Ruthenium(III) S-methylisothiosemicarbazone Schiff base complexes bearing PPh3/AsPh3 coligand: Synthesis, structure and biological investigations, including antioxidant, DNA and protein interaction, and in vitro anticancer activities

Article abstract of DOI:10.1016/j.jphotobiol.2014.04.019

New Ru(III) isothiosemicarbazone complexes [RuCl(EPh₃)L ^1-4] (E = P or As) were obtained from the reactions between [RuCl₃(EPh₃)₃] and bis(salicylaldehyde)-S-methylisothiosemicarbazone (H₂L ^1-3)/bis(2-hydroxy-naphthaldehyde)-S- methylisothiosemicarbazone (H₂L⁴) ligands. The new complexes were characterized by using elemental analyses and various spectral (UV-Vis, IR, ¹H NMR, FAB-Mass and EPR) methods. The redox properties of the complexes were studied by using cyclic voltammetric method. The new complexes were subjected to various biological investigations such as antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical and hydrogen peroxide, DNA/protein interaction studies and in vitro cytotoxic studies against human breast cancer cell line (MCF-7). New complexes showed excellent free radicals scavenging ability and could bind with DNA via intercalation. Protein binding studies using fluorescence spectroscopy showed that the new complexes could bind strongly with bovine serum albumin (BSA). Photo cleavage experiments using DNA of E-coli bacterium exhibited the DNA cleavage ability of the complexes. Further, the in vitro anticancer activity studies on the new complexes against MCF-7 cell line exhibited the ability of Ru(III) isothiosemicarbazone complexes to suppress the development of malignant neoplastic disease cells.

Full text of DOI:10.1016/j.jphotobiol.2014.04.019

Accepted Manuscript
Ruthenium(III) S-methylisothiosemicarbazone Schiff base complexes bearing
PPh3/AsPh3 coligand: Synthesis, structure and biological investigations, includ-
ing antioxidant, DNA and protein interaction, and in-vitro anticancer activities
Govindan Prakash, Rajendran Manikandan, Periasamy Viswanathamurthi,
Krishnaswamy Velmurugan, Raju Nandhakumar
PII:
S1011-1344(14)00127-4
DOI:
http://dx.doi.org/10.1016/j.jphotobiol.2014.04.019
JPB 9728
Reference:
Journal of Photochemistry and Photobiology B: Bi-
ology
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 March 2014
16 April 2014
21 April 2014
Please cite this article as: G. Prakash, R. Manikandan, P. Viswanathamurthi, K. Velmurugan, R. Nandhakumar,
Ruthenium(III) S-methylisothiosemicarbazone Schiff base complexes bearing PPh3/AsPh3 coligand: Synthesis,
structure and biological investigations, including antioxidant, DNA and protein interaction, and in-vitro anticancer
activities, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/
j.jphotobiol.2014.04.019
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Ruthenium(III) S-methylisothiosemicarbazone Schiff base complexes bearing
PPh3/AsPh3 coligand: Synthesis, structure and biological investigations,
including antioxidant, DNA and protein interaction, and in-vitro anticancer
activities
Govindan Prakasha, Rajendran Manikandana, Periasamy Viswanathamurthia*,
Krishnaswamy Velmuruganb, Raju Nandhakumarb
aDepartment of Chemistry, Periyar University, Salem-636011, Tamilnadu, India
bDepartment of Chemistry, Karunya University, Karunya nagar, Coimbatore 641 114, India.
* Corresponding author. Fax: +91 427 2345124.
E-mail address: viswanathamurthi72@gmail.com (P. Viswanathamurthi)
1

ABSTRACT
New Ru(III) isothiosemicarbazone complexes [RuCl(EPh3)L1-4] (E = P or As) were obtained
from
the
reactions
between
[RuCl3(EPh3)3]
/
and bis(salicylaldehyde)-S-
methylisothiosemicarbazone
(H2L1-3)
bis(2-hydroxy-naphthaldehyde)-S-
methylisothiosemicarbazone (H2L4) ligands. The new complexes were characterized by using
1
elemental analyses and various spectral (UV-Vis, IR, H NMR, FAB-Mass and EPR) methods.
The redox properties of the complexes were studied by using cyclic voltammetric method. The
new complexes were subjected to various biological investigations such as antioxidant assays
involving DPPH radical, hydroxyl radical, nitric oxide radical and hydrogen peroxide,
DNA/protein interaction studies and in-vitro cytotoxic studies against human breast cancer cell
line (MCF-7). New complexes showed excellent free radicals scavenging ability and could bind
with DNA via intercalation. Protein binding studies using fluorescence spectroscopy showed that
the new complexes could bind strongly with bovine serum albumin (BSA). Photo cleavage
experiments using DNA of E-coli bacterium exhibited the DNA cleavage ability of the
complexes. Further, the in-vitro anticancer activity studies on the new complexes against MCF-7
cell line exhibited the ability of Ru(III)isothiosemicarbazone complexes to suppress the
development of malignant neoplastic disease cells.
Keyword:
S-methylisothosemicarbazones
Ru(III) Schiff base complexes
Spectral studies, Free radical scavenging
DNA/protein interaction
Anticancer studies
2

