NHC-Ir(I) complexes derived from 5,6-dinitrobenzimidazole. Synthesis, characterization and preliminary evaluation of their in vitro anticancer activity

Article abstract of DOI:10.1016/j.ica.2019.119061

The design, synthesis, characterization and in vitro anticancer activity of a series of Ir(I) NHC complexes derived from 5,6-dinitrobenzimidazole is reported. The evaluation was performed in five human cancer cell-lines, namely glioblastoma (U-251), prostatic adenocarcinoma (PC-3), colorectal adenocarcinoma (HCT-15), mammary adenocarcinoma (MCF-7) and lung adenocarcinoma (SKLU-7), including healthy cells of African green monkey kidney (COS-7) for comparative purposes. The complexes exhibited better activity in comparison with the corresponding NHC ligand precursors. In particular, complex (4a) exhibited a good performance against PC-3 and SKLU-1 with IC₅₀ values of 10.6 ± 0.9 μM and 10.4 ± 1.5 μM, respectively.

Full text of DOI:10.1016/j.ica.2019.119061

InorganicaChimicaActa496(2019)119061
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Research paper
NHC-Ir(I) complexes derived from 5,6-dinitrobenzimidazole. Synthesis,
characterization and preliminary evaluation of their in vitro anticancer
activity
Arturo Sánchez-Moraa, Hugo Valdésa, María Teresa Ramírez-Apana, Antonio Nieto-Camachoa,
Simón Hernández-Ortegaa, Daniel Canseco-Gonzálezb, David Morales-Moralesa,⁎
a Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Ciudad de México C.P. 04510, Mexico
b CONACYT-Laboratorio Nacional de Investigación y Servicio Agroalimentario y Forestal, Universidad Autónoma Chapingo, Texcoco de Mora, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cancer
The design, synthesis, characterization and in vitro anticancer activity of a series of Ir(I) NHC complexes derived
from 5,6-dinitrobenzimidazole is reported. The evaluation was performed in five human cancer cell-lines,
namely glioblastoma (U-251), prostatic adenocarcinoma (PC-3), colorectal adenocarcinoma (HCT-15), mam-
mary adenocarcinoma (MCF-7) and lung adenocarcinoma (SKLU-7), including healthy cells of African green
monkey kidney (COS-7) for comparative purposes. The complexes exhibited better activity in comparison with
the corresponding NHC ligand precursors. In particular, complex (4a) exhibited a good performance against PC-
Cytotoxic activity
N-heterocyclic carbene
Iridium complexes
Fluorinated complexes
Antioxidant properties
3 and SKLU-1 with IC50 values of 10.6
0.9 μM and 10.4
1.5 μM, respectively.
1. Introduction
(Fig. 1), where NHC corresponds to a N-heterocyclic carbene ligand,
more specifically they prepared the imidazolylidene and triazolylidene
derivatives [41–43]. These compounds showed IC50 values in the mi-
Cancer is one of the main causes of death around the world, pro-
ducing 9.6 million deaths in 2018, only. Furthermore, each year there
are 18.1 million new cases of cancer worldwide. Besides the incalcul-
able human cost of cancer, its economical cost was estimated in $ 1.16
trillion USD. The estimation includes some important issues such as,
treatment cost, productivity lost due to premature death and disability
among others [1,2].
Patients with cancer are commonly treated with platinum deriva-
tives, such as cisplatin, carboplatin and oxaliplatin [1–4]. In fact, nearly
half of patients undergoing chemotherapy are treated with platinum
drugs [5]. However, their use has been related to several side effects,
such as infertility, alopecia, and anemia. In particular, cisplatin is also
nephrotoxic and ototoxic [6,7]. In order to overcome these drawbacks,
many research groups have focused their efforts on developing new
potential anticancer compounds based on other transition metals, such
as Au, Cu, Ru, Os, etcetera [8–26]. Recently, Ir has attracted much
attention for the design of metallodrugs because of its antiproliferative
and luminescent properties, especially when its oxidation state is + III
[27–37]. However, there are only a few examples of Ir(I) complexes for
this purpose [3,4,38–40]. Metzler-Nolte and co-workers described the
cytotoxic activity of a series of complexes of the type [(NHC)IrCl(COD)]
cromolar scale, ranging from 10.3
MCF-7.
2.9 to 46.920
0.085 μM for
In the past two decades, NHC ligands have become important motifs
for the design of catalysts [44–46], as well as biological active com-
pounds [8–26,47]. They form strong bonds with practically any tran-
sition metal, and can be easily prepared and functionalized, allowing
the fine tuning of their electronic and steric properties. Based on the
aforementioned reasons, herein we report the synthesis and cytotoxic
activity of a series of Ir(I)–NHC complexes derived from 5,6-dini-
trobenzimidazole (Fig. 1). The presence of the nitro groups at the NHC
ligand may provide interesting biological properties to their related
complexes. The nitro group can interact with biological nucleophiles
such as proteins, amino acids, nucleic acids, and enzymes through non-
covalent interactions and can be found in some antineoplasic, anti-
biotic, and antiparasitic agents [48,49]. Furthermore, the eNO2 groups
can be bioactivated by enzymatic reduction, producing reactive species
that may serve as prodrugs, ultimately inducing the desired or un-
desired biological effects [48].
⁎ Corresponding author.
E-mail address: damor@unam.mx (D. Morales-Morales).
https://doi.org/10.1016/j.ica.2019.119061
Received 19 May 2019; Received in revised form 22 July 2019; Accepted 8 August 2019
Availableonline09August2019
0020-1693/©2019ElsevierB.V.Allrightsreserved.

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
Fig. 1. Cytotoxic NHC-Ir(I) complexes.
Scheme 1. Synthesis of 5,6-dinitrobenzimidazolium salts and Ir(I)–NHC complexes.
2. Results and discussion
by a transmetallation reaction (Scheme 1). The reaction of Ag2O with
the corresponding azolium salt in acetonitrile, affords the Ag(I)–NHC
derivatives (not isolated). Then, the addition of [IrCl(COD)]2 to the
solution produces the immediately precipitation of AgBF4 and the for-
mation of the desired product. The 1H and 13C{1H} NMR spectra of the
complexes were in agreement with the symmetry loss of the azolium
salts. The most characteristic signal of the complexes corresponds to the
metallated carbon that appears at 202.9 ppm. The mass spectra of the
complexes were very clean, exhibiting peaks for the molecular ions at
648.0 m/z ([M]+) for (4a) and 738.0 m/z ([M]+) for (4b) respectively.
The molecular structure of complex (4a) was unequivocally de-
termined by single crystal X-ray diffraction studies (Table 1, Fig. 2).
