Synthesis, X-ray structure, physicochemical properties and anticancer activity of: Mer and fac Ru(iii) triphenylphosphine complexes with a benzothiazole derivative as a co-ligand

Article abstract of DOI:10.1039/c9dt01803d

Two mononuclear ruthenium(iii) mer- and fac-isomers of the formula [RuCl₃(PPh₃)(dmpbt)] (where PPh₃ = triphenylphosphine, dmpbt = 2-(3,5-dimethylpyrazoll-yl)benzothiazole) have been synthesised from the reaction of [RuCl₃(PPh₃)₃] with a bidentate ligand-dmpbt. Appropriate reaction conditions allowed obtaining the two isomers separately without separation techniques. X-ray crystallography has determined the crystal and molecular structures of the new complexes. mer-Ru(iii) (1) crystallised in the monoclinic P2(1)/n group, and fac-Ru(iii) (2, 2′) in the triclinic P1 space group. The composition of the ruthenium coordination sphere was confirmed and characterised using spectroscopic techniques (FT-IR, UV-vis and EPR), elemental analysis and mass spectrometry (MS-FAB). The structures of the complexes obtained were analysed using X-ray and other spectroscopic methods (IR and UV-vis). The electrochemical properties of the ligand and the complex compound were identified using cyclic voltammetry, determining the potential and charge of faradaic processes. Both isomers are redox active and display quasi-reversible metal centered redox processes for the Ru(iii)/Ru(ii) pair. Moreover, preliminary tests of their biological activity were performed. The cytotoxicity of these compounds has been tested for human lung carcinoma (A549), chronic myelogenous leukemia (K562), human cervix carcinoma (HeLa) cells, acute lymphoblastic leukemia (MOLT-4), human breast adenocarcinoma cell line (MCF-7) and normal human umbilical vein endothelial cells (HUVEC). The ability to induce apoptosis has been demonstrated in caspase 3/7 activity assay. In addition, the lipophilicity of both isomers was described by a partition coefficient, logP, values of which were estimated by the shake-flask method. The interesting and promising preliminary results of the biological and chemical activities of the new octahedral mer/fac Ru(iii) complexes motivate further in vitro and in vivo studies.

Full text of DOI:10.1039/c9dt01803d

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Synthesis, X-ray structure, physicochemical
properties and anticancer activity of mer and fac
Ru(III) triphenylphosphine complexes with a
benzothiazole derivative as a co-ligand
*
a
b
*c
c
Monika Richert, Mariusz Walczyk, Marcin Janusz Cieślak, Julia Kaźmierczak-Barańska,
c
b
b
b
Karolina Królewska-Golińska, Grzegorz Wrzeszcz, Tadeusz Muzioł, Stanisław Biniak
a.
Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in
Toruń; Jurasza 2; 85-094 Bydgoszcz, Poland;
E-mail: monika.richert@cm.umk.pl, phone: +48 52 585-38-03, Fax: +48 52 585-38-04
b.
Faculty of Chemistry, Nicolaus Copernicus University in Toruń; Gagarina 7, 87-100 Toruń,
Poland
c.
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza
12, 90-363 Lódź, Poland
1
Corresponding Authors
1

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DOI: 10.1039/C9DT01803D
*
*
E-mail: monika.richert@cm.umk.pl
E-mail: marcin@cbmm.lodz.pl – biological part
KEYWORDS: mer- and fac-Ru(III) complexes, benzothiazole derivative, X-ray structures, cyclic
voltammetry, cytotoxic effect, log P
ABSTRACT
Two mononuclear ruthenium(III) mer- and fac- isomers of the formula [RuCl (PPh )(dmpbt)]
3
3
(
where PPh = triphenylphosphine, dmpbt = 2-(3,5-dimethylpyrazoll-yl)benzothiazole) have been
3
synthesised from the reaction of [RuCl (PPh ) ] with a bidentate ligand – dmpbt. Appropriate
3
3 3
reaction conditions allowed to obtain the two isomers separately without separation techniques.
X-ray crystallography has determined the crystal and molecular structures of the new complexes.
Mer-Ru(III) (1) crystallised in the monoclinic P2(1)/n, and fac-Ru(III) (2, 2’) in the triclinic P–1
space group. The composition of the ruthenium coordination sphere was confirmed and
characterised using spectroscopic techniques (FT–IR, UV–vis and EPR), elemental analysis and
mass spectrometry (MS–FAB). The structures of the complexes obtained were analysed using X-
ray and other spectroscopic methods (IR, UV–vis). The electrochemical properties of the ligand
and the complex compound were identified using cyclic voltammetry, determining the potential
and charge of faradic processes. Both isomers are redox active and display quasi-reversible metal
centered redox processes for the Ru(III)/Ru(II) pair. Moreover, preliminary tests of their biological
activity were performed. The cytotoxicity of these compounds has been tested for human lung
carcinoma (A549), chronic myelogenous leukemia (K562), human cervix carcinoma (HeLa) cells,
acute lymphoblastic leukemia (MOLT-4) and human breast adenocarcinoma cell line (MCF-7)
and normal human umbilical vein endothelial cells (HUVEC). The ability to induce apoptosis has
2

