Synthesis, antiproliferative activity and apoptosis-promoting effects of arene ruthenium(II) complexes with N, O chelating ligands

Article abstract of DOI:10.1016/j.jorganchem.2018.01.022

New half sandwich arene ruthenium(II) complexes of the type [Ru(arene)Cl(L)] (where arene = benzene and p-cymene, L = thiophene benzhydrazone ligands) have been synthesized from the reactions of the neutral precursor [Ru(arene) (μ-Cl) Cl]₂ and

Full text of DOI:10.1016/j.jorganchem.2018.01.022

Accepted Manuscript
Synthesis, antiproliferative activity and apoptosis-promoting effects of arene
ruthenium(II) complexes with N, O chelating ligands
Nanjan Mohan, Mohamed Kasim Mohamed Subarkhan, Rengan Ramesh
PII:
S0022-328X(18)30022-6
10.1016/j.jorganchem.2018.01.022
JOM 20260
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 31 October 2017
Revised Date: 8 January 2018
Accepted Date: 16 January 2018
Please cite this article as: N. Mohan, M.K. Mohamed Subarkhan, R. Ramesh, Synthesis, antiproliferative
activity and apoptosis-promoting effects of arene ruthenium(II) complexes with N, O chelating ligands,
Journal of Organometallic Chemistry (2018), doi: 10.1016/j.jorganchem.2018.01.022.
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Synthesis, antiproliferative activity and apoptosis-promoting effects of
arene ruthenium(II) complexes with N, O chelating ligands
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Nanjan Mohan, Mohamed Kasim Mohamed Subarkhan and Rengan Ramesh*
Centre for Organometallic Chemistry, School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024,
Tamil Nadu, India
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A B S T R A C T
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New half sandwich arene ruthenium(II) complexes of the type [Ru(arene)Cl(L)]
(where arene= benzene and p-cymene, L = thiophene benzhydrazone ligands) have been
synthesized from the reactions of the neutral precursor [Ru(arene) (µ-Cl) Cl]2 and the
corresponding benzhydrazone ligand. All the complexes were completely characterized by
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elemental analysis and additionally by IR, UV-Vis, H NMR and ESI-MS spectroscopic
methods. The solid state structures of the complexes 6 and 7 were determined by single-
crystal X-ray diffraction analysis, which exhibit typical pseudo-octahedral geometry around
the metal center. The antiproliferative activity of the complexes was evaluated on cancerous
(HeLa, MDA-MB-231, and Hep G2) and noncancerous (NIH3T3) cell lines. In general,
complexes containing electron releasing OCH3 substituent have potential anticancer activity
than those incorporating H, Cl and Br substituents. Moreover, the p-cymene complexes show
more cytotoxicity than benzene derivatives, suggesting that the substituent at arene plays a
vital role in the biological activity of the compounds. Further, an apoptotic mechanism of
cell death in MDA-MB-231 was confirmed by AO-EB, Hoechst 33258 staining and annexin-
V/PI double-staining techniques. In addition, the extent of DNA fragmentation in cancer cells
was studied by comet assay.
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Keywords:
Benzhydrazone; η6-arene ruthenium(II) complex; Crystal structure; Cytotoxicity; Apoptosis
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* Corresponding author. Tel.: +91 431 2407053; fax: +91 431 2407045.
E-mail address: ramesh_bdu@yahoo.com, rramesh@bdu.ac.in (R. Ramesh).
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1. Introduction
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Although platinum-based drugs cisplatin, carboplatin and oxaliplatin have been
widely used for anticancer agents for the past few decades, the problems of high toxicity,
platinum resistance and undesirable side-effects are appealing the search for different
transition metal anticancer drugs. It is to note that ruthenium-complexes have attracted
significant attention among the other various metal complexes for their potential anticancer
activity. In this regard ruthenium complexes exhibit evidence of low toxicity compared to
traditional cisplatin agents. The ruthenium(III) complexes particularly [imiH2] [trans-Ru(N-
imiH)(S-dmso)Cl4] NAMI-A and [indH2][trans-Ru(N-indH)2Cl4] KP1019 [1,2] and its
sodium analogue Na[trans-Ru(N-indH)2Cl4] (NKP-1339 or IT-139) are the most promising
ruthenium complexes reaching clinical trials [3]. Notably, the activation method depends on
the redox potential of the Ru(III)/Ru(II) oxidation states, which in turn strongly depends on
the ligands coordinated to the metal centre. The activation by reduction results in a reactive
ruthenium(II) complex, which can react with numerous biomolecules [4-7].
