H O), 8.31 (C–NH), 8.55 (CH@N);
NMR (DMSO-d ,ppm): 30.62 (CH ), 55.56 (O–CH ), 109.90–138.92
(C-aromatic), 150.51 (C@N), 153.02 (C–S).
(
6
3
3
13
2.5.3. Synthesis of N-methyl-2-(5-methyl-2-hydroxybenzylidene)-
2
C
hydrazinecarbothioamide (3)
solution of 5-methyl-2-hydroxybenzaldehyde (0.64 g,
.75 mmol) in ethanol (20 ml) was added to a solution of 4-
methyl-3-thiosemicarbazide (0.5 g, 4.75 mmol) in ethanol
20 ml). The resulting colorless solution was refluxed with stirring
6
3
3
A
4
2.6.3. Synthesis of N-methyl-2-(5-methyl-2-hydroxybenzylidene)-
hydrazinecarbothioamide dioxomolybdenum(VI) (3a)
(
for 2 h. A white product was formed when the solution cooled to
room temperature. The solution was then filtered, washed with
ethanol, and then air-dried. M.p: 227–229 °C, (0.77 g, 73%). Anal.
2 2
A solution of MoO (acac) (0.146 g, 0.447 mmol) in methanol
(25 ml) was added to a solution of N-methyl-2-(5-methyl-2-
hydroxybenzylidene)hydrazinecarbothioamide (0.1 g, 0.447 mmol)
in methanol (25 ml). The resulting red solution was refluxed for
2 h and then filtered. The filtrate was left to stand at room temper-
ature for one week, subsequently obtaining orange, plate-like crys-
Calc. for C10
H, 5.84; N, 18.99%; IR(KBr) (
H
13
N
3
OS: C, 53.73; H, 5.82; N, 18.80. Found: C, 53.29;
À1
m
max/cm ): 3358 (s, NH), 1603 (m,
1
C@N), 1556 (s, CaroO), 1276 (m, C@S); H NMR (DMSO-d
6
, ppm):
2
8
d
.22 (H
.38 (CH@N), 9.71 (OH), 11.37 (N–NH);
,ppm): 20.04 (CH –Ph), 30.63 (CH ), 115.90–131.70 (C-aro-
3
C–Ph), 3.01 (CH
3
), 6.75–7.71 (H-aromatic), 8.33 (CS–NH),
3 4
tals. M.p: 222–224 °C, (0.11 g, 70%). Anal. Calc. for C11H15MoN O S:
C, 34.62; H, 3.93; N, 11.01; Mo, 25.16. Found: C, 34.65; H, 3.12; N,
13
C
NMR (DMSO-
À1
6
3
3
10.89; Mo, 25.02%. IR(KBr) (
m
max/cm ): 3320 (m, NH), 1590 (m,
1
matic), 154.18 (C@N), 177.41 (C@S).
C@N), 1565 (s, CaroO), 1275 (m, C-S), 938,898 (s, Mo@O); H NMR
DMSO-d , ppm): 2.22 (H C–Ph), 2.86 (alcoholic-CH ), 3.01 (CH ),
6.78, 7.24, 7.39 (H-aromatic), 7.46 (alcoholic-OH), 8.31 (C–NH),
(
6
3
3
3
2
.5.4. Synthesis of N-methyl-2-(5-ter-butyl-2-hydroxybenzylidene)-
hydrazinecarbothioamide (4)
solution of 5-ter-butyl-2-hydroxybenzaldehyde (0.84 g,
.75 mmol) in ethanol (20 ml) was added to a solution of 4-
methyl-3-thiosemicarbazide (0.5 g, 4.75 mmol) in ethanol
20 ml). The resulting colorless solution was refluxed with stirring.
1
3
8.50 (CH@N); C NMR (DMSO-d
(CH ), 48.56 (alcoholic-CH ), 117.59–139.55 (C-aromatic), 151.53
(C@N), 164.70 (C–S).