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been demonstrated in caspase 3/7 activity assay. In addition, the lipophilicity of both isomers was
described by a partition coefficient, log P, which values were estimated by the shake-flask method.
The interesting and promising preliminary results of the biological and chemical activities of the
new octahedral mer/fac Ru(III) complexes motivate for further in vitro and in vivo studies.
INTRODUCTION
1
The discovery of cisplatin and its anticancer properties significantly influences the development
of research concerning metallopharmaceuticals. Despite the large number of different d-electron
metals complexes already obtained, it is still necessary to search for new compounds with better
therapeutic properties, lower toxicity and greater specificity than cisplatin.2, 3 Ruthenium(III)
complexes seem to be the most attractive and promising prodrug. Ruthenium(III) ions, unlike
cisplatin, have a different coordination number and the ligands have other affinities. It caused
different kinetics of ligands substitution and redox activity than platinum(II) complexes . The
interest in ruthenium complexes as potential anticancer drugs results mainly from three of their
properties: a) the rate of ligand exchange comparable with that of platinum(II) compounds and the
possibility of controlling this rate through the coordination of ligands with suitably selected
properties; b) the wide range of oxidation states (II, III and IV) available under physiological
conditions and the possibility of controlling the redox potential and rate of electron transfer by the
selection of suitable ligands; and, c) the ability of ruthenium to mimic iron in its ways of binding
certain biological molecules like human transferrin.4-6 The intense study focused on the cytotoxic
effects and mechanism of action of ruthenium(III) compounds led to the selection of a few
complexes with the most promising properties.
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The anticancer activity of imidazolium trans-[tetrachloride-bis(1H-imidazole)ruthenate(III)],
7
(
Kp418) against murine P388 leukemia and B16 melanoma, as well as the ability to reduce a
8
tumour mass in rats, caused significant research attention of the analogue complexes. These
efforts led to identification of two the most promising ruthenium(III) complexes to indazolium
trans-[tetrachloride-bis(1H-indazole)ruthenate(III)], (KP1019), and imidazolium trans-
9
[
tetrachloride(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)] (NAMI-A). NAMI-A, the first
ruthenium anticancer compound to be studied on human beings, which demonstrated
1
0
antimetastatic and anticancer properties. The biological activity studies of NAMI-A proved that
1
1
it has numerous biological targets as DNA, RNA and proteins. Moreover, the clinical trial of
phase I/II showed that NAMI-A with gemcitabine is only moderately tolerated and less active in
Non-Small Cell Lung Cancer (NSCLC) patients after first line treatment than gemcitabine alone.12,
1
3
Currently, clinical trials include KP1019 (IndH[trans-RuCl (Ind) ], where Ind = indazole) and
4
2
1
4,
15. Despite the
NKP-1339 (Na[trans-RuCl (Ind) ]) and ruthenium(II)-arene complexes.
4
2
structural similarities of KP1019 and NAMI-A, they are characterised by different in vivo and in
vitro biological activities. KP1019 is devoid of side effects and causes in cancer cells apoptosis,
particularly of colorectal tumours. Anticancer activity studies KP1019 and NKP-1339 have shown
their affinity of donor atoms particularly in albumin and transferrin, which are the first potential
1
6
targets in the transport of ruthenium complexes to tumour cells. The cytotoxic effect is associated
1
7
with formation of reactive oxygen species (ROS). The group of the ruthenium-arene complexes
with PTA (RAPTA) (where PTA = 1,3,5-triaza-7-phosphoadamantane) exhibit excellent
1
8
antimetastatic and anti-angiogenic activity. Ru(II)–arene organometallic complexes interact with
1
9
DNA by coordination to the bases and intercalation. In contrast to platinum anticancer drugs,
NAMI-A is not very toxic towards a primary tumour, but the main effect of the compound is to
4

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stop tumours from spreading to other parts of a body.20, 21 NAMI-A is especially active against
tumour lung metastases and it is hypothesised that this compound interacts with a target on the
tumour cell membrane.22
NAMI-A, similarly to KP1019, interacts with DNA, but DNA does not seem to be its primary
target. NAMI-A forms intrastrand adducts (Ru-G and Ru-AG) in vitro, moreover, both in vitro and
in vivo studies have shown that NAMI-A also binds to the plasma proteins albumin and transferrin.
2
2, 23
Antimetastatic activity similar to that of NAMI-A was shown by RAPTA-T and various
different ruthenium organometallic14, 24, 25 for which the importance of the ruthenium and ligands
arrangement was emphasised.24, 26, 27 Based on the hypothesis of activation by reduction
mechanism, the reduction of NAMI-A was examined in the presence of ascorbic acid used at a
physiological concentration. This study has shown the possibility of the reduction of NAMI-A to
its corresponding Ru(II) via an outer-sphere mechanism.12
The discovery of antitumour and antimetastatic activity of ruthenium complexes, especially
KP1019 and NAMI-A, caused a considerable increase of the complexes synthesis with similar
2
8
structures to them. On the other hand, the inspiration came from the investigation of alternative
ruthenium(III) complexes with different N,N- and N,S-donor ligands. The choice of the ligand was
very often based on its well-known biological activity and an interesting pharmacological profile.
Examples of such ligands are pyrazole or benzothiazole and their derivatives, which play an
essential role in the design of new drugs. Pyrazole is known as an important and very attractive
2
9
pharmacophore with diverse therapeutic activities, including: antitumour, antibacterial, anti-
inflammatory,30 anticonvulsant, antidepressant,31 analgesic and anthelmintic ones.32
Benzothiazole is still one of the most versatile classes of compounds with antimicrobial activity33
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and its derivatives are characterised by varied biological activities, namely: antitumour,34
3
5
36
37
38
39
antitubercular, antimalarial, anticonvulsant, analgesic and anti-inflammatory.
In our work, we have focused on the synthesis of ruthenium(III) complexes with 2-(3,5-
dimethylpyrazoll-yl)benzothiazole (dmpbt) as a co-ligand. The rationale for choosing the dmpbt
4
0
41
ligand was its successful application in reactions with d-electron metals, such as cobalt, nickel
4
2
and molybdenum. Complexes of first two metals exhibited substantial cytotoxicity towards HL-
6
0 and NALM-6 leukemia cells and WM-115 melanoma cells, albeit lower as than cisplatin and
carboplatin.40, 41 Moreover, chlorido ligand and phosphine molecule used as ligands in the
coordination sphere of ruthenium(III) are intended to hydrolyses facilitate in vitro stabilisation of
the Ru(II) ions formed by the Ru(III) reduction respectively. The Ru(II) species (such as
glutathione in the cell and ascorbic acid in the blood in the human body) is expected to reactive in
the hypoxic regions of solid tumour tissues.43 44 Besides, the structural and spectroscopic
characterisation of the new ruthenium(III) complexes, we defined electrochemical properties using
the method of cyclovoltammetry to determine the ability of biological activation by a reduction
mechanism. Furthermore, obtained compounds showed some biological activities, their
lipophilicity was determined. The relationships between analysed properties will give a potential
mechanism of anticancer activity and possible further directions for in vitro and in vivo studies.
MATERIALS
Acetonitrile (MeCN) 99.99% HPLC grade (Sigma-Aldrich), as well as lithium perchlorate
(
LiClO , p.a., Acros organics) were used as received. Hydrated ruthenium(III) chloride,
4
triphenylphosphine, buffer PBS saline (pH=7.2), cisplatin and using solvents were purchased from
6

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Aldrich and used as supplied. The 2-(3,5-dimethylpyrazoll-yl)benzothiazole was prepared
according to literature procedures.45
Synthesis of complex compounds
mer-[RuCl (PPh )(dmpbt)] (1)
3
3
Triphenylphosphine (100 mg, 0.384 mmol) was added to a solution of ruthenium(III) chloride
0.096 mmol) in ethanol (10 ml) and hydrochloric acid (37%, 1ml), then the synthesis was carried
(
4
6
out according to in the literature. The dmpbt (20 mg, 0.096 mmol) was added to the brown-
colored trichlorotriphenylphosphineruthenium(III) ethanolic solution and refluxed for half an
hour. The solution became orange. After cooling to room temperature, the product started to
precipitate as an orange solid. Crystallisation from mother solution was carried out via vapor
diffusion technique to obtain a single crystal suitable for X-ray analysis. Diethyl ether was used as
a more volatile solvent. After three days yellow orange crystals of mer-[RuCl (PPh )(dmpbt)] (1)
3
3
were received. Yield: 0.17 g (75%). Analysis for RuC H N PSCl . Calculated (%): C, 51.55; H,
3
0
26
3
3
+
3
.75; N, 6.01. Found (%): C, 51.45; H, 3.86; N, 6.16. MS (FAB): m/z = 698.9 [M ], 399.1
+
+
+
+
[
[
(
RuClPPh ], 363.0 [RuPPh ], 262.1 [PPh ], 229.1 [dmpbt ], 135.0 [benzothiazole], 136.0
3 3 3
+
3
-1
-1
benzothiazoleH ]. UV-vis (acetonitrile, (λ, nm) (ɛ/dm mol cm ): 257sh (~21400); 275sh
-1
-1
13800); 305 (15600); 388 (3200); 465sh (~640). IR KBr pellet, cm and FTIR (CsI pellet, cm ):
1
579 (C=N), (pyr, dmpbt-Ru); 1512 (C=N), (btz, dmpbt-Ru); 442 ν(Ru-P); 420, 407, 391, 364
(Ru-N); 329, 291, 245, 193, 142 (Ru-Cl).

