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Synthesis, structure and anticancer activity of (η6-benzene) ruthenium(II) complexes containing aroylhydrazone ligands
Accepted Manuscript
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Synthesis, structure and anticancer activity of (η -benzene) ruthenium(II) complexes
containing aroylhydrazone ligands
Nanjan Mohan, Subramanian Muthumari, Rengan Ramesh
PII:
S0022-328X(16)30033-X
DOI:
10.1016/j.jorganchem.2016.01.033
Reference:
JOM 19384
To appear in:
Journal of Organometallic Chemistry
Received Date: 24 November 2015
Revised Date:
21 January 2016
Accepted Date: 28 January 2016
Please cite this article as: N. Mohan, S. Muthumari, R. Ramesh, Synthesis, structure and anticancer
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activity of (η -benzene) ruthenium(II) complexes containing aroylhydrazone ligands, Journal of
Organometallic Chemistry (2016), doi: 10.1016/j.jorganchem.2016.01.033.
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ACCEPTED MANUSCRIPT
New (η6-benzene) ruthenium(II) complexes containing aroylhydrazone ligands were
synthesized and characterized. The single crystal X- ray analysis of the complex 4
reveals a typical three leg piano stool structure. Complexes are more potent against
MCF-7 cells than cisplatin. Fluorescence staining methods confirm apoptosis induced
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cell death.
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Synthesis, structure and anticancer activity of (η6-benzene) ruthenium(II)
complexes containing aroylhydrazone ligands
Nanjan Mohan, Subramanian Muthumari and Rengan Ramesh*
School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
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ABSTRACT
New benzene ruthenium(II) aroylhydrazone complexes of general molecular formula [Ru(η6C6H6)Cl(L)] (where L = aroylhydrazone ligand) have been synthesized from the reaction of
the precursor [Ru(η6- C6H6)(µ-Cl)Cl]2 and aroylhydrazone ligands. The composition of the
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complexes has been accomplished by elemental analysis and spectral methods (FT-IR, UVVis, 1HNMR). The molecular structure of complex 4 has been established by single-crystal
X-ray structure analysis shows that the aroylhydrazone ligands are coordinated to ruthenium
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as a bidentate N, O donor and a typical piano stool geometry was observed around
ruthenium(II) metal center. All the complexes exhibit two consecutive irreversible oxidations
in the potential range +0.74 to +1.17 V (RuII/RuIII;RuIII/RuIV) Vs calomel electrode. Further,
in vitro anticancer activity of complexes 1-4 on human breast cancer cell line (MCF-7),
human cervical cancer cell line (HeLa) and non-cancerous NIH-3T3 cell line exhibit
It is also evident from IC50 values that the
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moderate to excellent cytotoxic activity.
complexes are more potent against MCF-7 cells than cisplatin. The superior activity of the
complex 4 assumes that presence of electron donating methoxy substituent which makes the
ring more reactive. Further, the morphological changes during cell death were investigated
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by Acridine Orange-Ethidium Bromide (AO–EB) and DAPI staining techniques, which
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confirm the complex 4 induces cell death only through apoptosis.
Keywords:
Aroylhydrazone, Benzene ruthenium(II) complex; Molecular structure; Redox behaviour;
Cytotoxicity; Apoptosis
* Corresponding author. Tel.: +91 431 2407053; fax: +91 431 2407045.
E-mail address: ramesh_bdu@yahoo.com (R. Ramesh).
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1. Introduction
The pioneering work on the anticancer properties of cisplatin by Rosenberg in 1965 [1]
has successfully resulted in using it as an effective drug against cancer for the past few
decades. However, this drug shows resistance, high toxicity and other side effects [2, 3]. For
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this reason ruthenium based drugs have been thought of as an attractive alternative due to
their fewer side effects, higher activity [4] and their similarity to iron in binding properties
[5]. In addition, most of the ruthenium complexes interact with DNA in vitro and display
binding modes and better activity when compared to those of platinum based drugs. In
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particular two ruthenium(III)-based drugs, KP1019 and NAMIA have already entered into
clinical trials [6-8], but differ considerably from cisplatin in their in vivo behaviour.
