Synthesis, characterization, crystal structure, DNA and BSA binding, molecular docking and in vitro anticancer activities of a mononuclear dioxido-uranium(VI) complex derived from a tridentate ONO aroylhydrazone

Article abstract of DOI:10.1016/j.jphotobiol.2016.03.001

A mononuclear dioxido-uranium(IV) complex [UO₂(L)(DMSO)₂], was prepared from the reaction of (2-hydroxy-3-methoxybenzylidene)benzohydrazide [HL] with UO₂(OAc)₂·2H₂O in DMSO. The obtained complex was fully characterized. Single crystal X-ray diffraction analysis of [UO₂(L)(DMSO)₂] revealed that U(VI) ion has been coordinated by ONO donor atoms of the dianionic ligand (L^{2 -}), oxo groups and two DMSO molecules in a pentagonal bipyramid geometry. In addition, interactions of the complex with salmon sperm DNA and bovine serum albumin (BSA) were thoroughly investigated using UV-vis absorption, voltammetry and molecular docking methods. The experimental studies showed an intercalative mode of interaction between the complex and DNA. Experiments on BSA interaction indicated a change in the polarity of the environment surrounded the complex as a result of the interaction between BSA and [UO₂(L)(DMSO)₂]. Finally, MTT assays indicated that the U(VI) complex had excellent cytotoxicity against human carcinoma cell lines of MCF-7, HPG-2, and HT-29, with IC₅₀ values of 8.4, 10.6 and 10.0 μM, respectively.

Full text of DOI:10.1016/j.jphotobiol.2016.03.001

Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, characterization, crystal structure, DNA and BSA binding,
molecular docking and in vitro anticancer activities of a mononuclear
dioxido-uranium(VI) complex derived from a tridentate ONO
aroylhydrazone
c
d
Maryam Mohamadi a,b, S. Yousef Ebrahimipour a, , Jesus Castro , Masoud Torkzadeh-Mahani
⁎
a
Department of Chemistry, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran
Department of Chemistry, Payame Noor University (PNU), 19395-4697 Tehran, Iran
b
c
Departamento de Química Inorgánica, Universidade de Vigo, Facultade de Química, Edificio de Ciencias Experimentais, 36310 Vigo, Galicia, Spain
Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advance Technology, Kerman, Iran
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 May 2015
Received in revised form 29 February 2016
Accepted 1 March 2016
Available online 4 March 2016
A mononuclear dioxido-uranium(IV) complex [UO2(L)(DMSO)2], was prepared from the reaction of (2-hydroxy-
3-methoxybenzylidene)benzohydrazide [HL] with UO2(OAc)2·2H2O in DMSO. The obtained complex was fully
characterized. Single crystal X-ray diffraction analysis of [UO2(L)(DMSO)2] revealed that U(VI) ion has been co-
ordinated by ONO donor atoms of the dianionic ligand (L2−), oxo groups and two DMSO molecules in a pentag-
onal bipyramid geometry. In addition, interactions of the complex with salmon sperm DNA and bovine serum
albumin (BSA) were thoroughly investigated using UV–vis absorption, voltammetry and molecular docking
methods. The experimental studies showed an intercalative mode of interaction between the complex and
DNA. Experiments on BSA interaction indicated a change in the polarity of the environment surrounded the com-
plex as a result of the interaction between BSA and [UO2(L)(DMSO)2]. Finally, MTT assays indicated that the U(VI)
complex had excellent cytotoxicity against human carcinoma cell lines of MCF-7, HPG-2, and HT-29, with IC50
values of 8.4, 10.6 and 10.0 μM, respectively.
Keywords:
U(VI) complex
X-ray crystal structure
DNA binding
BSA interaction
Molecular docking
MTT assays
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
reprocessing of nuclear wastes [8]. Among f-elements, uranium is partic-
ularly interesting since it exhibits both heavy metal and radioactive prop-
Schiff bases are versatile ligands widely used in coordination
chemistry due to their unique properties such as stability, multi-
denticity and easy synthesis [1]. Among these compounds, hydrazones
have attracted considerable attention not only because of their variety
of structure, but also for their various biological and chemical applica-
tions [2]. They have the ability to form complexes with metal ions in
different oxidation states [3].
Coordination chemistry of f-elements is rapidly developed because
their complexes show unique luminescent and magnetic properties
including relevance to luminescent systems with long lifetimes,
photostability and line-like emission bands [4] which make them appro-
priate options as diagnostic tools in biological sciences. For example, these
compounds have been used as markers for immunofluorescent assays or
paramagnetic contrast agents in magnetic resonance imaging [5], second-
order nonlinear optical (NLO) chromophores [6–7] as well as practical
erties [9–10]. Uranium is the heaviest naturally occurring earth element
with several oxidation states of II, III, IV, V, and VI [11].
Under physiological conditions, U(VI) is the most stable oxidation
state of uranium [12]. Hexavalent uranium has been reported to form
complexes with serum proteins, including albumin and transferrin
[11,13–15]. Accordingly, investigation of the interaction between
U(VI) compounds and serum proteins is of high importance with re-
spect to the coordination chemistry of U(VI) [16], nature of the proteins
[17] and quantitative description of the interaction [18].
In spite of the rapid development of novel anticancer drugs, drug re-
sistance and undesirable side effects have created many problems in
cancer therapy [19]. Thus it is necessary to identify new compounds
with better properties in this regard [20].
Generally, molecules which interact with DNA affect DNA replication
and transcription and ultimately, induce cell death and apoptosis. So,
study of the interaction mode and mechanism of such compounds is
of importance which helps us to provide new and even more efficient
anticancer drugs [21].
⁎
Corresponding author at: Department of Chemistry, Shahid Bahonar University of
Kerman, 76169-14111 Kerman, Iran.
E-mail addresses: Ebrahimipour@uk.ac.ir, Ebrahimipour@ymail.com
(S.Y. Ebrahimipour).
