New Ru(II)-dmso complexes with heterocyclic hydrazone ligands towards cancer chemotherapy

Article abstract of DOI:10.1016/j.poly.2008.02.036

The synthesis and characterization of ruthenium(II) complexes, [RuCl₂(dmso)₂(bfmh)] (1; dmso = dimethyl sulfoxide, bfmh = benzoic acid furan-2-ylmethylene-hydrazide), [RuCl₂(dmso)₂(btmh)](2; btmh = benzoic acid thiophen-2-ylmethylene-hydrazide), [RuCl₂(dmso)₂(bfeh)](3; bfeh = benzoic acid (1-furan-2-yl-ethylidene)-hydrazide) and [RuCl₂(dmso)₂(bpeh)](4; bpeh = benzoic acid (1-pyridin-2-yl-ethylidene)-hydrazide) are described. The ligands, when treated with either cis-[RuCl₂(dmso)₄] or trans(Cl)-[RuCl₂(dmso)₂(bpy)], resulted in the same products. This has been confirmed by IR spectra and single crystal X-ray diffraction studies. The redox behaviors of the complexes have been found to be strongly dependent on the electronic nature of the moieties present in the hydrazone ligands. The binding of the complexes to Herring sperm DNA has been studied by absorption titration and cyclic voltammetry. But, due to the random change in the absorption on the addition of DNA, only a qualitative result rather than a quantitative result has been obtained. All the complexes have been found to bind DNA through different modes to different extents. The antibacterial properties of the ligands and the complexes have been studied against five pathogenic bacteria and also the minimum inhibitory concentrations (MIC) of all the ligands and complexes 2 and 4 have been evaluated.

Full text of DOI:10.1016/j.poly.2008.02.036

Polyhedron 27 (2008) 1917–1924
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
New Ru(II)–dmso complexes with heterocyclic hydrazone ligands
towards cancer chemotherapy
Viswanathan Mahalingam a, Nataraj Chitrapriya a, Frank R. Fronczek b, Karuppannan Natarajan a,
*
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India
b Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and characterization of ruthenium(II) complexes, [RuCl2(dmso)2(bfmh)] (1; dmso = di-
methyl sulfoxide, bfmh = benzoic acid furan-2-ylmethylene-hydrazide), [RuCl2(dmso)2(btmh)](2;
btmh = benzoic acid thiophen-2-ylmethylene-hydrazide), [RuCl2(dmso)2(bfeh)](3; bfeh = benzoic acid
(1-furan-2-yl-ethylidene)-hydrazide) and [RuCl2(dmso)2(bpeh)](4; bpeh = benzoic acid (1-pyridin-2-yl-
ethylidene)-hydrazide) are described. The ligands, when treated with either cis-[RuCl2(dmso)4] or
trans(Cl)–[RuCl2(dmso)2(bpy)], resulted in the same products. This has been confirmed by IR spectra
and single crystal X-ray diffraction studies. The redox behaviors of the complexes have been found to
be strongly dependent on the electronic nature of the moieties present in the hydrazone ligands. The
binding of the complexes to Herring sperm DNA has been studied by absorption titration and cyclic vol-
tammetry. But, due to the random change in the absorption on the addition of DNA, only a qualitative
result rather than a quantitative result has been obtained. All the complexes have been found to bind
DNA through different modes to different extents. The antibacterial properties of the ligands and the
complexes have been studied against five pathogenic bacteria and also the minimum inhibitory concen-
trations (MIC) of all the ligands and complexes 2 and 4 have been evaluated.
Received 28 August 2007
Accepted 28 February 2008
Available online 12 April 2008
Keywords:
Ru(II)–dmso complexes
Hydrazone ligands
Trans influence
Cyclic voltammetry
Antibacterial activity
DNA-binding
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
doses [6]. Interaction with DNA, a rudimentary mechanism of ac-
tion responsible for cytotoxicity, has been suggested for some
Ruthenium complexes, launched two decades ago for antitumor
therapy, are believed to have great potential as alternative drugs to
cisplatin in view of their low toxicity and good selectivity for solid
tumor metastasis [1]. Similar to platinum anticancer drugs, the
ruthenium ion forms a covalent bond with DNA [2], affecting the
replication and transcription, and leads to cell death eventually.
One outstanding class of compounds which have been researched
since the seventies for cancer chemotherapy is that of halogen-
dimethylsulfoxide-ruthenium(II) complexes [3–6]. A well-known
compound of this class, cis-[RuCl2(dmso)4], has been shown to pos-
sess promising antineoplastic activity against several murine
metastasizing tumors [7]. Ruthenium–dmso complexes analogous
to NAMI-A (imidazolium trans-imidazole dimethylsulfoxide tetra-
chloro ruthenate) are prepared to ameliorate cisplatin activity, par-
ticularly on resistant tumors, or to reduce host toxicity at active
ruthenium complexes [8]. At the same time, the lability of the
chloro ligand in ruthenium complexes has been thought to be cru-
cial in enhancing covalent bond formation with DNA. This para-
doxical situation kindled us to synthesize new Cl–Ru–dmso
complexes and to study their DNA-binding ability, followed by
their anticancer activity, to arrive at a conclusion regarding their
mechanism of action. To achieve this, we chose heterocyclic hydra-
zones as secondary ligands, since they have demonstrated potent
activity against various tumor cell lines in preclinical studies, and
reacted them with cis-[RuCl2(dmso)4] and trans(Cl)–[RuCl2(dm-
so)2(bpy)]. The structure of the ligands is shown in Chart 1.
