Alteration of steric hindrance modulates glutathione resistance and cytotoxicity of three structurally related RuII-: P -cymene complexes

Article abstract of DOI:10.1039/c5dt04781a

The effect of steric hindrance on reactivity towards biomolecules while designing Ru^II-η⁶-p-cymene based anticancer agents seems to be an important parameter in improving the activity and inducing resistance against glutathione (GSH) deactivation. Herein we present the structure, hydrolysis, anticancer activity and the effect of steric hindrance on deactivation by glutathione for three complexes, [Ru^II(η⁶-p-cym)(L1)(Cl)](PF₆) (1), [Ru^II(η⁶-p-cym)(L2)(Cl)](PF₆) (2) and [Ru^II(η⁶-p-cym)(L3)(Cl)](PF₆) (3). The ligands L1-L3 are Schiff bases which show increasing substitution in a benzene ring, such that two ortho hydrogens are replaced by -methyl in 2 and by -isopropyl in 3. The cytotoxicity results strongly suggest that controlling the rate of hydrolysis through tuning of steric hindrance may be a feasible pathway to derive GSH resistant anticancer agents. The cellular studies show that all the three complexes show good blood compatibility (haemolysis <3%) and induce cellular death through caspase activation via the mitochondrial pathway. They have anti-angiogenic activity and prevent the healing of treated cells.

Full text of DOI:10.1039/c5dt04781a

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DOI: 10.1039/C5DT04781A
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Alteration of steric hindrance modulates glutathione resistance
and cytotoxicity of three structurally related RuII-p-cymene
complexes
Received 00th January 20xx,
Accepted 00th January 20xx
Kallol Purkait,‡ Saptarshi Chatterjee,‡ Subhendu Karmakar and Arindam Mukherjee*
DOI: 10.1039/x0xx00000x
The effect of steric hindrance on reactivity towards biomolecules while designing RuII-η6-p-cymene based anticancer agents
seems to be an important parameter in improving the activity and inducing resistance against glutathione (GSH)
deactivation. Here in we present the structure, hydrolysis, anticancer activity and the effect on steric hindrance on
deactivation by glutathione for three complexes, [RuII(η6-p-cym)(L1)(Cl)](PF6) (1), [RuII(η6-p-cym)(L2)(Cl)](PF6) (2) and
[RuII(η6-p-cym)(L3)(Cl)](PF6) (3). The ligands L1-L3 are Schiff bases which show increasing of substitution in a benzene
ring, such that two ortho hydrogens are replaced by –methyl in 2 and by -isopropyl in 3. The cytotoxicity results strongly
suggest that controlling rate of hydrolysis through tuning of steric hindrance may be a feasible pathway to derive GSH
resistant anticancer agents. The cellular studies show that all the three complexes show good blood compatibility
(haemolysis <3%) and induces cellular death through caspase activation via mitochondrial pathway. They have anti-
angiogenic activity and prevent the healing of treated cells.
www.rsc.org/
proteins11 and they may get reduced to RuII inside the cell in
presence of cellular reducing agents viz. glutathione, cysteine,
ascorbate.12-16 However, under hypoxic conditions of tumor17
Introduction
Metallo drugs have found wide use as anticancer agents.
Improving the design of a metal based anticancer agent has
certain advantages viz. higher number of accessible
coordination numbers, reversible mode of binding ability with
different kinds of electron donor species, accessible oxidation
states at normal physiological conditions1 and choice of
suitable ligands. All the above factors add up in tuning the
properties of the metal complexes.2-3 Platinum complexes are
the predominant contributors among the metal based drugs4
despite of several major drawbacks in the form of severe side
effects and induced resistance to several cancer types. During
the past few decades researchers have focused on non-
platinum complexes among which Ruthenium emerged as a
significant contributor.5-8 Ruthenium complexes are octahedral
or tetrahedral unlike the case of PtII complexes which are
generally square planar, and the tuning of electronic property
shows variation since they bear differences in electronic
configuration and geometries. Among the promising
ruthenium complexes, certain RuIII-complexes, NAMI-A,
NKP1339 have moved up to clinical trial phase-II.9-10 Ru-
complexes may be delivered to cells aided by plasma
these reducing agents can simultaneously inhibit those drugs
by forming stable bonds with the metal centre18-19 although
activation of the drugs due to glutathione is also reported.20
The literature data reveals that DNA is a potential site of action
for Ru complexes.21-22 While other possibilities like, inhibition
of the active site of an enzyme8, 23 is found to be an alternative
mode of action for RuII complexes. One reason of interest in
Ru-complexes is their relatively higher stability towards
aquation which is supposed to decrease the loss of effective
dosage due to formation of active species before reaching
target16, 24-25 and may minimize side effects compared to Pt-
complexes. In addition Ru is also thought to be interfering with
iron metabolism.11, 26
Several ruthenium(II) complexes are evaluated in vitro
against various cancerous cell lines, especially [RuII(η6-
arene)(L)Cl] class of complexes (where L = bidentate chelating
ligand) which show activity against primary tumour cells.2, 14, 27-
28
The library of work from Sadler et al. shows that activity of
these complexes can be tuned by changing the ligand (L) and
arene.28-29 To cite an example [RuII(η6-arene)(diamine)Cl]
family of complexes show that their anticancer activity is
related to the structure, hydrolytic ability,30 DNA binding31 and
also deactivation by thiolate sulfur.32 A different generation
but related to the above type of complexes is the RAPTA series
of complexes in which a phosphorus based monodentate
ligand developed by Dyson et. al. shows antimetastases
activity.33 Recent findings show that these RuII-anticancer
agents have strong affinity for the thiolate sulfur of cysteine,34
Department of Chemical Sciences, Indian Institute of Science Education and
Research Kolkata, Mohanpur campus, Pin 741246, India
Email: a.mukherjee@iiserkol.ac.in
‡These authors contributed equally.
†Electronic Supplementary Information (ESI) available: Table for crystal refinement
parameters, NMR, NMR and UV-visible spectra of hydrolysis, plots of cell viability,
cell cycle, JC-1. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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and glutathione32. Elevated levels of the glutathione (GSH) charges. All of the ligands and complexesVieww Aerrtiecle Ownleinlel
13
synthetase and other thiol based enzymes viz. γ- characterised by 1H, C, HMQC and DEPT 135 NMR, FT-IR, UV-
DOI: 10.1039/C5DT04781A
glutamylcysteine ligase, γ-glutamyl-transpeptidase are Vis spectra and single crystal X-ray structure. The bulk purity
observed in tumour cells35-36 which are rendered as major was confirmed by elemental analysis.
contributing factor for chemoresistance37 and their depletion
may increase the sensitivity of the drugs and oxidative stress.38
Since Ru has affinity for sulfur hence Ru-complexes may be
X-ray Crystallography
The single crystals of complexes 1-2 were obtained by
layering petroleum benzene over a dichloromethane solution
of the respective complex. They were structurally
characterized by single crystal X-ray diffraction method.
deactivated by the above enzymes, GSH and other cellular
thiols.32, 39-40
Hence complexes that are not deactivated by GSH or show
less affinity towards GSH and other cellular thiols would be of
high significance provided they are cytotoxic. We recently have
shown that complex 3, shows in vitro cytotoxicity in certain
Complex 1 crystallizes in space group P21/n (†ESI, Table S1).
The crystal structure shows that the RuII is bound with two
nitrogens of L1, one chloride and a 6-bonded p-cymene ring.
cancer cell lines and there is little deactivation in presence of
In the imidazole as usual the sp2 hybridized double bonded N-
glutathione under hypoxic condition.41 In addition no GSH
atom is the donor. The structure displays that the benzene and
binding was observed by NMR studies. We have synthesized
imidazole rings are not in same plane (Fig. 1). In 1, the methyl
group of p-cymene is on the same side as that of the chlorine,
two more isostructural complexes where the only difference
lies in the substitution at the vicinity of the metal centre so as
to correlate the effect of steric hindrance on the hydrolysis,
the isopropyl group of p-cymene seems to be moving away
from the benzene ring (Fig. 1). An octahedral [PF6]- is present
anticancer activity, glutathione binding and the pathways of
outside of coordination sphere and the Ru is in +2 oxidation
cytotoxicity which is strongly related to the breaking and
state. The average Ru-N bond distance is 2.097 ± 0.024 Å and
making of new bond with the metal centre leading to the
pattern of reactivity.
the Ru-Cl distance is 2.416(9) Å (†ESI,Table S2). The distances
between the carbons of the p-cymene and the RuII are not
uniform as described in †ESI,Table S2.
R
R
CHO
N
R
R
a
b (i, ii)
Complex 2 crystallizes with space group P-1 (†ESI, Table S1)
Similar to 1 the RuII in 2 is bound to two nitrogens from L2, a
chloride and the p-cymene (in a η6-fashion). However, unlike 1
the isopropyl group of the p-cymene is oriented towards the
chloride rather than the methyl group. The average Ru-N
distance is 2.105 ± 0.039 Å and the Ru-Cl bond shows a
distance of 2.410(7) Å (†ESI, Table S2). The bond distances
between the carbon atoms of the p-cymene ring and RuII are
not uniform (†ESI, Table S2). The structure of complex 3 which
has been described elsewhere showed similar Ru-N and Ru-Cl
distances however the orientation of the chloride was like 1.41
N
N
N
N
R
R
+
Cl
PF6
HN
RuII
NH2
HN
HN
R = H, Me, iPr
R = H (L1), Me (L2), iPr (L3)
R = H (1), Me (2), iPr (3)
Scheme 1. Synthetic scheme and chemical structure of ligands (L1-L3) and metal
complexes (1 - 3). (a) amine (1mmol), aldehyde (1mmol), EtOH, HCO2H (0.5%), reflux,
dark, 12 h. (b) i) Ligand (1.2 mmol), [Ru2(η6-p-cym)2Cl4] (0.5 mmol), MeOH, reflux, dark,
4 h. ii) NH4PF6.
Lipophilicity
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Fig. 1 Single crystal structures of complex 1 and 2 with 50% probability level. All
hydrogen atoms, counter anions and solvent molecules are omitted for clarity.
1
2
3
Results
Syntheses and Characterization
Fig. 2 Representative diagram of lipophilicity of 1–3 represented by a bar diagram
showing comparative log Do/w values of complexes in octanol–aqueous phosphate
buffer system, where error bars in the graph indicate the standard deviation in
measurement. For 3 data was obtained from reference [41].
The Schiff base ligands (L1-L2) were synthesized using
similar procedure as described by us earlier.41 The reaction of
II
one mole equivalent of [Ru2 (ƞ6-p-cym)2Cl4] precursor with ca.
