Synthesis, characterization, antimicrobial and cytotoxic activity of novel half-sandwich Ru(II) arene complexes with benzoylthiourea derivatives

Article abstract of DOI:10.1016/j.jinorgbio.2020.111164

Three new ruthenium(II)-arene complexes, [Ru(η⁶-p-cymene)(L¹)Cl₂] (C1) where L¹ is N-((4 methoxyphenyl)carbamothioyl)benzamide; [Ru(η⁶-p-cymene)(L²)Cl₂] (C2) where L²

Full text of DOI:10.1016/j.jinorgbio.2020.111164

Journal of Inorganic Biochemistry 210 (2020) 111164
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, characterization, antimicrobial and cytotoxic activity of novel
half-sandwich Ru(II) arene complexes with benzoylthiourea derivatives
a
b
c
c
d
Dragiša Obradović , Stefan Nikolić , Ivana Milenković , Marina Milenković , Predrag Jovanović ,
d
e
f
f
Vladimir Savić , Alexander Roller , Marija Đorđić Crnogorac , Tatjana Stanojković ,
a,⁎
Sanja Grgurić-Šipka
a
b
c
d
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Serbia
Innovative Centre Faculty of Chemistry Belgrade, University of Belgrade, Studentski trg 12-16, Serbia
Department of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Serbia
Department of Organic Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Serbia
e
Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Währinger Straße 42, Austria
f
Institute of Oncology and Radiology of Serbia, Pasterova, 14, Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
6
1
] (C1) where L1 is N-((4 methoxyphenyl)
2
Keywords:
Three new ruthenium(II)-arene complexes, [Ru(η -p-cymene)(L )Cl
2
6
2
Antimicrobial activity
Benzylthiourea derivatives
Cytotoxic activity
carbamothioyl)benzamide; [Ru(η -p-cymene)(L )Cl ] (C2) where L is 4-(3-benzoylthioureido)benzoic acid and
2
6
3
3
[
Ru(η -p-cymene)(L )Cl
2
] (C3) where L is methyl 4-(3- benzoylthioureido)benzoate have been synthetized,
characterized and evaluated for their antimicrobial and anticancer activity. Characterization was performed
Ruthenium(II) complexes
1
13
using H and C NMR, IR spectroscopy, mass spectrometry, electrical conductivity measurements and X-Ray
diffraction analysis. X-Ray diffraction analysis of C1 showed typical expected “piano-stool” geometry with ru-
thenium coordinated to ligand via nitrogen and sulfur atoms of benzoylthiourea derivatives. Interesting, in
herein described complex, upon coordination the four-membered ring was formed, instead of six-membered
chelate common for this type of ligands. Cytotoxic activity was determined in human cervix adenocarcinoma
(
HeLa) cell line and IC50 values ranged from 29.68 to 52.36 μM and the complexes were more active than related
ligands (except in case of C2 where it is found that IC50 value is close to IC50 value of related ligand). Complex
6
1
[
Ru(η -p-cymene)(L )Cl ] (C1) expressed the highest cytotoxic activity with IC50 value of 29.7 μM. Complexes
2
and ligands were tested against nine Gram-positive and Gram-negative bacteria and one yeast- Candida albicans.
Clinical Candida spp. strains from microbiological laboratories were included in testing processes as well.
Minimum inhibitory concentrations values ranged from 62.5 μg/ml for complexes against Candida albicans to
over 1000 μg/ml for several bacterial species.
1
. Introduction
Cancer caused about 9 million deaths worldwide in 2018 and it is
worldwide: the platinum complex of the second generation - carboplatin
and the third generation - oxaliplatin (Fig. 1). They were approved in
1993 and 2002 respectively [2,3].
estimated that by 2030 cancer mortality rate worldwide will rise up to
However, application of these compounds is limited due to acquired
drug resistance and toxicity to non-tumorigenic-cells causing undesir-
able side effects such as allergic reactions, nephrotoxicity, neurotoxi-
city, hepatotoxicity, hemolytic anemia, hair loss, disorder of electrolyte
concentrations, nausea, decrease immunity to infections, gastro-
intestinal disorders, hemorrhage, hearing loss and other side effects [1].
The search for more efficient and less toxic anticancer drug has
included many other metal based compounds. Among them, the ru-
thenium-based anticancer agents showed the greatest potential [4].
1
2 million annually. Official World Cancer Day, the February 4th was
declared with the aim to rise up awareness of cancer and to encourage
its prevention, detection and treatment. Growing problem in this area
of medicine initiated intensive research with goal to discover novel
anticancer drugs including metal-based ones. The most important
metal-based anticancer drug is cisplatin, discovered in 1965 [1]. Since
then, enourmous efforts of chemists have been put into metal com-
plexes, but, unfortunately, only two more compounds are used in clinics
⁎
Corresponding author.
E-mail address: sanjag@chem.bg.ac.rs (S. Grgurić-Šipka).
https://doi.org/10.1016/j.jinorgbio.2020.111164
Received 13 December 2019; Received in revised form 9 June 2020; Accepted 11 June 2020
Available online 26 June 2020
0162-0134/ © 2020 Elsevier Inc. All rights reserved.

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
Fig. 1. a) Cisplatin b) Carboplatin c) Oxaliplatin.
Fig. 2. Complexes KP1019, NAMI-A, RAED-C, and RAPTA.
Two ruthenium compounds, NAMI-A (imidazolium trans-[tetrachlorido
1H-imidazole)(S-dimethyl sulfoxide)ruthenate(III)]) and KP1019 (in-
hydrophobic aromatic benzene core, which prevents the oxidation of
Ru(II) and due to its lipophilicity enhances entering to cells. RAED-C
(
6
dazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) (also
studied as its more water soluble sodium salt NKP1339) (Fig. 2), have
been evaluated in clinical trials [5,6]. The combinatory therapy using
NAMI-A and gemcitabine is in phase II of clinical trial for the treatment
of non-small cell lung carcinoma [7]. KP1019 is effective against pri-
mary cancers [8] and is another Ru-based anticancer compound entered
clinical trials and the results of the early clinical development demon-
strated that five out of six evaluated patients experienced disease sta-
bilization with no severe side effects [9]. Since the mechanism of action
of those drug candidates included the reduction of metal to Ru(II) form
([Ru(η -p-cymene)Cl(en)]; en = ethylenediamine) unlike RAPTA-C
(Fig. 2) reacts with the DNA component of chromatin in the cell, and
shows high antitumor activity against primary cancer. Although the
structural similarity with RAPTA-C is noticeable, the mechanism of
cytotoxicity is quite different [15].