2. Experimental
2.1. Reagents and materials
All the chemicals used for the preparation of the ligands and
buffer solution are of analytical grade or chemically pure grade.
RuCl3 Á 3H2O, purchased from Himedia, was used as supplied. Pro-
tein free Herring sperm ds DNA, obtained from SRL chemicals, was
stored at 0–4 °C and its purity was checked by measuring its opti-
cal density before use. The tris-buffer solution was prepared with
Abbreviations: ds, double stranded; TBAP, tetra butyl ammonium perchlorate;
Tris, tris-(hydroxymethyl) aminomethane; UV–Vis, UV–Visible; cfu, colony forming
units; NCIM, National Centre for Industrial Microorganisms; MTCC, Microbial Type
Culture Collection; NCCLS, National Committee for Clinical Laboratory Standards
* Corresponding author. Tel.: +91 4222424655; fax: +91 4222422387.
E-mail address: k_natraj6@yahoo.com (K. Natarajan).
0277-5387/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2008.02.036

1918
V. Mahalingam et al. / Polyhedron 27 (2008) 1917–1924
Anal. Calc. for C17H25N2S2O4.5Cl2Ru (565.494): C, 36.10; H, 4.45;
O
S
O
C
O
O
C
N, 4.95; S, 11.34. Found: C, 35.92; H, 4.33; N, 4.71; S, 11.27%. IR
(KBr, cmÀ1): m(NH), 3110(w); m(C@O), 1639(s); (mC@N), 1598(m).
N
N
N
N
H
N
H
H
H
2.3.3. [RuCl2(dmso)2(btmh)] (2)
Cis-[RuCl2(dmso)4] (0.21 mmol) and the ligand btmh
(0.21 mmol) were heated under reflux in ethanol for 6–8 h. Slow
evaporation of the solvent after reduction to 50% gave orange crys-
tals suitable for single crystal XRD studies. Yield, 75 mg, 64%. Ele-
mental Anal. Calc. for C16H22N2S3O3Cl2Ru (558.531): C, 34.40; H,
3.97; N, 5.01; S, 17.22. Found: C, 34.28; H, 3.87; N, 5.32; S,
17.37%. IR (KBr, cmÀ1): m(NH), 3080(w); m(C@O), 1620(s); m(C@N),
1592(m). UV–Vis (dmso) k, nm (e, molÀ1 cmÀ1 L): 254 (25320),
313 (19320), 392 (5480). 1H NMR (dmso-d6, d ppm): 3.2–3.5
(group of peaks, dmso, 12H), 7.9 (s, H–C@N, 1H), 7.4–8.5 (multi-
plet, aromatic protons, 8H), 9.4 (s, –NH, 1H). The same reaction
when carried out with trans(Cl)–[RuCl2(dmso)2(bpy)] yielded a
crystalline product (2a) which conformed to the analytical and IR
data of 2 but was not structurally characterized.
bfmh
bfeh
O
C
O
N
N
H
C
N
N
H
btmh
bpeh
Chart 1. Structure of the hydrazone ligands.
double-distilled water and its pH was adjusted to 7.1 using 0.1 M
NaOH solution. DNA stock solutions were freshly prepared before
use with this buffer solution. Ethanol was purified following stan-
dard procedures for the preparation of the ligands and complexes.
Distilled dmso was used for the preparation of the starting com-
plex and for the preparation of solutions of complexes for DNA-
binding studies. Purified dry methanol was used to record the elec-
tronic spectra of the complexes. Commercially available TBAP was
properly dried and used as a supporting electrolyte for recording
cyclic voltammograms of the complexes (except for DNA-binding
studies). The starting complexes [9], cis-[RuCl2(dmso)4] (Ru1),
trans(Cl)–[RuCl2(dmso)2(bpy)] (Ru2) and the ligands [10] were
prepared according to the literature procedures.
2.3.4. [RuCl2(dmso)2(bfeh)] (3)
Cis-[RuCl2(dmso)4] (0.21 mmol) and the ligand bfeh
(0.21 mmol) were heated under reflux in ethanol for 6–8 h. Slow
evaporation of the solvent gave only an amorphous powder which
was recrystallised from hot dmso. Yield, 69 mg, 59%. Elemental
Anal. Calc. for C17H24N2S2O4Cl2Ru (556.491): C, 36.69; H, 4.34; N,
5.03; S, 11.52. Found: C, 36.28; H, 4.02; N, 5.39; S, 11.11%. IR
(KBr, cmÀ1): m(NH), 3073(w); m(C@O), 1650(s); m(C@N), 1593(m).
UV–Vis (dmso) k, nm (e, molÀ1 cmÀ1 L): 288 (9780), 354 (2620).
1H NMR (dmso-d6, d ppm): 3.1–3.4 (group of peaks, dmso, 12H),
2.2 (s, –CH3, 3H), 7.2–8.2 (multiplet, aromatic protons, 8H), 9.4
(s, –NH, 1H). The same reaction when carried out with trans(Cl)–
[RuCl2(dmso)2(bpy)] yielded an amorphous product (3a) which
conformed to the analytical and IR data of 3.
