Structures, DNA binding, DNA cleavage, and antitumor investigations of a series of molybdenum(VI) complexes with some N(4) methyl and ethyl thiosemicarbazone ligands

Article abstract of DOI:10.1080/00958972.2014.893430

Four dioxomolybdenum(VI) complexes were synthesized by reaction of [MoO₂(acac)₂] with thiosemicarbazones derived from 5-allyl-2-hydroxy-3-methoxybenzaldehyde (1), 2-hydroxynaphthaldehyde (2), 2,3-dihydroxybenzaldehyde (3), or 5-tert-

Full text of DOI:10.1080/00958972.2014.893430

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Structures, DNA binding, DNA cleavage,
and antitumor investigations of a
series of molybdenum(VI) complexes
with some N(4) methyl and ethyl
thiosemicarbazone ligands
Mouayed A. Husseina, Teoh S. Guana, Rosenani A. Haquea,
Mohamed B. Khadeer Ahamedb & Amin M.S. Abdul Majidb
a School of Chemical Science, Universiti Sains Malaysia, Minden,
Malaysia
b EMAN Research and Testing Laboratory, School of Pharmaceutical
Sciences, Universiti Sains Malaysia, Minden, Malaysia
Accepted author version posted online: 13 Feb 2014.Published
online: 20 Mar 2014.
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To cite this article: Mouayed A. Hussein, Teoh S. Guan, Rosenani A. Haque, Mohamed B.
Khadeer Ahamed & Amin M.S. Abdul Majid (2014) Structures, DNA binding, DNA cleavage,
and antitumor investigations of a series of molybdenum(VI) complexes with some N(4) methyl
and ethyl thiosemicarbazone ligands, Journal of Coordination Chemistry, 67:4, 714-727, DOI:
10.1080/00958972.2014.893430
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Journal of Coordination Chemistry, 2014
Vol. 67, No. 4, 714–727, http://dx.doi.org/10.1080/00958972.2014.893430
Structures, DNA binding, DNA cleavage, and antitumor
investigations of a series of molybdenum(VI) complexes with
some N(4) methyl and ethyl thiosemicarbazone ligands
MOUAYED A. HUSSEIN†, TEOH S. GUAN*†, ROSENANI A. HAQUE†,
MOHAMED B. KHADEER AHAMED‡ and AMIN M.S. ABDUL MAJID‡
†School of Chemical Science, Universiti Sains Malaysia, Minden, Malaysia
‡EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains
Malaysia, Minden, Malaysia
(Received 13 September 2013; accepted 15 January 2014)
Four dioxomolybdenum(VI) complexes were synthesized by reaction of [MoO2(acac)2] with
thiosemicarbazones derived from 5-allyl-2-hydroxy-3-methoxybenzaldehyde (1), 2-hydroxynaphthal-
dehyde (2), 2,3-dihydroxybenzaldehyde (3), or 5-tert-butyl-2-hydroxybenzaldehyde (4). The ligands
were coordinated to molybdenum as tridentate ONS donors. X-ray crystallography showed that the
distorted octahedral coordination of molybdenum is completed by methanol (D) in 1a, 3a, and 4a or
H2O in 2a. The molecular structures of 1, 3, and 4, and the complexes were determined by
single-crystal X-ray crystallography. Binding of the ligand and complexes with calf thymus DNA
were investigated by UV, fluorescence titrations, and viscosity measurements. Gel electrophoresis
revealed that all the complexes can cleave pBR322 plasmid DNA. The cytotoxic properties of the
complexes against human colorectal (HCT 116) cell line showed strong antiproliferative activities in
relative order 4a > 3a > 1a > 2a with IC50 values of 1.6, 4.0, 4.8, and 6.7 μM, respectively. The
complexes exhibited more activity than the standard reference drug, 5-fluorouracil (IC50 7.3 μM).
These studies show that dioxomolybdenum(VI) complexes have potential use in chemotherapy.
Keywords: Thiosemicarbazone; Molybdenum(VI) complexes; X-ray crystal structure; DNA binding;
DNA cleavage; Antiproliferative activity
*Corresponding author. Email: sgteoh@usm.my
© 2014 Taylor & Francis

