Synthesis and molecular structure of arene ruthenium(II) benzhydrazone complexes: Impact of substitution at the chelating ligand and arene moiety on antiproliferative activity

Article abstract of DOI:10.1039/c6nj01936f

A convenient method for the synthesis of ruthenium(ii) arene benzhydrazone complexes (1-6) of the general formula [(η⁶-arene)Ru(L)Cl] (arene-benzene or p-cymene; l-monobasic bidentate substituted indole-3-carboxaldehye benzhydrazone derivatives

Full text of DOI:10.1039/c6nj01936f

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Synthesis and molecular structure of arene
ruthenium(II) benzhydrazone complexes: impact
of substitution at the chelating ligand and arene
moiety on antiproliferative activity†
Cite this:DOI: 10.1039/c6nj01936f
Mohamed Kasim Mohamed Subarkhan,a Rengan Ramesh*a and Yu Liub
A convenient method for the synthesis of ruthenium(II) arene benzhydrazone complexes (1–6) of the
general formula [(Z6-arene)Ru(L)Cl] (arene-benzene or p-cymene; L-monobasic bidentate substituted
indole-3-carboxaldehye benzhydrazone derivatives) has been described. The complexes have been fully
characterized via elemental analysis, IR, UV-vis, NMR and ESI-MS spectral methods. The solid-state
molecular structures of the representative complexes were determined using a single-crystal X-ray
diffraction study and the results indicated the presence of a pseudo octahedral (piano stool) geometry.
All the complexes were thoroughly screened for their cytotoxicity against human cervical cancer cells
(HeLa), human breast cancer cell line (MDA-MB-231) and human liver carcinoma cells (Hep G2) under
in vitro conditions. Interestingly, the cytotoxic activity of complexes 3, 4 and 6 is much more potent
than cis-platin with low IC50 values against all the cancer cell lines tested. Furthermore, the mode of cell
death in the MDA-MB-231 cells was assessed via AO–EB staining, Hoechst 33258 staining, flow cytometry and
comet assay. Furthermore, the results of Western blot analyses suggest that complexes 3 and 6 accumulate
preferentially in the mitochondria of MDA-MB-231 cells and induce apoptosis via mitochondrial pathways by
up-regulating p53 and Bax, and down-regulating Bcl-2.
Received (in Montpellier, France)
21st June 2016,
Accepted 30th September 2016
DOI: 10.1039/c6nj01936f
www.rsc.org/njc
as platinum(II). A number of ruthenium compounds have
been shown to display promising anticancer activities and
Introduction
Over the past few decades, a large number of cisplatin analogs two ruthenium(III) complexes have entered clinical trials, trans-
have been screened as potential antitumor agents, but of these, [RuCl4(DMSO)(Im)]ImH (NAMI-A, where Im-imidazole),4 and
only two, carboplatin and oxaliplatin, have entered world-wide trans-[RuCl4(Ind)2]IndH (KP1019), where Ind-indazole.5
clinical use.1 Regardless of the achievements of the current
Several reports have been focused on the anticancer
platinum drugs, they are efficient only for a limited range of potential of half-sandwich Ru(II) arene complexes of the type
cancers, some tumors can posses acquired or intrinsic resistance to [(Z6-arene)Ru(YZ)(X)], where Y and Z are bidentate chelating
them and furthermore, they often cause severe side-effects.2,3 groups (NN, NO, OO, SO) or two monodentate ligands, and X is
Hence, there is a need for new approaches that are purposefully a monodentate moiety (often a leaving group e.g. Cl). These
designed to circumvent these drawbacks. In this regard, ruthenium complexes have been extensively studied as anticancer agents.6
compounds in the +2 or +3 oxidation state are considered to be These half-sandwich ‘‘piano-stool’’ complexes offer great scope
suitable candidates for anticancer drug design, since they exhibit a for design, with the potential to vary each of the building blocks
similar spectrum of kinetics for their ligand substitution reactions to allow modification of the thermodynamic and kinetic para-
meters. Indeed, it has been found that increasing the size of the
coordinated arene increases their activity in the human ovarian
cancer cell line. Changing the chelating ligand in these ruthenium
a Centre for Organometallic Chemistry, School of Chemistry,
Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India.
arene complexes also appears to have an enormous effect on their
kinetics and even changes their nucleobase selectivity.7
E-mail: rrameshbdu@gmail.com
b Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100 049,
The synthesis and antiproliferative activity of RuII(Z6-arene)
compounds carrying bioactive flavonol ligands have been
reported by Hartinger et al. (A).8 Wei Su et al. have described
the DNA binding properties and anticancer activities of ketone
N4 substituted thiosemicarbazones and their ruthenium(II)
China
† Electronic supplementary information (ESI) available: Selected crystal data and
structure refinement data and figures containing the 1H and 13C NMR, ESI-MS,
UV-vis spectrum and intermolecular interaction diagrams of complexes 3 and 6.
CCDC 1499166 (3) and 1498893 (6). For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c6nj01936f
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Fig. 1 Reported ruthenium(II) arene anticancer drugs.
arene complexes.9 A series of ruthenium(II) arene complexes (AO–EB staining, Hoechst staining, flow cytometry technique
with the 4-(biphenyl-4-carbonyl)-3-methyl-1-phenyl-5-pyrazolonate and comet assay). Furthermore, the apoptosis pathway was
ligand, and related 1,3,5-triaza-7-phosphaadamantane (PTA) confirmed by the change in the mitochondrial membrane
derivatives, have been reported along with their anticancer potential and through western blot analysis.
activities with low IC50 values (B).10 Furthermore, Dyson and
his co-workers have reported ruthenium(II)–arene complexes
Experimental section
Methods and instrumentation
with a perfluoroalkyl-modified ligand, which display remark-
able in vitro cancer cell selectivity (C).11 Recently, the inhibitory
activity of ruthenium(II) arene complexes of 2-phenylimid-
The microanalysis of carbon, hydrogen, nitrogen and sulphur
azole[4,5f ][1,10]phenanthroline against the migration and
was recorded on an analytical function testing Vario EL III CHNS
invasion of MDA-MB-231 breast cancer cells has been investi-
elemental analyser at the Sophisticated Test and Instrumenta-
gated (D) (Fig. 1).12
tion Centre (STIC), Cochin University, Kochi. Melting points
In recent years, much attention has been given to compounds
were recorded with a Boetius micro-heating table and were
with pharmacophore hydrazone moieties due to the identifi-
corrected. Thermal measurements (TGA/DTA) were carried out
cation of several hydrazone lead compounds showing anti-
on a Perkin Elmer Thermal Analyzer under a nitrogen atmo-
proliferative13 and antitumor activities.14 It has been found from
sphere with a heating rate of 10 1C minÀ1. FT-IR spectra were
the literature that only a few reports are available on the
recorded on KBr pellets using a JASCO 400 plus spectrometer.