2.4 mole equivalent of the respective ligand in methanolic
solution led to the formation of respective complexes (Scheme
1). The complexes were crystallized using 1 equivalent of
ammonium hexafluorophosphate due to their residual positive
To determine the distribution coefficient we used the
standard shake flask method.42 The log Do/w values as depicted
in Fig. 2, show that 3 has the highest lipophilicity (3.2 ± 0.1).
2 | J. Name., 2012, 00, 1-3
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Whereas, complex 1 and 2 shows log Do/w of 0.42 ± 0.12 and complexes up to 23 h (beyond which we did not check
View Article Online
0.66 ± 0.1 respectively. Since among complexes 1-3, complex 1 anymore) even when 7:3 v/v D2O - DMSDOO-Id:610m.10ix3t9u/Cre5DwT0it4h78n1oA
shows quicker hydrolysis hence, we use the term distribution chloride was used.
coefficient as a standard practice.
Binding studies
Table 1. Half-lives (t1/2) of complexes 1-3 at pH 7.4 and 6.7 and in various chloride
concentrations.
CT-DNA binding
The DNA binding ability was probed using UV visible
spectroscopy (†ESI, Fig. S17-S18). The complexes were
t1/2 (h)
2
Chloride
conc. (mM)
pH
hydrolyzed for 12 h before addition of DNA so that the spectral
variations due to hydrolysis are minimum, and in another case
fresh solution of complexes were used. We observe similar
results in both cases (†ESI, Table S4). Here, we present the
data which involves DNA binding after 12 h. The results show
that complex 2 binds more strongly than 1 or 3. The binding
ability shows the order 2>1>3. The DNA binding constants are
1
3
4
40
4
40
-
2.79 ± 0.66
3.91 ± 0.91
1.18 ± 0.36
2.55 ± 0.18
2.01 ± 0.24
Stable 10
days
5.67 ± 0.91
22.22 ± 2.13
4.37 ± 0.31
9.98 ± 2.90
7.56 ± 1.04
Stable 10
days
17.75 ± 0.02
49.40 ± 1.90
8.07 ± 1.1
23.96 ± 2.98
11.70 ± 2.48
Stable 10
days
7.4
6.7
Water
-
110a
3.04 (0.1)

104 and 4.11 (2.9)

103 M-1 for 1 and 3
105
aDetected by 1H NMR
respectively but for 2 it is higher in magnitude (1.11 (0.3)

M-1) (†ESI,Table S4, Fig. S17-S18).
Glutathione binding
1
The binding with reduced L-glutathione is monitored by H
NMR (†ESI, Fig. S19-S20) as well as ESI Mass Spectrometry, for
both the complexes 1 and 2 (Fig. 4, †ESI, Fig. S21-S22). 1H NMR
study of 1 and 2 with 2 molar excess of GSH was monitored up
to 8 hours and the results show no binding for each case. The
only new peaks that are identified are of the auto oxidation
product leading to GSH dimer and hydrolyzed species (†ESI,
Fig. S19-20).
But when we probed the concentration dependent binding
with excess GSH using ESI-MS, we found mono-GSH adduct of
the complexes at a higher concentration of GSH (at least 25
equivalent) for both 1 and 2. It appears that –SH groups of GSH
binds to the Ru-centre in the complexes rendering m/z of
714.12, 742.23 for 1 and 2 respectively (Fig. 4, †ESI, Fig. S21).
Another GSH bound fragmented peak with loss of p-cymene
from 2, at m/z 542.13, was identified. The isotopic distribution
ratio confirms the speciation is provided (†ESI, Fig. S21-22). At
this concentration it was not possible to obtain any significant
NMR data. The intensity of the mono-GSH adduct peak was
more for 1 compared to 2. In contrast even at 100 equivalent
concentration of GSH, no GSH bound peak was obtained for 3
even with ESI-MS.
Fig. 3 Hydrolysis study of complex 1-2 in 3:7 v/v DMSO-d6 and D2O mixture measured
by 1H NMR with time at 25°C. * indicate the peak corresponding to the hydrolysis
product.
Hydrolysis Study
To gain better insight into the properties of 1–3 we performed
hydrolysis studies at various conditions depicted in Table 1 (a
more elaborative Table is present in the †ESI, Table S3). The
results show that in acidic condition rate of hydrolysis
increases, leading to decrease in solution half-life and in the
presence of higher chloride concentration (40 and 110mM) the
half-life increases. At pH 6.7 the complexes show higher rate of
hydrolysis in 4 mM chloride containing buffer compared to
that observed for water (Table 1, †ESI,Table S3, Fig. S13-15).
1
The time dependent H NMR study in 7:3 v/v D2O - DMSO-d6
mixture shows the evidence of formation of hydrolyzed
species after 1 to 3 h (Fig. 3) for 1 and 2. The hydrolysis study
using 1H NMR ( 7:3 v/v D2O - DMSO-d6, 110 mM chloride)
shows excellent stabilities up to 10 days for all the complexes
(†ESI, Fig. S16). In the NMR studies 1-3 do not show any peak
corresponding to the dissociation of p-cymene from the metal
Fig. 4 ESI MS speciations of complex 1 and 2 upon treatment with excess glutathione
showing the proposed speciation as per the m/z peaks obtained. Complex 3 did not
provide any GSH bound species, hence not shown.
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Table 2. Cytotoxicity of the complex 1-3 with comparison to [RuII(η6-p-cym)(en)Cl](PF6) (C1) and cisplatin.
View Article Online
DOI: 10.1039/C5DT04781A
Hypoxia + glutathioned
IC50 (µM)  S.D.a
Normoxia
Hypoxiab
MCF-7
MCF-7
9 ± 1
A549
21 ± 1
HFF-1
10.2 ± 1
10.5 ± 1
6 ± 1
MIA PaCa-2
7 ± 1
Hep G2
6 ± 1
NIH 3T3
10 ± 1
11 ± 1
9.2 ± 1
N.D.c
A549
18 ± 1
MCF-7
16 ± 1
A549
37 ± 1
1
9 ± 1
11 ± 1
2
3
7 ± 1
19 ± 1
12 ± 1
10 ± 1
8 ± 1
17 ± 1
15 ± 1
27 ± 1
13.8 ± 1f
43.9 ± 3.6
15 ± 1
23.2 ± 1f
36.7 ± 2.3
24 ± 1
10 ± 1
9.1  1f
31.7 ± 1.7
19  2
15.6  1f
31.2 ± 1.5
27 ± 1
10.8  1 f
49.3 ± 1.8
29 ± 2e
16.7  1f
31.7 ± 2.6
40 ± 2e
C1f
N.D.c
N.D.c
N.D.c
Cisplatin
N.D.c
13.8 ± 5
14.3 ± 1
7 ± 1
aIC50 values were calculated by non linear curve fitting in dose response inhibition – variable slope model using Graph pad prism. S.D. = standard deviation. The data
presented are mean of three independent experiments, in a single experiment each concentration was assayed in triplicate. The statistical significance (p) of the data is
>0.001 to <0.05; bhypoxia (1.5% O2); cnot determined; dwith 1 mM of reduced L-glutathione; e20 molar equivalent of reduced L-glutathione used with respect to IC50
dosage of the respective cell line in hypoxia. f Data was obtained from reference [41].
Cellular studies
complexes. Multiple bands (i.e, ladder) were visible on agarose
gel electrophoresis of genomic DNA isolated from the MCF-7
cells treated with 1 and 2 (Fig. 5)
Cell viability assay
The cytotoxic activity of the synthesized compounds (1, 2
and 3) against various cell lines are presented in Table 2 in the
form of IC50 values. All the complexes show a potent
cytotoxicity profile against the tested cell lines when compared
to the in vitro activity of cisplatin. Under normoxic condition,
complex 1 and 3 are more promising against HepG2 while 2
has the best activity against MCF-7. Complex 1-3 are found to
be least effective against A549 and are in general efficient
towards MCF-7, MiaPaCa-2 or HepG2. To probe their relative
activity in hypoxia we used MCF-7 and A549. We found that
the toxicity profile of 1–3 shows variation dependent on the
conditions and cell lines. Although the toxicity of 1–3 is
relatively less in A549 but under hypoxic condition a definite
increase in activity is observed, except for the case of 2 which
exhibits deactivation for MCF-7 in hypoxia. The deactivation is
confirmed from the statistical significance of the normoxia and
hypoxia dataset. In addition, it is encouraging to find that 1
and 2 are marginally less toxic to the primary human foreskin
fibroblast (HFF-1). Based on hypoxia and normoxia data, 1
shows more cytotoxicity against MiaPaCa-2 and HepG2
compared to HFF-1. However their studies in presence of GSH
in hypoxia shows 1 and 2 are not as effective when excess GSH
is present, as the required dosage increases by 1.5 to 2 fold.
The complex C1 of Sadler et al. showed lesser activity in the
probed cell lines compared to 1-3, which was reported earlier
by us for comparison.41 C1 also exhibits better activity in
hypoxia although the activity is decreased in presence of
excess GSH in hypoxia.41
Table 3. Cell cycle inhibition of MCF-7 using 1 and 2.a
Sub G1
5.19
4.07
G0/G1
57.8
50.6
S
G2/M
15.96
19.69
24.02
26.03
24.91
DMSO control
1, 4 M
21.62
26.33
24.96
25.41
28.88
1, 6 M
2, 4 M
2, 6 M
1.66
2.42
3.62
50.09
46.71
43.39
aCells were treated for 24 h with 1 and 2. Cells were treated with propidium
iodide and analyzed by FACS. Cell populations were analyzed and expressed as
the percentage of cells in each phase. The data presented is an average of two
independent experiments.
Fig. 5 Agarose gel image of DNA ladder formation due to apoptosis using MCF-7 cell
line. (A) 50 bp step ladder, (B) DMSO control (C) 1 (8μM) (D) 1 (10μM) (E) 2 (8μM) (F) 2
(10μM) treated for 24 h.
Cell cycle analysis
The effect of complexes (1, 2) on cell cycle of MCF-7 was
probed with two different concentrations (4 and 6 μM) of each
complex and the observation shows that the percentage of cell
population in S and G2/M phase are comparatively higher with
respect to control (Table 3, †ESI, Fig. S28-S29). Whereas for 3,
we found that an increase in sub G1 population along with
G2/M phase arrest.41
Optical Microscopy Imaging
Cell swelling and nuclear fragmentation were observed for
both the cases upon treatment of complex 1 and 2 on MCF-7
(†ESI, Fig. S33). Our earlier reports on 3 suggest that the
observation is similar for all the three complexes.