Molybdenum(VI) thiosemicarbazone complexes
715
1. Introduction
Thiosemicarbazones have applications in biology and medicine [1, 2] with antifungal, anti-
bacterial, and anticancer activities [3, 4]. The biological activity is a result of their ability to
chelate with transition metal ions that bond through oxygen, nitrogen, and sulfur (ONS) [5]
or sulfur and two nitrogens [6]. Because thiosemicarbazone complexes are generally more
active than free ligands, the biological and pharmaceutical activities of ONS tridentate
systems and their metal complexes have received interest [7–9].
The coordination chemistry of molybdenum(VI) has biochemical significance [10–12];
ONS ligands around Mo(VI) in oxotransferase enzymes have led to interest in these types
of complexes and oxotransfer ability. Such complexes can serve as models that mimic oxo-
transferase molybdoenzymes [13–15]. Structural studies have shown that thiosemicarbazone
derivatives containing phenolic oxygen and acting as tridentate ONS donors coordinate
strongly [16–18]. In the present study, we report the synthesis and characterization of
cis-dioxomolybdenum(VI) complexes with 4-methyl (or ethyl) thiosemicarbazones shown
in scheme 1. The complexes were tested for their calf thymus DNA (CT DNA) binding,
cleavage of pBR322 plasmid DNA, and antiproliferative property against human colorectal
(HCT 116) cell line.
2. Experimental
2.1. Synthesis of ligands
The ligands were prepared by following a general procedure in which a solution of
corresponding aldehyde in ethanol (20 mL) was added to an ethanolic solution (20 mL) of
4-methyl-3-thiosemicarbazide (4.75 mM) or 4-ethyl-3-thiosemicarbazide (4.19 mM). The
mixture was refluxed with stirring for 2 h. The product was filtered, washed with ethanol,
and air-dried. Crystals for 1 were obtained by slow evaporation of DMF solution at room
temperature. Crystals for 3 and 4 were obtained by slow evaporation of ethanol solution at
room temperature.
Scheme 1. General synthetic procedure of ligands and complexes.

716
M.A. Hussein et al.
(1) M.p.: 176–178 °C (1.09 g, 83%). Anal. Calcd for C13H16N3O2S: C, 55.83; H, 5.72;
N, 15.03. Found: C, 55.77; H, 5.74; N, 14.96%. IR(KBr) (νmax/cm−1): 3317 (s, NH), 1609
1
(m, C=N), 1550 (s, CaroO), 1275 (s, C=S). H NMR (DMSO-d6, ppm): 3.01 (CH3), 3.30
(CH2–Ph), 3.79 (O–CH3), 5.02 (=CH), 6.02 (CH2=), 6.78–7.34 (H-aromatic), 8.37
(CS–NH), 9.09 (CH=N), 11.40 (N–NH). 13C NMR (DMSO-d6, ppm): 30.83 (CH3), 55.85
(O–CH3), 113.16 (CH2–Ph), 115.46 (H2C=), 117.49–144.28 (C-aromatic), 147.88 (C=N),
177.52 (C=S).
(2) M.p.: 213–215 °C (1.03 g, 90%). Anal. Calcd for C14H15N3OS: C, 61.45; H, 5.48; N,
15.36. Found: C, 61.33; H, 5.46; N, 15.35%. IR(KBr) (νmax/cm−1): 3405 (m, NH), 3149
(m, OH), 1600–1612 (m, C=N–N–C), 1537 (s, CaroO), 1264 (m, C=S). 1H NMR
(DMSO-d6, ppm): 1.17 (CH3), 3.61 (CH2), 7.20–7.89, 8.418 (H-aromatic), 8.37 (CS–NH),
9.06 (CH=N). 13C NMR (DMSO-d6, ppm): 14.49 (CH3), 30.62 (CH2), 109.77–142.61
(C-aromatic), 156.30 (C=N), 176.42 (C=S).
(3) M.p.: 201–203 °C (0.78 g, 78%). Anal. Calcd for C10H13N3O2S: C, 50.14; H, 5.43;
N, 17.55. Found: C, 48.01; H, 5.74; N, 18.79%. IR(KBr) (νmax/cm−1): 3399 (s, NH), 3146
1
(m, OH), 1612 (m, C=N). H NMR (DMSO-d6, ppm): 1.08 (CH3), 3.54 (CH2), 6.27–7.70
(H-aromatic), 8.25 (CS–NH), 8.30 (CH=N), 9.76, 9.79 (OH), 11.14 (N–NH). 13C NMR
(DMSO-d6, ppm): 14.68 (CH3), 37.64 (CH2), 102.28–157.87 (C-aromatic), 160.34 (C=N),
177.31 (C=S).
(4) M.p.: 210–212 °C (1.05 g, 90%). Anal. Calcd for C14H21N3OS: C, 60.12; H, 7.51; N,
15.03. Found: C, 59.72; H, 7.97; N, 15.04%. IR(KBr) (νmax/cm−1): 3352 (s, NH), 3275
1
(m, OH), 1603 (m, C=N), 1545 (s, CaroO). H NMR (DMSO-d6, ppm): 1.16 (CH3), 1.27
{C(CH3)3}, 3.60 (CH2), 6.80–7.73 (H-aromatic), 8.34 (CS–NH), 8.37 (CH=N), 9.69 (OH),
11.30 (N–NH). 13C NMR (DMSO-d6, ppm): 14.60 (CH3), 31.31 {(CH3)3}, 33.82
{C(CH)3}, 38.30 (CH2), 115.71–141.49 (C-aromatic), 154.24 (C=N), 176.43 (C=S).
2.2. Synthesis of complexes
Equimolar methanolic or ethanolic solutions (25 mL) of MoO2(acac)2 and the corresponding
ligand were refluxed for 2 h, and then filtered. Slow evaporation of a solvent at room
temperature gives a crystalline product.
Complex 2a was prepared by the following procedure in which a solution of
MoO2(acac)2 (0.119 g, 0.365 mM) in ethanol (25 mL) was added to a solution of 2
(0.1 g, 0.365 mM) 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 to obtain
purple block-shaped crystals.
(1a) M.p.: 167–169 °C (0.11 g, 71%). Anal. Calcd for C14H19MoN3O5S: C, 38.41; H,
4.34; N, 9.60; Mo, 21.94. Found: C, 37.08; H, 3.37; N, 9.79; Mo, 21.77%. IR(KBr)
(νmax/cm−1): 3324 (m, NH), 3437 (m, OH), 1589 (m, C=N), 1568 (m, CaroO), 1268 (s, C–
1
S), 932, 898 (s, Mo=O). H NMR (DMSO-d6, ppm): 3.01 (CH3), 2.86 (alcoholic-CH3),
3.30 (CH2–Ph), 3.79 (O–CH3), 5.02 (=CH), 5.79 (CH2=), 6.78, 7.34 (H-aromatic), 7.46
(alcoholic-OH), 8.37 (C–NH), 8.47 (CH=N); 13C NMR (DMSO-d6, ppm): 30.83 (CH3),
46.35 (alcoholic-CH3), 55.85 (O–CH3), 115.51 (CH2–Ph), 115.93 (=CH), 117.37 (H2C=),
120.26–147.05 (C-aromatic), 148.09 (C=N), 151.45 (C–S).
(2a) M.p.: 222–224 °C (0.11 g, 74%). Anal. Calcd for C14H15MoN3O4S: C, 40.25; H,
3.59; N, 10.06; Mo, 23.00. Found: C, 40.55; H, 2.81; N, 10.01; Mo, 22.96%. IR(KBr)
(νmax/cm−1): 3333 (s, NH), 1591 (m, C=N), 1552 (m, CaroO), 1281 (m, C–S), 938, 900 (s,
1
Mo=O). H NMR (DMSO-d6, ppm): 1.17 (CH3), 3.35 (CH2), 7.14, 7.45, 7.65, 7.89, 7.99,