Thiourea and its derivatives have various applications due to dif-
ferent atoms included in their functional group, such as antiviral [16],
herbicidal [17], anti-inflammatory [18] and fungicidal properties [19].
Bearing in mind problems with current metal drugs above described in
combination with fact that requirements for safer, new and more active
drugs are higher through years, specially taking into account the po-
tential of the combination of a Ru-arene complex with thiourea deri-
vatives as ligands, we have decided to conduct research for new metal-
based anticancer candidates. The structure and chemical properties of
[
10], the Ru(II)-arene complexes have emerged as highly promising
candidates [11–14].
6
RAPTA-C ([Ru(η -p-cymene)Cl
2
(pta)]; pta = 1, 3, 5-triaza-7-phos-
phaadamantane) is ruthenium-arene complex with
a
a
very
2

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
metal complexes with derivatives of thiourea as ligands have been
studied in recent years [20]. Derivatives of thiourea have more than
one potential atom for coordination, and this ability mark them as good
ligands in synthesis of complexes, as they can act as monodentate, bi-
dentate or polydentate ligands coordinated through oxygen, nitrogen
and sulfur atoms [21,22]. There are many papers that document great
anticancer and antimicrobial activity of this type of complexes [23,24].
In addition to antimicrobial and cytotoxic activity, these compounds
and complexes also exert various activities for instance antifungal,
antithyroid, rodenticidal, insecticidal, herbicidal and other activities
and properties [25,26].
concentration of DMSO in each well was 1%. The bacterial strains were
previously prepared by incubating for 24 h at 35 °C on Mueller-Hinton
agar and for Candida albicans on Sabouraud dextrose agar. Fresh
overnight cultures were suspended into sterile physiological saline
(0.9% w/w NaCl) and adjusted to 0.5 McFarland standard which is
8
approximately 2 × 10 CFU/ml (colony-forming unit/ml). The bac-
terial suspensions were further diluted in Mueller-Hinton broth to a
6
final suspension with 1 × 10 CFU/ml and yeast was diluted in Sa-
7
bouraud dextrose broth to a final suspension with 1 × 10 CFU/ml.
100 μl of each analyzed compound (complexes and ligands) and bac-
terial suspension were added in each well. To the bacterial suspensions
2, 3, 5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, USA) was
added as growth indicator. The final concentration of TTC after in-
oculation was 0.05%. Test compound stock solutions were prepared
several minutes before they were used for testing. Clinical Candida spp.
strains were procured from BeoLab laboratories and Clinical-Hospital
Centre Dragiša Mišović from Belgrade. Candida spp. strains identifica-
tions were managed using VITEK-2 system with YST ID cards. The MIC
results were determined by visual method and interpreted after in-
cubation of plates for 24 h at 35 °C. Each test (included clinical isolates)
was repeated three times, and all of the MIC determinations were
performed in duplicate, with two positive growth controls included
(wells containing only the microorganisms in the broth).
Synthetized Ru-arene complexes with thiourea derivatives as li-
gands are susceptible for additional exploration regarding therapeutic
activities like antithyroid, antimicrobial, anticancer, even as solution
for tuberculosis treatment, as well as sensors and other optical solutions
[
27], and it can unquestionably bring beneficial to different areas,
especially in medicine. Synthetized complexes are tested and evaluated
by their cytotoxicity against HeLa cells and antimicrobial activity
against several bacterial strains and yeast. By X-ray diffraction analysis,
a formation of four member ring between thiourea based ligand and
ruthenium metal center was revealed, as evidence that one ligand can
have two binding modes in the same complex, as already reported in
literature [22].
2
. Experimental
2.2.4. Cytotoxic activity
Cytotoxic activity of complexes and ligands were tested against
cervix adenocarcinoma cell line (HeLa) with the aim of determining in
vitro cytotoxic activity. The stock solutions of three investigated com-
plexes and three ligands were dissolved in DMSO (Sigma-Aldrich, St.
Louis, MO, USA) at the concentrations of 10 mM. Working concentra-
tions were diluted by culture nutrient medium RPMI-1640 (Sigma-Al-
drich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum,
2 mM L-glutamine, and 1% penicillin-streptomycin (Sigma). HeLa cells
2.1. Materials
All chemicals which were used for synthesis and testing anti-
microbial and cytotoxic activity were of reagent-grade quality or
higher, obtained from commercial suppliers (Sigma Aldrich) and were
used without further purification. Solvents were used as received or
2
dried over molecular sieves. RuCl
3
was purchased from I CNS.
were maintained in complete RPMI-1640 medium at 37 °C in humidi-
3
2.2. Instrumental and methods
fied atmosphere with 5% CO
2
and seeded at a density of 2 × 10 cells/
well in 96-well flat-bottomed microtitre plate. After 24 h, cells were
treated with different concentrations of investigated complexes and
related ligands. Control cells were grown into the culture medium only.
The final concentrations of treatments used in experiments were in the
range of 12.5 μM to 200 μM (12.5, 25, 50, 100 and 200 μM). As a po-
sitive control we used cisplatin with the final concentrations of 3.125,
6.25, 12.5, 25, and 50 μM. The final concentration of DMSO solvent
never exceeded 0.5%, which is a non-toxic concentration for the cells.