Two broad classes of non-covalent DNA-binding agents have been
identified: the intercalators and the groove binders. The intercalators
http://dx.doi.org/10.1016/j.jphotobiol.2016.03.001
1011-1344/© 2016 Elsevier B.V. All rights reserved.

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M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
bind via inserting a planar aromatic chromophore between adjacent
base pairs of DNA, whereas the groove binders fit into the DNA minor
grooves causing a little perturbation of the DNA Structure [22].
Herein, we have synthesized and fully characterized a new U(VI)
complex containing a tridentate ONO aroylhydrazone Schiff base. In
addition, the interaction of this complex with salmon sperm DNA and
BSA have been studied using spectroscopic and electrochemical tech-
niques. Finally, anticancer properties of the title complex against three
cancer cell line of MCF-7, HT-29 and HPG-2 have been evaluated with
MTT assay.
N, 4.14%. Molar conductance (10−3 M, DMSO) 3.7 Ω−1 cm2 mol−1. FT-
IR (KBr), cm−1: ν(CH) 2837–3059, ν(C_N) 1595, ν(C_Cring) 1461,
ν(C\\O) 1243, ν(NN) 1142, δoopb(CH) 712, ν(S_O) 1012, νasy(trans-
UO2) 949, νsy(trans-UO2) 891, ν(CSC) 639, ν(U\\O) 533, ν(U\\N) 470.
1H NMR (300 MHz, DMSO-d6, 25 °C, ppm): δ = 9.24 (s, 1H; CH_N),
6.61–8.38 (m, 8H, rings), 3.94 (s, 3H, OCH3), 3.36 (s, 6H, S(CH3)2). UV/
vis (DMSO) λmax, nm (logε, L mol−1 cm−1): 286 (4.14), 347 (4.16),
380 (4.45), 405 (4.34).
2.5. Crystal Structure Determination
2. Experimental
Crystallographic data were collected at room temperature using a
Bruker Smart 6000 CCD detector and Cu-Kα radiation (λ
=
2.1. Reagents
1.54178 Å) generated by an Incoatec microfocus source equipped with
Incoatec Quazar MX optics. The software APEX2 was used for collecting
frames of data, indexing reflections, and the determination of lattice pa-
rameters, SAINT for integration of intensity of reflections, and SADABS
for scaling and empirical absorption correction [24].
Crystallographic treatment was performed with the Oscail program
[25]. The crystal structure was solved with direct methods and refined
using a full-matrix least-squares method based on F2 [26]. Non-
hydrogen atoms were refined with anisotropic displacement parame-
ters. Hydrogen atoms were included in idealized positions and refined
with isotropic displacement parameters. It was found that one of the
DMSO molecules bonded to the uranium metal was disordered and oc-
cupancy factors of 0.866(5):0.134(5) were refined for two sulfur atoms.
Details of crystal data and structural refinement are given in Table 1.
Salmon sperm DNA was purchased from Sigma (St. Louis, USA). A
1.0 mg/mL stock solution of the DNA was prepared in TE buffer
(pH 7.4) and kept frozen. The concentration of the solution was de-
termined using the molar extinction coefficient of DNA bases at
260 nm (ε260) which was found to be 6600 L mol−1 cm−1 (per P or
nucleotide unit). Bovine serum albumin (BSA) was obtained from
Merck (Germany). Other chemicals and solvents were of analytical
reagent grade and used as received. [HL] was prepared according to
previous report [23].
2.2. Instrumentation
Cyclic voltammograms were obtained using an Autolab PGSTAT
302 electrochemical system from Metrohm (Herisau, Switzerland)
interfaced with a personal computer for data acquisition and potential
control. Electronic spectra were recorded on a double beam UV–vis–
NIR Varian Cary 500 spectrophotometer (Victoria, Australia). Micro
analyses for C, H, N were performed using a Thermo Finnigan Flash Ele-
mental Analyzer 1112EA. Melting points were Determined with the
help of an Electrothermal Apparatus-9200. FT-IR spectra were recorded
at a Bruker-Tensor 27 by embedding the material in KBr discs in the
range of 400–4000 cm−1. Molar conductance measurements were
made by means of a Metrohm 712 Conductometer in DMSO. 1H NMR
spectra were recorded at 25 °C on AVANCE BRX 500 and 300 MHz spec-
trometers. Diffraction data were measured using a Bruker Smart 6000
diffractometer.
2.6. UV–Vis Absorption Studies
Interaction of the U(VI) complex with DNA has been studied with
UV spectroscopy in order to investigate the possible binding mode.
Table 1
Crystal data and structure refinement for [UO2(L)(DMSO)2].
Empirical formula
C19 H24 N2 O7 S2 U
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
694.55
296(2) K
1.54178 Å
Monoclinic
P21/c
a = 15.5721(8) Å
b = 10.1274(5) Å
c = 16.7245(8) Å
α = 90°
2.3. Synthesis of (2-hydroxy-3-methoxybenzylidene) benzohydrazide [HL]
A mixture of benzohydrazide (0.14 g, 1 mmol) and 3-methoxy-2-
hydroxy benzaldehyde (0.15 g, 1 mmol) was refluxed in 10 mL
methanol. After 1 h, the resulted precipitate was filtered, washed with
cold ethanol and dried in vacuum over silica gel.
β = 115.5819(12)°
γ = 90°
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
2379.0(2) Å3
4
1.939 Mg/m3
Yield: 0.22 g, 83%. m.p.: 116 °C. FT-IR (KBr), cm−1: ν(NH) 3571,
ν(OH) 3367, ν(CHar) 2839–3062, ν(C_O) 1647, ν(C=N) 1610,
ν(C_Cring) 1470, ν(N\\N) 1149, ν(C\\O) 1296. 1H NMR (500 MHz,
DMSO-d6, 25 °C, ppm): δ = 12.10 (s, 1H; NH), 11.03 (s, 1H; OH), 8.67
(s, 1H; CH_N), 6.85–7.96 (m, 8H, rings), 3.82 (s, 3H, OCH3). UV/vis
(DMSO) λmax, nm (logε, L mol−1 cm−1): 307(4.61), 339(4.24),
423(2.90).