Molybdenum(VI) thiosemicarbazone complexes
717
8.28 (H-aromatic), 7.55 (H2O), 8.31 (C–NH), 8.45 (CH=N); 13C NMR (DMSO-d6,ppm):
14.47 (CH3), 30.61 (CH2), 112.16–142.93 (C-aromatic), 146.83 (C=N), 159.43 (C–S).
(3a) M.p.: 219–221 °C (0.14 g, 85%). Anal. Calcd for C11H15MoN3O5S: C, 33.22; H,
3.77; N, 10.57; Mo, 24.15. Found: C, 32.78; H, 3.0; N, 11.44; Mo, 24.11%. IR(KBr) (νmax
/
1
cm−1): 3379 (m, NH), 1592 (m, C=N), 1557 (m, CaroO), 936,897 (s, Mo=O). H NMR
(DMSO-d6, ppm): 1.08 (CH3), 3.16 (alcoholic-CH3), 3.25 (CH2), 6.20, 6.47, 7.41
(H-aromatic), 7.24 (alcoholic-OH), 8.25 (C–NH), 8.38 (CH=N) 10.45 (OH); 13C NMR
(DMSO-d6, ppm): 14.39 (CH3), 38.19 (CH2), 48.57 (alcoholic-CH3), 102.28–134.86
(C-aromatic), 151.72 (C=N), 162.64 (C–S).
(4a) M.p.: 180–182 °C (0.14 g, 93%). Anal. Calcd for C15H23MoN3O4S: C, 41.15; H,
5.25; N, 9.60; Mo, 21.94. Found: C, 40.64; H, 4.97; N, 9.69; Mo, 21.81%. IR(KBr) (νmax
/
1
cm−1): 3440 (s, NH), 1589–1607 (m, C=N–N–C), 1557 (s, CaroO), 936, 901 (s, Mo=O). H
NMR (DMSO-d6, ppm): 1.16 (CH3), 1.27 [(CH3)3], 3.16 (alcoholic-CH3), 3.29 (CH2),
6.79, 7.49, 7.59 (H-aromatic), 7.45 (alcoholic-OH), 8.31 (C–NH), 8.55 (CH=N); 13C NMR
(DMSO-d6, ppm): 14.35 (CH3), 31.04 [(CH3)3], 31.24 [C(CH)3], 33.77 (CH2), 48.56 (alco-
holic-CH3), 117.22–143.34 (C-aromatic), 151.82 (C=N), 156.68 (C–S).
2.3. DNA binding and cleavage
The binding of complexes with CT DNA was investigated in 6.3 mM Tris–HCl/50 mM
NaCl buffer (pH 7). The stock solution of DNA was prepared by dissolution of a suitable
amount of DNA in 6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7) at room temperature and
stored in a refrigerator for 2 days. A solution of CT DNA in the buffer gave a UV absor-
bance ratio at 260 and 280 nm of ca. 1.9 : 1, indicating that the DNA was sufficiently free
of protein. The DNA concentration was estimated by UV absorbance at 260 nm with a
known molar absorption coefficient of 6600 M−1 cm−1 [19]. The UV–Vis spectra were
scanned from 230 to 600 nm with a Tris/HCl buffer solution as reference.
Fluorescence emission assay was performed with the aforementioned method. The fluores-
cence spectra were scanned from 200 to 800 nm with a Tris/HCl buffer solution as reference.
Viscosity measurements were made with a Cannon-Manning Semi-Micro viscometer
immersed in a thermostatic water bath at 37 °C. Flow times were measured manually with a
digital stopwatch. The viscosity values were calculated from the observed flow time of
DNA-containing solutions (t) corrected for that of the solvent mixture used (t0), η = t – t0.
Viscosity data are presented as (η/η0)1/3 versus [complex]/[DNA], where η and η0 are the
viscosity of the complex in the presence of DNA and the viscosity of DNA alone,
respectively [20].
The ability of the ligands and complexes to cleave pBR322 plasmid DNA was studied by
agarose gel electrophoresis in Tris/EDTA buffer solution. The samples were incubated at
37 °C for 2 h, treated with loading dye, and electrophoresed for 1 h at 50 Von 1% agarose gel
consisting of 12 lanes: lane 1, pBR322 DNA (0.025 μM); lane 2, DNA (0.025 μM) + sample
(6 μM); lane 3, DNA (0.025 μM) + H2O2 (4.5 μM); lane 4, DNA (0.025 μM) + H2O2
(4.5 μM) + sample (1 μM); lane 5, DNA (0.025 μM) + H2O2 (4.5 μM) + sample (2 μM); lane
6, DNA (0.025 μM) + H2O2 (4.5 μM) + sample (3 μM); lane 7, DNA (0.025 μM) + H2O2
(4.5 μM) + sample (3.5 μM); lane 8, DNA (0.025 μM) + H2O2 (4.5 μM) + sample (4 μM); lane
9, DNA (0.025 μM) + H2O2 (4.5 μM) + sample (4.5 μM); lane 10, DNA (0.025 μM) + H2O2
(4.5 μM) + sample (5 μM); lane 11, DNA (0.025 μM) + H2O2 (4.5 μM) + sample (6 μM). The
gel was then stained with ethidium bromide before being photographed under UV light.

