New organometallic ruthenium(ii) complexes containing chelidonic acid (4-oxo-4H-pyran-2,6-dicarboxylic acid): Synthesis, structure and in vitro biological activity

Article abstract of DOI:10.1039/c3ra43865a

Two new bivalent organometallic ruthenium complexes [Ru(HL)(CH ₃CN)(CO)(PPh₃)₂] (3) and [Ru(HL)(CH ₃CN)(CO)(AsPh₃)₂] (4), (HL = 4-oxo-4H-pyran-2,6-dicarboxylic acid) were synthesized, structurally characterized and their biological activities (anti-microbial, DNA-protein interactions, antioxidant and cytotoxic activity studies (MTT, LDH release and NO release)) have been investigated and compared with that of appropriate precursor complexes [RuHCl(CO)(PPh₃)₃] (1), [RuHCl(CO)(AsPh₃)₃] (2) and the ligand H²L. The crystal structure of the complex 3 was solved by a single crystal X-ray diffraction technique, which revealed that it is a distorted octahedral with HL as a dibasic bidentate donor and the chelator was observed to undergo C-H activation at one of the ortho positions, leading to the formation of a five membered metallacycle. The in vitro antimicrobial activity was carried out using the well diffusion method against different species of pathogenic bacteria and fungi and complex 4 exhibited a better activity in inhibiting the growth of the tested organisms. DNA-protein interactions of the complexes have been examined by photophysical studies, which revealed that the complexes can bind with DNA through non-intercalation and the complexes strongly quench the intrinsic fluorescence of bovine serum albumin, through a static quenching process. The free radical scavenging ability, assessed by a series of in vitro antioxidant assays involving the DPPH radical, hydroxyl radical, nitric oxide radical, superoxide anion radical, hydrogen peroxide and a metal chelating assay showed that the new complexes 3 and 4 possess excellent radical scavenging properties over 1, 2, H²L, and the standard drugs, vitamin C and BHT. The in vitro cytotoxic activities of the compounds have been validated against A549 cells via an MTT assay, LDH release, NO release and the values were compared with that of the standard drug cisplatin. The results indicated that the new complexes, specifically the complex 3, displayed a higher cytotoxic activity in inhibiting the growth of A549 cells, outperforming by five fold, the standard drug cisplatin.

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New organometallic ruthenium(II) complexes
containing chelidonic acid (4-oxo-4H-pyran-2,6-
dicarboxylic acid): synthesis, structure and in vitro
biological activity†
Cite this: RSC Adv., 2014, 4, 2004
Thangavel Sathiya Kamatchi,a Palaniappan Kalaivani,a Paramasivan Poornima,b
b
c
a
*
Viswanadha Vijaya Padma, Frank R. Fronczek and Karuppannan Natarajan
Two new bivalent organometallic ruthenium complexes [Ru(HL)(CH3CN)(CO)(PPh3)2] (3) and
[Ru(HL)(CH3CN)(CO)(AsPh3)2] (4), (HL 4-oxo-4H-pyran-2,6-dicarboxylic acid) were synthesized,
¼
structurally characterized and their biological activities (anti-microbial, DNA–protein interactions,
antioxidant and cytotoxic activity studies (MTT, LDH release and NO release)) have been investigated and
compared with that of appropriate precursor complexes [RuHCl(CO)(PPh3)3] (1), [RuHCl(CO)(AsPh3)3] (2)
and the ligand H2L. The crystal structure of the complex 3 was solved by a single crystal X-ray diffraction
technique, which revealed that it is a distorted octahedral with HL as a dibasic bidentate donor and the
chelator was observed to undergo C–H activation at one of the ortho positions, leading to the formation
of a five membered metallacycle. The in vitro antimicrobial activity was carried out using the well
diffusion method against different species of pathogenic bacteria and fungi and complex 4 exhibited a
better activity in inhibiting the growth of the tested organisms. DNA–protein interactions of the
complexes have been examined by photophysical studies, which revealed that the complexes can bind
with DNA through non-intercalation and the complexes strongly quench the intrinsic fluorescence of
bovine serum albumin, through a static quenching process. The free radical scavenging ability, assessed
by a series of in vitro antioxidant assays involving the DPPH radical, hydroxyl radical, nitric oxide radical,
superoxide anion radical, hydrogen peroxide and a metal chelating assay showed that the new
complexes 3 and 4 possess excellent radical scavenging properties over 1, 2, H2L, and the standard
drugs, vitamin C and BHT. The in vitro cytotoxic activities of the compounds have been validated against
A549 cells via an MTT assay, LDH release, NO release and the values were compared with that of the
standard drug cisplatin. The results indicated that the new complexes, specifically the complex 3,
displayed a higher cytotoxic activity in inhibiting the growth of A549 cells, outperforming by five fold, the
standard drug cisplatin.
Received 24th July 2013
Accepted 14th October 2013
DOI: 10.1039/c3ra43865a
www.rsc.org/advances
carbon–hydrogen bonds represents one of the most powerful
tools in organic synthesis.1 Intramolecular C–H activation
1. Introduction
reactions are one of the major discoveries of organometallic
chemistry that provide access to metallacyclic derivatives of
transition metal complexes2,3 and cyclometallation is one of the
most commonly used methods in the activation of C–H bonds.4
Though nearly all transition metals have been successfully
employed for cyclometallation, the use of palladium,5,6 ruthe-
nium,7 rhodium,8 copper9 or iron10,11 complexes has set the stage
for chemo-, site-,12 diastereo-,13,14 and/or enantioselective15 C–H
bond functionalizations. Although palladium is, without any
doubt, the transition metal that has been the most studied
to promote the formation of metallacycles,16 ruthenium(II)
derivatives could efficiently complement their widely used pal-
ladium(II) congeners.17 In particular, in the recent years, cyclo-
ruthenated compounds have emerged as an extraordinarily
Due to development of transition metal mediated reactions
involving C–H bonds, the functionalization of traditionally inert
aDepartment of Chemistry, Bharathiar University, Coimbatore 641046, India. E-mail:
k_natraj6@yahoo.com; Fax: +91 422 2422387; Tel: +91 422 2428319
bDepartment of Biotechnology, Bharathiar University, Coimbatore 641 046, India
cDepartment of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
† Electronic supplementary information (ESI) available: ORTEP diagram of 3 with
hydrogen bonding interaction (Fig. S1). Packing diagram of the unit cell for
ꢀ
˚
complex 3: (Fig. S2). Selected bond lengths (A) and angles ( ) for 3 (Table S1).
The emission spectra of the DNA–EB system in the presence of H2L and
complexes 1–4 (Fig. S3). Synchronous spectra of BSA in the presence of
increasing amounts of H2L and complexes 1–4 (Fig. S4). Plausible mechanisms
for DPPH radical scavenging activity (Fig. S5). CCDC reference number: 900109
(3). For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3ra43865a
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versatile family of molecules, exhibiting interesting applications expected, we obtained new organometallic ruthenium(II)
in several elds. For example, their unique photophysical and complexes, with a new type of coordination mode for chelidonic
electrochemical properties have been exploited in the design of acid (Fig. 1, VIII). Herein, we report the synthesis, structure and
sensitizers for solar cells18 or electron shuttles in redox-catalyzed assessment of its biological properties, such as antimicrobial
processes.19 In addition, ruthenacycles have been found to and anticancer activity studies (DNA/protein binding, antioxi-
display an appealing potential as antitumor agents20 and are dant and cytotoxic activities (MTT, LDH release and NO
among the most active catalysts reported to date for transfer release)) of the new organometallic ruthenium(II) dicarboxylate
hydrogenation reactions.21 Various examples have appeared complexes.
recently in the literature dealing with the synthesis of ruthena-
cycles, implying the participation of a vast range of precursors
2. Results and discussion
2.1. Synthesis and characterization
and ligand settings.22 However, examples of cycloruthenated
complexes from heterocyclic rings is still scarce.23 In this area, it
would be interesting to synthesise ruthenium mediated cyclo-
metallated complexes comprising dicarboxylic acids and hence,
an attempt was made to study the reactions of chelidonic acid
(H2L, 4-oxo-4H-pyran-2,6-dicarboxylic acid) with ruthenium(II)
complexes. Chelidonic is one of the constituents of the greater
celandine Chelidonium majus, which exhibits a wide range of
therapeutic properties24 and its pKa value, of about 2.4, for both
carboxyl groups makes it an effective coordinating agent at a
physiological pH.25 The coordination chemistry of the chelido-
nate ion has been investigated with several metal ions26–31 in
which the chelidonate ligand showed diverse coordination
modes (Fig. 1, I to VII), which suggests great potential for the
construction of new coordination complexes. However, to
the best of our knowledge, its coordinative behaviour with
ruthenium(II) complexes containing triphenylphosphine/
triphenylarsine and the biological properties of new carboxy-
late complexes obtained have not been studied.
