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(Pyridyl)benzoazole ruthenium(III) complexes: Kinetics of ligand substitution reaction and potential cytotoxic properties
(Pyridyl)benzoazole ruthenium(III) complexes: kinetics of ligand substitution reaction
and potential cytotoxic properties
Reinner Ochola Omondi, 1 Deogratius Jaganyi*2 Stephen Otieno Ojwach, 1 Amos Akintayo
Fatokun3
1
School of Chemistry and Physics, University of KwaZulu-Natal, Private Bag X01, Scottsville,
Pietermaritzburg, 3209, South Africa
2
School of Science, College of Science and Technology, University of Rwanda, P.O. Box 4285,
Kigali, Rwanda
3
School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores
University, Liverpool L3 3AF, UK
*Deogratius Jaganyi:deojaganyi@gmail.com
Highlights
Ruthenium(III) complexes were successfully synthesised.
The substitution of the coordinated chloro ligands happened simultaneously.
Density functional theoretical calculations supports substitution kinetic results.
Complexes demonstrate low antineoplastic properties against Hela cancer cells.
Molecular docking of the compounds demonstrate good affinity for DNA molecule.
1
Graphical abstract
In all substitution kinetics reactions, a single step, considered to be the displacement of the
coordinated chlorides was seen with no solvation pathway. Due to the symmetrical nature of
the investigated ruthenium(III) complexes, the three chloride ligands are equally substituted
simultaneously. The docking experiments reveal that the binding energies of the complexes
have direct correlation to their kinetic stability.
Keywords: ruthenium(III) complexes; substitution kinetics; density functional theory;
molecular docking; cytotoxicity; anti-cancer activities
Abstract
The present work investigates the kinetics of ligand substitution reaction and anticancer
activities of the complexes, [{2-(2-pyridyl) benzimidazole} RuCl3] (C1), [{2-(2-pyridyl)
benzoxazole} RuCl3] (C2), [{2-(2-pyridyl) benzothiazole} RuCl3] (C3) and [{1-propyl-2(pyridin-2-yl)-H-benzoimidazole} RuCl3] (C4). The substitution kinetics reaction of the
complexes with the three bio-relevant nucleophiles, viz.: thiourea (TU), 1, 3-dimethyl-2thiourea (DMTU) and 1, 1, 3, 3-tetramethyl-2-thiourea (TMTU) was investigated under
2
pseudo first-order conditions as a function of concentration and temperature using UV-Visible
spectrophotometer. The substitution of the coordinated chloride was controlled by the
electronic effect. The order of reactivity of the complexes with the nucleophiles is in the form
C1 > C2 > C3 > C4 which is in line with the density functional theory (DFT) studies. The
complexes showed minimal anticancer activity against the HeLa cell line, which is in contrast
to the molecular docking experiments that exhibited stronger DNA binding affinities.
Introduction
The documented clinical limitations of cisplatin and its second and third generation analogues
have triggered the search for other anticancer agents containing metals other than platinum.
Metals such as iridium, gold, cobalt, ruthenium and silver have so far shown good cytotoxicity
[1-4]. These complexes appear to possess markedly lower toxicities, high tumour selectivity
and enhanced solubility. Among the non-platinum metal compounds, ruthenium complexes
seem to be the most promising class due to their structural and coordination properties. These
properties have been considered in a number of excellent reviews [5-8] and they include (i)
accessibility to multiple oxidation states at physiological conditions, which makes them
suitable for pharmaceutical use; (ii) octahedral geometry which differs from the square-planar
molecular geometry of platinum(II) compounds, this allows for tuning of the steric and
electronic properties of the complexes; (iii) a rate of ligand substitution kinetics that is close to
those of cellular processes, giving the drug high kinetic stability and hence reducing side
reactions; (iv) the ability of ruthenium metal to mimic iron in binding to serum transferrin and
albumin proteins, which transports and solubilises iron in cytoplasm and thereby exploiting the
body’s mechanism of nontoxic delivery of iron.
