Synthesis, crystal structure, deoxyribose nucleic acid interaction and antitumor activity of some thiosemicarbazonatomolybdenum(VI)

Article abstract of DOI:10.1016/j.ica.2014.04.005

Four dioxomolybdenum(VI) complexes were synthesized by reacting [MoO ₂(acac)₂] with thiosemicarbazone ligands derived from 5-chloro-2-hydroxybenzaldehyde (H₂L₁), 2-hydroxy-5-methylbenzaldehyde (H₂L

Full text of DOI:10.1016/j.ica.2014.04.005

Accepted Manuscript
Synthesis, crystal structure, Deoxyribose Nucleic Acid interaction and antitu-
mor activity of some thiosemicarbazonatomolybdenum(VI)
Mouayed A. Hussein, Teoh S. Guan, Rosenani A. Haque, Mohamed B. Khadeer
Ahamed, Amin M.S. Abdul Majid
PII:
S0020-1693(14)00209-6
http://dx.doi.org/10.1016/j.ica.2014.04.005
ICA 15944
DOI:
Reference:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
16 October 2013
13 March 2014
14 April 2014
Please cite this article as: M.A. Hussein, T.S. Guan, R.A. Haque, M.B. Khadeer Ahamed, A.M.S. Abdul Majid,
Synthesis, crystal structure, Deoxyribose Nucleic Acid interaction and antitumor activity of some
thiosemicarbazonatomolybdenum(VI), Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.
2014.04.005
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Synthesis, crystal structure, Deoxyribose Nucleic Acid interaction and
antitumor activity of some thiosemicarbazonatomolybdenum(VI)
Mouayed A. Hussein a, Teoh S. Guan a *, Rosenani A. Haque a, Mohamed B.
Khadeer Ahamedb, Amin M.S. Abdul Majidb
a
School of Chemical Science, Universiti Sains Malaysia, 11800 – Mindern, Pulau Pinang,
Malaysia
b
EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences,
Universiti Sains Malaysia, 11800 – Mindern, Pulau Pinang, Malaysia
*Corresponding author:
Dr. Teoh Siang Guan, PhD,
Professor
School of Chemical Sciences
Universiti Sains Malaysia
Penang-11800, Malaysia
E-mail: sgteoh@usm.my
H/P : (604) – 6577888
Fax : (604) - 6574854
Abstract
Four dioxomolybdenum(VI) complexes were synthesized by reacting [MoO2(acac)2]
with thiosemicarbazone ligands derived from 5-chloro-2-hydroxybenzaldehyde (H2L1),

2-hydroxy-5-methylbenzaldehyde (H2L2), 3-tert-butyl-2-hydroxybenzaldehyde (H2L3), or
2,3-dihydroxybenzaldehyde (H2L4). In all the complexes, the ligands were coordinated to
molybdenum as tridentate ONS donors. X-ray crystallography showed that the distorted
octahedral coordination of molybdenum atom is completed by methanol molecule (D) as in
[MoO2(L1D)], [MoO2(L2D)], and [MoO2(L3D)], or by an ethanol molecule as in
[MoO2(L4D)]. The molecular structures of H2L2, H2L3, and all synthesized complexes were
determined via single crystal X-ray crystallography. The binding properties of the ligand and
the complexes with calf thymus DNA were analyzed by UV, fluorescence titration, and
viscosity measurements. Gel electrophoresis shows that all complexes can cleave the pBR322
plasmid DNA. The cytotoxic properties of the complexes were studied against human
colorectal cell lines. All complexes showed strong antiproliferative activities in the relative
order [MoO2(L3D)] > [MoO2(L1D)] ~ [MoO2(L2D)] > [MoO2(L4D)] with IC50 values of 3.2,
4.5, 4.6, and 6.4 µM, respectively. The complexes exhibited greater pronounced activity than
the standard reference drug, 5-fluorouracil, did (IC50 = 7.3 µM). These studies demonstrate
the potential application of dioxomolybdenum (VI) complexes in chemotherapy.
Key words: Thiosemicarbazone, Molybdenum(VI) complexes, X-ray crystal structure, DNA
binding, DNA cleavage, Antiproliferative activity
1. Introduction
Thiosemicarbazones, which are important groups of compounds, have attracted much
research interest because of their substantial biological and pharmacological properties such
as antiviral, antibacterial, and antifungal activities [1,2]. The actions of thiosemicarbazones
display remarkable activities when coordinated with metal ions [3–5].