synthesis, characterisation and cytotoxicity of ruthenium(II)
Electronic spectra in chloroform solution were recorded using
complexes containing hydrazone ligands.15 Nevertheless, it
1
a CARY 300 Bio UV-visible Varian spectrometer. H NMR and
should be pointed out that, as far as we know, the biological
13C-NMR were spectra were recorded on a Bruker 400 MHz
properties of arene ruthenium complexes bearing aroylhydra-
instrument using tetramethylsilane (TMS) as the internal reference.
zones have not been studied so far. Therefore, in this study, we
A Micro mass Quattro II triple quadrupole mass spectrometer was
have combined a ruthenium unit with a benzhydrazone ligand
utilized for electrospray ionization mass spectrometry (ESI-MS). The
to generate a series of organometallic compounds with signifi-
theoretical calculations were performed using IsoPro software.16
cant anticancer activities, taking advantage of the synthetic
versatility of hydrazone derivatives and their promising bio-
logical activities.
We describe here the synthesis and characterization of Ru(II)
arene complexes containing bidentate indole-3-carboxaldehyde
Materials
The starting materials [(Z6-C6H6)RuCl2]2 and [(Z6-p-cymene)RuCl2]2
were prepared according to methods reported in the literature.17
benzhydrazone ligands and chlorine. All the synthesized
complexes have been characterized via elemental analysis, IR,
UV-vis and NMR and ESI-MS spectroscopy techniques. The
Procedure for the preparation of the indole-3-carboxaldehyde
benzhydrazone ligands
molecular structures of complexes 3 and 6 were confirmed The ligands L1–L3 were prepared according to the methods
through single crystal X-ray diffraction. The in vitro cytotoxicity reported in the literature.18 A mixture of 4-substituded benz-
of complexes 1–6 against HeLa, MDA-MB-231, Hep G2 and NIH hydrazide (R = H, Cl or OMe derivatives) (1 mmol) and indole-3-
3T3 cells were screened using an MTT assay. The morphological carboxaldehyde (1 mmol) in ethanol (10 mL) containing a drop
changes were investigated using various apoptosis assays of glacial acetic acid was refluxed for 30 min. The separated
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solid was filtered and dried in air. The ligands were further 5.43 (d, 1H, p-cym-H), 5.40 (d, 1H, p-cym-H), 5.32 (d, 1H, p-cym-H),
purified via recrystallisation from methanol. Yield: 67–92%.
2.85 (m, 1H, p-cym CH(CH3)2), 2.31 (s, 3H, p-cym CCH3), 1.28 (d, 3H,
p-cym CH(CH3)2), 1.23 (d, 3H, p-cym CH(CH3)2). 13C NMR (400 MHz,
CDCl3) (d ppm) 164.63, 151.89, 147.82, 146.37, 131.61, 131.06,
129.65, 129.38, 128.68, 127.39, 125.25, 123.62, 123.13, 117.2,
Procedure for the synthesis of the ruthenium(II) arene
benzhydrazone complexes
A mixture containing [(Z6-arene)RuCl2]2 (arene-benzene or 116.06, 113.01, 32.25, 29.31, 27.07 ppm. ESI-MS: displays a peak
p-cymene) (0.05 mmol), the indole-3-carboxaldehyde benzhydra- at m/z 497.62 (M À Cl)+ (calcd m/z 498.12).
zone ligand (0.1 mmol) and triethylamine (0.3 mL) in benzene
[Ru(g6-p-cymene)(Cl)(L2)] (5). Brown solid. Yield = 0.269 g
(20 ml) was created and stirred at room temperature for 2 h. The (82%); m.p.: 176 1C (with decomposition); calculated:
orange brown precipitate was filtered, washed with hexane and C26H25Cl2N3ORu: C, 55.03; H, 4.44; N, 7.40%. Found: C, 55.06;
dried in vacuo. The reaction progress was monitored using thin H, 4.41; N, 7.42%. IR (KBr, cmÀ1): 1532 n(CQN–NQC), 1481 n(NQC–O)
,
layer chromatography.
1376 n(C–O). UV-Vis (CH3CN, lmax/nm; e/dm3 mol1 cmÀ1): 410
[Ru(g6-C6H6)(Cl)(L1)] (1). Brown solid. Yield = 0.160 g (68%); (1237), 270 (6908), 232 (15 482). 1H NMR (400 MHz, CDCl3)
m.p.: 180 1C (with decomposition); Calculated: C22H18ClN3ORu: d (ppm): 11.86 (br, 1H, indole N–H), 9.39 (s, 1H, HCQN),
C, 55.40; H, 3.80; N, 8.81%. Found: C, 55.37; H, 3.79; N, 8.82%. 6.98–7.62 (m, 9H, aromatic), 5.63 (d, 1H, p-cym-H), 5.50 (d, 1H,
IR (KBr, cmÀ1): 1539 n(CQN–NQC), 1490 n(NQC–O), 1369 n(C–O)
.
p-cym-H), 5.45 (d, 1H, p-cym-H), 5.38 (d, 1H, p-cym-H), 3.10
UV-Vis (CH3CN, lmax/nm; e/dm3 mol1 cmÀ1): 418 (1143), 273 (m, 1H, p-cym CH(CH3)2), 2.34 (s, 3H, p-cym CCH3), 1.40 (d, 3H,
1
(6371), 227 (14 757). H NMR (400 MHz, CDCl3) (d ppm): 11.55 p-cym CH(CH3)2), 1.36 (d, 3H, p-cym CH(CH3)2). 13C NMR
(br, 1H, indole N–H), 9.24 (s, 1H, HCQN), 7.08–7.98 (m, 10H, (400 MHz, CDCl3) (d ppm) 164.15, 131.23, 129.73, 129.52, 129.20,
aromatic), 5.72 (s, 6H, CH-benzene). 13C NMR (400 MHz, CDCl3) 128.50, 127.50, 127.18, 125.05, 123.45, 122.45, 117.10, 116.82,
(d ppm) 164.15, 131.23, 129.73, 129.52, 129.20, 128.50, 127.50, 87.94 ppm. ESI-MS: displays a peak at m/z 531.21 (M À HCl)+
127.18, 125.05, 123.45, 122.45, 117.10, 116.82, 87.94 ppm. ESI-MS: (calcd m/z 532.08).
displays a peak at m/z 441.56 (M À Cl)+ (calcd m/z 442.05).