Culture medium without cells with different concentrations of com-
plexes and ligands was used as blank. Experiments were set up in tri-
plicate. The viability of cells was determined by microculture tetra-
zolium test (MTT) as described previously [29]. After 72 h of treatment
with complexes and ligands, 10 μL of MTT solution (3-(4,5-di-
methylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) (5 mg/ml) was
added to each well. After 4 h of incubation, the product of the con-
version of MTT dye by viable cells, formazan, was extracted by adding
100 μL of 10% SDS (100 g/L sodium dodecyl sulfate) into the wells. On
the next day, the intensity of the absorbance was measured at 570 nm
using Multiskan EX reader (Thermo Labsystems Beverly, MA, USA), and
it represented the number of viable cells in each well.
1
13
2
.2.1. H and C NMR spectroscopy
1
13
H and C NMR spectra were recorded in deuterated solvents on a
1
3
Bruker Ascend 400 (1H: 400 MHz, C: 100.6 MHz), MHz spectrometer
at room temperature internally referenced to TMS (tetramethylsilane).
The chemical shifts, δ, are reported in ppm (parts per million), and
coupling constants (J) in Hertz. The residual solvent peaks have been
used as an internal reference. The abbreviations for the peak multi-
plicities are as follows: s (singlet), d (doublet), dd (doublet of doublets),
t (triplet), q (quartet), m (multiplet), and br (broad).
2
.2.2. ESI mass and IR spectroscopy, elemental analysis
ESI (electrospray ionization) mass spectra were recorded on a LTQ
Orbitrap XL using direct injection of the complex solution in methanol.
Infrared spectra were recorded on a Nicolet 6700 FT-IR spectrometer
using the ATR (attenuated total reflection) technique. Elemental ana-
lysis was carried out with Elemental Vario EL III microanalyser.
2
.2.3. Antimicrobial activity
Antimicrobial activity was tested by broth microdilution method
according to guidelines of Clinical and Laboratory Standards Institute
(
CLSI) [28]. Minimum inhibitory concentrations (MIC) were de-
2.2.5. X-ray spectroscopy
termined as the lowest concentrations of tested chemical compounds
that inhibited visible growth of microorganisms. Antimicrobial assay
was carried out in 96-well polystyrene microtiter plates by using serial
dilutions of the tested compounds (62.5–1000 μg/ml) dissolved in
DMSO and further diluted in Mueller-Hinton broth for the bacterial
strains and in Sabouraud dextrose broth for Candida albicans. Final
The X-ray intensity data were measured on Bruker D8 Venture dif-
fractometer equipped with multilayer monochromator, Mo K/α INCO-
ATEC micro focus sealed tube and Oxford cooling system. The structure
was solved by direct methods. Non‑hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were inserted at
calculated positions and refined with riding model. The following
3

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
Table 1
software was used: Bruker SAINT software package [30] using a narrow-
frame algorithm for frame integration, SADABS [31] for absorption
correction, OLEX2 [32] for structure solution, refinement, molecular
diagrams and graphical user-interface, Shelxle [33] for refinement and
graphical user-interface SHELXS-2015 [34] for structure solution,
SHELXL-2015 [35] for refinement, Platon [36] for symmetry check.
Experimental data and CCDC-Codes Experimental data (Available on-
line: http://www.ccdc.cam.ac.uk/conts/retrieving.html) can be found
in Table S1. Crystal data, data collection parameters, and structure
refinement details are given in Tables 1 and 2. Crystal structure is vi-
sualized in Figs. 3 and 5.
Specified hydrogen bonds (with esds except fixed and riding H).
D-H
H…A
D…A
< (DHA)
0
0
.82
.82
2.10
2.63
2.662(3)
3.429(3)
125
163
N2-H2…O1
N2-H2…O1′
′
… [−x, −y+1, −z].
Table 2
Bond lengths in the four-membered ring on the Ru center.
Ru1-S1
S1-C1 C1-N1 N1-Ru1
CCDC codes
Å
Å
Å
Å
2.2.6. Electrical conductivity (EC)
Electrical conductivities of complexes were measured on Crison
MultiMeter MM41 conductometer. EC measurements were performed
using 1 mM solutions of the complexes in acetonitrile and acetone as
solvents at 25 °C.
C1
2.416(5)
2.460(3)
1.714
1.708
1.727
1.365
1.324
1.331
1.317
1.315
1.320
1.312
1.343
1.311
2.138(3) 1,959,425
Hang Zhu et al. [37]
2.209(6)
2.207(6)
2.109(4)
2.164(2)
2.165(2)
2.158(3)
2.097(8)
2.094(8)
813,250
813,250
776,948
854,737
854,738
854,739
150,372
150,372
2
.442(2)
Xi-Ying Wang et al. [38] 2.478(14) 1.729
Ai-Quan Jia et al. [39]
Ai-Quan Jia et al. [40]
Ai-Quan Jia et al. [41]
M.Aitali et al. [42]
2.495(8)
2.455(9)
2.438(9)
2.438(3)
1.714
1.722
1.720
1.730
1.724
2
.2.7. Synthesis of ligands
All three ligands N-([4-methoxyphenyl]carbamothioyl)benzamide
1
2
2
.429(3)
(
L ), 4-(3-benzoylthioureido)benzoic acid (L ) and methyl 4-(3-ben-
3
zoylthioureido)benzoate (L ) were synthesized according to a slightly
modified published procedure [43]. Potassium thiocyanate has been
Fig. 3. Two molecules of C1 associated into a centrosymmetric dimer via two bifurcated hydrogen bonds.
4

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
Scheme 1. Synthesis of aromatic derivatives of thiourea.
condensed with benzoyl chloride resulting of forming benzoyl-iso-
3. Results and discussion
3.1. Synthesis and characterization of Ru complexes
All three complexes were synthetized using a suspension of [(η -p-
thiocyanates and reacting with primary amines in the next phase
6
(
Scheme 1). The ruthenium dimer [(η -p- cymene)RuCl
2
] was prepared
2
according to a published procedure [44].