Over recent years, research in medicinal organometallic chemistry
has mainly focused on the preparation of organometallic
compounds for anticancer and antimicrobial purposes. Although
the organometallic pharmaceutical salvarsan was used for the
treatment of syphilis in the early 20th century,32 only a very few
organometallic compounds have reached the clinic in the mean-
time. Taking into account the urgent need for novel organome-
tallic chemotherapeutics, the present contribution describes the
synthesis of two new biologically active organometallic ruthenium
complexes, comprising 4-oxo-4H-pyran-2,6-dicarboxylic acid.
Straightforward reactions of H2L with [RuHCl(CO)(PPh3)3]/
[RuHCl(CO)(AsPh3)3] gave new complexes of the type
[Ru(HL)(CH3CN)(CO)(EPh3)2] (where E ¼ P/As), as depicted in
Scheme 1. The complexes are diamagnetic which corresponds to
the bivalent state of ruthenium (low-spin d6,
S
¼
0).
The ligand was observed to undergo C–H activation at one of the
ortho positions, leading to the formation of a ve-membered
metallacycle. The complexes were stable to air and light, non-
hygroscopic in nature and were remarkably soluble in meth-
anol, ethanol, chloroform, dichloromethane, DMF and DMSO.
With this background in mind, we attempted to synthesize
new organometallic complexes by reacting [RuHCl(CO)(PPh3)3]/
[RuHCl(CO)(AsPh3)3] with chelidonic acid monohydrate. As
Fig. 1 Observed and the new coordination mode of H2L found in the literature. I ¼ kO2, II ¼ kO3, III ¼ kO3:kO4, IV ¼ k3O1, O4, O6, V ¼ (m)
kO3:kO5, VI ¼ (m3) kO5, 2:3 kO4, VII ¼ (m4) O2:kO3:kO5:kO6, VIII ¼ k2 CO3.
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Scheme 1 General scheme for the synthesis of new organometallic ruthenium complexes.
submerged in the range of 1617 cmꢁ1, because the skeleton
vibrations of the n(C]O)ring of the aromatic rings appear at the
same range. The strong and sharp band at 1433 cmꢁ1 can
probably be assigned to the symmetric vibrational mode
nsy(CO2ꢁ) of the carboxylate group aer coordination to the
ruthenium ion. The difference between the asymmetric and
symmetric stretches of the carboxylate group is 184 cmꢁ1, which
suggests a monodentate binding of the carboxylate group to the
metal ion.33 The considerable decrement of the n(C]C) vibra-
tional frequency in the complex drives us to think that another
point of attachment of H2L to the ruthenium ion is one of the
pyrone carbons. The strong absorption at 1952 cmꢁ1 has been
assigned to the terminally coordinated carbonyl group. The
electronic spectrum of complex 3 presents absorption maxima
at 229, 243, 265 and 370 nm. The absorption observed at 229,
243 and 266 nm is most likely due to a transition involving only
ligand orbitals (p / p* and n / p*). The absorption at 370 nm
is probably due to metal-to-ligand charge transfer transitions.
These charge transfer transitions may be taking place from the
highest lled ruthenium t2 orbital (highest occupied molecular
orbital) to the vacant p* (–C]C– or –C]O–) orbital of H2L
(lowest unoccupied molecular orbital) or to the higher energy
vacant orbitals of another fragment of the ligand.
The NMR spectrum of the complex 3 contained one well
resolved, broad singlet, two sharp singlets and one multiplet
that were assigned as shown below (the numbering scheme can
be found in Fig. 2). A one proton sharp singlet, at d 11.29 ppm,
in complex 3 is due to the hydroxyl proton of the carboxylic acid.
Another one proton sharp singlet at d 7.87 ppm, is due to the
proton which is attached to carbon C4 of the pyrone ring. The
resonance around d 2.09 ppm is due to the methyl protons of
the coordinated acetonitrile solvent. A multiplet at the d 7.23–
7.70 ppm range corresponds to the aromatic ring protons of the
triphenylphosphine groups. The 13C-NMR spectrum of the
complex 3 displayed 14 signals. A downeld sharp, medium
intensity singlet at d 204.42 ppm is assigned to the terminal
carbonyl carbon, C8. The next downeld singlet at d 196.57 ppm
could be due to C2 and the very low eld appearance of this is
indicative of its coordination to ruthenium. A signal at d 119.18
ppm is due to the nitrile carbon C9^N. C10 (aliphatic methyl),
a shielded carbon of the coordinated acetonitrile resonated as a
singlet at d 13.62 ppm. The skeleton carbonyl carbon, C3,
appeared as a singlet at d 172.14 ppm. A singlet at d 93.57 ppm is
assigned to the C4 sp2 carbon. C5 (sp, low intensity band at d
153.44 ppm) is around 60 ppm downeld compared to that of
C4 (d 93.57 ppm). This might be due to the attachment of the
Fig. 2 Numbering scheme for the representative complex 3.
The synthesized complexes were then characterized by elemental
analysis, IR, 1H and 13C NMR spectroscopic techniques. The
complexes were analytically pure, as their microanalytical data
conform to the proposed molecular formula. It has been observed
that a molecule of 4-oxo-4H-pyran-2,6-dicarboxylic acid (H2L)
replaced a hydride and chloride ions from the precursor
complexes and acetonitrile, the solvent used for the reaction,
replaced one of the triphenylphosphines/triphenylarsines and
occupied one of the equatorial sites. The solid state structure of
one of the complexes (3) was determined by single crystal X-ray
crystallographic studies. It revealed that H2L behaved as a
dibasic bidentate donor.
2.2. Spectroscopic studies
The spectroscopic data for the new complexes 3 and 4 looked
very similar and hence, the spectroscopic data of complex 3 will
be described in detail here. The IR spectrum of the free 4-oxo-
4H-pyran-2,6-dicarboxylic acid (H2L) showed bands at 3575,
3476, 1790, 1730, 1644, 1587 and 1255 cmꢁ1 corresponding to
n(COOH), n(COOH)0, n(C]OOH), n(C]OOH)0, n(C]O)ring
,
n(C]C) and n(C–O) respectively. In the spectra of 3, the band
due to free COOH0 seems to be virtually unaltered, but slightly
broadened at 3476 cmꢁ1, indicating the non-participation of
one of the carboxylic acid groups. The presence of a medium
intensity band at 1600 cmꢁ1 is attributable to the C]O
stretching vibrations of the free carboxylic acid group
n(C]OOH)0. However, the complete disappearance of vibra-
tional responses corresponding to n(COOH) at 3575 cmꢁ1
evidences the coordination of another carboxylic acid oxygen
atom to a ruthenium ion aer deprotonation. It is to be noted
that the asymmetric carboxylate vibrations nas(CO2ꢁ) are
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Fig. 3 ORTEP diagram with atom numbering scheme for 3 as thermal ellipsoids at a 50% probability level. The hydrogen atoms (except one
carboxylic acid proton) and the solvent molecules of crystallization have been omitted for clarity.
electron withdrawing COOH. C1 (sp) resonated as a singlet at d molecule. A crystal fragment of the approximate dimensions of
168.52 ppm. One singlet appearing at d 163.38 is due to C7 of 0.35 ꢂ 0.30 ꢂ 0.25, was used for the diffraction data collection
the free carboxylic acid group and the other at d 181.91ppm is at 100 K. The molecule lies on a crystallographic mirror plane.
due to the other carboxylic acid group (C6), whose oxygen is The crystal structure of 3 consists of a six-coordinated ruthe-
bonded to ruthenium. Furthermore, four singlets appeared at d nium ion with a C2N1O1P2 core, where the equatorial plane is
130.52, 131.91, 132.00 and 132.52 ppm, for the carbon atoms constructed by a carbon and an oxygen atom of the bidentate
(a, b, c and d types) of the triphenylphosphine groups.
ligand HL and a carbonyl carbon. The nitrogen atom of the
acetonitrile (solvent used for the reaction) molecule completes
the equatorial plane. As is commonly observed for hexa-
coordinated complexes containing the {Ru(PPh3)2} unit, the
2.3. X-ray crystal structure
The molecular structure of 3 has been determined by single two bulky PPh3 molecules are above and below the equatorial
crystal X-ray analysis. ORTEPs diagram with the atom labeling plane. The ve membered chelate ring has a bite angle of
scheme, hydrogen bonding interaction and packing diagram of 78.52(7)ꢀ. The +2 oxidation state of the complex is compensated
the unit cell of 3 are displayed in Fig. 3, S1 and S2 (ESI†), by one of the carboxylate oxygens and a carbanion of the ligand
respectively. A summary of the structure renement is shown in HL. When ruthenium(II) is coordinated to two triphenylphos-
Table 1. The selected geometrical parameters (interatomic phine ligands, the relative disposition of them depends on the
distances and angles) and the hydrogen bond distances are competition of steric and electronic (p-back bonding) factors.