21.209 mm−1
1328
0.166 × 0.101 × 0.067 mm
3.15 to 67.84 °.
−18 ≤ h ≤ 18
−12 ≤ k ≤ 12
−19 ≤ l ≤ 19
62,993
4288 [R(int) = 0.0582]
4128
Reflections collected
Independent reflections
Reflections observed (N2σ)
Data Completeness
Absorption correction
Max. and min. Transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I N 2σ(I)]
2.4. Synthesis of Bis (dimethylsulfoxide) (3-Methoxy-2-oxidobenzylidene)
benzohydrazonato-dioxido-uranium(VI) [UO2(L)(DMSO)2]
0.994
Semi-empirical from equivalents
0.7530 and 0.2427
Full-matrix least-squares on F2
4288/0/295
1.048
R1 = 0.0246
A
DMSO solution (5 mL) of HL (0.1 mmol, 0.03 g) and
UO2(OAc)2·2H2O (0.1 mmol, 0.042 g) was stirred at room temperature
for 2 h. Suitable crystals for X-ray analysis were obtained after slow
evaporation of the solution for 5 days. The obtained crystals were fil-
tered off and dried in a desiccator over silica gel.
wR2 = 0.0667
R1 = 0.0253
R indices (all data)
wR2 = 0.0678
Yield: 0.042 g, 60%. m.p.:N300 °C. Anal. Calc. for C19H24N2O7S2U
Largest diff. peak and hole
2.021 and −0.872 e·Å−3
(694.56 g mol−1): C, 32.86; H, 3.48; N, 4.03. Found: C, 32.82; H, 3.79;

M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
221
Fig. 2. View of the twisting of the hydrazone ligand in [UO2(L)(DMSO)2].
compounds against each cell line can be concluded from dose–
response curves.
The cells were placed in 96-well micro-assay culture plates at a
density of 5 × 103 cells per well and grown at 37 °C in a humidified 5%
CO2 incubator for 24 h. Then the cells were treated with varying concen-
trations of the complex and ligand (0.31, 0.62, 1.25, 2.5, 5, 10, 20 and
40 μM) for 24 h. After that, 20 μL of MTT solution was added to each
plate and incubation was performed at 37 °C for 4 h. The metabolically
active cells reduced MTT to blue formazan crystals. The crystals formed
were then solubilized upon addition of DMSO and incubated at room
temperature for 20 min. Finally, absorbance (A) of the solution of each
well was measured with ELISA reader at 545 nm. The absorbance was
then converted to percentage of cell-growth inhibition according to
the following formula:
Fig. 1. ORTEP (30% probability level) drawn of [UO2(L)(DMSO)2]. Atom S3 is disorder over
to positions, with occupancy factors of 0.866(5):0.134(5). Last one was not drawn.
Absorption titration of DNA with the complex was carried out using
fixed concentration of DNA (4.5 × 10−5 M) in 0.02 M PBS (pH = 7.4)
while the concentration of the complex was gradually increased. In
the absorption titration of the complex with DNA, the UV–vis spectra
of the complex (6.0 × 10−5 M in PBS) in the presence of varying concen-
trations of DNA were recorded.
Â
À
ÁÃ
%Cell cytotoxicity ¼ 1− Adrug=Acontrol ꢀ 100:
All experiments were carried out at room temperature. In all exper-
iments, the incubation time for equilibrating the interaction was 5 min.
IC50 value defined as the compound concentration required reducing
the survival of cells by 50%, was also calculated.
2.7. Cyclic Voltammetric Measurements
2.9. Molecular Docking
Electrochemical techniques can be employed to study the interac-
tion of electro-active complexes with DNA in order to confirm the
binding mode suggested by spectroscopic studies [27]. Cyclic voltam-
mograms of [UO2(L)(DMSO)2] were recorded before and after adding
DNA to a fixed concentration of the complex in 0.1 M PBS at the scan
rate of 100 mV/s.
Molecular docking is a powerful technique to predict the binding
affinity and orientation of different drugs to their bio-targets such as
proteins and DNA. In the present study, molecular docking was used
to investigate the binding mode and intermolecular interaction of
[UO2(L)(DMSO)2] with DNA and BSA. Coordination sphere of the
U(VI) complex was generated from its X-ray crystal structure as a CIF
file. The CIF file was then converted to PDB format using Mercury soft-
ware (http://www.ccdc.cam.ac.uk/). The molecular docking study was
carried out using Autodock Vina software [28]. All molecular images
and animations were produced using Discovery Studio Visualizer 4.1
package.
2.8. Cell Proliferation and Viability Assay
Toxicity of the synthesized hydrazone ligand [HL] and its U(VI) com-
plex against three human carcinoma cell lines namely HPG-2, HT-29
and MCF-7 was investigated using MTT assay. MTT is transformed into
formazan by the enzyme hydrogenase in mitochondria. The surviving
cells can be determined by measuring their ability to reduce MTT
(yellow) to formazan product (purple). Cytotoxicity of the synthesized
2.10. Chemical Structures of DNA and BSA
The initial structure of BSA was taken from the Protein Data Bank
(PDB ID: 4F5S) at a resolution of 2.47 Å. Also, the DNA sequence
d(CGCGAATTCGCG)2 was obtained from the Protein Data Bank (PDB
ID: 1BNA) at a resolution of 1.90 Å.
Table 2
Selected bond lengths [Å] and angles [°] for [UO2(L)(DMSO)2].