Molybdenum has been broadly investigated recently because of its essential role in
almost all biological systems. Molybdenum participates in a wide range of metalloenzymes
called molybdoenzymes, in which molybdenum forms a part of the active sites of these
enzymes [6,7]. These enzymes utilize molybdenum as a cofactor that catalyzes the
transformation processes such as in sulfite oxidase [8], xanthine oxidase [9], and aldehyde
oxidase [10]. Molybdenum(VI) complexes with thiosemicarbazones serving as ONS donor
ligands have been regarded as excellent models for the molybdenum binding site of these
enzymes [11–15].
In this study, a series of cis-dioxomolybdenum(VI) complexes with thiosemicarbazone
ligands were synthesized and characterized (Scheme 1). The complexes and their respective
ligands were tested for DNA cleaving/binding by spectral and physical methods. Moreover,
the complexes were tested against human colorectal (HCT 116) cell lines. The structures of all
complexes were described in detail by X-ray crystallography.
2. Experimental
2.1.
Materials and methods
Melting point was measured by the Stuart Scientific SMP1 melting point apparatus. Infrared
(IR) spectra were recorded by the Perkin Elmer System 2000 spectrophotometer by using the
1
KBr disc method. The nuclear magnetic resonance (NMR) spectra of H and 13C were
recorded by a Bruker 500 MHz and 400 MHz, respectively, with tetramethylsilane serving as
an internal standard and DMSO-d6 serving as the solvent. Elemental analysis was conducted
by the Perkin Elmer 2400 Series-11 CHN analyzer. Electronic and fluorescent spectra were
recorded by Perkin Elmer Lambda-35 and Jasco FP-750 spectrophotometers, respectively.
X-ray crystallographic data were recorded on a Bruker SMART APEXII CCD area-detector
diffractometer using graphite monochromated Mo Kα radiation (λ = 0.71073 A0) at 100 K.

The data were collected and reduced using APEX2 and SAINT programs. The structure of all
compounds was solved using the SHELXS-97 program package, and refined using the
SHELXL-97 program package [16]. All non-hydrogen atoms were anisotropically refined.
The molecular graphics were created using SHELXTL-97. All chemicals, including
thiosemicarbazide, aldehydes, and the solvents, were purchased from Sigma-Aldrich.
2.2.
Synthesis of ligands
2.2.1. Synthesis of N-methyl-2-(5-chloro-2-hydroxybenzylidene) hydrazinecarbothioamide
(H2L1)
A solution of 5-chloro-2-hydroxybenzaldehyde (0.74 g, 4.75 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 yellow solution was refluxed with stirring. After 1 h, a pale yellow solid was
formed, which was then filtered, washed with ethanol, and air-dried. Mp: 234 °C to 236 °C,
(0.80 g, 70%). Anal. Calcd for C9H10ClN3OS: C, 44.41; H, 4.11; N, 17.27. Found: C, 44.21;
H, 4.13; N, 17.09 (%); IR(KBr) (νmax/cm−1): 3398 (s, NH), 3125 (m, OH), 1600–1615 (m,
C=N–N–C), 1553 (m, CaroO), 1271 (m, C=S); 1H NMR (DMSO-d6, ppm): 3.01 (s, CH3), 6.87,
7.25, 8.06 (d, d, s, H-aromatic), 8.30 (s, CS–NH), 8.58 (s, CH=N), 10.25 (OH), 11.47 (s, N–
NH). 13C NMR (DMSO–d6, ppm): 30.80 (CH3), 117.71–136.96 (C-aromatic), 155.06 (C=N),
177.66 (C=S).
2.2.2. Synthesis of N-ethyl-2-(5-methyl-2- hydroxybenzylidene) hydrazinecarbothioamide
(H2L2)
A solution of 5-methyl-2-hydroxybenzaldehyde (0.57 g, 4.19 mmol) in ethanol (20 ml) was
added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 ml). The
resulting colorless solution was refluxed with stirring. After 1 h, a white solid was formed,
which was then filtered, washed with ethanol, and air-dried. Colorless needle crystals were

obtained through the slow evaporation of ethanol solution at room temperature. Mp: 164 °C to
166 °C, (0.72 g, 73%). Anal. Calcd for C11H15N3OS: C, 55.61; H, 6.32; N, 17.69. Found: C,
54.16; H, 6.31; N, 17.91 (%); IR(KBr) (νmax/cm−1): 3351–3285 (s, NH), 1601 (m, C=N), 1548
1
(s, CaroO), 1273 (m, C=S); H NMR (DMSO-d6, ppm): 1.05 (t, CH3), 2.23 (s, H3C–Ph), 3.60
(dd, CH2), 6.76, 7.03, 7.70 (d, d, s, H-aromatic), 8.35 (s, CS–NH), 8.40 (s, CH=N), 9.65(OH),
11.31 (s, N–NH); 13C NMR (DMSO–d6, ppm): 14.62 (CH3), 20.07 (CH3–Ph), 30.62 (CH2),
115.94–141.53 (C-aromatic), 154.24 (C=N), 176.50 (C=S).
2.2.3. Synthesis of N-ethyl-2-(3-ter-butyl-2- hydroxybenzylidene) hydrazinecarbothioamide
(H2L3)
A solution of 3-ter-butyl-2-hydroxybenzaldehyde (0.74 g, 4.19 mmol) in ethanol (20 ml) was
added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 ml). The
resulting colorless solution was refluxed with stirring for 2 h and then filtered. The filtrate
was left to stand at room temperature for 2 d. Colorless block crystals were obtained. Mp:
171 °C to 173 °C, (0.93 g, 80%). 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): 3411 (m, NH), 3176 (m, OH),
1
1598 (s, C=N), 1544 (s, CaroO), 1268 (m, C=S); H NMR (DMSO-d6, ppm): 1.11 (t, CH3),
1.40 {s, (CH3)3}, 3.59 (dd, CH2), 6.86, 7.28 (t, dd, H-aromatic), 8.29 (s, CS–NH), 8.51 (s,
CH=N), 11.28 (s, N–NH); 13C NMR (DMSO-d6, ppm): 14.44 (CH3), 29.35 {(CH3)3}, 30.63
{C(CH)3}, 34.46 (CH2), 118.77–146.61 (C-aromatic), 155.19 (C=N), 176.49 (C=S).
2.2.4. Synthesis of N-methyl-2-(2,3-dihydroxybenzylidene) hydrazinecarbothioamide (H2L4)
A solution of 2,3-dihydroxybenzaldehyde (0.65 g, 4.75 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 pink solution was refluxed with stirring for 2 h. A white fluffy product was formed
when the solution was cooled down to room temperature. The resulting product was then