given in the Table S1 (ESI†). Single crystals of complex 3 were In our case, the former predominates and hence, the two
obtained as colorless needles from the slow evaporation of the phosphine groups are trans to each other. However, if the latter
reaction mixture for at least one week at room temperature. The dominates, a cis structure is expected. As expected, the two trans
crystal was a solvate, containing two acetonitriles and a water Ru–P bond lengths are much longer than the four equatorial
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Table 1 Crystallographic data for complex 3
CCDC deposit number
900109
C46H35NO7P2Ru.2(C2H3N)$H2O
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
976.88
100.0(5)
Orthorhombic
Cmc21
15.2613(3)
18.5194(4)
16.2330(3)
4587.75(16)
4
˚
a/Aꢁ1
˚
b/A
˚
c/A
Volume/A
3
˚
Z
rcalc/mg mmꢁ3
1.414
0.469
2008
Absorption coefficient mmꢁ1
F(000)
Crystal size/mm3
0.35 ꢂ 0.30 ꢂ 0.25
2q range for data collection
Index range
5.2–68.2ꢀ
ꢁ22 # h # 22, ꢁ27 # k # 27, ꢁ25 # l # 25
Reections collected
Independent reections
Data/restraints/parameters
Goodness-of-t on F2
Final R indexes [I $ 2s(I)]
Final R indexes [all data]
28 264
8448[R(int) ¼ 0.041]
8448/1/337
1.024
R1 ¼ 0.0292, wR2 ¼ 0.0692
R1 ¼ 0.0323, wR2 ¼ 0.0712
0.82/ꢁ0.90
ꢁ3
˚
Largest diff. peak/hole/e A
˚
bond lengths, indicating a large axial distortion. This length- be 2.06 A and the observed values span the range 1.96 to
34
˚
˚
ening is due to the strong trans inuence of the bulky triphe- 2.16 A. Furthermore, the Ru1–C(2) distance is 2.033 A, which is
nylphosphine ligands. The lengthening is evidenced by two very similar to that of the other structurally characterized
cyclometallated ruthenium(II) complexes.35 The Ru1–C(8) bond
0
˚
central bond lengths Ru1–P(1) [2.3897(3) A] and Ru1–P
˚
˚
[2.3897(3) A] that are longer when compared to that of the other distance (1.844(2) A) is in accordance with those found in other
2
four basal planar bonds Ru1–N(1) [2.119(2) A], Ru1–C(2) ruthenium(II) complexes.35,36 The Ru–C(2) (sp ) bond is 0.189(3)
˚
˚
˚
˚
˚
[2.033(2) A], Ru1–O(1) [2.143(2) A], Ru1–C(8) [1.845(2) A]. The A, longer than the Ru1–C(8) (sp) distance due to Ru–CO back
trans angle of P(1)–Ru1–P0(1) [175.48(3)ꢀ] is not close to the ideal bonding. The carbonyl group occupies a site trans to the O(1).
value of 180ꢀ, providing further evidence for the axial distortion. This may be a consequence of strong Ru(II) / CO back dona-
˚
However, the other trans angles of the basal planes, namely tion, as indicated by the short Ru–C bond (1.844(2) A) and low
C(2)–Ru1–N(1) [169.65(8)ꢀ] and O(1)–Ru1–C(8) [174.34(9)ꢀ], are CO IR stretching frequency (1952 cmꢁ1) preferring s or weak p
constrained, indicating clearly that the coordination geometry donor groups occupying a site opposite to CO to favor the d–p*
around the ruthenium metal centre is distorted octahedral. The back donation. The two Ru–P bond lengths are identical by
cis bond angles spread over the range from 78.52(7) to symmetry and are comparable to those reported for other
95.83(10)ꢀ. The P(1)–Ru–P0(1) is the largest (175.48(2)), while ruthenium(II) complexes containing coordinated triphenyl-
C(2)–Ru–N(1) is the smallest (169.65(8)) trans angle. From the phosphine.37,38 Ru(1)–N(1) (nitrogen from coordinated acetoni-
covalent radii values, the Ru(II)–C(2) (sp2) length is estimated to trile solvent) bond length (2.119(2) A) agrees well with those
˚
Table 2 Antibacterial and antifungal activity of the ligand, precursors and new ruthenium complexes – zone of inhibition in mm
Gram-positive
Gram-negative
Fungi
Compounds
S. aureusa
E. faecalisb
E. colic
K. pneumoniaed
A. nigere
C. albicansf
H2L
1
2
3
10
12
16
18
22
28
—
—
09
11
14
24
29
30
—
—
07
10
12
14
17
29
—
—
10
11
15
17
18
32
—
—
4
5
6
10
14
—
22
—
3
5
7
8
4
10
—
31
—
Ciprooxacin
Fluconazole
Control
a
d
Staphylococcus aureus. b Enterococcus faecalis. c Escherichia coli. Klebsiella pneumonia. e Aspergillus niger. f Candida albicans “—” no activity.
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Table 3 Minimum inhibitory concentration (MIC), mg mLꢁ1
Gram-positive
Gram-negative
Fungi
Compounds
S. aureus
E. faecalis
E. coli
K. pneumoniae
A. niger
C. albicans
H2L
1
2
3
100
75
50
25
25
100
75
50
25
25
100
100
50
75
50
100
75
75
50
25
>100
>100
>100
100
50
>100
>100
>100
100
50
4
Ciprooxacin
Fluconazole
12.5
—
12.5
—
12.5
—
12.5
—
—
12.5
—
12.5
reported for similar ruthenium complexes having the same against the same microorganisms under identical experimental
coordination sphere.35 The complex is stabilized by the inter- conditions. However, they behaved as better antibacterial
molecular hydrogen bonding networks with neighboring agents, rather than antifungal agents. The higher antibacterial
molecules. Though one of the carboxylic acid groups O(5) activity of the new ruthenium complexes is due to the chelation
(H-donor) does not take part in coordination, it is involved in of the metal ion, as has been explained by Tweedy’s chelation
intermolecular hydrogen bonding interactions with a neigh- theory.39
boring carboxylate oxygen O(2) (H-acceptor) atom. A water
(b) Among the new complexes tested, complex 4 was found to
molecule forms a hydrogen-bonded bridge, by serving as donor have higher reactivity against the different strains of bacteria
to both O(2) and O(4) of the same complex.
and fungi than complex 3.
(c) Complex 4 exhibited an almost equipotent activity in the
zone of inhibition (29 mm) with the standard drug ciprooxacin
(30 mm) against Enterococcus faecalis and 3 and 4 exhibited
good activity against all the other bacteria tested with the
inhibition zone of 14–29 mm. Complex 3 (17 mm) and 4
(18 mm) exhibited comparable antibacterial activities toward
the inhibition of growth of K. pneumoniae.
(d) The in vitro antifungal evaluation exhibited that the
precursors and the ligand were almost inactive and complexes 3
and 4 showed only a moderate activity. The new complexes 3
and 4 were found to be less active against the yeast C. albicans,
but moderate antifungal activity for both complexes was seen
against the pathogenic mould A. niger.
(e) The antimicrobial activity results displayed by the new
complexes are much better than that reported for other organ-
ometallic ruthenium(II) complexes.38
(f) In general, the deduced patterns of antimicrobial activity
of the newly synthesized organometallic complexes are in the
following order:
2.4. Antimicrobial activity
Infections due to different pathogenic bacteria and fungi are a
dreadful threat to the human race and modern healthcare.
Ruthenium based compounds exert an antimicrobial action
against a range of Gram-negative, Gram-positive bacteria and
fungi. A survey of the literature showed that ruthenium
complexes containing dicarboxylic acids are relatively
unstudied for their potential antimicrobial activity for exploi-
tation in medicine and industry. Here in our study, an attempt
was made to investigate the toxic effects of the ligand, ruthe-
nium precursor complexes and their corresponding newly
synthesized organometallic complexes on the antimicrobial
activity against four different bacterial species and two fungal
strains. As described in the materials and methods, different
concentrations of the compounds were used to identify their
minimum inhibitory concentration on two Gram-positive
bacteria (Staphylococcus aureus, Enterococcus faecalis), two
Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia)
and fungal strains (Aspergillus niger and Candida albicans) by an
agar well diffusion method. The effectiveness of an antimicro-
bial agent in sensitivity testing is based on the size of the
diameter zones of inhibition against Gram-positive, Gram-
negative bacteria and fungal strains. The diameter of the zone
is measured to the nearest millimetre and the data are given in
Table 2 and the minimum inhibitory concentration is repre-
sented in Table 3.