2.2. Physical measurements
FT-IR spectra (4000–400 cmÀ1) of the complexes and the free li-
gands were recorded as KBr pellets with a Nicolet Avatar Model FT-
IR spectrophotometer. Electronic spectra (800–250 nm) of the
complexes were obtained on a Systronics 119 UV–Vis spectropho-
tometer. 1H NMR spectra were recorded on a Varian-Australia
AMX-400. The absorption titrations were carried out with a JASCO
spectrophotometer. Micro analyses (C, H, N and S) were performed
on a Vario EL III Elementar analyzer. Cyclic voltammograms were
recorded on a CHI 1120A electrochemical analyzer with a three
electrode compartment consisting of a platinum disc working elec-
trode, a platinum wire counter electrode and an Ag/Ag+ reference
electrode.
2.3.5. [RuCl2(dmso)2(bpeh)] (4)
Cis-[RuCl2(dmso)4] (0.21 mmol) and the ligand bpeh
(0.21 mmol) were heated under reflux in ethanol for 6–8 h. Slow
evaporation of the solvent gave only an amorphous powder which
was recrystallised from hot dmso. Yield, 82 mg, 69%. Elemental
Anal. Calc. for C18H25N3S2O3Cl2Ru (567.518): C, 38.09; H, 4.40; N,
7.40; S, 11.30. Found: C, 38.44; H, 4.32; N, 7.12; S, 11.47%. IR
(KBr, cmÀ1): m(NH), 3056(w); m(C@O), 1689(s); m(C@N), 1598(m).
UV–Vis (dmso) k, nm (e, molÀ1 cmÀ1 L): 275 (7540), 304 (5700).
1H NMR (dmso-d6, d ppm): 3.1–3.4 (group of peaks, dmso, 12H),
2.2 (s, –CH3, 3H), 7.3–8.3 (multiplet, aromatic protons, 9H), 9.4
(s, –NH, 1H). The same reaction when carried out with trans(Cl)–
[RuCl2(dmso)2(bpy)] yielded an amorphous product (4a) which
conformed to the analytical and IR data of 4.
2.3. Synthesis
2.3.1. [RuCl2(dmso)2(bfmh)] (1)
Cis-[RuCl2(dmso)4] (0.21 mmol) and the ligand bfmh
(0.21 mmol) were heated under reflux in ethanol for 6–8 h. Slow
evaporation of the solvent after reduction to 50% gave crystals suit-
able for single crystal XRD studies. Yield, 70 mg, 61%. Elemental
Anal. Calc. for C16H22N2S2O4Cl2Ru (542.464): C, 35.42; H, 4.08; N,
5.16; S, 11.82. Found: C, 35.26; H, 4.12; N, 5.24; S, 11.37%. IR
(KBr, cmÀ1): m(NH), 3112(w); m(C@O), 1639(s); (mC@N), 1598(m)
(s, strong; m, medium; w, weak). UV–Vis (dmso) k, nm (e,
molÀ1 cmÀ1 L): 308 (14900), 359 (3950). 1H NMR (dmso-d6, d
ppm): 3.2–3.5 (group of peaks, dmso protons, 12H), 8.1 (s, H–
C@N, 1H), 7.5–8.1 (multiplet, aromatic protons, 8H), 9.2 (s, –NH,
1H).
2.4. Crystallographic structure determination
Single crystal X-ray diffraction measurements were performed
on a Nonius Kappa CCD (with Oxford Cryostream) diffractometer
with graphite monochromatized Mo Ka radiation. The structures
of 1 and 2 were solved by direct methods and refinements were
carried out by full-matrix least-square techniques. The hydrogen
atoms were treated by a mixture of independent and constrained
refinement. The following computer programs were used: struc-
ture solution SIR-97 [11], refinement SHELXL-97 [12], molecular dia-
grams ORTEP-3 [13] for windows.
2.3.2. [RuCl2(dmso)2(bfmh)] Á 0.5C2H5OH (1a)
trans(Cl)–[RuCl2(dmso)2(bpy)] (0.21 mmol) and the ligand bfmh
(0.21 mmol) were heated under reflux in ethanol for 6–8 h. Slow
evaporation of the solvent after reduction to 50% gave crystals suit-
able for single crystal XRD studies. Yield, 69 mg, 58%. Elemental
2.5. Anti-microbial screening
The bacterial strains, Escherichia coli NCIM 2831, Staphylococcus
aureus NCIM 2492, Staphylococcus epidermidis NCIM 2493,

V. Mahalingam et al. / Polyhedron 27 (2008) 1917–1924
1919
Klebsiella pneumoniae NCIM 5082 and Shigella sonnei MTCC 2957
3.1. IR and electronic spectra
were used in the study. The agar-well diffusion method [14] was
employed for the determination of anti-microbial activities. MICs
(minimum inhibitory concentration) of the compounds against test
organisms were determined by the broth micro dilution method.
All the tests were performed in duplicate and repeated twice. Mod-
al values were selected.