filtered, washed with ethanol, and air-dried. Mp: 240 °C to 242 °C, (0.89 g, 84%). Anal. Calcd
for C9H11N3O2S: C, 47.94; H, 4.88; N, 18.64. Found: C, 48.00; H, 4.68; N, 18.52 (%);
IR(KBr) (νmax/cm−1): 3451 (m, NH), 3147 (m, OH), 1610 (m, C=N), 1546–1529 (s, CaroO),
1
1267 (s, C=S). H NMR (DMSO-d6, ppm): 3.00 (s, CH3), 6.64, 6.84, 7.37 (t,d,d, H-aromatic),
13
8.37 (s, CS–NH), 8.90–9.55 (s, s, OH), 11.39 (s, N–NH). C NMR (DMSO–d6, ppm): 30.62
(CH3), 116.32–145.12 (C-aromatic), 145.54 (C=N), 177.56 (C=S).
2.3.
Synthesis of complexes
2.3.1. Synthesis of N-methyl-2-(5-chloro-2-hydroxybenzylidene) hydrazinecarbothioamide
dioxomolybdenum(VI) [MoO2(L1D)]
A solution of MoO2(acac)2 (0.134 g, 0.411 mmol) in methanol (25 ml) was added to a
solution of N-methyl-2-(5-chloro-2-hydroxybenzylidene) hydrazine carbothioamide (0.1 g,
0.411 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 3 d. Orange block crystals were
obtained. Mp: 244°C to 246 °C, (0.12 g, 75%). Anal. Calcd for C10H11ClMoN3O4S: C, 29.94;
H,2.74; N, 10.48 ; Mo, 23.94. Found: C, 29.08; H, 1.99; N, 10.52; Mo, 23.69 (%); IR(KBr)
(νmax/cm−1): 3397 (m, NH), 1595 (s, C=N), 1550 (m, CaroO), 938,902 (s, Mo=O).
2.3.2. Synthesis of N-ethyl-2-(5-methyl-2-hydroxybenzylidene) hydrazinecarbothioamide
dioxomolybdenum(VI) [MoO2(L2D)]
A solution of MoO2(acac)2 (0.137 g, 0.421 mmol) in methanol (25 ml) was added to a
solution of N-ethyl-2-(5-methyl-2-hydroxybenzylidene) hydrazine carbothioamide (0.1 g,
0.421 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 5 d. Brown needle crystals were
obtained. Mp: 211 °C to 213 °C, (0.13 g, 83%). Anal. Calcd for C12H17MoN3O4S: C, 36.42;

H, 4.30; N, 10.62; Mo, 24.27. Found: C, 35.93; H, 3.38; N, 10.58; Mo, 23.97 (%); IR(KBr)
(νmax/cm−1): 3382 (s, NH), 1590 (m, C=N), 1560 (s, CaroO), 934,896 (s, Mo=O).
2.3.3. Synthesis of N-ethyl-2-(3-ter-butyl-2- hydroxybenzylidene) hydrazinecarbothioamide
dioxomolybdenum(VI) [MoO2(L3D)]
A solution of MoO2(acac)2 (0.116 g, 0.357 mmol) in methanol (25 ml) was added to a
solution of N-ethyl-2-(3-ter-butyl-2-hydroxybenzylidene) hydrazine carbothioamide (0.1 g,
0.357 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 4 d. Orange block crystals were
obtained. Mp: 196 °C to 198 °C, (0.14 g, 90%). Anal. Calcd for C15H23MoN3O4S: C, 41.15;
H, 5.25; N, 9.60; Mo, 21.94. Found: C, 41.04; H, 5.06; N, 9.53; Mo, 21.75 (%); IR(KBr)
(νmax/cm−1): 3333 (m, NH), 1596 (m, C=N), 1284 (m, C-S), 934,875 (s, Mo=O).
2.3.4. Synthesis of N-methyl-2-(2,3-dihydroxybenzylidene) hydrazinecarbothioamide
dioxomolybdenum(VI) [MoO2(L4D)]
A solution of MoO2(acac)2 (0.144 g, 0.443 mmol) in ethanol (25 ml) was added to a solution
of N-methyl-2-(2,3-dihydroxybenzylidene) hydrazine carbothioamide (0.1 g, 0.443 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 5 d. Brown block crystals were obtained. Mp: 244
°C to 246 °C, (0.15 g, 89%). Anal. Calcd for C11H15MoN3O5S: C, 33.22; H, 3.77; N, 10.57;
Mo, 24.15. Found: C, 31.76; H, 3.37; N, 10.44; Mo, 24.02 (%); IR(KBr) (νmax/cm−1): 3386 (s,
NH), 1602 (s, C=N), 1572 (s, CaroO), 1266 (m, C-S), 932,902 (s, Mo=O).