Complex 4 > complex 3 > [RuHCl(CO)(AsPh3)3] (2)
> [RuHCl(CO)(PPh3)3] (1) > H2L
The arsenic containing precursor complex [RuHCl-
(CO)(AsPh3)3] (2) and the corresponding new complex
[Ru(HL)(CH3CN)(CO)(AsPh3)2] (4) were found to be more active
against the pathogens than the phosphine analogues. From
this, it presumed that arsenic plays a major role in the anti-
microbial activity.
Inhibition zones were measured and compared with the
current antimicrobial drugs ciprooxacin (antibacterial) and
uconazole (antifungal). A comparison of the antimicrobial
activities of the free ligand, precursors and the new ruthenium
complexes with standard antibiotics exhibit the following:
2.5. Anticancer activity studies
2.5.1. DNA binding studies. For evaluating the antitumor
(a) The new organometallic ruthenium complexes are more property of any new compound, DNA binding is the predom-
toxic than their parent ligand and the precursor complexes inant property looked for in pharmacology and hence, the
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Fig. 4 Absorption titration spectra of fixed concentration (10 mM) of H2L and complexes 1–4 with increasing concentrations (0–50 mM) of HS
DNA (Tris-HCl buffer, pH 7).
interaction between DNA and metal complexes is of para-
mount importance in understanding the mechanism. Thus,
the mode and propensity for binding of the ligand H2L,
ruthenium(II) starting materials 1, 2 and their corresponding
new ruthenium(II) complexes 3, 4 to HS DNA were studied with
the aid of electronic absorption and luminescence quenching
techniques.
2.5.1.1. Electronic absorption spectra. Electronic absorption
spectroscopy is commonly used technique to determine the
binding strength and binding mode of DNA with complexes.40
Hence, the ligand H2L, ruthenium(II) starting materials 1, 2 and
their corresponding new ruthenium(II) complexes 3, 4 were
subjected to DNA binding studies by taking HS DNA as a model
DNA. In the UV-vis spectra of the ligand and complexes 1–4,
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spectral changes of H2L and complexes 1–4 upon addition of
DNA are shown in Fig. 4. The observed hyperchromic effect with
a blue shi suggested that the new complexes bind to HS DNA
by external contact, possibly due to electrostatic binding.41
In order to compare quantitatively the binding strength of
the compounds, the intrinsic binding constants, Kb, of the
compounds with DNA were obtained by monitoring the changes
in absorbance around 259–269 nm with increasing concentra-
tion of DNA, using the following eqn (1):
Table 4 Binding constant for the interaction of compounds with
HS DNA
System
Kb (Mꢁ1
)
HS DNA + H2L
HS DNA + 1
HS DNA + 2
HS DNA + 3
HS DNA + 4
9.84 ꢂ 104
1.21 ꢂ 104
1.08 ꢂ 104
2.93 ꢂ 105
2.88 ꢂ 105
[DNA]/[3a ꢁ 3f] ¼ [DNA]/[3b ꢁ 3f] + 1/Kb[3b ꢁ 3f]
(1)
wherein, [DNA] is the concentration of DNA in base pairs, 3a, 3f,
and 3b are the apparent, free and bound-metal–complex
extinction coefficients, respectively. Kb is the equilibrium
binding constant of the compound binding to DNA. Each set of
data, when tted to the above equation, gave a straight line with
a slope of 1/(3b ꢁ 3f) and a y-intercept of 1/Kb(3b ꢁ 3f) and Kb was
determined from the ratio of the slope to the intercept (Table 4
and Fig. 5).
From the binding constant values (Table 4), it is inferred that
there are intense interactions of complexes 3, 4 with DNA,
which are stronger than that of H2L and their parent complexes
1 and 2.
2.5.1.2. Ethidium bromide displacement studies. Though
electronic spectral studies indicated the binding of the
compounds with DNA, it has to be conrmed by other methods
and hence, steady-state competitive binding experiments using
compounds H2L, 1, 2, 3 and 4 as quenchers were undertaken to
obtain nal proof for the mode of binding of the compounds to
DNA. For that, ethidium bromide (EB) was used as a uores-
cence probe. In general, the intrinsic uorescence intensity of
DNA is very low and that of EB in Tris-HCl buffer is also not
high, due to quenching by the solvent molecules. However, on
addition of DNA to EB, the uorescence intensity of EB will be
enhanced, because of its intercalation into the DNA. Thus, EB
can be used to probe the interaction of compounds with DNA.
The uorescence intensity of EB can be quenched by the addi-
tion of another molecule, due to decreasing of the binding sites
of DNA available for EB. In our experiment, the EB–DNA system
exhibits a strong emission band at a wavelength around
620 nm. By adding the H2L and complexes 1–4, the uorescence
emission of the EB–DNA system was enhanced rather than
quenched in the emission maximum, shown in Fig. S3 (ESI†),
indicating that they could not displace EB from the DNA–EB
complex. This observation indicates that the compounds bind
weakly to the DNA, probably by an electrostatic interaction.
2.5.2. BSA binding studies
Fig. 5 Binding isotherms of the H2L and complexes 1–4 with HS DNA.
Fig. 6 UV absorption spectra of BSA (1 mM) in the absence and pres-
ence of H2L and complexes 1–4 (10 mM).
absorption bands were observed at 230 and 270 nm. These are
assigned to the predominant p–p* and n–p* transitions,
2.5.2.1. UV absorption spectra of BSA. Serum albumins are
respectively. The electronic spectra of the compounds are major transport proteins42 found in the blood plasma and are
slightly different from each other only in their visible region. In capable of binding, transporting and delivering an extraordi-
order to study the binding of the test compounds with DNA, the narily diverse range of endogenous and exogenous compounds
change in the electronic spectrum was observed by titrating like fatty acids, nutrients, steroids, certain metal ions,
DNA with the test compounds. As the concentration of DNA was hormones and a variety of therapeutic drugs43ꢁ45 in the blood-
increased, the intensity of the absorption bands at 269, 267, stream to their target organs.46 Bovine serum albumin (BSA) has
259, 265 and 262 corresponding to H2L and complexes 1–4 was been one of the most extensively studied proteins, particularly
affected, resulting in hyperchromicity, with a blue shi. The because of its structural homology with human serum albumin
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Fig. 7 The emission spectra of BSA (1 mM; lexc ¼ 280 nm; lemi ¼ 346 nm) in the presence of increasing amounts of H2L and complexes 1–4 (0–35
mM). The arrow shows the emission intensity changes upon increasing complex concentration.
(HSA). Binding to these proteins may lead to the loss or constant at 1 mM and the concentration of H2L and the
enhancement of the biological properties of the original drug, complexes 1–4 was varied from 0–30 mM.
or provide paths for drug transportation. In order to investigate
Fig. 6 shows the absorption spectra of BSA in the absence
the binding of our compounds with BSA, the binding experi- and presence of H2L and complexes 1–4. On adding H2L and
ments were carried out using H2L and the complexes 1–4. In all complexes 1–4 to BSA, a progressive decrease in the absorbance
the experiments, the concentration of the BSA was kept of BSA was observed, except for the ligand and there was a small
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alone, because phenyl alanine has a very low quantum yield and
the uorescence of tyrosine is almost totally quenched if it is
ionized or near an amino group, a carbonyl group, or a trypto-
phan residue.48 Changes in the emission spectra of tryptophan
are common in response to protein conformational transitions,
subunit associations, substrate binding, or denaturation.
Therefore, the intrinsic uorescence of BSA can provide
considerable information on the structure and dynamics and is
oen utilized in the study of protein folding and association
reactions. Hence, the interaction of BSA with our compounds
(H2L and 1–4) was studied by uorescence measurement at
room temperature and the binding constants of the compounds
were calculated. In a typical experiment, a solution of BSA
(1 mM) was titrated with various concentrations of the
compounds (0–35 mM) and the uorescence spectra were
recorded in the range 290–500 nm, upon excitation at 280 nm.
The changes observed on the uorescence emission spectra of
BSA on the addition of increasing amounts of H2L or ruth-
enium(II) complexes 1–4 are shown in Fig. 7. Addition to BSA
produced a dramatic modication of the emission prole. The
uorescence of BSA was quenched effectively with a blue shi in
the emission maximum of H2L, complexes 3, 4 and with a red
shi in the starting complexes 1, 2 and this may be due to the
binding of compounds with the active site in BSA.49
Fig. 8 Plot of I0/I vs. log[Q].