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Particular attention has been paid to half sandwich arene ruthenium complexes
because of the π-ligated arene which confers great stability to Ru in the +2 oxidation state
and influences the hydrophobicity and interaction with biomolecules [8-10]. Substitutions at
arene moiety and variations in the chelating ligands will be able to fine tune their biological
properties [11]. Tocher et al. have reported that cytotoxicity was enhanced by coordinating
the antibacterial agent metronidazole [1-β-(hydroxyethyl)-2-methyl-5-nitro-imidazole] to a
benzene ruthenium dichloro fragment [12]. At first, the prototype arene ruthenium(II)
complexes [(p-MeC6H4Pri)RuCl2(P-pta)] (pta
=
1,3,5-triaza- 7-phospha-tricyclo-
[3.3.1.1]decane), termed RAPTA-C [13] which displays pH dependent DNA damage due to
the hypoxic (low pH) nature of cancer cells, and [(C6H5Ph)RuCl(N,N-en)][PF6] (en = 1,2-
ethylenediamine) exhibits selective binding to guanine bases on DNA, forming
monofunctional adducts [14] , though many various categories have since been reported [15].
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Hydrazones are versatile ligands with fascinating ligation properties with many transition
metals. Moreover, these ligands represent an important class of compounds for new drug
development because hydrazone moiety was selected for its high stability at physiological pH
and lability under strongly acidic and basic conditions as incontestable by drug delivery
agents in tumor targeting. Thus, all the hydrazones possess the azomethine (-CONHN=CH-)
group have been revealed to exhibit antiproliferative activities and act as cytotoxic agents
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with the ability to stop cell progression in cancerous cells through different mechanisms [16].
Aroylhydrazones are magnificent multidentate ligands for transition metals. They have been
exhibit to reveal a variety of biological e.g. antiamoebic activity [17] and DNA synthesis
inhibition or antiproliferative behaviour [18-20]. Herein, we present a systematic
investigation of half-sandwich Ru(II) complexes bearing benzhydrazone ligands (Fig. 1)
with respect to their antiproliferative activity on human cancer cells.
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Fig. 1 Design of arene ruthenium(II) benzhydrazone complexes.
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2. Results and discussion
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The benzhydrazones were obtained by condensation of equimolar amounts of
thiophene-2-carboxyaldehyde and substituted benzhydrazide [21]. The arene complexes of
the type [Ru(arene)Cl(L)] (arene= benzene and p-cymene and L = thiophene benzhydrazone
ligands) (Scheme 1) have been synthesised from the reactions of the ligands and ruthenium
arene dimers [Ru(arene) (µ-Cl)Cl]2 in a 2 : 1 molar ratio in benzene for 5h at reflux
temperature in the presence of triethylamine as a base. The isolated complexes were yellow,
brown in colour, air stable solids, partially soluble in water and completely soluble in polar
organic solvents like methanol, ethanol, acetone, chloroform, dichloromethane, acetonitrile,
dimethylformamide and dimethylsulfoxide. The elemental analysis of all the ruthenium(II)
complexes are in good agreement with the molecular formula of the proposed structure.
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Scheme 1. Synthesis of arene ruthenium(II) benzhydrazone complexes.
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FT-IR spectra of the ligands and the complexes (1-8) furnished significant
information about coordination of the ligand to metal. A medium to strong band in the range
3191-3280 cm-1 was assigned to the N-H functional group of the ligand. The ligands also
exhibit absorptions due to νC=N and νC=O within the range 1632-1649 cm-1. Upon
complexation the bands associated with νN–H and νC=O stretching vibrations are disappeared
and indicating that the ligands undergo tautomerization and consequent coordination of the
imidolate oxygen. The appearance of new bands in the range 1259-1272 and 1594-1620 cm-1
attributed to the C–O and C=N–N=C fragments which give further support for the
coordination of the ligand. Hence, the coordination through imine nitrogen and the imidolate
oxygen of the ligand to ruthenium was confirmed by IR spectra of all the complexes [22]. All
the complexes show three bands in the region 234-366 nm in acetonitrile at room
temperature. Bands due to ligand-centered (LC) transitions are appeared around 234-304 nm
and have been designated as π–π* and n–π* transitions. The lowest energy bands that
appeared in the region 360-366 nm were attributed to the charge transfer due to metal to
ligand transitions [23]. The pattern of the electronic spectra of all the complexes is very
similar to other previously reported octahedral complexes. Fig. S1-S8 (ESI†).
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The binding of the benzhydrazone ligand to the ruthenium(II) ion is further verified
by NMR spectra of the complexes. All the complexes show multiplets in the region δ 6.7- 8.1
ppm and have been assigned to the aromatic protons of benzhydrazone ligands. A sharp
singlet in the region δ 8.8-8.9 ppm is assigned to azomethine proton which shifted to
downfield on comparison with those of the free ligands, indicating deshielding of the
azomethine proton upon coordination to ruthenium. In addition, the absence NH proton of the
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free ligands in all the complexes confirmed the coordination to Ru(II) ion via imidolate
oxygen. An upfield shift of η6-C6H6 protons of 1-4 has been observed in the region of δ 5.5
ppm. Two sets of doublets have been observed in the region δ 1.0-1.3 ppm for the methyl
protons of isopropyl group in p-cymene moiety. The methine proton of the isopropyl group
appears as a septet in the range of δ 2.5-2.6 ppm. Further, a singlet at δ 2.2 ppm is attributed
to the methyl protons of the p-cymene moiety. Moreover, four sets of doublets in the range
of δ 4.6-5.3 ppm were assigned to the aromatic protons of the p-cymene ligand. In addition,
for complexes 4 and 8 the methoxy signals of the benzhydrazone ring were observed as a
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singlet at δ 3.8 and δ 3.7 ppm. Thus the H NMR spectra of all the complexes confirm the
coordination mode of the benzhydrazone ligand to the ruthenium(II) ion through the
azomethine nitrogen and the imidolate oxygen Fig. S9-S16 (ESI†).