[Ru(g6-p-cymene)(Cl)(L3)] (6). Orange-brown solid. Yield =
[Ru(g6-C6H6)(Cl)(L2)] (2). Brown solid. Yield = 0.0933 g (69%); 0.180 g (78%); m.p.: 183 1C (with decomposition); calculated:
m.p.: 172 1C (with decomposition); Calculated: C22H17Cl2N3ORu: C,
C27H28ClN3O2Ru: C, 57.59; H, 5.01; N, 7.46%. Found: C, 57.59;
51.67; H, 3.35; N, 8.22%. Found: C, 51.68; H, 3.36; N, 8.20%. IR H, 5.01; N, 7.47%. IR (KBr, cmÀ1): 1530 n(CQN–NQC), 1485 n(NQC–O)
,
(KBr, cmÀ1): 1531 n(CQN–NQC), 1487 n(NQC–O), 1378 n(C–O). UV-Vis 1372 n(C–O). UV-Vis (CH3CN, lmax/nm; e/dm3 mol1 cmÀ1): 427
(CH3CN, lmax/nm; e/dm3 mol1 cmÀ1): 419 (1044), 269 (4977), 233 (1576), 269 (7294), 229 (13 110). 1H NMR (400 MHz, CDCl3)
(10 051). 1H NMR (400 MHz, CDCl3) (d ppm): 11.45 (br, 1H, d (ppm): 11.88 (br, 1H, indole N–H), 9.49 (s, 1H, HCQN),
indole N–H), 9.35 (s, 1H, HCQN), 6.78–7.92 (m, 9H, aromatic), 6.74–8.58 (m, 9H, aromatic), 5.62 (d, 1H, p-cym-H), 5.47 (d, 1H,
5.72 (s, 6H, CH-benzene). 13C NMR (400 MHz, CDCl3) (d ppm) p-cym-H), 5.43 (d, 1H, p-cym-H), 5.36 (d, 1H, p-cym-H), 3.81 (s, 3H,
162.73, 159.67, 130.61, 130.43, 129.30, 128.17, 114.54, 114.12, OCH3), 2.89 (m, 1H, p-cym CH(CH3)2), 2.34 (s, 3H, p-cym CCH3),
88.57 ppm. ESI-MS: displays a peak at m/z 475.97 (M À Cl)+ (calcd 1.41 (d, 3H, p-cym CH(CH3)2), 1.37 (d, 3H, p-cym CH(CH3)2).
m/z 476.01).
13C NMR (400 MHz, CDCl3) (d ppm) 164.21, 147.73, 142.18, 132.99,
[Ru(g6-C6H6)(Cl)(L3)] (3). Orange brown solid. Yield = 0.268 g 131.73, 131.66, 131.40, 130.01, 129.12, 128.40, 127.65, 127.65, 127.12,
(92%); m.p.: 186 1C (with decomposition); Calculated 126.88, 124.94, 123.98, 117.44, 117.07, 32.16, 29.36, 27.14 ppm.
C
23H20ClN3O2Ru: C, 54.49; H, 3.98; N, 8.29%. Found: C, ESI-MS: displays a peak at m/z 527.86 (M À Cl)+ (calcd m/z 528.13).
54.48; H, 4.00; N, 8.29%. IR (KBr, cmÀ1): 1530 n(CQN–NQC), 1486 Single crystals suitable for X-ray diffraction were obtained via
n
(NQC–O), 1376 n(C–O). UV-Vis (CH3CN, lmax/nm; e/dm3 mol1 cmÀ1): recrystallisation in DCM and methanol solution.
429 (1496), 268 (4904), 236 (10 242). 1H NMR (400 MHz, CDCl3)
X-ray crystallography
(d ppm): 11.45 (br, 1H, indole N–H), 9.36 (s, 1H, HCQN),
6.78–8.02 (m, 9H, aromatic), 5.72 (s, 6H, CH-benzene), 3.86 Single crystals of [Ru(Z6-C6H6)(Cl)(L3)] (3) and [Ru(Z6-p-cymene)-
(s, 3H, OCH3). 13C NMR (400 MHz, CDCl3) (d ppm) 163.98, (Cl)(L3)] (6) were grown via slow evaporation of the dichloro-
136.49, 131.52, 128.90, 128.42, 126.99, 124.97, 12.69, 117.55, methane-methanol solution at room temperature. A single crystal
117.00, 114.27, 114.27, 105.82, 88.94, 56.08 ppm. ESI-MS: of suitable size was covered with Paratone oil, mounted on the top
displays a peak at m/z 471.99 (M À Cl)+ (calcd m/z 472.06). of a glass fiber, and transferred to a Bruker AXS Kappa APEX II
Single crystals suitable for X-ray diffraction were obtained via single crystal X-ray diffractometer using monochromated MoKa
recrystallisation in DCM and methanol solution.
radiation (l = 0.71073). Data were collected at 293 K. The structure
[Ru(g6-p-cymene)(Cl)(L1)] (4). Orange-brown solid. Yield = was solved with a direct method using SIR-97 and was refined via
0.240 g (80%); m.p.: 168 1C (with decomposition); Calculated: a full matrix least-squares method on F2 with SHELXL-97.19
C
26H26ClN3ORu: C, 58.59; H, 4.92; N, 7.88%. Found: C, 58.59; Non-hydrogen atoms were refined with anisotropic thermal
parameters. All hydrogen atoms were geometrically fixed and
H, 4.97; N, 7.85%. IR (KBr, cmÀ1): 1528 n(CQN–NQC), 1486 n(NQC–O)
,
1371 n(C–O). UV-Vis (CH3CN, lmax/nm; e/dm3 mol1 cmÀ1): 431 collected to refine using a riding model. Frame integration
(1044), 266 (4941), 228 (11 908). 1H NMR (400 MHz, CDCl3) and data reduction were performed using Bruker SAINT Plus
d (ppm): 11.86 (br, 1H, indole N–H), 9.29 (s, 1H, HCQN), (Version 7.06a) software. The multi scan absorption corrections
6.95–8.33 (m, 10H, aromatic), 5.58 (d, 1H, p-cym-H), were applied to the data using SADABS software. Fig. 1 was
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drawn with ORTEP20 and the structural data were deposited at MTT assay. The absorbance was monitored at 570 nm (measure-
the Cambridge Crystallographic Data Centre: CCDC 1499166 ment) and 630 nm (reference) using a 96 well plate reader
and 1498893.