Table 5 Binding constant and number of binding sites for interaction
of complexes with BSA
System
KSV ꢂ Mꢁ1
K ꢂ Mꢁ1
n
BSA + H2L
BSA + 1
BSA + 2
BSA + 3
BSA + 4
1.06 ꢂ 105
1.39 ꢂ 105
5.10 ꢂ 103
2.49 ꢂ 105
2.25 ꢂ 105
1.22 ꢂ 105
1.26 ꢂ 105
1.00 ꢂ 105
2.89 ꢂ 106
3.88 ꢂ 105
1.03
0.96
0.59
1.30
1.05
To study the quenching process further, uorescence
quenching data were analyzed with the Stern–Volmer equation
(2) and Scatchard equation (3). The ratio of the uorescence
intensity in the absence of (I0) and in the presence of (I) the
quencher, is related to the concentration of the quencher [Q] by
a coefficient KSV
.
I0/I ¼ 1 + KSV[Q]
(2)
The KSV value obtained from the plot of I0/I vs. [Q] was found
to be 1.06 ꢂ 105 Mꢁ1, 1.39 ꢂ 105 Mꢁ1, 5.10 ꢂ 103 Mꢁ1, 2.49 ꢂ
105 Mꢁ1 and 2.25 ꢂ 105 Mꢁ1 corresponding to H2L and
complexes 1–4, respectively. The observed linearity in the plots
(Fig. 8 and Table 5) indicated the ability of the complexes to
quench the emission intensity of BSA. From the KSV values, it is
seen that the complexes 3 and 4 exhibited a strong protein-
binding ability, with enhanced hydrophobicity compared to
H2L and their corresponding starting materials.
For the static quenching, when molecules bind indepen-
dently to a set of equivalent sites on a macromolecule, the
binding constant (K) and the number of binding sites (n) can be
determined by the Scatchard equation (3).
Fig. 9 Plot of log[(F0 ꢁ F)/F] vs. log[Q].
ꢀ
ꢁ
blue shi, of about 1, 3, 3, 1 and 3 nm for the ligand and
complexes 1–4, respectively. The observed changes indicate a
static quenching mechanism of BSA by H2L and the complexes
F0 ꢁ F
log
¼ log K þ n log½Qꢃ
(3)
F
(1–4), as the alternative dynamic quenching mechanism does where K is the binding constant of the quencher with BSA, n is
not change the electronic absorption spectrum.47
the number of binding sites, and F0 and F are the uorescence
2.5.2.2. Fluorescence quenching studies of BSA. In order to intensity in the absence and presence of the quencher. The
obtain more information on the binding of the compounds with value of K can be determined from the slope of log[(F0 ꢁ F)/F]
BSA, the uorescence spectrum of BSA was studied upon the versus log[Q], as shown in Fig. 9. The calculated value of the
addition of the test compounds. Though BSA contains three binding constant (K) and the number of binding sites (n) are
uorophores, namely, tryptophan, tyrosine and phenylalanine, listed in Table 5. The values of n at room temperature are
the intrinsic uorescence of BSA is mainly due to tryptophan approximately equal to 1, except for complex 2, which indicates
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Fig. 10 Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of H2L and complexes 1–4 (0–40 mM) for a wavelength
difference of Dl ¼ 60 nm. The arrow shows the emission intensity changes upon increasing concentration of compound.
that there is just one single binding site in BSA for the H2L and and hence, the synchronous uorescence spectra of BSA with
complexes 1, 3 and 4.
the compounds were studied. The uorescence of BSA is mainly
2.5.2.3. Synchronous uorescence spectra. Aer having due to the tryptophan and tyrosine residues. Among them,
obtained the binding constant and binding number of the tryptophan lies in the active site of the protein. When the D
compounds with BSA, it is important to know about the value (Dl) between the excitation and emission wavelength are
conformational change of the protein molecular environment stabilized at 60 nm, the synchronous uorescence gives the
in the vicinity of the uorophore functional groups.50 This can characteristic information of the tryptophan residues.51 The
be obtained from synchronous uorescence spectral studies variation in the tryptophan emission is the consequence of the
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Table 6 Antioxidant and metal chelating activity of the ligand, precursors, new complexes, vitamin C and BHT
IC50 (mM)
Compounds
DPPHc
OHc
NOc
O2ꢁc
H2O2
Metal chelation
H2L
1
2
3
96.3 ꢄ 2.9
199.7 ꢄ 3.4
243.5 ꢄ 4.5
41.6 ꢄ 2.8
37.4 ꢄ 1.9
147.6 ꢄ 4.2
86.2 ꢄ 1.8
129.9 ꢄ 3.6
234.9 ꢄ 1.5
289.8 ꢄ 5.8
54.2 ꢄ 2.3
57.7 ꢄ 0.8
232.8 ꢄ 1.9
163.4 ꢄ 0.7
151.7 ꢄ 0.4
212.2 ꢄ 4.8
256.3 ꢄ 5.9
66.2 ꢄ 1.3
62.9 ꢄ 3.5
215.8 ꢄ 2.7
154.3 ꢄ 2.4
187.3 ꢄ 5.2
267.4 ꢄ 3.6
294.2 ꢄ 4.8
68.1 ꢄ 3.1
67.3 ꢄ 0.9
221.4 ꢄ 1.2
131.6 ꢄ 1.5
144.2 ꢄ 1.7
288.5 ꢄ 3.2
302.2 ꢄ 2.7
135.8 ꢄ 2.0
146.9 ꢄ 4.1
238.5 ꢄ 3.6
149.8 ꢄ 4.3
51.6 ꢄ 2.4
387.9 ꢄ 1.8
413.1 ꢄ 2.3
49.7 ꢄ 0.8
45.2 ꢄ 2.7
—
4
Vitamin C
BHT
—
Fig. 13 Nitric oxide released by the human cancer cell line, A549, after
an incubation period of 48 hours with the complexes 3 (5 mM), 4
(50 mM) and cisplatin (25 mM). Results shown are mean ꢄ SEM, which
are from three separate experiments performed in triplicate.
Fig. 11 The IC50 value (50% inhibition of cell growth for 48 hours) for
complexes 3, 4 and cisplatin on human lung cancer cell line A549.
Results shown are mean ꢄ SEM, which are three separate experiments
performed in triplicate.
added compounds52 and that the complexes bind to the active
site in the protein, which makes them potential molecules for
biological applications. This result prompted us to further
explore the biological activities of the new organometallic
ruthenium(II) complexes.
2.6. Antioxidant activity
The antioxidant properties of ruthenium complexes have
attracted much interest and have been investigated mainly in in
vitro systems, but their radical scavenging activity is limited to
the hydroxyl radical.53 We carried out experiments to explore the
free radical scavenging ability of the new complexes, precursors
and the ligand, against a panel of free radicals, with a hope to
develop potential antioxidants and therapeutic reagents. The
IC50 values of all the precursor complexes, ligand and new
ruthenium complexes for the selected free radicals show inter-
esting results and are presented in Table 6. The IC50 values
Fig. 12 Percentage of lactate dehydrogenase released by human
cancer cell line, A549 after an incubation period of 48 hours with the
complexes 3 (5 mM) and 4 (50 mM) and cisplatin (25 mM). Results shown
are mean ꢄ SEM, which are from three separate experiments per-
formed in triplicate.
protein conformational changes. Therefore, to explore the obtained from the different types of assay experiments strongly
structural change of BSA, we measured the synchronous uo- support the view that the new complexes 3 and 4 presented in
rescence spectra at Dl ¼ 60 nm (Fig. 10) and Dl ¼ 15 nm (Fig. S4 this work possess excellent antioxidant activities, which are
ESI†) of BSA with H2L and the complexes 1–4.
better than those of the standard antioxidants including natural
The results showed that the uorescence of the tyrosine antioxidant, vitamin C and the synthetic antioxidant, BHT. The
residue was hyperchromic, and that of tryptophan residue was ligand displayed an almost comparable radical scavenging
hypochromic. However, the uorescence intensities of the activity with respect to the standard synthetic antioxidant
tryptophan residues changed signicantly, with a red shi, by (BHT), except in the case of the hydroxyl and superoxide anion
the addition of an increasing amount of the compounds. It radicals.
