Organometallic ruthenium(II) complexes: Synthesis ... · Organometallic ruthenium(II) complexes:...

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Chapter VI

Organometallic ruthenium(II) complexes: Synthesis, structure and

influence of substitution at azomethine carbon towards DNA/BSA

binding, radical scavenging and cytotoxicity

The coordination chemistry of ruthenium complexes with pyridine and its

derivatives is one of the most studied areas due to their catalytic, redox, photoreactivity,

biological and supramolecular properties. A small variation of the coordination

environment around ruthenium ion effected by alterations in the donor centre, ligand

structure and chelate ring size significantly vary the properties of the complexes.1,2

Because of the broad range of applications possessed by mixed ligand ruthenium

Abstract

Bivalent, ruthenium organometallics containing hydrazone ligands have been synthesised

from the reactions of [RuH2(CO)(PPh3)3] and benzoic acid pyridine-2-ylmethylene-hydrazide (HL1)

(1) / benzoic acid (1-pyridin-2-yl-ethylidene)-hydrazide (HL2) (2) / benzoic acid (phenyl-pyridin-2-yl-

methylene)-hydrazide (HL3) (3). The composition of the new complexes were found to be

[RuH(CO)(PPh3)2(L1)] (4), [RuH(CO)(PPh3)2(L

2)] (5) and [RuH(CO)(PPh3)2(L

3)] (6) based on the

elemental analysis, UV-visible, infrared and 1H NMR spectroscopic data. The X-ray crystal structure

of one of the above complexes, [RuH(CO)(PPh3)2(L3)] (6) demonstrated a distorted octahedral

coordination geometry around the metal center. An investigation on the effect of substitution at the

azomethine carbon of the hydrazone coordinated in these ruthenium chelates on the potential binding

with DNA/BSA, free radical scavenging and cytotoxicity is presented. The magnitude of the binding

constant (Kb) obtained from absorption spectral titration varies from 10-5

-10-7

M-1

depending upon the

nature of the substituent’s attached at the azomethine carbon atom and the same was found to decrease

in the order 6 > 5 > 4. Interaction of the above complexes 4-6 with bovine serum albumin (BSA)

suggested the occurrence of static quenching process between them and the number of binding sites

was found to be ~0.6-0.9 that proves single mode of binding. The conformational changes of BSA

upon the addition of metal chelates have also been monitored using synchronous fluorescence

measurements. Further, free radical scavenging and cytotoxicity of the compounds 4, 5 and 6 against

ABTS+, O2

- and OH radicals and HeLa and A341 cell lines under in vitro conditions proved their

pharmacological properties.

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Organometallic ruthenium(II) complexes: ….

P. Sathyadevi, 2011

complexes, there is a continuous endeavor to synthesize new complexes of it with an

objective to fine tune their properties.

Over the years, much attention has been paid to investigate the chemistry of

transition metal complexes with large number of hydrazones as co-ligand, in particular

pyridine containing systems. Hydrazone ligands create an environment similar to the one

present in biological systems usually by making coordination through oxygen and

nitrogen atoms. Various important properties of carbonic acid hydrazides, along with

their applications in medicine and analytical chemistry, have led to increased interest in

their complexation characteristics with transition metal ions.3 The hydrazone unit offers a

number of attractive features such as the degree of rigidity, a conjugated -system and an

NH unit that readily participates in hydrogen bonding and may be a site of protonation-

deprotonation. It is well established that the formation of metal complexes plays an

important role to enhance the biological activity of free hydrazones.4

Hydrazone ligands are very promising compounds from the view point of

coordination chemistry because of their ability towards complexation and involvement in

wide range of biological and non-biological properties.5,6

The chemistry of transition

metals with ligands from the hydrazine family has been of interest to coordination as well

as bioinorganic chemists due to their different bonding modes with both electron–rich

and electron-poor metals.7,8

The structural motif, –N=C–CH=N–NH–C=N–, present in

heterocyclic hydrazones is a remarkable tool for development of multifunctional organic

receptors that find applications in chemical, environmental and biological sciences.

Hydrazones of pyridine-2-carboxaldehyde is a well studied system in transition metal

coordination chemistry, providing metal complexes with important magnetic, redox,

photochemical, ion exchange, catalytic and biological activities.9-13

The metallochemistry

of biomolecules has been largely focused on proteins, amino acids, nucleic acids and

carbohydrates.14-17

Heterocyclic hydrazones constitute an important class of biologically

active drugs that have attracted the attention of medicinal chemists due to their wide

ranging pharmacological properties like antifungal, antibacterial and anticonvulsant

compounds.18-22

During the last two or three decades, there has been an increasing

attention on the binding study of small molecules to DNA, since it is an important genetic

substance in organisms.23-25

Errors in gene expression can often cause diseases and play a

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P. Sathyadevi, 2011

secondary role in the outcome and severity of human diseases.26

Hence, a complete

understanding of DNA-drug binding is significant in the rational design of DNA

structural probe, DNA footprinting, sequence-specific cleaving agents and potential

anticancer drugs.27-30

To design effective chemotherapeutic agents and better anticancer

drugs, it is essential to explore the interactions of metal complexes with DNA.

Serum albumin is the major soluble protein constituent in the circulatory system

of a wide variety of organisms and it has the ability to reversibly bind to a large variety of

endogenous and exogenous ligands such as fatty acids, drugs, and metal ions in the

bloodstream.31,32

The drug-protein complex not only strongly affected the absorption,

distribution, metabolism and excretion properties of drugs, but also influenced the drug

stability and toxicity during the chemotherapeutic process. Therefore, knowledge on the

mechanism of interaction between the selected drug and protein has become very vital to

design several new drugs with improved potential.33,34

In the current work, the authors

have chosen bovine serum albumin (BSA) as a model protein to investigate its interaction

with the newly synthesised ruthenium hydrazone chelates 4-6 owing to its similarity with

human serum albumin (HSA) in respect of approximately 76% sequence homology, and

3D structure.35

From the literature reports, it is understood that the variation in the bulkiness of

substituents present in the ligand that was coordinated to a transition metal in the

complexes containing them have an influence on the DNA binding of them. Based on the

above mentioned factors and in continuation of our interest on the studies related to

various hydrazone ligands towards transition metals, we herein, present the synthesis and

structural investigations of three new ruthenium hydrazone complexes containing the

azomethine carbon substituted with hydrogen or methyl or phenyl groups. Further studies

to explore the effect of these substitutions at the azomethine carbon on DNA / BSA

binding, free radical scavenging and cytotoxicity of these organometallic complexes were

also undertaken under in vitro conditions.

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Experimental section

Materials

Reagent grade chemicals were used without further purification in all the

synthetic work. All solvents were purified by standard methods. The compounds

RuCl2·6H2O, triphenylphosphine, benzhydrazide, pyridine-2-carbaldehyde, 2-acetyl

pyridine and 2-benzoyl pyridine were purchased from Sigma-Aldrich Chemie and Alfa

Aesar, respectively and used as received. Calf thymus (CT DNA) and bovine serum

albumin (BSA) were purchased from Himedia. The human cervical cancer cell line

(HeLa), human skin cancer cell line (A431) and non-cancerous NIH 3T3 mouse

embryonic fibroblasts was obtained from National Centre for Cell Science (NCCS),

Pune, India. All the other chemicals and reagents used for DNA binding, protein binding,

antioxidant and cytotoxicity assays were of high quality.

Physical measurements

Microanalyses (% C, H & N) were performed on a Vario EL III CHNS analyzer.

IR spectra of the samples were recorded using KBr pellets on a Nicolet Avatar instrument

in the frequency range of 400-4000 cm-1

. 1H NMR spectra of ligands and their metal

hydrazone complexes were recorded on a Bruker AMX 500 spectrometer operating at

500 MHz using DMSO as solvent and tetramethylsilane as an internal standard. The

electronic absorption and emission spectra were recorded in DMSO-buffer (5:95)

solution on a Jasco V-630 spectrophotometer and Jasco FP 6600 spectrofluorometer

respectively at room temperature.

A BRUKER APEX 2 X-ray (three-circle) diffractometer was employed for crystal

screening, unit cell determination, and data collection. The XRD data of the complex 6

was collected at 110 K with MoKα radiation λ = 0.70173Å. The structure was refined

(weighted least squares refinement on F2) to convergence.