U–O(1)
U–O(11)
U–O(31)
1.778(3)
2.210(3)
2.402(3)
U–O(2)
U–O(12)
U–O(21)
1.780(3)
2.333(3)
2.438(3)
U–N(12)
2.568(4)
Table 3
O(1)–U–O(2)
O(11)–U–N(12)
O(31)–U–O(21)
O(1)–U–O(11)
O(1)–U–O(12)
O(1)–U–O(31)
O(1)–U–O(21)
O(1)–U–N(12)
179.62(17)
70.25(11)
73.70(12)
92.05(15)
89.52(14)
85.17(14)
94.20(14)
88.29(14)
O(12)–U–N(12)
O(12)–U–O(21)
O(11)–U–O(31)
O(2)–U–O(11)
O(2)–U–O(12)
O(2)–U–O(31)
O(2)–U–O(21)
O(2)–U–N(12)
63.03(10)
71.13(11)
82.75(13)
88.02(16)
90.16(14)
95.21(15)
85.89(15)
91.38(15)
Hydrogen bonds for [UO2(L)(DMSO)2].
D–H…A
d(D–H)
d(H…A)
d(D…A)
b(DHA)
C(115)–H(11C)…O(2i)
C(32)–H(32B)…N(11ii)
0.96
0.96
2.57
2.57
3.489(7)
3.467(9)
159.3
155.5
Symmetry transformations used to generate equivalent atoms:
i, x, 3/2-y,z + 1/2; ii, x, y + 1, z.

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M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
Table 4
Summary of C–H⋯π interaction parameters, [Å] and [°].
Interaction (*)
H…Cg
[Å]
H-Perp
[Å]
γ
X–H…
Cg
X…Cg
[Å]
X–H⋯π
C(12)–H(12)⋯Ct1a
2.89
2.72
20.02
162
3.791(6)
65
(*) Code: Symmetry operations: a, x, 1/2-y, z-1/2; H-Perp, perpendicular distance of H to
ring plane; γ, angle between Ct-H vector and ring normal; X–H⋯π, angle of the X–H
bond with the π-plane.
3. Results and Discussions
The title complex was prepared from the reaction of the
aroylhydrazone Schiff base ligand (HL) and UO2(OAc)2.2H2O at 1:1 M
ratio. The complex was found to be stable in air and soluble in DMSO
and DMF while less soluble in methanol, chloroform, and acetonitrile
and insoluble in n-hexane and diethyl ether. Molar conductivity value
of the U(VI) complex was equal to 3.7 Ω1 cm2 mol1 in DMSO, indicating
the non-electrolytic behavior of this complex.
Fig. 4. Electronic absorption spectra obtained from the titration of 6.0 × 10−5
M
[UO2(L)(DMSO)2] in 20 mM PBS (pH 7.4) with increasing amounts of salmon sperm
DNA (0, 3.0 × 10−5, 1.3 × 10−4, 5.0 × 10−4 and 2.0 × 10−3, respectively from up to down).
showed some shifts compared with the transitions of free ONO ligand,
indicating enolization of the ligand followed by its deprotonation. In
the spectrum of the complex, absorption bands of the ligand-to-metal
charge transfer (LMCT) assigned to N → M and O → M transitions
were observed at 380 nm (4.45) and 430 nm (4.34), respectively [32].
3.1. Spectral Characterization
FT-IR spectrum of HL (Fig. S1) showed OH and NH vibrations at 3571
and 3367 cm−1 which were disappeared in the U(VI) complex (Fig. S2)
indicating that the enolic form of the ligand participates in the complex-
ation process [23]. Also, a 15-cm−1 red shift was observed in the
azomethine stretching vibration of the complex compared with that of
the free ligand HL which reveals the coordination takes place through
azomethine nitrogen [29–30]. In the [UO2(L)(DMSO)] spectrum, CO vi-
bration also shifted to lower frequencies supporting that the phenolic
oxygen contributes in the complexation [31]. The asymmetric and sym-
metric vibrations of trans-UO2 were appeared at 949 and 891 cm−1, re-
spectively [32].
3.2. X-ray Crystal Structure
Compound [UO2(L)(DMSO)2] consists of an uranium(VI) ion coordi-
nated by a tridentated dianionic (3-methoxy-2-oxidobenzylidene)
benzohydrazonato ligand (L2−), two oxo groups and two molecules of
dimethylsulfoxide leading a pentagonal bipyramid environment for the
actinide metal. ORTEP drawn of the compound is depicted in Fig. 1
along with the labeling scheme, and selected bond lengths. Also, corre-
sponding angles are set out in Table 2.
The 1H NMR spectrum of [UO2(L)(DMSO)] in DMSO is presented in
Fig. S4 and its spectral data are listed in the experimental section. In
the spectrum of the U(VI) complex, the methine proton is observed at
9.2 ppm which is at lower filed than that of its Schiff base ligand [HL]
(Fig. S3), indicating the coordination of the azomethine nitrogen to
the metal center. Protons of the aromatic rings are located in the
range of 6.6–8.4 ppm as multiplet signals. The singlet signal appeared
at 3.9 ppm corresponds to the protons of the methoxy group [23].