3
Over the years, most research groups have evaluated ruthenium complexes for their potential
to inhibit the growth of tumours. The key focus has been on the interaction between active
ruthenium compounds and their possible targets such as RNA and DNA [9-10]. It is widely
accepted that the antineoplastic properties of ruthenium(II) and ruthenium(III) compounds is
directly related to their ability to bind to DNA [11], although a few exceptions have been also
reported [12]. Contributing to this field of study, Sadler [13-14] and Dayson [15-16] have
developed structure-activity relationship using monofunctional compounds [Ru(η6C6H6)(en)(Cl)]+. The general mechanism for these half-sandwich ruthenium(II) arene
complexes encompasses rapid loss of the choro ligand with the formation of more reactive aqua
species, followed by the substitution kinetics of the labile aqua ligand by DNA and RNA.
In the recent years, Bugarčić and his co-workers [17-21] have examined the anticancer
activities of a series mononuclear ruthenium(II) chlorophenyl terpyridine complexes and
establishing their rate of substitution kinetics using L-histidine (L-His) and guanine derivatives
(i.e., 9-methylguanine (9MeG) and 5′-GMP). They found that the rates of substitution kinetics
of these complexes strongly depend on the nature of the inert chelating ligand on their inductive
effect and the steric hindrance including the charge of the entering nucleophile.
Two ruthenium(III) complexes, KP1019 and NAMI-A, are presently among the most
successful drug candidates to enter human clinical trials [22-23]. The preliminary
investigations of the two ruthenium compounds demonstrate that they might be promising in
the treatment of drug-resistant tumours, as they are effective against cancerous cell lines that
are highly resistant to other anticancer agents such as cisplatin and doxorubicin. Despite, the
success of these two ruthenium compounds, their mode of mechanism remains a matter of
debate. The in vitro cytotoxic properties of a series of ruthenium(III) complexes with
4
dithiocarbamato ligands towards a panel of human tumour cell lines have been investigated
[24-26]. The biological assays of most of these complexes on human tumour cell lines point
out intriguing selectivity and antiproliferative properties.
Building on the existing knowledge we have investigated the reactivity of mononuclear
ruthenium(III) complexes by varying the identity of the heteroatoms on the inert ligand
architecture. We have also examined their antineoplastic properties. Hence in this contribution
we report the substitution kinetics and anticancer activities of ruthenium(III) complexes
supported by 2-(2-pyridyl)benzoazole ligands with
thiourea nucleophiles viz; TU, DMTU,
TMTU. Thiourea nucleophiles act as good model compounds for thioether and thiolate which
are present in the cytoplasm. The binding of metal complexes with S-containing biomolecules
is accountable for the occurrence of toxic effects. The structures of the complexes and
nucleophiles used in this study are shown in Scheme 1 and 2, respectively. The experimental
work is supported by computational density functional theory (DFT) studies.
Scheme 1: Structures of the investigated mononuclear (pyridyl)benzoazole ruthenium(III)
complexes.
5
Scheme 2: Structures of the examined thiourea nucleophiles and its derivatives
Experimental
Chemicals
All synthetic procedures were carried out under a nitrogen atmosphere using standard Schlenk
techniques. Ruthenium(III) chloride hydrate (RuCl3.xH2O, 99.98%) was purchased from
Merck and used as supplied. Methanol was obtained from Merck and distilled over magnesium
prior to use. Sodium perchlorate monohydrate (NaClO4.H2O, 98%), lithium chloride (LiCl,
99%), thiourea (TU, 99%), N,N-dimethyl-2-thiourea (DMTU, 99%), N,N,N,N-tetramethyl-2thiourea (TMTU, 98%) were all obtained from Sigma-Aldrich and used as supplied.
Characterisations and instrumentations
Thermal Scientific Flash 2000 and a Carlo Erba Elemental Analyzer 1106. Mass spectrometry
measurements were recorded on an LC Premier micro-mass spectrometer. Kinetic experiments
were performed on Varian Cary 100 Bio UV-Visible spectrophotometer with an attached
Varian Peltier temperature-controller and an online kinetic applications and the temperature of
the instrument was controlled within ± 0.1 °C.