36 X-seed was employed for the

final data presentation and structure plots.37

Integrated intensity information for each

reflection was obtained by reduction of the data frames with APEX2.38

The integration

method employed a three dimensional profiling algorithm and all data were corrected for

Lorentz and polarization factors, as well as for crystal decay effects. Finally the data was

merged and scaled to produce a suitable data set. SADABS 39

was used to correct the data

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for absorption effects. A solution was obtained readily using XS as implemented in

SHELXTL. To possibly verify the solvated molecule and its occupancy, SQUEEZE was

applied using PLATON (after the removal of the ethanol molecule from refinement). All

non-hydrogen atoms were refined with anisotropic thermal parameters.

Synthesis of ruthenium(II) starting precursor complex

The precursor metal complex [RuH2(CO)(PPh3)3] was prepared according to the

literature method.40

Preparation of carbonyldihydridotris(triphenylphosphine)ruthenium(II)

[RuH2(CO)(PPh3)3]

A solution of 0.13 g (0.5 mmol) of ruthenium trichloride trihydrate (RuCl3·3H2O)

in ethanol (5 cm3), aqueous formaldehyde (5 cm

3, 40 %) and 0.15 g of potassium

hydroxide in ethanol (5 cm3) were added quickly and successively to a boiling solution of

0.79 g (3 mmol) of triphenylphosphine in ethanol (35 cm3). The solution was heated

under reflux for 15 minutes and then cooled. The resultant grey compound was separated

and washed successively with ethanol, water and n-hexane and dried in vacuo.

Yield: 72%. Colour; Grey; mp: 160 °C.

Synthesis of hydrazone ligands

The reactions involved in the synthesis of hydrazone ligands and its

corresponding ruthenium hydrazone complexes were given in scheme 6.1.

Synthesis of benzoic acid (pyridine-2-yl-methylene)-hydrazide HL1 (1)

Benzoic acid pyridine-2-ylmethylene-hydrazide ligand (1) was prepared by

refluxing a mixture of benzhydrazide (0.680 g; 5 mM) and pyridine-2-carboxaldehyde

(0.478 g; 5 mM) in 50 mL of absolute ethanol for 8 h as given in scheme 6.1. The

reaction mixture was cooled to room temperature and the solid obtained was filtered,

washed several times with distilled water and recrystallized from EtOH to afford the

ligand 1 in pure form with good yield.

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P. Sathyadevi, 2011

Yield: 90%. Colour: Creamy white; mp: 120 °C. Anal. Found (%) for C13H11N3O1 (Mol

wt = 225.246): C, 69.51; H, 5.09; N, 18.97. Calculated (%): C, 69.32C; H, 4.92; N, 18.66.

Selected IR bands (νmax in cm-1

): 3202 (N–H); 1661 (C=O); 1586 (C=N); 1072 (N–N).

1H NMR (500 MHz, [D6] DMSO): δ (ppm): 14.63 (s, 1H, enolic OH); 8.59 (s, 1H, NH);

8.43-7.38 (m, 9H, Ar–H); 7.53 (s, 1H, HC=N).

Synthesis of benzoic acid (1-pyridin-2-yl-ethylidene)-hydrazide HL2 (2)

Benzoic acid (1-pyridin-2-yl-ethylidene)-hydrazide ligand (2) was prepared by

refluxing a mixture of benzhydrazide (0.680 g; 5 mM) and 2-acetyl pyridine (0.605 g; 5

mM) in 50 mL of absolute ethanol for 8 h as given in scheme 6.1. The reaction mixture

was cooled to room temperature and the solid obtained was filtered, washed several times

with distilled water and recrystallized from EtOH to afford the ligand 2 in pure form with

good yield.

Yield: 88%. Colour: Pale yellow; mp: 125 °C. Anal. Found (%) for C14H13N3O1 (Mol wt

= 239.273): C, 70.36; H, 5.41; N, 17.41. Calculated (%): C, 70.28; H, 5.48; N, 17.56.

R

NN

O

Ru

PPh3

H

OC

NPh3P

R

NNH

O

NNR

O

H2NNH

O

EtOH

EtOH, MeOH

R

NN

HO

N+

[RuH2(CO)(PPh3)3]

Reflux, 8h

Reflux, 48h

Ligand R

HL1

HL2

HL3

H

CH3

C6H5

Scheme 6.1 Synthesis of hydrazone ligands and their ruthenium complexes.

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P. Sathyadevi, 2011


): 3178 (N–H); 1656 (C=O); 1540 (C=N); 1060 (N–N). 1H

NMR (500 MHz, [D6] DMSO): δ (ppm): 14.86 (s, 1H, enolic OH); 9.12 (s, 1H, NH);

8.46-7.23 (m, 9H, Ar–H); 2.52 (s, 3H, CH3C=N).

Synthesis of benzoic acid (phenyl-pyridin-2-yl-methylene)-hydrazide HL3 (3)

Benzoic acid (phenyl-pyridin-2-yl-methylene)-hydrazide ligand (3) was prepared

by refluxing a mixture of benzhydrazide (0.680 g; 5 mM) and 2-benzoyl pyridine (0.915

g; 5 mM) in 50 mL of absolute ethanol for 8 h as given in scheme 6.1. The reaction

mixture was cooled to room temperature and the solid obtained was filtered, washed

several times with distilled water and recrystallized from EtOH to afford the ligand 3 in

pure form with good yield.

Yield: 85%. Colour: Pale yellow; mp: 131 °C. Anal. Found (%) for C19H15N3O1 (Mol wt

= 301.342): C, 75.52; H, 5.15; N, 13.81. Calculated (%): C, 75.73; H, 5.02; N, 13.94.


): 3143 (N–H); 1678 (C=O); 1581 (C=N); 1075 (N–N).

1H NMR (500 MHz, [D6] DMSO): δ (ppm): 15.09 (s, 1H, enolic OH); 8.86 (s, 1H, NH);

8.01-7.43 (m, 14H, Ar–H).

Synthesis of ruthenium(II) hydrazone complexes

Synthesis of [RuH(CO)(PPh3)2(L1)] (4)

Complex 4 was synthesised by refluxing a methanolic solution of

[RuH2(CO)(PPh3)3] (0.917 g; 1 mM) and the ethanolic solution of ligand 1 (0.224 g; 1

mM) (40 mL) for 48 h (scheme 6.1). After cooling the reaction mixture to room

temperature, the precipitate formed was filtered, washed with a mixture of ethanol and

methanol and dried in vacuo. The purity of the complex was checked by TLC and

isolation of crystals suitable for single crystal XRD studies went unsuccessful.

Yield: 58%. Colour: Reddish brown; mp: 237 °C. Anal. Found (%) for Ru1C50H41N3O2P2

(Mol wt = 878.897): C, 68.49; H, 4.82; N, 4.61. Calculated (%): C, 68.33; H, 4.70; N,

4.78. Selected IR bands (νmax in cm-1

): 1598, 1480 (C=N–N=C); 1369 (C–O); 1092 (N–

N); 2023 (Ru–H); 1953 (Ru–CO). UV-visible (DMSO-buffer): λmax (nm): 266 & 305

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P. Sathyadevi, 2011

(ILCT); 376 (LMCT); 441 (MLCT). 1H NMR (500 MHz, [D6] DMSO): δ (ppm): 8.21 (s,

1H, HC=N); 8.02-6.32 (m, 39H, Ar–H); -10.09 (dt, 1H, Ru–H).

Synthesis of [RuH(CO)(PPh3)2(L2)] (5)





methanol and dried in vacuo. The purity of the complex was checked by TLC and

isolation of crystals suitable for single crystal XRD studies went unsuccessful.

Yield: 55%. Colour: reddish brown; mp: 243 °C. Anal. Found (%) for Ru1C51H43N3O2P2

(Mol. wt = 892.925): C, 68.89; H, 4.88; N, 4.61. Calculated (%): C, 68.60; H, 4.85; N

4.71. Selected IR bands (νmax in cm-1

): 1591, 1481 (C=N–N=C); 1366 (C–O); 1092 (N–

N); 2028 (Ru–H); 1961 (Ru–CO). UV-visible (DMSO-buffer): λmax (nm): 269 & 309

(ILCT); 442 (MLCT). 1H NMR (500 MHz, [D6] DMSO): δ (ppm): 2.77 (s, 3H,

CH3C=N); 8.12-6.26 (m, 39H, Ar–H); -10.56 (dt, 1H, Ru–H).