6 3
,ppm): 20.01 (CH –Ph), 30.66
A
3
3
4
(
2.6.4. Synthesis of N-methyl-2-(5-ter-butyl-2-hydroxybenzylidene)-
After 1 h, a white fluffy product was formed. The product was fil-
hydrazinecarbothioamide dioxomolybdenum(VI) (4a)
tered, washed with ethanol, and then air-dried. M.p: 221–223 °C,
2 2
A solution of MoO (acac) (0.123 g, 0.376 mmol) in methanol
(
1.15 g, 92%). Anal. Calc. for C13
5.82. Found: C, 58.57; H, 7.57; N, 15.81%; IR(KBr) (
H
19
N
3
OS: C, 58.78; H, 7.15; N,
(25 ml) was added to a solution of N-methyl-2-(5-ter-butyl-2-
hydroxybenzylidene)hydrazinecarbothioamide (0.1 g, 0.376 mmol)
in methanol (25 ml). The resulting red solution was refluxed for
2 h and then filtered. The filtrate was left to stand at room
temperature for four days, subsequently obtaining orange,
needle-like crystals. M.p: 231–233 °C, (0.14 g, 90%). Anal. Calc. for
À1
1
3
C
3
9
m
max/cm ):
384 (s, NH), 3186 (m, OH), 1598–1618 (m, C@N–N–C), 1550 (s,
aroO), 1263 (m, C@S); 1H NMR (DMSO-d
6 3 3
, ppm): 1.27 [C(CH ) ],
.03 (CH
.69 (OH), 11.35 (N–NH);
], 31.32 (CH , 33.83 (CH
54.22 (C@N), 177.41 (C@S).
3
), 6.79–7.76 (H-aromatic), 8.32 (CS–NH), 8.38 (CH@N),
13
C
6
NMR (DMSO-d ,ppm): 30.95
[
C(CH)
3
)
3 3
3
), 115.65–141.53 (C-aromatic),
C
14
3
H21MoN O
4
S: C, 39.68; H, 4.96; N, 9.92; Mo, 22.66. Found: C,
À1
1
38.79; H, 4.64; N, 9.92; Mo, 22.44%. IR(KBr) (
m
max/cm ): 3330

9
6
M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
(
m, NH), 1586–1605 (m, C@N–N–C), 1556 (m, CaroO), 932,902 (s,
in relation to N2 atom, as well as the cis geometries of N1 atom in
relation to N3 atom and C9 atom in relation to S1 atom. The C8–S1
bond distances are 1.7035(19) and 1.6874(13) Å as well as the N2–
C8 bond distances are 1.351(3) and 1.3691(17) Å for 1 and 2,
respectively, indicate the ligands are exist in the tautomeric thione
form. The ligands can divide into two planar moieties that are con-
nected by a single N1–N2 bond. The N-ethylthioureide plane
includes N2, S1, N3, and C8–C10 and, the salicylaldimine plane
includes N1, O1, and C1–C7. The atoms O2 and C10 from the meth-
oxy group in 2 form a coplanar from salicylaldimine fragment
which are inclined by 173.78(14)°. Dihedral angle between these
two planes is 13.8(3)° and 14.39(19)° for 1 and 2, respectively [23].
1
Mo@O); H NMR (DMSO-d
CH ), 3.05 (CH ), 6.81, 7.49, 7.65 (H-aromatic), 7.46 (alcoholic-
OH), 8.39 (C–NH), 8.61 (CH@N); C NMR (DMSO-d
(CH ], 31.30 [C(CH) ], 33.85 (CH ), 48.57 (alcoholic-CH
17.25–143.17 (C-aromatic), 152.02 (C@N), 156.67 (C–S).
6 3 3
, ppm): 1.29 [(CH ) ], 2.86 (alcoholic-
3
3
13
6
,ppm): 30.65
),
[
1
)
3 3
3
3
3
3
. Results and discussion
3
.1. Synthesis and characterization of complexes
Novel dioxomolybdenum(VI) complexes were obtained by
reacting [MoO (acac) ] with 4-methyl(ethyl)thiosemicarbazone
2
2
ligands, which were derived from 1, 2, 3, or from 4. In all the com-
plexes, Mo coordinated with the ligands via phenolic-oxygen,
imine-nitrogen, and thiol-sulfur bonds. X-ray crystallography
showed the distorted octahedral complexes of cis-dioxomolybde-
3
.2.2. Crystallographic description of 1a to 4a
The molecular structures of complexes 1a to 4a are shown in
Figs. 3–6. The general crystal data are listed in Table 1. The Mo sur-
roundings can be described as an equatorial plane posed by the
imine nitrogen, thiolate sulfur, phenolate oxygen, and one of the
num(VI), whose D site is occupied by CH
3
OH as in 1a, 3a, and 4a,
and H O as in 2a. The ligands and their respective complexes are
2
+
2
terminal oxo ligands of the MoO
2
unit, with the other terminal
all air-stable and are highly soluble in DMSO and DMF.