6
cymene)RuCl
2
] in absolute ethanol or toluene with dropwise addition
2
2
.2.8. General synthesis of the complexes C1–C3
of previously synthesized ligand dissolved in absolute ethanol with
constant stirring for several hours (Scheme 2). During the reaction
period, orange precipitates of complexes were formed. Precipitates
were filtered, and washed with cold ethanol and diethyl-ether and dried
in vacuo. The products were isolated in a moderate good and good
6
To a suspension of [(η -p-cymene)RuCl
2
] (0.053 mmol; 1 eq) in
2
absolute ethanol (toluene in case of C2) (8 ml) was added dropwise a
1
3
solution of ligand (L –L ; 0.105 mmol; 2 eq) in absolute ethanol (10 ml)
with constant stirring. The mixture was stirred at 60 °C for 4 h. During
this time orange precipitate of complex was formed. Precipitate was
filtered, and washed with cold ethanol (10 ml) and diethyl-ether
1
13
yields. Complexes were characterized by H and C NMR, IR, mass
spectrometry, and in the case of C1 the structure has been determined
1
13
(
10 ml) and dried in vacuo.
by X-ray analysis. In the H and C NMR spectra of the complexes, all
hydrogen and carbon atoms appear at chemical shifts expected for such
types of compounds. Collected data suggest monodentate coordination
of ligands, likely due to formation of kinetic reaction products. Similar
molecules as ligands used in this work, 1-acyl-thiourea derivatives, tend
to form intramolecular N-H⋯O=C hydrogen bond [45,46]. Formation
of intramolecular hydrogen bond results in making a six membered ring
within molecule. Distortion of molecule allows right conformation for
coordination of S and N atoms to the ruthenium center. This allows
formation of thermo-dynamic product of the complex, isolated as
monocrystal. Compex C1 showed in X-ray analysis piano-stool geo-
metry with a coordinated chloride ligand and four-membered ring
6
1
2
.2.8.1. [Ru(η -p-cymene)(L )Cl
2
] (C1). Yield: 48 mg (75%). Elemental
analysis, calculated for C25
H
28Cl
2
N
2 2
O
RuS: C 50.68; H 4.76; N 4.73; S
−
1
5
.41. Found: C 50.89; H 4.66; N 4.50; S 5.15. IR (ATR, cm ): 3133
1
(
m), 3028 (m), 2961 (m), 1656 (s), 1526 (s), 1271 (m), 1168 (m). H
NMR (400 MHz, DMSO‑d
6
) δ 12.79 (s, 1H), 11.68 (s, 1H), 8.01 (d,
J = 8.8 Hz, 4H), 7.92 (m, 2H), 7.68 (m, 1H), 7.55 (m, 2H), 5.78 (m,
4
6
1
8
H), 2.84 (dt, J = 13.8, 6.9 Hz, 1H), 2.59 (s, 3H), 2.09 (s, 3H), 1.19 (s,
13
H). C NMR (101 MHz, DMSO‑d ) δ 197.3, 179.4, 168.6, 142.6,
6
34.6, 133.7, 132.5, 129.4, 129.2, 128.9, 123.9, 106.8, 100.5, 86.8,
5.5, 30.4, 27.1, 21.9, 18.3. ESI-MS: m/z 521.08 ([M-Cl-HCL]+).
through N and S bidentate coordinaton of derivative of ben-
zoylthiourea. Complexes showed very good solubility in dimethyl
sulfoxide, good in acetonitrile and acetone, but moderate solubility in
ethanol and methanol.
6
2
2
.2.8.2. [Ru(η -p-cymene)(L )Cl
2
] (C2). Yield: 32 mg (48%). Elemental
analysis, calculated for C25
H
26Cl
2
N
2
O
3
RuS: C 49.51; H 4.32; N 4.62; S
IR spectra of ligands and corresponding complexes revealed all ex-
pected bands of interest- ν(NeH), ν(C=O), ν(CeN), ν(CeO), ν(C=S), v
(C=C)ar and v(OeH). Ligand bands of ν(NeH) can be identified in the
−
1
5
.29. Found: C 49.82; H 4.29; N 4.63; S 4.85. IR (ATR, cm ): 3054
1
(
(
(
m), 2971 (m), 1689 (s), 1537 (s), 1267 (m), 1172 (m). H NMR
−1
400 MHz, DMSO‑d
6
) δ 12.77 (s, 1H), 11.64 (s, 1H), 10.52 (s, 1H) 7.98
range of 3262–3225 cm . The strong ligand absorption bands of
m, 4H), 7.91 (m, 2H), 7.67 (m, 1H), 7.55 (m, 2H), 5.79 (d,
ν(C=S), ν(CeN), ν(C=O) and v(C=C)ar were observed in range of
1
3
−1
−1
−1
J = 11.9 Hz, 4H), 2.84 (s, 1H), 2.09 (s, 3H), 1.20 (s, 6H). C NMR
1160–1150 cm
,
1269–1259 cm
,
1
1689–1667 cm
and
−1
(
101 MHz, DMSO‑d
6
) δ 179.4, 168.6, 167.1, 142.4, 133.6, 132.5, 130.6,
1535–1522 cm , respectively. Ligand L as a derivative of benzoic
−1
1
8
30.4, 129.2, 128.9, 128.5, 128.2, 123.9, 119.9, 106.8, 100.5, 86.8,
acid has v(OeH) absorption band at 3004 cm . In addition, the ex-
istence of absorption bands for C]S can be observed in the range of
5.9, 65.3, 30.4, 21.9, 18.3, 15.6. ESI-MS: m/z 535.06 ([M-Cl-HCL]+).
−
1
7
05–687 cm
which it highly indicated that all ligands are indeed
thiourea derivatives. Upon coordination absorption bands of relevant
groups in the complexes were slightly shifted to the higher values.