fac-[RuCl (PPh )(dmpbt)]·H O (2)
3
3
2
The mother solution of the mer-[RuCl (PPh )(dmpbt)] (1) with orange crystals has remained for
3
3
the next three weeks. After this time, mer-isomer started to converse into dark shine brown needles
of the fac-[RuCl (PPh )(dmpbt)]·H O (2). Moreover, the mother solution containing isomers was
3
3
2
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left for next three months under the vapor diffusion conditions with diethyl ether. After this time,
it was observed that all orange crystals were converted into dark brown needles. Yield: 0.17 g
(
73%). Analysis for RuC H N OPSCl . Calculated (%): C, 50.25; H, 3.94; N, 5.86. Found (%):
30 28 3 3
C, 50.72; H, 4.12; N, 6.16%.
MS (FAB): m/z = 716.5 [M ], 699.0 [M –H O], 399.0 [RuClPPh ], 363.0 [RuPPh ], 262.1
+
+
+
+
2
3
3
+
+
+
[
(
(
PPh ], 229.1 [(dmpbt) ], 135.0 [benzothiazole], 136.0 [benzothiazoleH ]. UV-vis (acetonitrile,
3
3
-1
-1
-1
λ, nm) (ɛ/dm mol cm ): 257sh, 276sh, 308, 374sh, 476sh, 589. IR KBr pellet, cm and FTIR
-1
CsI pellet, cm ): 1583 (C=N), (pyr, dmpbt-Ru); 1508 (C=N), (btz, dmpbt-Ru); 448, 439 ν(Ru-
P); 414, 407, 391, 369 (Ru-N); 329, 291, 247, 194, 143 (Ru-Cl); 3451(O-H).
fac-[RuCl (PPh )(dmpbt)]·C H OH (2’)
3
3
2
5
Triphenylphosphine (100 mg, 0.384 mmol) was added to a solution of ruthenium(III) chloride
0.096 mmol) in ethanol (10 ml) and a mixture of sulfuric acid (95–98%, 1ml) and hydrochloric
(
4
6
acid (37%, 1 ml) and then further procedures described in the literature was followed. The dmpbt
20 mg, 0.096 mmol) was added to the brown-coloured trichlorotriphenylphosphineruthenium(III)
(
ethanolic solution and refluxed for about half an hour. The solution became a deep orange colour.
Crystallisation was carried out by a vapor diffusion technique. After three weeks, dark shine brown
needles of the fac-Ru(2’) were received, filtered off, washed with petroleum ether, and dried under
a vacuum (yield: 0.22 g, 85%). The structure was confirmed by X-ray, mass spectrometry and
elemental analyses.
Analysis for RuC H N OPSCl . Calculated (%): C, 51.58; H, 4.33; N, 5.64. Found (%): C,
3
2
32
3
3
+
+
+
5
3
1.60; H, 4.34; N, 5.66%. MS (FAB): m/z = 745.1 [M ], 699.1 [M –C H OH], 399.0 [RuClPPh ],
2 5 3
+
+
+
+
63.0 [RuPPh ], 262.1 [PPh ], 229.1 [(dmpbt) ], 135.0 [benzothiazole], 136.0 [benzothiazoleH ]
3
3
8

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EXPERIMENTAL METHODS
Magnetic measurements and EPR
Magnetic susceptibility was measured at room temperature by the Faraday method. The
4
7
magnetic field was calibrated with Hg[Co(NCS) ]. The molar susceptibilities were corrected for
4
4
8
6
6
3
1
diamagnetism using the Pascal’s constants (403  10 for 1 and 416  10 cm mol for 2).
½
The effective magnetic moments were calculated from the equation:   2.828 (
T) .
eff
Mcorr
EPR spectra of the powdered samples were recorded at room temperature with an X band (ca.
.33 GHz) Radiopan EPR SE/X-2541M spectrometer with a 100 kHz modulation. The microwave
9
frequency was monitored with a frequency meter. The magnetic field was measured with an
automatic NMR-type magnetometer.
UV–vis Spectroscopy
Electronic spectra were measured with a Metertech 8001 spectrophotometer in an acetonitrile
solution within the 200–1100 nm range.
IR Spectroscopy
IR spectra of the synthesised complexes were obtained using a Perkin-Elmer FTIR Spectrum
-1
2
000 spectrometer. Spectra were recorded from 4000 to 400 cm (MIR; KBr pellet) and 700 – 30
-1
-1
cm (FIR; CsI pellet) at a scan rate of 0.2 cm s , and the number of interferograms at a nominal
-1
resolution of 4 cm was fixed at 25. Before spectrum of a sample was recorded, the background
line was obtained arbitrarily and subtracted.
MS–FAB
+
The MS–FAB data were determined on Finnigan MAT 95 mass spectrometer (NBA, Cs gun
operating at 13 keV).
Elemental analyses
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Elemental analyses for the obtained complexes were performed on a Vario EL of Elementar
Analysensysteme GmbH.
X-ray diffraction data
Hereby, we report structures of two geometrical isomers, mer- and fac-, obtained in the single
synthesis formed as mer-[RuCl (PPh )(dmpbt)] (1), fac-[RuCl (PPh )(dmpbt)]·H O (2) and fac-
3
3
3
3
2
[
RuCl (PPh )(dmpbt)]·C H OH (2’). Suitable crystals were obtained using vapor diffusion
3 3 2 5
technique for all compounds. Diffraction data for the three complexes were collected on Oxford
4
9
Sapphire with CCD area detector, MoKα radiation λ  0.71073 Å. All structures were solved by
2
50
direct methods and refined by full-matrix least-squares techniques on F with SHELXL program.
The numerical absorption correction was applied for all crystals (RED171 package of programs,
4
9
Oxford Diffraction, 2000). Heavy atoms were refined with anisotropic thermal displacement
parameters. The positions of hydrogen atoms attached to carbon atoms were assigned at calculated
positions. All hydrogen atoms were refined with isotropic thermal displacement parameters fixed
to a value of 20% or 50% higher than those of the corresponding carbon atoms. For (2), partially
occupied water molecules were found, whereas in (2’), the ethanol molecule was found in the
crystal network. In (2), there are no hydrogen atoms attached to water molecules, and in (2’), we
found partially occupied oxygen atom (0.87/0.13) from ethanol, and in the minor position, the
5
1
52
hydrogen atom is missing. All figures were prepared in DIAMOND and ORTEP-3 and the
5
3
interactions were analysed using PLATON. The data for (1), and (2) are summarized in Table 1
and data for (2’) in Table S1 (Supporting Information). CCDC 1030325, CCDC 1030304 and
CCDC 1826278 contain supplementary crystallographic data for (1), (2) and (2’) respectively.
These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk.
1
0