Ligands 1 to 4 all exhibited FTIR, 1H NMR, and 13C NMR spectra
oxo group occupying the apical position. The coordination geome-
try around Mo has distorted the octahedral arrangement. The two
terminal oxygen atoms O3 and O4 are apart of a central cis-
and elemental analysis results that are consistent with their
À1
assigned structures. In FTIR, the bands in the 1598 to 1622 cm
+
2
[
MoO
2
]
unit. The molecular structure of 1a and 2a revealed that
range are attributed to the (C@N–N–C) group, those in the 1550
À1
the ligands 1 and 2 afford main changes upon coordinated with
to 1557 cm range are assigned to the (Caro–O) group, and the
+
2
À1
[MoO2] unit. The C10–C9–N3–C8–S1 set for 1 and, the C9–N3–
C8–S1 set for 2 rotates approximately 180° around the N2–C8 bond
as well as the phenyl ring moiety for both ligands has to flip around
the C6–C7 bond. Meanwhile, The trans geometry between N1 atom
and S1 atom, and the cis geometry between N1 atom and N3 atom
bands in the 1263 to 1276 cm range correspond to the (C@S)
1
group. In the H NMR spectra, the signals due to the (–CH
3
) group
of 2, 3, and 4 appeared in the 3.01 ppm to 3.03 ppm range, that of 1
appeared at 1.11 ppm, and that due to the (CS–NH) group appeared
in the 8.27–8.34 ppm range. Meanwhile, the signals located within
the 6.78–7.97 ppm range are attributed to the aromatic protons,
whereas the signal that appeared in the 11.30 ppm to 11.41 ppm
+2
are changing during the coordination of 1 and 2 with [MoO
2
]
unit
to cis and trans geometry, respectively. The same geometries are
observed for the complexes 3a and 4a in which the methanol
occupy the sixth position of their distorted octahedral geometry.
Interestingly, the cis geometry between C9 atom from the N-termi-
nal group and S1 atom, as well as the trans geometry between C9
and N2 atoms, are remained without changed during the coordina-
range is attributed to the (N–NH) group. In the 13C NMR, the signal
due to the (C@S) group appeared in the 176.36–177.56 ppm range,
that due to the (C@N) group appeared in the 152.35–154.97 ppm
range, and the signals due to the aromatic carbons appeared in
the 110.27–152.35 ppm range.
+
2
tion of 1 and 2 with [MoO
2
]
unit, unlike that shown for 3a and 4a,
Complexes 1a to 4a all presented single crystals. In addition, the
1
13
whereas the C9 atom is trans relative to S1 atom and the cis relative
to N2. The stereochemistry of the ligand is governed by steric effect
of the substituents in the thiosemicarbazone moiety [24]. In order
to increased the stability due to better electron delocalization in a
chelated ring system that resulting from the coordination with the
metal center, the molybdenum surroundings are take the appropri-
ate sites that serve this purpose. The bond distance of S1–C8 is
longer and N2–C8 is shorter than their value for free ligand of 1
and 2 (Table 2), which are indicate that the ligands in thiol form
upon coordinated with [MoO
with that for 3a and 4a. The strongly
and O4 result in the weak bonding if trans, as shown by Mo1–N1
and Mo–O2 [25].
FTIR, H NMR, C NMR spectra and elemental analysis results of
these complexes are consistent with their assigned structures. In
FTIR, two sharp bands attributed to the (Mo@O) groups appeared
in the 893–944 cmÀ1 range. In the 1H NMR spectra, the signals
belonging to phenolic OH protons and N–NH protons are absent,
whereas the signals belonging to the protons of the second ligand
that occupies the D site are present, which is due to formation of
the Mo complexes. The terminal N-ethyl complex of 1a in which
the CH
.25 ppm, whereas for the terminal N-methyl complexes of 3a
and 4a at 3.19 ppm, is attributed to the CH protons and, a broad
signal at 7.46 ppm is attributed to the OH proton. The complex
a in which the H O molecule occupies the D site show a broad sig-
nal at 7.46 ppm is attributed to the H
O protons. In the 13C NMR,
3
OH molecule occupies the D site show a singlet signal at
+
2
2
]
unit. These values are consistent
-bonded oxo-ligands O3
3
p
3
2
2
2
the signals belonging to the carbon of C@N and C@S groups are
shifted up to 4 and 18 ppm to upfield, upon complexation with
Mo, respectively. This shielding effects are the sum induced shift
and the formation of an imine bond in the complex instead of a
thiocarbonyl C@S bond in the ligand. The complexes 1a, 3a and
4
a show a signal at 48.56 ppm is attributed to the carbon of CH
3
group.