Suitable crystals for this analysis were obtained by slow diffusion of
hexane into a concentrated solution of the compound in 1,2-di-
chloroethane.
The structure reveals a distorted square planar geometry around the
metal center. The NHC ligand is coordinated to Ir(I), one chlorine and
one COD ligand complete the coordination sphere. The Ir(I)–NHC
length is 2.004 (3) Å, being typical for this kind of compounds. As a
consequence of the trans influence of the NHC ligand, the average
distance of the carbons trans to the carbene carbon (2.19 (4) Å) is
slightly longer than the distance of the carbons trans to the chlorine
ligand (2.11 (4) Å). The crystal packing of (4a) showed two close
contact interactions between pairs of molecules (Fig. 2), in which the
nitro moieties are involved. The C(14)⋯O(2′) distance is 3.33 Å, while
the C(7)⋯O(4′) bond distance is slightly longer, 3.41 Å.
2.1. Synthesis and characterization of NHC-Ir(I) complexes
The preparation of the 5,6-dinitrobenzimidazolium salts was per-
formed in three steps from benzimidazole (Scheme 1). First, the nitra-
tion of benzimidazole was carried out using a mixture of H2SO4/HNO3.
The reaction was heated at 130 °C for 12 h. After purification 5,6-di-
nitro-1H-benzo[d]imidazole was isolated in a 52% yield. Afterwards,
5,6-dinitro-1H-benzo[d]imidazole was reacted with either benzyl bro-
mide to afford (2a) (87%) or 2,3,4,5,6-pentafluorobenzyl bromide to
afford (2b) (86%). In our study we chose two different N-substituent
fragments, a benzyl and the fluorinated analog a pentafluorinated
benzyl. The presence of fluorine atoms may increase the lipophilicity of
the corresponding complex, and thus enhance its cytotoxic activity.
Finally, compound (2a) or (2b) were reacted with trimethyloxonium
tetrafluoroborate in 1,2-dichloroethane (Scheme 1). The 5,6-benzimi-
dazolium salts (3a) and (3b) were obtained in 81% and 87% yield,
respectively (Scheme 1).
All compounds were characterized by NMR spectroscopy, mass
spectrometry and elemental analysis. The NMR spectra of the azolium
salts (3a) and (3b) reveals a loss of symmetry. The most characteristic
signal in the 1H NMR spectra corresponds to the NCHN fragment at
10.06 and 10.14 ppm, respectively. In the 13C{1H} spectrum, the signals
of the procarbenic carbon can be found at 150.2 ppm for (3a) and
150.4 ppm for (3b). The mass spectra of the azolium salts provide
further information about their structures. The molecular ion
[M−BF4]+ of (3a) and (3b) was observed at 313.10 and 403.22 m/z,
respectively. All these data are in agreement with the proposed mole-
cular structures.
2.2. Cytotoxic evaluation of Ir(I)–NHC complexes
Preliminary cytotoxic evaluation of all compounds was performed
in five human cancer cell-lines; glioblastoma (U-251), prostatic
The preparation of the Ir(I) complexes (4a) and (4b) was performed
2

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
15 (11.2%), while for PC-3, MCF-7 and SKLU-1 were very good,
reaching a percentage of inhibition up to 100%. Fortunately, the per-
centage of inhibition growth for the healthy cell line COS-7 was low
(7.9%). In contrast, the fluorinated complex (4b) was very active in all
the evaluated cell lines, including COS-7.
Since compound (4a) exhibited low inhibition in healthy cells, we
decided to determine its IC50 in two different human tumor cell lines.
Thus, compound (4a) exhibited good cytotoxicity against PC-3
Table 1
Crystal data and structure refinement for (4a).
Empirical formula
C23H24ClIrN4O4
Formula weight
Temperature
Wavelength
Crystal system
Space group
648.11
298(2) K
0.71073 Å
Monoclinic
P21/c
Unit cell dimensions
a = 13.3172(5) Å α = 90°
b = 14.3965(5) Å β = 96.7621(11)°
c = 12.0061(4) Å γ = 90°
2285.81(14) Å3
(10.6
micromolar scale. These values are similar to those found for cisplatin,
8.4
0.9 μM) and SKLU-1 (10.4
1.5 μM), affording values in the
0.8 μM for PC-3 and 4.3
0.5 μM for SKLU-1, however with
Volume
Z
Density (calculated)
Absorption coefficient
F(0 0 0)
Crystal size
Theta range for data collection
Index ranges
lower toxicity against healthy cells.
4
1.883 Mg/m3
5.996 mm−1
2.3. Antioxidant properties
1264
0.290 × 0.087 × 0.065 mm3
2.218–25.392°
The production of reactive Oxygen Species (ROS) is an important
carcinogenic process through the promotion of cell proliferation-acti-
vating, growth-related signaling pathways [50]. Thus, given the re-
levance of this process and the fact that it may be possible that the
complexes may engage in relevant redox processes we evaluated the
antioxidant properties of both complexes (4a) and (4b), using the
thiobarbituric acid reactive substances (TBARS) technique, which in-
volves the production of ROS with FeSO4 in the presence of lipids ob-
tained from rat brain [51]. Finding that both species, do not produce
ROS but possess antioxidant properties (Table 3).
−16 ≤ h ≤ 12, −17 ≤ k ≤ 17,
−14 ≤ l ≤ 14
17,262
4201 [R(int) = 0.0227]
99.9%
Semi-empirical from equivalents
0.7452 and 0.5065
Full-matrix least-squares on F2
4201/0/299
Reflections collected
Independent reflections
Completeness to theta = 25.242°
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
1.123
Final R indices [I > 2sigma(I)]
R indices (all data)
Largest diff. peak and hole
R1 = 0.0233, wR2 = 0.0449
R1 = 0.0280, wR2 = 0.0471
0.671 and − 0.447 e.Å−3
For completeness the IC50 values for both complexes were also de-
termined and compared with those of butylated hydroxytoluene (BHT)
and α-tocopherol (Fig. 3) [52] two reference compounds with anti-
oxidant properties.
adenocarcinoma (PC-3), colorectal adenocarcinoma (HCT-15), mam-
mary adenocarcinoma (MCF-7) and lung adenocarcinoma (SKLU-1), as
well as healthy cells of African green monkey kidney (COS-7) for
comparative purpose. Tests were carried out using a single-dose of a
25 μM solution of the corresponding 5,6-dinitro compound derivative in
DMSO. Table 2 shows the results of the evaluation. The azolium salts
precursor (2a) was poorly active against all the cell lines, while com-
pound (2b) inhibited the growth of PC-3 (30.5%) and SKLU-1 (52.7%)
(Entry 2, Table 2). The only structural difference between (2a) and (2b)
being the presence of the fluorinated aromatic ring in (2b). Thus, it is
clear that the fluorine atoms play an important role in the behavior of
this compound.