1. Introduction
The alchemy of metal complexes offers enormous opportunities for the design and
development of compounds with excellent bioactivities due to the assortment of available metals
and the ability to tune the reactivity and structure of the metal complexes by their ligand sphere
[1,2]. The biological activities of the metal complexes are widely influenced by the nature of
metal ions, its oxidation state, the type and nature of bound ligands and isomers [3-5]. Hence, the
understanding of the factors affecting the biological activity of metal complexes should enable
the design of metal complexes with specific medicinal properties and also to overcome the
disadvantages of available medicines. For example, Cisplatin, a platinum(II) diamine complex
used in 70% of cancer treatment has some drawbacks like toxic side-effects and lack of activity
(drug resistance) against several types of cancer which are problems need to be overcome [6].
So, the search for anticancer activity amongst complexes of other metals has received much
interest. Currently, ruthenium complexes have been found as an attractive alternate for platinum
due to several favorable properties suited to rational anticancer drug design and biological
applications [7-9] such as their bio-compatible ligands exchange kinetics similar to platinum, a
higher coordination number, lower toxicity towards healthy tissues than their platinum counter
parts etc [10,11].
Experiments showed that the DNA interaction studies of small molecules can be used to
design more effective anticancer drugs [12,13]. Considerable interest has been generated in DNA
binding and DNA cleavage by metal complexes with redox and photoactive nature, in order to
explore the sequence specificities of DNA binding using a variety of intercalating ligands [14-
16]. In general, small molecules bind to DNA in non-covalent modes such as intercalation,
groove-binding, and external electrostatic binding [17,18] and the Intercalation ability of metal
complexes correlates to coordination geometry, metal ion type, its valence and the ligand donor
type [19,20]. The intercalation binding of metal complexes with DNA could induce cellular
degradation which is indispensable for the anticancer application of metal complexes [21].
Likewise, knowledge of interaction mechanisms between the drugs and plasma proteins is very
important to understand the pharmacodynamics and pharmacokinetics of a drug [22]. Serum
albumin is the most abundant soluble protein in animals, including the human circulatory system
that is in-charge for the transport, distribution and deposition of a sort of endogenous and
exogenous substances in the body [23]. This protein possesses a high homology and a similarity
3

to human serum albumin (HSA) in sequence and conformation [24] and is always chosen as a
model of general drug–protein interactions. Further, the uncontrolled production of free radicals
or reactive oxygen species such as OH• and hydrogen peroxide causes DNA damages in human
and results several diseases including cancer [25,26]. Various ruthenium complexes were
reported for their free radical scavenging ability against different harmful free radicals [27-30].
Thiosemicarbazones, their derivatives, as well as their transition metal complexes have
aroused considerable interest in the areas of chemistry and biology. These compounds present a
wide variety of biological activities such as antitumor [31-35], fungicidal [36,37], bactericidal
[38] or antiviral [33] and so on. The thiosemicarbazide fragment =N(1)-N(2)H-C(3)=S-N(4)-
NH2 is a bifunctional chelating agent, and can be coordinated in a bidentate mode to give five-
membered metal rings through the N(1) and S atoms (mode 1) or the N(1) and N(4) atoms (mode
2). The coordination of the thiosemicarbazide fragment of the ligand via mode 1 was found in
complexes of both 3d transition metals and platinum-group metals, namely, Pt(II) and Pd(II)
[39,40]. In some cases where this fragment is attached to a metal through the N(2) and S atoms
to create a four-membered metal ring was also reported [41]. It is significant to note that one
bond in all known structures is always through the sulfur atom [39-42]. Nevertheless the unusual
mode 2 is exceptionally observed in some molybdenum (VI) complexes. Thioalkylated
derivatives of this class are rather reactive; participate in interesting metal-promoted reactions,
which give rise to new sequences of atom and bond combinations and to unusual coordination
modes. Particularly, for S -methylisothiosemicarbazone of salicylaldehyde (H2L), it was shown
that thioalkylation results in both a change in the coordination mode from O,N,S to O,N,N and
modification of the acid–base properties of the thiosemicarbazide fragment.
As a part of our continuing research in ruthenium chemistry, here we have synthesized a
series of hexacoordinated ruthenium(III) S-methylisothiosemicarbazone complexes containing
triphenylphophine/triphenylarsine as co-ligands and characterized them by using elemental and
spectral methods (UV-Vis, IR, FAB-Mass and EPR). The radical scavenging ability (DPPH•,
OH•, NO•, H2O2), DNA and Protein interactions and the anticancer property against the MCF-7
cell line of the new complexes were investigated.
4

2. Experimental
2.1. Materials and Methods
All the reagents used were chemically pure and AR grade. The solvents were purified and
dried according to standard procedures [43]. RuCl3.H2O was purchased from Loba Chemie Pvt
Ltd. The starting complexes [RuCl3(PPh3)3] & [RuCl3(AsPh3)3], S-methyl thiosemicarbazide and S-
methylisothiosemicarbazone Schiff bases were prepared according to the literature procedures[44-47].
2.2. Physical measurements
Microanalyses (C, H, N, & S) were carried out on Vario EL III Elemental analyzer at
SAIF, Cochin, India. The IR spectra of the ligands and their complexes were recorded as KBr
pellets on a Nicolet Avatar model FT-IR spectrometer in 4000 - 400 cm-1 range. Electronic
spectra of the ligands and their complexes have been recorded in dichloromethane using a
1
Shimadzu UV - 1650 PC spectrophotometer at 800 - 200 nm range. H NMR spectra were
recorded in Jeol GSX - 400 nuclear magnetic resonance spectrometer using TMS as internal
reference. Electron paramagnetic resonance spectra (EPR) of the powdered samples were
recorded with a JEOL JES-FA200 electron spin resonance spectrometer with X-band frequencies
at room temperature using 2, 2’ - diphenyl-1-picrylhydrazyl radical (DPPH) as internal standard
at Pondicherry university, Pondicherry, India. The FAB-MS spectra were recorded by EI
technique using the JEOL JMS600H instrument in the HRMS facility NIIST,
Thiruvananthapuram. Magnetic moments were measured using an EG & G-PARC vibrating
sample magnetometer. Cyclic voltammetric measurements were carried out with a BAS CV-27
electrochemical analyzer in acetonitrile solution using tetrabutyl ammonium perchlorate (TBAP)
as supporting electrolyte. Three electrode cell was employed with glassy carbon working
electrode, a platinum wire as counter electrode and an Ag/AgCl as a reference electrode. Melting
points were recorded on a Technico micro heating table and are uncorrected.
2.3. Preparation of S-methylisothiosemicarbazone ligands
A mixture of thiosemicarbazide (10 mmol) and methyl iodide (10.6 mmol) in absolute
ethanol (20 mL) was refluxed for 45 min and allowed to cool at room temperature for 12 h.
White crystals of S-methylthiosemicarbazide hydrogen iodide separated which was filtered,
washed with ethanol and used for the next step without further purification. Yield: 68%. In the
5