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Table 1. Crystal data and structure refinement for (1) and (2)
Parameters
(1)
(2)
Empirical formula
C H Cl N PRuS
C H Cl N OPRuS
30 28 3 3
3
0
26
3
3
Formula weight
698.99
293(2)
0.71073
717.00
Temperature [K]
293(2)
Wavelength [Å]
0.71073
Crystal system, space group
Unit cell dimensions [Å] and []
monoclinic, P2(1)/n
a = 10.6546(3)
b = 17.3387(4)
c = 15.9083(4)
Triclinic, P –1
a = 8.5500(9)
b = 10.1649(11)
c = 17.926(2)
 = 92.169(10)

= 97.968(2)


= 97.822(10)
= 99.235(9)
Volume [Å3]
2910.49(12)
4, 1.595
1520.6(3)
2, 1.566
Z, Calculated density [mg/m3]
-1
Absorption coefficient [mm ]
0.967
0.930
F(000)
1412
726
Crystal size [mm]
0.42 x 0.13 x 0.08
2.17 to 26.73
0.34 x 0.07 x 0.04
2.03 to 25.02
Theta range for data collection []
Limiting indices
–12 ≤ h ≤ 13
–9 ≤ h ≤ 10
–12 ≤ k ≤ 11
–21 ≤ l ≤ 21
–
–
21 ≤ k ≤ 18
19 ≤ l ≤ 6
Reflections collected / unique
Completeness to theta
11504 / 6134 [R(int) = 0.0468] 8868 / 5358 [R(int) = 0.1162]
26.73 99.3 %
25.02 97.3 %
1
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Absorption correction
Max. and min. transmission
Refinement method
Numerical
Numerical
0.9266 and 0.6869
Full-matrix least-squares on F2
6134 / 0 / 354
0.9639 and 0.7434
Full-matrix least-squares on F2
5358 / 12 / 372
Data/restraints/parameters
Goodness-of-fit on F^2
Final R indices [I>2sigma(I)]
R indices (all data)
0.967
0.972
a
b
a
b
R1 = 0.0391, wR2 = 0.0919
R1 = 0.0844, wR2 = 0.1943
a
b
a
b
R1 = 0.0697, wR2 = 0.1050
----
R1 = 0.1810, wR2 = 0.2425
----
Extinction coefficient
-3
Largest diff. peak and hole [e.Å ]
1.127 and –0.465
1.842 and –0.756
a
b
2
2 2
2 2 1/2
R1 = F - F /F  wR2 = [w(F - F ) /(w(F ) )]
0
c
0
0
c
0
Electrochemical measurements
Cyclic voltammetric measurements were performed using an Autolab (Eco Chemie) modular
electrochemical system equipped with a PGSTAT128N potentiostat, controlled by NOVA
software and the typical three-electrode electrochemical cell. A platinum microelectrode served as
working electrode and another Pt wire and Ag wire were used as counter and reference electrodes,
5
4
respectively. Cyclic voltammetry measurements were carried out in 0.1 M LiClO /MeCN base
4
electrolyte solutions using ruthenium(III) chloride, ligands or ruthenium complex as a depolariser.
The approximate concentration of the dissolved depolariser was about 50 mM. The potentiometric
responses of the working electrode were measured in oxygen-free electrolyte solutions (usually
after 24 h). Cyclic voltammetric curves (CVs) were recorded for different sweep amplitudes once
electrochemical equilibrium had been established (no changes in repeated CV scans). The data
were collected under nitrogen atmosphere in the thermostated system at the room temperature (293
K). The scans start at 0.0 V in anodic direction as indicated by the arrows. From the CVs obtained,
1
2

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the anodic and cathodic peak potentials were determined with accuracy ±5 mV. All potentials are
quoted with respect to employed Ag-wire reference electrode.
Lipophilicity
The lipophilicity of mer-Ru(III) (1) and fac-Ru(III) (2) complexes was determined using the
shake-flask method.55, 56 Equal amounts of aqueous phosphate buffered saline and organic n-
octanol phases were mixed and saturated for 24 h. Ruthenium complexes and cisplatin were
dissolved at a concentration of 0.01 mg/mL in 10 mL of the saturated aqueous phase. An equal
volume of PBS was added to n-octanol, and solutions were shaken for 30 minutes, followed by
centrifugation (6000 rpm,15 min). After separation, the phases were analysed using UV-vis
spectroscopy to determine the amount of the compounds in each phase. The measurements were
performed using a SCHIMADZU UV-1800 UV-vis spectrophotometer. The absorption values
before and after shaking were compared. The partition coefficient (log P) in both phases for each
compound was calculated based on the Lambert–Beer Law to determine the log P values. The
procedures were repeated three times.
Cells and cytotoxicity assay
The HeLa (human cervix carcinoma), K562 (chronic myelogenous leukemia), A549 (human
lung carcinoma), MOLT-4 (acute lymphoblastic leukemia) and MCF-7 (human breast
adenocarcinoma) cells were cultured in RPMI 1640 medium supplemented with antibiotics and
3
1
9
0% fetal calf serum, in a 5% CO - 95% air atmosphere. 7x10 cells were seeded on each well on
2
6-well plate (Nunc). Twenty-four hours later, the cells were exposed to the test compounds. Stock
solutions (100 mM) of test compounds were freshly prepared in DMSO (dimethylsulfoxide). The
final concentrations of the compound (1), (2), and dmpbt in the cell culture were 1 mM, 1x10-1
-2
-3
-4
-5
mM, 1x10 mM, 1x10 mM, 1x10 mM and 1x10 mM. The concentration of DMSO in the cell
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culture medium was 1%. Cis-Pt stock solution of 5 mM was prepared in 0.9% NaCl. The final
-2
-2
-3
-3
concentration of the cis-Pt in the cell culture was 5x10 mM, 1x10 mM, 2.5x10 mM, 1x10
-4
-4
mM, 2.5x10 mM and 1x10 mM.
Human umbilical vein endothelial cells (HUVEC) were obtained from Life Technologies and
cultured in Medium 200 with low serum growth supplement (Life Technologies) according to
3
manufacturer’s instructions. 10x10 cells were seeded on each well on 96-well plate (Nunc).
The values of IC (the concentration of test compound required to reduce the cell survival
5
0
fraction to 50% of the control) were calculated from dose-response curves and used as a measure
of cellular sensitivity to a given treatment.
The cytotoxicity of all compounds was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-
2
,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay as described. 57 Briefly, after 48 h
of incubation with test compounds, the cells were treated with the MTT reagent for 2 h. MTT-
formazan crystals were dissolved in lysis buffer (20% SDS and 50% DMF, pH 4.7) and absorbance
was read at 570 and 650 nm on an ELISA-PLATE READER (FLUOstar Omega). As a control
(100% viability), we used cells grown in the presence of only vehicle (1% DMSO).
Caspase-3/7 assay
3
2
0x10 of HeLa cells were seeded on each well of 96-well plate in RPMI 1640 medium
supplemented with 10% fetal calf serum and antibiotics. The cells were grown for 24 h at 37°C
and 5% CO . The test compounds were dissolved in DMSO (except for cis-Pt which was dissolved
2
in 0.9% NaCl) and added to the cell culture. Untreated cells or cells treated with 1% DMSO served
as a negative control, while cells incubated with staurosporine (a potent inducer of apoptosis) were
used as a positive control. The cells were exposed to the test compounds for 18 h at 37°C and 5%
®
CO . Subsequently, the activity of caspase 3 and 7 was measured by Apo-ONE Homogeneous
2
1
4