Regarding the azolium salts (3a) and (3b), their cytotoxic activity
varies from 85.3% to 100%, being both more active than cisplatin
(Entry 7, Table 2). However, both compounds (3a, 86.3%) and (3b,
98.3%) were very active also for COS-7. Interestingly, the coordination
of Ir(I) decreased the inhibition of the growth of some tumor cell lines.
Compound (4a) exhibited low inhibition for U-251 (25.3%) and HCT-
3. Conclusions
In summary, we have synthesized and characterized a novel series
of Ir(I)–NHC complexes derived from 5,6-dinitrobenzimidazole. The
molecular structure of (4a) was unambiguously determined by single
crystal X-ray diffraction analysis. The structure shows the NHC co-
ordinated to Ir(I). The nitro groups favored H-bonds between pairs of
molecules. On the other hand, the in vitro anticancer activity experi-
ments showed that the presence of the fluorine atoms in the N-sub-
stituent increases the inhibition of the growth of human tumor cell
lines, however, in some cases decreases the selectivity, inhibiting the
growth of healthy cells (COS-7). The coordination of the NHC to IrCl
(COD) favors the selectivity towards cancer cell lines compared with
the NHC ligand precursor. Complex (4a) inhibited the growth of PC-3,
MCF-7 and SKLU-7 in 99.0%, 100% and 68.7%, respectively, and was
inactive for healthy cell (COS-7) representing the best case in this work.
Furthermore, the IC50 values of (4a) for PC-3 and SKLU-7 were in the
Fig. 2. a) Molecular structure of (4a). b)
Close contact interactions between pairs of
molecules. The ellipsoids are represented at
50% probability. Selected Bond Lengths
(Å): Ir(1)-C(2) 2.004(3); Ir(1)-C(18)
2.107(3), Ir(1)-C(19) 2.111(4), Ir(1)-C(22)
2.184(4), Ir(1)-C(23) 2.204(4), Ir(1)-Cl(1)
2.3641(9), O(1)-N(2) 1.210(4), N(1)-C(2)
1.362(4), N(1)-C(11) 1.468(4), N(3)-C(2)
1.357(4), N(3)-C(10) 1.455(5), N(4)-O(4)
1.202(5), N(4)-O(3) 1.204(5), N(4)-C(6)
1.473(5), O(2)-N(2) 1.213(5), N(2)-C(5)
1.469(5). Selected Bond Angles (o): C(2)-Ir
(1)-C(18)
89.12(14),
C(2)-Ir(1)-C(19)
92.44(15), C(18)-Ir(1)-C(19) 39.35(15),
C(2)-Ir(1)-C(22) 160.61(14), C(18)-Ir(1)-C(22) 96.70(15), C(19)-Ir(1)-C(22) 80.75(15), C(2)-Ir(1)-C(23) 162.20(14), C(18)-Ir(1)-C(23) 80.89(15), C(19)-Ir(1)-C(23)
89.13(15), C(22)-Ir(1)-C(23) 36.79(13), C(2)-Ir(1)-Cl(1) 90.98(10), C(18)-Ir(1)-Cl(1) 158.61(12), C(19)-Ir(1)-Cl(1) 161.85(12), C(22)-Ir(1)-Cl(1) 90.22(10), C(23)-Ir
(1)-Cl(1) 93.03(11), O(4)-N(4)-O(3) 124.8(4), N(3)-C(2)-N(1) 105.7(3), N(3)-C(2)-Ir(1) 126.7(3), N(1)-C(2)-Ir(1) 127.5(3), O(1)-N(2)-O(2)124.3(4), C(19)-C(18)-Ir
(1) 70.5(2), C(23)-C(22)-Ir(1) 72.4(2).
3

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
Table 2
Inhibition growth (%) of tumor cell lines at 25 μM by 5,6-dinitro compounds derivatives.
Entry
U-251
PC-3
HCT-15
MCF-7
6.4
SKLU-1
0.2
COS-7
2.1
1
6.8
10.5
–
2
–
30.5
6.6
–
52.7
1.1
3
4
100
100
85.3
100
100
100
100
100
86.3
98.3
100
92.7
5
6
25.3
75.0
99.0
100
11.2
100
100.0
68.7
99.0
7.9
100
93.2
7
Cisplatin
48.4
45.9
36.7
20.3
77.5
42.8
micromolar scale. Finally, the production of Reactive Oxygen Species
(ROS) by complexes (4a) and (4b )was tested showing that both species
do not produce ROS but instead have antioxidant properties even better
than α-tocopherol, being (4a) the best in these assays. Consequently,
this compound represents a good candidate to perform further studies,
some of which are currently under developing in our laboratory.
N = 16.26%, C = 41.81%, H = 4.71% and S = 18.62%). MS-
Electrospray determinations were recorded on a Bruker Daltonics-
Esquire 3000 plus Electrospray Mass Spectrometer. Melting points were
carried out on Mel-Temp® Digital Melting Point Apparatus using open
capillary tubes with a resolution of
1 °C.
4.1. Synthesis of 5,6-dinitro-1H-benzo[d]imidazole (1)
4. Experimental
A solution of benzimidazole (12 g, 101.6 mmol) in H2SO4 (40 mL)
was cooled at 0 °C in an ice bath. Then, fuming HNO3 (80 mL) was
added dropwise to the solution. The reaction was kept at 0 °C for 20 min
more. After this time, it was slowly heated until a temperature of 130 °C
was reached and was kept at this temperature for 12 h. After this time,
the resulting reaction mixture was cooled at 0 °C and 200 g of ice were
added. This solution was neutralized with NH4OH, and the yellow solid
obtained, filtered. This solid was purified by column chromatography
using ethyl acetate as eluent, a pale-yellow band was separated. The
resulting organic solution was concentrated until the precipitation of
the desired product, which was then filtered. Yield: (52%). The
All chemical compounds were commercially obtained from Aldrich
Chemical Co. and used as received without further purification. The 1
H
and 13C{1H} NMR spectra were recorded on a Bruker Ascend 500
spectrometer. Chemical shifts are reported in ppm down field of TMS
using the residual signals in the solvent as internal standard. Elemental
analyses were performed on a Perkin Elmer 240. CHNS analyses were
performed in Thermo Scientific Flash 2000 elemental analyzed, using a
Mettler Toledo XP6 Automated-S Microbalance and sulfanilamide as
standard (Thermo Scientific BN 217826, attained values N = 16.40%,
C = 41.91%, H = 4.65% and S = 18.63%; certified values
4

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
Table 3
Determination of Lipid peroxidation inhibition (rat brain).
Sample
Code
Concentration (μM)
TBARS (nmol/mg prot.)