second step, a mixture of S-methylthiosemicarbazide hydrogen iodide (5 mmol) and
corresponding aldehyde (10 mmol) in absolute ethanol was neutralized with an aqueous
Na2CO3.10H2O solution and kept overnight. The pale yellow crystals of the ligands obtained
were filtered off and washed with water and finally with ethanol. Yield: 85-90%.
2.3.1. Bis(salicylaldehyde) S-methylisothiosemicarbazone (H2L1)
H2L1 was prepared from the reaction of S-methylthiosemicarbazide (1.165 g, 5 mmol)
with salicylaldehyde (1.221 g, 10 mmol). Yield: 68%. Color: Yellow solid. M.p: 135 oC. Anal.
Calc (found). (C16H15N3O2S): C, 61.32(61.46); H, 4.82(4.71); N, 13.41(13.21); S, 10.23(10.18).
IR (KBr, cm-1): 3265(b) ν(O-H1); 3280 ν(O-H2); 1617(s) ν(C=N1); 1635(s) ν(C=N2); 1377 ν (C-
O). UV-Vis (λMax, nm (εmax, M-1 cm-1): 344(22,540), 242(48,763). 1H NMR (DMSO-d6, , ppm):
11.56, 10.83 (s, 1H each, -OH), 8.31(s, 1H, -CH=N1), 8.44 (s, 1H, -CH=N2), 7.54, 7.38, 7.25,
6.90(m, 1H each, Aromatic)
2.3.2. Bis(5-chloro-salicylaldehyde) S-methylisothiosemicarbazone (H2L2)
H2L2 was prepared from the reaction of S-methylthiosemicarbazide (1.165 g, 5 mmol)
with 5-chlorosalicylaldehyde (1.556 g, 10 mmol) Yield: 73%. Color: Yellow solid. M.p: 147 oC.
Anal. Calc (found). (C16H13N3O2SCl2): C, 50.27(50.12); H, 3.43(3.15); N, 10.99(10.74); S,
8.39(8.14). IR (KBr, cm-1): 3270(b) ν(O-H1); 3283 ν(O-H2); 1660(s) ν(C=N1); 1681(s) ν(C=N2);
1
1377 ν (C-O). UV-Vis (λMax, nm (εmax, M-1 cm-1): 334(22,797), 263(51,564). H NMR (DMSO-
d6, , ppm): 11.63, 10.75 (s, 2H each, -OH), 8.32 (s, 1H, -CH=N1), 8.42 (s, 1H, -CH=N2), 7.78,
7.52, 7.22, 6.88 (m, 1H each, Aromatic)
2.3.3. Bis(o-vanilin) S-methylisothiosemicarbazone (H2L3)
H2L3 was prepared from the reaction of S-methylthiosemicarbazide (1.165 g, 5 mmol)
o
with o-vanilin (1.521 g, 10 mmol) Yield: 61%. Color: Yellow solid. M.p: 186 C. Anal. Calc
(found). (C18H19N3O4S): C, 57.89(57.66); H, 5.13(5.11); N, 11.25(11.07); S, 8.59(8.16). IR (KBr,
cm-1): 3220(b) ν(O-H1); 3252 ν(O-H2); 1665(s) ν(C=N1); 1686(s) ν(C=N2); 1355 ν (C-O). UV-
1
Vis (λMax, nm (εmax, M-1 cm-1): 338(16,753), 262(40,867). H NMR (DMSO-d6, , ppm): 11.22,
10.76 (s, 1H each, -OH), 8.23 (s, 1H, -CH=N1), 8.56 (s, 1H, -CH=N2), 7.87,7.64, 7.23, 6.90 (m,
1H each, Aromatic), 3.67 (s, 3H, -OCH3)
6

2.3.4. Bis(2-hydroxynapthaldehyde) S-methylisothiosemicarbazone (H2L4)
H2L4 was prepared from the reaction of S-methylthiosemicarbazide (1.165 g, 5 mmol)
with 2-hydroxynapthaldehyde (1.722 g, 10 mmol) Yield: 61%. Color: Yellow solid. M.p: 194 oC.
Anal. Calc (found). (C24H19N3O2S): C, 69.71(69.88); H, 4.63(4.24); N, 10.16(10.10); S,
7.75(7.34). IR (KBr, cm-1): 3222(b) ν(O-H1); 3312 ν(O-H2); 1621(s) ν(C=N1); 1651(s) ν(C=N2);
1365 ν (C-O). UV-Vis (λMax, nm (εmax, M-1 cm-1): 310(18,285), 250(40, 564). 1H NMR (DMSO-
d6, , ppm): 12.99, 12.67 (s, 1H each, -OH), 9.19 (s, 1H, -CH=N1), 9.28 (s, 1H, -CH=N2), 8.35,
8.28, 7.90, 7.55, 7.39, 7.22 (m, 1H each, Aromatic)
2.4. Synthesis of new ruthenium(III) S-methylisothiosemicarbazone complexes
To a solution of 1 mmol [RuCl3(EPh3)3] (E = P or As) in benzene was added 1 mmol
ligand (1:1 mole ratio) and the mixture was refluxed for 6 h. The reaction mixture was reduced to
2 mL on a water bath and the product was separated by the addition of a small amount of
petroleum ether at room temperature. The resulting complexes were recrystallized from
CH2Cl2/petroleum ether and dried under vacuum. The overall yield obtained for all the
complexes were 70-78 %.
2.4.1. [RuCl(PPh3)L1] (1)
Complex 1 was prepared from [RuCl3(PPh3)3] (0.497 g, 0.5 mmol) and H2L1 (0.157 g, 0.5
mmol) ligand. Yield: 72%; Color: Green solid; M.p: 212oC; Anal. Calc (found).
(RuC34H28N3O2SPCl): C, 57.50(57.32); H, 3.97(3.86); N, 5.92(5.72); S, 4.52(4.26). IR (KBr, cm-
1): 1598(s) ν(C=N1); 1614(s) ν(C=N2); 1397 ν (C-O). UV-Vis (λMax, nm (εmax, M-1 cm-1)):
496(12,198), 315 (18,167), 316(45,620). Magnetic moment (µeff, BM): 1.81. FAB-MS, m/z calc
(found): 710.1 (711.3) [M+H]+..
2.4.2. [RuCl(PPh3)L2] (2)
Complex 2 was prepared from [RuCl3(PPh3)3] (0.497 g, 0.5 mmol) and H2L2 (0.191 g, 0.5
mmol) ligand. Yield: 76%; Color: Green solid; M.p: 206oC; Anal. Calc (found).
(RuC34H26N3O2SPCl3): C, 52.42(52.48); H, 3.36(3.12); N, 5.39(5.14); S, 4.12(4.15). IR (KBr,
cm-1): 1599(s) ν(C=N1); 1647(s) ν(C=N2); 1384 ν (C-O). UV-Vis (λMax, nm (εmax, M-1 cm-1)):
486(8,900), 327(16,174), 240(32,679). Magnetic moment (µeff, BM): 1.70. FAB-MS, m/z calc
7