718
M.A. Hussein et al.
2.4. Antiproliferative assay
2.4.1. Preparation of cell culture. HCT 116 cells were grown under optimum incubation
conditions. Cells that reached 70–80% confluence were chosen for cell plating purposes.
The old medium was replaced by aspiration. Then, the cells were washed with sterile PBS
(pH 7.4) two to three times. The intact layer of attached cells was subjected to trypsiniza-
tion. The cells were incubated at 37 °C in 5% CO2 for 3–5 min. Then, the flasks containing
the cells were gently tapped to aid in cell segregation and observed under inverted micro-
scope (to confirm whether cell segregation was complete). Trypsin activity was inhibited by
the addition of 5 mL of fresh complete media supplied with 10% fetal bovine serum. The
cells were counted and diluted to obtain a final concentration of 2.5 × 105 cells/mL and
inoculated into wells (100 μL/well). Plates containing the cells were incubated at 37 °C with
an internal atmosphere of 5% CO2.
2.4.2. MTT assay. The cytotoxicity was evaluated with MTT assay [21] against HCT 116
cells. HCT 116 cells (1.5 × 105 cells/mL, 100 μL/well) were seeded in a 96-well microtiter
plate. The plate was incubated in a CO2 incubator overnight to allow cell attachment. We
added 100 μL of test substance into each well containing the cells. The test substance was
diluted with media into the desired concentrations from the stock. The plates were incu-
bated at 37 °C with an internal atmosphere of 5% CO2 for 72 h. After this treatment period,
20 μL of MTT reagent was added into each well and the plates were incubated for 4 h.
After this incubation period, 50 μL of MTT lysis solution (DMSO) was added into the
wells. The plates were further incubated for 5 min in a CO2 incubator. Finally, the plates
were read at 570 and 620 nm with the use of a standard ELISA microplate reader. Data
were recorded and analyzed to assess the effects of test substance on cell viability. The
percentage of growth inhibition was calculated from the optical density (OD) obtained from
the MTT assay, or the 100th multiple of the subtracted OD value of the control and
survived cells over the OD of the control cells.
3. Results and discussion
3.1. Chemistry
New dioxomolybdenum(VI) complexes were synthesized by reaction of [MoO2(acac)2] with
substituted thiosemicarbazones. The ligands were prepared by condensation of thiosemicar-
bazone with salicylaldehyde derivatives [22]. The X-ray crystal structure shows distorted
octahedral complexes, in which the complex surrounding the molecules consists of cis-
2+
MoO2 core to which the doubly deprotonated tridentate ligand is meridionally bonded via
the phenolate-oxygen, imine-nitrogen, and thiolate-sulfur. The sixth coordination site is
occupied by CH3OH or H2O. All the ligands and complexes are air-stable and highly
soluble in DMSO and DMF; the ligands and complexes are insoluble [23] and highly stable
1
in aqueous solutions. H NMR and UV spectra were recorded after preparation, 7 days, 30
days, 3 months, and 8 months, while kept at room temperature. All spectra confirmed the
stability of the ligand and complexes in aqueous solutions.