Half-sandwich arene ruthenium(II) compounds have been the subject of intense research
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in recent years because of their lipophilic and hydrophilic properties [9,10]. In addition, all
these complexes adopt a typical three-legged piano stool conformation, where the arene
ligand forms the seat and the chelating ligand along with auxiliary ligand are the legs of the
piano stool. It is to be considered that arene Ru(II) complexes often possess good aqueous
solubility along with satisfactory lipophilicity needed to cross the cell membrane. Moreover,
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the coordinated arene stabilizes ruthenium in the +2 oxidation state and different types of
substituent can be modified to tune the properties of the arene-ruthenium complexes. The
ligand exchange kinetics of Pt(II) and Ru(II) complexes are very similar in aqueous solution,
pivotal for anticancer activity [11]. The mechanism of cytotoxic involves the hydrolysis of
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the Ru-X bond giving rise to the active targeting species [12]. However, arene ruthenium
complexes of the type [Ru(η6-arene)(PTA)Cl2] (PTA = 1,3,5-triaza-7-phosphaadamantane)
(η6-arene)RuCl2(imidazole)
[14],
(η6-arene)RuCl2(DMSO)
[Ru(η6-
[15],
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[13],
arene)(YZ)Cl][PF6] [16] (YZ = chelating diamine) as well as dinuclear compounds [17], and
the tri [18] and tetranuclear clusters [19] such as [H3Ru3(η6–C6H6)(η6–C6Me6)2O]+ and
[H4Ru4(η6–C6H6)4]2+ have been studied in vitro and in some cases in vivo for their antitumor
activity. Recent studies have shown that metal complexes bind to the primary target DNA as
well as strongly interact with proteins [9, 13b, 20 &21]. Therefore, advancement of the
anticancer agents targeting both DNA and proteins are most sought after [13b, 20-22].
Aroylhydrazones are versatile ligands exhibiting amide-imidol tautomerism (Scheme 1)
and display interesting coordination modes in metal complexes. Depending on the acidity,
the reaction conditions and the nature of the metal ion, these ligands coordinate to the metal
ion via the azomethine nitrogen either in the neutral amide form or in the monobasic
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imidolate form, as bidentate N, O donor ligands forming five-membered chelate rings with
the metal [23]. Various studies have also shown that the azomethine group having a lone pair
of electrons in either a p or sp2 hybridized orbital on trigonally hybridized nitrogen has
considerable biological importance [24]. Though several hydrazone complexes of Cu(II),
Ni(II), Pd(II), Pt(II), Co(II), V(V) and Ru(II) [25-31] have been studied, the biological
O
R1
N
R
OH
N
R1
N
R
R2
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R2
N
H
Tautomerization
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applications of arene ruthenium(II) complexes with hydrazone ligands are not well explored.
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Scheme 1. Amide and imidol forms of hydrazone.
With the objective of promoting the applications of ruthenium(II) hydrazone complexes, we
report here the synthesis and spectral characterization of a series of benzene ruthenium(II)
complexes containing aroylhydrazone ligands.
The molecular structure of one of the
complexes was determined by single crystal X-ray diffraction. The redox property of the
complexes was examined by cyclic voltammetry. In vitro anticancer activity of these
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complexes against human cancer cell lines and the effect of the substituents present on the
ligand on the above said properties were described. Further, the mechanism of cancer cell
death was also investigated by AO–EB and DAPI staining techniques.
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2. Results and discussion
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The aroylhydrazone ligand derivatives were conveniently prepared in an excellent yield by
the condensation of acetophenone with substituted benzhydrazides in an equimolar ratio.
These ligands were allowed to react with the ruthenium(II) precursor, [Ru(η6- C6H6)(µCl)Cl]2 in a 2:1 molar ratio in the presence of triethylamine as the base and the new
complexes of the general formula, [Ru(η6-C6H6)Cl(L)] (Scheme 2) were obtained in
reasonable yields. The addition of triethylamine to the reaction mixture was used to remove a
proton from the imidol oxygen and to facilitate the coordination of the imidolate oxygen to
the ruthenium(II) ion. The synthesised benzene ruthenium(II) complexes are soluble in
solvent such as benzene, toluene, chloroform, dichloromethane, acetonitrile, dimethyl
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formamide, dimethyl sulphoxide as well as in water. The analytical data of all the benzene
ruthenium(II) complexes are in good agreement with the molecular structures proposed.
Cl
Cl
Ru
H3C
Ru
Ru
N
HN
Benzene, Et3N
O
Cl
Cl
R
Reflux, 5h
H3C
N
O
N
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Cl
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R
R = -H (L1), -Cl (L2), Br (L3), -OCH3 (L4)
2.1. Spectral characterization
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Scheme 2. Synthesis of benzene ruthenium(II) aroylhydrazone complexes.