2.4.
DNA binding study
The binding of complexes with calf thymus DNA (CT DNA) were conducted in a 6.3
mM Tris-HCl/50 mM NaCl buffer (pH 7). The DNA stock solution was prepared by
dissolving a suitable amount of DNA in 6.3 mM Tris-HCl/50 mM NaCl buffer (pH 7) at room
temperature, which was then stored in a refrigerator for 2 d. The CT DNA solution in the
buffer presented a ratio of 1.9:1 for UV absorbance at 260 and 280 nm, suggesting that the
DNA was sufficiently free of protein. The DNA concentration was estimated by UV
absorbance at 260 nm by using the known molar absorption coefficient value of 6600
M−1cm−1 [17]. The UV-Vis spectra were scanned in the wavelength range of 230 nm to 600
nm by using the Tris/HCl buffer solution as a reference.
Fluorescence emission assay was carried out by using the above method. Fluorescence
spectra were scanned in the wavelength range of 200 nm to 800 nm by using the Tris/HCl
buffer solution as a reference.
Viscosity measurements were obtained by the Cannon Manning Semi-Micro
viscometer immersed in thermostatic water bath at 37 °C. Flow times were measured
manually with a digital stopwatch. Viscosity values were calculated from the observed flow
time of DNA-containing solutions (t) corrected by observed flow time of the solvent mixture
used (t0), ɳ = t − t0. Viscosity data were presented as (ɳ/ɳ0)1/3 versus the ratio of the
concentration (r) of the ligand or complex-DNA solutions, where ɳ and ɳ0 represent the
viscosity of the complex in the presence of DNA and the viscosity of the DNA alone,
respectively [18].
2.5.
DNA cleavage study
The activity of the ligands and the complexes to cleave the pBR322 plasmid DNA was
studied by agarose gel electrophoresis in a 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 V on
1% agarose gel. The gel was then stained with ethidium bromide prior to being photographed
under UV light.
2.6.
Cytotoxicity study
2.6.1. Preparation of cell culture
HCT 116 cancer cells were grown under optimal incubator conditions. The cells that
reached a confluency of 70% to 80% were selected for cell plating purposes. The old medium
was replaced through aspiration. The cells were then washed with sterile phosphate buffered
saline (pH 7.4) for two to three times. The intact layer of the attached cells was then subjected
to trypsinization. The cells were incubated at 37 °C in 5% CO2 for 3 min to 5 min. The flasks
containing the cells were gently tapped to aid cell segregation and were observed under an
inverted microscope to determine cell segregation completeness. Trypsin activity was
inhibited by adding 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, which
were then inoculated into wells (100 µl/well). Finally, the plates containing the cells were
incubated at 37 °C with an internal atmosphere of 5% CO2.
2.6.2. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay
The cytotoxicity against HCT 116 cells was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT) assay [19]. HCT 116 cells (1.5 × 105 cells/ml, 100
µl/well) were seeded in a 96-well microtiter plate. The plate was incubated overnight in a CO2
incubator to stimulate cell attachment. Approximately 100 µl of test substance was added into
each well. The test substance was diluted with media into the desired concentrations from the
stock. The plates were incubated at 37 °C with an internal atmosphere of 5% CO2 for 72 h.
After treatment, 20 µl of MTT reagent was added into each well, and the plates were further

incubated for 4 h. Afterward, 50 µL of MTT lysis solution (DMSO) was added into each well,
and the plates were further incubated for 5 min in the CO2 incubator. Finally, the plates were
read at 570 and 620 nm wavelengths by a standard enzyme-linked immunosorbent assay
microplate reader. The data were recorded and analyzed to assess the effects of the test
substance on the cell viability percentage of growth inhibition. Growth inhibition was
calculated from the optical density (OD) obtained from the MTT assay, i.e., the hundredth
multiple of the subtracted OD value of control and survived cells over the OD of control cells.
Statistical differences between the treatments and the control were evaluated using ANOVA,
followed by Tukey’s multiple comparison tests. The differences were considered significant
at p<0.05 and p<0.01.
3. Results and discussion
3.1.
Synthesis of complexes
New dioxomolybdenum(VI) complexes were obtained by reacting [MoO2(acac)2] with 4-
methyl(ethyl)thiosemicarbazone ligands derived from 5-chloro-2-hydroxybenzaldehyde
(H2L1), 2-hydroxy-5-methylbenzaldehyde (H2L2), 3-tert-butyl-2-hydroxybenzaldehyde
(H2L3), or 2,3-hydroxybenzaldehyde (H2L4). In all complexes, molybdenum coordinated to
the ligands as tridentate ONS donors through phenolic-oxygen, imine-nitrogen, and thiol-
sulphur. The sixth coordination site (D) required to complete the octahedral coordination of
molybdenum was occupied by the solvent molecule. X-ray crystallography demonstrated that
the D site of the distorted octahedral complexes of cis-dioxomolybdenum(VI) was occupied
by CH3OH when methanol was used as solvent, such as in [MoO2(L1D)], [MoO2(L2D)], and
[MoO2(L3D)], and by CH3CH2OH when ethanol (97%) was used as solvent, such as in
[MoO2(L4D)]. All ligands and their respective complexes are air-stable and are highly soluble
in DMSO and DMF.