(Bio-Rad, Hercules, CA, USA). Data were collected for four
replicates each and used to calculate the respective means.
The percentage of inhibition was calculated, from this data,
Stability studies
The stabilities of complexes 1–6 were checked by recording using the formula: Percentage inhibition = 100 Â {Mean OD of
their UV-visible spectra by dissolving them in a minimum untreated cells (control) À Mean OD of treated cells}/{Mean OD
amount of 1% DMSO and then diluting the sample with PBS of untreated cells (control)}. The IC50 value was determined as
buffer. The hydrolysis profiles of these complexes were the complex concentration that is required to reduce the
recorded by monitoring the electronic spectra for the resulting absorbance to half that of the control.
mixture over 24 h.
Acridine orange and ethidium bromide staining experiment
Partition coefficients determination
We observed the changes in chromatin organization in the
The hydrophobicity values of complexes 1–6 were measured MDA-MB-231 cells after treatment with IC50 concentrations of
using the ‘‘Shake flask’’ method in octanol–water phase partitions complexes 3 and 6 by using acridine orange (AO) and ethidium
as reported earlier. Complexes 1–6 (1 mg mLÀ1) were dissolved in a bromide (EB). Briefly, about 5 Â 105 cells were allowed to
mixture of water and n-octanol (2, 4, 6, 8, 10 mg mLÀ1) followed by adhere overnight on a coverslip placed in each well of a
shaking for 1 hour. The mixture was allowed to settle over a period 12-well plate. The cells were allowed to recover for 1 h, washed
of 30 minutes and the resulting two phases were collected thrice with DPBS, stained with an AO and EB mixture (1 : 1, 10 mM)
separately without cross contamination of one solvent layer into for 15 min, and observed with an epifluorescence microscope
another. The concentration of the complexes in each phase (Carl Zeiss, Germany).
was determined using UV-Vis absorption spectroscopy at
room temperature. The results are given as the mean values
Hoechst 33258 staining method
obtained from three independent experiments. The sample Hoechst 33258 staining was done using the method described
solution concentration was used to calculate log P. The parti- earlier with slight modifications. 5 Â 105 MDA-MB-231 cells
tion coefficients for 1–6 were calculated using the equation were treated with IC50 concentrations of complexes 3 and 6 for
log P = log[(1–6)oct./(1–6)aq.].
24 h in a 6-well culture plate and were fixed with 4% para-
formaldehyde followed by permeabilization with 0.1% Triton X-100.
Cells were then stained with 50 mg mLÀ1 Hoechst 33258 for 30 min
Cell culture and inhibition of cell growth
Cell culture. HeLa (human cervical cancer cell line), MDA- at room temperature. The cells undergoing apoptosis, repre-
MB-231 (triple negative breast carcinoma cell line), Hep G2 sented by the morphological changes of apoptotic nuclei, were
(human liver carcinoma cell line) and NIH 3T3 (noncancerous observed and imaged using an epifluorescence microscope
cell line, mouse embryonic fibroblast) were obtained from the (Carl Zeiss, Germany).
National Centre for Cell Science (NCCS), Pune. These cell lines
were cultured as a monolayer in RPMI-1640 medium (Biochrom
Apoptosis evaluation – flow cytometry
AG, Berlin, Germany), supplemented with 10% fetal bovine serum The MDA-MB-231 cells were grown in a 6-well culture plate and
(Sigma-Aldrich, St. Louis, MO, USA), and with 100 U mLÀ1 exposed to IC50 concentrations of complexes 3 and 6 for 24 h.
penicillin and 100 mg mLÀ1 streptomycin as antibiotics (Himedia, The Annexin V-FITC kit uses annexin V conjugated with fluor-
Mumbai, India), at 37 1C in a humidified atmosphere of 5% CO2 escein isothiocyanate (FITC) to label the phosphatidylserine
in a CO2 incubator (Heraeus, Hanau, Germany).
sites on the membrane surface of apoptotic cells. Briefly, the
cells were trypsinised and washed with Annexin binding buffer
and incubated with Annexin V-FITC and PI for 30 minutes and
Inhibition of cell growth
The IC50 values, which are the concentrations of the tested immediately analysed using flow cytometer FACS Aria-II. The
compounds that inhibit 50% of cell growth, were determined results were analysed using DIVA software and the percentage
using a 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium of positive cells was calculated.
bromide (MTT) assay. Cells were plated in their growth medium
Cellular DNA damage quantified using the comet assay
at a density of 5000 cells per well in 96 flat bottomed well plates.