The FT-IR spectra of the ligands showed a medium to strong band in the region 31803196 cm-1 which is characteristic of the ν(N-H) functional groups respectively. The free ligand
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also display ν(C=N) 1539-1576 cm-1 and ν(C=O) absorptions in the region 1610-1653 cm-1. The
bands due to N-H and C=O stretching vibrations are also not observed in the complexes
indicating that the ligand undergo tautomerisation and subsequent coordination of the
imidolate oxygen to the ruthenium(II) ion. This is further supported by the appearance of
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new bands in the region 1524-1530 cm-1 which may be attributed to the C=N-N=C fragments
[32] and disappearance of ν(C=O)and appearance of ν(N=C-O) and ν(C-O) in the region 1474-1486
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and 1369-1396 cm-1 respectively [33], in all the metal (II) complexes therefore ascertain the
coordination mode of the aroylhydrazone ligand to ruthenium(II) ion via the azomethine
nitrogen and the imidolate oxygen. (Fig. S1-S4, Supporting information)
The electronic spectra of all the benzene ruthenium(II) complexes were recorded in dry
chloroform solution in the range 200-800 nm. All the complexes display three intense
absorptions in the region 400-230 nm. The absorption spectra of the benzene ruthenium(II)
aroylhydrazone complexes exhibited very intense band around 266-288 nm and 241-244 nm
are assigned to ligand-centered (LC) π-π* and n-π* transitions respectively. The lowest
energy absorption bands in the electronic spectra of the complexes in the visible region 315326 nm are ascribed to metal to ligand charge transfer MLCT transitions. Based on the
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pattern of the electronic spectra of all the complexes an octahedral environment around the
ruthenium(II) ion has been proposed similar to that of the other octahedral ruthenium(II)
complexes.
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2.2. 1H NMR spectra
The bonding arrangement is further supported by 1H NMR spectra (Table 1). The 1H
NMR spectra of all the complexes were recorded in CDCl3. The multiplets observed in the
region around δ 6.8-8.1 ppm in all the complexes have been assigned to the aromatic protons
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of the aroylhydrazone ligand. The singlet in the region δ 2.8 ppm is due to methyl protons
nearer to coordinated azomethine group. In addition, peaks around δ 8.9-9.2 ppm for –NH
disappeared in all the complexes due to enolisation followed by deprotonation on imidole
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oxygen indicating that the ruthenium is coordinated through imidolate oxygen. The singlet
around δ 3.8 ppm is due to methoxy protons in the complex 4 and singlet in the region δ 5.0
ppm corresponding to the protons of the arene ligand. (Fig. S5-S8, Supporting information)
Table 1
H NMR data of benzene ruthenium(II) aroylhydrazone complexes
H NMR data δ/ppm
Ar-H
C6H6
-CH3
-OCH3
1
7.2–8.1
5.0
2.8
--
2
7.2–8.0
5.0
2.8
--
3
7.2–8.0
5.0
2.8
--
4
6.8–8.1
5.0
2.8
3.8
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complexes
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2.3. X-ray structure determination
The molecular structure of one of the benzene ruthenium(II) complexes [Ru(η6C6H6)Cl(L4)] 4 has been confirmed by a single – crystal X-ray diffraction analysis in order to
confirm the coordination mode of the ligand and geometry of the complex. The single
crystals of the complex 4 were obtained from slow evaporation of dichloromethanepetroleum ether solution at room temperature. The ORTEP view of complex 4 is shown in
Fig. 1. The summary of the data collection and refinement parameters are given in (Table 2)
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and selected bond lengths and angles are given in (Table 3). The complex 4 crystallize in the
monoclinic space group P2(1)/n. The benzene ligand is bonded to the ruthenium atom in η6
fashion with ruthenium centroid. The complex adopt a typical three-legged piano stool
conformation with N, O and Cl atoms as the legs and evident by the nearly 90⁰bond angles
for N(1)-Ru(1)-Cl(1) 84.64(6) and O(1)-Ru(1)-Cl(1) 85.81(7). The aroylhydrazone ligand
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bind to the metal center at N and O forming the five membered chelate ring with bite angle
76.64(8) O-Ru-N and bond length of Ru-N and Ru-O are 2.107(2) and 2.0420(18)
respectively. The Ru-Cl bond length is found to be 2.3907(8). The ruthenium atom is π
bonded to the benzene ring with an average Ru–C distance of 2.170(3) Å, whereas average
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distance of ruthenium to the chelating nitrogen and oxygen atoms is 2.074 Å. The average
C–C bond length in the benzene ring is 1.391 Å with alternating short and long bonds. As all
the four benzene ruthenium(II) complexes show similar spectral properties, the other three
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complexes are considered to have similar structure as that of complex 4.