Inhibition (%)
IC50 (μM)
4b (n = 3)
MMD5
Basal
Control
3
3.5
4
4.5
5
5.5
6
0.273
8.305
6.751
5.328
2.171
0.852
0.665
0.430
0.402
0.005
0.055
0.099
0.592**
1.062**
0.251**
0.078**
0.028**
0.025**
18.7
35.77
73.7
89.71
91.98
94.81
95.16
1.14
7.39
13.01
3.1
0.99
0.37
0.31
3.74
0.12
4a (n = 3)
MMD6
Basal
Control
3
3.5
4
4.5
5
5.5
6
0.273
8.305
6.848
6.966
5.812
5.511
3.289
2.512
1.094
0.005
0.055
0.068
0.233
0.238*
0.330*
1.034**
1.082**
0.435**
17.54
16.09
29.98
33.59
60.24
69.58
86.76
0.35
3.08
3.31
4.31
12.63
13.2
5.35
4.99
0.19
BHT (n = 3)
Basal
Control
0.56
0.75
1
1.33
1.78
2.37
0.268
7.384
6.098
5.559
4.457
3.228
1.315
0.487
0.053
0.630
0.353
0.294*
0.283**
0.572**
0.489**
0.075**
16.64
23.92
37.14
53.59
81.59
93.16
2.86
2.69*
7.44**
8.93**
6.89**
1.16**
1.22
0.44
α-Tocopherol (n = 4)
Basal
Control
0.32
1
3.16
10
0.200
6.589
6.048
5.211
3.676
2.725
1.849
1.408
0.011
0.213
0.242
0.332*
0.569**
0.335**
0.319**
0.364**
8.26
1.31
6.78
2.16
21.13
44.84
59.00
72.30
79.09
2.56*
6.74**
3.71**
3.87**
4.79**
31.62
100
Homogenized in: PBS; Vehicle: DMSO; Peroxidation: induce with FeSO4 10 μM, 1 h incubation; EDTA: 2 μM. The values are the average of three or four independent
experiments standard error of the mean (x¯ ES). Data were analyzed by one-way ANOVA followed by Dunnett’s test for comparison against control. Values of
p ≤ 0.05 (*) and p ≤ 0.01 (**) were considered statistically significant. The inhibitory concentration 50 (IC50), was estimated by means of a linear regression.
spectroscopic data were similar to those reported in the literature [53].
temperature and filtered through celite®. All the volatiles were removed
under high vacuum and the crude product was washed several times
with diethyl ether.
4.2. General procedure for the synthesis of 1-substituted-5,6-dinitro-1H-
benzo[d]imidazole
4.3. 1-benzyl-5,6-dinitro-1H-benzo[d]imidazole (2a)
To
a solution of 5,6-dinitro-1H-benzo[d]imidazole (1 eq.) and
K2CO3 (1.5 eq.) in acetonitrile (100 mL) was added the corresponding
benzyl bromide (1.2 eq.). The reaction was stirred and heated at 80 °C
for 48 h. After this time, the resulting solution was cooled to room
For the synthesis of (2a), benzyl bromide (1.3 mL, 10.6 mmol), 5,6-
dinitro-1H-benzo[d]imidazole (2.0 g, 9.6 mmol) and K2CO3 (1.9 g,
14.4 mmol) were used. Yield: 2.5 g (87%). 1H NMR (500 MHz, CDCl3) δ
Fig. 3. IC50 values for complexes (4a) and (4b).
5

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
8.35 (s, 1H, CHBIm), 8.28 (s, 1H, CHBIm), 7.86 (s, 1H, CHBIm), 7.43–7.40
(m, 3H, CHAr), 7.22–7.20 (m, 2H, CHAr), 5.46 (s, 2H, eCH2e); 13C{1H}
NMR (125 MHz, CDCl3) δ 149.4 (NCHN), 145.1 (CBIm), 139.8 (CBIm),
139.7 (CBIm), 134.8 (CBIm), 133.3 (CAr), 129.8 (CHAr), 129.6 (CHAr),
127.4 (CHAr), 118.3 (CHBIm), 108.4 (CHBIm), 50.2 (eCH2e). MS (EI+):
m/z 298 [M]+. Elem. Anal. Calcd. for C14H10N4O4: C, 56.38; H, 3.38; N,
18.78. Found: C, 56.10; H, 3.35; N, 18.70. Melting Point: 173–174 °C.
purified by column chromatography. Elution with 1,2-dichloroethane
affords a yellow band, which contains the desired product.
4.9. Complex (4a)
For the synthesis of (4a), Ag2O (29 mg, 0.13 mmol), (3a) (100 mg,
0.25 mmol) and [IrCl(COD)]2 (49 mg, 0.13 mmol) were used. Yield:
32.4 mg (20%). 1H NMR (500 MHz, CDCl3) δ 7.84 (s, 1H, CHBIm), 7.43
2
4.4. 5,6-dinitro-1-((perfluorophenyl)methyl)-1H-benzo[d]imidazole (2b)
(s, 1H, CHBIm), 7.40–7.37 (m, 5H, CHAr), 6.36 (d, JH-H = 15.6 Hz, 1H,
eCH2e), 5.91 (d, 2JH-H = 15.6 Hz, 1H, eCH2e), 4.96 (br s, 2H, CHCOD),
4.35 (s, 3H, eCH3), 3.08–3.00 (m, 1H, CHCOD), 2.91–2–86 (m, 1H,
CHCOD), 2.41–2.23 (m, 3H, CH2 COD), 2.19–2.09 (m, 1H, CH2 COD),
1.99–1.87 (m, 2H, CH2 COD), 1.87–1.78 (m, 1H, CH2 COD), 1.73–1.65
(m, 1H, CH2 COD). 13C{1H} NMR (125 MHz, CDCl3) δ 202.9 (Ir-Ccarbene),
139.1 (CBIm), 138.9 (CBIm), 136.8 (CBIm), 135.5 (CBIm), 133.7 (CAr),
129.5 (CHBIm), 129.0 (CHBIm), 127.4 (CHBIm), 107.7 (CHBIm), 106.4
(CHBIm), 91.4 (CHCOD), 91.2 (CHCOD), 53.8 (CHCOD), 53.6 (CHCOD), 53.5
(eCH2e), 35.4 (eCH3), 33.7 (CH2 COD), 33.1 (CH2 COD), 29.4 (CH2 COD),
28.9 (CH2 COD). MS (FAB+): m/z 648.0 [M]+. Elem. Anal. Calcd. for
C23H24ClIrN4O4: C, 42.62; H, 3.73; N, 8.64. Found: C, 42.80; H, 3.68; N,
8.63. Melting Point: 201–206 °C. SiO2 TLC Rf (1,2-dicloroethane): 0.15
For the synthesis of (2b), 2,3,4,5,6-pentafluorobenzyl bromide
(1.3 mL,
8.4 mmol),
5,6-dinitro-1H-benzo[d]imidazole
(1.5 g,
7.2 mmol) and K2CO3 (1.49 g, 10.8 mmol) were used. Yield: 2.4 g
(86%). 1H NMR (500 MHz, DMSO‑d6) δ 8.83 (s, 1H, CHBIm), 8.60 (s, 1H,
CHBIm), 8.59 (s, 1H, CHBIm), 5.89 (s, 2H, eCH2e). 13C{1H} NMR
(126 MHz, DMSO-d6)