(found): 779.0 (779.6) [M+]
2.4.3. [RuCl(PPh3)L3] (3)
Complex 3 was prepared from [RuCl3(PPh3)3] (0.497 g, 0.5 mmol) and H2L3 (0.186 g, 0.5
mmol) ligand. Yield: 68%; Color: Green solid; M.p: 214oC; Anal. Calc (found).
(RuC36H32N3O4SPCl): C, 56.14(56.02); H, 4.19(4.11); N, 5.46(5.40); S, 4.16(4.03). IR (KBr, cm-
1): 1658(s) ν(C=N1); 1674(s) ν(C=N2); 1394 ν (C-O). UV-Vis (λMax, nm (εmax, M-1cm-1)):
508(7,682), 333(16,890), 247(38,781). Magnetic moment (µeff, BM): 1.81. FAB-MS, m/z calc
(found): 770.2 (771.3) [M+H]+.
2.4.4. [RuCl(PPh3)L4] (4)
Complex 4 was prepared from [RuCl3(PPh3)3] (0.497 g, 0.5 mmol) and H2L4 (0.207 g, 0.5
mmol) ligand. Yield: 70%; Color: Green solid; M.p: 198oC; Anal. Calc (found).
(RuC42H32N3O2SPCl): C, 62.26(62.36); H, 3.36(3.31); N, 5.39(5.47); S, 4.12(4.17). IR (KBr, cm-
1): 1624(s) ν(C=N1); 1642(s) ν(C=N2); 1400 ν (C-O). UV-Vis (λMax, nm (εmax, M-1cm-1)):
516(6,720), 320(15,616), 235(28,197). Magnetic moment (µeff, BM): 1.68. FAB-MS, m/z calc
(found): 810.2 (810.9) [M+].
2.4.5. [RuCl(AsPh3)L1] (5)
Complex 5 was prepared from [RuCl3(AsPh3)3] (0.563 g, 0.5 mmol) and H2L1 (0.157 g,
0.5 mmol) ligand. Yield: 74%; Color: Brown solid; M.p: 190oC; Anal. Calc (found).
(RuC34H28N3O2SAsCl): C, 54.15(53.47); H, 3.74(3.27); N, 5.57(5.18); S, 4.25(4.10). IR (KBr,
cm-1): 1603(s) ν(C=N1); 1617(s) ν(C=N2); 1391 ν (C-O). UV-Vis (λMax, nm (εmax, M-1cm-1)):
484(10,540), 317(20,375), 297(42,569). Magnetic moment (µeff, BM): 1.75. FAB-MS, m/z calc
(found): 751.9 (751.0) [M+].
2.4.6. [RuCl(AsPh3)L2] (6)
Complex 6 was prepared from [RuCl3(AsPh3)3] (0.563 g, 0.5 mmol) and H2L2 (0.191 g,
0.5 mmol) ligand. Yield: 81%; Color: Brown solid; M.p: 212oC; Anal. Calc (found).
(RuC34H26N3O2SAsCl3): C, 49.62(49.42); H, 3.18(3.33); N, 5.11(5.02); S, 3.90(3.66). IR (KBr,
cm-1): 1644(s) ν(C=N1); 1659(s) ν(C=N2); 1394 ν (C-O). UV-Vis (λMax, nm (εmax, M-1cm-1)):
8

482(8,916), 335(17,916), 262(48,200). Magnetic moment (µeff, BM): 1.72. FAB-MS, m/z calc
(found): 823.9(824.8) [M+H]+
2.4.7. [RuCl(AsPh3)L3] (7)
Complex 7 was prepared from [RuCl3(AsPh3)3] (0.563 g, 0.5 mmol) and H2L3 (0.186 g,
0.5 mmol) ligand. Yield: 70%; Color: Brown solid; M.p: 234oC; Anal. Calc (found).
(RuC36H32N3O4SAsCl): C, 53.11(53.04); H, 3.96(3.66); N, 5.16(5.24); S, 3.94(3.67). IR (KBr,
cm-1): 1642(s) ν(C=N1); 1659(s) ν(C=N2); 1395 ν (C-O). UV-Vis (λMax, nm (εmax, M-1cm-1)):
505(11,400), 275(15,990), 242(30,168). Magnetic moment (µeff, BM): 1.86. FAB-MS, m/z calc
(found): 814.1 (815.5) [M+H]+
2.4.8. [RuCl(AsPh3)L4] (8)
Complex 8 was prepared from [RuCl3(AsPh3)3] (0.563 g, 0.5 mmol) and H2L4 (0.207 g,
0.5 mmol) ligand. Yield: 78%; Color: Brown solid; M.p: 231oC; Anal. Calc (found).
(RuC42H32N3O2SAsCl): C, 59.05(59.11); H, 3.78(3.62); N, 4.92(4.76); S, 3.75(3.51). IR (KBr,
cm-1): 1632(s) ν(C=N1); 1648(s) ν(C=N2); 1395 ν (C-O). UV-Vis (λMax, nm (εmax, M-1cm-1)):
486(10,673), 310(16,832), 236(40,142). Magnetic moment (µeff, BM): 1.58. FAB-MS, m/z calc
(found): 854.23 (854.31) [M+H]+.
2.5. Antioxidant assays
The ability of ruthenium complexes to act as hydrogen donors or free radical scavengers
was explored by conducting a series of in vitro antioxidant assays involving DPPH radical,
hydroxyl radical, nitric oxide radical, hydrogen peroxide radical and comparing the outcomes
with standard antioxidants, including the natural antioxidant vitamin C and the synthetic
antioxidant BHT.
2.5.1. DPPH• scavenging assay
The DPPH radical scavenging activity of the compounds was measured according to the
method of Blios [48]. The DPPH radical is a stable free radical. Because of the odd electron,
DPPH shows a strong absorption band at 517 nm in the visible spectrum. As this electron
becomes paired off in the presence of a free radical scavenger, the absorption vanishes and the
9