ICP-MS studies
To know the amount of ruthenium inside the cells we used
ICP-MS study. We obtained the ruthenium concentration
inside cell after 24 h incubation of the complex treated MCF-7
cells (with the respective complex) and the results show that
DNA ladder assay
Genomic DNA fragmentation known as the hallmark of
apoptosis,43 was observed in ladder assay for all of the three
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the concentration of Ru inside the cells for complex 3 is more >90% healing at 24 h of incubation. While for 1-3 the wide
View Article Online
than that for 1 and 2 (Fig. 6). Complex 3 also happens to be denuded area remained un-healed indicating
DOI: 10.1039/C5DT047t8h1Ae
antiproliferative nature of the cells, attained by the treatment
most lipophilic and slowest in hydrolysis among the three.
with the respective complexes (1-3).
Fig. 6 Plot shows the amount of ruthenium present inside the cells (in ppb) with respect
to control experiment of complex 1 - 3. Each data is the mean of three independent
experiments.
Caspase-7 activation
The caspase-7 activation study of all the complexes (1, 2
and 3) shows activation of caspase-7 in MCF-7 upon the
treatment of the respective complexes (Fig. 7). The
colorometric data based on the cleavage of the p-nitrophenol
from the DEVD sequence shows that 1 and 2 has almost similar
activity and the caspase-7 activity appears to be more in case
of complex 3 as the absorbance obtained is higher.
Fig. 8 Representative diagram for change of mitochondrial membrane potential upon
treatment of complexes (1-3) on MCF-7 stained by JC-1.
Fig. 7 Representative diagram for caspase 7 activation of MCF-7 after 24 h treatment of
1- 3 with three different concentrations (4, 6 and 8 μM) of each. Activities were shown
in terms of pNA released. Error bars show standard deviations in data.
Mitochondrial membrane potential change
When MCF-7 cells were treated with four different
concentrations of 1-3, we observed a significant change in
mitochondrial transmembrane potential (MMP). The change is
maximum for complex 3 and minimum for 2 (Fig. 8, †ESI, Fig.
S30-S32) using same concentration of each complex although
the dose dependence on IC50 is not same for the complexes
rather complex 3 requires higher dosage to achieve IC50.
Fig. 9 Analysis of migration of MCF-7 after treated with corresponding complexes. The
images were taken after 12h and 24h of drug loading.
Haemolysis assay
Interaction of drug with the blood components particularly
RBCs is an important and inevitable phenomenon, thus
assessing the haemolysis becomes crucial in evaluating the
blood compatibility of drugs.44-45 The results (†ESI, Fig. S34,
Table S5) shows that the compound 1, 2 and 3 show good
compatibility with RBCs. The haemolysis was found to be least
Wound Assay (Migration)
The effects of 1-3 on cancer cell migration in MCF-7 cell
line were assessed using scratch wound healing assay. From
Fig. 9 it can be seen that the untreated control cells rapidly
migrate over time, resulting in almost 45% healing in 12 h and
for complex 1 it was ca. 1% at a concentration of 15 M. For
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complex 2 at same concentration the haemolysis was ca. 1.5%
The hydrolysis is also affected by the pH and cVoienwcAerntictleraOtniolinne
whereas it was highest for complex 3 but less than 3% (†ESI, of the leaving anion (e.g. Cl¯) as wellDaOsI: 1t0h.1e03o9v/Ce5rDaTll04io78n1iAc
Fig. S34).
strength of a solution (Table 1). Although, the complexes
hydrolyze faster in pH 6.7 but due to the steric hindrance the
trend remains similar as mentioned earlier. The effect of
chloride concentration change seems to be least on the half-
life of 1 in both the pH. The trend in hydrolysis does not show
uniform increase with increase in steric hindrance. However,
the effect of better stabilization at higher chloride
concentration is prominent. It is encouraging to find that at
extracellular concentration of chloride (~110 mM), all the
three complexes are stable for at least 10 d (we did not probe
beyond 10 d). In case where there was no chloride added viz.
the hydrolysis performed in 7:3 (v/v) D2O, DMSO-d6, by 1H
NMR, showed that the complexes started to hydrolyze after 1-
3 h but none of them looses any other ligand than Cl– upto 23
h (beyond which it was not probed) (Fig. 3). The ESI-MS data
also supported the loss of chloride since we could obtain the
peaks corresponding to the general formulation [RuII(η6-p-
cym)(L-H)]+ at m/z values of 407.09, 436.18 and 490.15 for 1-3
respectively which matches well with the simulated values of
407.09, 436.13 and 490.18 for 1-3 respectively. The CT-DNA
binding studies showed that the strongest interaction is
exhibited by complex 2 (Kb = 1.11(3) × 105 M-1), although 2 is
not the least hindered complex. This suggests that the
presence of the ortho methyl groups on the benzene ring of
the L2 leads to enhanced complex DNA interaction,46 which
increases the DNA binding ability. The similarity in the DNA
binding constants for 12 h hydrolyzed sample and fresh
solution of complexes may be attributed to the slow
hydrolysis, since the timescale of the DNA binding experiment
is smaller (1.5 h). The p-cymene complexes of RuII show similar
binding constants with DNA as reported by Garcia et al.47-50
Aquation is thought to be a major step which may be
involved in activation of this type of metal complexes.
However, extracellular aquation may prevent the
internalization of the complexes due to their di-positive charge
and higher hydrophilicity. Since, the lipid bi-layered cell
membrane has a hydrophobic interior and hydrophilic exterior
surface, hence if a complex has to enter the cell through
diffusion pathway then the lipophilicity is an important
parameter. The partition co-efficient values of 1 and 2 (0.42
and 0.66 respectively) are significantly less than 3 (log D = 3.2),
which clearly tells us that the complexes (1 and 2) may not be
very efficient to cross cell membrane by simple diffusion
process. However, the cytotoxicity studies described later
shows that both 1 and 2 are in general more cytotoxic than 3.
The cellular internalization of 3 is the highest in the series as
per the ICP-MS studies, which signifies that the effective
cytotoxic concentration is lower for 1 and 2. The results
suggest that the steric hindrance may also prevent 3 to react
with the relevant biomolecules resulting in relatively less
cytotoxicity.
Chick embryo angiogenesis assay (CEA)
The anti-angiogenic potential of the compounds were
evaluated by the chick embryo angiogenesis (CEA) assay (Fig.
10). The results indicate the anti-angiogenic property of 1-3 as
degenerated and damaged blood vessels were observed on
the chick embryo (affected zone is marked with white box and
damaged blood vessels are marked with black arrows) after 4h
of exposure to 1-3. No new blood vessels were formed during
the period of treatment. However the control shows healthy
blood vessels and new blood vessels were formed during the
4h incubation period.
Fig. 10 Ex-ovo Chick Embryo Angiogenesis (CEA) assay depicting the anti-angiogenic
potential of complex 1-3. The zoomed area is of the dotted white box upon treatment
after 4h.
Discussions
Half sandwich RuII-complexes bearing the formulation
[RuII(η6-p-cym)(L1)(Cl)](PF6)
(1)
and
[RuII(η6-p-
cym)(L2)(Cl)](PF6) (2) along with some new studies on our
previously reported [RuII(η6-p-cym)(L3)(Cl)](PF6) (3) are
discussed here. All the complexes are well characterized by
NMR and single crystal X-ray structure. The X-ray structures of
the complexes show that all of them have a chloride and one
6-bonded p-cymene ring. The geometric orientation rendered
shows differences in terms of steric bulkiness in the vicinity of
the metal centre (isopropyl to methyl to hydrogen). Complex 3
displays maximum steric hindrance and 1 shows the least. The
hydrolysis results show that the solution half-life has increased
with increase in steric hindrance (Table 1; t1/2 trend: 3>2>1)
rendering better stability and changes the cytotoxicity pattern
as discussed later. (Table 2).
However, we have reported earlier that complex 3 is very
effective in hypoxia under an excess concentration of GSH. The
dose dependence in hypoxia is also better than the normoxic
IC50 dosage as per the in vitro studies. Cytotoxicity data of all
6 | J. Name., 2012, 00, 1-3
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the three complexes show that 3 is the most effective one when we probed the GSH adduct formation by ESI-MS using
View Article Online
under hypoxia in presence of excess GSH. Complex 1 and 2 are more excess of GSH (around 25-100 eDqOuIi:v1a0l.e1n03t9/eCx5cDeTs0s4)78w1Ae
very effective under normal circumstances without added found that with ca. 25 equivalent of excess GSH, there is GSH
excess GSH and also in hypoxia, except in MCF-7 for 2 (Table adduct formation by complex 1 and 2. Under same conditions
2). When excess GSH is present in the surroundings, which of experimentation, the signal intensity of the GSH adduct is
may be thought of as the case similar to many resistant forms much higher for 1 as compare to 2. However using up to 100
of cancer, than the dose dependence worsens for 1 and 2. The equivalent of GSH we did not get any ESI-MS signal of GSH
required dosage in presence of ca. 750-1000 equivalent excess adduct with 3 supporting our earlier reported NMR studies and
GSH escalates to almost double as compared to the general the cytotoxicity data.41
normoxia/hypoxia IC50 dose.