Electronic spectra of HL and its U(VI) complex have been recorded
in DMSO. In the spectrum of the ligand, the absorption bands related
to n→ π* and π → π* transitions of the azomethine moiety, and aromatic
rings of the hydrazone ligand were located between 307 nm and
423 nm [33]. For the U(VI) complex, these intra-ligand transitions
Disposition of oxo groups are trans, with an O–U–O angle of
179.62(17)°, as is usual for the so-called uranyl compounds [UO22+
]
[34–37]. Sum of the angles in the equatorial plane was 360.86°. Howev-
er, this value cannot be used as a probe of the planarity since oxygen
atoms from DMSO ligands are clearly deviated, one 0.140(2) below
and the other 0.167(2) over the plane (with a root-mean-square devia-
tion from a plane of 0.123 Å), in such a way that if the most deviated
atom is eliminated from the calculations, the best fitted plane, formed
by O(11), O(12), O(21) and N(12) (root-mean-square deviation value
of 0.003 Å) leaves the O(31) atom at 0.459(6) Å and the Uranium
atom at 0.094(2) Å. A three-fused system is found in the complex struc-
ture, the methoxyphenolate ring and two metallacycles, one of them is
six-membered and the other is five-membered. The metallacycles form
dihedral angles of 4.2(3)° and 1.9(3)°, respectively for the six- and five-
Fig. 3. Electronic absorption spectra obtained from the titration of 4.5 × 10−5 M salmon
sperm DNA in 20 mM PBS (pH 7.4) with increasing amounts of [UO2(L)(DMSO)2] (0,
9.4, 18.8, 28.2, 37.6, 47.1, 56.5 and 65.9 μM respectively from down to up).
Fig. 5. Cyclic voltammograms of [UO2(L)(DMSO)2] and [UO2(L)(DMSO)2]-DNA adduct at
the scan rate of 100 mV/s.

M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
223
Fig. 6. Molecular docking perspective of the U(VI) complex with DNA (left) three-dimensional interactions generated by Discovery Studio 4.1 (right).
membered rings The planarity of the three-fused system is such that the
root-mean-square deviation for the best plane (13 atoms including
uranium) is 0.0507 Å. The phenyl ring on the carbohydrazone is, howev-
er, twisted 22.4(2)° (dihedral angle between the planes) due to the free
rotation of the C(16)-C(17) bond (Fig. 2). The pentagonal bipyramid
polyhedron around the metal is also distorted due to the presence of
the five-membered metala-ring with a chelate angle of 63.0(1)° while
the six-membered metala-ring produces a chelate angle of 70.3(1)°.
The other cis angles in the equatorial plane are, 71.1(1), 73.7(1) and
82.7(1)°, where the differences can be due to the sterical requirements
pentagonal bipyramid uranium atoms in an NO6 environment, where
the nitrogen atom belongs to a hydrazone ligand resulted in similar
bond distances in the complex structure. Other uranyl complexes with
salicylaldehyde benzoylhydrazone described in the literature [36]
contain monodeprotonated ligands, so the lengths of their U\\O
bonds are not comparable with the complex synthesized here.
Some non-classical hydrogen interactions, C\\H⋯X (X_N,O) con-
struct the supramolecular network. The C(115)⋯O(2i) interactions
lead to the growth of the crystal in the c axis, while the C(32)⋯N(11ii)
ones result in the growth of the crystal in the b axis. The parameters
of these interactions are given in Table 3, Figs. S5 and S6. There are
also a CH⋯π interaction (Table 4) that reinforce the growth in the c
axis as is show in Fig. S7. A presentation of the unit cell is shown in
Fig. S8.
of the DMSO ligand. The bond distances in the uranyl group [UO22+
]
are 1.778(3) and 1.780(3) Å which are slightly longer than those
found for the binuclear compound [(UO2)2(H6pyr2oxdihyd)(DMSO)4]
[37], 1.778(7) and 1.766(7) Å. However, the U\\O (from DMSO)
bond distances are longer than those of similar compounds
[2.402(3) and 2.438(3) Å vs. 2.387(7) and 2.391(7) Å]. A similar U–O
distance of 2.395(3) Å was reported in [2-({(ethylsulfanyl)[2-(2-
oxidobenzylidene)hydrazinylidene]methyl}iminomethyl)phenolato]
dioxidouranium(VI) [35]. Coordination of the aroylhydrazone
ligand can be compared with the binuclear compound
3.3. Absorption Titration of DNA With the Complex
As Fig. 3 shows, a broad band is observed in the UV spectrum of DNA
with a maximum (λmax) placed at about 260 nm corresponding to pu-
rine and pyrimidine bases of DNA [38]. Upon addition of varying con-
centrations of the synthesized U(VI) complex to the DNA solution, the
intensity of the absorption band shows a significant increase with a
[(UO2)2(H6pyr2oxdihyd)(DMSO)4] [37],
a compound with two
red shift of 7 nm in the λmax
.
This observation exhibits that purine and pyrimidine bases of DNA
have been exposed due to the binding of the complex to DNA. So, an in-
tercalation interaction of the complex through the stacking interaction
of the aromatic rings of the ligand HL and the base pairs of DNA is pro-
posed [39]. On the other words, interaction between the complex and
DNA causes some changes in the conformation of DNA [40].
3.4. Absorption Titration of the Complex with DNA
Interaction of metal complexes with DNA leads to electronic pertur-
bations in the complexes, so electronic absorption spectroscopy can be
used to investigate the interaction characteristics [41].
The absorption spectra of the U(VI) complex in the absence and
presence of varying concentrations of DNA are given in Fig. 4. As is
seen, the complex exhibits a broad absorption band at about 320 nm
Fig. 7. Electronic absorption spectra obtained from the titration of 3.0 × 10−5
[UO2(L)(DMSO)2] in PBS (pH 7.4) in the absence and presence of increasing
concentrations of BSA.
M

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M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
Fig. 8. A molecular docking perspective of [UO2(L)(DMSO)]with BSA (left) and Three-dimensional interactions generated by Discovery Studio 4.1 (right).
with ε = 8.4 × 103 cm−1 M−1 assigned to the π–π* transition of the ar-
omatic chromophore. When titrated by the DNA, the intensity of the ab-
sorption peak decreases by more than 30% accompanied with a red shift
of 5 nm at [DNA]/[complex] ratio of 33. These high hypochromic and
bathochromic effects suggest that this complex possesses a high pro-
pensity for DNA binding. According to the spectral characteristics, an
intercalative mode of interaction is suggested for the U(VI) complex-
DNA system. Due to the intercalation, π* anti-bonding orbital of the
complex couples with π bonding orbital of the DNA base pairs which
reduces the π–π* transition energy and results in a bathochromic
shift [42].