Molybdenum(VI) thiosemicarbazone complexes
3.2. Crystal and molecular structure
719
The crystal and molecular structures of the complexes were obtained by single-crystal
X-ray diffraction. Crystal data of complexes are collected in table 1. The relevant bond
distances and angles are collected in table 2.
In all the complexes, molybdenum has a distorted octahedral coordination. The Schiff
2+
base is bonded to cis-MoO2 through phenolate oxygen O1, imine nitrogen N1, and thio-
late sulfur S1; oxo O3 is trans to N1, whereas the other oxo {O2 in 2a} lies trans to the D
site, which is occupied by CH3OH in 1a, 3a, and 4a or H2O in 2a. The molecular structure
of 2a is depicted in figure 1. Despite the complex being prepared using ethanol (99%), H2O
occupies the D site. There are two significant differences in conformation geometries
between 2a and the other complexes, where CH3OH occupies the D site. For 2a, C13 from
the N-ethyl group is cis to S1 and trans to N2, whereas for 3a (figure 2) and 4a C9 from
N-ethyl is trans to S1 and cis to N2. For 1a, C14 from N-methyl is trans to S1 and cis to
N2. The stereochemistry is governed by the steric effect of the substituents in the thiosemi-
carbazone [24]. To increase stability due to better electron delocalization, in a chelated ring
system resulting from coordination the metal coordinates with a suitable ligand for this
purpose. Accordingly, 2a coordinates with H2O rather than CH3OH. Substitution by water
1
and methanol was determined using H NMR and UV spectroscopy.
The Mo–O2 bond distances of 2.3613(14) and 2.3604(8) Å in 1a and 4a, respectively,
are longer than that of 2.3389(16) Å in 3a, showing the substituent effect of the aromatic
ring and the repulsion between O3 and O4 oxo groups. The double bonds to molybdenum
have bond distances ranging from 1.7141(12) to 1.7258(14) Å and 1.6953(13) to 1.6986(9)
Å for Mo–O3 and Mo–O4, respectively. In 2a, the Mo–O1W bond distance of 2.3208(13)
2+
Å exhibits strong bonding with MoO2 compared with CH3OH.
Table 1. Crystallographic data for 1a–4a.
1a
2a
3a
4a
Chemical formula
Formula weight
Crystal system
Crystal description
C
14H19MoN3O5S
C14H15MoN3O4S
417.30
Triclinic
Block purple
P-1
C11H15MoN3O5S
397.27
Triclinic
Needle brown
P-1
C15H23MoN3O4S
437.37
Monoclinic
Block orange
P21/c
437.33
Triclinic
Plate orange
P-1
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α (°)
7.0017(1)
10.9150(2)
11.7132(2)
76.564(1)
78.085(1)
89.197(1)
851.36(3)
2
8.3956(1)
9.5796(1)
10.5286(1)
67.264(1)
84.266(1)
85.028(1)
776.006(15)
2
7.3557(1)
9.5561(2)
10.9126(2)
95.558(1)
109.129(1)
92.071
719.42(2)
2
1.834
7.3552(1)
16.2793(3)
15.3487(3)
90
104.219(1)
90
1781.51(5)
4
1.631
β (°)
γ (°)
Volume (Å3)
Z
DCalcd (g/cm3)
1.706
1.786
Crystal size (mm)
Temperature (K)
0.05 × 0.19 × 0.41
100
0.11 × 0.19 × 0.35
100
0.08 × 0.09 × 0.44
100
0.13 × 0.19 × 0.58
100
Total data
Unique data
19,993
6219
20,471
5618
19,563
5214
25,336
6508
Rint
0.027
0.019
0.019
0.024
Observed data [I > 2σ(I)]
5682
5230
4844
6039
R1
wR2
S
0.0282
0.0671
1.11
0.0222
0.0588
1.05
0.0220
0.0754
1.22
0.0213
0.0518
1.04