1
The Fourier transform infrared spectroscopy (FTIR), H NMR, and 13C NMR spectra, as
well as the elemental analysis, exhibited by the ligands (H2L1–H2L4) were all consistent with
the assigned structures. In FTIR, the bands in the 1598 cm−1 to 1610 cm−1 range can be
attributed to the (C=N) group, the bands in the 1544 cm−1 to 1553 cm−1 range can be
attributed to the (Caro–O) group, and the bands in the 1267 cm−1 to 1273 cm−1 range can be
attributed to the (C=S) group. In the 1H NMR, the signal caused by the (–CH3) group of H2L1
and H2L4 emerged at 3.00 ppm, whereas that of H2L2 and H2L3 emerged in the 1.05 ppm to
1.11 ppm range. The signal caused by the (CS–NH) group emerged in the 8.29 ppm to
8.37 ppm range. The signals that emerged in the 6.64 ppm to 8.06 ppm range can be attributed
to the aromatic protons, whereas the signals in the 11.28 ppm to 11.47 ppm range can be
attributed to the (N–NH) group. In the 13CNMR, the signal caused by the (C=S) group
appeared in the 176.49 ppm to 177.66 ppm range. The signal caused by the (C=N) group
appeared in the 145.45 ppm to 155.54 ppm range. The signals caused by the aromatic carbons
appeared in the 115.94 ppm to 146.61 ppm range.
All complexes ([MoO2(L1D)]–[MoO2(L4D)]) presented single crystals. The FTIR
spectra and elemental analysis were consistent with the assigned structures. In FTIR, a very
sharp two bands attributed to the (Mo=O) groups appeared in the 875 cm−1 to 938 cm−1 range.
The data for all compounds are given in the experimental section.
3.2. Crystallographic studies
The crystal and molecular structure of two ligands (H2L2 and H2L3) and all complexes
([MoO2(L1D)]–[MoO2(L4D)]) were obtained by using the single crystal X-ray diffraction
method. The general crystal data for ligands and complexes are listed in Tables 1 and 2, the
relevant bond distances and angles are presented in Tables 3 and 4, respectively, and the
molecular structures are presented in Figs. 1–6.

The molecular structures of H2L2 and H2L3 (Figs. 1 and 2) show the trans configuration of
the S1 atom relating to N1, which is in the cis configuration relating to N3. In the free ligand,
the S1–C8 bond lengths of 1.702 (7) A0 (H2L2) and 1.6840 (18) A0 (H2L3) and the N2–C8
bond lengths of 1.337 (8) A0 (H2L2) and 1.358 (3) A0 (H2L3) were observed, indicating that
the ligands were in the tautomeric thione form –NH–C=S. By contrast, the relative
molybdenum complexes [MoO2(L2D)] (Fig. 4) and [MoO2(L3D)] (Fig. 5) demonstrate that
the meridional coordination plane alters the S1–C8 bond lengths, which become 1.7545 (15)
A0 and 1.7528 (14) A0, and the N2–C8 bond lengths, which become 1.3156 (19) A0 and
1.3192 (18) A0, with respect to the tautomeric thione form –N=C–S. Molybdenum has a
distorted octahedral coordination in all complexes (Figs. 3–6). The schiff base ligand bonded
+2
to the cis-MoO2 ion through the phenolate oxygen O1 {O4 in [MoO2(L3D)]}, the imine
nitrogen N1, and the thiolate sulfur S1. An oxo group O3 lay trans to N1, whereas the other
oxo group O2 {O4 in [MoO2(L1D)] and [MoO2(L4D)]} lay trans to the D group occupied by
CH3OH or CH3CH2OH in [MoO2(L4D)]. The bond lengths of the doubly bonded terminal oxo
ligands were in the range of 1.6975 (13) A0 to 1.7157(8) A0. These ligands had a high Ooxo–
Mo–Ooxo angle of 105.58(6)° because of the repulsion force between them, which is
responsible for the elongation of the Mo–D bond from 2.3207 (9) A0 to 2.3364 (11) A0 and
for the rather weak coordination to the MoO2+2 unit [17].
3.3. Interaction with DNA
3.3.1. Electronic absorption studies
Electronic absorption spectroscopy is normally used to investigate the binding of
compounds with DNA [20, 21]. Absorption spectroscopic analysis was conducted by a
spectrophotometer (Perkin Elmer Lambda-35) by using fixed concentrations of ligand or
complex (50 µM) with increasing amounts of DNA in the range of 28.9 µM to 173.4 µM in a