(Epc = 0.098 V). Furthermore, the potential of peak II (Epa) shows a
shift to a more positive potential (0.467 V) with a small decrease in
the peak current. In addition, peak III disappears in the complex-DNA
system. The observed decreases in the peak currents are related to the
interaction between the [UO2(L)(DMSO)2] complex and DNA which
can be explained by the slow diffusion of the complex-DNA adduct to
the electrode surface.
The interaction mode can be found from the changes of peak
separation (ΔEp) and half-wave potential (E1 ) due to the interaction.
=
2
ΔEp and E1 are defined as the difference and the average of Epa and
=
2
Epc, respectively.
According to the obtained results, interaction with DNA decreases
3.5. Electrochemical Studies
E1 of the complex from 0.327 V to 0.282 V while increases ΔEp from
=
2
0.261 V to 0.369 V.
Cyclic voltammetry is a useful technique to study the interaction of
electro-active complexes with DNA in order to confirm the DNA binding
mode suggested by spectroscopic studies [43].
Generally, if the interaction mode is of an electrostatic binding
nature, the half-wave potential shifts to a more negative value, while in-
tercalation binding results in the shift of half-wave potential to a more
positive value [44]. So, electrochemical studies confirm the intercalative
mode of the interaction between the title U(VI) complex and DNA
which has been proposed by spectroscopic studies.
Cyclic voltammograms of [UO2(L)(DMSO)] in the absence and pres-
ence of DNA are depicted in Fig. 5. Cyclic voltammogram of the U(VI)
complex in the absence of DNA exhibits a quasi-reversible redox behav-
ior. In the forward wave, a peak (peak I) is observed at 0.197 V with the
peak current of 0.232 μA, corresponding to U(VI)/U(IV) redox couple. In
the reverse scan, two successive waves are observed at 0.458 V (peak II)
and 0.620 V (peak II) with the peak currents of 0.22 μA and 0.143 μA, re-
spectively which can be assigned to U(IV)/U(V) and U(V)/U(VI)
couples.
3.6. Molecular Docking of the U(VI) Complex With DNA
In order to determine the site of DNA involved in its interaction with
[UO2(L)(DMSO)2], blind docking was performed on a DNA duplex with
the sequence of d(CGCGAATTCGCG)2. The obtained conformations were
ranked based on the lowest free binding energy. Accordingly, the most
stable complex formed between the DNA and [UO2(L)(DMSO)2] was
found to possess −6.9 kcal mol−1 binding energy which indicates
that the U(VI) complex has a good binding affinity to DNA and this bind-
ing is spontaneous. Fig. 6 depicts the energy-minimized docked pose of
[UO2(L)(DMSO)2]. There are several categories of hydrophobic contacts
between the complex atoms and bases of DNA, including, DT 119, DA
118, DA 117, DG 116, DC 115, DG 114, DG 112, DC 111, DG 110, DC 99
and DT 88 (Fig. 6). The resulting docking model reveals that the U(VI)
complex partially intercalates into the DNA duplex through its minor
groove.
Addition of DNA to the complex solution causes a decrease in the
current of peak I by 7.7% and a negative shift in the peak potential
3.7. BSA Interaction
3.7.1. UV–Vis Absorption Studies
BSA is an extensively studied serum albumin protein, due to its
structural similarity with human serum albumin [45]. Fig. 7 shows the
absorption spectra of [UO2(L)(DMSO)2] in the presence of varying
Fig. 9. Anticancer activities of different concentrations of [UO2(L)(DMSO)2] against HPG-2,
HT-29 and MCF-7 cell lines.

M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
225
Fig. 10. Cytotoxicity percentage of [UO2(L)(DMSO)2] and HL in the concentration range of 0.31–40 μM against HPG-2 (A), HT-29 (B) and MCF-7 (C) cell lines.
Fig. 11. Microscopic photographs of HPG-2, HT-29 and MCF-7 cancer cells in the absence and presence of 40 μM [UO2(L)(DMSO)2].

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M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
concentrations of BSA. As can be seen, the intensity of the absorption
peak of the complex observed at about 320 nm decreases with a red
shift of 7 nm. These observations indicate an interaction between BSA
and the title U(VI) complex resulted in some changes in the polarity
of the environment surrounded the complex [46] and π–π stacking in-
teraction between the aromatic rings of the complex and those of the ar-
omatic amino acid residues [47].
As shown by spectroscopic and electrochemical studies, the complex
can intercalate into DNA duplex. The changes observed in the absorp-
tion spectrum of [UO2(L)(DMSO)2] in the presence of BSA can be related
to the interaction with BSA. Molecular docking studies on the interac-
tion of [UO2(L)(DMSO)2] with DNA and BSA have confirmed the exper-
imental results. MTT assays has revealed the anticancer activity of the
title complex against MCF-7, HPG-2, and HT-29, with IC50 values of
8.4, 10.6 and 10.0 μM, respectively.
3.7.2. Molecular Docking of the U(VI) Complex With BSA
The existing results of biological activity of the title complex as a
DNA- and BSA-binding compound as well as an anticancer agent are
promising and can open a new window for the use of this compound
as a potential metallodrug.
To obtain useful information on the preferred binding location
and to understand the mechanism of [UO2(L)(DMSO)2]-BSA interac-
tion, molecular docking technique has been used to dock the com-
plex into the protein. Two principal binding sites have been found
in BSA in the proximity of Trp134 and Trp213 [48]. The probable
binding poses obtained from blind docking have been ranked based
on the lowest binding free energy. According to the obtained results,
the title U(VI) complex prefers the binding pocket of domain I con-
taining Trp134 with the relative binding energy of −7.1 kcal mol1.