The bulkiness of the ligands is in the order of 3>2>1 which
It is well known that A549 expresses higher GSH levels than corroborates well with the glutathione mediated deactivation
normal lung fibroblasts.51 All the complexes also show poorest under hypoxic condition (1>2>3). Complex 3 having the
activity in A549 (Table 2). However, 3 although requires higher isopropyl, most bulky group appears to exhibit higher
dosage but does exhibit better activity in hypoxia in presence resistance to GSH. Slowest rate of hydrolysis may be the
of excess extracellular GSH suggesting its resistance. However, reason behind such activity. As a trend, complex 3 is least
it should be noted that inside the cell there is glutathione-S- deactivated by GSH under hypoxia, followed by complex 2 and
transferase which catalyzes the conjugation of GSH to similar 1. Complex 1 showed 77% and 100% increase in IC50 values in
complexes for detoxification hence the higher dosage MCF7 and A549 respectively as a result of GSH mediated
required. However, in hypoxia 3 seem to overcome that and deactivation under hypoxia. The deactivation by extracellular
show better IC50 in the entire family which is encouraging.
glutathione is encouraging and suggests that steric hindrance
Complex 1 may be considered to be the most effective exhibits a significant role against deactivation by GSH. The
candidate against HepG2 and MIA PaCa-2 followed by MCF-7. steric hindrance also affects the partition coefficient of the
However, it is most susceptible to GSH deactivation. Complex 2 complexes rendering 3 more lipophilic hence ICP-MS studies
although shows greater DNA binding constant but is less show more internalization of 3 inside cells (Fig. 6) but yet 1 is
effective than 1 in most of the cell lines except for MCF-7, more effective emphasizing that 1 may be a better anticancer
where it shows the best activity in the series (Table 2). The agent for low GSH expressing hypoxic tumors.
cytotoxicity data of the complexes suggests that by change of
The cell cycle inhibition studies show that there may be
two ortho substitutions in L1-L3 (H in L1, methyl in L2 and variation in the mechanistic pathway of action since, 1 and 2
isopropyl in L3) the activity of the complexes gets affected in arrest MCF-7 cells at G2/M and S phase whereas 3 arrests in
terms of mechanism and pathways as described later. During G2/M and sub G1 phase. The arrest in sub G1 phase is
our previous work, we found that the effect of hypoxia may indicative of apoptotic pathway for cytotoxicity by 3.54-55
also be applicable to other known potential RuII based However, it should be noted that apoptosis does not always
anticancer agent41 viz. the [(RuII(η6-p-cymene)(en)Cl)(PF6)] render arrest of subG1 phase.56-57 The DNA ladder assay which
designed by Sadler et al.52 [(RuII(η6-p-cymene)(en)Cl)(PF6)] provides a qualitative indication to apoptosis confirms that all
showed better activity in hypoxia against MCF-7 and A54941 the complexes follow apoptotic pathway. In addition, the
but the required IC50 dosage is much higher for these cell lines. microscopy images also suggest apoptosis since there is
However, the encouraging part here is that RuII may have membrane blebbing, cell swelling as well as nuclear
potential as hypoxia active anticancer agent. Based on the fragmentation for all the complexes (†ESI, Fig. S33). Apoptotic
higher degree of aquation at acidic pH, it may be speculated cell killing involves extrinsic and intrinsic pathways. The
that the complexes 1-3 have potency to be more active inside intrinsic pathways lead to greater change in mitochondrial
tumors due to their lower interstitial pH.53
membrane potential. Complex 3 in spite of a poorer IC50 shows
The activity comparison of complexes 1-3 against primary highest change in MMP followed by complex 1 and the least
cell lines, primary human foreskin fibroblast (HFF-1) and change in MMP is observed with 2. Hence, the mitochondria
mouse embryonic fibroblast (NIH 3T3) show that complex 1 is mediated intrinsic apoptotic pathway may be least feasible in
more active against the HepG2 and MIA PaCa-2 than the HFF-1 2. In addition, the caspase activation trend by 2 and 1 although
and NIH 3T3 which is encouraging (Table 2). The clinical drug less than 3 but is still high and suggests that may be extrinsic
cisplatin is twice more cytotoxic to the primary cell line NIH pathways of apoptosis are active for them. The overall trend of
3T3 than any of the probed cancer cell lines in vitro. However, caspase 7 activations and MMP change shows that, it cannot
the in vitro cytotoxicity of complex 3 against NIH 3T3 is be ruled out that there are multiple pathways active, especially
comparable to its activity against HepG2 or MIA PaCa-2, which in case of 1 and 2.
is still better than the cytotoxicity pattern depicted by cisplatin
in these cell lines.
Inhibition of migration of cancer cells is taken as a positive
property to prohibit invasion and metastasis.58 The effects of
The NMR studies to probe the binding of GSH by 1-3 due to 1-3 on cancer cell migration in MCF-7 cell line showed that for
the known inhibitory property of GSH32 showed that under complexes 1-3 the wide denuded area remained un-healed
NMR conditions, where less excess of GSH is used (to get good after 24 h of exposure indicating the antiproliferative nature of
signal to noise ratio for both bound and unbound species), the cells, attained by the treatment of respective drugs.
there is no binding of GSH by any of the complexes. However Whereas, the untreated control cells rapidly migrated over
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time due to metastatic property resulting in >90% healing. The respective amine to the reaction mixture and refluxed. After
View Article Online
DOI: 10.1039/C5DT04781A
chick embryo angiogenesis (CEA) assay is a well known 12 h the reaction mixture was cooled at room temperature
angiogenesis assay.59,60 The results showed degenerated blood and evaporated to dryness, an off white coloured solid mass of
vessels with 1-3 treated embryos compared to normal product was formed. Finally the purification was done by
development of blood vessels in control, which is indicative of washing the obtained mass several times with diethyl ether.
the anti-angiogenic potential of the complexes.
This procedure produced ligands in satisfactorily pure form
which was used for complex syntheses. The analytical data of
L1 and L2 are given below. L3 has been reported earlier by
us.41
Experimental Section
Synthesis of N-((1H-imidazol-2-yl)methylene)aniline (L1)
L1 was synthesized using the above mentioned general
procedure. Yield: 0.164 g (95 %), Anal. Calc. for C10H9N3: C,
70.16; H, 5.30; N, 24.54; Found: C, 70.05; H, 5.33; N, 24.50%,
Materials and Methods
The solvents used for this work were dried and distilled as
per literature mentioned procedure.61 Aniline (Merck), 2,6-
dimethylaniline (Sigma-Aldrich), Imidazole-2-carboxaldehyde
(Sigma-Aldrich), Formic acid (Merck), Ruthenium(III) Chloride
Mp: 126°C, 1H NMR (400 MHz, CDCl3, 25
C): δ 8.54 (s, 1H,
CH=N), 7.43 (t, J = 6.44 Hz, 2H, Ar-H), 7.33 (t, J = 5.36 Hz, 1H,
Ar-H), 7.27 (d, J = 12.96 Hz, 2H, Ar-H), 7.25 (s, 2H, Imi-H) ppm
(Precious
metal
online,
Australia),
Ammonium
hexafluorophosphate (Sigma-Aldrich), Ethidium bromide (SRL,
India), agarose (molecular biology grade) (SRL, India), MTT [(3-
(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)]
(USB) and other cell growth media and their supplements
(Gibco), Reduced L-Glutathione (GSH) (Aldrich), were used
without further purification. [Ru2 (
(†ESI, Fig. S1); 13C NMR: (100MHz, CDCl3, 25
C): δ 149.9 (Imine-
C), 149.5 (Imi-C), 145.2 (Ar-C), 129.5 (Imi-CH), 127.1 (Ar-CH),
121.1 (Ar-CH), 115.4 (Ar-CH) ppm (†ESI, Fig. S2); ESI-HRMS
(Methanol) m/z (calc.): 194.07 (194.07) [C10H9N3.Na+], FT-IR
(KBr pellets, cm-1): 2914, 1625, 1440; UV-Vis: [CH3CN,
λmax,nm(ε/dm3mol−1cm−1)]: 220 (6967), 296 (11262), 317
(11669).
II
ƞ
6-p-cym)2(Cl)4] was
synthesized using a known procedure.62 The spectroscopy
grade solvents were used for spectroscopy and lipophilicity,
and the NMR solvents were purchased from Cambridge
Isotope Laboratories, Inc. (CIL). Fertilizable eggs were
purchased from a recognized local poultry farm and incubated
at 37˚C at 60% humidity level to grow embryo.
Synthesis
of
N-((1H-imidazol-2-yl)methylene)-2,6-
dimethylaniline (L2)
L2 was synthesized using the above mentioned general
procedure. Yield: 0.180 g (90 %), Anal. Calc. for C12H13N3: C,
72.33; H, 6.58; N, 21.09; Found: C, 72.25; H, 6.62; N, 21.02%,
SECOR India melting point apparatus was used to detect
melting point and the average value of three data are reported
without standard deviation. Perkin Elmer lambda 35
Mp: 121°C, 1H NMR (400 MHz, CDCl3, 25
C): δ 8.24 (s, 1H,
CH=N), 7.13 (s, 2H, Imi-H), 7.08 (d, J = 6 Hz, 2H, Ar-H), 7.02 (t, J
= 5.2 Hz, 1H, Ar-H), 2.13 (s, 6H, CH3) ppm (†ESI, Fig. S3); 13C
spectrophotometer
and
cary
300
UV-Visible
NMR: (100MHz, CDCl3, 25C): δ 153.2 (Imine-C), 135.8 (Imi-C),
spectrophotometer were used for UV-Visible experiments. FT-
IR spectra were measured in Perkin-Elmer SPECTRUM RX I
spectrometer in solid state using KBr pellets. Either JEOL ECS
400 MHz or Bruker Avance III 500 MHz spectrometer were
128.6 (Ar-C), 128.4 (Imi-CH), 127.9 (Ar-CH), 125.3 (Ar-CH),
118.2 (Ar-CH), 18.4 (CH3) ppm (†ESI, Fig. S4); ESI-HRMS
(Methanol) m/z (calc.): 222.10 (222.10) [C12H13N3.Na+], FT-IR
(KBr pellets, cm-1): 2910, 1638, 1442; UV-Vis: [CH3CN,
λmax,nm(ε/dm3mol−1cm−1)]: 282 (18744), 323 (3871).
1
used to obtained H, proton decoupled 13C, HMQC and DEPT
135 NMR spectra, each spectrum was recorded at 25°C and
the values are reported in ppm. Perkin-Elmer 2400 series II
CHNS/O analyzer was used for elemental analysis. Electro-
spray ionization mass spectra were recorded in +ve mode
electrospray ionization using a Q-Tof micro™ (Waters) mass
spectrometer. The recrystallization yields of isolated products
are only reported. The synthesized ligands and complexes
were dried in vacuum and stored in desiccators in dark under
nitrogen atmosphere. Synthetic procedure of 3 was recently
reported by our group, hence not described here.41 The well
known [RuII(η6-p-cym)(en)Cl](PF6) (C1) reported by Sadler et al.
was synthesized according to the reported procedure and the
analytically pure complex41 was used to study the cytotoxicity
in normoxia and hypoxia due to its known toxicity profile.52
Human blood was collected from a volunteer and all the work
was performed by following institute ethical guidelines.
Synthesis
General Procedure for synthesis of complexes 1 and 2
The complexes were synthesized according to a procedure
similar to that reported by us earlier.41 Precisely, 2.4 mmol
solution of the respective ligand in methanol (MeOH) was
II
added to the methanolic solution of [Ru2 (η6-p-cym)2(Cl)4]
(0.612 g, 1 mmol) in dark under nitrogen atmosphere at room
temperature. Then the reaction mixture was stirred for 10 h.
NH4PF6 (0.326 g, 2 mmol) was added to the resultant reaction
mixture and stirred for another 2 h. Evaporation of the
reaction mixture led to an orange coloured mass which was
washed twice with diethyl ether. The crude product was
purified by crystallization from a dichloromethane solution
layered with petroleum benzene.