Fig. 1. ORTEP diagram of the complex [Ru(η6_C6H6)Cl(L4)] (4), showing 50% probability level.
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Table 2
Crystal data and structure refinement for complex 4
Compound
4
C22 H21Cl N2 O2Ru
Formula weight
481.93
Temperature
296(2) K
Wavelength
0.71073 A
Crystal system, space group
Monoclinic, P21/n
Unit cell dimensions
a = 10.0868(7) Å alpha = 90 deg.
b = 19.0613(14) Å beta = 98.585(3) deg.
c = 10.4898(7) Å
gamma = 90 deg.
Volume
1994.2(2) Å3
Z, Calculated density
4, 1.605 Mg/m3
Absorption coefficient
0.940 mm-1
F(000)
976
Crystal size
0.35 x 0.30 x 0.30 mm
Theta range for data collection
2.24 to 28.55 deg.
Limiting indices
-13 ≤ h ≤ 10, -25 ≤ k ≤ 25, -13 ≤ l ≤ 12
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Formula
16583 / 4908 [R(int) = 0.0217]
Completeness to theta = 28.55
96.7 %
Absorption correction
Semi-empirical from equivalents
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Reflections collected / unique
0.7656 and 0.7343
Refinement method
Full-matrix least-squares on F2
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Max. and min. transmission
Data / restraints / parameters
4908 / 0 / 255
Goodness-of-fit on F2
1.097
Final R indices [I>2sigma(I)]
R1 = 0.0306, wR2 = 0.0832
R indices (all data)
R1 = 0.0411, wR2 = 0.0968
Largest diff. peak and hole
0.480 and -0.600 e.Å-3
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Table 3
Selected bond lengths (Å) and angles (⁰) for complex 4
Bond lengths (Å)
N(1)-N(2)
1.402(3)
N(2)-N(1)-Ru(1)
113.69(14)
N(1)-Ru(1)
2.107(2)
C(7)-N(2)-N(1)
111.10(2)
O(1)-Ru(1)
2.042(18)
C(7)-O(1)-Ru(1)
113.12(16)
Cl(1)-Ru(1)
2.390(8)
O(1)-Ru(1)-N(1)
76.64(8)
C(7)-O(1)
1.288(3)
O(1)-Ru(1)-Cl(1)
85.81(7)
C(7)-N(2)
1.309(3)
N(1)-Ru(1)-Cl(1)
84.64(6)
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ESD in parenthesis.
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Bond angles (⁰)
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2.4. Electrochemical property
Electrochemical study was carried out for all the free ligands and the benzene
ruthenium(II) aroylhydrazone complexes in degassed acetonitrile at room temperature in the
potential range of 0 to +1.5.
The supporting electrolyte used was 0.05 M
tetrabutylammonium perchlorate (TBAP) and the concentration of the complex was 10-3M.
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The resulting data are summarised in (Table4). All the complexes exhibit two consecutive
one electron irreversible oxidations in the potential range of 0.74 to +1.17 V at the scan rate
of 100 mV s-1with reference to saturated calomel electrode (Fig. S9-S12, Supporting
information). The comparison of its current height with that of the standard
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ferrocene/ferrocenium couple under identical conditions reveals the one electron transfer
process. The first oxidative response with Epa in the range +0.74 to +0.79 V is assigned to
RuII → RuIII oxidation whereas the second one in the range +1.12 to +1.17 V is assigned to
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RuIII → RuIV oxidation. These potentials are comparable with RuII→ RuIII and RuIII → RuIV
oxidation potential of other mononuclear ruthenium complexes [34, 35].
In addition,
irreversibility observed for the oxidative responses may be due to the fast dissociation of
chloride ligand from ruthenium(II) complexes [36].