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Caspase-3/7 Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Briefly, the cells were lysed and incubated for 1.5 h with profluorescent substrate for caspase 3
and 7. Next, the fluorescence was read at an excitation wavelength of 485 nm and emission of
5
20 nm with FLUOStar Omega (BMG-Labtech, Germany) plate reader.
RESULTS AND DISCUSSION
Synthesis
One of the ligands used in the described synthesis was bidentate chelating 2-(3,5-
dimethylpyrazoll-yl)benzothiazole. Dmpbt with two non-equivalent nitrogen atoms, chlorido
ligand and triphenylphosphine, after coordinating to the ruthenium ion formed two geometric
stereoisomers, mer and fac of the [RuCl (PPh )(dmpbt)] formula. The dmpbt ligand was prepared
3
3
4
5
by the described method and characterized by elemental analysis, IR (Supporting Information,
1
Figure S1) and H NMR data (Supporting Information, Figure S2).
Mer-[RuCl (PPh )(dmpbt)] (1) complex was synthesised by the reaction of [RuCl (PPh ) ] and
3
3
3
3 3
dmpbt in the M:L=1:1 molar ratio, (where M – metal, L – ligand) in refluxing with ethanolic
4
6
solution. [RuCl (PPh ) ] was prepared according to the literature methods. The fac-Ru(III) (2)
3
3 3
isomer was obtained in the isomerisation reaction of (1) or in similar reaction as a mer, but with
the use of 1:1 (v/v) sulphuric and hydrochloric acids mixture. According to the literature, 58, 59
synthesis of ruthenium complex isomers kinetically favours a mer isomer as the first product. The
electronic trans effect explains the thermal conversion of a mer into a fac isomer. Another way for
this conversion is photochemical isomerisation. For example fac-[RuCl (NO)(dppb)], (dppb = 1,4-
3
bis(diphenylphosphino)butane) was obtained from mer, which was refluxed for 4 h under argon.60
However, the same authors have shown that fac-[RuCl (NO)(P–P)] isomers can be obtained from
3
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a similar reaction but with different diphosphines ((P–P) = R P(CH ) PR (n = 1–3) and
2
2 n
2
R P(CH )POR , PR –CH=CH–PR , where R = C H and (C H ) P–(CH ) –P(C H ) ), which has
2
2
2
2
2
6
5
6
11 2
2 2
6
11 2
been explained by steric reasons. The isomer mer-[RuCl (NO)(P–P)] was received from the
3
6
1
corresponding fac-isomer by using white light. Another example of photochemical isomerisation
is the conversion of fac-[RuCl (NO)(P–N)] (P–N o-[(N,N-
dimethylamino)phenyl]diphenylphosphine) to mer-[RuCl (NO)(P–N)]. One of the main
=
3
6
2
3
6
3
problems of the mer and fac isomers synthesis is their separation. Fletcher and co-workers carried
out the separation of both Ru(II) complexes isomers using a stepwise templated synthesis or
through the separation by preparative plate chromatography.59, 64 It is known in the literature that
the formation of the mer isomer more likely than fac. The last studies have shown that the change
6
4
in the solvent and reaction conditions can lead to the pure fac isomer, which was confirmed in
our studies. The first synthesis product, the mer-Ru(III) (1) isomer, was formed as orange crystals
(in the mother solution under conditions of the diffusion process). The mer isomer slowly
converted to fac as dark shiny brown needle crystals. The observed colour change was assigned to
structural conversion. It can be a comfortable and straightforward method for the detection of
structural transformation.65, 66 Whereas, pure fac-Ru(III) (2’) isomer was obtained from a similar
reaction as mer-Ru(III) (1) but with the use of mixture HCl and H SO . The structural change of
2
4
the mer to fac isomer is probably caused by structural transformations–“single crystal-to-single
crystal” (SCSC), which is often difficult.65, 66 Among many external factors (e.g. temperature,
pressure, irradiation, solvent, concentration), the solvent probably plays a significant role in the
mer-Ru(III) (1) into fac-Ru(III) (2) transformation.66, 67 Change of the fac-Ru(2’) synthesis
conditions (use of 1:1 (v/v) sulphuric and hydrochloric acids mixture) probably caused formation
of the fac-isomer as more thermodynamically stable product (2’) and the presence of the ethanol
1
6