Bands of interest for the complexes are the same bands as for ligands
ν(NeH), ν(C=O), ν(CeN), ν(C=S), v(C=C)ar and v(OeH). They were
6
3
2
.2.8.3. [Ru(η -p-cymene)(L )Cl
2
] (C3). Yield: 42 mg (70%). Elemental
analysis, calculated for C26
H
28Cl
2
N
2
O RuS: C 50.32; H 4.55; N 4.51; S
3
−1
5
.17. Found: C 50.54; H 4.62; N 4.49; S 5.04. IR (ATR, cm ): 2963
1
−1
−1
−1
(
m), 1671 (s), 1544 (s), 1277 (m), 1170 (m). H NMR (400 MHz,
found
in
range
of
3207–2963 cm
,
1689–1656 cm
−1
,
,
−
1
−1
DMSO‑d
6
) δ 12.46 (s, 1H), 11.50 (s, 1H), 7.98 (d, J = 7.6 Hz, 2H), 7.66
1277–1267 cm 1172–1163 cm , 1535–1522 cm , and 2971 cm
(
m, J = 7.3 Hz, 1H), 7.55 (dd, J = 13.5, 8.0 Hz, 4H), 6.98 (m,
respectively. Existence of absorption bands for C]S can be observed in
−1
J = 8.7 Hz, 2H), 5.80 (m, J = 17.4, 6.0 Hz, 4H), 3.78 (s, 3H), 2.84
the range of 755–701 cm and indicate that all complexes are indeed
thiourea derivatives.
1
3
(
dt, J = 13.6, 6.9 Hz, 1H), 2.05 (d, J = 33.1 Hz, 3H), 1.18 (m, 6H).
C
1
NMR (101 MHz, DMSO‑d
6
) δ 179.6, 168.7, 157.9, 133.5, 132.6, 131.3,
The H NMR spectra contain a characteristic pattern for the p-
1
29.1, 128.9, 126.4, 114.3, 106.8, 100.5, 86.8, 85.9, 55.8, 21.9, 18.3.
cymene moiety. A methyl group singlet is observed at 2.09 ppm, the
+
ESI-MS: m/z 549.07 ([M-Cl-HCL] ).
resonance signal of –CH(CH
3
) appears as a multiplet in a range of
2
5

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
Scheme 2. Synthesis of C1–C3.
2
.82–2.85 ppm and -CH(CH
3
)
2
as a doublet in range of 1.09–1.19 ppm.
Acetonitrile has been used as complex solvent bearing in mind that
acetonitrile possesses high dielectric constant and low viscosity relative
to other solvents which are preferred for conductivity measurements.
Conductivity analysis with acetone as complex solvent were performed
as well. Conductivity values for 1 mM solutions of C1 in acetonitrile and
The signals of the arene ring protons were found in a range of
1
5
.77–5.83 ppm. Aromatic region of the H NMR spectra of the com-
plexes C1–C3 was found in a range of 7.51–8.03 ppm. Interestingly,
that protons from two NeH groups in thiourea are at very high che-
mical shifts of 11.68–11.5 ppm and 12.79–12.46 ppm, suggesting sig-
−1
2
−1
acetone are 15.49 and 0.53 Ω cm mol , respectively, while the
conductivity values for C2 are 17.79 and 3.26, respectively. Result for
C3 in acetonitrile is 10.92 while result in acetone is
1
nificant acidity. Change in proton shifts are noticeable from L to C1
where aromatic protons have different pattern. Also NeH group pro-
tons in this case, were shifted from 9.1 ppm and 12.5 ppm in the free
ligand to 11.7 ppm and 12.8 ppm in the complex, respectively. Similar
observation of different pattern is visible in aromatic protons of free
−1
2
−1
1.36 Ω cm mol . Based on the very low values and literature data
[47] we concluded that all complexes are a neutral species, as expected
values for 1:1 electrolyte type in acetone and acetonitrile are in range
3
−1
2
−1
ligand L and corresponding complex, with the less shift change in NeH
100–140, and 120–160 Ω cm mol , respectively.
group protons (stacked spectra in SI). In the case of C2, the difference in
ligand and complex proton shifts are much less obvious.
1
3
The C NMR spectra of synthesized complexes didn't suffer sig-
nificant changes comparing to spectra of uncoordinated ligand except
in case of C1 and C3 in thiourea group. Chemical shifts correspond to
3.2. X-ray analysis
In contrast to the data obtained by NMR and elemental analysis,
coordination of thiourea ligand has changed from monodentate to bi-
dentate of isolated crystal, corresponding to complex C1. Similar find-
ings are already reported in literature [22,48]. Piano-stool geometry for
the compound consists of coordinated chlorido ligand and four-mem-
bered ring through N and S bidentate coordinaton of derivative of
benzoylthiourea. The results of X-ray diffraction studies are shown in
Fig. 4. Geometrical environment around ruthenium atom has the ty-
pical “three leg piano-stool” geometry, which is common for a large
number of ruthenium(II) arene species. Although it is well known that
the most stable chelate ring is a five-membered one, in a case of ligands
used in this work, formation of five-membered ring is not possible.
In Fig. 3, two different hydrogen bonds could be detected, one in-
tramolecular (N2-H2…O1), one intermolecular (N2-H2…O1′). With the
help of the intramolecular hydrogen bond a six membered pseudo ring
is formed. Those rings contribute to the stabilization of the complex
1
3
the already described complexes of this type. The C NMR spectra
display chemical shift at 18.3 ppm from the methyl group attached to
the p-cymene moiety, 21.9 ppm from –CH(CH
3
) , while the chemical
2
shift at 30.4 ppm is due to the CH(CH
3
) group. The aromatic carbon
2
atoms from cymene display chemical shifts within range of
1
00.5–142.6 ppm. Carbon atoms signals from double bond C]S are
located in complexes around 179 ppm, and carbon atom chemical shifts
from C]O bond in range 167.1–168.7 ppm. Most changes in this region
is visible of thiourea group of complex C1 and C3.
Mass spectra of the complexes were recorded in a positive ion mode.
+
Molecular ions were detected at 521.8 ([M-Cl -HCL] ) for C1, 535.06
+
+
(
[M-Cl-HCL] ) for C2, and 549.07 ([M-Cl-HCL] ) for C3.
[
49]. In this context Platon detects one short interaction between O1
and O1′ (2.79 Å). Details are presented in Table 1 and Fig. 4.
Complex corresponding to C1 is similar to all listed XRD results. We
can classify the bond S1-C1 as single bond and C1-N1 as delocalised
double bond which is in good accordance to the planar geometry along
the bonds of L1 in the four membered ring, see Fig. 4. Therefore, we can
interpret L1 as mono-anion. The Ru1-S1 bond seems to be short in re-
lation to the listed results but M. Aitali et al. with identical ligands of
chlorido and p-cymene as in C1 shows also shorter bond length. Also
listed experiments were performed at room temperature, whereas C1
was recorded at 100 K. The center of gravity ring distance of the p-
cymene is 1.6698 Å. Finally, the ring slippage of the p-cymene with
0
.036 Å is detected and is not uncommon.