It is worth noting that correlation
between the Epa values and IC50 values has been observed for arene ruthenium compounds
with anticancer properties [37]. The values between +0.74 and +0.79 V, the RuII/RuIII redox
potentials for complex 1–4 are lower than those for the more cytotoxic complexes [(η6arene)RuCl2(NC5H4OOCC5H4FeC5H5)], the Epa values ranging from +0.91 to +1.00 V [38],
but significantly higher than the RuII/RuIII redox potentials for the complex [(η6-arene)Ru(SC5H4NH)3]2+, the Epa values ranging from +0.58 and +0.67 V [37].
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Table 4
Electrochemical data of benzene ruthenium(II) aroylhydrazone complexes
1
2
3
4
RuIII / RuIIRuIV / RuIII
Epa (V)
+0.76
+0.78
+0.79
+0.74
Epa (V)
+1.14
+1.16
+1.17
+1.12
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Complexes
Solvent = Acetonitrile; [Complex] = 1x10-3M; Supporting electrolyte [Bu4N] (ClO4) (0.05 M); Scan rate:
100mVs-1; All potentials referenced to SCE; Epa = anodic peak potential.
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2.5. In vitro cytotoxic activity
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The potential of the complexes to inhibit cancer cell growth was evaluated using the MTT
assay. The in vitro cytotoxicity of the metallic precursors, ligand and benzene ruthenium(II)
complexes 1-4, was evaluated against human breast cancer (MCF-7) cells, human cervix
carcinoma (HeLa) cells and non-cancerous NIH-3T3 mouse embryonic fibroblasts cell lines
using MTT assay after 24 hours of inhibition, which measures mitochondrial dehydrogenase
activity as an indication of cell viability. For comparison, the cytotoxicity of known anti-
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cancer drug cisplatin has also assessed against all the above cell lines. The results were
analysed by means of cell inhibition of cancer cell growth at the 50% level expressed as IC50
values and the values of the four benzene ruthenium(II) complexes 1-4 are listed in (Table 5).
It is to be noted that the precursor and the ligand did not show any inhibition of the cell
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growth even up to 100 µM and clearly indicates chelation of the ligand with metal ion is
responsible for the observed cytotoxicity properties of the complexes. All the complexes
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have moderate to excellent cytotoxic activity against MCF-7, HeLa and NIH-3T3 cell line
used. It is found that all the complexes are more potent against MCF-7 cells as compared to
other cell lines. Among them, complex 4 exhibit excellent IC50 against MCF cell lines. The
superior activity of the complex 4 assumes that presence of electron donating methoxy
substituent which makes the ring more reactive and thereby increases of the lipophilic
character of the metal complex which favours its permeation through the lipid layer of the
cell membrane [39]. Hence, the cytotoxicity increases in the order H ˂ Br ˂ Cl ˂ OCH3. It
should be noted that the observed IC50 values of the reported complexes are considerably
better against MCF-7 cell line than those characteristic of cisplatin. Further, the IC50 values
of the complexes are much better than those previously reported arene ruthenium(II)
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complexes [40]. The excellent cytotoxic activity observed for 4 against the tested cell lines
may be due to better cellular uptake inside the cells which is evident by fluorescence images.
Table 5
a
MCF-7
IC50 values (µM)
HeLa
NIH-3T3
15.8 ± 0.4
12.8 ± 0.7
13.5 ± 0.3
10.9 ± 0.3
15.2 ± 0.5
48.7 ± 0.9
38.4 ± 0.9
41.5 ± 0.6
34.3 ± 1.3
11.7 ± 0.7
192.4 ± 2.1
178.7 ± 1.0
152.6 ± 1.9
182.8 ± 0.9
240.9 ± 1.9
Complexes
a
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2
3
4
Cisplatin
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The cytotoxic activity of benzene ruthenium(II) aroylhydrazone complexes
IC50 = concentration of the drug required to inhibit growth of 50% of the cancer cells (µM).
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2.6. Morphological changes in AO/EB and DAPI fluorescence study
Cell apoptosis is an important phenomenon responsible for destroying undesirable cells
during the development and homeostasis of cellular organisms [41]. As such, the ability to
kill tumor cells through the induction of apoptosis has been used as a marker for the
identification of antitumor drugs [42].
Cells undergoing apoptosis are characterized by
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morphological and biochemical changes including cell shrinkage, chromatin condensation
and DNA fragmentation [43].