6.3 mM Tris-HCl/50 mM NaCl buffer (pH 7). Each addition was left to stand at 25 °C for 10
min to reach equilibrium. The samples were then scanned from 230 nm to 600 nm. All ligands
and their complexes revealed two absorption bands attributed to the transitions of π–π*and
n–π* except for the H2L4 ligand and its complex [MoO2(L4D)], which only revealed the π–π*
transitions at 308 nm. The electronic absorption spectra of the complexes (Figs. 7 and 8 )
show the n–π* transitions at 338 nm and the π–π* transitions at 296, 304, and 306 nm for
[MoO2(L1D)], [MoO2(L2D)], and [MoO2(L3D)], respectively. The π–π* absorption bands
were utilized to observe the interaction of CT DNA with ligands and complexes. The changes
in absorbance were characterized as hypochromism, which confirm that the complexes bind to
the DNA through intercalation [22]. The values of these changes with increasing amounts of
CT DNA were used to estimate the intrinsic binding constant Kb through the following
equation [23]:
[DNA] / (εa − εf) = [DNA] / (εb − εf) + 1/Kb ( εb − εf)
where the absorption coefficients εa, εf, and εb correspond to Aabs/[DNA], the extinction
coefficient for the free complex, and the extinction coefficient for the complex in fully bound
form, respectively. The plot of [DNA]/(εa- εf) versus [DNA] gives a slope 1/(εa − εf) and
intercept 1/Kb (εb − εf) as shown in Fig. 9. The binding constants (Table 5) suggest that all
ligands and complexes were bound to the CT DNA. The complexes revealed stronger
interaction with the DNA compared with their ligands. The highest Kb values were recorded
for H2L3 and [MoO2(L3D)] (3.174 × 105 M−1 and 1.559 × 106 M−1, respectively), whereas the
lowest values were recorded for H2L4 and [MoO2(L4D)] (1.342 × 104 M−1 and 1.00 × 105
M−1), respectively. Furthermore, the binding of the complexes to CT DNA resulted in
isosbestic spectral changes with an isosbestic point at 289 nm.

3.3.2. Fluorescence emission studies
Fluorescence spectroscopic analysis was conducted by a spectrophotometer (Jasco FP-
750) at 25 °C in a 6.3 mM Tris-HCl/50 mM NaCl buffer (pH 7). Except for the complex
[MoO2(L3D)], the fluorescence spectra of all complexes ( Figs. 10 and 11 ) show two
emission bands at 380 and 500 nm. The changes in fluorescence intensity with increasing
amounts of CT DNA were characterized as hypochromism at 500 nm and as hyperchromism
at 380 nm. These changes can be attributed to the isosbestic spectral changes with an
isosbestic point at 408 nm. The complex [MoO2(L3D)] revealed one emission band at 380 nm
accompanied by a hypochromism in fluorescence emission intensity. These fluorescence
emission intensities confirm the strong bound of the complexes to DNA.
3.3.3. Viscometric studies
Viscometric studies play an essential role in confirming the DNA binding pattern in
the solution. The intercalation of a compound into DNA is known to increase viscosity
because of the extension and stiffing of the DNA helix [24]. Viscometric analysis was
performed by adding increasing amounts of the ligands and the complexes to CT DNA at
37 °C in a 6.3 mM Tris-HCl/50 mM NaCl buffer (pH 7). Results are presented in Fig. 12. In
general, the addition of CT DNA results in an increase in the viscosity of the solutions of the
complexes. These results confirm the intercalative binding and support the spectrometric
results.
3.4.
DNA cleavage studies
Agarose gel electrophoresis experiments with pBR322 circular plasmid DNA were
performed to investigate the effect of various micro-concentrations (1.0µM to 6.0µM) of
ligands and complexes on DNA cleavage. The assay was conducted in an aqueous buffer
solution of 6.3 mM Tris-HCl/50 mM NaCl buffer (pH 7) with a fixed amount (4.5 µM ) of

30% H2O2 serving as oxidant. The solution was incubated at 37 °C for 2 h. As shown in Figs.
13 and 14, lane 4 of the agarose gel electrophoresis reveals that H2L1 and H2L4 exhibit DNA
cleavage in the plasmid DNA with typical two forms: the fast migration form related to the
closed circular supercoiled form (SC, form I) and the slow migration form related to the open
circular relaxed form (OC, form II) [25]. H2L2 and H2L3 can niked both strands of the
plasmid DNA, consequently showing the linear form (form III) at a concentration of 5 µM
(lane 10) accompanied by the disappearance of the second form (OC, form II) when the
concentration increases to 6 µM (lane 11) Thus, H2L2 exhibits all three forms of plasmid
DNA, whereas the other ligands show the inhibition of migration of the plasmid DNA at this
concentration.
The complexes exhibited SC form I, OC form II, and linear form III. For the complex
[MoO2(L1D)], forms I and III were exhibited at a concentration of 4 µM. However, form I
disappeared and form II reappeared with form III (lane 9) when the concentration increased to
4.5 µM. When the concentration further increased to 5 µM, only form III remained (lane 10).
For the complex [MoO2(L2D)], forms I and III were exhibited at a concentration of 4.5 µM
(lane 9). When the concentration increased to 5 µM, form I disappeared and only form III
remained (lane 10). Given that the complex [MoO2(L3D)] can niked both strands of the
plasmid DNA, forms I and III were exhibited at a concentration of 3 µM (lane 6). When the
concentration increased to 3.5 µM, all three forms were exhibited (lane 7). The complex
[MoO2(L4D)] exhibited forms II and III at a concentration of 4.5 µM (lane 9) and only form
II at a concentration of 5 µM (lane 10). The inhibitory action of the complexes on the plasmid
DNA were demonstrated at a concentration of 6 µM for [MoO2(L1D)], [MoO2(L2D)], and
[MoO2(L4D)] and at a concentration of 4 µM for [MoO2(L3D)]. On the basis of the size
fragments of the ladder DNA (lane L), the size of form II increased as the concentrations of
[MoO2(L1D)], [MoO2(L2D)], and [MoO2(L4D)] increased, which are consistent with their