The IR spectra of the free ligands and the new Ru(II) complexes
were compared in order to ascertain the probable mode of coordi-
nation of the ligands. Vibration bands of primary importance of the
free ligands were observed around 3250, 1650, 1600–1620 and
1080 cmÀ1 for m(NH), m(CO), m(CN) and m(NN) respectively. The pres-
ence of the m(NH) band confirms that the free ligands are in the
keto form. The IR spectra of the complex pairs 1 and 1a, 2 and
2a, 3 and 3a, and 4 and 4a were identical, suggesting the formation
of the same complexes when the ligands are treated with either
Ru1 or Ru2. None of the complexes obtained from Ru2 displayed
a pyridine ring breathing band around 995 cmÀ1, which implies
the replacement of bpy from Ru2 by the ligands. This directed us
to formulate the complexes in general as [RuCl2(dm-
so)2(L)](L = bfmh, bfeh, btmh or bpeh). In all of the complexes,
the observed significant decrement of m(CO) and m(CN) does evi-
dence the neutral bidentate nature of the ligands. Being so, the li-
gands have two possibilities for chelation with the metal, either
replacing two S-bonded dmso ligands or one S-bonded and one
O-bonded dmso ligand when treated with Ru1. Scrutinizing the
IR spectra of the precursor cis-[RuCl2(dmso)4] and that of the
new complexes was useful in determining the elimination of one
of the above possibilities. In the IR spectrum of cis-[RuCl2(dmso)4],
the S=O stretch of the S-bonded dmso appears at a higher fre-
quency (1090 cmÀ1) in comparison with that of the O-bonded
dmso (915 cmÀ1) due to the increase in the S@O bond order in
the former case. The desertion and retention of the bands at
915 cmÀ1 and around 1090 cmÀ1 respectively in the new com-
plexes indicate the replacement of one O-bonded and one S-
bonded dmso ligand from the precursor by the Schiff base chelate.
This is consistent with the previous observation that chelating li-
gands will replace first the weakly held O-bonded dmso and then
the S-bonded dmso cis to it [9].
2.5.1. Agar-well diffusion method
The compounds were weighed and dissolved in dimethyl sulf-
oxide (dmso), then the solutions were filter sterilized using a
0.45 lm membrane filter. Each microorganism was suspended in
sterile saline and diluted at ca. 106 colony forming units (cfu/
ml). They were flood inoculated onto the surface of Mueller–Hin-
ton Agar (MHA). The wells (8 mm in diameter) were cut from the
agar and 0.1 mL of solution was delivered into them. After incuba-
tion for 24 h at 37 °C, all the plates were examined for any zones of
growth inhibition, and the diameter of these zones were measured
in millimeters.
2.5.2. Determination of minimum inhibitory concentration (MIC)
A broth microdilution susceptibility assay was used as recom-
mended [15] by NCCLS for determination of the MIC. All the tests
were performed in Mueller–Hinton Broth (MHB) supplemented
with Tween 80 detergent (final concentration 0.5% (v/v)). Bacterial
strains were cultured overnight at 37 °C in MHA. Test strains were
suspended in MHB to give a final density of 5 Â 105 cfu/ml and
these were confirmed by viable counts. Geometric dilutions of
the compounds were prepared in a 96-well microtiter plate,
including one growth control (MHB + Tween 80) and one sterility
control (MHB + Tween 80 + compound). The plates were incubated
under normal atmospheric conditions at 37 °C for 24 h. The MIC of
kanamycin and oxytetracycline were individually determined in
parallel experiments in order to control the sensitivity of the test
organisms. The bacterial growth was indicated by the presence of
a white ‘‘pellet” on the well bottom.
The absorption spectra of the diamagnetic Ru(II) complexes
were recorded as 10À4 M dmso solutions in the range 800–
250 nm using a quartz cuvette of 1 cm path length. All the new
ruthenium(II) complexes, except 4, exhibit bands in both UV and
visible regions. Based on the position and nature of the peaks, all
the bands are assigned to either n ? p* or p ? p* intraligand tran-
sitions (data given in experimental section).
3. Results and discussion
The reactions of either Ru1 or Ru2 with the hydrazone ligands
in a 1:1 mol ratio in ethanol has yielded complexes of the type
[RuCl2(dmso)2(L)] (L = hydrazone ligand), as illustrated in Scheme
1. All the complexes are stable to air and light, and are soluble in
organic solvents such as dmso, dmf and dichloromethane. The
hydrazone ligands act as neutral bidentate donors replacing two
dmso ligands from Ru1. In the precursor Ru2, there is a bipyri-
dine chelate and two S-bonded dmso ligands in the equatorial
plane. Although the chelate effect should hinder the replacement
of the bpy ligand from Ru2, the two mutually cis S-bonded dmso
ligands that are capable of accepting p-back donated electrons
from the metal, along with the larger trans influence, weakens
the metal-N bonds, facilitating the replacement of the chelate
[16].
3.2. X-ray crystallography
Though slow evaporation of the respective reaction mixtures
yielded X-ray quality single crystals of 1, 1a, 2 and 2a, only struc-
tures of the first three complexes were solved. Structure determi-
nation of 1 and 1a showed that the two compounds are one and
the same, as the unit cell parameters of the two complexes were
very close and other structural features and geometric parameters
were similar. The crystal data and data collection procedure are
supplied in Table 1. Selected geometrical parameters are listed in
Table 2. Figs. 1 and 2 show the ORTEP diagrams of 1 and 2 respec-
tively. In both of the complexes, there are two independent
O
O
N
Cl
Ru
Cl
C
Me2OS
Reflux 4-6 h Me2OS
cis - [RuCl2(dmso)4] /
trans(Cl)-[RuCl2(dmso)2(bpy)]
Ethanol
R'
N
C
+
N
NH
H
R
R'
R
R = H; R’ = C4H3O, R = CH3; R’ = C4H3O, R = H, C4H3S, R = CH3; R’ = C4H5N
Scheme 1. Synthesis of the new Cl–Ru(II)–dmso complexes

1920
V. Mahalingam et al. / Polyhedron 27 (2008) 1917–1924
Table 1
complexes (A and B) in the asymmetric units and a disordered sol-
Crystal structure data of the complexes 1 and 2
vent molecule. Refinement of the disordered regions of both 1 and
2 has been carried out, but the fit to the data did not really im-
proved in either case, nor were there any significant changes in
the interesting parts of the structure.