Synthesis of [RuH(CO) (PPh3)2(L3)] (6)





methanol and dried in vacuo. The purity of the complex was checked by TLC following

which the sugar like reddish brown crystals of complex 6 suitable for single crystal X-ray

diffraction studies were obtained in a mixture of ethanol and methanol.

Yield: 59%. Colour: Reddish brown; mp: 244 °C. Anal. Found (%) for

Ru1C56H45N3O2P2 (Mol wt = 954.993): C, 70.69; H, 4.88; N, 4.51. Calculated (%): C,

70.43; H, 4.75; N, 4.40. Selected IR bands (νmax in cm-1

): 1588, 1479 (C=N–N=C); 1365

(C–O); 1091 (N–N); 2024 (Ru–H); 1952 (Ru–CO). UV-visible (DMSO-buffer): λmax

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P. Sathyadevi, 2011

(nm): 265 (ILCT); 428 (MLCT). 1H NMR (500 MHz, [D6] DMSO): δ (ppm): 8.16-6.21

(m, 44H, Ar–H); -10.02 (dt, 1H, Ru–H).

DNA binding studies

Electronic absorption experiments

Electronic absorption titration experiments were performed with a fixed

concentration of metal complex (25 μM) but variable nucleotide concentration ranging

from 0 to 25 μM and after each addition of DNA to the metal complex, the readings were

noted.

Competitive binding measurements

A proof for the binding of complexes 4, 5 and 6 with DNA via intercalation was

obtained through the emission quenching experiment involving the competitive DNA

binding between them and ethidium bromide carried out in the buffer by keeping

[DNA]/[EtBr] = 2; [DNA] = 10 μM, [EtBr] = 5 μM and varying the concentrations of the

metal complexes (0-80 μM). The buffer used in the binding studies was 50 mM Tris HCl,

pH 7.2, containing 10 mM NaCl. For all fluorescence measurements, the entrance and

exit slits were maintained at 5 and 10 nm, respectively. The excitation wavelength was

545 nm, and the emission range was set between 530 and 750 nm and the experimental

data were measured at room temperature. In this method, fixed amounts of DNA and

EtBr were titrated with increasing amounts of complex over a range of complex

concentrations from 0-80 μM. The experiments were conducted at 20 °C in a Tris buffer.

EB was non-emissive in Tris-HCl buffer solution (pH, 7.2) due to fluorescence

quenching of free EB by the solvent molecules. In the presence of DNA, EB showed

enhanced emission intensity due to its intercalative binding to DNA. A competitive

binding of the metal complexes to CT DNA resulted in the displacement of the bound EB

thereby decreasing its emission intensity. The quenching constant (Kq) was calculated

using the classical Stern-Volmer equation,41

I0/I = Kq [Q] + 1

where, I0 and I are the respective emission intensities in the absence and presence of

quencher, Kq is the quenching constant and [Q] is the quencher concentration. Kq is the

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P. Sathyadevi, 2011

slope, obtained from the plot of I0/I vs [Q]. The apparent binding constant (Kapp) has been

calculated from the equation,

KEB [EB] = Kapp [complex]

The complex concentration was obtained from the value at a 50% reduction of the

fluorescence intensity of EB and KEB = 1.0×107 M

-1.

Protein binding studies

Binding of metal hydrazone complexes with bovine serum albumin (BSA) was

studied from the fluorescence spectra recorded with an excitation at 280 nm and

corresponding emission at 345 nm assignable to that of bovine serum albumin (BSA).

The excitation and emission slit widths and scan rates were kept constant for all the

experiments. Sample solutions were carefully degassed using pure nitrogen gas for 15

minutes by using quartz cells (4×1×1 cm) with high vacuum teflon stopcocks. Stock

solution of BSA was prepared in 50 mM phosphate buffer (pH, 7.2) and stored in the

dark at 4 °C for further use. Concentrated stock solutions of the ligands and its copper

complexes were prepared by dissolving them in DMSO:phosphate buffer (5:95) and

diluted suitably with phosphate buffer to required concentrations. 2.5 mL of BSA

solution (1 μM) was titrated by successive additions of a 5 μL stock solution of metal

hydrazone complexes (10-4

M) using a micropipette. Synchronous fluorescence spectra

was also recorded using the same concentration of BSA and complexes as mentioned

above with two different λ (difference between the excitation and emission wavelengths

of BSA) values such as 15 and 60 nm.

Antioxidant studies

The superoxide, hydroxyl and ABTS radical scavenging activities of the

ruthenium hydrazone complexes were determined by the methods described by

Beauchamp et al , Nash and Re et al, respectively.42-44

Superoxide radical scavenging activity

The superoxide (O2–) radical scavenging assay was done based on the capacity of

the compounds to inhibit formazan formation by scavenging the superoxide radicals

generated in riboflavin-light-NBT system. Each 3 mL reaction mixture contained 50 mM

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P. Sathyadevi, 2011

sodium phosphate buffer (pH, 7.6), 20 µg riboflavin, 12 mM EDTA and 0.1 mg NBT.

Reaction was started by illuminating the reaction mixture with different concentration of

the test compounds (10-50 µM) for 90 seconds. Immediately after illumination, the

absorbance was measured at 590 nm. The entire reaction assembly was enclosed in a box

lined with aluminum foil. The above reaction mixture without test sample was used as

control.

Hydroxyl radical scavenging activity

The hydroxyl (OH) radical scavenging activity of complexes 4-6 have been

investigated using the Nash method. In vitro hydroxyl radicals were generated by Fe

3+ /

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 (ferrous ammonium sulphate (0.331 mM) and EDTA (0.698

mM), 0.5 mL of EDTA solution (0.048 mM) and 1.0 mL of DMSO (10.83 mM) DMSO

(v/v) in 0.1 M phosphate buffer, pH 7.4) were sequentially added to the test tubes

containing the test compounds with different concentrations in the range of 10-50 µM.

The reaction was initiated by adding 0.5 mL of ascorbic acid (1.25 mM) and 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 TCA (107 mM). Subsequently, 3.0 mL of Nash reagent

was added to each tube and left at room temperature for 15 min. The reaction mixture

without sample was used as control. The intensity of the colour formed was measured

spectrophotometrically at 412 nm against reagent blank.

ABTS cationic radical scavenging activity

Total antioxidant activity assay using ABTS cationic radical was studied

according to the following procedure. ABTS (2,2'-Azino-3-ethylbenzthiazoline-6-

sulfonic acid diammonium salt) was dissolved in water to a 5 mM concentration and its

cationic radical was produced by reacting with 5 mM potassium persulfate. The resulting

mixture was kept in dark at room temperature for 12-16 h before use. Prior to assay, the

solution was diluted in ethanol (about 1:79 v/v) and equilibrated to 30 °C to give an

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absorbance of 0.70±0.02 at 734 nm. After the addition of 2 mL of diluted ABTS cationic

radical solution to different concentration (10-50 µM) of the compounds, the absorbance

was taken at 30 °C exactly 30 min after the initial mixing and the reaction mixture

without test sample was used as control.

For each of the above assay, tests were run in triplicate by varying the

concentration of complexes 4, 5 and 6 ranging from 10-50 μM. The percentage activity

was calculated by using the formula,

% activity = [(A0-AC)/A0] × 100

where, A0 and AC represent the absorbance in the absence and presence of the test

compounds, respectively. The 50% activity (IC50) can be calculated from the result of

percentage activity.

Cytotoxicity

The in vitro cytotoxicity assay (IC50) was performed on the human cervical cancer

cell line HeLa, human skin cancer cell line A431 and the NIH 3T3 mouse embryonic cell

line by MTT assay.45

The HeLa tumour cell lines used in this work were grown in Eagles

Minimum Essential Medium containing 10% fetal bovine serum (FBS) and the NIH 3T3

fibroblasts were grown in Dulbeccos Modified Eagles Medium (DMEM) containing 10%

FBS. For the screening experiments, the cells were seeded into 96 well plates in 100 mL

of the respective medium containing 10% FBS, at a plating density of 10,000 cells/well.