δ 151.2 (NCHN), 146.3–145.8 (m, CFAr),
144.2–144.1 (m, CFAr), 144.0 (CBIm), 142.5–141.5 (m, CFAr),
140.3–139.6 (m, CFAr), 138.7 (CBIm), 138.3 (CBIm), 138.2–138.0 (m,
CFAr), 136.7–135.7 (m, CFAr), 134.5 (CBIm), 117.5 (CHBIm), 109.7
3
(CHBIm), 109.1 (t, JC-F = 17.4 Hz, CAr), 36.5 (eCH2e). MS (EI+): m/z
388 [M]+. Elem. Anal. Calcd. for C14H5F5N4O4: C, 43.31; H, 1.30; N,
14.43. Found: C, 43.69; H, 1.21; N, 14.44. Melting Point: 212–213 °C.
4.10. Complex (4b)
4.5. General procedure for the synthesis of azolium salts.
For the synthesis of (4b), Ag2O (24 mg, 0.10 mmol), (3b) (100 mg,
0.20 mmol) and [IrCl(COD)]2 (40 mg, 0.10 mmol) were used. Yield:
36.8 (24%). 1H NMR (500 MHz, Acetone-d6) δ 8.68 (s, 1H, CHBIm), 8.46
To a solution of (2a) or (2b) (1 eq.) in 1,2-dichloromethane (50 mL),
trimethyloxonium tetrafluoroborate (1.6 eq) was added. The resulting
solution was stirred at room temperature for 48 h and then cooled at
0 °C, to produce a solid that was filtered.
2
2
(s, 1H, CHBIm), 6.17 (d, JH-H = 16.0 Hz, 1H, eCH2e), 5.99 (d, JH-
H = 16.0 Hz, 1H, eCH2e), 4.60–4.42 (m, 2H, CHCOD), 4.41 (s, 3H,
eCH3), 3.26–3.19 (m, 1H, CHCOD), 3.05–3.01 (m, 1H, CHCOD),
2.45–2.31 (m, 2H, CH2 COD), 2.30–2.21 (m, 1H, CH2 COD), 2.19–2.08
(m, 1H, CH2 COD), 2.01–1.92 (m, 1H, CH2 COD), 1.88–1.82 (m, 1H, CH2
COD), 1.73–1.65 (m, 1H, CH2 COD), 1.65–1.55 (m, 1H, CH2 COD). 13C{1H}
NMR (125 MHz, Acetone-d6) δ 202.9 (Ir-Ccarbene), 147.0–146.8 (m,
CFAr), 145.1–149.8 (m, CFAr), 142.5–142.1 (m, CFAr), 140.5–139.9 (m,
CFAr), 139.1 (CBIm), 138.9 (CBIm), 138.9–138.5 (m, CFAr), 136.9 (CBIm),
136.9–136.6 (m, CFAr), 136.1 (CBIm), 111.–110.6 (m, CAr), 108.1
(CHBIm), 107.9 (CHBIm), 89.6 (CHCOD), 89.3 (CHCOD), 54.6 (CHCOD),
51.2 (CHCOD), 40.0 (eCH2e), 35.6 (eCH3), 34.3 (CH2 COD), 32.0 (CH2
4.6. Azolium salt (3a)
For the synthesis of (3a), (2a) (1.2 g, 4.0 mmol) and trimethylox-
onium tetrafluoroborate (1.0 g, 6.4 mmol) were used. Yield: 1.3 g
(81%). 1H NMR (500 MHz, Acetone-d6) δ 10.06 (s, 1H, NCHN), 9.08 (s,
1H, CHBIm), 8.98 (s, 1H, CHBIm), 7.65 (m, 2H, CHAr), 7.48–7.46 (m, 3H,
CHAr), 6.08 (s, 2H, eCH2e), 4.46 (s, 3H, eCH3). 13C{1H} NMR
(125 MHz, Acetone-d6) δ 150.2 (NCHN), 142.5 (CBIm), 142.4 (CBIm),
135.1 (CBIm), 133.7 (CBIm), 133.5 (CAr), 130.4 (CHAr), 130.3 (CHAr),
129.9 (CHAr), 114.1 (CHBIm), 113.9 (CHBIm), 52.7 (eCH2e), 35.5
(eCH3). MS (ESI+): m/z 313.1 [M−BF4]+. Elem. Anal. Calcd. for
COD), 29.3 (CH2 COD), 27.6 (CH2 COD). MS (FAB+): m/z 738.0 [M]+
.
Elem. Anal. Calcd. for C23H19ClF5IrN4O4: C, 37.43; H, 2.59; N, 7.59.
Found: C, 37.55; H, 2.57; N, 7.60. Melting Point: 223–227 °C. SiO2 TLC
Rf (1,2-dicloroethane): 0.15.
C
15H13BF4N4O4: C, 45.03; H, 3.28; N, 14.00. Found: C, 44.94; H, 3.25;
N, 13.94. Melting Point: 200–201 °C.
4.7. Azolium salt (3b)
4.11. Cytotoxic evaluation
For the synthesis of (3b), (2b) (1.0 g, 2.6 mmol) and trimethylox-
onium tetrafluoroborate (0.6 g, 4.1 mmol) were used. Yield: 1.1 (87%).
1H NMR (500 MHz, DMSO‑d6) δ 10.14 (s, 1H, NCHN), 9.20–9.18 (m,
2H, CHBIm), 6.08 (s, 2H, eCH2e), 4.15 (s, 3H, eCH3). 13C{1H} NMR
4.11.1. Cell lines culture and culture medium
The compounds were screened in vitro against human cancer cell
lines: HCT-15 (human colorectal adenocarcinoma), MCF-7 (human
mammary adenocarcinoma), U-251 (human glioblastoma), PC-3
(human prostatic adenocarcinoma), SKLU-1 (human lung adenocarci-
noma), as well as COS-7 (healthy cells of African green monkey
kidney). Cell lines were supplied by National Cancer Institute (USA).