Also, docking studies shows that the distance between Trp134 resi-
due and the U(VI) complex is 4.146 Å. The energetically favorable
docked image is shown in Fig. 8. As can be seen, there are interactions
between the U(VI) complex and Leu 138, Trp 134, Gly 135, Asp 129,
Lys 132, Leu 24, Val 40, Asp37, Phe 36, Lys 131, Gly 21, Glu 17, Glu 16,
Val 43 and Asn 44.
Acknowledgment
We are grateful to the Shahid Bahonar University of Kerman for the
financial support of this work.
Appendix A. Supplementary data
Crystallographic data for the [UO2(L)(DMSO)2] has been deposited
with the Cambridge Crystallographic Data Centre, CCDC No. 1403371.
Copies of this information may be obtained from the Director, CCDC,
12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336033; e-
mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). Supple-
mentary data associated with this article can be found in the online
version, at http://dx.doi.org/10.1016/j.jphotobiol.2016.03.001.
3.8. Cytotoxicity Study
The synthesized Schiff base ligand and its U(VI) complex have been
tested for their in-vitro anti-proliferative activity against MCF-7, HPG-2
and HT-29 cancer cell lines. Fig. 9 reveals the effect of different
concentrations of [HL] ligand and [UO2(L)(DMSO)2] on the cell lines.
As is seen, the viability of all cell lines decreases in both ligand- and
complex-treated ones as a function of concentration indicating a dose-
dependent growth inhibitory effect. For all cell types and in all tested
concentrations, it is clear that toxicity of the U(VI) complex is consider-
ably higher than that of the ligand. Especially in low concentrations, free
ligand [HL] shows less than 30% of the toxicity of [UO2(L)(DMSO)]. On
the other words, complexation with U(VI) significantly improves the
anticancer effect of the parent ligand.
In addition, viability of MCF-7, HPG-2 and HT-29 cancer cell lines in
the presence of different concentrations of the U(VI) complex has been
compared and revealed in Fig. 10. As can be seen, HPG-2 is the most sen-
sitive cell line to the complex at concentrations ≤5 μM. At higher con-
centrations, proliferation of MCF-7 cells is affected more than those of
the other cell lines. It is worth mentioning that, at high concentrations
the side effect of the compound should be also considered. Fig. 11
shows the photos taken from the cell lines before and after treating by
[UO2(L)(DMSO)2]. It is clear that the complex has cytotoxicity effect
against all three cell lines.
References
[1] S.Y. Ebrahimipour, I. Sheikhshoaie, A.C. Kautz, M. Ameri, H. Pasban-Aliabadi, H.A.
Rudbari, G. Bruno, C. Janiak, Polyhedron 93 (2015) 99–105.
[2] I. Sheikhshoaie, S.Y. Ebrahimipour, M. Sheikhshoaie, H.A. Rudbari, M. Khaleghi, G.
Bruno, Spectrochim. Acta, Part A 124 (2014) 548–555.
[3] N.A. Mangalam, S. Sivakumar, S. Sheeja, M.P. Kurup, E.R. Tiekink, Inorg. Chim. Acta
362 (2009) 4191–4197.
[4] R. Krishnan, J. Thirumalai, A. Kathiravan, Electron. Mater. Lett. 11 (2015) 24–33.
[5] N. Hildebrandt, H.-G. Lohmannsroben, Curr. Chem. Biol. 1 (2007) 167–186.
[6] A. Valore, E. Cariati, S. Righetto, D. Roberto, F. Tessore, R. Ugo, I.L. Fragala, M.E.
Fragala, G. Malandrino, F. De Angelis, J. Am. Chem. Soc. 132 (2010) 4966–4970.
[7] J.P. Morrall, G.T. Dalton, M.G. Humphrey, M. Samoc, Adv. Organomet. Chem. 55
(2007) 61–136.
[8] S.M. Cornet, I. May, M.P. Redmond, A.J. Selvage, C.A. Sharrad, O. Rosnel, Polyhedron
28 (2009) 363–369.
[9] Y. Zhang, I. Karatchevtseva, J.R. Price, I. Aharonovich, F. Kadi, G.R. Lumpkin, F. Li, RSC
Adv. 5 (2015) 33249–33253.
[10] H. Moll, A. Rossberg, R. Steudtner, B.R. Drobot, K. Müller, S. Tsushima, Inorg. Chem.
53 (2014) 1585–1593.
[11] J.D. Van Horn, H. Huang, Coord. Chem. Rev. 250 (2006) 765–775.
[12] A.E. Gorden, J. Xu, K.N. Raymond, P. Durbin, Chem. Rev. 103 (2003) 4207–4282.
[13] W. Stevens, F. Bruenger, D. Atherton, J. Smith, G. Taylor, Radiat. Res. 83 (1980)
109–126.
[14] S. Scapolan, E. Ansorborlo, C. Moulin, C. Madic, Radiat. Prot. Dosim. 79 (1998)
505–508.
Finally, IC50 values for the complex against MCF-7, HPG-2 and HT-29
cell lines were calculated to be 8.4, 10.6 and 10.0 μM, respectively which
are smaller than those reported for the well-known anticancer drug, cis-
platin [49,50].
[15] C. Vidaud, A. Dedieu, C. Basset, S. Plantevin, I. Dany, O. Pible, E. Quéméneur, Chem.
Res. Toxicol. 18 (2005) 946–953.
[16] C. Vidaud, S. Gourion-Arsiquaud, F. Rollin-Genetet, C. Torne-Celer, S. Plantevin, O.
Pible, C. Berthomieu, E. Quéméneur, Biochemistry 46 (2007) 2215–2226.
[17] H. Huang, S. Chaudhary, J.D. Van Horn, Inorg. Chem. 44 (2005) 813–815.
[18] M. Sutton, S.R. Burastero, Chem. Res. Toxicol. 17 (2004) 1468–1480.
[19] B.C. Baguley, D.J. Kerr, Anticancer Drug Development, Academic Press, 2001.