3
3
.2. Crystallographic study
.2.1. Crystallographic description of 1 and 2
The molecular structures of both ligands (Figs. 1 and 2) show
the trans geometries of the S1 atom in relation to N1 and C9 atom
Fig. 1. Molecular structure of 1.

M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
97
Fig. 2. Molecular structure of 2.
Fig. 5. Molecular structure of 3a.
3
3
.3. Interaction with DNA
.3.1. Absorption spectroscopic studies
Absorption spectroscopy is one of the most useful methods in
DNA binding studies [26]. These studies show that the compounds
can bind to DNA either through covalent bonding, such as in com-
plexes that contain ligands that can be substituted with the nitro-
gen base of DNA [27], or by noncovalent interactions such as
intercalation and electrostatic or groove binding [28]. The ligands
and their complexes all show two absorption bands, which are
⁄
⁄
attributed to
p–p and n–p transitions. These transitions occur at
different wavelengths: for 1a, at 295.75 and 337.10 nm; for 2a, at
Fig. 3. Molecular structure of 1a.
2
98.28 and 345.63 nm; and for 3a and 4a, at 303.15 and 332 nm
⁄
(Fig. 7). The
p–p
absorption bands were chosen to determine
the interaction of CT DNA with the ligands and complexes. The
increasing additions of DNA lead to a hypochromism of the absorp-
tion bands of all ligands and complexes concomitant with a blue
shift in 2a and a red shift in 3a. On the other hand, no apparent
shift is observed for 1a and 4a. The observed spectral behavior sug-
gests that intercalative binding to DNA, since intercalation leads to
hypochromism in the spectral bands [29]. Moreover, the existence
of the red shift is evidence of stabilization of the new DNA duplex
due to an intimate association between 3a and DNA, whereas the
existence of blue shift suggests the weak association between 2a
and DNA [26]. Furthermore, the binding of the complexes to CT
DNA leads to isosbestic spectral changes, with the isosbestic points
at 297 nm for 1a, 3a, and 4a and at 300 nm for 2a. The presence of
the isosbestic points in these titrations suggested that chemical
equilibria exist between the bound and unbound of the complexes
with DNA [26].
Fig. 4. Molecular structure of 2a.
21 3 4
The asymmetric unit of 4a contains two Mo (C14H N O S) com-
plexes (A and B). the crystal structure of 4a which reveals that the
A and B are enantiomer complexes. Accordingly, the 4a(A) shows
the syn conformation of C2A with respect to C11A [torsion angle
C2A–C3A–C4A–C11A = 173.72(19)°] and anti conformation of C3A
with respect to C13A [torsion angle C3A–C4A–C11A–
C13A = À101.2(2)°], whereas the corresponding conformations of
The binding strengths of the ligands and complexes to CT DNA
were determined by calculating the binding constant (K ) using the
b
equation [30]:
[
DNA]/(e
a
–
e
f
) = [DNA]/(
e
b
–
f b b f
e ) + 1/K (e – e )
4
a(B) show the anti [torsion angle = À178.57(18)°] and syn [torsion
angle = 147.99(19)°] conformation, respectively. Moreover, The
complex 4a(A) shows the syn conformations of Mo1A atom with
respect to C8A atom [torsion angle Mo1A–N1A–N2A–
C8A = 13.6(2)°], Mo1A atom with respect to C2A [torsion angle
Mo1A–O1A–C1A–C2A = 177.85(15)°] and N1A atom with respect
to C1A [torsion angle N1A–Mo1A–O1A–C1A = 3.63°], whereas the
corresponding conformations of 4a(B) show the anti conformations
The absorption coefficients
e
a
,
e
f, and e correspond to Aabs/
b
[DNA], the extinction coefficient for the free complex, and the
extinction coefficient for the complex in the fully bound form,
respectively. The plot of [DNA]/(
e
a
–
e
f
) versus [DNA] gives a slope
). The binding constants
of 1/( ) and an intercept of 1/K
e
a
–
e
f
b
(
e
b
–
e
f
(Table 3) indicate that all the ligands and complexes bind to CT
DNA. The complexes exhibit strong interaction compared with
their corresponding ligands: the binding strengths of the com-
plexes follow the order, 4a > 1a > 3a > 2a, whereas those of the
ligands follow the order, 4 > 1 > 2 > 3. The presence of the terminal
[
[
torsion angle = À6.3(2)° ], [torsion angle = À146.16(15)°], and
0
torsion angle = À42.77(16) ], respectively. These conformations
adopt by 4a(A) and 4a(B) are necessary for stability requirements.