2.1 Crystal structures
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Single crystal X-ray diffraction analysis of the complexes 6 and 7 were grown from
CH2Cl2/ Pet .ether by slow evaporation method. The ORTEP diagrams for the two structures
are shown in Fig. 2, crystallographic data and selected bond parameters are listed in Table 1
and 2. Both complexes 6 and 7 crystallize in the monoclinic space group P21/c. In the
complex 6, the (ɳ6-p-cymene) ligand occupying three coordination sites in ɳ6-fasion and the
remaining coordination sites are occupied by N, O donor atoms from chelating ligand and
one chloride. Thus the crystallographic structure of complex confirms pseudo octahedral
geometry around the ruthenium metal [24]. The Ru-N, Ru-O and Ru–Cl bond lengths are
2.107(4), 2.056(3) and 2.398(13) Å, respectively. The Ru-C (p-cymene) bond lengths ranging
from 2.157-2.221 Å and p-cymene ring C-C bond lengths ranging from 1.398-1.432 Å. Bond
angles of 86.18(11)⁰, 85.73(11)⁰ and 76.23(13)⁰ are observed for Cl-Ru-O, Cl-Ru-N, and N-
Ru-O respectively. A similar structural feature has been found in complex 7 with marginal
changes in bond lengths and bond angles.
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Fig. 2 Molecular structures of complexes 6 and 7; thermal ellipsoids are drawn at the 30% probability level. All
hydrogen atoms were omitted for clarity.
Table 1 Selected Bond Lengths (Å) and Angles (deg) for the Complexes 6 and 7
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Bond lengths (Å)
N(1)-N(2)
N(1)-Ru(1)
O(1)-Ru(1)
Cl(1)-Ru(1)
C(7)-O(1)
C(7)-N(2)
C(8)-N(1)
1.413(5)
2.107(4)
2.056(3)
2.398(13)
1.305(5)
1.299(6)
1.288(6)
N(1)-N(2)
N(2)-Ru(1)
O(1)-Ru(1)
Cl(1)-Ru(1)
C(7)-O(1)
C(7)-N(1)
C(8)-N(2)
1.410(6)
2.107(4)
2.053(3)
2.400(15)
1.300(6)
1.300(6)
1.296(6)
Bond angles (⁰)
N(2)-N(1)-Ru(1)
C(7)-N(2)-N(1)
C(7)-O(1)-Ru(1)
O(1)-Ru(1)-N(1)
O(1)-Ru(1)-Cl(2)
N(1)-Ru(1)-Cl(2)
113.5(3)
N(1)-N(2)-Ru(1)
C(7)-N(1)-N(2)
C(7)-O(1)-Ru(1)
O(1)-Ru(1)-N(2)
O(1)-Ru(1)-Cl(1)
N(2)-Ru(1)-Cl(1)
113.7(3)
110.9(4)
112.6(3)
76.23(13)
86.18(11)
85.73(11)
110.9(4)
112.9(3)
76.09(15)
85.76(12)
85.80(12)
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ESD in parenthesis.
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Table 2 Crystal data and structure refinement for complexes 6 and 7
Compound
6
7
Empirical formula
Formula weight
C22 H22 Cl2 N2 O2 Ru S
550.45
C22 H22 Br Cl N2 O2 Ru S
594.91
Temperature
Wavelength
Crystal system
296(2) K
296(2) K
0.71073Å
Monoclinic
0.71073 Å
Monoclinic
Space group
P21/c
P21/c
Unit cell dimensions
a = 13.9604(5)Å alpha = 90 deg.
a = 13.9337(6)Å alpha = 90 deg.
b = 17.0717(6) Å beta = 100.359(2)
deg.
b = 17.2743(8)Å beta = 101.267(2)
deg.
c = 10.2549(4)Å gamma = 90 deg.
c = 10.3593(4) Å gamma = 90 deg.
2445.38(18)Å3
Volume
2404.19(15) Å3
Z, Calculated density
Absorption coefficient
4, 1.521Mg/m3
0.981 mm-1
4, 1.616 Mg/m3
2.490 mm-1
F(000)
1112
1184
Crystal size
0.30 x 0.30 x 0.25 mm
0.35 x 0.30 x 0.30mm
Theta range for data collection 1.48 to 28.35 deg.
1.49 to 28.32 deg.