After 24 h, the benzhydrazone ligands and Ru(II) arene benz- DNA damage was quantified by means of the comet assay as
hydrazone complexes 1–6 were added at different concentrations described. Assays were performed under red light at 4 1C. Cells
(1–250 mM) for 24 h to study the dose dependent cytotoxic effects. used for the comet assay were sampled from a monolayer
To each well, 20 mL of 5 mg mLÀ1 MTT in phosphate-buffer (PBS) during the growing phase, 24 h after seeding. MDA-MB-231
was added. The plates were wrapped with aluminium foil and cells were treated with complexes 3 and 6 at the IC50 concen-
incubated for 4 h at 37 1C. The purple formazan product was tration, and the cells were harvested using a trypsinization
dissolved by the addition of 100 mL of 100% DMSO to each well. process at 24 h. A total of 200 mL of 1% normal agarose in PBS at
The quantity of formazan formed gave a measure of the number 65 1C was dropped gently onto a fully frosted microslide,
of viable cells. HeLa, MDA-MB-231 and Hep G2 were used for the covered immediately with a coverslip, and placed over a frozen
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ice pack for about 5 min. The coverslip was removed after the gel appropriate amounts of the cell lysates (25 mg protein) were
had set. The cell suspension from one fraction was mixed with 1% resolved over a 10% Tris-glycine polyacrylamide gel, and then
low-melting agarose at 37 1C in a 1 : 3 ratio. A total of 100 mL of this transferred onto the PVDF membrane. The blots were blocked
mixture was applied quickly on top of the gel, coated over the using 5% non-fat dry milk and probed using p53, Bcl-2 and Bax
microslide, and allowed to set as before. A third coating of 100 mL primary monoclonal antibodies in blocking buffer overnight at
of 1% low-melting agarose was placed on the gel containing the 4 1C. The membrane was then incubated with the appropriate
cell suspension and allowed to set. Similarly, slides were prepared secondary antibody-horseradish peroxidase conjugate (Amersham
(in duplicate) for each cell fraction. After solidification of the Life Sciences Inc., IL, USA), followed by detection using a chemi-
agarose, the coverslips were removed, and the slides were luminescence ECL kit (Amersham Life Sciences Inc., IL, USA). To
immersed in an ice-cold lysis solution (2.5 M NaCl, 100 mM ensure equal loading of the protein, the membrane was stripped
Na2EDTA, 10 mM Tris, NaOH; pH 10, 0.1% Triton X-100) and and reprobed with anti-b-actin antibody (Sigma Aldrich, USA).
placed in a refrigerator at 4 1C for 16 h. All of the above operations
were performed in low-lighting conditions in order to avoid addi-
tional DNA damage. Slides, after removal from the lysis solution,
Results and discussion
were placed horizontally in an electrophoresis tank. The reservoirs
were filled with an electrophoresis buffer (300 mM NaOH and
1 mM Na2EDTA, pH 4 13) until the slides were just immersed in
it. The slides were allowed to stand in the buffer for about 20 min
(to allow DNA unwinding), after which electrophoresis was carried
out at 0.8 V cmÀ1 for 15 min. After electrophoresis, the slides were
removed, washed thrice in a neutralization buffer (0.4 M Tris,
pH 7.5), and gently dabbed to dry. Nuclear DNA was stained with
20 mL of EB (50 mg mLÀ1). Photographs were taken using an
epifluorescence microscope (Carl Zeiss).
Synthesis of the ruthenium(II) arene benzhydrazone complexes
The hydrazone ligand derivatives were conveniently prepared in
an excellent yield via condensation of indole-3-carboxaldehyde
with 4-substituted benzhydrazides (H, Cl and OMe derivatives)
in an equimolar ratio.17 These ligands were allowed to react
with the ruthenium(II) arene precursor [(Z6-arene)RuCl2]2
(arene-benzene or p-cymene) in a 2 : 1 molar ratio in the
presence of triethylamine as the base and the new complexes
of the general formula, [(Z6-arene)Ru(L)Cl] (arene-benzene or
p-cymene; L-substituted indole-3-carboxaldehye benzhydrazone
derivatives) (Scheme 1) were obtained in high 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.
All complexes are air-stable and are highly soluble in most
organic solvents. The analytical data of all the ruthenium(II)
arene benzhydrazone complexes are in good agreement with
the molecular formula proposed.
Mitochondrial membrane potential assay
Mitochondrial membrane potential, Dcm is an important para-
meter of mitochondrial function used as an indicator of cell
health. MDA-MB-231 cells treated overnight with IC50 concentra-
tions of complexes 3 and 6 in 6-well plates were incubated for 1 h
with 2 mg mLÀ1 of JC-1 in the culture medium. The adherent cell
layer was then washed three times with PBS and dislodged with
250 mL of trypsin–EDTA. Cells were collected in PBS/2% bovine
serum albumin (BSA), washed twice via centrifugation, resuspended
in 0.3 mL of PBS/2% BSA, mixed gently, and examined using a
fluorescence microscope (Carl Zeiss, Jena, Germany).
Characterization of the complexes
The IR spectra of the free ligands displayed a medium to strong
band in the region of 3180–3196 cmÀ1 which is characteristic of
the N–H functional group. The free ligands also displayed nCQN
and nCQO absorptions in the region of 1548–1576 cmÀ1 and
Western blot analysis
For Western blot analysis, MDA-MB-231 cells were treated with 1610–1653 cmÀ1 respectively, which indicate that the ligands
complexes 3 and 6 at the IC50 concentrations for 24 h, and exist in the amide form in the solid state. Bands that are due to
Scheme 1 Synthesis of ruthenium(II) arene indole-3-carboxaldehyde benzhydrazone complexes.
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nN–H and nCQO stretching vibrations were not observed with the Stability of the complexes (time-dependent spectra)
complexes, which indicates that the ligands underwent tauto-
merization and subsequent coordination of the imidolate
enolate form during complexation. Coordination of the ligand
to the ruthenium(II) ion through an azomethine nitrogen is
expected to reduce the electron density in the azomethine link,
and thus lower the absorption frequency upon complexation
to 1528–1539 cmÀ1 which indicates the coordination of azo-
methine nitrogen to the ruthenium(II) ion. The band in the
region of 1369–1378 cmÀ1 is due to the imidolate oxygen, which
is coordinated to the metal. The IR spectra of all the complexes
therefore confirm the mode of coordination of the benzhydra-
zone ligand to the ruthenium(II) ion via the azomethine nitro-
gen and imidolate oxygen.21
Stability in solution is an important requirement for drug
candidates. The stability of the most cytotoxic complexes 1–6,
was studied using UV-Vis spectroscopy in a solution of 1%
DMSO in PBS. All the ruthenium(II) arene benzhydrazone com-
plexes showed characteristic peaks in the region of 200–800 nm
and did not exhibit any significant changes during a 24 hour
period. The absence of significant changes in the peak absorp-
tions and spectral characteristics for the tested complexes over
time may suggest that no structural alternations occurred in
buffer solution. The data for all the studied complexes are
presented in Fig. S5 (ESI†). Furthermore, the composition of
the complexes has been studied via ESI-MS spectral studies.