6
Preparation of (pyridyl)benzoazole ligands
The following ligands, namely, 2-(2-pyridyl)benzimidazole (L1), 2-(2-pyridyl)bezoxazole
(L2), 2-(2-pyridyl)bezothiazole (L3) and 1-propyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole
(L4) were synthesised for this investigation. The ligands, L1-L3 were all synthesised using a
method previously described in literature [27]. For the preparation of L4, this method was
slightly modified as described by Ogweno et al [28].
Synthesis of (pyridyl)benzoazole ruthenium(III) complexes
Synthesis of [{2-(2-pyridyl) benzimidazole}RuCl3] (C1)
Complex C1 was synthesised according to our published literature procedure [28]. A mixture
of ligand L1 (0.09 g, 0.48 mmol) and RuCl3.3H2O (0.10 g, 0.48 mmol) was refluxed in absolute
ethanol (20 mL) for 4 h. A brown precipitate was collected by filtration, washed thoroughly
with excess ethanol and dried to obtain complex C1 as a brown solid. Yield = 0.08 g (38%).
TOF MS-ES, m/z (%) 402.07, (M+ + H). Anal. Calcd (%) for C12H9Cl3N3Ru: C, 35.80; H, 2.25;
N, 10.44. Found (%): C, 35.98; H, 2.19; N, 10.66.
Synthesis of [{2-(2-pyridyl)benzoxazole}RuCl3] (C2)
Complex C2 was prepared following the synthetic procedure for C1, using ligand L2 (0.21 g,
0.98 mmol) and RuCl3·3H2O (0.20 g, 0.98 mmol) to afford a brown solid. Yield = 0.395 g
(31%). TOF MS-ES, m/z (%) 404.12, (M+ + H). Calcd (%) for C12H8Cl3N2ORu: C, 35.71; H,
2.00; N, 6.94; O, 3.96. Found (%): C, 35.41; H, 1.78; N, 7.03; O, 4.06.
Synthesis of [{2-(2-pyridyl)benzothiazole}RuCl3] (C3)
The procedure used in the synthesis of complex C1 was used in the preparation of C3, using
ligand L3 (0.21 g, 0.98 mmol) and RuCl3·3H2O (0.20 g, 0.98 mmol) to yield a light brown
7
solid. Yield = 0.411g (76%). TOF MS-ES, m/z (%) 420.09 (M+ + H). Calcd (%) for
C12H8Cl3N2RuS: C, 34.34; H, 1.92; N, 6.67; S, 7.64. Found (%): C, 34.63; H, 1.02; N, 6.92; S,
7.47.
Synthesis of [{1-propyl-2-(pyridin-2-yl)-H-benzoimidazole}RuCl3] (C4)
This complex was synthesised following the synthetic procedure described for complex C1,
using ligand L4 (0.21 g, 0.98 mmol) and RuCl3·3H2O (0.20 g, 0.98 mmol) to produce complex
C4 as a brown solid. Yield = 0.09 g (43%). TOF MS-ES, m/z (%) 408, (M+ + -Cl). Calcd (%)
for C15H15Cl3N3Ru: C, 40.51; H, 3.40; N, 9.45. Found (%): C, 40.09; H, 3.08; N, 9.64.
Preparation of nucleophile solutions for kinetic measurements
The stock solutions of nucleophiles, viz. thiourea (TU), 1,3-dimethyl-2-thiourea (DMTU) and
1,1,3,3-tetramethyl-2-thiourea (TMTU) were prepared shortly before use by dissolving known
amounts of the nucleophiles in 0.1 M (adjusted with NaClO4 and LiCl) methanol solutions to
afford a concentration of approximately 150 times greater than that of the metal complex. The
other nucleophile solutions were prepared by subsequent dilutions of the same stock solutions
to obtain a series of standards of 120, 90, 60 and 30 times the concentration of the metal
complex. The NaClO4 was used because perchlorate ions do not bind to the ruthenium(III)
metal centre. In addition, LiCl was added to prevent possibility of any spontaneous solvolytic
reactions.