Limiting indices
-18 ≤ h ≤ 12,
-22 ≤ k ≤ 22,
-13 ≤ l ≤ 13
-17 ≤ h ≤ 18,
-19 ≤ k ≤ 22,
-13 ≤ l ≤ 10
Reflections collected / unique 19987 / 5955 [R(int) = 0.0291]
19409 / 5975 [R(int) = 0.0324]
Completeness to theta = 28.44 99.2 %
98.3 %
Absorption correction
Semi-empirical from equivalents
Semi-empirical from equivalents
Max. and min. transmission
Refinement method
0.7915 and 0.7573
Full-matrix least-squares on F2
0.5221 and 0.4761
Full-matrix least-squares on F2
Data / restraints / parameters
5955 / 0 / 271
5975 / 0 / 271
Goodness-of-fit on F2
1.124
1.088
Final R indices [I>2sigma(I)]
R1 = 0.0505, wR2 = 0.1655
R1 = 0.0505, wR2 = 0.1631
R indices (all data)
R1 = 0.0679, wR2 = 0.1888
2.583 and -0.677 e.Å-3
R1 = 0.0765, wR2 = 0.1816
2.342 and -0.753 e.Å-3
Largest diff. peak and hole
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2.2 Stability of the complexes (time-dependent spectra)
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Stability of compounds in solution is an essential requirement for drug candidates.
The stability of complexes (1-8) in a solution of buffer-DMSO was explored using UV-Vis
spectroscopy Fig.S9-S16 (ESI†). The spectra did not exhibit any noticeable changes during a
period of 24 hour indicate the stability of the complexes. Further, ESI-MS spectral studies of
the complexes confirm the composition. All the complexes showed the characteristic peaks at
m/z 410.00 (1, M-Cl+), 444.96 (2, M−Cl+), 486.89 (3, M – Cl+), 439.00 (4, M−Cl+), 465.05
(5, M−Cl+), 499.06 (6, M−Cl+), 544.96 (7, M−Cl+), and 495.06 (8, M−Cl+) Fig. S25-S32
(ESI†). The results strongly indicate that the chlorine atom in these complexes is highly labile
and the resulting species easily interacts with biomolecules [25].
2.3 Partition Coefficient Determination
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Hydrophobicity is the basic physiochemical parameters in the design of drugs and
their biological processes [26] and is determined by the n-octanol/water partition coefficient
(P) method [27]. Moreover, Log P, were measured to explain the permeability of complexes
(1-8) through a biological system [28] based on solubility of a given compound in a two-
phase system [29]. The log P results are presented in Table S1(ESI†). The partition
coefficient values (log P) of the complexes suggested that hydrophobicity can be arranged in
the order 8 > 4 > 6 > 7 > 5 > 2 > 3 > 1.
2.4 Cytotoxicity studies
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The cytotoxicities of the metallic precursors, ligands and complexes were
determined by spectrofluorimetric MTT assay. The plot of percentage of cell death versus
concentration is illustrated in Fig. S33&34 (ESI†). The cytotoxicity of the complexes was
expressed by IC50 values and are reported in Table 3. It is to be noted that the precursor and
the ligand did not show any inhibition even up to 100 µM and the observed cytotoxicity of
the complexes is mainly due to chelation of the ligand to ruthenium. The in vitro anticancer
activity of the Ru-arene complexes 1-8 towards several human cancer cell lines (HeLa,
MDA-MB-231, and Hep G2) and a normal human cell line (NIH3T3) were determined after
24 h inhibition and cisplatin was used as a positive control. Based on IC50 values obtained, in
vitro anticancer activity of the complexes follows the order: 8>4>6>7>5cisplatin = 1>2>3.
These results are also consistent with hydrophobicity of the complexes [30]. Complexes 1–8
show markedly increased cytotoxic potencies compared with the respective hydrazone
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ligands. A comparison of the IC50 values of these complexes against MDA-MB-231cells
indicates that complexes 4 and 8 exhibits comparatively better than the other complexes
under same experimental conditions. The complexes containing methoxy substituent exhibit
higher hydrophobicity and enables permeation of complexes across cell membranes [31].
Further, the arene group plays significant role in the antiproliferative activity of these
complexes. In general p-cymene complexes show higher cell killing activities which may be
due to the higher hydrophobic interactions between p-cymene complexes and the
biomolecules. Thus, the in vitro anticancer activity of the complex towards NIH-3T3 (non-
cancerous cells) was determined to be above 221 µM, confirms that these complexes are
specific for cancer cells.