indicates that the hydrophobicity of the microenvironment
The reactivity of free radicals can be neutralized by the
around the tryptophan residues decreases in the presence of the donation of an electron or proton. Lower the IC50 increases the
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hydrogen donating ability and thus the antioxidant activity of crystals, was undertaken. The cells were treated with different
the free radical scavengers. Consistent with the above fact, 3 concentrations of the test compounds. The compounds were
and 4 showed better a radical scavenging activity than the dissolved in DMSO and blank samples containing the same
precursor complexes and the ligand. This might be due to the volume of DMSO were taken as controls, to identify the activity
presence of a free carboxylic acid group in 3 and 4, thus making of the solvent in this cytotoxicity experiment. In parallel, the
those complexes efficient hydrogen donors to stabilize the inuence of the widely used anticancer drug, cisplatin, was
unpaired electrons and thereby scavenging the free radicals. also assayed, as a positive control. The cell viability of the
The appropriate attachment of a COOH group and a metal human lung cancer cells was assessed and the results were
fragment might only be the responsible factor which makes 3 expressed as the % cell viability. It is to be noted that the
and 4 superior to the ligands and the precursor complexes in all ruthenium precursor complexes 1, 2 and the ligand did not
the radical scavenging assays. Out of the six radical species show any signicant activity, even up to a 100 mM concentra-
chosen for examination, the DPPH radical scavenging power of tion, on the A549 cell growth in the MTT assay, LDH release
the tested complexes was the greatest (37.4 ꢄ 1.9 mM) and the and NO release, conrming that the chelation of the ligand
hydrogen peroxide scavenging ability was the least (146.9 ꢄ with the ruthenium(II) ion might be the only responsible factor
4.1 mM). A plausible mechanism for the DPPH scavenging for the observed cytotoxic properties of the new complexes.
activity of the complexes is given in Fig. S5 (ESI†). The DPPH The new complexes exhibited high cytotoxic effects on lung
radical scavenging ability of the new complexes is better than cancer cells with low IC50 values indicating their efficiency in
that of those reported by us for other ruthenium(II) complexes, inhibiting the growth of lung cancer cells even at low
comprising dicarboxylic acids.37
concentrations. The results also showed that the new
The antioxidant capacity of any compound, besides being complexes inhibited A549 cell proliferation in a dose depen-
related with the hydrogen atom transfer reaction, could also be dent manner. The IC50 values were found to be 5 ꢄ 0.29, 50 ꢄ
due to its capacity to chelate metal ions and/or inhibit oxidative 2.39 and 25 ꢄ 1.68 mM for complexes 3, 4 and cisplatin,
enzymes. Some investigators have proposed the participation of respectively. On comparing the IC50 values of the complexes, it
a perferryl complex (ADP–Fe3+–O2ꢁc) in the initiation and was found that 3 was more effective in inducing cell death than
propagation of lipid peroxidation, which also require the pres- 4 (Fig. 11). The IC50 value of the complex 3 was found to be ve
ence of oxygen and free iron.54 So, the presence of molecules times lower than the standard anticancer drug cisplatin, sug-
with the ability to chelate metal ions could reduce the reactive gesting that these complexes can be explored for their poten-
species and, consequently, protect lipid membranes against tial use as medicines for cancer.
peroxidation. Hence, we studied the metal chelating ability of
2.7.2. Lactate dehydrogenase release. LDH is a stable
our compounds with the Fe2+ ion, which showed that the cytoplasmic enzyme that is released into the culture medium,
complexes (3 and 4) have a high metal chelating activity, which following the loss of membrane integrity resulting from
might be due to the chelation by the uncoordinated COOH apoptosis. LDH activity, therefore, can be used as an indicator
group. It is to be noted that no signicant radical scavenging of the cell membrane integrity and serves as a general means to
activities were observed in all the experiments carried out with assess cytotoxicity resulting from chemical compounds or
the ruthenium precursor complexes under the same experi- environmental toxic factors.55,56 Hence, in the present study,
mental conditions. From the above results, it can be concluded LDH leakage into the culture medium of the compounds treated
that the scavenging effects of the precursor complexes and free cells was analyzed. It was observed that the new complexes
ligands is signicantly less when compared to their corre- could signicantly induce the release of LDH into the culture
sponding new ruthenium(II) complexes, which is mainly due to medium, which indicates that the complexes could rupture the
the chelation of the organic ligand with the ruthenium(II) ion. plasma membrane (Fig. 12). Among the compounds examined,
Although the radical scavenging mechanism of the complexes 3 was found to be more potent in inducing LDH leakage into the
under study remains unclear, the experimental results are culture than the rest. Even though there was signicant increase
helpful in designing more effective antioxidant agents against in the LDH leakage in the complex treated cells than the control
free radicals.
cells, they could not induce LDH leakage as high as that of
cisplatin.
2.7.3. Nitric oxide release. A major challenge in the treat-
ment of cancer is improving the response to chemotherapy.
2.7. Cytotoxic activity studies
2.7.1. MTT assay. Since the results of the antioxidant Nitric oxide (NO) has great potential as an agent to augment the
activity experiments revealed that the compounds can exhibit effects of radiation therapy in several cancers. Hence, we
a good radical scavenging activity, we focussed our attention focussed our study on the release of NO by the treatment of our
on the anticancer activity evaluation studies, because it is complexes on A549 cells and the results are shown in Fig. 13.
strongly suspected that cancer may be one of those degener- The results of the NO release assay authenticate the results
ative disease induced by free radicals. Hence, the tumour obtained by MTT and LDH leakage assays indicating that
inhibiting capacity of the compounds against A549 cells (a complex 3 is more effective than the complex 4. Though it is
model cell line for lung cancer) by the MTT assay, that is based slightly lower than that of cisplatin, it is much better than those
on the mitochondrial reduction of the tetrazolium salt by reported for other ruthenium(II) complexes containing
actively growing cells to produce blue insoluble formazan triphenylphosphines.57
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monohydrate was purchased from Acros Organics. The melting
points were determined with a Lab India instrument. The
elemental analyses of carbon, hydrogen and nitrogen were per-
formed on a Vario EL III Elementar elemental analyzer. The
electronic absorption spectra of the compounds were recorded
using a JASCO 600 spectrophotometer and the emission
measurements were carried out by using a JASCO FP-6600
spectrouorometer. A Nicolet Avatar Model FT-IR spectropho-
tometer was used to record the IR spectra (4000–400 cmꢁ1) of the
3. Conclusion
The present contribution describes the synthesis of two new
organometallic ruthenium complexes comprising chelidonic
acid. Both the complexes have been extensively characterized
and their biological activities were compared with the activities
their respective precursor complexes and the ligand. The
dibasic bidentate behavior of the ligand, the metal mediated
C–H activation and ortho metallation along with the molecular
structure of 3 were conrmed by single crystal X-ray crystallo-
graphic studies. Such an ortho metallation is quite surprising in
this case, though an electron rich donor (6 oxygen atoms) is
present in the ligand. The antimicrobial tests showed that the
new complexes 3 and 4 exhibited antimicrobial properties, but
the activity of 4 is better than that of 3 and both were found to be
more active against Gram-positive than Gram-negative bacteria
and fungi. While comparing the binding ability of ligand and
complexes 1–4 with HS DNA/BSA, the new ruthenium(II)
complexes (3 and 4) had a better binding ability than the ligand
and their parent complexes (1 and 2). The phosphine analogues
(1 and 3) showed a greater binding affinity over the arsine
analogues (2 and 4). This might be due to the presence of bulky
AsPh3 groups trans to each other, which causes steric
hindrance. It revealed that the effective binding strength of the
chelating ligand (H2L) towards the double helical DNA would be
reduced by the steric clashes from the six phenyl rings of the
trans AsPh3 with the DNA surface. The steric clash involving the
co-ligands and the DNA polymer would hinder the placing of
the chelating rings in between the DNA helix. These steric
clashes also appeared in the triphenylphosphine complexes,
but the potency was small when compared to the tripheny-
larsine analog. The details obtained from the various antioxi-
dant assays showed that the new complexes, which contain an
uncoordinated COOH group displayed an excellent radical
scavenging ability and the deduced patterns of antioxidant
activity decreased in the following order 3–4 > H2L > 1, 2. The
cytotoxic effects of the compounds examined on A549 cells by
an MTT assay, LDH and NO release demonstrated that complex
3 showed a promising tumour cell growth inhibiting activity,
outperforming that of cisplatin. This signicant activity of 3
might be due to the presence of the phosphine ligands, which is
supposed to provide a better cytotoxicity by enhancing lip-
ophilicity and consequently permeability through the cell
membrane. At this juncture, it is notable to mention that the
cyclometallation reaction presented in this paper took place
under neutral conditions (without auxiliaries).
1
free ligand and the complexes as KBr pellets. The H NMR and
13C NMR spectra were recorded on a Bruker AV 500 (500 MHz
(1H) and 125 MHz (13C)) spectrometer using tetramethylsilane
(TMS) as an internal reference. The chemical shis are expressed
in parts per million (ppm). Protein free herring sperm ds DNA
obtained from SRL Chemicals was stored at 0–4 ꢀC. Bovine
serum albumin (BSA) and ethidium bromide (EB) were obtained
from Sigma-Aldrich. The antioxidant activity measurements
were done using a UV spectrophotometer (UV-1800, Shimadzu).