resulting decolorization is stoichiometric with respect to the number of electrons taken up.
Various concentrations of the experimental complexes were taken and the volume was 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 made 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 min at 27◦ C. The absorbance of the sample was
quantified at 517 nm against the blank (methanol).
2.5.2. OH• scavenging assay
The hydroxyl radical scavenging activity of the complex has been investigated by using
the Nash method [49]. In vitro hydroxyl radicals were generated by a Fe3+/ascorbic acid system.
The detection of hydroxyl radicals was carried out by measuring the amount of formaldehyde
formed from the oxidation reaction with DMSO. The formaldehyde produced was detected
spectrophotometrically at 412 nm. 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. 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. After 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
Nash reagent was added to each tube and left at room temperature for 15 min. The intensity of
the color formed was measured spectrophotometrically at 412 nm against the reagent blank.
2.5.3. NO• scavenging assay
The assay of nitric oxide scavenging activity is based on a method [50] in which sodium
nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide,
which interacts with oxygen to produce nitrite ions. These can be estimated using the Greiss
reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite
ions. For the experiment, sodium nitroprusside (10 mM) in phosphate buffered saline was mixed
with a fixed concentration of the complex and standards and incubated at room temperature for
150 min. After the incubation period, 0.5 mL of Griess reagent containing 1% sulfanilamide,
2%H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride was added. The
10

absorbance of the chromophore formed was measured at 546 nm.
2.5.4. H2O2 scavenging assay
The ability of the complexes to scavenge hydrogen peroxide was determined using the
method of Ruch et al. [51]. A solution of hydrogen peroxide (2.0 mM) was prepared in
phosphate buffer (0.2 M, pH 7.4) and its concentration was determined spectrophotometrically
from absorption at 230 nm with molar absorptivity 81 M-1cm-1.The complexes (100 µgmL-1),
BHT and vitamin C (100 µgmL-1) were added to 3.4mL of phosphate buffer together with
hydrogen peroxide solution (0.6 mL). An identical reaction mixture without the sample was
considered as negative control. Absorbance of hydrogen peroxide at 230 nm was determined
after 10 min against the blank (phosphate buffer).
For the four assays described above, all the tests were run in triplicate and the percentage
of scavenging activity was calculated using the following formula
Percentage of scavenging activity = [(A0-Ac)/A0] X 100
(A0 and Ac are the absorbance in the absence and presence of the compounds tested). The
effective concentration of sample required to scavenge the corresponding radical by 50% (IC50
value) was obtained by linear regression analysis of the curve plotting between % inhibition and
concentrations.
2.6. DNA binding experiments
Luminescence measurement was performed to clarify the binding affinity of
ruthenium(III) complexes by emissive titration at room temperature. The complexes were
dissolved in mixed solvent of 5% DMSO and 95% Tris–HCl buffer (5 mM Tris–HCl/50 mM
o
NaCl buffer for pH = 7.2) for all the experiments and stored at 4 C for further use and used
within 4 days. Tris–HCl buffer was subtracted through baseline correction. The excitation
wavelength was fixed by the emission range and adjusted before measurements. Emissive
titration experiments were performed with a fixed concentration of metal complexes (25 µM).
While gradually increasing the concentration of DNA (0–25 µM), the emission spectra were
monitored by keeping the excitation of the test compounds at 400 nm. MB-DNA experiments
were conducted by adding the complexes to the Tris–HCl buffer of MB-DNA. The change in the
fluorescence intensity was recorded. The excitation and emission wavelengths were 605 nm and
11

684 nm, respectively.
2.7. Protein binding studies
The emission of BSA at 280 nm was monitored at the wavelength 344 nm for the protein
binding studies. 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 5 mM Tris–HCl/50 mM
o
NaCl buffer (pH = 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.
Titrations were manually done by using a micropipet for the addition of the complexes.
2.8. DNA cleavage experiment
DNA cleavage experiments were carried out according to reported procedure [52]. For
the gel electrophoresis experiment, supercoiled DNA of Escherichia coli was treated with the
ruthenium(III) complexes in TAE buffer (10 mmol Tris acetate, 10 mmol EDTA, PH 8.0 ) and the
solution was then incubated at 37oC for 2 h. Loaded 20 μl of DNA sample (mixed with
bromophenol blue dye, 1:1 ratio), carefully into the wells, along with standard DNA marker. The
samples were analyzed by electrophoresis for 30 min at 50V on a 0.8% agarose gel in TAE (4.84
g Tris-acetate, 0.5 mol EDTA/1 ltr, PH 8.0). The gel was stained with 10 µg/ml ethidium bromide
and observes the bands under illuminator.
2.9. In vitro anticancer activity evaluation by MTT assay
The human breast cancer cell line (MCF-7) was obtained from National Centre for Cell
Science (NCCS), Pune, and grown in Eagles Minimum Essential Medium containing 10% fetal
bovine serum (FBS). Cells were maintained at 370C, 5% CO2, 95% air and 100% relative
humidity. Maintenance cultures were passaged weekly, and the culture medium was changed
twice a week.
The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA)
to make single cell suspensions and viable cells were counted using a hemocytometer and diluted
with medium containing 5% FBS to give final density of 1x105 cells/ml. One hundred microlitres
per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well
12