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Table 3 The cytotoxic activity of arene ruthenium(II) benzhydrazone complexes after 24 h exposure
aIC50 values (µM)
Complexes
HeLa
MDA-MB-231
Hep G2
>100
NIH3T3
L1
>100
>100
>100
>100
>100
L2
>100
>100
L3
>100
>100
>100
>100
L4
>100
>100
>100
>100
[(benzene)RuCl2]2
>100
>100
>100
>100
[(p-Cymene)RuCl2]2
>100
>100
>100
>100
1
32.5 ± 0.3
28.6 ± 0.4
31.2 ± 0.2
10.2 ± 0.5
22.9 ± 0.5
15.4 ± 0.3
17.2 ± 0.5
9.4 ± 0.2
22.6 ± 0.8
19.6 ± 0.3
18.7 ± 0.2
19.1 ± 0.3
9.8 ± 0.2
16.8 ± 0.2
10.5 ± 0.1
11.8 ± 0.2
8.3 ± 0.4
14.9 ± 0.5
26.9 ± 0.1
21.3 ± 0.5
22.0 ± 0.2
12.0 ± 0.3
21.9 ± 0.6
18.4 ± 0.3
18.5 ± 0.2
10.9 ± 0.2
21.3 ± 0.9
223.7 ± 0.8
232.4 ± 0.3
236.1 ± 0.3
272.9 ± 0.4
261.3 ± 0.9
242.6 ± 0.2
243.6 ± 0.3
288.0 ± 0.5
221.3 ± 0.6
2
3
4
5
6
7
8
Cisplatin
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aIC50 = concentration of the drug required to inhibit growth of 50% of the cancer cells (µM).
The sign (>) indicates that IC50 value was not obtained up to given concentration.
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2.5 Morphological changes in AO and EB dual staining
An Acridine Orange–Ethidium Bromide (AO–EB) dual fluorescent staining method
was used to investigate apoptosis in a MDA-MB-231cell line treated with complex 4 and 8.
After treatment of cells with the complexes 4 and 8 for 24 h and irradiated with visible light
showed significant reddish-orange emission with condensed chromatin and membrane
blebbing. In the control, the cells of MDA-MB-231 were stained bright green in spots.
Henceforth, the morphological changes clearly indicate that the complexes induce cell death
through apoptosis.
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Fig. 3 Morphological assessment of AO and EB dual staining of MDA-MB-231cells treated with complex 4 & 8
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(IC50 concentration) for 24 h. The scale bar 20 mm.
2.6 Morphological changes in Hoechst 33258 staining
To investigate the nuclear morphologic characteristics, MDA-MB-231 cells were
stained with Hoechst 33258 and treated with complexes 4 and 8 using fluorescence
microscopy. After 24 h, complexes treated cells showed fragmented nuclei and chromatin
condensation which are features of apoptosis different from control cells (Fig.4).
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Fig. 4 Morphology of the nuclei of MDA-MB-231 cells observed by fluorescence microscopy (Hoechst 33258
staining, 24 h incubation at IC50 concentrations) after treatment with control complexes 4 and 8.
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2.7 Evaluation of apoptosis – Flow cytometry
As shown in Fig. 5 and 6, MDA-MB-231cells were treated with complex 4 and 8 at
two different concentrations for 24 h. The increase of annexin V+/PI+ (Q2) population from
3.7% to 6.7% for 4 and 5.0% to 8.2% for 8 at 50 and 100 µM concentrations of the
complexes respectively represent cells undergoing apoptosis. Taken together, these results
indicate that cell death induced by complexes is mainly caused by induction of apoptosis.
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Fig. 5 Annexin V/propidium iodide assay of MDA-MB-231cells treated by complex 4 (50 and 100 µM
concentration) measured by flow cytometry.
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Fig. 6 Annexin V/propidium iodide assay of MDA-MB-231cells treated by complex 8 (50 and 100 µM
concentration) measured by flow cytometry.
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2.8 Comet assay
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The comet assay was used to detect the DNA strand breaks with high sensitivity at the
single-cell level [32]. As shown in Fig. 7, MDA-MB-231cells treated with IC50 concentration
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of the complexes 4 and 8 for 24h show the increase in the length of the comet tail and
illustrate that the complexes induce a remarkable DNA damage in a time-dependent manner,
the percentage of DNA damage presented in Fig.S35 (ESI†). Further, the results of comet
assay demonstrate that the complexes are capable of eliciting DNA damaging effects, as
evidenced by the comet assays on MDA-MB-231 cells
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Fig. 7 Comet assay of EB-stained control, complex 4 and 8 treated breast cancer cells at 24h incubation.
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3. Conclusions
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In summary, we have described the synthesis of a series of arene ruthenium(II)
benzhydrazone complexes. All the complexes have been completely characterized by
analytical techniques and spectroscopic methods. Crystallographic studies of the complexes 6
and 7 have shown that the benzhydrazone ligands are coordinated to Ru(II) in a bidentate
fashion via azomethine nitrogen and imidolate oxygen atoms. Besides, all the complexes
were tested for anticancer activity against HeLa, MDA-MB-231, and Hep G2 cancer cell
lines, and they were found to show excellent cytotoxicity to cancer cells without affecting the
normal NIH 3T3 cells. Remarkably, complexes 4 and 8 display high cytotoxicity against
cancer cell lines tested with very low IC50 values. Moreover, fluorescence staining
techniques, flow cytometry and comet assays demonstrated that complexes induce apoptosis
in MDA-MB-231 cells. Hence, confirming that these arene ruthenium(II) benzhydrazone
complexes have promising biological properties and are worth investigating further.
4. Experimental
4.1 Reagents and materials
RuCl3.3H2O was purchased from Loba Chemie Pvt. Ltd. and used as received.