Fig. 4. Mono-anion L1.
6

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
Fig. 5. X-ray structure of complex C1.
Fig. 6. The survival of HeLa cells incubated for 72 h with different concentrations of synthesized ligands and Ru complexes.
3
.3. Antimicrobial activity
9111), Salmonella abony (ATCC 6017), Pseudomonas aeruginosa (ATCC
2
7853) and one strain of yeast Candida albicans (ATCC 24433).
Growing need for efficient antibacterial and antifungal agent is
Results indicate that the investigated complexes and related ligands
caused by rapid emergence of multi resistant bacterial agents [50–53].
For the first time, the antimicrobial activity of synthesized complexes
was investigated against nine bacterial strains: Staphylococcus aureus
were less active than the standard antimicrobial and antifungal drugs
(Table S6). In most antimicrobial assays complexes expressed better
antimicrobial and antifungal activity than their related ligands. All
complexes expressed low to moderate antimicrobial activity.
Complexes C1 and C3 expressed significant antimicrobial activity
against C. albicans (MIC = 62.5 μg/ml for both complexes) among
(
ATCC 6538), Staphylococcus epidermidis (ATCC 1228), Enterococcus
faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633), MRSA (clinical
isolate), Escherichia coli (ATCC 10536), Klebsiella pneumoniae (NCIMB
7

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
Table 3
Antimicrobial activity of complexes and theirs related ligands against Candida spp. clinical strain isolates (MIC values are given in μg/ml)
Type of infection
MIC (μg/ml)
Type of sample
Strain number
Ligand
L1
Complex
C1
L2
L3
C2
C3
Local infections strains
Vaginal swab
Vaginal swab
Vaginal swab
Cervical swab
Vaginal swab
Cervical swab
Vaginal swab
Sputum
2
3
> 1000
> 1000
> 1000
500
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
125
250
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
125
1000
500
1000
7
250
1000
a
8
n.t.
1000
1000
1000
62,5
125
> 1000
> 1000
> 1000
62,5
20
250
n.t.
1000
21
> 1000
> 1000
> 1000
1000
n.t.
> 1000
> 1000
500
22
1000
Systemic infections strains
670
712
719
791
805
826
1067
> 1000
> 1000
> 1000
> 1000
500
1000
1000
Sputum
> 1000
> 1000
1000
500
1000
> 1000
> 1000
1000
Sputum
1000
62,5
62,5
250
> 1000
> 1000
1000
Sputum
250
Sputum
125
500
250
Tracheal swab
Chemoculture
> 1000
62,5
> 1000
n.t.
> 1000
> 1000
250
> 1000
> 1000
1000
250
> 1000
a
n.t. - not tested.
tested. For that reason, we tested clinical Candida spp. strain isolates as
well. The results are shown in Table 3. Complex C1 expressed higher
antimicrobial activity then other complexes and ligands, especially on
systemic infection isolates (MIC ranged between 62.5 and 500 μg/ml).
4. Conclusion
In this study three half-sandwich ruthenium complexes have been
synthesized and characterized with several instrumental methods and
evaluated for antimicrobial and cytotoxic activity. All complexes were
isolated as neutral complexes with monodentate coordination of
thiourea ligand. X-ray structure analysis revealed the piano stool geo-
metry around ruthenium center with the formation of a four-membered
ring between ruthenium ion and sulfur and nitrogen atoms from ligand.
Antimicrobial activity has been evaluated against nine Gram-positive
and Gram-negative bacterial strains and one yeast - Candida albicans.
Complexes C1 and C3 exerted moderate antibacterial activity against S.
aureus (MIC = 500 μg/ml for complex C1 and MIC = 250 μg/ml for
complex C3) and the best antimicrobial activity against C. albicans
(MIC = 62.5 μg/mlfor both complexes). At the same time, complex C1
expressed higher antimicrobial activity against Candida spp. clinical
isolates then other complexes and ligands, especially on systemic in-
fection isolates (MIC ranged between 62,5–500 μg/ml). Corresponding
ligands had MIC values ranged from 250 to 1000 μg/ml in the most
analyzed examples. The anticancer activity of investigated ruthenium
complexes and related ligands was examined against human cervix
adenocarcinoma (HeLa) cell line. Compounds showed moderate antic-
ancer activity, with complex C1 being the most effective one. All
compounds had less efficient cytotoxic activity against HeLa cells in
comparison to cisplatin. In the expanding field of the rational drug
discovery, ruthenium complexes represent promising class of small
molecules that can be optimized to more specifically target tumor cells.
Future study will be focused toward synthetizing complexes with higher
activity and selectivity.
3.4. Cytotoxic activity
The anticancer activity of investigated ruthenium complexes and
related ligands was examined against human cervix adenocarcinoma
(
HeLa) cell line by MTT test incubated for 72 h with different con-
centrations of complexes or ligands. The positive control was cisplatin.
The results for IC50 concentrations are shown in Table 4.
1
2
3
IC50 concentrations of ligands L , L and L and complexes C1, C2
and C3 were ranging from 29.7 to 69.6 μM. Comparative dose-response
survival curves of HeLa cells treated with different concentrations of
synthesized ligands and their complexes are presented on graphs in
Fig. 6. Complexes C1 and C3 showed significant lower IC50 values than
1
3
IC50 values of ligands L and L , respectively. The most efficient an-
ticancer activity had complex C1 with IC50 value of 29,7 μM but the
biggest difference in IC50 values and anticancer activity compared with
2
its ligand showed complex C3. Complex C2 and related ligand L had
the similar values of IC50 concentration. Graphs are showing that
1
complexes C1, C2 and C3 have better cytotoxic activity than ligands L ,
2
3
L
and L at concentrations higher than IC50 concentrations. Also, this
trend of cytotoxicity activity can also be explained by differences in
lipophilicity of the complex, where complex C1 is the most lipophilic,
and C2 is the most polar. All examined compounds had higher IC50
values when comparing with positive control, cisplatin.