720
M.A. Hussein et al.
Table 2. Selected bond distances (Å) and angles (°) for 1a–4a.
1a
2a
3a
4a
Bond distances (Å)
Mo–S1
Mo–O1
Mo–O2
Mo–O3
Mo–O4
Mo–N1
S1–C8
N2–C8
2.4268(5)
1.9198(13)
2.3613(14)
1.7258(14)
1.6953(13)
2.2689(14)
1.761(2)
2.4337(4)
2.4243(6)
1.9250(15)
2.3389(16)
1.7206(15)
1.6973(16)
2.2657(16)
1.757(2)
2.4417(3)
1.9324(8)
2.3604(8)
1.7120(8)
1.6986(9)
2.2864(9)
1.7555(4)
1.3437(15)
1.9585(11)
2.3208(13)*
1.7141(12)
1.6966(13)*
2.2559(12)
1.7423(14)*
1.3154(19)
1.308(2)
1.312(3)
Bond angles (°)
S1–Mo–O1
S1–Mo–O2
S1–Mo–O3
S1–Mo–O4
S1–Mo–N1
O1–Mo–O2
O1–Mo–O3
O1–Mo–O4
O1–Mo–N1
O2–Mo–O3
O2–Mo–O4
O2–Mo–N1
O3–Mo–O4
O3–Mo–N1
O4–Mo–N1
152.06(4)
79.17(3)
89.49(5)
98.80(5)
76.63(4)
77.66(5)
104.35(6)
100.62(6)
82.85(5)
86.41(6)
168.00(6)
76.17(5)
105.45(6)
159.39(6)
91.84(6)
153.49(3)
81.37(3)*
92.66(4)
97.58(4)*
76.84(3)
79.26(5)
102.67(5)
98.72(6)
81.59(4)
82.87(5)
170.98(5)
78.18(4)
106.14(6)
159.47(5)
92.84(5)
153.53(5)
82.62(4)
89.51(5)
97.96(6)
75.73(4)
77.61(6)
105.03(7)
99.22(7)
83.65(6)
82.61(6)
171.87(7)
80.19(6)
105.49(7)
158.58(7)
92.07(7)
155.58(2)
83.52(2)
90.94(3)
95.06(3)
75.62(2)
80.60(3)
104.90(4)
98.52(4)
82.59(3)
81.67(3)
173.21(4)
76.05(3)
105.01(4)
155.04(4)
97.17(4)
*For 2a: O2 = O1W, O4 = O2.
Figure 1. ORTEP diagram of 2a.

Molybdenum(VI) thiosemicarbazone complexes
721
Figure 2. ORTEP diagram of 3a.
3.3. Interaction with DNA
3.3.1. Electronic absorption studies. Electronic absorption spectroscopy is used to inves-
tigate the binding of compounds with DNA [25, 26]. Complexes can bind to DNA through
covalent bonding [27] or non-covalent interactions such as intercalation, electrostatic, or
groove binding [28]. Absorption spectroscopic studies were conducted on a Perkin Elmer,
Lambda 35 spectrophotometer using a fixed concentration of ligand or complex (50 μM)
with increasing amounts of DNA from 28.9 to 173.4 μM in 6.3 mM Tris–HCl/50 mM NaCl
buffer (pH 7). Each addition was left for equilibrium at 25 °C for 10 min and scanned from
230 to 600 nm. The electronic absorption spectra (figure 3) showed one absorption for 1a at
305 nm, which is attributed to π–π* transition. Complex 2a showed three absorptions at
321 nm attributed to π–π* transition, and the 331 and 368 nm attributed to n–π* transitions.
3a and 4a showed two absorptions at 308 and 338 nm attributed to π–π* and n–π*
transitions, respectively. All the ligands showed the same transitions as their complexes.
The π–π* absorptions were chosen to study the interaction of CT DNA with ligands and
complexes. Increasing additions of DNA resulted in hypochromism in the absorption bands
with no apparent shifts observed. This spectral behavior suggests intercalative binding to
DNA [29].
The binding strengths of ligands and complexes to CT DNA were determined from the
calculated binding constant (Kb) with the following equation [30]:
½DNAꢀ=ðea ꢁ ef Þ ¼ ½DNAꢀ=ðeb ꢁ ef Þ þ 1=Kbðeb ꢁ ef Þ
The absorption coefficients εa, εf, and εb correspond to Aabs/[DNA], the extinction coeffi-
cient for the free complex, and the extinction coefficient for the complex in fully bound
form, respectively. A plot of [DNA]/(εa − εf) versus [DNA] yields a slope of 1/(εa − εf) and
intercept of 1/Kb (εb − εf). The binding constants (table 3) suggest that the complexes
exhibit strong interactions compared with the ligands. The highest Kb of 3.698 × 106 M−1
was observed for 4a, whereas the lowest Kb of 1.033 × 105 M−1 was observed for 2a. By