respective ligands H2L1, H2L2, and H2L4. However, the complex [MoO2(L3D)] exhibited a
different behavior from the other complexes. The size of form II decreased as the
concentrations increased, which is inconsistent with the behavior of its respective ligand
H2L3. The agarose gel electrophoresis patterns show only the naturally occurring supercoiled
form (SC, form I) for DNA control and buffer (lane 1), DNA and ligand or complexes (lane
2), and DNA, H2O2, and buffer (lane 3). This finding indicates that the activity of ligands and
complexes to cleave DNA can only be accomplished in the presence of H2O2 acting as
oxidant (lanes 4–11) by adding a fixed amount (4.5 µM) of H2O2 to different amounts of
ligands and complexes (1 µM to 6 µM).
The activity to cleave DNA was greatly enhanced in complexes compared with their
related free ligands, which proves that the complexes can produce hydroxyl radical from
H2O2. This capability to generate hydroxyl radicals from metal ions is well reported [26, 27].
These free radicals share the oxidation of deoxyribose moiety, followed by the hydrolytic
cleavage activity of the sugar phosphate backbone [28].
3.5.
Antitumor activity
Similar to the DNA binding and the DNA cleavage activity, the complexes
demonstrated higher efficiency in anti-proliferation activity compared with their respective
ligands. Anti-proliferation tests of the complexes against HCT 116 cancer cells were
performed in vitro. The complexes exhibited greater pronounced activity than the standard
reference 5-fluorouracil did (IC50 = 7.3 µM). The median inhibitory concentrations (IC50) of
the tested compounds are presented in Table 6. Results show that the anti-proliferative
efficiency of the complexes are in the following order: [MoO2(L3D)] > [MoO2(L1D)] ~
[MoO2(L2D)] > [MoO2(L4D)]. These results justify the importance of substituents in the
aromatic ring on anti-proliferation efficiency, which are in the following order: C(CH3)3 > Cl

~ CH3 > OH. These results are consistent with the binding capabilities of the complexes with
CT DNA. The presence of the terminal N-ethyl and tert-butyl groups provide strong lipophilic
properties that enable the complex [MoO2(L3D)] to move into the hydrophobic medium of
DNA. The terminal N-methyl and N-ethyl groups as well as the chlorine and methyl moieties,
are provide good lipophilic properties that enable the complex [MoO2(L1D)] and
[MoO2(L2D)] to move into DNA, respectively. The polarized chlorine moiety increases the
London dispersion forces, which are well increase the lipophilic properties of [MoO2(L1D)].
The presence of hydroxyl group adjacent to O(1) in [MoO2(L4D)] can form a strong
intrahydrogen bonds between them, which decreases the lipophilic properties while increasing
the hydrophilic properties, consequently decreasing its efficiency to move between the DNA
bases. Meanwhile, the coordination with MoO2+2 unit leads to geometrical and conformational
changes which in turn lead to an extent of planarity which increases the affinity between the
complexes and DNA, consequently leads to increases the apoptosis ability of complexes
against the cell proliferation. Furthermore, the inhibition activity of complexes showed that
100 µM of [MoO2(L3D)], [MoO2(L1D)], [MoO2(L2D)], and [MoO2(L4D)] is sufficient to
inhibit 95.6%, 91.3%, 76.9%, and 48.9% of cell proliferation, respectively (Fig. 15). Figure
16 shows the photomicrographs of HCT 116 cell images after 48 h treatment with the
complexes. These images were taken under an inverted phase-contrast microscope at ×200
magnification with a digital camera after 48 h of treatment with the samples. The
photomicrograph of the untreated (control) group showed aggressively growing cells with full
confluence and compact monolayer of HCT 116 cells. A closer examination of the images of
untreated and treated cells reveals the strong cytotoxic effect of the complexes against the
HCT 116 cell line. The analysis of the photomicrographs shows that the complexes generally
show apoptotic features in the affected cells. The blabbing of the cell membrane, the nuclear

condensation, and the formation of vesicles in the treated cells clearly indicate the unique
characteristics of apoptosis.
4. Conclusions
The synthetic and structural descriptions, as well as the biological evaluation, of four
molybdenum(VI) complexes ([MoO2(L1D)]–[MoO2(L4D)]) were studied. The complexes
show that the thiosemicarbazonato ligands (L1–L4) of N-methyl-2-(5-chloro-2-
hydroxybenzylidene)
hydroxybenzylidene)
hydroxybenzylidene)
hydrazinecarbothioamide
hydrazinecarbothioamide
hydrazinecarbothioamide
(H2L1),
(H2L2),
(H2L3),
N-ethyl-2-(5-methyl-2-
N-ethyl-2-(3-ter-butyl-2-
and
N-methyl-2-(2,3-
dihydroxylbenzylidene) hydrazinecarbothioamide (H2L4) are coordinated to molybdenum as
tridentate ONS-donors. On the basis of the X-ray crystallography results, all complexes show
the distorted octahedral coordination. The complexes reveal good efficiency for CT DNA
binding, which is supported by the high values of the binding constant Kb in the range 1.00 ×
105 M−1 to 1.559 × 106 M−1, the UV hypochromism of the absorption bands at 296 and
305 nm, the increase in viscosity with increasing CT DNA concentrations, and the efficient
cleavage activity of the plasmid pBR322 DNA. The complexes exhibit higher activity
compared with their respective ligands.
The anti-proliferative activities of the complexes against HCT 116 cell lines were
investigated. All complexes exhibit high in vitro anticancer activities in the relative order
[MoO2(L3D)] > [MoO2(L1D)] ~ [MoO2(L2D)] > [MoO2(L4D)] according to their IC50 values
of 3.2, 4.5, 4.6, and 6.4 µM, 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.
Supplementary material
CCDC 931643 and 925646 contain the supplementary data for H2L2 and H2L3. CCDC
891975, 902962, 902960, and 906634 contain the supplementary data for [MoO2(L1D)]–
[MoO2(L4D)], respectively. These data can be obtained free of charge from
http://www.ccdc.cam.ac.uk/data_request/cif.
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Graphical abstract