Apoptotic cells reveal increased plasma membrane
permeability to certain fluorescent dyes, e.g., AO/EB, Hoechst, AO/PI, DAPI etc. In this
To investigate the
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study, we have carried out AO/EB and DAPI staining methods.
morphological changes, the most active compound 4 in MCF-7 cells were further studied
using Acridine Orange/Ethidium Bromide (AO/EB) staining technique to perceive whether
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the inhibition is due to apoptotic induction or nonspecific necrosis and the resulting images of
the control and treated MCF-7cells are depicted in Fig. 2 and 3. Acridine Orange permeates
the intact cell membrane and stains the nuclei green, whereas Ethidium Bromide is excluded
from the cells having intact plasma membrane and stains the DNA of dead cells, showing
orange fluorescence. Furthermore, in DAPI staining the control cells shows clear intensity
but the treated cells with the complex shows strong fluorescence intensity. The cells treated
with the complex in the dark did not show any significant nuclear morphological change.
The microscopic image Fig. 2 and 3 gives clear evidence of the formation of more apoptotic
bodies, characterized by the fragmentation of nuclei with condensed chromatin, upon the
treatment of MCF-7 with compound 4.
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Fig.2. Morphological changes in human breast cancer MCF-7cell treated with compound 4 for 24 h.
Fig.3. Morphological changes in human breast cancer MCF-7 cell treated with compound 4 for 24 h.
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3. Conclusions
A series of benzene ruthenium(II) aroylhydrazone complexes has been synthesised and
characterized by analytical and spectroscopic methods. The molecular structure of 4 has been
studied by single-crystal X-ray structure analysis indicates that the aroylhydrazone ligands
are coordinated to ruthenium as a bidentate N, O donor and a typical piano stool structure
was observed around ruthenium(II) metal center. All the complexes (1-4) show moderate to
excellent cytotoxic activity against MCF-7 and HeLa cell line used.
Particularly the
complexes are more active against MCF-7 cells as compared to other cell lines. Among all
the complexes, 4 is found to have higher cytotoxicity (10.9 µM against MCF-7 cancer cell
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lines). The microscopic images give clear evidence of the formation of more apoptotic
bodies, characterized by the fragmentation of nuclei with condensed chromatin, upon the
treatment of MCF-7 with compound 4.
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4. Experimental
4.1. Reagents and materials
RuCl3.3H2O is commercially available and was used as supplied from Loba chemie Pvt.
received.
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Ltd. Ketones and benzhydrazide derivatives were purchased from Aldrich and were used as
The supporting electrolyte, tetrabutylammonium perchlorate (TBAP) was
purchased from Aldrich and dried in vacuum prior to use. All other chemicals were obtained
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from commercial sources and were used as received. The Solvents were distilled following
the standard procedures [44] and degassed before use. The precursor [Ru(η6-C6H6)(µ-Cl)Cl]2
complex was prepared by reported literature method [45].
The human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and
non-cancerous NIH-3T3 mouse embryonic fibroblasts cell line were obtained from the
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National Center for Cell Science (NCCS), Pune, India.
4.2. Physical measurements
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FT-IR spectra were recorded in KBr pellets with JASCO 400 plus spectrometer. The
microanalysis of carbon, hydrogen, nitrogen and sulphur were recorded by an analytical
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function testing Vario EL III CHNS elemental analyser at the Sophisticated Test and
Instrumentation Centre (STIC), Cochin University, Kochi. Electronic spectra in chloroform
solution were recorded with a CARY 300 Bio UV- visible Varian spectrometer.
1
H NMR
spectra were recorded on a Bruker 400 MHz instruments using tetramethylsilane (TMS) as an
internal reference. Electrochemical measurements were made using a CH Instruments, Inc.
Model 600E SN: I1140 Electrochemical Analyser using a glassy carbon working electrode,
Pt wire as counter electrode and all the potentials were reference to saturated calomel
electrode.
Melting points were recorded with a Boetius micro-heating table and are
corrected.
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4.3. Preparation of aroylhydrazone ligand
A mixture of 4-substituded benzhydrazide (0.01 mmol) and acetophenone (0.01 mmol) in
methanol (30 mL) was refluxed for 30 min. The separated solid was filtered and dried in air.
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Yield: 87-91%.
4.4. Synthesis of benzene ruthenium(II) aroylhydrazone complexes
The complexes were prepared using a general procedure in which [Ru(η6-C6H6)(µ-Cl)Cl]2
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(25mg, 0.04 mmol) with aroylhydrazone ligand (19.4-25.8 mg, 0.08 mmol) in the presence of
Et3N (0.5 mL) in benzene 30 mL. The resulting solution was refluxed for 5h and the progress
of the reaction was monitored by TLC.