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molecule (guest molecule) in the crystal lattice of (2’). The ability to control the reaction by
maintaining appropriate conditions gives us the selectivity and purity of the products reactions and
is undoubtedly a significant advantage.
The differences in the cell parameters and symmetry crystals of the fac isomers (2 and 2’) do not
influence the behaviour in solution (UV-vis analysis), which is essential for biological studies.
Analysis of IR spectra
-1
Spectra analysis was limited mainly to the 160–1600 cm range, in which the presence of
benzothiazole, pyrazole, phosphine groups and their connection with metal are studied. The
-1
presence of a water molecule was confirmed in the region of 3000–1600 cm for the fac-Ru(III)
2). Characteristic IR frequency bands of the mer-Ru(III) (1), and fac-Ru(III) (2) complexes and
(
their assignments are given in Table S2 (Supporting Information).
Comparison of the infrared spectra of the 2-(3,5-dimethylpyrazoll-yl)benzothiazole, and its
complexes indicated the coordination of the ligand to the metal through tertiary nitrogens on the
benzothiazole and pyrazole group. It is confirmed by the presence of (C=N) (benzothiazole)
-1
-1
-1
-1
vibrations at 1579 cm , 1583 cm and (C=N)(pyrazole) at 1512 cm , 1508 cm for (1) and (2)
-1
isomers respectively. Additional founded bands in the 360 to 420 cm range of the Ru–N bond.
6
8
-1
-1
According to literature data, bands of (Ru–N) appear in the 370–380 cm and 450–475 cm
-1
69
range. The letter region is a very close range (440–455 cm ) of the bands Ru–P. The (Ru–P)
-1
bond vibrations in spectra of (1) and (2) complexes were found at 442 and 448 cm , respectively.
Frequency positions of the (Ru–N) infrared bands located in the lower frequency and lower
intensity than (Ru–P). It is probably caused by the high electron density on the metal atom of the
Ru–P bond. The other characteristic bands related to triphenylphosphine (around 1432, 1089, 741,
−
1
6
95, 519 cm ) are also noticeable in both isomers spectra and confirm the presence of PPh in the
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ruthenium coordination sphere.69-71 The presence chloride (σ/π donor ligand), decreases the
electron density on the ruthenium centre and, therefore, the (Ru–Cl) vibrations appeared in the
-1
71
region of 130–350 cm , the lowest frequencies of the metal-ligand bonds in (1), (2) complexes.
Moreover, the presence of water molecules in the crystal structure of the (2) complex is confirmed
by the broad O–H stretching band at 3451 cm and at 1741 cm-1 72, confirmed.
-1
Magnetism and EPR
At room temperature (295 K), the mer-[RuCl (PPh )(dmpbt)] (1) and fac-
3
3
[
RuCl (PPh )(dmpbt)]·H O (2) complexes have a magnetic moment equal to 1.99 and 2.02 B.M.,
3 3 2
respectively, which are typical of one unpaired electron systems, and the values are consistent with
5
low-spin d ruthenium(III) in an octahedral environment. The μ are somewhat higher than the
eff
2
expected spin-only value of 1.73 B.M. The orbitally degenerate T (O ) ground state is split due
2
g
h
to spin-orbit coupling and low symmetry ligand field effects.
The EPR spectrum of (1) in the solid state (Figure S3) exhibited three distinct resonances
characteristic of a rhombic symmetry S  ½ spin system with g  2.48, g  2.26, and g  1.77.
1
2
3
The EPR spectrum of (2) exhibited two distinct resonances characteristic of an axial symmetry S
½ spin system with g  2.27 and g  1.82. However, because of its poorer quality, the small



rhombic component cannot be excluded. The difference between the EPR spectra of (1) and (2)
isomers is similar to that observed in mer- and fac-[RuCl (As(Ph ) ], which show rhombic and
3
3 3
axial spectra, respectively. 73 Hence, the EPR data are characteristic of ruthenium(III) present in a
distorted octahedral ligand environment as observed in the crystal structure of both isomers with
larger deformation in the mer isomer.
UV–vis
1
8

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The electronic spectra of mer-[RuCl (PPh )(dmpbt)] (1) and fac-[RuCl (PPh )(dmpbt)]·H O (2)
3
3
3
3
2
isomers and ligands were recorded in an acetonitrile solution. The mer-[RuCl (PPh )(dmpbt)]
3
3
isomer exhibits intense high-energy absorption overlapping bands in the range from 200 to 280
nm with shoulders at 257 and 275 nm, weaker absorptions at 305 and 388 nm with a low-energy
3
1
shoulder at 465 nm (molar coefficients are 21400, 13800, 15600, 3200, and 640 dm mol

1
cm , respectively). The (1) isomer shows strong high-energy absorptions in the range from 200
to 290 nm with shoulders at 257 and 276 nm (spectra were collected for a saturated solution and
molar coefficients are not calculated.). Weaker bands with peak maxima at 308 and 589 nm with
shoulders at 374 and 476 nm are also observed. For both isomers the absorptions in the ultraviolet
region below 350 nm are very similar and are attributed to the transitions within the ligand orbitals
(
* and n*), of the PPh and dmpbt ligands. The absorptions observed in the region of 388–
3
3
74 nm are probably due to ligand-metal charge transfer transitions taking place from the filled
ligand orbitals to the singly-occupied ruthenium orbital. The low energy absorptions in the region
of 465–589 nm are attributed to dipole-forbidden d–d transitions of the ruthenium(III) ion in a
distorted octahedral environment.74
As expected, the electronic spectrum of fac-[RuCl (PPh )(dmpbt)]·C H OH (2’) recorded in
3
3
2
5
acetonitrile solution is identical, within experimental error, to the spectrum of (2).
X-ray
Hereby, we report the structures of the two geometrical isomers formulated as mer-
[
RuCl (PPh )(dmpbt)] (1) and fac-[RuCl (PPh )(dmpbt)]·H O (2). Their corresponding selected
3 3 3 3 2
bond lengths and angles are given in Table 2.
The fac isomer was also obtained as fac-[RuCl (PPh )(dmpbt)]·C H OH (2’), structural data for
3
3
2
5
which, description and short comparison with (2) are given in the Supporting Information (Table
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9

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S3, Table S4, and Figure S4). The minor product, mer-[RuCl (PPh )(dmpbt)] (1), crystallised in
3
3
the monoclinic P2 /n space group with all atoms in general positions and the whole molecule given
1
by the formulae in the asymmetric unit (Figure 1). The crystal date and refinement for (1) and (2)
are presented in Table 1.
Fig. 1 Molecule of mer-[RuCl (PPh )(dmpbt)] (1) with the numbering scheme and the thermal ellipsoids at
3
3
2
0% probability.
Table 2. Selected bonds lengths (Å) and angles (º) for (1) and (2)
Complex
(1)
(2)
Distances (Å)
Ru1–N1
Ru1–N14
Ru1–Cl4
Ru1–P1
Ru1–Cl2
2.105(3)
Ru1–N14
Ru1–N1
Ru1–Cl2
Ru1–Cl3
Ru1–P1
2.083(9)
2.118(8)
2.326(3)
2.326(3)
2.374(4)
2.172(3)
2.3247(10)
2.3368(9)
2.3413(9)
2
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Ru1–Cl3
2.3458(9)
Ru1–Cl1
2.438(4)
Angles (º)
N1–Ru1–N14
N1–Ru1–Cl4
N14–Ru1–Cl4
N1–Ru1–P1
75.78(13)
171.39(9)
95.90(10)
103.82(9)
174.92(9)
84.67(4)
84.56(8)
90.93(9)
93.47(4)
94.07(3)
87.92(8)
84.04(9)
93.48(4)
90.90(3)
171.80(3)
N14–Ru1–N1
N14–Ru1–Cl2
N1–Ru1–Cl2
N14–Ru1–Cl3
N1–Ru1–Cl3
Cl2–Ru1–Cl3
N14–Ru1–P1
N1–Ru1–P1
77.5(4)
172.3(3)
95.6(2)
96.5(3)
N14–Ru1–P1
Cl4–Ru1–P1
N1–Ru1–Cl2
N14–Ru1–Cl2
Cl4–Ru1–Cl2
P1–Ru1–Cl2
N1–Ru1–Cl3
N14–Ru1–Cl3
Cl4–Ru1–Cl3
P1–Ru1–Cl3
Cl2–Ru1–Cl3
173.9(2)
90.39(12)
91.3(3)
93.1(3)
Cl2–Ru1–P1
Cl3–Ru1–P1
N14–Ru1–Cl1
N1–Ru1–Cl1
Cl2–Ru1–Cl1
Cl3–Ru1–Cl1
P1–Ru1–Cl1
92.60(13)
86.09(12)
85.3(3)
89.1(3)
91.06(13)
91.30(12)
175.51(13)
In (1) the ruthenium coordination sphere adopts a slightly distorted octahedral environment. It
consists of two nitrogen atoms from the organic ligand, a phosphorous atom from
triphenylphosphine and three chloride ligand occupying mer positions. The N14 pyrazole atom is
positioned trans to the phosphorous atom, whereas the N1 atom is positioned trans to the Cl4
chloride anion. The angles between these trans positioned atoms are close to 180º and range from
1
1
71.39(9) to 174.92(9)º. The cis positioned atoms form angles ranging from 75.78(13) to
03.82(9)º. Distances in the coordination sphere fall into two ranges: shorter bonds were found for
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nitrogen atoms [2.105(3) and 2.172(3) Å] and longer for phosphorous [2.3368(9) Å] and chlorine
atoms [from 2.3247(10) to 2.3458(9) Å]. All rings remain flat within an rms deviation of 0.028 Å
observed for the N1 benzothiazole ring. The chelate ring is the only folded ring with an rms of
0
.118 Å. The C2 and N1 rings form the smallest dihedral angle of 5.3(2)º. However, four fused
rings (C2, N1, chelate and N10) are relatively coplanar with the biggest angle being 15.51(18)º.
The three phenyl rings are twisted and the angles between them are 52.13(19), 70.6(2) and 83.0(2)º.
This mutual orientation of these phenyl rings results from intramolecular C–H…Cl hydrogen
bonds and steric hindrance. Additionally, this conformation is influenced by numerous
intramolecular interactions formed between aromatic rings, mainly involving the C31 phenyl
group. In the packing the shortest two Ru(III)–Ru(III) contacts (7.436 and 8.367 Å) occur for
zigzags running along the a axis (Figure 2).
Fig. 2 Packing of (1) along a axis with marked C–H…Cl hydrogen bonds (dashed green lines).
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2