Kinetic procedure
The rate of substitution of the coordinated chloride from each of the four compounds of
ruthenium(III) was investigated using a series of neutral sulphur-donor nucleophiles viz. TU,
DMTU and TMTU using UV-Vis spectrophotometer. All kinetic investigations were
8
conducted under pseudo-first order conditions, in which the concentration of the incoming
nucleophile was at least 30-fold excess of the complexes so as to force the substitution reactions
to completion [29]. The working wavelengths were pre-determined by recording spectra of the
reaction mixture over the wavelength range 200–850 nm to establish suitable wavelengths at
which their kinetic measurements could be investigated.
Computational modelling
Computational modelling was performed by the Gaussian09 program package to gain a clear
understanding of the electronic and structural differences of the investigated compounds [30].
The geometry optimisations, frequency calculations, and mapped molecular orbital diagrams
were carried out at density functional theoretical (DFT) level based on B3LYP/LanL2DZ (Los
Alamos National Laboratory 2 double ξ) level theory [31-32]. The singlet state was adopted
due to the low electronic spin of (pyridyl)benzoazoles ruthenium(III) compounds. The
compounds were computed with methanol solvent, considering the solvolysis effects by means
of the Conductor Polarizable Continuum Model [33-34].
Cytotoxicity assay
The human cervical carcinoma HeLa cells were used for the cytotoxicity experiments as
previously reported [35]. They were grown in 75 cm2 tissue culture flasks using Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% Foetal Calf Serum, 2 mM Lglutamine, 1% non-essential amino acid and 1% antibiotic-antimycotic solution. They were
incubated at 37 oC in a humidified atmosphere of 5% CO2. At about 80-90% confluency, the
medium was removed and cultures were rinsed with pre-warmed PBS, trypsinised and seeded
into micro-clear, flat-bottom 96-well plates at a density of 5 x 104 cells per ml (100 µl/well).
After 24 h, a range of concentrations up to 400 µM was prepared from the DMSO stock
9
concentration of each tested compound and added to the cultures, each in triplicate (the final
highest concentration (v/v) of DMSO in the growth medium that the cultures were exposed to
was confirmed not to affect cell viability), and plates were incubated for 48 h, after which
viability was assessed using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide [MTT] assay [36]. This was done by adding to each well 10 µl of the MTT solution
(5 mg/ml, prepared in sterile PBS) before incubating the plates for 3 h. Thereafter the content
of each well was aspirated and 100 µl of DMSO was added to dissolve the insoluble formazan.
The absorbance of each well was then read at a wavelength of 570 nm using the CLARIOstar
plate reader (BMG Labtech). Changes to the morphology of the cells caused by the treatments
were monitored on an Olympus CKX41 microscope fitted with an Olympus DP71 U-TVIX-2
camera. Images were captured using the Olympus cellSens entry software.
For each treatment the mean of the triplicate values was calculated. The mean of the control
cultures (cultures not exposed to treatment with any compound) was then set to 100% and the
mean viability for each concentration normalised to it. Values are represented as Mean ± SEM
(standard error of the mean). Statistical analyses were done using GraphPad (Version 7.03,
GraphPad Software, Inc., CA, USA). Statistically significant differences between the means
were determined using analysis of variance (ANOVA) followed by a post-hoc test for multiple
comparisons (Tukey test). A P-value of less than 0.05 was considered statistically significant.
IC50 for each compound was determined using GraphPad by fitting the data to the non-linear
regression of log [inhibitor] versus response (3 parameters or 4 parameters (variable slope)).
Molecular docking experiment
Molecular docking studies were performed using HEX 8.0.0 software, which uses spherical
polar Fourier correlations to accelerate the calculations. All calculations were carried out on an
10
Intel Core i3-4170 CPU using the following parameters; correlation type: (shape + electro);
search order: 25; receptor/ligand: 180; step sizes: 7.5; grid dimension: 0.6; number of solutions:
2000. The structures of the compounds were sketched by CHEMSKETCH [37] and converted
to PDB format from MOL format using Mercury software [38]. The crystal structure of the BDNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was downloaded from the protein
data bank [39]. All water molecules were removed before docking calculations with the
examined compounds. The docked poses were visualised using UCSF Chimera software [40].