At the end of the reaction the solution was
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concentrated to about 3 mL and petroleum ether was added whereby solid separated out. The
obtained solid was recrystallized from CH2Cl2-petroleum ether at room temperature. Yield:
70-81%.
4.4.1. [Ru(η6-C6H6)(Cl)(L1)]
Colour: Brown; Yield: 81%; M.p.: 170 ⁰C (with decomposition); Anal. Calc. for C21H19
1
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ClN2ORu: C, 55.81; H, 4.23; N, 6.19%. Found: C,55.71; H, 4.17; N, 6.25%. IR (KBr, cm):1530 ν(C=N-N=C), 1486 ν (N=C-O), 1376 ν(C-O). UV–Vis (CH3CN, λmax/nm; ε/dm3 mol-1 cm-1):
315(7261), 266(10,825), 243(12,234).
1
H NMR (400 MHz, CDCl3) (δ ppm): 7.2–8.1 (m,
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10H, aromatic), 5.0(s, 6H), 2.8 (s, 3H, CH3).
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4.4.2. [Ru(η6-C6H6)(Cl)(L2)]
Colour: Brown; Yield: 76%; M.p.: 165 ⁰C (with decomposition); Anal. Calc. for C21H18
Cl2N2ORu: C, 51.86; H, 3.73; N, 5.75%. Found: C,51.81; H, 3.77; N, 5.70%. IR (KBr, cm1
):1524 ν(C=N-N=C), 1482 ν (N=C-O), 1376 ν(C-O). UV–Vis (CH3CN, λmax/nm; ε/dm3 mol-1 cm-1):
320(2494), 267(4293), 242(4817). 1H NMR (400 MHz, CDCl3) (δ ppm): 7.2–8.0 (m, 9H,
aromatic), 5.0(s, 6H), 2.8 (s, 3H, CH3).
4.4.3. [Ru(η6-C6H6)(Cl)(L3)]
Colour: Brown; Yield: 70%; M.p.: 162 ⁰C (with decomposition); Anal.Calc. for C21H18
ClN2BrORu: C, 47.51; H, 3.41; N, 5.27%. Found: C,47.51; H, 3.47; N, 5.25%. IR (KBr, cm-
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1
):1529 ν(C=N-N=C), 1477 ν (N=C-O), 1369 ν(C-O). UV–Vis (CH3CN, λmax/nm; ε/dm3 mol-1 cm-1):
320(4913), 268(830), 244(8673).
1
H NMR (400 MHz, CDCl3) (δ ppm): 7.2–8.0 (m, 9H,
aromatic), 5.0(s, 6H), 2.8 (s, 3H, CH3).
4.4.4. [Ru(η6-C6H6)(Cl)(L4)]
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Colour: Brown; Yield: 72%; M.p.: 159 ⁰C (with decomposition); Anal. Calc. for C22H21
ClN2O2Ru: C, 54.82; H, 4.39; N, 5.81%. Found: C,54.88; H, 4.36; N, 5.85%. IR (KBr, cm1
):1525 ν(C=N-N=C), 1474 ν (N=C-O), 1396 ν(C-O). UV–Vis (CH3CN, λmax/nm; ε/dm3 mol-1 cm-1):
326(4061), 288(5749), 241(6109). 1H NMR (400 MHz, CDCl3) (δ ppm): 6.8–8.1 (m, 9H,
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aromatic), 5.0(s, 6H), 2.8 (s, 3H, CH3), 3.8 (s, 3H, OCH3).
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4.5. X-ray crystallography
Single crystal of [Ru(η6-C6H6)Cl(L4)] (4) were grown by slow evaporation of
Dichloromethane-Petroleum ether solution at room temperature. A single crystal of suitable
size was covered with Paratone oil, mounted on the top of a glass fiber, and transferred to a
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Bruker AXS Kappa APEX II single crystal X-ray diffractometer using monochromated MoKα radiation (λ=0.71073). Data were collected at 293K. The structure was solved with
direct method using SIR-97 [46] and was refined by full matrix least-squares method on F2
with SHELXL-97[47].
Non-hydrogen atoms were refined with anisotropy thermal
riding model.