[RuII(η6-p-cym)(L1)(Cl)](PF6) (1)
Yield: 0.352 g (60 %), Anal. Calc. for C20H25ClF6N3OPRu: C,
39.71; H, 4.17; N, 6.95; Found: C, 39.67; H, 4.12; N, 7.02%, Mp:
General Procedure for synthesis of L1 and L2
206°C, 1H NMR (500 MHz, DMSO-d6, 25
C): δ 8.46 (s, 1H,
1 mmol of imidazole-2-carboxaldehyde was dissolved in
methanol at 30C followed by addition of 1 mmol of the
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Imine-H), 8.15 (s, 1H, Imi-H), 7.83 (d,1H, J = 0.5 Hz, Imi-H), 7.75 deposited to https://deposit.ccdc.cam.ac.uk. The CCDC
View Article Online
(d, 2H, J = 7 Hz, Ar-H), 7.59 (t, 2H, J = 7.5 Hz, Ar-H), 7.52 (t, 1H, numbers are 1431153 for 1 and 1431154DfoOrI:210. .1039/C5DT04781A
J = 7.5 Hz, Ar-H), 6.00 (d, 1H, J = 6 Hz, p-cym-H), 5.65 (d, 1H, J The ORTEP diagrams were processed through POV-ray with
= 6 Hz, p-cym-H), 5.58 (d, 1H, J = 6 Hz, p-cym-H), 5.47 (d, 1H, J 50% probability level. All the non-hydrogen atoms were
= 6 Hz, p-cym-H), 2.46 (m, 1H, J = 7 Hz, iPr-H), 2.09 (s, 3H, p- refined with anisotropic displacement parameters.
cym-CH3), 0.99 (d, 3H, J = 7 Hz, iPr-CH3), 0.83 (d, 3H, J = 7 Hz,
iPr-CH3) ppm (†ESI, Fig. S5); 13C NMR (125 MHz, DMSO-d6,
Lipophilicity
25C): δ 154.8 (Imine-C), 151.9 (Imi-C), 146.2 (Ar-C), 133.7
Distribution coefficient (log D) was determined using
standard shake-flask method42 for all ligands and complexes in
octanol–aqueous phosphate buffer (20 mM, pH 7.4) system.
After overnight pre-equilibration of octanol and aqueous
phosphate buffer (equal volume, 3mL each)67 the solid
samples (1.2 mg each) were added to the solvent mixture and
shaken constantly for 10 hours at 37°C on a shaking incubator
to get full distribution between the two layers. Followed by
this, all the tubes were centrifuged and left undisturbed for an
hour. Aliquots of the aqueous and octanol layers were
pipetted out separately and the absorbances were measured
with UV-Vis spectrophotometer using proper dilution. Each set
was performed in triplicate, concentration of the substances in
each layer was calculated using the respective molar extinction
coefficients and the distribution coefficient values (log D) were
obtained from the ratio.
(Imi-CH), 129.3 (Ar-CH), 129.0 (Ar-CH), 123.9 (Imi-CH), 122.4
(Ar-CH), 103.7 (p-cym-C-CH3), 101.3 (p-cym-C-iPr), 85.2 (p-cym-
CH), 84.6 (p-cym-CH), 83.7 (p-cym-CH), 83.0 (p-cym-CH), 30.4
(iPr-CH), 21.9 (iPr-CH3), 21.3 (iPr-CH3), 18.2 (p-cym-CH3) ppm
(†ESI, Fig. S6); ESI-HRMS (Methanol) m/z (calc.):
442.06(442.06) [C20H23ClN3Ru+], 406.10(406.10) [C20H22N3Ru+];
FT-IR (KBr pellets, cm-1): 3436, 2972, 2929, 1627, 1439, 841.
UV-vis: [CH3CN, λmax,nm(ε/dm3mol−1cm−1)]: 318 (12301), 385
(2962).
[RuII(η6-p-cym)(L2)(Cl)](PF6) (2)
Yield: 0.340 g (55 %), Anal. Calc. for C22H27ClF6N3PRu: C,
42.97; H, 4.43; N, 6.43; Found: C, 42.85; H, 4.38; N, 6.49%; Mp:
>250°C, 1H NMR (500 MHz, DMSO-d6, 25
C): δ 8.51 (s, 1H,
Imine-H), 8.17 (d, 1H, J = 1 Hz, Imi-H), 7.84 (d, 1H, J = 1.5 Hz,
Imi-H), 7.31 (d, 2H, J = 5.5 Hz, Ar-H), 7.27 (t, 1H, J = 5.5 Hz, Ar-
H), 5.73 (d, 1H, J = 6.5 Hz, p-cym-H), 5.52 (d, 2H, J = 7 Hz, p- Aquation studies: Dependence on pH and chloride concentration
cym-H), 5.27 (d, 1H, J = 6 Hz, p-cym-H), 2.64 (m, 1H, J = 3.5 Hz,
iPr-H), 2.31 (s, 3H, p-cym-CH3), 2.17 (s, 3H, Ar-CH3), 2.06 (s, 3H,
The pH dependent aquation of 1, 2 and 3 were measured
by UV-Vis absorption spectroscopy at pH 7.4 and 6.7 (using 1%
methanol-20 mM phosphate buffer in 40 mM or 4 mM NaCl)
for 12 h. The hydrolysis of the complexes follow the pseudo
first order kinetics with respect to water and the half-life as
well as the rate were obtained from monoexponential decay
fitting at a constant wavelength with time.
Ar-CH3), 1.06 (d, 3H, J = 5 Hz, iPr-CH3), 1.04 (d, 3H, J = 4.5 Hz,
iPr-CH3) ppm (†ESI, Fig. S9); 13C NMR (125 MHz, DMSO-d6,
25C): δ 160.0 (Imine-C), 150.7 (Imi-C), 145.9 (Ar-C-N), 133.4
(Imi-CH), 131.1 (Ar-C-CH3), 129.0 (Ar-C-CH3), 128.8 (Ar-CH),
128.5 (Ar-CH), 127.6 (Ar-CH), 124.4 (Imi-CH), 105.4 (p-cym-C-
CH3), 99.0 (p-cym-C-iPr), 85.7 (p-cym-CH), 84.7 (p-cym-CH),
83.4 (p-cym-CH), 83.4 (p-cym-CH), 30.5 (iPr-CH), 22.2 (Ar-CH3),
21.4 (Ar-CH3), 19.7 (p-cym-CH3), 18.2 (iPr-CH3), 17.9 (iPr-CH3)
ppm (†ESI, Fig. S10); ESI-HRMS (Methanol) m/z (calc.): 470.09
(470.09) [C22H27ClN3Ru+], 434.12(434.12) [C22H26N3Ru+], FT-IR
(KBr pellets, cm-1): 3430, 2971, 2927, 1634, 1436, 846. UV-vis:
[CH3CN, λmax,nm(ε/dm3mol−1cm−1)]: 276 (6006), 303 (7635),
342 (3871).
Hydrolysis of respective complexes was also monitored by
1H NMR by taking spectra at different intervals of time in
DMSO-d6 and D2O mixture at 25C (3:7 v/v).
Stability in saline solution
The stability of each complex in extracellular chloride
1
concentration (110 mM) was observed by H NMR at 25C in
D2O: DMSO-d6 (7:3 v/v), 110 mM saline solution. 2 mg of each
complex was dissolved in 640 µL of 7:3 (v/v) D2O/DMSO-d6
solution containing 3.5 mg of NaCl and the spectra were
recorded upto ten days.
X-ray Crystallography
Single crystals of 1 and 2 suitable for diffraction were
coated with Fomblin oil and fixed on a loop and mounted on
the goniometer under flow of nitrogen. The data were
collected in SuperNova, Dual, Cu at zero, Eos diffactometer,
Agilent using monochromatic Mo Kα source (λ = 0.71073 Å)
using detector distance of 75 mm at 100K with the exposure of
6 sec. CrysAlisPro, Agilent Technologies, Version 1.171.37.31
(release 14-01-2014 CrysAlis171.NET) software was used for
data collection and data reduction. All the structures were
solved with Superflip63 structure solution programme Charge
Flipping and refined with the ShelXL64 refinement package
using Least Squares minimisation, using Olex2.65 All the
hydrogen atoms were calculated and fixed using ShelXL after
hybridization of all non-hydrogen atoms.66 Crystals were
Interaction with CT DNA
Deoxyribonucleic acid Sodium salt isolated from Calf
Thymus (CT DNA) was dissolved in 50mM Tris-NaCl solution at
pH 7.4 and kept for 10 h at 4C. UV-Visible spectrum showed
two absorption bands at 260nm and 280nm and their
absorption ratio was 1.9, which indicate that the DNA was
apparently free from protein.68 The concentration of the DNA
was determined from the absorption value at 260 nm (Molar
extinction coefficient is 6600 dm3mol-1cm-1)69 and the average
value of the concentration was taken after three independent
measurements of the same stock solution. The stock solution
of the DNA was stored at 4°C and used within four days.
This journal is © The Royal Society of Chemistry 20xx
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Interaction of 1 and 2 with CT DNA was measured with the was maintained at 1.5% and other afoVrieewmAertnictleioOnnleinde
DOI: 10.1039/C5DT04781A
help of UV-Vis spectroscopy in Tris-HCl buffer (50mM, pH 7.4) parameters were kept unaltered.
– acetonitrile (99:1 v/v) media. Acetonitrile was used for
Cell viability assay
preparation of stock solution of complexes. Spectroscopic
titrations were carried out at room temperature (25°C). The
concentration of 1 and 2 for the binding experiments was fixed
to 1 × 10−4 M and incubated for 12 h at 25°C for pre-
hydrolysis. In another set of experiments the concentrations
were fixed to 5 × 105 and 4.4 × 105 M for 1 and 2 respectively
and binding titration started instantaneously after preparing
the complex solution. The change in absorbance at 311 and
300 nm for 1 and 2 respectively were monitored with
subsequent addition of an aliquot of 5 μL (concentration of 1 ×
10-2 M and 5.3 × 10-3 M for 12 h and 0 h of hydrolysis
respectively) of CT DNA in the sample and also in the reference
cuvette after 5 min of equilibration. The titration was
continued until there was no significant change in absorbance
for at least three successive additions.