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Weak interactions usually maintain the crystal network. There are only two intermolecular
hydrogen bonds: C15–H15B…Cl4(1/2–x, –1/2+y, 1/2–z) and C35–H35…Cl2(–1/2+x, 1/2–y,
1
/2+z) formed with molecules from adjacent zigzags translated along the c axis. Apart from these
hydrogen bonds we found numerous – interactions. The closest ruthenium ions can interact via
– interactions formed by the significantly inclined C31 phenyl ring (PPh ) and rings from dmpbt.

3
In contrast, the interactions between molecules related by (1–x, –y, 1–z) are assured by stacking
interactions formed by organic ligands. Interactions between adjacent zigzags are maintained by

– interactions between edge-to-face or edge-to-edge oriented rings.
Fac-[RuCl (PPh )(dmpbt)]·H O (2), crystallised in the triclinic P–1 space group with all atoms
3
3
2
in general positions and the whole molecule is given by the formulae in the asymmetric unit
Figure 3). The refinement details are presented in Table 1.
(
Fig. 3 Molecule of (2) with the numbering scheme and the thermal ellipsoids at 20% probability. O51 and
O52 partially occupied crystallization water molecules are omitted for clarity.
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3

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In (2) the ruthenium coordination sphere adopts a slightly distorted octahedral environment. It
consists of two nitrogen atoms from the organic ligand, a phosphorous atom from
triphenylphosphine and three chlorido ligands occupying fac positions. Cl1, Cl2 and Cl3 chlorine
atoms are trans positioned to P1, N14 and N1 atoms, respectively. The angles between the trans
positioned atoms range from 172.3(3) to 175.51(13)º. The cis positioned atoms form angles
ranging from 77.5(4) to 96.5(3)º. The smallest value is observed for the chelate ring. Distances in
the coordination sphere fall into two ranges: shorter bonds were found for nitrogen atoms [2.083(9)
and 2.118(8) Å] and longer for phosphorous [2.374(4) Å] and chlorine atoms [from 2.326(3) to
2
.438(4) Å]. The benzothiazole ligand is flat within an rms deviation of 0.085 Å and even the
chelate ring remains flat within rms deviation of 0.049 Å. The C2 and chelate rings form the
smallest dihedral angle of 1.09º. The whole ligand remains planar and the biggest angle between
its rings is 12.52º for N10 and S8 rings. Three phenyl rings from triphenylphosphine are planar
within 0.012 Å. They are twisted and the angles between them are 49.98, 73.16 and 89.27º. The
smallest value is found for C21 and C41 phenyl rings, whereas C21 and C31 rings are oriented
perpendicularly. Mutual orientation of the phenyl rings results from intramolecular C–H…Cl
hydrogen bonds and steric hindrance as well as numerous intramolecular interactions formed
between aromatic rings, involving mainly the C41 phenyl group. In the packing we observe ab
layers composed of zigzags running along the b axis. Ru–Ru distances in the zigzag are 8.057 and
8
.705 Å, whereas the closest Ru–Ru distance between zigzags is 8.550 Å (Figure 4).
2
4