9
8
M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
Fig. 6. Molecular structure of 4a.
Table 1
Crystallographic data for complexes 1a–4a.
1
a
2a
3a
4a
Chemical formula
Formula weight
Crystal system
Crystal description
Space group
Unit cell dimensions
a (Å)
C
11
H
3
14ClMoN O
4
S
C
10
H
13MoN
3
O
5
S
C
11
H
15MoN
3
O
4
S
14 3 4
C H21MoN O S
423.35
monoclinic
needle orange
415.71
triclinic
plate orange
P1ꢀ
383.24
triclinic
needle brown
P1ꢀ
381.27
monoclinic
plate orange
P21/n
P21/c
7.8380(2)
9.5122(3)
10.2364(3)
92.665(2)
95.721(2)
92.297(2)
757.80(4)
2
7.7992(1)
8.4445(1)
10.4722(1)
74.599(1)
84.278(1)
85.684(1)
660.767(14)
2
7.8225(1)
18.0343(3)
10.5669(2)
90
110.510
90
1396.21(4)
4
1.814
20.3171(4)
12.6805(3)
13.2331(3)
90
96.423(1)
90
3387.86(13)
8
1.660
b (Å)
c (Å)
a
(°)
b (°)
(°)
c
3
V (Å )
Z
D
calc (g/cm3)
1.822
1.926
Crystal size (mm)
T (K)
0.08 Â 0.18 Â 0.36
0.04 Â 0.12 Â 0.39
0.07 Â 0.12 Â 0.2
0.11 Â 0.17 Â 0.48
100
100
100
100
Total data
Unique data
14826
4010
0.040
17756
4800
0.023
19972
5065
0.029
39181
9980
0.028
R
int
Observed data [I > 2
r
(I)] 3172
4459
4306
8722
R
wR
S
1
0.0564
0.1377
1.07
0.0213
0.0571
1.08
0.0304
0.0592
1.07
0.0295
0.0671
1.08
2
Table 2
Selected bond length (Å) and bond angles (°) for complexes 1a–4a.
1
a
2a
3a
4a
Bond distances (Å)
Mo–O3
Mo–O4
Mo–S1
Mo–O1
Mo–N1
C8–S1
N2–C8
C1–O1
1.698(4)
1.699(3)
2.4304(19)
1.926(4)
2.300(4)
1.738(7)
1.293(16)
1.337(6)
1.7141(11)
1.7007(12)
2.4323(4)
1.9408(10)
2.2858(12)
1.7453(14)
1.3190(18)
1.3570(18)
1.7184(13)
[1.7112(15),1.7071(16)]
[1.6995(15), 1.7033(15)]
[2.4234(5), 2.4377(6)]
[1.9327(14), 1.9414(14)]
[2.2736(16), 2.2769(16)]
[1.749(2), 1.748(2)]
1.6978(15)
2.4257(5)
1.9280(14)
2.2724(14)
1.7531(18)
1.318(2)
[1.323(3), 1.312(3)]
[1.338(2), 1.349(2)]
1.343(2)
Bond angels (°)
O3–Mo–O4
S1–Mo–N1
N1–Mo–O1
N1–Mo–O2
S1–Mo–O1
O1–Mo–O2
N1–Mo–O2
O3–Mo–O1
105.58(19)
75.47(11)
81.96(14)
77.92(13)
152.46(11)
78.46(14)
159.34(16)
105.36(16)
105.78(5)
76.21(3)
83.10(4)
79.37(4)
154.45(3)
79.97(4)
160.41(5)
102.80(5)
105.02(6)
75.87(4)
83.59(5)
79.81(5)
153.68(4)
76.52(6)
157.95(6)
106.72(6)
[105.74(7), 105.72(7)]
[76.16(4), 75.29(4)]
[83.29(6), 82.79(6)]
[75.58(6), 77.65(5)]
[155.07(5), 153.53(5)]
[78.52(6), 77.41(6)]
[156.85(7), 156.96(7)]
[104.57(7), 105.92(7)]

M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
99
Fig. 7. UV spectra of complexes 1a–4a in 6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7) in presence of CT DNA at increasing amounts.[complex] = 50
l
M, [DNA] = (28.9–173.4)
lM. The arrows show the changes in absorbance upon increasing amounts of CT DNA.