Results
Kinetic analyses
All substitution kinetics experiments showed that all reactions were characterised by a single
step that could be studied using UV-Visible spectroscopy. A typical Spectral changes and
kinetic trace obtained by monitoring the reaction between TU and C3 as shown in Figure 1.
Figure 1: Spectral changes and kinetic trace obtained from the UV-Visible spectrometer with
a single exponential fit for the reaction between C3 (5.0 x 10-4 mol dm-3) and TU (1.5 x 10-2
11
mol dm-3) in methanol followed at 310 nm, I = 0.1 M (0.01 M LiCl, 0.09 M NaClO4), T = 298
K.
All the kinetic traces for the substitution reactions provided excellent fits to first-order
exponential decay to produce, pseudo-first-order rate constants, kobs, at specific concentrations
of the nucleophiles and temperature. The kobs, were established by a nonlinear least square fit
of the absorbance-time data to Equation 3.1 using Origin 7.5® software graphical analysis
software [41].
At = Ao + (Ao - A∞) e (-kobs.)t
(1)
where Ao, At and A∞ represent absorbance of the mixture of the reaction at the start of the
reaction at time, t and at the end of the reaction, respectively. The dependence of the rate
constant on the concentration of the incoming nucleophiles was analysed for all the examined
nucleophiles while keeping the temperature at 25 °C. The kobs was calculated as a mean of at
least three independent kinetic experiments. The second-order rate constant, k2, for the reactions
of the ruthenium(III) complexes with the incoming nucleophiles were attained from the slopes
of the graphs of kobs versus the concentration of the incoming nucleophile using Origin 7.5®
software. Representative plots for complex C1 are shown in Figure 2. In all cases, the reactions
displayed a first-order dependence on the concentration of the incoming nucleophile.
12
0.00025
kobs, s-1
0.00020
TU
DMTU
TMTU
0.00015
0.00010
0.00005
0.00000
0.000
0.002
0.004
0.006
0.008
0.010
[Nu], M
Figure 2: Concentration dependence of kobs for the substitution of chlorides from C1 (1.0 x 104
M) by thiourea nucleophiles in methanol solution, I = 0.1M (0.01 M LiCl, 0.09 M NaClO4)
at 298 K.
The plots gave straight line fits with zero intercept for each of the investigated nucleophiles.
Therefore, the proposed mechanism of substitution is as shown in Scheme 3 and the
corresponding rate law is represented by Equation 1, showing that the kinetic pathway is
irreversible and does not depend on the solvent. The values of the second-order rate constant
k2, which were obtained from the slopes of these plots at 25 °C are summarised in Table 1.
Scheme 3: Proposed substitution mechanism for the ruthenium complexes supported by 2-(2pyridyl)benzoazole ligands
13
kobs = k2 [nucleophile]
(1)
Table 1: Summary of the rate constants and activation parameters for the substitution of
chloride by TU, DMTU and TMTU in methanol solution, I = 0.1 M (0.01 M LiCl, 0.09 M
NaClO4) at 298 K
Complex
Nu
TU
k2/M-1s-1
(2.47 ± 0.02) ×10-2
ΔH≠/kJmol-1
65 ± 1
ΔS≠/Jmol-1K-1
-43 ± 3
DMTU
(1.67 ± 0.03) ×10-2
76 ± 1
-11 ± 4
TMTU
(9.7 ± 0.05) ×10-3
78 ± 1
-20 ± 3
TU
(1.33 ± 0.04) ×10-2
68 ± 1
-48 ± 3
DMTU
(9.0 ± 0.02) ×10-4
83 ± 2
-25 ± 5
TMTU
(7.0 ± 0.03) ×10-4
84 ± 1
-24 ± 3
TU
(4.6 ± 0.02) ×10-3
64 ± 1
-60 ± 3
DMTU
(5.0 ± 0.02) ×10-4
84 ± 2
-27 ± 6
TMTU
(2.0 ± 0.01) ×10-4
85 ± 1
-37 ± 4
TU
(2.0 ± 0.01) ×10-3
69 ± 1
-74 ± 3
DMTU
(4.0 ± 0.01) ×10-4
88 ± 1
-13 ± 3
TMTU
(1.0 ± 0.01) ×10-4
90 ± 1
-19 ± 3
C1
C2
C3
C4
The second order rate constant k2, was also determined as a function of temperature, over a
temperature range of 25-45 °C at intervals of 5 °C to obtain the activation parameters for
substitution of the coordinated chlorides from ruthenium(III) complexes. The enthalpy of
activation (ΔH≠) and entropy of activation (ΔS≠), were calculated using Eyring equation [42].