Crystal data
1
2
Formula
Formula
C
16H22Cl2N2O4RuS2 Á 0.5(C2H6O) C16H22Cl2N2O3RuS3 Á 0.5(C2H6O)
565.48
581.54
weight
In complex 1, the ruthenium(II) ion is in a distorted octahedral
environment equatorially coordinated by an ON chelate of the
hydrazone ligand and two sulfur atoms of the dmso ligands at cis
positions. A pair of chlorine atoms at the axial positions completes
the octahedral coordination. The Ru–N (Ru1a–N1a = 2.107(2) and
Ru1b–N1b = 2.098(3) Å) and Ru–O (Ru1a–O1a = 2.123(2) and
Ru1b–O1b = 2.101(2) Å) bond lengths observed in the two inde-
pendent complexes are comparable to literature values [17–23].
The two Ru–S bond lengths observed in the independent com-
plexes A and B are comparable with that of similar Ru(II) com-
plexes [17] and with that of the precursor, cis-[RuCl2(dmso)4]
[24]. As expected and previously observed [24,25] there is a change
in the S–O bond length of dmso after coordination in comparison
with the free ligand.
Color, habit
Dimensions
Space group
colorless, fragment
0.23 Â 0.20 Â 0.12 mm
Pıꢀ
orange, parallelepiped
0.25 Â 0.10 Â 0.05 mm
Pıꢀ
´
a (Å)
11.2248(11)
11.0411(15)
´
b (Å)
14.1644(15)
14.2763(16)
´
c (Å)
14.663(2)
92.762(4)
98.201(3)
103.233(8)
15.1254(16)
92.617(7)
100.003(7)
104.928(5)
a (°)
b (°)
c (°)
3
´
V (Å )
Z
2238.3(4)
4
1.678
2258.4(5)
4
1.710
Dcalc
(Mg mÀ3
)
T (K)
F000
110
1148
1.154
115
1180
1.232
l(Mo Ka)
(mmÀ1
Rint
Goodness of
fit (S)
The substitution of one O-bonded dmso and one S-bonded
dmso in the precursor cis-[RuCl2(dmso)4] by the ON chelating li-
gand has yielded 1 in which the two chloro ligands take mutual
trans positions, which is in accordance with a previous study
[17]. The two Cl ligands in the axial positions are faintly bent to-
)
0.075
1.077
0.035
1.024
hmax (°)
32.5
30.6
Table 2
Selected geometrical parameters for 1 and 2
´
Bond distances (Å)
Bond angles (°)
1
2
1
2
Ru1a–N1a
Ru1a–O1a
Ru1a–S1a
Ru1a–S2a
Ru1a–Cl1a
Ru1a–Cl2a
Ru1b–N1b
Ru1b–O1b
Ru1b–S1b
Ru1b–S2b
Ru1b–Cl1b
Ru1b–Cl2b
S1a–O3a
2.107(2)
2.123(2)
2.2242(9)
2.2584(8)
2.4174(8)
2.3959(8)
2.098(3)
2.101(2)
2.2297(9)
2.2453(9)
2.3934(8)
2.4091(8)
1.489(2)
1.486(2)
1.493(3)
1.487(3)
2.107(2)
2.115(2)
2.2304(8)
2.2487(7)
2.4054(8)
2.3964(8)
2.083(2)
2.088(2)
2.2264(8)
2.2547(8)
2.4037(7)
2.4095(8)
1.485(2)
1.490(2)
1.497(2)
1.490(2)
Cl1a–Ru1a–Cl2a
S1a–Ru1a–S2a
N1a–Ru1a–O1a
Cl1b–Ru1b–Cl2b
S1b–Ru1b–S2b
N1b–Ru1b–O1b
C13a–S1a–C14a
C15a–S2a–C16a
C13b–S1b–C14b
C15b–S2b–C16b
C13a–S1a–O3a
C14a–S1a–O3a
C15a–S2a–O4a
C16a–S2a–O4a
C13b–S1b–O3b
C14b–S1b–O3b
C15b–S2b–O4b
C16b–S2b–O4b
174.30(3)
94.50(3)
77.31(9)
172.28(3)
94.14(3)
77.86(9)
100.09(16)
99.1(2)
99.17(17)
98.7(2)
104.07(15)
106.34(16)
106.28(16)
106.75(17)
107.37(17)
105.56(17)
107.18(17)
106.3(2)
173.95(3)
94.69(3)
77.12(8)
172.06(3)
94.52(3)
77.54(9)
99.55(16)
98.67(18)
99.54(15)
99.71(17)
104.78(16)
106.63(16)
106.03(15)
106.43(16)
106.71(14)
104.90(14)
106.73(16)
104.91(13)
S2a–O4a
S1b–O3b
S2b–O4b
Fig. 1. Asymmetric unit of 1 with anisotropic displacement ellipsoids at 50% probability. Hydrogen atoms are omitted for clarity.