Table 3
DNA binding constants (K
ing strength of complexes [31]. Moreover, the complex 3a revealed
binding strength more than the complex 2a despite the binding
strength of the ligand 2 more than the ligand 3, this difference
attributed to the presence of water molecule which occupied the
b
) of ligands and complexes.
À1
À1
Ligand
K
b
(M
)
Complex
b
K (M )
5
5
4
5
6
5
5
6
1
2
3
4
1.83 Â 10
1.58 Â 10
6.30 Â 10
4.59 Â 10
1a
2a
3a
4a
1.43 Â 10
4.87 Â 10
6.50 Â 10
3.75 Â 10
+
2
D upon coordinated 2a with MoO
2
unit, consequently leads to
decreases the lipophilic properties for it.
3.3.2. Fluorescence spectroscopic studies
N-methyl and tert-butyl groups provide strong lipophilic proper-
ties that enable the complex 4a to move into the hydrophobic
medium of DNA. The terminal N-ethyl and N-methyl groups as
well as the chlorine and methyl moieties, are provide good lipo-
philic properties that enable the complex 1a and 3a to move into
DNA, respectively. The polarized chlorine moiety increases the
London dispersion forces, which are well increase the lipophilic
The fluorescence spectra (Fig. 8) show that the 1a and 3a com-
plexes have two emission bands at 500 and 380 nm, which are
accompanied by hypochromism and hyperchromism, respectively.
These changes are attributed to isosbestic spectral changes with
the isosbestic points at 408 nm as a result of the increased DNA
concentrations (28.9 to 173.4
lM). DNA was added at 10 min inter-
vals to a fixed concentration (50
lM) of the complex. The increased
+
2
properties of 1a. Meanwhile, the coordination with MoO
2
unit
concentrations also lead to a hypochromism in the emission spec-
tra of 2a at 500 nm and of 4a at 380 nm without isosbestic spectral
changes. The emission spectra of the ligands show behaviors sim-
ilar to those of the complexes when CT DNA was added. The fluo-
leads to geometrical and conformational changes which in turn
lead to an extent of planarity that increases the affinity between
the complexes and DNA, consequently leads to increases the bind-

1
00
M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
Fig. 8. Fluorescence spectra of complexes 1a–4a in 6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7) in presence of CT DNA at increasing amounts.[complex] = 50
DNA] = (28.9–173.4) M. The arrows show the changes in fluorescence intensity upon increasing amounts of CT DNA.
lM,
[
l
rescence emission intensities confirm the strong binding between
the ligands or complexes with DNA.
migration form is related to the open circular relaxed form (OC,
form II) [33]. The ligand 4 exhibits a significant cleavage activity
to plasmid DNA at a concentration of 4.5 lM (lane 10). This ligand
3
.3.3. Viscometric studies
The interaction of the ligands and complexes to CT DNA was
can nick both plasmid DNA strands, indicating that the linear form
(form III) is accompanied by the disappearance of the SC form I.
Based on the size fragments of the ladder DNA (lane L), the size
of the SC form increases with increasing 1, 2, and 4 concentrations.
By contrast, the size of the SC form decreases with increasing 3
further confirmed via viscometric studies. The intercalation of a
compound into DNA increases the viscosity because of the length-
ening of the DNA helix as the base pairs are pushed apart [32]. In
general, the viscosities of the solutions increase with the addition
of CT DNA as shown in Fig 9. These data are consistent with the
spectrometric results.
concentration. At the highest concentration (6.0
inhibit the plasmid DNA (lane 11).