The cells were incubated at 37 °C in 5% CO2 and 95% air at a relative humidity of 100%

for 24 h prior to the addition of the complexes. The complexes were solubilized in

dimethylsulfoxide and diluted in the respective serum free medium. After 24 h, 100 mL

of the medium containing the test compounds with various concentrations (e.g. range of

15-500 µM for all the three complexes to HeLa and NIH 3T3 normal cell lines whereas

7.5-250 µM of complexes 4 and 5 and 3.12-100 µM concentration of complex 6 to the

A431 cell lines) was added and incubated at 37 °C in an atmosphere of 5% CO2 and 95%

air with 100% relative humidity for 48 h. All measurements were made in triplicate and

the medium containing no test complexes served as the control. After 48 h, 15 mL of

MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and

incubated at 37 °C for 4 h. The medium with MTT was then flicked off and the formazan

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crystals that had formed were solubilized in 100 mL of DMSO and the absorbance at 570

nm was measured using a micro plate reader. The % cell inhibition was determined using

the following formula,

% cell Inhibition = 100 - Abs(sample) / Abs(control) × 100.

The IC50 values were calculated from the graph plotted between % cell inhibition

and concentration.

Results and discussion

The reactions of [RuH2(CO)(PPh3)3] with the hydrazone ligands benzoic acid

(pyridine-2-yl-methylene)-hydrazide (HL1) (1), benzoic acid (1-pyridin-2-yl-ethylidene)-

hydrazide (HL2) (2) and benzoic acid (phenyl-pyridin-2-yl-methylene)-hydrazide (HL

3)

(3) yielded complexes of the type [RuH(CO)(PPh3)2(L1)] (4), [RuH(CO)(PPh3)2(L

2)] (5)

and [RuH(CO)(PPh3)2(L3)] (6) (scheme 6.1). The analytical data of the above said

complexes are in good agreement with the proposed molecular formulae with 1:1 metal

to ligand stoichiometries (given in the experimental part). All the synthesised compounds

are quite stable in air and light and soluble in most of the common organic solvents and

are well characterised using several physico-chemical techniques.

Infrared spectra

To identify the mode of coordination of the free ligands to the ruthenium ion, the

IR spectra of the metal hydrazone complexes were compared with that of the free

hydrazone ligands in the region 4000-400 cm-1

. The spectra of the free hydrazone ligands

1, 2 and 3 displayed the characteristic absorption bands in the range of 3143-3202, 1656-

1678, 1540-1586 and 1060-1075 cm-1

due to ν(N–H), ν(C=O), ν(C=N) and ν(N–N) vibrations,

respectively. The bands due to the ν(N–H) and ν(C=O) vibrations of the free ligand were

absent in the spectra of complexes 4-6 thus indicating that enolization and deprotonation

had taken place prior to coordination. This was further confirmed by the detection of two

new bands in the ranges 1598-1480 cm-1

and 1365-1369 cm-1

that are assigned to ν(C=N–

N=C) and ν(C–O) stretching vibrations, respectively. The band attributed to ν(N–N) stretching

underwent a positive shift of 17-32 cm-1

in comparison with that of the free ligand, thus

implying that coordination involves the nitrogen atom of the azomethine group. Further,

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the presence of Ru–H and Ru–CO has been identified from the absorptions found in the

range of 2023-2028 and 1952-1961 cm-1

respectively. Taken collectively, the foregoing

spectral data indicate that the hydrazone behaves as a monobasic bidentate (NO)

chelating ligand in each of the complexes 4-6.

Electronic spectra

The electronic absorption spectra of complexes 4, 5 and 6 consist of two to four

well-resolved bands in the range of 250 to 450 nm. Complex 4 exhibited absorptions at

266, 305, 376 and 441 nm. Similarly complex 5 showed its absorptions at 269, 309, 442

nm and complex 6 displayed at 265 and 428 nm. The high energy absorption bands

appeared in the spectra of respective complexes at 266, 269 and 265 nm are assigned to

π→π* intra ligand charge transfer transitions and the band that were found at 305 and

309 nm for complexes 4 and 5 can be attributed to n→π* intra ligand charge transfer

transitions of the imine group. Further, the band that was present at 376 nm for the

complex 4 was due to ligand to metal charge transfer (LMCT) transitions and the bands

that were present at 441, 442 and 428 nm for complexes 4, 5 and 6 respectively can be

assigned to metal to ligand charge transfer (MLCT) transitions.

1H NMR spectra

1H NMR spectrum of the free hydrazone ligands and their complexes were

assigned on the basis of observed chemical shift. The spectra of the ligands 1, 2 and 3

displayed a sharp singlet respectively at 14.63, 14.86 and 15.09 ppm due to enolic OH

proton. Another singlet observed at 8.59, 9.12 and 8.86 ppm are attributed to NH proton

of the respective ligands. But, the NMR spectrum of complexes 4-6 did not register any

signals corresponds to either NH or enolic OH and indicated that the ligands adopted enol

form followed by deprotonation prior to coordination with the metal ion. In addition, a

sharp singlet observed in the spectrum of ligand 1 owing to azomethine (HC=N) proton

at 7.53 ppm underwent downfield shift and aroused its peak at 8.21 ppm in the spectrum

of complexes. Further, ligand 2 showed a singlet at 2.52 ppm for (CH3C=N) which upon

complexation exhibited its peak at 2.77 ppm. The signal due to Ru–H for all the

complexes appeared as doublets of triplet at -10.09, -10.56 and -10.02 ppm respectively

202


P. Sathyadevi, 2011

that are due to coupling to the two phosphorus nuclei.46,47

The signals corresponding to

the protons of aromatic moieties of the ligands were observed as multiplets in the range

of 6-8.1 ppm.

Fig. 6.1 1H NMR spectra of complexes 4 and 5.

[RuH(CO)(PPh3)2(L2)] (5)

[RuH(CO)(PPh3)2(L1)] (4)

203


P. Sathyadevi, 2011

X-ray crystallography

From the elemental analyses, IR, electronic and 1H NMR spectroscopic studies it

is understood that both the complexes 4-6 are not structurally similar to each other.

Hence, the exact structures of the complexes have been studied by using single crystal X-

ray diffraction method.

Crystallographic study of [RuH(CO)(PPh3)2(L3)] (6)

An ORTEP representation of the structure of [RuH(CO)(PPh3)2(L3)] (6) inclusive

of the atom numbering scheme is shown in Fig. 6.3. Crystal and structure refinement data

and the bond parameters associated with the metal centers are listed in Table 6.1 and

Table 6.2. Complex 6 features the coordination of HL3 (3) ligand to the metal ion thus

forming a fused five membered chelate rings. Systematic reflection conditions, and

statistical tests of the data suggested that the crystals of 6 are triclinic with

centrosymmetric space group P-1, Z = 2 and unit cell dimensions a = 11.9691(14) Å, b =

Fig. 6.2 1H NMR spectrum of complex 6.

[RuH(CO)(PPh3)2(L3)] (6)

204


P. Sathyadevi, 2011

12.0656(14) Å, c = 17.618(2) Å; V = 2387.0(5) Å3 Dc = 1.385 Mg/m

3. Ruthenium centre

is in distorted octahedral H(CO)NOP2 coordination sphere with meridionally spanning

imine N and deprotonated amide O donor bidentate ligand and the hydrogen atom and

carbonyl molecule of the starting metal precursor form an H(CO)NO square plane around

the metal centre. The bulky PPh3 molecules from the precursor complex are trans

oriented to occupy the remaining two axial positions of the coordination pockets. As

expected, the two trans Ru–P bond lengths are much longer than the four equatorial bond

lengths indicating a large axial distortion. This lengthening is most likely due to the

strong trans effect of the PPh3 ligands. The lengthening is evidenced by two central bond

lengths Ru1–P1 [2.3736(3) Å] and Ru1–P2 [2.3469(3) Å] that are longer when compared

to that of the other four basal planar bonds Ru1–O2 [2.1011(2) Å], Ru1–N1 2.1946(3)

Å], Ru1–H1 [1.4438(2) Å], Ru1–C1 [1.8274(2) Å]. The trans angle of P1–Ru1–P2

[169.02(7)°] is not close to the ideal value of 180°, that provides further evidence for the

lengthening of axial substituent. However, the other trans angles of the basal planes

namely C1–Ru1–N1 [105.85(6)°], N1–Ru1–O2 [75.66(6)°], O2–Ru1–H1[92.47(6)°] and

H1–Ru1–C1 [86.02(6)°], are constrained by the meridional ligands that clearly indicates

Fig.6.3 Molecular structure of complex 3, with displacement ellipsoids drawn at 50% probability level.