4.2. Synthesis of complexes
4.2.1. Synthesis of [Ru(HL)(CH3CN)(CO)(PPh3)2] (3). The
ligand 4-oxo-4H-pyran-2,6-dicarboxylic acid (0.021 g, 0.105
mmol) and [RuHCl(CO)(PPh3)3] (0.100 g, 0.105 mmol) were
dissolved in dry CH3CN (20 mL) and reuxed for 6 h. The
resulting solution was allowed to stand for at least one week
under air, until colorless crystals of the target complex 3 were
formed. These were lteꢀred, washed with n-hexane and dried.
Yield: 76%. Mp: 246 C. Anal. Calcd for C46H35NO7P2Ru: C,
63.01; H, 4.02; N, 1.60%. Found: C, 63.12; H, 4.01; N, 1.60%. FT-
IR (cmꢁ1) in KBr: 3436 n(free COOH), 1600 n(free C]OOH), 1617
n(pyrone ring (C]O) + asymm. COO), 1433 n(symm. COO), 1188
n(C–O), 1952 n(C^O), 695, 1090, 1395 n(PPh3). UV-vis (DMSO),
lmax, nm (3, dm3 molꢁ1 cmꢁ1): 229 (77890), 243 (31110), 265
(11200) (intraligand transitions); 370 (1670) (MLCT p*(H2L) )
dp(Ru)). 1H NMR (DMSO-d6, ppm): 11.29 (bs, 1H, –COOH), 7.87
(s, 1H, ]C–H), 2.09 (s, 1H, –CH3), 7.23–7.70 (m, 30H, aromatic
PPh3). 13C NMR (DMSO-d6, ppm): 204.42 (C^O), 119.18 (C^N),
196.57 (C–Ru), 13.62 (CH3), 163.38 (free COOH), 181.91
(COORu), 168.52 (C]C–COO), 130.52, 131.91, 132.00, 132.52
(aromatic PPh3), 172.14 (pyrone ring C]O), 93.57 (C]C–H),
153.44 (C–COOH).
4.2.2. Synthesis of [Ru(HL)(CH3CN)(CO)(AsPh3)2] (4). This
compound was synthesized using the same procedure as
described for 3, with the ligand (H2L) (0.021 g, 0.106 mmol) and
the precursor complex, [RuHCl(CO)(AsPh3)3] (0.115 g, 0.106
mmol). The purity of the complex was checked by TLC ((99 : 5)
chloroform–methanol) and attempts to isolate crystals suitable
for single crystal XRD studies were unsuccessful.
4. Experimental section
4.1. Materials and instrumentation
Yield: 71%. Mp: 227 ꢀC. Anal. Calcd for C46H35NO7As2Ru: C,
All the chemicals were of reagent grade and were used as 57.27; H, 3.66; N, 1.45%. Found: C, 57.31; H, 3.62; N, 1.40%. FT-
received from commercial suppliers, unless otherwise stated. All IR (cmꢁ1) in KBr: 3189 n(free COOH), 1611 n(free C]OOH), 1644
the solvents were degassed and distilled according to standard n(pyrone ring (C]O) + asymm. COO), 1431 n(symm. COO), 1254
procedures.58 Commercially available RuCl3$3H2O (Himedia) n(C–O), 1955 n(C^O), 697, 1086, 1388 n(AsPh3). UV-vis (DMSO),
was used to prepare the starting complexes. The starting lmax, nm (3, dm3 molꢁ1 cmꢁ1): 235 (55460), 246 (53460), 262
complexes [RuHCl(CO)(PPh3)3],59 [RuHCl(CO)(AsPh3)3]60 were (43830) (intraligand transition); 360 (3400) (MLCT p*(H2L) )
prepared as reported earlier. The ligand chelidonic acid dp(Ru)). 1H NMR (DMSO-d6, ppm): 12.32 (bs, 1H, –COOH), 7.79
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(s, 1H, ]C–H), 2.37 (s, 1H, –CH3), 7.17–7.26 (m, 30H, aromatic chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid
AsPh3). 13C NMR (DMSO-d6, ppm): 205.47 (C^O), 121.53 at room temperature. A solution of herring sperm DNA (0.1 g) in
(C^N), 199.41 (C–Ru), 16.87 (CH3), 163.90 (free COOH), 181.35 tris-HCl buffer (10 mL) gave a ratio of UV absorbance at 260
(COORu), 169.87 (C]C–COO), 132.12, 132.21, 132.87, 132.94 and 280 nm of ca. 1.7–1.8 : 1 indicating that the DNA was
(aromatic AsPh3), 172.68 (pyrone ring C]O), 93.99 (C]C–H), sufficiently free of protein. The DNA concentration per nucleo-
153.59 (C–COOH).
tide was determined by absorption spectroscopy, using the
molar absorption coefficient (6600 Mꢁ1 cmꢁ1) at 260 nm. The
compounds were dissolved in a mixed solvent of 5% DMSO and
95% tris-HCl buffer. The absorption spectra were recorded aer
equilibrium at 20 ꢀC for 10 min. The absorption titration
experiments were performed with a xed concentration of the
compounds (10 mM) while gradually increasing the concentra-
tion of DNA (0–50 mM). While measuring the absorption
spectra, an equal amount of DNA was added to both the test
solution and the reference solution, to eliminate the absor-
bance of DNA itself.
In order to know the exact mode of attachment of the
compounds with HS DNA, uorescence quenching experiments
of ethidium bromide (referred to as EB)–DNA complex were also
carried out by increasing the concentration of the ruthenium(II)
complexes (0–50 mM) to the samples containing 10 mM EB,
10 mM DNA, and tris buffer. Before the measurements, the
system was shaken and incubated at room temperature for
ꢅ5 min. The emission was recorded at 530–750 nm. The uo-
rescence spectra in the uorimeter were obtained at an excita-
tion wavelength of 522 nm and an emission wavelength of
620 nm.
4.3. Single crystal X-ray crystallography
The X-ray diffraction measurements were performed on a
Nonius Kappa CCD diffractometer equipped with graphite
monochromated Mo Ka radiation and an Oxford Cryostream
cryostat. The structure of the complex was solved by direct
methods and the renements were carried out by the full matrix
least-squares technique. The hydrogen atoms were generally
visible in the difference maps and were placed in idealized
positions and treated as riding in the renements, except for
those on the water molecule, for which the coordinates were
rened. The crystal was an inversion twin, with A rened Flack
parameter x ¼ 0.67(16). The following computer programs were
used: structure solution SIR-97,61 renement SHELXL-97,62
molecular diagrams and ORTEP-363 for Windows.
4.4. In vitro antimicrobial assay
The synthesized compounds were evaluated for their antimi-
crobial activity by the agar well diffusion method.64,65 The
bacterial pathogens used in the present study included Staph-
ylococcus aureus, Enterococcus faecalis, Escherichia coli and
Klebsiella pneumonia and the fungi Aspergillus niger and Candida
albicans, which were procured from the PSG Hospital, Coim-
batore, India. The required nutrient broth and sabouraud
dextrose broth were prepared and sterilized at 121 ꢀC. The
bacterial strains were inoculated onto nutrient broth (108 cells
per mL) and fungal strains were inoculated onto sabouraud
dextrose broth (10 spores per mL) and incubated overnight.
About 30 mL of a sterilized agar medium were transferred
aseptically in to each sterilized petri plate. The plates were le at
room temperature for solidication. The overnight grown
bacterial cultures and fungal spores were swabbed onto the
solidied media to achieve a lawn of conuent bacterial/fungal
growth. A well of 6 mm diameter was made using a sterile cork
borer. The test drugs were added at concentrations of
0 (control), 25, 50, 75, 100 mg mLꢁ1. Ciprooxacin and uco-
nazole were used as positive control drugs for the antibacterial
and antifungal activities, respectively. The antibacterial assay
4.6. Protein binding studies
The binding of the compounds with bovine serum albumin
(BSA) was studied from the uorescence spectra recorded with
an excitation wavelength of 280 nm and the corresponding
emission at 345 nm, assignable to that of BSA. The excitation
and emission slit widths and scan rates were maintained
constant for all of the experiments. A stock solution of BSA was
prepared in 50 mM phosphate buffer (pH ¼ 7.2) and stored in
the dark at 4 ꢀC for further use. A concentrated stock solution of
the compounds was prepared as mentioned for the DNA
binding experiments, except that the phosphate buffer was used
instead of a Tris-HCl buffer for all of the experiments. The
titrations were manually done by using a micropipette for the
addition of the compounds. For the synchronous uorescence
spectra also, the same concentrations of BSA and the
compounds were used, and the spectra were measured at two
different Dl values (difference between the excitation and
emission wavelengths of BSA), such as 15 and 60 nm.