Abbreviations
Table 4
Concentrations of compounds that induced a 50% decrease in HeLa cell survival
MIC
minimum inhibitory concentration
deoxyribonucleic acid
rate expressed as IC50 (μM).
Class of compound
DNA
Compounds
IC50(μM)
HeLa
KP1019 indazolium-trans-tetrachlorobis(1H-indazole)ruthenate(III)
KP1339 sodium-trans-tetrachlorobis(1H-indazole)ruthenate(III)
NAMI
sodium-trans-imidazoledimethylsulfoxidetetra-
chlororuthenate(III)
Complexes
Ligands
C1
C2
C3
29.7 ± 0.9
52.4 ± 4.0
32.4 ± 3.2
46.2 ± 0.5
56.6 ± 6.9
69.6 ± 7.3
4.6 ± 0.1
HeLa
FemX
A549
MRC-5
ESI
human cervix adenocarcinoma cells
human melanoma cells
1
L
2
3
lung adenocarcinoma cells
human fetal fibroblast cells
electrospray ionization
L
L
Positive control
Cisplatin
8

D. Obradović, et al.
Journal of Inorganic Biochemistry 210 (2020) 111164
DFT
CLSI
TTC
density functional theory
B. Stefano, G. Gabriele, W. Małgorzata, S. Marta, Eur. J. Med. Chem. 101 (2015)
1
11.
Clinical and Laboratory Standards Institute
2, 3, 5-triphenyltetrazolium chloride
[
18] S. Sohail, R. Naghmana, G.J. Peter, H. Rizwan, H.B. Moazzam, Cent. Eur. J. Chem. 8
(
2010) 550–558.
RPMI-1640 Roswell Park Memorial Institute
[19] L. Qiao, Y. Zhang, W. Hu, J.J. Guo, W.L. Cao, Z.M. Ding, Z.W. Guo, A. Fan,
J.R. Song, J. Huang, J. Mol. Struct. 1141 (2017) 309–321.
DMSO
DMSO‑d
L1
dimethyl sulfoxide
[
20] J. Akhtar, J.C. Bruce, M.A. Malik, K.R. Koch, M. Afzaal, P. O'Brien, Mater. Res. Soc.
Symp. Proc. 1148E (2009) 1148.
6
deuterated dimethyl sulfoxide
N-((4-methoxyphenyl)carbamothioil)benzamide
4-(3-benzoylthioureido)benzoic acid
methyl-4-(3-benzoyltioureido)-benzoate
N-acetylglucosamine
[21] A. Rilak, R. Puchta, Ž.D. Bugarčić, Polyhedron 91 (2015) 73–83.
L2
[22] B.N. Cunhaa, L. Colina-Vegas, A.M. Plutín, R.G. Silveira, J. Honorato, K.M. Oliveira,
M.R. Cominetti, A.G. Ferreira, E.E. Castellano, A.A. Batista, J. Inorg. Biochem. 186
L3
(
2018) 147–156.
NAG
NAM
MTT
[
23] A. Molter, S. Kathrein, B. Kircher, F. Mohr, Dalton Trans. 47 (2018) 5055–5064.
N-acetylmuramic acid
[24] R.S. Correa, K.M. de Oliveira, F.G. Delolo, A. Alvarez, R. Mocelo, A.M. Plutin,
M.R. Cominetti, E.E. Castellano, A.A. Batista, J. Inorg. Biochem. 150 (2015) 63–71.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
mide
bro-
[
25] M.M. Ghorab, M.S. Alsaid, M.S.A. El-Gaby, M.M. Elaasser, Y.M. Nissan, Chem Cent
J. 11 (2017) 32.
IC50
inhibitory concentration
[26] R.-Z. Huang, B. Zhang, X.-C. Huangb, G.-B. Lianga, J.-M. Qina, Y.-M. Pana, Z.-
X. Liao, H.-S. Wang, RSC Adv. 7 (2017) 8866–8878.
[
27] R.K. Mohapatra, P.K. Das, M.K. Pradhan, M.M. El-Ajaily, D. Das, H.F. Salem,
U. Mahanta, G. Badhei, P.K. Parhi, A.A. Maihub, Md.K.E Zahan, Comment Inorg
Chem 39 (2019) 127–187.
Declaration of competing interest
[
28] Clinical and Laboratory Standardads Institute (CLSI), Performance standards for
antimicrobial susceptibility testing, 26th Informational Supplement. Approved
Standard. CLSI Document M100S. Wayne, PA, USA, 2016.
There are no conflicts to declare.
[
[
29] I.Z. Matić, I. Aljančić, Ž. Žižak, V. Vajs, M. Jadranin, S. Milosavljević, Z.D. Juranić,
BMC Complement. Altern. Med. 13 (2013) 36.
Acknowledgements
30] Bruker SAINT v8.38B Copyright © 2005–2019 Bruker AXS.
This work was supported by the Ministry of Education, Science and
Technological Development of Republic of Serbia Contract number:
[31] G.M. Sheldrick, SADABS, University of Göttingen, Germany, 1996.
[
[
32] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, OLEX2,
J. Appl. Crystallogr. 42 (2009) 339–341.
4
51-03-68/2020-14/200288.
33] C.B. Huebschle, G.M. Sheldrick, B. Dittrich, ShelXle: a Qt graphical user interface
for SHELXL, J. Appl. Crystallogr. 44 (2011) 1281–1284.
Appendix A. Supplementary data
[34] G.M. Sheldrick, SHELXS v 2016/4, University of Göttingen, Germany, 2015.
[
[
[
35] G.M. Sheldrick, SHELXL v 2016/4, University of Göttingen, Germany, 2015.
36] A.L. Spek, Acta Cryst D65 (2009) 148–155.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2020.111164.
37] Hang Zhu, Qing Ma, Ai-Quan Jia, Qun Chen, Wa-Hung Leung, Qian-Feng Zhang,
CCDC 813250: Experimental Crystal Structure Determination, (2013), https://doi.
org/10.5517/ccw97w8.