205


P. Sathyadevi, 2011

that the coordination geometry around the ruthenium metal center is in distorted

octahedral. In addition, a molecule of ethanol was found solvated in the unit cell. Further,

there exists a hydrogen bonding between the solvated ethanol molecule and pyridine

nitrogen (N3) of the ligand moiety. The unit cell packing diagram along with hydrogen

bonding is given in Fig. 6.4.

Fig. 6.4 Unit cell packing diagram of the complex 6 with hydrogen bonding.

206


P. Sathyadevi, 2011

Table 6.1 Crystal and structure refinement data.

Description Complex 6

Formula C57.74H50.23N3O2.87P2Ru Formula weight 995.11

Temperature (K) 110(2)

(Å) 0.71073 Å

Crystal system Triclinic

Space group P-1

Cell dimensions a (Å)

b (Å)

c (Å)

(°)

(°)

(°)

11.9691(14)

12.0656(14)

17.618(2)

95.5210(10) 107.1440(10)

97.3580(10)

Z 2 hkl limits -15<=h<=15

-15<=k<=15

-22<=l<=22

Dcalcd (Mg/m3) 1.385 F(000) 1029

Crystal size (mm3) 0.10×0.05×0.03

Independent reflections 10758 [R(int) = 0.0249]

Data / restraints / parameters 10758 / 0 / 594

Goodness of fit on F2 1.067 Final R indices [I>2sigma(I)] R1 = 0.0355, wR2 = 0.0855

R indices (all data) R1 = 0.0419, wR2 = 0.0894

Table 6.2 Selected bond lengths (Ǻ) and bond angles (°).

Complex 6

Bond lengths (°) Bond angles (Å)

Ru1–H1 1.4438(2) P1–Ru1–O2 84.19

Ru1–C1 1.8274(2) H1–Ru1–O2 92.47

Ru1–O2 2.1011(2) H1–Ru1–P2 83.91

Ru1–N1 2.1946(3) P2–Ru1–C1 87.44

Ru1–P1 2.3736(3) C1–Ru1–N1 105.85

Ru1–P2 2.3469(3) N1–Ru1–P1 95.64

N1–N2 1.4047(1) P1–Ru1–P2 169.02

N2–C2 1.3110(1) C1–Ru1–O2 178.49

C2–O2 1.2872(1) H1–Ru1–N1 167.78

P1–C27 1.8268(1) C3–C2–N2–N1 177.86

P2–C45 1.8357(2) C9–N1–N2–C2 173.35

N1–C9 1.3038(1) N2–N1–C9–C15 -173.96

207


P. Sathyadevi, 2011

Biological properties

DNA binding studies

Electronic absorption measurements

The electronic absorption spectra of complexes 4, 5 and 6 consist of two to four

well-resolved bands in the range of 250 to 450 nm. Complex 4 exhibited absorptions at

266, 305, 376 and 441 nm. Similarly complex 5 showed its absorptions at 269, 309, 442

nm and complex 6 displayed at 265 and 428 nm. These bands were assigned to ILCT,

LMCT and MLCT transitions that were discussed in previous section. Upon the addition

of DNA, all the absorption bands corresponding to complex 4 showed significant

increase in its absorption intensity at the beginning followed by hypochromism together

Fig. 6.5 Electronic absorption spectra of complexes 4-6 (25 μM) in the absence and presence of increasing amounts of CT DNA (2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 and 20.0, 22.5 and 25 μM). Arrows show the changes in

absorbance with respect to an increase in the DNA concentration.

6

250 300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

Wavelength (nm)

250 300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

Wavelength (nm)

4

250 300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

Wavelength (nm)

5

208


P. Sathyadevi, 2011

with a blue shift of about 1, 1, 5 and 0 nm respectively. In the case of complex 5 and 6 we

observed the same phenomenon of hyperchromism followed by hypochromism

respectively at 269, 309, 442, 265 and 428 nm together with a blue shift of 1, 1, 0, 2 and

1 nm. These results are similar to those reported earlier for various metallointercalators48

suggesting that the complexes were bound to DNA in an intercalative mode. The

electronic absorption spectra of all the complexes with or without CT DNA are shown in

Fig. 6.5.

Competitive binding studies

Fluorescence quenching of EB-DNA complex is used to monitor the binding of

metal complexes to DNA regardless of their binding modes and only measures their

ability to influence the EB luminescence intensities in the EB-DNA complex.49

It has

been previously reported that the fluorescence intensity of EB-DNA could be decreased

by addition of the complexes as quenchers, indicating the competition between the

complexes and EB in binding to DNA that proved the intercalation of metal complexes to

the base pairs of DNA.50,51

The fluorescence emission spectra of EB bound to DNA in the absence and

presence of the three complexes are shown in Fig. 6.6. From the figure, it is clear that an

appreciable reduction in the fluorescence intensity of about 40.98, 57.21 and 88.43%

together with bathochromic shift of 1, 1 and 14 nm, respectively, was observed on

addition of Ru(II) carbonyl hydrazone complexes 4, 5 and 6 to DNA pre-treated with EB,

indicating the replacement of EB molecules accompanied by intercalation of the

complexes with DNA.52,53

The fluorescence quenching spectra of DNA-bound EB by

complexes 4, 5 and 6 illustrated that the quenching of EB bound to DNA by the test

complexes are in good agreement with the linear Stern-Volmer equation. The ratio of the

slope to the intercept obtained by plotting I0/I vs [Q] (as insets in Fig. 6.6) yielded the

value of quenching constant (Kq) corresponding to the three complexes 4, 5 and 6 as

8.9016×103

M-1

, 1.4302×104

M-1

and 1.8834×105

M-1

, respectively. From the plot of

intensity against complex concentrations furnished in Fig. 6.6, the values of the apparent

DNA binding constant (Kapp) were calculated using the equation, KEB [EB] = Kapp

[complex] and found to be 4.2061×105

M-1

, 6.5184×105

M-1

and 2.5984×106

M-1

,

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P. Sathyadevi, 2011

respectively. This study strongly supports the nature of binding of DNA with complex is

intercalation mode.54

Also, it was assessed that the complex 6 showed higher quenching efficiency than

the other complexes 4 and 5 that reflected the strong binding of complex 6 with DNA to

leach out more number of EB molecules originally bound to DNA. The binding affinity

of the complexes towards DNA increased in the order 4 < 5 < 6. The observed strong

binding efficiency of complex 6 is attributed to the presence of a phenyl ring at the

azomethine carbon that extended the delocalization of electrons in the aromatic ring than

the complexes those are either unsubstituted (complex 4) or substituted with a methyl

group in the above said position (complex 5).

5

550 600 650 700 750

0

200

400

600

800

1000

Inte

ns

ity

Wavelength (nm)

0 20 40 60 80

1.0

1.2

1.4

1.6

1.8

2.0

2.2

I 0/I

[Q] x 10-6M

4

550 600 650 700 750

0

200

400

600

800

1000

Inte

ns

ity

Wavelength (nm)

0 20 40 60 80

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

I 0/I

[Q] x 10-6 M

6

550 600 650 700 750

0

200

400

600

800

1000

Inte

ns

ity

Wavelength (nm)

0 20 40 60 80

1

2

3

4

5

6

7

8I 0

/I

[Q] x 10-6 M

Fig 6.6 Emission spectra of DNA-EB, in the presence of 0, 10, 20, 30, 40, 50, 60, 70 and 80 µM of complexes 4-6. Arrow indicates the change in the emission intensity as a function of complex concentration. Inset: Stern-Volmer

plot of the fluorescence titration data corresponding to the complexes.