Aldehydes and benzhydrazide derivatives were obtained from Aldrich. All other chemicals
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were purchased from commercial sources and used without further purification. The
Solvents were distilled following the standard procedures [33] and degassed prior to use.
[Ru(arene) (µ-Cl)Cl]2 (arene= benzene and p-cymene) was prepared by reported procedure
[34].
4.2 Physical measurements
FT-IR spectra in KBr pellets were recorded on a JASCO 400 plus spectrometer.
Microanalysis of carbon, hydrogen, nitrogen and sulphur were carried out by Vario EL III
CHNS elemental analyzer. UV- visible spectra was recorded on a CARY 300 Bio UV- Vis
1
spectrometer. The H NMR spectra were carried out with Bruker 400 MHz instruments.
Melting points were determined on a Boetius micro-heating table and are corrected. ESI-MS
spectra were obtained by micro mass Quattro II triple quadrupole mass spectrometer. The
annexin V-FITC kit (APOAF-20TST) from Sigma-Aldrich was used based on manufacturer
instructions.
4.3 Preparation of thiophene benzhydrazone ligands
A solution of thiophene-2-carboxyaldehyde (5 mmol) in ethanol (10mL) was added
drop wise to the ethanol solution (10 mL) of 4-substituted benzhydrazide (5 mmol) and the
reaction mixture was refluxed for about 3 h. The solution was concentrated to 5 ml and
cooled to room temperature. The cream or pale brown solid formed was filtered, washed
with cold methanol (5mL) and dried in air. Yield 83-88%.
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4.4 Synthesis of arene ruthenium(II) benzhydrazone complexes
A mixture of [Ru (ɳ6-C6H6) (µ-Cl)Cl]2 or [Ru (ɳ6-p-cymene) (µ-Cl)Cl]2 (0.04 mmol)
and benzhydrazone ligand (0.08 mmol) was refluxed in benzene in the presence of
triethylamine (0.5 mL) for 5 h. After removing the triethylammonium chloride by filtration,
the solution was concentrated and light petroleum ether (bp 60-80 ⁰C) was added whereby the
solid separated out. The resulted solids were recrystallized from CH2Cl2/petroleum ether and
dried under vacuum.
4.4.1 [Ru(η6-C6H6)(Cl)(L1)] (1). Colour: Brown; Yield: 80%; M.p.: 165 ⁰C; Anal. Calc. For
C18 H15 Cl N2 O Ru S: C, 48.70; H, 3.40; N, 6.31; S, 7.22%. Found: C, 48.52; H, 3.45; N,
6.30; S, 7.25%. IR (KBr, cm-1):1598 ν(C=N-N=C), 1265 ν(C-O). UV–Vis (CH3CN, λ max/nm;
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ε/dm3 mol-1 cm-1): 354(3415), 274(4254), 236(6652). H NMR (400 MHz, CDCl3) (δ ppm):
8.9 (s, 1H, N=CH), 7.1–8.1 (m, 8H, aromatic), 5.5(s, 6H). ESI-MS (CH3CN): calcd for C18
H15 Cl N2 O Ru S m/z 443.96; found [M - Cl]+ :410.00.
4.4.2 [Ru(η6-C6H6)(Cl)(L2)] (2). Colour: Brown; Yield: 77%; M.p.: 163 ⁰C; Anal. Calc. For
C18 H14 Cl2 N2 O Ru S: C, 45.19; H, 2.94; N, 5.85; S, 6.70%. Found: C, 45.35; H, 2.90; N,
5.88; S, 6.72%. IR (KBr, cm-1):1620 ν(C=N-N=C), 1260 ν(C-O). UV–Vis (CH3CN, λ max/nm;
ε/dm3 mol-1 cm-1): 366(1855), 281(2394), 238(4473). 1H NMR (400 MHz, CDCl3) (δ ppm):
8.9 (s, 1H, N=CH), 7.1–8.1 (m, 7H, aromatic), 5.5 (s, 6H). ESI-MS (CH3CN): calcd for C18
H14 Cl2 N2 O Ru S m/z 477.92; found [M - Cl]+ :444.96.
4.4.3 [Ru(η6-C6H6)(Cl)(L3)] (3). Colour: Brown; Yield: 74%; M.p.: 161 ⁰C; Anal. Calc. For
C18 H14 Br Cl N2 O Ru S: C, 41.35; H, 2.69; N, 5.35; S, 6.13%. Found: C, 41.58; H, 2.67; N,
5.36; S, 6.19%. IR (KBr, cm-1):1612 ν(C=N-N=C), 1256 ν(C-O). UV–Vis (CH3CN, λ max/nm;
1
ε/dm3 mol-1 cm-1): 361(7045), 273(8836), 244(12972). H NMR (400 MHz, CDCl3) (δ ppm):
8.9 (s, 1H, N=CH), 7.1–8.1 (m, 7H, aromatic), 5.5(s, 6H). ESI-MS (CH3CN): calcd for C18
H14 Br Cl N2 O Ru S m/z 521.87; found [M - Cl]+ :486.89.