The human tumor cytotoxicity was determined using the protein-
binding dye sulforhodamine B (SRB) in microculture assay to measure
cell growth, as described in the protocols established by the NCI1.54 The
cell lines were cultured in RPMI-1640 medium supplemented with 10%
fetal bovine serum, 2 mM L-glutamine, 10,000 units/mL penicillin G
sodium, 10,000 l g/ml streptomycin sulfate and 25 μg/mL amphotericin
B (Gibco) and 1% non-essential amino acids (Gibco). They were
maintained at 37 °C in humidified atmosphere with 5% CO2. The via-
bility of the cells used in the experiments exceeds 95% as determined
with trypan blue.
(125 MHz, DMSO-d6)
δ 150.5 (NCHN), 146.8–147.1 (m, CFAr),
144.9–145.1 (m, CFAr), 142.8–143.1 (m, CFAr), 141.1 (CBIm), 140.9
(CBIm), 138.3–138.7 (m, CFAr), 136.4–136.7 (m, CFAr), 133.7 (CBIm),
132.5 (CBIm), 113.9 (CHBIm), 113.5 (CHBIm), 107.7–107.3 (m, CAr), 39.0
(eCH2e), 35.1 (eCH3). MS (MALDI-TOF): m/z 403.22 [M−BF4]+
.
Elem. Anal. Calcd. for C15H8BF9N4O4: C, 36.76; H, 1.65; N, 11.43. C,
36.86; H, 1.55; N, 11.41. Melting Point: 248–250 °C.
4.8. General procedure for the synthesis of the Ir(I)–NHC complexes
A solution of Ag2O (0.5 eq) and the corresponding azolium salt
(1 eq.) in acetonitrile (50 mL) was stirred under the exclusion of light at
room temperature for 12 h. After this time [IrCl(COD)]2 (0.5 eq) was
added in one portion to the solution. The reaction was further stirred
for 2 h. After this time, the solution was filtered through celite®. All the
volatiles were removed under high vacuum and the crude product was
4.11.2. Cytotoxic assay
Cytotoxicity after treatment of the tumors cells and normal cell with
6

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
the test compounds was determined using the protein-binding dye
sulforhodamine B (SRB) in microculture assay to measure cell growth
[54]. The cells were removed from the tissue culture flasks by treatment
with trypsin, and diluted with fresh media. Of this cell suspension,
100 μL containing 5000–10,000 cell per well, were pipetted into 96
well microtiter plates (Costar) and the material was incubated at 37 °C
for 24 h in a 5% CO2 atmosphere. Subsequently, 100 μL of a solution of
the compound obtained by diluting the stocks were added to each well.
The cultures were exposed for 48 h to the compound at concentrations
25 μM. After the incubation period, cells were fixed to the plastic sub-
stratum by addition of 50 μL of cold 50% aqueous trichloroacetic acid.
The plates were incubated at 4 °C for 1 h, washed with tap H2O, and air-
dried. The trichloroacetic-acid-fixed cells were stained by the addition
of 0.4% SRB. Free SRB solution was the removed by washing with 1%
aqueous acetic acid. The plates were then air-dried, and the bound dye
was solubilized by the addition of 10 mM unbuffered tris base (100 μL).
The plates were placed on and shaken for 10 min, and the absorption
was determined at 515 nm using a ELISA plates reader (Bio-Tex In-
struments).
All data were represented as mean
were analyzed by one-way ANOVA followed by Dunnetts test for
comparison against control. Values of p ≤ 0.05 (*) and p ≤ 0.01 (**)
were considered statistically significant. The inhibitory concentration
50 (IC50), was estimated by means of a linear regression.
standard error (SEM). Data
́
4.13. Data collection and refinement for compounds 4a
A yellow and prism crystal of (4a), was grown from CH2Cl2/diethyl
ether and mounted on glass fibers, then placed on a Bruker Smart Apex
II diffractometer with a Mo-target X-ray source (λ = 0.71073 Å). The
detector was placed at a distance of 5.0 cm from the crystal frames were
collected with a scan width of 0.5 in ω and exposure time of 5 s/frame.
17,262 reflections were collected and integrated with the Bruker SAINT
software package using
a narrow-frame integration algorithm.
Systematic absences and intensity statistics were used in monoclinic
system and P21/c space group. The structure was solved using Patterson
methods using SHELXS-2014/7 program [60]. The remaining atoms
were located via a few cycles of least squares refinements and difference
Fourier maps. Hydrogen atoms were input at calculated positions and
allowed to ride on the atoms to which they are attached. Thermal
parameters were refined for all hydrogen atoms using a Ueq = 1.2 Å.
The final cycle of refinement was carried out on all non-zero data using
SHELXL-2014/7 [60]. Absorption correction was applied using SADABS
program.
The crystallographic analysis was performed using PLATON [61]
program, the figures of the molecular structures are represented using
ellipsoids model while the supramolecular arrays were done using the
ball and stick model, both representations were elaborate using the
DIAMOND [62] program.
4.12. Lipid peroxidation inhibition
4.12.1. Animals
Adult male Wistar rat (200–250 g) was provided by the Instituto de
Fisiología Celular, Universidad Nacional Autónoma de México (UNAM).
Procedures and care of animals were conducted in conformity with
Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-
1999). They were maintained at 23
2 °C on a 12/12 h light–dark
cycle with free access to food and water.
4.12.2. Rat brain homogenate preparation
Acknowledgments
Animal sacrifice was carried out avoiding unnecessary pain. Ten
rats were sacrificed with CO2 to carry out all experiments. The cerebral
tissue (whole brain), was rapidly dissected and homogenized in phos-
phate buffered saline (PBS) solution (0.2 g of KCl, 0.2 g of KH2PO4, 8 g
of NaCl, and 2.16 g of NaHPO4·7 H2O/l, pH adjusted to 7.4) as reported
elsewhere [55,56] to produce a 1/10 (w/v) homogenate. Then, the
homogenate was centrifuged for 10 min at 800 rcf (relative centrifugal
field) to yield a pellet that was discarded. The supernatant protein
content was measured using the Folin and Ciocalteu’s phenol reagent
[57] and adjusted with PBS at 2.666 mg of protein/ml.
We would like to thank Chem. Eng. Luis Velasco Ibarra, Dr.
Francisco Javier Pérez Flores, Q. Eréndira García Ríos, M.Sc. Lucia del
Carmen Márquez Alonso, M.Sc. Lucero Ríos Ruiz, M.Sc. Alejandra
Núñez Pineda (CCIQS), Q. María de la Paz Orta Pérez, Q. Roció Patiño-
Maya and Ph.D. Nuria Esturau Escofet for technical assistance. H. V.
would like to thank Programa de Becas Posdoctorales-DGAPA-UNAM
for postdoctoral scholarships (Oficio: CJIC/CTIC/4751/2018). A. S.-M.
would like to thank CONACYT (No. de becario: 629466) for M.Sc.
scholarship. J.D.C-G would like to thank Programa de Cátedras
CONACYT-PRY for generous support. The financial support of this re-
search by PAPIIT-DGAPA-UNAM (PAPIIT IN207317) and CONACYT
A1-S-33933 is gratefully acknowledged.