210


P. Sathyadevi, 2011

Protein binding studies

Fluorescence quenching measurements

Serum albumins are proteins that are amongst others involved in the transport of

metal ions and metal complexes with drugs through the blood stream. Binding to these

proteins may lead to loss or enhancement of the biological properties of the original drug,

or provide paths for drug transportation. Hence, the binding experiments with BSA were

carried out using the newly synthesised ruthenium hydrazone complexes 4, 5 and 6.

Generally known fact is that the fluorescence of a protein is caused by three intrinsic

characteristics of the protein, namely tryptophan, tyrosine, and phenylalanine residues.

Fluorescence quenching refers to any process that decreases the fluorescence intensity of

a fluorophore due to a variety of molecular interactions including excited state reactions,

molecular rearrangements, energy transfer, ground state complex formation and collision

quenching. Qualitative analysis of ruthenium hydrazones bound to BSA has been

undertaken by examining the respective fluorescence spectra. The intensity of

fluorescence band of BSA observed at 345 nm was quenched to an extent of about 42.44,

43.90 and 51.58% from its initial intensity upon the addition of metal hydrazone chelates

4-6 together with a hypsochromic shift of 5 to 7 nm due to the formation of a ruthenium

hydrazones-BSA complex. Fig. 6.7 showed the effect of increase in the concentration of

test compounds on the emission intensity of BSA.

The fluorescence quenching is described by Stern-Volmer relation: I0/I = 1 + KSV

[Q]; where I0 and I are the fluorescence intensities of the fluorophore in the absence and

presence of quencher, respectively, KSV is the Stern-Volmer quenching constant and [Q]

is the quencher concentration. KSV value obtained from the plot of I0/I vs [Q] (as insets

in Fig. 6.7) was found to be 6.201×105

M-1

,

7.339×105

M-1

and 8.832×105

M-1

corresponding to the respective ruthenium hydrazone complexes 4, 5 and 6. The observed

linearity in the plot supported the fact that the quenching of BSA by the test complexes

are in good agreement with the linear Stern-Volmer equation. The calculated KSV values

for the test compounds exhibits their strong protein-binding ability.

211


P. Sathyadevi, 2011

UV-visible absorption measurements

UV-visible absorption measurement is a simple method to explore the structural

changes and is useful to distinguish the type of quenching exist i.e., static or dynamic

quenching. Dynamic quenching only affects the excited states of the fluorophore and

there are no changes in the absorption spectra. However, ground-state complex formation

will frequently result in perturbation of the absorption spectrum of the fluorophore. From

the absorption spectra of pure BSA and BSA-complex 4-6 shown in Fig. 6.8, it can be

said that, upon addition of ruthenium complexes to a fixed concentration of BSA led to a

gradual increase in BSA absorption, while keeping the position of the peak unchanged.

Fig 6.7 Emission spectra of BSA (1×10-6 M; λexi = 280 nm; λemi = 345 nm) as a function of concentration of the

complexes 4-6 (0, 2, 4, 6, 8 and 10×10-7 M). Arrow indicates the effect of metal complexes on the fluorescence

emission of BSA.

300 350 400 450

0

200

400

600

800

Inte

ns

ity

Wavelength (nm)

0 2 4 6 8 10

1.0

1.2

1.4

1.6

I 0/I

[Q] x 10-7 M

4

300 350 400 450

0

200

400

600

800

Inte

ns

ity

Wavelength (nm)

0 2 4 6 8 10

1.0

1.2

1.4

1.6

1.8

I 0/I

[Q] x 10-7 M

5

300 350 400 450

0

200

400

600

800

Inte

ns

ity

Wavelength (nm)

0 2 4 6 8 10

1.0

1.2

1.4

1.6

1.8

2.0

Inte

nsit

y

[Q] x 10-7 M

6

212


P. Sathyadevi, 2011

The result implies that an interaction between test compounds and BSA occurs. In other

words, the fluorescence quenching between 4-6 and BSA is mainly ascribed to be static

quenching.55

The above results can be rationalized in terms of strong interaction between

ruthenium chelates and BSA may lead to a change in the conformation of BSA.

Binding analysis

For the static quenching interaction, if it is assumed that there are similar and

independent binding sites in the biomolecule, the binding constant (Kb) and the number

of binding sites (n) can be determined according to the method described by using the

following equation56

: log [(F0-F)/F] = log [K] + n log [Q], where in the present case, K is

the binding constant for the metal hydrazones-BSA complex and n is the number of

250 275 300 325 350

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

Wavelength (nm)

BSA

BSA + Complex 5

240 260 280 300 320 340

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

Wavelength (nm)

BSA

BSA + Complex 4

250 275 300 325 350

0.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rban

ce

Wavelength (nm)

BSA

BSA + Complex 6

Fig. 6.8 Absorption spectra of BSA (1×10-5 M) and BSA-complexes 4-6 (BSA= 1×10-5 M and complexes 4-6 =

1×10-6 M).

213


P. Sathyadevi, 2011

binding sites per albumin molecule that can be determined from the slope and intercept of

the double logarithm regression curve of log [(F0-F)/F] versus log [Q] (Fig. 6.9). The

binding constants obtained from the plot corresponding to complexes 4, 5 and 6 were in

the magnitude of 1.0247×104

M-1

, 1.0726×105 M

-1 and 1.9533×10

5 M

-1, respectively. The

values of n found are 0.6906, 0.8480 and 0.9159 which indicates that there is a single

mode of binding in BSA for the ruthenium hydrazones.

The significantly high Kb value for the ruthenium hydrazone complex 6, in

comparison to that of its 4 and 5 analogue could be due to the presence of extended

delocalization of electrons by the aromatic ring ie., phenyl group substituted at the

azomethine carbon which facilitate its BSA binding propensity as observed in the DNA

binding experiments discussed in the previous section.

Synchronous fluorescence spectroscopic studies of BSA

Synchronous fluorescence spectroscopy is a very useful method to study the

microenvironment of amino acid residues by measuring the emission wavelength shift

and have several advantages such as sensitivity, spectral simplification, spectral

bandwidth reduction and avoiding different perturbing effects. Vekshin57

suggested a

useful method to study the environment of amino acid residues by measuring the possible

shift in the wavelength of emission maximum, the shift in position of emission maximum

-6.0 -6.2 -6.4 -6.6 -6.8

-0.8

-0.6

-0.4

-0.2

0.0

log

[F

0-F

/F]

log [Q]

Complex 4

Complex 5

Complex 6

Fig. 6.9 Plot of log [(F0-F)/F] vs log [Q].

214


P. Sathyadevi, 2011

280 300 320 340 360

0

50

100

150

200

250

300

Inte

ns

ity

Wavelength (nm)

6A

280 300 320 340 360

0

50

100

150

200

250

300

Inte

ns

ity

Wavelength (nm)

5A

300 320 340 360 380

0

50

100

150

200

250

300

350

Inte

ns

ity

Wavelength (nm)

5B

280 300 320 340 360

0

50

100

150

200

250

300

350

Inte

ns

ity

Wavelength (nm)

4A

300 320 340 360 380

0

50

100

150

200

250

300

Inte

nsity

Wavelength (nm)

4B

300 320 340 360 380

0

50

100

150

200

250

300

350

Inte

ns

ity

Wavelength (nm)

6B

Fig 6.10 Synchronous spectra of BSA (1×10-6 M) as a function of concentration of the complexes 4-6 (0, 2, 4, 6, 8 and 10×10-7 M) with wavelength difference of Δλ = 15 nm (A) and Δλ = 60 nm (B). Arrow indicates the change in

emission intensity w.r.t various concentration of complexes 4-6.

215


P. Sathyadevi, 2011

corresponding to the changes of the polarity around the chromophore molecule. As is

known, synchronous fluorescence spectra show tyrosine residues of BSA only at the

wavelength interval Δλ of 15 nm whereas tryptophan residues of BSA at Δλ of 60 nm.

The synchronous fluorescence spectra of both tyrosine and tryptophan residues present in

BSA were shown in Fig. 6.11 with respect to various concentrations of ruthenium

hydrazone complexes added.

After the addition of complex 4 to BSA, a slight increase in the intensity of

tyrosine residue along with a bathochromic shift of 1 nm was observed in the

fluorescence spectra. Similar changes were also observed in the case of complex 5.

However, addition of complex 6 to the BSA solution caused a very slight decrease in the

intensity of tyrosine residue but without any change in the wavelength of emission.