4.4.4 [Ru(η6-C6H6)(Cl)(L4)] (4). Colour: Brown; Yield: 72%; M.p.: 157 ⁰C; Anal. Calc.
For C19 H17 Cl N2 O2 Ru S: C, 48.15; H, 3.61; N, 5.91; S, 6.76%. Found: C, 48.25; H, 3.67;
N, 5.95; S, 6.71%. IR (KBr, cm-1):1594 ν(C=N-N=C), 1272 ν(C-O). UV–Vis (CH3CN, λ max/nm;
1
ε/dm3 mol-1 cm-1): 360(5095), 279(5742), 253(7314). H NMR (400 MHz, CDCl3) (δ ppm):
8.9 (s, 1H, N=CH), 6.8–8.1 (m, 7H, aromatic), 5.5(s, 6H), 3.8 (s, 3H, OCH3). ESI-MS
(CH3CN): calcd for C19 H17 Cl N2 O2 Ru S m/z 473.97; found [M + H]+ :474.98, [M - Cl]+
:439.00.
4.4.5 [Ru(η6-p-cymene)(Cl)(L1)] (5). Colour: Yellow; Yield: 85%; M.p.: 188 ⁰C; Anal.
Calc. For C22 H23 Cl N2 O Ru S: C, 52.84; H, 4.63; N, 5.60; S, 6.41%. Found: C, 52.67; H,
4.60; N, 5.64; S, 6.44%. IR (KBr, cm-1):1596 ν(C=N-N=C), 1259 ν(C-O). UV–Vis (CH3CN, λ
max/nm; ε/dm3 mol-1 cm-1): 364(5516), 280(6007), 234(6622). H NMR (400 MHz, CDCl3)
1
(δ ppm): 8.8 (s, 1H, N=CH), 7.1–8.0 (m, 8H, aromatic), 5.3 (d, J = 5.6 Hz, 1H, cymene Ar-
H), 5.3 (d, J = 6 Hz, 1H, cymene Ar-H), 5.0 (d, J = 5.6 Hz, 1H, cymene Ar-H), 4.6 (d, J =
5.6 Hz, 1H, cymene Ar-H), 2.5 (m, 1H, CH of p-cymene), 2.2 (s, 3H, CH3 of p-cymene), 1.0-
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1.3 (dd, J = 94.8 Hz, J = 7.2 Hz, 6H, 2CH3 of p-cymene). ESI-MS (CH3CN): calcd for C22
H23 Cl N2 O Ru S m/z 500.02; found [M + H]+ :501.03, [M - Cl]+:465.05.
4.4.6 [Ru(η6-p-cymene)(Cl)(L2)] (6). Colour: Yellow; Yield: 82%; M.p.: 180 ⁰C; Anal.
Calc. For C22 H22 Cl2 N2 O Ru S: C, 49.43; H, 4.14; N, 5.24; S, 5.99%. Found: C, 49.28; H,
4.15; N, 5.22; S, 5.98%. IR (KBr, cm-1):1599 ν(C=N-N=C), 1262 ν(C-O). UV–Vis (CH3CN, λ
max/nm; ε/dm3 mol-1 cm-1): 364(3579), 282(3771), 245(5264).1H NMR (400 MHz, CDCl3)
(δ ppm): 8.8 (s, 1H, N=CH), 7.1–8.0 (m, 7H, aromatic), 5.3 (d, J = 6.4 Hz, 1H, cymene Ar-
H), 5.3 (d, J = 6 Hz, 1H, cymene Ar-H), 5.0 (d, J = 5.6 Hz, 1H, cymene Ar-H), 4.6 (d, J =
5.6 Hz, 1H, cymene Ar-H), 2.5 (m, 1H, CH of p-cymene), 2.2 (s, 3H, CH3 of p-cymene), 1.0-
1.3 (dd, J = 100.8 Hz, J = 14.4 Hz, 6H, 2CH3 of p-cymene). ESI-MS (CH3CN): calcd for C22
H22 Cl2 N2 O Ru S m/z 533.98; found = [M - Cl]+ :499.02.
4.4.7 [Ru(η6-p-cymene)(Cl)(L3)] (7). Colour: Yellow; Yield: 78%; M.p.: 178 ⁰C; Anal.
Calc. For C22 H22 Br Cl N2 O Ru S: C, 45.64; H, 3.83; N, 4.83; S, 5.53%. Found: C, 45.43; H,
3.85; N, 4.81; S, 5.54%. IR (KBr, cm-1):1607 ν(C=N-N=C), 1260 ν(C-O). UV–Vis (CH3CN, λ
1
max/nm; ε/dm3 mol-1 cm-1): 360(6761), 304(7395), 246(10158). H NMR (400 MHz, CDCl3)
(δ ppm): 8.8 (s, 1H, N=CH), 7.1–8.0 (m, 7H, aromatic), 5.3 (d, J = 6 Hz, 1H, cymene Ar-H),
5.3 (d, J = 6 Hz, 1H, cymene Ar-H), 5.0 (d, J = 5.6 Hz, 1H, cymene Ar-H), 4.6 (d, J = 5.6
Hz, 1H, cymene Ar-H), 2.5 (m, 1H, CH of p-cymene), 2.2 (s, 3H, CH3 of p-cymene), 1.0-1.3
(dd, J = 104 Hz, J = 7.2 Hz, 6H, 2CH3 of p-cymene). ESI-MS (CH3CN): calcd for C22 H22 Br
Cl N2 O Ru S m/z 577.93; found [M - Cl]+ :544.96.