and incubated to allow for cell attachment at 37oC, 5% CO2, 95% air and 100% relative
humidity. After 24 h, the cells were treated with serial concentrations of the test samples. They
were initially dissolved in neat dimethylsulfoxide (DMSO) to prepare the stock (200 mM) and
stored frozen prior to use. At the time of drug addition, the frozen concentrate was thawed and an
aliquot was diluted to twice the desired final maximum test concentration with serum free
medium. Additional three, 10 fold serial dilutions were made to provide a total of four drug
concentrations. Aliquots of 100 µl of these different drug dilutions were added to the appropriate
wells already containing 100 µl of medium, resulted the required final drug concentrations.
Following drug addition the plates were incubated for an additional 48 h at 37oC, 5% CO2, 95%
air and 100% relative humidity. The medium containing without samples were served as control
and triplicate was maintained for all concentrations.
MTT is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells,
succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble
purple formazan. Therefore, the amount of formazan produced is directly proportional to the
number of viable cells. After 48h of incubation, 15µl of MTT (5mg/ml) in phosphate buffered
saline (PBS) was added to each well and incubated at 37oC for 4h. The medium with MTT was
then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then
measured the absorbance at 570 nm using micro plate reader [53,54].
The % cell inhibition was determined using the following formula.
% cell Inhibition = 100- Abs (sample)/Abs (control) x100.
Nonlinear regression graph was plotted between % Cell inhibition and Log10
concentration and IC50 was determined using GraphPad Prism software.
3. Results and discussion
The reactions of [RuX3(EPh3)3] (E = P or As; X = Cl or Br) with S-
methylisothiosemicarbazone Schiff base ligands at 1:1 molar ratio in dry benzene afforded new
hexacoordinated low-spin ruthenium(III) Schiff base complexes (Scheme 1). All the complexes
are air-stable, non-hygroscopic in nature, insoluble in water and highly soluble in common
organic solvents such as chloroform, dichloromethane, acetonitrile and dimethylsulphoxide. Our
efforts to grow single crystals of the complexes went unsuccessful. However, the analytical data
and FAB-Mass spectral data of the complexes are in good agreement with the calculated values
13

and thus confirming the proposed general molecular formula for all the complexes.
3.1. Infrared Spectral analyses
The IR spectra of the complexes have been examined in comparison with that of the
ligands. The spectra of free ligands showed two intense bands in the region 1635-1686 cm-1 and
1617-1665 cm-1 characteristic of two υC=N imine groups. These bands shifted towards lower
frequencies 1614-1672 cm-1 and 1598-1658 cm-1 respectively in complexes indicate the
coordination of ligands with metal through the two azomethine nitrogens [55]. The two broad
bands appeared in ligand spectra very close to each other in the region 3265-3320 cm-1 were due
to the stretching frequencies of two phenolic -OH groups. These bands disappeared in complexes
which showed the binding of phenolic oxygen with metal via deprotonation. This was further
supported by the increase of phenolic C-O stretching to 1384-1400 cm-1 in complexes which
appeared at 1355-1377 cm-1 region in their corresponding ligands.
3.2. Electronic Spectroscopic Analysis
The electronic spectra of all the ligands and the complexes were recorded in
dichloromethane solution at 800–200 nm region. The spectra of the free ligands showed two
bands at 344-310 nm and 263-242 nm regions which are attributed to л→ л* and n→ л*
transitions of aromatic rings and imine groups. However, in the complexes, the electronic spectra
have shown only the charge transfer transitions in the region 516-426 nm though there is a
possibility for d-d transitions. We have assigned these bands as being due to charge transfer
transitions based on the molar extinction coefficient values (ε = 12,198-4700 dm3 mol-1cm-1).
Since the d5 system in ruthenium(III) has relatively high oxidizing properties, the charge transfer
of the type Lπy→t2g are prominent in the low energy region which obscure the weaker d-d
transitions [56]. The other peaks appeared in the 336-240 nm regions have been assigned to intra
ligand charge transition in all the complexes. The pattern of electronic spectra of all the
complexes indicates an octahedral environment around the ruthenium(III) ion [57,58].
3.3. 1H NMR spectral analysis
1H NMR spectra of ligands were recorded in order to confirm their structures. The spectra
have shown two azomethine proton (-CH=N-) signals in the range 8.23-9.28 ppm and two
14

hydroxyl proton (-OH) signals in the range 10.75-12.99 ppm. The aromatic protons have
exhibited their signals at 6.84-8.35 ppm range. A signal at 3.67 ppm in the spectra of L3 ligand
corresponds to –OCH3 proton. The spectral data obtained have supported the formation and the
structure of S-methylisothiosemicarbazone ligands.
3.4. Magnetic moments
Magnetic susceptibility measurements were carried out at room temperature for the all
the complexes. The µeff values were obtained in the range of 1.58-1.81 BM which corresponds to
a single unpaired electron in low-spin d5 configuration and confirms the +3 oxidation state of
5
ruthenium in the new complexes. The µeff values suggest a low spin t2g configuration for
ruthenium(III) ion in an octahedral environment [59-61].
3.5. EPR Spectroscopic Analysis
The EPR spectra of the complexes have been recorded in the solid state at room
temperature. The nature of spectra showed the absence of any hyperfine splitting due to
interaction with any other nuclei present in the complexes. All the complexes exhibit a single
isotropic resonance with ‘g’ values ranging from 2.47 - 2.59. Although the complexes have some
distortion in their octahedral geometries, the observation of isotropic lines in the EPR spectra
may be due to intermolecular spin exchange and due to the occupancy of the unpaired electron in
a degenerate orbital [62]. The observed EPR spectra are in good agreement with that of
previously reported ruthenium(III) complexes [63,64].
3.6. Redox properties
The electron transfer properties of the new ruthenium(III) complexes were studied by
cyclic voltammetry in acetonitril (Table 1). Voltammograms of these complexes were recorded
from -2.0 to 2.0 V for RuIII/RuIV and RuII/RuIII redox cycles and the results were presented in
table 1. The voltammograms showed a redox wave for these complexes at -1.19 to -1.49 V,
which corresponds to RuIII/RuII (reduction). The ∆Ep values for the above reduction process
ranging from 260 to 380 mV which shows a quasi-reversible reduction. Quasi-reversible process
observed for the above RuIII/RuII reduction is due to slow electron transfer and absorption of the
complexes on the electrode surface. The oxidations of RuIII/RuIV cycle were observed at 0.54-
15