Regarding the effect of adding ruthenium hydrazone complexes to the BSA solution, a

significant decrease in the intensity of tryptophan fluorescence emission was observed by

the addition of all the three complexes without any change in the position of emission

wavelength. These observations do indicate that the complexes 4, 5 and 6 did not affect

the microenvironment of tyrosine residues during the binding process significantly but

the tryptophan microenvironment to a larger extent. The interaction of ruthenium

hydrazone chelates with tryptophan residue led to a decrease in the polarity of the

fluorophore by an increasing the hydrophobicity around it.

Antioxidant studies

It is well documented in the literature that transition metal hydrazone complexes

displayed significant antioxidant activity.58

Therefore, we undertook a systematic

investigation on the antioxidant potential of the newly synthesised ruthenium(II)

hydrazone organometallics 4, 5 and 6 against O2-, OH and ABTS cationic radicals as a

function of concentration of the test compounds ranging from 0 to 50 µM and the results

were shown in Fig. 6.11. IC50 values of the complexes 4-6 against superoxide radicals

were found to be 30.09, 26.57 and 23.34 μM, respectively. In the case of OH and ABTS

radical scavenging activities, the IC50 values of the above set of three complexes were in

the magnitude of 40.13, 34.88, 29.98, 28.72, 26.21 and 22.64 μM, respectively.

216


P. Sathyadevi, 2011

Results of the antioxidant activity data of bivalent ruthenium complexes against

the free radicals i.e., ABTS cationic, O2- and OH revealed that the activity decreased in

the order of 6 > 5 > 4. The superior free radical scavenging potential of complex 6 could

be related to the presence of 6 -electron delocalized phenyl ring at the azomethine

carbon than the complexes 4 and 5 that are having H or CH3 at the same position. A

similar effect due to the presence of phenyl substitution at the azomethine carbon was

also observed in DNA/BSA experiments. Out of the three radical species chosen to

examine the potential towards ABTS cationic radicals than others. The radical

scavenging ability of the new complexes were compared with that of the standard

antioxidant butylated hydroxyl anisole (BHA) in which case the titled complexes shown

better performance than the standard indicating that they have more scope to apply for in

vitro trials.

In vitro cytotoxicity

It is commonly believed that DNA is the main target of many antitumour agents59

and most of the drugs act through binding to DNA. From the DNA binding experiments

discussed elsewhere in the manuscript it can be understood that all the three ruthenium

complexes did interact with DNA. In order to determine their anticancer properties, in

vitro cytotoxicity tests were conducted utilising all the three ruthenium hydrazone

complexes against a pair of selected human tumour cell lines HeLa and A341 and NIH

3T3 normal cell lines by means of a colorimetric assay (MTT assay) that measures

mitochondrial dehydrogenase activity as an indication of cell viability after an exposure

10 20 30 40 50

20

30

40

50

60

70Hydroxyl radical

Scaven

gin

g a

cti

vit

y (

%)

Concentration ( M)

Complex 4

Complex 5

Complex 6

10 20 30 40 50

30

40

50

60

70

80

90ABTS Cationic radical

Scaven

gin

g a

cti

vit

y (

%)

Concentration ( M)

Complex 4

Complex 5

Complex 6

10 20 30 40 50

20

40

60

80Superoxide radical

Scaven

gin

g a

cti

vit

y (

%)

Concentration ( M)

Complex 4

Complex 5

Complex 6

Fig. 6.11 Trends in the inhibition of ABTS cationic, hydroxyl and superoxide radicals by the ruthenium(II)

hydrazone complexes 4, 5 and 6 at various concentrations.

217


P. Sathyadevi, 2011

period of 48 h in the respective concentration range of 15-500 µM for all the three

complexes to HeLa and NIH 3T3 normal cell lines whereas 7.5-250 µM of complexes 4

and 5 and 3.12-100 µM concentration of complex 6 to the A431 cell lines. Upon

increasing the concentration of complexes, the results of MTT assay revealed that the

complexes 4, 5 and 6 exhibited excellent cytotoxic potencies with IC50 values generally

in the low micromolar concentrations. The activities corresponding to inhibition of cancer

cell growth at maximum level were tested and figures are not presented here. In parallel,

the influence of widely used anticancer drug, cisplatin has been also assayed as

standard.60

The IC50 values of all the three complexes ranged from 18-122 μM, indicating

that all of these complexes exhibited antitumour activity against both the tumour cell

lines in different degrees (Fig. 6.12). A simple structure activity relationship (SAR)

analysis suggest that among the three types of the complexes under investigation, the

complex 6 posses phenyl substitution at azomethine carbon showed potential activity

than rest of the complexes 4 and 5 with H or CH3 groups at the same position of the

ligands respectively. Hence, we realized that the substitution at the azomethine carbon

played a vital role on the pharmacophore of the metal chelates and hence, the cytotoxic

activities of the present ruthenium complexes are mainly governed by those ligands that

are highly cytotoxic themselves, while complexation to metal ions rather serves to

modulate their mode of action and activity. These investigations clearly said that the

449

122

54

382

109

48

347

94

18

0

50

100

150

200

250

300

350

400

450

IC50 V

alu

es (

M)

Complex 4 Complex 5 Complex 6

NIH 3T3

HeLa

A431

Fig. 6.12 Comparison of IC50 values between complexes 4, 5 and 6 on the inhibition of NIH 3T3, HeLa and A431 cell lines.

218


P. Sathyadevi, 2011

complexes in particular are effective against A341 cells than HeLa cell lines under

identical experimental conditions without causing significant damage to the normal NIH

3T3 cells. As evident from the IC50 values, it was also observed that the activity of

cisplatin overcomes the tested complexes 4 and 5 to some extent but the complex 6

showed a comparable activity to cisplatin under in vitro conditions.

Conclusion

Three new bivalent ruthenium carbonyl complexes with hydrazone ligands HL1,

HL2 and HL

3 have been synthesized and well characterized in detailed by elemental

analysis and spectral techniques (UV-visible, IR and 1H NMR). The molecular structure

of one of the complexes 6 investigated through X-ray crystallography demonstrated an

octahedral geometry around the metal ion. All the newly synthesised complexes have

been subjected to examine their biological property like DNA binding, protein binding,

antioxidant and cytotoxicity under in vitro experimental conditions. The DNA binding

ability of the above complexes assessed by absorption and emission spectra suggested an

intercalative mode of binding with different binding affinities. Results of BSA binding

experiments revealed that the quenching mechanism found between the protein and tested

ruthenium hydrazone complexes is a static type. The synchronous fluorescence spectral

measurements confirmed the occurrence of conformational changes at tryptophan micro

environment of BSA. The antioxidant activity showed that all the metal complexes can

serve as potential antioxidants. The results of cytotoxicity experiments revealed that the

complexes synthesised in this work possess moderate activity against both the of HeLa

and A341 cell lines with a preference to inhibit the proliferation of later thereby proved

that the selected compounds could serve as promising candidates in antitumour

applications. At this juncture, it is notable to mention that the major chemical and

biological findings of this study throw some light on the potential of these complexes in a

reasonable range of concentrations under in vitro conditions. In our opinion, the

significant outcome of the present investigation regarding the abilities of ruthenium

organometallic hydrazone complexes towards various biological evaluations is that the

substitution of the phenyl ring at the azomethine carbon of the ligand led to an increased

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interaction with biomolecules such as DNA/BSA, free radicals and tumour cell lines than

the rest of the complexes without such a phenyl ring in that position.

220


P. Sathyadevi, 2011

Reference

1. K. Kalyansundaram and M. Gratzel, Coord. Chem. Rev., 1998, 177, 347-414.

2. E.Y. Li, Y.-M. Cheng, C.-C. Hsu, P.-T. Chou, G.-S. Lee, I.-H. Lin, Y. Chi and C.-S. Liu, Inorg. Chem., 2006, 45, 8041-8051.

3. M. Katual and G. Dutt, Talanta, 1972, 22, 151-166.

4. B. Singh, R. Shrivastava and K.K. Narang, Synth. React. Inorg. Met. Org. Chem., 2000, 30, 1175-1192.

5. C.M. Amstrong, P.V. Bernhardt, P. Chin and D.R. Richardson, Eur. J. Inorg.

Chem., 2003, 1145-1156.