722
M.A. Hussein et al.
Figure 3. UV spectra of 1a and 2a. The arrows show the changes in absorbance upon increasing amounts of CT
DNA.
contrast, Kb of 7.568 × 105 M−1 was observed for ligand 4, whereas the lowest Kb was
recorded for 3. The ability to interact with DNA depends on the substituent on the aromatic
ring, which is in the following order C(CH3)3 > OH > CH2CH=CH2, OCH3 >
. These
groups facilitate intercalation between the complex and DNA because of changes in the
planarity of complexes. The binding of the complexes to CT DNA resulted in isosbestic
spectral changes with an isosbestic point at 297 nm in 1a, 3a, and 4a and at 309 nm in 2a.
3.3.2. Fluorescence spectroscopic studies. Fluorescence spectroscopic studies were per-
formed on a spectrophotometer (Jasco FP-750) at 25 °C and in 6.3 mM Tris–HCl/50 mM
NaCl buffer (pH 7). Fluorescence spectra (figure 4) show that the complexes have emission
bands at 380 nm with hypochromism in 2a and 4a, and with hyperchromism in 1a and 3a.
Complexes 1a and 3a have other emission bands at 525 and 500 nm, respectively, accompa-
nied by hypochromism, which changed to hyperchromism at 380 nm because of the isos-
bestic spectral changes at 410 and 408 nm, respectively. No apparent shifts were observed
Table 3. DNA binding constants (Kb) of ligands and complexes.
Ligand
Kb (M−1
)
Complex
Kb (M−1
)
1
2
3
4
9.10 × 104
4.68 × 104
3.08 × 104
7.56 × 105
1a
2a
3a
4a
2.14 × 105
1.03 × 105
2.24 × 105
3.69 × 106

Molybdenum(VI) thiosemicarbazone complexes
723
during the addition of CT DNA to the ligands or complexes. The fluorescence emission
intensities show the complexes have strong interactions with DNA.
3.3.3. Viscometric studies. Interactions of the ligands and complexes with CT DNA were
confirmed via viscometric studies. The intercalation of compounds in DNA increases
viscosity because the DNA helix is lengthened as the base pairs are pushed apart [31].
Viscometric studies were performed by the addition of increasing amounts of CT DNA to
the ligands and complexes at 37 °C in 6.3 mM Tris–HCl/50 mM NaCl buffer (pH 7). The
results are shown in figure 5. The viscosity of solutions generally increases with the addi-
tion of CT DNA. These data support the results of spectrometric analysis.
3.4. DNA cleavage study
Agarose gel electrophoresis experiments that use pBR322 circular plasmid were performed
for micro concentrations (1.0‒6.0 μM) of ligands and complexes in aqueous 6.3 mM Tris–
HCl/50 mM NaCl buffer (pH 7) in the presence of a fixed amount (4.5 μM) of 30% H2O2
as an oxidant. The solution was then incubated at 37 °C for 2 h. In figure 6, lane 4 of the
agarose gel electrophoresis reveals that the ligands exhibit DNA cleavage with two forms
of typical patterns; the fast migration form is related to the closed circular supercoiled form
(SC, form I) and the slow migration form is related to the open circular relaxed form (OC,
form II) [32]. Complexes 1 and 4 show only the SC form at 5 μM (lane 10). At 6 μM, both
ligands can nick both strands of plasmid DNA, and this results in the linear form (form III)
and the disappearance of SC form I (lane 6), whereas 2 and 3 denature the DNA. 3 can nick
both DNA strands at 4.5 (lane 9) and 5 μM (lane 10), resulting in linear and SC forms and
disappearance of the OC form. Complexes exhibit higher cleavage activity to plasmid DNA
than their ligands. The complexes show SC, OC, and linear, and all of them denature
plasmid DNA at 6 μM (lane 6). Complex 1a shows the three forms at 4.5 μM (lane 9).
However, at 5 μM (lane 10), the OC form disappears and only the SC and linear forms
remain. Complex 2a shows the SC and linear forms at 4 μM (lane 8), whereas the OC form
accompanies the SC and linear forms at 4.5 μM (lane 9). At 5 μM, the SC form disappears
Figure 4. Fluorescence spectra of 1a and 2a. The arrows show the changes in fluorescence intensity upon increas-
ing amounts of CT DNA.