MTT assay was used to determine the cytotoxicity of the
compounds against the cancer cells based on cell viability as an
indicator for the sensitivity of the cells to the individual
compound. Briefly, exponentially grown cancer cells were
seeded in 96-well microplate (Nunc) at a density of 6

103
viable cells per well. Cells were incubated at 37°C (5% CO2
atmosphere) for 48 h to resume exponential growth followed
by removal and replacement of fresh media containing desired
concentration of test compounds. Each concentration was
loaded in triplicate. The stock solutions of compounds were
prepared in DMSO such that concentration of DMSO in well
does not exceed 0.2%. After 48 h of subsequent incubation,
compound containing media was removed, followed by
addition of 200 µL of fresh medium and 20 µL of a 1 mg.ml-1
MTT in PBS (pH 7.2) in each well. On incubation of the plates
for 3 h at 37°C in a humidified 5% CO2 atmosphere, MTT was
allowed to form formazan crystals in metabolically active cells.
Finally media was removed after incubation and replaced with
200 µL of DMSO in each well that would solubilize the
formazan crystals. The absorbance for each well was recorded
at 515 nm using a BIOTEK ELx800 plate reader.
IC50 values represent the drug concentration at which 50%
cells are inhibited compared to control, which were calculated
by fitting nonlinear curves in GraphPad Prism 5, constructed by
plotting cell viability (%) versus log of drug concentration in
µM or in nM. Each independent experiment was carried out in
triplicate.
For experiment in hypoxic condition the oxygen percentage
of the CO2 incubator (ESCO cell culture CO2 incubator, model:
CCL-170T-8-UV) was maintained at 1.5%. Compounds were
loaded in 96 well micro plates in level-II bio safety cabinet
under atmospheric conditions which took ca. 10 minutes and
then the drug loaded 96 well micro plate were placed in the
incubator programmed to attain 1.5% oxygen concentration.
The incubator takes ca. 30-40 minutes to reach the 1.5%
Binding studies with reduced L-glutathione (GSH)
The binding studies of 1 and 2 with reduced L-glutathione
1
were monitored by H NMR. The samples were prepared in a
degassed a D2O/ DMSO-d6 (7:3 v/v) mixture at 25C under
nitrogen atmosphere to minimize the auto oxidation of
glutathione. Each experiment involved 0.002 mmol of complex
and 0.004 mmol (2 equiv.) of GSH dissolved in the solution.
The binding was studied up to 29 hours at different intervals of
time although after 8 h total auto oxidation process was
completed.
Binding studies were also observed in ESI Mass
spectrometry. Lower equivalents of GSH viz. upto 10 equiv. did
not produce any m/z peak corresponding to adduct with the
respective complexes. Hence, higher amounts viz. at least 25
equiv. and up to 100 equivalent of GSH with complex was
incubated for half an hour at 37°C under stirring condition,
diluted with water and ESI mass spectrometry was performed.
Cell lines and culture
The Human breast adenocarcinoma (MCF-7), human lung oxygen level.
adenocarcinoma (A549) and mouse embryonic fibroblast (NIH
Cell cycle analysis
3T3) were kindly donated from Department of Biological
Sciences, IISER Kolkata (originally purchased from ATCC);
Hepatocarcinoma (Hep G2) and human pancreatic carcinoma
(MIA PaCa-2) were obtained from NCCS, Pune, and Primary
Human Foreskin Fibroblast (HFF-1) were donated to us by Dr.
Rupak Dutta, Department of Biological Sciences, IISER Kolkata.
Cell lines (MCF-7, A549, MIA PaCa-2) were maintained in the
logarithmic phase in Dulbecco's Modified Eagle Medium
(DMEM) while HFF-1 and Hep G2 were grown in Minimum
Essential Medium (MEM). Both the media were supplemented
with 10% fetal bovine serum (GIBCO) and antibiotics (100 unit
ml-1 penicillin and 100 mg ml-1 streptomycin). The cell culture
MCF-7 cells (5

105 per plate) were seeded in a 90 mm dia
petri-dish in 12 mL DMEM and incubated for 48 h at 37°C in a
5% carbon dioxide atmosphere. After 48 h, media was
removed, followed by addition of fresh media containing
appropriate concentrations of test compounds and incubated
for another 24 h under same conditions as above. After 24 h of
incubation with test compounds, cells were harvested by
trypsinization and washed twice with cold 1X PBS (pH 7.2).
Cells were fixed using 70% ethanol and stored at 4°C for 12 h.
In order to stain DNA for determining the cell cycle
distribution, cell pellets were transferred and resuspended in
PBS solution containing RNase (100 μg/mL) and propidium
iodide (55 μg/mL). The resulting solution was then incubated
for 30 min at 37°C in dark. Finally, homogenized cell samples
condition used was 95% humidity and 5% CO2 at
a
temperature of 37°C. For hypoxic cell culture the oxygen level
10 | J. Name., 2012, 00, 1-3
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Page 11 of 15
PleaDsealdtoonnTortaandsjuascttimonarsgins
Journal Name
were analyzed by flow-cytometry using
activated cell sorter (FACS) (FACS Calibur, Becton Dickinson,
CA). The resulting DNA histograms were quantified using the
CellQuestPro software (BD).
ARTICLE
a
fluorescence- Ruthenium accumulation study in cancer cells
View Article Online
DOI: 10.1039/C5DT04781A
Briefly, 1 × 106 MCF-7 Cells were seeded in each 90 mm dia
petri-dish and incubated for 48 h. After incubation, equi-molar
concentrations (3 µM) of each complex were added and a
further 24 h of drug treatment was allowed. Following this,
cells were washed with PBS (pH 7.2), treated with trypsin-
DNA Ladder Assay for Apoptosis Detection
DNA ladder assay was used to detect the cellular apoptosis EDTA, counted and 1 × 106 number of cells were collected as
induced by the test compound. MCF-7 cells (5

105 per plate) cell pellets using centrifugation. Cell pellets were digested
were seeded in a 90 mm dia petri-dish in 12 mL DMEM and overnight in concentrated nitric acid (70% v/v) at 65˚C; the
incubated for 48 h at 37°C in a 5% carbon dioxide atmosphere. resulting solutions were diluted using double-distilled water to
Subsequently the media was replaced with fresh media give a final concentration of 5% HNO3 and the amount of Ru
containing required concentration of test compounds and taken up by the cells was determined by ICP–MS (Thermo
incubated at 37°C in a 5% carbon dioxide atmosphere for 24 h. Scientific XSERIES2). The standard solutions of ruthenium was
Cells were then harvested by trypsinization followed by freshly prepared and analyzed while analyses for the samples
centrifugation at 2000 rpm for 5 minutes. Cells were subjected were carried out in triplicates and the standard deviations
to PBS (pH=7.2), washed and re-suspended in 500 μL lysis were calculated.
buffer [20 mM Tris-HCl (pH 7.4), 0.4 mM EDTA, 0.25% Triton-X
Caspase 7 activation
100] and incubated for 15 minutes at room temperature.
Lysed cells were centrifuged at 14000 rpm for 10 minutes and
the supernatant was collected. This was mixed with 1:1 (v/v)
phenol-chloroform mixture (1 mL) and the aqueous layer was
carefully separated to which 55 μL of 5 M NaCl and 500 μL
isopropanol were added respectively following incubation for
overnight at -20°C. After that the solution was centrifuged at
14000 rpm and washed with 70% ice cold ethanol and dried in
air which was again resuspended in 1X TE solution [10 mM
Tris-HCl (pH 8.0), 1 mM EDTA] containing RNase (150 μg/mL)
and finally it was incubated at 37°C for 20 minutes. The
supernatant, containing isolated DNA was mixed with
bromophenol blue dye and loaded in 1.6% agarose gel
containing EtBr (1.0 μg/mL) and run at 60 V for around 3 h in
1X TBE (Tris-borate-EDTA) buffer. Untreated cells were used
as controls whereas 50bp DNA ladder was used to track the
migration of bands and fragmentation sizes on the agarose gel.
Visible bands on gel were observed and picture taken on
exposure to UV light through gel documentation system of Bio-
Rad.
A quantitative estimation of caspase activity was studied
using MCF-7 cells by colorimetric Caspase Assay kit from
Sigma. The technique principally detects the free chromophore
(p-nitroaniline, pNA), following its cleavage from the Ac-DEVD-
pNA substrate. Though the substrate Ac-DEVD-pNA is cleaved
by both caspase-3 and caspase-7, yet this experiment would
indicate the presence of caspase 770-74 since MCF-7 cells are
already known not to have caspase-3.75 Briefly, 1 × 106 MCF-7
Cells were seeded in 90 mm dia petri-dish and incubated for 48
h followed by treatment with test compounds with an
additional incubation of 24 h. Then the cells were harvested,
lysed and treated as per the kit manufacturer's (Sigma)
standard protocol. All the test samples were done in triplicate
including the standard curve of pNA and data were recorded
after 24h of incubation with cell lysate at 405 nm using ELISA
plate reader.
The data is graphically represented as the absolute
concentration of pNA released (nmol/min.ml) in y-axis and
concentration of treated drugs in x-axis. Each concentration
has been performed in triplicate.
Optical Microscopy Imaging

103 per well in a 6-well
Detection of Mitochondrial Membrane Potential
MCF-7 cells were seeded at 15
1 × 106 MCF-7 Cells were seeded in each 90 mm dia petri-
dish and incubated for 48 h. Media was then replaced with
fresh media containing desired sub IC50 concentrations (4, 6
plate and were grown with 3 mL of DMEM. Existing media was
replaced after 48 h of incubation and cell were further
incubated with fresh medium containing required
concentrations of metal complex for 24 h. After removal of
drug containing media, cells were treated using 4% para
formaldehyde solution in 1X PBS (pH 7.2) and incubated at 4°C
for half an hour. The cells were then washed two times with 1X
and 8 M) of each compound. After further incubation of 24
h, the cells were harvested by removing media and
subsequently washed with 1X PBS solution. The entire
washings were collected in falcon tubes and centrifuged at
2500 rpm for 10 minutes. Supernatants were discarded and
the pellets were washed with 1X PBS solution. Washed cells
were suspended in the 1X PBS buffer containing 10 µg/mL of
JC-1 and supplemented with 10% FBS followed by incubation
for 30 minutes at 37°C under dark condition. Finally the
supernatant was removed by centrifugation and the cell pellet
was further re-suspended in 1X PBS buffer. The stained cells
were analyzed using BD Biosciences FACS Calibure flow
PBS (pH 7.2) and stained with DAPI (1

g.mL-1) for a few
seconds. The cells were washed several times using 1X PBS (pH
7.2). The optical microscopy images of MCF-7 cells were
acquired using OLYMPUS IX 81 epifluorescence inverted
microscope at 60X magnification. Both DIC and fluorescence
microscopy images were taken and processed using OLYMPUS
Cell P software. Merged images were produced for better
understanding.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Page 12 of 15
ARTICLE
Journal Name
cytometer measuring the red and green fluorescent placed on sterile petri-dish. Precautions were taken to prevent
View Article Online
intensities.
puncture of any of the blood vessels whDilOeI:t1r0a.n10sf3e9r/Cri5nDgT.0S4t7o8c1Ak
solution of complexes (1-3) was prepared in 10% DMSO–PBS
(v/v) solution and further diluted with PBS to achieve complex
concentration of 10 µM (final DMSO concentration 0.2%).