6. A. Basoglu, S. Parlayan, M. Ocak, H. Alp, H. Kantekin, M. Ozdemir and U. Ocak, Polyhedron, 2009, 28, 1115-1120.

7. A.A.R. Despaigne, J.G. Da Silva, A.C.M. Do Carmo, O.E. Piro, E.E. Castellano and H. Beraldo, J. Mol. Struct., 2009, 920, 97-102.

8. S. Pal, J. Pushparaju, N.J. Sangeetha and S. Pal, Trans. Met. Chem., 2000, 25, 529-

533.

9. A. Wood, W. Aris and D.J.R. Brook, Inorg. Chem., 2004, 43, 8355-8360.

10. V. Sadus and M. Angelica, Trans. Met. Chem., 1995, 20, 256-261.

11. M. Mohan, M.P. Gupta and L. Chandra, Inorg. Chim. Acta, 1988, 151, 61-68.

12. E. Mihkelson, J. Inorg. Nucl. Chem., 1981, 43, 127-136.

13. C. Mirco, P. Paolo, P. Corrado and R. Dominga, J. Mol. Catal. A Chem., 2002, 178, 21-26.

14. W. Kaim, B. Schewederski, O. Heilmann and F.M. Hornung, Coord. Chem. Rev., 1999, 182, 323-342.

15. W. Kaim and B. Schwederski, Bioinorganic Chemistry, Wiley, Chichester, 1994.

16. B. Lippert, BioMetals, 1992, 5, 195-208.

17. D.M. Whitfield, S. Stoijkowski and B. Sarkar, Coord. Chem. Rev., 1993, 122, 171-

225.

18. Y. Kim, Y. Kang and D. Baek, Bull. Korean Chem. Soc., 2001, 22, 141-144.

19. H. Beraldo, W.F. Nacif and D.X. West, Spectrochim. Acta A, 2001, 57, 1847-1854.

20. J.R. Dimmock, R.N. Puthucode, J.M. Smith, M. Hetherington, J.W. Quail and U. Pugazhenthi, J. Med. Chem., 1996, 39, 3984-3997.

21. J.R. Dimmock, S.N. Pandeya, J.W. Quail, U. Pugazhenthi, T.M. Allen and G.I. Kao, Eur. J. Med. Chem., 1995, 30, 287-301.

22. J.R. Dimmock, S.N. Pandeya, J.W. Quail, U. Pugazhenthi, T.M. Allen and G.I.

Kao, Eur. J. Med. Chem., 1995, 30, 303-314.

221


P. Sathyadevi, 2011

23. K.E. Erkkila, D.T. Odom and J.K. Barton, Chem. Rev., 1999, 99, 2777-2795.

24. J.K. Barton and A.L. Raphael, J. Am. Chem. Soc., 1984, 106, 2466-2468.

25. A. Chouai, S.E. Wicke, C. Turro, J. Bacsa, K.R. Dunbar, D. Wang and R.P. Thummel, Inorg. Chem., 2005, 44, 5996-6003.

26. J. Hooda, D. Bednarski, L. Irish and S.M. Firestine, Bioorg. Med. Chem., 2006, 14,

1902-1909.

27. F. Liang, P. Wang, X. Zhou, T. Li, Z.Y. Li, H.K. Lin, D.Z. Gao, C.Y. Zheng and C.T. Wu, Bioorg. Med. Chem. Lett., 2004, 14, 1901-1904.

28. J.M. Kelly, A.B. Tossi, D.J. McConnel and C. O. Huigin, Nucleic Acids Res., 1985, 13, 6017-6034.

29. V. Uma, V.G. Vaidyanathan and B.U. Nair, Bull. Chem. Soc. Jpn., 2005, 78, 845-

850.

30. W. Szczepanik, J. Ciesiolka, J. Wrzesinski, J. Skala and M. Jezowska-Bojczuk, Dalton Trans., 2003, 1488-1494.

31. Y.P. Wang, Y.L. Wei and C. Dong, J. Photochem. Photobiol. A Chem., 2006, 177, 6-11.

32. J.N. Tian, J.Q. Liu, Z.D. Hu and X.G. Chen, Bioorg. Med. Chem., 2005, 13, 4124-

4129.

33. L.B. Qu, X.L. Chen, R. Yang, L. Wang and H.J. Zeng, Chin. J. Chem., 2007, 25, 1151-1155.

34. Y.J. Hu, H.G. Yu, J.X. Dong, X. Yang and Y. Liu, Spectrochim. Acta A, 2006, 65, 988-992.

35. X.M. He and D.C. Carter, Nature, 1992, 358, 209-215.

36. G.M. Sheldrick, Acta Cryst., 2008, A64, 112-122.

37. L.J. Barbour, J. Supramol. Chem., 2001, 1, 189-191.

38. APEX2 “Program for Data Collection on Area Detectors” BRUKER AXS Inc.,

5465, East Cheryl Parkway, Madison, WI 53711-5373, USA.

39. G.M. Sheldrick, SADABS, Program for Absorption Correction of Area Detector

Frames, BRUKER AXS Inc., 5465, East Cheryl Parkway, Madison, WI 53711-5373, USA.

40. R.A.S. Pelgado, W.Y. Lee, S.R. Choi and Y.Cho, M.J.Jun, Trans. Met. Chem.,

1991, 16, 241-244.

41. P. Krishnamoorthy, P. Sathyadevi, K. Senthil Kumar, P. Thomas Muthiah, R. Ramesh and N. Dharmaraj, Inorg. Chem. Comm., 2011, 14, 1318-1322.

42. C. Beauchamp and I. Fridovich, Anal. Biochem., 1971, 44, 276-287.

43. T. Nash, Biochem. J, 1953, 55, 416-421.

222


P. Sathyadevi, 2011

44. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans, Free Radic. Bio. Med., 1999, 26, 1231-1237.

45. M. Blagosklonny and W.S. EI-diery, Int. J. Cancer, 1996, 67, 386-392.

46. J.D.E.T. Wilton-Ely, Ming Wang, Sanaz J. Honarkhah, Derek A and Tocher, Inorg.

Chim. Acta, 2005, 358, 3218-3226.

47. J.G. Małecki and R. Kruszynski, Polyhedron, 2010, 29, 1023-1028.

48. P. Sathyadevi, P. Krishnamoorthy, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvanesh and N. Dharmaraj, Dalton Trans., 2011, DOI: 10.1039/C1DT10767D.

49. W.J. Mei, J. Liu, H. Chao and L.N. Ji, Trans. Met. Chem., 2003, 28, 852-857.

50. G.Y. Bai, K.Z. Wang, Z.M. Duan and L.H. Gao, J. Inorg. Biochem., 2004, 98, 1017-1022.

51. S.A. Tysoe, R. Kopelman and D. Schelzig, Inorg. Chem., 1999, 38, 5196-5197.

52. H. Mansuri-Torshizi, R. Mital, T.S. Srivastava, H. Parekh and M.P. Chitnis, J.

Inorg. Biochem., 1991, 44, 239-247.

53. E.J. Gao, X.M. Zhao, Q.T. Liu and R. Xiu, Acta Chim. Sin., 2004, 62, 593-597.

54. V. Rajendiran, M. Murali,E. Suresh, S. Sinha, K. Somasundaramc and M. Palaniandavar, Dalton Trans., 2008, 148-163.

55. D. Senthil Raja, G. Paramaguru, N.S.P. Bhuvanesh, J.H. Reibenspies, R. Renganathan and K. Natarajan, Dalton Trans., 2011, 4548-4559.

56. X.Z. Feng, Z. Yang, L.J. Wang and C. Bai, Talanta, 1998, 47, 1223-1229.

57. N.L. Vekshin, Biophysics, 1996, 1176-1182.

58. Y. Li, Z.Y. Yang and M.F. Wang, Eur. J. Med. Chem., 2009, 44, 4585-4595.

59. F. Gao, H. Chao, F. Zhou, Y.X. Yuan, B. Peng and L.N. Ji, J. Inorg. Biochem.,

2006, 100, 1487-1494.

60. P. Krishnamoorthy, P. Sathyadevi, A.H. Cowley, R.R. Butorac and N. Dharmaraj, Eur. J. Med. Chem., 2011, 46, 3376-3387.

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