0.66 region and indicate the irreversible nature. This may be due to the short-lived oxidation state
of the RuIV metal ion or due to oxidative degradation followed by deposition [65]. These
observations indicate that the charge transfer process for RuIV-RuIII couple is not in general as
rapid as in the case of RuIII-RuII couple. Further, a quasi- reversible ligand based reduction in a
range of
-0.72-0.81 V with ∆Ep(mV) value 260-420 mV was also observed. Hence, it has
been clearly seen that the present ligand system is ideally suitable for stabilizing the
ruthenium(III) oxidation state.
3.7. Antioxidant activity
We carried out experiments to explore the free radical scavenging ability of the
complexes, with the hope of developing potential antioxidants and therapeutic reagents for
respiratory diseases, such as asthma, emphysema and asbestosis [66]. Free radicals can cause
DNA damage in humans. The damage to DNA has been suggested to contribute to aging and
various diseases including cancer and chronic inflammation [67].
The principle for the reduction in DPPH• free radicals is that the antioxidant reacts with
stable free radical DPPH and converts it to 1,1-diphenyl-2-picrylhydrazine. The ability to
scavenge the stable free radical DPPH is measured by a decrease in the absorbance at 517nm.
Hydrogen peroxide itself is not very reactive, but sometimes it is toxic to cells because it may
give rise to hydroxyl radicals in the cells [68]. Nitric oxide or reactive nitrogen species, formed
+
during their reaction with oxygen or with superoxide, such as NO2, N2O4, N3O4, NO3 - and NO2
are very reactive. These species are responsible for altering the structural and functional behavior
of many cellular components. Further, the scavenging activity may also help to arrest the chain
of reactions initiated by the excess generation of NO that is detrimental to human health. Among
all free radicals, the hydroxyl radical is a major product arising from the high-energy ionization
of water, the most important biological sources, of which are the Haber–Weiss and Fenton
reactions, involving the superoxide anion, hydrogen peroxide and transition metals [69]. This is
the most aggressive oxidant known and therefore the most dangerous oxygen metabolite, which
is capable of attacking any biological molecule and can be implicated in the tissue damage
associated with inflammation, therefore elimination of this radical is one of the major aims of
antioxidant administration [70].
Scavenging activity (%) = [(A without sample(control) – A with sample(test) ) / A without sample(control)
]
16

The suppression ratio of all radicals increases with the increasing amount of the complexes
concentration in the range of 10µg to 50µg. The average suppression ratio of complexes is
comparable under the same condition [71]. The IC50 values of all the complexes (Table 2)
obtained from different types of assay experiments strongly supports that they possess good
antioxidant activities, which are better than those of standard antioxidants, including the natural
antioxidant vitamin C and the synthetic antioxidant BHT (butylated hydroxytoluene) (Figure 1).
The higher antioxidant activities of the complexes can be explained by the availability of an odd
electron in the low spin ‘d5’ electronic configuration and Ru(III) ion, which increases the
capacity to stabilize the unpaired electrons and thereby scavenge the free radicals. The electron
deficient metal centers in Ru(III) complexes might increase the acidity of the available protons
on ligands which also increasing the antioxidant activity in terms of H+ donors. The OH•
scavenging power of the tested complexes was greater than other radicals. Being a potent
scavenger of hydroxyl radicals, the complexes could capture superoxide anion radicals and
hydrogen peroxide, as apparent from the IC50 values [34]. Substituents on the ligands have
shown a considerable effect on the antioxidant activity of the complexes. With respect to the
ligands, the antioxidant activity is in the order L3> L2≈ L1>L4. The high antioxidant activity of
L3 may be due to the more activated phenyl ring.
3.8. DNA and protein binding studies
DNA & Protein binding ability of the complexes were examined using fluorescence
spectroscopy. Triphenylarsine complexes showed better activity in antioxidant evaluation and in
optimization binding studies. Hence three complexes containing triphenylarsine colignds and
with different substitutions on ligand moiety (ie., 5, 6 and 7) have been chosen for DNA &
protein interaction studies.
3.8.1. DNA binding using fluorescence spectral studies
All the new Ruthenium(III)isothiosemicarbazone complexes could emit luminescence at
ambient temperature with a maximum excitation wavelength at 450 and emission wavelength at
490 nm containing Tris–HCl buffer (5 mM Tris–HCl/50 mM NaCl buffer for pH = 7.2). While
the complexes bound to DNA through intercalation show a decrease in intensities
(hypochromism) and a red shift of their emission bands due to the strong stacking interaction
17

between the aromatic chromophores of the complex and base pairs of DNA [72]. Upon
increasing concentration of CT-DNA the emission intensities of complexes of both 5, 6 and 7
exhibited hypochromism ranging from 19 to 22 % with red shift up to 3 nm (Fig. 2). The
intrinsic binding constants were obtained according to the following Scatchard equation.
CF = CT [(I/I0)  P] / (1  P)
Where, CT is the concentration of the probe (complex) added, CF is the concentration of
the free probe, and I0 and I are its emission intensities in the absence and in the presence of
DNA, respectively. P is the ratio of the observed emission quantum yield of the bound probe to
the free probe. The value of P was obtained from a plot of I/I0 versus 1 / [DNA] such that the
limiting emission yield is given by the y-Intercept. The amount of bound probe (CB) at any
concentration was equal to CT − CF. The Scatchard plots of r/CF versus ‘r’ for complexes 5–7
with increasing concentration of CT-DNA shown in fig. 3. A plot of r/CF verses (= CB/ [DNA])
gave the binding isotherm and the best fit of the data resulted in the intrinsic binding constant
(Kb) values was calculated from the ratio of the slope and the intercept [73]. The observed Kb
vales of complexes 5, 6 and 7 were 4.7 x 105 M-1, 3.6 x 104 M-1 and 3.2 x 104 M-1 respectively,
which showed that the complexes bind with DNA via intercalative mode [74].
3.8.2. Competitive binding with methylene blue
Methylene Blue (MB) is a planar cationic dye which is widely used as a sensitive
fluorescence probe for native DNA. MB emits intense fluorescent light in the presence of DNA
due to its strong intercalation between the adjacent DNA base pairs. The displacement technique
is based on the decrease of fluorescence resulting from the displacement of MB from a DNA
sequence by a quencher, and the quenching is due to the reduction of the number of binding sites
on the DNA that are available to the MB. The fluorescence quenching spectra of DNA-bound
MB for complexes 5, 6 and 7 were shown in Fig. 4. This illustrates that, as the concentration of
the complexes increases, the emission band at 684 nm exhibited hypochromism of 17.18%,
22.61% and 15.76% of the initial fluorescence intensities respectively. The observed reduction in
the fluorescence intensity clearly indicates that the MB molecules are displaced from their DNA
binding sites and are replaced by the complexes under investigation [75]. Quenching data were
analyzed according to the following Stern–Volmer equation:
18