[20] H. Vogel, Drug Discovery and Evaluation: Pharmacological AssaysSpringer Science &
Business Media 2007.
4. Conclusion
[21] W.P. Roos, B. Kaina, Trends Mol. Med. 12 (2006) 440–450.
[22] J.B. Chaires, Curr. Opin. Struct. Biol. 8 (1998) 314–320.
[23] S.Y. Ebrahimipour, H. Khabazadeh, J. Castro, I. Sheikhshoaie, A. Crochet, K.M. Fromm,
Inorg. Chim. Acta 427 (2015) 52–61.
[24] A. Bruker, Smart, Saint, Bruker AXS Inc., Madison, Wisconsin, USA, 2007.
[25] P. McArdle, J. Appl. Crystallogr. 28 (1995) 65.
[26] G.M. Sheldrick, Acta Crystallogr. Sect. A: Found. Crystallogr. 64 (2007) 112–122.
[27] F. Arjmand, M. Aziz, Eur. J. Med. Chem. 44 (2009) 834–844.
[28] O. Trott, A.J. Olson, J. Comput. Chem. 31 (2010) 455–461.
[29] S.Y. Ebrahimipour, Z.R. Ranjabr, E.T. Kermani, B.P. Amiri, H.A. Rudbari, A. Saccá, F.
Hoseinzade, Transit. Met. Chem. 40 (2015) 39–45.
Herein, we report the synthesis and characterization of a new
dioxido-uranium(IV) complex as well as the results of
investigating its interaction with DNA and BSA and anticancer ac-
tivity. The reaction of UO2(OAc)2·2H2O and (2-hydroxy-3-
methoxybenzylidene)benzohydrazide [HL] in DMSO has resulted
in the formation of the complex with the formula of [UO2(L)(DMSO)2].
According to the results of X-ray crystallography, U(VI) ion has been co-
ordinated by ONO donor atoms of the dibasic aroyl hydrazone ligand,
oxo groups and two DMSO molecules in a pentagonal bipyramid
geometry.
[30] S.Y. Ebrahimipour, I. Sheikhshoaie, A. Crochet, M. Khaleghi, K.M. Fromm, J. Mol.
Struct. 1072 (2014) 267–276.

M. Mohamadi et al. / Journal of Photochemistry & Photobiology, B: Biology 158 (2016) 219–227
227
[31] S. Yousef Ebrahimipour, M. Mohamadi, J. Castro, N. Mollania, H. Amiri Rudbari, A.
Saccá, J. Coord. Chem. 68 (2015) 632–649.
[32] S. Ebrahimipour, I. Sheikhshoaie, J. Castro, M. Dusek, Z. Tohidiyan, V. Eigner, RSC
[40] J.-Q. Sha, X. Li, H.-B. Qiu, Y.-H. Zhang, H. Yan, Inorg. Chim. Acta 383 (2012) 178–184.
[41] M. Navarro, E.J. Cisneros-Fajardo, A. Sierralta, M. Fernández-Mestre, P. Silva, D.
Arrieche, E. Marchán, JBIC, J. Biol. Inorg. Chem. 8 (2003) 401–408.
[42] A. Terenzi, G. Barone, A. Silvestri, A.M. Giuliani, A. Ruggirello, V.T. Liveri, J. Inorg.
Biochem. 103 (2009) 1–9.
Adv. 5 (2015) 95104–95117.
[33] S.Y. Ebrahimipour, I. Sheikhshoaie, M. Mohamadi, S. Suarez, R. Baggio, M. Khaleghi,
M. Torkzadeh-Mahani, A. Mostafavi, Spectrochim. Acta A 142 (2015) 410–422.
[34] M. Azam, S.I. Al-Resayes, G. Velmurugan, P. Venuvanalingam, J. Wagler, E. Kroke,
Dalton Trans. 44 (2015) 568–577.
[43] M. Mohamadi, S.Y. Ebrahimipour, M. Torkzadeh-Mahani, S. Foro, A. Akbari, RSC Adv.
5 (2015) 101063–101075.
[44] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901–8911.
[45] M.R. Eftink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199–227.
[46] G. Zhang, Y. Ma, L. Wang, Y. Zhang, J. Zhou, Food Chem. 133 (2012) 264–270.
[47] J. Zhu, L. Wu, Q. Zhang, X. Chen, X. Liu, Spectrochim. Acta A 95 (2012) 252–257.
[48] D. Lu, X. Zhao, Y. Zhao, B. Zhang, B. Zhang, M. Geng, R. Liu, Food Chem. Toxicol. 49
(2011) 3158–3164.
[35] R. Takjoo, S.W. Ng, E.R. Tiekink, Acta Crystallogr. Sect. E: Struct. Rep. Online 68
(2012) m244–m245.
[36] C. Gatto, E. Schulz Lang, A. Kupfer, A. Hagenbach, U. Abram, Z. Anorg. Allg. Chem. 630
(2004) 1286–1295.
[37] D.F. Back, G.M. de Oliveira, D. Roman, M.A. Ballin, R. Kober, P.C. Piquini, Inorg. Chim.
Acta 412 (2014) 6–14.
[38] K.C. Skyrianou, V. Psycharis, C.P. Raptopoulou, D.P. Kessissoglou, G. Psomas, J. Inorg.
Biochem. 105 (2011) 63–74.
[49] Y. Chen, M.-Y. Qin, J.-H. Wu, L. Wang, H. Chao, L.-N. Ji, A.-L. Xu, Eur. J. Med. Chem. 70
(2013) 120–129.
[50] N. Desbois, D. Pertuit, J. Moretto, C. Cachia, B. Chauffert, F. Bouyer, Eur. J. Med. Chem.
69 (2013) 719–727.
[39] A. Silvestri, G. Barone, G. Ruisi, D. Anselmo, S. Riela, V.T. Liveri, J. Inorg. Biochem. 101
(2007) 841–848.