Mass spectrometric measurements were carried out under a
positive ion ESI mode using acetonitrile as the solvent. Their
positive ESI mass spectra 1–6 showed major peaks due to the
cationic fragment [(Z6-arene)Ru(L)Cl]+ generated by loss of the
ClÀ. The ESI spectra of complexes 1–6 display m/z found (calcd):
[441.56 (442.05) (1, M À Cl)+], [475.97 (476.01) (2, M À Cl)+],
[471.99 (472.06) (3, M À Cl)+], [497.62 (498.12) (4, M À Cl)+],
[531.21 (532.08) (5, M À HCl)+] and [527.86 (528.13) (6, M À Cl)+],
respectively confirming the presence of a monomeric entity in
the solution phase. The mass spectrometry results are in good
agreement with the proposed molecular formulae of the com-
plexes and suggest that the chloro (ClÀ) group is labile and
possibly replaced by the targeted biomolecules. The experimen-
tally observed and theoretically calculated isotopic distributions
were in excellent agreement with each other as shown in Fig. S3
and S4 (ESI†). Furthermore, the thermal stability of the synthe-
sized ruthenium(II) arene complexes 3 and 6 was determined
using thermogravimetric analysis (TGA) and differential thermal
analysis (DTA) as shown in Fig. S6 (ESI†). The synthesized
complex is stable up to 180 1C. The results are in good agreement
with the formulae suggested from the analytical data.
The absorption spectra of the ruthenium(II) arene benz-
hydrazone complexes in chloroform exhibited very intense bands
around 266–273 nm and 227–236 nm, which are assigned to the
ligand-centered (LC) p–p* and n–p* transitions, respectively.
The lowest energy absorption bands in the electronic spectra
of the complexes in the visible region 410–431 nm are ascribed
to MLCT (metal to ligand charge transfer) transitions. Based on
the pattern of the electronic spectra of all the complexes, an
octahedral environment around the ruthenium(II) ion has been
proposed similar to that of other octahedral ruthenium(II)
arene complexes.22
1
The H NMR spectra of all the complexes were recorded in
CDCl3 to confirm the bonding of the benzoylhydrazone ligand
to the ruthenium(II) ion. The multiplets observed in the region
d 6.74–8.61 ppm in the complexes have been assigned to the
aromatic protons of the benzhydrazone ligands. The signal due
to the azomethine proton appears in the region d 9.24–9.49 ppm.
The position of the azomethine signal in the complexes is slightly
downfield in comparison with that of the free ligand, suggesting
deshielding of the azomethine proton due to its coordination to
ruthenium. The singlet due to the –NH proton of the free ligand
in the region d 11.22–11.60 ppm is absent in the complex, further
supporting enolisation and coordination of the imidolate oxygen
X-ray crystallographic studies
to the Ru(II) ion. Therefore, the 1H NMR spectra of the complexes Attempts were made to grow single crystals for all the com-
confirm the bidentate coordination mode of the benzhydrazone plexes to confirm the coordination mode of the ligand to metal
ligands to the ruthenium(II) ion. In all the complexes, the and the geometry of the complex. However, we obtained
indole N–H protons are observed as singlets in between d single crystals for complexes [Ru(Z6-p-cymene)(Cl)(L3)] (3) and
11.41–11.88 ppm. The cymene protons appeared in the region [Ru(Z6-C6H6)(Cl)(L3)] (6). Crystals of 3 and 6 grew from the slow
of d 5.32–5.62 ppm.23 In addition, the two isopropyl methyl diffusion of dichloromethane into methanol solutions and
protons of the p-cymene appeared as two doublets in the region crystallized in the monoclinic system with a P2(1)/n space
of d 1.23–1.41 ppm, and the methine protons appeared in the group. The selected bond lengths and bond angles are given
region of d 2.31–3.10 ppm as septets. Furthermore, the methyl in Table 1, whereas the crystallographic data and structural
group of the p-cymene appeared as a singlet around the region of refinement parameters are gathered in Table S1 (ESI†). The
d 2.31–2.34 ppm. Additionally, the methoxy protons are observed ORTEP views of the molecules with atom numbering are shown
as singlets for complexes 3 and 6 at d 3.81–3.86 ppm. On the in Fig. 2 and 3. The molecular structure of complex 3 shows
other hand, the benzene arene protons displayed an upfield shift clearly that the benzhydrazone ligand coordinates in a bidentate
relative to complex 4–6 in the region d 5.72–5.73 ppm (Fig. S1, manner to the ruthenium ion via the azomethine nitrogen and
ESI†). The 13C NMR of the Ru(II) arene complexes showed imidolate oxygen in addition to one chlorine and one arene
resonance in the expected regions (Fig. S2, ESI†), and the group. The complex adopts the commonly observed piano-stool
complex revealed a downfield shift of the azomethine carbon geometry as reported in many half-sandwich arene ruthenium(II)
relative to the free ligands, indicating coordination of the complexes.24 In this case, the arene ring forms the seat of the
azomethine nitrogen to the metal centre.
piano-stool, while the bidentate benzhydrazone N, O and Cl
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Paper
Table 1 Selected bond lengths (Å) and angles (1) in 3ÁH2O and 6
are 2.069(3) and 2.069(3) Å, respectively. The Ru–Cl bond length is
found to be 2.4233(13) Å and the bond length is in agreement with
other structurally characterized p-cymene ruthenium complexes.25
The ruthenium atom is p bonded to the arene ring with an average
Ru–C distance of 2.156(7) Å, whereas the average C–C bond length
in the arene ring is 1.425(8) Å, with alternating short and long
bonds. It was observed that complex 6 also adopts a similar
geometrical environment as in complex 3 with slight variation in
the bond angles and bond distances. The crystal structures of 3
and 6 revealed the presence of extensive intermolecular hydrogen
bonding interactions as shown in Fig. S7, ESI†.