The values of ΔS≠ and ΔH≠ were calculated from the intercepts and slopes of Eyring plots,
14
respectively. These parameters are tabulated in Table 1. Representative Eyring plots obtained
for the reactions of C1 with the three nucleophiles are shown in Figure 3.
-5
TU
DMTU
TMTU
-6
In(k2/T)
-7
-8
-9
-10
0.00310
0.00315
0.00320
0.00325
0.00330
0.00335
0.00340
1/T, K-1
Figure 3: Eyring plot obtained for the substitution of chloride from C1, by thiourea
nucleophiles I = 0.1 M (0.01 M LiCl, 0.09 M NaClO4) in methanol at various temperatures in
the range 25 - 45 °C.
Computational calculations
Computational analyses of the ruthenium(III) complexes were determined in order to help
explain the kinetic trends observed and the influence of the molecular structures as well as the
electronic properties of the complexes on the observed reactivity. Table 2 shows an extract of
the geometry optimised structures, LUMO and HOMO frontier molecular orbitals for the
complexes while Table 3 shows a summary of the corresponding properties of the frontier
molecular orbitals: HOMO-LUMO, NBO atomic charges and bond lengths.
15
Table 2: DFT frontier molecular orbitals and minimum energy structures of ruthenium(III)
complexes C1-C4 as established by density functional theory using B3LYP level of theory
and LANL2DZ basis set
Geometry-optimised
structure
C1
HOMO map
LUMO map
C2
C3
C4
16
Planarity
Table 3: Summary of the data obtained from the DFT calculations for the ruthenium(III)
complexes
C1
C2
C3
C4
LUMO /eV
HOMO /eV
ΔE /eV
Chemical hardness (η)
Electronic chemical potential (μ)
Electrophilicity index (ω)
NBO Charges
Ru
X
N1
Bond Length ( Å)
Ru-Cl bond length a
-3.67
-6.79
3.12
3.12
-5.23
4.38
-3.82
-6.96
3.14
3.14
-5.39
4.63
-3.80
-6.97
3.16
3.16
-5.39
4.60
-2.90
-6.74
3.83
3.83
-4.82
3.03
0.354
-0.394
-0.382
0.348
-0.251
-0.347
0.344
0.358
-0.313
0.316
-0.170
-0.310
2.43
2.42
2.40
2.38
Ru-N1
2.06
2.07
2.11
2.30
a
NBO = natural bond orbital, average Ru-Cl bond length of three chlorides.
In vitro cytotoxicity testing to assess potential anti-cancer activity
All the complexes were screened for their in vitro cytotoxicity against the HeLa cell, with
doxorubicin used as a standard reference. The effects of doxorubicin (up to 20 µM) and the
complexes (each up to 400 µM) on cell viability were determined after cells were exposed to
them for 48 h. Doxorubicin decreased HeLa cell viability in a concentration-dependent manner,
with an IC50 of 0.8 ± 0.2 µM, while the complexes did not cause significant reductions in cell
viability at the concentrations tested, except that C2 at 400 µM reduced viability to 82.6 ± 0.3%
(P<0.05) (Table 4).
17
Table 4: In vitro cytotoxic activity results (IC50 µM) of ligands and complexes against Hela
cella
Compound
IC50 (in µM)
b
Doxorubicin
0.8
>200
C1
>200
C2
>200
C3
>200
C4
a
IC50 values obtained by MTT assay results after 48 h drug exposure at various concentrations.
Results are presented as mean ± SD of three independent experiments. bReference drug.
Molecular docking analyses
The investigated compounds were docked within the DNA duplex of sequence
d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1 BNA) to determine the binding site inside the
DNA. Docked images and relative binding energies of the ruthenium(III) complexes are
summarised in Table 4.