V. Mahalingam et al. / Polyhedron 27 (2008) 1917–1924
1921
Fig. 2. Asymmetric unit of 2 with anisotropic displacement ellipsoids at 50% probability. Hydrogen atoms and disorder in thiophene ring are omitted for clarity.
Table 3
wards the chelate (the angle Cl2a–Ru1a–Cl1a is 174.30(3)° and
Cl2b–Ru1b–Cl1b is 172.28(3)°) due to the pushing by the
Hydrogen-bonding distances (Å) and angles (°)
Complex
D–HÁ Á ÁA
D–H
HÁ Á ÁA
DÁ Á ÁA
D–HÁ Á ÁA
somewhat bulky cis dmso ligands. The Ru–Cl distances in 1
(Ru1a–Cl1a = 2.4174(8), Ru1a–Cl2a = 2.3959(8) Å; Ru1b–Cl1b =
2.3934(8), Ru1b–Cl2b = 2.4091(8) Å) are shorter than that of Ru1
(mean Ru–Cl distance of the different forms is 2.425 Å) and fairly
comparable with that of Ru2 (2.402 Å). This is in agreement with
the greater trans influence of dmso [24].
The angles O–S–C and C–S–C in A and B of 1 are comparable
with that of the S-bonded dmso ligands in cis- and trans-
[RuCl2(dmso)4]. But these angles of O-bonded dmso in one of the
precursors, Ru1, is relatively narrow when compared with that of
S-bonded dmso ligands. This is due to the different electronic situ-
ation of the two types of sulfur atom and can be attributed to the
compression effect of the bulkier lone pair of the pyramidal sulfur
atoms on the bonding pairs [25]. The bite angles of the ON chelate
in A and B are 77.31(9) and 77.86(9)° respectively, and these are
comparable with that of a similar chelate [17].
1
N2a–H2NaÁ Á ÁO2a
N2b–H2NbÁ Á ÁO4a
N2b–H2NbÁ Á ÁO2b
N2a–H2NaÁ Á ÁO3bi
N2b–H2NbÁ Á ÁO4a
0.77(4)
0.87(4)
0.87(4)
0.85(4)
0.85(3)
2.28(4)
2.14(4)
2.21(4)
2.18(4)
1.96(3)
2.750(3)
2.917(3)
2.682(3)
2.988(3)
2.803(3)
120(3)
149(3)
114(3)
159(3)
167(3)
2
i
Symmetry codes used to generate identical atoms: x, y À 1, z.
2a, 3 and 3a, and 4 and 4a will be mentioned henceforth as 2, 3 and
4 respectively assuming that they have same chemical composi-
tion (based on the microanalyses) although no structural evidence
is currently available for the other complexes.
3.3. 1H NMR spectra
The structure of 2 shares similar properties and the same
discussion goes well with that for 1 except in a few aspects. The
heterocyclic ring in 1 is furan whereas in 2 it is thiophene and there
is a slight distortion in the thiophene moiety in one of the two
independent complexes. Further refinement, using a number of
restraints, of the disordered thiophene led to a slight improvement,
most notably in the reduction of residual electron densities. How-
ever, full anisotropic refinement did not lead to realistic displace-
ment parameters. The independent complexes in the asymmetric
units of 1 are stabilized by intramolecular N(amide)–HÁ Á ÁO(furan)
bonds and intermolecular N(amide)–HÁ Á ÁO(dmso) bonds in addi-
tion to bifurcated weak interactions, O(disordered ethanol)–HÁ Á ÁCl
and O(disordered ethanol)–HÁ Á ÁO(furan). In contrast to 1, intermo-
lecular hydrogen-bonding, N(amide)–HÁ Á ÁO(dmso) appears to be
predominant in complex 2. The geometric parameters pertaining
to hydrogen-bonding are provided in Table 3.
With the single crystal X-ray structures of 1 and 1a, it is clear
that the chelating ligands replace the coordinated ligands that
are trans to the two S-bonded dmso from either of the precursors
Ru1 and Ru2. This is due to the strong trans influence of the S-
bonded dmso ligands. Also, in Ru2, the chelating bpy ligands are
replaced by the ON hydrazone chelate relatively easily compared
to the two S-bonded dmso ligands because of the strong p-acceptor
character of the two sulfur atoms. Hence, the complex pairs 2 and
The 1H NMR spectra of the complexes recorded in dmso showed
narrow signals typical for diamagnetic Ru(II) complexes. A compar-
ison of the 1H NMR data [9] of the precursor cis-[RuCl2(dmso)4]
with that of the new complexes seems indispensable in order to
ascertain the bonding mode of the two dmso ligands, as evidenced
by the single crystal X-ray diffraction studies (for 1 and 2), and to
predict the same in 3 and 4. The resonance for methyl protons of
free dmso is ꢀd 2.5 ppm. The complex cis-[RuCl2(dmso)4] (contains
three S-bonded dmso and one O-bonded dmso) displayed six sing-
lets at d 3.50, 3.48, 3.43, 3.32, 2.72 and 2.66 ppm due to six sets of
chemically different protons. The peak at d 2.66 ppm has been as-
signed to free dmso which resulted from the 10% dissociation of O-
bonded dmso. Similarly the peak at d 2.72 ppm has been attributed
to the O-bonded dmso since the methyl resonance is close to that
of free dmso. The other peaks appearing in a group have been as-
signed to S-bonded dmso but the integration of these peaks had
not been possible owing to their proximity and their sharing of
the same broad base line. The three S-bonded dmso ligands in
the complex are trans to either O-bonded dmso, S-bonded dmso
or Cl ligands. This, along with some degree of methyl inequivalen-
cy, has been thought of as responsible for the complexity of the
spectra. Keeping all these in mind, the group of peaks appearing
in the new complexes around ꢀd 3.2–3.4 ppm is attributed to S-
bonded dmso resonances. Here no resonance was observed at d

1922
V. Mahalingam et al. / Polyhedron 27 (2008) 1917–1924
2.7 ppm for any of the complexes, suggesting that there is no O-
bonded dmso in the complexes which is consistent with the single
crystal X-ray diffraction results obtained for 1 and 2, and predicts
the same for 3 and 4. Thus the inequivalency in the methyl groups
is due to the chelating ligand. The resonance around d 2.5 ppm in
all the complexes is due to the solvent. The azomethine proton
in 1 and 2 appears as a singlet at d 8.1 and 7.9 ppm respectively.