Figure captions
Scheme 1. General synthetic procedure of ligands and complexes
Fig 1. Molecular structure of H2L2
Fig 2. Molecular structure of H2L3
Fig 3. Molecular structure of [MoO2(L1D)]
Fig 4. Molecular structure of [MoO2(L2D)]
Fig 5. Molecular structure of [MoO2(L3D)]
Fig 6. Molecular structure of [MoO2(L4D)]
Fig. 7. UV spectra of complexes [MoO2(L1D)] and [MoO2(L2D)] in 6.3 mM Tris-HCl/50
mM NaCl buffer (pH= 7) in presence of CT DNA at increasing amounts.[complex] = 50 µM
, [DNA] = (28.9 - 173.4) µM . The arrows show the changes in absorbance upon increasing
amounts of CT - DNA .
Fig. 8. UV spectra of complexes [MoO2(L3D)] and [MoO2(L4D)] in 6.3 mM Tris-HCl/50
mM NaCl buffer (pH= 7) in presence of CT DNA at increasing amounts.[complex] = 50 µM
, [DNA] = (28.9 - 173.4) µM . The arrows show the changes in absorbance upon increasing
amounts of CT-DNA .
Fig. 9. Plots of [DNA]/(εa – εf) vs [DNA] for A= [MoO2(L1D)], B= [MoO2(L2D)], C=
[MoO2(L3D)] and D= [MoO2(L4D)].
Fig. 10. Fluorescence spectra of complexes [MoO2(L1D)] and [MoO2(L2D)] in 6.3 mM
Tris-HCl/50 mM
NaCl buffer (pH= 7) in presence of CT - DNA at increasing

amounts.[complex] = 50 µM , [DNA] = (28.9 - 173.4) µM. The arrows show the changes in
fluorescence intensity upon increasing amounts of CT - DNA.
Fig. 11. Fluorescence spectra of complexes [MoO2(L3D)] and [MoO2(L4D)] in 6.3 mM
Tris-HCl/50 mM
NaCl buffer (pH= 7) in presence of CT DNA at increasing
amounts.[complex] = 50 µM , [DNA] = (28.9 - 173.4) µM. The arrows show the changes in
fluorescence intensity upon increasing amounts of CT - DNA.
Fig. 12. Viscometric results of ligands (A) and complexes (B) , in 6.3 mM Tris-HCl/50 mM
NaCl buffer (pH= 7) upon addition increasing amounts of CT DNA.
Fig. 13. Agarose gel electrophoresis patterns of pBR 322 (0.025 µM) increasing
concentrations (1.0 µM – 6.0 µM) of ligands H2L1 and H2L2 and their complexes
[MoO2(L1D)] and [MoO2(L2D)] in 6.3 mM Tris-HCl/50 mM
NaCl buffer (pH= 7)
0
incubated at 37 C for 2h , using H2O2 (4.5 µM) and ladder 1 KbDNA as a marker (lane L);
lane 1, pBR 322 DNA ; lane 2, DNA + Sample (ligand or complex)(6 µM); lane 3, DNA+
H2O2 ; lane 4, DNA +H2O2+ Sample (1 µM);lane 5, DNA +H2O2+ Sample(2 µM); lane 6,
DNA +H2O2+ Sample (3 µM); lane 7 , DNA +H2O2+ Sample(3.5 µM); lane 8, DNA +H2O2+
Sample(4 µM); lane 9, DNA +H2O2+ Sample(4.5 µM); lane 10, DNA +H2O2+ Sample (5
µM); lane 11, DNA +H2O2+ Sample(6 µM).
Fig. 14. Agarose gel electrophoresis patterns of pBR 322 (0.025 µM) increasing
concentrations (1.0 µM – 6.0 µM) of ligands H2L3 and H2L4 and their complexes
[MoO2(L3D)] and [MoO2(L4D)].
Fig. 15. Cell proliferation inhibition by complexes in concentration range 3.0-100 µM
using 5-FU as a reference.

Fig. 16. Photomicrographs of the HCT 116 cell lines before and after treatment with
complexes ; image control = HCT 116 cell before treatment , the images after treatment ;
A=[MoO2(L1D)] (IC50=4.5 µM), B=[MoO2(L2D)] (IC50=4.6 µM),
C=[MoO2(L3D)]
(IC50=3.2 µM), D=[MoO2(L4D)] (IC50=6.4 µM) and 5-FU= standard reference (IC50=7.3
µM) .

Scheme 1. General synthetic procedure of ligands and complexes

Fig 1. Molecular structure of H2L2
Fig 2. Molecular structure of H2L3 (A and B)

Fig 3. Molecular structure of [MoO2(L1D)]

Fig 4. Molecular structure of [MoO2(L2D)]
Fig 5. Molecular structure of [MoO2(L3D)]