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Fig. 4 Packing of (2) along b axis with marked intermolecular C–H…Cl hydrogen bonds (dashed green
lines).
The separation between ruthenium ions coming from different layers is much longer and exceeds
1
1 Å. In the zigzag molecules are connected by numerous – interactions, mainly by
benzothiazole moieties (S8 and C2 rings) which are almost coplanar forming stacking interactions.
They seem to be strong due to the mutual orientation of the rings and its small separation (in a
consequence the slippage is also very small). Additionally, the moieties in the chain are connected
with the C2 ring via C15–H15… interactions. The layer is formed by the only intermolecular
C15–H15A…Cl1[–1+x, y, z] hydrogen bond and – interactions between almost perpendicularly
oriented C21 and C31 phenyl rings from the triphenylphosphine molecule. Interactions between
adjacent layers are assured by – interactions between two edge-to-edge oriented C21 phenyl
rings and stacking interactions between C31 phenyl rings. In the latter case these rings form a
zipper running along the b axis. Water molecules are loosely packed in the space between adjacent
layers. There are no hydrogen atoms belonging to the O51 and O52 crystallisation water
molecules. However, it seems they are not involved in essential network interactions.
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Both isomers reveal similar geometry. However, careful inspection of Table 1 shows that there
are significant differences, such as inversion of Ru–N distances. In the mer form the Ru–N14
(pyrazole) bond is much longer than the Ru–N1band in this complex as well as much shorter than
the analogous bond in the fac isomer. The difference between the Ru–N14 bonds is 0.09 Å. In the
fac-Ru(III) (2), N14 is positioned trans to a chlorine atom, whereas in the mer-Ru(III) (1) isomer
it is positioned trans to phosphorous. The difference between the Ru–N1 bonds is much smaller
(0.013 Å) but in both structures these atoms are trans to chlorine atoms. In the mer (1) isomer we
observe Ru–Cl/P bonds ranging from 2.32 to 2.34, whereas in the fac (2) form two Ru–Cl distances
are 2.32 Å, but the third chlorine (Cl1) forms a very long bond (2.44 Å). This atom is positioned
trans to phosphorous. It results in mutual elongation of the Ru–Cl1 and Ru–P1 bonds, because the
latter was found to be 2.37 Å, revealing elongation by 0.04 Å according to the mer isomer. It might
be related to lower the lipophilicity of the fac (2) isomer than mer-isomer (expressed as log P). We
observed better solubility of complex (2) and can expect faster ligand substitution in the
coordination sphere. We hypothesise that longer Ru–Cl is more susceptible to splitting and this
might result from the trans effect of the phosphorous ligand (if the influence of the trans ligands
is the same as in four-coordinated square planar complexes). The chloride substitution would be
much slower for the mer (1) isomer because the trans effect expected for phosphorous is stronger.
Hence, dissociation of such a ligand is more difficult because cleavage of one bond is not enough,
the second bond still exists and the broken bond can be reformed. We found also that the fac (2)
isomer shows a slightly smaller surface (507.75 vs. 523.97 Å) and asphericity (a measure of
anisotropy, 0.025 vs. 0.037), whereas globularity is bigger (0.766 vs. 0.741). These parameters
seem to confirm the lower lipophilicity of the fac (2) isomer. The valence angles also show
significant differences, especially for atoms in the cis positions. In the fac isomer, they range from
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7
7.5(4) to 96.5(3)º and in the mer from 75.78(13) to 103.82(9)º. In the latter case the biggest value
was observed for N1–Ru1–P1. These differences might be ascribed to two factors: the trans effects
of different donor atoms and steric hindrance which should be bigger in case of fac (2) isomer
because two huge ligands (triphenylphosphine and dmpbt) – are close to each other – phosphorous
is cis to both nitrogen, whereas in the mer form it is trans to N14 and cis to N1 atoms. We can
observe this effect in the mutual orientation of rings coming from both organic ligands, whereas
the angles between rings from the same ligand are similar in both structures. The dihedral angles
between the sulphur ring and phenyl rings of triphenylphosphine are 25.89, 27.18 and 71.48º for
the fac (2) structure and 36.33, 50.47 and 61.7º for the mer (1)isomer.
Electrochemical behaviour
It is commonly believed that ruthenium(III) complexes may be active as prodrugs, which are
activated by reduction in vivo to the usually less inert Ru(II) species than ruthenium(III) forms.4,
7
5
Therefore, the reduction of ruthenium(III) to ruthenium(II) is preferable in a tumour where there
is a relatively low electrochemical potential and low pH as well as less oxygenation than in normal
tissue.75 Electrochemical properties of ruthenium compounds were defined using a cyclic
voltammetry to determine the possibility of the “activation by reduction” mechanism. The half-
+
wave potentials (E ) vs. Ag-wire reference electrode of the ferrocene (Fc)/ferrocenium (Fc )
1
/2
couple in the investigated organic solvent media have been obtained. Ferrocene undergoes a
reversible one-electron transfer process in the investigated solvent media.76 The half-wave
+
potential (E ) of the ferrocene (Fc)/ferrocenium (Fc ) couple is 0.25 V vs. Ag-wire in the 0.1 M
1
/2
LiClO /MeCN base electrolyte solutions.
4
2
7

Dalton Transactions
Page 28 of 42
View Article Online
DOI: 10.1039/C9DT01803D
The electrochemical properties of the ruthenium(III) chloride, ligands or ruthenium complex
-1
were investigated by cyclic voltammetry in an acetonitrile solution at a scan rate of 2 mV s using
0
.1 M LiClO as the supporting electrolyte. The quasi-reversible character of the electrode reaction
4
is characteristic of the Ru(III)/Ru(II) and Ru(IV)/Ru(III) couples. The anodic (E ) and cathodic
a
(
E ) potentials (V vs Ag-wire) as well as the anodic/cathodic peaks currents and charge ( i and
c
a
/
c
Q , respectively) are listed in Table 3. Additionally, the half-wave potentials characteristic (E )
a/c
1/2
and anodic/cathodic peak current relation (abs(i /i )) of all of the redox pairs were calculated and
a
c
are also presented in Table 3.
Table 3. Selected chemical properties of the ligands and complexes (CV measurements)
compound E , V ia, A g-1 ic, A g-1
Q , C E , V Q , C
E , V E, V i /i
0.063 2.62E-05 1.87E-06 –0.107 –5.45E-05 2.63E-06 –0.02 –0.17 2.08
a
a
c
c
1/2
a c
RuCl3
PPh3
-
-
-
-
-
-
-
-
-
dmpbt
mer (1)
0.066 9.68E-07 6.27E-08 –0.183 –4.24E-07 2.59E-08 –0.06 –0.25 2.28
0.049 5.71E-06 7.05E-07 –0.127 –1.20E-05 5.07E-07 –0.04 –0.18 0.47
0
.476 2.91E-06 5.48E-07 0.391 –3.01E-06 7.09E-07 0.43 –0.09 0.97
fac (2)
0.054 2.02E-05 2.27E-06 –0.142 –3.46E-05 1.76E-06 –0.04 –0.20 0.58
The electrochemistry of ligands both PPh and dmpbt undergo no or little activity in the solvent
3
limit (dashed lines in Figure 5).
2
8

Page 29 of 42
Dalton Transactions
View Article Online
DOI: 10.1039/C9DT01803D
Fig. 5 Cyclic voltammogram of RuCl and PPh and dmpbt ligands in 0.2 M n-Bu NPF in acetonitrile at a
3
3
4
6
–
1
platinum working electrode (scan rate 0.2 V s ).
mer-[RuCl (PPh )(dmpbt)] (1)
3
3
The electrochemical data are listed in Table 3. Figure 6 shows a typical cyclic voltammetry
curve recorded in the presence of the (1) complex, where two redox couples exist in the potential
range from –0.6 to 0.8 V vs. Ag wire. The anodic peak at approximately 0.05 V and cathodic peak
at a potential of approximately –0.13 V correspond to the oxidation of Ru(II) to Ru(III) and the
reduction of Ru(III) to Ru(II), respectively. This couple represents a quasi-reversible with an
anodic to cathodic peak separation (equal to 0.18 V). The second redox couple is situated in a more
positive potential range, and is associated with the reduction and oxidation of the Ru(IV)/Ru(III)
couple (cathodic peak 0.39 V and anodic peak 0.48 V). The reactions of these couples are
reversible. However, in the case of the first scan, only the second redox couple was observed,
namely a Ru(IV)/Ru(III) pair, and a poorly formed cathodic peak (E = –0.15 V) of the reduction
c
of Ru(III) to Ru(II).77
2
9