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parameters. All hydrogen atoms were geometrically fixed and collected to refine using a
Frame integration and data reduction were performed using the Bruker
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SAINT-Plus (Version 7.06a) software. The multiscan absorption corrections were applied to
the data using SADABS software. CCDC reference number is 1064013.
4.6. In vitro cytotoxic activity. maintenance of cell lines
The assays were carried out in monolayer cells were detached with trypsinethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells
were counted using a hemocytometer and diluted with medium containing 5% FBS to give
final density of 1x105 cells/ml. One hundred microliters per well of cell/well and incubated
to allow for cell attachment at 37 ⁰C, 5% CO2, 95% air and 100% relative humidity. After 24
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hours the cells were treated with serial concentrations of the test samples. They were initially
dissolved in neat DMSO to prepare the stock (200 µM) and stored frozen prior to use. At the
time of drug addition, the frozen concentrate was thawed and an aliquot was diluted to twice
the desired final maximum test concentration with serum free medium. Additional three, 10
fold serial dilutions were made to provide a total of four drug concentrations. Aliquots of
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100 µL of these different drug dilutions were added to the appropriate wells already
containing 100 µL of medium, resulted the required final drug concentrations. Following
drug additions the plates were incubated for an additional 24 h at 37 ⁰C, 5% CO2, 95% air
and 100% relative humidity. The medium containing no samples served as a control and
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triplicate was maintained for all concentrations.
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4.7. Evaluation by MTT assays
MTT is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells,
succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble
purple colour formazan. Therefore, the amount of formazan produced is directly proportional
to the number of viable cells after 24 h of plating benzene ruthenium(II) complexes 1-4 were
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added at various concentration (1 to 100 µM for 24 h, with a final volume in the well of 300
µL) for 24 h to study the dose dependent cytotoxic effect. To each well, 15 µL of MTT (5
mg/mL) in phosphate buffered saline was added to each well and incubated at 37 ⁰C for 4 h.
The medium with MTT was then flicked off and the formed formazan crystals were
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solubilized in 100 µL of DMSO and then the absorbance at 570 nm was using micro plate
reader. Nonlinear regression graph was plotted with the percentage of cell inhibition versus
From this, the IC50 value was calculated by using the following
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log10 concentration.
formula.
% cell inhibition= 1-Abs (sample)/Abs (control) x100.
4.8. Fluorescent dual staining experiment
Acridine Orange and Ethidium Bromide (AO and EB) staining was performed as follows:
the cell suspension of each sample containing 5 x 105 cells, was treated with 25 µL of AO
and EB solution (1 part of 100 µg mL-1 AO and 1 part of µg mL-1 EB in PBS) and examined
in a laser scanning confocal microscope LSM 710 (Carl Zeiss, Germany) using an UV filter
(450–490 nm). Three hundred cells per sample were counted in triplicate for each dose point.
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The cells were scored as viable, apoptotic or necrotic as judged by the staining, nuclear
morphology and membrane integrity.
Morphological changes were also observed and
photographed.
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4.9. DAPI staining method
DAPI (4ˈ, 6ˈ-diamidino−2−phenylindole) staining was done using the following
procedure: 5 × 105 cells were treated with the complex 4 (100 µg mL-1) for 24 h in a 6-well
culture plate and were fixed with 4% paraformaldehyde followed by permeabilization with
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0.1% Triton X-100. Cells were then stained with 50 µg mL-1 DAPI for 30 min at room
temperature. The cells undergoing apoptosis, represented by the morphological changes of
confocal microscope LSM 710 (Zeiss).
Acknowledgments
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apoptotic nuclei, were observed and imaged from ten eye views at under a laser scanning
One of the authors (N. M) thanks University Grants Commission (UGC), New Delhi, for
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the award of UGC-RFSMS. We express sincere thanks to DST-FIST, India for the use of
Bruker 400 MHz spectrometer at the School of Chemistry, Bharathidasan University,
Tiruchirappalli. We also thank the CSIR, New Delhi for Electrochemical Analyser through
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research project [Ref. No: 02 (0142) /13/EMR-II].
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Appendix A. Supplementary material
CCDC 1064013 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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New (η6-benzene) ruthenium(II) aroylhydrazone complexes have been synthesized
X- ray analysis of the complex reveals a typical three leg piano stool structure
Complexes are more potent against MCF-7 cells than cisplatin
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Fluorescence staining methods confirm apoptosis induced cell death