lM), all ligands
The complexes exhibit higher plasmid DNA cleavage activities
compared with the ligands. The plasmid DNA strands all exhibit
SC form I, OC form II, and linear form III. The ability of the 1a com-
3.3.4. DNA cleavage
Agarose gel electrophoresis experiments using a pBR322 circu-
plex to nick both plasmid DNA strands was investigated at 3.5
(lane 7) and 4.0 M (lane 8), concomitant with the disappearance
of the OC prior to the loss of DNA activity as a result of inhibitory
action at 4.5 M (lane 9). The 2a complex shows the three forms at
4.0 M (lane 8) and 4.5 M (lane 9). At 5.0 M (lane 10), the SC
lM
lar plasmid were performed using microconcentrations (1.0–
.0 M) of the ligands and complexes in an aqueous buffer solution
6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7)] in the presence of a
fixed amount (4.5 M) of 30% H as an oxidant. The mixture
was incubated at 37 °C for 2 h. All the ligands and complexes
Fig. 10, starting from lane 4 of the agarose gel) cleave the plasmid
l
6
[
l
l
l
2
O
2
l
l
l
form disappears, and only the OC and linear forms are present
before DNA loses its activity as a result of the inhibitory action at
(
DNA into two forms. The naturally fast migration form is related to
the closed circular supercoiled form (SC, form I), whereas the slow
6.0
l
M (lane 11). Meanwhile, 3a and 4a show the SC and linear
forms at 3.5
l
M (lane 7) and 4.0 M (lane 8), respectively. The
l

M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
101
Fig. 9. Viscometric results of ligands (A) and complexes (B), in 6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7) in presence of CT DNA at increasing amounts.[complex] = 50 lM,
[
DNA] = (28.9–173.4) lM, at 37 °C.
Fig. 10. Agarose gel electrophoresis patterns of pBR 322 (0.025
0 mM NaCl buffer (pH 7) incubated at 37 0C for 2 h, using H
complex) (6 M); lane 3, DNA + H lane 4, DNA + H
M); lane 8, DNA + H + Sample (4
l
M) increasing concentrations (1.0–6.0
(4.5 M) and ladder 1 KbDNA as a marker (lane L); lane 1, pBR 322 DNA; lane 2, DNA + Sample (ligand or
M);lane 5, DNA + H + Sample (2 M); lane 6, DNA + H + Sample (3 M); lane 7,
2 2
M); lane 9, DNA + H + Sample (4.5 M); lane 10, DNA + H + Sample (5 M); lane 11, DNA + H O +
lM) of ligands 1–4 and their complexes 1a–4a in 6.3 mM Tris–HCl/
5
2
O
2
l
l
2
O
2
;
2
O
2
+ Sample (1
l
l
2
O
2
l
2
O
2
l
DNA + H
2
O
2
+ Sample (3.5
l
2
O
2
2
O
2
l
2
O
2
l
Sample (6
lM).
OC form reappears at 4.0
Furthermore, 4a still show the OC and linear forms at 5.0
l
M 3a (lane 8) and at 4.5
l
M 4a (lane 9).
M (lane
M (lane 11), whereas 4.5
a (lane 9) is sufficient for the inhibition of the pBR322 plasmid.
The cleavage mechanism of pBR322 DNA induced by the ligands
l
and complexes was investigated in the presence of the hydroxyl
radical scavenger DMSO. The presence of DMSO leads to inhibit
the nuclease activity of the complexes and their respective ligands.
These observations suggest the possibility of the formation of OH
radicals as the reactive species [34]. The hydroxyl radicals gener-
ated by ligands may either cleave the DNA by abstraction of the
hydrogen atom from the deoxyribose sugar [35], or the interca-
lated ligands react directly on the DNA strand to cleave it [36].
The nuclease behavior of the ligands suggests that the second path-
way is more probable than the first pathway. The strong cleavage
activity of the ligand 4 attributed to the intimate association to
the hydrophobic environment of DNA and, unlike the ligands 1, 2
and 4, the ligand 3 showed that the size fragments of SC form
decreases with increases the concentration. This different behavior
confirms the hypothesis of the cleavage efficiency depends on the
1
0) before its inhibitory action at 6.0
l
lM
3
The agarose gel electrophoresis patterns show that the size of the
SC form fragments increases with increasing 2a, 3a, and 4a concen-
trations but decreased with increasing concentrations of 1a. 1a and
3
a inhibit the actions of their free ligands.
The agarose gel electrophoresis patterns all show only the nat-
urally occurring SC form I as well as the DNA control and buffer
lane 1), DNA and the ligands or complexes (lane 2), and DNA,
, and buffer (lane 3). This result indicates that the ligands
(
2 2
H O
and complexes can cleave DNA only in the presence of H
an oxidant (lanes 4 to 11). A fixed amount (4.5 M) of H
added to different amounts of the ligands and complexes (1
to 6 M).
2
O
O
2 2
2
as
is
l
lM
l

1
02
M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
Table 4
IC50 M) of complexes 1a–4a.
(l
Complex
IC50 (lM)
1
2
3
4
5
a
a
a
a
3.5
6.9
6.4
3.4
7.3
-FU
N-terminal moieties and the aromatic ring moieties. Moreover, the
[
(
ligand/H
1:1), the ligand 4 showed the maximal DNA cleavage at concen-
M and, all the ligands causes the DNA denaturation at
concentration 6 M. These observations confirm the concentra-
tion-dependent DNA cleavage by the ligands but in presence of
2 2
O ] mol ratio is constant and approximately equal to
tration 5
l
l
2 2
H O .