4.12.3. Induction of lipid peroxidation and thiobarbituric acid reactive
substances (TBARS) quantification
As an index of lipid peroxidation, TBARS levels were measured
using rat brain homogenates according to the method described by Ng
et al. [58] with some modifications. Supernatant (375 µL) was added
with 50 µL of 20 µM EDTA and 50 µL of each sample concentration
solved in DMSO (50 µL of DMSO for control group) and incubated at
37 °C for 30 min. Lipid peroxidation was started adding 50 µL of freshly
prepared 100 µM FeSO4 solution (final concentration 10 µM) and in-
cubated at 37 °C for 1 h. The TBARS content was determined as de-
scribed by Ohkawa et al. [59] with some modifications. 500 µL of TBA
reagent (1% 2-thiobarbituric acid in 0.05 N NaOH and 30% tri-
chloroacetic acid, in 1:1 proportion) was added at each tube and the
final suspension was cooled on ice for 10 min, centrifugated at 13,400
rcf for 5 min and heated at 80 °C in a water bath for 30 min. After
cooling at room temperature, the absorbance of 200 µL of supernatant
was measured at λ = 540 nm in a Bio-Tek Microplate Reader Synergy
HT. Concentration of TBARS was calculated by interpolation in a
standard curve of tetra-methoxypropane (TMP) as a precursor of MDA.
Results were expressed as nmoles of TBARS per mg of protein. The
inhibition ratio (IR[%]) was calculated using following formula IR = (C-
E)*100/C, where C is the absorbance of control and E is the absorbance
of the test sample. Butylated hydroxytoluene (BHT) and αtocopherol
were used as positive standards.
Appendix A. Supplementary data
Supplementary data for compound 4a were deposited at the
Cambridge Crystallographic Data Centre. Copies of this information are
available free of charge on request from The Director, CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (Fax: +44-1223-336033; e-mail de-
posit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk) quoting
the deposition numbers CCDC 1913480. Supplementary data to this
article can be found online at https://doi.org/10.1016/j.ica.2019.
119061.
References
[1] a) B. W. Stewart and C. P. Wild, Eds., World Cancer Report 2014, International
Agency for Research on Cancer, Lyon, 2014.
[2] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, CA Cancer J.
Clin. 68 (2018) 394–424.
[3] R.G. Kenny, C.J. Marmion, Chem. Rev. 119 (2018) 1058–1137.
[4] J.J. Wilson, S.J. Lippard, Chem. Rev. 114 (2014) 4470–4495.
[5] a) T.C. Johnstone, G.Y. Park, S.J. Lippard, Anticancer Res. 34 (2014) 471–476;
b) A. Ramos-Espinosa, H. Valdés, M.T. Ramírez-Apan, S. Hernández-Ortega,
B.A. Aguilar-Castillo, R. Reyes-Martínez, J.M. Germán-Acacio, D. Morales-Morales,
7

A. Sánchez-Mora, et al.
InorganicaChimicaActa496(2019)119061
Inorg. Chim. Acta. 466 (2017) 584–590;
Inorg. Chem. 56 (2017) 12232–12247.
c) S. Ramírez-Rave, M.T. Ramírez-Apan, H. Tlahuext, D. Morales-Morales,
R.A. Toscano, J.-M. Grévy, J. Organomet. Chem. 814 (2016) 16–24.
[6] A.M. Breglio, A.E. Rusheen, E.D. Shide, K.A. Fernandez, K.K. Spielbauer,
K.M. McLachlin, M.D. Hall, L. Amable, L.L. Cunningham, Nat. Commun. 8 (2017)
1654.
[37] C.C. Konkankit, S.C. Marker, K.M. Knopf, J.J. Wilson, Dalton Trans. 47 (2018)
9934–9974.
[38] T. Giraldi, G. Sava, G. Mestroni, G. Zassinovich, D. Stolfa, Chem.-Biol. Interact. 22
(1978) 231–238.
[39] G. Sava, S. Zorzet, L. Paressin, Inorg. Chim. Acta 137 (1987) 69–71.
[40] E. Maftei, C.V. Maftei, P.G. Jones, M. Freytag, M.H. Franz, G. Kelter, H.-H. Fiebig,
M. Tamm, I. Neda, Helv. Chim. Acta 99 (2016) 469–481.
[41] Y. Gothe, T. Marzo, L. Messori, N. Metzler-Nolte, Chem. Commun. 51 (2015)
3151–3153.
[7] D.W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Pharmacol. Rev. 64 (2012)
706–721.
[8] S.M. Meier-Menches, C. Gerner, W. Berger, C.G. Hartinger, B.K. Keppler, Chem. Soc.
Rev. 47 (2018) 909–928.
[9] L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao, Z.-S. Chen, Chem. Soc. Rev. 46
[42] Y. Gothe, T. Marzo, L. Messori, N. Metzler-Nolte, Chem. Eur. J. 22 (2016)
(2017) 5771–5804.
12487–12494.
[10] C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391–401.
[11] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 114
(2014) 815–862.
[43] Y. Gothe, I. Romero-Canelón, T. Marzo, P.J. Sadler, L. Messori, N. Metzler-Nolte,
Eur. J. Inorg. Chem. 2018 (2018) 2461–2470.
[44] E. Peris, Chem. Rev. 118 (2018) 9988–10031.
[12] B. Englinger, C. Pirker, P. Heffeter, A. Terenzi, C.R. Kowol, B.K. Keppler, W. Berger,
[45] M. Poyatos, J. Mata, E. Peris, Chem. Rev. 109 (2009) 3677–3707.
[46] J. Mata, M. Poyatos, E. Peris, Coor. Chem. Rev. 251 (2007) 841–859.
[47] a) J.M. Alfaro, A. Prades, M. del Carmen Ramos, E. Peris, J. Ripoll-Gómez,
M. Poyatos, J.S. Burgos, Zebrafish 7 (2010) 13–21;
Chem. Rev. 119 (2019) 1519–1624.
[13] K.D. Mjos, C. Orvig, Chem. Rev. 114 (2014) 4540–4563.
[14] A. John, P. Ghosh, Dalton Trans. 39 (2010) 7183–7206.
[15] W. Liu, R. Gust, Chem. Soc. Rev. 42 (2013) 755–773.
[16] F. Hackenberg, M. Tacke, Dalton Trans. 43 (2014) 8144–8153.