724
M.A. Hussein et al.
Figure 5. Viscometric results of ligands (A) and complexes (B) at 37 °C.
Figure 6. Agarose gel electrophoresis patterns of pBR322 (0.025 μM) with increasing concentrations (1.0–
6.0 μM) of 1 and 2 and their complexes 1a and 2a.
and only the OC and linear forms remain. Complexes 3a and 4a show only SC and linear
forms at 4 and 4.5 μM (lanes 8 and 9). At 5 μM (lane 10), 3a shows only the linear form,
whereas 4a shows the OC and linear forms.
The agarose gel electrophoresis patterns show only the naturally occurring SC form (form
I) for DNA control and buffer (lane 1), DNA and ligand or complexes (lane 2), and DNA,
H2O2, and buffer (lane 3). Thus, the ligands and complexes cleave DNA only in the
presence of H2O2 as an oxidant (lanes 4–11) (4.5 μM) to different amounts of ligands and
complexes (1–6 μM).
DNA cleavage enhanced in complexes over ligands indicates that these complexes may
produce a hydroxyl radical from H2O2. Generation of hydroxyl radicals from metal ions has
been well reported [33, 34]. These free radicals share in the oxidation of deoxyribose
moiety, followed by hydrolytic cleavage of the sugar-phosphate backbone [35].
3.5. Antitumor activity
Similar to the DNA binding and cleavage activities, the complexes demonstrated higher
efficiency in antiproliferative activity than their respective ligands. Antiproliferation tests

Molybdenum(VI) thiosemicarbazone complexes
725
were performed in vitro on human colorectal cancer (HCT 116) cell line. The complexes
exhibited more pronounced activity than the standard reference, 5-fluorouracil (median
inhibitory concentration [IC50] = 7.3 μM). The IC50 values of the tested compounds (table 4)
show that the order of antiproliferative efficiency was 4a > 3a > 1a > 2a, consistent with
DNA binding, and confirm that complex activity is dependent on the substituent of the
aromatic ring in the order (CH3)3 > OH > CH2CH=CH2, OCH3 >
. The inhibition activi-
ties of the complexes are listed in table 5, which shows that 100 μM 4a, 1a, 3a, and 2a is
sufficient to inhibit 92.6, 69.4, 62.0, and 43.9% cell proliferation, respectively (figure 7).
HCT 116 cell images after 48 h of treatment with the complexes is shown in
Table 4. IC50 (μM) of 1a–4a.
Complex
IC50 (μM)
1a
2a
3a
4a
4.8
6.7
4.0
1.6
7.3
5-FU
Table 5. % inhibition of cell proliferation by complexes using 3–100 μM.
(% inhibition of cell proliferation)
Conc. (μM)
1a
2a
3a
4a
5-FU
3
6
12
25
50
100
6.3
17.1
25.7
39.8
50.6
69.4
6.5
11.4
25.6
34.3
36.5
43.9
0.0
4.0
7.3
17.0
32.2
62.0
32.2
57.5
72.3
81.1
86.0
92.6
34
42
67
70
82
95
Figure 7. Cell proliferation inhibition by complexes in concentration range 3.0–100 μM using 5-FU as a reference.

726
M.A. Hussein et al.
Supplementary material (see online supplemental material at http://dx.doi.org/10.1080/
00958972.2014.893430). These images were taken with inverted phase-contrast microscopy
at×200 magnification and a digital camera at 48 h after treatment of the samples. The
photomicrograph of the untreated (control) group showed aggressively growing cells with
full confluence and a compact monolayer of HCT 116 cells. A close look at these images
of untreated and treated cells shows strong cytotoxic effects of the complexes against HCT
116 cell line. Analysis of the photomicrographs shows that the complexes generally showed
apoptotic features in the affected cells. The blabbing of the cell membrane, nuclear
condensation, and vesicle formation in the treated cells indicate the unique characteristics of
apoptosis.
4. Conclusion
The synthesis, structural description, and biological evaluation of molybdenum(VI) com-
plexes 1a–4a were studied. The complexes showed that the thiosemicarbazones coordinate
to molybdenum as tridentate ONS donors. X-ray crystallography showed a distorted octahe-
dral coordination. The complexes have high efficiency of CT DNA binding, as indicated by
the high values of binding constant Kb, UV hypochromism of the absorption bands, fluores-
cence emission, and increase in viscosity because of the addition of CT DNA. The
complexes also demonstrated a cleavage of plasmid pBR322 DNA with higher activity than
their respective ligands.
The antiproliferative activities of the complexes were investigated against HCT 116 cell
line in the order 4a > 3a > 1a > 2a corresponding to their IC50 values of 1.6, 4.0, 4.8, and
6.7 μM, respectively.
Supplementary material
CCDC 885705, 969007, and 919582 contain the supplementary data for 1, 3, and 4. CCDC
895506, 905156, 913192, and 913194 contain the supplementary data for 1a–4a,
respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/
data_request/cif.
Acknowledgements
We thank the Malaysian Government for a Research University Grant which partly
supported this work. We also thank the University of Basrah, Iraq, for the study leave to
Mouayed A. Hussein.
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