Distances/angles
3ÁH2O
2.069(3)
6
Ru1–N2
2.077(4)
2.065(4)
2.4060(16)
2.153(5)
1.389(6)
1.326(6)
1.289(6)
75.71(16)
83.85(13)
115.7(3)
112.8(3)
110.6(4)
104.81(16)
86.90(12)
Ru1–O1
Ru1–Cl1
Ru1–C22
N1–N2
N2–C7
2.069(3)
2.4233(13)
2.156(7)
1.391(5)
1.326(6)
1.289(5)
76.18(12)
84.26(11)
116.0(2)
112.8(2)
110.5(3)
106.2 (3)
84.46(10)
O2–C9
O1–Ru1–N2
N2–Ru1–Cl1
N2–N1–Ru1
C7–O1–Ru1
C7–N2–N1
C19–Ru1–Cl1
O1–Ru1–Cl1
Partition coefficient determination
Lipophilicity is an important factor for the cellular accumula-
tion and oral bioavailability of drugs. It is often expressed as
the n-octanol/water partition coefficient (log P), which is also
a central parameter in many in silico medicinal chemistry
approaches, such as the determination of the drug likeliness
of a new drug. This was investigated by the partition coefficient,
P, a parameter which indicates the hydrophobic character of
molecules and their ability to cross lipid bilayers.26 The calculated
log P values for complexes 1–6 are 2.59, 2.72, 2.48, 2.23, 2.35 and
1.99 respectively. It has been observed that complex 6 with
a p-cymene group shows a higher potency than the rest of the
complexes (Table 2).
In vitro antiproliferative activity
All the ruthenium complexes and the free benzhydrazone
ligands were evaluated for their cytotoxic activity against HeLa,
MDA-MB-231 and Hep-G2 along with NIH 3T3 cell lines by
using a colorimetric assay (MTT assay) that measures mito-
chondrial dehydrogenase activity as an indication of cell viability.
The effects of the ruthenium(II) arene complexes to arrest the
proliferation of cancer cells were evaluated after exposure for 24 h.
It is to be noted that the ligands did not show any inhibition of
the cell growth even up to 100 mM and clearly indicates that
chelation of the ligand with the metal ion is responsible for the
observed cytotoxicity properties of the complexes. The results of
the MTT assays revealed that the complexes showed notable
Fig. 2 ORTEP drawing of complex 3ÁH2O at 30% probability level.
Table 2 Cytotoxicity (IC50, mM) of the ligands and complexes 1–6. (n.e.:
no effect) and their calculated partition coefficients (log P)
IC50 values (mM)
Complex HeLa
MDA-MB-231 Hep G2
NIH3T3
log P
Complex 1 20.8 Æ 0.2 18.2 Æ 0.8
Complex 2 25.9 Æ 0.8 19.9 Æ 0.1
Complex 3 19.4 Æ 0.3 15.3 Æ 0.3
Complex 4 13.6 Æ 0.4 11.2 Æ 0.3
Complex 5 17.9 Æ 0.3 12.8 Æ 0.2
Complex 6 11.4 Æ 0.7 4.1 Æ 0.4
14.2 Æ 0.3 223.9 Æ 0.7 2.59 Æ 0.4
16.8 Æ 0.5 215.3 Æ 0.6 2.72 Æ 0.3
13.4 Æ 0.4 235.4 Æ 0.3 2.48 Æ 0.3
11.6 Æ 0.4 230.4 Æ 0.5 2.23 Æ 0.2
12.8 Æ 0.1 224.3 Æ 0.8 2.35 Æ 0.3
9.1 Æ 0.3 241.3 Æ 0.4 1.99 Æ 0.2
Fig. 3 ORTEP drawing of complex 6 at 30% probability level.
ligands form the three legs of the stool. Therefore, the
ruthenium(II) ion is sitting in a NOCl (Z6-arene) coordination
environment. The benzhydrazone ligand binds to the metal
centre at N and O forming the five membered chelate ring with
a bite angle of 76.18(12)1 O(1)–Ru(1)–N(2) and 84.26(11)1
L1
L2
L3
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
91.7 Æ 0.5
94.7 Æ 0.9 n.e.
Cisplatin 19.2 Æ 1.1 12.9 Æ 0.6
20.1 Æ 1.2 212.3 Æ 0.6
The ligands L1–L3 were added at different concentrations (1–250 mM)
N(2)–Ru(1)–Cl(1). The bond lengths of Ru(1)–N(2) and Ru(1)–O(1) for 24 h.
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activity against the cell lines HeLa, MDA-MB-231 and Hep-G2 with by cell shrinkage, blebbing of the plasma membrane and
respect to the IC50 values (Table 2). From the IC50 values obtained, chromatin condensation. To identify apoptosis, at a basic level,
it was inferred that complexes 3, 4 and 6 are highly active against we adopted AO–EB staining to visualize and quantify the
all the cell lines with very low IC50 values compared with the number of viable and apoptic cells. According to the difference
values for the well-known anticancer drug cisplatin. In addition, in membrane integrity between necrotic and apoptosis, AO can
the in vitro cytotoxic activity studies of the complexes against the pass through a cell membrane, but EB cannot. The apoptotic
mouse embryonic fibroblast cell line NIH 3T3 (normal cells) was effect after the treatment of MDA-MB-231 cells with complexes
undertaken and the IC50 values are above 215 mM, which confirms 3 and 6 for 24 h at IC50 concentrations is shown in Fig. 4. The
that the complexes are very specific to cancer cells.
cells incubated with complexes 3 and 6 for 2 h and irradiated
These ruthenium(II) arene benzhydrazone complexes 1–6 with visible light showed the significant reddish-orange
posses significant cytotoxicity over the ligands, which may be emission characteristic of apoptotic cells. In the control, the
due to the presence of extended p conjugation resulting from cells of MDA-MB-231 were stained bright green in spots.
the chelation of Ru(II) ions with the ligand. Furthermore, the Additionally, complexes 3 and 6 treated MDA-MB-231 cells
observed higher activity of complexes 4 and 6 is correlated to were stained with Hoechst 33258, and apoptotic features such
the nature of the chelating benzoylhydrazone ligand and arene as nuclear shrinkage and chromatin condensation were also
moiety. Additionally, the observed higher activity of complexes observed (Fig. 5). Hence the results of AO–EB and Hoechst
3 and 6 is correlated to the nature of the chelating benzhydra- staining assays suggest that complexes 3 and 6 induce apoptosis
zone ligand and arene moiety. In complexes 3 and 6, the in MDA-MB-231 cells.28,29
presence of an electron donating methoxy substituent at the
Evaluation of apoptosis – flow cytometry
phenyl ring of the ligand increases the lipophilic character of
the metal complex, which favours its permeation through the
lipid layer of a cell membrane.