[17] L. Oehninger, R. Rubbiani, I. Ott, Dalton Trans. 42 (2013) 3269–3284.
[18] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 54 (2011) 3–25.
[19] G. Gasser, N. Metzler-Nolte, Curr. Opin. Chem. Biol. 16 (2012) 84–91.
[20] T. Zou, C.T. Lum, C.-N. Lok, W.-P. To, K.-H. Low, C.M. Che, Angew. Chem. Int. Ed.
53 (2014) 5810–5814.
b) H. Valdes, D. Canseco-Gonzalez, J.M. German-Acacio, D. Morales-Morales, J.
Organomet. Chem. 867 (2018) 51–54;
c) S. Ray, R. Mohan, J.K. Singh, M.K. Samantaray, M.M. Shaikh, D. Panda,
P. Ghosh, J. Am. Chem. Soc. 129 (2007) 15042–15053;
d) L. Oehninger, R. Rubbiani, I. Ott, Dalton Trans. 42 (2013) 3269–3284;
e) J.L. Hickey, R.A. Ruhayel, P.J. Barnard, M.V. Baker, S.J. Berners-Price,
A. Filipovska, J. Am. Chem. Soc. 130 (2008) 12570–12571;
f) A. Gautier, F. Cisnetti, Metallomics 4 (2012) 23–32.
[21] J. Lemke, A. Pinto, P. Niehoff, V. Vasylyeva, N. Metzler-Nolte, Dalton Trans. 107
(2009) 7063–7070.
[48] K. Nepali, H.-Y. Lee, J.-P. Liou, J. Med. Chem. (2018), https://doi.org/10.1021/acs.
[22] A. Gutiérrez, M.C. Gimeno, I. Marzo, N. Metzler-Nolte, Eur. J. Inorg. Chem. 2014
(2014) 2512–2519.
jmedchem.8b00147.
[49] M. Silva Lopes, C. Filizzola de Andrade Sena, B. Leonardo Silva, C. Maria de Souza,
J. Pereira Ramos, G. Dantas Cassali, E. Maria de Souza-Fagundes, R. Jose Alves,
M. Cristina de Oliveira, R. Barbosa de Oliveira, Anticancer Agents Med. Chem. 15
(2015) 206–216.
[50] S.J. Dixon, B.R. Stockwell, Nat. Chem. Biol. 10 (2014) 9–17.
[51] A. Lukaszewicz-Hussain, J. Moniuszko-Jakoniuk, J. Rogalska, Pol. J. Environ. Stud.
16 (2007) 233–236.
[52] T. Ohsima, V.V. Yankah, H. Ushio, C. Kiozumi, Adv. Exp. Med. Biol. 434 (1998)
181–188.
[53] K. Kincaid, J. Beckman, A. Zivkovic, R.L. Halcomb, J.W. Engels, R.D. Kuchta,
Nucleic Acids Res. 33 (2005) 2620–2628.
[54] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose,
J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo,
M. Boyd, J. Natl. Cancer Ins. 83 (1991) 757–766.
[55] M. Dominguez, A. Nieto, J.C. Marin, A.S. Keck, E. Jeffery, C.L. Cespedes, J. Agric.
Food Chem. 53 (2005) 5889–5895.
[23] R. Rubbiani, E. Schuh, A. Meyer, J. Lemke, J. Wimberg, N. Metzler-Nolte, F. Meyer,
F. Mohr, I. Ott, Med. Chem. Commun. 4 (2013) 942–948.
[24] R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can, A. Kitanovic, L.A. Onambele,
M. Stefanopoulou, Y. Geldmacher, W.S. Sheldrick, G. Wolber, A. Prokop, S. Wölfl,
I. Ott, J. Med. Chem. 53 (2010) 8608–8618.
[25] W. Liu, R. Gust, Coor. Chem. Rev. 329 (2016) 191–213.
[26] P.O. Wagers, M.J. Panzner, M.R. Southerland, M.A. DeBord, M.C. Deblock,
C.A. Tessier, C.L. Cannon, W.J. Youngs, Biologically Active N-Heterocyclic Carbene-
Metal Complexes in From Laboratory Curiosities to Efficient Synthetic Tools, 2nd edn.,
Royal Society of Chemistry, Cambridge, 2016, pp. 567–595.
[27] K.K.-W. Lo, K.Y. Zhang, RSC Adv. 2 (2012) 12069–12083.
[28] Y. Geldmacher, M. Oleszak, W.S. Sheldrick, Inorg. Chim. Acta 393 (2012) 84–102.
[29] M. Graf, Y. Gothe, N. Metzler-Nolte, R. Czerwieniec, K. Sünkel, J. Organomet.
Chem. 765 (2014) 46–52.
[30] C.-H. Leung, H.-J. Zhong, D.S.-H. Chan, D.-L. Ma, Coor. Chem. Rev. 257 (2013)
1764–1776.
[56] J.I. Rossato, L.A. Ketzer, F.B. Centuriao, S.J. Silva, G. Lueni, A.L. Braga, M.A. Rubin,
B.T. Rocha, Neurochem. Res. 27 (2002) 297–303.
[57] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951)
[31] Z. Liu, P.J. Sadler, Acc. Chem. Res. 47 (2014) 1174–1185.
[32] Q. Du, L. Guo, M. Tian, X. Ge, Y. Yang, X. Jian, Z. Xu, Z. Tian, Z. Liu,
Organometallics 37 (2018) 2880–2889.
265–275.
[33] P. Štarha, Z. Trávníček, H. Crlíková, J. Vančo, J. Kasparkova, Z. Dvořák,
[58] T.B. Ng, F. Liu, Z.T. Wang, Antioxidative activity of natural products from plants,
Life Sci. 66 (2000) 709–723.
Organometallics 37 (2018) 2749–2759.
[34] J. Li, M. Tian, Z. Tian, S. Zhang, C. Yan, C. Shao, Z. Liu, Inorg. Chem. 57 (2018)
1705–1716.
[59] H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 (1979) 351–358.
[60] G.M. Sheldrick, Acta Cryst. C71 (2015) 3–8.
[35] A. Petrini, R. Pettinari, F. Marchetti, C. Pettinari, B. Therrien, A.N. Galindo,
R. Scopelliti, T. Riedel, P.J. Dyson, Inorg. Chem. 56 (2017) 13600–13612.
[36] R.P. Paitandi, S. Mukhopadhyay, R.S. Singh, V. Sharma, S.M. Mobin, D.S. Pandey,
[61] A.L. Spek, Acta. Crystallogr. D65 (2009) 148–155.
[62] K. Brandenburg, M. Berndt, J. Appl. Cryst. 32 (1999) 1028–1029.
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