F0/F = Kq [Q] + 1
Where, F0 is the emission intensity in the absence of the compound, F is the emission
intensity in the presence of the compound, Kq is the quenching constant, and [Q] is the
concentration of the compound. The Kq value is obtained as a slope from the plot of F0/F versus
[Q]. In the Stern–Volmer plot (Fig. 5) of F0/F versus [Q], the quenching constant (Kq) is obtained
from the slope and which was 5.8 x 103 M-1, 5.4 x 104 M-1 and 3.2 x 103 M-1 for the complexes 5,
6 and 7 respectively. The results have suggested the intercalation binding mode for complex 6
and further confirm the higher DNA binding ability of the same compared to 5 and 7
3.9. Fluorescence quenching of BSA with ruthenium(III) complexes
The interaction of BSA with ruthenium complexes was studied by fluorescence
measurement at room temperature. A solution of BSA (1 µM) was titrated with various
concentrations of the complexes (0–50 µM). Fluorescence spectra were recorded in the range of
290–450 nm upon excitation at 280 nm. The effects of the compound on the fluorescence
emission spectrum of BSA are shown in Fig. 6. The addition of the above complexes to the
solution of BSA resulted in a significant decrease of the fluorescence intensity of BSA at 344
nm, by a hypsochromic shift up to 3 nm. The observed blue shift is mainly due to the fact that
the active site in protein is buried in a hydrophobic environment. This result suggested a definite
interaction of all of the complexes with the BSA protein [76,77]. The fluorescence quenching
data obtained were analyzed with the Stern–Volmer and Scatchard equations. From the plot of
I0/I versus [Q], the quenching constant (Kq) can be calculated. If it is assumed that the binding of
compounds with BSA occurs at equilibrium, the equilibrium binding constant can be analyzed
according to the Scatchard equation:
log [(I0/I) / I0] = log Kbin + n log [Q]
where, Kbin is the binding constant of the compound with BSA and n is the number of
binding sites. The number of binding sites (n) and the binding constant (Kbin) were found from
the plot of log [(I0/I) / I0] versus log [Q] (Fig. 7). The calculated Kq, Kbin, and n values are
19

given in table 3. Among the three complexes, the complex 6 has better interaction with BSA
than the other two complexes. The order of efficiency of the tested complexes to bind with BSA
was observed as 6 > 5 > 7.
3.10. DNA cleavage studies by gel electrophoresis
When circular plasmid DNA is subjected to gel electrophoresis, relatively fast migration
will be observed for the intact supercoil form (Form I). If scission occurs on one strand (nicking),
the supercoil will relax to generate a slower-moving open circular form (Form II). If both strands
are cleaved, a linear form (Form III) that migrates between Forms I and II will be generated [78]
Fig. 8 shows the interaction of complexes with DNA at different concentrations (20, 30, 40
µg/mL) and it is evident that the disappearance of supercoiled form at all concentrations. This
shows that the super coiled form (Form I) converted to nicked form (form II) and confirms the
scission that occurs by the complex. Both the complexes 1 and 2 cleaved the DNA of E-coli
species completely at all concentrations.
3.11. Cytotoxic activity evaluation by MTT assay
MTT assay was performed on human breast cancer cell line (MCF-7) to check the
anticancer activity of triphenylarsine complexes containing different ligands (5, 6, 7 and 8).
Ruthenium(III) species have a high propensity for ligand exchange reactions with S-
methylisothiosemicarbazone ligands and may therefore interact with target molecules more
rapidly [79,80]. Upon increasing the concentration of complexes from 0.1µm to 100µm, the %
cell inhibition also increased. It is evident from the figure 9, that the number of cells decreased
with an increase in the concentration of the complexes. The comparison of the effect of
complexes over the cell inhibition tendency clearly reveals that complex containing ligand L3
exhibit higher inhibition capacity compared to others. This may be due to the more activated
phenyl ring in L3 ligand. Among the complexes tested, 6 and 7 have shown comparatively higher
activity than the standard drug cisplatin (Table 4). Complex 5 has shown activity almost closer to
cisplatin, whereas complex 8 showed very low activity among all complexes (higher IC50). The
plots of % growth inhibition of skin carcinoma cells against different concentrations have been
exhibited in figure 10.
20

4. Conclusion
A new series of ruthenium(III) S-methylisothiosemicarbazone complexes (1-8) were
prepared and the structure of the complexes were established by analytical and spectral methods.
Drug-like properties of the new complexes were analyzed by a series of biological studies,
including antioxidant, DNA interaction, protein binding and in-vitro anticancer activity. The
results have shown that the new complexes were better antioxidants than standard available
antioxidants like vitamin C and BHT. DNA binding studies of complexes (5, 6 and 7) have
revealed that the complexes are capable of binding with DNA via intercalative mode. Protein
binding investigations revealed that these complexes also have the tendency to bind with BSA.
Methoxy group substitution on the ligand moiety considerably enhances both the DNA and
protein interaction nature of the complexes. Further, DNA photocleavage experiments on
complexes (1 and 5) showed the characteristic DNA cleavage ability of the new complexes, even
at their very low concentrations (10 µM). The new complexes (5, 6, and 7) also showed
anticancer activity on human breast cancer cell line (MCF-7). The complex 6 has found to
exhibit better biological activity compared to all other complexes.
Acknowledgement
We are thankful to the Department of Chemistry, Pondicherry University, Pondicherry,
SAIF, Cochin, HRMS facility NIIST, Thiruvananthapuram, KMCH, Coimbatore and Biogenics,
Hubli for providing assistance in various studies. One of the authors (G. Prakash) thanks Periyar
University, Salem, for the financial support in the form of University Research Fellowship.
21

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