ꢀ
plates were incubated at 37 ꢄ 2 C for 24 h and the antifungal
assay plates were incubated at 28 ꢄ 2 ꢀC for 48 h. Aer the
incubation period, the diameter of the inhibition zone was
measured as an indicator for the activity of the compounds. 4.7. Antioxidant assays
Dimethyl sulphoxide was used both as a solvent and as a
negative control. No inhibition zone was observed in the control
(i.e. for DMSO). Each experiment was performed in triplicate.
The ability of the ligand and ruthenium complexes to act as
hydrogen donors or free radical scavengers was tested by con-
ducting a series of in vitro antioxidant assays, involving the
DPPH radical, hydroxyl radical, nitric oxide radical, hydrogen
peroxide, superoxide anion radical, metal chelating assay and
4.5. DNA interaction studies
All of the experiments involving the binding of the compounds the results were compared with that of standard antioxidants
with HS DNA were carried out in deionised water with including natural antioxidant vitamin C and the synthetic
tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium antioxidant BHT.
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4.7.1. DPPHc scavenging assay. The DPPH radical scav-
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spectrophotometrically from the absorption at 230 nm with a
molar absorptivity of 81 Mꢁ1cmꢁ1. The compounds (100 mg
mLꢁ1), BHT and vitamin C (100 mg mLꢁ1) were added to 3.4 mL
of a phosphate buffer prepared above, together with a hydrogen
peroxide solution (0.6 mL). An identical reaction mixture
without the sample was taken as a negative control. The
absorbance of hydrogen peroxide at 230 nm was determined
aer 10 min against the blank (phosphate buffer).
enging activity of the compounds was measured according to
the method of Blois.66 The DPPH radical is a stable free radical
and due to the presence of an odd electron, it shows a strong
absorption band at 517 nm in the visible spectrum. If this
electron becomes paired off in the presence of a free radical
scavenger, this absorption vanishes resulting in decolorization
stoichiometrically with respect to the number of electrons taken
up. Various concentrations of the experimental compounds
were taken and the volumes were adjusted to 100 mL with
methanol. About 5 mL of a 0.1 mM methanolic solution of
DPPH was added to the aliquots of samples and standards (BHT
and vitamin C) and shaken vigorously. A negative control was
prepared by adding 100 mL of methanol in 5 mL of 0.1 mM
methanolic solution of DPPH. The tubes were allowed to stand
for 20 minutes at 27 ꢀC. The absorbance of the sample was
measured at 517 nm against the blank (methanol).
4.7.2. OHc scavenging assay. The hydroxyl radical scav-
enging activity of the compounds was investigated using the
Nash method.67 In vitro hydroxyl radicals were generated by an
Fe3+–ascorbic acid system. The detection of the hydroxyl radicals
was carried out by measuring the amount of formaldehyde
formed from the oxidation reaction with DMSO. The formalde-
hyde produced was detected spectrophotometrically at 412 nm.
In a typical experiment, a mixture of 1.0 mL of iron–EDTA
solution (0.13% ferrous ammonium sulfate and 0.26% EDTA),
0.5 mL of EDTA solution (0.018%), and 1.0 mL of DMSO (0.85%
DMSO (v/v) in 0.1 M phosphate buffer, pH 7.4) were sequentially
added in the test tubes, which contained a xed concentration
of the test compounds. The reaction was initiated by adding
0.5 mL of ascorbic acid (0.22%) and was incubated at 80–90 ꢀC
for 15 min in a water bath. Aer incubation, the reaction was
terminated by the addition of 1.0 mL of ice-cold trichloroacetic
acid (17.5% w/v). Subsequently, 3.0 mL of the Nash reagent was
added to each tube and le at room temperature for 15 min. The
intensity of the colour formed was measured spectrophoto-
metrically at 412 nm against the reagent blank.
4.7.5. O2ꢁcscavenging assay. The superoxide anion radical
(O2ꢁc) scavenging assay is based on the capacity of the
compounds to inhibit formazan formation by scavenging the
superoxide radicals generated in the riboavin–light–NBT
system.70 In a typical experiment, a 3 mL reaction mixture
contained 50 mM sodium phosphate buffer (pH 7.6), 20 mg
riboavin, 12 mM EDTA, 0.1 mg NBT and 1 mL complex solu-
tion (20–100 mg mLꢁ1). The reaction was started by illuminating
the reaction mixture with different concentrations of the
complex for 90 s. Immediately aer illumination, the absor-
bance was measured at 590 nm. The entire reaction assembly
was enclosed in a box lined with aluminium foil. Identical tubes
with the reaction mixture kept in the dark served as blanks.
4.7.6. Metal chelating activity. The chelation with ferrous
ions by the experimental compounds was estimated by the
method of Dinis et al.71 Initially, about 100 mL of the samples
and the standards were added to a 50 mL solution of 2 mM
FeCl2. The reaction was initiated by the addition of 200 mL of
5 mM ferrozine and the mixture was shaken vigorously and le
standing at room temperature for 10 minutes. The absorbance
of the solution was then measured spectrophotometrically at
562 nm against the blank (deionized water).
For the above six assays, all the tests were run in triplicate
and the percentage activity was calculated using the following
equation:
Scavenging activity (%) ¼ [(A0 ꢁ A1)/A0] ꢂ 100
where A0 is the absorbance of the control, A1 is the absorbance
of the complex/standard. When the inhibition of the tested
4.7.3. NOc scavenging assay. The assay of nitric oxide (NOc)
scavenging activity is based on the method,68 where sodium
nitroprusside in an aqueous solution at a physiological pH
spontaneously generates nitric oxide, which interacts with
oxygen to produce nitrite ions. This can be estimated using the
Griess reagent. Scavengers of nitric oxide compete with oxygen,
leading to a reduced production of nitrite ions. For the experi-
ment, sodium nitroprusside (10 mM) in phosphate buffered
saline was mixed with a xed concentration of the compounds,
standards and incubated at room temperature for 150 min.
Aer the incubation period, 0.5 mL of the Griess reagent con-
taining 1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl)
ethylenediaminedihydrochloride was added. The absorbance of
the chromophore formed was measured at 546 nm.
compounds is 50%, the tested compound concentration is IC50
.
4.8. Anticancer activity studies
4.8.1. MTT assay. The effects of the compounds on the
viability of human lung cancer cells (A549) was assayed by the
3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazoliumbromide
(MTT) assay.72 The cells were seeded at a density of 10 000 cells
per well in 200 mL DMEM (Dulbecco’s Modied Eagle’s
Medium) and were allowed to attach overnight in a CO2 incu-
bator. Then the ligand and the complexes dissolved in DMSO
were added to the cells at a nal concentration of 1, 10, 25 and
50 mM in the cell culture media. Aer 48 h, 20 mL of MTT (5 mg
mLꢁ1 PBS) was added and incubated at 37 ꢀC for 4 h. The purple
formazan crystals formed were dissolved in 200 mL of DMSO
and read at 570 nm in a micro plate reader.
4.7.4. H2O2 scavenging assay. The ability of the compounds
to scavenge hydrogen peroxide was determined using the
method of Ruch et al.69 In a typical experiment, a solution of
hydrogen peroxide (2.0 mM) was prepared in a phosphate buffer
(0.2 M, pH-7.4) and its concentration was determined
4.8.2. Release of lactate dehydrogenase (LDH). The LDH
activity was determined by the linear region of a pyruvate
standard graph, using regression analysis and expressed as the
percentage (%) leakage, as described previously.73 Briey, to a
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set of tubes, 1 mL of buffered substrate (lithium lactate) and
0.1 mL of the media or cell extract were added and the tubes
were incubated at 37 ꢀC for 30 min. Aer adding 0.2 mL of NAD
(nicotinamide adenine dinucleotide) solution, the incubation
was continued for another 30 min. The reaction was then
7 L. Ackermann and R. Vicente, Top. Curr. Chem., 2010, 292,
211–229.
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15 min at 37 C. Aer this, 0.1 mL of media or cell extract was 11 C. L. Sun, B. J. Li and Z. J. Shi, Chem. Rev., 2011, 111, 1293–
4109.
ꢀ
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Acknowledgements
The Department of Science and Technology, New Delhi for
funds for the research and the Council of Scientic and
Industrial Research, New Delhi, India, for the award of a Senior
Research Fellowship to T. Sathiya Kamatchi are gratefully
acknowledged. We would also like to thank Mr T. Sajeesh and
Dr T. Parimelazhagan, Department of Botany, Bharathiar
University, India for their help in the radical scavenging assays.
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