Sterile filter paper discs (6 mm dia) were soaked in solution of
respective complexes and placed in 3 different positions (10
µM; 10 µL) over the generating blood vessels. Sterile filter
paper disks (6 mm dia) soaked with 10 µL of 0.2% DMSO in PBS
were also used in triplicate as control. Image of blood vessels
were captured using a camera attached stereomicroscope
(Leica M80) at initial (i.e. 0 h) and after 4 h of incubation.
Wound Assay (Migration)
To evaluate the effect of drugs against cancer cell
migration, wound healing assay was used as a tool.76-77 The
migration capacities of drug treated cells were compared to
control in MCF-7 cell lines. Briefly, 1
106 cells were seeded in
each well of a 6 well plate and incubated at 37°C humidified
incubator with 5% CO2. Sufficient time was provided for the
formation of uniform monolayer in the plate. The monolayer
of the cells were then carefully scratched using a sterile
microtip to form a denuded area across the diameter of each
well. Each well was carefully washed with 1X PBS followed by
replenishment of fresh medium and addition of sub IC50
Statistical Analysis
All the IC50 data are expressed as mean ± standard
deviation of three independent experiments carried out in
each cell line and in each experiment each concentration was
assayed in triplicate. The statistical analyses were performed
using Graph pad prism® software 5.0 with student’s t-test.
Differences with *P < 0.05 were considered statistically
significant.
concentration (6
M) of 1-3. The experiments have been
carried out in duplicate. The images were captured in Phase
contrast microscope (OLYMPUS IX 81 epifluorescence inverted
microscope) at variable time points (0 h i.e. initial, 12 h and 24
h). The wound area was quantified using Image J software.
Haemolysis assay
The haemolysis assay was performed following standard
method.44 Briefly, blood was collected in EDTA containing vials,
followed by centrifugation at 3000 rpm for 10 min.
Erythrocytes were subsequently washed with cold PBS (pH 7.4)
for 3 times by centrifugation at 3000 rpm for 10 min. The cells
were re-suspended in the same buffer to a final concentration
of 20% (v/v) and stored at 4°C. Various concentrations of 1, 2
and 3 were prepared in DMSO and diluted in PBS as such that
the final concentration of compound remains 5, 10 and 15μM
in the reaction vial, while the final DMSO concentration is kept
at 0.2%. The reaction vial contains 2% (final concentration) cell
suspension and required concentrations of compound. The
vials were incubated at 37°C for 1 h in a shaking water bath.
After incubation the vials were centrifuged at 3000 rpm for 10
min and the supernatants were collected. The releases of
Conclusions
In summary, we have shown that design of RuII-p-cymene
complexes (1-3) by variation of the steric hindrance in the
ligand (L1-L3) may result in change of cytotoxicity and the
mechanism of action towards various cancer cells. Faster
hydrolysis does lead to higher cytotoxicity but it appears that
the pathway of action also changes as found from the cell cycle
arrest, MMP change and caspase activation data of the
complexes. All our complexes are stable against 110 mM
chloride concentration. None of the complexes loose the arene
moiety upto 24 h or more. All the complexes arrest the cell
cycle in G2/M phase. However, 1 and 2 also exhibits arrest in
S-phase whereas 3 shows significant arrest in the sub G1
phase. Although lipophilicity of complex 1 and 2 are less, yet
they exhibit higher cytotoxicity in spite of lower accumulation
inside cells. The cellular toxicity data suggests that higher
steric hindrance can prohibit deactivation by GSH and also
alter the pathway of action. Hence by proper tuning of steric
effect it may be possible to avoid direct inhibition by cellular
thiols, in RuII-arene complexes, in those forms of cancers
where GSH is overexpressed. It is encouraging to see that none
of the complexes exhibit binding to GSH until they are
incubated with at least 25 equivalent excess GSH and 3 shows
no GSH adduct even up to 100 equivalent of GSH. Complex 3
shows maximum change in the MMP and highest caspase 7
activation, suggesting an intrinsic pathway of apoptosis
involving the mitochondria may be active. Whereas in the case
of 1 and 2, other extrinsic pathways may be playing a
significant role in cell killing, especially in complex 2 which has
lowest MMP change but higher activation of caspase than 1. In
addition complexes 1-3 exhibit potential anti-metastatic and
anti-angiogenic activity.
haemoglobin
in
the
supernatant
were
assessed
spectrophotometrically at 540 nm. Positive control in which
complete haemolysis takes place was achieved using 0.2%
(final concentration) Triton X-100. Appropriate DMSO
containing PBS was used as negative control. The experiments
were run in triplicate and the percentages of haemolysis were
calculated as follows:
Haemolysis percentage (%) = (As-An)/(Ap-An)
 100
Where, As, An, Ap are absorbance of sample, negative control
and positive control respectively at 540 nm.
Chick embryo angiogenesis assay (CEA)
The chick embryo angiogenesis assay (CEA) was used to
investigate the anti-angiogenic activity of 1-3 using a reported
protocol.76 To briefly mention the used procedure, specific
pathogen free (SPF) fertile chicken eggs were obtained from a
recognized local poultry farm which was incubated at 37 °C at
a humid atmosphere. After the fourth day of incubation, the
shells of the eggs were cautiously broken using forceps and
12 | J. Name., 2012, 00, 1-3
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ARTICLE
21 M. Groessl, Y. O. Tsybin, C. G. Hartinger, B. K. KVeiepwpAlertriclaenOdnlinPe.
Acknowledgements
DOI: 10.1039/C5DT04781A
J. Dyson, JBIC, J. Biol. Inorg. Chem., 2010, 15, 677-688.
We sincerely acknowledge DST for financial support vide
project no SB/S1/IC-02/2014. We also thank IISER Kolkata for
the annual research funding and infra-structural support,
including NMR, single crystal X-ray, microscopy and FACS
facilities. K.P. and S.K. thanks UGC, S.C. thanks IISER Kolkata for
providing research fellowship. We sincerely thank Prof.
Subhransu Pan, West Bengal University of Animal and Fishery
Sciences for useful discussions on CEA assay.
22 R. Fernandez, M. Melchart, A. Habtemariam, S. Parsons and
P. J. Sadler, Chem. - Eur. J., 2004, 10, 5173-5179.
23 S. M. Smalley Keiran, R. Contractor, K. Haass Nikolas, N. Kulp
Angela, G. E. Atilla-Gokcumen, S. Williams Douglas, H.
Bregman, T. Flaherty Keith, S. Soengas Maria, E. Meggers and
M. Herlyn, Cancer Res, 2007, 67, 209-217.
24 J. Reedijk, Platinum Met. Rev., 2008, 52, 2-11.
25 W.-H. Ang, A. Casini, G. Sava and P. J. Dyson, J. Organomet.
Chem., 2011, 696, 989-998.
26 C. A. Smith, A. J. Sutherland-Smith, B. K. Keppler, F. Kratz and
E. N. Baker, JBIC, J. Biol. Inorg. Chem., 1996, 1, 424-431.
27 S. H. van Rijt, A. J. Hebden, T. Amaresekera, R. J. Deeth, G. J.
Clarkson, S. Parsons, P. C. McGowan and P. J. Sadler, J. Med.
Chem., 2009, 52, 7753-7764.
28 T. Bugarcic, O. Novakova, A. Halamikova, L. Zerzankova, O.
Vrana, J. Kasparkova, A. Habtemariam, S. Parsons, P. J. Sadler
and V. Brabec, J. Med. Chem., 2008, 51, 5310-5319.
29 A. Habtemariam, M. Melchart, R. Fernandez, S. Parsons, I. D.
H. Oswald, A. Parkin, F. P. A. Fabbiani, J. E. Davidson, A.
Dawson, R. E. Aird, D. I. Jodrell and P. J. Sadler, J. Med.
Chem., 2006, 49, 6858-6868.
30 F. Wang, A. Habtemariam, E. P. L. van der Geer, R.
Fernandez, M. Melchart, R. J. Deeth, R. Aird, S. Guichard, F.
P. A. Fabbiani, P. Lozano-Casal, I. D. H. Oswald, D. I. Jodrell, S.
Parsons and P. J. Sadler, Proc. Natl. Acad. Sci., 2005, 102,
18269-18274.
31 H. Chen, J. A. Parkinson, S. Parsons, R. A. Coxall, R. O. Gould
and P. J. Sadler, J. Am. Chem. Soc., 2002, 124, 3064-3082.
32 F. Wang, J. Xu, A. Habtemariam, J. Bella and P. J. Sadler, J.
Am. Chem. Soc., 2005, 127, 17734-17743.
33 S. Chatterjee, S. Kundu, A. Bhattacharyya, C. G. Hartinger and
P. J. Dyson, JBIC, J. Biol. Inorg. Chem., 2008, 13, 1149-1155.
34 F. Wang, H. Chen, J. A. Parkinson, P. d. S. Murdoch and P. J.
Sadler, Inorg. Chem., 2002, 41, 4509-4523.
35 J. Estrela, A. Ortega and E. Obrador, Crit. Rev. Clin. Lab. Sci.,
2006, 43, 143-181.
36 M. L. O'Brien and K. D. Tew, Eur. J. Cancer, Part A, 1996, 32A,
967-978.
37 N. Traverso, R. Ricciarelli, M. Nitti, B. Marengo, A. L. Furfaro,
M. A. Pronzato, U. M. Marinari and C. Domenicotti, Oxid.
Med. Cell. Longevity, 2013, 972913, 972910 pp.
38 M. Benlloch, A. Ortega, P. Ferrer, R. Segarra, E. Obrador, M.
Asensi, J. Carretero and J. M. Estrela, J. Biol. Chem., 2005,
280, 6950-6959.
39 Y. Lin, Y. Huang, W. Zheng, F. Wang, A. Habtemariam, Q. Luo,
X. Li, K. Wu, P. J. Sadler and S. Xiong, J. Inorg. Biochem.,
2013, 128, 77-84.