[
[
[
[
[
[
38] Xi-Ying Wang, Hua-Tian Shi, Fang-Hui Wu, Qian-Feng Zhang, CCDC 776948:
Experimental Crystal Structure Determination, (2011), https://doi.org/10.5517/
ccv2gv6.
References
39] Ai-Quan Jia, Qing Ma, Qun Chen, Hua-Tian Shi, Wa-Hung Leung, Qian-Feng Zhang,
CCDC 854737: Experimental Crystal Structure Determination, (2012), https://doi.
org/10.5517/ccxpf55.
[
[
[
1] https://www.who.int/en/news-room/fact-sheets/detail/noncommunicable-
diseases.
2] R. Canetta, K. Bragman, L. Smaldone, M. Rozencweig, Cancer Treat. Rev. 15
40] Ai-Quan Jia, Qing Ma, Qun Chen, Hua-Tian Shi, Wa-Hung Leung, Qian-Feng Zhang,
CCDC 854738: Experimental Crystal Structure Determination, (2012), https://doi.
org/10.5517/ccxpf66.
(
1988) 17.
3] J.P. Caussanel, F. Levi, S. Brienza, J.L. Misset, M. Itzhaki, R. Adam, G. Milano,
B. Hecquet, G. Mathe, J. Natl. Cancer Inst. 82 (1990) 1046.
41] Ai-Quan Jia, Qing Ma, Qun Chen, Hua-Tian Shi, Wa-Hung Leung, Qian-Feng Zhang,
CCDC 854739: Experimental Crystal Structure Determination, (2012), https://doi.
org/10.5517/ccxpf77.
[
[
4] N. Muhammad, Z. Guo, Curr. Opin. Chem. Biol. 19 (2014) 144–153.
5] A. Bergamo, C. Gaiddon, J.H. Schellens, J.H. Beijnen, G. Sava, J. Inorg. Biochem.
1
06 (2012) 90–99.
42] M. Aitali, M.Y. Ait Itto, A. Hasnaoui, A. Riahi, A. Karim, S. Garcia-Granda,
A. Gutierrez-Rodriguez, CCDC 150372: Experimental Crystal Structure
Determination, (2000), https://doi.org/10.5517/cc51gqc.
[
[
6] R. Trondl, P. Heffeter, C.R. Kowol, M.A. Jakupec, W. Berger, B.K. Keppler, Chem.
Sci. 5 (2014) 2925–2932.
7] S. Leijen, S.A. Burgers, P. Baas, D. Pluim, M. Tibben, E. van Werkhoven, E. Alessio,
G. Sava, J.H. Beijnen, J.H. Schellens, Investig. New Drugs 33 (2015) 201–214.
8] P.J. Dyson, G. Sava, Dalton Trans. 1 (2006) 1929–1933.
43] T.O. Brito, A.X. Souza, Y.C.C. Mota, V.S.S. Morais, L.T. De Souza, A. de Fatima,
F. Macedo Jr., L.V. Modolo, RSC Adv. 5 (2015) 44507.
[
[
[
[
44] S.B. Jensen, S.J. Rodger, M.D. Spicer, J. Organomet. Chem. 556 (1998) 151–158.
45] A. Saeed, A. Khurshid, J.P. Jasinski, C.G. Pozzi, A.C. Fantoni, M.F. Erben,
Chem.Phys. 431-432 (2014) 39–46.
9] C.G. Hartinger, S. Zorbas-Seifried, M.A. Jakupec, B. Kynast, H. Zorbas, B.K. Keppler,
J. Inorg. Biochem. 100 (2006) 891–904.
[
[
[
[
10] W.H. Ang, P.J. Dyson, Eur. J. Inorg. Chem. 20 (2006) 8153.
11] H.-K. Liu, P.J. Sadler, Acc. Chem. Res. 44 (2011) 349–359.
12] W.M. Motswainyana, P.A. Ajibade, Adv. Chem. (2015) ID 859730.
13] A.A. Nazarov, C.G. Hartinger, P.J. Dyson, J. Organomet. Chem. 751 (2014)
[
[
[
[
[
46] A. Saeed, U. Flörke, M.F. Erben, J. Sulfur Chem. 35 (2013) 318–355.
47] W.J. Geary, Coord. Chem. Rev. (1971) 81–122.
48] K.R. Koch, Coord. Chem. Rev. 216-217 (2001) 473–488.
49] K.B. Wiberg, Angew. Chem. Int. Ed. 25 (1986) 312–322.
50] European Centre for Disease Prevention and Control, Antimicrobial resistance
surveillance in Europe 2011, Annual Report of the European Antimicrobial
Resistance Surveillance Network (EARS-Net), ECDC, Stockholm, 2012.
51] M.A. Kohanski, D.J. Dwyer, J.J. Collins, Nat. Rev. Microbiol. 8 (2010) 423–435.
52] R.E.W. Hancock, A. Nijnik, D.J. Philpott, Nat. Rev. Microbiol. 10 (2012) 243–254.
53] K.M. O'Connell, J.T. Hodgkinson, H.F. Sore, M. Welch, G.P. Salmond, D.R. Spring,
Angew. Chem. Int. Ed. 52 (2013) 10706–10733.
2
51–260.
[
[
[
[
14] B.S. Murray, M.V. Babak, C.G. Hartinger, P.J. Dyson, Coord. Chem. Rev. 306 (2016)
8
6–114.
15] A. Weiss, R.H. Berndsen, M. Dubois, C. Muller, R. Schibli, A.W. Griffioen,
P.J. Dyson, P. Nowak Sliwinska, Chem. Sci. 5 (2014) 4742–4748.
[
[
[
16] L. Qiao, J. Huang, W. Hu, Y. Zhang, J.J. Guo, W.L. Cao, K.H. Miao, B.F. Qin,
J.R. Song, J. Mol. Struct. 1139 (2017) 149–159.
17] B. Anna, S. Joanna, S. Karolina, N. Agnieszka, A.K. Ewa, S. Giuseppina, M. Silvia,
9