The protons of the methyl group in 3 and 4 gave a resonance at d
2.2 ppm. The multiplet around d 7.2–8.5 ppm is due to aromatic
and heterocyclic ring protons. The appearance of a singlet in all
the complexes around d 9.2–9.4 ppm is due to the NH proton
and this suggests that the chelating ligand coordinates in its keto
form to the metal and supports a neutral bidentate coordination
of the hydrazone ligands.
allowed to stand for 5 min before recording their absorption. The
titration process was repeated until no further change was ob-
served in the spectra.
The binding of the ruthenium complexes to the DNA helix was
characterized by following the changes in the absorbance and shift
in wavelength upon each addition of DNA solution to the complex.
Representative absorption spectra are given in Figs. 3 and 4. The
ruthenium(II) complexes in dmso-buffer mixture exhibit an in-
tense intraligand transition in the region around 315–330 nm. On
the titration of Herring sperm DNA with the complexes, a consid-
erable increase or decrease in the absorption along with a small
red or blue shift was observed. The initial increase in absorption
for complexes 2 and 3 is due to the electrostatic interaction of
DNA and the complexes [28]. In complexes 2 and 4 remarkable
hypochromicity is noted after the ratio [DNA]:[Ru] becomes 1. In
complex 1 the absorption decreases and increases at random with
a small red shift (2–4 nm) on titration with the DNA solution.
These trends show that the complexes bind to the DNA double-he-
lix in different modes to different extents [29]. As can be perceived
from Figs. 3 and 4, the shift in absorption and kmax are random and
this tendency frustrated the calculation of intrinsic binding con-
stants of the complexes using the equation valid for perfect
intercalators.
3.4. Cyclic voltammetric studies
The electrochemical behavior of the complexes was studied
with the help of cyclic voltammetry using dmso solutions of the
complexes and TBAP as a supporting electrolyte. All the potentials
were referenced to the Ag/AgCl electrode and scanned at a rate of
0.1 V/s. The complexes show different redox behavior in the cho-
sen potential window. Complexes 1 and 4 show only a RuIII/RuII
oxidation couple with the formal potentials 0.63 and 0.50 V respec-
tively. Complex 2 shows only an irreversible oxidation wave with
Epa = 0.49 V. This irreversibility may be anticipated due to the
transient lifetime of the reduced state. The peak-to-peak separa-
tion value DEp for 1 and 4 is 240 and 80 mV suggesting a quasire-
versible and reversible one electron transfer process respectively. A
reversible reduction (RuII/RuI) is observed for 3 with a formal po-
tential of À0.39 V (DEp = 80 mV). These different conducts of the
complexes can be accredited to the different electronic nature of
the substituents present in the hydrazone ligands. When the Ef val-
ues for the oxidation couple for 1 and 4 are compared, a negative
shift in the potential is observed. This shift is due to the presence
of an electron releasing CH3 group and pyridine moiety in 4 which
favor easier oxidation of the central metal ion. The more electron
withdrawing furan ring of 3 renders the molecule prone to reduc-
tion of the ruthenium centre [26]. In summary it can be drawn
that electron releasing substituents stabilize the +3 oxidation
state whereas electron withdrawing substituents stabilize the +2
oxidation state of ruthenium.
3.5. DNA-binding studies
Fig. 3. Absorption bands of 1 on titration with DNA at different [DNA]/[Ru] ratios
(R). R1 = 0, R2 = 0.25, R3 = 0.50, R4 = 0.75, R5 = 1 and R6 = 1.5.
Most ruthenium complexes bind to DNA by non-covalent inter-
actions such as electrostatic binding, groove binding, intercalative
binding and partial intercalative binding [27]. A quantitative
understanding of the factors that determine the recognition of
DNA sites would be valuable in the rational drug design of new
DNA targeted molecules for application in chemotherapy. The
binding of the new ruthenium complexes to the Herring sperm
ds DNA has been monitored through absorption titration and cyclic
voltammetry.