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Table 5: Molecular docking results of ruthenium(III) complexes
Complex
Docked images of complexes
Relative
binding
energy of complexes
(kJ/Mol)
C1
-216.57
C2
-220.63
C3
-226.14
C4
-244.03
Discussion
Comparing the rates of displacement of the coordinated chloride from the ruthenium(III)
complexes, a clear general trend is observed from the reactivity data in Table 1. Using the rate
constant of C1 with TU as a reference point, the ratio of the rate of substitution of the chloro
from the complexes is 10:5:2: 1 for C1: C2: C3: C4, respectively. Therefore, the reactivity for
the substitution of the chloride ligands by the thiourea nucleophiles decreases in the order of
19
C1 > C2 > C3 > C4. A similar order of reactivity trend was observed for all the investigated
nucleophiles. The difference in reactivity is ascribed to the electronic effects of the heteroatoms
of benzoazole of the ligand architecture.
The identity of the heteroatom NH (C1); O (C2); S (C3); and N-propyl (C4) on the inert ligand
framework affects the rate of substitution of the coordinated chlorides from ruthenium(III)
complexes. The electronegativity of the heteroatoms aids in the withdrawal of electron density
from the ligand, enhancing π-back bonding and thereby creating a more electrophilic metal
centre [43]. Thus increasing the rate of the substitution kinetic reaction. Evidence of π-back
bonding is shown by the magnitude of the negative charge on the nitrogen atom attached to the
ruthenium(III) metal centre. There is an increase of the negative NBO charge of the N1 with
increase in electronegativity of the heteroatom, which is in the order NH > O > S > N-propyl.
DFT data in Table 3 show that the NBO charges of N1 decrease from C1-C4. The back donation
of electron density towards the ligand from the metal centre due to the influence of the
electronegativity of the heteroatom on the inert ligand shortens the Ru-N1 bond length, thereby
lengthening the Ru-Cl bond and thus enhancing reactivity as shown by the trend in the bond
lengths (Table 3).
Higher rate of displacement of coordinated chloride in C2 (O-atom) in comparison to C3 (Satom) is because oxygen is more electronegative than sulphur [44]. The DFT data show that
the negative NBO charges of the heteroatoms decrease from C1-C3, leading to a decrease in
the removal of electrons from the metal centre, making it more electrophilic and hence
decreasing the rate of substitution reaction. This observation is in line with the observed second
order rate constant, k2, values. The high rate of substitution of the chlorides observed for C1
(N-H) is associated with the presence of the acidic amine proton which is electron deficient
20
and hence withdraws electrons from the nitrogen atom enhancing its electron withdrawal
ability; this then increases the π-back donation effect of the ligand, thereby increasing the
electrophilicity of the metal centre and thus the reactivity.
The rate of displacement of coordinated chlorides in C4 (N-propyl) by the nucleophiles
decreased by a factor of about 10 compared to C1. Addition of the alkyl group (N-propyl in
C4), which is an electron donor, reduces electron withdrawing ability of the nitrogen atom and
consequently lowering π-back donation. This argument is supported by the DFT data that show
that the positive NBO charges of the ruthenium(III) metal centre decrease from C1-C4. The
DFT data shows that the N1-Ru bond length follows the order C1 < C2 < C3 < C4, which is
opposite to the Ru-Cl bond lengths that follow the order, C1 > C3 > C2 > C4. This observation
affirms the argument that back donation of electron density from ruthenium(III) to the ligand
shortens the N1-Ru bond and lengthens the Ru-Cl bond.
DFT calculated frontier orbitals (Table 2) demonstrate that the electron density of the HOMO
orbitals mostly lies on the ruthenium(III) metal centre and the chloride ligands while the
LUMO electron density is largely located on the ligand framework, an indication of the
influence from the metal centres due to their strong π-acceptor ability. This therefore creates a
high electron density condition in both the occupied and unoccupied orbitals, and thus the
ΔE(gap) decreases with the increase in the electronegativity of the atoms on the framework of
the ligand. The observed comparable ΔE /eV of 3.12, 3.14, 3.16 and 3.83 for C1, C2, C3 and
C4, respectively.