40 F. Wang, J. Xu, K. Wu, S. K. Weidt, C. L. MacKay, P. R. R.
Langridge-Smith and P. J. Sadler, Dalton Trans, 2013, 42,
3188-3195.
41 K. Purkait, S. Karmakar, S. Bhattacharyya, S. Chatterjee, S. K.
Dey and A. Mukherjee, Dalton Trans, 2015, 44, 5969-5973.
42 C. Zhang, Y. Wang and F. Wang, Bull. Korean Chem. Soc.,
2007, 28, 1183-1186.
Notes and references
Glutathione (GSH), Mitochondrial Membrane Potential
(MMP), Parts per million (ppm),
1
2
E. Meggers, Curr. Opin. Chem. Biol., 2007, 11, 287-292.
C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38,
391-401.
3
4
G. Suess-Fink, Dalton Trans, 2010, 39, 1673-1688.
M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger and B.
K. Keppler, Dalton Trans, 2008, 183-194.
V. Brabec and O. Novakova, Drug Resist. Updates, 2006, 9,
111-122.
A. Levina, A. Mitra and P. A. Lay, Metallomics, 2009, 1, 458-
470.
E. S. Antonarakis and A. Emadi, Cancer Chemother.
Pharmacol., 2010, 66, 1-9.
A. Bergamo, C. Gaiddon, J. H. M. Schellens, J. H. Beijnen and
G. Sava, J. Inorg. Biochem., 2012, 106, 90-99.
J. M. Rademaker-Lakhai, D. Van Den Bongard, D. Pluim, J. H.
Beijnen and J. H. M. Schellens, Clin. Cancer Res., 2004, 10,
3717-3727.
5
6
7
8
9
10 C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, B. Kynast,
H. Zorbas and B. K. Keppler, J. Inorg. Biochem., 2006, 100,
891-904.
11 M. Pongratz, P. Schluga, M. A. Jakupec, V. B. Arion, C. G.
Hartinger, G. Allmaier and B. K. Keppler, J. Anal. At.
Spectrom., 2004, 19, 46-51.
12 P. Schluga, G. Hartinger Christian, A. Egger, E. Reisner, M.
Galanski, A. Jakupec Michael and K. Keppler Bernhard,
Dalton Trans, 2006, 1796-1802.
13 M. Matczuk, M. Przadka, S. S. Aleksenko, Z. Czarnocki, K.
Pawlak, A. R. Timerbaev and M. Jarosz, Metallomics, 2014, 6,
147-153.
14 M. J. Clarke, Coord. Chem. Rev., 2003, 236, 209-233.
15 A. D. Kelman, M. J. Clarke, S. D. Edmonds and H. J. Peresie, J.
Clin. Hematol. Oncol., 1977, 7, 274-288.
16 R. Trondl, P. Heffeter, C. R. Kowol, M. A. Jakupec, W. Berger
and B. K. Keppler, Chem. Sci., 2014, 5, 2925-2932.
17 M. J. Clarke, S. Bitler, D. Rennert, M. Buchbinder and A. D.
Kelman, J. Inorg. Biochem., 1980, 12, 79-87.
18 Y. Han, Q. Luo, X. Hao, X. Li, F. Wang, W. Hu, K. Wu, L.
Shuang and P. J. Sadler, Dalton Trans, 2011, 40, 11519-
11529.
19 J. Reedijk, Chem. Rev., 1999, 99, 2499-2510.
20 K. Mitra, S. Patil, P. Kondaiah and A. R. Chakravarty, Inorg.
Chem., 2015, 54, 253-264.
43 M. M. Compton, Cancer Metastasis Rev., 1992, 11, 105-119.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 14 of 15
ARTICLE
Journal Name
44 H.-W. An, S.-L. Qiao, C.-Y. Hou, Y.-X. Lin, L.-L. Li, H.-Y. Xie, Y. 67 J. Sangster and A. D. Pelton, J. Phys. Chem. Ref. Data, 1987,
View Article Online
DOI: 10.1039/C5DT04781A
Wang, L. Wang and H. Wang, Chem. Commun., 2015, 51,
16, 509-561.
13488-13491.
68 J. Marmur, J. Mol. Biol., 1961, 3, 208-218.
45 B. Naeye, H. Deschout, M. Roding, M. Rudemo, J. Delanghe, 69 M. E. Reichmann, C. A. Rice, C. A. Thomas and P. Doty, J. Am.
K. Devreese, J. Demeester, K. Braeckmans, S. C. De Smedt
and K. Raemdonck, Biomaterials, 2011, 32, 9120-9127.
46 S. Ramakrishnan, E. Suresh, A. Riyasdeen, M. A. Akbarsha
and M. Palaniandavar, Dalton Trans, 2011, 40, 3245-3256.
47 N. Busto, J. Valladolid, C. Aliende, F. A. Jalon, B. R. Manzano,
Chem. Soc., 1954, 76, 3047-3053.
70 X.-H. Yang, T. L. Sladek, X. Liu, B. R. Butler, C. J. Froelich and
A. D. Thor, Cancer Res., 2001, 61, 348-354.
71 U. Janicke Reiner, Breast Cancer Res Treat, 2009, 117, 219-
221.
A. M. Rodriguez, J. F. Gaspar, C. Martins, T. Biver, G. Espino, 72 R. U. Janicke, M. L. Sprengart, M. R. Wati and A. G. Porter, J.
J. M. Leal and B. Garcia, Chem-Asian J., 2012, 7, 788-801. Biol. Chem., 1998, 273, 9357-9360.
48 J. Valladolid, C. Hortigueela, N. Busto, G. Espino, A. M. 73 H. Kurokawa, K. Nishio, H. Fukumoto, A. Tomonari, T. Suzuki
Rodriguez, J. M. Leal, F. A. Jalon, B. R. Manzano, A. Carbayo
and B. Garcia, Dalton Trans, 2014, 43, 2629-2645.
49 M. Martinez-Alonso, N. Busto, F. A. Jalon, B. R. Manzano, J.
and N. Saijo, Oncol. Rep., 1999, 6, 33-37.
74 D. Twiddy, G. M. Cohen, M. MacFarlane and K. Cain, J. Biol.
Chem., 2006, 281, 3876-3888.
M. Leal, A. M. Rodriguez, B. Garcia and G. Espino, Inorg. 75 N. A. Thornberry, T. A. Rano, E. P. Peterson, D. M. Rasper, T.
Chem., 2014, 53, 11274-11288.
Timkey, M. Garcia-Calvo, V. M. Houtzager, P. A. Nordstrom,
S. Roy, J. P. Vaillancourt, K. T. Chapman and D. W. Nicholson,
J. Biol. Chem., 1997, 272, 17907-17911.
50 N. Busto, M. Martinez-Alonso, J. M. Leal, A. M. Rodriguez, F.
Dominguez, M. I. Acuna, G. Espino and B. Garcia,
Organometallics, 2015, 34, 319-327.
51 A. Russo, W. DeGraff, N. Friedman and J. B. Mitchell, Cancer
Res., 1986, 46, 2845-2848.
76 P. Nagababu, A. K. Barui, B. Thulasiram, C. S. Devi, S.
Satyanarayana, C. R. Patra and B. Sreedhar, J. Med. Chem.,
2015, 58, 5226-5241.
52 R. E. Morris, R. E. Aird, P. d. S. Murdoch, H. Chen, J. 77 G. V. M. Sharma, A. Ramesh, A. Singh, G. Srikanth, V.
Cummings, N. D. Hughes, S. Parsons, A. Parkin, G. Boyd, D. I.
Jodrell and P. J. Sadler, J. Med. Chem., 2001, 44, 3616-3621.
53 B. A. Webb, M. Chimenti, M. P. Jacobson and D. L. Barber,
Nat. Rev. Cancer, 2011, 11, 671-677.
Jayaram, D. Duscharla, J. H. Jun, R. Ummanni and S. V.
Malhotra, MedChemComm, 2014, 5, 1751-1760.
54 W. Xia, S. Spector, L. Hardy, S. Zhao, A. Saluk, L. Alemane and
N. L. Spector, Proc. Natl. Acad. Sci.., 2000, 97, 7494-7499.
55 M. Kajstura, H. D. Halicka, J. Pryjma and Z. Darzynkiewicz,
Cytometry, Part A, 2007, 71A, 125-131.
56 I. Romero-Canelon, L. Salassa and P. J. Sadler, J. Med. Chem.,
2013, 56, 1291-1300.
57 R. Pettinari, C. Pettinari, F. Marchetti, B. W. Skelton, A. H.
White, L. Bonfili, M. Cuccioloni, M. Mozzicafreddo, V.
Cecarini, M. Angeletti, M. Nabissi and A. M. Eleuteri, J. Med.
Chem., 2014, 57, 4532-4542.
58 P. Friedl and K. Wolf, Nat. Rev. Cancer, 2003, 3, 362-374.
59 P. Nowak-Sliwinska, C. M. Clavel, E. Paunescu, M. T. te
Winkel, A. W. Griffioen and P. J. Dyson, Mol. Pharmaceutics,
2015, 12, 3089-3096.
60 A. K. Barui, V. Veeriah, S. Mukherjee, J. Manna, A. K. Patel, S.
Patra, K. Pal, S. Murali, R. K. Rana, S. Chatterjee and C. R.
Patra, Nanoscale, 2012, 4, 7861-7869.
61 D. D. Perrin and W. L. F. Armarego, Purification of Laboratory
Chemicals. 3rd Ed, 1988.
62 M. A. Bennett, T. N. Huang, T. W. Matheson and A. K. Smith,
Inorg. Synth., 1982, 21, 74-78.
63 L. Palatinus and G. Chapuis, J. Appl. Crystallogr., 2007, 40,
786-790.
64 G. M. Sheldrick, Acta Crystallogr., Sect. A Found. Crystallogr.,
2008, 64, 112-122.
65 O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard
and H. Puschmann, J. Appl. Crystallogr., 2009, 42, 339-341.
66 G. M. Sheldrick, Int. Union Crystallogr., Crystallogr. Symp.,
1991, 5, 145-157.
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DOI: 10.1039/C5DT04781A
Table of Contents
Alteration of steric hindrance modulates glutathione resistance and
cytotoxicity of three structurally related RuII-p-cymene complexes
Kallol Purkait, Saptarshi Chatterjee, Subhendu Karmakar and Arindam Mukherjee
Three structurally related RuII-p-cymene complexes of imidazole based Schiff bases show steric
hindrance influences hypoxia reactivity, cell cycle arrest and resistance to glutathione. The complexes
show anti-metastatic and anti-angiogenic effect.