The ability to cleave DNA is significantly enhanced in the com-
plexes compared with the related free ligands. This finding proves
that the complexes can produce hydroxyl radicals from H . The
2 2
O
formation of hydroxyl radicals from this type of metal ion is well
reported [37,38].
Fig. 12. Photomicrographs of the HCT 116 cell lines before and after treatment with
complexes; image control = HCT 116 cell before treatment, the images after
The suggested cleavage mechanism of pBR322 DNA induced by
the complexes as follows:
treatment; A = 1a (IC50 = 3.5
l
M), B = 2a (IC50 = 6.9
l
M), C = 3a (IC50 = 6.4
M) .
lM),
+
2
]+ + OH + H
Å
+
[
MoO
Generation of a HO radical adduct by the molybdenum(VI)–
system resembles the vanadyl (IV)–H [39,40]. The
observed DNA cleavage results suggest the intercalated com-
plexes-H react directly on the DNA strand to cleave it. The
2
]
+ H
2
O
2
? [MoO
3
D = 4a (IC50 = 3.4 M) and 5-FU = standard reference (IC50 = 7.3 l
l
H
2
O
2
2 2
O
DNA, consequently leads to increases the apoptosis ability of com-
plexes against the cell proliferation, which is consistent with their
binding capabilities with CT DNA. Furthermore, the inhibition
2 2
O
highly cleavage efficiency of complexes attributed to the intimate
association with DNA which consistent with that for DNA binding
results.
activities of the complexes show that 100
lM of 1a, 4a, 2a, and
3
a is sufficient to inhibit 95.6%, 70.2%, 46.6%, and 44.5% of cell pro-
liferation, respectively (Fig. 11). Furthermore, the cytotoxicity effi-
ciencies of the complexes against HCT 116 cell lines were
confirmed by the microscope images (Fig. 12). These images show
the HCT 116 cell lines before and after the addition of complexes.
Results were compared with those of 5-fluorouracil (5-FU), which
is commonly used as a reference in antitumor studies because of its
high activity against cancer [41].
3
.4. Antitumor activity
The complexes show considerable activities in binding or cleav-
ing DNA compared with their free ligands. Thus, only the com-
plexes were investigated for antitumor activities. Cytotoxicity
tests were performed in vitro against HCT 116 cell lines using 5-flu-
orouracil (IC50 = 7.3 lM) as the standard reference. The maximal
inhibition concentrations (IC50) listed in Table 4 show that the
cytotoxicity efficiencies of the complexes follow the order,
4
. Conclusions
4
a > 1a ꢀ 3a > 2a. These results show that the cytotoxicity effi-
Four molybdenum(VI) complexes, namely, 1a, 2a, 3a, and 4a,
ciency is determined by the substituent on the aromatic ring of
have been synthesized. The molecular structures of these com-
plexes and of their two ligands have been described in detail by
X-ray crystallography. The interaction of the complexes and
ligands with CT DNA was investigated. The cleavage activities of
the ligands and complexes against pBR322 circular plasmid DNA
were also determined. The complexes show high efficiency to
DNA binding and DNA cleavage compared with the free ligands.
The interaction with DNA has been investigated by electronic
absorption, fluorescence emission studies, and viscosity measure-
ments. The reaction of the ligands and complexes with pBR322 cir-
cular plasmid DNA was investigated by agarose gel electrophoresis.
All complexes show cytotoxic activities against HCT 116 cell lines
in the order, 4a > 1a ꢀ 3a > 2a, according to their IC50 values (3.4,
the complex in the order, [C(CH
3
2
)
3
] > Br ꢀ CH
3 3
> OCH respec-
+
tively. The coordination with MoO
2
unit leads to an extent of pla-
narity which increases the affinity between the complexes and
3
.5, 6.4, and 6.9 lM, respectively).
Acknowledgements
We thank the Malaysian Government for a Research University
Grant which partly supported this work. We also thank University
of Basrah, Iraq, for the study leave to Mouayed A. Hussein.
Fig. 11. Cell proliferation inhibition by complexes in concentration range 3.0–
1
00 lM using 5-FU as a reference.

M.A. Hussein et al. / Polyhedron 85 (2015) 93–103
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