In all of these studies a reverse reaction was not observed. Only a single step, considered to be
the substitution of the coordinated chlorides was observed with no solvation pathway. Due to
21
the symmetrical nature of the mononuclear ruthenium(III) complexes, the three chloride
ligands are equally susceptible to substitution which happens simultaneously. Comparing the
rates of substitution by nucleophiles, TU, DMTU and TMTU these followed their steric
hindrance ability with TMTU being the most sterically hindered nucleophile hence being the
least reactive [45-46]. The observed relatively low ΔH‡ and negative ΔS‡ values is in line with
the associative mode of activated process [47-48]. The negative ΔS‡ values signifies a decrease
in disorder due to the bond formation in the transition state [49-50].
A preliminary investigation of complexes C1-C4 as potential anticancer drugs was carried out
in vitro using HeLa cells (Table 4). The anti-cancer drug, doxorubicin, which was used as
standard (reference drug), was potent in killing the cells, whereas the complexes had little
cytotoxic effect. In anti-cancer drug discovery and design, inhibition of target biomolecules
and selectivity of action are very essential. While current results have not established a
potential for the tested complexes as anti-cancer agents, our future studies will examine the
effects of the complexes on a wider panel of cancer cells and on normal cells, in order to
understand whether there exist any differential cytotoxic sensitivities to the complexes of a
variety of cells representing different cancers, consistent with the heterogeneous nature of the
pathology and also with a number of reports suggesting that HeLa cells could be resistant to
(pyridyl)benzoazole ligands and their respective ruthenium(III) complexes [52-54]. The
inactivity of the complexes C1-C4 could also be largely attributable to the low aromaticity
(low number of the conjugated rings) of the (pridyl)benzoazole ligands, as found in a previous
anti-cancer study of the ruthenium complexes [20], or to potential difficulty in their penetrating
the cells to access and overwhelm target survival machineries to induce cell death. Poor
cytotoxicity of the complexes could also be due to their relatively lower rate of substitution
kinetics [17-20].
22
The docking experiments show the best possible intercalation between DNA groove structure
and metal complexes. The most stable docked poses suggest that all investigated complexes
interact in a parallel manner with respect to the minor groove of the DNA backbone (Table 5).
The molecular docking results reveal that the investigated ruthenium(III) complexes approach
the DNA site forming stable complexes through non‐covalent interactions like hydrogen
bonding and hydrophobic and van der Waals interactions. The relative binding energies of the
complexes, summarised in Table 5, show the significant binding pattern of the complexes with
the DNA [55]. The more negative the relative binding energy, the stronger the binding between
DNA and complexes. Therefore, C4 has a stronger binding ability compared to other
complexes, which may be due to its electronic property that favours the stacking interactions
between DNA base pairs leading to hydrogen bonding and van der Waals and hydrophobic
interactions. It was observed that the binding energies are directly proportional to the kinetic
stability of the investigated complexes (the most kinetically stable complex having the lowest
second-order rate constant, k2).
Conclusion
The study clearly demonstrated that the reactivity of ruthenium(III) complexes anchored on
bidentate (pyridyl)benzoazole ligands can be systematically be tuned by varying the identity of
the heteroatoms. The order of reactivity for the substitution of the chloride ligands by the
thiourea ligands decreased in the order of C1 > C2 > C3 > C4 and this trend is due to electronic
effect which iis linked to the electronegativity of the heteroatoms. This observation is supported
by the DFT DATA. The displacement of the coordinated chlorides was also found to be
sensitive towards the steric hindrance of the incoming nucleophiles. The observed ΔH‡ and ΔS‡
supported an associative mode of activation. The studied compounds exhibit poor cytotoxicity
when compared to the reference compound, doxorubicin. The binding energies of the studied
23
complexes are inversely proportional to the rates of kinetic substitution. This study is of great
importance for the development of new ruthenium anticancer drugs with reduced side effects.
The authors gratefully acknowledge financial support from the University of KwaZulu-Natal
and the research bursary awarded to Reinner Omondi. The authors wish to thank Meshack
Sitati and Arumugam Jayamani for kinetic and molecular docking analyses, respectively.
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