4.4.8 [Ru(η6-p-cymene)(Cl)(L4)] (8). Colour: Yellow; Yield: 76%; M.p.: 168 ⁰C; Anal.
Calc. For C23 H25 Cl N2 O2 Ru S: C, 52.11; H, 4.75; N, 5.28; S, 6.04%. Found: C, 52.35; H,
4.70; N, 5.25; S, 6.08%. IR (KBr, cm-1):1592 ν(C=N-N=C), 1259 ν(C-O). UV–Vis (CH3CN, λ
1
max/nm; ε/dm3 mol-1 cm-1): 361(4930), 291(5508), 246(7594). H NMR (400 MHz, CDCl3)
(δ ppm): 8.8 (s, 1H, N=CH), 6.7–7.9 (m, 9H, aromatic), 5.3 (d, J = 6 Hz, 1H, cymene Ar-H),
5.3 (d, J = 6 Hz, 1H, cymene Ar-H), 5.0 (d, J = 5.6 Hz, 1H, cymene Ar-H), 4.6 (d, J = 5.6
Hz, 1H, cymene Ar-H), 3.7 (s, 3H, OCH3), 2.5 (m, 1H, CH of p-cymene), 2.2 (s, 3H, CH3 of
p-cymene), 1.0-1.3 (dd, J = 101.6 Hz, J = 7.2 Hz, 6H, 2CH3 of p-cymene). ESI-MS
(CH3CN): calcd for C23 H25 Cl N2 O2 Ru S m/z 530.04; found [M + H]+ :531.04, [M - Cl]+
:495.06.
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4.5 X-ray crystallography
A Single crystal of [Ru(η6- p-cymene)Cl(L2)] (6) and [Ru(η6- p-cymene)Cl(L3)] (7)
were obtained Dichloromethane-Petroleum ether solution at room temperature by slow
evaporation technique. X-Ray data were collected with a Bruker AXS Kappa APEX II single
crystal X-ray diffractometer using monochromated Mo-Kα radiation (λ=0.71073). The
structure solution was obtained by direct methods (SIR-97) [35] and refined using (SHELXL-
97) full matrix least-squares calculations on F2 [36]. All non-hydrogen atoms were refined
anisotropically, hydrogen atoms were fixed geometrically and refined by riding model. The
Bruker SAINT-Plus (Version 7.06a) software were used to analyse the Frame integration and
data reduction. The multiscan absorption corrections were applied using SADABS software.
CCDC reference number is 1449681-1449682.
4.6 Stability Studies
The stability of the complexes were carried out as described previously [37].
4.7 Partition Coefficient Determination
Partition coefficients (P) between n-octanol and water phases were carried out as
described previously [27,38].
4.8 Cell culture
HeLa human cervical cancer cell line, MDA-MB-231 Triple negative breast
carcinoma, Hep G2 human liver carcinoma cell line and NIH 3T3 noncancerous cell,
mouse embryonic fibroblast were supplied by the National Centre for Cell Science
(NCCS), Pune. The cell lines were cultured as a monolayer in RPMI-1640 medium
(Biochrom AG, Berlin, Germany), supplemented with 10% fetal bovine serum (Sigma-
Aldrich, St. Louis, MO, USA) and with 100 U mL-1 penicillin and 100 µg mL-1 streptomycin
as antibiotics (Himedia, Mumbai, India), at 37 oC in a humidified atmosphere of 5% CO2 in a
CO2 incubator (Heraeus, Hanau, Germany).
MTT assay, AO-EB staining, Hoechst 33258 staining, Flow cytometry and
comet assay were evaluated as described previously [39-42].
Acknowledgments
One of the authors (N. M) thanks University Grants Commission (UGC), New Delhi,
for the award of UGC-RFSMS. We express sincere thanks to DST-FIST, India for the use of
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Bruker 400 MHz spectrometer at the School of Chemistry, Bharathidasan University,
Tiruchirappalli. We thank Dr T. R. Santhosh Kumar for the flow cytometry analysis.
Appendix A. Supplementary material
1H spectra of the complexes (1-8), The ESI-MS of the complexes (1-8) and log P
Values for Complexes 1-8. CCDC 1449681-1449682 contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
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‹ Synthesis and characterization of arene ruthenium(II) benzhydrazone complexes.
‹ The single-crystal X-ray structure analysis of two complexes is depicted.
‹ The complexes have been screened for their in vitro antiproliferative activities.
‹ The mechanism of action of the most potent complexes was evaluated.