3.5.1. Absorption titration
The experiments were carried out in Tris–HCl buffer (50 mM,pH
7.2) using a solution of Herring sperm DNA which gave a ratio of
UV absorbance at 260 and 280 nm in the ratio 1.8:1, indicating that
the DNA is sufficiently free from protein. So no further effort was
made to purify the commercially obtained DNA. The concentration
of DNA was determined by absorption spectroscopy using the e va-
lue of 6600 MÀ1 cmÀ1 at 260 nm. Absorption titrations were per-
formed using
a fixed ruthenium concentration to which
increments of the DNA stock solutions were added in different ra-
tios ranging from 0 to 3 [DNA]/[Ru]. Complex–DNA solutions were
Fig. 4. Absorption bands of 4 on titration with DNA at different [DNA]/[Ru] ratios
(R). R1 = 0, R2 = 0.25, R3 = 0.50, R4 = 0.75, R5 = 1, R6 = 1.5, R7 = 2 and R8 = 3.

V. Mahalingam et al. / Polyhedron 27 (2008) 1917–1924
1923
Table 4
Antibacterial activity data for the ligands and complexes
Compound
Escherichia coli
Shigella sonnei
Staphylococcus aureus
Staphylococcus epidermidis
Klebsiella pneumoniae
WDa
MICb
WD
MIC
WD
MIC
WD
MIC
WD
MIC
2
4
NA
NA
15
NA
30
20
30
32
136
121
25
85
24
23
15
18
17
NA
20
16
33
25
37
48
74
82
28
97
26
34
8
12
16
16
NA
26
NA
32
40
31
153
18
44
21
137
7
10
14
15
12
14
20
14
44
60
46
32
26
87
23
92
8
NA
NA
15
NA
30
20
30
35
144
140
47
124
28
52
8
10
bfmh
btmh
bfeh
bpeh
Oxytetracycline
Kanamycin
9
11
NA – not active.
a
WD, disc diffusion method as recommended by NCCLS. Diameter of zone of inhibition (mm) including well diameter 8 mm. 100 lg of both compound and antibiotics were
used.
b
MIC, minimum inhibitory concentration.Values given as lg/ml for both compounds and antibiotics.
3.5.2. Cyclic voltammetry
chemical and biochemical factors are responsible for the process
of inhibition of the growth of the bacteria.
Since all the new complexes are electroactive, electrochemical
methods such as cyclic voltammetry can be used effectively to
monitor their binding to DNA as a complement to the absorption
spectral technique. In a typical cyclic voltammetric titration, a
fixed concentration of the complex was taken and DNA solution
in buffer was added in different ratios as done in the absorption
titration, and the voltammetric response was recorded. As ob-
served in the UV experiments, an increase or decrease of the peak
current was observed for all the complexes. The peak current in-
creased initially and then decreased. The initial increase in the
peak current is due to the absorption of the DNA bound complex
onto the electrode surface [30]. The decrease in peak current on
the addition of DNA to the complex is suggestive of an interaction
between the complex and DNA [31]. Except for complex 1, a de-
crease in the peak-to-peak separation was observed, which is con-
sistent with non-coordinating intercalative binding of the
complexes through the planar aromatic rings between the DNA
base pairs [32]. The formal potential Ef shifts slightly towards the
positive side and is attributed to characteristic behavior of interca-
lation of the complexes into the DNA double-helix [33], and sug-
gests that Ru(II) and Ru(I) forms bind to DNA at different rates
[34,35].
4. Conclusions
The present study demonstrates a simplistic synthesis of a
family of new Cl–dmso–Ru(II) complexes. The greater trans influ-
ence of mutually cis S-bonded dmso ligands in comparison to the
bipyridine ligand has been proved by the observed replacement
of bipyridine from Ru2 by the hydrazone ligands, with crystallo-
graphic evidence. The electrochemical properties of the com-
plexes are very sensitive to the electronic nature of the
substituents present in the hydrazone ligands. The new com-
plexes have been shown to possess DNA-binding abilities. The
in vitro antineoplastic activity of the complexes against human
breast cancer cell lines is under investigation and the results will
be published shortly.
Appendix A. Supplementary data
CCDC 613460 and 632085 contain the supplementary crystallo-
graphic data for 1 and 2. These data can be obtained free of charge
via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: depos-
it@ccdc.cam.ac.uk. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.poly.2008.02.036.
3.6. Antibacterial activity
All the ligands and their ruthenium complexes were screened
in vitro for their growth inhibitory activity against the pathogenic
bacteria E. coli, S. aureus, S. epidermidis, K. pneumoniae and S. sonnei.
The inhibition zone (in mm) and MIC of the ligands and their
ruthenium complexes are given in Table 4. From the figures it is
perceived that the ligands bfmh and btmh inhibit the growth of
all the test organisms to a moderately comparable extent to that
of standard antibiotics and they can be said to be resistive to the
organisms. The ligand btmh is resistive only to S. sonnei and S. epi-
dermidis since no inhibition zone is found for the other organisms.
Except for S. aureus, the ligand bpeh is resistive to all other patho-
genic bacteria. All the complexes except 3 are resistive to at least
one of the organisms under study. Complex 2 shows considerable
resistivity to S. aureus, S. epidermidis and S. sonnei. Similarly 4 is ac-
tive against S. aureus and S. epidermidis and 1 is active against only
S. sonnei. None of the complexes are active against the other two
organisms. Thus all the ligands and complexes 2 and 4 alone have
been studied further to evaluate the MIC because only these two
complexes demonstrate activity against at least two of the five
organisms. A low value of MIC implies high resistivity against the
particular organism. No serious attempts were made to correlate
the activity to the structures of the compounds since too many
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