The potential to induce apoptosis in cancer cells by the addition
of synthesized complexes can be quantitatively investigated using
flow cytometry analysis and the Annexin V protocol, with the help
of Annexin V-FITC Apoptosis Detection Kit to perform double-
staining with propidium iodide and Annexin V-FITC. Annexin V,
a Ca2+ dependent phospholipid-binding protein with a high
affinity for the membrane phospholipid phosphatidylserine
(PS), is quite helpful for identifying apoptotic cells with exposed
PS. Propidium iodide is a standard flow cytometric viability probe
used to distinguish viable from non-viable cells (Fig. 6). The
MDA-MB-231 cells were treated with complexes 3 and 6 at IC50
concentrations for 24 h. The cell death induced by the complexes
follows a pathway from the lower left quadrant to the upper right
quadrant (Annexin V+/PI+) which represents cells undergoing
apoptosis.30
On the other hand, the arene groups also play an important
role in the antitumor activity of these ruthenium complexes. It
has been observed that complex 6 with a p-cymene group shows
higher potency than those with a benzene group in complex 3,
which may be attributed to the stronger hydrophobic inter-
actions between the Ru(II)–cymene complex and the biomole-
cular targets as evidenced by the partition coefficient value.27
Complex 6 shows a high cytotoxic activity with very low IC50
values of 11.4 Æ 0.7, 4.1 Æ 0.4 and 9.1 Æ 0.3 mM toward HeLa,
MDA-MB-231 and Hep-G2. Furthermore, the IC50 values are
much better than those previously reported for other Ru(II)
arene arylazo, 2-thiosalicylic acid, phenanthroimidazole or poly-
pyridyl complexes.10,28 These excellent results suggest further
investigation of the underlying mechanism accounting for the
antiproliferative action of these ruthenium arene benzhydrazone
complexes is warranted.
Comet assay
The comet assay (single-cell gel electrophoresis) in an agarose
gel matrix was used to study DNA fragmentation. When the
comet assay was performed with treated MDA-MB-231 cancer
AO–EB and Hoechst staining assays
Acridine orange and ethidium bromide (AO and EB) dual staining cells with IC50 concentrations of complexes 3 and 6, large and
followed by fluorescence microscopy revealed apoptosis from the well-rounded comets were observed, while the control cells
perspective of fluorescence emission. Apoptosis is characterized failed to show a comet like appearance (Fig. 7). The comet
Fig. 4 Morphological assessment of AO and EB of MDA-MB-231 cells treated with complexes 3 and 6 (at IC50 concentrations) for 24 h. The scale bar 20 mm.
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Fig. 5 Morphological assessment of complexes 3 and 6 (at IC50 concentrations) and MDA-MB-231 cells for 24 h. The scale bar 20 mm.
Fig. 6 AnnexinV/propidium iodide assay of MDA-MB-231 cells treated with complexes 3 and 6 (at IC50 concentrations) measured using flow cytometry.
Fig. 7 Comet assay of staining of the EB control (untreated) treated with complexes 3 and 6 (at IC50 concentrations) for 24 h. The scale bar 40 mm.
score for complexes 3 and 6 shows a significant number of MDA-MB-231 cells were treated with the complexes, JC-1 displays
nucleoids with larger comet tails, indicative of higher levels of a green fluorescence. The changes from red to green fluorescence
DNA single-strand breaks.31
indicate the decrease of mitochondrial membrane potential
(Fig. 8). These results suggest that complexes 3 and 6 can
induce a decrease in mitochondrial membrane potential.32
Mitochondrial membrane potential detection
Mitochondria act as a point of integration for apoptotic signals
originating from both extrinsic and intrinsic apoptotic path-
Western blot analysis
ways. Mitochondria play important roles in apoptosis through To reveal the underlying mechanism behind the antiproliferative
the release of proapoptotic factors such as cytochrome c activity of the Ru(II) benzhydrazone complexes, the Western blot
and other apoptosis-inducing factors. The changes in the technique was utilized. It is established that apoptic proteins like
mitochondrial membrane potential were detected using the p53, Bax and anti-apoptotic protein Bcl-2 play a pivotal role
fluorescent probe JC-1. It exhibits potential-dependent accumu- during the induction of apoptosis. The expression levels of p53,
lation in mitochondria, indicated by a fluorescence emission Bax and Bcl-2 proteins were analyzed in the 3 and 6 treated
shift from red (B590 nm) to green (B525 nm). As shown in MDA-MB-231 cells and control cells. It was observed that the
Fig. 8, in the control, JC-1 emits red fluorescence. When the expression level of the Bcl-2 protein decreases suggesting that
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Fig. 8 MDA-MB-231 cells were treated with complexes 3 and 6 (at IC50 concentrations) for 24 h. The scale bar 20 mm.
techniques and flow cytometry using the annexin-V assay revealed
that complexes 3 and 6 induce apoptosis in MDA-MB-231 cancer
cells. Furthermore, alkaline comet assays confirmed the single-
strand break of DNA. The results of mitochondrial membrane
potential and Western blot analysis demonstrated that the
complexes with potent antiproliferative activity are able to
induce mitochondria-mediated apoptosis in human cancer
cells. On the basis of these results, we suggest that ruthenium
arene benzhydrazone complexes may be the best candidates for
further evaluation as chemopreventive and chemotherapeutic
agents for human cancers.
Fig. 9 Western blot of p53, Bax and Bcl-2 proteins in MDA-MB-231 cells.
Lane-1 control samples, lanes-2 and 3 are samples treated with complexes
3 and 6 (at IC50 concentrations) respectively. b-Actin was used as the
loading control.
Acknowledgements
One of the authors (MKMS) thanks the University Grants
Commission (UGC), New Delhi for financial assistance through
the UGC-BSR fellowship (Ref. no. F.7–22/2007(BSR)). We express
sincere thanks to DST-FIST, India for the use of Bruker 400 MHz
spectrometer at the School of Chemistry, Bharathidasan Uni-
versity, Tiruchirappalli-24.
apoptosis by 3 and 6 could be mediated through the downregula-
tion of the antiapoptotic protein Bcl-2. The p53 and Bax protein
levels in the MDA-MB-231 cancer cell lines are remarkably
increased upon treatment with the complexes indicating that
the complexes induce apoptosis (Fig. 9). Hence, the upregulation
of proapoptotic protein Bax, p53 and the downregulation of
antiapoptotic protein Bcl-2 caused by complexes 3 and 6 could
possibly activate mitochondria-mediated apoptosis.33
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